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Quantitation of proteins using HPLC-detector response rather than standard curve comparison by G.A. Eberlein is a Chemistry article available to read on EtoBox.
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Modern high performance liquid chromatography (HPLC)-diode array detectors with features such as multiple wavelength monitoring are capable of maintaining a high degree of response reproducibility over extended periods of time. This reproducibility suggests that detector response factors, rather than dilution based standard curves, might be used to measure concentrations of proteins and pharmaceuticals. Four different HPLC methods were used to analyze a single protein and to test the accuracy and precision of measurements using response factors. These results were compared to the accuracy and precision obtained using fitting to a standard curve. Protein solutions were analyzed by HPLC after the concentration was determined by quantitative amino acid analysis. The extinction coefficient at 277 nm of these protein solutions was determined by UV spectroscopy as well as calculated based on the known amino acid composition. The theoretical extinction coefficient calculated by summing the extinction coefficient of the individual amino acids was within 2% of the experimental value. Response factors at 215 and 277 nm were calculated using the peak area produced by the injection of a known
Who reads Quantitation of proteins using HPLC-detector response rather than standard curve comparison?
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- Author
- G.A. Eberlein
- Publisher
- Elsevier Science; Elsevier ; Elsevier BV (ISSN 0731-7085)
- Published
- 1995
- Language
- EN
- Field
- Chemistry (Physical Sciences)