Agilent HPLC Column Selection Guide

Columns for Bioanalytical ChromatographyZORBAX Strategy for Reversed-PhaseMethod Development of Proteins and PeptidesThis ZORBAX column selection strategy for proteins and peptides provides some critical details on method development for proteins orpolypeptides. For small peptides, Molecular Weight (MW) < 2000, please follow the method development strategy for small and largemolecules in the reference section of this guide. For efficient separations of large molecules, columns with a wide-pore size (300Å) arerequired. For method development of larger peptides and proteins, review the suggested guidelines outlined below. Wide-pore columnchoices are described in the following section of this Column Selection Guide.Choose the Initial Column and Conditions for Proteins and PeptidesPeptides, Polypeptides, ProteinsMW < 50 kDAPeptides, Polypeptides, ProteinsMW < 1,000 kDAInitial Bonded Phase ChoiceStableBond 300SB-C8300 SB columns are wide-pore columns with unbeatable lifetime inTFA-containing mobile phases. This makes them an ideal first choicefor separations of peptides and proteins.• C8 is an excellent starting bonded phase because of its moderatehydrophobicity.• C18 and C8 are generally selected for peptides and protein digestsbut can also be used for proteins.• C3, C4 and CN are generally selected for larger, hydrophobicpolypeptides and proteins but can also be used for peptides.Poroshell 300SB-C18Poroshell 300SB columns use an innovative particle technology todeliver rapid protein separations. Short analysis times with efficientpeaks are easily obtained with Poroshell columns.• C18 is a good starting bonded phase choice with Poroshell formost peptides, polypeptides and proteins because the retention ismaximized.• C8 is generally selected for moderate size proteins but can be usedwith polypeptides or very large proteins.• C3 is generally selected for antibodies or large proteins but can beused for peptides and polypeptides.Initial Separation ConditionsColumn:Mobile Phase:Gradient:Temperature:Flow Rate:StableBond 300SB-C84.6 x 150 mm, 3.5 or 5 µm883995-906863973-906A: 95% H 2 O:5% ACN with 0.1% TFAB: 5% H 2 O:95% ACN with 0.085% TFA0-60% B in 60 min35-40°C1 mL/minColumn:Mobile Phase:Gradient:Temperature:Flow Rate:Poroshell 300SB-C182.1 x 75 mm, 5 µm660750-902A: 95% H 2 O:5% ACN with 0.1% TFAB: 5% H 2 O:95% ACN with 0.085% TFA0-60% B in 10 min35-40°C2 mL/min106 Order online at www.agilent.com/chem/store