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Resonance Rayleigh scattering for detection of proteins in HPLC
Author(s) -
Lu Xin,
Luo Zhihui,
Liu Chengwei,
Zhao Shulin
Publication year - 2008
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200800242
Subject(s) - chromatography , chemistry , high performance liquid chromatography , detection limit , lysozyme , fluorescence , rayleigh scattering , analytical chemistry (journal) , linear range , size exclusion chromatography , biochemistry , optics , enzyme , physics
An HPLC–resonance Rayleigh scattering (RRS) (HPLC–RRS) detection system is described for separation and detection of proteins. This system is based on the modification of a commercial HPLC instrument involving the addition of a pump and a T‐shaped interface, and a common fluorescence detector was used for detection. The detection principle is based on the change of RRS intensity of the ion‐association complex formed from biebrich scarlet (BS) and protein. The RRS signal was detected at λ ex = λ em = 376 nm. The utility of the presented method was demonstrated by the separation and determination of four proteins involving cytochrome (Cyt‐ c ), lysozyme (Lys), HSA, and γ‐globulin (γ‐Glo). An LOD of 0.2–1.0 μg/mL was reached and a linear range was found between peak area and concentration in the range of 0.20–3.0 μg/mL for Cyt‐ c , 0.25–2.5 μg/mL for Lys, 1.5–10 μg/mL for HSA, and 2.0–15 μg/mL for γ‐Glo, with linear regression coefficients all above 0.99. The method presented has been applied to determine HSA and γ‐Glo in human serum samples synchronously.