peptides... Discovery C18, C8, RP-AmideC16

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1 US $ PLC Column Selection: Small Molecules (<000 mw) PLC: Small Molecules Column Selection This chart is meant to be a starting point for choosing an PLC column. In some cases, more than one column can be used for an analysis. Although general rules apply, you should first consult the technical literature, official methodology, or our Technical Service chemists for definitive recommendations. For column choices for peptides, proteins, and other biopolymers, see pages -. AALYTE CARACTERISTICS TECIQUE AALYTE CLASS CLUM GEERAL (p -7.)... Discovery C8, C8, RP-AmideC6; Discovery Cyano, Discovery S C8, S F, S PEG GEERAL (p -)... SUPELCGEL DP-0, TPR-00 acids, organic... Discovery C8, C8, RP-AmideC6 acids, amino... Discovery C8, RP-AmideC6; SUPELCSIL LC-DABS amines, organic... Discovery C8, RP-AmideC6; Discovery Cyano, Discovery S F; SUPELCGEL TPR-00 amines, quaternary (p>7.)... SUPELCGEL TPR-00, DP-0 chelating compounds... Discovery C8, C8, RP-AmideC6; SUPELCGEL TPR-00, DP-0 drugs, basic... Discovery C8, C8, RP-AmideC6; Discovery Cyano, Discovery S F reversed drugs in serum... isep; Discovery C8, RP-AmideC6, C8; Discovery S F phase explosives... Discovery C8, C8, RP-AmideC6; Discovery Cyano nucleosides... SUPELCSIL LC-8-S; Discovery C8, RP-AmideC6 nucleotides... SUPELCSIL LC-8-T; Discovery C8, RP-AmideC6 peptides... Discovery C8, C8, RP-AmideC6 nonionic sugars... SUPELCSIL LC- taxol, taxanes... SUPELCSIL LC-F tricyclic antidepressants... SUPELCSIL LC-PC; Discovery C8; Discovery Cyano vitamins, water soluble... Discovery C8, C8, RP-AmideC6 water hydrophobic soluble interaction peptides... TSK-GEL size oligosaccharides... SUPELCGEL Ag, Ag; TSK-GEL ligo-pw exclusion polymers/oligomers, hydrophilic... TSK-GEL PW XL organic soluble ion anions, organic (- charge)... SUPELCSIL SAX exchange cations, organic (+ charge)... SUPELCSIL LC-SCX ionic ion pairing acids, organic... Discovery C8, Discovery S C8 amines, organic... Discovery C8, Discovery S C8 ion acids, organic... SUPELCGEL, C60 exclusion carbohydrates, sugars... SUPELCGEL C6, K, Pb, Ag, Ag, Ca normal phase reversed phase size exclusion GEERAL... Discovery Cyano, LC- -P, LC-Si, Discovery S PEG aflatoxins... SUPELCSIL LC-Si steroids... SUPELCSIL LC-Diol vitamins, fat soluble... SUPELCSIL LC- -P fatty acids... SUPELCSIL LC-8 polyaromatic hydrocarbons... SUPELCSIL LC-PA polymers, hydrophobic... SUPELCSIL LC-8 triglycerides... SUPELCSIL LC-8 vitamins, fat soluble... Discovery C8, C8, RP-AmideC6, SUPELCSIL LC-8 polymers/oligomers, hydrophilic... TSK-GEL R rder: Technical Service: Web: Liquid Chromatography SUPELC

2 Liquid Chromatography PLC: Small Molecules Discovery Reversed-Phases Comprehensive Suite of Alkyl and Functionalized Phases S C8 C8 C Si C C Si (C ) 7 C (C ) 7 C Valuable, Different Separations versus C8 US $ While C8 s from different sources can provide significant differences in retention and selectivity, these differences are frequently small and not sufficient to produce really valuable, improved separations. The Discovery suite of reversed-phases is designed to be complimentary to C8 by combining polar functionality with the standard alkyl/hydrophobic phase. The result It s much more likely to achieve an improved, valuable separation with a polar functionalized reversed-phase than with another C8. C SUPELC rder: Technical Service: Web: RP-AmideC6 C Si (C ) C-(C ) C C C8 C Si (C ) 7 C C S F F F C Si (C ) F C F F Cyano C Si (C ) C C S PEG = n Ideal for all small molecule PLC applications Designed to meet the exacting requirements of pharmaceutical analysis and purification, Discovery columns are also ideal for all other application segments requiring reversed-phase PLC, including: - food and beverage - agriculture - environmental - consumer products - clinical - industrial chemical - petrochemical - and more. Characteristics of Discovery Reversed-Phases Coverage Pore Diameter Surface Area Phase µmole/m %C Endcapped Angstroms m /g S C8.8 0% yes 0 00 C8.0 % yes RP-AmideC6.6 % yes C8. 7.% yes S F.0 % yes 0 00 Cyano..% yes S PEG.8 % no 0 00 n the next page, begin to rediscover method development...

3 US $ Take Advantage of the Discovery Suite of Reversed-Phases Whether you re developing a separation that must be perfect, or you re just not satisfied with your reversed-phase separation... Turn to Discovery! a suite of PLC columns featuring functionalized reversed-phases designed to provide differentiated separations vs. C8 based on unique combinations of polar and hydrophobic retention. The Discovery suite of reversed-phases enables you to optimize your separation..., e.g. - retention - selectivity - resolution - analysis time...while minimizing method development time. ydrophobicity: Discovery Reversed-Phases S PEG Cyano S F C8 RP-AmideC6 C8 S C k Butylbenzene (0:60 water:c C) PLC: Small Molecules Rediscover Method Development Discovery RP-AmideC6 Delivers Better Peak Spacing and Faster Analysis! Scientists have long known polar embedded phases such as Discovery RP- AmideC6 provide unique retention and selectivity compared to C8 s. Taking advantage of Discovery RP-AmideC6 s unique retention and selectivity results in a better separation vs. C8. - Unique selectivity - peak reversal of compounds and - Better peak spacing - less dead space between peaks and - igher assay throughput - 0% reduction in analysis time - Improved resolution of critical peak pair and Column: cm x.6mm ID, µm particles Mobile Phase: 90:0, mm Potassium Phosphate (p.0): Me Flow Rate:.0mL/min Temp.: 0 C Det.: UV at nm Inj.: 0µL Discovery C G000 Discovery RP-AmideC G000 Sample:. izatidine. Ranitidine. Famotidine. Cimetidine rder: Technical Service: Web: Liquid Chromatography SUPELC

4 6 Liquid Chromatography SUPELC rder: Technical Service: Web: PLC: Small Molecules Rediscover Method Development Deliver Better Separations in Less Time Unique retention and selectivity of Discovery S F enables rapid development of simple impurity assay where C8 fails! Impurity methods requiring retention and resolution of vastly differing analytes may not be suitably obtained using simple C8-based systems. By simply changing the stationary phase the method development scientist can avoid: - complicated or forbidden gradients - complex mobile phases - long, drawn-out method development n Discovery S F, it took just a few hours to develop an excellent separation. -Aminopyridine Piroxicam S C C -Aminopyridine (-AMP) is Unretained on C8 Under Mobile Phase Conditions Used to Assay Piroxicam Column: Discovery C8 cm x.6mm ID, µm particles Mobile Phase: :, 0 mm Potassium Phosphate (p.): C C (v/v) Flow Rate:.0mL/min Det.: UV at 0nm Inj.: µl Sample:. -Aminopyridine (0µg/ml in 90:0 buffer:c C). Piroxicam (00µg/ml in 90:0 buffer:c C). -Aminopyridine. Piroxicam 0 US $ Decreasing the % Acetonitrile Results in Excessive Piroxicam Retention and -AMP is Still Unretained C8 Fails Same buffer but with lower organic: 8:, 0 mm Potassium Phosphate (p.): C C (v/v) Increasing p to 6.8 Retains the -AMP but Piroxicam Retention is Still Excessive C8 Fails Same %organic, but changing the p to 6.8: 8:, 0 mm Potassium Phosphate (p 6.8): C C (v/v) The Unique Retention and Selectivity of Discovery S F Produces Excellent Separation at Both p s F Delivers Excellent Separation! 8:, 0 mm Potassium Phosphate (p.): C C (v/v) 8:, 0 mm Potassium Phosphate (p 6.8): C C (v/v)

5 PLC: Small Molecules Rediscover Method Development 7 Different Retention is a General and Valuable Characteristic of Functionalized Reversed-Phases Unique retention vs. C8 Retention of several basic drugs is compared on C8, RP-AmideC6 and F. (F mobile phase %organic is +0% vs. C8 and RP-AmideC6.) - Both F and RP-AmideC6 deliver valuable, different retention compared to C8. - F is significantly more retentive for most compounds tested vs. both C8 and RP- AmideC6 - Retention on functionalized reversedphases is almost always different than retention on C8! TE: k ratios not equal to.0 mean different retention. Retention on RP-AmideC6 and F Relative to C8 k X/k C8 k C8/k RP-Amide C Procainamide izatidine orfluoxetine. Acetaminophen. Methyl Paraben. Ethyl Paraben. aproxen. Ketoprofen 6. Propyl Paraben 7. Butyl Paraben 8. Quinidine 6 7 Fluoxetine 8 ydrochlorothiazide Quinidine Retention on C8 Relative to RP-AmideC6 Diphenhydramine Amitriptyline RP-AmideC6 S F Quinine 0. Desacetyldiltiazem. Clonazepam. Cimetidine. Diltiazem. Strychnine. izatidine 6. Clorazepate 7. Ranitidine 8. Desmethyldiltiazem 9. Famotodine 0. Codeine. Diazepam. Papaverine. oscapine rder: Technical Service: Web: Liquid Chromatography SUPELC

6 8 PLC: Small Molecules Rediscover Method Development Liquid Chromatography SUPELC rder: Technical Service: Web: Different Selectivity is a General and Valuable Characteristic of Functionalized Reversed-Phases Key to interpreting results When a color aligns, the selectivity is similar. When a color does not align, the selectivity is different. Aniline Phenol Toluene,-Dimethylaniline Ethylbenzoate Ethylbenzene Mobile phase: : mm Potassium Phosphate (p 7.0):Me (All columns except S PEG run at 7: mm Potassium Phosphate (p 7.0):Me). Flow Rate:.0mL/min Similar Phases (C8 s and C8 s) - Similar Selectivities The near-perfect alignment of the colors clearly illustrates the similar selectivities of the C8 and C8 phases tested k compared to k toluene Unique, Functionalized Phases - Different Selectivities k compared to k toluene S C8 The functional group containing solutes - Aniline, Phenol,,-Dimethylaniline (, DMA) and Ethylbenzoate - clearly illustrates the very different selectivities of the functionalized reversed-phases vs. C8. bserve the colors representing solutes containing polar groups dramatically change positions from phase to phase. Also observe the changing hydropbobic selectivity by looking at the Ethylbenzene bar. Both polar and hydrophobic selectivities are different on the different phases. C8 C8 S C8 RP-AmideC6 S F S PEG

7 PLC: Small Molecules Rediscover Method Development 9 Tips for Getting Started: Good Method Development Practices Tip ne: Column Selector Valve Automated PLC + Column Selector Valve - While screening of functionalized reversedphases can be done with a simple, manual PLC system, an automated, multi-solvent system with programmable, temperature controlled column selector valve is highly recommended. Liquid Chromatography Tip Two: Simple Column Screening Guidelines for Rapid Screening of Functionalized Reversed-Phases - Step : Scout for best mobile phase on C8 - Step : Initial screening runs - Chromatograph sample on Discovery RP-AmideC6 and F using best C8 mobile phase - Chromatograph sample on PEG using 0% lower organic than best C8 mobile phase - Step : Evaluate screening runs - Retention K? If no, adjust % organic and rerun (ote: F sometimes requires stronger mobile phase than C8) -Step : ptimize separation on most promising or columns using standard reversedphase mobile phase adjustment techniques Tip Three: Always screen several functionalized reversed-phases and a C8. Tip Four: ptimize your separation on the or most promising phases! Scout Evaluate ptimize Screen rder: Technical Service: Web: SUPELC

8 0 PLC: Small Molecules Alkyl Reversed-Phases Liquid Chromatography Discovery C8, C8, S C8... C8 and C8 - Excellent performance - Similar selectivities Barbiturates Column: cm x.6mm columns, µm particles Mobile Phase: : Water:Me Flow Rate:.0mL/min Det.: UV at nm Temp.: ambient Inj.: µl (Discovery C8) or 0µL (Discovery C8) Sample: µg/ml of each. Barbital. Aprobarbital. Phenobarbital. Butabarbital. Mephobarbital 6. Pentobarbital 7. Secobarbital - C8 generally more retentive than C8 Discovery C8 Discovery C8 SUPELC rder: Technical Service: Web: Surface Area: (m /g) Pore Size: (Å) Coverage: (µmoles/m ) Endcapped %C C Yes 7.% C Yes % S C Yes 0% G00090 Anticonvulsants Column: cm x.6mm columns, µm particles Mobile Phase: 70:0 Water:C C Flow Rate:.0mL/min Det.: UV at nm Temp.: 0 C Inj.: 0µL G Alhanoic / Aryloxyalhanoic Acid Using Isocratic Elution Column: cm x.6mm columns, µm particles Mobile Phase: 60:0 mm Potassium Phosphate (p.):c C Flow Rate:.0mL/min Det.: UV at nm Temp.: 0 C Inj.: µl Discovery C8 Discovery C G G Sample:. Clonazepam. Clorazepate. Diazepam Discovery C G00078 Discovery C8 Sample:. Solvent. Dalapon.,-D.,-DB.,,-T 6.,-D Methyl ester 7.,,-T Methyl ester 8.,-DB Methyl ester G000 8

9 PLC: Small Molecules Alkyl Reversed-Phases...Excellent Chromatography for a Wide Range of Compounds S C8 and C8 - C8 most general, st choice - Similar selectivities - S C8 generally more retentive than C8 - S C8 is more acidic Mobile phase additive can improve chromatography of bases Conditions for Discovery C8 - no additive Column: cm x.6mm columns µm particles Mobile Phase: : mm Ammonium Phosphate (p7.0):c C Flow Rate:.0mL/min Temp.: 0 C Det.: UV at nm Inj.: 0µL Sample: 00µg/mL of each Conditions for Discovery S C8 - TEA added Column: cm x.6mm columns µm particles Mobile Phase: : mm TEA phosphate (p7):c C Flow Rate:.0mL/min Temp.: 0 C Det.: UV at nm Inj.: 0µL Sample: 00µg/mL of each rganic Acids Column: cm x.6mm columns Mobile Phase: 60:0 0.%TFA in Water:Me Flow Rate:.0mL/min Temp.: 0 C Det.: UV at nm Inj.: 0µL 0 Antibiotics (Fluoroquinolones from Tablets) Column: cm x.6mm columns Mobile Phase: (A) mm Potassium Phosphate (p.0) Discovery C8 (B) C C Flow Rate:.0mL/min Temp.: C Det.: UV at 0nm Inj.: 0µL G00...S C8 is More Acidic... Discovery C8 6 Discovery C8 no additive G00 Sample:. ordoxepin. Protriptyline/Desipramine. ortriptyline G0066 Sample:. omovanillic acid (0.06µg/mL). Sorbic acid (0.006µg/mL). Salicylic acid (0.06µg/mL). p-toluic acid (0.006µg/mL) Discovery S C8 0 Gradient: %A %B Discovery S C G00. Doxepin. Imipramine 6. Amitriptyline 7. Trimipramine Discovery S C8 - TEA added 6 6 G00 Sample:. Levofloxacin. Ciprofloxacin. Lomefloxacin. Sparfloxacin. Grepafloxacin 6. Trovafloxacin G0066 rder: Technical Service: Web: Liquid Chromatography SUPELC

10 PLC: Small Molecules Discovery S F Liquid Chromatography SUPELC rder: Technical Service: Web: Unique Retention and Selectivity Enables Better Separations C Si C - Excellent performance - Unique selectivity - Similar retention to C8 (sometimes requires stronger mobile phase strength than C8) Surface Area: (m /g) (C ) Pore Size: (Å) Coverage: (µmoles/m ) Endcapped %C S F Yes F F F F F Excellent Retention of Multifunctional Compounds The Discovery S F shows greater retention, versus C8, of the multifunctional compounds shown in these chromatograms Compounds that elute too closely to the void volume (peak ) on C8 columns are sufficiently retained by Discovery S F. Column: Discovery S F and Conventional C8, cm x.6mm, µm particles Mobile Phase: (A) 0mM ammonium acetate, 0.% formic acid; (B) Me Flow Rate:.mL/min Temp.: C Det.: UV at nm Inj.: 0µL Discovery S F: Good Retention G006 F Provides Excellent Separation - Solutes Are ot Retained on C8 Column: Discovery S C8, cm x.6mm, µm particles Mobile Phase: 0:70 0mM Ammonium Acetate (p=6.98): C C Flow Rate:.0mL/min Temp.: C Det.: Photodiode Array Inj.: µl 0, Gradient: %A %B Conventional C8 Phase: Elutes at void volume Discovery S C8 Column: Discovery S F, cm x.6mm Conditions same as Discovery S C8 above Discovery S F Sample:. p-aminophenol (00µg/mL). Acetaminophen (0µg/mL). Acetanilide (0µg/mL). Phenacetin (0µg/mL) G007 Sample:. Methcathinone (00µg/mL). (+/-) Ephedrine (00µg/mL) G0067 G0067

11 PLC: Small Molecules Discovery S F F, a unique, functionalized reversed-phase uncovers a trace impurity missed by C8. Pure Quinidine was assayed on C8 under a variety of mobile phase conditions. Conditions C and D produced a single peak suggesting the Quinidine was pure. The peak resulting from condition B might be showing a partially resolved front shoulder. A quick screen of % organic was unable to resolve the possible impurity. n F (chromatogram A) the impurity is clearly resolved. During method development a quick screen using unique, functionalized reversedphases such as F greatly increases the chances of finding trace impurities early, before they can cause big problems. elpful int Routinely screen separations on several, complimentary functionalized reversed-phases (e.g. F, PEG, RP-AmideC6) early in method development. At lower organic, Discovery S F exhibits reversed-phase behavior. At higher organic it exhibits normal phase behavior. At high % organic retention actually increases as % organic increases for some analytes. Benefits include: - Improve LC/MS detection by using higher % organic mobile phase. - Use mobile phase selectivity to develop valuable, different separations at high % organic Retention Time F Resolves Trace Impurity in Quinidine C8 Does ot! Column: Discovery S F and Conventional C8, cm x.6mm, µm particles Mobile Phase: mm Ammonium Phosphate (p 7.0):C C. Varying Ratios: (A) :6, (B) 70:0, (C) 76:, (D) 80:0 Flow Rate:.0mL/min Temp.: 0 C Det.: UV at nm Inj.: 0µL (A) Discovery S F G00666 G00667,, (C) C G00668 (D) C8 (B) C G00669 F Exhibits U Shaped Retention Creating Interesting Possibilities F - Retention vs. % rganic Partially resolved front shoulder % C C methcathinone ephedrine Sample:. Quinidine (0µg/mL). Impurity rder: Technical Service: Web: Liquid Chromatography SUPELC

12 PLC: Small Molecules Discovery RP-Amide C6 Liquid Chromatography SUPELC rder: Technical Service: Web: Unique Retention and Selectivity Enables Better Separations - Excellent performance - Unique selectivity - Similar retention to C8 (typically requires similar mobile phase strength as C8) Surface Area: (m /g) Pore Size: (Å) Endcapped Coverage: (µmoles/m ) %C RP-AmideC Yes % Discovery RP-AmideC6 Gives Better Resolution and Faster Analysis - faster analysis from lower hydrophobicity - better peak spacing (RP-AmideC6) - better resolution of small impurities (RP-AmideC6) Column: cm x.6mm, µm particles Mobile Phase: 80:0 mm Potassium Phosphate (p.0):me Flow Rate:.0mL/min Temp.: C Det.: UV at nm Inj.: 0µL Discovery C Discovery RP-AmideC6 G G Sample:. Codeine. Strychnine. Quinidine. Quinine. oscapine 6. Papaverine 6

13 PLC: Small Molecules Discovery RP-Amide C6 Faster analysis and different selectivity. Antitussive/Antihistamine/Antipyretic Mix Dramatic differences in peak order and run time are demonstrated by different Discovery reversed-phase stationary phases. Column: cm x.6mm, µm particles Mobile Phase: (A) mm Potassium Phosphate (p.) (B) C C Flow Rate:.0mL/min Temp.: ambient Det.: UV at nm Inj.: 0µL Gradient: %A %B Sample: (µg/ml of each). Acetaminophen. Doxylamine. Pseudoephedrine. Codeine. Chlorpheniramine Liquid Chromatography But... different selectivity is not always better! ere C8 provides a better separation. Discovery RP-AmideC6 Barbiturates Elution rder Changes! G00078 G Different Discovery column chemistries show different separations of barbiturates under identical conditions. Column: cm x.6mm columns, µm particles Mobile Phase: : Water:Me Flow Rate:.0mL/min Temp.: ambient Det.: UV at nm Inj.: 0µL Discovery RP-AmideC6, Sometimes C8 is better! G0009 Discovery C8 Discovery C8 Sample: (µg/ml of each). Barbital. Aprobarbital. Phenobarbital. Butabarbital. Mephobarbital 6. Pentobarbital 7. Secobarbital G00090 rder: Technical Service: Web: SUPELC

14 6 PLC: Small Molecules Discovery Cyano Liquid Chromatography Unique Retention and Selectivity Enables Better Separations C Si C - Excellent performance - Unique selectivity (C ) C Faster Analysis - Eliminate Wasted Time Urea Pesticides Using Isocratic Elution Column: cm x.6mm columns, µm particles Mobile Phase: 60:0 Water:C C Flow Rate:.0mL/min Temp.: 0 C, Det.: UV at nm Inj.: µl Discovery C8 Discovery Cyano Sample:. Fenuron. Monuron. Diuron. Linuron SUPELC rder: Technical Service: Web: - Significantly less retentive than C8 (therefore typically requires greater % mobile phase) Surface Area: (m /g) Pore Size: (Å) Coverage: (µmoles/m ) Endcapped %C Cyano Yes G00070 Faster Analysis - Different Selectivity rganophosphorous Pesticides Using Isocratic Elution Column: Discovery C8, cm x.6mm, µm particles Mobile Phase: 0:70 Water:Me Flow Rate:.0mL/min Temp.: 0 C Det.: UV at nm Inj.: µl Column: Discovery Cyano, cm x.6mm, µm particles Mobile Phase: 7: Water:C C Flow Rate:.0mL/min Temp.: 0 C Det.: UV at nm Inj.: µl Discovery C G0007 Discovery Cyano G Sample:. Dichlorvos. Guthion. Methyl parathion. Ethoprophos. Disulfoton 6. Fenchlorvos 7. Chlorpyrifos 8. Prothiophos G0000

15 PLC: Small Molecules Discovery S PEG 7 Unique Retention and Selectivity Enables Better Separations = - Excellent performance - Unique selectivity - Significantly less retentive than C8 (therefore typically requires greater % mobile phase) Surface Area: (m /g) Pore Size: (Å) Coverage: (µmoles/m ) Endcapped %C S PEG o n Flavones Demonstrate Dramatic Selectivity Differences Between S PEG and C8 Column: cm x.6mm columns, µm particles Mobile Phase: : 0.% Formic Acid in Water : 0.% Formic Acid in Me Flow Rate:.0mL/min Temp.: 0 C, Det.: UV at nm Inj.: 0µL Discovery S C8 6, G0066 Chlorzoxazone - Excellent Separation on PEG; Excessive Retention and Resolution on S C8 Column: cm x.6mm columns, µm particles Mobile Phase: 70:0 0mM Acetic Acid in Water (p. with Ammonium ydroxide):c C Flow Rate:.0mL/min Temp.: 0 C, Det.: UV at 8 nm Inj.: 0µL Column: cm x.6mm columns, µm particles Mobile Phase: 7: 0mM Acetic Acid in Water (p. with Ammonium ydroxide):c C Flow Rate:.0mL/min Temp.: 0 C, Det.: UV at 8 nm Inj.: 0µL 8 Discovery S PEG 6 8 Sample: 0µg/mL of each. Myricetin. Quercetin. Luteolin. Baicalein. 7-ydroxyflavone 6. Flavone 7. Chrysin 8. -ydroxyflavone G G G0067 Sample: 00µg/mL of each. 6-ydroxychlorzoxazone. Chlorzoxazone Discovery S PEG Sample: 00µg/mL of each. 6-ydroxychlorzoxazone. Chlorzoxazone Discovery S C8 rder: Technical Service: Web: Liquid Chromatography SUPELC

16 8 PLC: Small Molecules Discovery C8 and Discovery S C8 Columns Discovery C8 Discovery S C8 Liquid Chromatography SUPELC rder: Technical Service: Web: LEGT X ID PARTICLE SIZE (cm x mm) CAT.. PRICE DISCVERY C8 (80Å, % CARB) µm x. 097 µm 0 x. 690-U µm. x. 699-U µm x. 09 µm x µm 0 x.0 69-U µm. x U µm x µm x µm x µm 0 x.0 69-U µm. x.0 69-U µm x µm x µm x µm 0 x.6 69-U µm. x.6 69-U µm x.6 09 µm x µm x U µm x. 696-U DISCVERY C8 VALIDATI PACKS* µm x. 700-U µm 0 x U µm x. 70-U µm x U µm 0 x U µm x.6 70-U µm x.6 70-U DISCVERY C8 SUPELGUARD CARTRIDGES µm x. (/pk) 088 µm x. kit ** 06 µm x.0 (/pk) 976-U µm x.0 kit ** 97-U µm x.0 (/pk) 07 µm x.0 kit ** 09 * Packs include columns, each from a different lot of bonded phase. ** Kits include one cartridge, a stand-alone holder, a piece of tubing, nuts and ferrules. DISCVERY C8 PRPERTIES: Bonded Phase: ctadecylsilane, endcapped Silica: Spherical, high purity (Fe <0; A <7; Ca <7; Ti <; Al <; Mg <ppm) Particle Size: µm Pore Size: 80Å Surface Area: 00m²/g %C: ~% Coverage: ~µmoles/m² LEGT X ID PARTICLE SIZE (cm x mm) CAT.. PRICE DISCVERY S C8 (0Å, 0% CARB) µm x. 69-U µm 7. x. 69-U µm x. 69-U µm x U µm 7. x.6 69-U µm x.6 69-U µm x U µm 0 x. 680-U µm x. 680-U µm x. 680-U µm x U µm 0 x.0 68-U µm x.0 68-U µm x.0 68-U µm x U µm 0 x.6 68-U µm x.6 68-U µm x.6 68-U µm x U µm 0 x U µm x U µm x U µm x. 680-U µm 0 x. 68-U µm x. 68-U µm x. 68-U 0µm x U 0µm 0 x U 0µm x U 0µm x U 0µm x U 0µm 0 x. 686-U 0µm x. 686-U 0µm x. 686-U DISCVERY S C8 SUPELGUARD CARTRIDGES µm x. (/pk) 6976-U µm x. kit ** 6977-U µm x.0 (/pk) 697-U µm x.0 kit ** 697-U µm x. (/pk) 6870-U µm x. kit ** 687-U µm x.0 (/pk) 687-U µm x.0 kit ** 687-U µm x U 0µm x U ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. DISCVERY S C8 PRPERTIES: Bonded Phase: ctadecylsilane, endcapped Silica: Spherical, high purity (<0 ppm metals) Particle Size:,, 0µm Pore Size: 0Å Surface Area: 00m²/g %C: ~0% Coverage: ~.8µmoles/m²

17 PLC: Small Molecules Discovery RP-AmideC6 and Discovery S F Columns 9 Discovery RP-AmideC6 Discovery S F LEGT X ID PARTICLE SIZE (cm x mm) CAT.. PRICE DISCVERY RP-AMIDEC6 (80Å, % CARB) µm x. 000 µm 0 x. 690-U µm. x. 699-U µm x. 00 µm x µm 0 x.0 69-U µm. x U µm x µm x µm x µm 0 x.0 69-U µm. x.0 69-U µm x µm x µm x µm 0 x.6 69-U µm. x.6 69-U µm x.6 00 µm x µm x U µm x. 696-U DISCVERY RP-AMIDEC6 VALIDATI PACKS * µm x. 70-U µm 0 x U µm x. 707-U µm x.6 70-U µm 0 x U µm x U µm x U DISCVERY RP-AMIDEC6 SUPELGUARD CARTRIDGES µm x. (/pk) 00 µm x. kit ** 00 µm x.0 (/pk) 978-U µm x.0 kit ** 977-U µm x.0 (/pk) 0099 µm x.0 kit ** 0080 * Packs include columns, each from a different lot of bonded phase. ** Kits include one cartridge, a stand-alone holder, a piece of tubing, nuts and ferrules. DISCVERY RP-AMIDEC6 PRPERTIES: Bonded Phase: Palmitamidopropylsilane, endcapped Silica: Spherical, high purity (Fe <0; A <7; Ca <7; Ti <; Al <; Mg <ppm) Particle Size: µm Pore Size: 80Å Surface Area: 00m²/g %C: ~% Coverage: ~.6µmoles/m² LEGT X ID PARTICLE SIZE (cm x mm) CAT.. PRICE DISCVERY S F (0Å, % CARB) µm x U µm 0 x. 670-U µm x. 670-U µm x U µm 0 x.0 67-U µm x.0 67-U µm x U µm 0 x U µm x U µm x U µm 0 x. 670-U µm x. 67-U µm x. 67-U µm x.0 67-U µm 0 x.0 67-U µm x.0 67-U µm x U µm x.6 67-U µm 0 x.6 67-U µm x U µm x U µm x U µm 0 x U µm x U µm x U µm x. 67-U µm 0 x. 679-U µm x. 67-U µm x. 67-U 0µm x U 0µm 0 x U 0µm x U 0µm x U 0µm x. 677-U 0µm 0 x. 670-U 0µm x. 678-U 0µm x. 679-U DISCVERY S F SUPELGUARD CARTRIDGES µm x. (/pk) 6770-U µm x. kit ** 677-U µm x.0 (/pk) 677-U µm x.0 kit ** 677-U µm x. (/pk) 677-U µm x. kit ** 677-U µm x.0 (/pk) 6776-U µm x.0 kit ** 6777-U µm x U 0µm x U ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. DISCVERY S F PRPERTIES: Bonded Phase: Pentafluorophenylpropylsilane, endcapped Silica: Spherical, high purity (<0 ppm metals) Particle Size:,, 0µm Pore Size: 0Å Surface Area: 00m²/g %C: ~% Coverage: ~µmoles/m² rder: Technical Service: Web: Liquid Chromatography SUPELC

18 0 PLC: Small Molecules Discovery S PEG and Discovery C8 Columns Discovery S PEG Discovery C8 Liquid Chromatography SUPELC rder: Technical Service: Web: LEGT X ID PARTICLE SIZE (cm x mm) CAT.. PRICE DISCVERY S PEG (0Å, % CARB) µm x U µm 0 x. 670-U µm x. 670-U µm x U µm 0 x.0 67-U µm x.0 67-U µm x U µm 0 x U µm x U µm x U µm 0 x. 670-U µm x. 67-U µm x. 67-U µm x.0 67-U µm 0 x.0 67-U µm x.0 67-U µm x U µm x.6 67-U µm 0 x.6 67-U µm x U µm x U µm x U µm 0 x U µm x U µm x U µm x. 67-U µm 0 x. 679-U µm x. 67-U µm x. 67-U 0µm x U 0µm 0 x U 0µm x U 0µm x U 0µm x. 677-U 0µm 0 x. 670-U 0µm x. 678-U 0µm x. 679-U DISCVERY S PEG SUPELGUARD CARTRIDGES µm x. (/pk) 6770-U µm x. kit ** 677-U µm x.0 (/pk) 677-U µm x.0 kit ** 677-U µm x. (/pk) 677-U µm x. kit ** 677-U µm x.0 (/pk) 6776-U µm x.0 kit ** 6777-U µm x U 0µm x U ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. DISCVERY S PEG PRPERTIES: Bonded Phase: Polyethyleneglycol, endcapped Silica: Spherical, high purity (<0 ppm metals) Particle Size:,, 0µm Pore Size: 0Å Surface Area: 00m²/g %C: ~% Coverage: ~.8µmoles/m² LEGT X ID PARTICLE SIZE (cm x mm) CAT.. PRICE DISCVERY C8 (80Å, 7.% CARB) µm x. 9-U µm 0 x. 690-U µm. x. 69-U µm x. 9-U µm x.0 9-U0 µm 0 x.0 69-U µm. x.0 69-U µm x.0 9-U0 µm x.0 9-U0 µm x.0 9-U0 µm 0 x.0 69-U µm. x U µm x.0 9-U0 µm x.0 9-U0 µm x.6 9-U µm 0 x.6 69-U µm. x U µm x.6 9-U µm x.6 9-U DISCVERY C8 VALIDATI PACKS * µm x. 70-U µm 0 x U µm x. 7-U µm x.6 70-U µm 0 x U µm x.6 7-U µm x.6 7-U DISCVERY C8 SUPELGUARD CARTRIDGES µm x. (/pk) 988-U µm x. kit** 987-U µm x.0 (/pk) 980-U µm x.0 kit** 979-U µm x.0 (/pk) 990-U µm x.0 kit** 989-U * Packs include columns, each from a different lot of bonded phase. ** Kits include one cartridge, a stand-alone holder, a piece of tubing, nuts and ferrules. DISCVERY C8 PRPERTIES: Bonded Phase: ctylsilane, endcapped Silica: Spherical, high purity (Fe <0; A <7; Ca <7; Ti <; Al <; Mg <ppm) Particle Size: µm Pore Size: 80Å Surface Area: 00m²/g %C: ~7.% Coverage: ~.µmoles/m²

19 PLC: Small Molecules Discovery Cyano Columns, Discovery Selectivity Packs Discovery Cyano LEGT X ID PARTICLE SIZE (cm x mm) CAT.. PRICE DISCVERY CYA (80Å,.% CARB) µm x. 9-U µm 0 x. 69-U µm. x. 69-U µm x. 96-U µm x.0 9-U0 µm 0 x.0 69-U µm. x.0 69-U µm x.0 96-U0 µm x.0 97-U0 µm x.0 9-U0 µm 0 x.0 69-U µm. x U µm x.0 96-U0 µm x.0 97-U0 µm x.6 9-U µm 0 x U µm. x U µm x.6 96-U µm x.6 97-U DISCVERY CYA VALIDATI PACKS* µm x. 7-U µm 0 x U µm x. 77-U µm x.6 7-U µm 0 x U µm x.6 77-U µm x.6 79-U DISCVERY CYA SUPELGUARD CARTRIDGES µm x. (/pk) 98-U µm x. kit** 98-U µm x.0 (/pk) 697-U µm x.0 kit** 6970-U µm x.0 (/pk) 986-U µm x.0 kit** 98-U * Packs include columns, each from a different lot of bonded phase. ** Kits include one cartridge, a stand-alone holder, a piece of tubing, nuts and ferrules. DISCVERY CYA PRPERTIES: Bonded Phase: Cyanopropylsilane, endcapped Silica: Spherical, high purity (Fe <0; A <7; Ca <7; Ti <; Al <; Mg <ppm) Particle Size: µm Pore Size: 80Å Surface Area: 00m²/g %C: ~.% Coverage: ~.µmoles/m² Discovery Selectivity Packs You can conveniently order the four Discovery column chemistries RP-AmideC6, C8, C8, and Cyano in your choice of column dimensions, in a single kit. Quickly evaluate mobile phases on all four columns to find the optimum combination of chemistries for your separation. The Discovery PLC Column Selectivity Pack gives you a powerful tool for rapid, efficient, simple pharmaceutical method development. LEGT X ID PARTICLE SIZE (cm x mm) CAT.. PRICE µm x. 70-U µm 0 x. 698-U µm x. 7-U µm x.0 70-U0 µm 0 x U µm x.0 7-U0 µm x.0 7-U0 µm x.0 70-U0 µm 0 x U µm x.0 7-U0 µm x.0 7-U0 µm x.6 70-U µm 0 x U µm x.6 7-U µm x.6 7-U rder: Technical Service: Web: Liquid Chromatography SUPELC

20 PLC: Small Molecules Special Purpose SUPELCSIL Columns Liquid Chromatography SUPELC rder: Technical Service: Web: Special Purpose Columns: Amino Acids SUPELCSIL LC-DABS columns feature a specially treated PRPERTIES Particles: spherical silica, µm and tested octadecylsilane bonded phase, for reversed-phase Pore Size: 0Å Bonded Phase: octadecylsilane separations of precolumn derivatized dabsyl amino acids. More Surface Area: 70m²/g than 0 amino acids and ammonia can be separated in less than Pore Volume: 0.6mL/g one hour. p Range: - 7. Amino Acid -p-ser -p-thr D -p-tyr C E C C Q S Met-Sox T G A R Met-Son P V M I L W F C K Y o-phosphoserine o-phosphothreonine Aspartic acid o-phosphotyrosine Cysteic acid Glutamic acid S-Carboxymethylcysteine S-Sulfocysteine Asparagine Glutamine Serine Methionine sulfoxide Threonine Glycine Alanine Arginine Methionine sulfone Proline Valine Methionine Isoleucine Leucine Tryptophan Phenylalanine Ammonia Cystine Lysine istidine Tyrosine Dabsyl Amino Acids Column: SUPELCSIL LC-DABS cm x.6mm ID, µm particles Cat. o.: 97 Mobile Phase: A = mm K P, p7.0 B = acetonitrile:methanol (70:0) Flow Rate:.mL/min Det.: VIS, 6nm Inj.: µl, approx. 0pM each derivative Gradient Program Temp. (min) % B p-ser -p-thr -p-tyr C D LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC-DABS (0Å,.% CARB) µm x.6 97 SUPELCSIL LC-8-T Supelguard Cartridges (use for LC-DABS) µm x.0 (/pk) 96 µm x.0 kit ** ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. E C C Q Met- Sox S T Met-Son G A R P V M RELATED IFRMATI Literature References Stocchi, V., et al., Anal. Biochem.78: 07-7 (989). Stocchi, V., et al., Amino Acids: 0-09 (99). References not available from Supelco. I L W F C K Y 7-0

21 PLC: Small Molecules Special Purpose SUPELCSIL Columns Special Purpose Columns: Basic Columns SUPELCSIL LC-8-DB andsupelcsil LC-8-DB phases are deactivated (DB) for basic compounds. These columns provide shorter retention, better peak shape, and higher efficiency for organic bases than can be obtained on conventional reversed-phase columns. DB columns have become standards in the industry for analyses of difficult basic compounds, particularly in the pharmaceutical industry. An example of the performance of the LC-8-DB column versus that of a conventional C8 column is shown. The deactivated column allows the use of a simpler mobile phase to elute trimethoprim with good peak shape. To obtain similar results from the LC-8 column would require the addition of competing base to the mobile phase PRPERTIES Silica: Spherical Particle size: µm and µm Pore size: 0Å Pore Volume: 0.6mL/g Surface Area: 70m /g p Range: -7 Improved Peak Symmetry for Antibacterials Conventional Column SUPELCSIL LC-8 ELPFUL ITS 0. Amoxicillin. Cephalexin. Trimethoprim. Ampicillin Column:.cm x.6mm, µm Cat. o.: 8977 (LC-8), 8978 (LC-8-DB) Mobile Phase: methanlo:0mm K P, p.0 (:77) Flow Rate: ml/min Det.: UV, nm Inj.: 0µL,,, and 06µg/mL 6 Deactivated Column SUPELCSIL LC-8-DB Relative Strengths for Different Solvents The graph provides for the interconversion of reversed-phase mobile phases having the same strength. Vertical lines in this figure intersect mobile phases having the same strength. For example, 0% Acetonitrile has the same strength as 0% Methanol or 0% TF. ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules , 709 LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC-8-DB (0A,.0% CARB) µm x. 79 µm. x C0 µm 7. x.0 899C0 µm x.0 899C0 µm x.0 899C0 µm. x µm 7. x µm x µm 0 x µm x. 790 µm x.0 8C0 µm 0 x.0 908C0 µm x.0 88C0 µm x.0 8C0 µm x.0 88C0 µm x.0 8C0 µm 0 x.0 96 µm x.6 8 µm 0 x µm x.6 88 µm x.6 8-U µm x SUPELCSIL LC-8-DB Supelguard Cartridges µm x. (/pk) 967 µm x. kit ** 966 µm x.0 (/pk) 96C0 µm x.0 (/pk) 96 µm x.0 kit ** 9 SUPELCSIL LC-8-DB (0Å, 6.0% CARB) µm 7. x C0 µm x.0 899C0 µm x.0 899C0 µm. x µm 7. x U µm x µm x. 79 µm x.0 87C0 µm x.0 87C0 µm x.0 8C0 µm x.6 8 µm x.6 87 µm x.6 8 µm x SUPELCSIL LC-8-DB Supelguard Cartridges µm x. (/pk) 969 µm x. kit ** 968 µm x.0 (/pk) 96 µm x.0 kit ** 9 AC/ Me/ AC = Acetonitrile Me = Methanol TF = Tetrahydrofuran TF/ rder: Technical Service: Web: Liquid Chromatography SUPELC

22 PLC: Small Molecules Special Purpose SUPELCSIL Columns Liquid Chromatography SUPELC rder: Technical Service: Web: Special Purpose Columns: Carbohydrates Within the different classes of sugars, chemical and physical and sugar alcohols. As with resins containing a single cation, properties vary only slightly. PLC separations of carbohydrates di-,tri-, and oligosaccharides are separated by class. Galactose and depend on differences in conformation, configuration, and mannose are well separated. column type. Because of this complexity, no single PLC columnsupelcgel Ag columns provide rapid separations of or method is capable of separating every carbohydrate. To choose oligosaccharides. Glycerol and ethanol are well resolved. the best column for your carbohydrate analysis, consult Table (below), the Retention Time Index (next page), and the Applications section of this catalog. For more information, request Bulletin 887 (PLC Carbohydrate Column Selection Guide). SUPELCGEL K columns separate raffinose, sucrose, glucose, fructose, and betaine, a trimethylammonium zwitterionic compound found in beet and cane sugars and widely distributed in other plants. The lead-form resin insupelcgel Pb columns provides the highest resolution and best selectivity for monosaccharides. SUPELCGEL Pb columns provide excellent separation of xylose, galactose, and mannose, which are not completely resolved on calcium-form resin columns. SUPELCGEL Ca columns separate monosaccharides and sugar alcohols. Di-, tri-, and oligosaccharides are separated by class. A frequent application for this column is the separation of sugars in high fructose corn syrup (FCS). SUPELCGEL C-60 and columns are ideal for separating carbohydrates, alcohols, and organic acids present in the same sample: wines and other fermentation products, fruit juices, biological samples, etc. SUPELCGEL C-6 columns contain a unique ion exchange resin containing two divalent cations, rather than one. This provides different selectivities for separating monosaccharides Table. Carbohydrate Column Applications and Mobile Phases Silica-basedSUPELCSIL LC- columns separate monosaccharides, disaccharides, and some trisaccharides, with elution generally in order of increasing molecular weight. Retention decreases as the proportion of water:acetonitrile in the mobile phase is increased. For more information, request T976 (Sugars on SUPELCSIL LC- Columns). SUPELCGEL CARBYDRATE CLUM CARACTERISTICS Particles: sulfonated polystyrene/divinylbenzene, spherical, 9µm Counter Ion: varies (see Table ) p Range: - rganic Compatibility: <0% in mobile phase Maximum Temperature: varies (see Table ) Maximum Flow Rate: 7.8mm ID columns:.ml/min.6mm ID columns: 0.mL/min Maximum Pressure: 000psi (70 bar) SUPELCSIL LC- CLUM CARACTERISTICS Particles: spherical silica, µm Bonded Phase: aminopropylsilyl p Range: -7. rganic Compatibility: no limits (avoid aldehydes and ketones) Maximum Flow Rate: ml/min (.6mm ID columns) Maximum Pressure: 6000psi (0 bar) TYPICAL MAX. TEMP. CLUM APPLICATI FRM MBILE PASE ( C) SUPELCGEL K beet sugar, cane sugar, potassium 0mM P K 90 molasses, corn syrup SUPELCGEL Pb monosaccharides, lead deionized water 90 xylose/galactose/mannose SUPELCGEL Ca high fructose corn syrup, calcium deionized water 90 monosaccharides, sugar alcohols, oligosaccharides SUPELCGEL C-60 organic acids hydrogen 0.% S or P 60 SUPELCGEL organic acids hydrogen 0.% S or P 90 SUPELCGEL C-6 mono-, di-, and trisaccharides, divalent cations 0 a 8 galactose/mannose SUPELCGEL Ag beer, dark corn syrup silver deionized water 90 SUPELCGEL Ag oligosaccharides, glycerol/ethanol, silver deionized water 90 corn syrup, hydrolyzed starch SUPELCSIL LC- mono-, di-, some trisaccharides aminopropyl silica 7% C C in water 70 See Applications pages.

23 PLC: Small Molecules Special Purpose SUPELCSIL Columns Table. Retention Time Index for Carbohydrate Columns SUPELCGEL CLUMS SUPELCSIL Ca C-60 Pb C-6 K Ag LC- Cat. o.: 90-U 90-U 90-U U Dimensions (mm): 00 x x x x.6 00 x x x x x.6 Temp.: 80 C 0 C 0 C 0 C 8 C 60 C 8 C 8 C ambient Mobile Phase : D 0.% P 0.% P 0.% P D 0 a mm K P D AC: D (:) Flow Rate (ml/min): Det.: refractive index Compound Retention Times (min) Arabinose Arabitol Betaine D D D D R D.0 D D Dulcitol Erythritol Ethanol 9..6 D D D.0 D 8. R Fructose Galactose Glucose Glycerol R Inositol D Isomaltose D D D.8 D.6 9. Isomaltotriose D D D.6 D 9.8 R Lactitol D D D 0.6 D D Lactose Maltitol Maltoheptaose R Maltohexaose R Maltopentaose R Maltose Maltotetraose R Maltotriose Mannitol Mannose Melezitose Psicose Raffinose Ribitol D Ribose Sorbitol Stachyose Sucrose Xylitol Xylose R - not recommended D - no data available For optimal separations, allow at least minute between compounds. For resins used in processing sugars and foods, refer to the low pressure LC media section. SUPELCGEL and SUPELCSIL Carbohydrate Columns and Guard Columns LEGT ID SUPELGUARD CLUM (cm) (mm) CAT.. PRICE GUARD CLUM CAT.. PRICE SUPELCGEL K K* 9 SUPELCGEL Pb Pb* 9 SUPELCGEL Ca U Ca* 906-U SUPELCGEL C U * 99 SUPELCGEL U * 99 SUPELCGEL.6 96 * 99 SUPELCGEL C U Ca* 906-U SUPELCGEL Ag U Ag* 97-U SUPELCGEL Ag Ag* 96 SUPELCSIL LC LC-, (kit)** 98 LC- ( cartridges) 968 * cm x.6mm guard column, does not include tubing, nuts or ferrules. ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. rder: Technical Service: Web: Liquid Chromatography SUPELC

24 6 PLC: Small Molecules Special Purpose SUPELCSIL Columns Liquid Chromatography SUPELC rder: Technical Service: Web: CLUM CARACTERISTICS Particles: µm spherical silica Bonded Phase: aminopropylsilyl Pore Size: 0Å Surface Area: 70m /g Pore Volume: 0.6mL/g p Range: α-tocopherol. β-tocopherol. γ-tocopherol. δ-tocopherol Figure A. Tocopherols Column: SUPELCSIL LC- -P, cm x.6mm ID, µm particles Cat. o.: 9 Mobile Phase: hexane:ethyl acetate, 70:0 Flow Rate:.0mL/min Temp.: 0 C Det.: UV, 9nm Inj.: 0µL hexane,.0mg/ml each analyte 0 0 Figure B. Vitamin K Isomers Column: SUPELCSIL LC- -P, cm x.6mm ID, µm particles Cat. o.: 9 Mobile Phase: hexane:ethyl acetate, 99: Flow Rate:.mL/min Temp.: 0 C Det.: UV, nm Inj.: 0µL hexane, 0.mg/mL each analyte ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules.. cis-k. trans-k. K. K Special Purpose Columns: Dedicated ormal Phase SUPELCSIL LC- -P is an amino phase dedicated to normal phase chromatography. By employing special bonding technology, and avoiding water in manufacturing and testing the column, we have dramatically reduced the retention variation that is characteristic of normal phase chromatography. SUPELCSIL LC- -P columns: show stable retention in normal phase separations are less sensitive to small or varying amounts of water in the mobile phase, relative to unmodified silica provide excellent separations of fat-soluble vitamins ormal phase chromatography is especially useful when the analytes are not water soluble for example, the fat-soluble vitamins A, D, E, and K. Figure A shows a separation of tocopherols (vitamin E) on a SUPELCSIL LC- -P column. The various isomers of vitamin K are separated in Figure B. These columns should be used with non-aqueous mobile phases only. LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC- -P (0Å,.0% CARB) µm x.6 9 SUPELCSIL LC- -P Supelguard Cartridges µm x.0 (/pk) 96 µm x.0 kit ** 9 ELPFUL ITS Reversed-phase versus ormal Phase Reversed-phase - is characterized by strong interactions between analytes and the polar mobile phase. Interactions between analytes and the nonpolar stationary phase are weak. Mobile phases typically consist of water / organic solvent combinations. Reversed-phase columns include: Amide-C6, C8, C8, Phenyl, C, Pentafluorinated Phenyl (F), Cyano, C, DP-0, and TPR-00. ormal phase is characterized by strong interactions between analytes and the polar stationary phase. Interactions between analytes and the nonpolar mobile phase are weak. Mobile phases consist of organic solvents. ormal phase columns include: Cyano,, and Silica.

