HICHROM. Chromatography Columns and Supplies. LC COLUMNS Inertsil. Catalogue 9. Hichrom Limited

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1 HICHROM Chromatography Columns and Supplies LC COLUMNS Inertsil Catalogue 9 The Markham Centre, Station Road Theale, Reading, Berks, RG7 PE, UK Tel: + (0) Fax: + (0) sales@hichrom.co.uk

2 Inertsil Series Enhanced sensitivity High resolution Excellent for acidic, basic and chelating compounds Compatible with up to 00% aqueous eluents Inertsil Series Phases Phase Endcapped Particle Size (μm) Pore Size (Å) Surface Area (m /g) Carbon Load (%) ODS- and ODS-V Yes,, 00 0 C8- Yes,, 00 0 The Inertsil series is based on enhanced deactivation techniques which produce highly inert silica, enabling successful analysis of trace levels of the most demanding basic and acidic compounds. Strictly classified particle size, with narrow distribution width and an ideal carbon loading, enables high efficiency columns to be guaranteed. Figure illustrates the high sensitivity LC-MS/MS separation of haloacetic acids, harmful substances generated when tap water is chlorinated. Inertsil ODS- and C8- columns also show excellent stability during the analysis of strong metal chelating compounds. Figure shows the separation of tetracyclines, veterinary antibiotics with very strong chelating characteristics. The μm phases are also available in High Pressure (HP) format. Please enquire for details. Column: Inertsil ODS- (μm, 0 x.mm) Eluent: A: CH OH B: 0.% HCOOH Gradient: A/B = /9.min /9.min 9/ min 9/ Flow rate: 0.ml/min Temperature: 0 C 6 Detection: LC-MS/MS (ESI, negative, MRM). Bromoacetic acid. Dibromoacetic acid. Tribromoacetic acid. Chloroacetic acid. Dichloroacetic acid 6. Trichloroacetic acid 7. Bromochloroacetic acid Column: Inertsil ODS- (μm, 7 x.mm) Eluent: CH OH B: 0.% HCOOH Gradient: A/B = 0/80.min 0/80 min 9/.min 9/ Flow rate: 0.ml/min Temperature: 0 C Detection: LC-MS/MS (ESI, positive, MRM). Tetracycline (TC). Oxytetracycline (OTC). Chlortetracycline (CTC). Doxycycline (DC) Mins Mins Figure. Analysis of haloacetic acids by LC-MS/MS Figure. Analysis of tetracyclines by LC-MS/MS Ordering Information Inertsil Series Column Dimensions (mm) Guard Cartridges (/pk) 0 x. 0 x. 7 x. 00 x. 0 x. 0 x. (0 x.mm) μm ODS C μm ODS C ODS-V μm ODS C Column Dimensions (mm) Guard Cartridges (/pk) 0 x.6 0 x.6 7 x.6 00 x.6 0 x.6 0 x.6 (0 x.0mm) μm ODS C ODS-V μm ODS C ODS-V Other dimensions available Use with guard cartridge holder ( 98) sales@hichrom.co.uk Tel:

