Question

Extra Credit: In a biopharmaceutical process Proteins A and B were separated by the HPLC with elution times labeled in the chromatogram. Determine Retention Factors for proteins A and B and Selectivity between them. (4 marks)

• Extra Credit: In a biopharmaceutical process Proteins A and B were separated by the HPLC with elution times labeled in the chromatogram. Determine Retention Factors for proteins A and B and Selectivity between them. (4 marks) 0.14 0.12 Buffer components Peak B AU Peak A Gradient spike TTTTT 10.00 20.00 50.00 60.00 3000 1 40.00 Mnutes TA: 23 min Te: 36 min To: 2.5 min

Answer 1

RETENTION FACTORS

RetentionFactor(R.F.) = (TR - T0) / T0 = TR' / T0, where TR' is the time that component reacts with the stationary phase and T0 is the time of the component iun mobile phase. Therefore,

RetentionFactor(R.F.) of PROTEIN A  = (TA - T0) / T0 = (23-2.5) / 2.5 = 8.2

RetentionFactor(R.F.) of PROTEIN B = (TB - T0) / T0 = (36-2.5) / 2.5 = 13.4

Selectivity from the R.F. value of Protein A and B indicates Protein B has spent more time in stationary phase than in the mobile phase where as Protein A has spent less time in stationary phase than that of Protein B. So Protein A is extracted/seperated out the column much time before Protein B, which indicates Protein B is highly selective in Nature than Protein A .