TABLE OF CONTENTS - BGB Analytik AG

LC and LC/MS
Column Selection
Column Configuration
The column sizes most often recommended for analytical method development are 4.6 x 150 mm
or 4.6 x 75 mm. If more resolution is needed, use a longer column, 4.6 x 250 mm or the same
size column with a smaller particle size. During method development, choose the column internal
diameter (e.g., 2.1, 3.0 mm) to accommodate additional application objectives (e.g., sensitivity,
solvent usage) or compatibility with certain instrument types (capillary, nano, or prep).
Silica Type and Bonded Phase
Silica Type
Agilent ZORBAX reversed phase columns use three different types of porous silica
microspheres, the original ZORBAX SIL, ZORBAX Rx-SIL and modified ZORBAX Rx-SIL.
ZORBAX Rx-SIL and modified ZORBAX Rx-SIL are highly purified and less acidic than the
original ZORBAX SIL. Less acidic silica means less potential for interaction between the
analyte and silanol groups on the silica surface, especially if the solutes are basic, and
contributes to improved peak shape. For new method development, we strongly recommend
using reversed-phase products based on modified ZORBAX Rx-SIL (Eclipse Plus) and ZORBAX
Rx-SIL (Eclipse, StableBond etc.). However, many excellent methods have been developed on
reversed phase columns based on ZORBAX SIL and we continue to manufacture these high
quality, reliable products.
Bonded Phase
A good first choice for bonded phase is C18 or C8. If the sample solutes of interest are not
adequately separated on these columns, CN and Phenyl columns may offer significant
differences in selectivity from the straight-chain alkyl phases to effect the separation.
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In general, larger solutes, such as proteins, are best separated on short-chain reversed-phase
columns (C3, CN) and peptides and small molecules are separated on longer-chain columns
(C8, C18). There are many cases, however, where this conventional wisdom does not apply.
For example, peptides can also be effectively separated using short-chain columns, and
hydrophobic peptides can show better recovery on longer-chain phases. Therefore, it is best
to initially select a phase in the middle of the hydrophobic spectrum (e.g., C8), then change to
a more hydrophobic phase or more hydrophilic phase depending on initial results and solubility
properties of your sample.