peak split

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fresher's picture
peak split





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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?

Dr. Analytical
Dr. Analytical's picture
Your problem is caused by

Your problem is caused by your injection solvent.

In a perfect LC experiment, the sample is dissolved in mobile phase (85:15 MeOH:H2O in your case) and injected.  With this approach, the injection solvent does not affect the chromatography in any way.

In your reversed phase (RP) system, when the mobile phase contains more organic solvent (methanol), it is a "stronger" eluting solvent, and causes retention to decrease.  The organic solvent is more non-polar, and so is more like the non-polar (C18) stationary phase.  When the mobile phase becomes more like the stationary phase, it is better able to pull the analytes away from the stationary phase and cause them to elute earlier.  If you experimented with other alcohols, like ethanol and propanol, you would find that as you added carbons to the alcohol, it becomes a stronger solvent - propanol is a stronger solvent than methanol.

Now we must discuss your method.

Using hexane as a sample solvent means that you are using a very strong solvent.  When this solvent enters the column, for the first few mm of the column, the samples are experiencing a stronger solvent because of the hexane.  This causes some of the analytes to move faster through the column, and you see this as a shoulder.  As the hexane moves through the column it becomes more dilute and no longer affects retention.  But since some of the analyte molecules started moving faster at the beginning, they will elute before the other molecules.

Heptane is a stronger solvent than hexane, and it caused so many molecules to move faster that you see it as a split peak.  Toluene is also a strong solvent, but not as strong as heptane.

The best solution is to change your injection solvent so that it is more like the mobile phase.  You could evaporate all the hexane and dissolve in mobile phase, or even just 100% methanol.  If you can not remove all the hexane, then you should dilute it with a solvent like acetonitrile or 2-propanol (methanol and hexane do not like to mix).

I suggest that you consider attending a course on HPLC to help you understand these issues and other problems that are common in HPLC.  The Meeting Annoucements section has a list of some courses.

Thank you for posting your question on  Please come back often.

Santosh@India's picture
Advise you to have basic

Advise you to have basic training on HPLC. Definately you will come to know what creats the proble.