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Are Split LC Peaks Giving you a Splitting Headache?

20 Apr 2022

Are split peaks in your method keeping you up at night? Below you will find some reasons that could be causing these split peaks, and how to troubleshoot the problem.

There are a few questions that should be answered to determine where to start investigating the root cause of the peak splitting. Are all of the peaks in the method splitting or are there only one or a few peaks splitting? Have you always observed split peaks in your method or if they seemed to start happening out of nowhere? Figuring out these answers first allow you to know where to start with troubleshooting.

If only the earliest eluting peaks are splitting, this can be attributed to diluent mismatch. This can be resolved by using a weaker solvent to dilute samples or matching the sample diluent with the starting mobile phase conditions. This can also be fixed by injecting a smaller amount or using a larger column. See examples of mismatched diluents below in Figure 1 and Figure 2.

Figure 1: Method conditions and chromatogram for the analysis of 2 acylcarnitines using methanol as the diluent.

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Figure 2: Method conditions and chromatogram for the analysis of 2 acylcarnitines using mobile phase A as the diluent.

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If only one or a few of the peaks in the method are splitting, the next problem to rule out is if it is actually one peak splitting into two or if they are coeluting peaks. There could be a matrix interference coeluting with your analytes. In this case, try injecting a blank matrix sample to rule out any issues with coelution. Another cause could be too high of a concentration or injection volume of your samples could be causing sample overloading, leading to split peaks. Try injecting a smaller sample volume and if you notice that there are still two distinct peaks, then it may be time to take a look back at your method and work on adjusting the conditions to improve selectivity.  

Another variable that could be causing split peaks in your method is if the pH of the mobile phase is too close to the pKa of the analyte. It is a general rule of thumb to adjust the pH of the mobile phase 2 or more units from the analyte’s pKa. Split peaks can be caused by the ionization states of analytes at a certain pH. If the pH is too close to the pKa of the analyte, there will be multiple ionization states that could result in multiple peaks for the analyte.  In these instances, try adjusting your mobile phase pH by +/- 2 and run a few samples to see if this helps with the peak splitting.

Insolubility is another factor that could cause peaks to split in your method. To avoid this, it is important to make sure that the sample is soluble with both the diluent and the mobile phase. If it is not soluble in one of these, it could cause split or distorted peaks.

If split peaks are normally not a problem in your method and suddenly you are noticing split peaks, this could indicate a couple of issues. Contamination is another factor than can cause split peaks. In terms of mobile phases, it is also important that you are using fresh solutions, and if you are using MS detection, you should be using LC-MS grade solvents. It is also important to only use mobile phases that the manufacturer recommends on the column, and to wash it and store it under the proper conditions.

Additionally, contamination or a void in the column packing can also cause split peaks. Some steps can be taken to ensure that this is less likely, such as filtering your sample with Thomson SINGLE StEP Standard Filter Vials and by using your column within the pressure recommendation limits of your manufacturer.

You could also try using a guard column or precolumn filter to protect your column. If you are using a guard column, remember to change it regularly, and also make sure that the phase matches the phase of your column.

If none of the resolutions above seem to fix your split peak issue, then it might be time to replace your column.

Hopefully these tips helped and be sure to check back for more tips and tricks from Restek!