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I'm facing a strange case (at least to me).
The problem occurred when I was optimizing an extraction method followed by HPLC determination of some pesticides.
At the beginning I used a (150, 4.6, 5 ) column, the mobile phase was MeOH:H2O 85:15, the extraction solvent was HPLC hexane which was directly injected into the column. I got well-shaped and well separated peaks.
After that I switched to another column (250, 4.6,5) to check the effect of the length on resolution. I used the same extraction method but in addition to n-hexane I used also n-heptane and toluene. After column equilibration, the first injection was with n-heptane but I noticed that my peaks (three original peaks) have shoulders on them. I repeated the injection thrice but the problem persists although the degree of peak splitting differs from run to another. I suspected the solvent and in the next runs I used n-hexane but the problem occurred again ( although I had'nt this problem earlier with hexane). Surprisingly, when I used toluene for extraction (and then directly injeted) the peak splitting disappeared and well-shaped and well separated peaks were obtained. The first thing I thought about was that column equilibration was not well performed and only it was reached when toluene was used. So, I repeated using n-hexane and n-heptane for extraction but the problem occurred again.
Can anybody extend his help in this regard?