What is a Spectrophotometer?

- Jul 18, 2019-

A spectrophotometer is one of the scientific instruments commonly found in many research and industrial laboratories. Spectrophotometers are used for research in physics, molecular biology, chemistry, and biochemistry labs. Typically, the name refers to Ultraviolet-Visible (UV-Vis) Spectroscopy.

The energy of light is dependent on its wavelength, usually designated as lambda. Although the electromagnetic spectrum extends over a tremendous range of wavelengths, most laboratories can only measure a small fraction of them. UV-Vis Spectroscopy measures between 200 and 400 nanometers (nm) for UV light measurements, and up to approximately 750 nm in the visible spectrum.

For UV-Vis Spectroscopy, samples are usually contained and measured in small containers called cuvettes. These can be plastic if used in the visible spectrum, but need to be quartz or fused silica if used for UV measurements. There are some machines that can utilize glass test tubes.

Visible Spectroscopy is often used industrially for colorimetry. Using this method, samples are measured at multiple wavelengths from 400-700 nm, and their profiles of absorbance are compared to a standard. This technique is frequently used by textile and ink manufacturers. Other commercial users of UV-Vis Spectroscopy include forensic laboratories and printers.

In biological and chemical research, solutions are often quantified by measuring their degree of light absorption at a particular wavelength. A value called the extinction coefficient is used to calculate the concentration of the compound. For instance, molecular biology laboratories use spectrophotometers to measure the concentrations of DNA or RNA samples. They sometimes have an advanced machine called a NanoDrop™ spectrophotometer that uses a fraction of the amount of sample compared to that used by traditional spectrophotometers.

For quantification to be valid, the sample must obey the Beer-Lambert Law. This requires that the absorbance be directly proportional to the path length of the cuvette and the absorption of the compound. There are tables of extinction coefficients available for many, but not all, compounds.

Many chemical and enzymatic reactions change color over time, and spectrophotometers are very useful for measuring these changes. For instance, the polyphenol oxidase enzymes that cause fruit to brown oxidize solutions of phenolic compounds, changing clear solutions to ones that are visibly colored. Such reactions can be assayed by measuring the increase in absorbance as the color changes. Ideally, the rate of change will be linear, and one can calculate rates from this data. A more advanced spectrophotometer will have a temperature-controlled cuvette holder to carry out the reactions at a precise temperature ideal for the enzyme.

Microbiological and molecular biology laboratories frequently use a spectrophotometer to measure the growth of cultures of bacteria. DNA cloning experiments are often done in bacteria, and researchers need to measure the growth stage of the culture to know when to carry out certain procedures. They measure the absorbance, which is known as the optical density (OD), on a spectrophotometer. One can tell from the OD whether the bacteria are actively dividing or whether they are starting to die.

Spectrophotometers use a light source to shine an array of wavelengths through a monochromator. This device then transmits a narrow band of light, and the spectrophotometer compares the light intensity passing through the sample to that passing through a reference compound. For instance, if a compound is dissolved in ethanol, the reference would be ethanol. The result is displayed as the degree of absorbance of the difference between them. This indicates the absorbance of the sample compound.

The reason for this absorbance is that both ultraviolet and visible light have enough energy to excite the chemicals to greater energy levels. This excitation results in a higher wavelength, which is visible when the absorbance is plotted against wavelength. Different molecules or inorganic compounds absorb energy at different wavelengths. Those with maximum absorption in the visible range are seen as colored by the human eye.

Solutions of compounds can be clear, but absorb in the UV range. Such compounds usually have double bonds or aromatic rings. Sometimes there are one or more detectable peaks when the degree of absorption is plotted against wavelength. If so, this can aid in the identification of some compounds by comparing the shape of the plot against that of known reference plots.

There are two types of UV-Vis spectrophotometer machines, single-beam and double-beam. These differ in how they measure the light intensity between the reference and test sample. Double-beam machines measure the reference and test compound simultaneously, while single-beam machines measure before and after the test compound is added.