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:
- 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.
- A poor quality HPLC method. Poor quality methods which do not use mobile phase solutions which are at an appropriate pH (*If the pH of the mobile phase is close to the pKa of the sample, then split peaks may result); which does not dissolve the sample in (should be fully soluble) or are unstable, show sample or mobile phase precipitation can cause this effect. Always check solubility before starting.
- 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,
- 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).
- A poorly packed column, void at column inlet, a dirty frit or poor mechanical connection (i.e. improperly swaged fitting). These types of structural or mechanical 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. Connections should always be double checked.
- 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.
