Method or System?

Many of us use liquid chromatography (LC) methods supplied to us by others. These can be compendial methods — those that come from one of the pharmacopoeia — or they can come from the scientific literature or from another laboratory within our company. Such methods can be very specific in terms of the column and mobile phase to be used or they might be more general, so that the end user has some flexibility in adapting the method to his or her needs. One of the most common traits of such methods, however, is that the person who developed the method is not available to field our questions about the method. And the method usually does not have any background on why certain conditions were chosen. This month's "LC Troubleshooting" discusses the type of problems one can encounter when using such methods.

The method in question specified a 150 mm × 4.6 mm, 5-μm particle C18 column (brand not listed) and a mobile phase of 57% methanol and 43% 10 mM ammonium carbonate buffer, pH 9.6, run at 1.5 mL/min. The column temperature was not specified. The sample is prepared by dissolving a drug tablet in water, filtering it, and injecting 20 μL. The separation is simple, and with typical retention times of 2.5 and 3.7 min for the two components of interest, the requirement of resolution, Rs, of more than 2 is met easily. However, the required tailing factor, TF, of ≤1.5 is more of a challenge. Typically, for the second peak, TF = 1.4–1.45, but for the first peak, TF = 1.4–1.5 with a new column, but degrades to TF > 1.5 after 1000–1500 injections. Installation of a new column solves the problem. I was asked how to fix this method.