Thus, for a fixed path length, UV/Vis spectroscopy can be used toĭetermine the concentration of the absorber in a solution. The concentration of the absorbing species in the solution and the path
States that the absorbance of a solution is directly proportional to While charge transfer complexes also give rise to colours, the colours are often too intense to be used for quantitative measurement.Tyrosine,įor example, increases in absorption maxima and molar extinctionĬoefficient when pH increases from 6 to 13 or when solvent polarity PH can affect the absorption spectrum of an organic compound. UV absorption not all solvents are suitable for use in UV spectroscopy.Įthanol absorbs very weakly at most wavelengths.) Solvent polarity and The solvents for these determinations are often water for water-soluble compounds, or ethanolįor organic-soluble compounds. Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum.For instance, the colour of a dilute solution of copper sulfate is a very light blue adding ammonia intensifies the colour and changes the wavelength of maximum absorption (λ max). Presence of other species, such as certain anions or ligands. The colour of metal ion solutions is strongly affected by the Within the metal atoms can be excited from one electronic state toĪnother. Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons.Spectroscopic analysis is commonlyĬarried out in solutions but solids and gases may also be studied. UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, highly conjugated organic compounds,Īnd biological macromolecules. However, if after making the solutions a few times the calibration is still poor, something may be wrong with the instrument for example, the lamps may be going bad. If the correlation coefficient is lower than that, try making the solutions again as the problem may be human error. The correlation coefficient of an acceptable calibration is 0.9 or better. To make a calibration curve, the value for the absorbances of each of the spectral curves at the highest absorbing wavelength, is plotted in a graph similar to that in Figure 6 of absorbance versus concentration. An example of absorbance spectra of calibration solutions of Rose Bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein, Figure 4) can be seen in Figure 5. The calibration solutions should be spaced relatively equally apart, and they should be made as accurately as possible using digital pipettes and volumetric flasks instead of graduated cylinders and beakers. The concentrations should start at just above the estimated concentration of the unknown sample and should go down to about an order of magnitude lower than the highest concentration. To make a calibration curve, at least three concentrations of the compound will be needed, but five concentrations would be most ideal for a more accurate curve.