25 PLC: Small Molecules Special Purpose SUPELCSIL Columns 7 Special Purpose Columns: Direct Serum Injection Figure A. isep Columns Perform The isep column uses a shielded hydrophobic phase (SP) for Consistently for Many Injections its mechanism of separation. The silica-based material is covered with a thin polymer consisting of hydrophobic regions in a hydrophilic network. Small analytes, such as drugs, penetrate the hydrophilic network and are retained by the hydrophobic moieties. The hydrophilic network shields protein molecules from Third 0µL After a Total Serum Injection of 0mL Serum contact with the surface and the hydrophobic groups, and thus these molecules are not retained. Direct injection of biological samples eliminates time-consuming cleanup steps and increases analytical accuracy. Excellent column stability and an ability to exclude proteins over a wide p range set isep columns apart from other direct serum injection columns and techniques. Stability is demonstrated in Figure A no significant change is seen in the chromatography of trimethoprim after injecting 0mL of serum, in 0µL increments, onto a isep column. A low p application is shown in Figure B. The profile of the excluded serum peak will change with p due to differing protonation states of the serum proteins. Trimethoprim Trimethoprim PRPERTIES Particles: spherical silica, µm Pore Size: 0Å Surface Area: 70m²/g Pore Volume: 0.6mL/g p range: -7. SUPELCSIL isep LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL ISEP (0Å) µm x. 79 µm x.6 9 µm x.6 89 µm x SUPELCSIL isep Supelguard Cartridges µm x.0 (/pk) 960-U µm x.0 kit ** 969 RELATED IFRMATI Request free literature by phone or fax, or see our website. o. Subject T97 isep columns for drugs Literature References Feibush, B., C. Santasania, J. Chromatogr.: -9 (99). Gisch, D.J., B.T. unter, B. Feibush, J. Chromatogr.: 6-68(988). Wong, S..Y., L.A. Bretts, A.C. Larson, J. Liq. Chromatogr.: (988). References not available from Supelco. ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. Column: isep, cm x.6mm ID, µm particles Cat. o.: 89 Mobile Phase: acetonitrile:80mm ammonium acetate (:8), p 7 Flow Rate: ml/min Det.: UV, nm Inj.: 0µL spiked serum (µg/ml trimethoprim) Figure B. isep Columns Are Compatible with Low p Mobile Phases Phenytoin Column: isep, cm x.6mm ID, µm particles Cat. o.: 89 Mobile Phase: acetonitrile:80mm ammonium acetate (p.6) Flow Rate: ml/min Det.: UV, nm Inj.: µl phenytoin-spiked serum rder: Technical Service: Web: Liquid Chromatography SUPELC

26 8 PLC: Small Molecules Special Purpose SUPELCSIL Columns Liquid Chromatography Gradient Program Time (min) % B aphthalene. Acenaphthylene. Acenaphthene. Fluorene. Phenanthrene 6. Anthracene 7. Fluoranthene 8. Pyrene 9. Benzo(a)anthracene 0. Chrysene. Benzo(b)fluoranthene. Benzo(k)fluoranthene. Benzo(a)pyrene. Dibenzo(a,h)anthracene. Benzo(ghi)perylene 6. Indeno(,,-cd)pyrene 6 Special Purpose Columns: Polyaromatic ydrocarbons SUPELCSIL LC-PA columns were designed specifically for analyses of the priority pollutant PAs listed in US EPA Method 60 (Figure A)..mm and.0mm columns save solvent and improve sensitivity when sample mass is limited. µm columns provide extremely rapid, highly efficient analyses (Figure B), while retaining the durability of porous silicas. They are excellent and economical substitutes for.µm nonporous silicas. CLUM CARACTERISTICS Particles: spherical silica, µm, µm Pore Size: 0Å Bonded Phase: octadecylsilane Surface Area: 70m²/g Pore Volume: 0.6mL/g p Range: - 7. SUPELC rder: Technical Service: Web: Figure A. ptimum PA Resolution: µm Particles Column: SUPELCSIL LC-PA, cm x.6mm ID, µm particles Cat. o.: 89 Mobile Phase: A = water, B = acetonitrile Flow Rate:.mL/min Det.: UV, nm Inj.: µl LC-PA Test Mix (Cat. o. 87), diluted :0 with acetonitrile Gradient Program Time (min) % B aphthalene. Acenaphthylene. Acenaphthene. Fluorene. Phenanthrene 6. Anthracene 7. Fluoranthene 8. Pyrene Figure B. Rapid Analyses: µm Particles Column: SUPELCSIL LC-PA, cm x.6mm ID, µm particles Cat. o.: 9 Mobile Phase: A = water, B = acetonitrile Flow Rate:.0mL/min Det.: UV, nm Benz(a)anthracene 0. Chrysene. Benzo(b)fluoranthene. Benzo(k)fluoranthene. Benzo(a)pyrene. Dibenz(a,h)anthracene. Benzo(ghi)perylene 6. Indeno (,,-cd)pyrene ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC-PA (0Å) µm x.0 9C0 µm 0 x.0 9C0 µm x.6 9 µm 0 x.6 9 µm x. 79 µm x.0 88C0 µm x.6 88 µm x.6 89 SUPELCSIL LC-8 Supelguard Cartridges (use for LC-PA) µm x. (/pk) 96 µm x. kit ** 96 µm x.0 (/pk) 96C0 µm x.0 (/pk) 96 µm x.0 kit ** 9 ELPFUL ITS Reversed-phase versus ormal Phase Reversed-phase - is characterized by strong interactions between analytes and the polar mobile phase. Interactions between analytes and the nonpolar stationary phase are weak. Mobile phases typically consist of water / organic solvent combinations. Reversed-phase columns include: Amide-C6, C8, C8, Phenyl, C, Pentafluorinated Phenyl (F), Cyano, C, DP-0, and TPR-00. ormal phase is characterized by strong interactions between analytes and the polar stationary phase. Interactions between analytes and the nonpolar mobile phase are weak. Mobile phases consist of organic solvents. ormal phase columns include: Cyano,, and Silica.

27 PLC: Small Molecules Special Purpose SUPELCGEL Columns 9 Special Purpose Columns: ucleosides SUPELCSIL LC-8-S columns are designed for reliable separations of deoxyribonucleosides and ribonucleosides. Each column is tested to ensure performance. PRPERTIES Silica: Spherical Particle Size: µm Pore Size: 0Å Bonded Phase: octadecylsilane Surface Area: 70m /g Pore Volume: 0.6mL/g p Range: -7. C dc U m dc hm U G dg du dt l dl m dc m da '-da 0 0 Figure provided by Dr. C. W. Gehrke and K.C. Kuo, University of Missouri- Columbia, Experimental Station Chemical Laboratories, Columbia, M USA Deoxyribonucleosides and Ribonucleosides on a SUPELCSIL LC-8-S Column Column: SUPELCSIL LC-8-S, cm x.6mm ID, µm particles Cat. o.: 89 Mobile Phase: 0mM K P /K P (p.0): methanol A = 97.:., B = 80:0 Flow Rate:.0mL/min Temp.: 0 C Det.: UV, nm Inj.: nucleoside standards in water LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC-8-S (0A,.0% CARB) µm 0 x µm x. 799 µm x.0 89C0 µm x.6 89 µm x U SUPELCSIL LC-8-S Supelguard Cartridges µm x.0 (/pk) 960 µm x.0 kit ** 969 ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. A da Gradient Program Time (min) % A ATP m 6 da 7-09 Special Purpose Columns: ucleotides SUPELCSIL LC-8-T columns feature an octadecylsilane bonded phase and a special surface treatment, for efficient separations of nucleotides. Each batch of packing material is tested to ensure good peak shape for a representative nucleotide, adenosine diphosphate (ADP). Chromatography of other compounds that exhibit metal chelating properties also can be improved by using this phase. PRPERTIES Silica: Spherical Particle Size: µm, µm Pore Size: 0Å Bonded Phase: octadecylsilane Surface Area: 70m /g Pore Volume: 0.6mL/g p Range: -7. ADP AMP ADP AD 8 Gradient Program Time (min) % B ucleotides on a SUPELCSIL LC-8-T Column Column: SUPELCSIL LC-8-T, cm x.6mm ID (µm particles) Cat. o. 897 Mobile Phase: A = 0.M K P, p 6 B = A:methanol, 90:0 gradient program shown on figure Flow Rate:.mL/min Det: UV, nm LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC-8-T (0Å,.% CARB) µm x C0 µm x U µm x.0 897C0 µm x SUPELCSIL LC-8-T Supelguard Cartridges µm x.0 (/pk) 96C0 µm x.0 (/pk) 96 µm x.0 kit ** 960 rder: Technical Service: Web: Liquid Chromatography SUPELC

28 0 PLC: Small Molecules Special Purpose SUPELCGEL Columns Liquid Chromatography SUPELC rder: Technical Service: Web: Special Purpose Columns: rganic Acids SUPELCGEL C-60 columns are prepared specifically for analyses of organic acids. Acetic, propionic, butyric, formic, malic, citric, succinic, lactic, and other acids are easily separated on these columns, using a simple isocratic mobile phase and minimal sample preparation. Retention times for many acids are shown in the table. Separation is based on ion exclusion the analytes selectively partition between the resin phase and the external aqueous phase. Analyses are best performed at low p (0.% P often is used as the mobile phase); the same mobile phase conditions can be applied to a wide variety of sample matrices. CLUM CARACTERISTICS Particles: sulfonated polystyrene/divinylbenzene, spherical, 9µm Counter Ion: + p Range: - rganic Compatibility: <0% in mobile phase Maximum Flow Rate:.mL/min Maximum Pressure: 000psi (70 bar) SUPELCGEL columns have the same particle composition, retention mechanism, performance, sensitivity, and applications as SUPELCGEL C-60 columns. owever, particle improvements have made it possible to pack the SUPELCGEL packing material efficiently into conventional.6mm ID columns, to improve detection and reduce solvent consumption relative to 7.8mm ID columns. CLUM CARACTERISTICS Particles: sulfonated polystyrene/divinylbenzene, spherical, 9µm Counter Ion: + p Range: - rganic Compatibility: <0% in mobile phase Maximum Flow Rate:.mL/min (7.8mm), 0.mL/min (.6mm) Maximum Pressure: 000psi (70 bar) LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCGEL C-60 9µm 0 x U SUPELCGEL Guard Column (use to protect C-60) 9µm x.6 * 99 SUPELCGEL 9µm x µm 0 x U SUPELCGEL Guard Column 9µm x.6 * 99 * cm x.6mm guard column does not include tubing, nuts or ferrules. rganic Acids. xalic. Citric. Tartaric. Malic. Succinic 6. Formic 7. Acetic 8. Fumaric Column: SUPELCGEL C-60, 0cm x 7.8mm ID Cat. o.: 90 Mobile Phase: 0.% P Flow Rate: 0.mL/min Temp.: 0 C Det.: UV, 0nm Inj.: 0µL Typical Retention Times for rganic Acids on SUPELCGEL C-60 and Columns CLUM: C-60 LEGT: 0cm 0cm cm ID: 7.8mm 7.8mm.6mm CAT..: Acid Typical Retention Time (min) Acetic Adipic..0. Ascorbic... Benzoic Butyric Citric Formic Fumaric Gluconic.0.0. Isobutyric Isocitric Lactic Maleic Malic.9..0 Malonic..7. xalic Phytic Propionic.. 0. Quinic..0.8 Shikimic Succinic Tartaric Mobile Phase: 0.% P, 0.mL/min (0.7mL/min for cm x.6mm column), Temperature: 0 C, Detection: UV, 0nm As sodium benzoate

29 PLC: Small Molecules Special Purpose SUPELCGEL Columns CLUM CARACTERISTICS Packing Composition : polyvinylalcohol polymer, spherical Functional Groups: C8 Particle Size: µm Pore Size: 0Å Surface Area: 0m /g p: - p. Procainamide. Procaine Separation of Basic Drugs Is Improved at igh p Column: SUPELCGEL DP-0, cm x.0mm ID, µm particles Cat. o.: 907 Mobile Phase: acetonitrile:mm phosphate, 0:60 Flow Rate: 0.mL/min (80psi/.8MPa) Temp.: 0 C Det.: UV, nm p 7 p ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules Special Purpose Columns: Resin Based SUPELCGEL DP-0: the selectivity of a C8 column, with a wider p range Because silica-based bonded phase packings are not stable above p 7, many basic compounds are analyzed on these columns as positively charged compounds. The charged compounds often interact with residual silanol groups on the packing surface, giving low efficiency and tailing peaks. Resin-based reversedphase columns are stable over almost the entire p range, but traditionally have shown low efficiency for most analytes, compared to silica-based columns. Resin-based SUPELCGEL DP-0 columns behave like silica C8 reversed phase columns, but enable you to operate at basic p (recommended p range: -). ctadecyl functional groups are covalently attached to the hydroxyl groups of spherical µm polyvinylalcohol polymer particles, giving a high density C8 coverage (7% carbon). Separation characteristics are similar to conventional C8 reversed-phase columns even at high p. The improved processes used to manufacture SUPELCGEL DP-0 particles ensure column efficiencies and mechanical stability that rival those of silica-based packings. The figure shows the benefits of high p separations in analyses of basic drugs at higher p, the compounds lose their charge and interact more strongly with the packing, prolonging their retention times. Selectivity for most compound pairs is significantly improved, and some pairs reverse their elution order. LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCGEL DP-0 (0Å, 7.0% CARB) µm x U SUPELCGEL DP-0 Supelguard Cartridges µm x.0 (/pk) 9C0 µm x.0 kit** 9-U rder: Technical Service: Web: Liquid Chromatography SUPELC

30 PLC: Small Molecules Special Purpose SUPELCGEL Columns Liquid Chromatography SUPELC rder: Technical Service: Web: Special Purpose Columns: Resin Based CLUM CARACTERISTICS SUPELCGEL TPR-00: a high efficiency resin-based column. Particles: poly(divinylbenzene/methacrylate), spherical, µm Pore Size: 00Å Unique combination of hydrophilic and hydrophobic (aromatic Surface Area: 0m /g and aliphatic) monomers p: - Crosslinked for mechanical stability arrow pore size and particle size distributions for high efficiency and good peak symmetry SUPELCGEL TPR-00 Column o micropores. Uracil o bleed from residual porogens. Aniline.,6-Diethylaniline.,-Dimethylaniline Use with 00% aqueous or 00% organic mobile phases without swelling or shrinking Relative to silica, supports composed of organic polymers or resins offer superior p stability and chemical inertness. Choose a resinbased column when resolution is improved by using a p above p 7 or below p, or when silanol or metal ion interactions cause peak tailing or irreproducible retention on a silica-based column. SUPELCGEL TPR-00 poly(divinylbenzene/methacrylate) resin has unique selectivity less hydrophobic than a pure divinylbenzene resin, but less hydrophilic than a methacrylate resin. The resin also offers excellent efficiency, peak symmetry, and mechanical stability. Repeated gradients do not change the bed structure, and the columns can be used with 00% aqueous orconventionally Deactivated C8 Column 00% organic mobile phases without shrinking or swelling. We recommend using these columns at pressures below 000 psi. The figure demonstrates the inertness of SUPELCGEL TPR- 00 resin. Although,-dimethylaniline tails severely on the silica-based C8 column, its peak shape on the SUPELCGEL column is very good, without a competing base modifier in the mobile phase. LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCGEL TPR-00 (00Å) µm x.0 9C0 µm x.0 9C0 µm x.6 9 SUPELCGEL TPR-00 Supelguard Cartridges µm x.0 (/pk) 97C0 µm x.0 (/pk) 97 µm x.0 kit ** 970-U ELPFUL ITS Do not use SUPELCGEL TPR-00 columns with methanol or methanol-containing mixtures. ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules Better Chromatography of Anilines than on a Deactivated Silica-Based Column Columns: cm x.6mm ID, µm particles Cat. o.: 9 (SUPELCGEL TPR-00 Column) Mobile Phase: acetonitrile:water, 60:0 Flow Rate: ml/min Temp.: ambient Det.: UV, nm Inj.: 0µL , 007

31 PLC: Small Molecules Special Purpose SUPELCSIL & SUPELCGEL Columns Special Purpose Columns: Taxols/Taxanes SUPELCSIL LC-F columns contain a pentafluorophenyl functional group/endcapped packing material. These columns offer selectivities different from traditional reversed phase columns for: alogenated compounds, esters, ketones, and Taxanes, including taxol Taxol, a chemically and pharmacologically unique taxane diterpene amide found in the bark and needles of the Pacific yew tree, has been approved by the US Food and Drug Administration for treatment of ovarian cancer. In isocratic analyses of taxol, the crude and complex sample matrix shortens the lifetimes of traditional reversed phase columns. Analysis of a crude taxol mixture on a SUPELCSIL LC-F is shown. Excellent resolution of the compounds of interest is obtained by using a mobile phase of acetonitrile, tetrahydrofuran, and water. Small adjustments in the percentage of tetrahydrofuran compensate for normal variation among samples. CLUM CARACTERISTICS Particles: spherical silica, µm Pore Size: 0Å Bonded Phase: pentafluorophenyl Surface Area: 70m /g Pore Volume: 0.6mL/g p Range: - 7. LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC-F (0Å,.0% CARB) µm x.0 98C0 µm x.6 98 SUPELCSIL LC-F Supelguard Cartridges µm x.0 (/pk) 9 µm x.0 kit ** 90 ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. Tetrahydrofuran Concentration Affects Taxol Analysis Column: SUPELCSIL LC-F, cm x.6mm ID, µm particles Cat. o.: 98 Mobile Phase: acetonitrile:tetrahydrofuran:water, A: :0:, B: 0::, C: 7:8: Flow Rate:.mL/min Det.: UV, 7nm Inj.: 0µL crude taxol mix ELPFUL IT: PRPERTIES F RGAIC SLVETS CMMLY USED I PLC MISCIBLE WIT UV REFRACTIVE IDEX SLVET STREGT, VISCSITY SLVET PLARITY WATER? CUTFF AT 0 C Î (SILICA) AT 0 C, CP exane nonpolar no Isooctane no Carbon tetrachloride no Chloroform no Methylene chloride no Tetrahydrofuran yes Diethyl ether no Acetone yes Ethyl acetate poorly Dioxane yes Acetonitrile yes Propanol yes Methanol yes Water polar yes.8 > Typical values Cephalomannine. Pretaxol. Taxol A R pre/ceph =.7, α pre/ceph =. R tax/pre = 0.8, α tax/pre =.07 B R pre/ceph =., α pre/ceph =.09 R tax/pre =., α tax/pre =.0 C R pre/ceph =., α pre/ceph =. R tax/pre =., α tax/pre = Sample provided by S. Richheimer, auser Chemical Research Company , 0080, 008 rder: Technical Service: Web: Liquid Chromatography SUPELC

32 PLC: Small Molecules SUPELCSIL Columns Liquid Chromatography SUPELC rder: Technical Service: Web: SUPELCSIL PLC Columns ur SUPELCSIL silica-based PLC column line includes nearly 0 bonded phase chemistries in a range of particle sizes, mnand colu configurations from microbore to preparative scale. When developing a new method, see Supelco s new Discovery suite - of reversed phase PLC columns in this catalog. LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL ABZ+PLUS (0Å,.0% CARB) µm. x. 99 µm x. 99 µm 0 x. 797 µm. x.0 99C0 µm 7. x.0 99C0 µm x.0 99C0 µm x.0 99C0 µm. x.6 99 µm x.6 99-U µm 7. x.6 99 µm x.6 99 µm 0 x µm x. 99 µm 0 x. 79 µm x. 796 µm x. 797 µm x.0 99C0 µm x.0 996C0 µm x.0 997C0 µm x.0 99C0 µm x.0 996C0 µm x.0 997C0 µm x.6 99-U µm x µm x µm x µm 0 x. 98 µm x. 8 µm x.6 96 µm x. 97 SUPELCSIL ABZ+Plus Supelguard Cartridges µm x. (/pk) 960 µm x. kit ** 960 µm x.0 (/pk) 9C0 µm x.0 (/pk) 9-U µm x.0 kit ** 9-U SUPELCSIL LC-ABZ (0Å,.0% CARB) µm 0 x U µm x. 796 µm x.0 9C0 µm x.0 90C0 µm x.0 9C0 µm x.0 9C0 µm x.6 9 µm x.6 90-U µm x.6 9 µm x SUPELCSIL LC-ABZ Supelguard Cartridges µm x. (/pk) 96 µm x. kit ** 960 µm x.0 (/pk) 9C0 µm x.0 (/pk) 9-U µm x.0 kit ** 9-U ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL SUPLEX pkb-00 (0Å,.% CARB) µm x. 797 µm x.0 89C0 µm x.0 89C0 µm x.6 89-U µm x.6 89 µm x.6 89 µm x SUPELCSIL Suplex pkb-00 Supelguard Cartridges µm x. (/pk) 9609 µm x. kit ** 9608 µm x.0 (/pk) 9-U µm x.0 kit ** 9-U SUPELCSIL LC-8 (0Å,.0% CARB) µm x. 79 µm. x C0 µm x.0 897C0 µm x.0 898C0 µm 7. x.0 898C0 µm x.0 898C0 µm. x µm x µm 7. x µm x µm 0 x µm x. 79 µm x. 79 µm 0 x.0 909C0 µm x.0 80C0 µm x.0 898C0 µm x.0 89C0 µm 0 x.0 909C0 µm x.0 80C0 µm x.0 898C0 µm 0 x.0 96 µm x.6 89 µm 0 x µm x.6 80-U µm x µm x µm x. 89 µm x.6 98 µm x. 98 SUPELCSIL LC-8 Supelguard Cartridges µm x. (/pk) 96 µm x. kit ** 96 µm x.0 (/pk) 96C0 µm x.0 (/pk) 96 µm x.0 kit ** 9

33 PLC: Small Molecules SUPELCSIL Columns LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC-8-DB (0Å,.0% CARB) µm x. 79 µm. x C0 µm 7. x.0 899C0 µm x.0 899C0 µm x.0 899C0 µm. x µm 7. x µm x µm 0 x µm x. 790 µm x.0 8C0 µm 0 x.0 908C0 µm x.0 88C0 µm x.0 8C0 µm x.0 88C0 µm x.0 8C0 µm 0 x.0 96 µm x.6 8 µm 0 x µm x.6 88 µm x.6 8-U µm x SUPELCSIL LC-8-DB Supelguard Cartridges µm x. (/pk) 967 µm x. kit ** 966 µm x.0 (/pk) 96C0 µm x.0 (/pk) 96 µm x.0 kit ** 9 SUPELCSIL LC-8-S (0Å,.0% CARB) µm 0 x µm x. 799 µm x.0 89C0 µm x.6 89 µm x U SUPELCSIL LC-8-S Supelguard Cartridges µm x.0 (/pk) 960 µm x.0 kit ** 969 SUPELCSIL LC-8-T (0Å,.% CARB) µm x C0 µm x U µm x.0 897C0 µm x SUPELCSIL LC-8-T Supelguard Cartridges µm x.0 (/pk) 96C0 µm x.0 (/pk) 96 µm x.0 kit ** 960 SUPELCSIL LC-DABS (0Å,.% CARB) µm x.6 97 SUPELCSIL LC-8-T Supelguard Cartridges (use for LC-DABS) µm x.0 (/pk) 96 µm x.0 kit ** 960 ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC-PA (0Å) µm x.0 9C0 µm 0 x.0 9C0 µm x.6 9 µm 0 x.6 9 µm x. 79 µm x.0 88C0 µm x.6 88 µm x.6 89 SUPELCSIL LC-8 Supelguard Cartridges (use for LC-PA) µm x. (/pk) 96 µm x. kit ** 96 µm x.0 (/pk) 96C0 µm x.0 (/pk) 96 µm x.0 kit ** 9 SUPELCSIL LC-8 (00Å, 6.0% CARB) µm x.6 88 µm x SUPELCSIL LC-8 Supelguard Cartridges µm x.0 (/pk) 9 µm x.0 kit ** 90 SUPELCSIL LC-8 (0Å, 6.0% CARB) µm. x.0 897C0 µm 7. x.0 898C0 µm x.0 898C0 µm x.0 898C0 µm. x µm 7. x µm x µm 0 x µm x. 799 µm x.0 80C0 µm x.0 897C0 µm x.0 80C0 µm x.0 897C0 µm x.6 88 µm x.6 80-U µm x µm x µm x. 8 µm x.6 98 µm x. 98 SUPELCSIL LC-8 Supelguard Cartridges µm x. (/pk) 96 µm x. kit ** 96 µm x.0 (/pk) 96C0 µm x.0 (/pk) 96 µm x.0 kit ** 9 rder: Technical Service: Web: Liquid Chromatography SUPELC

34 6 PLC: Small Molecules SUPELCSIL Columns SUPELCSIL PLC Columns (cont d) Liquid Chromatography SUPELC rder: Technical Service: Web: LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC-8-DB (0Å, 6.0% CARB) µm 7. x C0 µm x.0 899C0 µm x.0 899C0 µm. x µm 7. x U µm x µm x. 79 µm x.0 87C0 µm x.0 87C0 µm x.0 8C0 µm x.6 8 µm x.6 87 µm x.6 8 µm x SUPELCSIL LC-8-DB Supelguard Cartridges µm x. (/pk) 969 µm x. kit ** 968 µm x.0 (/pk) 96 µm x.0 kit ** 9 SUPELCSIL LC-08 (00Å,.% CARB) µm x.6 88 µm x SUPELCSIL LC-08 Supelguard Cartridges µm x.0 (/pk) 9-U µm x.0 kit ** 90 SUPELCSIL LC-DP (0Å, 6.0% CARB) µm x.0 90C0 µm x.0 88C0 µm 0 x µm x.6 88 µm 0 x.6 9 µm x.6 90-U µm x.6 88 SUPELCSIL LC-DP Supelguard Cartridges µm x.0 (/pk) 966C0 µm x.0 (/pk) 966 µm x.0 kit ** 96 SUPELCSIL LC-DP (00Å,.0% CARB) µm x SUPELCSIL LC-DP Supelguard Cartridges µm x.0 (/pk) 9 SUPELCSIL LC-F (0Å,.0% CARB) µm x.0 98C0 µm x.6 98 SUPELCSIL LC-F Supelguard Cartridges µm x.0 (/pk) 9 µm x.0 kit ** 90 SUPELCSIL LC-0 (00Å,.7% CARB) µm x.6 88 µm x.6 88 SUPELCSIL LC-0 Supelguard Cartridges µm x.0 (/pk) 99 µm x.0 kit ** 99 ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL LC-C (0Å,.0% CARB) µm. x C0 µm 7. x C0 µm. x µm 7. x µm x.0 8C0 µm x.0 8C0 µm x.0 8C0 µm x.0 8C0 µm x.6 8 µm x.6 8-U µm x.6 8 µm x SUPELCSIL LC-C Supelguard Cartridges µm x.0 (/pk) 967C0 µm x.0 (/pk) 967 µm x.0 kit ** 97 SUPELCSIL LC-PC (0Å,.0% CARB) µm x.0 877C0 µm x.0 878C0 µm x.0 877C0 µm x µm 0 x µm x SUPELCSIL LC-PC Supelguard Cartridges µm x.0 (/pk) 9 µm x.0 kit ** 90 SUPELCSIL LC- (0Å,.0% CARB) µm x.0 80C0 µm x.0 80C0 µm x.6 87 µm x.6 80-U µm x SUPELCSIL LC- Supelguard Cartridges µm x.0 (/pk) 96C0 µm x.0 (/pk) 96 SUPELCSIL LC- (0Å,.0% CARB) µm 7. x C0 µm x C0 µm 7. x µm x µm x.0 88C0 µm x.0 88C0 µm x.6 88 SUPELCSIL LC- Supelguard Cartridges µm x.0 (/pk) 968C0 µm x.0 (/pk) 968 µm x.0 kit ** 98 SUPELCSIL LC- -P (0Å,.0% CARB) µm x.6 9 SUPELCSIL LC- -P Supelguard Cartridges µm x.0 (/pk) 96 µm x.0 kit ** 9

35 PLC: Small Molecules SUPELCSIL & SUPELCGEL Columns 7 SUPELCGEL resin-based PLC Columns LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCSIL ISEP (0Å) µm x. 79 µm x.6 9 µm x.6 89 µm x SUPELCSIL isep Supelguard Cartridges µm x.0 (/pk) 960-U µm x.0 kit ** 969 SUPELCSIL LC-Si (0Å) µm 7. x C0 µm x.0 898C0 µm x.0 898C0 µm. x µm 7. x U µm x µm 0 x U µm x. 790-U µm 0 x.0 90C0 µm x.0 800C0 µm x.0 800C0 µm x.0 89C0 µm 0 x µm x.6 86 µm 0 x.6 90-U µm x U µm x.6 89 µm x µm x. 8 µm x U µm x. 98 SUPELCSIL LC-Si Supelguard Cartridges µm x.0 (/pk) 960C0 µm x.0 (/pk) 960 µm x.0 kit ** 90 SUPELCSIL LC-Si (00Å) µm x SUPELCSIL LC-Si Supelguard Cartridges (use for LC-Si) µm x.0 (/pk) 960 µm x.0 kit ** 90 SUPELCSIL LC-DIL (0Å,.% CARB) µm x.0 80C0 µm x.0 80C0 µm x.6 80 SUPELCSIL LC-Diol Supelguard Cartridges µm x.0 (/pk) 969 µm x.0 kit ** 99 SUPELCSIL SAX (0Å) µm x.0 98C0 µm x.0 98C0 µm x.6 98 SUPELCSIL SAX Supelguard Cartridges µm x.0 (/pk) 97-U µm x.0 kit ** 96-U SUPELCSIL LC-SCX (0Å) µm x C0 µm x SUPELCSIL LC-SCX Supelguard Cartridges µm x.0 (/pk) 99 µm x.0 kit ** 909 ** Kits include one cartridge, stand-alone holder, a piece of tubing, and nuts and ferrules. **** Does not include tubing, nuts or ferrules. For reversed-phase separations at high p or low p, we offer SUPELCGEL TPR-00 and SUPELCGEL DP-0 resinbased PLC columns. SUPELCGEL resin-based ion exclusion PLC columns contain sulfonated divinylbenzene resins in six cationic forms, each offering a unique selectivity for analyses of saccharides or organic acids. LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE SUPELCGEL TPR-00 (00Å) µm x.0 9C0 µm x.0 9C0 µm x.6 9 SUPELCGEL TPR-00 Supelguard Cartridges µm x.0 (/pk) 97C0 µm x.0 (/pk) 97 µm x.0 kit ** 970-U SUPELCGEL DP-0 (0Å, 7.0% CARB) µm x U SUPELCGEL DP-0 Supelguard Cartridges µm x.0 (/pk) 9C0 µm x.0 kit ** 9-U SUPELCGEL AG 9µm 0 x U SUPELCGEL AG Guard Column 9µm x.6 **** 97-U SUPELCGEL AG 9µm 0 x SUPELCGEL AG Guard Column 9µm x.6 **** 96 SUPELCGEL C-60 9µm 0 x U SUPELCGEL Guard Column (use to protect C-60) 9µm x.6 **** 99 SUPELCGEL C-6 9µm 0 x U SUPELCGEL Ca Guard Column (use to protect C-6) 9µm x.6 **** 906-U SUPELCGEL CA 9µm 0 x U SUPELCGEL Ca Guard Column 9µm x.6 **** 906-U SUPELCGEL 9µm x µm 0 x U SUPELCGEL Guard Column 9µm x.6 **** 99 SUPELCGEL K 9µm 0 x SUPELCGEL K Guard Column 9µm x.6 **** 9 SUPELCGEL Pb 9µm 0 x SUPELCGEL Pb Guard Column 9µm x.6 **** 9 rder: Technical Service: Web: Liquid Chromatography SUPELC

36 8 PLC: Small Molecules ther Columns ther PLC Columns - Small Molecules Liquid Chromatography SUPELC rder: Technical Service: Web: In addition to our own product lines, we offer name brand silica-based columns: alphabond, ypersil, Kromasil, LiChrosorb, LiChrospher, ucleosil, TSK-GEL, and Waters Spherisorb. alphabond LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE ALPABD (Å) C8 0µm x µm 0 x alphabond Guard Cartridges C8 0µm x.6 (/pk) 790-U ypersil LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE YPERSIL (0Å) DS (C8) µm 0 x. 9 µm 0 x.6 Z67 µm x. 9 µm x. 9 µm x.6 Z6 µm x.6 Z6 BDS-C8* µm x. 9-U µm x. 96 µm x.6 78 µm x MS (C8) µm 0 x. 9 µm 0 x.6 Z6 µm x.6 Z678 µm x.6 Z686 BDS-C8* µm x µm x Phenyl µm 0 x. 9 µm 0 x.6 Z69 µm x. 9 µm x.6 Z667 µm x.6 Z67 CPS (Cyano) µm 0 x. 90 µm 0 x.6 Z6 µm x. 9 µm x.6 Z6 µm x.6 Z60 SAS (C) µm 0 x.6 Z68 µm x. 97-U µm x.6 Z69 µm x.6 Z60 * BDS = Base Deactivated Silica. ** Deactivated C8. LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE YPERSIL (0Å) CT D Silica µm 0 x.6 Z697 µm x.6 Z600 µm x.6 Z69 APS ( ) µm 0 x. 97 µm 0 x.6 Z69 µm x. 98 µm x. 99 µm x.6 Z608 µm x.6 Z66 ypersil Guard Cartridges DS (C8) µm x.6 (/pk) Z706 BDS-C8* µm x.6 (/pk) 787 MS (C8) µm x.6 (/pk) Z707 BDS-C8* µm x.6 (/pk) 067 Phenyl µm x.6 (/pk) Z70 CPS (Cyano) µm x.6 (/pk) Z7099 SAS (C) µm x.6 (/pk) Z70 Silica µm x.6 (/pk) Z706 APS ( ) µm x.6 (/pk) Z7080 Inertsil LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE IERTSIL (0Å) DS-** µm x µm x Inertsil Guard Cartridges DS-** µm x.6 (/pk) 06087

37 PLC: Small Molecules ther Columns 9 Kromasil LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE KRMASIL (00Å) C8 µm x.6 8 µm x.6 7 0µm x.6 9 C8 µm x.6 µm x.6 0-U 0µm x.6 C µm x.6 -U µm x.6 60-A (Silica, 60Å) µm x.6 6 µm x.6 6 0µm x A (Silica) µm x.6 9 0µm x.6 6 µm x.6 6-U 0µm x.6 8-U Kromasil Guard Cartridges C8 µm x.6 (/pk) 6-U C8 µm x.6 (/pk) 66 C µm x.6 (/pk) A (Silica, 60Å) µm x.6 (/pk) 70-U Lichrosorb LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE LICRSRB (00Å) RP-8 (C8) µm x. 9 µm x. 90-U µm x.6 9 µm x.6 99 RP-8 (C8) µm x.6 9-U µm x.6 9-U Si-60 (Silica, 60Å) µm x. 96 µm x.6 96-U µm x.6 96 µm x. 98-U µm x.6 99 µm x.6 97-U LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE LiChrosorb Guard Cartridges RP-8 (C8) µm x.6 (/pk) 96-U RP-8 (C8) µm x.6 (/pk) 966 Si-60 (Silica, 60Å) µm x.6 (/pk) 968 µm x.6 (/pk) 967 Licrospher LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE LICRSPER (00Å) RP-8 (C8) µm x. 77 µm x. 777 µm x.6 77 µm x RP-8 (C8) µm x. 779 µm x µm x C (Cyano) µm x µm x Si-60 (Silica, 60Å) µm x. 79-U µm x U µm x.6 79 µm x. 78 µm x. 78 µm x.6 78 µm x.6 78 LiChrospher Guard Cartridges RP-8 (C8) µm x.6 (/pk) 79 C (Cyano) µm x.6 (/pk) 798 Si-60 (Silica, 60Å) µm x.6 (/pk) 797-U µm x.6 (/pk) 796-U rder: Technical Service: Web: Liquid Chromatography SUPELC

38 0 PLC: Small Molecules ther Columns ucleosil Liquid Chromatography LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE UCLESIL (00Å) C8 µm 0 x. 97 µm 0 x.6 Z66 µm x. 98 µm x. 99 µm x.6 Z67 µm x.6 Z68 C8 µm 0 x. 90-U µm 0 x.6 Z60 µm x. 9 µm x. 9 µm x.6 Z6 µm x.6 Z68 Phenyl 7µm x.6 Z66 C (Cyano) µm x. 9 µm x. 9-U µm x.6 Z6 µm x.6 Z66 Silica µm x. 9 µm x.6 Z69 µm x.6 Z67 µm x. 96 µm x. 97 µm x.6 Z689 ucleosil Guard Cartridges C8 µm x.6 (/pk) Z77 C8 µm x.6 (/pk) Z7 Phenyl 7µm x.6 (/pk) Z7 C (Cyano) µm x.6 (/pk) Z76 Silica µm x.6 (/pk) Z79 µm x.6 (/pk) Z788 LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE TSK-GEL C8 CLUMS, CT D Super-DS (0Å) µm x.6 88 µm 0 x TSK-GEL Guard Cartridges and Filters DS-80Ts (80Å) µm. x. (/pk) 87 DS-0T (0Å) µm. x. (/pk) 8 Filter (/pk) 8807 SUPELC rder: Technical Service: Web: TSK-GEL C8 LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE TSK-GEL C8 CLUMS DS-80Tm (80Å) µm 7. x µm x µm x DS-80Ts (80Å) µm 7. x µm x µm x DS-0A (0Å) µm x µm x DS-0T (0Å) µm x µm x Waters Spherisorb LEGT X ID PARTICLE SIZE (cm X mm) CAT.. PRICE WATERS SPERISRB (80Å) DS- (C8) µm 0 x. 90 µm 0 x.6 Z60 µm x. 90 µm x. 90 µm x.6 Z60 µm x.6 Z6068 ctyl (C8) µm 0 x.6 Z6076 µm x. 908 µm x.6 Z608 µm x.6 Z609 Phenyl µm x.6 Z606 Cyano µm x. 90 µm x.6 Z6 Silica µm 0 x.6 Z6009 µm x. 90 µm x. 90 µm x.6 Z607 µm x.6 Z60 Amino ( ) µm x. 9-U µm x.6 Z6 SAX µm x.6 Z60 Waters Spherisorb Guard Cartridges DS- (C8) µm x.6 (/pk) Z697 ctyl (C8) µm x.6 (/pk) Z6998 Phenyl µm x.6 (/pk) Z700 Cyano µm x.6 (/pk) Z70 Silica µm x.6 (/pk) Z696 Amino ( ) µm x.6 (/pk) Z70 SAX µm x.6 (/pk) Z708

39 PLC: Small Molecules Guard Column olders U 6799-U CLUM T RECMMEDED 987 BE PRTECTED PELLIGUARD CLUM CAT.. PRICE Silica LC-Si Kit 96 Cartridges (pk. of ) 96 Guard Column olders P0008 Cyano LC-C Kit 96-U Cartridges (pk. of ) 96 Use these guard column holders with the guard cartridges listed Amino LC- Kit 966 Cartridges (pk. of ) 966 on the previous pages. The Direct-Connect holders allow a guard cartridge to attach to a Supelco modular column with no dead C8 LC-8 Kit 96 Cartridges (pk. of ) 96 volume. The Direct-Connect holders can only be used with C8 LC-8 Kit 96 Supelco modular columns. The Swivel-type holders allow the Cartridges (pk. of ) 96 tubing to move independently of the holder, reducing the risk of leaks caused by crimped tubing. The Stand-Alone holders include the necessary tubing, nuts and ferrules for connecting to Pellicular Packing Kit any analytical columns. 80 DESCRIPTI CAT.. PRICE FR SUPELC SUPELGUARD CARTRIDGES Direct-Connect (Swivel-type) older* 06 Direct-Connect (Swivel-type) older** 0 Direct-Connect older** 0 Stand-Alone older*** 9660-U Stand-Alone older**** 6799-U FR SUPELC PELLIGUARD CARTIDGES Stand-Alone older 000 FR TER CARTRIDGES Stand-Alone older (includes PEEK Column Coupler) 987 Replacement PEEK Column Coupler 986 FR TSK-GEL CARTRIDGES AD FILTERS Stand-Alone older for TSK-GEL Cartridges 800 Stand-Alone older for TSK-GEL Filters 8806 * For.mm ID Supelco columns ** For.0,.0 and.6mm ID Supelco columns *** For.,.0,.0 and.6mm ID Supelco columns **** For 0.0mm ID Supelco columns Pelliguard Guard Cartridges For µm, 0µm, or µm SUPELCSIL and other silica-based ELPFUL ITS Before flushing a reversed-phase PLC column that contains a buffer (salt), flush with warm (60 C) DI thoroughly to remove salts. ot following this general rule may result in salt 89 precipitation when returned to 00% organic for long-term Guard Column ardware Kit, storage. For more information, refer to literature T00, Buffer Funnel and Tubing Solubility section. Kit includes cm x.6mm ID column, endfittings, frits (.0µm pores), and "/cm of 0.0" ID x /6" D SS tubing. Funnel connects to column with tygon tubing (included) for easier column filling. P PLC columns, where samples are especially dirty, and a small loss of efficiency is acceptable. Each kit contains one cartridge (cm x.6mm ID) filled with 0µm Pelliguard packing, a reusable stand-alone column holder, and hardware for connecting the holder to /6" tubing. Replacement cartridges come in packages of four. Bulk Pellicular Packing Kits Reusable cm x.6mm ID guard column hardware and 0µm pellicular packing, for protecting 0µm columns. Each column kit contains an empty cm x.6mm ID column, 0g of Pelliguard packing, 0 frits, and hardware for connecting the column to /6" tubing. About. grams of packing is needed to pack one cm x.6mm column. CLUM T RECMMEDED BE PRTECTED PELLIGUARD CLUM CAT.. PRICE Silica LC-Si Kit 80 LC-Si Packing, 0g 89 Cyano LC-C Kit 8 LC-C Packing, 0g 8 C8 LC-8 Kit 8-U LC-8 Packing, 0g 89 C8 LC-8 Kit 8 LC-8 Packing, 0g 89 P0006 DESCRIPTI CAT.. PRICE Guard Column ardware Kit 89 Replacement Frits (pk. of 0) 86 Funnel and Tubing 090-U rder: Technical Service: Web: Liquid Chromatography SUPELC

40 PLC: Small Molecules PLC Column Test Mixes Liquid Chromatography SUPELC rder: Technical Service: Web: PLC Column Test Mixes Performance evaluation mixes for PLC columns. Well defined test mixes enable you to troubleshoot chromatographic problems, optimize system efficiency, and evaluate nder columns u conditions where their performance is understood. We ship these test mixes in amber ampuls to prevent photodegradation, and we include instructions for proper use and interpretation of results. Choose from column-specific or application-specific mixes. All mixes except the amino phase test mix (Cat. o. 8) UV call for detection; the amino phase test mix (sugars) calls for refractive index detection. We recommend PLC our Troubleshooting Guide (Bulletin 86) for additional information about using test mixes. ml unless otherwise specified. CMPETS TEST MIX USE T TEST SLVET (CC./mL) CAT.. PRICE Amino Phase LC- columns acetonitrile:water, D-fructose (mg) 8 :7 α-d-glucose (mg) sucrose (mg) maltose (mg) lactose (mg) Cyano Phase LC-C, LC-PC, acetonitrile:water, uracil (7µg) 899 ABZ + Plus columns, :7 acetophenone (7µg) any weakly benzene (70µg) hydrophobic phase toluene (77µg) ormal Phase Mix LC-Si (silica) columns methylene chloride benzene (600µg) 88 benzanilide (0µg) acetanilide (0µg) ormal Phase Mix LC-Si, LC-C, LC- ethanol:hexane, toluene (mg) 760-U columns :9 diethyl phthalate (mg) dimethyl phthalate (mg) ucleosides LC-8-S columns water, sodium nucleosides (0-00µg) 70-U formate (0mg/mL) (see page 6) LC-PA LC-PA columns methanol: 6 PAs (00-000µg) 87 methylene chloride, 0:0 (see page 0) Peptide Standard reversed phase none Gly-Tyr (~0.mg) 06-VL columns used (dried film) Val-Tyr-Val (~0.mg) for peptide Met enkephalin (~0.mg) separations Leu enkephalin (~0.mg) (e.g., 00Å phases) angiotensin II (~0.mg) Reversed Phase Mix hydrophobic RP methanol:water, uracil (7µg) 878 columns 60:0 acetophenone (7µg) (e.g., LC-8, LC-8) benzene (70µg) toluene (77µg) Reversed Phase Mix hydrophobic RP acetonitrile:water, uracil (µg) 76-U columns 8: phenol (700µg) (e.g., LC-8, LC-8),-diethyl-m-toluamide (600µg) toluene (mg) Chiral chiral phases hexane:ethyl toluene (70µg) 80-U acetate, 80:0 (+) TFAE (µg) ( ) TFAE (µg) Chiral chiral phases chloroform benzene (00µg) 8 (+) -PDBA (0µg) ( ) -PDBA (0µg)),,-trifluoro--(9-anthryl)ethanol -(-phenylethyl)-,-dinitrobenzamide Custom Test Mixes For information on made-to-order standards and test mixes, call our Technical Service chemists, or request our Custom Chemical Reference brochure (Publication o. 9690).

41 PLC: Small Molecules PLC Column Test Mixes Restore Column Performance Properly performing column (A) is exposed to water and alcohol (B), then treated with Silica Column Regeneration Solution (C).. Methyl paraben (0.9 min). Ethyl paraben (. min). Propyl paraben (.7 min). Butyl paraben (.9 min) Flow Rate: ml/min Det.: UV, nm Inj.: 0µL. Uracil (0. min). Phenol (.6 min). Methyl paraben (.8 min). Ethyl paraben (. min). Propyl paraben (.0 min) 6. Butyl paraben (. min) 7. eptyl paraben (6. min) Isocratic Evaluation Mix A B C 6 Flow Rate: ml/min methanol:water, 0:90 to 90:0 in min Det.: UV, nm Inj.: 0µL. Benzene. Benzanilide. Acetanilide Column: SUPELCSIL LC-Si, cm x.6mm, µm particles Cat. o.: 898 Mobile Phase: A = methylene chloride:methanol:water (99.:0.:0.) B = -propanol:water (0:0) C = Silica Column Regeneration Solution, ml/min for 0 min, then methylene chloride:methanol:water (99.:0.:0.), ml/min for 0 min Det.: UV, nm Inj.: 0µL Gradient Evaluation Mix , 77, PLC Column Test Mixes System diagnostics: test kits and column regeneration solutions. Silica Column Regeneration Solution This - solution effectively regenerates a silica column that has come into contact with very strongly polar solvents, such as water or alcohols. Simply flush the column with regeneration solution for 0 minutes, then reequilibrate with mobile phase for 0 minutes. Column performance usually is restored to that obtained before exposure to the polar solvent. System Diagnostic Kit - Take a systematic approach to diagnosing problems in an PLC system. This kit consists of: cm x.6mm SUPELCSIL LC-8SD column 6 x ml Isocratic Evaluation Mix 6 x ml Gradient Evaluation Mix four.8ml screwcap vials with hole caps and septa When you need to determine the cause of a problem, install the cm column, prepare a simple methanol:water mobile phase, and inject 0µL of Isocratic Evaluation Mix onto the column. Compare your chromatogram with that from a properly performing system and use the information sheet included with the kit to help isolate the source of the problem. If necessary, make injections with the gradient mix. We recommend our PLC Troubleshooting Guide (Bulletin 86, available free on request) to help you interpret the results you obtain. Evaluation Test Mixes - Six ml ampuls of test compounds in methanol:water, 60:0. These formulations are designed for evaluating how reliably a chromatographic system is providing such fundamentally important parameters as flow rate, proportioning, and mixing. DESCRIPTI QTY. CAT.. PRICE Silica Column Regeneration Solution 00mL 7 LC-8SD System Diagnostic Kit 8 Isocratic Evaluation Mix 6 x ml 870-U Gradient Evaluation Mix 6 x ml 87 RELATED IFRMATI Request free literature by phone or fax, or see our website. o. Subject T0086 PLC troubleshooting guide T9690 custom chemicals brochure rder: Technical Service: Web: Liquid Chromatography SUPELC

42 PLC: Biopolymers Column Selection: ucleic Acid Separations Liquid Chromatography SUPELC rder: Technical Service: Web: AMERSAM MCI GEL BISCIECES TSK-GEL SUPELC SAMPLE SEPARATI PLC FPLC PLC PLC TYPE MDE (SEE PAGE) (SEE PAGE) (SEE PAGE) (SEE PAGE) DA/RA Gel Filtration Superdex 00 G-DA-PW (6) (6) Ion Exchange ProtEx-DEAE Mono Q DEAE-PR (60) (60) (6) PCR Fragments Ion Exchange ProtEx-DEAE Mono Q DEAE-PR (60) (60) (6) ligonucleotides Ion Exchange ProtEx-DEAE Mono Q DEAE-PW (60) (60) (6) DEAE-PR (6) Reversed ligo-da RP SUPELCSIL LC-8 Phase (6) (6) Discovery C8 (8) ucleotides Reversed- SUPELCSIL LC-8-T Phase (9) Discovery C8 (8) Discovery RP-AmideC6 (9) Ion Exchange DEAE-SW SUPELCSIL SAX (6) (7) ucleosides Reversed- SUPELCSIL LC-8-S Phase (9) Discovery C8 (8) Discovery RP-AmideC6 (9) ucleic Acid Reversed- SUPELCSIL LC-8 Bases Phase () SUPELCSIL LC-8-DB () Discovery C8 (8) Discovery RP-AmideC6 (9) Ion Exchange SUPELCSIL LC-SCX (7) RELATED IFRMATI Request free literature by phone or fax, or see our website. o. Subject T008 Discovery BI Wide Pore PLC Columns and Capillaries: Solutions to Protein and Peptide Separation Challenges McClung, G., W.T. Frankenburger, Jr. Comparison of Reversed Phase igh Performance Liquid Chromatographic Methods for Precolumn Derivatized Amino Acids, J. Liq. Chromatogr. : 6-66 (988). Reference not available from Supelco.

