Chromatographers frequently have to identify and rectify problems that can be divided in different categories. In this guide, we will discuss some of the most common issues that may appear and how to solve them. Emphasis is on reversed phase separation. Often problems can be avoided by routine maintenance (e.g. planned replacement of worn out parts). Simple rules are useful for classifying deficiencies and can help in avoiding follow-up mistakes.
Retention
Small differences in mobile phase composition may cause huge differences in retention time when the column is overloaded and this also changes with temperature. However, even if the mobile phase is buffered and the pump is working properly, the retention times may fluctuate if the pH is too close to the pK of the sample substance. The pH of the mobile phase should therefore be chosen to be at least one pH unit above or below the pK value of the analytes being separated. Retention time drift indicates insufficient column conditioning. With increasing column life, the retention times may shift towards less retentivity, especially if the user is working at acidic pH (≤ pH 2). Abrupt changes in retention time are usually due to errors in the system.
Problem: Changing retention times
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