Rapid peptide separation by supercritical fluid chromatography.

BACKGROUND: Supercritical fluid chromatography (SFC) is continually gaining attention in the separation sciences as demand increases for higher throughput isolations and purifications. The higher flow rates associated with SFC provide a significant decrease in analysis time and increase in sample-throughput efficiency. Peptides are of particular interest for SFC due, in part, to the rather extensive analysis time required by HPLC. RESULTS: This work explored a wide range of peptides not only for detection, but also for separation using SFC. A separation of five peptides ranging in molecular weights from 238.2 to 1046.2 was achieved by SFC in less than 12 min, compared with 50 min using HPLC. CONCLUSION: This research further illustrates the ever-expanding applicability of SFC to a wider variety of compound classes. The rapid analysis time associated with SFC, as seen in this work, provides a nearly fivefold decrease in analysis time when compared with HPLC.

Ionisation in the absence of high voltage using supercritical fluid chromatography: a possible route to increased signal.

Supercritical fluid chromatography (SFC) is fast becoming a technique of choice for the analysis of a wide range of compounds and has been found to be complementary to high-performance liquid chromatography (HPLC). The combination of SFC and mass spectrometry (MS) affords a very useful tool in the separation and analysis of compounds. In this study the ionisation of samples in the absence of an applied electrospray voltage has been observed when using SFC/MS, with some compounds showing increased sensitivity when all ionisation source high voltage (HV) is removed. In an attempt to understand the mechanism of ionisation, a series of test compounds were analysed using standard electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) source configurations and also different API source designs. In both cases, data were acquired with the applied high voltage on (normal conditions) or with the high voltage off, i.e. no voltage spray (novo-spray). The standards were analysed with a range of pressure and modifier percentage conditions. To understand the nature of the ionisation process observed, this was compared with three established liquid-to-gas ionisation mechanisms. These were thermospray (TSP), charge residue model (CRM) of ESI and sonic spray ionisation (SSI). Experiments were undertaken in an attempt to explain this ionisation phenomenon and quantify any observed change in sensitivity. The most important point to note is that enhanced ionisation was observed under novo-spray conditions in a SFC/MS configuration, which in certain cases provides a lowering in the overall limit of detection (LOD).