3 Inertsil Series High purity base silica (99.999%) Large surface area Exceptional stability and column lifetime Validated ODS- column Compared with Inertsil Series, Inertsil Series phases are based on a purer, higher surface area silica which is specially manufactured to provide maximum phase coverage. The result is a series of columns which provide excellent peak shapes using simple eluents while operating at low pressure. Inertsil Series Phases Phase Endcapped Particle Size (μm) Pore Size (Å) Surface Area (m /g) Carbon Load (%) Recommended ph Range* SIL -, ODS- Yes,,,, ODS-V Yes, ODS-P No, ODS-EP No Peptide C8 Yes ODS-Sprint Yes, Sulfa C8 Yes, C8- Yes,, Ph- No,, CN- No, NH No, Diol No, HILIC No, Amide No, AX No CX No *Inertsil phases are known to provide excellent results at ph levels of 9 0. However, optimum column life may be achieved at a ph between and 7.. Inertsil ODS- Peak tailing of basic components in reversed-phase is frequently the result of residual silanol groups. GL Sciences developed a proprietary bonding endcapping technology to eliminate these silanols. To ensure maximum inertness, each batch of Inertsil ODS- must pass a QC test using 9 Si NMR. After ODS bonding, the magnitude of the isolated silanols is greatly decreased. After endcapping, the isolated silanols have been completely eliminated, thereby assuring excellent inertness and optimum performance of ODS- (see Figure ). Isolated silanol Si-OH Silica-gel structure Si-(O-Si) ODS group C 8 H 7 -Si-O- Remaining silanol Endcapped groups (CH ) -Si-O- Geminal silanol Si-(OH) For free technical advice and support technical@hichrom.co.uk Bare silica gel support After ODS bonding After ODS bonding and endcapping Figure. Measurement of residual silanol groups by 9 Si NMR Inertsil ODS-V a validated Inertsil column Inertsil ODS-V is a highly efficient, validated HPLC column, developed to provide optimum separation for a wide range of acidic and basic compounds. Each lot of packing undergoes a series of critical QC analytical tests, consistent with the demands of GLP/GMP compliance, to confirm: ) the absence of surface silanols ) minimal metallic residues ) inertness to basic compounds ) reproducible separations. Norephedrine. Nortriptyline. Toluene. Imipramine. Amitriptyline Column dimensions: 0 x.6mm Flow rate: ml/min. Eluent: CH OH - mm KH PO, ph 6 (60:0) Temperature: C Figure 6. Separation of antidepressant drugs on Inertsil ODS-V Mins 08

4 Inertsil Series (continued) Inertsil ODS-P Polymerically bonded C8 Improved separation of planar compounds Useful for carotenoid analysis Separation of the 6 EPA PAHs Inertsil ODS-P is a polymerically bonded phase which is densely bonded with octadecylsilane groups. It shows unique separation capabilities and offers an alternative selectivity to Inertsil ODS-. Inertsil ODS-P is particularly suited to the separation of polycyclic aromatic hydrocarbons and shows additional retentivity for planar compounds. Figure 7 shows the separation of terphenyl isomers on three types of ODS phases. On the polymeric Inertsil ODS-P phase, the planar p-terphenyl is retained longer than the non-planar o- and m- isomers. Figure 8 shows the separation of components from toad venom on the ODS-P phase. o-terphenyl (non-planar) m-terphenyl (non-planar) p-terphenyl (planar) m- m- m- Column dimensions: 0 x.6mm Eluent: CH CN - H O (8:) Flow rate: ml/min Temperature: 0 C Detection: UV, nm o- p- o- p- o- p Mins Monomeric (Inertsil ODS-) Intermediate (Inertsil ODS-) Polymeric (Inertsil ODS-P) Figure 7. Separation of terphenyl isomers Column: Inertsil ODS-P (0 x.6mm) Eluent: CH CN - 0.% H PO (:) Flow rate: ml/min Temperature: 0 C. Bufalin. Resibufogenin. Cinobufagin. Indomethacin Mins Figure 8. Separation of toad venom Inertsil ODS-EP C8 with polar embedded group Compatible with 00% aqueous eluents Alternative selectivity to C8 Ideal for LC-MS Inertsil ODS-EP Uracil Column dimensions: 0 x.6mm Eluent: CH CN - H O (70:0) Flow rate: ml/min Column temperature: 0 C Figure 9. Comparison of Inertsil ODS-EP and C8 columns* *The comparative separations presented here may not be representative for all applications. Inertsil ODS-EP contains a polar functional group embedded between the silica surface and the C8 group. This creates a stable phase, which is resistant to phase collapse (or pore dewetting) in 00% aqueous eluents. It provides highly reproducible separations for acids and bases in organic eluents as well as acidified eluents typically used in LC-MS. Figure 9 shows a comparison of the selectivity of Inertsil ODS-EP with that of a C8 phase. Ethylbenzene Naphthalene Propylbenzene Butylbenzene Anthracene Uracil Naphthalene Ethylbenzene Propylbenzene Alkyl-C8 Anthracene Butylbenzene sales@hichrom.co.uk Tel:

