TheReporter VOLUME 3, ISSUE THE TECHNICAL NEWSLETTER FROM SUPELCO The Alumina Chloride PLOT for the Analysis of Freons Katherine Stenerson kstenerson@sial.com Traditionally, the analysis of Freon has been done by packed column GC. However, the limited availability of packings able to do this application has created a problem. Alumina PLOT columns provide a better alternative to traditional packings, including the popular Fluorcol materials, for the analysis of Freon gases. Alumina PLOT columns are prepared using submicron, granular activated alumina (aluminum oxide). The polarity of the aluminum oxide provides a unique selectivity, for example, eluting small, unsaturated hydrocarbons after larger (or similar molecular sized) saturated hydrocarbons. Figure A. Separation of Freons on the 50m x 0.53mm ID Alumina Chloride PLOT column Column: Alumina Chloride PLOT, 50m x 0.53mm ID, 0µm Cat. No.: 28329-U Oven: 35 C ( min), 2 C/min to 80 C Inj.: 200 C Det.: FID, 230 C Carrier: He, 3mL/min @ 35 C Injection: µl, gas syringe Sample: Freons @ ~20% each (custom standard). Freon 2 2. Freon 22 3. Freon 4 4. Freon 4a 5. Freon 6. Freon 2 4 G00228 Figure A illustrates the separation of a typical Freon mixture on the Alumina chloride PLOT column. The column exhibits peak shape and resolution that is superior to the same application on Fluorcol packing. In addition, the polarity/selectivity of this column allows for the separation of the Freon 4 and 4a impurity. For more information request the PLOT Column Packet, T40355 (GHX). Did you know...? Moisture present in the carrier gas can alter the retention characteristics of activated Alumina PLOT columns. To prevent moisture in the carrier gas, install traps capable of removing moisture, such as the Mol Sieve 5A, the High Capacity Gas Purifier, or the OMI -2, on the carrier gas line to the GC. RELATED PRODUCTS Cat. No. Alumina Chloride GC Capillary PLOT Column 50m x 0.53mm ID, 0µm... 28329-U Molecular Sieve 5A Water Vapor Trap 200cc, /8 Fittings... 2069 High Capacity Gas Purifier 0VAC, /8 Fittings (CE approved)... 23800-U OMI Indicating Purifiers OMI-2 Purifier Tube... 23906 OMI-2 Tube Holder, /8 fittings... 2392 Seal Kit for OMI-2 Tube Holder (includes 2 Teflon seals and tool)... 2397 First time users must order both purifier and corresponding holder. Holder is reusable. For a complete listing of all Sigma-Aldrich products, log on to our website: sigma-aldrich.com Gas Chromatography The Alumina Chloride PLOT for the Analysis of Freons... Minimize Instrument Downtime by Reducing Moisture in Carrier Gas Streams... 2 Thermogreen LB-2 Septa... 2 Analysis of Permanent Gases... 3 Reagents, Standards & Accessories SCOTTY Gas Cylinders... 4 Supelco Custom Standards... 4 Analytical Standards... 4 Improve Stability of LC/MS Baselines by Using High-Quality Solvents... 5 Liquid Chromatography Isocratic Elution of Analytes Differing Significantly in Polarity on Discovery HS F5... 6 The Importance of Ion-Exchange Capacity in Mixed-Mode SPE... 7! How to Obtain Related Information To obtain any of the Related Information mentioned in this issue, check off the appropriate box on the back of the cover ad, call 800-359- 0682, or visit our web site: sigma-aldrich.com/thereporter sigma-aldrich.com/supelco LEADERSHIP IN LIFE SCIENCE, HIGH TECHNOLOGY AND SERVICE SUPELCO 595 NORTH HARRISON ROAD BELLEFONTE, PA 6823-0048 USA TheReporter Vol. 3, Issue
