BioHPLC Column Selection Guide Cover - Agilent Technologies

Method Development
Ionic Strength:
Certain ionic strength is required to sustain the function of columns. Usually, a minimal concentration of 10-20 mM salt is required. However,
greater than 20 mM strength may prevent the absorption of biomolecules onto the column. Commonly used salts are sodium and potassium
chloride and acetate. For elution, a typical salt concentration is 400-500 mM.
Note: Never use water alone for washing columns as it causes a significant increase in backpressure.
Selection of Buffers and pH:
Buffers play a key role in the optimization of separations. Phosphate buffers are typically
used for antibodies and many biomolecules. The following are also recommended:
MES, Tris, and ACES buffers. Use buffers of pH 5.0-6.5. pH can be adjusted usually
by +/- 0.2 units. For some specific proteins, buffers with higher pH (>pH 6.5) may
be needed. Phosphoric acid, acetic acid, HCl and NaOH can be used to adjust pH.
pH gradients can also be used for elution.
Optimize Conditions
Some separations may require a specific buffer, ionic strength, pH, and/or temperature
Additives
Organic Solvents:
Acetonitrile, ethanol, methanol, and other similar
solvents can be used up to 50%.
Detergents:
Non-ionic, anionic, and zwitterionic detergents
can be used. Cationic detergents are not
recommended.
Temperature:
Selection of Buffers and pH:
For anion-exchange, acetate and phosphate
buffers of pH 8.0-9.0 are recommended. pH
can be adjusted usually by +/- 0.2 units. For
some specific proteins, buffers with higher or
lower pH may be needed. Phosphoric acid,
acetic acid, HCl and NaOH can be used to
adjust pH.
pH gradients can also be used for elution.
Additives
Organic Solvents:
Acetonitrile, ethanol, methanol, and other similar
solvents can be used up to 50%.
Detergents:
Non-ionic, cationic, and zwitterionic detergents
can be used. Anionic detergents are not
recommended.
Agilent Bio MAb and IEX columns are stable up to 80 ºC. However, many proteins and biomolecules are heat labile. Be sure to establish the
temperature stability of your sample before routinely using high temperature for separation.
UHPLC/HPLC
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