Method Development for the Separation of a Sample of Chymotrypsin, Lysozyme, Bovine Serum Albumin, and Ovalbumin and Quantitation of Bovine Serum Albumen Using Reverse-Phase Liquid Chromatography

Reversed-phase liquid chromatography (RPLC) has been demonstrated to be a promising separation technique in separating proteins and peptides. The efficiency obtained with the RPLC is generally superior to other separation techniques such as size-exclusion chromatography, ion-exchange chromatography, hydrophilic interaction chromatography, and hydrophobic interaction chromatography. Another advantage of RPLC is its coupling with mass spectrophotometry detection in forced-degradation studies. A simple reversed-phase liquid chromatographic method was developed to separate a sample of chymotrypsin, lysozyme, bovine serum albumin, and ovalbumin. The method was developed on a 25 mm Phenomenex Column C4, Particle size: 5 µm, I.D. 4.6 mm under 50oC column temperature. Mobile phase used consisted of 1.0% trifluoroacetic acid at pH 2.0 and Acetonitrile at a flow rate of 1.0 ml/min with UV-Vis detection at 210, 220 and 280 nm using DAD detector. The separation was conducted under gradient elution with 30 to 70% ACN gradient rate for 20 min. The developed method was validated for robustness, linearity, accuracy, precision, detection limit and quantitation to quantify bovine serum albumin. The method proved simple, accurate and precise with over 97% average recovery of bovine serum albumin