HPLC Useful HPLC-Tip of the month

Why Is It That Peaks Appear Later With the New Column?


The new column you ordered has just arrived. Although it has the same stationary phase and the same dimensions and you have used the same supplier, the peaks come later, however constant you kept the chromatographic conditions. Why?


Suppose the chromatographic conditions (temperature, eluent incl. pH value, ionic strength etc) and sample preparation have indeed remained constant. Now check whether the breakthrough  (front peak, dead time) is also delayed.  If that is the case, the flow rate has probably changed. Check your pump and look out for leakages.  Once you have made sure with the help of a stopwatch and a measuring cylinder that the flow is okay, look at your column as the likely cause for the discrepancy. The density of packing may vary from one column to the next if your supplier does not follow consistent packing procedure. You now have a column that contains a greater amount of material than its predecessor, e.g. 0.90 g instead of 0.82 g. It is no surprise that all peaks – including the front peak – elute later. Let us now look at the second case where the peaks elute later, but the dead time tM, remains constant. If tM remains constant while tR changes, so too does the retention factor k. Now this means that there is a change in the interaction between the components and the stationary phase, so a chemical change is taking place. If you are certain that the chemistry (temperature, eluent composition, pH value, ionic strength, modifier concentration) have remained constant the problem must lie with the supplier. Although the stationary phase carries the same label, its properties are not identical with the previous one, which shows in the differences between the various batches. However, it is also possible that you have unintentionally changed the surface of the previous column, perhaps by injecting a substance that was irreversibly sorbed, thus modifying the properties of the stationary phase and its interaction with the components.


Is tM constant or not? This is the detail that points you to the cause of your problem – be it physical changes in your system (flow), at the supplier’s end (density of packing) or chemical problems either at your end (temperature, eluent) or at the supplier’s end (properties of stationary phase) − unless you have tampered with the chemistry of the stationary phase.

©  Dr. Stavros Kromidas