Tips to obtain reliable UV-Vis spectra

UV-Vis spectroscopy seems to be one of the simplest forms of optical spectroscopy. However, most of us have learned at some point of time or another that what looks simple can be highly complex (like many other things in life!). some truly simple tips that might help you to obtain and analyze your spectra effectively.

1. Filter your solutions!

Spectroscopic signals, which result from the interaction of matter with light, can be very sensitive to solution components.In particular, for samples with low absorption, the presence of dust particles can give misleading signals.This is because the dust particles scatter light, which Appears as signals in the UV spectrum. Depending on the size of the dust particles, scattering can occur at all wavelengths.

2. Clean your cuvettes!

Cleaning of cuvettes, although a tiring task in many cases, is an important step in acquiring reliable data. The presence of dirt or residual solutions/particles on cuvette walls (remaining from previous experiments) will alter the amount of light reaching the detector and hence , give inaccurate results. Using clean cuvettes is particularly important when the spectrum of a new sample is being acquired or when related experiments are performed. Clean cuvettes also ensure good reproducibility. Results obtained with an unclean cuvette may not be any good even if you use a filtered solution!

On a similar note, avoid bubble formation in the sample inside the cuvette, and do not use cuvettes with cracks on their absorbing and reflecting surfaces.

3. Choose the right type of cuvettes!

Two opposite sides of a UV-Vis cuvette are opaque (and the remaining two are transparent), whereas all four sides of a fluorescence cuvette are transparent. This is because the detector is placed at 90° with respect to the direction of the incident light in a fluorescence spectrometer, whereas the light source, sample, and detector lie along a straight line in a UV-Vis spectrometer. Since the opposite sides of a fluorescence cuvette are transparent (just like that in a UV-Vis cuvette), does this mean a fluorescence cuvette can be used for UV-Vis spectroscopy? Well, it can be used, but it should not be! The two opposite sides of a UV-Vis cuvette are typically designed to be opaque in order to prevent losses due to scattering and to allow maximum light to reach the detector. The choice of cuvettes can alter your readings!

4. Is an absorbance greater than 1 reliable?

While learning to derive Beer’s law, we are often taught that an absorbance of 1 is not reliable because particles may not exist independently of each other at high concentrations (i.e., they might interact with each other), giving rise to an absorbance that is not directly related to the solution concentration. However, in real scenarios, absorbance greater than 1 is acceptable for qualitative/trend-based studies. For example, in a series of samples with analytes at different (increasing or decreasing) concentrations, an absorbance greater than 1 is acceptable if the absorbance of all samples increases/decreases with a regular trend.

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On the other hand, if you are conducting a quantitative study to measure the analyte concentration, and the absorbance is greater than 1, dilute your solution! Absorbance greater than 1 is not acceptable in this case.

5. UV-Vis spectrometers also measure “scattering”

Turbidity studies are often conducted using UV-Vis spectrometers, and the “absorbance” in such cases is often recorded at 650, 600, 550, or 500 nm. Did you know, however, that it is actually the scattering that is measured in these cases? The y-axis of such plots should ideally be labeled as “Scattering at 600 nm” and not “Absorbance at 600 nm.” Often, these scattering signals are referred to as optical density, represented as “OD₆₀₀.”