Optimizing the peak capacity per unit time in one-dimensional and off-line two-dimensional liquid chromatography for the separation of complex peptide samples

To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional (2D) liquid chromatography (I-C). column technology and operating conditions were optimized. The effects of gradient time, flow rate. column temperature. and column length were investigated in one-dimensional reversed-phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit time for peptide separations An off-line two-dimensional LC approach was developed using a micro-fractionation option of the autosampler, which allowed automatic fractionation of peptides after a first-dimension ion-exchange separation and re-injection of the fractions onto a second-dimension RP nano-LC column Under the applied conditions. which Included a preconcentration/desalting time of 5 min, and a column equilibration time of 12.5 min, the highest peak capacity per unit time in the 2D-LC mode was obtained when applying a short (10 mm) first-dimension gradient and second-dimension RP gradients of 20 min duration. For separations requiring a maximum peak capacity of 375. one-dimensional LC was found to be superior to the off-line strong cation-exchange/x/RPLC approach in terms of analysis time Although a peak capacity of 450 Could be obtained in one-dimensional LC when applying 120-min gradients on 500-mm long columns packed with 3-mu m particles, for separations requiring a peak capacity higher than 375 2D-LC experiments provide a higher peak capacity per unit time Finally, the potential of off-line 2D-LC coupled to tandem mass spectrometry detection is demonstrated with the analysis of a tryptic digest of a mixture of nine proteins and an Escherichia coli digest. (C) 2009 Elsevier B.V All rights reserved.