PERFORMANCE TIP PERFORMANCE TIP PERFORMANCE TIP PERFORMAN GC Minimize Instrument Downtime by Reducing Moisture in Carrier Gas Streams To insure proper operation, Alumina PLOT columns must be regenerated after exposure to moisture. Limiting moisture exposure will help to minimize instrument downtime resulting from the regeneration procedure. Typically, moisture is introduced onto the column via the sample injection or through impure carrier gas. Unfortunately, the user has limited control over the amount of moisture present in a particular sample. Moisture in the carrier gas, however, can be controlled by following these steps: ) Use carrier gas that is UHP (Ultra-High Purity) Grade or better. Typically, this means a minimum purity level of 99.9995%. Routinely check the cylinder volume and change out cylinders when the pressure gauge reaches 25psi. 2) Use ultra-high purity gas regulators. These regulators feature 36 grade stainless steel diaphragms, metal-tometal diaphragm seals, and nickel-plated brass bodies. + Related Products Thermogreen LB-2 Septa We test every lot of Thermogreen LB-2 silicone septa to ensure low bleed and high puncture tolerance. We also condition them so they are ready to use, right from the container. Lowest bleed of any septum we have tested. Extremely low bleed over a wide range of inlet temperatures 00 C to 350 C Already conditioned, ready to use Bleed after 25 punctures Easier needle penetration Temperature: 40 C to 250 C and high puncture tolerance ideal for auto- Injector Set Point: 350 C samplers Rubber formulation exclusive to Supelco Coated Gray Blue Brick Red Burnt Orange Thermogreen LB-2 93-032 These features eliminate diffusion of contaminates (such as moisture) from the room into the carrier gas. Additionally, these regulators are sealed to a helium leak rate of less than 2x0-8 cc/second. 3) Use gas regulators equipped with purge valves. When gas cylinders are changed, the gas regulator must be removed from the gas cylinder. A small amount of room air will fill the cylinder connector fitting. Once the valve on the newly attached full gas cylinder is opened, this slug of room air (containing moisture) is forced into the carrier gas line. The solution is to use a gas regulator that is equipped with a purge valve. A purge valve allows this small volume of gas to be expelled into the room instead of into the carrier gas line. 4) Use purifiers designed to remove moisture. A purifier that contains Molecular Sieve 5A adsorbent material will remove the trace amounts of moisture that exist in the gas cylinder. To determine when the purifier needs to be replaced, position an indicating purifier downstream. Thermogreen LB-2 Septa Diameter (mm) Diameter (In.) Qty. Cat. No. 5.0 3/6 50 20638 6.0 /4 50 2065 9.0 /32 50 2802-U 9.5 3/8 50 20652 9.5 3/8 250 20666 9.5 3/8 000 20677 0.0 3/32 50 20653-U 0.0 3/32 250 2356 0.0 3/32 000 2357.0 7/6 50 20654.0 7/6 250 2363.0 7/6 000 2364.5 /24 50 2354 2.5 /2 50 20660-U 2.5 /2 250 20678 4.0 9/6 50 20662-U 6.0 5/8 50 20663 7.0 2/32 50 2359 Cylindrical, for Shimadzu instruments Plug Type 0 20608 Plug Type 50 20633 Drilled, for Solid Phase Microextraction 9.5 3/8 25 236 9.5 3/8 50 2362-U.0 7/6 25 2367.0 7/6 50 2368 We recommend a 9.5mm (3/8") septum to those who previously used the 9mm size. 2 TheReporter Vol. 3, Issue
ARTICLE ARTICLE ARTICLE ARTICLE Analysis of Permanent Gases Katherine Stenerson kstenerson@sial.com The separation of permanent gases can be easily achieved on a PLOT column without using sub ambient oven conditions. The Mol Sieve 5A PLOT column is made from a granular zeolite that possesses strong adsorption strength. This makes it effective for the separation of oxygen, nitrogen, hydrogen, carbon monoxide, and methane. In addition, the separation of oxygen and argon can be achieved at ambient temperature if a thick film (>50µm) PLOT column is used. Figure A illustrates a typical permanent gas separation on the Mol Sieve 5A PLOT column. At 65 C, oxygen and nitrogen are baseline resolved. The presence of both water and carbon dioxide, however, is detrimental to the performance of this column, because both are adsorbed under normal operating conditions. Figure A. Separation of Permanent Gases on the Mol Sieve 5A PLOT Column Column: Mol Sieve 5A PLOT, 30m x 0.53mm ID, 30µm Cat. No.: 25463 Oven: 65 C isothermal Inj.: 200 C Det.: TCD, 230 C Carrier: He, 0mL/min Injection: 0µL, direct valve Sample: permanent gases 0 Min 2 2 3. Oxygen (.0%) 2. Nitrogen (bulk) 3. Methane (.0%) 4. Carbon monoxide (.0%) 