Spectrophotometry

What is spectrophotometry used for?

Spectrophotometry presents a quantitative way of determining the concentration of molecules in a solution, where light absorbance is directly linked to the composition of the substance. It can also be used to monitor the progression of enzymatic reactions based on changes in absorbance. Such enzymatic tests are common in food analysis, where spectrophotometry is used to determine the concentrations of lactose and phytic acid.

Spectrophotometry can also be used for quality control purposes to ensure that the visible color of a product stays within an appropriate range even after time passes or the product is exposed to external influences. This is useful in determining the yellowness and lightfastness of paper and board.

How does a spectrophotometer work?

Inside the spectrophotometer, a light source and a monochromator produce a light beam with a controlled wavelength. This light beam is passed through the sample, which is usually a solution contained inside a transparent cuvette. The sample will absorb specific wavelengths of light, depending on its chemical and physical properties, and transmit or reflect the rest of the spectrum. The remaining light is picked up by a photodetector and recorded. 

Data collected with the spectrophotometer can then be used to produce a plot showing which wavelengths were absorbed and by how much, which provides information on the concentrations of chemicals present, as well as quantitative data on the colors they appear. Information on some chemical aspects, such as levels of conjugation and conformation, can also be obtained.

UV-Vis spectrophotometry

The most common form of spectrophotometry is specific to the ultraviolet and visible sections of the electromagnetic spectrum and is commonly known as UV-Vis spectrophotometry. Colored samples inherently absorb sections of visible and UV light, which makes this analysis applicable to a wide range of chemical species. Some white sample types also absorb light in the UV region, especially conjugated organic compounds.

Suitable samples and sample preparation

Spectrophotometry has traditionally focused on testing solutions, which have to be placed in suitable cuvettes for analysis. The type of cuvette depends on the wavelength of light used (i.e. quartz for ultraviolet, glass for visible). Solid samples can also be tested using special spectrophotometers, such as a color spectrophotometer. In such cases, the sample should usually have a relatively flat surface for light to reflect off of.

Advantages and limitations

Spectrophotometry is a straightforward technique that provides consistently accurate results on a wide variety of sample types. Once samples are prepared, they can be tested relatively quickly, and the analysis is generally non-destructive.

Limitations of spectrophotometry include a relatively low sensitivity and selectivity. It may thus be difficult to detect very low concentrations of an analyte or distinguish the analyte from other substances that absorb light on the same wavelength.

Spectrometry vs spectrophotometry

Spectrometry is a commonly used term that describes a variety of different analytical techniques. Specifically, it refers to any method of analysis that collects a spectrum of data. This means that while it can be used to describe spectrophotometry, it is also used to describe unrelated techniques like high-resolution mass spectrometry and nuclear magnetic resonance spectrometry.

Spectrophotometry, on the other hand, specifically refers to techniques where a sample is exposed to varying wavelengths of electromagnetic radiation, most commonly in the visible and adjacent parts of the spectrum.

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