Chromatography with Higher Pressure, Smaller Particles and Higher Temperature: A Bioanalytical Perspective

Abstract

Recent years have seen widespread use of ultra-high-pressure liquid chromatography (UHPLC) in biological fluids. Most commonly the emphasis is on developing high throughput assay methods to reduce the analysis time and cost. Particle size and temperature are chromatographic parameters that can be changed to improve efficiency and obtain rapid separations. UHPLC and high-temperature liquid chromatography (HTLC) are two techniques that reduce the analysis time by decreasing the size of the column packing material and increasing the column temperature, respectively. Both of these techniques have advantages and limitations. In this article we have summarized the history, theoretical background of UHPLC and HTLC and the various advantages and limitations of sample preparation techniques and the detection systems (mass spectrometry and ultraviolet) used for the bioanalytical assays. In addition, selected bioanalytical applications of the two techniques have been reviewed and tabulated.

A review of high performance liquid chromatographic-mass spectrometric urinary methods for anticancer drug exposure of health care workers

“…Reduced particle size and smaller column diameters generally decrease analyte dispersion, enhance peak resolution and allow for decreased sample loading. These improvements can be expected to increase the number of theoretical plates [54]. This in turn may increase sensitivity by increasing the signal-to-noise ratio of the detector based on use of the tandem mass spectrometer.…”

“…These improvements led to shorter columns, faster analysis times, and shorter equilibration periods [54]. As described earlier, Fabrizi et al, used improved separation media and a shorter column to create a multi-analyte method [41].…”

Mercapturic acids: recent advances in their determination by liquid chromatography/mass spectrometry and their use in toxicant metabolism studies and in occupational and environmental exposure studies

“…Automated sample preparation has become popular and two forms using SPE media have come into common use (Varma et al, 2010). Popular 96-multi-well sample plate format has been adapted to high-throughput SPE (Mallet et al, 2003).…”

“…Sub-2µm particles produce sharper peaks while decreasing sample loading over conventional HPLC. These improvements in column efficiency and linear velocity can be expected to increase the number of theoretical plates (Varma et al, 2010). This in turn may increase sensitivity by increasing the signal to noise ratio of the detector.…”

“…These fundamental aspects and practical requirements of UHPLC have been reviewed (Wu & Clausen, 2007). Application of UHPLC specifically to bioanalysis has been reviewed (Varma et al, 2010). UHPLC analysis of structurally similar mercapturates found in the urine of smokers (Wu et al, 2012, Pluym et al, 2015) and multiple mercapturates in workers exposed to multiple volatile organic chemicals (Alwis et al, 2012) will be considered in later sections of this review.…”