4 795-0367 Figure B. Transformer Gas Analysis for ASTM Method D362-96 on the Carboxen-00 PLOT. Hydrogen (500ppm) 2. Oxygen (500ppm) 3. Nitrogen (500ppm) 4. Carbon monoxide (500ppm) 5. Methane (500ppm) 6. Carbon dioxide (500ppm) 7. Acetylene (500ppm) 8. Ethylene (500ppm) 9. Ethane (500ppm) Column: Carboxen-00 PLOT, 30m x 0.53mm ID, 30µm Cat. No.: 25467 Oven: 35 C (7 min), 24 C/min to 250 C Inj.: 200 C Det.: TCD/methanizer-FID, 230 C Carrier: Argon, 3.0 ml/min Injection: 0µL, valve injection Sample: transformer gas for ASTM method D362-96 As a superior alternative to the Mol Sieve 5A PLOT, the Carboxen -00 PLOT will separate the permanent gases, including CO 2, as well as larger molecules (up to C3). This is achieved due to a combination of the Carboxen-00 s higher surface area, larger 7Å pore diameter, and improved access of the analytes to the microporous region of the material. Figure B illustrates the effective strength and range of this synthetic, microporous carbon molecular sieve. In addition, due to the hydrophobic nature of the carbon material, the performance of the Carboxen-00 PLOT is not affected by the presence of water. For more information request the PLOT Column Packet, T40355 (GHX). Did you know...? The separation characteristics of molecular sieve columns, including the Mol Sieve 5A PLOT, can be adversely affected by both water and carbon dioxide. The hydrophilic nature of this material results in the strong adsorption of water and/or carbon dioxide to the inner pore walls. This will reduce the pore capacity of the PLOT, resulting in a gradual decrease in retention times. The column can be regenerated by extended thermal conditioning at a temperature of 280 C to 320 C. The surface of carbon molecular sieves, such as Carboxen-00, is hydrophobic. As a result, the strong adsorption of water is not a factor, and retention time shifts, due to moisture, should not occur. Therefore, thermal conditioning to restore performance is not necessary. G002209 GC TheReporter Vol. 3, Issue 3
PERFORMANCE TIP PERFORMANCE TIP PERFORMANCE TIP PERFORMAN Tips for Working With SCOTTY Gas Cylinders Reagents, Standards, & Accessories SCOTTY transportable gas cylinders provide high-purity and accurate calibration gases in a wide variety of non-returnable cylinder sizes and volumes. Their high reliability and convenient size make them a cost-effective alternative to returnable, high-pressure cylinders whenever portability or limited gas quantities are needed. The non-refillable, non-returnable design of SCOTTY gas cylinders results in substantial cost savings by eliminating cylinder rental and two-way shipping charges associated with refillable cylinders. Convenient cylinder sizes also allow you to purchase only the amount of gas your application requires. SCOTTY gas cylinders are easy to use. The following tips will assist the user in getting the best performance. ) Use a gas cylinder stand. Stabilizing your gas cylinder on a bench top or other surface will free up both your hands for manipulating a syringe, the regulator, or cylinder valve as needed. Additionally, a stand will securely hold the gas cylinder from tipping over when not in use. 2) Use a gas regulator with a stainless steel or brass body. Gas regulators manufactured from these materials have superior durability over gas regulators manufactured from aluminum. 3) Use a gas regulator with an integrated syringe adapter. A syringe adapter allows calibration gas to be withdrawn through a silicone rubber septum with a syringe. A vent at the septum permits purging prior to filling the syringe to insure a clean sample of the cylinder gas. + Related Products Supelco Custom Standards Sigma-Aldrich offers over 8,000 standards and reference materials. If you still can t find exactly the standard you require, Supelco can prepare a custom chemical standard for your application. Every custom standard is shipped with a material safety data sheet (MSDS) and a certificate of composition. To obtain a custom quote, please call 800-359-304 or email techservice@sial.com for assistance. Analytical Standards Supelco offers over 50 different petroleum and specialty gas standards to meet your everyday calibration requirements. Three standards that relate to the GC applications shown elsewhere in this Reporter are listed to the right. For a complete listing see the Supelco catalog or visit us on the web at sigma-aldrich.com/supelco. 