April 18, 2022
March 17, 2022
Though spectrophotometry applications are used in industries for solids and gases, this technique was initially developed for liquid analysis. Liquid spectrophotometry is a non-destructive, rapid method of liquid analysis that can replace many cumbersome techniques. Liquid spectrophotometry is popular not only for food research, but throughout the food industry. An emphasis on quality and accountability requires a non-destructive, quick, and precise analysis method to get objective and comparable data to comply with accepted standards in global supply chains. Growing use of this technique can be additionally attributed to the availability of small, portable spectrophotometers, which have improved in recent years.
Spectrophotometry measures absorbance, reflectance, and transmittance of light, just like spectroscopy. However, while spectroscopy measures the interaction of matter with different wavelengths, spectrophotometry measures the intensity of light in the wavelengths interacting with matter.
Spectrophotometry estimates the number of photons in the light spectrum, which is absorbed and transmitted by matter. The light spectra usually used in spectrophotometers are visible (400-700nm), ultra-violet (185-400nm), and infrared (760nm to 1mm). Both inorganic and organic compounds can be analyzed by spectrophotometry. In the food industry the following bands are ordinarily used:
A spectrophotometer consists of two parts: one that produces light of a selected wavelength and a part that measures the light intensity of transmitted light. As Figure 1 shows, the liquid sample is placed in a cuvette between the light source and the photometer that measures light intensity.
Figure 1: Principle of liquid spectrophotometry, Rahman et al. (2020). (Image credits: DOI:10.31479/jtek.v1i8.62)
Light from a source is focused by a lens system to create a parallel beam that is directed to a monochromatic grating, which acts as a prism and separates the white light into its component wavelengths. By rotating the monochromatic grating, it is possible to choose a wavelength that is then passed through an exit slit, which directs the light to the sample in a cuvette. The light passing through the cuvette is measured by a photometer, which sends an electrical signal to a galvanometer that displays the results.
The amount of light that is absorbed by a liquid depends on the concentration and type of compounds in it. Hence, it is necessary to know which light color or wavelength will be absorbed by the sample. For example, liquid spectrophotometry is used for estimating hemoglobin, which absorbs blue and green light, making blood look red. So, absorbance of green or blue light is used to determine the concentration of blood in a sample. The difference in intensity of wavelengths that are transmitted or are passed through the solution is used to identify the components and their concentrations.
In transparent and translucent liquids, transmission is used in spectrophotometry. Turbid liquids, which are opaque, need reflectance to analyze samples.
In liquid analysis, the sample has to be held in a cuvette/container and often requires a reagent. Light from the exit slit, while traveling through the sample, will also have to pass through the cuvette and the reagent. Light will react with these two associated materials and their influence must be accounted for, as they are not fixed parts of the device but are changed frequently.
Water: In liquid spectrophotometry, water is important, as it is commonly used as a reagent. Water is also used as a blank, standard, or in-sample treatment. If water purity is not ensured, the accuracy of the analysis will be compromised.
Impurities in water could be organic compounds, ions, particulates, or even microbes. The purity of water that can be used for liquid spectrophotometry is, therefore, regulated by strict requirements, especially for sensitive measurements. Purity is quantified by levels of compounds and electrical resistance.
Containers: The cuvettes used for liquid spectrophotometry must have a standard width, as this is the length of the light path through the sample. This is usually 10 millimeters. The shape and size of the cuvettes will differ, depending on the minimum sample that is to be used. Similarly, the containers can be made of plastic or glass. The material will depend on the instrument and the wavelengths selected. For example, UV light needs cuvettes made of quartz glass.
Liquid spectrophotometry is used to determine the following sample traits:
By definition, opacity means the ability of a material to obstruct the transmission of light. At times, this can be interesting without delving deeper to find the compounds causing the opacity. The amount of light allowed to pass through depends on the concentration of the solute.
Figure 2: A non-contact imaging spectrophotometer is used to test the color of foundation on a participant’s foundation-coated cheek, applied with a brush applicator, Yan et al. (2020). (Image credits: https://doi.org/10.1002/col.22584)
Liquid spectrophotometers also measure color. This is used in scientific studies and industries for analyses of raw materials and finished products.
Color is also important in water quality or wastewater analysis. It can indicate the presence of microbes or pollutants like organic matter, oil, grease, industrial waste, or metals in water and wastewater.
Spectrophotometry can detect the structure of a chemical compound and can also detect microbes. This helps in content determination in liquids and has applications in the food industry, forensics, and water quality testing.
Of the traditional alternatives to liquid spectrophotometry, devices and methods to measure color and content are more abundant than opacity.
In the following traditional methods, before spectrophotometry became an established practice, color in liquids were measured by comparing samples with a standard:
Liquid spectrophotometry is far more sophisticated and objective than even colorimeter measurement as it gives direct spectral data. It is, therefore, also suitable for research and product development.
