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HICHROMChromatography Columns and Supplies
Hichrom Limited1 The Markham Centre, Station RoadTheale, Reading, Berks, RG7 4PE, UK
The Inertsil® 4 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 3 illustrates the high sensitivity LC-MS/MS separation of haloacetic acids, harmful substances generated when tap water is chlorinated.
Inertsil ODS-4 and C8-4 columns also show excellent stability during the analysis of strong metal chelating compounds. Figure 4 shows the separation of tetracyclines, veterinary antibiotics with very strong chelating characteristics.
The 3μm phases are also available in High Pressure (HP) format. Please enquire for details.
Please contact Hichrom for current prices. Tel: +44 (0)118 930 3660 Email: [email protected]
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• High purity base silica (99.999%)• Large surface area• Exceptional stability and column lifetime• Validated ODS-3 column
Compared with Inertsil® 2 Series, Inertsil 3 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.
Phase Endcapped Particle Size (μm) Pore Size (Å)Surface Area
(m2/g)Carbon Load (%)
Recommended pH Range*
SIL - 3, 5 100 450 - 2 - 7.5
ODS-3 Yes 2, 3, 4, 5, 10 100 450 15 2 - 7.5
ODS-3V Yes 3, 5 100 450 15 2 - 7.5
ODS-P No 3, 5 100 450 29 2 - 7.5
ODS-EP No 5 100 450 9 2 - 7.5
Peptide C18 Yes 5 100 450 15 2 - 7.5
ODS-Sprint Yes 3, 5 100 450 8.5 2 - 7.5
Sulfa C18 Yes 3, 5 100 450 15 2 - 7.5
C8-3 Yes 2, 3, 5 100 450 9 2 - 7.5
Ph-3 No 2, 3, 5 100 450 9.5 2 - 7.5
CN-3 No 3, 5 100 450 14 2 - 7.5
NH2 No 3, 5 100 450 8 2 - 7.5
Diol No 3, 5 100 450 22 2 - 7.5
HILIC No 3, 5 100 450 20 2 - 7.5
Amide No 3, 5 100 450 18 2 - 7.5
AX No 5 100 450 17 2 - 7.5
CX No 5 100 450 14 2 - 7.5
Inertsil 3 Series Phases
*Inertsil phases are known to provide excellent results at pH levels of 9 – 10. However, optimum column life may be achieved at a pH between 2 and 7.5.
Inertsil ODS-3Peak 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-3 must pass a QC test using 29Si 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-3 (see Figure 5).
Inertsil ODS-3V – a validated Inertsil columnInertsil ODS-3V 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:
1) the absence of surface silanols2) minimal metallic residues3) inertness to basic compounds4) reproducible separations
Figure 5. Measurement of residual silanol groups by 29Si NMR
Figure 6. Separation of antidepressant drugs on Inertsil ODS-3V
Column dimensions: 250 x 4.6mm Flow rate: 1ml/min.Eluent: CH
• Polymerically bonded C18• Improved separation of planar compounds• Useful for carotenoid analysis• Separation of the 16 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-3. 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.
Inertsil® ODS-P
• C18 with polar embedded group• Compatible with 100% aqueous eluents• Alternative selectivity to C18• Ideal for LC-MS
Inertsil ODS-EP
Inertsil ODS-EP contains a polar functional group embedded between the silica surface and the C18 group. This creates a stable phase, which is resistant to phase collapse (or pore dewetting) in 100% 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 C18 phase.
*The comparative separations presented here may not be representative for all applications.
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• Balanced retention of polar and non-polar compounds• Compatible with 100% 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 10 the hydrophobic compounds are eluted in approximately two-thirds the time of Inertsil ODS-3, 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.
Inertsil® ODS-Sprint
Inertsil Peptide C18The Inertsil Peptide C18 column is specifically suited to the analysis of peptide digests. Figure 11 shows the application of this column to the analysis of cytochrome C tryptic digest.
Inertsil Sulfa C18Inertsil Sulfa C18 (3 and 5μm) 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 C18 is tested for the effective separation of sulpha drugs, as shown in Figure 12.
Figure 10. Polar and non-polar mixture Figure 11. Analysis of cytochrome C tryptic digest on Inertsil Peptide C18
• 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 13 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 14).
Inertsil® HILIC
• Unique selectivity in both reversed-phase and normal-phase• Applications for amino acids, water-soluble vitamins and sugars
Inertsil Diol
The Inertsil Diol phase is a dihydroxypropyl bonded silica which can be used in both reversed-phase and normal-phase modes. Figure 15 shows the analysis of free amino acids on an Inertsil Diol column.
