Ultra-High-Pressure LC

Section II covers the latest trends in reducing sample preparation time, including direct sample infusion/injection and on-line solid phase extraction (SPE). In Section III, we focus on newer trends in stationary phases and how these phases hope to offer different selectivities compared to current CIS-based phases. Section IV briefly provides a few observations on how new detectors are increasing the versatility of HPLC. Finally, in Section V we examine monolithic columns, small particles packed in short columns, high-temperature LC, ultra high-pressure LC, and parallel injection techniques. 

A commercial HPLC system and columns capable of performing ultra high-pressure LC were recendy introduced at PITTCON 2004 (ACQUITY Ultra Performance LC System by Waters). This HPLC system was designed to take full advantage of the potential of novel, sub-2-micron particles to give scientists chromatographic run times that are up to 9 times shorter than current fast HPLC systems, up to 2 times better peak capacity or resolution, and 3 times better routine sensitivity. 

As described in more detail in Section 13.3.2, the main analytical techniques that are employed for metabonomic studies are based on nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The latter technique requires a preseparation of the metabolic components using either gas chromatography (GC) after chemical derivatization or liquid chromatography (LC), with the newer method of ultra-high-pressure LC (UPLC) being used increasingly. The use of capillary electrophoresis (CE) coupled to MS has also shown promise. Other more specialized techniques such as Fourier transform infrared spectroscopy and arrayed electrochemical detection have been used in some cases. 

Beside column dimension the size of stationary phase particles is a matter of recent progress. More traditional columns are packed with 3.0-5 pm particles enabling satisfying resolution and reasonable column back pressure of solvent suitable to be processed by conventional HPLC pumps. In contrast, sub 2-pm particles (e.g. 1.7 and 1.8 pm) as applied in rapid or fast LC or ultra high-performance LC (UHPLC) allow better resolved separations in shorter run times. Column back pressure (>12,000 psi) is remarkably high demanding more robust solvent pumps. 

The use of ultra-high pressure in LC was found to have beneficial effects on protein recovery [44]. By using pressures >1600 bar, protein recovery was enhanced and carry-over from run to run was reduced and in some cases eliminated. While the mechanism of recovery is not known, it was postulated that ultra-high pressures improve desorption from the stationary phase surface by causing partial unfolding or deaggregation of the proteins. 

Parallel, ultra-high-pressure, micro and nano-LC. Multi-dimensional, Lab-on-a-Chip... 

Low dispersion, ultra-high-pressure HPLC systems, multidimensional LC and parallel analysis... 

ICAT is an emerging technique for differential expression proteomics, and its full potential remains to be fully evaluated. Advances in sample fractionation at the protein level, sample fractionation at the peptide level, and improved data acquisition schemes, will all be required for the full potential of ICAT to be realized. New separation systems, such as ultra-high pressure nanoscale capillary LC will improve the peak capacity for ICAT... 

The use of ultra-high-pressure regimes and/or flow rates in the micro- or nanoliters per minute range imposes unique requirements on LC system hardware, such as pumps, valves, injectors, connecting tubing, column construction, stationary phases, column heating systems, detection flow cells, and the detector s data collecting speed. 

The use of LC with conventional LC columns (5 jm) is a common and well-established technique in food analysis. However, the limitations of the use of this type of column do not solve all analytical problems. This fact is especially critical in food metabolomics because of the complexity of food matrices and the high number of compounds of interest. Therefore, the use of ultra-high-pressure liquid chromatography (UHPLC) can overcome the limitations of conventional LC, providing... 

Most conventional pumps operate at pressures up to 400 bar, while some ultra-high pressure systems may operate at as high as 1000-1200bar. Ultrahigh-pressure LC (UPLC or UHPLC) requires special made columns and connections for the high pressures. The high pressures are a result of columns packed with very small particles. 

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