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addition to outstanding performance, Supelco is promoting very com- petitive prices, which start at $300.
BIOshell columns from Supelco are packed with core-shell particles for proteins and peptides. For peptides the core is 1.7 μm diam. The shell is 0.5 μm thick with a 160-Å pore. Surface chemistry is either nitrile or C18. For proteins the core is 3.0 μm with 0.2 μm shell. Pore size is 400 Å, surface chemistry is C4, Pmax
is 600 bar, and Tmax is 90–100 °C. BIOshell col-
umns offer about 50% higher peak capacity for peptides than is realized with similar columns packed with totally porous particles.
Columns for HPLC, UHPLC, and SFC The chemists at ES industries (West Berlin, NJ) have been busy ex- tending their line of stationary phases, particularly in fluorophases. FluoroSep-RP HILIC is a fluorinated diol phase showing preferential retention of polar halogenated analytes using HILIC mode. FluoroSep-RP XP is a fluorophenyl surface chemistry for aromatic halogenated analytes including isomers. FluoroSep-RP R XF has a perfluorinated alkyl surface chemistry designed for general-purpose columns. FluoroSep-RP Octyl has a perfluoro octyl group bonded to silica. Compared to C18 and C8, the perfluoro C8 is less hydrophobic with different selectivity. In favor- able cases, it provides isocratic separations when other phases require gradient elution. Epic HILIC-FL sub-2-μm is a fluorophase for UHPLC HILIC separations. It is especially favored for LC/MS since separations are achieved with higher organic content mobile phases.
ES also added several new stationary phases to its more traditional col- umn lines. GreenSep Basic 3 μm is a new basic phase for SFC. GreenSep Basic PFP 3 μm offers unique selectivity for SFC due to the pentafluoro surface chemistry, again for SFC.
Chromaga Chiral CCC from ES Industries is a new column packing for chiral separations with 3-chloro-4-methylphenylcarbamate and 3,5-di- chlorophenylcarbamate bonded to cellulose. This phase shows promise in separating previously unresolved mixtures.
MacroSep C4 columns from ES Industries have a 300-Å pore. They are designed for separation of biomolecules, including glycoproteins, hemo- globin variants, and membrane proteins.
Raptor™ columns
RPLC covers a wide range of applications, niches, and conditions. Restek’s (Bellefonte, PA) new Raptor SSP column line started with a bi- phenyl surface chemistry to the core-shell base. At Pittcon 2014, Restek added the ARC-18 phase, which bonds steric protection groups to the base of the C18 brush to increase stability at the extremes of low pH.
SEC columns for antibodies and aggregates For more than three decades, Tosoh’s SW series of columns have dominated the modern market for columns for steric exclusion chro- matography columns. Initially, the particle size was a nominal 10 μm or larger. A few years ago, Tosoh introduced columns packed with nominal 4 or 5 μm diameter. This year the company focused on monoclonal antibodies with 3- or 4-μm particles. The TSKgel UltraSW Aggregate, 3 μm, is designed for high-resolution separation of antibody multimers and aggregates. The TSKgel SuperSW mAB HTP, 4 μm, is designed for
high-throughput analysis of antibody monomer and dimer under UHPLC conditions. Run times are typically cut in half. TSKgel SuperSW mAB HR, 4 μm, is recommended for antibody monomers, dimers, and fragments under HPLC conditions. Tosoh has developed a pore control technology that gives a shallow molecular weight calibration curve in the region where antibody fragments to dimers elute.
CSH C18 columns
LC/MS of peptides with C18 columns often involves use of ion pairing agents such as trifluoroacetic acid (TFA), but TFA suppresses ionization with ESI interfaces. Waters has developed a charged-surface hybrid C18 (called CSH C18) that obviates the need for TFA or other ion pairing agents. One example was a peptide map of trastuzumab, a therapeutic antibody.
Zenix® SEC columns Sepax Technologies (Newark, DE) introduced Zenix SEC columns packed with 3-μm-diam porous particles with a choice of 100-, 150-, and 300-Å nominal pore diameter. Sepax developed a proprietary surface chemistry to reduce nonspecific adsorption with biological and indus- trial water-soluble polymers.
Applications-focused columns GlycanPac AXR-1 columns
Glycan structure of proteins is very complex and even more important since it affects the safety and efficacy of protein therapeutics. Until now, determining the glyco heterogeneity involved many steps with specific enzymes that differentially responded to glycan structure. This year, Thermo Fisher Scientific introduced Glycan Pac AXR-1 columns for HPLC separation of free glycans by sialylation number (from 0 to 5) plus high resolution of hetero forms within the sialylation cluster. Further, the mobile phases are MS compatible, which has not been true with other separation modes.
The stationary phase has a proprietary mixed-mode surface chemis- try (WAX and RPLC) bonded to nominal 1.9-μm or 3.0-μm-diam silica (175-Å pore diameter). With the 1.9-μm particles, a mixture of 135 gly- cans produced 73 peaks. MS detection was effective in resolving many coelutions. Most examples utilize a 150-mm-long column, but if more resolution is required, a 250 × 2.1 mm column is available. One example showed separation of 105 fluorescently labeled glycans in 70 min.
CarboPac SA 10-4 μm column Thermo’s Dionex CarboPac SA10-4 μm is designed for rapid assay of mono- and disaccharides in food, beverages, and biofuels. The speed comes from reducing the particle size from 6 to 4 μm. Both are composite phases. The stationary phase starts with a macroporous polystyrene- divinyl benzene copolymer that has 6 µm diam agglomerated with 55-nm microbead difunctional quaternary ammonium ion. Run time is usually less than 8 min. The column and mobile phase are compatible with the Dionex pulsed amperometric detector (above).
AMERICAN LABORATORY • 6 • SEPTEMBER 2014
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