43 PLC: Biopolymers Column Selection: Peptide and Protein Separations AMERSAM MCI GEL BISCIECES TSK-GEL SUPELC SEPARATI PLC FPLC PLC PLC MDE (SEE PAGE) (SEE PAGE) (SEE PAGE) (SEE PAGE) Gel Filtration Superdex Peptide G000SW, G000SW XL Superdex 7 G000SW, G000SW XL Superdex 00 G000SW, G000SW XL (6) Super 000 SW Fast Desalting R 0/0 (6) (6) Strong Anion ProtEx-DEAE Mono Q Exchange (60) (60) Weak Anion Mono P DEAE-PW, DEAE-PR Exchange (60) (6) Chromatofocusing Mono P (60) Strong Cation ProtEx-SP Mono S SP-PW, SP-PR Exchange (60) (60) (6) Weak Cation CM-PW Exchange (6) ydrophobic Interaction Ether-PW Phenyl-PW Butyl-PR (6) Reversed Phenyl-PW RP Discovery BI Wide Phase ctadecyl-pw Pore C8 ctadecyl-pr (7) Super-DS Discovery BI Wide (6) Pore C8 (8) Discovery BI Wide Pore C (9) Discovery RP-AmideC6 (9) Discovery C8 (8) Discovery C8 (0) Affinity ote: Products in italics are used under low pressure conditions. ABA-PW Boronate-PW Chelate-PW eparin-pw Tresyl-PW (6) Columns for Amino Acid Separations DERIVATIZED SUPELCSIL DIMESIS SUPELGUARD AMI ACID PLC (cm x mm ID) CAT.. PRICE GUARD CLUM CAT.. PRICE Dabsyl-AA LC-DABS (µm) x. 97 LC-8-T 96 DABT-AA LC-8 (µm) x LC-8 96 PA-AA LC-8 (µm) x.6 80-U LC-8 96 PTC-AA LC-8-DB (µm) x.6 8-U LC-8-DB 96 PT-AA LC-8-DB (µm) x. 79 LC-8-DB 96 LC-8-DB (µm) x.6 8-U LC-8-DB 96 Alternative to µm LC-8-DB. rder: Technical Service: Web: Liquid Chromatography SUPELC

44 6 PLC: Biopolymers Discovery BI Wide Pore Columns Liquid Chromatography Discovery BI Solutions to Protein and Peptide Separation Challenges Discovery BI PLC columns and capillaries provide sensitive, stable, efficient, reproducible separations of proteins and peptides. The different phase chemistries provide unique selectivity increasing your resolution options. Separations are completely scalable from analytical to prep. The low-bleed feature and microbore and capillary dimensions make them ideal for proteomics and LC/MS applications. Discovery BI Wide Pore C8 Ideal for high resolution peptide mapping and synthetic and native peptide separations. Mixture of Synthetic Peptides on Discovery BI Wide Pore C8 and a Leading Competitive Column Columns: (A) Discovery BI Wide Pore C8, cm x.6mm, µm (Cat. o. 68-U) (B) Competitive protein and peptide C8, cm x.6mm, 00Å, µm Mobile Phase: (A) 80:0, (0.% TFA in water): (0.% TFA in C C); (B) 66:, (0.% TFA in water): (0.% TFA in C C) Flow Rate:.0mL/min Temp: 0 C Detection: 0nm Injection: Gradient: 0µL, ~0.µg each peptide (Sigma Peptide Mix, Cat. o. P 69 containing Arg 8 -vasopressin, bradykinin (fragment -), oxytocin, luteinizing hormone releasing hormone, Metenkephalin, bradykinin, Leu-enkephalin, bombesin, Substance P) in 0.%TFA. See sequence in Figure. 0-00%B in min after minute delay (A) Discovery BI Wide Pore C8 SUPELC rder: Technical Service: Web: Discovery BI Wide Pore C8 Also ideal for high resolution peptide mapping and synthetic and native peptide separations. The intermediate hydrophobicity of the C8 phase makes it very useful for method development. Discovery BI Wide Pore C The short-chain C phase is used for protein and hydrophobic peptide separations. Discovery BI Wide Pore C is more stable 0 than the commonly-used C phases. (B) Competitor Capillary and Microbore Dimensions With the rapid growth of proteomics, the demand for detailed characterization on smaller sample volumes is increasing. So is the utility of smaller ID PLC columns. Traditional column IDs of.6mm and even.mm are being replaced by microbore (mm ID) and capillary (< mm ID) dimensions. The benefits are twofold: increased sensitivity and decreased sample consumption. Discovery BI C8 and C is available in capillary and microbore dimensions to meet these criteria. Go to to see the entire Discovery BI line, including new additions, and download the most recent literature and applications. Discovery column resolves synthetic peptides better than the competitive column G008 Suggestions for Choosing a Discovery BI Wide Pore Column: APPLICATIS BDED PASES Proteins BI Wide Pore C ydrophobic peptides or proteins (e.g. membrane proteins) BI Wide Pore C Peptide mapping BI Wide Pore C8 Proteomics BI Wide Pore C8 Scouting BI Wide Pore C8 (because of its intermediate hydrophobicity between a C8 and C) APPLICATI SILICA PARTICLE SIZES LC/MS micron or micron Fast analysis, or highthroughput applications micron Peptide mapping micron or micron Analytical PLC micron or micron Preparative 0 micron APPLICATI CLUM ID LC/MS.mm or smaller Peptide mapping.6mm,.0mm,.mm Analytical PLC.0mm,.6mm Preparative 0mm,.mm Low level detection or limited sample volume 0.mm, 0.mm,.0mm G00

45 PLC: Biopolymers Discovery BI Wide Pore C8 Columns 7 Discovery BI Wide Pore C8 Peptide maps generated by RP-PLC provide valuable information about protein structure, stability, and purity. To be effective, the RP-PLC column must be able to resolve a high percentage of the peptides in the sample. The more peptides, the better the information. Discovery BI Wide Pore C8 gives unsurpassed RP-PLC resolution of peptide maps from tryptic digests. The improvements in silica and bonded phase chemistry we ve incorporated into the Discovery BI Wide Pore line improve resolution by increasing efficiency and reducing the peak tailing. An added benefit to this is the ability to analyze peptides without TFA in the mobile phase thereby increasing the LC/MS signal. 00 % Peptide : ac-rgagglglgk-amide Peptide : ac-rgggglglgk-amide Peptide : ac-rgagglglgk-amide Peptide : ac-rgvgglglgk-amide Peptide : ac-rgvvglglgk-amide 0 G % % 0 0 0% TFA (mm formic acid), µl injection 0.0% TFA, µl injection 0.% TFA, µl injection Effect of Chromatographic Conditions on MS Signals of Peptides Column: Discovery BI Wide Pore C8, cm x.mm, µm Cat. o.: 670-U Mobile Phase A: mm formic acid in water Mobile Phase B: 0:0 (mm formic acid in water) : (0mM formic acid in MeC) Mobile Phase C: 0.0 or 0.% TFA in water Mobile Phase D: 0:0 (0.0 or 0.% TFA in water) : (0.0 or 0.% TFA in MeC) Flow Rate: 0.08mL/min Det.: +ES Temp.: ambient Inj.: µl or µl Sample: RP Peptide Performance Standard, p/n RPS-P000 (Alberta Peptide Institute) 0 0. molarity of formic acid adjusted to provide minimum baseline drift.. linear velocity equal to ml/min on.6mm ID columns. G0069 Gradient %C %D ,: TE: Coelution of peptides and G Gradient %A %B Gradient %C %D LEGT x ID PARTICLE SIZE (cm x mm) CAT.. PRICE DISCVERY BI WIDE PRE C8 (00Å, 9.% CARB) µm x U µm 0 x U µm x U µm 0 x U µm x.0 60-U µm 0 x U µm x U µm 0 x. 670-U µm x. 670-U µm x U µm 0 x U µm x U µm x U µm x U µm x U µm x U µm x U µm 0 x. 680-U µm x. 680-U µm x. 680-U µm x U µm 0 x.0 68-U µm x.0 68-U µm x.0 68-U µm x U µm 0 x.6 68-U µm x.6 68-U µm x.6 68-U µm x U 0µm x U 0µm x U 0µm x U 0µm x U 0µm x. 670-U 0µm x. 67-U 0µm x. 67-U Supelguard Cartridges µm x. (/pk) 6770-U µm x. kit ** 677-U µm x.0 (/pk) 677-U µm x.0 kit ** 677-U µm x. (/pk) 6870-U µm x. kit ** 687-U µm x.0 (/pk) 687-U µm x.0 kit ** 687-U 0µm x U ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. PRPERTIES: Bonded Phase: Covalently-bonded octadecylsilane, endcapped Silica: Spherical, high purity (<0ppm metals) Particle Size:,, and 0mm Pore Size: 00Å Surface Area: 00m²/g %C: ~9% Coverage: ~ µmoles/m² rder: Technical Service: Web: Liquid Chromatography SUPELC

46 8 PLC: Biopolymers Discovery BI Wide Pore C8 Columns Liquid Chromatography SUPELC rder: Technical Service: Web: LEGT X ID PARTICLE SIZE (cm x mm) CAT.. PRICE DISCVERY BI WIDE PRE C8 (00Å,.0% CARB) µm x. 67-U µm 0 x. 67-U µm x. 67-U µm x U µm 0 x U µm x U µm x U µm 0 x. 680-U µm x. 680-U µm x. 680-U µm x U µm 0 x.0 68-U µm x.0 68-U µm x.0 68-U µm x U µm 0 x.6 68-U µm x.6 68-U µm x.6 68-U µm x U 0µm x U 0µm x U 0µm x U 0µm x U 0µm x. 67-U 0µm x. 67-U 0µm x. 67-U Supelguard Cartridges µm x. (/pk) 677-U µm x. kit ** 677-U µm x.0 (/pk) 6776-U µm x.0 kit ** 6777-U µm x. (/pk) 6870-U µm x. kit ** 687-U µm x.0 (/pk) 687-U µm x.0 kit ** 687-U 0µm x U ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules.. Angiotensin II (.67g/L) (DRVYIPF). Angiotensin III (.67g/L) D(RVYIPF). Angiotensin I (.67g/L) (DRVYIPFL) Resolution of Angiotensins at eutral p Column: Discovery BI Wide Pore C8, cm x.6mm, µm Cat. o.: 68-U Mobile Phase: (A) 0mM P /, p 7; (B) 0:0, (0mM P /, p 7):MeC Flow Rate: ml/min Temp: 0 C Det.: nm Inj.: 6µL in water Gradient: 0-60% B in min Discovery BI Wide Pore C8 Discovery BI Wide Pore C8 is an RP-PLC phase for proteins and peptides that has hydrophobicity intermediate between the Discovery BI Wide Pore C and the Discovery BI Wide Pore C8. The difference in hydrophobicity gives it unique selectivity relative to these other phases. It is ideal for peptide mapping because it provides complementary information compared to a C8 separation. Because of its intermediate hydrophobicity we also recommend it for method development or scouting work. As with all Discovery BI Wide Pore phases, the C8 phase gives efficient, symmetrical peaks, exceptional stability, long column lifetime, and LC/MS compatibility. PRPERTIES: Bonded Phase: Covalently-bonded octylsilane, endcapped Silica: Spherical, high purity (<0ppm metals) Particle Size:,, and 0mm Pore Size: 00Å Surface Area: 00m²/g %C: ~ Coverage: ~ µmoles/m² Discovery BI Wide Pore C Discovery BI Wide Pore C8 Discovery BI Wide Pore C Each Discovery BI Wide Pore Phase Gives Unique Elution Profiles of Carboxymethylated Apohemoglobin Peptide Fragments Columns: (A) Discovery BI Wide Pore C (Cat. o. 68-U), (B) Discovery BI Wide Pore C8 (Cat. o. 68-U), or (C) Discovery BI Wide Pore C8 (68-U), each cm x.6mm, µm Mobile Phase: (A) 9:, (0.% TFA in water):(0.% TFA in C C); (B) 0:0, (0.% TFA in water):(0.% TFA in C C) Flow Rate:.0mL/min Temp: 0 C Detection: nm Injection: 0µL carboxymethylated apohemoglobin tryptic digest in 0mM C Gradient: 0-00%B in 6 min G000-6

47 PLC: Biopolymers Discovery BI Wide Pore C Columns 9 Discovery BI Wide Pore C Discovery BI Wide Pore C was designed for the efficient and reliable separation of proteins and peptides, especially hydrophobic peptides, by RP- PLC. Long-chain phases, like C8 or C8, are often too hydrophobic for proteins and can cause excessively long retention time or even irreversible binding to the column. For this reason short-chain phases, typically C or C, are often used for RP-PLC of proteins. owever, these short-chain phases are susceptible to hydrolysis resulting in short column lifetime, especially at low p. The Discovery BI Wide Pore C gives elution order similar to a conventional C, yet has enhanced p stability for longer column lifetime. Generally, higher efficiency separations are achievable on the Discovery BI Wide Pore C because of the improvements we have made to the silica and bonded phase chemistry. PRPERTIES: Bonded Phase: Covalently-bonded pentylsilane, endcapped Silica: Spherical, high purity (<0ppm metals) Particle Size:,, and 0mm Pore Size: 00Å Surface Area: 00m²/g %C: ~% Coverage: ~ µmoles/m² mau 0nm mau 0nm Discovery BI Wide Pore C Impurity in Lysozyme Impurity in Myoglobin Conventional C Excellent Performance for Proteins Column: Discovery BI Wide Pore C, cm x.6mm, µm Cat. o.: 68-U Mobile Phase: (A) 0.% (v/v) TFA in : MeC (7:), (B) 0.% (v/v) TFA in : MeC (:7) Flow Rate: ml/min Temp: 0 C Det.: 0nm Inj.: 0µL Sample: Protein Test Mix in 0.% TFA Gradient: 0-00% B in min. G0088 G00 LEGT X ID PARTICLE SIZE (cm x mm) CAT.. PRICE DISCVERY BI WIDE PRE C (00Å,.% CARB) Efficiency: % of first injection µm x 0. 6-U µm 0 x 0. 6-U µm x U µm 0 x 0. 6-U µm x.0 6-U µm 0 x.0 6-U µm x. 676-U µm 0 x. 677-U µm x. 678-U µm x U µm 0 x U µm x.6 67-U µm x 0. 6-U µm x 0. 6-U µm x.0 6-U µm x U µm 0 x. 680-U µm x. 680-U µm x. 680-U µm x U µm 0 x.0 68-U µm x.0 68-U µm x.0 68-U µm x U µm 0 x.6 68-U µm x.6 68-U µm x.6 68-U µm x U 0µm x.6 67-U 0µm x U 0µm x U 0µm x U 0µm x. 676-U 0µm x. 677-U 0µm x. 678-U Supelguard Cartridges µm x. (/pk) 6778-U µm x. kit ** 6779-U µm x.0 (/pk) 6780-U µm x.0 kit ** 678-U µm x. (/pk) 6870-U µm x. kit ** 687-U µm x.0 (/pk) 687-U µm x.0 kit ** 687-U 0µm x U ** Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. Increased Stability of Discovery BI Wide Pore C over Conventional C Efficiency on Discovery BI Wide Pore C is stable even after,000 column volumes ( gradient cycles). G0088 Under the same conditions, there was significant loss of efficiency on competitive C. rder: Technical Service: Web: Liquid Chromatography SUPELC

48 60 PLC: Biopolymers Ion Exchange Chromatography Liquid Chromatography SUPELC rder: Technical Service: Web: MCI GEL Ion Exchange Columns Cytochrome c Species Variants by Ion Exchange PLC In ion exchange purifications and analyses of biopolymers, ProtEx-DEAE columns (diethylaminoethyl functionality) and ProtEx-SP columns (sulfopropyl functionality) offer significant benefits: Cytochrome c Source. Bovine Excellent separations of protein isoforms. orse. Rabbit igh resolution at low sample load Quantitative recovery a hydrophilic surface eliminates protein adsorption igh efficiency (plate number) ProtEx columns are.0cm x.6mm ID polyetheretherketone (PEEK) hardware packed with high performance, monodisperse, µm, 000Å macroporous polymethacrylate beads with a chemically bonded, crosslinked hydrophilic surface. Physical characteristics of these columns are summarized below. Reproduced with permission of Mitsubishi Chemical Corp. We especially recommend ProtEx columns for applications that Column: ProtEx-SP,.0cm x.6mm ID, µm particles Cat. o.: 70-U require high resolution, such as separating protein isoforms, Mobile Phase: A = 0mM Bis-Tris Cl, p 7.0; B = A + 0.M acl isozymes, or DA. % B 69% B in 0 min Flow Rate: 0.mL/min CLUM CARACTERISTICS Det.: UV, 80nm Dimensions:.0cm x.6mm ID Inj.: 0µg each isoform Bed Volume: 0.8mL Flow Rate: 0.-.mL/min Maximum Backpressure: 7psi (.9MPa) Temperature: C - 0 C p: - 7 Maximum Loading Capacity: 0mg/injection Proteins at Low Loading Concentrations. Myoglobin, µg. Conalbumin, µg. Trypsin inhibitor, µg Reproduced with permission of Mitsubishi Chemical Corp. Column: ProtEx-DEAE,.0cm x.6mm ID, µm particles Cat. o.: 7 Mobile Phase: A = 0mM Tris Cl, p 8. B = A + 0.M acl 0% B 00% B in 0 min Flow Rate: 0.mL/min Det.: UV, 80nm RELATED IFRMATI Request free literature by phone or fax, or see our website. o. Subject T9677 ProtEx ion exchange PLC columns T988 Mobile phases for ion exchange and chromatofocusing T907 Mono Q and Mono S columns P00009 PART. LEGT x ID DESCRIPTI SIZE (cm x mm) CAT.. PRICE ProtEx-SP µm x.6 70-U ProtEx-DEAE µm x.6 7 Amersham Biosciences Ion Exchange Columns Mono columns are highly efficient, p-stable columns designed for high performance ion exchange separations of proteins, peptides, and polynucleotides, in applications including peptide mapping and monoclonal antibody purification. The unique properties of these columns are based on MonoBeads support a beaded hydrophilic material with the narrowest particle size distribution of any chromatographic support. This monodispersity permits high flow rates at relatively low backpressures. DESCRIPTI QTY. CAT.. PRICE AMERSAM BISCIECES I EXCAGE CLUMS Mono Q R/ 807 Mono S R/ 808 Mono P R/ 809 CRMATFCUSIG REAGETS Pharmalyte 8-0. ml P7-ML Polybuffer 7 00mL P96-00ML Polybuffer 7 0mL P96-0ML Polybuffer 96 ml P9777-ML Polybuffer 96 00mL P ML Polybuffer 96 0mL P9777-0ML

49 PLC: Biopolymers Ion Exchange Chromatography 6 TSK-GEL Ion Exchange Columns TSK-GEL ion exchange columns are highly efficient and combine sample purification with excellent recovery rates. Anion- ionexchangers are available on porous polymer-based and silica-based matrices, and in nonporous resin (PR) columns. The various and cat column types are described in the table below. TSK-GEL PW and PR ion exchange columns are stable between p -; TSK-GEL SW columns can be used from p -7.. TSK-GEL SW ion exchange columns (Å pores) are best suited for small molecular weight solutes, such as nucleotides. Larger biomolecules, including peptides and small proteins, can be analyzed TSK-GEL on SW ion exchange columns (0Å pores). The wide-pore (000Å), polymer-based PW columns are suitable for analyses and purifications of large proteins and nucleic ample acids. S capacity for a 7.cm x 7.mm PW ion exchange column is approximately mg. Proteins and nucleic acids can be analyzed - times faster on a nonporous TSK-GEL PR column. The sample capacity of these columns for proteins is, however, 0-00 times smaller. TSK-GEL DEAE-PR columns are commonly used to separate DA fragments, particularly those obtained from the polymerase chain reaction (PCR). We strongly recommend using a DEAE-PR guard column to protect the analytical column when analyzing PCR fragments. SP-PR columns can provide fast results in hemoglobin Ac screening. Due to their small particle size (.µm), packings in TSK-GEL PR columns must be protected by using a precolumn ter fil containing a 0.µm frit (Rheodyne in-line filter or Sigma-Aldrich precolumn filter). Liquid Chromatography CARACTERISTIC DEAE-PW DEAE-SW DEAE-SW DEAE-PR AI EXCAGE CLUMS Matrix hydrophilic resin silica silica hydrophilic resin Particle Size (µm) 0 0. Pore Size (Å) nonporous Functional Group -C C + (C ) -C C + (C ) -C C + (C ) -C C + (C ) Counter Ion Cl Cl Cl Cl p Range Exclusion Limit (PEG, Dalton),000,000 0,000 0, Capacity (mg BSA/mL) 0 0 not available Small Ion Capacity >0.meq/mL >0.meq/g >0.meq/mL >0.meq/mL pka.... CARACTERISTIC SP-PW SP-PR CM-PW CM-SW CM-SW CATI EXCAGE CLUMS Matrix hydrophilic resin hydrophilic resin hydrophilic resin silica silica Particle Size (µm) Pore Size (Å) 000 nonporous Functional Group -C C C S -C C C S -C C - -C C - -C C - Counter Ion a + a + a + a + a + p Range Exclusion Limit (PEG, Dalton),000,000 00,000,000 0,000 0,000 Capacity (mg b/ml) 0 0 Small Ion Capacity >0.meq/mL >0.meq/g >0.meq/mL >0.meq/mL >0.meq/mL pka..... TSK-GEL Ion Exchange (Anion) Columns PART. LEGT x ID DESCRIPTI SIZE (cm x mm) CAT.. PRICE TSK-GEL I EXCAGE (AI) CLUMS DEAE-PR.µm. x DEAE-PW 0µm 7. x DEAE-SW µm x Guard Columns and Kits DEAE-PR µm 0. x DEAE-PW 0µm x 6.0 kit 8070 DEAE-SW 0µm x 6.0 kit Kits include one cartridge, a stand-alone holder, ml packing, cm of /6 tubing, and nuts and ferrules. TSK-GEL Ion Exchange (Cation) Columns PART. LEGT x ID DESCRIPTI SIZE (cm x mm) CAT.. PRICE TSK-GEL I EXCAGE (CATI) CLUMS CM-PW 0µm 7. x SP-PR.µm. x SP-PW 0µm 7. x CM-SW µm x CM-SW 0µm 7. x Guard Column Kits CM-PW 0µm x 6.0 kit 8069 SP-PW 0µm x 6.0 kit 807 CM-SW 0µm x 6.0 kit RELATED IFRMATI Request free literature by phone or fax, or see our website. o. Subject T9077 TSK-GEL Ion Exchange Columns T0986 TSK-GEL PR Columns rder: Technical Service: Web: SUPELC

50 6 PLC: Biopolymers Gel Filtration Chromatography Liquid Chromatography SUPELC rder: Technical Service: Web: Amersham Biosciences Gel Filtration Columns TSK-GEL Gel Filtration Columns (Silica-Based) For use in an PLC system, use unions described under Accessories for R and iload Columns. Fast Desalting Column -Ready for use in an FPLC system. Superdex R Column -Filter Kit R 0, a filter tool, wrench, and instructions are included with Superdex R columns. i-load Superdex Column - A dismantling tool, support screen, increases in proportion with column length. 0mm net ring, -ring, and domed nut (in an accessory bag) are included. SEPARATI DESCRIPTI EXCLUSI LIMIT RAGE (PRTEIS) Fast Desalting Column >,000 Da,000-,000 Superdex Peptide >0,000 Da 00-7,000 Superdex 7 >00,000 Da,000-70,000 Superdex 00 >00,000 Da 0, ,000 DESCRIPTI QTY. CAT.. PRICE FAST DESALTIG CLUMS Fast Desalting R0/0 80-U PD-0 Columns 0 80 Empty PD GEL FILTRATI CLUMS Superdex Peptide R0/0 06 Superdex 7 R0/0 800-U Superdex 00 R0/0 80-U iload 6/60 Superdex 7 PG 80-U ACCESSRIES FR R AD ILAD CLUMS Filters, R U Filter Tool 86 Unions, /6", female to M6 female Waters 866 Valco Adapter, male 0-/female M6 868 Column End Plugs (Domed ut) for M6 male 86 Capillary Tubing, PTFE m x.8mm D x 0.mm ID 870 Syringe barrel sample preparation columns; cannot be connected to any system. Amersham Biosciences columns are supplied ready for installation into an FPLC system. To connect these columns to an PLC system, use one of the three listed unions. RELATED IFRMATI Request free literature by phone or fax, or see our website. o. Subject T9076 TSK-GEL SW and SW XL Columns TSK-GEL SW andtsk-gel SW XL columns contain silicabased, hydrophilic bonded phase packings that minimize interaction with proteins. Properties of these popular columns are summarized in the table below. A 0cm TSK-GEL SW XL column and a 60cm TSK-GEL SW column provide similar resolution, but the SW XL column requires half the time. Sample capacity Because TSK-GEL SW XL and TSK-GEL SW columns are silicabased, they must be operated within the recommended p range of.-7.. Detailed operating conditions are described in the information accompanying the columns. We recommend protecting these columns with the appropriate SW XL or SW guard column. TSK-GEL QC-PAK columnsprovide fast, high resolution analyses, especially in quality control applications. The cm glass or stainless steel columns are packed with µm SW XL materials. TSK-GEL PARTICLE SIZE PRE SIZE SAMPLE MW CLUM (µm) (Å) (GLBULAR PRTEIS) Super SW000-0 x 0 G000SW XL -0 x 0 G000SW 0-00 x 0 Super SW x 0 G000SW XL x 0 G000SW x 0 G000SW XL ,000 x 0 G000SW 0 0-0,000 x 0 Mobile Phase: 0.0M acl in 0.M phosphate buffer, p 7.0 PART. LEGT x ID DESCRIPTI SIZE (cm x mm) CAT.. PRICE TSK-GEL GEL FILTRATI CLUMS G000SW XL µm 0 x G000SW XL µm 0 x G000SW XL 8µm 0 x Super SW000 µm 0 x Super SW000 µm 0 x G000SW 0µm 0 x G000SW 0µm 60 x G000SW 0µm 0 x G000SW 0µm 60 x G000SW µm 0 x G000SW µm 60 x TSK-GEL QC-PAK GEL FILTRATI CLUMS GFC 00 µm x GFC 00 µm x GFC 00GL µm x GUARD CLUMS SW XL 7µm x SW 0µm 7. x Glass.

51 PLC: Biopolymers Gel Filtration Chromatography 6 TSK-GEL Gel Filtration Columns (Polymer-Based) PARTICLE PRE TSK-GEL PW and TSK-GEL PW SIZE SIZE SAMPLE MW XL columns are used in high CLUM (µm) (Å) PEGs/PEs DEXTRAS performance gel filtration separations of water-soluble polymers G-ligo-PW 6 <000 and oligosaccharides. The hydrophilic polymer matrix has G000PW 0 <000 excellent chemical and mechanical stability. Although commonly G00PW XL 6 <00 <000 used with aqueous solvents, the polymer is compatible with up tog00pw 0 <00 <000 0% organic solvent. G000PW XL 6 00 <0,000 <60,000 G000PW 0 00 <0,000 <60,000 TSK-GEL GMPW and TSK-GEL GMPW XL columns are mixed G000PW XL , ,000 bed columns with calibration curves that are linear over a wide G000PW , ,000 range of molecular weights. Because the pore volume of a mixedg000pw XL ,000,000 0,000-7,000,000 bed column is the same as that for a narrow pore size column, the G000PW ,000,000 0,000-7,000,000 slope of the calibration curve is much steeper, which limits G6000PW XL >000 0,000-8,000,000 00,000-0,000,000 resolution. Mixed bed columns are ideal for preliminary investiga-g6000ptions, when the molecular weight composition of a sample is G-DA-PW ,000-8,000,000 7 >000 0,000-8,000,000 00,000-0,000,000 GMPW XL < ,000,000 <0,000,000 unknown. Then, unless the molecular weight distribution of the GMPW 7 < ,000,000 <0,000,000 sample is very broad, one selects a second column (or series of Mobile Phase: Polyethylene glycols/polyethylene oxides - distilled water columns) with a pore size (or range of pore sizes) that can provide Mobile Phase: Dextrans - 0.M phosphate buffer, p 6.8 optimum resolution. TSK-GEL G-ligo-PW columnsare specially prepared for separating noncharged or cationic oligomers. A small residual positive charge makes G-ligo-PW and G000PW columns unsuitable for analyses of anionic oligomers. PART. LEGT x ID DESCRIPTI SIZE (cm x mm) CAT.. PRICE TSK-GEL GEL FILTRATI CLUMS G-ligo-PW 6µm 0 x G00PW XL 6µm 0 x G000PW XL 6µm 0 x G000PW XL 0µm 0 x G000PW XL 0µm 0 x G6000PW XL µm 0 x GMPW XL µm 0 x G-DA-PW 0µm 0 x G000PW 0µm 0 x G00PW 0µm 0 x G000PW 0µm 0 x G000PW 7µm 0 x G000PW 7µm 0 x G6000PW 7µm 0 x GMPW 7µm 0 x GUARD CLUMS G-ligo-PW µm x PW XL µm x PW µm 7. x RELATED IFRMATI Request free literature by phone or fax, or see our website. o. Subject T907 TSK-GEL PW and PW XL Columns Molecular Weight Elution Volume (ml). G00PW XL. G000PW XL. G000PW XL. GMPW XL. G000PW XL 6. G6000PW XL PEG/PE Calibration Curves on TSK-GEL PW XL Columns Column : G00PW XL, 0cm x 7.8mm, 6µm particles Cat. o.: Column : G000PW XL, 0cm x 7.8mm, 6µm particles Cat. o.: 8080 Column : G000PW XL, 0cm x 7.8mm, 0µm particles Cat. o.: 8080 Column : GMPW XL, 0cm x 7.8mm, µm particles Cat. o.: 8080 Column : G000PW XL, 0cm x 7.8mm, 0µm particles Cat. o.: 8080 Column 6: G6000PW XL, 0cm x 7.8mm, µm particles Cat. o.: 8080 Mobile Phase: DI water Flow Rate: ml/min Det.: refractive index Inj.: polyethylene glycols and polyethylene oxides 708 rder: Technical Service: Web: Liquid Chromatography SUPELC

52 6 PLC: Biopolymers Reversed-Phase, Affinity, ydrophobic Interaction Chromatography Liquid Chromatography SUPELC rder: Technical Service: Web: SUPELCSIL Wide Pore (00Å) Reversed-Phase Columns TSK-GEL Reversed-Phase Columns SUPELCSIL Wide Pore reversed-phase columns continue to be PART. LEGT X ID available. Discovery BI Wide Pore reversed-phase columns DESCRIPTI SIZE (cm x mm) CAT.. PRICE provide enhanced performance and are recommended as the first choice. The Discovery BI Wide Pore columns can be found on the preceding pages. LEGT x ID PARTICLE SIZE (cm x mm) CAT.. PRICE SUPELCSIL LC-8 (00Å, 6.0% CARB) µm x.6 88 µm x SUPELCSIL LC-8 Supelguard Cartridges µm x.0 (/pk) 9 µm x.0 kit 90 SUPELCSIL LC-08 (00Å,.% CARB) µm x.6 88 µm x SUPELCSIL LC-08 Supelguard Cartridges µm x.0 (/pk) 9-U µm x.0 kit 90 SUPELCSIL LC-DP (00Å,.0% CARB) µm x SUPELCSIL LC-DP Supelguard Cartridges µm x.0 (/pk) 9 SUPELCSIL LC-0 (00Å,.7% CARB) µm x.6 88 µm x.6 88 SUPELCSIL LC-0 Supelguard Cartridges µm x.0 (/pk) 99 µm x.0 kit 99 SUPELCSIL LC-Si (00Å) µm x SUPELCSIL LC-Si Supelguard Cartridges (use for LC-Si) µm x.0 (/pk) 960 µm x.0 kit 90 RELATED IFRMATI Request free literature by phone or fax, or see our website. o. Subject T0079 Separating Proteins and Peptides using SUPELCSIL columns. T9079 TSK-GEL Reversed-Phase Columns T9080 TSK-GEL Affinity Columns T9078 TSK-GEL ydrophobic Interaction Columns SILICA-BASED CLUMS ligo-da RP µm x.6 8 Super DS µm x.6 88 Super DS µm 0 x Guard Filters Filter (/pk) 8807 Stand-Alone older for Filters 8806 RESI-BASED CLUMS C8-PR.µm. x C8-PW 7µm x.6 8 Phenyl-PW RP 0µm 7. x Guard Filters Filter (/pk) 8807 Stand-Alone older for Filters 8806 TSK-GEL Affinity Columns Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules. Kits include one cartridge, a stand-alone holder, ml packing, cm of /6 tubing, and nuts and ferrules. PART. LEGT X ID DESCRIPTI SIZE (cm x mm) CAT.. PRICE ABA-PW 0µm 7. x Boronate-PW 0µm 7. x Chelate-PW 0µm 7. x Guard Column Kits Chelate-PW 0µm x 6.0 kit eparin-pw 0µm x 6.0 kit 8 TSK-GEL ydrophobic Interaction (IC) Columns PART. LEGT X ID DESCRIPTI SIZE (cm x mm) CAT.. PRICE Butyl-PR.µm. x Ether-PW 0µm 7. x Phenyl-PW 0µm 7. x Guard Column Kits Phenyl-PW 0µm x 6.0 kit 8076

53 PLC: Industrial Polymers Gel Permeation Chromatography 6 TSK-GEL Gel Permeation Columns Using Calibration Curves TSK-GEL R gel permeation columns are stable in solvents GPC is widely used for fingerprinting molecular weights of having a wide range of polarities. The particles do not swell or industrial polymers. For compounds of similar molecular shape, a shrink as the solvent is changed from toluene through methanol. sigmoidal calibration curve is obtained by plotting the logarithm owever, these columns cannot be used with polar solvents, such of molecular weight (MW) versus the elution volume e )(Vfor as water or water:methanol mixtures. Spherical µm polystyrene/ molecules of known weight. The optimal separation range is divinylbenzene particles provide a minimum of 6,000 plates perdefined by the linear portion of this curve. nce a calibration 0cm x 7.8mm ID column. Eight pore sizes are available (Figure curve is prepared, the elution volume for a polymer of similar A), ranging from an exclusion limit of about 000 Dalton for shape, but unknown weight, can be used to determine the MW. G000 R columns to more than 0,000,000 Dalton for Results are most accurate when the investigator prepares the G7000 R columns. The four mixed bed columns (, L, M, ) calibration curve and determines the molecular weight of the feature extended linear molecular weight operating ranges for unknown molecule on the same day, with the same mobile sample screening or more formal analyses. phases, etc. Liquid Chromatography AALYTE MLECULAR CLUM WEIGT RAGE (DALT) G000 <00 G000 <000 G00 <. x 0 G000 <.0 x 0 G000 <. x 0 G000 <. x 0 6 G6000 <~ x 0 7 G7000 <~ x 0 7 GM- <~ x 0 7 GM-L <.0 x 0 GM-M <.0 x 0 6 PART. LEGT X ID DESCRIPTI SIZE (cm x mm) CAT.. PRICE TSK-GEL R GEL PERMEATI CLUMS G000 R µm 0 x G000 R µm 0 x G00 R µm 0 x G000 R µm 0 x G000 R µm 0 x G000 R µm 0 x G7000 R µm 0 x GM R -L µm 0 x GM R -M µm 0 x GM R - µm 0 x Guard Columns R -L 7µm x TSK-GEL XL GEL PERMEATI CLUMS G000 XL µm 0 x G000 XL µm 0 x G00 XL µm 0 x G000 XL 6µm 0 x G000 XL 6µm 0 x G000 XL 9µm 0 x G6000 XL 9µm 0 x GM XL 9µm 0 x GM XL -T µm 0 x Guard Columns XL -L 6µm x XL - µm x Use to protect G000 R G000 R and GM R -L columns. Use to protect G000 XL G000 XL columns. Use to protect G000 XL G6000 XL columns. Molecular Weight (dalton) G000 R. G000 R. G00 R. G000 R. G000 R 6. G000 R 7. G6000 R 8. G7000 R Elution Volume (ml) 8 Molecular Weight (dalton) Sample Elution by Molecular Weight LGM R -L GM R - MGM R -M GM R - L Elution Volume (ml) Columns: TSK-GEL R, 0cm x 7.8mm ID, µm particles Mobile Phase: tetrahydrofuran Temp.: ambient Flow Rate: ml/min Det.: UV, nm Inj.: polystyrene standards RELATED IFRMATI Request free literature by phone or fax, or see our website. o. Subject T9608 TSK-GEL R Columns 79-07, 076 rder: Technical Service: Web: SUPELC

54 66 PLC Custom, Endfittings, Connectors Liquid Chromatography SUPELC rder: Technical Service: Web: Custom Prepared PLC Columns If the column of your choice is not listed as a stock product in our catalog, we may be able to prepare it for you as Listed a custom. below is our current listing of available packings. For the most current listing of hardware, and a price quotation, ntact please co Technical Service. Delivery: We typically ship custom prepared analytical PLC columns within to 7 business days to anywhere in the world. Larger sizes (preparative and special requests) may take longer. Performance Testing: Supelco s custom prepared columns are fully tested for efficiency and symmetry. Please let us know if you have special iteria. test cr PASE PARTICLE SIZE (µm) DISCVERY BI WIDE PRE PASES RP-AmideC6 C8 C8 Cyano DISCVERY S PASES C8 C8 C8 0 F F F 0 PEG PEG PEG 0 DISCVERY BI WIDE PRE PASES C8 C8 C8 0 C8 C8 C8 0 C C C 0 SUPELCSIL PASES ABZ + Plus ABZ + Plus ABZ + Plus LC-ABZ Suplex pkb-00 LC-8-DB LC-8-DB LC-8 LC-8 LC-8 Replacement Discovery/SUPELCSIL Column Endfittings Use when fittings become damaged or with replacement cartridges. (pk. of ) 00-U DESCRIPTI CAT.. PRICE For.mm columns 0-U For.0mm,.0mm,.6mm columns 00-U PASE PARTICLE SIZE (µm) SUPELCSIL PASES, CT D LC-8 LC-DABS LC-PA LC-PA LC-8-S LC-8-T LC-8-T LC-8-DB LC-8-DB LC-8 LC-8 LC-8 LC-08 LC-DP LC-DP LC-F LC-0 LC-C LC-C LC-PC LC- LC-Si LC-Si LC-Si LC- LC- LC- -P LC-Diol LC-Diol SAX LC-SCX isep SUPELCGEL PASES TPR-00 DP-0 Ion Exclusion 9 Column Connector Connects two Discovery or SUPELCSIL analytical columns. Zero dead volume design. DESCRIPTI CAT.. PRICE Column Connector

55 Pharmaceutical Applications PLC and SPE Products for Pharmaceutical Analysis and Purification SUPELC Discovery Pharmaceutical Applications Book

56 TABLE F CTETS Analyte Page PLC Column SPE Tube Acetaminophen and Caffeine from Serum Discovery DSC-8Lt a-ydroxy Aliphatic Acids Discovery RP-AmideC6 Alkaloids Discovery RP-AmideC6, C8, and C8 Aniline omologs Discovery C8 Antibiotics (ß-Lactam) 6 Discovery RP-AmideC6, C8, C8, and Cyano Antibiotics (Cephalosporins) 6 Discovery RP-AmideC6, C8, and C8 Antibiotics (Cephalosporins from Serum) 7 Discovery DSC-8Lt Antibiotics (Fluoroquinolone from Tablets) 7 Discovery RP-AmideC6, C8, and S C8 Antibiotics (Peptides) 8 Discovery RP-AmideC6 and C8 Antibiotics (Sulfa Drugs) 8 Discovery C8 Antibiotics (Sulfa Drugs from Serum) 9 Discovery DSC-8 Anticonvulsants 9 Discovery RP-AmideC6, C8, and C8 Anticonvulsant Compounds from Serum 0 Discovery DSC-8Lt Anticonvulsants/Anxiolytics from Serum 0 Discovery DSC-8 Antidepressants (Tricyclic) Discovery C8 and C8 Antidepressants (Tricyclic) from Serum Discovery DSC-8 Antihypertensive-Diuretic Combination Discovery RP-AmideC6 Antihypertensive ACE Inhibitors Discovery RP-AmideC6 Antihypertensive Drugs Discovery RP-AmideC6 Antihypertensive/Antiarrhythmic Compounds from Serum Discovery DSC-8 Antipyretics/Analgesics/Antifungals Discovery RP-AmideC6 Antitussives/Antihistamines/Antipyretics Discovery RP-AmideC6, C8, and C8 Antiulcer Compounds Discovery RP-AmideC6, C8, C8, and S C8 Antiulcer Compounds from Serum Discovery DSC-8 Barbiturates 6 Discovery RP-AmideC6, C8, and C8 Barbiturates from Serum 6 Discovery DSC-8Lt Benzalkonium Chlorides 7 Discovery Cyano Bronchodilator (Caffeine Metabolites) from Serum 7 Discovery DSC-8 Catecholamines 8 Discovery C8 Chlorophyll from Methanolic Plant Extracts 8 Discovery DPA-6S Cold Remedy Ingredients 9 Discovery RP-AmideC6, C8, C8, and Cyano Fluoxetine (Prozac) 9 Discovery RP-AmideC6, C8, C8, and Cyano umic Acids from Water 0 Discovery DPA-6S onsteroidal Antiinflammatory Drugs 0 Discovery RP-AmideC6 onsteroidal Antiinflammatory Drugs from Serum Discovery DSC-8 rganic acids Discovery C8 and S C8 Parabens Discovery RP-AmideC6, C8, C8, and Cyano Peptides (Angiotensins) Discovery C8 Peptides (Carboxyamidomethylated BSA Tryptic Digest) Discovery RP-AmideC6 and C8 Peptides (Cytochrome c Tryptic Digest) Discovery RP-AmideC6 and C8 Peptides (Substance P and Fragments) Discovery C8 Phloroglucinol from Water Discovery DPA-6S Proteins (Bovine Insulin) Discovery C8 Tannic Acid from Water or Methanol Discovery DPA-6S Vitamins (Fat Soluble, A and E) 6 Discovery C8 and C8 Vitamins (Fat Soluble, D and D ) 6 Discovery C8 and S C8 Vitamins (Water Soluble) 7 Discovery RP-AmideC6, C8, and C8 SUPELC Discovery Pharmaceutical Applications Book

57 PLC CLUMS PLC column Icon Features PLC column Icon Features RP-AMIDE RP-AmideC6 excellent reproducibility unique selectivity C excellent retention and resolution of polar Si (C)C-(C) C compounds C less hydrophobicity than C8 phases C Si (C ) 7 C different elution profiles compared to C8 phases C C8 SLID PASE EXTRACTI TUBES compatible with low organic/highly aqueous mobile phases C8 excellent reproducibility faster separation of strongly hydrophobic analytes C exceptional peak shapes for basic and acidic Si (C ) 7 C compounds C excellent stability from p to p 8 suitable for LC/MS applications SPE C8 C8 excellent reproducibility exceptional peak shape for basic and acidic analytes S C8 greater (Discovery S C8) and lesser (Discovery C8) hydrophobic phases available resolution of geometrical isomers and other structurally closely related compounds separation of peptides and small proteins (especially Discovery C8) suitable for LC/MS applications (especially Discovery S C8) CYA Cyano excellent reproducibility C low hydrophobicity, for rapid elution of hydrophobic molecules C Si (C ) C retention and separation of strongly basic analytes, including quaternary ammonium salts, with excellent peak shapes compatible with highly aqueous mobile phases exceptional stability and column lifetime SPE tube features SPE tube features DSC-8 Polymerically bonded octadecyl (C8)-bonded silica isolates, purifies and concentrates pharmaceuticals and related compounds from aqueous media, like biological fluids 8% typical %carbon loading for high hydrophobic capacity Careful quality-control testing ensures consistent lot-to-lot and tube-totube chemical and physical properties DSC-8Lt A moderate carbon loading bonded silica (% carbon) with carbon loading lower than that of its sister product, DSC-8 (8% carbon) Lower carbon loading is useful when high hydrophobic capacity is not desired, or for extracting polar compounds that need secondary interactions with the silica backbone for efficient extraction Ideal for eluting hydrophobic compounds with small volumes of solvent, relative to volumes that are needed when using high-carbon loading silicas DPA-6S Polyamide, used to adsorb polar compounds such as those with hydroxyl groups, especially phenolic substances Polyamide has been used for extracting tannis, chlorophyll, humic acid, pharmacologically active terpenoids, flavonoids, gallic acid, catechol A protocatechuic acid, and phloroglucinol A from aqueous or methanolic solutions Carboxylic acids, especially aromatic carboxylic acids and compounds with multiple carboxylic acid groups, also can bind with the polyamide sorbent, forming hydrogen bonds SUPELC Discovery Pharmaceutical Applications Book

58 Acetaminophen and Caffeine from Serum PLC Conditions: Discovery C8, cm x 6mm, µm particles, Me: mm K P, p7 0 (:97) 0mL/min 0 C UV, 0nm 0µL Discovery SPE Tube: DSC-8Lt 00mg/mL SPE Procedure, Using Zymark RapidTrace SPE Workstation Solvent/ Volume Flow Rate Step Solution (ml) (ml/min) Comments Condition Me 0 0 conditions sorbent Condition 0 0 conditions sorbent Load spiked porcine serum 0 A 0 7 applies serum sample Rinse % Me 0 0 washes sample Purge-Cannula cleans sample cannula 6 Rinse vent 0 0 positions SPE tube over waste port 7 Dry Time = 0 min dries sorbent 8 Purge-Cannula Me cleans sample cannula 9 Collect Me 0 0 elutes analytes into collection vessel 0 Collect vent pushes residual eluent into vessel B Purge-Cannula cleans sample cannula A ml porcine serum spiked with 0 µg/ml acetaminophen and 0µg/mL caffeine, then diluted with ml water B SPE Eluent evaporated to dryness with nitrogen stream at 0 C, using a Zymark TurboVap LV Workstation, then reconstituted with 00µL of water Acetaminophen and Caffeine from Serum eutral antipyretic/analgesic compounds like acetaminophen can be extracted from serum, using Discovery DSC-8Lt SPE tubes and a generic methanol and waterbased SPE method This method is applicable to many other neutral compounds Recoveries for acetaminophen and caffeine are about 9% with this method Acetaminophen CC Efficiency of Recovery C C Compound Concentration %Recovery %RSD (n=6) Acetaminophen 0 0µg/mL 9 ±0 9 Caffeine 0µg/mL 9 ± Caffeine C G000, G a-ydroxy Aliphatic Acids Discovery RP-AmideC6 cm x 6mm, µm particles, mm K P, p 0 ml/min C UV, 0nm 0µL, µg/ml each analyte Tartaric acid Malic acid Lactic acid Citric acid RP-AMIDE a-ydroxy Aliphatic Acids a-ydroxy aliphatic carboxylic acids are strongly acidic compounds, with pka values around 0 and can significantly interact with residual silanols This chromatogram shows the separation of four such hydroxy acids on a Discovery RP- AmideC6 column under buffered acidic mobile phase conditions This example demonstrates the applicability of the amide-functionalized bonded phase for separations requiring 00% aqueous mobile phases without chain collapse as observed for C8 columns Tartaric acid C C C C Lactic acid Malic acid C C C C Citric acid C C C C C C C C C Time (min) G00, G00 G00, G00 SUPELC Discovery Pharmaceutical Applications Book

59 Alkaloids Alkaloids are naturally occurring bases with complex multicyclic ring structures They are difficult candidates for separation based on RP-PLC as they are amenable to hydrophobic as well as polar and ionic interactions with a silica-based reversed phase packing material They can tail significantly due to ion exchange with residual silanols, and mobile phase additives are commonly employed to alleviate this problem They can be analyzed on a Discovery column under isocratic conditions, without any additive, and show excellent peak symmetry The RP-AmideC6 column shows the best separation characteristics for the test mixture, with all components eluting within minutes Under the methanol/ buffer conditions used, papaverine and noscapine coelute on the C8 column All columns provide excellent separation for quinine and quinidine, which differ only in stereochemistry at the exocyclic carbon to the C carbon of the quinoline ring Papaverine and noscapine, which differ in the number of methoxyls and the absence/presence of a lactone group, also are best resolved on the RP- AmideC6 column C C=C Codeine Strychnine Quinidine C C Time (min) RP-AMIDE, 6 C8 G Ti ( i ) 6 C8 G00069 Alkaloids cm x 6mm columns, µm particles, Me/mM K P, p 0 (0:80) ml/min C UV, nm 0µL 6 Codeine Strychnine Quinidine Quinine oscapine Papaverine Quinine C C=C oscapine C C C Papaverine C C C G000, G000, G0000, G000, G0008, G0009 C C 0 0 Ti ( i ) G Aniline omologs Aniline derivatives are weak bases if the amine moiety is primary, but progressively become more basic with alkyl substitution on the nitrogen atom Thus, aniline has a pka of 6, while -ethylaniline and,-diethylaniline have values of and 6 6, respectively These bases can interact with acidic silanols through ionic interaction The excellent peak symmetries obtained for six aniline derivatives on a Discovery C8 column indicate the absence of any such reactions under unbuffered mobile phase conditions Aniline -Ethylaniline -Propylaniline,-Dimethylaniline -Butylaniline,-Diethylaniline C C C C 7 C 7 (C ) 6 C8 Aniline omologs Discovery C8, cm x 6mm, µm particles, Me: (60:0) ml/min 0 C UV, nm 0µL, µg/ml each analyte Aniline -Ethylaniline -Propylaniline,-Dimethylaniline -Butylaniline 6,-Diethylaniline G00079, G00, G006, G00009, G007, G008 SUPELC Discovery Pharmaceutical Applications Book G0009 Time (min)

60 Antibiotics (ß-Lactam): cm x 6mm columns, µm particles, A: 0 0% TFA in B: 0 0% TFA in MeC ml/min Gradient (RP-AmideC6, C8, C8) min %B 0 0 Gradient (Cyano) min %B Amoxicillin Ampicillin Piperacillin Penicillin G Penicillin V Cloxacillin Time (min) Time (min) 6 6 RP-AMIDE C8 G0078 G Time (min) G Time (min) 6 6 C8 CYA G008 Antibiotics (ß-Lactam): b-lactam antibiotics contain 6-aminopenicillanic acid as the basic skeleton and structurally vary from each other with respect to the 6-acyl substituent These antibiotics are weakly acidic and are rapidly inactivated by strong acids or bases Amoxicillin is the most polar of the series investigated, due to the phenolic hydroxyl and amino moieties on the acyl chain Ampicillin, which carries an amino group on its acyl chain, is less polar than amoxicillin Penicillin G and V are much less polar than amoxicillin or ampicillin, as they do not contain any polar functionality on the acyl chain Cloxacillin consists of a chlorophenyl-isoxazole moiety on the acyl chain, and is the most hydrophobic due to this feature The interaction of the stationary phase with the acyl chain appears to be the governing factor in the RP-PLC of these antibiotics The carboxylic group is sterically hindered by the dimethyl group on the adjoining carbon of the thiazolidine ring Since TFA is a strong protonating agent, the functional groups on ampicillin and amoxicillin are protonated, hence these two are less retained o significant selectivity differences among the Discovery bonded phases are observed under the gradient conditions utilized The Discovery Cyano column requires a more gradual gradient for optimal resolution Amoxicillin CC Penicillin G C C S S C C C C C C Ampicillin CC Penicillin V C C S S C C C C C C C Cl Piperacillin G0076, G0077, G00099, G007, G007, GG007 Cloxacillin C C S S C C C C C C Antibiotics (Cephalosporins) conditions for RP-AmideC6: cm x 6mm column, µm particles, Me:mM K P, p 0 (0:90) ml/min, 0 C, nm, µl conditions for C8, C8: cm x 6mm column, µm particles, Me:mM K P, p 0 (0:80) ml/min, 0 C, nm, µl Cefadroxil Cefaclor Cephalexin G00077 Ti ( i ) RP-AMIDE C8 C8 0 6 G00076 Ti ( i ) Antibiotics (Cephalosporins) Cephalosporins are weakly acidic antibiotics based on the -thia--aza bicyclo [,,0] oct--ene--carboxylic acid skeleton, with pka values of and 7 Cefadroxil and cephalexin differ only by a phenolic hydroxyl on the acyl chain of the former Cefaclor and cephalexin differ in the -substituent on the thiazine nucleus, with chlorine the functionality in the former and methyl in the latter The C8 and C8 columns show similar elution patterns, with the most hydrophobic component, cephalexin, eluting last A more aqueous mobile phase is needed for the RP-AmideC6 column to separate cefaclor from cephalexin Cefadroxil CC S C C Cephalexin S CC C C CC Cefaclor S C Cl 0 6 Ti ( i ) G G00068, G00068, G SUPELC Discovery Pharmaceutical Applications Book

61 Antibiotics (Cephalosporins) from Serum These closely related sulfur- and nitrogen-containing compounds can be extracted from serum using Discovery DSC-8Lt SPE tubes and a simple SPE method The recoveries for all compounds are about 90% S Cefuroxime C C C C G0006, G0007, G0008, G0009 Cefotaxime C C Efficiency of Recovery S S C C C CC C C Compound Concentration %Recovery %RSD (n=6) Cefotaxime µg/ml 89 0 ± Cefazolin µg/ml 9 9 ± Cefuroxime 0µg/mL 89 6 ± 7 Cefmetazole µg/ml 9 ± C C C S C SC C C S C C C S Cefazolin Cefmetazole C S C S C SPE Procedure, Using Zymark RapidTrace SPE Workstation Solvent/ Volume Flow Rate Step Solution (ml) (ml/min) Comments Condition Me 0 0 conditions sorbent Condition 0 0 conditions sorbent Load sample A 0 7 applies serum sample Rinse 0 0 washes sample Purge-Cannula clean sample cannula 6 Rinse vent 0 0 positions SPE tube over waste port 7 Dry Time = min dries sorbent 8 Purge-Cannula Me cleans sample cannula 9 Collect Me 0 0 elutes analytes into collection vessel 0 Purge-Cannula Me cleans sample cannula Purge-Cannula cleans sample cannula A ml porcine serum spiked with each analyte, then diluted with ml of 0 m K P (p 7 0) B SPE Eluent was evaporated to dryness with a nitrogen stream at C, using a Zymark TurboVap LV Workstation The residue was reconstituted with 00µL water prior to PLC analysis Antibiotics (Cephalosporins) from Serum PLC Conditions: Discovery C8, cm x 6mm, µm particles, preceded by C8 guard column and 0 µm frit filter MeC:0mM K P p 0 (adjusted with 0% phosphoric acid) (:86) ml/min, 0 C, UV, nm 0µL reconstituted porcine serum SPE Tube: DSC-8Lt 00mg/mL Antibiotics (Fluoroquinolones from Tablets) Fluoroquinolone antibiotics consist of the,-dihydro--oxo--quinoline carboxylic acid skeleton (nalidixic acid), along with a 6-fluoro substituent They differ from each other in the substitution pattern on the quinolone ring nitrogen and on the piperazinyl moiety at the 7- position of the quinoline The lone exception is trovafloxacin, which has a pyridino-pyridone skeleton Fluoroquinolone antibiotics are amphoteric, due to the acidic carboxyl group and the basic quinoline and piperazine nitrogens Their separation under RP-PLC conditions is governed by the differences in substitution patterns Thus, the elution order ciprofloxacin<lomefloxacin<sparfloxacin stems from the gradual increase in methyl substitution on the piperazine moiety Grepafloxacin, with a -methyl substituent on the quinoline ring, is retained longer than lomefloxacin Trovafloxacin, due to the difluorophenyl group on the quinoline nitrogen, is the most hydrophobic of the analytes studied and is eluted last Under the gradient conditions employed, the only significant difference among the RP-AmideC6 and C8 columns is the longer retention of ciprofloxacin on the former, possibly due to -bonding interactions The S (high surface area) C8 provides somewhat better resolution than the other C8 phase due to more retention (higher k ) C Levofloxacin Ciprofloxacin Lomefloxacin F C C F C Sparfloxacin Grepafloxacin Trovafloxacin C F F C C F Time (min) G RP-AMIDE S C8 6 C Time (min) G00 Antibiotics (Fluoroquinolones from Tablets) cm x 6mm columns, (A) mm phosphate buffer, p 0 (B) MeC 0% B to % B in min ml/min C UV, 0nm 0µL 6 Levofloxacin Ciprofloxacin Lomefloxacin Sparfloxacin Grepafloxacin Trovafloxacin C G00097, G00097, G000970, G00097, G000968, G00097 C F F C C F C C F F C 0 0 Time (min) G00 SUPELC Discovery Pharmaceutical Applications Book 7

62 Antibiotics (Peptides) cm x 6mm columns, µm particles, MeC:mM K P, p 0 (0:60) ml/min C UV, 0nm 0µL, 0µg/mL each analyte RP-AMIDE C8 Antibiotics (Peptides) Bacitracin A is a cyclic decapeptide, carrying an aminoalkylthiazoline substituent on the terminal leucine moiety Virginiamycin M is a macrocyclic molecule containing an oxazole and a pyrroline ring as part of the cyclic skeleton, and does not contain any amino acid in its structure wing to the presence of multiple amide linkages, bacitracin A is much more polar than Virginiamycin M and is eluted first Virginiamycin M is retained longer on the RP-AmideC6 column, since it potentially undergoes dipolar interactions through the keto and ester functionalities with the amide moiety of the stationary phase Bacitracin A Virginiamycin M G G0009 Ti ( i ) Ti ( i ) D-Phe L-Ile D-rn Bacitracin A L-is D-Asp L-Asn C Virginiamycin M C C L-Lys L-Ile D-Glu L-Leu C C S G0009, G00096 Antibiotics (Sulfa Drugs) Discovery C8 cm x 6mm column, µm particles, Me: with % acetic acid (0:80) ml/min 0 C UV, nm 0µL, µg/ml each analyte Sulfanilamide Sulfathiazole Sulfamerazine Sulfamethazine C8 Antibiotics (Sulfa Drugs) These drugs are based on a -aminobenzenesulfonamide skeleton and carry a thiazole or pyrimidine system on the sulfonamide nitrogen The parent sulfanilamide exhibits pka values of 0 and 7, and the heterocylic derivatives show values of 7 and 6 The sulfonamide moiety is acidic and forms salts readily, while the aromatic amino group imparts basic properties to these drugs These polar functionalities make the PLC separation of sulfa drugs difficult The longer retention of sulfamerazine and sulfamethazine may be attributable to the hydrophobic effect of the methyl substituents Sulfanilamide S Sulfathiazole S S Sulfamerazine Sulfamethazine S C C C S Time (min) G009, G00097, G00098, G00099 SUPELC Discovery Pharmaceutical Applications Book

