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How is UV - Vis used for Quantitative Analysis? Routine quantitative analysis can be performed easily and rapidly using UV-Vis spectroscopy, and is used by a number of industries for such a task. Quantification is possible only if a calibration is performed and the standard analyte concentration range spreads over all possible concentrations of the real world samples. If this is not the case, extrapolation must be performed, and at high concentrations this is problematic as Beer-Lambert's law applies for low concentrations only. Step 1: Take a full UV-vis scan of your analyte. You will need an analytically pure sample of the material you wish to analyse. A typical scan range is from 600nm to 200nm.
Fig.1:Full UV scan of pure analyte. Above the full scan shows that 360nm is a good candidate for calibration, so the instrument is switched from scanning mode to target mode and set to 360nm. The samples to be tested are going to be in the range of 0.5 to 1.0mM, so a set of calibration standards are produced, from 0.4 to 1.2mM (to cover any outlier samples.) and run with several repetitions of each concentration (the mean averages being taken as the result). Step 2: A calibration curve is produced from the resultant data (shown below).
Fig.2: Calibration of analyte at 360nm A line of best fit can be applied to the calibration, and an equation from that line will link your absorbance to the concentrations (alternatively you cant do it less accurately by drawing your sample absorbance on your calibration where the absorbance crosses the calibration line, to find the concentration.) Step 3: Thus now you run all of your test samples, and determine their actual concentrations from the calibration plot. This method for quantification (via a calibration model) is applicable to many techniques (not just UV-vis), and thus is a very important technique for analytical chemistry.
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