4) Make sure you close the valve on top of the gas cylinder when not in use. Since a feature of a syringe adapter is the constant purge, the only way to keep gas in the cylinder is by closing the valve on top of the gas cylinder. 5) Use Gastight Sample Lock syringes for transferring gas from the gas cylinder to a GC or another vessel. These syringes allow you to transfer gas without loss or contamination. Following these tips will help the user get the most from their SCOTTY gas cylinders. Specifications Contents Pressure Outlet Fitting Dimensions Weight SCOTTY4 4 Liters 240psig CGA-60 3" x ".50lb. SCOTTY48 48 Liters 300psig CGA-65 4" x 6.25".75lb. RELATED PRODUCTS Freon Solution Description Qty Cat. No. Freon Mix 5 analytes, 0,000µg/mL each in ethyl acetate ml 48420-U Freon Freon 2 Freon 4/4A Freon 2 Freon 22 (mixed isomers) Gas Standards Cat. No. SCOTTY Model 24 Single-Stage Regulator with CGA-60 (for SCOTTY 4)... 5079 with CGA-65 (for SCOTTY 48)... 50395 Syringe Adapter for SCOTTY 4 and 48 Cylinders Syringe Adapter... 60900 Additional Septa (pk. of 0)... 60800 For a complete listing of all Sigma-Aldrich products, log on to our website: sigma-aldrich.com Description Cylinder Cat. No. Carbon monoxide, carbon dioxide, methane, ethane, ethylene, & acetylene each at % in nitrogen SCOTTY 4 23437 SCOTTY 48 23462 Methane, carbon monoxide, carbon dioxide, hydrogen, & oxygen each at % in nitrogen SCOTTY 4 2256 SCOTTY 48 23463 4 TheReporter Vol. 3, Issue
PERFORMANCE TIP PERFORMANCE TIP PERFORMANCE TIP PERFORMAN Improve Stability of LC/MS Baselines by Using High-Quality Solvents Using LC-MS CHROMASOLV eliminates impurities, reducing background ion counts. LC/MS is fast becoming the dominant analytical tool for researchers in a wide range of industries and applications. LC/MS combines the advantages of a chromatographic separation with mass spectrometry. Low detection limits and qualified analysis of molecular structures is increasing the popularity of this technique, especially in the field of biotechnology and within the analytical departments of the pharmaceutical industry. Besides the column and instrument, the solvent can seriously affect the performance of an LC/MS system. Impurities arising from the solvent can mask low levels of target ions, or give false peak responses, resulting in difficulty in spectra interpretation. Sigma-Aldrich s Riedel-de Haën brand of LC/MS CHROMASOLV solvents is manufactured specifically to meet the needs of modern LC/MS instruments because they are designed to have low amounts (max. 00ppb) of alkaline impurities, such as calcium, magnesium, potassium and sodium, which can form ion adducts with the analyte, especially when Table. Product Specifications using electro spray ionization. All LC/MS CHROMASOLV solvents undergo a battery of specific UV-spectroscopic quality control tests to guarantee the traditional Riedel-de Haën CHROMASOLV specifications and to ensure their reproducibility. They are also checked for LC/MS use by a special test and for particles, which can clog up Nano-LC inlet filters. Existing solvents have been purified to meet tighter specifications, except ethyl acetate, which is used only for sample preparation. (See Table ) For more information request T40408 (GND) For a complete listing of all Sigma-Aldrich products, log on to our website: sigma-aldrich.com LC-MS Solvent Water Acetonitrile Methanol 2-Propanol Ethyl Acetate Catalog Number 39253 34967 34966 34965 34972 Size L-R L / 2.5L L / 2.5L L / 2.5L L-R / 2.5L-R