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How is a UV-vis spectrum obtained? Below is a general explanation of how a UV-vis spectrum is obtained. It may vary from instrument to instrument, and also depending on what techniques are being used, however the basic ideas are the same. The instrument is set to scanning mode, meaning that it scans through monochromatic UV light, one wavelength at a time (typically 10 wavelengths per second). A routinely used scan range is between 600 and 200nm. The sample dissolved in appropriate solvent (one with very little UV absorbance, unless using a double beam instrument, or taking blanks) and is placed in a clean, dry quartz cuvette (note that a UV cuvette has 2 clear sides, and 2 hatched sides. Hold the cuvette only by the hatched sides, as any fingerprints or residues that get on the clear side will influence your scan). The cuvette is placed into the UV-vis spectrophotometer sample compartment with the clear sides inline with the light beam. on a double beam instrument, place a blank (a cuvette filled with just the solvent) in the blank beam slot. If using a single beam device, you may be asked to put a blank sample on before putting in your actual sample, so the computer can do the necessary spectral subtractions. Close the compartment and press run... the following occurs:
Fig.1: the UV-vis irradiation Above the spectrophotometer supplies a beam of UV and visible light, across the wavelength range requested, at a constant intensity (above denoted Q). As the light goes through the cuvette full of sample, some frequencies are absorbed by the molecules to promote electrons to higher orbitals (as already discussed). This means the UV that permeates the other side of the cuvette which is collected by the detector is less intense than the incident beam at certain wavelengths (as demonstrated in the right hand image). the spectrophotometer simple notes how much of each wavelength is missing compared to the incident ray, and converted that into an absorption plot (as below)
Fig.2: the absorbance spectrum for your sample The absorbance plot is simply the inverse of the plots in Fig.1 (sometimes called the transmission plots). Lambda max (λmax) is the wavelength of the greatest absorbance, and is used for the calibration of the sample for quantitative analysis.
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