UV/Vis spectroscopy serves multiple scientific and industrial purposes, including:
These capabilities support applications in quality control for pharmaceuticals and food products, as well as research in fields such as chemistry, biology, and materials science. This helps in studying material properties and understanding how light interacts with various substances.
When UV/Vis light interacts with a sample, it can either be absorbed or transmitted. Absorbance measures the amount of light the sample absorbs at a specific wavelength, indicating how much light is retained. Transmittance (T = I/I₀) represents the fraction of light that passes through the sample. Understanding these properties is fundamental to analyzing a substance's interaction with light.
Absorbance (A) is a dimensionless quantity commonly reported in Absorbance Units (AU) for clarity. It is mathematically related to transmittance through the equation: A = -log₁₀(T).
The UV/Vis spectroscopy results are displayed in a UV/Vis spectrum, a graph that typically plots absorbance or transmittance against wavelength (190-780 nm). This spectrum reveals which wavelengths were absorbed and to what extent.
Peaks in this spectrum indicate the wavelengths of maximum absorption (λmax), which are specific to the molecule's structure and serve as a unique 'fingerprint' for the substance being analyzed. The intensity of these peaks (or the absorption intensity) is directly related to the concentration of the absorbing species, as described by the Beer-Lambert Law.
This core technique involves passing UV/Vis light through a sample and measuring the transmitted light. We can determine the sample's absorbance characteristics across the UV/Vis range by comparing the initial light intensity to the transmitted light intensity.
The Beer-Lambert Law (A = ϵdc) is essential for quantitative analysis. It states that absorbance is directly proportional to the concentration (c) of the absorbing substance and the path length (d) of the light beam through the sample. The molar absorption coefficient (ϵ) is a constant specific to the substance. This law allows for the accurate determination of analyte concentrations.
A calibration curve is a graphical tool used to ascertain the concentration of a substance within a sample. Here’s a step-by-step overview of the process:
To determine the concentration of an unknown sample:
Calibration curves are vital for transforming absorbance measurements into accurate concentration values, thereby facilitating precise quantitative analysis.
UV/Vis spectroscopy can also provide insights into the electronic properties of materials, such as semiconductors. By analyzing the absorption edge in the spectrum, the band gap energy can be estimated using techniques like the Tauc plot. This analysis helps characterize the energy required to excite electrons within the material.
Curious about the inner workings of a UV/Vis spectrophotometer? This video breaks down how these instruments operate, showcasing METTLER TOLEDO's FastTrack technology for rapid, precise, and reliable results.
Dissolve your sample in a suitable solvent and put it in a special transparent container (cuvette). Also, prepare a reference cuvette (called blank) with pure solvent.
The machine shines a beam of UV and visible light through both the sample and the reference.
A part of the machine (monochromator) acts like a filter, selecting one specific color (wavelength) of light at a time to pass through the sample. It then repeats this for all the wavelengths in the UV/Vis range.
A detector measures how much of the light passes through the sample and how much passes through the reference at each wavelength.
The machine compares the amount of light that passed through the sample to the amount that passed through the reference. This tells us how much light the sample absorbed at each wavelength.
The machine then creates a graph (the UV/Vis spectrum) showing how much light the sample absorbed at each different wavelength. This helps identify and quantify the substances in the sample
A typical UV/Vis spectrophotometer consists of several key components:
The detector is a crucial component responsible for converting the light energy that has passed through the sample and reference into a measurable electrical signal. The transmitted light intensity is inversely proportional to the sample's absorbance at a specific wavelength. The detector measures these intensities to calculate absorbance values for the UV/Vis spectrum, and its sensitivity and wavelength range influence the spectrophotometer's performance and applicability.
These instruments measure light transmittance one wavelength at a time. A diffraction grating separates the light into its component wavelengths, and a single detector measures the light that passes through the sample at each selected wavelength. This process is repeated across the desired range to construct the full spectrum.
These instruments illuminate the sample with the entire spectrum of UV/Vis light simultaneously. The sample absorbs different wavelengths at the same time, and the transmitted light is diffracted by a grating onto an array of multiple detectors (such as photodiodes). Each detector in the array measures the intensity of a specific narrow band of wavelengths concurrently. This design allows for much faster acquisition of the complete UV/Vis spectrum compared to scanning instruments.