Assay (GC) (min) - 99.9% 99.9% 99.9% 99.7% Fluorescence at 254nm (max) ppb 0.5ppb ppb ppb - Fluorescence at 365nm (max) ppb 0.5ppb ppb ppb - Chloride (Cl) (max) 0.00000% - - - - Fluoride (F) (max) 0.00000% - - - - Nitrate (NO 3 ) (max) 0.0000% - - - - Sulfate (SO 4 ) (max) 0.0000% - - - - Free acid (max) - 0.00% 0.00% 0.00% - Free alkali (as NH 3 ) (max) - 0.0002% 0.0005% 0.0005% 0.0005% Non-volatile matter (max) 0.00 % 0.0002% 0.0005% 0.0005% 0.0005% Water (Karl Fischer) (max) - 0.0% 0.02% 0.05% 0.03% Transmittance at 200nm (min) 95% 95% - - - Transmittance at 230nm (min) 99% 99% 75% 75% - Transmittance at 260nm (min) - - 98% 98% 50% HPLC gradient (254nm) (max) mau 0.2mAU 2 mau 2 mau - Silver (Ag) (max) 0.ppm 0.ppm 0.ppm 0.ppm - Aluminum (Al) (max) 0.5ppm 0.5ppm 0.5ppm 0.5ppm - Barium (Ba) (max) 0.ppm 0.ppm 0.ppm 0.ppm - Calcium (Ca) (max) 0.ppm 0.ppm 0.ppm 0.ppm 0.ppm Cadmium (Cd) (max) 0.05ppm 0.05ppm 0.05ppm 0.05ppm - Cobalt (Co) (max) 0.02ppm 0.02ppm 0.02ppm 0.02ppm - Chromium (Cr) (max) 0.02ppm 0.02ppm 0.02ppm 0.02ppm - Copper (Cu) (max) 0.02ppm 0.02ppm 0.0ppm 0.02ppm - Iron (Fe) (max) 0.ppm 0.ppm 0.ppm 0.ppm - Potassium (K) (max) 0.ppm 0.ppm 0.ppm 0.ppm 0.ppm Magnesium (Mg) (max) 0.ppm 0.ppm 0.ppm 0.ppm 0.ppm Manganese (Mn) (max) 0.02ppm 0.02ppm 0.0ppm 0.02ppm - Sodium (Na) (max) 0.ppm 0.ppm 0.ppm 0.ppm 0.ppm Nickel (Ni) (max) 0.02ppm 0.02ppm 0.02ppm 0.02ppm - Lead (Pb) (max) 0.ppm 0.ppm 0.02ppm 0.ppm - Tin (Sn) (max) 0.ppm 0.ppm 0.ppm 0.ppm - Zinc (Zn) (max) 0.ppm 0.ppm 0.ppm 0.ppm - Particle test + + + - - LC/MS suitability test + + + + + Reagents, Standards, & Accessories TheReporter Vol. 3, Issue 5
ARTICLE ARTICLE ARTICLE ARTICLE Isocratic Elution of Analytes Differing Significantly in Polarity on Discovery HS F5 LC Dave Bell dbell@sial.com Role of Stationary Phase Chemistry Toward Retention Retention and selectivity observed in chromatographic processes are the manifestation of the multiple types of interactions between the analytes and the stationary phase. Traditional alkyl phases such as C8 are designed to operate primarily by hydrophobic interactions and exhibit minimal polar interactions. These properties often provide for predictable method development and smooth operation, however, in many cases analytes that vary significantly in polarity require gradients to perform the analysis efficiently. Although gradient elution is acceptable practice for some applications, isocratic solutions are still favored. Polar reversed-phase columns, by design, impart polar interactions of retention as well as the hydrophobic interactions available on traditional C8 phases. The additional polar interactions can be utilized to alter the selectivity for a given set of analytes. In this report, such manipulation of the polar and hydrophobic interactions provided by a pentafluorophenylpropyl stationary phase (Discovery HS F5) is demonstrated using the antiparkinsonian drug, selegiline, and two of its polar metabolites. Retention and Separation on Discovery HS F5 Selegiline and two of its metabolites (amphetamine and methamphetamine) vary significantly in polarity (Figure A). Using alkyl stationary phase chemistries, low percentages of organic solvents are required to retain the polar amphetamines. Under the same conditions the relatively non-polar selegiline solute is highly retained resulting in an overall run time of about 30 minutes. Due to the polar interactions available on the Discovery HS F5 phase, the amphetamines can be retained using greater percentages of organic modifier. Under these conditions, selegiline and the amphetamine analytes elute in less than 5 minutes as shown in Figure A. Conclusion The availability of polar interactions provided by modern stationary phases such as the Discovery HS F5 provides new opportunities for optimal HPLC method development. In those cases where C8 phases provide too much resolution or inadequate retention of polar analytes, polar reversed-phases are likely to provide