TY - JOUR
T1 - Simplified gradient generator for micro- and nano-liquid chromatography
AU - Murata, Kaoru
AU - Mano, Nariyasu
AU - Asakawa, Naoki
AU - Ishihama, Yasushi
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2006/8/4
Y1 - 2006/8/4
N2 - We have developed a simple device to generate gradient elution profiles using split tubing array (STAR) units for micro- and nano-HPLC. This gradient device consists of a delivery pump, a splitter, tubes in parallel, and a relatively large-volume mixing chamber. In the mixing chamber, an initially filled water-rich solvent is adjusted to an organic-rich solvent by delivery of appropriate components through the split paths, in which the flow rate and residence time are controlled by the sizes of the tubes employed. A program was developed to describe the output gradient profiles, and the predicted gradient profiles were highly consistent with the observed ones. A linear gradient generated by this STAR system was successfully applied to micro-HPLC systems for separation of digested peptides derived from serum albumin with sufficient reproducibility. Further miniaturization of STAR systems for nano-HPLC coupled with tandem mass spectrometry was accomplished to separate digested peptides from serum albumin with a reproducibility of retention times of better than 0.2%, and the obtained spectra from the well-separated chromatographic peaks allowed protein identification with high confidence by protein database searching. We believe that this simple and reproducible gradient system using an isocratic pump will be especially advantageous for nanoLC-MS, instead of flow-controlled gradient pumps.
AB - We have developed a simple device to generate gradient elution profiles using split tubing array (STAR) units for micro- and nano-HPLC. This gradient device consists of a delivery pump, a splitter, tubes in parallel, and a relatively large-volume mixing chamber. In the mixing chamber, an initially filled water-rich solvent is adjusted to an organic-rich solvent by delivery of appropriate components through the split paths, in which the flow rate and residence time are controlled by the sizes of the tubes employed. A program was developed to describe the output gradient profiles, and the predicted gradient profiles were highly consistent with the observed ones. A linear gradient generated by this STAR system was successfully applied to micro-HPLC systems for separation of digested peptides derived from serum albumin with sufficient reproducibility. Further miniaturization of STAR systems for nano-HPLC coupled with tandem mass spectrometry was accomplished to separate digested peptides from serum albumin with a reproducibility of retention times of better than 0.2%, and the obtained spectra from the well-separated chromatographic peaks allowed protein identification with high confidence by protein database searching. We believe that this simple and reproducible gradient system using an isocratic pump will be especially advantageous for nanoLC-MS, instead of flow-controlled gradient pumps.
KW - Gradient generator
KW - LC-MS
KW - Micro-HPLC
KW - Nano-HPLC
KW - Proteomics
KW - Split tubing array
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U2 - 10.1016/j.chroma.2006.05.004
DO - 10.1016/j.chroma.2006.05.004
M3 - Article
C2 - 16714026
AN - SCOPUS:33745726212
VL - 1123
SP - 47
EP - 52
JO - Journal of Chromatography A
JF - Journal of Chromatography A
SN - 0021-9673
IS - 1
ER -