Peak Fronting . . . Some of the Time

Dec 01, 2013

Why do some peaks front and others don't in the same method?

I often get asked how I come up with the topics that are covered in my "LC Troubleshooting" column every month. Most of the time, the source is either questions I receive via e-mail (see instructions at the end of this column if you have a question to ask me) or in live classes that I teach around the world. This month, the topic originates in the latter category. One course attendee had observed a problem with her liquid chromatography (LC) method in which a peak fronted. Most of us are familiar with tailing peaks, in which the peak rises from the baseline rapidly, but returns to the baseline more slowly, resulting in a distorted peak, where the second half of the peak is broader than the first half, forming a "tail." In the present case, injections of the reference standard gave normal, nearly symmetric peaks, but when the samples were injected, the peaks rose more slowly from the baseline than normal, resulting in a fronting peak, where the first half of the peak is broader than the second half. The question, of course, was what was going on and how to correct the problem.

Divide and Conquer

One of the troubleshooting principles that I frequently mention is what I call the "divide-and-conquer" technique. This is simply the process of sequentially dividing the problem into (usually) two major parts by changing some variable and observing the result. The goal is to find changes that either do or do not make a difference in the observed problem. This helps to eliminate possible causes of the problem. As we go through a series of such steps, we progressively eliminate potential problem sources until we are left with the root cause of the problem. For example, in the first step, we may eliminate half of the possible causes, in the second, half of the remaining causes, and so on. It doesn't take long to be left with just one or two things that may have to be sorted out to correct the problem.

A second principle of troubleshooting is to use the scientific method and change just one variable at a time. In this manner, we will know which variables influence the problem and which do not. Often it is tempting to change several items together, just to get the problem solved and get back to work, but in the long run, this approach doesn't help us identify the real problem source so that we can avoid or minimize future problems. For example, for a problem observed in the chromatogram, we might change the column, guard column, in-line filter, and make a fresh batch of mobile phase. This may fix the problem, but it does not get to the root cause of the problem — for example, use of the wrong pH for the mobile phase — and therefore may waste money (we may have discarded a perfectly good column) and invite future problems (perhaps we should add a step to double-check the mobile-phase pH).

Usually, we apply the divide-and-conquer technique along with changing just one thing at a time during troubleshooting. This may be a conscious or subconscious process. Let's try this approach with the present problem.

What's Different and What's the Same?

The first round of troubleshooting has already been done for us; the remaining ones we'll have to speculate on, but they will help to define experiments that will help us identify the root cause of the problem. We know that injection of the reference standards results in acceptable peak shapes, but sample injections give fronting peaks. We also know that the two injection types consistently give distinctly different results (our first divide-and-conquer experiment). What do these two items have in common, and what is different between them?

Both the standards and samples use the same mobile phase, instrument, and column, so it is unlikely that any of these items is at fault. As a side observation, peak tailing is a much more common problem with LC separations. Tailing is most commonly attributed to unwanted secondary interactions between the analyte molecule and the column. Usually this results from strong interactions between acidic silanol groups on the column packing and basic functional groups on the analyte. When this happens, usually the analyte in both the reference standards and samples gives the same tailing peak shape. The different peak shape between the two cases in the present example is further evidence that the column is not at fault. The most common cause of peak fronting that is observed today is the collapse of the column bed, but when column collapse occurs, both standards and sample will generate the same fronting peak shape, so we know this is not the source of the current problem. However, because column collapse is the most common cause of fronting peaks, it is covered briefly at the end of the present discussion.

The present method was used to determine the potency, or concentration of the analyte, in a formulated drug product. We will assume that during method development and validation it was found that acceptable precision and accuracy were obtained with the reference standard prepared in a water–organic solution, as often is the case for this type of method. The most likely source of the problem is something that is different between the standard and the sample. Let's make a list of some possible differences, then we can devise experiments to isolate the root cause. Here are some possibilities:

  • injection volume
  • aqueous–organic ratio of the injection solvent
  • pH of the injection solvent
  • presence or absence of matrix components
  • presence or absence of other analytes.

I'm sure I've missed something obvious in this list — you readers are always helpful to point this out! Let's look at each item in turn.