superior performance. Figure A. Retention of Selegiline and Polar Metabolites on Discovery HS F5 00 % 0 00 % 0 00 % Column: Discovery HS F5, 5cm x 4.6mm, 5µm particles Cat. No.: 56753-U Mobile Phase: 30:70, 0mM ammonium acetate (ph 4.0 w/ acetic acid):ch 3 CN Flow Rate: ml/min Temp.: 35 C Det.: MS, ESI (+) Inj.: 5µL Sample: 0µg/mL each component in CH 3 OH Selegiline LogD, ph 7 = 2.32 Methamphetamine LogD, ph 7 = -0.3 Amphetamine LogD, ph 7 = -0.24 For more information request T40354 (GMR), Solute Attributes and Molecular Interactions Contributing to U-Shape Retention on Fluorinated Stationary Phases. G00232 0 0 5 0 Min RELATED PRODUCTS NH 2 CH 3 HN CH 3 CH 3 G002037 G002035 m/z 88 m/z 50 m/z 36 Cat. No. Selegiline, R-( )-Deprenyl hydrochloride (Sigma)... M-003 Amphetamine (Sigma)... A-2262 Methamphetamine (Sigma)... M-5260 Ammonium Acetate, ReagentPlus (Aldrich)... 43,3 Acetic Acid, glacial (Aldrich)... 33,882-6 Acetonitrile, LC-MS CHROMASOLV (Riedel-de Haën)... 34967 Methanol, HPLC Grade (Aldrich)... 27,047-4 For a complete listing of all Sigma-Aldrich products, log on to our website: sigma-aldrich.com Did you know...? Due to the additional interactions available using polar reversedphase columns such as Discovery HS F5, the choice and concentration of buffer salts is critical to adequately control retention and separation compared to alkyl phases. For instance, the use of ammonium formate versus formic acid often results in improved peak shape and selectivity. 6 TheReporter Vol. 3, Issue
ARTICLE ARTICLE ARTICLE ARTICLE The Importance of Ion-Exchange Capacity in Mixed-Mode SPE An Trinh atrinh@sial.com Introduction Most researchers who use SPE to process biological samples rely on traditional single-mode retention mechanisms to recover, clean up, and concentrate their compounds of interest prior to LC or GC analysis. Although single-mode SPE chemistries are often adequate, problems can easily arise. For example, reversed-phase chemistries (e.g., C8) can result in insufficient cleanup. Although selectivity can be improved when using ion-exchange phases, the technology is often very sensitive to sample matrix effects. In this report, we will discuss the utility of mixed-mode SPE technology, and how ion-exchange capacity can play a critical role in many bio-analytical applications. How Mixed-Mode SPE Works Discovery DSC-MCAX (Mixed-Mode Cation Exchange) SPE utilizes the dual retention mechanisms of both hydrophobic and electrostatic interaction to retain basic, acidic, neutral, and zwitterionic compounds from aqueous sample matrices such as biological fluids. Its broad affinity for such a wide range of compounds is due to the dual bondings (octyl, C8 & benzene sulphonic acid, SCX) contained within the packed-bed. Although the technology was designed to drastically improve the selectivity/sample clean up of basic and zwitterionic compounds, DSC-MCAX is useful in other applications as well. Through the careful manipulation of ph and organic strength, the user can also fractionate basic and zwitterionic from acidic and neutral compounds, or adjust the protocol to permanently retain any unwanted basic and zwitterionic interfering components while eluting acidic and neutral compounds of interest. Table summarizes the generic protocol recommended for DSC-MCAX use. The Importance of Ion-Exchange Capacity Most bio-analytical applications provide analytical support for drug metabolism/ pharmacokinetic studies. In some cases, parent drug compounds can metabolize into very polar compounds that are difficult to retain via reversed-phase SPE. As a result, two different retention mechanisms are required to retain both parent drug and metabolite(s). Table. Recommended Generic Protocol for Discovery DSC-MCAX. Condition and equilibrate with methanol and 0mM potassium phosphate, ph 3-6 2. Load Sample 3. Wash off hydrophilic compounds/interferences with 0mM potassium phosphate, ph 3-6; and/or M acetic acid 4. Wash off hydrophobic