Peak Shape Problems

Jul 01, 2008

John W. Dolan
Most of us are quite familiar with tailing peaks in liquid chromatography (LC) separations. In my experience, it is a rare chromatogram that is free of peak tailing. Peak tailing is understood reasonably well by most workers. In reversed-phase separations, it is due primarily to unwanted secondary interactions — especially basic and acidic compounds that undergo ion exchange or interaction with metal contaminants in the silica-based stationary phase. These tailing problems usually affect just one or a few peaks in a chromatogram. What about those chromatograms in which all the peaks have severely tailing peaks, or split or doubled peaks? Then there are peaks that front badly — a rare event for most workers — but common under certain conditions. This month's "LC Troubleshooting" column will consider these last two problem areas.

Against the Flow

Figure 1
Recently, a reader sent me the two chromatograms shown in Figure 1. He observed that when he ran his samples for analysis, all the peaks in the chromatogram appeared as if in double vision, as seen in Figure 1a. Reinjection of the same sample gave similar results, so he injected another sample, which also gave doubled peaks. This led him to wonder if the problem was associated with all injections, so he injected the analytical standard and observed the chromatogram shown in Figure 1b. The laboratory had limited resources, with only one LC system and no spare column. This restricted the use of one of the most powerful troubleshooting techniques — substituting a known good part for a questionable one. Nevertheless, he did what he could. A new batch of mobile phase was made and new autosampler wash solvent was used with no improvement. The entire system was flushed thoroughly with acetonitrile in an effort to clean the column and wash the pump, autosampler, and detector; this did not appear to help, either. Finally, in desperation, he reversed the column, and was surprised to see the standard coalesce from a double peak to a single one. An injection of a sample also showed single peaks for each of the sample components. Because he was nervous about running the column in the opposite direction of the flow arrow, he returned the column to the normal direction, but found that the problem reappeared. It was at that point that he e-mailed me for help.

A Classic Chromatogram

Figure 2
The reader had encountered one of the most easily recognizable problems with a chromatogram. In fact, this is so stereotypical that I often refer to this as a "signature chromatogram." It is characteristic of one of two types of column failure — a partially blocked column frit or a void at the head of the column. Other example chromatograms as a result of this are shown in Figure 2. The common symptom is that every peak in the chromatogram shows the same type of peak distortion — doubling, tailing, or another unexpected shape.