Help on: Developing a HPLC Method for Veterinary Drugs

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Question:
“I am trying to develop a HPLC method for some veterinary drugs. I am using a C18 column 250mm x 4.6 mm, 5u particle size. The mobile phase I’m using has a composition MeOH: 5% GAA in water, 98:2 (v/v). During the analysis I'm observing that between analyses of the same sample, the RT for the drug shifts in each run. The RTs for 5 successive injections are: 8.1 min, 8.2 min, 8.3 min, 8.4 min and 8.2 min.”

Answer:
“The retention time shifts that you are experiencing could be due to a number of different factors, the most common three being variation in temperature (are you controlling the column temperature?), insufficient column equilibration (prior to any injections and between injections), and changes in mobile phase composition (how are you mixing the mobile phase? If it is premixed this is unlikely to be the source of the retention time variation).

Regarding your choice of mobile phase, the proportion of organic is very high at 98% methanol. The analytes must interact very strongly with the column. Have you considered using a less retentive phase, such as C8? This may reduce your consumption of organic solvent and give you more room to manoeuvre when selecting optimum conditions for your method.”