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Spectrophotometry principle

The spectrophotometer is set only to measure at a certain wavelength; this wavelength can be adjusted so that the optimal wavelength for measuring the specific compound is used. The spectrophotometer displays the so-called absorbance (A), which is calculated as log(It/I0), where I0 is the intensity of the incident light, and It I 0 is the intensity of the incident light, and I t is the intensity of the light that passes through the solution and is measured by the spectrophotometer. There is a linear relationship between absorbance and concentration; this relationship is described by the Lambert-Beer Law as follows:

A = log (I0 / It) = ε • c • l Absorbance is equal to the logarithm of I 0 divided by I t, which is also equal to epsilon times c times l

where c is the concentration of the solution, l is the length of the solution (through which the light has to pass) and ε is the extinction coefficient, which is specific for a compound. [1]

A spectrophotometer consists of the following (Fig. 1):

  • a light source

  • a wavelength selector

  • a sample container (e.g., a cuvette)

  • radiation transducers

  • a detector

The black straight line goes from the light source, presented as a bulb, towards the tip of a blue pyramid, named monochromator. From there, the same straight line, now named I zero, bends to be horizontal, and goes through a thick, black, vertical line, named adjustable aperture. Behind the aperture, the vertically aligned rectangle named cuvette, with green solution, named sample, is placed. The I zero line passes through the cuvette with the sample, changing name to I, and ending at an elliptical structure, named photoresistor. Photoresistor is connected to another triangle, named amplifies, which in turn is connected to a yellow rectangle, inside which the output is displayed, equal to 0.260 A.

Fig. 1. Light emitted from the source passes through the slit, letting only one particular wavelength through. This light will pass through the sample placed in a cuvette and will be measured by the detector.

For example, we will measure concentrations of NADH. NADH has an absorption maximum at 340 nm; therefore, the spectrophotometer is set to measure at this wavelength. The extinction coefficient for NADH is calculated as follows: ε =6220 M-1cm-1. The length of the solution is usually set to 1 cm.

There are certain limitations of the Beer-Lambert's Law that the investigator needs to take into consideration. Some of these are related to technical issues; however, the law does have a real limitation, because it only applies to dilute solutions. When the concentration of an absorbing species increases, so does the physicochemical interactions among the molecules. Thus, at a given concentration, the molecules will begin to affect the charge distribution of the neighboring molecules; when this occurs, the relationship between absorbance and concentration is no longer linear. As a rule of thumb, one should stay below an absorption value of 1 when doing measurements.

Notably, absorbance is inversely proportional to what is known as transmittance (see a textbook for details); at an absorbance value of 1, 10% of light is transmitted through the sample; at 2, 1% of light is transmitted, and so on in a logarithmic trend.

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Spectrophotometry