UV Absorbance

Absorbance is the logarithm of the ratio of the intensities of the incident light (Io) and the transmitted light (I). It is related according to the Beer-Lambert Law to the molar absorptivity (molar extinction coefficient, e), the thickness of the substance (i.e., the path length of the cell, b) and the molar concentration of the substance (c):

In HPLC, the photodetector measures transmitted light I, but the electronics converts this signal to a logarithmic relationship (A) which is proportional to concentration.

The ordinate of the chromatogram represents the detector signal, which in general, is proportional to the analyte concentration in the cell. Since chromatographic systems permit the quantitative analysis of sample components representing many orders of magnitude - from ppm to percent concentrations - one may select, various amplification ranges so that the visual display of components (both small and large).

Table. Molar Absorptivity (e) Values of Various Compound Types at Specified Wavelengths.
Name Chromophore Wavelength [nm] Molar extinction, e
acetylide -C=C 175-180 6,000
Aldehyde -CHO 210 1,500
amine -NH2 195 2,800
azo -N=N- 285-400 3-25
bromide -Br 208 300
carboxyl -COOH 200-210 50 - 70
disulphide -S-S- 194 5,500
ester -COOR 205 50
ether -O- 185 1,000
ketone >C=O 195 1,000
nitrate -ONO2 270 12
nitrile -C=N 160 -
nitrite -ONO 220 - 230 1000-2000
nitro -NO2 210 strong

In UV detection, one expresses the detector range in absorbance units (A). One absorbance unit correspond to the depreciation of the light intensity by 90% of the incident light.

Molar Absorptivity. This term (e) - also called the molar extinction coefficient - corresponds to the absorbance for a molar concentration of the substance with a path length of 1 cm. Molar adsorptivity is dependent on the wavelength and chromatographic conditions, (solvent, pH and temperature). It is a constant at a specified wavelength.

Table above lists the molar absorptivities of a number of compounds at specified wavelengths. The knowledge of these values is important because they indicate the wavelengths to be selected for maximum response.