5 Inertsil Series (continued) Inertsil ODS-Sprint Balanced retention of polar and non-polar compounds Compatible with 00% aqueous eluents Low back pressure Inertsil ODS-Sprint is super base deactivated and optimally bonded to retain polar compounds without excessive retention of non-polar compounds. This phase shows excellent peak shapes for a wide range of polar analytes, including both basic and acidic compounds. In Figure 0 the hydrophobic compounds are eluted in approximately two-thirds the time of Inertsil ODS-, whilst maintaining retention of polar compounds. Inertsil ODS-Sprint operates at low back pressures, even at high flow rates and shows excellent robustness and reproducibility. Column: Inertsil ODS-Sprint and Inertsil ODS- Column dimensions: 0 x.6mm Eluent: CH OH - H O (80:0) Flow rate: ml/min Temperature: 0 C Inertsil ODS-Sprint 6 7. Uracil. Caffeine. Phenol. n-butylbenzene. o-terphenyl 6. n-amylbenzene 7. Triphenylene Column: Inertsil Peptide C8 (00 x.mm) Eluent: A: H O - CH CN (9:) + 0.0% TFA B: CH CN - H O (80:0) + 0.0% TFA Gradient: - 0% B in 0 mins Flow rate: 0.ml/min Sample: 60pmol cytochrome C tryptic digest 7 Inertsil ODS Mins Mins Figure 0. Polar and non-polar mixture Figure. Analysis of cytochrome C tryptic digest on Inertsil Peptide C8 Inertsil Peptide C8 The Inertsil Peptide C8 column is specifically suited to the analysis of peptide digests. Figure shows the application of this column to the analysis of cytochrome C tryptic digest. Inertsil Sulfa C8 Inertsil Sulfa C8 ( and μm) is designed for the analysis of sulpha drugs. In addition, it is effective in the screening of synthetic antibacterial agents and residues in products of animal origin, as directed by food safety investigative guidelines. Each batch of Inertsil Sulfa C8 is tested for the effective separation of sulpha drugs, as shown in Figure. For free technical advice and support technical@hichrom.co.uk Figure. Analysis of sulfa reagents Mins 0 Column: Inertsil Sulfa C8 (µm, 0 x.6mm) Eluent: A: CH CN CH CO H - (777.66:., w/w) B: CH CO H H O (.:997, w/w) 00% B to 67% B in mins then to 0% B in mins Flow rate: ml/min Temperature: 0 C Sample: ppm each. Sulfanilamide. Sulfacetamide. Sulfadiazine. Sulfathiazole. Sulfapyridine 6. Sulfameradine 7. Sulfacymidine 8. Sulfamethoxypyridazine 9. Sulfamonomethoxine 0. Sulfachlorpyridazine. Sulfamethoxazole. Sulfabenzamide. Sulfadimethoxine. Sulfaquinoxaline. Sulfanitran 0

6 Inertsil Series (continued) Inertsil HILIC Unique selectivity for polar compounds High sensitivity LC-MS analyses Low back pressure Applications for amino acids, water-soluble vitamins and drug metabolites Inertsil HILIC is a propyl alcohol bonded silica phase, suitable for the analysis of polar compounds in the hydrophilic interaction (HILIC) mode. It has also shown promise in SFC applications. Unlike traditional reversed-phase materials, HILIC columns retain highly polar compounds with only small amounts of water in the eluent. The high organic solvent composition of the eluent will lead to high sensitivity LC-MS analyses, due to a decrease in ion suppression. It also results in low back pressure and the ability to run at high flow rates. Figure shows the separation of a mixture of oligosaccharides on Inertsil HILIC. Inertsil HILIC has also been used in the development of a fast and sensitive LC-MS/MS method for the analysis of melamine and cyanuric acid contaminants in pet food samples (see Figure ). Column: Inertsil HILIC (µm, 0 x.6mm) Eluent: CH CN - H O (6:) Flow rate: ml/min Temperature: 0 C. Maltose. Maltotriose. Maltotetraose. Maltopentaose. Maltohexaose 6. Maltoheptaose Column: Inertsil HILIC (0 x.0mm) Eluent: A: 0mM ammonium acetate in CH CN B: 0mM ammonium acetate in H O Gradient: T(mins) %A %B Flow rate: 0.ml/min Temperature: 0 C Detection: MS/MS (ESI) 6 Cyanuric Acid (Negative ESI) Melamine (Positive ESI) Mins Mins Figure. Analysis of oligosaccharides on Inertsil HILIC Figure. Analysis of melamine and cyanuric acid on Inertsil HILIC Inertsil Amide Excellent for polar compounds in HILIC mode Ideal for LC-MS/MS Long column lifetime Inertsil Amide is a carbamoyl bonded silica phase designed for the separation of highly polar compounds by HILIC. Inertsil Amide columns offer significantly increased retentivity and a unique selectivity in separating compounds that were unresolvable on other columns. Column: Inertsil Diol (0 x.6mm) Eluent: CH CN - H O (80:0) Flow rate: ml/min Column temperature: 0 C Inertsil Diol Unique selectivity in both reversed-phase and normal-phase Applications for amino acids, water-soluble vitamins and sugars 6. Phenylalanine. Methionine. Valine. Dopa. Alanine 6. Serine 7. Citrulline 7 The Inertsil Diol phase is a dihydroxypropyl bonded silica which can be used in both reversed-phase and normal-phase modes. Figure shows the analysis of free amino acids on an Inertsil Diol column. Figure. Analysis of free amino acids sales@hichrom.co.uk Tel:

