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Fluorescent detection of peptides and amino acids for capillary electrophoresis via on-line derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole

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Abstract

An in-capillary derivatization of amino acids and peptides with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed for their subsequent capillary electrophoretic analysis with laser-induced fluorescence detection (λ ex=488 nm). The in-capillary derivatization was achieved in zone-passing mode by introducing successive plugs of sample and NBD-F into a fused silica capillary previously equilibrated with an alkaline borate buffer. To prevent NBD-F hydrolysis and to achieve a reliable derivatization, NBD-F was prepared daily in absolute ethanol and a plug of absolute ethanol was introduced between the sample and NBD-F reagent plugs. Various parameters influencing the derivatization efficiency were investigated and the optimum conditions were as follows: background electrolyte (BGE), 20 mM borate buffer (pH 8.8); introduction time, 4 s for sample and 2 s for NBD-F; molar ratio of NBD-F/sample, above 215; temperature, 45 °C for amino acids and 35 °C for peptides; applied voltage, +15 kV. The validation of the in-capillary derivatization method under optimal conditions showed a good linearity between the heights of the derivative peaks and the concentrations of the amino acids. The intra-day relative standard deviations of the migration times and the peak heights were less than 1.3% and 4.6%, respectively. The efficient derivatization and separation of a mixture of valine, alanine, glutamic acid and aspartic acid were achieved using this technique. Peptides such as buccaline and β-protein fragment 1–42 could also be derivatized using the developed in-capillary derivatization procedure.

In‑capillary derivatization and separation of amino acids with different concentrations.

From the top to bottom the concentrations are 1.11×10−5 M, 5.55×10−6 M, 2.78×10−6 M, 6.95×10−7 M. for valine; 1.26×10−5 M, 6.30×10−6 M, 3.15×10−6 M, 7.88×10−7 M for alanine; 3.78×10−5 M, 1.89×10−5 M, 9.45×10−6 M, 2.36×10−6 M for glutamic acid;, 4.27×10−5 M, 2.14×10−5 M, 1.07×10−5 M, 2.68×10−6 M for aspartic acid.

Experiment conditions: injection order: 4s for sample, 1s for absolute ethanol, and then 2s for 5.24×10−2 M NBD‑F; BGE: 20 mM borate pH 8.77; Applied voltage: 15 kV.

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Acknowledgements

Hongyi Zhang gratefully acknowledges Faculté de Pharmacie, Université Paris-Sud 11 for providing the experimental space, instrument, and reagents, the partial financial support from the National Natural Science Foundation of China (20575016), the Science Foundation of Hebei Province (B2006000953).

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Authors and Affiliations

  1. College of Chemistry and Environmental Science, Hebei University, Baoding, 071002, People’s Republic of China

    Hongyi Zhang

  2. Faculté de Pharmacie, Université Paris-Sud 11, JE 2495, IFR 141, Châtenay-Malabry cedex, 92296, France

    Isabelle Le Potier, Claire Smadja, Jiyou Zhang & Myriam Taverna

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  1. Hongyi Zhang
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  2. Isabelle Le Potier
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Correspondence to Hongyi Zhang.

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Zhang, H., Le Potier, I., Smadja, C. et al. Fluorescent detection of peptides and amino acids for capillary electrophoresis via on-line derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole. Anal Bioanal Chem 386, 1387–1394 (2006). https://doi.org/10.1007/s00216-006-0709-6

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  • DOI: https://doi.org/10.1007/s00216-006-0709-6

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