HPLC Analysis of Biomolecules Technical Guide - Interscience

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HPLC Analysis of Biomolecules
RP-LC/MS of Biomolecules
HPLC with mass spectrometric detection (LC/MS) is fast becoming
the method of choice for many biomolecule analyses. A full discussion
of LC/MS, the instruments, ionization techniques, and interfaces, is
beyond the scope of this guide. However, the focus of this guide, the
HPLC column, is more than a MS filter. The chemistry of the particle
and the configuration of the hardware significantly affect the relevance
and reliability of the data that is obtained from the LC/MS instrument.
In addition to the column, the mobile phase conditions have a profound
influence on the quality of LC/MS data. Mass spectrometric detection
has high compound specificity and selectivity which lessens the requirements
for high selectivity separations; however, it brings limitations
to the method development process. For instance, the choice of mobile
phase composition is restricted since mobile phase needs to balance
the chromatographic requirements with the ionization efficiency.
Figure 23: LC/MS method selection guidelines
Column Selection for RP-LC/MS: Column ID
Usually the aim of an LC/MS experiment is to maximize sensitivity;
to be able to see compounds that exist at very low concentration in
the sample or that may have adequate concentration but are not
easily detected (ionized) by the MS. When samples are limited, as
in most proteomics applications, it is essential that maximum LC/MS
information is obtained from the smallest injection volume. To
increase sensitivity, the concentration of the compound entering the
ionization source must be maximized. The more concentrated the
analyte, the stronger the signal and the higher the sensitivity. Using
concentration to increase sensitivity is especially important for
compounds that are not readily ionizable. The ID (internal diameter) of
the HPLC column affects the concentration of the sample. Samples
are diluted in proportion to the cross-sectional area of the column.
Therefore, smaller ID columns yield less dilution. Sensitive LC/MS
analyses are typically run on very small ID columns or capillaries.
To demonstrate the effect of column ID on sensitivity, Figure 22
shows the same test solution (same injection volume and mass) on
three columns with decreasing IDs (1 mm, 0.5 mm and 0.32 mm).
The flow rate was adjusted to obtain the same linear velocity and
retention time on each column. The improvement in signal-to-noise
ratio obtained with the smaller ID columns is obvious from the
normalized chromatograms.
The ideal flow rate in LC/MS varies depending on the ion
source type and design. Typical flow rate ranges and the column ID
that will be required to match it are shown in Figure 23.
Figure 22: Effect of column ID on sensitivity