63 Antibiotics (Sulfa Drugs) from Serum These four antibacterial agents in the sulfa drug family can be extracted from serum After acidifying a diluted serum sample, recoveries for the compounds are greater than 90%, using a simple methanol and water-based SPE method S S G00097, G00098, G00099, G Sulfathiazole Sulfamerazine Efficiency of Recovery S C Compound Concentration %Recovery %RSD (n=6) Sulfathiazole 0µg/mL 90 ± 7 0µg/mL 97 7 ± Sulfamerazine 0µg/mL 9 8 ± 8 0µg/mL 97 ± Sulfamethazine 0µg/mL 9 9 ± 8 0µg/mL 96 ± Sulfamethizole 0µg/mL 88 7 ± 0µg/mL 98 ± C C Sulfamethazine S S Sulfamethizole S C SPE Procedure, Using Zymark RapidTrace SPE Workstation Solvent/ Volume Flow Rate Step Solution (ml) (ml/min) Comments Condition Me 0 0 conditions sorbent Condition 0 0 conditions sorbent Load spiked porcine serum 0 A 0 7 applies serum sample Rinse % Me in 0 0 washes sorbent Purge-Cannula clean sample cannula 6 Rinse vent 0 0 positions SPE tube over waste port 7 Dry Time = 0 min dries sorbent 8 Purge-Cannula Me cleans sample cannula 9 Collect Me 0 0 elutes analytes into collection vessel 0 Collect vent pushes residual eluent into vessel B Purge-Cannula cleans sample cannula A ml porcine serum spiked 0µg/mL or 0µg/mL, diluted with ml water, then acidified with 0µL P B SPE Eluent evaporated to dryness with a nitrogen stream at 0 C, using a Zymark TurboVap LV Workstation, then reconstituted with ml mobile phase Antibiotics (Sulfa Drugs) from Serum PLC Conditions: Discovery C8, cm x 6mm, µm particles, preceded by cm C8 guard column and 0 µm frit filter Me: % acetic acid (8:9) 0mL/min (8 min) then 0 6mL/min 0 C UV, nm 0µL reconstituted porcine serum extract SPE Tube: Discovery DSC-8 00mg/mL Anticonvulsants These drugs contain a benzodiazepine nucleus Clonazepam is the most polar, due to the presence of the nitro substituent, and shows pka values of and 0 Clorazepate remains as the potassium salt under the neutral mobile phase conditions and is moderately acidic Diazepam is the most hydrophobic of the three and is a weaker acid with a pka of Clonazepam and clorazepate contain an moiety capable of -bonding with the amide group of the RP- AmideC6 stationary phase and hence is retained longer on this phase Diazepam is retained longer on the C8 phase, as expected from its hydrophobic interactions Clonazepam (IS) Clorazepate Diazepam Cl C Cl C G00077 G00076 Ti ( i ) Ti ( i ) RP-AMIDE C8 C8 Anticonvulsants cm x 6mm columns, µm particles, MeC: (0:70) ml/min 0 C UV, nm 0µL Clonazepam (IS) Clorazepate Diazepam Cl G00, G00099, G00000 SUPELC Discovery Pharmaceutical Applications Book G00076 Ti ( i ) 9

64 Anticonvulsant Compounds from Serum PLC Conditions: Discovery C8 cm x 6mm, µm particles, preceded by a cm C8 guard column and 0 µm frit filter Me: (:) 0mL/min 0 C UV, nm 0µL SPE Tube: DSC-8Lt 00 mg/ml SPE Procedure, Using Zymark RapidTrace SPE Workstation Solvent/ Volume Flow Rate Step Solution (ml) (ml/min) Comments Condition Me 0 0 conditions sorbent Condition 0 0 conditions sorbent Load spiked porcine serum 0 A 0 7 applies serum sample Rinse 0% Me in 0 0 washes sample Purge-Cannula cleans sample cannula 6 Rinse vent 0 0 positions SPE tube over waste port 7 Dry Time = 0 min dries sorbent 8 Purge-Cannula Me cleans sample cannula 9 Collect Me 0 0 elutes analytes into collection vessel 0 Collect vent pushes residual eluent into vessel B Purge-Cannula cleans sample cannula A ml porcine serum spiked with 0 µg/ml or 0 µg/ml each analyte and clonazepam (IS), then diluted with 0mL water B SPE Eluent evaporated to dryness with a nitrogen stream at 0 C, using a Zymark TurboVap LV Workstation, then reconstituted with 00µL methanol Anticonvulsant Compounds from Serum These anticonvulsant compounds, similar in structure, can be extracted from serum using Discovery DSC-8Lt SPE tubes, then analyzed on a Discovery C8 PLC column Recoveries range from 9 to 99% Cl Clorazepate C Efficiency of Recovery Diazepam Compound Concentration %Recovery %RSD (n=6) Clorazepate 0 0µg/mL 9 ± 0 0 0µg/mL 99 7 ± Diazepam 0 0µg/mL 9 8 ± 7 0 0µg/mL 98 ± 0 Cl C G00, G00099 Anticonvulsants/ Anxiolytics from Serum PLC Conditions: Discovery C8, cm x 6mm, µm particles, preceded by cm C8 guard column and 0 µm frit filter MeC:Me: mm K P (p 7 0 with triethylamine) (::) 0mL/min, ambient temp UV, nm 0µL reconstituted porcine serum extract SPE Tube: Discovery DSC-8 00mg/mL SPE Procedure, Using Zymark RapidTrace SPE Workstation Solvent/ Volume Flow Rate Step Solution (ml) (ml/min) Comments Condition Me 0 0 conditions sorbent Condition 0 0 conditions sorbent Load spiked porcine serum 0 A 0 7 applies serum sample Rinse MeC/Me/ 0 0 washes sorbent ::70 Purge-Cannula cleans sample cannula 6 Rinse vent 0 0 positions SPE tube over waste port 7 Dry Time = 0 min dries sorbent 8 Purge-Cannula Me cleans sample cannula 9 Collect Me 0 0 elutes analytes into collection vessel 0 Collect vent pushes residual eluent into vessel B Purge-Cannula cleans sample cannula A ml porcine serum spiked with 0 0µg/mL or 0 µg/ml each analyte and, then diluted with ml water B SPE Eluent evaporated to dryness with a nitrogen stream at room temp, using a Zymark TurboVap LV Workstation, then reconstituted with 00µL mobile phase Anticonvulsants/Anxiolytics from Serum Five closely related benzodiazepine derivatives were extracted from serum using Discovery DSC-8 SPE tubes and an automated SPE method igh recoveries and low RSD s for these compounds are observed when using these SPE tubes and the Zymark RapidTrace SPE Workstation xazepam Cl Temazepam Efficiency of Recovery Cl C Desmethyldiazepam Cl Medazepam Compound Concentration %Recovery %RSD (n=6) xazepam 0 0µg/mL 98 ± 8 0 0µg/mL 99 ±0 9 Temazepam 0 0µg/mL 98 6 ± 0 0µg/mL 97 7 ± Desmethyldiazepam 0 0µg/mL 98 6 ± 0 0µg/mL 0 7 ±0 8 Diazepam 0 0µg/mL 0 ± 0 0µg/mL 0 7 ±0 8 Medazepam 0 0µg/mL 9 9 ± 0 0 0µg/mL 97 0 ± 8 Diazepam Cl C Cl C G0008, G0008, G00086, G00000, G00087 C C 0 SUPELC Discovery Pharmaceutical Applications Book

65 Antidepressants (Tricyclic) These drugs are comprised of a dibenzo-cycloheptene or oxepin or azepin skeleton and are strongly basic in nature They tail significantly under RP-PLC conditions and mobile phase additives are frequently used to overcome this problem A Discovery C8 column can separate multi-component mixtures of these drugs without any mobile additive This chromatogram illustrates typical therapeutic levels of these compounds, including two internal standards, Chlorimipramine and orchlorimipramine ordoxepin Desipramine ortriptyline C8 Antidepressants (Tricyclic) cm x 6mm column, with C8 guard column, µm particles, MeC:mM K P, p 7 0 (:) ml/min C UV, 8nm 0µL, 0ng/mL of each analyte CC C C or-chlorimipramine (IS) (C ) C Cl C C C C Doxepin CC C C C Imipramine Amitriptyline Chlorimipramine (IS) CC C C ordoxepin Desimipramine ortriptyline or-chlorimipramine (IS) Doxepin Imipramine Amitriptyline Chlorimipramine (IS) C C C C C CC C (C ) G00, G00060, G00068, G00, G00069, G00060, G0009, G Cl (C ) (C ) Time (min) G006 Antidepressants (Tricyclic) from Serum This application shows the recoveries for four common tricyclic antidepressant compounds These basic compounds can be extracted and analyzed with a simple SPE method, using Discovery DSC-8 SPE tubes Recoveries for all are 9 to 00% ortriptyline CC C C G00068, G00069, G00060, G0009 Doxepin Efficiency of Recovery CC C C C SUPELC Discovery Pharmaceutical Applications Book Imipramine C C C C C Amitriptyline CC C (C ) Compound Concentration %Recovery %RSD (n=6) ortriptyline 0 0µg/mL 0 6 ± 0 0µg/mL 97 ± Doxepin 0 0µg/mL 0 ± 0 0 0µg/mL 00 8 ± 8 Imipramine 0 0µg/mL 9 0 ± 0 0µg/mL 97 ± 7 Amitriptyline 0 0µg/mL 9 6 ± 0 0µg/mL 9 7 ± SPE Procedure, Using Zymark RapidTrace SPE Workstation Solvent/ Volume Flow Rate Step Solution (ml) (ml/min) Comments Condition Me 0 0 conditions sorbent Condition 0 0 conditions sorbent Load spiked porcine serum 0 A 0 7 applies serum sample Rinse 0% Me in 0 0 washes sorbent Purge-Cannula cleans sample cannula 6 Rinse vent 0 0 positions SPE tube over waste port 7 Dry Time = 0 min dries sorbent 8 Purge-Cannula Me cleans sample cannula 9 Collect Me 0 0 elutes analytes into collection vessel 0 Collect vent pushes residual eluent into vessel B Purge-Cannula cleans sample cannula A ml porcine serum spiked with 0 µg/ml or 0 µg/ml each analyte basified with µl 0 K, then diluted with ml water B SPE 0µL water added per ml methanolic eluent before analysis Antidepressants (Tricyclic) from Serum PLC Conditions: Discovery C8 column, cm x 6mm, µm particles, preceded by cm C8 guard column and 0 µm frit filter MeC:Me: mm K P (p 7 0 with triethylamine) (::0) 0mL/min, ambient temp UV, nm 0µL diluted porcine serum extract SPE Tube: Discovery DSC-8 00mg/mL

66 Antihypertensive- Diuretic Combination Discovery RP-AmideC6 cm x 6mm column, µm particles, Me: mm K P, p 7 0 (:78) ml/min 0 C UV, nm 0µL, µg/ml of each analyte RP-AMIDE Antihypertensive-Diuretic Combination This mix consists of three diuretics, amiloride (a pyrazine derivative), hydrochlorothiazide (a benzothiadiazine derivative) and caffeine (a xanthine analog), along with a b-blocker, atenolol They are basic compounds capable of interactions with the silica surface through ionic interaction or hydrogen bonding in the presence of exposed siloxide ions A Discovery RP-AmideC6 column completely separates these compounds in approximately 6 minutes under neutral mobile phase conditions Atenolol Amiloride Atenolol Amiloride ydrochlorothiazide Caffeine C C C C CC CC ydrochlorothiazide Cl CC Caffeine S S C C Cl C G00080 Time (min) G0006, G00, G00, G Antihypertensive ACE Inhibitors Discovery RP-AmideC6 cm x 6mm column, µm particles, MeC:mM K P, p (:67) 0 6mL/min C UV, nm µl, µg/ml of each analyte Enalapril Lisinopril Captopril RP-AMIDE Antihypertensive ACE Inhibitors ACE inhibitors are amino acid derivatives containing an L-proline skeleton and incorporating highly polar functionalities such as thiol (captopril), secondary amine (enalapril and lisinopril), primary amine (lisinopril) and carboxyl (all three) They exhibit a wide range of pka values (e g, 0, 6 7 and 0 for lisinopril) Their PLC on reversed phase columns can be difficult, yet a complete separation of all compounds can be achieved in less than minutes on a Discovery RP-AmideC6 column C C Enalapril C CC C C C C C C Lisinopril C C (C ) C C C Captopril C C SC C C G00079 Time (min) G00, G00,G00 SUPELC Discovery Pharmaceutical Applications Book

67 Antihypertensive Drugs (calcium channel blockers, b-blocker and ACE inhibitor) Five antihypertensive drugs of different classes are resolved in a single separation on a Discovery RP-AmideC6 column They include the calcium channel blockers diltiazem, verapamil and nifedipine, with a benzothiazepinone, valeronitrile, and dihydropyridine skeleton, respectively, pindolol, a b-blocker with an indole skeleton, and captopril, an ACE inhibitor with an L-proline skeleton Desmethyldiltiazem is a metabolite of diltiazem and is commonly encountered in fluids of patients taking diltiazem These strongly basic molecules are difficult candidates for PLC A Discovery RP-AmideC6 column demonstrates excellent separation of these compounds at neutral p, and provides good peak characteristics SC Antihypertensive/Antiarrhythmic Compounds from Serum Basic drugs like procainamide and atenolol can be extracted from serum, using Discovery DSC-8 SPE tubes The results shown here are approximately 00%, recovery C G00009, G0006 Captopril Procainamide C C C C C C Efficiency of Recovery Pindolol ifedipine Diltiazem Verapamil C C C C C C C C CC CC CC(C) G00, G006, G000, G007, G000, G008 S CCCC(C) Desmethyldiltiazem Atenolol C C C C CC CC Compound Concentration %Recovery %RSD (n=6) Procainamide 0 µg/ml 98 6 ± 0 0µg/mL 99 7 ± Atenolol 0 µg/ml 0 7 ± 7 0 0µg/mL 00 ± C C C C S CC CCC C(C)CC C(C) C C C C G000 Time (min) SPE SPE Procedure, Using Visiprep SPE Vacuum Manifold Condition: ml Me, then ml mm K P (p 9 0) Apply Sample: Wash and Dry: Elute: ml porcine serum spiked with 0 µg/ml or 0 0µg/mL each analyte, basified with µl 0M K ml % Me in mm K P (p 9 0): dry tube 0 min with nitrogen stream ml Me; evaporate to dryness with nitrogen stream at room temperature; reconstitute in 00µL 6 RP-AMIDE Antihypertensive Drugs (calcium channel blockers, b-blocker and ACE inhibitor) Discovery RP-AmideC6 cm x 6mm column, µm particles, Me:mM K P, p 7 0 (0:60) ml/min C UV, nm µl, 0 µg/ml of each analyte 6 Captopril Pindolol Desmethyldiltiazem ifedipine Diltiazem Verapamil Antihypertensive/ Antiarrhythmic Compounds from Serum PLC Conditions: Discovery C8, cm x 6mm column, µm particles, preceded by a cm C8 guard column and 0 µm frit filter Me:mM K P p 7 0 (0:90) ml/min 0 C UV, 0nm 0µL reconstituted porcine serum extract SPE Tube: Discovery DSC-8 00mg/mL SUPELC Discovery Pharmaceutical Applications Book

68 Antipyretics/Analgesics/ Antifungals Discovery RP-AmideC6 cm x 6mm column, µm particles, MeC:, 0 % TFA (:7) ml/min 0 C UV, nm 0µL, µg/ml of each analyte RP-AMIDE Antipyretics/Analgesics/Antifungals These test probes consist of weakly acidic (acetaminophen) and strongly acidic (salicylic and benzoic acids and aspirin) benzene derivatives and an aliphatic dienoic acid (sorbic acid) These compounds may undergo hydrogen bonding under acidic mobile phase conditions if silanol groups are present on the bonded phase surface A Discovery RP-AmideC6 column provides excellent separation with good peak shapes for this application Acetaminophen Aspirin Sorbic acid C C C CC C C=CC=CC Acetaminophen Aspirin Sorbic acid Benzoic acid Salicylic acid Benzoic acid C Salicylic acid C Time (min) G G009, G0060, G00009 G006, G Antitussives/ Antihistamines/ Antipyretics cm x 6mm columns, µm particles, A) mm K P, p B) MeC 0% (min) to 0% B in min, hold min, to 0% B in 0 min ml/min, ambient temp UV, nm 0µL, µg/ml of each analyte S Solvent Acetaminophen Doxylamine Pseudoephedrine Codeine Chlorpheniramine G00078 G Ti ( i ) Ti ( i ) S RP-AMIDE S C8 C8 Antitussives/Antihistamines/Antipyretics Many cold remedies contain various combinations of acetaminophen, pseudoephedrine, and an antihistamine as ingredients The separation of a five component mix on Discovery columns is shown in this figure It is striking that the RP- AmideC6 column elutes doxylamine, pseudoephedrine and codeine before acetaminophen, while the C8 and C8 columns display the opposite behavior This selectivity difference may be exploited for method development of those cold remedies which contain large amounts of acetaminophen with minor quantities of other ingredients Acetaminophen CC C Codeine C Doxylamine C C C C (C ) C 6 Chlorpheniramine Pseudoephedrine C C (C ) C Cl C C.Cl G Ti ( i ) G000, G00, G00087, G000, G00 SUPELC Discovery Pharmaceutical Applications Book

69 Antiulcer Compounds These molecules are complex in structure, with a core heterocyclic ring (furan, imidazole or thiazole) and a side chain carrying a sulfide and secondary amine functionalities ther polar groups such as nitro, cyano or iminosulfonamide also are present on the side chain The hetero ring carries a tertiary amine or guanidine moiety These compounds are strongly basic and are not only difficult to separate, but also produce severe tailing from silanol interactions They require highly aqueous mobile phases for sufficient retention needed for achieving good separation A notable selectivity difference between the RP-AmideC6 and C8 columns can be seen in the reversal of the elution order of cimetidine and famotidine This reversal may be attributable to the -bonding interactions of the guanidine and/or sulfonamide groups on famotidine with the stationary phase amide moiety of RP- AmideC6, leading to its longer retention ote the better resolution of the S (high surface area) C8 compared to the other C8 column, attributable to the former column s greater retention of the analytes (C ) C S izatidine Ranitidine C Time (min) G007 RP-AMIDE C8 C Time (min) G008 S C8 Antiulcer Compounds cm x 6mm columns, Me:mM P /K, p 0 (:8) 0 ml/min ambient temp nm µl, 6µg/mL of each analyte izatidine Ranitidine Famotidine Cimetidine Cimetidine C C SC C -C-C (C ) C C SC C -C-C Famotidine S C C C -C-C C C -S -C C C C -S C G0007, G0008, G0009, G0000 S Time (min) G Time (min) G000 Antiulcer Compounds from Serum These basic, sulfur-containing compounds can be extracted from serum using Discovery DSC-8 SPE tubes under basic conditions The compounds can be analyzed subsequently on a Discovery C8 PLC column This method provides recoveries in the range of 9-98% for these compounds Ranitidine (C ) C C SC C -C-C G0008, G0007, G0009 Efficiency of Recovery C Cimetidine C -S C C C -C-C -C (C ) C Compound Concentration %Recovery %RSD (n=6) Ranitidine 0 µg/ml 9 ± 0 0µg/mL 9 ± Cimetidine 0 µg/ml 9 ± 0 0µg/mL 98 ± izatidine 0 µg/ml 97 0 ± µg/mL 9 8 ± S izatidine C C SC C -C-C SPE SPE Procedure, Using Visiprep SPE Vacuum Manifold Condition: ml Me, then ml mm K P (p 9 0) Apply Sample: Wash and Dry: Elute: ml porcine serum spiked with 0 µg/ml or 0 0µg/mL each analyte, basified with µl 0M K ml % Me in mm K P (p 9 0): dry tube 0 min with nitrogen stream ml Me; evaporate to dryness with nitrogen stream at room temperature; reconstitute in 00µL mobile phase Antiulcer Compounds from Serum PLC Conditions: Discovery C8, cm x 6mm, µm particles, preceded by cm C8 guard column and 0 µm frit filter Me: mm K P, p 7 0 (0:80) 0mL/min, ambient temp UV, nm 60µL reconstituted porcine serum extract SPE Tube: Discovery DSC-8 00mg/mL SUPELC Discovery Pharmaceutical Applications Book

70 Barbiturates cm x 6mm columns, µm particles, Me:, (:) ml/min ambient temp UV, nm µl (Discovery C8) or 0µL (Discovery RP-AmideC6, Discovery C8) µg/ml each of analyte 6 7 Barbital Aprobarbital Phenobarbital Butabarbital Mephobarbital Pentobarbital Secobarbital Barbiturates from Serum PLC Conditions: Discovery C8 cm x 6mm, µm particles, preceded by cm C8 guard column and 0 µm frit filter Me: (0:60) 0mL/min 0 C UV, nm 0µL SPE Tube: DSC-8Lt 00mg/mL Ti ( i ) G00079 Ti ( i ) SPE Procedure, Using Zymark RapidTrace SPE Workstation Solvent/ Volume Flow Rate Step Solution (ml) (ml/min) Comments Condition Me 0 0 conditions sorbent Condition 0 0 conditions sorbent Load sample 0 A 0 7 applies serum sample Rinse % Me in 0 0 washes sample Purge-Cannula cleans sample cannula 6 Rinse vent 0 0 positions SPE tube over waste port 7 Dry Time = 0 min dries sorbent 8 Purge-Cannula Me cleans sample cannula 9 Collect Me 0 0 elutes analytes into collection vessel 0 Collect vent pushes residual eluent into vessel B Purge-Cannula cleans sample cannula A 0 ml porcine serum spiked with 0 µg/ml or 0µg/mL each analyte, then diluted with 0 ml water B SPE, RP-AMIDE C G0009 G00090 Ti ( i ) Eluent evaporated to dryness with a nitrogen stream at 0 C, using a Zymark TurboVap LV Workstation, then reconstituted with 00µL water C8 Barbiturates These sedative drugs are good candidates for comparing the hydrophobic selectivity of reversed phase columns A mixture of seven barbiturates differing from each other in the extent of alkyl substitution were screened The RP- AmideC6 column coelutes phenobarbital and aprobarbital under these conditions, while the C8 and C8 show good baseline separation of these two C C Barbital Barbiturates from Serum Six drugs from this class of compounds were extracted from serum, using DSC-8Lt SPE tubes, and were analyzed subsequently on a Discovery C8 PLC column Recoveries for these compounds are greater than 9% using this simple methanol and water-based SPE method Efficiency of Recovery (C ) C C =CC Mephobarbital Ph C C Aprobarbital Phenobarbital For chemical structures, see above Compound Concentration %Recovery %RSD (n=6) Aprobarbital 0 µg/ml 98 ± 0µg/mL 00 8 ±0 8 Phenobarbital 0 µg/ml 96 ± 6 0µg/mL 9 9 ± 7 Butabarbital 0 µg/ml 97 ± 9 0µg/mL 98 7 ± 8 Mephobarbital 0 µg/ml 99 7 ± 0µg/mL 0 0 ± 0 Pentobarbital 0 µg/ml 96 ± 7 0µg/mL 96 ± 9 6 Secobarbital 0 µg/ml 98 ± 7 0µg/mL 97 7 ± 8 C Ph Pentobarbital C C C C C C a Butabarbital C C C C C Secobarbital C =CC C C C C C a G0000, G0000, G0000, G0000 G0000, G00006, G SUPELC Discovery Pharmaceutical Applications Book

71 Benzalkonium Chlorides These compounds are mixtures of alkyl benzyldimethylammonium salts with the alkyl moiety ranging from C 8 to C 8 They are used as topical antiseptives and preservatives These are retained for long periods on conventional reversed phase columns, and are not well resolved Because of its low hydrophobic retention, the Cyano column is best suited for this separation and elutes all components within 6 minutes The sample contained components of undefined alkyl chain length Benzalkonium Chlorides + CYA Benzalkonium Chlorides Discovery Cyano cm x 6mm column, µm particles, MeC:acetate buffer, p, (60:0) ml/min ambient temp UV, nm C C R C Cl Time (min) G00076 Bronchodilator (Caffeine Metabolites) from Serum Theophylline and other caffeine metabolites can be extracted from serum with Discovery DSC-8 SPE tubes, using the method shown here Recoveries are greater than 9% using a Visiprep SPE vacuum manifold Theobromine C C Paraxanthine C Efficiency of Recovery C Compound Concentration %Recovery %RSD (n=6) Theobromine 0 µg/ml 97 ±6 8 0 µg/ml 96 ±8 0µg/mL 96 ± 0 Paraxanthine 0 µg/ml 96 ±8 0 µg/ml 9 ±8 7 0µg/mL 9 0 ±8 7 Theophylline 0 µg/ml 97 8 ±8 0 µg/ml 97 8 ±8 8 0µg/mL 98 ± 7 Caffeine 0 µg/ml 98 8 ± 9 0 µg/ml 9 6 ±6 7 0µg/mL 97 6 ± 8 SUPELC Discovery Pharmaceutical Applications Book Theophylline C C Caffeine C C C G00088, G00089, G00090, G SPE SPE Procedure, Using Visiprep SPE Vacuum Manifold Condition: Apply Sample: Wash and Dry: Elute: ml Me, then ml ml porcine serum spiked with 0 µg/ml, 0 µg/ml, or 0µg/mL each analyte ml % Me in : dry tube 0 min with nitrogen stream ml Me; evaporate to dryness with nitrogen stream at room temperature; reconstitute in 00µL mobile phase Bronchodilator (Caffeine Metabolites) from Serum PLC Conditions: Discovery RP-AmideC6 column, cm x 6mm, µm particles, preceded by cm RP-AmideC6 guard column and 0 µm frit filter Me:% acetic acid (7:8) 0mL/min 0 C UV, 7nm 0µL reconstituted porcine serum extract SPE Tube: DSC-8 00mg/mL 7

72 Catecholamines Discovery C8 cm x 6mm column, µm particles, MeC:0mM K P / P, p 0 00mg/L EDTA, 00mg/L -octane-sulfonic acid (:9) ml/min 0 C UV, nm 0µL, µg/ml of each analyte C8 Catecholamines These dihydroxyphenylethylamine derivatives are very difficult candidates for PLC separations since they carry both acidic and basic functionalities in their structures A Discovery C8 column demonstrates excellent selectivity for the separation of catecholamines, using an acidic mobile phase with ion-pairing additives orepinephrine CC Epinephrine CC C orepinephrine Epinephrine,-Dihydroxybenzylamine Dopamine,-Dihydroxybenzylamine Dopamine C C C Time (min) G0009, G006, G006, G006 Chlorophyll from Methanolic Plant Extracts SPE Tube: DPA-6S 00mg/mL Breakthrough Analysis The eluents that were collected during sample application were tested by UV absorption at 660nm using a cm cell Breakthrough was defined as the point at which the absorbance of the fraction was greater than that of a standard % solution of chlorophyll in methanol SPE SPE Procedure, Using Visiprep SPE Vacuum Manifold Sample Prep : Condition: Apply Sample: 0g of freshly cut green leaves of Philodendron were blended for min with 80mL Me After centrifugation for min at 000rpm, the green supernatant was decanted and used as the sample ml Me ml aliquots of the methanolic plant extract were applied using a flow rate of 0 7mL/min; fractions were collected after each ml of sample was applied Breakthrough Results Fraction Absorbance at 660nm (AU) Appearance 0 08 Clear 0 78 Clear 0 Clear 0 86 Clear 0 0 Clear Slightly green Slightly green % chlorophyll standard 0 99 Slightly green Breakthrough of chlorophyll occurred while fraction 6 was applied to the DPA-6S SPE tube This result indicates that Discovery DPA-6S products may be used to remove chlorophyll from plant extracts, while allowing unretained species to pass through, free of chlorophyll Chlorophyll from Methanolic Plant Extracts Chlorophyll is a green pigment that is present in all photosynthetic plants, as well as in some bacteria It occurs in three forms, all of which are magnesium-centered porphyrins containing a hydrophilic carbocyclic ring with a lipophilic phytyl tail Chlorophyll is a photoreceptor up to a wavelength of 700nm; it is sparingly soluble in alcohols; and its solutions are blue-green in color Chlorophyll often interferes with the analysis of bioactives in natural product research or in pesticide analysis Discovery DPA-6S polyamide SPE products can be used to remove chlorophyll from aqueous or methanolic extracts in these applications In this experiment, 00mg of bulk DPA-6S sorbent was packed into ml SPE tubes and plant extracts were processed through the tubes Chlorophyll A C CC Mg C C CC C CPhytyl R C C C G007 8 SUPELC Discovery Pharmaceutical Applications Book

73 Cold Remedy Ingredients The separation of acetaminophen and pseudoephedrine on Discovery columns at biological p is shown in this figure The RP-AmideC6 column displays unique selectivity for this pair of drugs, and elutes acetaminophen after pseudoephedrine This may be due to the fact that the phenolic hydroxyl on acetaminophen can undergo hydrogen bonding with the amide moiety on the stationary phase and this interaction contributes to longer retention This order of elution can be exploited in analyzing over-the-counter cold remedies that contain large amounts of acetaminophen and much smaller amounts of pseudoephedrine If pseudoephedrine elutes after acetaminophen in such cases, quantitation of pseudoephedrine becomes a problem as the large acetaminophen peak can overlap or swamp the smaller pseudoephedrine peak Pseudoephedrine Acetaminophen 0 Ti ( i ) RP-AMIDE C8 G Time (min) C8 G CYA Cold Remedy Ingredients cm x 6mm columns µm particles, MeC:mM K P p 7 0 (:8) ml/min 0 C UV, nm µl Pseudoephedrine Acetaminophen C C.Cl CC G00087, G000 0 Ti ( i ) G Ti ( i ) G00089 Fluoxetine (Prozac) This drug is a phenyl benzyl ether with a methylaminoethyl chain attached to the benzylic carbon It shows longer retention on the RP-AmideC6 column than on C8 or C8, possibly due to the -bonding interactions of the secondary amine functionality on the drug with the amide of the stationary phase In fact, the RP- AmideC6 forms a stronger interaction than the Cyano phase, as evidenced by the longer retention of fluoxetine on the former F C Fluoxetine (Prozac ) C C C RP-AMIDE 0 6 G00090 Ti ( i ) C8 0 Ti ( i ) C8 G CYA Fluoxetine (Prozac) cm x 6mm columns, µm particles MeC:mM K P p 7 0 (0:60) ml/min 0 C UV, nm µl G Ti ( i ) G G Ti ( i ) SUPELC Discovery Pharmaceutical Applications Book 9

74 umic Acids from Water SPE Tube: DPA-6S 00mg/mL and 600mg/mL Breakthrough Analysis The eluents that were collected during sample application were tested by UV absorption over the range of 0nm to 700nm, in nm intervals, using a cm cell Breakthrough was defined as the point at which the total absorbance of the fraction was greater than that of a standard % solution of humic acids in water SPE SPE Procedure, Using Visiprep SPE Vacuum Manifold Condition: Apply Sample: ml Me, then ml ml aliquots of 0 mg/ml and 0mg/mL humic acid in were applied using a flow rate of 0 7mL/min; fractions were collected after each ml of sample was applied Breakthrough Results umic Acids DPA-6S Sorbent Fraction Absorbance of Fraction Conc (mg/ml) Mass (mg) Versus % Standard Lower igher (breakthrough) Lower 7 igher (breakthrough) 0 00 Lower igher (breakthrough) Lower igher (breakthrough) These results indicate that Discovery DPA-6S products can be used to remove humic acids from aqueous solutions, while allowing unretained species to pass through, free of humic acids The capacity of the sorbent for humic acids depends on the concentration of the sample, and these studies indicate that the relationship may be linear Perform breakthrough studies for your sample, to select the best SPE tube size umic Acids from Water umic acids are found in soils, coals, and peat They are brown-colored, mixtures of not-well-defined macromolecules with polymeric phenolic and heterocyclic structures containing carboxyl groups and nitrogen functionalities They are soluble in water and bases, but insoluble in mineral acids and alcohols umic acids may interfere with the analysis of bioactives in natural product research or in pesticide analysis Discovery DPA-6S polyamide SPE products can be used to remove humic acids from aqueous solutions in these applications In this experiment, 00mg and 600mg of bulk DPA-6S sorbent was packed into ml SPE tubes and aqueous humic acid solutions were processed through the tubes onsteroidal Antiinflammatory Drugs cm x 6mm column, µm particles MeC:mM K P, p 0 (0:60) ml/min 0 C UV, 0nm 0µL, µg/ml of each analyte Piroxicam Sulindac Tolmetin Ketoprofen aproxen 6 Diflunisal 7 Indomethacin 8 Ibuprofen 9 Diclofenac 0 Mefanamic acid RP-AMIDE 0 onsteroidal Antiinflamatory Drugs SAIDs are strongly acidic molecules possessing diverse structural features They typically present problems during RP-PLC separations/analyses A Discovery RP- AmideC6 column provides an isocratic separation of these drugs within a short time, including the baseline separation of nine compounds within minutes Piroxicam Sulindac Tolmetin S Ketoprofen aproxen Diflunisal Indomethacin C C C C C C Ibuprofen Diclofenac Mefanamic acid C C C C C S F C CC Cl C C F C C C F C C C C C C C C C C Cl C Cl C C Time (min) G00006 G006, G0066, G0067, G0000, G0068, G0069, G007, G00060, G00060, G0070 SUPELC Discovery Pharmaceutical Applications Book

75 onsteroidal Antiinflammatory Drugs from Serum Ketoprofen, ibuprofen, and diclofenac are commonly used antiinflammatory drugs Serum levels for these acidic compounds can be monitored using this SPE method, after acidification of the serum sample Recoveries of the compounds are greater than 90% using this method Ketoprofen C G0000, G00060, G00060 C C C Efficiency of Recovery Cl Diclofenac C C Cl C C C C Ibuprofen C Compound Concentration %Recovery %RSD (n=6) Ketoprofen 0 µg/ml 9 0 ±7 7 0µg/mL 90 8 ± 7 Ibuprofen 0 µg/ml 9 ±8 0µg/mL 00 7 ± 9 Diclofenac 0 µg/ml 0 ±7 0µg/mL 97 ± 0 C C SPE SPE Procedure, Using Visiprep SPE Vacuum Manifold Condition: Apply Sample: Wash and Dry: Elute: ml Me, then ml ml porcine serum spiked with 0 µg/ml or 0µg/mL each SAID, acidified with 0µL P ml % Me in : dry tube 0 min with nitrogen stream ml Me; evaporate to dryness with nitrogen stream at room temperature; reconstitute in 00µL mobile phase onsteroidal Antiinflammatory Drugs from Serum PLC Conditions: Discovery RP-AmideC6 column, cm x 6mm, µm particles, preceded by cm RP-AmideC6 guard column and 0 µm frit filter MeC: mm K P p 0 (0:0) 0mL/min 0 C UV, 0nm 0µL reconstituted porcine serum extract SPE Tube: DSC-8 00mg/mL rganic Acids Aromatic carboxylic acids have pka values ranging from to They can interact with residual silanols on a reversed phase packing material through hydrogen bonding under acidic mobile phase conditions In addition, chelating molecules like salicylic acid can bind to metallic impurities, if present, on the silica surface This chromatogram of four organic acids on the C8 columns indicates the absence of silanol and metal chelating interactions The longer retention of Discovery S (high surface area) C8 may be desirable for serum or urine samples, where matrix peaks appear just after the void volume (not shown) omovanillic acid C C C Salicylic acid Sorbic acid C C=CC=CC p-toluic acid 0 Time (min) C8 G00 0 Time (min) S C8 G00 rganic Acids cm x 6mm column, Me:, 0 % TFA (0:60) 0mL/min 0 C UV, nm 0µL omovanillic acid 0 06µg/mL Sorbic acid 0 006µg/mL Salicylic acid 0 06µg/mL p-toluic acid 0 006µg/mL C C C G007, G00009, G000098, G007 SUPELC Discovery Pharmaceutical Applications Book

76 Parabens conditions for RP-AmideC6, C8, and C8 MeC:, (0:60), ml/min, 0 C, UV, nm, 0µL conditions for Cyano MeC:, (0:70), ml/min, 0 C, UV, nm, 0µL Methyl paraben Ethyl paraben Propyl paraben Butyl paraben RP-AMIDE Time (min) C8 G00 C Time (min) CYA G00 Parabens Parabens are alkyl esters of -hydroxybenzoic acid and are commonly used in formulations of a variety of drugs The lower members of the sequence are very polar, and hydrophobicity increases in the order methyl<ethyl<propyl<butyl A significant feature in the RP-PLC of these molecules under unbuffered mobile phase conditions is that all four parabens are retained longer on the RP-AmideC6 column with either acetonitrile or methanol as organic component, in comparison with C8 or C8 columns This behavior may be attributable to the strong - bonding capability of the phenolic hydroxyl with the amide moiety of the RP- AmideC6 stationary phase The Cyano column requires more aqueous mobile phase conditions for resolution of the four parabens and its low hydrophobic retention is reflected in the elution of butyl paraben in about 6min even at this 0:70 acetonitrile/water ratio Methyl paraben CC Ethyl paraben CC Propyl paraben Butyl paraben CC 7 CC Time (min) G Time (min) G007 G0009, G0009, G00096, G00097 Peptides (Angiotensins) Discovery C8 cm x 6mm column, µm particles, (A) mm ( ) P /, p 7 0 (B) mm ( ) P /, p 7 0: MeC (0:0) 0-60% B in min ml/min C Angiotensin I Angiotensin II Angiotensin III I DRVYIPFL II DRVYIPF III DRVYIPF C8 Peptides (Angiotensins) Angiotensins are peptide hormones that effect blood pressure and aldosterone release from adrenal glands uman angiotensins I, II, & III contain a conserved seven amino acid sequence exemplified by angiotensin III Two additional C-terminal amino acids, histidine and leucine, of angiotensin I confer greater retention on RPC columns compared to angiotensins II & III Angiotensin II differs from angiotensin III only by the addition of an -terminal aspartate Under conditions typical for peptide chromatography (p 0), angiotensins II & III are not resolved since the aspartate contains no charge and the retention coefficient (p 0) is only 0 Usually, resolution of these two angiotensins is performed at alkaline p, but with Discovery C8, baseline resolution is achieved at neutral p At p 7 0 the retention coefficient of aspartate is - 6 and thus significantly diminishes retention Consequently, separating the angiotensins at neutral p on Discovery C8 provides baseline resolution of all three components G00008 Time (min) SUPELC Discovery Pharmaceutical Applications Book

77 Peptides (Carboxyamidomethylated BSA Tryptic Digest) Discovery columns provide high resolution of very complex peptide mixtures Selectivity can be conveniently altered by use of different bonded phases RP-AmideC6, with its embedded polar group, provides a unique selectivity compared to conventional alkyl bonded phases typically employed for peptide mapping RP-AMIDE C8 Peptides (Carboxyamidomethylated BSA Tryptic Digest) cm x 6mm columns, µm particles, (A) 0 % TFA, % MeC in (B) 0 % TFA, 0% MeC ml/min C 0-00% B in 0 min Time (min) G Time (min) G00086 Peptides (Cytochrome c Tryptic Digest): Unique selectivity of bonded phases is illustrated with complex mixtures The chromatograms illustrate unique selectivity for certain sample components on RP-AmideC6, compared to C8 RP-AMIDE C8 Peptides (Cytochrome c Tryptic Digest) cm x 6mm columns, µm particles, (A) 0 % TFA, % n-propanol in (B) 0 % TFA, % n-propanol in MeC - 7% B in 7 min 0mL/min C Time (min) G Time (min) G00007 SUPELC Discovery Pharmaceutical Applications Book

78 Peptides (Substance P and Fragments) Discovery C8 cm x 6mm column µm particles, (A) 0 % TFA in (B) 0 % TFA in MeC - 9% B in 6 min, then to % B in min ml/min C 6 7 C8 Peptides (Substance P and Fragments) Substance P is a vasoactive peptide which among other things, induces vasodilation and increases capillary permeability Several features can be discerned from the chromatogram of this undecapeptide Presence of an additional phenylalanyl residue in fragment 7 makes it substantially more hydrophobic than fragment, which lacks this additional residue Presence of an additional phenylalanyl residue in fragment also imparts significant hydrophobicity relative to fragment The difference between fragment and the holopeptide, the lack of the C-terminal amino acids leucine and methionine in the former, make the former much less hydrophobic, thereby resulting in earlier elution This example demonstrates that in general, at acidic p ( 0), -terminal variants impact retention less than C-terminal variants, because of the a-amino group s positive charge, while the C-terminal carboxylate is neutral Substance P: RPKPQQFFGLM Fragments olopeptide Time (min) G000 Phloroglucinol from Water Discovery C8 column, cm x 6mm, µm particles, Me: (60:0) ml/min ambient temp UV, nm 0µL of each fraction SPE Tube: DPA6-S 600mg/mL Breakthrough Analysis Breakthrough was defined as the point at which the concentration of phloroglucinol in the eluent from the tube was greater than that of a % solution of phloroglucinol in water Concentrations were determined via PLC-UV and by comparing peak heights of the phloroglucinol peak from each fraction to peak heights of phloroglucinol in standards SPE SPE Procedure, Using Zymark RapidTrace SPE Workstation Condition: Apply Sample: ml Me, then ml ml aliquots of mg/ml phloroglucinol in water were applied using a flow rate of 0 7mL/min; fractions were collected after each ml of sample was applied Breakthrough Results Fraction Peak eight (mau) % phloroglucinol standard 9 Breakthrough of phloroglucinol occurred while fraction 7 was applied to the DPA-6S SPE tube This result corresponds to a loading capacity of about mg of phloroglucinol in water per gram of DPA-6S sorbent in this tube configuration Phloroglucinol from Water Phloroglucinol (,,-trihydroxybenzene or phloroglucine) was used as a representative compound for polyhydroxybenzene adsorption on Discovery DPA- 6S polyamide sorbent 600mg of bulk DPA-6S sorbent was packed into ml SPE tubes and samples were processed through the tubes Phloroglucinol G007 SUPELC Discovery Pharmaceutical Applications Book

79 Proteins (Bovine Insulin) Insulin is a heterodimeric protein hormone that directly affects uptake and metabolism of glucose The native protein contains three disulfides, two of which serve to crosslink the two subunits Buffered mobile phases atypical of peptide chromatography are required to dramatically reduce peak tailing of subunit A G00076 Time (min) C8 A Time (min) G B C8 Proteins (Bovine Insulin) cm x 6mm columns, µm particles, (A) 0mM ap, 0mM a S, p 0 (w/ P ): MeC (9:) (B) 0mM ap, 0mM a S, p 0 (w/ P ): MeC (0:0) % to 7 % B (0% to 8% MeC) in 8 min ml/min, C, UV, 0nM Subunit A, oxidized (sulfonic acids; Sigma I6) oloenzyme (Sigma I00) Subunit B, oxidized (sulfonic acids; Sigma I68) oloenzyme (Sigma I00) reduced and carboxy-methylated A Subunit A B Subunit B Tannic Acid from Water or Methanol Tannic acid (gallotannic acid) is a naturally occurring substance found in tree barks, fruits, and other plant parts Tannins are derivatives of gallic acid or flavanols, and comprise a broad group of plant-derived phenolic compounds which have the ability to precipitate proteins Some tannins may be more toxic than others, depending upon the source Tannins derived from nutgalls are believed to be carcinogens, while those found in tea and coffee are considered non-toxic These compounds are soluble in water and alcohols and can be extracted from plant matter Tannins may interfere with the analysis of bioactives in natural product research Discovery DPA-6S polyamide SPE products can be used to remove tannins from aqueous and methanolic solutions in these applications In this experiment, 00mg or 600mg of bulk DPA-6S sorbent was packed into ml SPE tubes and tannic acid solutions were processed through the tubes SUPELC Discovery Pharmaceutical Applications Book SPE SPE Procedure, Using Zymark RapidTrace SPE Workstation Condition: ml Me for methanolic samples ml Me followed by ml for aqueous samples Apply Sample: ml aliquots of 0mg/mL tannic acid in Me or were applied using a flow rate of 0 7mL/min; fractions were collected after each ml of sample was applied Breakthrough Results DPA-6S Sorbent Fraction Methanolic sample Aqueous Sample Mass (mg) Peak eight (mau) Peak eight (mau) =breakthrough > > > =breakthrough breakthrough - - breakthrough % acid standard 70 Equivalent results were obtained when using a slower flow rate (0 6mL/min) during sample application, indicating that capacity of DPA-6S for tannic acid does not depend heavily on flow rate Capacity also seems to be linear with respect to sorbent mass These results indicate that Discovery DPA-6S products can be used to remove tannic acid and other forms of the compound from aqueous or methanolic solutions, while allowing unretained species to pass through, free of these interfering species Tannic Acid from Water or Methanol PLC Analysis Conditions Discovery C8 column, cm x 6mm, µm particles, Me: (60:0) ml/min, ambient UV, nm 0mL of each fraction SPE Tube: DPA-6S 00mg/mL and 600mg/mL Breakthrough Analysis Breakthrough was defined as the point at which the concentration of tannic acid in the eluent from the tube was greater than that of a % solution of tannic acid in methanol or water Concentrations were determined via PLC-UV and by comparing peak heights of the tannic acid peak from each fraction to peak heights of tannic acid in the appropriate standard

80 Fat Soluble Vitamins (A and E) cm x 6mm columns, µm particles, ml/min 0 C UV, 90nm 0µL Discovery C8 Me: (9:) Discovery C8 MeC: (90:0) Retinol acetate (Vitamin A acetate), 0µg/mL d-tocopherol, 6µg/mL g-tocopherol, 00µg/mL a-tocopherol (Vitamin E), 9µg/mL a-tocopherol acetate (Vitamin E acetate), 0µg/mL Ti ( i ) C8 G0009 C G0009 Ti ( i ) Fat Soluble Vitamins (A and E) The three isomeric tocopherols (alpha, gamma, and delta) differ in the substitution pattern on the aromatic ring of the,-dimethylchroman skeleton The delta isomer has one methyl, the gamma analog has two, in the 7, 8 positions, and the alpha isomer has three, in the, 7, 8 positions The hydrophobicity-based elution of the three tocopherols follows the same order Alpha tocopheryl acetate, being much less polar than the tocopherols, is eluted last Vitamin A acetate (retinol acetate) contains a series of five conjugated double bonds and is evidently the most polar of all the components, resulting in its fast elution on both C8 and C8 columns C C C Retinol acetate C C C C CC g-tocopherol C C (CCCC) C C CC C a-tocopherol acetate C C C (CCCC) C C C C C d-tocopherol C C (CCCC) C a-tocopherol C C (CCCC) C G00, G0009, G0009, G00096, G00097 Fat Soluble Vitamins (D and D ) Discovery C8 cm x 6mm column, 00% MeC 0 8mL/min 0 C UV, 90nm 0µL, 0µg/mL of each analyte C8 Fat Soluble Vitamins (D and D ) Vitamin D (ergocalciferol) is 9,0-seco-ergosta-,7,0(9),-tetraen--ol and the D analog is 9,0-secocholesta-,7,0-trien--ol They differ in the number of double bonds (one more in D ) and methyl groups (one more in D in the form of a branched chain) Thus, they are closely related structures The versatility of the C8 column in separating this pair in less than minutes is evident The D analog, being more polar, is eluted first The S C8 column provides better resolution with slightly longer run times Ergocalciferol (Vitamin D ) Cholecalciferol (Vitamin D ) Time (min) G008 S C8 Ergocalciferol C C C C C Cholecalciferol C C C C C C Time (min) G009 G00098, G SUPELC Discovery Pharmaceutical Applications Book

81 Water Soluble Vitamins The separation of seven water soluble vitamins on three Discovery columns under gradient conditions is shown in this figure Selectivity differences among the columns is evident from the reversal of thiamine and nicotinic acid peaks Ascorbic acid C C C Folic acid C C C C C C C G00, G006, G00, G007, G008, G007, G000 icotinic acid Pyridoxine C C C C icotinamide C Thiamine C C C S C C C Riboflavin C Time (min) G RP-AMIDE Time (min) G C Time (min) G Water Soluble Vitamins RP-AmideC6 cm x 6mm column, µm particles, Me:0mM K P in, p (0:70), C, UV, 0nm, 0µL C8 and C8 cm x 6mm columns, µm particles, 0mM K P, p to Me:0mM K P, p (0:70) min (Discovery C8) or 8 min (Discovery C8) ml/min, ambient temp, UV, nm, µl, µg/ml of each analyte Ascorbic acid icotinic acid Thiamine Pyridoxine icotinamide 6 Folic acid 7 Riboflavin Discovery SPE-96 Well Plates & Accessories 96-Well Plates Discovery DSC-8 SPE-96 Plate, mg/well U Discovery DSC-8 SPE-96 Plate, 0mg/well U Discovery DSC-8 SPE-96 Plate, 00mg/well U Discovery DSC-8Lt SPE-96 Plate, mg/well U Discovery DSC-8Lt SPE-96 Plate, 0mg/well U Discovery DSC-8Lt SPE-96 Plate, 00mg/well U Discovery DSC-Si SPE-96 Plate, mg/well U Discovery DSC-Si SPE-96 Plate, 0mg/well U Discovery DSC-Si SPE-96 Plate, 00mg/well U Discovery DSC-PS/DVB SPE-96 Plate, mg/well U Discovery DSC-PS/DVB SPE-96 Plate, 0mg/well U 96-Well Plate Accessories 96 Sq. Well Collection Plates, 0.mL, PP, 0/pk U 96 Sq. Well Collection Plates, ml, PP, 0/pk U 96 Sq. Well Collection Plates, ml, PP, 0/pk U Disposable Reservoir/Waste Tray, PVC, /pk U 96 Sq. Well Piercable Cap Mats, 0/pk U Reagent Reservoir... R99-00ea. Cluster Tube Rack... Z76 - pak PlatePrep Manifold and Manifold Replacement Parts 96 Well Plate Starter Kit with Manifold U Contents of kit: Plate Prep Manifold; 96 Sq. Well Collection Plates, ml, PP; Disposable Reservoir/Waste Trays, PVC; 96 Sq. Well Piercable Cap Mats; Reagent Reservoirs; Cluster Tube Rack PlatePrep Vacuum Manifold U Acrylic Clear Top for Manifold U Polypropylene Base for Manifold U Gasket Kit for Manifold U Vacuum Gauge/Bleed Valve for Manifold U SUPELC Discovery Pharmaceutical Applications Book Improve your sample preparation with Discovery SPE-96 Well Plates! igher throughput and more... consistent bedweight and flow rates for automated or robotic processing reduced height for larger volumes without leak-prone extensions standard dimensions that are common to most square well extraction plate designs ideal for fulfilling IS, GMP, GLP requirements compatible with most vacuum manifolds and collection plates works with most robotic and automated sample processing systems reproducible from lot to lot, plate to plate, and well to well consistent capacities, recoveries and flow rates certificate of analysis included with each plate Trademarks Discovery, Sigma, Visiprep - Sigma-Aldrich Co Prozac - Dista Products Company RapidTrace, TurboVap, Zymark - Zymark Corp 7

