Fast LC separation of a myoglobin digest: a case study using monolithic and particulate RP 18 silica capillary...

A method was developed for the fast separation of a myoglobin digest using a monolithic RP 18 silica capillary column of 100 microm I.D. The results were compared with those obtained with a particulate RP 18 silica capillary column of 100 microm I.D. at a flow-rate between 0.6 and 1.2 microl/min. The digest was analyzed at the monolithic column at a flow-rate up to 2.8 microl/min. This high flow-rate could not be applied to the particulate column due to the high back-pressure. When the starting composition of the gradient was changed from 0 to 20% and a gradient steepness of 16%/min was used, the analysis time was less than 4 min. A positive Mascot identification score of 115 was achieved for the MS-MS data. When a lower gradient steepness was employed, the chromatographic resolution and the peak capacity did not increase for most compounds. The intraday repeatability for the retention time of the monolithic column was better than 1.5% at 2.8 microl/min and even less than 0.5% using a flow-rate of 0.6 or 1.0 microl/min. For the particulate column, it was between 0.5 and 1.4% for a flow-rate of 0.6 microl/min, probably due to the high column back-pressure. The interday reproducibility for the retention time of the monolithic column was less than 0.9% using a flow-rate of 1.0 microl/min.