Resonance Rayleigh scattering for detection of proteins in HPLC.
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 lambdaex=lambdaem=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 gamma-globulin (gamma-Glo). An LOD of 0.2-1.0 microg/mL was reached and a linear range was found between peak area and concentration in the range of 0.20-3.0 microg/mL for Cyt-c, 0.25-2.5 microg/mL for Lys, 1.5-10 microg/mL for HSA, and 2.0-15 microg/mL for gamma-Glo, with linear regression coefficients all above 0.99. The method presented has been applied to determine HSA and gamma-Glo in human serum samples synchronously.
Lu
Xin
X
College of Chemistry and Chemical Engineering, Guangxi, Normal University, Guilin, China.
Luo
Zhihui
Z
Liu
Chengwei
C
Zhao
Shulin
S
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D045304:Cytochromes c / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000032:analysis; D013057:Spectrum Analysis / Q000295:instrumentation / Q000379:methods; D013997:Time Factors; D005719:gamma-Globulins / Q000032:analysis
D011506:Proteins; D012709:Serum Albumin; D005719:gamma-Globulins; D045304:Cytochromes c; D009113:Muramidase