An integrated gradient capillary HPLC/MS system incorporating photodiode array detection
Recently, the popularity of capillary separations has been
rapidly expanding, largely driven by the need to analyze increasingly
smaller sample amounts. Capillary HPLC (CLC) is an attractive choice
for these tasks, giving high resolution separations with flow rates and
sample capacity suited for facile interfacing to mass spectrometers.
CLC also has sufficient sample capacity to serve as a micropreparative
technique for subsequent offline analyses. The challenges of designing
an optimized CLC system, however, are numerous. Each component of the
system, (solvent delivery, autosampler, fluidic connections, detection
and interfacing) must be appropriately engineered. Component
integration is essential to limit extra-column bandbroadening. Here we
describe results obtained with an integrated CLC system consisting of a
binary solvent delivery system, autosampler and photodiode array (PDA)
detector suitable for analysis with columns 0.18 - 0.5 mm ID diameter.
Key improvements in flow control technology have been incorporated.
Each solvent is delivered by a pair of high resolution syringe pumps,
where one syringe refills and is brought up to system pressure during
the delivery cycle of the other. This allows for continuous, positive
displacement flow with excellent flow accuracy and reproducibility. The
solvent delivery system is thus capable of submicroliter per minute
flow rates without need for problematic stream splitting schemes.
Highly reproducible gradient separations are achieved with low
microliter per minute flow rates. Typical relative standard deviations
for retention time are less than 0.5%, even with rapid gradient
analysis. Tight control over system bandspread typically yields peak
volumes less than 5 microliters. A unique detector design with an
extended path flow cell permits very high sensitivity detection
(estimated detection limits less than 50 fmol for many peptides). Some
unique design features include a fiber optic based light path, in
conjunction with a flow cell design that provides for nearly
quantitative light throughput. The low flow rates permit easy
interfacing with an electrospray mass spectrometer without need for
sample splitting, thus maximizing sensitivity. Detection by mass
spectrometry has similar sensitivity to the PDA detector, and thus each
detector provides confirmation of the presence of components observed
by the other. Instrument performance will be illustrated with examples
of peptide and drug analysis.