The pK1, pK2, and pKR of the amino acid lysine are 2.2, 9.1, and 10.5, respectively. The pK1, pK2, and pKR of the amino acid arginine are 1.8, 9.0, and 12.5, respectively. A student at SDSU wants to use ion exchange chromatography to separate lysine from arginine. What pH is likely to work best for this separation?
10.5
Ion exchange chromatography separates molecules based on their net charge, which is determined by the pH of the mobile phase. In this case, lysine and arginine have different pK values, which means that they have different net charges at different pH values.
At a pH below 2.2, lysine will be mostly in the protonated form (+NH3CH2CH2COOH) and thus will have a net positive charge. At the same time, arginine will be in the doubly protonated form (+NH3CH2CH2CH(NH3)+COOH) and will have an even higher net positive charge. At high pH values above 10.5, both lysine and arginine will be in their unprotonated form (NH2CH2CH2COOH and NH2CH2CH2CH(NH2)COOH, respectively) with a net zero charge. Thus, neither lysine nor arginine will bind to the ion exchange resin.
Therefore, the best pH for separating lysine and arginine would be within the range of their pK values. At a pH between 9.1 and 10.5, lysine will be in the partially deprotonated form (NH3+CH2CH2COO-) and will have a lower net positive charge. At the same time, arginine will still be in the doubly protonated form (+NH3CH2CH2CH(NH3)+COOH) and will have a higher net positive charge. This difference in charge will allow the two amino acids to be separated from each other using ion exchange chromatography.
Therefore, a pH between 9.1 and 10.5 is likely to work best for this separation.
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