The cuvette is a key component of a spectrophotometer, holding the liquid sample in the light path. It must be made of material transparent to the wavelengths used, ensuring accurate measurements. The path length, or distance light travels through the sample, is also important, with standard cuvettes typically having a 10 mm path length, though it can range from 1 mm to 50 mm depending on the application.
These are the most common type and are suitable for most samples. They typically have external dimensions of 12.5 mm x 12.5 mm and a height of 45 mm, with internal dimensions of 10 mm x 10 mm, resulting in a standard path length of 10 mm.
These cuvettes have a path length greater than 10 mm and are used when a sample is too diluted, requiring a longer path length to increase the absorbance signal and improve sensitivity. They are also useful when a sample may vaporize or undergo a chemical change during measurement, as the longer path allows for more interaction with the light beam.
Cuvettes with a path length of less than 10 mm are used when the absorbance of a sample is very high and dilution is either difficult or undesirable. The shorter path length helps keep the absorbance within the instrument's measurable (linear) range.
These cuvettes are specifically designed for analyzing very small sample volumes. For example, some micro cuvettes feature an optical path length of 10 millimeters and are made of fused quartz glass. They are suitable for measurements in the ultraviolet and visible range, covering wavelengths between 200 nm – 2500 nm, and can handle sample volumes around 700 µL.
They are suitable for measurements in the ultraviolet and visible range, covering wavelengths between 170 nm – 2700 nm, and require a small sample volume of 440 µL. These cells are reliable and reusable.
For accurate and reliable UV/Vis measurements, particularly in clinical, pharmaceutical, and industrial quality control, a spectrophotometer must operate within its specified performance limits. Consequently, regular performance verification is essential, and all calibration results should be meticulously documented and stored.
Calibration involves checking and adjusting the accuracy of the instrument in the following areas. These checks are conducted regularly, and adjustments are made if the readings fall outside acceptable limits, making it essential to keep records of these calibrations.
Ensuring that the selected color of light is correct by using standard reference materials.
Confirming that the instrument accurately measures the amount of light absorbed or transmitted through standard solutions.
Checking for any unwanted light that could lead to measurement errors.
Verifying the instrument's ability to distinguish between closely spaced colors of light.
Ensuring a stable and accurate zero reading.
Performance test | Certified reference material (CRM) | Instrument Test Parameter | Acceptance criteria | |
USP 42 NF 37 | Ph. Eur. 10 | |||
Wavelength accuracy & repeatability | Ho(ClO4)3: 4 % Ho2O3 in 10 % v/v HClO4 Blank: Air | 14 wavelengths (240 nm – 650 nm) Xe: 2 wavelengths (260.6, 528.6 nm) | UV (200 – 400 nm): ± 1 nm Vis (400 – 780 nm): ± 2 nm (S.D.) < 0.5 nm | UV (< 400 nm): ± 1 nm Vis (> 400 nm): ± 3 nm |
Photometric accuracy & repeatability** | K2Cr2O7 in 0.001 M HClO4 Blank: 0.001 M HClO4 | 60 mg/L 0 A – 2 A, 235, 257, 313, 350 nm | For absorbance ≤ 1A Accuracy : ± 0.010A Repeatability: S.D. ≤ 0.005 A For absorbance > 1A Accuracy: ± 1% Repeatability: S.D. ≤ 0.5% | Accuracy: ± 0.010 A or ± 1 %, whichever is greater |
Nicotinic acid in 0.1 M HCl Blank: 0.1 M HCl | 12 mg/L 0.26 A – 1.6 A 213, 261 nm | |||
Photometric linearity | K2Cr2O7 in 0.001 M HClO4 Blank: 0.001 M HClO4 | 6 – 200 mg/L, up to 3.0 A, 235, 257, 313, 350 nm | All measured filters fulfill photometric accuracy acceptance criteria | R2> 0.999 |
Nicotinic acid in 0.1 M HCl Blank: 0.1 M HCl | 6 – 60 mg/L, up to 2.5 A 213, 261 nm | |||
Stray light according to procedure A (SFRM) | 1.2 % w/v KCl/H2O; 10 mm path length Blank: 1.2 % w/v KCl/H2O, 5 mm path length | Amax at 198 nm | ≥ 0.7 A | (NA) |
Stray light according to procedure B (SWM) | 1.2 % w/v KCl/H2O; 10 mm path length Blank: H2O, 10 mm path length | Amax at 198 nm | ≥ 2.0 A | ≥ 2.0 A |
Resolution | 0.02 % v/v toluene in n-hexane Blank: n-hexane/ n-heptane (Ph. Eur. 10) | Amax,269/Amin,267 | >1.3 | Levels are stated in the respective monograph |
** No specification of Photometric Repeatability (Precision) in Ph. Eur. S.D. - Standard deviation | ||||
Ensures consumer safety by assessing food quality and composition, focusing on attributes like color, flavor, and aroma. It also employs analytical techniques to identify contaminants and adulterants.
