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1 EXPERIMENT 10 PROCESSING LIQUID CHROMATOGRAPHIC DATA A. PURPOSES Principal purposes of this experiment are a) to illustrate peak-height and peak-area data-processing options for liquid chromatography and b) to illustrate the use of internal standards to compensate for changes in experimental conditions. B. SIGNIFICANCE Analytical problems involving foods, body fluids, pharmaceutical preparations, etc. involve complex mixtures of compounds. Invariably it is necessary to separate components of interest from other components before making measurements. Liquid chromatography is one of the most powerful separation methods available for both qualitative and quantitative applications. This experiment focuses on quantitative aspects of liquid chromatography. The two most common data-processing options for quantitative applications of liquid chromatography involve peak heights and peak areas, with each having some potential advantages over the other. This experiment illustrates these two data-processing options. A common problem with complex samples involves uncontrolled effects of sample matrices on peak-height and peak-area responses. One method commonly used to compensate for differences between samples and standards involves the use of an internal standard. Applications of internal standards usually involve addition of a known amount of a component similar to the component of interest. If the internal standard responds to changes in experimental conditions in the same way as the component of interest, then the internal standard can be used to reduce effects of changes in experimental conditions. The usual procedure is to use ratios of responses rather than absolute responses to calculate concentration. C. ILLUSTRATIVE DATA Two groups of chromatographic data are provided. One data group involves chromatograms for six concentrations of tartaric acid separated from synthetic mixtures of several other carboxylic acids. The other group of data is for tartaric and oxalic acids separated from synthetic mixtures of several carboxylic acids. The first group of data is used to illustrate calibration plots of peak height and peak area vs. concentration. To obtain peak-height data, it will be necessary only to identify the position (time) of each peak and to read peak heights directly from data for absorbance vs. time. To obtain peak area, it will be necessary to integrate the data and extract peak area from the results. The second group of data is used to illustrate the use of an internal
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This note was uploaded on 02/16/2012 for the course CHEM 125 taught by Professor Na during the Fall '11 term at Purdue University-West Lafayette.

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