Introduction Polymeric monolithic
stationary phases offer an alternative to the classical microparticulate
sorbents, bringing important advantages to sample analysis. In contrast
to the traditional stationary phases that consist of packed particles,
the monolithic separation medium is made of a continuous, rigid
polymeric rod with a porous structure. The lack of intraparticular void
volume improves mass transfer and separation efficiency. Monolithic
nanocolumns, like their 200-µm i.d. capillary counterparts, allow for
extremely fast separations of proteins and peptides.
Figure 1. A Monolithic nanocolumn
in protective housing, 100-µm i.d. × 5 cm.
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Instrumentation
All experiments were performed on the UltiMate™ Plus Nano and
Capillary LC System equipped with a microchip flow sensor for flow
adjustment and a 3-nL ultraviolet (UV) flow cell. The cytochrome c
digest was injected using a FAMOS™ Micro Autosampler. The Monolithic
nanocolumn, 100-µm i.d. by 5 cm (Figure 1), made of polystyrene-divinylbenzene
polymer (PS-DVB), was thermostatted at 60 °C using the UltiMate column
oven. UV detection was performed at 214 nm. The peptides were eluted
with a fast gradient at a flow rate of 1 µL/min.
Figure 2. Capillary LC separation
of cytochrome c sample (800 fmol/µL). Injected amount was 0.3
µL.
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Fast LC Analysis
Figure 2 shows the fast separation of a cytochrome c digest
sample. A gradient from 0 to 45% acetonitrile in acidified water (0.04%
TFA) is performed in 8 min., resulting in a fast separation of each
peptide with baseline resolution. Figure 2 demonstrates the good
selectivity of the polymeric bed. The mobile phase composition is
ideally suited for direct coupling to electrospray ionization mass
spectrometry (ESI-MS).
Experimental Conditions
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Monolithic columns allow for the
separation of proteins with widely differing properties ranging from
small and hydrophilic (cytochrome c) to large and rather
hydrophobic proteins (not shown). By extension of the gradient, the
separation of tryptic peptides and (hydrophobic) proteins is possible in
the same run.
The major advantage of the
monolithic material, based on the PS/DVB copolymer, is the excellent
chromatographic separation efficiency for proteins and peptides. This
technology allows for a high peak capacity in a short analysis time.
Fast and high-resolution separations make these columns ideally suited
for on-line coupling to ESI-MS applications in proteomics.
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