7 Inertsil Series (continued) Inertsil Ion-Exchange Phases Silica based ion-exchange columns High reproducibility High efficiency Exceptional stability and long column lifetime Inertsil AX and CX are silica based anion- and cation-exchange phases respectively, based on high purity silica. They show good reproducibility, exceptional stability and long column lifetime. Figure 6 shows the separation of nucleotides on Inertsil AX and Figure 7 shows the analysis of nucleic acid bases by cation-exchange chromatography on Inertsil CX. Column: Inertsil AX (0 x.6mm) Eluent: 0.0M KH PO (ph.0 with H PO ) Flow rate: ml/min Temperature: 0 C N(CH CH ) C 6 H SO - H + AX bonded phase structure CX bonded phase structure Column: Inertsil CX (0 x.6mm) Eluent: 0.M ammonium formate, ph.0 Flow rate: ml/min Temperature: 0 C. CMP. AMP. UMP. GMP. XMP. Uracil. Thymine. Guanine. Cytosine. Adenine Mins Mins 0 Figure 6. Analysis of nucleotides on Inertsil AX Figure 7. Analysis of nucleic acid bases on Inertsil CX Ordering Information Inertsil Series Inertsil ODS- (Hichrom Manufactured Columns) Column Dimensions (mm) 00 x.6 0 x.6 0 x.6 ODS- μm INODS--00D 06 INODS--0D 9 INODS--0D 60 ODS- μm INODS-00D INODS-0D 7 INODS-0D 06 Please contact Hichrom for availability of other Series phases and alternative dimension columns. Microbore (.mm i.d.) Columns (GL Sciences Manufactured) Column Dimensions (mm) 0 x. 0 x. 7 x. 00 x. 0 x. μm C ODS Ph For free technical advice and support technical@hichrom.co.uk Column Dimensions (mm) Guard Cartridges (/pk) 0 x. 00 x. 0 x. 0 x. (For.mm i.d. Columns) μm C ODS ODS-P ODS-Sprint Ph CN NH Diol SIL HILIC Amide Other column dimensions available Use with free standing holder ( 98)

8 Ordering Information Inertsil Series (continued) Microbore (.mm i.d.) Columns (GL Sciences Manufactured) (continued) Column Dimensions (mm) 0 x. 00 x. 0 x. 0 x. Guard Cartridges (/pk) (For.mm i.d. Columns) μm ODS-V Sulfa C μm ODS μm C ODS ODS-P ODS-EP ODS-Sprint Ph CN NH Diol SIL HILIC Amide Sulfa C AX CX Peptide C Medium bore (.0mm i.d.) Columns (GL Sciences Manufactured) Column Dimensions (mm) 0 x.0 0 x.0 7 x.0 00 x.0 0 x.0 μm C ODS Ph Column Dimensions (mm) Guard Cartridges (/pk) 0 x.0 00 x.0 0 x.0 0 x.0 (For.0mm i.d. Columns) μm C ODS ODS-P ODS-Sprint Ph CN NH Diol SIL HILIC Amide ODS-V Sulfa C μm ODS Other column dimensions available Use with free standing holder ( 98) sales@hichrom.co.uk Tel:

