Extra Chromatographic Peaks — A Case Study

May 01, 2012

A common liquid chromatography (LC) problem that I get e-mails about from readers of this column relates to unexpected and unwanted peaks that appear in the chromatogram. This month's discussion centres around an e-mail discussion I had with a reader of the electronic version of LCGC (available on-line in countries where the paper editions are not available) about a problem they were experiencing. Because this problem relates to a pharmaceutical product and contains proprietary information, I've exercised some obfuscation privileges to hide some of the specifics, but retain the key information.

The problem relates to the analysis of an antibiotic for impurities. It is a fairly simple method, comprising a phosphate–acetonitrile gradient on a reversed-phase column. The method had been validated and worked quite well to determine a mixture of eight antibiotics and impurities. After successful use in the development laboratory, it was transferred to another laboratory for routine analysis. For identification, I'll refer to these as the R&D laboratory and the production laboratory.

Extra Peak


Figure 1
When the method was transferred to the production laboratory, it appeared to work well, with one exception — a broad peak appeared at a retention time that overlapped the elution of antibiotic 2. In Figure 1, I've shown a section of the baseline including peaks for antibiotics 1 (13.4 min) and 2 (15.6 min) from a chromatogram obtained in the R&D laboratory. This was a typical result for the injection of these two compounds. When the same sample was run in the production laboratory, the results of Figure 2(a) were observed. The peak for antibiotic 1 appears as normal, but there is a broad peak that all but obscures antibiotic 2.

An extra peak, that is not related to the sample itself, in a gradient run typically arises from one of three sources:

  • late elution from a previous chromatogram
  • carryover from a previous injection
  • contamination of the mobile phase (ghost peaks).

Many people call all three kinds of peaks ghost peaks, but I like to distinguish between them because, although they may look similar in the chromatogram, their sources are different, and so the process of eliminating them will be different. Let's look briefly at my definitions of each of these peak types.

Late elution of a peak from a previous chromatogram results when you don't allow enough time for all the peaks to be eluted following sample injection. As a result, the problem peak continues to travel through the column, but shows up in a later chromatogram, instead of the one in which it belongs.

Carryover from a previous injection is caused by sample that is actually injected, but its injection is unintentional. For example, you make an injection and get a normal run, then you make a blank injection with no analyte present, but the peak appears anyway, usually much smaller than the original.

Ghost peaks tend to be unique to gradient elution, whereas late elution and carryover are common for isocratic separations, as well. Ghost peaks appear when a contaminant is present in the mobile phase (usually the weak, A-solvent), is concentrated on the column and then is released as the gradient runs. Ghost peaks will appear in sample injections when a blank is injected, and even when a gradient is run with no injection at all.