BioHPLC Column Selection Guide Cover - Agilent Technologies

Protein Column Selection Guide
Application Technique Agilent Columns Notes
Primary structure
analysis
Aggregation
analysis
Charge variant
analysis
UHPLC/HPLC
reversed-phase
separations
Size exclusion
separations
Ion-exchange
separations
Biomolecule Separations
The environment of the protein can influence, stabilize, or disrupt the structure of the protein. Factors
to consider include pH, temperature, salt concentrations, aqueous or organic solvent content, and for
some proteins, the presence of a stabilizing small molecule or metal ion. Protein structure can also be
disrupted by the use of sulfhydryl reducing agents to break -S-S- bonds or chaotropic agent, like urea
or guanidine HCL. With the complexity of proteins and the intramolecular interactions that determine
the three-dimensional structure, you can also expect that there will be intermolecular associations
between protein molecules and other molecular entities and the surfaces with which they come into
contact. This can result in protein complexes, aggregation (with possible precipitation), and
deposition on surfaces, including those of the HPLC column and system. Therefore, you should
consider the handling and environment in which the protein is maintained.
ZORBAX 300Å
Poroshell 300Å
ZORBAX 300Extend-C18
PLRP-S
Bio SEC-3
Bio SEC-5
ProSEC 300S
ZORBAX GFC
Agilent Bio IEX
Agilent Bio MAb
PL-SAX
PL-SCX
Reversed-phase separations require (or cause) denaturing of the protein to
obtain detailed information about the amino acid sequence and/or amino acid
modifications (including post-translational modifications).
Aggregates in protein biopharmaceuticals are of major concern as they can
induce an immunogenic response and can influence the composition of
the final formulation.
The ratio of individual amino acids determines the net charge of the protein
molecule. The pH at which the net charge is zero is called the isoelectric point (pI).
When the solution pH is less than the pI, the protein will be positively charged
(acidic), and when the solution pH is greater than the pI, the protein is negatively
charged (basic). For ion-exchange analysis, we recommend the eluent pH be
at least one pH unit away from its pI. Protein analysis using ion-exchange
columns requires buffered mobile phase and either salt gradients or pH
gradients for elution.
BioHPLC COLUMNS
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