Improving new on-line method aim of detection in determined proteins with HPLC and applying analyzed food and drug

After searching UV-Vis spectrophotometric methods used for protein determination, development of an on-line HPLC-Protein determination system (on-line HPLC-Biuret, on-line HPLC-Ninhydrine, on-line HPLC-Bradford) is sought for by adapting the methods having better validation properties to protein separation with post-column reagent injection. On-line-HPLC-Bradford method, being superior in terms of detection, provided better quantitative determination at 595 nm after protein separation in comparison to the semi-quantitative protein determination at 280 nm. By using various standard protein samples (CA and BSA proteins) the new method was optimized, limit of detection and limit of quantification values were determined, and quantitative determination of food (gelatin) and biopharmaceutic drug (insulin and immunoglobulin G) polypeptides were achieved by using size-exclusion column. In addition organic solvent systems (methanol, ethanol, acetonitrile) were evaluated spectrophotometrically by changing % compositions in order to show a model for the interaction with the determination of proteins (CA, BSA, immunglobuline G, insuline) and Bradford reagent in the on-line HPLC system for isocratic or gradient applications and different column systems. Optimization results were obtained as 0.3 mL/min reagent flow rate, 70 mg/L reagent concentration, 30 °C temperature and 2.5 m tubing length. It was also shown that all organic solvents below 10% mixing ratio can be used.