9 For free technical advice and support Ordering Information Inertsil Series (continued) Medium bore (.0mm i.d.) Columns (GL Sciences Manufactured) (continued) Column Dimensions (mm) Guard Cartridges (/pk) 0 x.0 00 x.0 0 x.0 0 x.0 (For.0mm i.d. Columns) μm C ODS ODS-P ODS-EP ODS-Sprint Ph CN NH Diol SIL HILIC Amide ODS-V Sulfa C AX CX Peptide C Analytical (.6mm i.d.) Columns (GL Sciences Manufactured) Column Dimensions (mm) Guard Cartridges (/pk) 0 x.6 00 x.6 0 x.6 0 x.6 (For.6mm i.d. Columns) μm C ODS ODS-P ODS-Sprint Ph CN NH Diol SIL HILIC Amide ODS-V Sulfa C ( ) ( 6) ( 8) ( 09) μm ODS μm C ODS ODS-P ODS-EP ODS-Sprint Ph CN NH Diol SIL HILIC Amide ODS-V Sulfa C ( 6) AX CX Peptide C Other column dimensions available Use with free standing holder ( 98)

10 Inertsil Wide Pore Phases High purity 00Å silica phases Excellent reproducibility Good physical and chemical stability Inertsil Wide Pore Phases Inertsil WP00 Particle Size Surface Area Carbon Load Bonding Endcapped Pore Size (Å) Phase (μm) (m /g) (%) ph Range SIL C Polar embedded butyl Yes C8 Octyl Yes C8 Octadecyl Yes Diol Propyldiol No The Inertsil WP00 series of columns has been developed for the analysis and purification of proteins and peptides. It currently consists of five phases, which are available in column dimensions ranging from capillary to preparative. Inertsil WP00-C8 and C8 show high separation efficiency for peptides and proteins of medium molecular size (recommended <,000 Da for C8 phase and <0,000 Da for C8 phase). Inertsil WP00-C can elute hydrophobic proteins >,000 Da with excellent peak shape due to the shielding effects of embedded polar groups. Figure 8 shows the separation of a polypeptide mixture on Inertsil WP00-C8. Please contact Hichrom for ordering information for Inertsil wide pore phase columns. Column: Inertsil WP00-C8 (0 x.6 mm) Eluent: A: H O (0.% TFA) B: CH CN-H O (90:0) (0.% TFA) Gradient: A:B 80:0 to 0:60 over mins Temperature: 0 C. Ribonuclease. Insulin. Cytochrome. Lysozyme. BSA 6. Trypsin Inhibitor from Soybean 7. Ovalbumin Mins Figure 8. Analysis of polypeptide mixture Capillary and Nano Columns Excellent efficiency and column-to-column reproducibility Easy installation Easy coupling with MS GL Sciences produce a range of capillary and nano columns for high sensitivity trace analyses. These columns are available for InertSustain, Inertsil Series, Inertsil Series and Inertsil wide pore materials as well as Titansphere products, which are designed specifically for applications involving phosphopeptide extractions (see page 8). The Capillary EX columns are available in 0., 0. and 0.7mm i.d. and 0 or 0mm lengths. These columns are packed using glass-lined stainless steel tubing of / o.d. and come with stainless steel end fittings. Capillary EX-Nano columns are available with 0.0, 0.07, 0. and 0.mm i.d. with lengths of 0, 0 and 0mm. These columns are packed using fused silica tubing and inserted into / 6 o.d. PEEK tubing for protection, with stainless steel end fittings. Please contact Hichrom for ordering information for Inertsil Capillary EX and Capillary EX-Nano columns. sales@hichrom.co.uk Tel:

