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A researcher may choose fluorescence spectroscopy to take advantage of the specificity of the technique. When a UV-Vis absorption spectrum of a mixture is analysed, all the molecules in the mixture that absorb light will contribute to the absorbance observed. With fluorescence spectroscopy, generally the excitation wavelength of a specific molecule can be used so that the emission profile that is observed is specific for that molecule.


The benefit of UV-Vis spectroscopy is in the low sample preparation requirement, particularly when analysing a pure sample, such as a purified protein. Many fluorescence assays exist for detecting protein concentration or amount, but these generally rely on the use of a fluorescent dye and can be inaccurate. UV-Vis spectroscopy is preferred for analysing pure proteins as it is a simple and highly accurate technique. UV-Vis spectroscopy can also measure higher concentration samples that might not be measurable using fluorescence due to quenching or inner filter effects.


Synergy: Combining UV-Vis and fluorescence


In many biological laboratories a UV-Vis spectrophotometer will be found next to a Fluorescence spectrophotometer as the techniques are complementary. The combination of the two techniques can provide greater insight for many biological applications and combining detection modes into a single instrument provides researchers with some efficiency gains especially with high throughput applications where microplate readers are used. Multimodal plate readers provide the ability to employ diverse labelling strategies around a particular application area. For example, in the realm of studying kinases, a hybrid microplate reader combing both UV-Vis absorbance and fluorescence (as well as other detection modalities) can be used.


While UV-Vis spectroscopy is ideal for quantitative analysis or characterising enzymes, fluorescence can provide additional information on how molecules interact. Two examples of this are polarisation assays providing fast and quantitative information on molecular interactions, and fluorescence resonance energy transfer (FRET) assays that also take advantage of fluorescent properties of molecules to analyse interactions.


If using the techniques together, the main application and benefit is in the optimisation of both methods. When developing methods, the combined use of UV-Vis and Fluorescence spectroscopy can ensure optimal results. For example, UV-Vis spectroscopy can be used to confirm an excitation wavelength or the concentration of a molecule before it is analysed by fluorescence spectroscopy so that useful data is obtained.


Using the techniques together can also benefit sample optimisation. For lower concentration samples or if the sample is scarce, fluorescence will be a more sensitive technique that requires less initial sample. However, not every molecule fluoresces, and some fluorescence assays can be cost-prohibitive so the simplicity and low sample preparation requirements of UV-Vis spectroscopy may be preferred.


Practical tips for researchers


Sample preparation matters UV-Vis:


• Ensure sample purity: Contaminants can interfere with absorption measurements. Purify your samples to minimise unwanted signals.


• Use appropriate cuvettes: Choose quartz cuvettes for UV measurements and glass cuvettes for visible range.


• Blank correction: Always measure a blank (solvent) to subtract its absorbance from sample measurements.


Fluorescence:


• Avoid auto-fluorescent materials: Some plastics, glass, or impurities emit fluorescence. Use low-fluorescence materials for cuvettes and containers.


• Dilution effects: High concentrations can lead to inner filter effects or quenching. Optimise sample concentrations for accurate results.


Instrument calibration and validation


UV-Vis: • Calibrate wavelength accuracy: Regularly check the instrument’s wavelength calibration using standard solutions (e.g., holmium oxide filter).


• Validate linearity: Measure known concentrations of standard solutions to verify linearity within the Beer-Lambert range [2].


Fluorescence:


• Check lamp intensity: Fluorescence lamps degrade over time. Monitor and replace them as needed.


• Validate sensitivity: Use known fluorophores (e.g., quinine sulphate) to validate sensitivity and linearity.


Spectral overlap and multicomponent analysis


UV-Vis:


• Be aware of overlapping spectra: Some molecules have similar absorption profiles. Deconvolute overlapping peaks using mathematical methods.


• Use derivative spectroscopy: Derivative spectra enhance peak resolution, making it easier to identify components.


Fluorescence:


• Spectral unmixing: When multiple fluorophores are present, use spectral unmixing algorithms to separate their contributions.


• Lifetime-based analysis: Fluorescence lifetime measurements provide additional information beyond intensity.


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