Improvement of the liquid-chromatographic analysis of protein tryptic digests by the use of long-capillary monolithic columns with UV and MS detection

Optimisation of peak capacity is an important strategy in gradient liquid chromatography (LC). This can be achieved by using either long columns or columns packed with small particles. Monolithic columns allow the use of long columns at relatively low back-pressure. The gain in peak capacity using long columns was evaluated by the separation of a tryptic bovine serum albumin digest with an LC–UV–mass spectrometry (MS) system and monolithic columns of different length (150 and 750 mm). Peak capacities were determined from UV chromatograms and MS/MS data were used for Mascot database searching. Analyses with a similar gradient slope for the two columns produced ratios of the peak capacities that were close to the expected value of the square root of the column length ratio. Peak capacities of the short column were 12.6 and 25.0 with 3 and 15 min gradients, respectively, and 29.7 and 41.0 for the long column with 15 and 75 min gradients, respectively. Protein identification scores were also higher for the long column, 641 and 750 for the 3- and 15-min gradients with the short column and 1,376 and 993 for the 15- and 75-min gradients with the long column. Thus, the use of long monolithic columns provides improved peptide separation and increased reliability of protein identification

Photoionisation detection in packed-capillary liquid and supercritical-fluid chromatography.

A photoionisation detector (PID) was coupled to packed-capillary liquid and supercritical-fluid chromatography to study its performance. Several mobile phases were tested to evaluate the potential of liquid chromatography with photoionisation detection, LC-PID. The behaviour of the PID was not as good as in gas chromatography (GC), due to the absorption of photons by the mobile phase vapour. Therefore, the minimum detection limits (MDLs) were high compared to those in GC-PID, being at the low nanogram level for, e.g., ketones, aldehydes and amides. Coupling of the PID with supercritical-fluid chromatography (SFC) using modified carbon dioxide gave more satisfactory results. For aromatic compounds like phenanthrene and pyrene MDLs were found to be 12 and 20 pg, respectively. These values were almost the same as found in GC-PID. Separation and detection of aliphatic C4-Q3 carboxylic acids and some organosulphur/phosphorus pesticides (disulfoton, ethion and sulfotepp) showed the possibility to detect several classes of compounds at the low nanogram level using methanol-modified carbon dioxide. © 1995, Taylor & Francis Group, LLC. All rights reserved

Monolithic and Small Particle Column Materials for Application in Proteomics

In this thesis the influence of the capillary liquid chromatography separation on the identification of protein digests is studied. In the first part the chromatographic parameters for silica monolithic columns are optimized to obtain a high throughput or a high separation performance. in the second part different column materials, monolithic and ultra-small particulate columns, are compared. In the last part trends and suggestions for future research are given