Methacrylate monolithic capillary columns for gradient peptide separations

Abstract

For the separation of peptides with gradient-elution liquid chromatography a poly(butyl methacrylate- co-ethylene dimethacrylate) (BMA) monolithic capillary column was prepared and tested. The conditional peak capacity was used as a metric for the performance of this column, which was compared with a capillary column packed with C18-modified silica particles. The retention of the peptides was found to be smaller on the BMA column than on the particulate C18 column. To obtain the same retention in isocratic elution an approximately 15% (v/v) lower acetonitrile concentration had to be used in the mobile phase. The retention window in gradient elution was correspondingly smaller with the BMA column. The relation between peak width and retention under gradient conditions was studied in detail. It was found that in shallow gradients, with gradient times of 30 min and more, the peak widths of the least retained compounds are strongly increased with the BMA column. This was attributed to the fact that these compounds migrate and elute with an unfavorable high retention factor. More retained compounds are eluted later in the gradient, but with a lower effective retention factor. With shallow gradients the peak capacity of the BMA column (≈90) was clearly lower than that of a conventional packed column (≈150). On the other hand, with steep gradients, when components elute with a low effective retention factor, the performance of the BMA column is relatively good. With a gradient time of 15 min similar peak widths and thus similar peak capacities (≈75) were found for the packed and the monolithic column. Two strategies were investigated to obtain higher peak capacities with methacrylate monolithic columns. The use of lauryl methacrylate (LMA) instead of butyl methacrylate (BMA) gave an increase in retention and narrower peaks for early eluting peptides. The peak capacity of the LMA column was ≈125 in a 60 min gradient. Another approach was to use a longer BMA column which resulted in a peak capacity of ≈135 could be obtained in 60 min.