The Impact of Column Peak Capacity on the Multidimensional Chromatography of Complex Peptide Mixtures

Library Number:
WA31777
Part Number:
WA31777
Author(s):
Martin Gilar*, Amy E. Daly, Mariana Kele, Uwe D. Neue, Tad Dourdeville, John C. Gebler [Waters]
Source:
ISPPP 2003, Delray Beach, Florida, USA, November 9-12
Content Type:
Posters
Content Subtype:
ISPPP
Compounds:
Peptides, Peptide Mapping
Column:
Symmetry300(™) C18 300 Å 3.5 µm Steel 4.6 mm 150 mm Symmetry300(™) C18 300 Å 5 µm Steel 4.6 mm 50 mm Symmetry300(™) C18 300 Å 5 µm Steel 4.6 mm 150 mm Symmetry300(™) C18 300 Å 7 µm Steel 4.6 mm 150 mm Symmetry300(™) C18 300 Å 5 µm Steel 4.6 mm 300 mm
Related Products:
Two-dimensional chromatography-mass spectrometry (2D HPLC-MS) is a useful tool for the separation of complex peptide mixtures. Since the samples may contain hundreds of thousands of peptides of various abundances, lengths, and hydrophobicity, high separation power is required. The currently used 2D HPLC approach employs ion-exchange (IE) HPLC followed by reversed-phase (RP) HPLC and MS analysis. The peak capacity of IE-HPLC is relatively low; only 10-20 fractions are typically collected using a step gradient elution. This puts a great demand on resolution in the second dimension. We have evaluated the practical column peak capacity for RP-HPLC. The number of separated peaks reaches an upper limit of several hundred (for currently available columns). Doubling the column efficiency (length) improves the peak capacity by 40 %, and proportionally increases either the separation time or backpressure. Similarly, extremely shallow gradients improve peak capacity, but analysis becomes unacceptably long. The experimental data were compared with theoretical peak capacity prediction.

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