Chapter 4. Spectrophotometry and protein concentration measurements

by András Málnási-Csizmadia

Table of Contents

4.1. Photometry
4.2. The UV-VIS photometer
4.3. Other possible uses of photometry
4.4. Frequently arising problems in photometry
4.5. Determination of protein concentration
4.5.1. Biuret test
4.5.2. Lowry (Folin) protein assay
4.5.3. Bradford protein assay
4.5.4. Spectrophotometry based on UV absorption
4.6. Spectrophotometry in practice: some examples
4.6.1. Absorption spectrum of ATP
4.6.2. Hyperchromicity of DNA
4.6.3. Absorption spectra and molecular structure of NAD and NADH
4.6.4. Absorption spectrum of proteins
4.6.5. Determination of the purity of DNA and protein samples
4.7. Fluorimetry
4.7.1. Physical basis of fluorescence
4.7.2. The fluorimeter
4.7.3. Fluorophores
4.8. Appendix
4.8.1. Fluorescence, phosphorescence and chemiluminescence
4.8.2. Photobleaching
4.8.3. Fluorescence anisotropy and circular dichroism
4.8.4. Quenching and FRET

4.1. Photometry

Spectrophotometry is one of the most widely used analytical procedures in biochemistry. The technique is well suited for simple routine determination of small quantities of materials. These measurements require that the examined material have an absorption maximum at one point of the spectrum. If the absorption maximum falls into the visible part of the spectrum, the material is coloured. Analyses can be performed in the visible spectrum also if the material in question has no colour by itself, but a chemical reaction can be performed that leads to the formation of a coloured product. Analyses in the ultraviolet spectrum are widespread, too, since many colourless materials exhibit intense absorption in this range (190-320 nm).

Quantitative measurements in spectrophotometry are based on the Lambert-Beer law of light absorption by solutions. The amount of light absorbed by the sample is defined as the ratio of the intensities of the incident and the transmitted light (Figure 4.1).

Incident and transmitted light

Figure 4.1. Incident and transmitted light. Light absorbed by the sample is characterised by the ratio of the intensity of the incident and transmitted light.

From the degree of absorption, the concentration of the absorbing component can be deduced by using the following equation. If the intensity of light, I0, diminishes to I after passing through a path of length L in a medium, then, according to the Lambert-Beer law:


The expression log I0/I is called extinction (E), absorbance (A) or optical density (OD). At a wavelength where the solvent does not absorb, according to the Lambert-Beer law, E will be proportional to the concentration of the solution (c) if the solute does not undergo molecular changes during dilution. The law is valid only for monochromatic light of a given wavelength. The degree of the absorption is characterised by ε, named extinction coefficient, a constant independent of the concentration of the solute. If the concentration is given in mol/L, then we speak of a molar extinction coefficient or molar absorptivity. Molar absorptivity gives the extinction of a solution of 1 mol/L (M) at the given wavelength and 1 cm path length; its unit is thus M-1cm-1. Its value is characteristic of the material in question, but also depends on the solvent and the temperature.

Using the above equation, the concentration can be calculated from the extinction as:


If ε is the molar extinction coefficient, then c will be molar concentration. L is the optical path length of the cuvette (a transparent cell that holds the sample, usually made of glass, plastic or quartz), given in centimetres.

If the material to be measured does not follow the Lambert-Beer law exactly, a calibration curve can be established using a series of samples of increasing concentration, established using another exact method of measurement. The technique can then be used with the appropriate corrections.

Older photometers may measure the transmission (transmittance):

T = I/I0 or T % = I/I0x 100


The relation of transmittance to extinction is (see also Table 4.I below):



T %


98 %


79 %


32 %


10 %


1 %


0.1 %

Table 4.I. Some examples of the relation of extinction (E) to transmittance (T %). Note that an extinction value of 2 means that the sample absorbs 99 % of the incident light. At such a high level of absorption, the intensity of the light reaching the detector is very small. Thus, the accuracy of the measurement diminishes in the case of highly absorbent samples.