Question

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 was a dector wavelength of 284 nm chosen? How would changing the dector wavelength to 190 nm impact the sensitivity of the method for caffeine?

Answer 1

Ans:

High performance liquid chromatography (HPLC) makes use of a high pressure pump to deliver a mobile phase solvent at a uniform rate at pressures.

The most obvious advantage of HPLC over gravity liquid chromatography is that samples can be separated much more quickly. In addition, samples that are not volatile or that would thermally decompose in gas chromatography can be rapidly and routinely separated in HPLC.

HPLC allows for rapid separation and quantitation of caffeine from the many other substances found in these beverages including tannic acid, caffenic acid, amino acids, sucrose etc.

In the extract of caffeinated beverages around 176 peaks may be identified and these compounds respond to wavelength of 284 nm.

In case of changing the detector wavelength to 190 nm total peaks may not be identified which will result wrong concentration of caffeine in caffeinated beverages.