In the field of laboratory analysis, the spectrophotometer plays a central role. This instrument is essential for identifying and quantifying chemical substances present in a solution by measuring their ability to absorb light. Thanks to its efficiency, precision, and versatility, it is widely used in diverse sectors such as food processing , cosmetics , animal health, the environment, and biomedical research. Understanding the operation, role, and applications of the spectrophotometer allows one to grasp its strategic importance in quality control, regulatory validation, and product development processes.
YesWeLab supports manufacturers in implementing these spectrophotometric analyses by relying on a network of specialized laboratories that comply with current standards.
Table of Contents
What is a spectrophotometer?
Definition and fundamental principles
A spectrophotometer is an analytical instrument used to measure the absorbance of a substance dissolved in a liquid. Absorbance corresponds to the amount of light absorbed by the solution when a beam of light of a specific wavelength passes through it. This measurement is directly related to the concentration of the substance being analyzed, according to a fundamental principle of analytical chemistry: Beer-Lambert's law .
Unlike a simple spectrometer that can analyze the entire light spectrum, a spectrophotometer is designed to perform a precise measurement at one or more selected wavelengths. It converts the intensity of the transmitted light into a numerical absorbance value, which allows for a direct correlation with the amount of molecule present in the sample.
Difference between a spectrophotometer and a spectrometer
These two terms are sometimes used interchangeably, but they refer to different devices. A spectrometer is a more general instrument used to analyze light based on its spectral components. It is used to determine the composition of a sample by identifying the wavelengths emitted or absorbed. A spectrophotometer, on the other hand, is a specialized instrument that precisely measures the absorbance (or transmittance) of a sample at a specific wavelength. It is therefore more suited to quantitative analyses.
An evolving technology at the service of laboratories
Since its development at the beginning of the 20th century, the spectrophotometer has undergone numerous technical improvements. Modern models are capable of automatically scanning a wide range of wavelengths, storing data, transferring it to an information system, or even operating in dual-beam mode to improve measurement accuracy. Detection technologies have also diversified: photodiodes, photomultiplier tubes (PMTs), and CCD sensors offer varying levels of sensitivity and resolution.
Today, spectrophotometers are available for every use: compact devices for field use, automated systems for serial analysis, or high-precision models for scientific research.
An indispensable tool in laboratory analysis
The spectrophotometer is used daily in many analytical laboratories . It allows for the quantification of active substances, the identification of contaminants, the validation of the concentration of an active ingredient, and the monitoring of chemical reactions. In the food industry, it is used to analyze the content of colorants, vitamins, acids, or sugars. In cosmetics, it serves to verify the concentration of active ingredients in a formulation. In environmental science, it allows for the monitoring of water pollution by measuring nitrates, phosphates, or dissolved metals.
Its ability to provide reliable, reproducible and rapid results makes it an essential tool in any quality control or product development strategy, especially in an increasingly demanding regulatory context.
What is the role of the spectrophotometer?
Measuring the absorbance of a solution
The primary function of a spectrophotometer is to measure the absorbance of a solution at a specific wavelength. Absorbance is a dimensionless physical quantity that quantifies the loss of intensity of a light beam as it passes through a medium. The more light a solution absorbs, the higher its measured absorbance will be.
This measurement is based on the principle that certain molecules absorb light at very specific wavelengths. For example, DNA has a maximum absorbance around 260 nm, while many proteins absorb at 280 nm. By analyzing the absorbance at these specific wavelengths, the spectrophotometer can detect the presence of these compounds in a sample.
Quantifying a substance using Beer-Lambert's law
One of the major advantages of the spectrophotometer is its ability to quantify a substance in solution. This quantification is based on Beer-Lambert's law, which establishes a direct relationship between absorbance (A), the concentration of the substance (C), the molar absorption coefficient (ε), and the optical path length (l), generally 1 cm in standard cuvettes:
A = ε × l × C
Thus, if the molar absorption coefficient of a given substance at a specific wavelength is known, it becomes possible to calculate its concentration in a sample simply by measuring the absorbance. This principle is commonly used in laboratories to perform colorimetric assays, that is, to measure the concentration of an analyte based on its color or its ability to absorb light.
For example, in the context of the determination of malic acid, used in the food and cosmetic sectors, spectrophotometry makes it possible to evaluate the concentration of the acid in a jam or a skincare cream, with great precision.