82 PLC and SPE Products for Pharmaceutical Analysis and Purification Discovery PLC Special Application Kits DESCRIPTI CAT.. DISCVERY SELECTIVITY PACKS cm x.mm ID Columns 70-U 0cm x.mm ID Columns 698-U cm x.mm ID Columns 7-U cm x.0mm ID Columns 70-U0 0cm x.0mm ID Columns 698-U cm x.0mm ID Columns 7-U0 cm x.0mm ID Columns 7-U0 cm x.0mm ID Columns 70-U0 0cm x.0mm ID Columns 698-U cm x.0mm ID Columns 7-U0 cm x.0mm ID Columns 7-U0 cm x.6mm ID Columns 70-U 0cm x.6mm ID Columns 6980-U cm x.6mm ID Columns 7-U cm x.6mm ID Columns 7-U DISCVERY RP-AMIDEC6 VALIDATI PACKS cm x.mm ID Columns 70-U 0cm x.mm ID Columns 6980-U cm x.mm ID Columns 707-U cm x.6mm ID Columns 70-U 0cm x.6mm ID Columns 6980-U cm x.6mm ID Columns 707-U cm x.6mm ID Columns 709-U DESCRIPTI CAT.. DISCVERY C8 VALIDATI PACKS cm x.mm ID Columns 700-U 0cm x.mm ID Columns U cm x.mm ID Columns 70-U cm x.6mm ID Columns 700-U 0cm x.6mm ID Columns 6980-U cm x.6mm ID Columns 70-U cm x.6mm ID Columns 70-U DISCVERY C8 VALIDATI PACKS cm x.mm ID Columns 70-U 0cm x.mm ID Columns 6980-U cm x.mm ID Columns 7-U cm x.6mm ID Columns 70-U 0cm x.6mm ID Columns 6980-U cm x.6mm ID Columns 7-U cm x.6mm ID Columns 7-U DISCVERY CYA VALIDATI PACKS cm x.mm ID Columns 7-U 0cm x.mm ID Columns U cm x.mm ID Columns 77-U cm x.6mm ID Columns 7-U 0cm x.6mm ID Columns U cm x.6mm ID Columns 77-U cm x.6mm ID Columns 79-U cm Supelguard Cartridges with µm Discovery Packings Kits include one cartridge, a stand-alone holder, a piece of tubing, and nuts and ferrules..0mm ID CARTRIDGE.0mm ID CARTRIDGE.mm ID CARTRIDGE KIT PK. F KIT PK. F KIT PK. F DISCVERY PASE CAT.. CAT.. CAT.. CAT.. CAT.. CAT.. Discovery C U 976-U Discovery RP-AmideC U 978-U Discovery C8 989-U 990-U 979-U 980-U 987-U 988-U Discovery S C8 697-U 697-U U 6976-U Discovery Cyano 98-U 986-U 6970-U 697-U 98-U 98-U Four columns of equal dimensions, one of each Discovery phase (C8, RP-AmideC6, C8, Cyano). Identical columns made from three different bonded phase lots. For.0mm ID or.6mm ID analytical columns. Discovery SPE Tubes DESCRIPTI QTY.PKG. CAT.. DSC-8 0mg/mL U 00mg/mL U 00mg/mL 60-U 00mg/6mL 0 60-U g/6ml U g/ml U g/0ml U 0g/60mL U bulk packing 00g 600-U DSC-8Lt 0mg/mL U 00mg/mL 08 6-U 00mg/mL 6-U 00mg/6mL 0 6-U g/6ml 0 66-U g/ml 0 68-U g/0ml 0 6-U 0g/60mL 6 6-U bulk packing 00g 6-U DPA-6S 0mg/mL 08 6-U 0mg/mL 6-U 0mg/6mL 0 66-U 00mg/6mL 0 67-U g/ml 0 69-U g/0ml 0 6-U g/60ml 6 6-U 0g/in 800mL Büchner funnel 6-U bulk packing 0g 6-U Discovery PLC Columns ID LEGT (mm) (cm) CAT.. DISCVERY RP-AMIDEC U U U U U U U U DISCVERY C U U U U U U U U DISCVERY S C8. 69-U U. 69-U U U.6 69-U ID LEGT (mm) (cm) CAT.. DISCVERY C8. 9-U U.. 69-U. 9-U.0 9-U U U.0 9-U0.0 9-U0.0 9-U U U.0 9-U0.0 9-U0.6 9-U U U.6 9-U.6 9-U DISCVERY CYA. 9-U U.. 69-U. 96-U.0 9-U U U.0 96-U U0.0 9-U U U.0 96-U U0.6 9-U U U.6 96-U.6 97-U Call for availability on prep columns. The SIGMA-ALDRIC Family 8 SUPELC, Supelco Park, Bellefonte, PA phone: / 8-9- Biochemicals and Reagents for Life Science Research fax: / internet: [email protected] rganics and Inorganics for Chemical Synthesis Specialty Chemicals and Analytical Reagents for Research Chromatography Products for Analysis and Purification Laboratory Chemicals and Reagents for Research and Analysis Sigma-Aldrich Research is the research sales and marketing division of Sigma-Aldrich, Inc. Sigma-Aldrich Fine Chemicals is the bulk division of Sigma-Aldrich, Inc. 00 Sigma-Aldrich Co. Printed in USA. Supelco brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip. T997C SUPELC CJW Discovery Pharmaceutical Applications Book

83 Ascentis PLC Columns Elevated PLC Performance from a Wide Range of Phases Wide range of chromatographic selectivities Enhanced polar compound retention Ideal for LC-MS and all modern applications

84 Ascentis: Elevated PLC Performance We have placed heavy emphasis on optimizing Ascentis phases with relation to the three terms of the resolution equation: efficiency, retention and selectivity. owever, our strongest emphasis has been on the most powerful term, selectivity. Together, Ascentis bonded phases have a wide range of selectivities. It is likely that one or more Ascentis phase will accomplish any small molecule PLC separation. Packed in micro- to preparative hardware dimensions, Ascentis products cover all PLC application areas, including the most sensitive trace-level analyses. The General Features of the Ascentis Family Include: igh purity, type B silica for inertness, reproducibility and stability Modern bonding processes that optimize bonded phase coverage and maximize stability, while minimizing bleed and unwanted secondary interactions Wide selection of bonded phase chemistries and bare silica Phases with enhanced polar compound retention Compatible with LC-MS and all of today s sensitive instruments and methods Scalable selectivity from analytical to preparative igh surface area silica for high preparative loading capacity Ascentis Characteristics Phase USP Code Key Competitive Feature Modes Primary Uses Page Ascentis C8 L igh surface area, inert surface Reversed-phase Small molecules and peptides 9 Ascentis RP-Amide L60 Phase stability, low bleed Reversed-phase Excellent first choice alternative to C8 for routine 0 RP method development. Polar molecules, especially phenolics and other -bond donors, acids, bases (uncharged), anilines Ascentis Phenyl L Phase stability, low bleed Reversed-phase, Ring systems and strong dipoles, -acids, -electron ILIC acceptors, heteroaromatics, nitroaromatics Ascentis ES Cyano L0 Phase stability Reversed-phase, Polar compounds, strong dipoles, ILIC, 00% Aqueous tricyclic antidepressants Ascentis Silica L igh loading capacity, controlled and ormal phase on-polar compounds (in P mode), highly-polar 6 uniform surface activity (non-aqueous), ILIC compounds (in ILIC mode), nucleosides, amino acids Ascentis C8 L7 igh surface area, inert surface Reversed-phase Small molecules and peptides 8 Discovery S F* L rthogonal selectivity to C8, ours is Reversed-phase, All electron and -electron donors, bases (charged), 0 well-characterized ILIC, ion-exchange positional isomers * We have chosen to include Discovery S F in this brochure because of its complementary selectivity to the Ascentis phases and its benefits for certain analytes. Ascentis PLC columns represent a continuum of improvement through innovations in PLC technology. sigma-aldrich.com/ascentis ordering: (US only) / 8-9-

85 Developing PLC Methods on Ascentis Selecting An Ascentis Column Column Screening: The Ascentis & Discovery Method Development Tool Kit We recommend every PLC method developer have these five columns in their arsenal. Ascentis C8 Classic C8 selectivity will achieve most reversed-phase separations Ascentis RP-Amide For enhanced retention and performance of polar compounds, especially bases (uncharged) and compounds with -bond potential Ascentis Phenyl For enhanced retention and performance of polar compounds, especially ring systems, dipoles, and nitroaromatics Ascentis ES Cyano Extra stable for low p mobile phases due to sterically protected phase. Useful for polar selectivity in reversed-phase mode. Also useful as an alternative to silica in ILIC mode Discovery S F For enhanced retention and performance of polar compounds, especially bases (charged) and when the sample contains a mixture of non-polar and polar compounds Simply screen these five columns in your desired mobile phase, using your preferred column. Choosing an Ascentis or Discovery Phase Based on Compound Class and Separation Challenge or bjective Typically, Ascentis C8 is the first choice for starting a new method. owever, when a C8 doesn t give the desired separation or your sample contains compounds that are known to be difficult to retain or resolve on a C8, consider changing the stationary phase. The range of selectivity provided by Ascentis and Discovery phases makes this easy. The flow chart below helps guide the selection of Ascentis or Discovery phase based on the particular compound type or separation challenge. For more information about each phase, and the other Ascentis phases, please refer to their dedicated pages in this brochure. Desire to Improve Retention or Resolution on C8 on-polar Compounds Mixture of Polar and on-polar Compounds Polar Compounds Very Polar Compounds Geometric Isomers Too Much Retention Poor Peak Spacing: Too much retention of non-polar compounds and/or too little retention of polar compounds Retention Too Short or Inadequate Separation Poor Peak Shape (Basic Compounds) ot Enough RP Retention, LC-MS ot Enough Separation Basic Compounds -Bonding, Phenolic, Acidic or ydroxylated Compounds Electron- Acceptors itroaromatics, eteroaromatics Strong Dipoles Electron- Donors ILIC Mode Reversed- Phase ormal Phase Charged Uncharged Ascentis C8 Discovery Ascentis Ascentis Discovery Ascentis Ascentis Discovery Ascentis S F RP-Amide Phenyl S F RP-Amide Silica S F Silica or or or or Ascentis ES Cyano Ascentis ES Cyano Ascentis ES Cyano Ascentis ES Cyano Column selection guidelines for Ascentis and Discovery phases based on compound class and separation challenge or objective. TRADEMARKS: ACE - Advanced Chromatography Technologies; Ascentis, Discovery, CIRBITIC, CYCLBD, CIRALDEX, CRMASLV - Sigma-Aldrich Biotechnology LP; Fused-Core - Advanced Materials Technology, Inc.; ucleosil - Machery-agel; Prodigy - Luna; Symmetry, XTerra - Waters Corporation; Zorbax - Agilent Technologies Chromatography Products for Analysis and Purification

86 arnessing the Power of Chromatographic Selectivity Chromatographic resolution is a function of column efficiency (), retention (k) and selectivity ( ). It is usually written in the form of the resolution equation: Figure. Affect of Selectivity on Improving Resolution When resolution is plotted vs. these three parameters in Figure, it becomes apparent that selectivity has the greatest affect on resolution. The Power to Accomplish Difficult Separations ne of the most important reasons why selectivity is leveraged in PLC is to resolve closely-eluting compounds. A good example of this is the need to quantify a compound that elutes in the tail of a more abundant compound, perhaps a low-level impurity in the presence of the parent compound, like shown in the Figure. By altering the stationary phase, in this case going from a C8 to an RP-Amide, the impurity can be eluted before the main peak, thereby allowing more sensitive and reliable quantification. Figure. Impurity Eluted on the Downslope of the Main Peak on C8 Difficult to Quantify I C8 Impurity Eluted Before the Main Peak on Ascentis RP-Amide Better Quantification Ascentis RP-Amide I The power of chromatographic selectivity is demonstrated in this example. The C8 column elutes the impurity in the downslope of the major peak, limiting the ability to detect and quantify the impurity. By using a column with different selectivity, in this case an Ascentis RP- Amide, the impurity peak is eluted before the main peak. sigma-aldrich.com/ascentis technical service: (US and Canada only) / 8-9-0

87 Key Ascentis Application Areas Polar Analytes: Enhanced Retention, Selectivity, and Compatibility with ighly-aqueous Mobile Phases Polar compounds are difficult to analyze by traditional reversed-phase because they lack the high proportion of hydrophobic character necessary for retention. Since most pharmaceutically- and biologically-active compounds are highly polar, this has presented a continual problem in PLC. From the beginning of our commitment to PLC innovation, we have focused on bonded phase to enhance polar compound retention. Figure. - Interactions (Ascentis Phenyl) column: Ascentis C8, cm x.6 mm I.D., μm particles (8-U) Ascentis Phenyl, cm x.6 mm I.D., μm particles (866-U) mobile phase: 0:60, acetonitrile:water flow rate:.0 ml/min. temp.: 0 C det.: UV at 0 nm injection: μl sample: nitrobenzene, m-dinitrobenzene,,,-trinitrobenzene Ascentis Phenyl Stationary Phases with Enhanced Polar Compound Retention Compared to C8 Rather than relying solely on dispersive forces to achieve retention, our column portfolio contains bonded phases that lend additional retentive character toward analytes with specific polar functional groups. Ascentis C8 G0007 Figure. -bonding (Ascentis RP-Amide) column: Ascentis RP-Amide & C8, cm x.6 mm I.D., μm particles mobile phase: 70:0, water with 0.% TFA:acetonitrile flow rate: ml/min. -ydroxybenzoic Acid temp.: C. Acetylsalicylic Acid det.: UV at 0 nm. Benzoic Acid injection: 0 μl. -ydroxybenzoic Acid. Ethyl paraben Ascentis RP-Amide Ascentis Phenyl exhibits strong π-π interactions with the π-acidic nitroaromatics. Addition of each nitro group increases retention. Figure. Ion Exchange (Discovery S F) column: Discovery S F, cm x.6 mm I.D., μm particles (676-U) conventional C8, cm x.6 mm I.D., μm particles mobile phase: 0:70, 0 mm ammonium acetate (p 6.98): C C flow rate:.0 ml/min. temp.: C det.: photodiode array injection: μl G0007 C8 G0009. Methcathinone. Ephedrine Discovery S F G0067, C8 G0009 Ascentis RP-Amide exhibits strong retention relative to C8 for phenols and organic acids G0067 Discovery S F exhibits enhanced retention from ion exchange interactions with ionized analytes. Chromatography Products for Analysis and Purification

88 ILIC Mode: Enhanced Retention and igh MS-Suitability ighly-polar compounds, like underivatized amino acids, nucleosides and nucleotides, are not well-retained by reversed-phase PLC. owever, under ILIC (hydrophilic interaction chromatography) conditions, they can be retained. ILIC is a variation of normal phase PLC where the mobile phase contains high percentages of organic modifier. It is also called aqueous normal phase or AP. Under high organic conditions, polar interactions become prominent which can lead to increased retention. Ascentis Phenyl, Ascentis ES Cyano, Ascentis Silica and Discovery S F exhibit ILIC character under highly-organic mobile phases. An added benefit of ILIC mobile phases is the high organic (often >90%) is amenable to MS detection. Stability in ighly-aqueous Mobile Phases Unless working in ILIC mode, mobile phases for polar compounds are often highly-aqueous, with only small percentages of organic modifiers. Under such conditions, C8 phases are not wetted, which causes two problems. First, the bonded phase molecules coalesce resulting in phase collapse and subsequent loss of hydrophobic retention. Second, analytes have an unpredictable approach to the silica surface, resulting in irreproducible and unstable retention run-to-run and column-to-column. Ascentis Phenyl, Ascentis ES Cyano, Ascentis RP-Amide, and Discovery S F are completely aqueous-compatible, and will not undergo phase collapse even in mobile phases that contain 00% water. Figure 6. Separation of ucleosides on Ascentis Phenyl column: Ascentis Phenyl, cm x. mm I.D., μm particles (86-U) mobile phase: 0 mm ammonium formate (p.0 with formic acid) flow rate: 0. ml/min temp.: C det.: UV at 70 nm injection: μl Ascentis Phenyl 00% Aqueous- Compatible 6 7. Cytidine (0. g/l). Deoxycytidine (0. g/l). Uridine (0. g/l). Guanosine (0. g/l). Adenosine (0. g/l) 6. Deoxyguanosine (0. g/l) 7. Deoxyadenosine (0. g/l) 8. Thymidine (0. g/l) 8 Figure 7. Separation of rganic Acids on Ascentis RP-Amide column: Ascentis RP-Amide, cm x.6 mm I.D., μm particles (6-U) mobile phase: mm monobasic potassium phosphate (p.0 with phosphoric acid) flow rate: ml/min. temp.: C det.: UV at 0 nm injection: 0 μl. Dehydroascorbic acid. Ascorbic acid Ascentis RP-Amide 00% Aqueous- Compatible G00078 G007 Selecting the Right Buffer A partial list of common buffers and their corresponding useful p range is supplied. Perhaps the most common buffer in PLC is the phosphate ion. Although, with the growth of LC-MS, volatile buffers such as TFA, acetate, formate, and ammonia are becoming more frequently used. Remember, the purpose of a buffer in the mobile phase is to inhibit a p change in the mobile phase after the introduction of a sample. When developing a method, it is important to select a mobile phase with a final p at least one p unit away from any analytes pk value. As a rule of thumb, one should work within a ± p unit of the buffer pka. Typical buffer concentrations for PLC tend to be 0-00 millimolar level. Buffer C Useful p Range Trifluoroacetic acid (TFA) 0. <. Phosphate.. -. Formate Acetate Phosphate Ammonia Phosphate sigma-aldrich.com/ascentis ordering: (US only) / 8-9-

89 LC-MS Compatibility Through Phase Stability, Retentivity and Inertness In today s laboratory, PLC columns and bonded phases must be compatible with mass spectrometric detection. Complete MS compatibility is an important design input for all Ascentis phases. egligible Phase Bleed Loss of stationary phase can contribute to high background interference in all forms of detection, but it is most notable in MS detection where phase bleed can also lead to fouling of the instrument and subsequent downtime for cleaning and repair. Modern bonding procedures and an intelligent selection of the Ascentis phase chemistry combine to give all Ascentis phases low detectable bleed under MS and sensitive UV detection. Amide Chemistry Avoids the eed for TEA and TFA Additives Silanol-suppressing mobile phase additives, like TEA and TFA, are required for good peak shape on traditional PLC phases. owever, because they suppress the MS signal they should be avoided. Ascentis RP-Amide, by virture of the embedded amide group, does not require silanol-suppressing additives for good peak shape. Formic acid is a suitable acidic modifier for use with Ascentis RP-Amide columns. Ascentis p Stability: Extending the Working p Range ccasionally, PLC mobile phases outside the normal p -7 range are desired to control sample stability, solubility or ionization state, or for compatibility with detection methods. A limitation of most silica-based PLC phases is their instability outside this range where hydrolysis of the bonded phase and dissolution of the underlying silica can occur. Ascentis columns have excellent stability compared to competitive silica-based columns. The high bonded phase coverage and proprietary endcapping combine to increase resistance to hydrolysis and dissolution. As a result, Ascentis columns can be used successfully between p.-0 under certain conditions. ote, however, that it is important to avoid storing Ascentis columns, and any silica-based column, in harsh mobile phases. Ascentis Provides Scalable Separations from Microbore to Preparative Time and precious samples are wasted during scale-up if the analytical and preparative columns do not give the same elution pattern. The high surface area of the underlying Ascentis silica provides high loading capacity to purify larger quantities of material. Additionally, bonded phase and silica chemistry are uniform across,, and 0 μm particle sizes. These features combine to ensure that analytical separations that are developed on Ascentis or μm particles are completely scalable to preparative separations on Ascentis 0 μm particles and larger columns. Additionally, separations developed on or 0 μm particles can be scaled down for fast analysis on Ascentis μm particles. Ascentis 0 μm particles in large column dimensions are ideal for isolating and purifying mg to gram amounts of compounds for further characterization. Ascentis μm particles in short columns are ideal for rapid analysis and LC-MS applications. Guidelines for Preparing Mobile Phases It should be understood that slight variations in p and buffer concentration could have a dramatic affect on the chromatographic process; consistent and specific techniques should be a regular practice in the preparation of mobile phases. A common practice is to place a sufficient amount of pure water into a volumetric flask and add an accurate amount of buffer. The p of the solution should be adjusted, if necessary, and then dilute to final volume of water prior to adding or blending of organic solvents. Then, add a volumetrically measured amount of organic solvent to obtain the final mobile phase. Thorough blending, degassing, and filtering prior to use is also recommended. To view a listing of suitable PLC and LC-MS additives and solvents, visit sigma-aldrich.com/lc-ms-solvents Chromatography Products for Analysis and Purification 7

90 Ascentis C8 The First Choice for Classic C8 Retention and Selectivity ptimization of silica and bonded phases make Ascentis C8 a true workhorse for the vast majority of PLC separations. igh surface area and phase stability give it perfect character for demanding LC-MS and preparative separations. Features: Classic C8 selectivity igh non-polar retentivity Symmetric peak shape ighly reproducible and stable Ideal for LC-MS Key Applications: General reversed phase, hydrophobic and polar compounds Properties: USP code: L Bonded phase description: ctadecyl Endcapped: Yes Particle composition: Type B silica gel Particle purity: < ppm metals Particle shape: Spherical Particle size:, and 0 μm Pore size: 00 Å Surface area: 0 m/g Carbon load: % p range (recommended): -8 Extended p range*:.-0 Characterization of Chromatographic Performance on Ascentis C8 Acidic Ion Exchange Capacity at p.7 Ion Exchange Capacity at p 7.6 ydrogen Bonding Capacity Shape Selectivity ydrophobic Selectivity ydrophobic Retention All competitive data obtained from M. R. Euerby, P. Perterson, J. Chromatogr. A, 99 (00) -6; or M. R. Euerby, P. Perterson, W. Campbell, W. Roe, J. Chromatogr. A, (007) 8-. Ascentis data developed by Supelco/Sigma-Aldrich scientists using the Euerby methodology or obtained directly from cited references. Data for Aromatic selectivity is calculated as atb/b/atb/dt. Use The classic reversed-phase column, Ascentis C8 is suitable for any method that specifies a C8-type column. Its high surface area gives Ascentis C8 strong hydrophobic retention and high loading capacity for preparative applications. LC-MS Implications Ascentis C8 is low-bleed for clean ESI and APCI traces. The high retentivity means that the mobile phase can contain high levels of organic modifier that are more readily desolvated. otes Modern C8 columns have very similar selectivity, even for basic compounds, because silica quality and bonding techniques have improved to the point that silanol effects are minimal. Unless quality with your current column is the issue, the main reasons for evaluating different brands of C8 columns are for improved peak shape and for slight changes in selectivity. * Under certain conditions, the Ascentis family can be operated in the extended p range. For more information, request an electronic file of Acid/Base Stability of Silica Based on C8, C8, and Amide PLC Columns (T0608) 8 sigma-aldrich.com/ascentis technical service: (US and Canada only) / 8-9-0

91 Ascentis C8 is one of the most retentive reversed-phase columns available. igh retentivity extends the linear range of loading capacity, making Ascentis C8 ideal for separations that are or might be used for preparative applications. igh retentivity also means Ascentis C8 can accommodate the highly-organic mobile phases encountered in LC-MS. Ascentis C8: Top of Its Class in ydrophobic Retentivity Ascentis C8 Prodigy DS- Zorbax Extend-C8 Symmetry C8 Luna C8 () ucleosil C8 ACE C8 XTerra MS C8 Discovery C k Pentylbenzene All competitive data obtained from M. R. Euerby, P. Perterson, J. Chromatogr. A, 99 (00) -6; or M. R. Euerby, P. Perterson, W. Campbell, W. Roe, J. Chromatogr. A, (007) 8-. Ascentis data developed by Supelco/Sigma-Aldrich scientists using the Euerby methodology or obtained directly from cited references. Data for Aromatic selectivity is calculated as atb/b/atb/dt. Analysis of basic compounds at neutral p often gives longer retention compared to acidic mobile phases, but sometimes causes poor peak shape due to silanol interactions. The highly-inert surface of Ascentis C8, as with all Ascentis phases, permits analysis in neutral p mobile phases. In this example, a mix of tricyclic antidepressants at p 7 shows excellent peak shape on Ascentis C8. Figure 8. Symmetrical Peaks for Basic Compounds Indicative of igh Surface Deactivation of Ascentis Phases column: Ascentis C8, cm x.6 mm I.D., μm particles (6-U) mobile phase: 0:70; mm ammonium phosphate, p 7.0: methanol flow rate:. ml/min. temp.: C det.: UV at nm inj.: 0 μl. Desmethyl doxepin. Protriptyline. Desimpramine. ortriptyline. Doxepin 6. Impramine 7. Amitriptyline 8. Trimpramine G0069 Ascentis C8 provides the selectivity and retention for a range of compounds including steroids. Ascentis C8 is a reliable first choice PLC column that gives symmetric peak shape and excellent retention for even difficult compounds. Figure 9. Estrogenic Steroids on Ascentis C8 column: Ascentis C8, cm x.6 mm I.D., μm particles (8-U) mobile phase: :, water:acetonitrile flow rate: ml/min. temp.: C det.: UV at 0 nm inj.: 0 μl. Estriol. -Estriol. -Estriol. Estrone Chromatography Products for Analysis and Purification 9

92 Ascentis RP-Amide Ultra-Low Bleed Alkyl Amide Phase that Rivals C8 as a Generic Scouting Column. Excellent Peak Shape and Resolution, Especially for Polar Compounds or Mixtures of Compound Polarity As pioneers in embedded polar group (EPG) phases for PLC, Supelco is pleased to offer Ascentis RP-Amide, which has all the benefits of enhanced polar compound retention and selectivity, without any of the disadvantages of competitive EPG phases. Features Improved peak shape for bases compared to C8 Different selectivity than C8 or C8 for wide range of polar compounds (especially acids) Lower bleed than competitive EPG phases 00% aqueous compatible Key Applications Small, water soluble molecules and peptides, -bond donors, acids, phenols, basic compounds, polar compounds Properties USP code: L60 Bonded phase description: Stable amide group embedded in an 8-carbon chain Endcapped: Yes Particle composition: Type B silica gel Particle purity: < ppm metals Particle shape: Spherical Particle size:, and 0 μm Pore size: 00 Å Surface area: 0 m/g Carbon load: 9.% p range (recommended): -8 Extended p range*:.-0 Characterization of Chromatographic Performance on Ascentis RP-Amide Acidic Ion Exchange Capacity at p.7 Ion Exchange Capacity at p 7.6 ydrogen Bonding Capacity Shape Selectivity ydrophobic Selectivity ydrophobic Retention All competitive data obtained from M. R. Euerby, P. Perterson, J. Chromatogr. A, 99 (00) -6; or M. R. Euerby, P. Perterson, W. Campbell, W. Roe, J. Chromatogr. A, (007) 8-. Ascentis data developed by Supelco/Sigma-Aldrich scientists using the Euerby methodology or obtained directly from cited references. Data for Aromatic selectivity is calculated as atb/b/atb/dt. Use Ascentis RP-Amide can be used for many of the same separations as a C8 while avoiding some of the disadvantages of C8 such as poor wettability in high aqueous mobile phases. In addition, it is much more retentive for those molecules that can interact by hydrophobic interactions and also by -bonding with the amide group. Compared to alkyl-only phases, Ascentis RP-Amide has enhanced retention and selectivity for phenols, organic acids and other polar solutes due to strong -bonding between the amide carbonyl (-bond acceptor) and -bond donors, like phenols and acids. Compared to other EPG phases, like carbamates, ureas, sulfonamides and ethers, Ascentis RP-Amide gives retention comparable to C8 and C8 for easy column comparison without the need to change mobile phase conditions. LC-MS Considerations Unlike other amide-based phases, Ascentis RP-Amide uses an amidosilane reagent and a one-step bonding method similar to C8. Polymeric reagents are also employed to achieve maximum stability and low bleed with all modern PLC detectors. otes Generally, acids are retained more and bases retained less on RP-Amide compared to C8 and C8 columns. Methanol can be comparable in elution strength to acetonitrile when compounds are retained by -bonding mechanism on the RP-Amide phase. * Under certain conditions, the Ascentis family can be operated in the extended p range. For more information, request an electronic file of Acid/Base Stability of Silica Based on C8, C8, and Amide PLC Columns (T0608) 0 sigma-aldrich.com/ascentis ordering: (US only) / 8-9-

93 Figure 0. Structure of Ascentis RP-Amide G0077 Figure. Enhanced Phenolic Compound Retention via -bonding on Ascentis RP-Amide column: Ascentis RP-Amide, cm x.6 mm I.D., μm particles (6-U) mobile phase: 7:, 0 mm phosphoric acid:acetonitrile flow rate:. ml/min temp.: 0 C det.: UV at 70 nm Resorcinol injection: μl Because the amide group lies near the silica surface, it is believed to suppress tailing of basic solutes by electrostatic shielding (repulsion) or by interacting preferentially with the silanols (-bonding between the phase and the substrate). The absence of unwanted silanol and other secondary interactions gives the Ascentis RP-Amide excellent peak shape for both acids and bases. This example of internal deactivation with acetaminophen and pseudoephedrine (Figure ) shows the dramatic effect of changing stationary phase: not only are the peaks more symmetrical, but elution order is reversed.. Resorcinol. Catechol. -Methyl resorcinol. -Methyl catechol.,-dimethyl resorcinol 6. -Methyl catechol 7. -itro catechol Catechol Ascentis RP-Amide 6 G009 G0090 Figure. Improved Efficiency, Selectivity and Resolution of Ascentis RP-Amide vs. C8 column: Ascentis RP-Amide, cm x.6 mm I.D., μm particles (6-U) Ascentis C8, cm x.6 mm I.D., μm particles (8-U) mobile phase: :8, acetonitrile: mm potassium phosphate flow rate: ml/min. temp.: C det.: UV at nm inj.: 0 μl 7 G009 Ascentis C8. Acetaminophen. Pseudoephedrine C G Ascentis RP-Amide G G00789 An Ascentis RP-Amide column is more retentive and selective for phenolic compounds, like catechols and resorcinols, compared to a C8 and an ether-type polar embedded phase (Figure ). The ether phase does not have the ability to -bond with phenolic groups like the amide group does. Although comparing an ether phase to a C8 phase may be useful if only slightly different selectivity is needed, the most dramatic results for acids such as phenols and carboxylic acids will be obtained with amide-based phases, like Ascentis RP-Amide. 0 Competitor EPG G00790 Chromatography Products for Analysis and Purification

94 Ascentis Phenyl Ultra-low Bleed Phenyl Phase with Enhanced Phenyl Selectivity Phenyl-based reversed-phases were one of the first alternatives to C8 selectivity. ur Ascentis Phenyl has been improved to offer exceptional phase stability and enhanced phenyl retention. Ascentis Phenyl offers versatility by also operating in the ILIC mode. Features Low-bleed for MS or UV gradient applications due to the use of trifunctional bonding reagent utstanding phenyl selectivity due to high phase loading and short butyl spacer 00% aqueous-compatible for highly-polar compounds Key Applications Small, water soluble molecules and peptides, π-acceptors, nitroaromatics, polar compounds, dipoles, heterocyclics, ILIC mode Properties USP code: L Bonded phase description: Phenyl ring with short butyl spacer Endcapped: Yes Particle composition: Type B silica gel Particle purity: < ppm metals Particle shape: Spherical Particle size:, and 0 μm Pore size: 00 Å Surface area: 0 m/g Carbon load: 9.% p range (recommended): -8 Extended p range*:.-0 Characterization of Chromatographic Performance on Ascentis Phenyl Acidic Ion Exchange Capacity at p.7 Ion Exchange Capacity at p 7.6 ydrogen Bonding Capacity Shape Selectivity ydrophobic Selectivity ydrophobic Retention All competitive data obtained from M. R. Euerby, P. Perterson, J. Chromatogr. A, 99 (00) -6; or M. R. Euerby, P. Perterson, W. Campbell, W. Roe, J. Chromatogr. A, (007) 8-. Ascentis data developed by Supelco/Sigma-Aldrich scientists using the Euerby methodology or obtained directly from cited references. Data for Aromatic selectivity is calculated as atb/b/atb/dt. Use Phenyl phases are -basic (electron donating) and are similar in overall retention to alkyl and EPG phases for easy column screening. The alternate selectivity of phenyl phases is often explained by the - interactions available through the phenyl ring. Compounds that appear to exhibit differential selectivity on the phenyl phase include: hydrophobic bases (TCAs, tetracyclines) moderate bases (anesthetics and narcotic analgesics) benzodiazepines some acidic compounds such as ACE inhibitors and quinoline antibiotics nucleosides (e.g. cytidine) nitro, azide and sulfonyl compounds otes Methanol can be a more selective mobile phase component than acetonitrile. Activate ILIC mode by using highly-aqueous (>90%) mobile phases. * Under certain conditions, the Ascentis family can be operated in the extended p range. For more information, request an electronic file of Acid/Base Stability of Silica Based on C8, C8, and Amide PLC Columns (T0608) sigma-aldrich.com/ascentis technical service: (US and Canada only) / 8-9-0

95 Figure. Structure of Ascentis Phenyl Si G009 Most commercially-available phenyl phases actually show a great deal of C8-like selectivity, negating their impact on improving the separation. With the highest level of true phenyl character among all phases tested, Ascentis Phenyl has excellent aromatic selectivity, making it a true alternative to traditional C8. The short, butyl spacer of Ascentis Phenyl does not dilute the phenyl character as conventional hexyl spacers do. Figure shows the stronger contribution of -electron interactions from the phenyl ring on Ascentis Phenyl compared to the phenylhexyl phase allowing it to resolve buspirone and trazadone. The phenyl and alkyl selectivity tend to cancel each other out on the competitive phenylhexyl phase in this case. Aromatic Selectivity - Phenyl Ascentis Phenyl Luna Phenyl exyl XBridge Phenyl Ace Phenyl Inertsil Phenyl Zorbax SB Phenyl Gemini Phenyl exyl ypersil (BDS) Phenyl Betabasic Phenyl XTerra Phenyl ypersil Phenyl Figure. Better Phenyl Selectivity on Ascentis Phenyl Compared to Competitive Phases column: Ascentis Phenyl, cm x.6 mm I.D., μm particles (866-U) mobile phase: 0:60, 0 mm ammonium acetate (p. with acetic acid):acetonitrile flow rate: ml/min. temp.: C det.: UV at nm injection: μl All competitive data obtained from M. R. Euerby, P. Perterson, J. Chromatogr. A, 99 (00) -6; or M. R. Euerby, P. Perterson, W. Campbell, W. Roe, J. Chromatogr. A, (007) 8-. Ascentis data developed by Supelco/Sigma-Aldrich scientists using the Euerby methodology or obtained directly from cited references. Data for Aromatic selectivity is calculated as atb/b/atb/dt. Ascentis Phenyl Trazadone. Trazadone. Buspirone G G000, Phenyl exyl type Buspirone G G000 Chromatography Products for Analysis and Purification

96 Ascentis ES Cyano Extra stable for low p mobile phases due to sterically protected phase Useful for polar selectivity in the reversed-phase mode, including - and dipole-dipole interacting compounds. Can also be used in ILIC mode and normal phase chromatography. Features: Enhanced stability at low p perates in reversed-phase, ILIC and normal phase modes of chromatography Low MS bleed 00% aqueous compatible Available as μm and μm particles Key applications: polar compounds, nitroaromatics, tricyclic antidepressants, steroids Properties: USP Code: L0 Bonded phase description: diisopropyl cyano propyl Endcapped: Yes Particle composition: Type B silica gel Particle purity: < ppm metals Particle shape: Spherical Particle size: and μm Pore size: 00 Å Surface area: 0 m /g Carbon load: 0 % p range recommended: -8 Use Cyano phases have become very popular because of their unique selectivity for polar groups and double bonds. Their potential for dipole/dipole and dipole/induced-dipole interaction made them one of the earliest stationary phase functional groups when alternate selectivity was needed. In the past, stability of the cyano phase under reversed-phase and ILIC conditions has been poor. Exposure to aqueous organic mobile phases, acidic p and elevated temperatures can create gradual retention and selectivity loss that is reportedly due to stationary phase hydrolysis. A new Ascentis ES Cyano column, based on and μm porous silica substrate, has been developed. This new phase compares very favorably in stability to C8, C8, Amide and Phenyl. otes Can be used in reversed, ILIC and normal phase modes It is best to dedicate a specific column to one mode of chromatography mentioned above. Methanol gives more selectivity than acetonitrile in the reversed phase mode. Cyano phases are used in EPA Method 80 () for the analysis of explosives and nitroaromatics. Figure. Structure of Ascentis ES Cyano G US EPA Method 80A, itroaromatics and itramines by igh Performance Liquid Chromatography (PLC) Revision (February 007), obtained from the web site. sigma-aldrich.com/ascentis ordering: (US only) / 8-9-

97 Cyano columns have been used for the analysis of tricyclic antidepressants for some time. A comparison of the most popular competitor column and the Ascentis ES Cyano phase is shown to the right. Better resolution is seen using the ES Cyano than the competitor column for three critical peak pairs. In addition, note the selectivity change of the trimipramine and doxepin (peaks and ) between these two phases under these conditions. Figure 6. Comparison of Tricyclic Antidepressants on Ascentis ES Cyano and a Competitor Cyano Phase column: as indicated; cm x.6 mm, μm particle mobile phase A: 0 mm potassium phosphate, dibasic (p 7.0 with phosphoric acid) mobile phase B: acetonitrile mobile phase C: methanol mixing proportions: A:B:C, :60: flow rate:.0 ml/min temp.: C det.: nm injection: μl sample: 00 μg /ml in 70:0, water: methanol Ascentis ES Cyano. Trimipramine. Doxepin. Amitriptyline. Imipramine. ordoxepin 6. ortriptyline 7. Desipramine 8. Protriptyline G0097 Competitor Cyano G0097 Figure 7. Structures of Tricyclic Antidepressants Trimipramine Doxepin Amitriptylin G0097 G0097 G0097 ortriptyline ordoxepin Protriptyline G00979 Imipramine G00977 Desipramine G00978 G00976 G00980 Chromatography Products for Analysis and Purification

98 Ascentis Silica igh Surface Area and igh Surface Deactivation Combine to Give Ascentis Silica Exceptional Performance as a ormal Phase, ILIC and Preparative PLC Material Besides being the underlying support for all Ascentis phases, Ascentis Silica has applications in its own right. Silica is widely used to separate positional isomers in normal phase mode, and polar compounds in ILIC (aqueous normal phase modes). Silica is also used in organic synthesis to purify reaction mixtures. In each case, a high purity, controlled and uniform surface is necessary to impart the desirable chromatographic performance. Features igh-loading capacity perates in both normal-phase and ILIC modes Tested in both modes and shipped in ethanol, Ascentis Silica is ready to use in either mode Ultra-pure, spherical silica Available in,, and 0 μm Key Applications Small molecular weight positional (geometric) isomers, non-polar compounds (in P mode), vitamins, steroids, polar compounds (in ILIC mode) Properties USP code: L Bonded phase description: one (surface comprises silanol, -Si-, and siloxane, -Si--Si-, groups) Endcapped: o Particle composition: Type B silica gel Particle purity: < ppm metals Particle shape: Spherical Particle size:, and 0 μm Pore size: 00 Å Surface area: 0 m/g Carbon load: 0% p range (recommended): -6 Use ormal phase and ILIC PLC modes Preparative chromatography Purification (organic synthesis) LC-MS The classic use of silica columns is for normal phase PLC. The rigid structure of the silica surface, as opposed to the flexible nature of bonded phases, allows it to distinguish between molecules with different footprints that may have the same hydrophobicity. Geometric isomers and closely-related substances, like the steroids shown in Figure, can be separated on Ascentis Silica under normal phase conditions. ormal phase is also widely used in preparative chromatography because the mobile phase is more easily removed by evaporation than the water-containing reversed-phase mobile phases. Figure 8. Ascentis Silica: ormal Phase Separation of Geometric Isomers and Closely-Related Compounds column: Ascentis Si, cm x.6 mm I.D., μm particles (8-U) mobile phase: 88:, hexane:ethanol flow rate:.0 ml/min. temp.: C det.: UV at nm injection: 0 μl sample: 0 μg/ml in 8:, hexane: -propanol Cortisone. Cortisone. Prednisone. Prednisolone G00087 Prednisone Prednisolone G00086 G0008 G sigma-aldrich.com/ascentis technical service: (US and Canada only) / 8-9-0

99 Ascentis Silica is used successfully in the aqueous normal-phase or ILIC mode. In this mode, water is the strong modifier and the organic is the weak modifier of the mobile phase. Like reversed-phase, ILIC offers the flexibility of using p and ionic strength to control retention. ILIC is ideal for very polar compounds and is highly compatible with LC-MS. Figure 9. Ascentis Silica in ILIC Mode Gives Better Peak Shape and Retention of Basic Drugs than Competitive Silicas column: cm x.6 mm I.D., μm particles mobile phase: 0:90, 0.% ammonium acetate in water (p unadjusted): 0.% ammonium acetate in acetonitrile temp.: C flow rate: ml/min. det.: UV at nm. eroin. xycodone. Codeine. Morphine Ascentis Si Elution order is generally opposite to that obtained under reversedphase conditions. Figure 6 compares Ascentis Silica with two competing silicas, demonstrating better resolution by virtue of excellent peak shape and high retentivity G00089 Inertsil Si G00090 Waters ILIC Si G0009 Polar biomolecules, like amino acids, nucleotides and nucleosides, typically require derivatization for their analysis by reversed phase PLC. The ILIC mode offered by Ascentis Silica permits the retention and resolution of these compounds without derivatization, eliminating a time-consuming sample preparation step (Figure 7). Figure 0. Ascentis Silica in ILIC Mode: Amino Acids column: Ascentis Si, cm x. mm I.D., μm particles (809-U) mobile phase A: 00 mm ammonium formate (p.0, with formic acid) mobile phase B: water mobile phase C: acetonitrile temp.: C flow rate: 0. ml/min det.: ESI (+), full scan inj.: μl sample: in 0:90, (0 mm ammonium formate/ formic acid, p.0):acetonitrile Gradient %A %B %C Threonine. Serine. Asparagine. Glutamine. Glutamatic acid G00088 Chromatography Products for Analysis and Purification 7

100 Ascentis C8 ne of The Most ydrophobic C8 Phases Available Leveraging the improvements to silica and bonded phase properties that made Ascentis C8 so useful, its shorter alkyl chain cousin, Ascentis C8, is also suitable for routine PLC and LC-MS. Features: Selectivity similar to C8 for non-polar compounds Different selectivity for polar compounds Less hydrophobic than C8, more hydrophobic than other C8 phase Symmetric peak shape ighly reproducible and stable Ideal for LC-MS Key Applications: Small, water soluble molecules and peptides, less hydrophobic retention than C8 but comparable selectivity, LC-MS Properties: USP code: L7 Bonded phase description: ctyl Endcapped: Yes Particle composition: Type B silica gel Particle purity: < ppm metals Particle shape: Spherical Particle size: and μm Pore size: 00 Å Surface area: 0 m/g Carbon load: % p range (recommended): -8 Extended p range*:.-0 Characterization of Chromatographic Performance on Ascentis C8 Acidic Ion Exchange Capacity at p.7 Ion Exchange Capacity at p 7.6 ydrogen Bonding Capacity Shape Selectivity ydrophobic Selectivity ydrophobic Retention All competitive data obtained from M. R. Euerby, P. Perterson, J. Chromatogr. A, 99 (00) -6; or M. R. Euerby, P. Perterson, W. Campbell, W. Roe, J. Chromatogr. A, (007) 8-. Ascentis data developed by Supelco/Sigma-Aldrich scientists using the Euerby methodology or obtained directly from cited references. Data for Aromatic selectivity is calculated as atb/b/atb/dt. Use Ascentis C8 is suitable for any method that specifies a C8-type column. Although C8 columns often show similar selectivity to C8 columns, shorter alkyl chains sometimes show different selectivity toward polar compounds because they can solvate differently with the mobile phase and interact differently due to the size and shape of certain molecules. Also, C8 reagents are smaller than C8 reagents and have improved primary phase coverage, thereby requiring less end-capping. Ascentis C8 has excellent peak shape and very high phase stability. Ascentis C8: Top of Its Class in ydrophobic Retentivity Ascentis C8 Symmetry C8 ucleosil C8 D RP Select B C8 Genesis C8 Zorbax SB-C8 ypurity C8 YMC Basic C8 XTerra MS C8 Discovery C8 Data cited from same source as above. 0 k Pentylbenzene Among commercially available C8 columns, Ascentis C8 has the highest degree of hydrophobic retention. This permits the use of higher percentages of organic modifier, a benefit to LC-MS users. * Under certain conditions, the Ascentis family can be operated in the extended p range. For more information, request an electronic file of Acid/Base Stability of Silica Based on C8, C8, and Amide PLC Columns (T0608) 8 sigma-aldrich.com/ascentis ordering: (US only) / 8-9-

101 Ascentis C8 is an excellent choice for fast gradient analysis. Ascentis C8 is typically more retentive at low organic composition that C8 and less retentive at high organic composition. Furthermore, Ascentis C8 has better aqueous compatibility for gradients that start at 00% aqueous composition. Figure. Fast Gradient Analysis on Ascentis Columns column: Ascentis C8, cm x.0 mm I.D., μm particles (80-U) mobile phase A: 9:, 0 mm ammonium phosphate:acetonitrile, p. mobile phase B: 0:60, 0 mm ammonium phosphate:acetonitrile, p. flow rate:. ml/min temp.: C det.: 0 nm inj.: 0 μl gradient: 0% B to 00% B in min p-toluidine. p-itroaniline. p-itrophenol. p-cresol. p-itrobenzoic acid 6. p-methylbenzoic acid 7. p-itrotoluene 8. p-xylene 6 G0077 Ascentis C8 often yields enhanced retention than Ascentis C8 for small polar molecules under highly aqueous conditions. Greater retention for Ascentis C8 may be related to greater wettability of Ascentis C8 as compared to Ascentis C8. Figure. Polar Molecules Under ighly Aqueous Conditions column: Ascentis C8, cm x.6 mm I.D., μm particles (8-U) mobile phase: :97, methanol:water with 0.% TFA flow rate: ml/min. temp.: C det.: UV at 0 nm inj.: 0 μl. L-tartaric. Lactic Acid. Citric Acid. Fumaric Acid. Acrylic Acid G0068 Analysis of bases at neutral p often yields enhanced retention over acidic mobile phase but sometimes causes poor peak shape with C8 and C8 columns due to silanol interaction. In this example, a mix of tricyclic antidepressants at p 7 shows excellent peak shape on Ascentis C8. Figure. Pharmaceutical Bases at p 7 column: Ascentis C8, cm x.6 mm I.D., μm particles (8-U) mobile phase: 0:70; mm ammonium phosphate, p 7.0: methanol flow rate:. ml/min. temp.: C det.: UV at nm inj.: 0 μl Desmethyl Doxepin. Protriptyline. Desimpramine. ortriptyline. Doxepin 6. Impramine 7. Amitriptyline 8. Trimpramine G0070 Chromatography Products for Analysis and Purification 9

102 Discovery S F Unique Reversed-phase Selectivity Compared to C8 and C8 Discovery S F provides reversed-phase separations that are distinctly different from C8 columns. owever, compounds will generally elute within the same retention time window, making most C8 methods easily transferable. Features Unique (orthogonal) selectivity compared to C8 and C8 Stable, low-bleed LC-MS separations Both reversed-phase and ILIC modes Possesses multiple types of interactions: dispersive, dipole-dipole, π-π, charge-transfer Key Applications Small, water soluble molecules and peptides, polar compounds, basic compounds, positional isomers Properties USP code: L Bonded phase description: Pentafluorophenylpropyl Endcapped: Yes Particle composition: Type B silica gel Particle purity: <0 ppm metals Particle shape: Spherical Particle size:, and 0 μm Pore size: 0 Å Surface area: 00 m/g Carbon load: % p range (recommended): -8 Characterization of Chromatographic Performance on Discovery S F Acidic Ion Exchange Capacity at p.7 Ion Exchange Capacity at p 7.6 ydrogen Bonding Capacity Shape Selectivity ydrophobic Selectivity ydrophobic Retention All competitive data obtained from M. R. Euerby, P. Perterson, J. Chromatogr. A, 99 (00) -6; or M. R. Euerby, P. Perterson, W. Campbell, W. Roe, J. Chromatogr. A, (007) 8-. Ascentis data developed by Supelco/Sigma-Aldrich scientists using the Euerby methodology or obtained directly from cited references. Data for Aromatic selectivity is calculated as atb/b/atb/dt. otes Compared to a C8: Generally, bases are retained longer on the S F than on a C8, hydrophobic compounds are retained less. Increasing the organic content of a C8 separation to 0 percent will generally provide similar retention on a S F. Also, as a general rule, solutes with log P o/w values less than. will be retained longer on S F compared to a C8. Compared to a phenyl phase: Although aromatic in nature, the pentafluorophenylpropyl (F) phase does not resemble a phenyl phase in retention or selectivity. The F is a strong Lewis acid due to the electron withdrawing effects of five fluorine groups; the F ring is electron deficient whereas the phenyl ring is electron rich. Figure. Structure of Discovery S F G000 0 sigma-aldrich.com/ascentis technical service: (US and Canada only) / 8-9-0

103 Guidelines for Transferring a C8 Method to Discovery S F Generally, bases are longer retained on the S F than on a C8. Increasing the organic content of a C8 separation to 0 percent will generally provide similar retention on a S F. Results with other compounds are highly variable. owever, it is generally true that solutes with logp o/w values less than. will be retained longer on S F compared to a C8. The degree of difference is highly solute dependent. Figure. S F Provides Excellent Separation - Solutes Are ot Retained on C8 column: Discovery S F, cm x.6 mm I.D., μm particles (676-U) Conventional C8, cm x.6 mm I.D., μm particles mobile phase: 0:70, 0 mm Ammonium Acetate (p 6.98): C C flow rate:.0 ml/min temp.: C det.: Photodiode Array inj.: μl Discovery S F G0067,. Methcathinone (00 μg/ml). (+/-) Ephedrine (00 μg/ml) Conventional C8 o Retention G0067 In Figure, cytidine and related compounds provide another example of the power of S F to provide unique and valuable separations compared to a C8. An added benefit of the S F is its resistance to phase collapse under 00% aqueous conditions. Figure 6. Unique Selectivity of S F Resolves Compounds Better than C8 column: Discovery S F, cm x.6 mm I.D., μm particles (676-U) Conventional C8, cm x.6 mm I.D., μm particles mobile phase: 0 mm K P, p.0 with P, (C8 separation has % C C) flow rate: ml/min temp.: 0 C det.: UV at 80 nm inj.: 0 μl, each compound 00 μg/ml. Cytidine. Cytosine. -Deoxycytidine Discovery S F 0 6 G009 Conventional C8 0 6 G009 Chromatography Products for Analysis and Purification

104 Extreme Performance on Any LC System The demand for increased sample throughput and speed of results has driven PLC users to search for breakthroughs in PLC instrument and column technology. Although improvements have been realized, setbacks have been encountered. Reductions in column ruggedness, costly replacements of existing instrumentation, and difficulties in transferring methods to new systems have often made these past improvements unappealing to analysts. Ascentis Express has changed all of that. Ascentis Express with Fused-Core Particle Technology provides the ultimate solution for today s separation demands - high speed and high efficiency with low backpressure. By simply changing to Ascentis Express Columns, sample throughput can be improved by 00%! o longer will you have to make changes to: sample prep flow rate system pressure And no new instrumentation is required! For more information on this exciting new technology, visit our website: sigma-aldrich.com/express Do More Work in Less Time without Changing your Method With identical conditions, the Ascentis Express C8 column performs X as many separations as a standard C8 column in less time. CDITIS Theoretical Standard C8 Ascentis C8 Sample Prep Flow Rate System Pressure PLC System Plates Throughput Throughput SAME SAME SAME SAME SAME X X C8, cm x.6 mm I.D., μm particles mobile phase: 6:, water:acetonitrile flow rate: ml/min. temp.: ambient det.: UV at nm. xazepam: FW = 86. Alprazolam: FW =. Cloazepam: FW =. -desmethyldiazepam: FW = 70. Diazepam: FW = 8 X Ascentis Express C8, 0 cm x.6 mm I.D.,.7 μm particles (87-U) X 9.. X 9. X 8.. X sigma-aldrich.com/ascentis ordering: (US only) / 8-9-

