Reversed- Phase Liquid Chromatographic Method Development for Separation of Lysozyme, Chymotrypsin, Ovalbumin, and Human Serum Albumin, Including Validation of Lysozyme Separation
Location
SU-217
Department
Chemistry
Abstract
Analytical characterization of biomolecules such as proteins is inherently more complex than its traditional counterpart, small molecule pharmaceuticals. A combination of reversed-phase, ion exchange, gel electrophoresis, and size exclusion chromatography are typically used. A reversed-phase liquid chromatographic method was exploited to separate a sample of four proteins, including human serum albumin (HSA), ovalbumin (OVA), Lysozyme (Lyso), and chymotrypsin (Chymo). Agilent 1100 series HPLC system with Diode Array Detector was used with Jupiter 5 um C4 300 A° size 25 x 0.46 cm column and a mobile phase of 0.08% TFA inwater solvent A and 0.08% TFA in 100% ACN solvent B. A DryLab simulation software was used to assist in developing the method. An optimum conditions of column temperature of 50°C, injection volume of 10 uL, a flow rate of 1.0 mL/min, detection wavelength 280 nm, a gradient range of 30 to 70% solvent B, and a gradient time of 13 minutes were found to be the optimal conditions for the separation. To confirm the method's robustness towards Lysozyme, the developed method was validated in terms of the FDA validation parameters, including robustness, specificity, linearity, accuracy, and precision. The proposed method proved to be simple, accurate, and precise, with over 98.0 % recovery of the Lysozyme protein.
Faculty Sponsor
John Sargon Albazi
Reversed- Phase Liquid Chromatographic Method Development for Separation of Lysozyme, Chymotrypsin, Ovalbumin, and Human Serum Albumin, Including Validation of Lysozyme Separation
SU-217
Analytical characterization of biomolecules such as proteins is inherently more complex than its traditional counterpart, small molecule pharmaceuticals. A combination of reversed-phase, ion exchange, gel electrophoresis, and size exclusion chromatography are typically used. A reversed-phase liquid chromatographic method was exploited to separate a sample of four proteins, including human serum albumin (HSA), ovalbumin (OVA), Lysozyme (Lyso), and chymotrypsin (Chymo). Agilent 1100 series HPLC system with Diode Array Detector was used with Jupiter 5 um C4 300 A° size 25 x 0.46 cm column and a mobile phase of 0.08% TFA inwater solvent A and 0.08% TFA in 100% ACN solvent B. A DryLab simulation software was used to assist in developing the method. An optimum conditions of column temperature of 50°C, injection volume of 10 uL, a flow rate of 1.0 mL/min, detection wavelength 280 nm, a gradient range of 30 to 70% solvent B, and a gradient time of 13 minutes were found to be the optimal conditions for the separation. To confirm the method's robustness towards Lysozyme, the developed method was validated in terms of the FDA validation parameters, including robustness, specificity, linearity, accuracy, and precision. The proposed method proved to be simple, accurate, and precise, with over 98.0 % recovery of the Lysozyme protein.