Rigorous analysis is vital for verifying the purity, concentration, and identity of drugs. Monitoring the stability of medications is also essential to ensure efficacy over time under varying environmental conditions.
Evaluates product safety and effectiveness by analyzing the photostability of UV filters, characterizing particles, and measuring color indices. It also detects adulteration and quantifies dyes and antioxidants to meet consumer expectations.
Characterizes crude oil, calculates asphaltene fractions, formulates aromatic content indices, determines sulfur content, and calculates solubility factors.
Determines chemical properties, assesses final product quality, studies polymer composition, qualifies water, determines purity and dyeing efficiency, analyzes photocatalytic degradation and pesticide residues.
Determines concentration and purity of nucleic acids and proteins, monitors microbial cell cultures, studies protein denaturation and kinetics, and analyzes biological samples like blood plasma and serum.
UV/Vis spectroscopy is used in a myriad of applications that span across diverse industries, from pharmaceuticals to environmental monitoring.
METTLER TOLEDO's market support group offers a library of downloadable UV/Vis applications. These proven and tested applications include notes and methods that can be directly imported to your instruments.
Whether you are working in research and development, quality control, or manufacturing, our web page is the go-to resource for all your UV/Vis spectrophotometry needs. Browse our collection today and discover the latest standards and protocols to ensure the highest level of precision and accuracy in your measurements.
Microvolume spectrophotometers facilitate the analysis of extremely small sample volumes, as low as 1 μL, which is essential in fields such as biomolecular analysis. These instruments use fiber optic technology to direct light through a tiny sample droplet placed on a pedestal. The short path length enables accurate measurements of concentrated samples without the need for dilution.
Specifically, microvolume spectrophotometry accurately measures nucleic acid purity and concentration by assessing absorbance at 260 nm and calculating the A260/A280 ratio, which indicates protein contamination. It also supports protein quantification via absorbance at 280 nm, enabling reliable quality control in molecular biology workflows such as PCR and sequencing.
Advantages | Disadvantages |
Simple to operate and experiments are quick. | Limited detailed structural information. |
Relatively low cost for instrumentation and analysis. | Can lack specificity in complex mixtures due to overlapping bands. |
Applicable to a wide range of analytes and fields. | Sensitivity may be insufficient for some analytes. |
Useful for quantitative analysis with calibration curves. | Susceptible to matrix effects. |
Can provide some qualitative information (λmax) | Deviations from Beer-Lambert Law can occur. |
Often non-destructive to the sample. | Provides limited information about the molecular environment. |
Good sensitivity for strongly absorbing analytes. | Not all substances absorb strongly in the UV/Vis region. |
Allows for process integration as e.g., in multiparameter analysis. | Only works with translucent, non-turbid samples. |
Low cost per sample/analysis | |
Small footprint |
Achieving good accuracy and precision in UV/Vis measurements require taking precautions to avoid errors. Here are some typical sources of error that should be considered:
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Achieve more reliable results with GUVP™ by minimizing potential errors. Learn how in our "Trustworthy Results for UV/VIS Spectroscopy" brochure.
