I recently received a question from a reader regarding the impact of a flow-rate change on a validated method. The method was for the analysis of a multivitamin product using gradient elution with a reversed-phase liquid chromatography (LC) separation. The method ran over a 35 min period at 0.7 mL/min. The reader had observed small column-to-column changes over the several years that the method had been in use, but the main concern was that the retention times had all increased recently. It was found that an increase in the flow-rate to 0.8–0.85 mL/min adjusted the retention times sufficiently that the retention times specified in the system suitability parameters could be attained. The concern was how much variation in flow-rate was allowed (changes in flow-rate had not been investigated during method validation) and what could have caused the problem in the first place.

This month's "LC Troubleshooting" instalment will examine the possible reasons the retention times could have changed. If the root cause can be identified and corrected, no adjustment in flow-rate will be necessary. After reliable system operation is assured, we will look at the influence of flow-rate and the allowable changes, specifically for gradient methods.

Possible Method Changes

I like to approach LC problems using a "divide-and-conquer" technique. One of the most common questions when problems occur is whether the problem is related to a change in the method itself or the LC system hardware. In each of these two categories there are several possible causes of problems. I have listed a few of these in Table 1, and I am sure that other potential problem sources exist, as well.

Table 1:

Let's look first at possible changes in the method, then at changes in the hardware. As listed in Table 1, the most probable causes of retention time differences caused by method changes are variations in the mobile phase composition, the column chemistry, or the column temperature. We'll look at each of these in a little more detail next.

Mobile phase composition changes are an obvious potential source of retention time changes. Make the easy checks first — I would replace the mobile phase with a fresh batch. Take special care if the A-solvent (aqueous phase) or B-solvent (organic phase) is premixed (for example, 5% acetonitrile in water for A and 5% water in acetonitrile for B). Buffers can be especially problematic if they are not prepared correctly. Some methods are very sensitive to small changes in buffer pH. This should have been discovered during method development and validation or from experience using the method in routine analysis. The buffer should be used in its buffering range (±1 pH unit from the pK_a), should be of sufficient concentration (generally 10–20 mM is adequate), and the pH should be adjusted before organic solvent is added. If a fresh mobile phase does not correct the problem, move on to examine the column.

Column chemistry changes: Chemistry changes between columns are much less common with today's high-purity silica columns (also called Type-B silica) than with the lower-purity columns (Type-A) that were predominant 20 years ago. However, no matter how well a manufacturer works to minimize column-to-column variation, it seems as though users always come up with a special compound that shows up minor differences between columns. For existing methods, the easiest way to check for column chemistry changes is to replace the column with a new one. You can check the influence of batch-to-batch changes by ordering columns from different batches. If changes in the column as a result of manufacturing differences are the source of the retention time problems, you should see step-wise changes in retention with a change in columns. Double-check the observed changes by running each column at least twice in a series of experiments. For example, column 1 then column 2, then column 1, then column 2, and so forth. You should get reproducible results from each column and a reproducible change between columns if the column packing is the source of your problem.