Microchip HPLC of Peptides and Proteins

David S. Reichmuth, Timothy J. Shepodd, and Brian J. Kirby*
Sandia National Laboratories, P.O. Box 969 MS 9291, Livermore, California 94551
Anal. Chem., 2005, 77 (9), pp 2997–3000
DOI: 10.1021/ac048358r
Publication Date (Web): March 29, 2005
Copyright © 2005 American Chemical Society
*

 To whom correspondence should be addressed. Current address:  Sibley School of Mechanical and Aerospace Engineering, 238 Upson Hall, Cornell University, Ithaca, NY 14853. E-mail:  bk88@cornell.edu.

Abstract

Rapid microchip reversed-phase HPLC of peptides and proteins at pressure gradients of 12 bar/cm (180 psi/cm) has been performed using a microdevice that integrates subnanoliter on-chip injection and separation with a miniaturized fluorescence detector. Proteins and peptides were separated on a C18 side-chain porous polymer monolith defined by contact lithography, and injection was achieved via a pressure-switchable fluoropolymer valve defined using projection lithography. Preliminary separations of peptide standards and protein mixtures were performed in 40−200 s, and switching between samples with no detectible sample carryover has been performed. The injections and separations were reproducible; the relative standard deviation (RSD) for retention time was 0.03%, and peak area RSD was 3.8%. Sample volumes ranging from 220 to 800 pL could be linearly metered by controlling the pressure injection pulse duration with conventional timing and valving. The current prototype system shows the potential for rapid and autonomous HPLC separations with varying modalities and the potential for direct connection to mass spectrometers at nanospray flow rates.

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History

  • Published In Issue May 01, 2005
  • Received for review November 5, 2004. Accepted January 31, 2005.

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