UV-Vis spectrophotometry and its use in the laboratory
— Equipo JP Selecta
Spectrophotometry is one of the most versatile and widespread analytical methodologies in the scientific and industrial world. It makes it possible to identify, quantify and analyse various types of substances through their capacity to absorb or transmit electromagnetic radiation.
In practice, understanding what spectrophotometry and UV-Vis spectrophotometry are, and how modern instruments are used, is essential in order to optimise quality control processes, chemical research or clinical analysis.
What is spectrophotometry and what is it used for?
Spectrophotometry is an analytical technique based on optical principles that quantitatively measures how a sample absorbs light as a function of wavelength. Its main purpose is to determine the concentration of an analyte present in a solution or to characterise the molecular properties of a compound.
Unlike other inspection methodologies, it stands out for being a fast technique, non-destructive (in most cases) and extremely precise. Its relevance in the laboratory lies in the fact that it makes everything possible, from monitoring chemical reactions to verifying the purity of reagents or synthesised compounds.
How does spectrophotometry work?
The basic operating principle consists of generating a beam of light with a specific wavelength, passing it through a liquid sample placed in a suitable container and recording the amount of light that manages to pass through it.
By comparing the intensity of the initial light with that of the emerging light, the system translates the response into usable values through a series of physical and instrumental principles.
The interaction of light with matter
When radiation of a given wavelength strikes the atoms or molecules of a substance, they absorb energy if that radiation matches the difference between their molecular energy levels. This absorption is selective and reproducible. Since every molecule has a unique absorption pattern, this phenomenon is the key to unequivocally identifying the type of compound being analysed.
Absorbance and transmittance
To understand the instrument's readings, it is necessary to distinguish between these two closely linked terms:
- Transmittance (T): Indicates the fraction of incident light that passes through the sample without being absorbed (T = I/I0).
- Absorbance (A): This is the amount of light retained or absorbed by the analyte. It is inversely logarithmically related to transmittance through the expression A = -log(T)
The Beer-Lambert law
This law establishes the mathematical basis that turns a purely optical measurement into a direct quantitative figure:
A = ε · c · l
Where:
- A is the absorbance (dimensionless).
- ε represents the molar extinction coefficient, characteristic of each substance at a given wavelength.
- c is the concentration of the compound in the solution.
- l corresponds to the optical path length travelled by the light (usually the width of the cuvette).
What is UV-Vis spectrophotometry?
Ultraviolet-visible spectrophotometry (UV-Vis spectrophotometry) is the modality most widely used in the laboratory and covers radiation between 200 and 800 nm:
- Ultraviolet region (UV): Covers 200 to 400 nm. It is used for molecules that do not absorb in the visible spectrum, such as nucleotides or amino acids.
- Visible region (Vis): Covers between 400 and 700-800 nm. This is the range in which coloured or pigmented solutions interact.
This wavelength range is crucial because it covers the electronic transitions of the vast majority of molecules of interest in chemistry, biochemistry, the pharmaceutical industry and environmental control.
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Key laboratory equipment: the UV-Vis spectrophotometer
To carry out these determinations specific to UV-Vis spectrophotometry, a high-precision instrument called a spectrophotometer is required. Understanding the parts of the spectrophotometer makes it possible to grasp how it works as a whole:
- Light source: Emits stable radiation. In the Vis range, halogen/tungsten lamps are used, whereas the UV range requires deuterium or xenon lamps.
- Monochromator: Isolates and selects the specific wavelength (by means of a diffraction grating) that will be passed through the medium.
- Cuvette / sample compartment: Transparent container that holds the sample. These are usually made of glass (for the visible range) or quartz (essential for UV readings).
- Detector: Device (such as a silicon photodiode or a CCD array) that captures the transmitted photons and converts them into an electrical signal.
- Readout system: Processes the electronic signal and displays the results translated directly into units of absorbance, transmittance or concentration.
Knowing what a spectrophotometer is means recognising the importance of its components in obtaining reproducible analyses. For example, in advanced or molecular biology applications where very small samples are handled, there are specialised variants such as the microvolume UV-Vis spectrophotometer (e.g. Nano-Drop), capable of assessing drops of barely 0.5 to 2 µl without the need for prior dilution.
UV-Vis spectrophotometry is an essential technique in sectors such as biological research, the pharmaceutical industry, clinical analysis and industrial quality control. For this reason, having reliable, precise equipment adapted to the operational needs of each centre is crucial in order to guarantee optimal analytical results.
Frequently asked questions
What types of samples is UV-Vis spectrophotometry used for?
It is normally used with homogeneous liquid samples (aqueous or organic solutions), although with special accessories it is also possible to measure transparent solids, gels or cell suspensions.
What is the difference between spectrophotometry and spectrometry?
Spectrometry is the general term covering any study of the interaction between electromagnetic radiation and matter. Spectrophotometry is a specific quantitative branch that makes direct use of a photometer to measure light intensity as a function of wavelength.
What advantages does spectrophotometry have over other methods of analysis?
It offers a fast response, excellent sensitivity and precision, a low operating cost per analysis and it does not generally require the analysed sample to be altered or destroyed.
What limitations does spectrophotometry have?
It is not suitable for turbid solutions or those with suspended particles that scatter light, nor does it easily allow different compounds that absorb at the same wavelength to be distinguished without prior chemical separation.
How do you choose a suitable spectrophotometer?
You must assess the spectral range required (Visible only or UV-Vis), the desired bandwidth, the type of samples (standard 10 mm path cuvettes or microvolumes) and whether the applications call for a single-beam or double-beam system for greater photometric stability.