Sept. 1, 2021
Aug. 17, 2021
Spectrophotometers have come a long way from the first model invented in 1940. Miniaturized and combined with other modern technology, these devices now have a wide range of research and practical applications in food, medical, industrial, and environmental fields. This article focuses on the reasons for the popularity of this technique.
Light consists of electromagnetic radiation with a wide range of frequencies, wavelengths, and energies that all travel at the same speed. This includes the visible spectrum, as well as radiation of longer and shorter wavelengths on either side of the band; see Figure 1.
Light is split when it strikes an object, as some parts of it are absorbed and the rest is reflected or transmitted. The kind of interaction depends on the wavelengths of the light and also the chemical composition of the object. The resultant distribution in terms of frequency and wavelengths is called a spectrum. The spectrum of light that objects emit gives us information about their makeup. For example, Figure 2 shows the different spectra produced by common elements.
You can see this phenomenon occurring in everyday life. Leaves look green because chlorophyll absorbs blue and red light, and it emits the remaining light made of green wavelengths.
Spectrophotometry is defined as the quantitative measurement of the intensity of light of various wavelengths in the spectrum emitted by matter.
The quantitative measurement of the interaction of matter with specific wavelengths has important applications in many fields of science, including physics, chemistry, astronomy, and biochemistry.
Spectrophotometry may seem similar to spectroscopy.
However, there is a vital difference between spectroscopy vs. spectrophotometry. Spectroscopy is used to analyze matter based on the wavelengths produced in the spectrum, while spectrophotometry analyzes matter based on the intensity of light in each wavelength involved in light’s interaction with matter (absorbance, reflectance, transmittance).
Thus, spectrophotometry is based on spectroscopy and can be called an application of the latter.
Spectrophotometry applications are useful to measure the absorbance, reflectance, and transmission of light by gases, liquids, and solids.
A spectrophotometer measures the number of photons emitted to estimate the intensity of light spectra absorbed and transmitted by a sample. This provides information on the amount of a compound in the sample. For example, clear water will allow more light to pass through than a solution colored with pigments, which will absorb more of the light in many wavelengths.
The light band that each compound will absorb will differ. For example, Figure 3 shows that chlorophyll a and b absorb many wavelengths, but chlorophyll a absorbs more violet and orange light, while chlorophyll b absorbs more blue and yellow wavelengths.
While the size and design of a spectrophotometer can differ, they each consist of a few basic parts, (see Figure 4):
There are two spectrophotometer types based on the light band chosen for analysis:
There are two kinds of spectrophotometers: single beam and double beam. See Figure 4 for schematic diagrams.
Double beam spectrophotometers compare the light intensity of the spectrum from a sample to a reference beam. Applications that require stability, speed, and automation rely on double beam spectrophotometers and are expensive. These typically have similar or better precision than single beam spectrophotometers.
Single beam spectrophotometers are cost-effective compared to double beam variants and have the potential to perform better, as they do not need to expend energy splitting the beam. However, these devices are less stable than their double beam counterparts. Moreover, they require more work, as users must provide a reference to standardize the device before using it.
Besides the wavelength of light, the absorbance of a spectrophotometer is influenced by the amount of a compound in a solution, as well as the size of the cuvette. Two laws define these two aspects: Beer's Law and Lambert's Law. These laws are considered the principles of spectrophotometry.
Lambert’s Law states that there is a direct but non-linear relationship between the length of the light path through the cuvette/sample (l) and the intensity of light transmitted, as shown in Figure 5. Io is the intensity of light before it enters the sample, and It is the intensity of light after it has passed through the sample.
So Transmittance (T) = It / Io
Beer’s law states that the light absorbance depends on solute concentration (c).
The combination of the two laws called the Beer-Lambert Law states that absorbance depends on solute concentration (c), its molar absorptivity or absorption coefficient (ϵ), and length of light path (l).
So, Absorbance (A) = ϵlc
Absorbance and transmittance have no spectrophotometric units of measurement.