• Excellent for polar compounds in HILIC mode• Ideal for LC-MS/MS• Long column lifetime
Inertsil Amide
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 Sulfa C18 (3μm, 150 x 4.6mm)Eluent: A: CH
3CN – CH
3CO
2H - (777.66:3.15, w/w)
B: CH3CO
2H – H
2O (3.15:997, w/w)
100% B to 67% B in 33mins then to 10% B in 4minsFlow rate: 1ml/minTemperature: 40°CSample: 1ppm each
Figure 13. Analysis of oligosaccharides on Inertsil HILIC
• 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 16 shows the separation of nucleotides on Inertsil AX and Figure 17 shows the analysis of nucleic acid bases by cation-exchange chromatography on Inertsil CX.
• High purity 300Å silica phases• Excellent reproducibility• Good physical and chemical stability
Inertsil® Wide Pore Phases
The Inertsil® WP300 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 WP300-C8 and C18 show high separation efficiency for peptides and proteins of medium molecular size (recommended <5,000 Da for C18 phase and <20,000 Da for C8 phase). Inertsil WP300-C4 can elute hydrophobic proteins >5,000 Da with excellent peak shape due to the shielding effects of embedded polar groups. Figure 18 shows the separation of a polypeptide mixture on Inertsil WP300-C8.
Please contact Hichrom for ordering information for Inertsil wide pore phase columns.
Inertsil Wide Pore PhasesInertsil WP300 Phase
Bonding EndcappedParticle Size
(μm)Pore Size (Å)
Surface Area (m2/g)
Carbon Load (%)
pH Range
SIL - - 5 300 150 - 2 - 7.5
C4 Polar embedded butyl Yes 5 300 150 3 2 - 7.5
C8 Octyl Yes 5 300 150 8 2 - 7.5
C18 Octadecyl Yes 5 300 150 9 2 - 7.5
Diol Propyldiol No 5 300 150 9 2 - 7.5
Figure 18. Analysis of polypeptide mixture
Column: Inertsil WP300-C8 (150 x 4.6 mm)Eluent: A: H
2O (0.1% TFA)
B: CH3CN-H
2O (90:10) (0.1% TFA)
Gradient: A:B 80:20 to 40:60 over 25 minsTemperature: 30°C
• Excellent efficiency and column-to-column reproducibility• Easy installation• Easy coupling with MS
Capillary and Nano Columns
GL Sciences produce a range of capillary and nano columns for high sensitivity trace analyses. These columns are available for InertSustain, Inertsil 4 Series, Inertsil 3 Series and Inertsil wide pore materials as well as Titansphere products, which are designed specifically for applications involving phosphopeptide extractions (see page 118).
The Capillary EX columns are available in 0.3, 0.5 and 0.7mm i.d. and 50 or 150mm lengths. These columns are packed using glass-lined stainless steel tubing of 1/ 4˝ o.d. and come with stainless steel end fittings.
Capillary EX-Nano columns are available with 0.05, 0.075, 0.1 and 0.2mm i.d. with lengths of 50, 150 and 250mm. These columns are packed using fused silica tubing and inserted into 1/ 16˝ 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.
0 10 20 Mins
1
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4
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• Spherical porous silica• Excellent for acidic and basic molecules• Well referenced in the scientific literature
The well established Inertsil® 2 Series phases are based on high purity silica which is robust and shows high reproducibility from batch-to-batch and column-to-column.
Inertsil® 2 Series and Inertsil ODS
Inertsil Phase Functional Group Endcapped Particle Size (μm) Pore Size (Å)Surface Area
(m2/g)Carbon Load (%)
ODS-2 Octadecyl Yes 5 150 320 18.5
C8 Octyl Yes 5 150 320 10.5
C4 Butyl Yes 5 150 320 7.5
Phenyl Phenyl Yes 5 150 320 10
ODS* Octadecyl Yes 5 100 350 14
Inertsil 2 Series and Inertsil ODS Phases
* From original Inertsil series
Ordering Information – Hichrom Manufactured ColumnsMicrobore (1.0mm i.d.) Columns
1 Other column dimensions available – please enquire 2 Use with free-standing holder HI-161 (£45) and column coupler HI-081 (£26) – see p. 24 3 Starter kits (£145) also available – see p. 25
Semi-Preparative and Preparative (7.75 – 21.2mm i.d.) ColumnsPlease contact Hichrom for further details of 7.75 – 21.2mm i.d. Inertsil columns.
Please contact Hichrom for current prices. Tel: +44 (0)118 930 3660 Email: [email protected]