Department of Chemistry
HPLC with in-capillary optical fiber laser-induced fluorescence detection of picomolar amounts of amino acids by precolumn fluorescence derivatization with fluorescein isothiocyanate
In this paper, a fluorescein isothiocyanate (FITC) precolumn derivatization technique in conjunction with an HPLC-in-capillary optical fiber laser-induced fluorescence (HPLC-ICOF-LIF) detection method has been developed for determination of amino acids. The HPLC separation of FITC-labeled amino acids and the ICOF-LIF detection system are studied and optimized. Optimum separation conditions were obtained with a gradient elution program of acetonitrile and phosphate buffer (10 mM, pH 6.8). The ICOF-LIF detection system comprises a 530-μm capillary and a 380-μm optical fiber. The analyses of amino acids display excellent linear relationship between peak area and concentration with correlation coefficients greater than 0.999 and the method also provides good repeatability with RSD < 3%. The detection limits for FITC-tagged amino acids are very low and the lowest LOD for tyrosine is 51 pM. The proposed method has been successfully applied to determination of amino acids in human serum. Our developed HPLC-ICOF-LIF system is cheap, simple, stable, and sensitive which is potentially useful for the formulation analysis and bioanalysis. © 2011 Springer-Verlag.
Amino acids, Column liquid chromatography, FITC, In-capillary optical fiber laser-induced fluorescence
Source Publication Title
He, Yunyun, Lijun Zhao, Hongyan Yuan, Zemin Xu, Ying Tang, Dan Xiao, and Martin M. F. Choi. "HPLC with in-capillary optical fiber laser-induced fluorescence detection of picomolar amounts of amino acids by precolumn fluorescence derivatization with fluorescein isothiocyanate." Chromatographia 74.8-7 (2011): 541-547.