Type of Spectroscopy | Type of Radiation | Interactions | Wavelength |
ϒ-ray spectroscopy | Y-rays | Atomic nuclei | < 0.1 nm |
X-ray fluorescence spectroscopy | X-rays | Inner shell electrons | 0.01 – 2.0 nm |
Vacuum UV spectroscopy | Ultraviolet (UV) | Ionization | 2.0 – 200 nm |
UV/Vis spectroscopy | UV/Vis | Valance electrons | 200 – 800 nm |
Infrared & Raman spectroscopy | Infrared | Molecular vibrations | 0.8 – 300 mm |
Microwave spectroscopy | Microwaves | Molecular rotations | 1 mm – 30 cm |
Electron spin resonance spectroscopy | Electron spin | ||
Nuclear magnetic resonance spectroscopy | Radio waves | Nuclear spin | 0.6 – 10 |
The different spectroscopic techniques are mainly differentiated by the radiation they use, the interaction between the energy and the material, and the type of material and applications they are used for. The spectroscopic techniques commonly used for chemical analysis are atomic spectroscopy, ultraviolet and visible spectroscopy (UV Vis spectroscopy), infrared spectroscopy, Raman spectroscopy and nuclear magnetic resonance.
To read a UV/Vis spectrum, you analyze the plot of absorbance (or sometimes transmittance) versus wavelength. Key points to focus on include:
By interpreting these features, you can identify compounds, determine concentration, and study molecular properties.
UV/Vis spectroscopy typically covers the wavelength range from 190 nm to 780 nm.
More specifically:
METTLER TOLEDO’s UV/VIS Excellence spectrophotometers extend further into the near-infrared region, reaching 1100 nm.
UV/Vis spectroscopy is important because it enables both qualitative and quantitative analysis of substances by measuring their absorption of ultraviolet and visible light.
This method helps determine the concentration of analytes, study chemical kinetics, assess purity, perform objective color measurements, and analyze molecular structures. Its applications cover a wide range of fields, including chemistry, biology, environmental science, and materials science, making it a versatile and essential tool for analysis.
UV/Vis spectroscopy measures the light absorbed by a sample to determine its concentration and identify compounds. This process involves the removal of light.
In contrast, fluorescence spectroscopy measures the light emitted by a sample after it absorbs light, usually at a longer wavelength. This technique focuses on the re-emission of light, providing much higher sensitivity for specific fluorescent molecules.
To prepare a sample for UV/Vis spectroscopy, you'll need to handle your cuvettes and solutions carefully:
To determine the concentration of an unknown solution using UV/Vis spectroscopy, follow these steps:
This method relies on Beer-Lambert Law, which states that absorbance A=εbc, where ε is the molar absorptivity, b is the path length, and c is the concentration.
The absorption of UV light results in electronic transitions from lower energy levels to higher energy levels. Absorption of ultraviolet radiation in organic molecules is restricted to certain functional groups (chromophores) that contain valence electrons of low excitation energy. The molecular transitions/interactions that take place due to UV absorption are:
These transitions need an unsaturated group in the molecule to provide the π electrons.
σ (bonding) to σ* (anti-bonding) transitions require higher energy and therefore cannot be detected using UV Vis spectroscopy.
Consider a functional group containing atoms with one or more lone pairs of electrons that do not absorb ultraviolet/visible radiation. However, when this functional group is attached to a chromophore, it alters the intensity and wavelength of absorption. This phenomena is called an auxochrome or a color-enhancing group.
The presence of an auxochrome causes the position shift of a peak or signal to a longer wavelength, which is called a bathochromic or red shift. The functional groups contributing to bathochromic groups are substituents such as methyl, hydroxyl, alkoxy, halogen and amino groups.
The auxochrome that causes position shift of a peak or signal to shorter wavelength is called a hypsochromic or blue shift. Actually, the combination of chromophore and auxochrome behaves like a new chromophore having a different absorption maxima (λmax). For example, benzene shows λmax at 256 nm, whereas aniline shows λmax at 280 nm. Hence, the NH2 group acts as an auxochrome and causes the shift of λmax to a larger value.
| Instrumen | Spectral resolution | Equivalent SBW (nm) |
| UV5 | > 1.5 | < 2.0 |
| UV5Bio | > 1.5 | < 2.0 |
| UV5Nano | > 1.7 | < 1.5 |
| UV7 | > 1.9 | ≤ 1.0 |
The table shows the resolution of METTLER TOLEDO's UV/VIS Excellence spectrophotometers, which is measured using toluene in hexane, and the equivalent SBW.
The spectral bandwidth (SBW) of a spectrophotometer is related to the physical slit-width and optical dispersion of the monochromator system. Resolution is the ability of an instrument to separate light into finite, distinct wavelength regions and to distinguish each finite region. Spectral bandwidth is typically used for scanning instruments, whereas resolution is typically used for array instruments.
