In addition to protein analysis, reversed-phase chromatography also provides insight into mechanism of interaction between protein and hydrophobic surface and whether it includes processes such as change of protein conformation. I studied influence of temperature on retention behaviour of four proteins (insulin, cytochrome c, lysozyme and BSA) on reversed-phase C18 column. Analysis of insulin in six combinations of organic (ACN, MeOH, EtOH) and aqueous phases (0.1 % TFA, 0.1 M KCl) indicates that choice of organic component of mobile phase has greatest influence on temperature dependence of retention time. I compared retention behaviour of insulin and cytochrome c in two mobile phases, 0.1 % TFA-ACN (70:30) and 0.1 % TFA-MeOH (44:56) at constant flow and pressure. Van't Hoff plots and thermodynamic quantities calculated from them indicate that significant conformational changes in insulin molecule in TFA-ACN begin at ~ 53 °C, while these are already present at lower temperatures in TFA-MeOH. Similarly, significant conformational changes of cytochrome c in TFA-ACN are present at lower temperatures, but do not occur in TFA-MeOH. Lysozyme and BSA were analysed with gradient method 0.1 % TFA-ACN. Trends in retention times indicate that conformational changes occur in these two proteins as well.
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