105 rdering Information Particle ID Length Ascentis Ascentis Ascentis Ascentis Ascentis Ascentis Discovery Size (mm) (cm) C8 RP-Amide Phenyl ES Cyano Silica C8 S F μm 8-U 609-U inquire inquire inquire 8-U inquire 0 86-U 689-U 8600-U inquire 80-U 8-U inquire 86-U 666-U 860-U inquire 8-U 86-U inquire. 8-U 6-U inquire inquire inquire 8-U inquire. 8-U 6-U 860-U inquire 8-U 8-U 670-U. 800-U 600-U 860-U 7708-U 800-U 800-U 6700-U U 60-U 860-U 7709-U 80-U 80-U 670-U. 80-U 60-U 860-U 770-U 80-U 80-U 670-U 8-U 6-U inquire inquire inquire 8-U inquire 80-U 60-U 8606-U inquire 8-U 80-U 670-U 807-U 6-U inquire inquire inquire 80-U inquire U 6-U 8607-U inquire 80-U 80-U 678-U.6 8-U 66-U inquire inquire inquire 86-U inquire.6 86-U 67-U inquire inquire inquire 87-U 6709-U.6 80-U 60-U 8608-U 77-U 80-U 806-U 670-U U 6-U 8609-U 77-U 80-U 807-U 670-U.6 8-U 6-U 860-U inquire 806-U 808-U 6707-U μm. 868-U 69-U inquire inquire inquire 89-U inquire. 87-U 6-U inquire inquire inquire 80-U inquire. 80-U 60-U 86-U 7700-U 807-U 80-U 6708-U U 60-U 86-U 770-U 808-U 89-U 670-U. 80-U 60-U 86-U 770-U 809-U 8-U 67-U. 80-U 606-U 86-U inquire 80-U 8-U 67-U 88-U 6-U inquire inquire inquire 8-U inquire 869-U 69-U inquire inquire inquire 80-U inquire 89-U 6-U inquire inquire inquire 8-U inquire.6 80-U 6-U inquire inquire inquire 8-U inquire.6 8-U 66-U inquire inquire inquire 8-U inquire.6 8-U 6-U 86-U 770-U 8-U 8-U 67-U.6 0 inquire 68-U inquire 770-U inquire inquire 67-U.6 8-U 6-U 866-U 7706-U 8-U 8-U 676-U.6 8-U 6-U 867-U 7707-U 8-U 8-U 677-U 0 80-U 60-U inquire inquire inquire inquire 678-U U 6-U inquire inquire inquire inquire 677-U 0 8-U 6-U inquire inquire inquire inquire 679-U 0 8-U 6-U 868-U inquire 8-U inquire 670-U. 8-U 6-U inquire inquire inquire inquire inquire. 87-U 68-U 869-U inquire 8-U inquire 67-U 0 μm.6 80-U 6-U inquire inquire inquire inquire inquire.6 8-U 6-U inquire inquire 8-U inquire inquire 0 8-U 6-U inquire inquire inquire inquire inquire U 6-U inquire inquire inquire inquire inquire 0 8-U 66-U inquire inquire inquire inquire inquire 0 8-U 67-U inquire inquire 86-U inquire inquire. 86-U 68-U inquire inquire inquire inquire inquire U 69-U inquire inquire inquire inquire inquire. 88-U 660-U inquire inquire inquire inquire 678-U. 89-U 66-U inquire inquire 87-U inquire 679-U ID Length Particle Size Ascentis Ascentis Ascentis Ascentis Ascentis Discovery (mm) (cm) (μm) C8 RP-Amide Phenyl Silica C8 S F Ascentis Supelguard Cartridges Kit. 876-U inquire inquire inquire inquire 677-U Pack of. 877-U inquire inquire inquire inquire 6770-U Pack of. 870-U 67-U inquire inquire inquire 677-U Kit. 87-U 67-U inquire inquire inquire 677-U Pack of 87-U 67-U inquire inquire inquire inquire Kit 87-U 67-U inquire inquire inquire inquire Kit 878-U inquire inquire inquire inquire 677-U Pack of 879-U inquire inquire inquire inquire 677-U Pack of 87-U 670-U 860-U 88-U 86-U 6776-U Kit 87-U 67-U 86-U 89-U 87-U 6777-U Kits include one cartridge, stand alone holder, a piece of tubing, nuts and ferrules. Ascentis Validation Packs U 69-U 869-U inquire inquire inquire.6 89-U 69-U 8696-U inquire inquire inquire Validation Packs include columns, each from a different bond lot. Chromatography Products for Analysis and Purification

106 Argentina Free Tel: Tel: (+) 6 7 Fax: (+) 698 Australia Free Tel: Free Fax: Tel: (+6) 98 0 Fax: (+6) Austria Tel: (+) Fax: (+) Belgium Free Tel: Free Fax: Tel: (+) Fax: (+) 899 Brazil Free Tel: Tel: (+) 7 00 Fax: (+) 989 Canada Free Tel: Free Fax: Tel: (+) Fax: (+) Chile Tel: (+6) 9 79 Fax: (+6) China Free Tel: Tel: (+86) 6 66 Fax: (+86) 6 67 Czech Republic Tel: (+0) Fax: (+0) Denmark Tel: (+) Fax: (+) Finland Tel: (+8) Fax: (+8) France Free Tel: Free Fax: Tel: (+) Fax: (+) Germany Free Tel: Free Fax: Tel: (+9) Fax: (+9) ungary Ingyenes telefonszám: Ingyenes fax szám: Tel: (+6) 906 Fax: (+6) India Telephone Bangalore: (+9) ew Delhi: (+9) Mumbai: (+9) 70 6 yderabad: (+9) Kolkata: (+9) Fax Bangalore: (+9) ew Delhi: (+9) Mumbai: (+9) yderabad: (+9) Kolkata: (+9) Ireland Free Tel: Free Fax: Tel: (+) Fax: (+ ) 0 07 Israel Free Tel: Tel: (+97) Fax: (+97) Italy Free Tel: Tel: (+9) 0 70 Fax: (+9) Japan Tel: (+8) Fax: (+8) Korea Free Tel: (+8) Free Fax: (+8) Tel: (+8) Fax: (+8) Malaysia Tel: (+60) 6 Fax: (+60) 6 6 Mexico Free Tel: Free Fax: Tel: (+) Fax: (+) The etherlands Free Tel: Free Fax: Tel: (+) Fax: (+) ew Zealand Free Tel: Free Fax: Tel: (+6) 98 0 Fax: (+6) orway Tel: (+7) Fax: (+7) Poland Tel: (+8) Fax: (+8) Portugal Free Tel: Free Fax: Tel: (+) 9 Fax: (+) 9 60 Russia Tel: (+7) Fax: (+7) Singapore Tel: (+6) Fax: (+6) Slovakia Tel: (+) 7 6 Fax: (+) 7 6 South Africa Free Tel: Free Fax: Tel: (+7) Fax: (+7) Spain Free Tel: Free Fax: Tel: (+) Fax: (+) Sweden Tel: (+6) Fax: (+6) 8 7 Switzerland Free Tel: Free Fax: Tel: (+) Fax: (+) United Kingdom Free Tel: Free Fax: Tel: (+) Fax: (+) 77 8 United States Toll-Free: Toll-Free Fax: Tel: (+) Fax: (+) Vietnam Tel: (+8) 6 80 Fax: (+8) 68 8 Internet Life Science, igh Technology Service rder/customer Service (800) -00 Fax (800) -0 Technical Service (800) -8 sigma-aldrich.com/techservice Development/Custom Manufacturing Inquiries (800) -7 Safety-related Information sigma-aldrich.com/safetycenter World eadquarters sigma-aldrich.com LV T0D

107 Ascentis Express PLC Columns with Fused-Core Technology Extreme Performance on Any LC System yper-fast Separations igh Definition Resolution Super-Sensitive Super-Rugged

108 A Breakthrough in PLC Performance The Fused-Core Advantage Ascentis Express provides the high speed and high efficiency of sub- μm particles, but at approximately half the backpressure for the same column length. This lower pressure means that Ascentis Express can be run on conventional PLC and LC-MS systems, as well as mid-pressure, UPLC and other ultra-high pressure systems. Lower pressure also means longer columns can be used for additional resolving power. Ascentis Express offers these benefits over sub- μm particles, along with excellent column lifetime. At the heart of Ascentis Express is the.7 μm Fused- Core particle which comprises a.7 μm solid core and a 0. μm porous shell (Figure ). Compared to totally porous particles, the Fused-Core particles have a much shorter diffusion path because of the solid core. This partial porosity reduces axial dispersion of solutes and minimizes peak broadening. ther features, such as a very tight particle size distribution and high packing density, result in Ascentis Express columns that are capable of 0,000 /m. This is comparable to the efficiency of sub- μm particle columns and nearly twice the efficiency possible with μm particles. While the Ascentis Express efficiency is as high as sub- μm columns, the larger particle size delivers approximately half the backpressure for the same column dimensions and conditions. This allows Ascentis Express to turn any PLC system into an extreme performance workhorse for your lab Figure. Fused-Core Structure of Ascentis Express Compared to Totally Porous Particles Ascentis Express Particle Totally Porous Particle.7 μm 0. μm 0. μm Diffusion Path. μm.7 μm G0088 Table of Contents Ascentis Express Topics... Page Speed... Resolution... Sensitivity...6 Ruggedness...7 Alternate Selectivity Ascentis Express Topics... Page Throughput Fast PLC... - Column Coupling... rdering Information... sigma-aldrich.com/express ordering: (US only) / 8-9-

109 Ascentis Express FAQs What is unique about Ascentis Express? Ascentis Express columns provide a breakthrough in PLC performance. Based on Fused-Core particle technology, Ascentis Express provides the benefits of sub- μm particles but at much lower backpressure. These benefits include the capability of providing fast PLC and higher resolution chromatography. The Fused-Core particle consists of a.7 μm solid core and a 0. μm porous shell. A major benefit of the Fused-Core particle is the small diffusion path (0. μm) compared to conventional fully porous particles. The shorter diffusion path reduces axial dispersion of solutes and minimizes peak broadening. Do I need special fittings and tubing to connect Ascentis Express columns? While operating pressures may not exceed the 00 bar (6,000 psi) capability of your traditional instruments, sustained pressures of about 00 bar (,000 psi) will exceed the recommended pressure for conventional PEEK tubing and fittings at the column inlet. We recommend changing to stainless steel fittings in all high pressure locations and have designed special low-dispersion connectors (pg. ) that will stay tight at pressures of,000 bar (,000 psi) or greater, even when elevated column temperatures are employed. What phases are available in Ascentis Express? Currently, C8, C8, RP-Amide, and ILIC (bare silica) phases are available for Ascentis Express. When are additional phases expected? Additional phases are being developed. The best way to track new products is to visit sigma-aldrich.com/express for the latest updates. Can I use Ascentis Express on any type of PLC system? Ascentis Express PLC columns are capable of use on standard PLC systems as well as UPLC systems. Columns are packed in high pressure hardware capable of withstanding the pressures used in UPLC systems. Is there anything I need to do to my PLC system to use Ascentis Express? othing special is required to use Ascentis Express PLC columns. To obtain the full benefits of Ascentis Express, one should minimize dispersion or instrument bandwidth in the PLC system (tubing, detector flow cell) as well as confirm the detector response system is set at a fast level. For more information, request Guidelines for ptimizing Systems for Ascentis Express Columns (T070) or visit sigma-aldrich. com/express and download. ow can I measure my instrument bandwidth (IBW) and determine what columns can be used with minimal efficiency loss created by too much internal instrument volume? For simple instructions on how to measure IBW, request Guide to Dispersion Measurement (T08) or visit our website sigma-aldrich.com/express and download. Can I use Ascentis Express on a UPLC system? Yes. Ascentis Express columns are packed in a way making them suitable for these ultra high pressure instruments. In fact, Ascentis Express outperforms sub- μm columns on many applications since Ascentis Express provides the benefits of sub- μm particles but at much lower backpressure. Can Ascentis Express columns be used for LC-MS? Ascentis Express Fused-Core particles were designed with LC-MS in mind. Even extremely short column lengths exhibit sufficient plate counts to show high resolving power. The flat van Deemter plots permit resolution to be maintained at very high flow rates to maximize sample throughput. All Ascentis stationary phases have been evaluated for MS compatibility during their development, and the Express phases are no exception. A bonus of Ascentis Express columns for high throughput UPLC and LC-MS is that they are extremely rugged and highly resistant to plugging, a very common failure mode for competitor columns. What flow rate should I use with Ascentis Express columns? Based on the minimum in the van Deemter curves, higher flows than μm particle columns are required in order to maximize Ascentis Express column efficiency. Ascentis Express Suggested Starting Point PLC Column ID for Flow Rate.6 mm I.D..6 ml/min.0 mm I.D. 0.8 ml/min. mm I.D. 0. ml/min Are guard columns available? Guard columns packed with Ascentis Express are currently not available. Ascentis Express columns are rugged and almost all users prefer operation of Ascentis Express columns without a guard column. If you would like to use a guard column, we recommend the Ascentis guard columns. technical service: (US and Canada only) / 8-9-0

110 yper-fast Separations Double the Speed Designed for high flow rates alf the backpressure of sub- μm particles Compared to sub- μm particles, the.7 μm Ascentis Express particles generate approximately half the backpressure. This permits both longer columns and faster flow rates. Figure shows the separation of a steroid mixture on Ascentis Express (top) and a conventional sub- μm column (lower) of the same dimensions. Because higher flow rates on Ascentis Express even doubled in this example generate similar backpressure, hyper fast separations are possible that have efficiency and resolution equal to the sub- μm particle column. Shown in Figure is a comparison against a traditional cm, μm column and a cm, Ascentis Express. The chromatograms further illustrate the high-speed capabilities of Ascentis Express at backpressures manageable by all PLC systems. igh flow rates are quite amenable to Ascentis Express PLC columns due to the Fused-Core particle. Figure. yper-fast Separations on Ascentis Express: Twice the Speed at Equivalent Pressure columns: Ascentis Express C8, 0 cm x. mm I.D.,.7 μm particles (8-U) and sub- μm particle column (same dimensions) mobile phase: 9: or :, water:acetontrile flow rate: 0. or 0. ml/min. temp.: ambient det.: UV at 00 nm injection: μl. Estradiol Ascentis Express C8 0. ml/min flow rate C8 Sub- μm 0. ml/min flow rate G psi 000 psi. -Estradiol. Impurity. Estrone. Estrone degradant TWICE TE SPEED AT EQUAL PRESSURES Figure. yper-fast Separations on Ascentis Express: Eight Times the Speed of Traditional Columns column: Ascentis Express C8, cm x.0 mm I.D. and μm particle column, cm x.0 mm I.D. mobile phase: :69, water:acetonitrile flow rate:. ml/min or 0. ml/min temp.: C inj.: 0. μl Ascentis Express C8. ml/min flow rate mv EIGT TIMES FASTER. Uracil. Phenol. Acetophenone. Benzene. Toluene 6. aphthalene 0 0 Sec 0 0 G008 C8 μm 0. ml/min flow rate 6 6 G00976 mau G007 sigma-aldrich.com/express ordering: (US only) / 8-9-

111 igh Definition D -Resolution Double the Efficiency Short analyte diffusion path Twice the efficiency of μm particles Longer columns permit doubling the plates over sub- μm particles Compared to conventional μm and μm particles, Ascentis Express PLC columns provide sharper peaks under the same conditions. By simply swapping in an Ascentis Express PLC column of the equivalent dimensions to your current μm and μm particle PLC columns, an improvement in resolution can be achieved. This improvement is shown in Figure. ote: Remember Ascentis Express PLC column recommended flow rates are higher than that for conventional μm and μm particles. Ascentis Express and sub- μm columns of the same dimensions give approximately the same number of Figure. D-Resolution on Ascentis Express: Sharper Peaks than Traditional Columns Ascentis Express μm theoretical plates (efficiency). owever, because Ascentis Express columns are more permeable and exhibit half the backpressure, you can use longer columns for even more resolving power. The high backpressure generated by the sub- μm particles precludes the use of longer columns, even on ultra-high pressure systems under ambient conditions. An example of the D-Resolution is shown in Figure where the additional theoretical plates on the 0 cm Ascentis Express column provided significantly better resolution of -estradiol and the impurity compared to the cm sub- μm column at comparable backpressures. Figure. D-Resolution on Ascentis Express Compared to Sub- μm Columns columns: Ascentis Express C8, 0 cm x. mm I.D.,.7 μm particles (8-U) and sub- μm particle column, cm x. mm I.D. mobile phase: : or :6, water:acetontrile flow rate: 0. ml/min. 00 psi temp.: ambient det.: UV at 00 nm injection: μl Ascentis Express C8 0 cm length. Estradiol. -Estradiol. Impurity. Estrone. Estrone degradant TWICE TE RESLUTI AT EQUAL PRESSURE G0097 SARPER PEAKS TA REGULAR CLUMS C8 Sub- μm cm length 60 psi G0097 G008 technical service: (US and Canada only) / 8-9-0

112 Super-Sensitive igh Sample Loading Capacity and Signal/oise for Trace Analysis igh column efficiency for high S/ igh sample loading from thick, porous shell layer Trace analysis benefits from high column efficiency. Efficient peaks are taller and provide higher S/ ratios. As discussed in earlier sections of this brochure, the Ascentis Express columns can provide higher efficiency than any traditional particle. The added sensitivity of the higher efficiency Ascentis Express particles is visualized as the sensitivity gap in Figure 6. Figure 6. igher Efficiency of Ascentis Express Provides Better Sensitivity than Traditional Columns columns: as indicated; 0 cm x.6 mm I.D. mobile phase : acetonitrile:water flow rate:.8 ml/min temp.: C det.: nm injection: μl. Uracil. Acetophenone. Benzene. Toluene Ascentis Express Traditional μm Traditional μm BETTER SESITIVITY TA REGULAR CLUMS Sensitivity Gap Sensitivity Gap Figure 7. igher Loading Capacity of Ascentis Express Compared to Sub- μm Particles Peak Symmetry as a Measure of verload Sub- μm Quickly verloads Ascentis Express C8 AS IG LADIG CAPACITY Concentration (μl/ml) Figure 8 shows how Ascentis Express extends the dynamic range. It has the high efficiency of sub- μm particles needed for trace analysis, and the high surface area of totally porous particles needed for high sample capacity. Figure 8. Extended Dynamic Range of Ascentis Express IG G000 Although they have a solid core, the 0. μm-thick shell of the Fused-Core particles provides roughly 7% of the surface area as a totally porous particle of the same diameter. nly the pores with very long diffusion paths are fused in the Ascentis Express PLC columns. The resulting particles have effective surface areas of ~ m²/g; comparable to totally porous particles. The higher surface area gives higher sample loading capacity compared to sub- μm particles, as evidenced by the symmetry vs. concentration relationship in Figure 7. Above ppm, the sub- μm experiences sample overload and subsequent loss of peak shape. Dynamic Range (Analyte Concentration) LW Sub- μm Particles μm Particles Ascentis Express Particles ave igh Surface Area and igh Efficiency for Wide Dynamic Range 6 sigma-aldrich.com/express ordering: (US only) / 8-9-

113 Super-Rugged Columns Extended Column Lifetime Compared to Both μm and sub- μm Columns arrow particle size distribution allows use of μm frits Dense particles for more stable bed Figure 9. arrow Particle Size Distribution of Ascentis Express Fused-Core particles are produced in a way that yields an extremely narrow particle size distribution (Figure 9). This narrow particle size distribution permits the use of frits with nominal μm pores, the same as used on most columns packed with μm particles. In comparison, sub- μm particles require frits with much smaller pore size 0. μm or smaller that are prone to fouling, lead to peak-splitting and high backpressure, and ultimately shorten the column lifetime. Another feature of the Fused-Core particles that contributes to their ruggedness is that they are denser than totally porous particles and form highly stable beds in the packed column. Ascentis Express ARRW PARTICLE SIZE DISTRIBUTI Absence of Fine Particles in Ascentis Express Allows Larger Pore Size Frits Porous Particle - Broad Particle Size Distribution Sample Prep Simplicity Combines the simplicity of protein precipitation and the selectivity of SPE for the targeted removal of phospholipids and proteins from biological samples EW! ybridspe Precipitation Technology Reduce ion-suppression 00% removal of phospholipids & precipitated proteins imal to no method development Available in 96-well and ml cartridge dimensions To learn more, visit our website: sigma-aldrich.com/hybridspe-ppt or contact Supelco Technical Service at /8-9-0 and [email protected] technical service: (US and Canada only) /

114 Alternative Selectivity with Ascentis Express RP-Amide Ascentis Express RP-Amide can solve Co-eluting peaks Unresolved components Poor retention of polar compounds Peak tailing of basic compounds Silanol interactions causing poor reproducibility While the Ascentis Express C8 provides classic reversed-phase selectivity, the Ascentis Express RP-Amide provides increased selectivity for polar compounds, especially those that can act as a hydrogen-bond donor. ther attributes of the RP-Amide include improved peak shape for bases, 00% aqueous compatibility, and low bleed for LC-MS applications. The amide group provides enhanced selectivity with analytes that have hydrogen bonded to a heteroatom. Phenols, carboxylic acids, amines and, to a lesser extent, alcohols show enhanced retention on the RP-Amide phase when compared to neutral non-polar analytes. An example of the power of the hydrogen bonding mechanism is shown in Figure 0. The Ascentis Express C8 and RP-Amide columns are compared. The analyte mixture contains neutral, non-polar analytes (benzene and toluene) and protic analytes (p-methoxyphenol, p-nitrobenzoic acid, and p-chlorophenol). As observed from the chromatograms in Figure, the neutral molecules show slightly reduced retention on the RP-Amide, but the protic molecules show greatly enhanced retention yielding a chromatogram with very different selectivities and even a change in elution order. The potential for solving separations difficulties is tremendous. Two other points should be noted. The Ascentis Express RP-Amide has the same high efficiency as the Ascentis Express C8 with the same low back-pressure. Secondly, both separations were carried out in the same mobile phase. This is important since it simplifies method development. If a separation is not adequate on an Express C8, there is no need to change mobile phase to optimize the separation, simply switch to the Ascentis Express RP Amide and if protic moieties are present, a change in selectivity will be achieved. Figure 0. Alternative Selectivity Provided by Ascentis Express RP-Amide Compared to C8 columns: Ascentis Express RP-Amide, 0 cm x.6 mm I.D. Ascentis Express C8, 0 cm x.6 mm I.D. mobile phase: 0:60, 0.% formic acid in water:methanol flow rate:.0 ml/min temp.: C det.: nm injection: μl. Uracil. p-methoxyphenol. Acetophenone. p-itrobenzoic acid. p-chlorophenol 6. Benzene 7. Toluene Ascentis Express RP-Amide Ascentis Express C G00 7 G006 Ascentis Express RP-Amide Applications atural products Phenolics Bases Metabolites Polar Compounds 8 sigma-aldrich.com/express ordering: (US only) / 8-9-

115 Polar Compound Retention with Ascentis Express ILIC Benefits of ILIC Separation Retention of highly polar analytes like metabolites Complimentary selectivity to reversed-phase chromatography Increased MS sensitivity Quick transfer from final steps of sample prep (SPE, protein precipitation, etc.) ILIC chromatography is gaining popularity due to increased retention of polar compounds. Many classes of polar compounds can be retained in ILIC. These include polar neutrals, polar acids, and polar and non-polar basic amines. Both polar and ionic interactions can contribute to retention and selectivity in this mode of chromatography. Figure. Comparison of the Analysis of Polar Molecules on Ascentis Express ILIC and Ascentis Express C8 columns: Ascentis Express ILIC, 0 cm x. mm ID,.7 μm particles (99-U) Ascentis Express C8, 0 cm x. mm ID,.7 μm particles (8-U) mobile phase: 0:90; 00 mm ammonium formate, p.0 with concentrated formic acid:acetonitrile flow rate: 0. ml/min temp.: C det.: UV at nm injection: μl. Acenaphthene, 80 μg/ml in mobile phase. Adenosine, μg/ml in mobile phase. Cytosine, 7 μg/ml in mobile phase Acenaphthene ow ILIC works ILIC separates compounds by using a mostly organic mobile phase across a polar stationary phase, causing solutes to elute in order of increasing polarity-the opposite of Reversed-Phase. Retention in ILIC is likely to be a combination of hydrophilic interaction, ion-exchange, and some reversed-phase retention. A typical mobile phase consists of 60-9% acetonitrile and an aqueous buffer. 0-0 mm ammonium acetate or ammonium formate are useful due to volatility and solubility. The sample solvent should be similar in type and strength as the mobile phase. The sample solvent can contain a higher amount of organic than the mobile phase, but should not contain more water than the mobile phase. Ascentis Express ILIC Ascentis Express C8, Adenosine G008 G0060 G008 Shown in Figure is a comparison of the analysis of highly polar molecules on Ascentis Express ILIC and Ascentis Express C8. Cytosine Ascentis Express ILIC Applications Amino acids Small, polar acids (metabolomics) Biogenic amines (neurotransmitters, contaminants in food & beverage) Phosphates (pesticides, herbicides) Sugars Drug metabolites and conjugates G000 0 G008 technical service: (US and Canada only) /

116 Improving PLC Sample Throughput Do More Work in Less Time Without Changing Your Method The demand for increased sample throughput and speed of results has driven PLC users to search for breakthroughs in PLC instruments and column technology. Although improvements have been realized, setbacks have been encountered. Reduction in column ruggedness, costly replacements of existing instrumentation, and difficulties in transferring methods to new systems have often made these past improvements unappealing to analysts. The Fused-Core PLC particle technology behind Ascentis Express permits - to 6-fold reduction in analysis time, with a subsequent increase in sample throughput compared to conventional PLC columns, without sacrificing resolution or column ruggedness and without the need to change systems or sample prep procedures The current high resolution column for traditional PLC methods is a cm column packed with μm particles. Until now, this dimension provided the most efficiency within the pressure limit of a conventional PLC system. With the high efficiency Ascentis Express, one can now achieve the same number of plates as a cm column packed with μm particles with a 0 cm column or even more efficiency and resolution with a cm Ascentis Express column. Therefore, by simply changing columns and keeping all other conditions the same, you can reduce the runtime and increase the resolution of your method. Figure compares the resolution of a five-component sample on cm, μm C8 and 0 cm Ascentis Express C8 columns. Each column has approximately the same number of theoretical plates and hence the same resolving power. owever the shorter Ascentis Express column delivers this separation in a much shorter time, in this case less than one-fourth the time as the cm column. Figure. Increase Sample Throughput by Using Ascentis Express C8, cm x.6 mm I.D., μm particles mobile phase: 6:, water:acetonitrile flow rate: ml/min temp.: ambient det.: UV at nm. xazepam: FW = 86. Alprazolam: FW =. Cloazepam: FW =. -desmethyldiazepam: FW = 70. Diazepam: FW = 8 X G0009 Ascentis Express C8, 0 cm x.6 mm I.D.,.7 μm particles (87-U) X 9.. X 9. X 8.. X G0000 Ready to do more work in less time? See the flow chart on the next page. 0 sigma-aldrich.com/express ordering: (US only) / 8-9-

117 Selecting the ptimal Ascentis Express Column Is your column a cm ( μm), cm ( μm), or 0 cm ( μm)? YES Do you want to MAXIMIZE SPEED or IMPRVE RESLUTI & SPEED? Convert to Ascentis Express PLC Columns for Both! Supelco Technical Service to assist in selecting an optimal Ascentis Express Column [email protected] MAXIMIZE SPEED A.: Conversion Table Current Ascentis Column Express cm ( μm) 0 cm cm ( μm) 7. cm 0 cm ( μm) cm IMPRVE RESLUTI & SPEED B.: Conversion Table Current Ascentis Column Express cm ( μm) cm cm ( μm) 0 cm 0 cm ( μm) 7. cm : Maximize flow rate within system pressure constraints * Ascentis Express Suggested Starting Point PLC Column ID for Flow Rate.6 mm I.D..6 ml/min.0 mm I.D. 0.8 ml/min. mm I.D. 0. ml/min : Confirm method suitability parameters.: Is resolution maintained? YES.: Is additional throughput or resolution desired? YES Improved sample throughput with Ascentis Express and your existing PLC system.: Select longer column.: Select shorter column or faster flow rate *Read Guidelines for ptimizing Systems for Ascentis Express Columns (T070) and Guide to Dispersion Measurement (T08). technical service: (US and Canada only) / 8-9-0

118 Fast PLC for Rapid Screening of Pharmaceutical Compounds Ideal for Walk-up LC-MS Systems PLC is critical to the discovery, development and eventual commercialization of pharmaceutical products. PLC is the benchmark analytical method in the pharmaceutical industry due to its ability to score such high marks in analytical validation characteristics including accuracy, precision, limit of detection, specificity, linearity and range, and ruggedness. o other analytical techniques can consistently score high in all characteristics on compounds and matrices that are of interest to the pharmaceutical industry. Furthermore, it has been generally accepted that a typical PLC analysis takes -0 minutes with some as great as an hour. When multiplied by the number of samples to be analyzed either in discovery or product release, the total instrument time required is staggering. This overwhelming amount of instrument time has resulted in a growing number of instruments, around-theclock analysis, and a push for faster methods. Fast PLC, using short columns (-0 cm) packed with small particles (< μm) and high flow rates has recently become an effective means to reduce analysis time. This is primarily due to the improved quality of sub- μm particle columns and the introduction of new instrumentation to meet the requirements of higher column backpressure and low instrument dispersion. The reasons for using sub- μm particle columns in fast PLC are evident by examining Van Deemter plots for various particle sizes. The smaller particles yield lower ETP or higher efficiency per unit length. Furthermore, the optimum flow rate is higher for smaller particles. Smaller particle columns have less efficiency loss at high flow rates because mass transfer is less sensitive to velocity changes as illustrated by flatter Van Deemter plots. Unfortunately, column backpressure increases at a greater rate than column efficiency as you decrease particle size. This increase in backpressure is so great for sub- μm particle columns that they are practically unusable using standard PLC systems. Shown in Figures - are the chromatograms for the separation of three sets of closely related pharmaceutical compounds. These examples include both basic and neutral as well as polar and non-polar compounds. While each example utilizes. mm I.D. columns, three different flow rates and three unique mobile phase conditions are presented to demonstrate the versatility of fast PLC with Fused-Core particle columns. Figure. TCAs on Ascentis Express column: Ascentis Express C8, 0 cm x. mm ID (8-U) instrument: Jasco X-LC mobile phase A: 00 mm ammonium acetate (p 7.0; titrated with ammonium hydroxide) mobile phase B: water mobile phase C: methanol mobile phase ratios: A:B:C = 0:0:60 flow rate: 0. ml/min temp.: C det.: Thermo LCQ Advantage; ESI(+), m/z 0-0 injection: μl. ordoxepin. Desipramine. ortiptyline. Doxepin. orclomipramine 6. Imipramine 6 G0006 sigma-aldrich.com/express ordering: (US only) / 8-9-

119 Shown in Figure is the separation of six tricyclic antidepressants (TCAs). The separation of these closely related compounds was performed under isocratic mobile phase conditions with mass spectrometric (MS) detection. Baseline resolution was achieved with a total separation time of minutes demonstrating not only the potential speed of the Ascentis Express columns but also the resolving power. ote the MS compatible mobile phase and flow rate. Furthermore, the use of. mm I.D. columns provides a reduction in solvent consumption compared to typical flow rates for.6 mm I.D. or monolithic columns. Data in Figure further illustrates the speed in which closely related compounds can be resolved using the Fused-Core particle. In this example, four -blockers are resolved in less than one minute under isocratic conditions utilizing MS detection. While a 0 cm column was utilized for the TCAs separation, a cm column was used for the -blockers example. The separation of three steroids as well as a related impurity and degradant is shown in Figure. A high mobile phase flow rate of 0.6 ml/min was utilized and is suitable for Ascentis Express columns due to the Van Deemter curve associated with these columns. Isocratic mobile phase conditions were utilized as well as UV detection at 00 nm, a common detection wavelength for impurity profiling. Again, baseline resolution was achieved for all compounds with a total runtime of less than two minutes. It should be noted that the isocratic conditions used in these examples further enhances sample throughput versus gradient conditions due to no need for column re-equilibration. With a backpressure of just 00 psi, this analysis could be performed on almost any PLC system. A similar separation was attempted using a sub- μm particle column but was not possible given the same instrument constraints put on the Ascentis Express column. Figure. -Blockers on Ascentis Express column: Ascentis Express C8, cm x. mm ID (8-U) instrument: Agilent 00 mobile phase A: 0.% acetic acid in water mobile phase B: 0.% acetic acid in acetonitrile mobile phase ratios: A:B = 7:6 flow rate: 0. ml/min temp.: C det.: ABI 00 QT; ESI(+), MS/MS injection: μl. Atenolol. Pindolol. Timolol. Metoprolol Figure. Steroids on Ascentis Express column: Ascentis Express C8, 0 cm x. mm ID (8-U) instrument: Jasco X-LC mobile phase: : water:acetonitrile flow rate: 0.6 ml/min temp.: ambient det.: 00 nm injection: μl. Estriol. -Estradiol. Impurity. Estrone. Estrone degradant G0006 G0006 technical service: (US and Canada only) / 8-9-0

120 Ultra-igh Resolution PLC: Column Coupling Maximize the Resolution of UPLC systems Column coupling in PLC is gaining interest since LC systems are being designed to withstand column back pressures of up to,000 psi. Column coupling is a simple and practical way to increase resolution by simply increasing column length. Because Ascentis Express PLC columns provide higher efficiencies at any pressure compared to μm and sub- μm particles, the coupling of Ascentis Express columns enables significantly higher resolution than any other column on any commercial PLC system. Efficiencies greater than 0,000 plates/column are possible and demonstrated in the isocratic separation of benzene and toluene with various deuterium substitutions. Figure 6 shows the efficiency obtained by coupling Ascentis Express cm columns together. Figure 6. Column Coupling of Ascentis Express Provides over 00,000 Plates per Separation column: Ascentis Express C8, cm x mm I.D., x mobile phase: 6:, water:acetonitrile flow rate: 0.6 ml/min temp.: 60 C pressure: 00 psi (90 bar) d 6 -Benzene. Uracil. Acetophenone. Benzene. Toluene d -Benzene d -Toluene d -Toluene Benzene d 8 -Toluene Toluene Column Coupling Applications atural product chemistry Tryptic digests Synthetic peptide mapping Stress studies of APIs LC-MR 60 igh Performance PLC Fittings/Interconnects Improve PLC performance with these fittings only from Supelco. mau 0 nm 0 0 Key Benefits Body Eliminate dead volume Use o Tools! that contributes to peak broadening and decreased resolution Sliding ferrule design allows for use in any port Fingertight fittings, no tools required Rated to,000 psi For a complete list, visit sigma-aldrich.com and enter the keywords: igh Performance Fitting P G00 TRADEMARKS: Ascentis, ybridspe Sigma-Aldrich Biotechnology LP Fused-Core Advanced Materials Technology UPLC Waters Associates, Inc. sigma-aldrich.com/express ordering: (US only) / 8-9-

121 rdering Information Analytical ID Length Ascentis Ascentis Ascentis Ascentis (mm) (cm) Express C8 Express C8 Express RP-Amide Express ILIC. 80-U 89-U 90-U 9-U. 8-U 8-U 9-U 9-U U 8-U 9-U 98-U. 0 8-U 8-U 9-U 99-U. 8-U 8-U 9-U 96-U.0 80-U 8-U 9-U 96-U.0 8-U 88-U 96-U 967-U U 89-U 97-U 969-U U 8-U 98-U 970-U.0 86-U 8-U 99-U 97-U.6 88-U 87-U 9-U 97-U.6 86-U 86-U 9-U 97-U U 88-U 9-U 977-U U 87-U 99-U 979-U.6 89-U 88-U 9-U 98-U Capillary Ascentis Express C8 Ascentis Express C8 Length Length cm cm cm cm 7 μm I.D. 98-U 9-U 98-U 9-U 00 μm I.D. 98-U 6-U 987-U 60-U 00 μm I.D. 989-U 6-U 99-U 6-U 00 μm I.D. 99-U 7-U 997-U 7-U 00 μm I.D. 998-U 7-U 999-U 7-U Ascentis Express Properties Stationary Phase Support Ultra-pure, Type B silica.7 μm solid core particle with 0. μm porous silica shell (effective.7 μm) 0 m /gram surface area (comparable to ~ m /g porous particle) 90 Å pore size Bonded Phase Coverage μmoles/m p Range Endcapping C8. -9 Yes C8.7-9 Yes RP-Amide.0-9 Yes ILIC n/a -8 o technical service: (US and Canada only) / 8-9-0

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123 Ascentis Express PLC Resource Guide Application Articles Choosing an Alternate Phase Bibliography of Fused-Core Publications Practical Recommendations for Success Listing of Available Technical Literature

124 Table of Contents The Fused-Core Advantage... Introducing Ascentis Express... Practical Recommendations for Success... Rapid, Sensitive, General-Purpose Cleaning Validation Using Ascentis Express PLC Columns...6 Ascentis Express Peptide ES-C8 Expands the Fused-Core Particle Platform into Bioseparations...8 Improving the Current USP Method for the Analysis of Lansoprazole Drug Substance Using PLC Columns Based on Fused-Core Particle Technology...9 Selecting an Ascentis Express Phase... Profiling of Stevia rebaudiana Extract by Accurate Mass Using ILIC and Reversed-Phase Chromatography... Bibliography of Fused-Core Publications... Colors of the World: Fast Separation of Dyes with Ascentis Express...7 Ascentis Express RP-Amide...0 Ascentis Express Phenyl-exyl... Ascentis Express FAQs...6 arrow Particle Size Distribution More Consistent Bed The Fused-Core Advantage Extreme performance on any LC system alf the backpressure of sub- μm columns Twice the performance of μm columns Fused-Core Particles Traditional Porous Particles The innovative manufacturing process for Fused-Core particles produces a very narrow particle size distribution. A narrow particle size distribution allows for the use of large porosity frits that resist clogging, resulting in a more rugged column. Traditional porous particles are not manufactured in a way to yield extremely narrow particle size distributions. The A term in the van Deemter equation accounts for the effects of inhomogeneities in the packed bed of an PLC column. arrow particle size distributions form a more consistent packed bed and a consistent path length, minimizing analyte diffusion through the column. This eddy diffusion is effectively independent of mobile phase velocity. Available Technical Literature...6 rdering Information...7 Available Resources US Technical Service phone: (US and Canada only) fax: / [email protected] EU Technical Services [email protected] Internet: sigma-aldrich.com/express Webinars: updates: Twitter: sigma-aldrich.com/videos (available hours/day) Fused-Core Report (register at sigma-aldrich.com/express) twitter.com/plcsessions Shorter Diffusion Path The short diffusion path of the Fused-Core particle yields sharper peaks than traditional porous particle columns. The minimized resistance to mass transfer, the C term in the van Deemter equation, of the Fused-Core particle provides sharper peaks than traditional porous particles. The short diffusion path also permits the use of higher flow rates without peak broadening. sigma-aldrich.com/express ordering: (US only) / 8-9-

125 Introducing Ascentis Express ow, igh Speed and igh Efficiency PLC Separations are Possible on Any LC System Increasing speed and resolution of PLC analyses are drivers for innovation in both PLC column and hardware design. While columns packed with μm particles have been the standard, reducing particle size has been the strategy of many column manufacturers and users alike. Smaller particles result in faster chromatography. The cost for the improved speed is higher column backpressures. To obtain the benefit of the small particles, instrumentation beyond conventional PLC is required. Ascentis Express columns provide a breakthrough in PLC column performance. Based on Fused-Core particle technology, Ascentis Express provides the benefits of high speed of much smaller particles but at a backpressure suitable to conventional PLC systems. Due to this fundamental performance advantage, Ascentis Express can benefit both conventional PLC users as well as UPLC or other ultra pressure system users. yper-fast Super-Sensitive Extreme Performance on AY LC System Super-Rugged igh Definition D Resolution Introduction Exceeding the Performance of ther Fast PLC Particles Designed to deliver speed and resolution on all LC systems, Ascentis Express meets and exceeds the benefits of competitive particles, including μm and sub- μm particles. Under the same conditions, Ascentis Express columns deliver the same efficiencies at half the backpressure of sub- μm particles and nearly twice the efficiency of μm particles. Compared to Sub- μm Particles: Advantage: Ascentis Express columns can be run successfully on conventional, mid-pressure and ultra high pressure PLC and LC-MS instruments. Advantage: Double the flow rate. Run Ascentis Express columns at higher flow rates for faster analyses. Advantage: Double the column length. Longer Ascentis Express columns can be used, giving additional resolving power. Compared to μm Particles: Advantage: Double the efficiency. Ascentis Express columns have nearly twice the column efficiency of μm particles. Practical Recommendations for Success Use 0.00 in. I.D. inlet and outlet tubes. Broadening is much less sensitive to the tube length than to the I.D. imize lengths of the inlet and outlet tubes for best performance, but do not worry about having a few extra centimeters of length if it makes maintenance or column installation easier. If high pressure becomes a problem, then use acetonitrile as modifier and elevate the column temperature whenever possible. If methanol, TF, or another more viscous modifier is required, then elevating the temperature becomes even more beneficial. Even a modest temperature increase will greatly reduce the mobile phase viscosity and the required pressure while improving mass transfer. Use only.6 mm diameter Fused-Core columns on conventional PLC systems to minimize broadening problems from the remaining system components. Extra column broadening worsens as the column diameter is decreased. Keep the sample volumes small μl or less if the peaks of interest elute early (k = ). Up to 0 μl is acceptable if k exceeds 0. Avoid sample solvents that are stronger than the mobile phase. Use data rates of 0 z or greater, and watch out for bunching factors. technical service: (US and Canada only) / 8-9-0

126 Figure. Fused-Core Structure of Ascentis Express Compared to Totally Porous Particles Ascentis Express Particle Totally Porous Particle 0. μm 0. μm Introduction.7 μm.7 μm Diffusion Path. μm G0088 The Particle Platform Innovations Behind Ascentis Express Like most modern PLC particles, Ascentis Express particles are high surface area spheres made from high purity silica gel. The total particle diameter is.7 μm. owever, here the comparison ends. What sets apart Ascentis Express from conventional PLC particles is the patent pending Fused-Core technology. Ascentis Express particles comprise a solid.7 μm diameter silica core that is encapsulated in a 0. μm thick layer of porous silica gel. There are five distinct properties of Ascentis Express particles that account for their high performance and are worth emphasizing:. The solid core Because of the solid core, analytes cannot diffuse as deeply into the particle, resulting in less band broadening, and hence higher efficiency and sensitivity, compared to totally porous particles of the same diameter.. The 0. μm porous shell surrounding the solid core The porous shell gives the particles a surface area comparable to totally porous particles for excellent phase loading and sample capacity.. The total particle diameter (.7 μm) Compared to sub- μm porous particles, Ascentis Express yields half the column backpressure, allowing longer columns and faster flow rates (Figures and ). Compared to μm porous particles, Ascentis Express yields nearly twice the efficiency (Figure ).. The narrow particle size distribution. Compared to both sub- μm and μm particles, Ascentis Express provides longer column lifetime because the narrow particle size distribution allows us to use larger pore size frits ( μm vs. 0. μm) that are less susceptible to fouling.. The high particle density By virtue of the solid core, Ascentis Express particles yield a more densely packed bed for added stability and long column lifetime. Figure. yper-fast Separations on Ascentis Express column: Ascentis Express C8, 0 cm x. mm I.D.,.7 μm particles (8-U) and sub- μm particle column (same dimensions) mobile phase: 9: or :, water:acetontrile flow rate: 0. or 0. ml/min. temp.: ambient det.: UV at 00 nm injection: μl. Estradiol Ascentis Express C8 0. ml/min flow rate Sub- μm competitor 0. ml/min flow rate G psi 000 psi. -Estradiol. Impurity. Estrone. Estrone degradant TWICE TE SPEED AT EQUAL PRESSURES G00976 sigma-aldrich.com/express ordering: (US only) / 8-9-

127 Ascentis Express: igh Speed, igh Efficiency Separations Adaptable Equally to R&D and Routine Analysis Settings The recent introduction of UPLC and other ultra high pressure LC systems addressed the need for high throughput separations. owever, speed is not the only important criteria: the need for more sensitivity, more resolution and improved ruggedness of the technique has lead to a continual stream of new LC and LC-MS instruments. Coupled with the large installed base of conventional PLC instruments, the result is that most laboratories have a mixture of instruments, old and new. Whereas columns packed with sub- μm particles require ultra high pressure instruments, Ascentis Express columns can be run on any LC system. Methods developed on Ascentis Express can be Figure. D-Resolution on Ascentis Express Compared to μm Particles column: Ascentis Express C8, cm x.6 mm I.D.,.7 μm particles (89-U) and C8, cm x.6 mm I.D., μm particles mobile phase: :6 or 7.:7., water:acetontrile flow rate:. ml/min. temp.: ambient det.: UV at 0 nm injection: μl. aphthalene. p-xylene. Biphenyl Ascentis Express C8 EARLY TWICE TE EFFICIECY 6,800,700 Introduction Figure. D-Resolution on Ascentis Express Compared to Sub- μm Columns column: Ascentis Express C8, 0 cm x. mm I.D.,.7 μm particles (8-U) and sub- μm particle column, cm x. mm I.D. mobile phase: : or :6, water:acetontrile flow rate: 0. ml/min. temp.: ambient 00 psi det.: UV at 00 nm injection: μl Ascentis Express C8 0 cm length. Estradiol. -Estradiol. Impurity. Estrone. Estrone degradant TWICE TE RESLUTI AT EQUAL PRESSURE C8, μm 7,00,700,600 G00979,00 G00980 G0097 readily and reliably validated and transferred from R&D to routine analysis labs, whether across the building or across the world. C8 Sub- μm cm length 60 psi We hope this article has sparked an interest in Ascentis Express and the benefits it can bring to your laboratory. Subsequent articles will develop the Ascentis Express message by focusing on specific features and application areas. G0097 technical service: (US and Canada only) / 8-9-0

128 Rapid, Sensitive, General-Purpose Cleaning Validation Using Ascentis Express PLC Columns Cleaning Validation Contributed Article The following was generated by an outside source using Sigma-Aldrich products. Technical content provided by: S. Bannister, M. Talbott, F. anciles Xcelience LLC, Tampa, FL Verification of the removal of drug residue from multiproduct manufacturing equipment is required by GMP regulations and the suitability of applied analytical methods is judged with a combination of sensitivity, selectivity, and because the release of equipment is dependent - speed. The FDA does not set quantitative acceptance specifications, but the commonly used limit is based on not more than 0.% of a dose carried over into a single dose of the next product. Translation of this into an analytical limit combines the total product contact area, the mass (or volume) of product contacting the surface, the mass (or volume) of each dose unit, the sampled area, the rinse volume and the fraction of the rinse sample used for analysis. The requisite limits are commonly measured in ng/ ml of injected sample. The ubiquity of PLC in drug analysis makes it an attractive choice for cleaning validation. Methods qualified for cleaning validation are often adaptations of drug-substance methods. The original methods are capable of determining the drug and its related impurities, but the ability to simultaneously measure multiple closely related analytes comes at the expense of run time and is not needed in cleaning validation. This work was undertaken to investigate the use of rapid gradients using recently introduced FCP columns on conventional instrumentation in the development of general-purpose methods for cleaning validation. The benefits include high sensitivity and reductions in the time needed to set up and run the method. Resolution, limits of detection and quantitation, and run time in PLC analyses are improved by reducing the width of eluted bands. Contributions to bandwidth include both column (particle size, packing structure and resistance to mass transfer in the stationary and mobile phases) and extracolumn volumes (injection, unswept and tubing). Columns packed with μm fully porous particles have been the standard for conventional PLC for twenty-five years. Smaller-particle packings ( μm) have been available almost as long and offer higher efficiency (lower band dispersion) Figure. Acidic and eutral Drug Panel column: Ascentis Express C8, 0 cm x.6 mm I.D. (87-U) mobile phase A: water with 0.% phosphoric acid mobile phase B: acetonitrile with 0.% phosphoric acid temp.: ambient flow rate:.76 ml/min det.: UV at nm inj.: 00 μl gradient: %A %B G00 Figure. Basic Drug Panel. ydrochlorothiazide (9). Chlorthalidone (). Prednisolone (). Pravastatin (). Carbamazepine () 6. Diclofenac () 7. Ibuprofen () 8. Progesterone () column: Ascentis Express C8, 0 cm x.6 mm I.D. (87-U) mobile phase A: water with 0.0M potassium phosphate and 0.% TEA and 0.6% SA-a at p =.9 mobile phase B: acetonitrile temp.: ambient flow rate:.76 ml/min det.: UV at nm inj.: 00 μl gradient: %A %B G Quinidine (). Dipyridamole (9). Propranolol (). aloperidol (8). Amlodipine (9) 6. Fluoxetine () 6 sigma-aldrich.com/express ordering: (US only) / 8-9-

129 dispersion) on conventional instrumentation, but require higher pumping pressures due to lower bed permeability. Efficiency can be further increased by the use of particles smaller than μm but only with the use of instrumentation optimized with respect to both pressure and extra-column effects. Supelco has recently introduced reversed-phase packings based on.7 μm silica particles in which a 0. μm layer of 90-Å porous silica has been deposited onto a.7 μm solid spherical core. Advantages of columns packed with these particles include high efficiency, lower backpressure due to a very narrow particle size distribution, and smaller efficiency losses with increasing velocity due to improved mass-transfer kinetics in the shallow porous layer. The narrow particle size distribution allows the use of larger pore column frits, which combined with the greater stability of the packed bed should produce longer column lifetimes in routine use. The high resolving power of gradient elution in the analysis of closely related substances is the result of the reduction of peak width as a band moves through the column. The back of the band is accelerated by the stronger solvent. A broad gradient will elute a wide range of substances and a steep gradient will elute them quickly. Versatile Separations To judge the utility of Ascentis Express columns in cleaning validation, an Agilent 00 component system with standard components (including a 0 mm/ μl flow cell) was used to develop a short gradient separation using Ascentis Express C8, 0 cm x.6 mm for each of two panels: eight acidic or neutral drugs (A) and six basic drugs (B). For each separation, the flow rate was.76 ml/min, detection was at nm, and 00 μl injections were made of aqueous solutions representing the final equipment rinse. The separations are shown in Figures &. Limits of detection (ng/ml) are listed next to each analyte in Figures and. These separations demonstrate the capabilities of Ascentis Express columns on conventional, robust, instrumentation in rapid analyses of multiple drugs at low ppb levels suitable for development as methods for cleaning validations in multiproduct manufacturing facilities. Cleaning Validation Selecting the Right Buffer A partial list of common buffers and their corresponding useful p range is supplied. Perhaps the most common buffer in PLC is the phosphate ion. Although, with the growth of LC-MS, volatile buffers such as TFA, acetate, formate, and ammonia are becoming more frequently used. Remember, the purpose of a buffer in the mobile phase is to inhibit a p change in the mobile phase after the introduction of a sample. When developing a method, it is important to select a mobile phase with a final p at least one p unit away from any analytes pk value. As a rule of thumb, one should work within a ± p unit of the buffer pka. Typical buffer concentrations for PLC tend to be 0-00 millimolar level. Buffer C Useful p Range Trifluoroacetic acid (TFA) 0. <. Phosphate.. -. Formate Acetate Phosphate Ammonia Phosphate.. -. Guidelines for Preparing Mobile Phases It should be understood that slight variations in p and buffer concentration could have a dramatic affect on the chromatographic process; consistent and specific techniques should be a regular practice in the preparation of mobile phases. A common practice is to place a sufficient amount of pure water into a volumetric flask and add an accurate amount of buffer. The p of the solution should be adjusted, if necessary, and then dilute to final volume of water prior to adding or blending of organic solvents. Then, add a volumetrically measured amount of organic solvent to obtain the final mobile phase. Thorough blending, degassing, and filtering prior to use is also recommended. To view a listing of suitable PLC and LC-MS additives and solvents, visit sigma-aldrich.com/lc-ms-solvents technical service: (US and Canada only) /