Figure 3: The opacity developed by Holker (1921). (Image credits: https://doi.org/10.1042/bj0150216)
A hundred years ago, a device called an opacimeter was developed by pathologist J. Holker to measure the opacity of liquids. It consisted of a long glass tube with a millimeter-scale etched on it, starting from the bottom at 20 to 250 at the top. At the bottom was a copper wire, which was placed over a light source.
When a turbid liquid was sampled, the wire was not visible from the top of the glass tube. Then, the tap was opened to lower the level of liquid in the glass tube. The millimeter reading, when the copper wire was again visible, was noted as the opacity expressed as 1/20 to 1/250 or 500x 10-4 to 40x 10-4.
Finding compounds, metals, or elements in a liquid usually requires complicated chemical experiments and assays in laboratories. The methods vary depending on the compound being tested, and the range is too vast to cover here. Microbes, on the other hand, require investigation through microscopes.
A single liquid spectrophotometer could identify many such compounds and microbes if the wavelengths it reacts with are known. All of these traditional methods are tedious, time-consuming, and require skilled staff.
Liquid spectrophotometry offers non-destructive, rapid, and precise analysis in real-time, making it suitable for repetitive and large-scale quantitative and qualitative analysis that is necessary these days in the food industry. Liquid spectrophotometry applications in the food industry use the technique to do the following:
The main liquid spectrophotometry applications are for
Controlling quality by liquid spectrophotometry uses all three properties of the technique and is replacing more complex analysis techniques, such as mass spectrometry and liquid and gas chromatography.
Liquid spectrophotometry is used to analyze the content of drinks and oils for quality control by ensuring the following:
Many fruit juices on the market are made by mixing concentrates with water. Using opacity, it is possible to judge if the drink has the correct and stated concentration of fruit juice. This quality control is carried out not only by producers, but also by suppliers and retailers.
Figure 4: Fruit juice color matters, Brunei, Pixabay. (Image credits: https://pixabay.com/photos/juice-water-drink-fruit-fresh-1228276/)
Color is a crucial parameter not only for fresh produce, but also for processed products to establish the following:
Producers take pains to see that the color of their beverages remains desirable, so objective color measurement is important. The color of juices, milk, or other beverages has been difficult to measure objectively with traditional methods due to opacity, and this is where spectrophotometry is invaluable. Transparent and translucent liquids’ color is measured through light transmission, while opaque liquids' color is analyzed through reflectance.
Producers of non-alcoholic and alcoholic beverages use liquid spectrophotometers, analyzing production in real-time to finetune the process and as an objective means of quality control of the final product. Monitoring production by frequent and quick analysis helps to consistently achieve the proper mix of ingredients and maintain flavor. In wine and beer production, spectrophotometry can also save money and increase quality.
Drinks are made in many ways. Some drinks, such as energy drinks, are made by mixing ingredients. Using spectrophotometry makes it possible to measure if the beverage has the correct combination during production to maintain consistency in the final product. In the case of energy drinks, it is necessary to monitor the concentrations of key ingredients, like caffeine.
Where drinks are developed by mixing different fruit juices, spectrophotometric analysis shows the percentage of each kind of juice present in the mix, based on the difference in spectra, and helps in creating the selected flavor. Measuring color can also be used to perfect the flavor or color to meet consumer expectations or brand requirements.
Winemaking is a complex and intricate business. Even small wineries are investing in liquid spectrophotometers to produce quality wine, as the technology saves time by avoiding tedious laboratory analysis and waiting for results. Spectrophotometers are used to monitor and predict fermentation throughout the process. Tracking the products of fermentation, like L-malic acid, acetic acid, and residual reducing sugars, can help winemakers adjust the sensory attributes like aroma and mouthfeel. Similarly, quantifying phenols can enhance flavor, color, and mouthfeel perception.
Beer brewing relies on fermentation by yeast to convert cereal sugars into ethanol and CO2. Additional measurable compounds formed in the process give the beer its aroma, taste, and color. Color can be essential for beer, as each type or brand is associated with a certain color, and this must be maintained to prove quality.
Liquid spectrophotometry is used to detect fraud perpetrated by the following:
A single spectrophotometer that analyzes a range of compounds is an investment worth making, and each test works out to be cost-effective. Moreover, the degree of accuracy and depth of knowledge it provides, which is necessary for research and product development, is not possible from most traditional methods. Since liquid spectrophotometers come in a range of sizes and types, their use is no longer confined to laboratories; instead, they have vital applications in many industries, and with modern advancements, can increasingly be operated by laypersons with little technical training.
Science Writer, CID Bio-Science
Ph.D. Ecology and Environmental Science, B.Sc Agriculture
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