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Split Peaks — A Case Study
Mar 1, 2005
By: John W. Dolan
LCGC North America

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John W. Dolan
Case studies are good ways to look at specific examples of common liquid chromatography (LC) problems and to draw general conclusions that can be applied to prevent similar problems from happening for other workers. The example in this month's installment of "LC Troubleshooting" comes from a reader who works in the pharmaceutical industry. The sample is a cold-cough syrup analyzed with an ion-pairing LC method. I have disguised the details somewhat to protect the proprietary nature of the method, but there should be sufficient information to help us gain some knowledge of the peak-splitting problem experienced by the user.


Figure 1: Codeine peak from a sample of cold-cough syrup (a) after approximately 40 injections, (b) three injections later, and (c) after eight more injections. See text for details.
The Problem The analyst was developing an assay for stability and purity of the product. After approximately 40 injections, she noticed that the codeine peak had deteriorated to a peak with a shoulder on it, as illustrated in Figure 1a. The peak splitting increased as the run continued, with a distinct doublet appearing after three more injections (Figure 1b) and showed further deterioration (Figure 1c) after another eight injections. When one is developing a stability-indicating assay, the appearance of new peaks can indicate that additional decomposition has occurred. The analyst suspected this and checked the spectra across the entire peak with a photodiode-array detector. All of the spectra were identical to that of codeine, which suggests that it is peak splitting, not chemical degradation of the sample or appearance of a new compound. Other peaks in the sample showed similar distortion.


Figure 2: Codeine peak for injection of reference standard obtained (a) using a new column, (b) soon after the run of Figure 1(c), (c) using a replacement column, and (d) after failure of the replacement column. See text for details.
The next step taken was to see if reference standards behaved the same as the sample. The peak for the reference standard shown in Figure 2a was obtained when the column was working well, whereas the peak in Figure 2b was obtained from a run made soon after that of Figure 1c. It is clear that the problem is not unique to the sample.

The Method When I am presented with a problem such as this, I like to look carefully at the analytical method to see if any red flags appear. The method was ion pairing, using one of the sulfonic acid ion-pairing reagents at a concentration of 10 mM as the A solvent, with the pH adjusted to 2.8 with phosphate buffer. The B solvent was methanol. The isocratic portion of the run was 20% B. At the end of the run, the column was flushed with 60% methanol to remove late-eluted materials. The column was a 250 mm × 4.6 mm C18 "aqueous phase" column packed with 5-mm particles and thermostated to 40 °C with a flow rate of 1 mL/min.

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Source: LCGC North America, 3/1/2005
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