Identify the presence of a specific compound
In addition to quantification, the spectrophotometer also allows for the identification of a compound. Each molecule exhibits a absorbance spectrum , much like an optical fingerprint. By scanning different wavelengths and recording the absorbance, it is possible to obtain a spectrum that allows for the identification of a compound by comparison with databases of reference spectra.
This capability is particularly useful for verifying the identity of an active ingredient, detecting contamination, or validating a production batch in regulated sectors.
Control the quality and conformity of the products
In an industrial context, the spectrophotometer plays a crucial role in quality control . It ensures that products meet specified concentrations, that the formulation is homogeneous, and that the raw materials used are compliant. For example, in the cosmetics sector, it is used to control the concentration of sunscreens or brightening agents and guarantee batch consistency.
Furthermore, some regulations require verification of maximum permitted concentrations, particularly for active substances, preservatives, or colorants. The spectrophotometer is therefore essential for documenting regulatory compliance, especially with regard to ISO 17025 standards or COFRAC accreditations required for analyses performed by partner laboratories such as those of YesWeLab.
Measuring the stability of formulations over time
In an industrial context, the spectrophotometer plays a crucial role in quality control . It ensures that products meet specified concentrations, that the formulation is homogeneous, and that the raw materials used are compliant. For example, in the cosmetics sector, it is used to control the concentration of sunscreens or brightening agents and guarantee batch consistency.
Furthermore, some regulations require verification of maximum permitted concentrations, particularly for active substances, preservatives, or colorants. The spectrophotometer is therefore essential for documenting regulatory compliance, especially in relation to ISO 17025 standards or COFRAC accreditations required for analyses performed by laboratories such as those in the YesWeLab .
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What is the purpose of spectrophotometry?
A precise quantitative analysis method
The main goal of spectrophotometry is to provide a simple, rapid, and reliable method for quantitative analysis. By measuring the amount of light absorbed by a solution at a given wavelength, this technique allows for the precise determination of the concentration of one or more chemical compounds. It is used to analyze solutions containing organic or inorganic substances, dyes, proteins, nucleic acids, metals, or even environmental pollutants.
This ability to quantify very low concentrations, sometimes at the nanogram scale, makes it an ideal method in sectors requiring high sensitivity. This is the case in molecular biology, agri-food quality control, cosmetic formulation, and environmental analysis.
A non-destructive and reproducible method
Spectrophotometry has the advantage of being a non-destructive technique . The analyzed sample is not significantly altered by the passage of the light beam, allowing it to be reused or stored for further testing. This is particularly valuable when the available quantity is limited, or when multiple analyses are planned for the same sample.
Furthermore, the method is highly reproducible when analytical conditions are standardized: fixed wavelength, clean cuvette, controlled temperature. This reproducibility is essential for ISO 17025 accredited laboratories, which must guarantee the consistency and reliability of their results over time.
An aid in the identification of compounds
Beyond quantification, spectrophotometry also enables qualitative analysis through the study of absorption spectra. Each molecule exhibits a unique optical signature, with absorbance peaks at specific wavelengths. By scanning a sample across a broad wavelength range (from 190 to 1100 nm for some UV-visible models), a complete spectrum can be obtained, which can be used to identify an unknown compound by comparison with spectral databases.
This feature is notably used in analytical chemistry to characterize substances in complex mixtures, or to check the conformity of an active ingredient in an industrial formulation.
A technique used for quality control
The purpose of spectrophotometry in industrial laboratories is often related to quality control of raw materials, finished products, or packaging . For example, in the food industry, it allows for monitoring the concentration of colorants, additives, or vitamins. In cosmetics, it is used to measure UV filters or skin-brightening agents. In environmental analysis, it serves to assess the content of nitrates or organic substances in water.
By carrying out regular measurements, quality teams can validate batch conformity, identify manufacturing deviations, or even anticipate formulation drifts.
An essential tool in regulatory studies
Spectrophotometry is also used in regulatory compliance studies . European (INCO, EC 1935/2004) and American (FDA) regulations impose strict limits on the concentration of certain substances in consumer products. Spectrophotometry can demonstrate, with supporting documentation, that these limits are not exceeded.
For example, food packaging must be tested to ensure the absence of migration of undesirable compounds. Spectrophotometry is one of the methods used to detect the presence of potentially released organic acids, dyes, or metals. YesWeLab supports its clients with these issues by offering migration analyses that comply with CE and FDA standards.
A technique integrated into multi-analytical protocols
Finally, spectrophotometry is frequently used in conjunction with other analytical techniques . It is often integrated into protocols combined with high-performance liquid chromatography (HPLC) , mass spectrometry (MS), or rheological analysis. For example, in the analysis of malic acid , laboratories may use spectrophotometry for a rapid initial measurement before validating the results with a more precise method such as HPLC.
This complementarity between techniques makes it possible to strengthen the reliability of the results and to adapt the methods to the specific requirements of the industrial sectors concerned.
How a spectrophotometer works: scientific and technical principles
The principle of absorbance
The spectrophotometer operates on a fundamental principle: certain molecules absorb light when exposed to a specific wavelength. This absorption results in a decrease in the measured light intensity after the beam passes through the sample. The spectrophotometer quantifies this loss as a value called absorbance .
Absorbance is related to transmittance (the ratio between the intensity of transmitted light and that of incident light), according to the following relationship:
A = −log10(I / I₀)
Or :
- A is the absorbance,
- I is the light intensity transmitted through the sample,
- I₀ is the initial light intensity.
High absorbance means that the solution absorbs a lot of light, which generally corresponds to a higher concentration of the substance being analyzed.
Beer-Lambert's Law
The scientific basis of quantitative analysis by spectrophotometry rests on Beer-Lambert's law , which expresses the relationship between absorbance and the concentration of a solution:
A = ε × l × C
Or :
- A is the measured absorbance,
- ε is the molar absorption coefficient (expressed in L·mol⁻¹·cm⁻¹),
- l is the length of the optical path (usually 1 cm),
- This is the concentration of the solution (mol/L).
This equation shows that absorbance is proportional to concentration, provided that ε is known and a constant cuvette length is maintained. The law is valid for dilute solutions (generally less than 0.01 mol/L), making it a preferred method for laboratory analyses at low concentrations.
The role of wavelength
Choosing the wavelength is a crucial parameter for ensuring the sensitivity and selectivity of the measurement. Each molecule has an absorbance spectrum , with peaks corresponding to the wavelengths where absorption is greatest. The spectrophotometer must therefore be calibrated to emit a light beam centered on this specific wavelength.
For example, malic acid exhibits significant absorption in the UV range, as do nucleic acids (DNA at 260 nm). In contrast, proteins are often measured at 280 nm. This setting improves measurement accuracy by limiting interference from other compounds present in the solution.
In enzymatic analyses, a specific wavelength is sometimes selected where only one of the reaction products absorbs. This is the case for NADH, measured at 340 nm, while its precursor NAD⁺ does not absorb at this wavelength. This allows for precise monitoring of biochemical reactions.
The steps of a spectrophotometric analysis
Spectrophotometric analysis typically follows these steps:
- Sample preparation : possible dilution, addition of reagents, homogenization.
- Choice of wavelength : determined from the literature or a prior spectrum.
- Setting up the blank : cuvette containing only the solvent or mixture without analyte, to calibrate the instrument.
- Absorbance measurement : reading the absorbance value for the sample at the chosen wavelength.
- Concentration calculation : application of Beer-Lambert's law or comparison with a calibration curve.
This standardized procedure is fast (a few minutes per sample), inexpensive, and reproducible, which explains its widespread adoption in quality control laboratories.
Types of spectrophotometers and available technologies
Single-beam spectrophotometer
The single-beam spectrophotometer is the simplest and most common model in teaching, quality control, and production laboratories. In this type of instrument, a single beam of light is used to perform measurements. The operator begins by inserting a cuvette containing the "blank" (solvent or matrix without analyte) to calibrate the instrument, then replaces this cuvette with the one containing the sample to be analyzed.
This model offers several advantages:
- relatively low acquisition cost,
- ease of use,
- compact size.
However, it is more sensitive to fluctuations in the light source or variations in ambient temperature, as no simultaneous measurements are taken on a reference sample. For routine applications where absolute reproducibility is not critical, the single-beam spectrophotometer remains an excellent choice.
Dual-beam spectrophotometer
The double-beam spectrophotometer is more sophisticated. It splits the light beam into two parts: one passes through the sample, the other through the reference solution (the blank). The two signals are measured simultaneously, allowing for instantaneous correction of fluctuations in the light source and significantly improving the stability and accuracy of the measurements.
