HPLC Peak Splitting. Common Reasons For It

True "Split" HPLC peaks 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. If you dissolve and inject your sample in a solution that is stronger than your mobile phase, peak splitting can result. 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.