Chromatography Forum

Hi all,
May anyone explain this mobile phase composition for HPLC analysis of adenine?


Time (min) Buffer solution(%) Acetonitrile (%) Water (%)

0 5 5 90
20 5 5 90
20.1 10 10 80
30 10 10 80
30.1 5 5 90
40 5 5 90
Thank all in advance

An USP method: always a challenge
Gradient run: You start with high amount of water to get higher retention times.
And what is your question?

Based on the USP monograph, you'll need three lines for your three mobile phases. Line A is the buffer (ammonium phosphate, pH 6.2), Line B is acetonitrile and Line C is water.

Initial conditions are 5% A, 5% B, 90% C. This stays isocratic for 20 minutes. At 20.1 minutes the proportion shifts to 10% A, 10% B and 80% C. This stays isocractic until 30 minutes. At 30.1 minutes you revert back to 5% A, 5% B, 90% C and stays isocratic until 40 minutes; this is a re-equilibration step.

does it mean the run time for 1 sample analysis is 40 min?
Thank you all.

Yes.

Gradient methods often look something like what you posted in your question.

is that method for assay or determination of the related substances because if it is for the related substance i think that increase in pH and % of the organic solvent after 30 min was for resolution of the impurities from the principle molecules