11 Inertsil Series and Inertsil ODS Spherical porous silica Excellent for acidic and basic molecules Well referenced in the scientific literature Inertsil Series and Inertsil ODS Phases Functional Group Endcapped Particle Size (μm) Pore Size (Å) Surface Area (m /g) Carbon Load (%) ODS- Octadecyl Yes C8 Octyl Yes C Butyl Yes Phenyl Phenyl Yes ODS* Octadecyl Yes 00 0 * From original Inertsil series The well established Inertsil Series phases are based on high purity silica which is robust and shows high reproducibility from batch-to-batch and column-to-column. Ordering Information Hichrom Manufactured Columns Microbore (.0mm i.d.) Columns Column Dimensions (mm) Guard Cartridges 0 x.0 00 x.0 0 x.0 0 x.0 (For.0mm i.d. Columns) ODS- INODS-0M 0 INODS-00M INODS-0M 0 INODS-0M 70 INODS-0CE (/pk) C INC-0M 70 INC-00M 90 INC-0M 00 INC-0M 0 INC-0CE (/pk) Phenyl INP-0M 0 INP-00M INP-0M 0 INP-0M 80 INP-0CE (/pk) Microbore (.mm i.d.) Columns Column Dimensions (mm) Guard Cartridges 0 x. 00 x. 0 x. 0 x. (For.mm i.d. Columns) ODS- INODS-0AM 8 INODS-00AM 00 INODS-0AM INODS-0AM INODS-0CM (/pk) C INC-0AM INC-00AM 60 INC-0AM 7 INC-0AM 0 INC-0CM (/pk) Phenyl INP-0AM 9 INP-00AM 0 INP-0AM INP-0AM INP-0CM (/pk) Medium Bore (.mm i.d.) Columns Column Dimensions (mm) 0 x. 00 x. 0 x. 0 x. Guard Cartridges (For.mm i.d. Columns) ODS- INODS-0AS 60 INODS-00AS 7 INODS-0AS 90 INODS-0AS 0 INODS-0C (/pk) C INC-0AS 0 INC-00AS INC-0AS 0 INC-0AS 80 INC-0C (/pk) Phenyl INP-0AS 70 INP-00AS 8 INP-0AS 00 INP-0AS 0 INP-0C (/pk) Analytical (.0mm i.d.) Columns For free technical advice and support technical@hichrom.co.uk Column Dimensions (mm) Guard Cartridges 0 x.0 00 x.0 0 x.0 0 x.0 (For.0mm i.d. Columns) ODS- INODS-0AF 60 INODS-00AF 7 INODS-0AF 90 INODS-0AF 0 INODS-0C (/pk) C INC-0AF 0 INC-00AF INC-0AF 0 INC-0AF 80 INC-0C (/pk) Phenyl INP-0AF 70 INP-00AF 8 INP-0AF 00 INP-0AF 0 INP-0C (/pk) Analytical (.6mm i.d.) Columns Column Dimensions (mm) Guard Cartridges 0 x.6 00 x.6 0 x.6 0 x.6 (For.6mm i.d. Columns) ODS- INODS-0A 60 INODS-00A 7 INODS-0A 90 INODS-0A 0 INODS-0C (/pk) C INC-0A 0 INC-00A INC-0A 0 INC-0A 80 INC-0C (/pk) Phenyl INP-0A 70 INP-00A 8 INP-0A 00 INP-0A 0 INP-0C (/pk) Other column dimensions available please enquire Use with free-standing holder HI-6 ( 60) and column coupler HI-08 ( 0) see p. Starter kits ( ) also available see p. Semi-Preparative and Preparative (7.7.mm i.d.) Columns Please contact Hichrom for further details of 7.7.mm i.d. Inertsil columns. 6

12 Ordering Information GL Sciences Manufactured Columns (Inertsil Series and Inertsil ODS) Microbore (.mm i.d.) Columns Column Dimensions (mm) Inertsil μm Phase 0 x. 0 x. Guard Cartridges (/pk) (For.mm i.d. Columns) ODS C Phenyl C ODS Medium Bore (.0mm i.d.) Columns Inertsil μm Phase Column Dimensions (mm) Guard Cartridges (/pk) 0 x.0 0 x.0 (For.0mm i.d. Columns) ODS C Phenyl C ODS Analytical (.0 and.6mm i.d.) Columns Inertsil μm Phase Column Dimensions (mm) Guard Cartridges (/pk) 0 x.0 0 x.0 0 x.6 0 x.6 (For.0 and.6mm i.d. Columns) ODS C Phenyl C ODS Other column dimensions available Use with free-standing holder ( 98) sales@hichrom.co.uk Tel: