Spectrophotometric assay: Principles and Applications
Spectrophotometry is the science behind spectrophotometer, an instrument used to measure how much light a chemical substance absorbs, transmits, or reflects across a given range of wavelengths. It is one of the most widely used methods of quantitative analysis in chemistry, physics, biochemistry, materials science, chemical engineering, and clinical diagnostics. From analysing proteins and nucleic acids to testing dyes, pigments, and industrial materials, spectrophotometry underpins both academic research and practical quality control.
Why are Spectrophotometers Important?
When a beam of light passes through a sample solution, part of it is absorbed by the molecules while the rest is transmitted or reflected. The unique way each compound interacts with light produces a measurable "fingerprint" that can be used to identify chemicals and determine their concentration. The power of spectrophotometry lies in its precision and versatility. Because molecules absorb and transmit light at unique wavelengths, the technique allows researchers to:
- Identify unknown compounds by their absorption spectrum.
- Quantify the concentration of dissolved chemicals in solution.
- Monitor enzyme activity and biochemical reactions.
- Measure the optical properties of industrial products such as dyes, inks, LEDs, and coatings.
In production facilities, spectrophotometers are essential for ensuring consistency and quality. For example, checking textile colour accuracy or monitoring light emission in LED manufacturing. In laboratories, they are invaluable for measuring sample concentrations, analysing reaction kinetics, and determining purity.
Principle of Spectrophotometry
At its core, a spectrophotometer measures light intensity as a function of wavelength. A light source is passed through a monochromator (prism or diffraction grating) to separate it into specific wavelengths. These are then directed onto the sample in a cuvette. The instrument measures how much light is transmitted and absorbed, displaying the results as a spectrum.
Components
A typical spectrophotometer includes the following key components:
- Light source - commonly tungsten, deuterium, or hydrogen lamps
- Monochromator - a prism or grating that separates light into different wavelengths
- Collimators and lenses - focus the light beam
- Cuvette holder - where the sample is placed
- Detector - usually a photocell that converts light into an electrical signal
- Digital display - outputs the absorbance or transmittance values
Mechanism
The process follows five main steps:
- Light from the source is directed onto the monochromator.
- A single wavelength is selected and focused onto the sample solution.
- Transmitted light passes through the cuvette to the detector.
- The detector converts light into an electrical signal.
- The signal is amplified and displayed as absorbance or transmittance.
This principle is based on the Beer-Lambert Law, which states that the amount of light transmitted from the solution is inversely proportional to the absorption of light, provided the system behaves linearly. In other words, as the concentration of a solution increases, it absorbs more light and transmits less.
- A = absorbance of light
-
I0 = intensity of incident light
- I = Intensity of transmitted light
-
ɛ = absorption coefficient
- C = concentration of the absorption material
- l = path length (cm)
To ensure accuracy, the spectrophotometer must first be calibrated using a reference solution. This process is also known as "zeroing". Once calibrated, it can measure absorption spectra with a high degree of sensitivity, often detecting concentration changes as subtle as a fraction of a percent.
Applications of Spectrophotometry
Because of its versatility, spectrophotometry is used across both scientific and industrial fields.
Biochemistry - quantifying proteins, enzymes, nucleic acids, and blood glucose
Chemistry - quantifying the concentration of chemicals in solution
Molecular biology - quantifying DNA, RNA, and protein samples
Clinical applications - enzyme assays, metabolic activity measurements, and drug testing
Industrial quality control - ensuring consistent dye colours, paint formulation, and LED emission
Material science - studying pigments, coatings, polymers, and nanomaterials
Enzyme kinetics - tracking changes in absorbance to monitor reaction rates
Education and research - providing and inexpensive, reliable, and highly accurate method for laboratory experiments
Quantum yield measurement - determining the quantum yield of fluorescence for fluorescent compounds, which is crucial for fluorescence-based experiments and assays
Ossila's spectrophotometer operates in the visible range (380-1000 nm), making them ideal for colour-based analysis of dyes, pigments, and materials, though not suitable for DNA and RNA measurements that require UV ranges.
Spectrophotometric Assay
There are several spectrophotometric assay procedures varying depending on the specific type of substance. Nonetheless, they all share general steps that are outlined below:
- Preparation
- Zero calibration
- Calibration (if you are performing quantitative analysis)
- Sample measurement
- Data analysis (quantitative)
- Data recording
- Quality control
- Data presentation
- Data analysis (qualitative)
One of the most common applications is the spectrophotometric enzyme assay. These assays monitor changes in absorbance or transmittance as an enzyme catalyses reaction. For example, cofactors such as NADH and NADPH absorb UV light at 340 nm in their reduced form but not when oxidised, making it possible to track enzyme activity in real time.
Both UV/visible and colorimetric assays are widely used because they are inexpensive, reproducible, and sensitive. The well-known MTT assay uses a tetrazolium dye that changes colour to assess cell viability and enzyme activity. Similar assays using dyes like XTT, MTS, and WTS are frequently employed in biomedical research.
The reliability of these tests depends on the stability and sensitivity of the spectrophotometer, making high-quality instruments essential for reproducible results.
Spectrophotometric assay of ascorbic acid oxidase
L-ascorbate oxidase is an enzyme that belongs to the family of oxidoreductases. Its function involves acting as a donor with oxygen as an acceptor in chemical reactions involving diphenols and related substances.
A spectrophotometric assay for the ascorbic acid oxidase is a method that consists of enzymatic reaction and a blank solution for accurate measurement. It involves measuring the enzyme's activity by observing the oxidation of ascorbic acid to dehydroascorbic acid. This is quantified by the absorbance of a specific wavelength, which usually occurs at 358 nm.
Spectrophotometric assay of polyphenol oxidase activity
Polyphenol oxidase (PPO) is an enzyme involved in fruit browning. It is classed as tetramer because it contains four atoms of copper per molecule. The enzyme's function is based on catalysing the o-hydroxylation of monophenol molecules in which the benzene ring contains a single hydroxyl substituent to o-diphenols.
While there are many ways to measure its activity, a common one is the UV spectrophotometric assay, which directly looks at the formation of o-quinones. Alternatively, a spectrophotometric assay involving the coupling of o-quinones with nucleophilic reagents can also be used.
Spectrophotometric assay for measuring catalase activity in biological tissues
Catalase is an important enzyme that acts to dissociate hydrogen peroxide (H2O2) into molecular oxygen (O2) and water (H2O). The protein is produced by a wide spectrum of prokaryotic and eukaryotic organisms, including aerobic and anaerobic bacteria. There has been quite a good progress in the development of efficient methods for assessing catalase activity in the fields of clinical pathology, biotechnology, and applied microbiology because of the spread of microbial populations in variety of foods. The most common way to measure the catalase activity is to use the UV spectrophotometric method. This method depends on monitoring the change of 240 nm absorbance at high level of hydrogen peroxide solution (≥30 mM).
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