A) When you try separating an unknown mixture by reversed-phase chromatography u

Question

A) When you try separating an unknown mixture by reversed-phase chromatography using 50% acetonitrile for the mobile phase, the peaks eluted between 1 and 3 minutes and are too close together to be well resolved for quantitative analysis. Should you use a higher or lower percentage of acetonitrile in the next run?

B) After the isocratic elution has been optimized for an aqueous solvent B after several different solvents were tried, the chromatogram still has a resolution of 1.2 between the two closest peaks. How might you increase the resolution without changing the identity of solvents or type of stationary phase? Suggest two possible directions.

Explanation / Answer

ANSWER
(A) In reverse PHASE CHROMATOGRAPHY the more polar component is eluted first. For polar components we need polar solvents. Hence in the next run we will increase polarity of solvent for efficient separation. So percentage of of acetonitrile will be INCREASED.

(B) Two possible directions to increase the resolution is to (i) Increase the TIME of elution and (ii) Change the temperature.

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