Comprehensive Off-Line, Two-Dimensional Liquid Chromatography. Application to the Separation of Peptide Digests

Abstract

The separation of the peptide digests of myoglobin and bovine serum albumin was performed with an off-line combination of two commercial, conventional HPLC columns. The first column was packed with a strong ion exchanger and eluted with a KCl gradient. The second column was packed with particles of C18-bonded silica and eluted with an acetonitrile gradient. The conditional peak capacities of the 2D separations achieved exceed 7000 under the experimental conditions investigated. This performance is achieved at the cost of an analysis time of the order of 28 hours. Possible improvements to the separation method described here are discussed.

Tryptic digest analysis by comprehensive reversed phase×two reversed phase liquid chromatography (RP‐LC×2RP‐LC) at different pH's

“…Next to the online approaches, various reports describe offline combinations [40,41]. SCX-LC separations are typically operated at low pH to promote the interaction of the peptides with the SCX resin, which conditions are ideal for coupling to RP-LC separations and subsequent MS detection.…”

Comprehensive liquid chromatography: Fundamental aspects and practical considerations—A review

“…The potential of off-line LC should in this respect not be neglected [32,104,[153][154][155]. For the analysis of stable compounds, this approach is by far superior to comprehensive LC in terms of peak capacity, since there are no restrictions to column formats, flow rates, fractionation times, compatibility nor analysis time.…”

“…Due to this column technology, the efficiency and separation speed rivals that of sub-2 m columns with the advantage of lower pressure drops, through which conventional instrumentation can be employed [9][10][11][12][13]. This type of stationary phase has been used in the second dimension of offline MD analyses of peptides [104] as well as in comprehensive separations for the analysis of polyphenolic antioxidants [105].…”

Optimal gradient operation in comprehensive liquid chromatography×liquid chromatography systems with limited orthogonality

“…Most of the success and popularity it has met is due to the fact that, in LC × LC, total peak capacity is ideally given by the product of the peak capacity in the first and in the second dimension [2]. The resolving power obtainable in LC × LC has been successfully exploited for analyzing complex peptide mixtures [3,4]. For these samples the limited separation obtained by one-dimensional liquid chromatography (1D-LC) can make data interpretation difficult, even when mass spectrometry (MS) is used for detection, due to coelution.…”

“…Off-line LC × LC is the technique which can provide analysts with the highest resolving power because the fractions collected after the first-dimension ( 1 D) separation can be subsequently run on a long column. If this is the case, however, the technique becomes very time consuming [4]. On-line LC × LC analysis requires an automated set-up.…”