A spectrophotometer measures the amount of light that a sample absorbs when a beam of light is made to pass through it. The intensity of light is measured by a detector that is placed after the sample. The beam of light consists of a stream of photons that have chance of getting absorbed by the sample, thus reducing the number of photons in the beam of light, thereby reducing the intensity of the light beam that reaches the detector.

A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected wavelength, and a photometer for measuring the intensity of light. The instruments are arranged so that sample liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. The photometer converts light into a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes.

There are two major kinds of spectrophotometers- single beam spectrophotometer and double beam spectrophotometer. A singe beam spectrophotometer measures the ratio of absolute light intensity and the double beam spectrophotometer measures the ratio of the light intensity of two different light paths. Although ratio measurements are easier, single beam instruments have advantages for they can have a larger range.

To use a single beam spectrophotometer, the machine is zeroed first, the wavelength is set, the blank is adjusted and then the sample is inserted and read. The wavelength is then adjusted by some determined interval, the zero is checked, the blank re-inserted and adjusted, and the sample re-inserted and read. This procedure continues until all wavelengths to be scanned have been read. In this procedure, the sample remains the same, but the wavelength is adjusted. Compounds have differing absorption coefficients for each wavelength. Thus, each time the wavelength is altered, the instrument must be recalibrated.

A dual beam spectrophotometer divides the light into two paths. One beam is used to pass through a blank, while the remaining beam passes through the sample. Thus, the machine can monitor the difference between the two as the wavelength is altered. These instruments usually come with a motor driven mechanism for altering the wavelength, or scanning the sample.

The newer version of an instrument scans a blank, and places the digitalized information in computer memory. It then rescans a sample and compares the information from the sample scan to the information obtained from the blank scan. Since the information is digitalized, manipulation of the data is possible. These instruments usually have direct ports for connection to personal computers, and often have built in temperature controls as well.  In these the voltage meter scale has given way to a CRT display, complete with graphics and built in functions for statistical analysis.

The most common spectrophotometer is used in the UV and visible regions of the spectrum and some of these instruments also operate into the near-infrared region. One major factor is the type of photo sensors that are available for different spectral regions. Usually, spectrophotometers use a monochromator to analyze the desired spectrum but there are also spectrophotometers that can use an array of photosensors.

The steps required in a spectrophotometer are as follows:

  • The light is projected into a monochromator.
  • A particular wavelength is selected and beamed at the sample in a cuvette.
  • The sample absorbs light.
  • The photo detector behind the sample responds to the light stimulus and converts it into an electronic current which is then transformed into a usable format.
  • The results are either plotted straight away or fed into a computer and manipulated in different presentable forms.

An infrared spectrometer directs infrared radiation through a sample and records the relative amount of energy absorbed by the sample as a function of the wavelength or frequency of the infrared radiation. The method is applicable particularly to organic materials, because the vibrational frequencies of the constituent groups within the molecules coincide with the electromagnetic frequencies of the infrared radiation. Therefore, the infrared radiation is selectively absorbed by the material to produce an absorption spectrum.

The spectrum produced is compared with correlation spectra from known substances. A sample cell for infrared spectrophotometry comprises a sample holder for holding a sample to be analyzed by infrared spectrophotometry, a cool air passageway and a vortex tube. The sample holder includes a primary optical surface through which infrared radiation is directed to a sample contained in the holder, and the cool air passageway is adjacent to the primary optical surface of the sample holder for directing a cool air stream across the primary optical surface. The vortex tube has a cool air outlet connected to the cool air passageway for supplying cool air to the passageway. Infrared spectrophotometry is most commonly used in studying the molecular structures of complex organic compounds.

The UV-Visible spectrophotometer uses two light sources, a deuterium (D2) lamp for ultraviolet light and a tungsten lamp for visible light. After bouncing off a mirror, the light beam passes through a slit and hits a diffraction grating. The grating can be rotated allowing for a specific wavelength to be selected. At any specific orientation of the grating, only monochromatic (single wavelength) successfully passes through a slit. A filter is used to remove unwanted higher diffractions. The light beam hits a second mirror before it gets split by a half mirror in which half of the light is reflected, while the other half passes through.

One of the beams is allowed to pass through a reference cuvette which contains the solvent only, while the other beam passes through the sample cuvette. The intensities of the light beams are then measured at the end. UV/Vis spectroscopy is routinely used in the quantitative determination of solutions of transition metal ions and highly conjugated organic compounds. Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible regions of the electromagnetic spectrum. The solvents for these determinations are often water for water soluble compounds, or ethanol for organic-soluble compounds. Ultraviolet spectrophotometry is particularly useful in detecting colourless substances in solution and measuring their concentration.

 Laws of absorption of energy

Two laws express the relationship between the absorption of radiant energy and the absorbing medium. According to Bouguer’s (or Lambert’s) law, each layer of equal thickness of the medium absorbs an equal fraction of the energy traversing it. According to Beer’s law, the absorptive capacity of a dissolved substance is directly proportional to its concentration in a solution.

The change in the intensity of light after passing through a sample should be proportional to the following:

  • Path length – the longer the path, more photons should be absorbed.
  • Concentration of sample – more molecules absorbing means more photons absorbed.
  • Intensity of the incident light – more photons means more opportunity for a molecule to see a photon.