You must separate three different compounds with reversed phase HPLC. Compound 1 is an acid (pKa = 4.3), compound 2 is neutral compounds and compound 3 is a base (pKa = 9.6). As eluent, use 30/70 (v / v)% methanol / pH 3.0 formic acid buffer. The retention time and the top width at the base of the top for the three components are presented below. You can assume the dead time to 1.2 minutes in your calculations. Compound Retention time [minutes] Peak width [seconds] 1 10.1 28 2 5.0 14 3 6.1 17 a) Calculate the efficiency (number of theoretical bases) for component 1. b) Calculate the selectivity and resolution between components 1 and 2. c) When you increase the pH of your eluent to pH 6, how will the retention be affected (increase, decrease or unchanged) for the different associations (1, 2 and 3) ?, justify your answer.
You must separate three different compounds with reversed phase HPLC. Compound 1 is an acid (pKa = 4.3), compound 2 is neutral compounds and compound 3 is a base (pKa = 9.6). As eluent, use 30/70 (v / v)% methanol / pH 3.0 formic acid buffer. The retention time and the top width at the base of the top for the three components are presented below. You can assume the dead time to 1.2 minutes in your calculations.
Compound Retention time [minutes] Peak width [seconds]
1 10.1 28
2 5.0 14
3 6.1 17
a) Calculate the efficiency (number of theoretical bases) for component 1.
b) Calculate the selectivity and resolution between components 1 and 2.
c) When you increase the pH of your eluent to pH 6, how will the retention be affected (increase, decrease
or unchanged) for the different associations (1, 2 and 3) ?, justify your answer.
d) How retention factors are generally affected by an increase in the proportion organically
mobile phase solvents for reversed phase HPLC separations ?, justify your answer.
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