This type of device is recommended for research laboratories or for analyses requiring high reliability, such as those performed in the pharmaceutical or cosmetics industries. It allows, in particular:
- improved kinetic tracking,
- measures that are more reproducible over the long term,
- partial automation in the series of analyses.
UV-visible spectrophotometer
The UV-visible spectrophotometer is the most common model in modern laboratories. It allows for analyses to be performed in two complementary spectral ranges:
- Ultraviolet (190 to 380 nm), used for the analysis of nucleic acids, proteins, food additives or pesticide residues.
- Visible (400 to 800 nm), used for colorimetric assays, pigments, or cosmetic colorants.
UV-visible instruments are versatile and suitable for a wide variety of samples. They allow for spot or serial analyses and are compatible with numerous software programs for data processing, concentration calculation, and report generation.
Visible Spectrophotometer
Some models are limited to visible light (400 to 800 nm). These visible spectrophotometers are sufficient for basic colorimetry applications or for analyzing substances that absorb light in the visible spectrum (dyes, fruit juices, textile dyes, etc.).
They are generally more compact and economical, but their scope of application is limited by the impossibility of performing measurements in the UV.
Portable Spectrophotometer
Portable or benchtop spectrophotometers are designed for in-situ analysis or in production environments. They are used, for example, for:
- measuring the color of a product directly on a production line,
- analyze water quality in the field,
- perform a quick check in the warehouse or collection center.
Compact, lightweight, and often equipped with rechargeable batteries, these devices enable rapid decision-making without waiting for samples to be sent to the laboratory. Their accuracy is slightly lower than that of laboratory models, but their responsiveness more than compensates for this difference in many industrial settings.
Criteria for choosing a spectrophotometer
The choice of a spectrophotometer depends on several factors, related to the specific needs of each laboratory:
- Spectral range : UV-visible or visible only?
- Beam type : single or dual?
- Sensitivity : desired detection level (expressed in A or ng/µl).
- Targeted applications : molecular biology analysis, cosmetics, food products, environment…
- Budget : some high-performance models can cost tens of thousands of euros.
- Ergonomics and connectivity : compatibility with analysis software, data export capability, touchscreen, etc.
YesWeLab works with laboratories equipped with various technologies, allowing the spectrophotometric method to be adapted to the sample type, matrix, and analytical objectives of the client. Thanks to this flexibility, manufacturers benefit from a customized solution that complies with current regulatory requirements.
Applications of spectrophotometry in laboratory analysis
In biology and biochemistry
Spectrophotometry is a fundamental tool in biology and biochemistry laboratories. It is used to quantify biomolecules, monitor enzymatic reactions, or assess the purity of biological extracts.
- Quantification of DNA, RNA, and proteins :
Absorbance measured at 260 nm determines the concentration of nucleic acids (DNA, RNA), while absorbance at 280 nm provides an estimate of protein concentration. The A260/A280 ratio is used to assess the purity of a DNA sample. A ratio of 1.8 to 2.0 indicates good purity, while a lower ratio suggests protein contamination. - Monitoring enzyme kinetics :
Many enzymatic reactions involve cofactors such as NADH, measurable at 340 nm. By monitoring the evolution of absorbance over time, it is possible to calculate the rate of the enzymatic reaction and determine kinetic parameters such as the Michaelis-Menten constant (Km) or the maximum velocity (Vmax). - MTT Assay :
Used to assess cell viability, this assay relies on the conversion of tetrazolium salt (yellow) to formazan (purple) by live cells. The amount of formazan is measured at 570 nm, which allows for an indirect estimation of the number of live cells in a sample.
In the agri-food sector
In the food industry, spectrophotometry is used for quality control of raw materials and finished products.
- Sugar, organic acid, and vitamin analysis :
Many analyses are performed using UV-visible light, such as measuring vitamin C at 265 nm or malic acid by UV reading after a chemical reaction. These measurements ensure product compliance with nutritional declarations and regulatory requirements. - Pigment and colorant monitoring :
The color intensity of products such as fruit juices, jams, or sauces is controlled by visible spectrophotometry. This ensures consistent presentation from batch to batch.
In cosmetics
Cosmetic laboratories use spectrophotometry to ensure the quality, stability and regulatory compliance of products.