However, the light path length (l) is measured in cm, and ϵ is measured in L·mol-1·cm-1.
Usually, the spectrophotometer cuvette width (or light path length) is 1 cm, and the molar absorptivity of a solute is known, so based on the absorbance reading, the device can calculate the concentration or amount of solute (c) in the sample.
Spectrophotometery’s availability in small, portable, and affordable devices, is expanding use of this precise technique in both scientific and practical applications.
Liquid spectrophotometry was the first application of the technique. However, it is now also common for spectrophotometers to analyze opaque solids, including glass, and various films, such as those used in semiconductor manufacturing. Similarly, gas spectrophotometers are used to analyze air pollutants.
Spectrophotometry analysis has several functions:
These spectrophotometry analyses of organic and inorganic compounds have applications in the following:
Several branches of science and industry make use of the applications of spectrophotometry analysis, with the notable ones being
One of the most important and rapidly increasing branches of spectrophotometry applications is in the food supply chain. The technique is ideal for the determination of the organic compounds in ingredients and food mixtures.
NIR spectrophotometers are used for food analysis, as this light band targets the organic bonds formed between elements of a compound. These tools are simple to use and give rapid measurements of several constituents simultaneously. Moreover, as water has lower absorbance, NIR spectrophotometry can be used for analyzing food and ingredients with high water content, such as wine.
All the main stakeholders of the food chain - farmers, processors, distributors, retailers, and restaurants - can benefit from spectrophotometry analysis.
Uses of spectrophotometry can be found in practically all categories of food at every stage of the supply chain, as discussed below:
UV-vis spectrophotometers are used in the qualitative and quantitative estimation of DNA, RNA, and proteins. These applications are useful in identifying species and monitoring enzymatic reactions to determine the products formed and estimate rates of reactions.
There are several applications of spectrophotometers in the medical field. They can be used to diagnose diseases and analyze blood. Trials have established that hand-held spectrophotometers can be used for non-destructive diagnostics, such as
Spectrophotometric measurements are also used extensively in the pharmaceutical industry.
Both UV-vis and IR spectrophotometers are used to trace evidence in forensics. Micro-spectrophotometry is used to analyze impurities that are present in a materials such as textiles, hair, oil, etc., that are too minute to be examined even by microscopes. Similarly, spectrophotometric measurements can also identify cosmetics, inks, drugs, pesticides, fungi, etc. collected from evidence or as evidence.
Another application of spectrophotometric measurements is to estimate the age of bruises by studying the degradation of blood.
Currently, there are several major sources of water pollutants: chemicals and animal wastes from farms, oil and plastics, as well as industrial and untreated sewage discharges. These affect the quality of water in rivers and groundwater. Air pollutants come from burning agriculture and fossil fuels such as oil, gas, and coal.
Specific pollutants or aggregates can be easily analyzed qualitatively and quantitatively by UV-vis spectrophotometers to control and monitor the quality of water and air.
There are several applications of spectrophotometric measurements in industries.
Spectrophotometry is used as an analytical technique to find failures in the aerospace, chemical, oil, and gas industry by analyzing the metal alloys, such as iron and aluminum. For example, aluminum alloys are used in structural components in aerospace, so spectrophotometry can detect faults and weaknesses in structures.
Spectrophotometry is also used in the quality control of paint systems and cement.
UV-vis spectrophotometers are suitable for colorimetry applications in industries that use pigments, such as printing, textiles, or ink production.
Several sensors, devices, probes, or instruments use spectrophotometry to analyze biochemical and physical characteristics of solids and liquids. There is a wide and growing range of spectrophotometers on the market today. For example, the CI-710s SpectraVue Leaf Spectrometer is a NIR spectrophotometer used to study many physiological processes and detect stress in plants and whose novel application is the non-destructive quantification of chemical concentrations and color analysis in plants.
Thus, the application of spectrophotometry continues to develop rapidly into many new spheres.
Science Writer, CID Bio-Science
Ph.D. Ecology and Environmental Science, B.Sc Agriculture
Feature image courtesy of Neal Fowler.
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