For most pharmacopeia quantitative purposes, a spectral bandwidth of less than 2 nm is sufficient and the acceptance criteria for the ratio is 1.3. Spectral resolution can be used for comparison with spectral bandwidth.
| Light Source | Wavelength Range (nm) | Region | Lifetime |
| Tungsten filament lamp | 350 – 2500 | Vis + IR | 3,000 hr |
| Deuterium arc lamp | 190 – 400 | UV | 1,000 hr |
| Hydrogen lamp | 190 – 400 | UV | 1,000 hr |
| Xenon flash lamp | 190 – 1100 | UV + Vis + NIR | 5,500 hr* |
* Corresponds to 50 Hz flashes at constant operation
The best light source would be one that provides good intensity with low noise across all ultraviolet and visible wavelengths and offers stability over a long period. There is a range of light sources which are commonly employed as mentioned above.
Diffraction gratings are generally better than prisms for splitting different wavelengths because:
Higher Spectral Resolution: Gratings can provide much higher spectral resolution due to their ability to produce multiple sharp diffraction orders, allowing finer separation of closely spaced wavelengths.
Linear Dispersion: The angular separation between wavelengths in a grating is more linearly related to wavelength, making it easier to analyze and calibrate spectra compared to the nonlinear dispersion of prisms.
No Material Dispersion Limitations: Prisms rely on material dispersion (variation of refractive index with wavelength), which can limit performance, especially in certain wavelength ranges. Gratings use interference effects, which are not limited by material properties.
Broad Wavelength Range: Gratings can work efficiently over a broader range of wavelengths, including ultraviolet and infrared, whereas prisms have absorption and dispersion limitations.
Overall, diffraction gratings provide more precise and versatile wavelength separation than prisms, which is why they are commonly used in spectrometers and optical instruments.
Molecules can be analyzed using UV/Vis spectroscopy if they possess any functional group or conjugation, or if they produce a color complex. As inorganic compounds do not contain any functional group or conjugation, the common method for analyzing them is by reaction with a suitable compound. This produces a color complex whose absorbance can be photometrically measured in the visible region and correlated with its actual concentration. For example, iron is commonly analyzed by a reaction with 1, 10-phenthroline to produce a red color complex. The absorbance of the complex is measured at 570 nm to estimate iron concentration.
The main difference between a single beam and double beam spectrophotometer follows.
Beam splitting in a double beam spectrophotometer is achieved in two ways:
The analysis of a solid sample is performed mainly by estimating its absorbance, transmittance and reflectance. Common parameters determined for solid polymers include % transmittance, cutoff wavelength, and yellowness index. The sample is mounted on a holder specifically designed for solid samples and readings are taken in the same manner as they are for liquid samples. A solid sample holder enables measuring of solid samples such as films or glass.
Temperature affects absorbance values. Different solvents undergo different interactions at different temperatures. Solution parameters that change due to temperature changes are:
Optical performance parameters such as photometric noise, wavelength accuracy/repeatability, photometric repeatability and stray light are not influenced by temperature within a range of 10 – 40 °C.
Whereas, optical parameters like photometric resolution (toluene/hexane ratio) and photometric accuracy wavelengths (K2Cr2O7 in HClO4) show a temperature dependency ranging from 0.014 to -0.034/unit within 10 – 40 °C.
Stray light is defined as light that reaches the detector which is not from the instrument's light source and does not follow the optical path, causing a deviation at the corresponding wavelength. Therefore, the light intensity measured by the detector is higher than it actually should be. Conversely, this also means that the measured absorbance is lower than the true absorbance because it is reduced by the contribution of stray light. This effect is more prominent at higher absorbance values (high sample concentrations).
Download the whitepaper to learn more about the origin and accurate measurement of stray light:
The sample compartment in UV/Vis array spectrophotometers is open due to the fact that array instruments use reverse optics and the simultaneous detection of all wavelengths of the spectrum.
Anika Adams is Senior Product Manager for UV/Vis Spectrophotometers at METTLER TOLEDO, where she focuses on driving innovation and optimizing customer workflows. With a degree in business administration and experience spanning software development, mechanical engineering, and production, Anika brings a multidisciplinary perspective to the laboratory business. Her expertise lies in automating and enhancing customer processes, ensuring both product excellence and customer satisfaction.
