Performance of HPLC/MS microchips in isocratic and gradient elution modes

Abstract

We analyzed the chromatographic performance of particle-packed, all-polyimide high-performance liquid chromatography/mass spectrometry (HPLC/MS) microchips in terms of their hydraulic permeabilities and separation efficiency under isocratic and gradient elution conditions. The separation channels of the chips (with ca 50 µm × 75 µm trapezoidal cross-section and a length of 43 mm) were slurry packed with either 3.5 or 5 µm spherical porous C18-silica particles. A custom-built holder enveloped the chip during packing to prevent channel deformation and delamination from high pressures. It is shown that the packing conditions significantly impact the packing density of the HPLC/MS chips, which determines their performance in both, isocratic and gradient elution modes. Even with steep solvent gradients, peak shape and chromatographic resolution for the densely packed HPLC/MS chips are much improved. Our data show that the analytical power of the HPLC/MS chip is limited by the quality of the chromatographic separation.

Improved particle‐packed HPLC/MS microchips for proteomic analysis

“…As a consequence, the number of identified proteins in complex biological samples should also increase. In the present study, which builds on (and in this regard complements) our previous work 12, 14, 22 we tested this hypothesis with a carefully selected batch of standard Agilent HPLC/MS chips (Fig. 1) by systematically varied packing conditions and particle sizes of the stationary phase.…”

“…The quality of the latter chip packings was thus comparable to those of the packed fused‐silica capillaries used in nano‐HPLC. Furthermore, in gradient elution separations of a simple pharmaceutical drug mixture, for example, a peak width decrease in ∼15% and a resolution increase in up to 33% were observed for chip packings with 5 μm particles at changing the packing conditions from 150–300 bar and ultrasound; with reduction of the particle size to 3.5 μm an additional decrease in the peak width of ∼15% and improved resolution up to 22% were achieved 22. The concomitantly reduced slope of the plate height curves toward higher linear flow velocities enabled the application of higher flow velocities without a significant loss of chromatographic resolution, i.e.…”

“…Steep gradients inevitably result in narrowed peaks and together with the resolving power of MS, especially MS/MS, the experimental conditions are often thought to compensate for a lack of packing quality. However, recently it was shown that even under these conditions peak shape and chromatographic resolution are much improved by dense chip packings 22: HPLC/MS‐chips packed with 5 μm particles at 150 bar without ultrasound had a bed porosity (interparticle void fraction) of 0.46 – which reflects a very loose packing – and a minimum isocratic reduced plate height of 2.9, whereas HPLC/MS‐chips packed with 5 or 3.5 μm particles at 300 bar with ultrasound had bed porosities of 0.41 and 0.39, respectively, and a reduced plate height of 2.1. The quality of the latter chip packings was thus comparable to those of the packed fused‐silica capillaries used in nano‐HPLC.…”

High-performance liquid chromatography on glass chips using precisely defined porous polymer monoliths as particle retaining elements

“…In this context the most common bead trapping approach is tapering of the channel to provoke particle stacking either by use of the key stone effect [9] or by creating channel segments smaller than the bead size [10]. A wide range of differently shaped retaining elements was investigated for this purpose such as multi-channel types (frit-like) [3,[11][12][13][14][15][16][17] or a confined single channel appearing in form of a weir [10,18,19], a step [7] or a smooth taper [9,20]. Particulate chromatographic beds in microfluidic devices are generated by slurry packing against these structures.…”

Polydimethylsiloxane microchannel coupled to surface acoustic wave nebulization mass spectrometry

“…In the case of LC/MS, microfluidics has facilitated the integration of pre-columns for enrichment, chromatographic columns for separation, and nanospray tip for ionization, with all components integrated onto a single chip. [12,13] This has reduced much of the fluidic dead volumes found in the junction interconnects and improved detection of lower abundance samples. Integration of microfluidic devices to MS has increased the need for robust microfabricated electrospray emitters on-chip.…”