130 Ascentis Express Peptide ES-C8 Expands the Fused-Core Particle Platform into Bioseparations Peptides Introduction Ascentis Express Peptide ES-C8 columns were specifically engineered to separate higher molecular weight compounds such as peptides and small proteins. These columns contain advanced Fused-Core particles that have bigger pores (60 Å versus 90 Å in standard Ascentis Express), which greatly expands the application range for Ascentis Express columns. Key Applications for Ascentis Express Peptide ES-C8: Pharmaceutical/therapeutic peptides Peptide mapping atural and synthetic peptide analysis ligonucleotide analysis Key Advantages: igher peak capacity providing greater resolution Amenable to higher flow rates for faster analysis Exceptional ruggedness providing long column lifetime Ascentis Express Peptide ES-C8 columns utilize a stericprotected C8 bonded-phase with extremely high resistance to acid-catalyzed hydrolysis of the siloxane bond that attaches the C8 chain to the surface. Thus, the combination of low p and elevated temperature operation of the column is well tolerated. Peptide separations are efficiently conducted using low p mobile phase modifiers, often at % concentration, most popularly employing trifluoroacetic acid (TFA), and the related perfluorocarboxylic acids, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (FBA). These acids exhibit desirable low UV transparency, volatility, and peptide ion-pairing properties. Additional opportunities for low p operation include the normal short chain carboxylic acids, formic acid and acetic acid, as well as mineral acids, such as phosphoric acid ( M). Shown in Figure is the chromatographic separation of a peptide mix. The peptide mix contains a range of peptides in terms of molecular weight, basicity, and hydrophobicity. Excellent peak shape and peak width are achieved with a standard acetonitrile gradient and 0.% TFA modifier. The resolution of small baseline impurities are shown in the inset, demonstrating the resolving power of the Ascentis Express Peptide ES-C8 column versus a traditional μm column. Figure. Comparison of Peptide Test Mix with Ascentis Express Peptide ES-C8 and Traditional Column column: Ascentis Express Peptide ES-C8, 0 cm x.6 mm I.D. (-U) mobile phase A: 0% acetonitrile / 90% water / 0.% trifluoroacetic acid mobile phase B: 7% acetonitrile / % water / 0.% trifluoroacetic acid gradient: 0% to 0% B in min flow rate:. ml/min. det: UV at 0 nm temp: 0 C injection: μl Peptide Test Mix. Gly-Tyr MW =. Val-Tyr-Val MW = 79. Met Enkephalin MW = 7. Angiotensin II MW = 0 Ascentis Express Peptide ES-C G009. Leu-Enkephalin MW =.6 6. Ribonuclease MW =, Bovine Insulin MW = G G0098 7, G Traditional μm Column 7 For more information on the Ascentis Express Peptide ES-C8, request brochure T00 (MII). 8 sigma-aldrich.com/express ordering: (US only) / 8-9-

131 Improving the Current USP Method for the Analysis of Lansoprazole Drug Substance Using PLC Columns Based on Fused-Core Particle Technology Contributed Article The following was generated by an outside source using Sigma-Aldrich products. Technical content provided by: Kai Li, Jiajie e, and Xiaoya Ding PPD - 8 Research Way, Suite 90, Middleton, WI 6 Introduction Compendial methods from the USP (United States Pharmacopeia) are widely used in pharmaceutical drug product and raw materials testing. owever, not all methods in the USP use modern technologies. In chromatographic methods, it is not uncommon that older brands of columns are specified. Therefore, the USP methods are under continuous revision to improve existing procedures or to allow the user to obtain better results. Due to the improved resolving power of Fused-Core particles, the method was optimized with a shorter runtime without sacrificing resolution. In an effort to improve the compliance of drug product, drug substance, and excipient monographs with current scientific/regulatory standards, USP is seeking the submission of proposals for improved methods. The intent is to replace the current procedures that may be deficient, flawed, or unsafe (e.g. Monograph/improveMon.html). Requests for revision of an existing monograph are encouraged by USP in light of advances in analytical technologies. Furthermore, ease of operation, suitability for automation, and potential for high-throughput analysis can be considered in a revision. To develop the best possible analytical test method for its intended use, a fully integrated method development process such as the selection of column, mobile phase, detection technology, and LC hardware by utilizing the most advanced technologies viable should be considered to ensure the methods are robust, consistent, and easy to use. In this study, the USP method for lansoprazole was considered for improvement. Several drawbacks in the current USP monograph for lansoprazole prompted the investigation. These drawbacks include sample solution instability, use of different columns and samples preparations for the evaluation of assay and impurity, the requirement of using internal standard for assay and a long PLC runtime (60 min). A new PLC column, Ascentis Express C8, based on Fused-Core particle technology was investigated for this study. The Ascentis Express PLC column claims high efficiencies as a result of a 0. μm layer of porous silica on a.7 μm solid silica core. An additional advantage to the column is that standard PLC instrumentation can be used as opposed to UPLC that is required for sub- μm columns. Results and Discussion Initially, a traditional μm C8 column as specified in the USP monograph was compared to the.7 μm Ascentis Express C8 using the standard USP conditions for chromatographic purity for lansoprazole (, ). Improved resolution and sensitivity were obtained using the Fused- Core column that allowed us to make several significant improvements to the method. Due to the improved resolving power of Fused-Core particles, the method was optimized with a shorter runtime without sacrificing resolution. The total run time was reduced from 60 min to 0 min (Table ). Moreover, the improved sensitivity allowed for the reduction in concentration of the test sample for chromatographic purity from 0 mg/ml to 00 mg/ml, the level required for assay in the USP monograph. Therefore, simultaneous evaluation of assay and chromatographic purity is achieved. Finally, a change of diluent p, was implemented to improve sample solution stability removing the requirement of injecting sample within 0 minutes after preparation. Table. Method Parameters for Improved Lansoprazole Method column: Ascentis Express C8, cm x.6 mm,.7 μm (89-U) mobile phase A: Water mobile phase B: Acetonitrile: 0.% Triethylamine in Water, p=7.0 [80:0] flow rate: 0.8 ml/minute column temp.: Ambient autosampler Temp.: C injector volume: μl detector wavelength: 8 nm run time: 0 min gradient: Time () %A %B USP Method technical service: (US and Canada only) /

132 Figure. Linearity Curve from 0 to 0% ominal Concentration Figure. Analysis of Lansoprazole Using Improved Method with Ascentis Express C8 PLC Column Unstressed Lansoprazole 0.8 USP Method AU Lansoprazole 0.0 Figure. Linearity Curve from 0.0 to.0% ominal Concentration G006 Aged Resolution Solution 0. Lansoprazole G009 AU Degraded Lansoprazole Exposed to 0. Cl 0. G0060 G006 The new method was shown to be linear from 0.0% to 0% of nominal concentration of 00 mg/ml, with quantitation limit less than 0.0%. The broad range of linearity allows for simultaneous impurity and assay analysis. The linearity data are shown in Figures and. The RSD of replicate injections of standard solution was 0.%. In additional experiments, the method was evaluated by analysis of degraded lansoprazole drug substance. Lansoprazole was stressed under four separate conditions by exposure to acid, base, heat and hydrogen peroxide. The chromatograms of the acid and peroxide exposed drug substance along with the unstressed drug substance are shown for reference. The resolving power of the Ascentis Express PLC column makes it very suitable for these types of studies. AU AU Lansoprazole Degraded Lansoprazole Exposed to 0.0% Lansoprazole G006 G006 0 sigma-aldrich.com/express ordering: (US only) / 8-9-

133 Conclusion The method developed using.7 μm Fused-Core C8 column provided significant improvements in comparison with the original USP method in terms of resolution, on, run time and sensitivity. As a result, the consolidated single method can be used for both assay and impurity quantitaation. The advantages of Fused-Core columns as an alternative for sub- μm columns without using new UPLC instruments could be appealing for pharmaceutical cal testing. Furthermore, this paper has presented one of the ways (a road map) that could be utilized by analytical scientists in the pharmaceutical field to improve USP monographs for their intended purposes using modern analytical technologies. USP Method References. USP Monograph: Lansoprazole, USP 0-F through second supple-plement.. Pharmacopeial Forum, In-Process Revision, Lansoprazole, Vol. (6), 70-7 (006) Selecting an Ascentis Express Phase Ascentis Express C8 is the first choice for starting a new method. owever, when a C8 doesn t give the desired separation or your sample contains compounds that are known to be difficult to retain or resolve on a C8, consider changing stationary phases. The range of selectivity provided by Ascentis Express makes this easy. The flow chart below helps guide in the selection of an Ascentis Express phase, based on the particular compound type or separation challenge. Desire to Improve Retention, Selectivity or Resolution vs. C8 on-polar Compounds Polar Compounds Very Polar Compounds Peptides Too Much Retention Retention Too Short or Inadequate Separation Poor Peak Shape (Basic Compounds) ot Enough RP Retention, LC-MS Basic Compounds -Bonding, Phenolic, Acidic or ydroxylated Compounds p Electron- Acceptors itroaromatics, eteroaromatics Strong Dipoles ILIC Mode C8 RP-Amide Phenyl- exyl RP-Amide ILIC Peptide ES-C8 technical service: (US and Canada only) / 8-9-0

134 Profiling of Stevia rebaudiana Extract by Accurate Mass Using ILIC and Reversed-Phase Chromatography atural Products Introduction There is growing public interest in low-calorie alternatives to carbohydrate-based sweeteners. Synthetic sweeteners are often regarded as having an undesirable aftertaste. Recent publications have shown a dramatic increase in attention toward natural extracts including the Stevia rebaudiana plant, not only for its sweetening effect but also for additional health benefits attributed to the plant. The major sweetening components are stevioside, rebaudioside A, rebaudioside C, and dulcoside A, each of which is over 00 times sweeter than sucrose-based sweeteners. The concern with the human consumption of the stevia leaf had been attributed to the possible mutagenic properties of steviol, but more recent studies conducted by the World ealth rganization have established the safety for steviol and its glycosides. In this study, an evaluation of the Stevia rebaudiana plant extract was conducted using modern chromatographic and mass spectrometry techniques for the determination of extracted components. The purpose was to evaluate the utility of performing two different modes of chromatographic separation for component identification. An accurate mass time of flight (TF) mass spectrometer was used in the detection and identification of components. A novel software package was then utilized for the determination of common components between the two chromatographic modes and to depict the impact of chromatographic selectivity. The concept behind the study was to utilize both reversedphase chromatography and ILIC chromatography for the determination of extract components. By using two different modes of selectivity, components that co-retain, do not retain, or do not elute under one chromatographic mode may be resolved under a separate mode. By resolving a component chromatographically, a more accurate assessment of the component can be made without relying specifically on accurate mass data. With traditional reversed-phase chromatography, analytes are primarily retained on an alkyl based stationary phase by partitioning interaction between the non polar stationary phase and the analyte. Though this mode of chromatography is widely accepted for separation of moderately polar to non-polar compounds, highly polar analytes often have minimal or no retention on these phases. More popular polar embedded stationary phases address this issue with the addition of a polar functional group within the alkyl chain. Polar embedded phases can enhance retention of polar compounds, but it is not a solution for all applications. ften highly polar analytes require alternative modes of chromatographic retention. In particular, ILIC chromatography allows for alternative selectivity by utilizing a highly polar stationary phase with a relatively non polar mobile phase. Under ILIC conditions, the partitioning of analytes is achieved through a preferential solvation of an aqueous environment on the polar surface. More polar analytes will partition more into the surface solvent and thus be retained longer than a less polar analyte. In addition to the partitioning, the polar surface of the stationary phase allows for adsorptive interactions via hydrogen bonding, dipole, etc. When ionic samples are separated, the potential for ion-exchange interactions also exists and in many cases becomes the dominant retention mechanism. Using silica-based stationary phases, ionized surface silanol groups may interact via ion-exchange with positively charged analytes. Experimental In this study, both reversed-phase and ILIC separations were conducted using the Ascentis Express RP-Amide and Ascentis Express ILIC. The polar embedded group of the Amide was chosen over traditional C8 phases to increase the retention of the polar analytes in the stevia Stevia rebaudiana sigma-aldrich.com/express ordering: (US only) / 8-9-

135 extract. The Ascentis ILIC allowed for alternative selectivities for polar analytes. Because of the large amount of unknown components in the stevia extract, using both reversed-phase and ILIC modes enabled orthogonal selectivity to resolve co-retained components and enable better determination of components in the extract with confirmation between the two modes. Stevia leaves were obtained from Sigma Aldrich (S8). Sample extraction of the stevia leaves was performed by weighing 00 mg of crushed stevia leaves into a 7 ml amber vial. A total of ml of 0:0 acetonitrile:water was added and the sample was vortexed and sonicated for minutes. The sample was then centrifuged for minutes at 000 rpm. The supernatant was then collected and analyzed directly. The sample extract was analyzed using a gradient elution profile for both ILIC and reversed-phase chromatographic modes. Analysis was conducted using an Agilent 00SL Rapid Resolution system in sequence with an Agilent 60 TF mass spectrometer. The TF enabled the use of accurate mass for determination of components. The acquired data was processed using the Mass unter software package. The data was pushed to the Mass Profiler package for statistical comparison of the two chromatographic modes. This software package enabled the identification of common components between the two chromatographic separations of the stevia extract. By performing this type of statistical comparison, the components attributed to the stevia extract were differenti- ated from components attributed to chromatographic anomalies. From this comparison the major components of the stevia extract were determined. Available standards were then used to confirm the identification of several of the components. Results and Discussion Figure and Figure represent the total ion chromatogram for the stevia extract under both ILIC and reversed-phase conditions. Both of these chromatographic separations demonstrate the complexity of the stevia extract. Table depicts the major components that were common in both the reversed-phase and ILIC separations of the stevia extract. More than 0 components were identified with this comparison, but only the major components were targeted in this study. The highlighted components in Table depict co-retention of analytes under reversed-phase conditions. A good example of using this orthogonal approach is observed in the case of steviobioside and ducloside A. Under the reversedphase separation, these components were co-retained. By performing the separation under ILIC conditions, steviobioside and ducloside A were well separated. ther unidentified major components that were unresolved under the reversedphase conditions were also separated under the ILIC conditions. The data in Table also depicts the selectivity difference between the two chromatographic modes. Polar components that were poorly retained in the reversed-phase conditions were (continued on page 6) atural Products Figure. Component Chromatogram of Stevia Extract on Ascentis Express ILIC column: Ascentis Express ILIC, cm x. mm I.D.,.7 μm with upchurch inline filter flow: 0. ml/min. mobile phase A: mm ammonium formate (98: acetonitrile:water) mobile phase B: mm ammonium formate (80:0 acetonitrile:water) temp.: C inj. vol.: μl system: Agilent 00SL 60 TF, ESI(+) datafile: 808.d gradient: Time %A %B Flow Figure. Component Chromatogram of Stevia Extract on Ascentis Express RP-Amide column: Ascentis Express RP-Amide, cm x. mm I.D.,.7 μm with Upchurch inline filter flow: 0. ml/min. mobile phase A: 0 mm ammonium formate water mobile phase B: 0 mm ammonium formate (9: acetonitrile:water) temp.: C inj. vol.: μl system: Agilent 00SL 60 TF, ESI(+) datafile: d gradient: Time %A %B Flow G G007 technical service: (US and Canada only) / 8-9-0

136 Bibliography of Fused-Core Publications Title: Comprehensive two-dimensional liquid chromatography to quantify polyphenols in red wines. Authors: Paola Dugo, Francesco Cacciola, Paola Donato, Diego Airado-Rodríguez, Miguel errero, Luigi Mondello Affiliation: Dept of Food & Environment, University of Messina, Messina, Italy; Dept of Pharmaceutical Chemistry, University of Messina, Messina, Italy; Dept of Analytical Chemistry, University of Extremadura Avda, Badajoz, Spain; Campus-Biomedico, Roma, Italy Journal: Journal of Chromatography A, 009 Title: Serial coupled columns reversed-phase separations in high-performance liquid chromatography Tool for analysis of complex real samples Authors: Luigi Mondello, Miguel errero, Francesco Cacciola, Paola Donata, Daniele Giuffrida, Giacomo, Dugo, Paola Dugo Affliation: Dept of Pharmaceutical Chemistry; University of Messina, Messina,Italy; Campus-Biomedico, Roma, Italy; Dept of Food & Environment, University of Messina, Messina, Italy Journal: Journal of Chromatography A, 008, 08- Title: Characterization of new types of stationary phases for fast liquid chromatographic applications Authors: Szabolcs Fekete, Jenö Fekete, Katalin Ganzler Affliation: Formulation Development, Gedeon Richter Plc, Budapest X, ungary; Dept of Inorganic & Analytical Chemistry; Budapest University of Technology & Economics Journal: Journal of Pharmaceutical and Biomedical Analysis, 009 Title: The use of partially porous particle columns for the routine, generic analysis of biological samples for pharmacokinetic studies in drug discovery by reversed-phase ultra-high performance liquid chromatography-tandem mass spectrometry Authors: David. Mallett, César Ramírez-Molina Affliation: GlaxoSmithKline Medicines Research Centre; Respiratory CEDD DMPK & Immuno-Inflammation CEDD DMPK; Stevenage, erts, United Kingdom Journal: Journal of Pharmacuetical and Biomedical Analysis, 009, Title: Shell and small particles; Evaluation of new column technology Authors: Szabolcs Fekete, Jenö Fekete, Katalin Ganzler Affiliation: Formulation Development, Gedeion Richter Plc, Budapest X, ungary; Dept. of Inorganic & Analytical Chemistry; Budapest University of Technology & Economics, Budapest, ungary Journal: Journal of Pharmaceutical and Biomedical Analysis, 008 Title: Modeling of the Mass-Transfer Kinetics in Chromatographic Columns Packed with Shell and Pellicular Particles Authors: Krzysztof Kaczmarski, Georges Guiochon Affiliation: Dept of Chemical & Process Engineering; Rzesnow University of Technology, Rzeszow, Poland; Dept of Chemistry, University of Tennessee, Knoxville, Tennessee; Div of Chemical Sciences, ak Ridge ational Lab, ak Ridge, Tennessee Journal: Analytical Chemistry 007, Title: Mass-Transfer Kinetics in a Shell Packing Material for Chromatography Authors: Georges Guiochon, Alberto Cavazzini, Fabrice Gritti, Krysztof Kaczmarski, icola Marchetti Affiliation: Dept of Chemistry, University of Tennessee, Knoxville, Tennessee; Div of Chemical Sciences, ak Ridge ational Lab, ak Ridge, Tennessee; Faculty of Chemistry, Rzeszow University of Technology, Rzeszow, Poland Journal: Analytical Chemistry, 007, Title: Comprehensive ff-line, Two-Dimensional Liquid Chromatography. Application to the Separation of Peptide Digests Authors: Georges Guiochon, icola Marchetti, Jacob Fairchild Affiliation: Dept of Chemistry, University of Tennessee, Knoxville, Tennessee, Div of Chemical Sciences, ak Ridge ational Lab, ak Ridge, Tennessee Journal: Analytical Chemistry 008, Title: Comparison between the loading capacities of columns packed with partially and totally porous fine particles. What is the effective surface area available for adsorption? Authors: Georges Guiochon, Fabrice Gritti Affiliation: Dept of Chemistry, University of Tennessee, Knoxville, Tennessee; Div of Chemical Sciences, ak Ridge ational Lab, ak Ridge, Tennessee Journal: Journal of Chromatography A, 007, 07- Title: Comparison between the efficiencies of columns packed with fully and partially porous C8-bonded silica materials Authors: Georges Guiochon, Fabrice Gritti, Alberto Cavazzini, icola Marchetti Affiliation: Dept of Chemistry, University of Tennessee, Knoxville, Tennessee; Div of Chemical Sciences, ak Ridge ational Lab, ak Ridge, Tennessee; Dept of Chemistry, University of Ferrara, Ferrara, Italy Journal: Journal of Chromatography A, 007, 89-0 Title: Gradient elution separation and peak capacity of columns packed with porous shell particles Authors: Georges Guiochon, icola Marchetti, Alberto Cavazzini, Fabrice Gritti Affiliation: Dept of Chemistry, University of Tennessee, Knoxville, Tennessee, Div of Chemical & Analytical Sciences, ak Ridge ational Lab, ak Ridge, Tennessee, Dept of Chemistry, University of Ferrara, Ferrara, Italy Journal: Journal of Chromatography A 007, 0- Title: Comparative study of the performance of columns packed with several new fine silica particles. Would the external roughness of the particles affect column properties? Authors: Georges Guiochon, Fabrice Gritti Affiliation: Dept of Chemistry, University of Tennessee, Knoxville, Tennessee; Div of Chemical Sciences, ak Ridge ational Lab, ak Ridge, Tennessee Journal: Journal of Chromatography A, 007, 0-6 Title: igh peak capacity separations of peptides in reversed-phase gradient elution liquid chromatography on columns packed with porous shell particles Authors: Georges Guiochon, icola Marchetti Affiliation: Dept of Chemistry, University of Tennessee, Knoxville, Tennessee; Div of Chemical Sciences, ak Ridge ational Lab, ak Ridge, Tennessee Journal: Journal of Chromatography A, 007, 06-6 Title: Evaluation of the properties of a superficially porous silica stationary phase in hydrophilic interaction chromatography Authors: David V McCalley Affiliation: Center for Research in Biomedicine, University of the West of England, Frenchay, Bristol, United Kingdom Journal: Journal of Chromatography A, 008, 8-9 Title: ptimization of two-dimensional gradient liquid chromatography separations Authors: Pavel Jandera, Petr Cesla, Tomás ájek Affiliation: Dept of Analytical Chemistry, University of Pardubice, Pardubice, Czech Republic Journal: Journal of Chromatography A, 008 Title: Comprehensive two-dimensional liquid chromatography separations of pharmaceutical samples using dual Fused-Core columns in the nd dimension Authors: Anthony J Alexander, Lianjia Ma Affiliation: Analytical R & D, Bristol Myers Squibb Co, ew Brunswick J Journal: Journal of Chromatography A, 009, 8- Title: Determination of fatty alcohol ethoxylates with diphenic anhydride derivatization and liquid chromatography with spectrophotometric detection. A comparative study with other anhydrides. Authors: G. Ramis-Ramos, A. Micó-Tormos, F. Bianchi, E. F. Simó-Alfonso Affiliation: Dept de Química Analitica, Universitat de València, Valencia, Spain Journal: Journal of Chromatography A, 009, 0-00 Title: Determination of fatty alcohol ethoxylates by derivatisation with maleic anhydride followed by liquid chromatography with UV-vis detection Authors: G. Ramis-Ramos, A. Micó-Tormos, C. Collado-Soriano, J. R, Torres-Lapasió, E. Simó-Alfonso Affiliation: Dept de Química Analitica, Universitat de València, Valencia, Spain Journal: Journal of Chromatography A, 008, - Title: n-column epimerization of dihydroartemisinin: An effective analytical approach to overcome the shortcomings of the International Pharmacopoeia monograph Authors: Francesco Gasparrini, Walter Cabri, Alessia Ciogli, Ilaria D Acquarica, Michela Di Mattia Affiliation: Analytical Development, R & D Dept.,Pomezia, Italy; Dept di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Rome, Italy Journal: Journal of Chromatography B, 008, 80-9 Title: Use of partially porous column as second dimension in comprehensive two-dimensional system for analysis of polyphenolic antioxidants Authors: Paola Dugo, Francesco Cacciola, Miguel errero, Paola Donato, Luigi Mondello Affiliation: Dipartimento de Scienze degli Alimenti e dell Ambiente, Facoltà di Scienze, Messina, Italy; Dipartimento Farmacia-chimco, Facoltà di Farmacia, Università di Messina, Messina, Italy; Campus-Biomedico, Roma, Italy Journal: Journal of Separation Science, 008, sigma-aldrich.com/express ordering: (US only) / 8-9-

137 Title: igh peak capacity separation of peptides through the serial connection of LC shell-packed columns Authors: Paola Donato, Paola Dugo, Francesco Cacciola, Giovanni Dugo, Luigi Mondello Affiliation: Dipartimento Farmacia-chimco, Facoltà di Farmacia, Università di Messina, Messina, Italy; Dipartimento de Scienze degli Alimenti e dell Ambiente, Facoltà di Scienze, Messina, Italy; Campus-Biomedico, Roma, Italy Journal: Journal of Separation Science, 009, 9-6 Title: Maximizing peak capacity and separation speed in liquid chromatography Authors: Patrik Petersson, Andre Frank, James eaton, Melvin R. Euerby Affiliation: AstraZeneca R&D Lund, Lund, Sweden; AstraZeneca, R&D Charnwood, Loughborough, Leicestershire, England, UK Journal: Journal of Separation Science, 008, 6-7 Title: Tryptic digest analysis by comprehensive reversed phase x two reversed phase liquid chromatography (RP-LC x RP-LC) at different p s Authors: Isabelle François, Deirdre Cabooter, Koen Sandra, Frédéric Lynen, Gert Desmet, Pat Sandra Affiliation: Separation Sciences Group & Pfizer Analytical Research Centre, Ghent University, Ghent, Belgium; Dept. of Chemical Engineering, Vrije Universiteit Brussel, Brussels, Belgium; Research Institute for Chromatography, Kortrijk, Belgium Journal: Journal of Separation Science, 009, 7- Title: Fused-core particle technology as an alternative to sub--μm particles to achieve high separation efficiency with low backpressure Authors: Jennifer M. Cunliffe, Todd D. Maloney Affiliation: Pfizer Global Research & Development, Michigan Laboratories, Ann Arbor Journal: Journal of Separation Science, 007 Title: igh specific activity tritium-labeled -(-methoxybenzyl)-,-dimethoxy--iodophenethylamine (IBMe): A high-affinity -TA receptor-selective agonist radioligand Authors: David E. ichols, Stewart P. Frescas, Benjamin R. Chemel, Kenneth S. Rehder, Desong Zhong, Anita. Lewin Affiliation: Dept. of Medicinal Chemistry & Molecular Pharmacology, School of Pharmacy & Pharmaceutical Sciences, Purdue University, West Lafayette, Indiana; Center for rganic & Medicinal Chemistry, Research Triangle Instutite, Durham, C Journal: Bioorganic & Medicinal Chemistry, 008, 66-6 Title: Fused Core Particles for PLC Columns Authors: Joseph J. Kirkland, Timothy J. Langlois, Joseph J. DeStefano Affiliation: Advanced Materials Technology Journal: American Laboratory, 007 Title: Sub--Micron Performance With a Conventional PLC Instrument Using.7-Micron Fused Core Particles Authors: Thomas L. Chester Affiliation: Dept. of Chemistry, University of Cincinnati, Cincinnati, Journal: American Laboratory, 009 Title: Fused-Core Silica Column igh-performance Liquid Chromatography/Tandem Mass Spectrometric Determination of Rimonabant in Mouse Plasma Authors: Yunsheng sieh, Christine J. G. Duncan, Jean-Marc Brisson Affiliation: Drug Metabolism & Pharmacokinetics Dept., Schering-Plough Research Institute, Kenilworth, J Journal: Anal. Chem., 007, 79 (), pp Title: Discovery of Pyridine-Containing Imidazolines as Potent and Selective Inhibitors of europeptide Y Y Receptor Authors: Makoto Ando, agaaki Sato, Tsuyoshi agase, Keita agai, Shiho Ishikawa, irobumi Takahashi Affiliation: Tsukuba Research Institute, Merck Research Laboratories, Banyu Pharmaceutical Co, Ltd, Tsukuba, Japan Journal: Bioorganic & Medicinal Chemistry, 009 Title: Discovery of potent and orally active -alkoxy--phenoxy--thiazolyl benzamides as novel allosteric glucokinase activators Authors: Tomoharu Iino, Daisuke Tsukahara, Kenji Kamata, Kaori Sasaki, Sumika hyama, ideka osaka Affiliation: Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co Ltd, Tsukuba, Japan Journal: Bioorg Med Chem. 009 Apr ;7(7):7- Title: Achiral Liquid Chromatography with Circular Dichroism Detection for the Determination of Carnitine Enantiomers in Dietary Supplements and Pharmaceutical Formulations Authors: Ángel Ríos, Fernando de Andrés, Gregorio Castañeda Affiliation: Dept. of Analytical Chemistry & Food Technology, University of Castilla-La Mancha, Ciudad Real, Spain Journal: Journal of Pharmaceutical and Biomedical Analysis, 009 Title: Characterization of new types of stationary phases for fast liquid chromatographic applications Authors: Szabolcs Fekete, Jenö Fekete, Katalin Ganzler Affiliation: Formulation Development, Gedeon Richter Plc, Budapest, ungary; Budapest University of Technology & Econmics, Dept. of Inorganic & Analytical Chemistry, Budapest, ungary Journal: Journal of Pharmaceutical and Biomedical Analysis, 009 Title: Comprehensive Two-Dimensional Liquid Chromatography Separations of Pharmaceutical Samples using Dual Fused-Core Columns in the Second Dimension Authors: Anthony J. Alexander, Lianjia Ma Affiliation: Analytical Research & Development, Bristol Myers Squibb Co, ew Brunswick, J Journal: Journal of Chromatography A, 009, Pages 8- Title: Curcumin Structure-Function, Bioavailability, and Efficacy in Models of euroinflammation and Alzheimer s Disease Authors: Aynun. Begum, Mychica R. Jones, Giselle P. Lim, Takashi Morihara, Peter Kim, Dennis D. eath Affiliation: Dept. of Medicine & eurology & Psychiatry & Biobehavioral Sciences and the Semel Institute, UCLA, Los Angeles, CA; Greater LA ealthcare System, Geriatric Research Education Clinical Center, Sepulveda, CA; Cancer Prevention & Control Program, Moores UCSD Cancer Center, UCSD, San Diego, CA; Dept. of Post-Genomics & Diseases, Division of Psychiatry & Behavioral Proteomics, saka University Graduate School or Medicine, saka, Japan Journal: J Pharmacol Exp Ther., 008, Title: Liposome-Encapsulated Curcumin Suppresses Growth of ead and eck Squamous Cell Carcinoma In vitro and in Xenografts through the Inhibition of uclear Factor kb by an AKT-Independent Pathway Authors: Dorothy Wang, Mysore S. Veena, Kerry Stevenson, Christopher Tang, Baran o, Jeffrey D, Suh Affiliation: Dept. of Surgery & Pathology, VA Greater LA ealthcare System; David Geffen School of Medicine, UCLA, Los Angeles, CA; Dept of Psychiatry & Biobehavioral Sciende, Semel Institute, UCLA, Los Angeles, CA; Dept. of Experimental Therapeutics, MD Anderson Cancer Center, University of Texas, ouston, TX Journal: Clinical Cancer Research, 008 Title: Analysis of Impurities in Streptomycin and Dihydrostrepomycin by ydrophilic Interaction Chromatography/Electrospray Ionization Quadrupole Ion Trap/Time-of-Flight Mass Spectrometry Authors: Shin-ichi Kawano Affiliation: Analytical Applications Dept., Shimadzu Corp., Kyoto, Japan Journal: Rapid Communications in Mass. Spectrometry, 009, Title: Determination of Fatty Alcohol Ethoxylates by Derivatization with Phthalic Anhydride followed by Liquid Chromatography with UV-vis Detection Authors: G. Ramis-Ramos, A. Micó-Tormos, E. F. Simó-Alfonso Affiliation: Dept de Química Analitica, Universitat de València, Valencia, Spain Journal: Journal of Chromatography A, 009, 7- Title: ighly Efficient Peptide Separations in Proteomics: Part. Unidimensional igh Performance Liquid Chromatography Authors: Koen Sandra, Mahan Moshir, Filip D hondt, Katleen Verleysen, Koen Kas, Pat Sandra Affiliation: Dept. of Chemical Engineering, Vrije Universiteit Brussel, Brussels, Belgium Journal: Journal of Chromatography B, 008, 8-6 Title: Multidimensional LCxLX analysis of Phenolic and Flavone atural Antioxidants with UV-electrochemical coulometric and MS Detection Authors: Tomás ájek, Veronika Skerikova, Petr Cesla, Katerina Vynuchalova, Pavel Jandera Affiliation: Dept of Analytical Chemistry, University of Pardubice, Pardubice, Czech Republic Journal: Journal of Separation Science, 008, 09-8 Title: Separation of atural Product using columns packed with Fused-Core Particles Authors: P. Yang, G.R. Litwinski, M. Pursch, T. McCabe, K. Kuppannan Affiliation: The Dow Chemical Company, Analytical Sciences, Midland, MI Journal: J. Sep Sci., 009, 86-8 technical service: (US and Canada only) / 8-9-0

138 atural Products (continued from page Table. Major Component Retention Comparison Between ILIC and Reversed-Phase Modes Accurate Ascentis Express Ascentis Express Component Mass ILIC RP-Amide RT RT Rebaudioside A/E Stevioside Steviolbioside Dulcoside A Steviol Figure. Stevia Extract on Ascentis Express RP-Amide, Extracted Ion Chromatogram for Steviol Steviol G007 strongly retained under ILIC conditions. In two cases, where known components were identified, it was necessary to use standards to confirm their retention. Figures and depict the extracted ion chromatogram for the accurate mass of steviol and stevioside. As can be seen in both chromatograms, multiple peaks are observed for each of the accurate masses. In the case of stevioside, it is isobaric with rebaudioside B making identification difficult. A stevioside standard (Sigma Aldrich) was used for positive identification. In addition, the reversed-phase separation of the extract resulted in multiple peaks observed for the accurate mass of steviol. This was due to fragments from additional glycosides that resulted in a steviol fragment ion, again it was necessary to confirm the steviol retention with a standard. Conclusions The profiling of the Stevia rebaudiana extract demonstrates the utility of performing orthogonal chromatographic modes when handling complex samples. The two modes of chromatography were complimentary for the determination of major components from the stevia extract. In most cases where coelution occurred in one chromatographic mode, the components were separated under the orthogonal mode. Though component identification was made easier through the accurate mass of the TF, it was still necessary to have good chromatographic resolution to confirm component identity. In both cases, the Fused-Core particle demonstrated the ability to perform complex matrix analysis in both ILIC and reversed-phase separations G007 Figure. Stevia Extract on Ascentis Express RP-Amide, Extracted Ion Chromatogram for Stevioside Stevioside G0070 Rebaudioside B G007 PLC Seminars on the Web - /7 PLC Column Fundamentals Ultra-high Performance using Fused-Core PLC Columns The Use of Alternate Selectivity in Reversed-Phase PLC Introduction to Ascentis PLC Columns PLC Instrument Bandwidth and ptimizing Systems for igh-performance igh-performance PLC Fittings and Interconnects go to sigma-aldrich.com/videos 6 sigma-aldrich.com/express ordering: (US only) / 8-9-

139 Colors of the World: Fast Separation of Dyes with Ascentis Express Dyes surround us everywhere every day. They can be found in common places like the printing ink in magazines or books and in plastics, textiles, and leather, but also in unusual places like diesel fuel and tattoo color. Most of these synthetic colors are based on aromatic ring structures containing heteroatoms and tend to have a high potential for causing cancer; as a result, they are not intended for use in food coloring. But since 00, there have been several incidents of Sudan I contamination in chili powder. This situation necessitates the analysis of spice mixtures to determine if they have been adulterated (, ). Further, a sensitive PLC method is needed for quality control testing of dyes and the identification of byproducts. Supelco s Ascentis Express PLC columns provide outstanding sensitivity and resolution for such applications. Method Development for Dyes Table contains a list of dyes added to one sample and dissolved in a mixture of methanol and acetonitrile. The sample was injected on an Ascentis Express C8 PLC column under varying mobile phase conditions to determine the best separation parameters. Temperature, injection volume, detector settings, and flow rate were kept constant. The chemical and physical properties of the dyes differ strongly, so the first step in developing a suitable PLC method was the use of a gradient run ranging from % acetonitrile to 00% acetonitrile (B) and 0.% formic acid in water as an aqueous counterpart (A). The UV chromatogram of the combined wavelengths 60, 0, and 60 nm showed good chromatography of all compounds except for the poor peak shape of Sudan 0 at 7. minutes (Figure A). To optimize that peak shape, methanol was added to the organic mobile phase (acetonitrile:methanol, 90:0); the gradient run was repeated, resulting in better peak shape for Sudan 0 (Figure B). In a final experiment, the gradient profile was changed and optimum conditions were attained (see Table ). Figure C shows the step-by-step improvements in the chromatography. nly three runs were needed to get the final method, showing how easy and fast it is to develop methods with Ascentis Express columns. Further, Ascentis Express columns contain Fused-Core particles that allow for faster run times; even separations performed on standard PLC systems can be sped up by up to 0% with Ascentis Express. Dyes Table. Structure and Mass of the Dyes in the Sample Mixture. Most of the Compounds are Detected as [M+] + Ions except (), which gives [M] + Ions Peak o. Structure ame / Exact Mass G00779 Parafuchsin C Peak o. Structure ame / Exact Mass G0078 Malachite Green C G00780 Basic Fuchsin C G0078 Sudan III C 6. G0078 Methylfuchsin C.77 7 G0078 Sudan 0 C G0078 ewfuchsin C technical service: (US and Canada only) /

140 Table. Initial and Final PLC Method Settings for Separation of the Seven Dyes Listed in Table, After ptimization Dyes Fixed Parameters column: Ascentis Express C8, 0 cm.6 mm I.D.,.7 μm particles flow rate: 0.8 ml/min temp: C UV DAD: nm MS: ESI(+), SPS target 00 m/z, stability 00%, trap lvl. 00%, optimize normal, range m/z, nebulizer 0 psi, dry gas L/min, dry temp. 6 C. injection volume: μl run time: min ( min posttime) Variable Parameters Initial Conditions Final Conditions solvents: (A) water with 0.% formic acid (A) water with 0.% formic acid (B) acetonitrile (B) acetonitrile:methanol (90:0) gradients: Time %A %B Time %A %B % %. 7 %. 7 % %.0 98% %.0 98%.0 7 %.0 7 % Figure. UV Chromatograms of Sudan III and Sudan 0. (A) Initial Conditions, (B) Addition of Methanol to Mobile Phase, (C) Final Conditions after Adjusting Gradient Figure A Figure B Figure shows UV chromatograms of Sudan III (Peak 6) and Sudan 0 (Peak 7) after three optimization steps. An organic phase mixture of methanol:acetonitrile (0:90) and a final gradient composition of 98% organic mobile phase resulted in the best overall peak shapes with a minimum of tailing of compounds (Peak 7). Figure shows the final chromatogram with very good separation of all analytes. Figure is a UV chromatogram of the final PLC method. Resolution, sensitivity, and peak symmetry were optimal for all analytes. The total run time on a standard PLC instrument (Agilent 00) was minutes, but the separation could easily be performed faster on ultra-performance instruments. Figure. UV Chromatogram of the Final PLC Method Figure C G00786 G sigma-aldrich.com/express ordering: (US only) / 8-9-

141 Ascentis Express PLC Columns: igh Efficiency and LC-MS Compatibility Using Ascentis Express columns on standard PLC, fast LC, and ultra-performance instruments can yield heightened sensitivity (Figure ). Mass detectors in LC-MS systems are very sensitive to contaminants in solvents and column bleed, both of which are very low with Ascentis Express columns combined with the right set of Fluka LC-MS solvents and additives. Both aspects, high efficiency and low column bleed, are basic requirements in trace analysis of small target analyte concentrations or in identification of byproducts which may influence dramatically the quality and application of dyes. Figure shows an example of the identification of low concentrations of byproducts even in very complex mixtures. The unknown substance at the retention time of 7.68 minutes shows a nearly identical UV spectrum to Malachite Green (Peak at 8.0 minutes), but the mass is Da lower. This may correspond to the exchange of a methyl residue with a proton at a position in the Malachite Green molecule that has no influence on the chromophore. nly the displayed molecular structures fit the UV and mass spectroscopic results. Figure. Expanded View of UV Chromatogram Showing Unknown Impurity at 7.7 min. and Malachite Green () The inset shows the UV spectra of malachite green (blue) and the unknown impurity (red). The mass spectra are of malachite green (top) and unknown impurity (bottom). To get optimal results from your LC-MS system and accurate UV and mass spectra of impurities with a high signal-to-noise level, it is best to use high purity LC-MS solvents from Fluka and high performance PLC columns such as Ascentis Express from Supelco. References. Commission Decision. fficial Journal of the European Union. L/. June 0, 00.. Rapid Alert System for Food and Feed (RASFF). 00. Annual Report. European Commission of ealth & Consumer Protection Directorate General. Dyes Ascentis Express Properties Stationary Phase Support Ultra-pure, Type B silica.7 μm solid core particle with 0. μm porous silica shell (effective.7 μm) 0 m /gram surface area (comparable to ~ m /g porous particle) 90 Å pore size, 60 Å for Peptide ES C-8 Bonded Phase Coverage μmoles/m p Range Endcapping C8. -9 Yes C8.7-9 Yes RP-Amide.0-9 Yes ILIC n/a -8 o Phenyl-exyl. -9 Yes Peptide ES-C8. -9 o G00788 technical service: (US and Canada only) /

142 Ascentis Express RP-Amide: Combining an Embedded Polar Group Stationary Phase and Fused-Core Particles Alternate Selectivity Ascentis Express RP-Amide PLC columns are the most recent product additions to the Supelco PLC product line. Combining an embedded polar group (EPG) stationary phase with the Fused-Core particles, Ascentis Express RP-Amide provides a host of useful benefits to the PLC chromatographer. The benefits come from both the phase technology and the particle technology and can be summarized as: Fused-Core Benefits igher efficiency than traditional PLC columns ( and μm) alf of the backpressure of sub micron columns Capable of UPLC performance on traditional PLC systems RP-Amide Benefits Alternative reversed-phase selectivity to C8 Improved peak shape for bases 00% aqueous compatible reversed-phase column At the heart of the Ascentis Express RP-Amide is the.7 μm Fused-Core particle that comprises a.7 μm solid core and a 0. μm porous shell. Compared to totally porous particles, the Fused-Core particles have a much shorter diffusion path because of the solid core. This partial porosity reduces dispersion of solutes and minimizes peak broadening. ther features, such as a very tight particle size distribution and high packing density, result in Ascentis Express columns capable of delivering extreme performance to any PLC system. In fact, there have been many reports on the vast improvements in efficiency and speed provided by Ascentis Express PLC columns versus traditional PLC columns. The improvements provide UPLC performance on traditional PLC systems. While the Ascentis Express C8 and C8 provide classic reversed-phase selectivity, the RP-Amide phase offers an alternative selectivity. Supelco first commercially introduced the EPG phase in 988. At that time, large tailing factors for basic analytes continued to plague conventional C8 and C8 bonded phases. The EPG phase was found to improve peak shape of basic analytes. The early generation EPG phases were based on a two-step bonding process (Figure ). The first step was the bonding of an aminopropylsilane to the bare silica surface creating a surface with amine functionality. In step two, palmitoyl chloride was reacted with the amine to create a long chain amide. ot all amines would be converted in the process, leaving a mixed system. These early generation EPG phases suffer from poor reproducibility. ext generation phases, including Ascentis Express RP-Amide, are produced using a one-step process (Figure ). In the single step process, no free amino ligands occur since the amide is introduced as a whole unit. This one-step bonding process yields excellent batch-to-batch reproducibility. Interestingly, not all EPG phases on the market use the modern, one-step bonding approach. Improved Peak Shape for Basic Compounds As previously mentioned, Ascentis Express RP-Amide phase reduces silanol interactions with basic analytes improving peak shape. A good test to demonstrate this effect is highly basic compounds using a mobile phase p of 7. At this p, many of the residual silanols are in the ionized form and the basic compounds are protonated. The protonated (charged) bases interact with the charged silanols via ion exchange and result in a tailing peak. A test Figure. Early Generation Two-step Bonding Process for EPG Amide Phases Step. Aminopropyl Phase Step. Amide Phase Incomplete converson to amide phase (C ) C (C ) C Si Si Si Si Si Si Si Si Si Si Si Si Si Si G00, Si Si Si Si Si Si 0 sigma-aldrich.com/express ordering: (US only) / 8-9-

143 Figure. ext Generation ne-step Bonding Process for EPG Amide Phases Base Silica Step. Amide Phase Si Si C Si Si Si (C ) C (C ) C Si Si (C ) C (C ) C (C ) C Si Si Si Si G00, 6, 7 Si Si Si Si Si Si Alternate Selectivity mix of tricyclic antidepressants was analyzed on Ascentis Express RP-Amide and a C8 column with a mobile phase p of 7 (Figure ). As shown in Figure, the RP-Amide produces more symmetrical peaks than the C8 for these difficult test probes. Asymmetry data is summarized in Figure for doxepin, imipramine, and amitriptyline. Alternative Selectivity Ascentis Express RP-Amide provides increased selectivity for polar compounds, especially those that can act as a hydrogen-bond donor. Phenols, carboxylic acids, amines, and to a lesser extent, alcohols show enhanced retention on the RP-Amide phase when compared to neutral, non-polar analytes. An example of the power of the hydrogen bonding mechanism is shown in Figure. The phenolic nature of catechols and resorcinols provides a good test for demonstrating enhanced selectivity of the RP-Amide phase. The RP-Amide phase shows complete baseline resolution of these related compounds while the C8 phase shows reduced retention, resolution, and selectivity for the phenolics. In comparing the Ascentis Express RP-Amide to the Waters BE Shield RP8, a competitive EPG phase, the selectivity is very similar. The difference in this example is the Ascentis Express RP-Amide yields a backpressure half of the.7 μm column. This difference in backpressure means the Ascentis Express column is suitable for traditional PLC systems while the.7 μm column is not. The selectivity differences between the RP-Amide and the C8 can be a useful tool in method development. In many cases, when peaks co-elute on a C8 phase, the RP-Amide can be substituted to achieve separation without a change in mobile phase. Figure. Separation of Tricyclic Antidepressants on Ascentis Express RP-Amide and Conventional C8 columns: as indicated; 0 cm x.6 mm I.D. mobile phase: :6, mm ammonium phosphate in water, p = 7 : methanol flow rate:. ml/min. temp.: C det.: nm injection: 0. μl 6 7 C G G Maleate. ordoxepin. Protripyline. Desipramine. ortriptyline 6. Doxepin 7. Imipramine 8. Amitriptyline 9. Trimipramine Figure. Asymmetry Factors for Difficult Bases on Ascentis Express RP-Amide and Conventional C8 Asymmetry.0.0 Ascentis Express RP-Amide Doxepin Imipramine Amitriptyline 9 C8 RP-Amide technical service: (US and Canada only) / 8-9-0

144 Alternate Selectivity Figure. Separation of Phenolics on Ascentis Express RP- Amide & C8, & Waters BE Shield RP8,.7 μm columns: as indicated; 0 cm x. mm I.D. mobile phase A: 0 mm phosphoric acid, p (unadjusted) mobile phase B: water mobile phase C: acetonitrile mobile phase ratios: A:B:C = 7::0 flow rate: 0. ml/min. temp.: C det.: 70 nm injection: μl sample: 0 mg/l ea. in 0 mm phosphoric acid Resorcinol G009 Catechol. Resorcinol. Catechol. -Methylresorcinol. -Methylcatechol.,-Dimethylresorcinol 6. -Methylcatechol 7. -itrocatechol G0090 G0008 G0009 Ascentis Express RP-Amide,.7 μm pressure: 00 psi 7 6 Ascentis Express C8,.7 μm pressure: 00 psi 6 Waters BE Shield RP8,.7 μm pressure: 80 psi Figure 6. Separation of Small rganic Acids Under 00% Aqueous Conditions column: Ascentis Express RP-Amide, 0 cm x. mm I.D. mobile phase: 0.% TFA (v/v) in water flow rate: 0. ml/min. temp.: C det.: 0 nm injection: μl sample: in mobile phase; tartaric acid, g/l; lactic acid, citric acid, g/l; acrylic acid, 0. g/l; fumaric acid, 0. g/l. Tartaric acid. Lactic acid. Citric acid. Acrylic acid. Fumaric acid 0 Aqueous Compatible Reversed-Phase Column G006 Ascentis Express RP-Amide provides stable and reproducible analyte retention in 00% aqueous mobile phases. Many C8 phases are known to suffer from phase collapse under highly aqueous mobile phase conditions causing loss of retention. Shown in Figure 6 is a mix of organic acids analyzed under 00% aqueous conditions. Excellent selectivity and peak shape is noted for all the test probes, even citric acid, which is a notoriously difficult analyte. Conclusion Ascentis Express RP-Amide is a blend of modern phase technology and innovative particle technology. The Fused- Core particle provides benefits in terms of speed, resolution, sensitivity, and ruggedness. The one-step RP-Amide bonding chemistry provides benefits in terms of selectivity, aqueous stability, and improved peak shape for bases. G0060 sigma-aldrich.com/express ordering: (US only) / 8-9-

145 Ascentis Express Phenyl-exyl: Combining the popular Phenyl-exyl Stationary Phase and Fused-Core Particles Even though new column technologies have more than doubled the plates per meter possible with traditional μm columns, resolution still cannot be routinely achieved in every case without the ability to adjust retention and selectivity by proper selection of column stationary and mobile phases. This article features Ascentis Express Phenyl-exyl phase, a new addition to the Fused-Core column family, and describes how column selectivity and higher efficiency can be coupled to achieve much faster separations than have previously been possible. Table. Brief verview of Ascentis Express Column Retention and Selectivity Ascentis Express Principle Principle Fused-Core Phase Retention Mode Solute Interaction C8 Reversed-Phase (RP) ydrophobic (dispersive) C8 Reversed-Phase (RP) ydrophobic (dispersive) RP-Amide RP with embedded ydrophobic and polarity -bonding Phenyl-exyl RP with pendant ydrophobic and - aromaticity ILIC (Silica) ILIC (or normal ydrophilic (dipole, phase) -bonding, ion exchange) Alternate Selectivity The vast majority of UPLC separations have been carried out with C8 columns in the classic reversed-phase (RP) mode; however, suppliers now offer many different phases. Although no one would dispute the fact that UPLC columns with different phases are needed, very little has been published yet on the performance that can be expected from UPLC columns having different, complementary selectivity to C8 and C8. Two of the most popular polar-rp phases are RP-Amide, which is often categorized as an embedded polar-group phase, and Phenyl, which can interact with solutes by - mechanisms. A brief retention and selectivity comparison for the Ascentis Express column family is given in Table. C8 and C8 phases are highly popular because they are stable, reproducible, and easy-to-use. Retention correlates closely with log P values, which have been established for many solutes. Solute ionization causes retention to decrease in a predictable manner and is relatively easy to control by adding dilute acids, bases, and buffers to the mobile phase. Changing the organic component of the mobile phase between acetonitrile and methanol (or other solvents) allows the user to tweak resolution because solvation affects phase structure and selectivity. Temperature is also a useful variable for optimizing phase selectivity. Columns with C8 and C8 phases will frequently give optimum resolution when solutes are nonpolar or slightly polar; however, columns with polar-rp phases such as RP-Amide or Phenyl-exyl will often show improved retention and selectivity for more polar solutes. It should be emphasized that even polar-rp phases have a significant alkyl phase character in addition to their polar character. The same mobile phase solvents and techniques may be employed with polar-rp phases, with comparable phase stability to C8. The RP-Amide phase is complementary to C8 because the amide group has several unique features: ) strong interaction by -bonding when solutes can donate or accept protons, ) effective shielding of silanols by internal -bonding between amide group and silica surface, and ) the ability to wet and operate well, even in 00% aqueous solvents. -bonding allows solutes with carboxyl and phenol groups to be retained much longer and separate much better on RP-Amide than on C8 or C8. Shielding prevents solutes with amino groups from interacting with silanols and can result in shorter retention and sharper peaks on amide phases. Another interesting feature of amide phases is that methanol and other alcohols become much stronger solvents when -bonding between phase and solute occurs. Except for the special situations listed above, an RP-Amide phase often performs similar to C8 due to the long alkyl chain extending away from the surface. The Phenyl phase has unique selectivity arising from solute interaction with the aromatic ring and its delocalized electrons. It is complementary (orthogonal) to both C8 and RP-Amide phases because of this unique aromaticity. An unsubstituted phenyl ring is a -donor or Lewis base, which interacts strongly with -acceptors and any electron-deficient Lewis acid. Phenyl phases also tend to exhibit good shape selectivity, which may originate from solute multipoint interaction with the planar ring system. More retention and selectivity will often be observed for solutes with aromatic electron-withdrawing groups (fluorine, nitro, etc.) or with a delocalized heterocyclic ring system such as the benzodiazepine compounds. technical service: (US and Canada only) / 8-9-0

