Translator for HPLC HINTS and TIPS for Chromatographers

Saturday, September 24, 2016

HPLC Peak Splitting. Common Reasons For It

True "Split" HPLC peaks, not resulting from co-elution of another peak, can be caused by a number of chromatography problems. Here are a few examples and their solutions:

  1. Sample overload. Sample overloading is one of the most common reasons for observing peak "splitting". Reduce the sample concentration by factors of ten to see if the peak shape improves. 
  2.  A poor quality HPLC method. Poor quality methods which do not use mobile phase solutions which are at an appropriate pH, dissolve the sample in (are fully soluble) are unstable or show sample or mobile phase precipitation can cause this effect. Always check solubility before starting.
  3. A partially plugged or fouled column. A dirty or fouled column (from not washing down properly with a solution which is STRONGER than the mobile phase). Analysis methods should be followed by separate wash methods to remove all bound material and any late eluters,
  4. Wrong injection solution. Peak splitting may be the result of dissolving and injecting your sample in a solution that is stronger than your mobile phase. Dissolve and inject samples in the mobile phase or in a solution which is a slightly weaker solution (not stronger).
  5. A poorly packed column, void at column inlet or a dirty frit. These types of structural defects can each result in peak "splitting" (all of these are less common today than in the past using modern HPLC columns). When present, a dirty inlet frit can be replaced with a new one, or the column can sometimes be backflushed to remove any accumulated material.
  6. Detector data rate set too low. Too few peaks collected over time may result in integration errors and inaccurate peak symmetry problems. Read more about how to determine the best data collection rate at this link.


  1. We recently started to see our API as two connected peaks instead of one with a dip in between the two peaks. We moved the method to a different HPLC system and could not see the sample anymore so we increased the injection from 10 ul to 100 ul and now see it, but as a split peak. What can we do to fix this?

    1. By increasing the sample concentration (and volume) by ten times, it is likely you have overloaded the HPLC column (see #1 above). To avoid overload, try and keep the injection volume below 3% of the column dead volume. You may want to perform a loading study to find out what the limits of the specific column and method are.

      The reason you experienced sensitivity problems when transferring the method to a second HPLC system is most likely that the two systems are not configured the same. Many differences could exist which can result in different response outputs.
      For example:
      (1) Difference in each detectors flow cell path length can have a dramatic effect on the observed signal (i.e. 6 mm vs 10 mm). For more information, please refer to the article:

      (2) For UV/VIS detectors, the wavelength bandwidth value also can have a large effect on the observed signal too. For more information, please refer to the article,

      *You need to carefully compare each system to first.

    2. BTW: Sample degradation could also explain the "extra" peak seen.