HPLC Experiment

EXPERIMENT VIII HIGH PERFORMANCE LIQUID CHROMATOGRAPHY SEPARATION AND ANALYSIS OF MIXTURES These files are in Adobe Acrobat format, if you are using Netscape Navigator or Internet Explore r and have Adobe Acrobat Reader instal led (If you do not; Acrobat Reader can be downloaded for free from Adobe) these files should open directly in your browser. INTRODUCTION Increasingly, the determination of low concentrations of active ingredients (either desired or undesired) in complex mixtures, sold for human consumption, has become more necessary. Federa l regula tio ns have imp ose d str ict limi ts on the type and concentra tions of a hos t of substances sold as foods or drugs. Such requirements demand analytical techniques t hat are fast and reliable and combine the separation (to alleviate interferences) and analysis steps in a single operation. Chromatography is the most widely used technique for the analysis of non-inorganic mi xt ures. Ga s chro ma togr aphy (where the sa mple must be vola ti li ze d) and li quid chromatography (where the sample can be determined in the liquid state) are the most common metho ds in general use. High Perfo rmanc e Liquid Chromat ography (HPLC) is the method of choice whenever the sample cannot easily be converted to the gas phase. In this la bor at or y, you wi ll use the techni que of HPLC to determine ei ther the concentration of caffeine in a soft drink, coffee or tea, or caffeine in one of a variety of analgesic (pain relief) formulations. THEORY Fi gu re I de pi cts th e ma in co mp onents of a modern HPLC sy ste m an d the ir interrelationships. In HPLC, a solution containing the compound(s) of interest is injected into a loop which has been calibrated to contain a specified volume (a 20 µL loop injector is a commonly used size). The valve switch is then rotated, allowing a sample stream of mobile phase (the eluent) to sweep the sample from the loop onto a column, packed with a suitable stationary phase, where the separation occurs. The eluent is deli vered from a pump at a constant rate, (on the order of 1 mL/min) at a pressure sufficiently high to overcome the backpressure of the column. Pressures of 1000-2000 psi are commonly necessar y. An upper limit of 4000 psi is normally set on the instrument. Recall that the separation efficiency is inversely proportional to the parti cle size of the column packing mate rial . High pres sures are requi red to force a liquid through a tightly-packed column filled with small particle material, and the availability of high pressure solvent delivery systems is di rectly responsible for the "high performance". Assuming that a suitable column has been chosen for the separation of interest, all components should pas s through the column and "el ute " at dif ferent times (di ff erential migration). This time differential is due to the differences in the distribution (partitioning) of the vario us compon ents between the mobil e phase (eluent) and stat ionar y phase (column packing), which arise from the physical/chemical differ ences among the components of the mixture. Thus, each component will pass through the detector and be identified separately.

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HPLC Experiment