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 (LC), 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 min) 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-mum 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.
Eeltink
Sebastiaan
S
Dionex, Abberdaan 114, 1046 AA Amsterdam, The Netherlands. sebastiaan.eeltink@dionex.com
Dolman
Sebastiaan
S
Swart
Remco
R
Ursem
Mario
M
Schoenmakers
Peter J
PJ
D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D008962:Models, Theoretical; D010455:Peptides / Q000032:analysis* / Q000302:isolation & purification
D010455:Peptides