There is standard of benzoic acid of 500ppm prepare in methanol: acetate buffer(55:45) during retention time in hplc there are two peaks what will be the reason for these two peaks
Hi Monika, here are a few possibilities:
1) Most likely reason - sample is impure, if both the peaks elute later on the gradient (assuming it's a reverse phase HPLC, see point 2). NMR (or even TLC with a good mobile phase) can tell whether your sample is pure.
2) If you are monitoring at lower wavelengths (210-240 nm) - what is the retention time? Are you sure it's another peak. Could it be the noise in the signal due to methanol/acetate buffer at the time when dead volume elutes. This can be easily tested by injecting a blank buffer and comparing the retention times with the peaks in the sample.
3) Highly unlikely - you have set up your hplc conditions such that benzoic acid is partially deprotonated. This would typically result in a broad, single peak. But since we are talking about possibilities, the protonated and deprotonated versions of benzoic acid can elute as two independent peaks. Including TFA in your mobile phase will reduce this and make your peaks sharper.
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