Spectroscopy Methods

The techniques related to these regions of the spectrum are probably the foremost widely used for analytic work.The molecular substructures that are liable for interacting with the electromagnetic wave are called chromophores. In proteins, the relevant types in UV/Vis spectroscopy are peptide bonds, certain aminoalkanoic acid side chains primarily tryptophan and tyrosine and certain prosthetic groups and coenzymes, porphyrin groups present in haem. Colorimetric assays require a calibration curve to be plotted concentration versus absorbance) which should be linear as long because the Beer–Lambert law applies. Using this, absorbance of unknowns is then measured and their concentrations are often interpolated from the linear region of the plot. Qualitative analyses are often wont to identify certain classes of compounds both as pure samples and in biological mixtures. This sort of spectroscopy is most ordinarily used for quantification of biological samples either directly or via colorimetric assays. In many cases, proteins are often directly quantified using their intrinsic chromophores, tyrosine and tryptophan. Protein spectra are acquired by scanning from 500 to 210 nm. The characteristic features during a protein spectrum are a band at 278/280 nm and another at 190 nm. The region from 500 to 300 nm provides valuable information about the presence of any prosthetic groups or coenzymes.