- Measurement of cosmetic active ingredients :
Ingredients such as fruit acids (AHAs), UV filters, or colorants can be measured using spectrophotometry. For example, an active ingredient like glycolic acid can be quantified in a cream using UV spectroscopy. - Stability tests :
By subjecting products to conditions of heat, light or humidity, laboratories measure the variations in absorbance to detect possible chemical degradation or loss of effectiveness. - Packaging control :
Spectrophotometry can also be used for migration tests of substances from containers to the cosmetic product, in connection with the requirements of EC Regulation No. 1223/2009 and EC Regulation No. 1935/2004 for contact materials.
In the environment
Spectrophotometry is a technique of choice for environmental analyses, particularly in the context of regulatory monitoring of water quality and industrial discharges.
- Analysis of nitrates, phosphates, and ammonium :
These compounds are measured by spectrophotometry after a colorimetric reaction with specific reagents. For example, the Griess method is used for the determination of nitrites. - Wastewater and effluent monitoring :
The absorbance of certain organic substances allows for monitoring the pollution of water or industrial discharges. This data is often required in environmental assessments or regulatory dossiers submitted to health authorities. - Detection of UV-active pollutants :
Certain pesticides, hydrocarbons, or aromatic compounds exhibit a characteristic spectral signature in the UV range. They can be detected even at low concentrations using highly sensitive spectrophotometers.
In industrial control and materials
In industrial control and materials
In industrial sectors such as automotive, plastics or printing, spectrophotometry is used to analyze solid or semi-solid materials.
- Measurement of transmittance and opacity :
Materials such as glass, plastics or films are tested to verify their transparency or their ability to block certain light radiation. - Colorimetric analysis :
In the textile or graphic sectors, spectrophotometry is used to control color fidelity according to colorimetric standards (e.g., L a b* system). - Formulation stability and material aging :
Materials are subjected to accelerated aging tests, and the evolution of their color or absorption is monitored to assess their resistance to conditions of use (UV, heat, humidity, etc.).
Thanks to its versatility, spectrophotometry can be integrated into all industrial sectors where the composition, color or stability of products must be measured, verified and documented accurately.
Example of spectrophotometric analysis in the laboratory: the case of malic acid
Malic acid is an organic acid naturally present in many fruits, including apples, grapes, and rhubarb. It is widely used in the food industry to adjust the acidity of finished products (confectionery, beverages, dairy products), and in cosmetics for its exfoliating and moisturizing properties, particularly as an alpha-hydroxy acid (AHA).
Its concentration in products must be precisely controlled for reasons of sensory quality , cosmetic efficacy , and above all, regulatory compliance . This is why it is subject to numerous laboratory analyses, particularly by UV-visible spectrophotometry , which is one of the most accessible and rapid methods for its quantification.
The analytical techniques used
Three main techniques are used to measure malic acid in the laboratory:
- UV-visible spectrophotometry :
This method relies on the ability of malic acid to absorb ultraviolet light after derivatization or enzymatic reaction. Absorbance is measured at a specific wavelength, often between 260 and 280 nm, depending on the chosen protocol. It allows for rapid and reliable quantification and is particularly well-suited for routine monitoring. - High-performance liquid chromatography (HPLC) :
More precise than spectrophotometry, HPLC is often used as a reference method to validate results. It allows for the separation of malic acid from other organic acids present in the sample (citric acid, lactic acid, etc.) and its quantification with very high precision. - Acid-base titration :
This more traditional method relies on the chemical neutralization of malic acid by a base. It provides an estimate of the total acid content, but does not allow for the differentiation of the various acids present.
Practical applications of spectrophotometry for malic acid
- In the food industry :
Spectrophotometry is used to verify that food products comply with regulatory acidity levels. For example, in jam making, the amount of malic acid must be adjusted to ensure proper product preservation and a balanced flavor. In winemaking, measuring malic acid allows for monitoring malolactic fermentation, which transforms this acid into the milder lactic acid. - In cosmetics :
Malic acid is used for its keratolytic (exfoliating) properties. It is an ingredient in creams, lotions, and masks. Its concentration must be rigorously measured to guarantee the product's effectiveness without risk of skin irritation. UV spectrophotometry allows for precise control of this concentration, particularly to comply with regulatory recommendations for AHAs (alpha-hydroxy acids).
