What is UV-VIS absorption spectroscopy?

Absorption spectroscopy is a widely used analytical technique in chemistry and life sciences for identifying and quantifying substances within a sample. It operates on the principle that molecules absorb light at specific wavelengths, depending on their electronic structure. By measuring the amount of light absorbed at these characteristic wavelengths, it is possible to determine not only whether a particular compound is present, but also to estimate its concentration with high accuracy.

One of the most commonly employed forms of this technique is Ultraviolet-Visible (UV-VIS) spectroscopy, which utilizes light in the UV (typically 200–400 nm) and visible (400–700 nm) ranges. UV-VIS spectroscopy is routinely used in applications such as pharmaceutical quality control, environmental monitoring, biochemical assays, and food safety testing.

The simplicity, speed, and non-destructive nature of absorption spectroscopy make it an indispensable tool in both research and industrial settings. With proper calibration and sample preparation, it can deliver precise quantitative data, often down to micromolar or nanomolar concentrations, enabling scientists and engineers to monitor chemical processes, detect contaminants, and validate product formulations with confidence.

There are many different approaches for measuring absorption spectra in spectrophotometry. The most common one is to point a beam of light at a sample and detect the intensity of the radiation that goes through it. The absorbance is defined as the negative of the logarithm (base 10) of the relative transmission. A 10% transmission corresponds to an absorbance of 1 absorption units (AU) and a transmission of 1% corresponds to 2 AU.

Especially for UV-VIS spectroscopy, it is common to use the Beer-Lambert Law to calculate the concentration of a particular molecule from the measured absorbance.

The UV-detector inside a spectrophotometer for absorption spectroscopy can be implemented in three fundamentally different ways

• Fixed wavelength detector
• Tunable wavelength detector
• Full spectrum detector

Below you can read some more detailed descriptions of each.

A fixed wavelength detector consists of an optical bandpass filter allowing a specific wavelength (for instance 254 nm) to pass through to a silicon photo-detector. All other wavelengths than the filter wavelength are blocked. The main benefits of this solution are it’s simplicity, robustness and relatively low cost. The main drawback is that the wavelength is fixed and you can only analyse at one specific wavelength at a time. This means that this kind of solution is best suited for systems where you are going to do the same analysis on the same material over and over again – like in a production set-up in a factory. Some systems will enable you to manually exchange the filter or include several filters in the UV-detector. In any case, you are still limited to a fixed set of analysis wavelengths. For this reason, it can be hard to troubleshoot if something goes wrong – for instance if your sample is contaminated and there is absorption at a different wavelength than the one you are measuring at.

In a tunable wavelength detector the filtering element is a so-called grating monochromator that can be adjusted (by the instrument control software) to select the wavelength you want to use for analysis. The benefit of this is clearly that you are free to choose any analysis wavelength. However, as with the fixed wavelength filter above it can be difficult to troubleshoot when you only have data at one wavelength.

The grating monochromator can also be used to scan through the entire wavelength range for each measurement. This means you have much data which can be used to troubleshoot or analyse more complex absorption spectra with many peaks at different wavelenghts. The scanning is however fairly slow – a full scan will often take around 1 minute. Therefore, this is often not a workable method for time critical experiments.

Grating mono-chromators can offer a large absorbance grange – even up to 5 AU for the more advanced ones.

A final thing to mention about monochromators is that the detector has movable parts inside that can be worn out over time.

A spectrometer with a detector array detects absorbance at all wavelengths in the spectrum simultaneously. This type of analysis is thus much more versatile than the two other methods because you always obtain absorbance data at all wavelengths in one “shot”. Typically, a full spectrum can be acquired in a few milliseconds. This is particularly useful if you are using your instrument for many different samples – like in development of new pharmaceuticals or for online process control in manufacturing. Even if you are only interested in absorbance at one wavelength, having access to the full spectrum will make it much easier to troubleshoot if something goes wrong.

A spectrometer with a diode array is also robust and compact since it doesn’t include any movable parts.

Compared to filters and mono-chromators, the main drawback of spectrometer using diode arrays and fixed gratings is that they can typically only measure absorbance levels up to 2.5 – 3 AU.