146 Alternate Selectivity UPLC Results with Ascentis Express Phenyl-exyl Low-pressure drop with high efficiency and a flat van Deemter curve have been confirmed for Phenyl-exyl, as shown in Figure. In general, more than twice the column efficiency of μm particles can be expected for all Ascentis Express Fused-Core columns at pressures that are easily managed with all PLC instruments. ote that 0,000 plates have been achieved for a 0 cm x mm I.D. column operating at optimum flow. A Jasco X-LC PLC instrument was used for the study. Figure illustrates the benzodiazepine chemical structures used in this study. As shown in Figure, the selectivity of Ascentis Express Phenyl-exyl is very similar to that of other commercial Phenyl columns, so methods can be readily transferred between columns. The difference in efficiency and pressure drop for the two porous μm columns can be explained by different particle size distributions. Figures - show comparisons of five benzodiazepines separated on the four Ascentis Express RP phases in water:acetonitrile and water:methanol mobile phases. o additives were employed in order to observe the interaction between these polar solutes and the different phases; however, a dilute buffer will normally be used for development of a validated method. The addition of 0-0 mm buffer at neutral p typically has little or no effect upon the separation with these highly deactivated column phases. Figure. Flow Performance of Ascentis Express Phenyl- exyl Column with eutral Probes 0 cm x mm I.D. column, water:acetonitrile, 0:0, C, 0 nm, μl Efficiency, Reduced Plate eight, h,000 0,000 8,000 6,000,000,000 0, ml/min G naphthalene, k = mm/sec G008 Reference. Kazakevitch, Y. V., et al. J. Chromatogr., A. 00, 08, 8 6. naphthalene, k =. Figure. Benzodiazepine Structures Diazepam -desmethyldiazepam Alprazolam Figure. Comparison of Phenyl Column Selectivity for Benzodiazepines column: as indicated, 0 cm x mm I.D. mobile phase A: water mobile phase B: acetonitrile mobile phase ratios: A:B = 6: flow rate: 0.6 ml/min. temp.: C det.: UV, 0 nm injection: μl Ascentis Express Phenyl-exyl Total = 000 psi Col = 60 psi Column A Phenyl-exyl, μ Total = 700 psi Col = 060 psi Column B Phenyl, μ Total = 70 psi Col = 0 psi Clonazepam G0088 G0067 G00670 G00879 xazepam Sample (0. g/l ea.). xazepam. Alprazolam. Clonazepam. -desmethyldiazepam. Diazepam = 8,900 k = 0. =,00 k =. = 0,600 k =. G006 G008 G G0087 ote that overall retention in acetonitrile is similar for the four bonded phases, but elution order is different. The two less polar compounds, diazepam and desmethyldiazepam, elute late and show the same order for all columns due to predominance of hydrophobic interactions. The more polar solutes, however, elute earlier and interact differently with Phenyl-exyl and the other phases. sigma-aldrich.com/express ordering: (US only) / 8-9-

147 With this test sample and operating conditions, three of the four Ascentis Express RP columns provide good resolution with different selectivity, however, Phenyl-exyl shows the best retention and selectivity. A switch to water:methanol in Figure shows a dramatic change in retention for Ascentis Express Phenyl-exyl. In water:methanol mobile phase, the phenyl group interacts much more strongly than the other phases with the solute heterocyclic ring system, presumably by a - mechanism. Kazakevitch () has published evidence that methanol forms only monolayer coverage on aromatic phases (and also thinly solvates other phases), which allows the aromatic selectivity to shine through more strongly. Elution order for the polar compounds also changes from that of water:acetonitrile conditions. For this test sample, Ascentis Express Phenyl- exyl selectivity is clearly superior in water:methanol to the other phases. Figure. Benzodiazepines in 60% Methanol Mobile Phase with no Additive column: Ascentis Express, as indicated0 cm x mm I.D. mobile phase A: water mobile phase B: methanol mobile phase ratios: A:B = 0:60 flow rate: 0.6 ml/min pressure: 00 psi total; 0 psi column temp.: C Phenyl-exyl C8 det.: UV, 0 nm injection: μl, Sample (0. g/l ea.). xazepam. Alprazolam. Clonazepam. -desmethyldiazepam. Diazepam G0088 Alternate Selectivity Figure. Benzodiazepines in % Acetonitrile Mobile Phase with no Additive column: Ascentis Express, as indicated, 0 cm x mm I.D.,.7 μm particles mobile phase A: water mobile phase B: acetonitrile mobile phase ratios: A:B = 6: flow rate: 0.6 ml/min. pressure: 000 psi total, 60 psi column Phenyl-exyl temp.: C det.: UV, 0 nm injection: μl Sample (0. g/l ea.). xazepam. Alprazolam. Clonazepam. -desmethyldiazepam. Diazepam C8 RP-Amide G0080 G0089 C8 G G008 C8 RP-Amide G00807 G G00809 Conclusion A new Phenyl-exyl phase has been paired with Fused- Core particles to complete the primary Ascentis Express column family. igh performance with lower pressure drop than other UPLC columns has been confirmed for all Fused-Core particle phases. Ascentis Express Phenyl-exyl correlates well to other Phenyl phases for easy method development or method transfer. Selectivity for benzodiazepine compounds has been compared to the other Ascentis Express RP phases in water:acetonitrile and water:methanol. The extra retention possible with Phenyl phases in water:methanol has been demonstrated for these heterocyclic aromatic compounds. The potential for faster, more sensitive assays using Ascentis Express Phenyl-exyl and all Ascentis Express phases has been shown. technical service: (US and Canada only) / 8-9-0

148 FAQs Ascentis Express FAQs What is unique about Ascentis Express? Ascentis Express columns provide a breakthrough in PLC performance. Based on Fused-Core particle technology, Ascentis Express provides the benefits of sub- μm particles but at much lower backpressure. These benefits include the capability of providing fast PLC and higher resolution chromatography. The Fused-Core particle consists of a.7 μm solid core and a 0. μm porous shell. A major benefit of the Fused-Core particle is the small diffusion path (0. μm) compared to conventional fully porous particles. The shorter diffusion path reduces axial dispersion of solutes and minimizes peak broadening. Can I use Ascentis Express on any type of PLC system? Ascentis Express PLC columns are capable of use on standard PLC systems as well as UPLC systems. Columns are packed in high pressure hardware capable of withstanding the pressures used in UPLC systems. Is there anything I need to do to my PLC system to use Ascentis Express? othing special is required to use Ascentis Express PLC columns. To obtain the full benefits of Ascentis Express, one should minimize dispersion or instrument bandwidth in the PLC system (tubing, detector flow cell) as well as confirm the detector response system is set at a fast level. For more information, request Guidelines for ptimizing Systems for Ascentis Express Columns (T070) or visit sigma-aldrich.com/express and download. Can I use Ascentis Express on a UPLC system? Yes. Ascentis Express columns are packed in a way making them suitable for these ultra high pressure instruments. In fact, Ascentis Express outperforms sub- μm columns on many applications since Ascentis Express provides the benefits of sub- μm particles but at much lower backpressure. Can Ascentis Express columns be used for LC-MS? Ascentis Express Fused-Core particles were designed with LC-MS in mind. Even extremely short column lengths exhibit sufficient plate counts to show high resolving power. The flat van Deemter plots permit resolution to be maintained at very high flow rates to maximize sample throughput. All Ascentis stationary phases have been evaluated for MS compatibility during their development, and the Express phases are no exception. A bonus of Ascentis Express columns for high throughput UPLC and LC-MS is that they are extremely rugged and highly resistant to plugging, a very common failure mode for competitor columns. What flow rate should I use with Ascentis Express columns? Based on the minimum in the van Deemter curves, higher flows than μm particle columns are required in order to maximize Ascentis Express column efficiency. Ascentis Express Suggested Starting Point PLC Column ID for Flow Rate.6 mm I.D..6 ml/min.0 mm I.D. 0.8 ml/min. mm I.D. 0. ml/min Key Technical Literature (available by request through technical service) Code Publication Title T09 Method ptimization using Alternative Selectivities in Fused-Core Particle PLC Column Technology T090 Increased Bioanalytical Throughput Using Fused-Core PLC with Selective Phospholipid Depletion T090 Extended Performance of LC Instruments with Fused-Core Particle Columns T08 Utilizing Fused-Core Technology for LC-MS Applications T08088 Transfer and Speedup of Methods to Fused-Core Particle Columns T08087 ptimization of PLC Instrumentation for igh Efficiency Separations T08077 Achieving Sub- μm LC-MS Performance at Moderate Pressures using Fused-Core Particle Technology T080 igh-resolution PLC Through Coupling Columns T080 igh Resolution PLC Performance Under Both Isocratic and Gradient Conditions T080 Achieving ptimum UPLC Column Performance by Measuring and Reducing verall System Dispersion T080 Achieving Ultra-PLC Column Performance with lder Instruments T077 Achieving Efficient Bioanalytical Separations at Moderate Pressures using Fused-Core Particle Technology T07078 ptimizing PLC Particles and Column Dimensions for Fast, Efficient Separations T070 Guidelines for ptimizing Performance with Ascentis Express PLC Columns T08 Guide to Dispersion Measurement 6 sigma-aldrich.com/express ordering: (US only) / 8-9-

149 rdering Information Analytical Ascentis Express Columns ID (mm) Length (cm) C8 C8 Phenyl-exyl ILIC RP-Amide Peptide ES -C U. 80-U 89-U -U 9-U 90-U 99-U. 8-U 8-U -U 9-U 9-U 0-U U 8-U -U 98-U 9-U 0-U. 0 8-U 8-U 6-U 99-U 9-U 06-U. 8-U 8-U 8-U 96-U 9-U 07-U.0 80-U 8-U -U 96-U 9-U 08-U.0 8-U 88-U -U 967-U 96-U -U U 89-U -U 969-U 97-U -U U 8-U -U 970-U 98-U -U.0 86-U 8-U 6-U 97-U 99-U -U.6 88-U 87-U 7-U 97-U 9-U 6-U.6 86-U 86-U 8-U 97-U 9-U 8-U U 88-U -U 977-U 9-U -U U 87-U -U 979-U 99-U -U.6 89-U 88-U -U 98-U 9-U 8-U rdering Information Capillary Ascentis Express Columns C8 C8 Length (cm) I.D. (μm) Cat. o U 7 9-U U 00 6-U U 00 6-U U 00 7-U U 00 7-U 7 98-U 7 9-U U U U 00 6-U U 00 7-U U 00 7-U Trademarks Ascentis, Fluka, ybridspe Sigma-Aldrich Biotechnology LP Fused-Core Advanced Materials Technology Agilent Agilent Technologies, Inc. UPLC Waters Corp. Ascentis Express Guard Columns Universal Guard older Description Cat. o. older w/exp Titanium ybrid Ferrule -- cartridge not included with holder 00-U Ascentis Express Guard Cartridges E000 Description I.D. (mm) Pkg. Size Cat. o. C8. 0-U C8.0 0-U C U C8. 09-U C8.0 -U C8.6 -U RP-Amide. -U RP-Amide.0 6-U RP-Amide.6 9-U ILIC. 0-U ILIC.0 -U ILIC.6 -U Phenyl-exyl. -U Phenyl-exyl.0 6-U Phenyl-exyl.6 -U Peptide ES-C8. 6-U Peptide ES-C8.0 7-U Peptide ES-C8.6 -U technical service: (US and Canada only) /

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151 Ascentis Express Peptide ES-C8 PLC Columns The Fused-Core Advantage for Bioseparations Improved Peak Shape on any PLC System Extreme Stability with TFA and ther Additives Rugged PLC Column Design

152 A Breakthrough in Bioseparations Performance Ascentis Express Peptide ES-C8 Ascentis Express Peptide ES-C8 is a high-speed, high-performance liquid chromatography column based on a new 60Å Fused- Core particle design. The Fused-Core particle provides a thin porous shell of high-purity silica surrounding a solid silica core. This particle design exhibits very high column efficiency for high MW solutes (up to 0 kda) due to Table. Specifications for Ascentis Express Peptide ES-C8 Silica igh Purity Type B Phase Sterically protected C8 p range 9 Temperature 00 C Average pore diameter 60 Å Surface area, nitrogen 80 sq.m/g Pore volume 0.0 ml/g Particle density. g/cc the shallow diffusion paths in the 0.-micron thick porous shell and the small overall particle size of.7-microns. The non-end-capped, sterically protected C8 bonded phase of Ascentis Express Peptide ES-C8 pro- vides a stable, reversed phase packing with a pore structure and pore size that is optimized for reversed-phase PLC separations of peptides and polypeptides, using typical acidic mobile phases favored for protein structure-function and proteomic applications. rdering Information Ascentis Express Peptide ES-C8 Columns I.D. Length (cm) (mm) U 0-U 0-U 06-U 07-U.0 08-U -U -U -U -U.6 6-U 8-U -U -U 8-U Applications The Ascentis Express Peptide ES-C8 columns are best utilized with mobile phases that are mixtures of acetonitrile and water or methanol and water. igher levels of the organic solvent component will typically reduce the retention of the sample compounds. Using elevated temperatures (e.g., 0 00 C) will reduce the viscosity of the mobile phase and allow the use of faster flow rates and lower column pressure for high sample throughput. Gradient-elution techniques using -0% organic component as the initial mobile phase and increasing to 00% organic component as the final mobile phase often can affect separations of complex sample mixtures in minimal time. Ionizable compounds, such as acids and bases, are generally best separated with mobile phases buffered at p of to. The use of 0-0 mm buffers is always recommended for optimum results and long-term stability when separating ionizable compounds. Ascentis Express Peptide ES-C8 columns utilize a steric-protected C8 bonded phase with extremely high resistance to acid-catalyzed hydrolysis of the siloxane bond that attaches the C8 chain to the surface. Thus, the combination of low p and elevated temperature operation of the column is well tolerated. Peptide separations are efficiently conducted using low p mobile phase modifiers, often at 0.% concentration. Most popularly employing trifluoroacetic acid (TFA), and the related perfluorocarboxylic acids, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (FBA). These acids exhibit desirable low UV transparency, volatility, and peptide ion pairing properties. Additional opportunities for UV detection at low p operation is with mineral acids such as phosphoric acid (-0 mm). For MS detection 0.% formic acid is most commonly employed (sometimes acetic acid), but significant benefit to peak shape can be realized with 0.% formic acid adjusted to p. (with ammonium hydroxide, for instance), especially with basic peptides. This is likely due to greater availability of formate anion for ion pairing. Pharmaceutical Peptides Many peptides have been investigated as therapeutic pharmaceutical drugs and are active vasodilators, vasoconstrictors, hormones, and neuropeptides. Using reversed-phase PLC, it is possible to solve the tasks of identification, purity monitoring, and quantitative analysis in many cases, including those where the application of other methods is impossible. Synthetic Peptides The difficulty with synthetic peptides involves the production of many deletion variants. A deletion may occur at any point in the peptides synthesis and so several versions of the peptide are produced which are absent one, two or three amino acids from the desired product. This makes for an interesting chromatographic problem because the resultant peptide mix contains peptides that are very similar in structure. Peptide Mapping Protein analysis and characterization has become more crucial due to many biopharmaceutical advances. Peptide mapping via LC-MS is one such technique that is commonly used today. A typical procedure involves the preparation of a tryptic digest from the protein, with subsequent characterization using reversed-phase, gradient PLC separation followed by mass spectral analysis and database search. sigma-aldrich.com/express-peptide

153 Difficult Separations on Ascentis Express Peptide ES-C8 Figure. Basic Peptides column: Ascentis Express Peptide ES-C8, 0 cm x. mm I.D. (06-U) mobile phase A: 0.% additive in water mobile phase B: :7, (0. % additive):acetonitrile additive: formic acid, p. (adjusted with ammonium hydroxide) gradient: initial = % B, slope = % MeC / column volume flow rate: 0. ml/min temp.: C det.: ESI(+)-TF injection: μl sample: mg/l peptide &, mg/l peptide, mg/l peptide Peptide probes (listed in order of elution):. ac-ggglggagglkg monoisotopic mass: 9.. ac-kyglggagglkg monoisotopic mass: 8.6. ac-ggavkalkglkg monoisotopic mass: 9.7. ac-kyalkalkglkg monoisotopic mass: 0.8 Figure. Peptide Test Mix column: Ascentis Express Peptide ES-C8, 0 cm x.6 mm I.D. (-U) mobile phase A: 90:0, (0. % TFA):acetonitrile mobile phase B: :7, (0.% TFA):acetonitrile gradient: initial = 0% B to 0% B in min. flow rate:. ml/min temp.: 0 C det.: UV at 0 nm injection: μl The test mix employed contains the following peptides. Gly-Tyr MW =. Val-Tyr-Val MW = 79. Met Enkephalin MW = 7. Angiotensin II MW = 0. Leu-Enkephalin MW = 6. Ribonuclease MW =, Bovine Insulin MW = G G009 Figure. Carbonic Anhydrase Tryptic Digest column: Ascentis Express Peptide ES-C8, 0 cm x.6 mm I.D. (-U) mobile phase A: 0. % TFA in water mobile phase B: 0:60, (0.% TFA):acetonitrile gradient: initial = % B to 00% B in min. flow rate:.0 ml/min temp.: 0 C det.: UV at nm injection: 0 μl Figure. Small Proteins column: Ascentis Express Peptide ES-C8, 0 cm x.6 mm I.D. (-U) mobile phase A: 90:0, (0.% TFA):acetonitrile mobile phase B: :7, (0.% TFA):acetonitrile gradient: initial = % B to 0% B in min.; then to 60% B at 0 min. flow rate:. ml/min temp.: 0 C det.: UV at 0 nm injection: μl. Ribonuclease MW =,700. Porcine Insulin MW =,780. Bovine Insulin MW =,70. uman Insulin MW =,800. Cytochrom C MW =,7 6. Lysozyme MW =, G G0097 TRADEMARKS: Ascentis is a registered trademark of Sigma-Aldrich Biotechnology LP. Fused-Core is a trademark of Advanced Materials Technologies, Inc.

154 Competitor Comparison Basic Peptides Columns run under equivalent conditions of gradient slope ( % MeC per column volume). column: C8, 0 cm x. mm I.D. mobile phase A: 0.% additive in water mobile phase B: :7, (0. % additive):acetonitrile additive: formic acid, p. (adjusted with ammonium hydroxide) gradient: initial = % B, slope = % MeC / column volume flow rate: 0. ml/min temp.: C det.: ESI(+)-TF injection: μl sample: mg/l peptide &, mg/l peptide, mg/l peptide Peptide probes (listed in order of elution):. ac-ggglggagglkg Monoisotopic mass: 9.. ac-kyglggagglkg Monoisotopic mass: 8.6. ac-ggavkalkglkg Monoisotopic mass: 9.7. ac-kyalkalkglkg Monoisotopic mass: 0.8 Peptide probes increase in basicity and hydrophobicity. Column Backpressure 6 Pressure (x000) 0 A B C Column D A. Ascentis Express Peptide ES-C8,.7μm C. Competitor V C8,.μm Avg Peak Width of Basic Peptides w (width at half-height). min A B C D Column Avg h/w of Basic Peptides 0 6 B. Competitor Z C8,.μm G D. Competitor W C8,.7μm G0090 h/w (x 000) A B C Column D 0 6 G G009 Avg Tailing Factor of Basic Peptides TF (0% of height) A B C D Column Experimental Setup: Columns and Elution Gradients Pore Grad slope Column μm (Å) ID (mm) L (cm) CV (ml)* start %B [ % MeC / CV ] A. Ascentis Express Peptide ES-C B. Competitor Z C C. Competitor V C D. Competitor W C * CV determined as follows: t 0 = r.t. of unretained component. d 0 = r.t. of unretained component with ZDV union in place of column. CV = (t 0 - d 0 ) x ml/min Some Quantitative Comparisons Peak Peak Peak Peak Column w ½ h/w ½ TF 0. w ½ h/w ½ TF 0. w ½ h/w ½ TF 0. w ½ h/w ½ TF 0. A. Ascentis Express Peptide ES-C8,.7 μm B. Competitor Z C8,. μm C. Competitor V C8,. μm D. Competitor W C8,.7 μm sigma-aldrich.com/express-peptide

155 The Fused-Core Advantage Fused-Core Particles Traditional Porous Particles arrow Particle Size Distribution The innovative manufacturing process for Fused-Core particles produces a very narrow particle size distribution. A narrow particle size distribution allows for the use of large porosity frits that resist clogging, resulting in a more rugged column. Traditional porous particles are not manufactured in a way to yield extremely narrow particle size distributions. More Consistent Bed The A term in the van Deemter equation accounts for the effects of inhomogeneities in the packed bed of an PLC column. arrow particle size distributions form a more consistent packed bed and a consistent path length, minimizing analyte diffusion through the column. This eddy diffusion is effectively independent of mobile phase velocity. Shorter Diffusion Path The short diffusion path of the Fused-Core particle yields sharper peaks than traditional porous particle columns. The minimized resistance to mass transfer, the C term in the van Deemter equation, of the Fused-Core particle provides sharper peaks than traditional porous particles. The short diffusion path also permits the use of higher flow rates without peak broadening.

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157 CIRBITIC Chiral by ature Columns for versatile, robust chiral PLC and LC-MS separations Aqueous and non-aqueous separations on the same column o solvent or additive memory effects Wide applicability, especially suited to polar and ionizable compounds Predictable scale-up from analytical to prep

158 CIRBITIC Versatile Chiral PLC and LC-MS Separations of Polar, Ionizable and eutral Compounds CIRBITIC CSPs (chiral stationary phases) interact with polar, ionizable and neutral analytes via multiple molecular interactions. This versatility means that the same CIRBITIC column can be successfully used in a variety of mobile phases, a significant benefit over CSPs that operate only in a single mode, normal or reversedphase, for example, and must be dedicated to those mobile phase systems. owever, the most interesting feature of CIRBITIC CSPs is the presence of ionic interactions, which allows them to be used in polar ionic and reversed-phase modes for sensitive LC-MS operation. Key application areas Drug Discovery igh enantioselectivity, fast screening protocols, scalability to prep, reproducibility for reliable methods, polar and non-polar analytes rganic Synthesis Compatible with all PLC solvents to optimize sample solubility, fully scalable to prep Bioanalytical, Drug Metabolism igh throughput, MS-compatibility, aqueous samples, short run times, rugged columns Amino Acid and Peptide Analysis Resolves underivatized natural and synthetic chiral amino acids and peptides, different selectivity and higher preparative capacity than C8 for achiral amino acids What is the CIRBITIC family? Developed originally by Advanced Separations Technologies (Astec), the CIRBITIC family comprises highly enantioselective CSPs based on macrocyclic glycopeptides that have been bonded through multiple covalent linkages to high purity silica particles. CIRBITIC CSPs offer flexibility in choice of mobile phase conditions, both aqueous and non-aqueous, and are ideal for analytical and preparative separations of neutral, polar and ionic compounds. ow do CIRBITIC CSPs separate enantiomers? CIRBITIC CSPs offer six different types of molecular interactions: ionic, -bond, -, dipole, hydrophobic and steric. They also possess multiple inclusion sites that influence selectivity based on the molecular shape of the analyte. The optimization of enantiomer resolution is achieved by changing the mobile phase to leverage the types and relative strengths of the various interactions. What makes CIRBITIC CSPs unique? The bonded macrocyclic glycopeptide itself (Figure ), in terms of its morphology, molecular composition and multiple covalent linkages to the silica surface, is what makes CIRBITIC CSPs unique and gives them significant and valuable benefits over other CSPs. The truly differentiating feature of CIRBITIC CSPs is the presence of ionic interactions. These interactions are unique to CIRBITIC CSPs and are responsible in large part for their desirable retention characteristics toward polar and ionizable analytes in aqueous and non-aqueous solvents. ow do the CIRBITIC CSPs differ? The various CIRBITIC phases share the benefits of robustness, flexibility in mobile phase options, ionic interactions, compatibility with polar compounds and LC-MS and preparative scalability. owever, CIRBITIC CSPs differ in selectivity, primarily because of their differing number and types of interaction sites, and the number, type and accessibility of ionic sites in the bonded macrocyclic glycopeptide. The CIRBITIC CSP Family CIRBITIC CSPs are based on, 0 or 6 μm, high purity, porous silica gel. They differ in the nature of the bonded macrocyclic glycopeptide and resulting enantioselectivity. CIRBITIC V and V* Vancomycin CIRBITIC T and T* Teicoplanin CIRBITIC R Ristocetin CIRBITIC TAG Teicoplanin Aglycone *CIRBITIC V and T differ from V and T, respectively, in their bonding chemistry that gives them different selectivity and preparative capacity for certain classes of analytes. sigma-aldrich.com/chiral ordering: (US only) / 8-9-

159 Key features of CIRBITIC CSPs: Aqueous and non-aqueous separations on the same column CIRBITIC CSPs have -bond, ionic, dispersive, -, dipole stacking, steric and inclusion mechanisms, usually multiple types of interactions per analyte. Wide applicability Applications cover a very broad range of compound classes, with the different CIRBITIC CSPs showing complementary selectivity. LC-MS compatibility The wide choice of mobile phases makes CIRBITIC CSPs ideal for LC-MS, where analyte ionization and detection sensitivity are of critical concern. o solvent or additive memory effect The same CIRBITIC column can be used alternately in polar, reversed-phase and normal phase solvents without damage, unlike cellulosic and amylosic phases that require dedicated operation. Robust columns with long lifetimes Each macrocyclic glycopeptide molecule is linked to the silica surface via four or five covalent bonds for exceptional stability and long column life. They are designed to withstand high pressure and flow rates, as well as rapid changes in mobile phase conditions. Solvent choices maximize sample solubility CIRBITIC CSPs operate in highly aqueous and non-aqueous polar mobile phases for polar compound solubility. They also operate in normal phase mobile phases to maximize solubility of non-polar compounds. CIRBITIC CSPs are compatible with all organic solvents. Excellent preparative scalability and capacity From narrowbore to prep, separations on CIRBI- TIC are fully scalable, even with polar analytes. By relying on primarily aqueous eluents, the use and disposal of toxic organic solvents are eliminated. Additionally, preparative methods in the non-aqueous polar ionic mode are just as easy to process as normal phase solvents. Fast kinetics for speed and efficiency The kinetics of the molecular interactions between the analyte and the CIRBITIC CSP are fast, providing efficient separations and relatively short retention times. rthogonal selectivity to other CSPs The six CIRBITIC CSPs are different from each other, and from other types of CSPs to offer choices in enantioselectivity, like reversal of elution order. Figure. Proposed Structure of Vancomycin-based CIRBITIC V and V CIRBITIC chiral PLC stationary phases are macrocyclic glycopeptides attached to the surface of high purity, porous silica by four or more covalent bonds. Vancomycin is shown here, but all four CIRBITIC CSPs possess multiple functional groups that can undergo different types of interactions. The CIRBITIC surface is essentially ionic, allowing the use of polar ionic and aqueous mobile phases. Additionally, the glycopeptide structure has several inclusion sites that provide selectivity based on the molecular structure of the analyte. ionic hydrogen bonding & dipole stacking sites -acceptor A B C ionic sugar molecules A, B, C are inclusion sites G00 technical service: (US and Canada only) / 8-9-0

160 Incorporating CIRBITIC CSPs into Your Chiral Column Screening Protocol We recommend that you incorporate CIRBITIC into your routine screening protocol. Experience has shown that one or more of the CIRBITIC CSPs, particularly V, T and TAG, will perform the majority of chiral separations. Even if other CSPs give adequate resolution, a CIRBITIC CSP may allow use of mobile phases that are better suited to your sample and detection method, or the CIRBITIC method may be faster, more efficient or more robust. A CIRBITIC method may also have advantages from a preparative standpoint in terms of solvent selection and sample capacity. For developing a new chiral PLC method, we have created and use routinely in our laboratories a simple and rapid chiral column screening protocol (Table ). It is important to keep in mind that a single CIRBITIC column possesses multiple types of molecular interactions that are different in each of the four distinct modes. The same column can be exposed to all of the conditions outlined in the screening protocol shown in Table without any change or loss of performance. This versatility is just one advantage that CIRBITIC CSPs have over other CSPs. The four CIRBITIC CSPs we recommend in the screening protocol are available in cm or 0 cm column kits. Also, you can further expand the screening field by incorporating the CYCLBD bonded cyclodextrin and P-CAP chiral polymer CSPs into your screening protocol to accommodate other types of compounds not covered by the routine screen. Table. The CIRBITIC Screening Protocol columns: CIRBITIC V, T, R and TAG procedure: Method development follows a simple strategy that tests polar ionic, polar organic, reversed-phase and normal phase modes. Separation Types of Screening Parameters Mode Description Compound Mobile Phase to ptimize Polar Ionic Polar organic solvents Acids, Bases, Zwitterions (00:0.:0., v:v:v) Change acid-base ratio, (C or C C) C :Acetic Acid: change the type of acid or containing small amounts Triethylamine base, add a volatile salt (test of acid and base or salt different ammonium salts) Reversed-Phase Typical RP eluents, water Acids, Bases, Zwitterions, (0:70) C C:0mM Change the % and type of or buffers with C eutrals Ammonium Acetate, p.0 organic modifier, adjust p, or C C buffer type and ionic strength Polar rganic Polar organic solvents eutrals 00% Ethanol Use other polar organic without ionic additives solvents or blends ormal Phase on-polar organic solvents eutrals (0:70) Ethanol:eptane Increase % of polar modifier, with polar solvent modifiers change both solvents Method ptimization: Acid-Base Ratio, Temperature and Flow Rate in Polar Ionic Mode Using CIRBITIC CSPs in the polar ionic mode has the highest probability of success. ptimizing resolution usually involves changing the contribution to retention of ionic interactions between the analytes and functional groups in the macrocyclic glycopeptide structure by: Changing the ratio of acid to base (Figure ) Adding a soluble, volatile salt (instead of the acid and base) directly to the methanol The acid, base or salt that is ultimately selected is based on its compatibility with the detection method (e.g. LC-MS), sample solubility and whether the separation will be scaled up to preparative. sigma-aldrich.com/chiral ordering: (US only) / 8-9-

161 Figure. Demonstration of Polar Ionic Mode Mechanism: Effect of Acid:Base Ratio Column Mobile Phase 00/0./0. Acid:base ratio=: CIRBITIC V C /Acetic Acid/Triethylamine Mianserin 00/0./0.0 Acid:base ratio=: 0. G00 G007.6 G00 igh acid: itrogen on mianserin is positively charged, while -C group on vancomycin is negatively charged: strong ionic interaction 00/0.0/0. Acid:base ratio=:..8 G006 igh base: itrogen on mianserin group is free amine, but -C group on vancomycin is fully charged: weak ionic interaction TRADEMARKS: CIRBITIC, CYCLBD, ybridspe, P-CAP Sigma-Aldrich Biotechnology LP technical service: (US and Canada only) / 8-9-0

162 CIRBITIC: Ideally Suited for LC-MS of Polar, Ionizable and eutral Compounds Each of the various ionization sources has an optimal set of mobile phase conditions. utside this set, ionization may be suppressed with resulting loss in sensitivity. CIRBITIC phases are uniquely able to operate across all mobile phase systems. CSPs that are limited to normal phase operation, like many cellulosic and amylosic CSPs, reduce the options in detection methods. ESI perate CIRBITIC CSPs in reversed-phase and unique polar ionic modes. APCI perate CIRBITIC CSPs in polar ionic mode. APPI perate CIRBITIC CSPs in normal phase mode. Typical polar ionic mobile phases are methanol with low concentrations ( %) of volatile salts like ammonium acetate or ammonium formate. Figures and show examples of CIRBITIC CSPs for LC-MS in reversedphase and polar ionic mode mobile phases, respectively. In addition to mobile phase compatibility, the allowable high flow rates and short columns make them ideally suited to fast MS applications. CIRBITIC columns can be used in conjunction with ybridspe -PPT plates to enhance sensitivity by completely removing endogenous proteins and phospholipids. This approach was used to resolve the enantiomers of clenbuterol on a CIRBITIC T column in Figure. Figure. ESI-MS of Ketoprofen on CIRBITIC R in Reversed-phase Mode column: CIRBITIC R, cm x. mm, μm particles (09AST) mobile phase: (0:70) C :0 mm ammonium acetate, p.6 flow rate: 0. ml/min. det.: ESI(-) temp.: C analyte: Ketoprofen 6 Ketoprofen CPS * G G00 Figure. ESI-MS of Clenbuterol Extracted from Plasma on CIRBITIC T in Polar Ionic Mode column: CIRBITIC T, 0 cm x. mm, μm particles (08AST) mobile phase: 0 mm ammonium formate in C flow rate: 0. ml/min. det.: ESI(+) temp.: 0 C analyte: Clenbuterol in rat plasma (0 ng/ml) Clenbuterol CPS 00 Clenbuterol * Phospholipids G G00 6 sigma-aldrich.com/chiral ordering: (US only) / 8-9-

163 Unique Polar Ionic Mode A valuable feature of CIRBITIC CSPs, the novel and very versatile polar ionic mode is popular because its mobile phases are polar organic solvents containing volatile additives that are ideally suited for preparative and LC-MS applications. An example is shown in Figure. Additionally, compared to normal phase separations, the polar ionic mode has speed, efficiency and sensitivity advantages, all valuable assets for LC-MS. Figure. Beta-Receptors on CIRBITIC T in Polar Ionic Mode column: CIRBITIC T, cm x.6 mm, μm particles (0AST) mobile phase: mm ammonium formate in C flow rate: ml/min. det.: UV at 0 nm temp.: C Clenbuterol Clenbuterol * 00 Metoprolol G00 Sotalol * * G00 Response Metoprolol Sotalol Atenolol G00 Atenolol * G G007 technical service: (US and Canada only) /

164 Multi-modal Interactions Permit Use in Aqueous and on-aqueous Solvents All CIRBITIC CSPs possess multiple interaction sites on the same column. Changing the mobile phase affects the relative strength of specific types of interactions. The power and flexibility of multi-modal CIRBI- TIC CSPs are demonstrated in Figures 6 through 0. The vancomycin-based CIRBITIC CSPs were used successfully in four different modes. Figure 6. Polar Ionic Mode column: CIRBITIC V, cm x.6 mm, μm particles (0AST) mobile phase: mm ammonium formate in C flow rate: ml/min. det.: UV at 0 nm temp.: C analyte: Fluoxetine Fluoxetine Polar Ionic Mode A valuable feature of CIRBITIC, the novel and very versatile polar ionic mode mobile phase system is desirable because of its high volatility and beneficial ionization effect for LC-MS (Figure 6) * G000 Reversed-phase Mode Also highly suitable for LC-MS and polar analytes, reversed-phase (RP) is a mode familiar to all chromatographers. CIRBITIC CSPs have RP character and can be used in a wide range of buffers and solvents (Figure 7). Polar rganic Mode Enantiomers of polar neutral analytes have been successfully separated on CIRBITIC in the polar organic mode where the mobile phase is typically a polar organic solvent or solvent blend. Reaction mixtures, even in pyridine, can be run on CIRBITIC in this mode (Figure 8). ormal Phase Mode ormal phase chiral separations are desirable to maintain solubility of hydrophobic compounds and when analyzing reaction mixtures in non-polar organic solvents. CIRBITIC CSPs have the flexibility to operate in normal phase mode. The same column can be used with normal phase and polar/aqueous solvents and additives without memory effects (Figure 9). Response Response 0 7 Figure 7. Reversed-phase Mode column: CIRBITIC V, cm x.6 mm, μm particles (0AST) mobile phase: (0:70) C C: mm ammonium acetate, p. flow rate: ml/min. det.: UV at nm Warfarin temp.: C analyte: Warfarin.6. * G00 G G00 8 sigma-aldrich.com/chiral ordering: (US only) / 8-9-

165 Figure 8. Polar rganic Mode column: CIRBITIC V, cm x.6 mm, μm particles (0AST) mobile phase: C flow rate: ml/min. det.: UV at 0 nm temp.: C analyte: Thalidomide Figure 9. ormal Phase Mode column: CIRBITIC V, cm x.6 mm, μm particles (0AST) mobile phase: (9:) hexane:ethanol flow rate: ml/min. det.: UV at 0 nm temp.: C analyte: Mephenytoin Thalidomide Mephenytoin * * G006 G G G00 Preparative Applications Using CIRBITIC CSPs Scalability across all CIRBITIC particle sizes Low retention times give high throughput CIRBITIC columns can be used in all preparative PLC techniques, including elution and recycle chromatography, mass-directed prep, SFC and simulated moving bed (SMB). Scale-up is highly predictable because the same bonded phase chemistry is employed across all particle sizes. Multiple covalent bonds attach the CIRBITIC macrocyclic glycopeptides to the silica surface, meaning no CSP ligand will contaminate the product. Preparative separations on CIRBITIC columns often have speed and efficiency benefits over other CSPs. In terms of loading capacity, a cm x. mm column has medium to high loadings, from a few mg to over 00 mg per injection. Prep separations on CIRBITIC are reproducible and scalable. Figure 0 shows the separation of phenylalanine isomers in reversed-phase mode on columns packed with, 0, and 6 μm particles of CIRBITIC T. Figure 0. Scalability Across CIRBITIC CSP Particle Sizes column: CIRBITIC T, cm x.6 mm mobile phase: (0:0) ethanol:water flow rate: 0.9 ml/min. det.: UV at 0 nm μm 0 μm 6 μm G00 G00 G00 technical service: (US and Canada only) /

166 Preparative Applications (contd.) A significant advantage of CIRBITIC for preparative applications is the fact that the mobile phase can be chosen to optimize sample solubility a critical preparative consideration. The examples here show preparative CIRBITIC separations in three different mobile phase systems. Preparative Reversed-phase and Polar Ionic Modes Preparative separations in reversed-phase and polar ionic mode solvents have benefits over normal phase preparative separations in terms of solvent safety and waste disposal costs. Figure shows the use of CIRBI- TIC TAG in a preparative separation in polar ionic mode. Preparative Polar rganic Mode Figure shows the analytical and preparative separations of thalidomide enantiomers on CIRBITIC T. The analytical scale gave an value of. in 00% methanol and a retention time under 0 minutes. owever, since thalidomide is fairly insoluble in pure methanol, it was possible to add 0% dioxane to the mobile phase to increase solubility.-fold while still achieving the necessary separation. Figure. Preparative Separation on CIRBITIC TAG in Polar Ionic Mode column: CIRBITIC TAG, cm x. mm, μm particles (0AST) mobile phase: 0.% ammonium acetate in C flow rate: ml/min. det.: UV at 00 nm throughput: 0 mg/g CSP/hr. load: 00 mg in 6 ml analyte: -Acetyl Tryptophan.86 Figure. Sample Solubility Considerations in Preparative Analytical Scale column: CIRBITIC V, cm x.6 mm, μm particles (0AST) mobile phase: C det.: UV at 9 nm flow rate: ml/min. analyte: Thalidomide.. G Prep Scale column: CIRBITIC V, cm x. mm, μm particles (0AST) mobile phase: (80:0) C :dioxane det.: UV at nm flow rate: 0 ml/min. load: 70 mg in ml analyte: Thalidomide..79 G008 G009 0 sigma-aldrich.com/chiral ordering: (US only) / 8-9-

167 CIRBITIC V and V Bonded Macrocylic Glycopeptide: Vancomycin Chiral Centers: 8 Sugar Moieties: Inclusion Cavities: Separates a wide variety of secondary and tertiary amines in the polar ionic mode. ave many of the separation characteristics of protein-based stationary phases but with exceptional stability and much higher sample capacity. CIRBITIC V and V differ in the chemistry used to bond the glycopeptide to the silica, which gives them differences in selectivity. CIRBITIC T and T Bonded Macrocylic Glycopeptide: Teicoplanin Chiral Centers: Sugar Moieties: Inclusion Cavities: These CSPs have resolved all of the known beta-blockers and dihydrocoumarins and many other compound classes. Generally reproduces chiral crown ether or ligand-exchange for amino acid separations. CIRBITIC T and T differ in the chemistry used to bond the glycopeptide to the silica, which gives them differences in selectivity. CIRBITIC TAG Bonded Macrocylic Glycopeptide: Teicoplanin Aglycone Chiral Centers: 8 Sugar Moieties: 0 Inclusion Cavities: The removal of the three sugar moieties enhances resolution of many of the amino acids (alpha, beta, gamma and cyclic). CIRBITIC TAG has shown remarkable selectivity for sulfur-containing molecules, such as sulfoxides and the amino acids methionine, histidine and cysteine. eutral molecules, like oxazolidinones, hydantoins and diazepines, have shown enhanced resolution and, more remarkably, in single-solvent systems like methanol, ethanol and acetonitrile. Some acidic molecules have also shown increased selectivity. CIRBITIC R Bonded Macrocylic Glycopeptide: Ristocetin A Chiral Centers: 8 Sugar Moieties: 6 Inclusion Cavities: The presence of amines in the ristocetin structure makes it a good choice when screening acidic compounds. CIRBITIC Product Listing For more information and to review our complete offering of CIRBITIC columns, please visit sigma-aldrich.com/chiral Method Development Kits ID (mm) Length (cm) Cat. o AST - ne each of CIRBITIC V, T, TAG and R.6 00AST - ne each of CIRBITIC V, T, TAG and R CIRBITIC Columns* V V T T TAG R ID (mm) Length (cm) Cat. o. Cat. o. Cat. o. Cat. o. Cat. o. Cat. o. μm. 09AST 09AST 09AST 609AST 09AST 09AST. 00AST 00AST 00AST 600AST 00AST 00AST.6 0AST 0AST 0AST 60AST 0AST 0AST.6 0 0AST 0AST 0AST 60AST 0AST 0AST.6 0AST 0AST 0AST 60AST 0AST 0AST.6 0AST 0AST 0AST 60AST 0AST 0AST 0 0AST 0AST 0AST 60AST 0AST 0AST AST 06AST 06AST 606AST 06AST 06AST. 0AST 0AST 0AST 60AST 0AST 0AST 0 μm.6 AST AST AST 6AST AST AST *ther column dimensions, including guard columns and preparative dimensions are available on our website or by inquiring to [email protected]. Chiral Services: Column Screening and Small-Scale Purification Consult Supelco to obtain a quotation for our expert services for chiral column screening (PLC and GC), method development and optimization, as well as isolation of up to 0 grams of purified enantiomer. The complete listing of our chiral PLC and GC columns can be found at sigma-aldrich.com/chiral, our corporate chiral web portal, where you can view our other products for chiral chemistry, like chiral catalysts, building blocks, mobile phase additives, derivatization reagents and more. technical service: (US and Canada only) / 8-9-0

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169 Astec P-CAP and P-CAP -DP Polycyclic amine polymer stationary phases for chiral PLC Stable, covalent chemistry Reversible elution order o solvent limitations or memory effects igh capacity for preparative applications MS and SFC-compatible

170 Chiral PLC for Chemists: Ultimate Solvent Choice with igh Capacity Using Astec s P-CAP and P-CAP-DP Useful for chiral PLC and SFC separations, Astec P-CAP and P-CAP-DP polymeric chiral stationary phases (CSPs) have a thin, ordered layer of chiral polymer covalently bonded to the silica surface. They offer high stability, high sample loadability, easy scale-up, and no memory effect. Today s chiral PLC columns too often give excellent enantioselectivity at the expense of solvent choice. Sample solubility and its link to preparative separations can mean that a compromise has to be reached between selectivity and solvent choice. Astec P-CAP and P-CAP-DP chiral PLC columns have no solvent restrictions, so the user can select a solvent that provides optimum enantioselectivity and analyte solubility. Astec P-CAP Bonded phase: Poly(trans-,-cyclohexanediyl-bis-acrylamide) Invented by Prof. Francesco Gasparrini (), P-CAP is made from a diacryloyl -trans-,-diphenylethylenediamine polymerization, and utilizes hydrogen bonding and steric effects as enantiomer separation mechanisms. Astec P-CAP-DP Bonded phase: Poly(diphenylethylenediamine-bis-acryloyl) or Poly-DPEDA Invented by Prof. Daniel Armstrong (), Astec P-CAP-DP introduces phenyl rings to add p-p interactions, giving it one additional type of interaction compared to P-CAP. P-CAP-DP is less polar than P-CAP. Solvent Choice and Reversal of Elution rder The P-CAP and P-CAP-DP polyamide CSPs feature a thin, ordered polymer layer chemically bonded to µm or. µm spherical silica using a patented radical polymerization. This gives the phases high permeability across the surface and, because they are synthetic, they can be identically manufactured in both R,R and S,S forms. This provides a predictable reversal of elution order in the same mobile phase (Figure ). The P-CAP phases have no solvent or additive memory effects, so the same column can be used in a number of different mobile phases without any detrimental effects. These phases form a new generation bridge between the traditional brush type CSPs and the conventional polymeric phases. Chiral method development is typically carried out either in normal phase (heptane/ipa or hexane/ethanol) or polar organic (acetonitrile/methanol) modes. For method optimization, a wide range of organic solvents can be used, from acetone to dichloromethane to dioxane, and many others. For acids and bases, the addition of 0.% TFA often mau mau Figure. Predictable Elution rder Reversal UV (nm) columns: cm x.6 mm I.D., µm mobile phase: 9:, methylene chloride:methanol flow rate: ml/min. Lorazepam analog Figure. igh-capacity Separations Analyte: furoin column: (R,R) P-CAP-DP, cm x.6 mm I.D., µm particles (0AST) mobile phase: 80:0, hexane:ethanol flow rate:.0 ml/min. temp.: C det.: UV at nm injection: (a) µl at mg/ml (8 µg total) (b) 0 µl at 0 mg/ml (000 µg total) 8 µg injection 000 µg injection * * G0000 CIRALYSER Polarimetric Detector ( ) (+) 0 Peak = 9. min. Peak = 0. min. Peak =. min. (+) (R,R) P-CAP (S,S) P-CAP ( ) G007 Peak =. min. G G000 sigma-aldrich.com/supelco ordering: technical service:

171 increases resolution and efficiency and decreases retention times. There are no known limitations on the kind of solvents that can be used with these phases. For MS detection, volatile acids and buffers such as ammonium acetate can be added to enhance peak efficiency or to enhance ionization when needed. igh Capacity for Preparative PLC Solvent flexibility and high loading capacity make P-CAP and P-CAP-DP CSPs ideal for analytical, preparative and process scale separations. The separation of furoin enantiomers is shown in Figure using an analytical cm x.6 mm Astec (R,R) P-CAP-DP column and mobile phase of 80:0 hexane:ethanol. Excellent separation is demonstrated with an injection of 8 µg of the furoin racemate. Increasing the load to mg on this analytical column demonstrates the phase s high loading capacity. MS-Compatible peration Astec P-CAP and P-CAP-DP operate in mobile phases that are amenable to MS-detection. Salt and/or acetic acid can be added to improve efficiency or enhance ionization and detection (Figure ). Applications Astec P-CAP CSPs have been used for a wide variety of molecular types and are ideal for medium to high polarity compounds. The mechanism of separation is either through hydrogen bonding for P-CAP, or through both hydrogen bonding (donor and acceptor) with additional π-π interaction for the P-CAP-DP. Both also use dipole-dipole and steric interactions. Examples of the separations completed to date are shown in Table. Table. Examples of P-CAP and P-CAP-DP Application Areas ydroxycarboxylic acids Alcohols Sulfoxides Esters Amides -blocked amino acids Complementary to ther Astec CSPs Benzene sulfonamides Binaphthols Benzodiazepines Phosphonic acids Bis-Sulfones Chromemones Astec P-CAP and P-CAP-DP are complementary to Astec CIRBITIC, CYCLBD and the polysaccharide-based CSPs. We suggest you incorporate them into your chiral column screening protocol. Figure. Improved Analytical Separation with MS-Compatible Mobile Phases column: (R,R) P-CAP, cm x.6 mm I.D., µm particles (0AST) flow rate:.0 ml/min. temp.: C det.: nm Separation of, -Bi--naphthol mobile phase: 9::0 mm ammonium acetate:methanol:acetate Peak = 7.9 min. Peak = 9. min. G008 xazepam mobile phase: 70:0:0 mm ammonium acetate:methanol:acetate Peak =.9 min. Peak = 7.7 min. G009 Lorazepam mobile phase: 70:0:0 mm ammonium acetate:methanol:acetate Summary Peak =.97 min. Peak = 8. min. G000 Astec P-CAP and P-CAP-DP are rugged chiral PLC phases, experience no memory effects, and can be run in a wide variety of solvents with speed and high efficiencies. For preparative applications, a combination of wide solvent choice and high capacity make them ideal for large-scale purification. References ) ew hybrid polymeric liquid chromatography chiral stationary phase prepared by surface initiated polymerization. Gasparrini, F.; Misiti, D.; Rompietti, R.; Villani, C. J Chromatogr, A. (00), 06(), -8. ) Chromatographic evaluation of poly(trans-,-cyclohexanediyl-bisacrylamide) as a chiral stationary phase for PLC. Zhong, Qiqing; an, Xinxin; e, Lingfeng; Beesley, Thomas E.; Trahanovsky, Walter S.; Armstrong, Daniel W. Department of Chemistry, Iowa State University, Ames, IA, USA. Journal of Chromatography, A (00), 066(-), -70. Chromatography Products for Analysis and Purification

172 rdering Information Sigma-Aldrich Worldwide ffices Particle Size (µm) Length (cm) I.D. (mm) Cat. o. Astec (R,R) P-CAP Argentina. France.6 00AST Free Tel: Free.6 Tel: AST Tel: (+).6 7 Free.6 Fax: AST Fax: (+) 698 Tel:.6 (+) AST 0 Fax:. (+) AST Australia 0.6 0AST Free Tel: Germany. 09AST Free Fax: Free.6 Tel: AST Tel: (+6) 98 0 Free. Fax: AST Fax: (+6) Tel:.6 (+9) AST Fax: 0 (+9) AST Austria. 0AST Tel: (+) ungary.6 AST Fax: (+) Ingyenes telefonszám: Guards* Ingyenes fax szám: Belgium Tel: (+6) 906 0AST Free Tel: Fax: (+6) AST Free Fax: Tel: Astec (+) (S,S) P-CAP India Fax: (+) 899 Telephone..6 0AST Brazil. 0 Bangalore:.6 (+9) AST 900 Free Tel: ew.6 Delhi: (+9) 8 0AST 8000 Tel: (+) 7 00 Mumbai:.6 (+9) AST Fax: (+) yderabad:. (+9) 0 008AST 88 0 Kolkata:.6 (+9) AST Canada Fax. 09AST Free Tel: Bangalore:.6 (+9) AST 90 Free Fax: 6 88 ew. Delhi: (+9) 8 00AST 800 Tel: (+) Mumbai:.6 (+9) AST Fax: (+) yderabad: 0 (+9) 0 0 0AST 88. 0AST Chile Kolkata: (+9) AST Tel: (+6) 9 79 Fax: Guards* Ireland (+6) Free Tel: AST China Free Fax: AST Free Tel: Tel: (+) Tel: (+86) 6 66 Fax: (+ ) 0 07 Fax: (+86) 6 67 Czech Republic Tel: (+0) Fax: (+0) Denmark Tel: (+) Fax: (+) Finland Tel: (+8) Fax: (+8) Israel Free Tel: Tel: (+97) Fax: (+97) Italy Free Tel: Tel: (+9) 0 70 Fax: (+9) Japan Tel: (+8) Fax: (+8) Particle Size (µm) Length (cm) I.D. (mm) Cat. o. Astec (R,R) P-CAP-DP Korea. Slovakia.6 0AST Free Tel: (+8) Tel: (+) AST Free Fax: (+8) Fax: (+) AST.6 0AST Tel: (+8) South 0 Africa 0AST Fax: (+8) Free Tel: AST Malaysia Free Fax: Guards* Tel: (+60) 6 Tel: (+7) AST Fax: (+60) 6 6 Fax: (+7) Mexico Spain Astec (S,S) P-CAP-DP Free Tel: Free Tel: Free Fax: Free Fax: AST Tel: (+) Tel: (+) AST Fax: (+) AST Fax: (+) Sweden.6 70AST The etherlands Tel: (+6) AST Free Tel: AST Fax: (+6) 8 7 Free Guards* Fax: Tel: (+) Switzerland 700AST Fax: (+) Free Tel: Free Fax: ew Zealand *Guard Column olders Tel: (+) Free Tel: Fax: (+) Guard olders for mm I.D. cartridges 0AST Free Fax: (holder not required for mm I.D. guards) United Kingdom Tel: (+6) 98 0 Free Tel: Fax: (+6) Free Fax: orway Tel: (+) Tel: Visit (+7) sigma-aldrich.com/chiral to Fax: see (+) our 77 complete 8 offering Fax: of (+7) chiral 7 chemistry, 60 0 separation products, United States and services. Toll-Free: Poland Toll-Free Fax: Tel: (+8) Tel: (+) Fax: (+8) Fax: (+) Portugal Free Tel: Free Fax: Tel: (+) 9 Fax: (+) 9 60 Russia Tel: (+7) Fax: (+7) Singapore Tel: (+6) Fax: (+6) Vietnam Tel: (+8) 6 80 Fax: (+8) 68 8 Internet sigma-aldrich.com CIRBITIC, CYCLBD and P-CAP are trademarks of Sigma-Aldrich Biotechnology LP Accelerating Customers Success through Innovation and Leadership in Life Science, igh Technology and Service rder/customer Service (800) -00 Fax (800) -0 Technical Service (800) -8 sigma-aldrich.com/techservice Development/Custom Manufacturing Inquiries (800) -7 Safety-related Information sigma-aldrich.com/safetycenter World eadquarters 00 Spruce St. St. Louis, M 60 () sigma-aldrich.com 00 Sigma-Aldrich Co. All rights reserved. SIGMA, SAFC, SIGMA-ALDRIC, ALDRIC, FLUKA, and SUPELC are trademarks belonging to Sigma-Aldrich Co. and its affiliate Sigma-Aldrich Biotechnology, L.P. Sigma brand products are sold through Sigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip. M T0060