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| - Influence of Pressure on the Retention andSeparation of Insulin Variants under LinearConditions
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| - The effect of pressure on the retention behavior of insulinvariants in RPLC on a YMC-ODS C18 column was investigated under linear conditions. The retention factors ofthese variants increase nearly 2-fold when the averagecolumn pressure is increased from 55 to 250 bar whiletheir separation factors remain nearly unchanged. Thiseffect is explained by a change of the partial molar volumeof the insulin variants associated with their adsorption thatdecreases from −99 to −80 mL/mol for mobile-phaseconcentrations of acetonitrile increasing from 29 to 33%(v/v). This volume change is much larger than the oneobserved with low molecular weight compounds. For thesame pressure variation, the average number Z of acetonitrile molecules displaced from the protein and thestationary phase upon adsorption increases from 22 to23.3. The pressure-induced relative increase of the termb[S]/[D0]Z (which corresponds to the initial slope of theadsorption isotherm) is approximately twice as large forLispro than for porcine insulin. Because the bindingconstant of insulin decreases with increasing pressure,this suggests that the number of binding sites on thestationary phase increases even faster. Finally, it wasobserved that the column efficiency at flow rates higherthan 0.6 mL/min increases slightly with increasing pressure. It is suggested that these observations are also validfor other proteins analyzed in RPLC.
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