compounds/interferences with methanol 5. Elute basic/zwitterionic compounds with 5% ammonium hydroxide in methanol In this application, piroxicam and piroxicam s highly polar metabolite, 2-aminopyridine, were spiked in human urine. Spiked samples were then extracted via Discovery DSC-MCAX using the recommended generic protocol, and analyzed via HPLC-UV. The results were then compared against two competitor mixed-cation phases run in parallel. Using the DSC-MCAX provided excellent clean up of biological samples such as urine (Figure A, Page 8). Although greater than 80% recovery was observed for piroxicam on all three mixed-cation phases, recovery suffered for 2-aminopyridine on the two competitor phases. Unlike piroxicam, 2-aminopyridine s polar nature required cation exchange to be the dominant mode of retention. Discovery DSC- MCAX s high ion-exchange capacity allowed for excellent retention of both piroxicam and its polar metabolite 2- aminopyridine. Conclusion The dual retention properties of Discovery DSC-MCAX provide a broad affinity for a wide range of compounds. By controlling ph and organic strength, the technology offers superior clean up and selectivity relative to most single-mode SPE phases. DSC-MCAX s high ion-exchange capacity also allows for the selective recovery of polar basic compounds. (article continued on page 8) For more information request the SPE Packet, T4004 (DUY). LC TheReporter Vol. 3, Issue 7
LC ordering: 800-247-6628 (US only) / 84-359-344 ordering: technical 800-247-6628 service: 800-359-304 (US only) / 84-359-344 (US only) / 84-359-304 technical service: 800-359-304 (US only) / 84-359-304 ARTICLE ARTICLE ARTICLE ARTICLE Figure A. Competitor Comparison of 2-Aminopyridine and Piroxicam from Human Urine SPE Tube: Discovery DSC-MCAX, 00mg/3mL Cat. No.: 52783-U HPLC Column: Discovery HS F5, 5cm x 4.6mm, 5µm particle size Cat. No.: 56756-U Mobile Phase: 0mM potassium phosphate, ph 6:MeCN (85:5) Flow Rate: 2mL/min T20400 GLC Temp.: 25 C Det.: UV, 220nm Inj. Vol.: 0µL. 2-Aminopyridine (4µg/mL spike) 2. Piroxicam (0µg/mL spike) Spiked Urine Samples Before DSC-MCAX G00220 Spiked Urine Samples After DSC-MCAX G002202 Efficiency of Absolute Recovery (n=4) % Recovery ± RSD 2-Aminopyridine Piroxicam Discovery DSC-MCAX 02 ± 3.5% 0 ±.2% Leading Competitor A 30 ± 52.5% 98 ± 3.2% Leading Competitor B 36 ± 24.2% 83 ± 4.3% sigma-aldrich.com/supelco Order/Customer Service 800-247-6628, 800-325-300 (US only) Fax 800-325-5052 (US only) E-mail supelco@sial.com Technical Service 800-359-304 (US only), 84-359-304 Fax 800-359-3044 (US only), 84-359-5468 E-mail techservice@sial.com SUPELCO 595 North Harrison Road, Bellefonte, PA 6823-0048 84-359-344 2 2 (continued from page 7) RELATED PRODUCTS Cat. No. 2-Aminopyridine (Aldrich)... A2880 Piroxicam... P5654 Potassium Phosphate, Monobasic... P0662 Acetonitrile, HPLC grade... 27,07-7 For a complete listing of all Sigma-Aldrich products, log on to our website: sigma-aldrich.com Did you know...? Strict ph control is critical when conducting ion-exchange SPE (e.g., mixed-mode, strong cation, weak anion, etc.). For example, when dealing with basic compounds, reducing the sample ph during loading will ionize the compounds basic functional groups. When both sorbent and compound functional groups are in their ionized state, electrostatic interaction (compound retention) can occur. Conversely, increasing the ph during elution will neutralize the basic compounds functional groups disrupting the compound and sorbent s electrostatic interaction. Trademarks Freon, Teflon - E.I. du Pont de Nemours & Co., Inc. Carboxen, CHROMASOLV, Discovery, Fluorcol, OMI, Thermogreen - Sigma-Aldrich Co. SCOTTY - Scott Specialty Gases, Inc. We are committed to the success of our Customers, Employees and Shareholders through leadership in Life Science, High Technology and Service. The SIGMA-ALDRICH Family 2004 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. 8 TheReporter Vol. 3, Issue