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In this lab, we are trying to determine the concentration of caffeine in caffeinated beverages using...

In this lab, we are trying to determine the concentration of caffeine in caffeinated beverages using high pressure-liquid chromatography (HPLC) and UV absorbance detector. Why is it important that the pressure remain constant during an HPLC run?

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HPLC involves separation of components of mixture by passing them through a column under high pressure. In order to achieve high and even separation the columns are made up of small particles. For passing mobile phase through these columns very high pressure is required. If this pressure is not constant during HPLC run, then the retention times obtained will be erratic. It will not be possible to obtain same retention time reproducibly. Further, baseline will be noisy and there will be spikes in chromatogram, resulting in poor chromatographic separation. Therefore, it is important to maintain constant pressure during HPLC run.

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