Mass Spectrometry & Spectroscopy


UV-Vis and fl uorescence spectroscopy illuminate biological applications Dr Ursula Tems, Pharma/BioPharma Marketing Director, Molecular Spectroscopy, Agilent Technologies


In the intricate landscape of biological research, understanding the behaviour and properties of biomolecules - those tiny building blocks of life - is paramount. UV-Vis (Ultraviolet-Visible) spectroscopy and fl uorescence spectroscopy are two powerful techniques that shed light on the otherwise invisible interactions within cells, tissues, and organisms. In this article, we delve into their applications, principles, and the advantages they offer over other methods.


About the technologies


UV-Vis spectroscopy involves shining light in the ultraviolet (UV) and visible (Vis) wavelength regions onto a sample and measuring how much light it absorbs, to provide information about the electronic transitions of molecules. As well as being non-destructive, UV-Vis is a well-established, easy, and fast technique that requires minimal consumables and so is also cost effi cient. UV-Vis is used in many biological applications for providing the concentration of a sample, or for characterisation of a sample. For example, protein concentration can be determined by observing the maximum absorbance at a specifi c wavelength, and the quality of the sample determined by observing the spectra to ensure that there are no contaminants.


The other key use is to measure changes that occur over time, or over different temperatures, for example, monitoring an enzyme assay or a thermal denaturation experiment. Recent advances in UV-Vis technology have enhanced its usability and accuracy, for example the Cary 3500 UV-Vis spectrophotometer can measure eight samples simultaneously, and at four different temperatures simultaneously, ideal for monitoring biological enzymes. In addition to this, some accessories exist that allow users to directly quantify very low volume samples of purifi ed nucleic acid or protein samples without the need for sample dilution.


Fluorescence spectroscopy takes advantage of the intrinsic fl uorescence of many biological molecules, such as proteins and nucleic acids. These molecules absorb light (excitation) and emit light (emission) at specifi c wavelengths. The strengths of fl uorescence spectroscopy are specifi city, sensitivity, and the ability to analyse molecular interactions, but fl uorescence spectroscopy has many additional biological applications and is a widely used workhorse in life science laboratories.


A recent and unique example of how fl uorescence spectroscopy can be used in biological laboratories is for screening monoclonal antibodies that are prone to aggregation.


A fl uorescence spectrophotometer is fl exible, able to be combined with many types of sample presentation accessories, and in this case a plate reader is used for high throughput. The properties of a fl uorescent dye are used to indicate which monoclonal antibody samples aggregate and are therefore not useful for further analysis [1].


The wavelengths at which many biomolecules fl uoresce is well characterised, and this can be used to measure the concentration of one type of molecule very specifi cally in a mixture.


Alternatively, fl uorescent molecular ‘tags’ have been designed that can specifi cally bind molecules of interest, such as proteins or other targets, and the known fl uorescence profi le of these tags, or how those change when molecules interact can provide a lot of information. Applications of this technique include looking at cell viability, apoptosis, enzyme kinetics and oxidative stress, for example.


Flexible fl uorescence spectroscopy with plate reader.


Choosing the right technique for biological applications


Applications of UV-Vis for biology


In many cases biological applications require working with nucleic acids or proteins and samples are in low volumes, are diffi cult to obtain or expensive. UV-Vis spectroscopy is a robust, informative technique that does not require a lot of sample and equally has a strong advantage of not requiring much sample preparation.


Temperature (°C) Tm


Figure 1. The plot represents a melting curve of a DNA sample.


Eight samples at four temperatures simultaneously with the Cary 3500 UV-Vis Multizone Peltier Spectrophotometer.


Protein Analysis: In biochemical and biophysical studies, UV-Vis spectroscopy measures the absorbance of proteins at approximately 280 nm due to the absorbance from tryptophan and tyrosine residues. For a pure protein solution, for example assessment of a protein drug, the presence of absorbance at 350 nm can


The melting profile of other molecules/compounds can also be analyzed using UV-Vis spectroscopy. Some examples include polymer cloud-point determination, drug-protein interactions, and protein thermal denaturation.


This document describes how to optimize experimental conditions for thermal melt measurements to achieve high-quality data with confidence. Here are our top tips:


Use the right cuvette


The choice of cuvette will depend on the volume of sample to be analyzed, the z-height of the UV-Vis spectrophotometer1 and the wavelengths used. The most widely used cuvette in UV-Vis spectroscopy has a pathlength of 10 mm and volume of 3.5 mL.


Figure 2. The highly focused beam of the Cary 35 LAB ASIA - JUNE 2024


If measuring more than one sample at a recommended to use the same type of


DNA and RNA Analysis: UV-Vis spectroscopy is commonly employed to verify the concentration and purity of DNA and RNA samples. This is particularly important for downstream applications such as sequencing. By assessing the absorbance patterns of these nucleic acids, researchers can ensure the quality and suitability of their samples for further analysis.


Figure 1: A wavelength scan of a DNA sample showing an absorbance level of around 0.5 Abs and 260 nm.


While taking measurements, the cuvett the cuvette holder such that the transpa with the beam path. If only a small amo available, consider a smaller volume cu the sample is in the beam path (see Tab


The Cary 3500 system is ideal for meas nucleic acid samples as the instrument beam of less than 1.5 mm width (see F instrument’s factory-aligned optics requ before taking measurements, even whe cuvettes at the same time.


Absorbance (A)


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