For accurate results, the instrument's response to the analyte in the unknown should be compared with the response to a standard this is very similar to the use of calibration curves. The presence of an analyte gives a response assumed to be proportional to the concentration. This can be taken from references (tables of molar extinction coefficients), or more accurately, determined from a calibration curve.Ī UV/Vis spectrophotometer may be used as a detector for HPLC. It is necessary to know how quickly the absorbance changes with concentration.

Thus, for a fixed path length, UV/Vis spectroscopy can be used to determine the concentration of the absorber in a solution. The Beer–Lambert law states that the absorbance of a solution is directly proportional to the concentration of the absorbing species in the solution and the path length.

While charge transfer complexes also give rise to colours, the colours are often too intense to be used for quantitative measurement.

Tyrosine, for example, increases in absorption maxima and molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity decreases. Ethanol absorbs very weakly at most wavelengths.) Solvent polarity and pH can affect the absorption spectrum of an organic compound. (Organic solvents may have significant UV absorption not all solvents are suitable for use in UV spectroscopy. The solvents for these determinations are often water for water-soluble compounds, or ethanol for 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). The colour of metal ion solutions is strongly affected by the presence of other species, such as certain anions or ligands.

Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons within the metal atoms can be excited from one electronic state to another.

Spectroscopic analysis is commonly carried 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, and biological macromolecules.

Chem draw uv spectra