Agilent HPLC Column Selection Guide
Agilent HPLC Column Selection Guide Agilent HPLC Column Selection Guide
Columns for Bioanalytical ChromatographyStarting Column Choices for Analytical Separationsof Peptides, Polypeptides, and ProteinsLow pHMid and High pHMW < 50 kDa300SB-C84.6 x 150 mm, 3.5 µm863973-906MW < 1000 kDaPoroshell 300SB-C182.1 x 75 mm, 5 µm660750-902MW < 25 kDa300Extend-C184.6 x 150 mm, 3.5 µm763973-902Separations of Proteins and Peptides Using Reversed-Phase LC/MS MethodsLC/MS of proteins and peptides is used to provide information for protein characterization, to accurately identify post-translationalmodifications of proteins, and to determine the molecular weight of synthetic and natural peptides. LC/MS is used to provide proteinidentification in 2-D separations for proteomics applications. Therefore, LC/MS of proteins and peptides is a critical separation area, whichrequires some special column and mobile phase recommendations. In general, smaller column sizes are used for LC/MS and TFA is generallynot used in mobile phase because of reduced sensitivity in the MS with this mobile phase additive.Initial Column Choices for LC/MS Separations of Proteins and PolypeptidesAnalytical LC/MS Applications – 2.1 mm ID columns will provide good sensitivitywhen sample size is not limited. With Poroshell columns, smaller column IDs are used.Low pHMid and High pHMW < 50 kDa300SB-C82.1 x 150 mm, 3.5 µm863750-906MW < 1000 kDaPoroshell 300SB-C181.0 x 75 mm, 5 µm661750-902MW < 25 kDa300Extend-C182.1 x 150 mm, 3.5 µm763750-902High Sensitivity/Proteomics ApplicationsCapillary columns are used for high sensitivity protein and peptide applications. The 0.5 mm ID columns are used for proteinand protein digest separations while the 0.3 mm ID columns are most often used for protein digests. These can be analyzed at high pHwith an ammonium hydroxide mobile phase. Nano columns (0.1 and 0.075 mm ID) are often used in 2-D LC/MS systems for proteomicsand the initial choice is a C18 bonded phase.108 Order online at www.agilent.com/chem/store
Columns for Bioanalytical ChromatographyHigh Sensitivity Capillary ColumnsLow pHMid and High pHMW < 50 kDa300SB-C180.5 x 150 mm, 3.5 µm5064-8268MW < 1000 kDaPoroshell 300SB-C80.5 x 75 mm, 5 µm5065-4468MW < 25 kDa300Extend-C180.3 x 150 mm, 3.5 µm5065-4464Nano Columns for Proteomics/2-D LC/MSSCX (1st Dimension)0.3 x 35 mm, 5 µm5065-9912Reversed-Phase (2nd Dimension)0.075 x 150 mm, 3.5 µm5065-9911Mobile Phase ConsiderationsLow pHMid and High pHTFA is generally not used for LC/MS separations of proteinsand peptides. The first step is normally to replace TFA with0.1-1.0% formic acid. Acetic acid, up to 1%, can also be used asan alternative mobile phase modifier. At low pH, the best separationmay still be obtained with TFA in the mobile phase. In some cases,the TFA can be displaced post column with an alternate acid,such as propionic acid.LC/MS can also be done at high pH with 10-20 mM NH 4 OHas a mobile phase additive.Order online at www.agilent.com/chem/store109
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<strong>Column</strong>s for Bioanalytical ChromatographyHigh Sensitivity Capillary <strong>Column</strong>sLow pHMid and High pHMW < 50 kDa300SB-C180.5 x 150 mm, 3.5 µm5064-8268MW < 1000 kDaPoroshell 300SB-C80.5 x 75 mm, 5 µm5065-4468MW < 25 kDa300Extend-C180.3 x 150 mm, 3.5 µm5065-4464Nano <strong>Column</strong>s for Proteomics/2-D LC/MSSCX (1st Dimension)0.3 x 35 mm, 5 µm5065-9912Reversed-Phase (2nd Dimension)0.075 x 150 mm, 3.5 µm5065-9911Mobile Phase ConsiderationsLow pHMid and High pHTFA is generally not used for LC/MS separations of proteinsand peptides. The first step is normally to replace TFA with0.1-1.0% formic acid. Acetic acid, up to 1%, can also be used asan alternative mobile phase modifier. At low pH, the best separationmay still be obtained with TFA in the mobile phase. In some cases,the TFA can be displaced post column with an alternate acid,such as propionic acid.LC/MS can also be done at high pH with 10-20 mM NH 4 OHas a mobile phase additive.Order online at www.agilent.com/chem/store109
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