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UV/Vis spectroscopy is an analytical technique applied for a wide range of the spectrum in various industries. Recent researches on life sciences, analytical chemistry, and more require high-performance UV/Vis spectroscopy for their applications.
Even the traditional application of spectroscopy such as optics, pharmaceutical, food and beverages, quality control, color analysis, material characterization has the requirement of more sophisticated spectroscopy that provides high quality and faster results.
There are many considerations taken into account while selecting the UV/Vis spectrophotometer for various applications in your lab. There are certain parameters that are specific and important to your applications such as wavelength range or sensitivity.
Other parameters such as speed of analysis, flexible sampling, durability, maintenance, and more are also taken into consideration. It is important to select the optimum parameters of UV-Vis spectroscopy measurements for accurate and precise measurements.
Apart from the instrument performance, there are some major sources of errors while sample handling. The factors that affect the absorption spectrum of the substance are
These variables of the sample should be realized so that the absorption of the sample will not vary in higher magnitudes. Some of the important effects that affect UV/ Vis spectrophotometer measurements are discussed as follows,
Spectrophotometer uses cuvette to measure sample. The material of the cuvette that is made up of, its geometry and position has a great influence on the UV/Vis spectroscopy measurements and affects the accuracy and precision of absorbance measurements.
It is a must to take care of the cuvette is to be positioned in the same direction of the light source placed in the cuvette holder for the measurements. This avoids the error that occurs due to optical effects and provides identical optical effects for both reference and sample.
It is important to use the same type of cuvettes for the measurement of both reference and sample. Quartz or suprasil glass cuvettes are best for UV measurements since these materials are UV transparent.
Using disposal cuvettes made of poly-methyl-methacrylate cannot be used in the UV range as it absorbs UV light and can be used only in the visible range.
Table 1. Different types of cuvettes with different usable wavelengths.
The cuvettes are fabricated with windows through which the light passes. This window is to be cleaned properly with a lint-free tissue.
The presence of dust or fingerprints on the cuvette windows results in additional absorbing components and hence provides inaccurate results. It is a must to take additional care to clean the cuvettes before and after use.
UV/Vis spectroscopy must have transparent solvent and dissolve the required quantity of samples. Every solvent has a specific absorbance cut-off wavelength i.e., below this cut-off wavelength the complete light rays are absorbed. It is important to choose the right solvent for UV/Vis measurements.
The solvent chosen should have a known absorbance cut-off wavelength. If the absorbance of the sample compound is close to the absorbance cut-off wavelength of solvent then there is no use in choosing that solvent.
It is required to take more care when working under 300nm as the absorbance of solvent is higher. The below table shows the list of common solvents and the lower limits of the wavelength range.
Table 2. UV absorbance cutoff wavelength of various common solvents
In the linear range of optical measurement results comply with Lambert-Beer law and the absorbance can be determined. In the linear range, it is best to limit the absorbance values that are not very high and very low otherwise it may lead to non-linearity.
UV/Vis measurements are used for samples with low concentrations such as 0.01mol/L. The absorption behavior changes when the sample dissolves in the solvent. It is important to use non-interacting solvent and low concentration analyte to reduce error in the absorbance values.
It is important to obtain the optimum wavelength range corresponding to the peak absorbance. The best wavelength ranges selected close to the higher molar absorptivity ?max as it provides no interfering substance of other components that absorbs at the same wavelength.
There are different components present in the sample, the total absorbance obtained is the sum of absorbance of different components present in the solution.
It is important to analyze the molar extinction of individual components at a specific wavelength as these coefficients of components differ significantly from the other. With these coefficients, the wavelength range is selected optimally.
The complete analysis was made within the wavelength range ?1 and ?2 where the mixture components have a high molar extinction coefficient. The concentration of the mixtures can be determined with the known absorbance and extinction component of the mixtures.
There is certain performance parameter that is to be tested for accurate, sensitive, and reproducible results are wavelength accuracy, photometric accuracy, stray light, resolution, photometric noise, photometric drift, and baseline flatness.
Wavelength accuracy is defined as the deviation of observed wavelength reading compared to that actual wavelength in the absorption band. When the deviation becomes higher it results in an error in both qualitative and quantitative analysis of UV/Vis measurement.
So, it is important for the spectrophotometer to maintain accurate wavelength accuracy to characterize the analyte accurately.
Wavelength reproducibility is defined as the deviation of repeated observed maximum wavelength readings(same wavelength reading). It is measured with the peak reference either the deuterium lamp or standard holmium oxide in perchloric acid.
Photometric accuracy is defined as the deviation between the measured absorbance value and the standard value. Photometric accuracy is the important parameter to be considered in spectral identification and purity control where extinction coefficients or factors are used.
Stray light radiation increases with the imperfections in the optical surfaces from diffraction effects or light scattering or from any other damaged components. Higher stray light will not provide accurate results and deviates from Lambert-Beer’s law. If you do a lot of work in UV spectrophotometer then it is a must to test the stray light parameter.
The stray light parameter will have a strong impact on the linearity between absorbance and concentration. Hence it is important for the spectrophotometer with very less stray light to get accurate results in UV/Vis spectroscopy measurements.
The resolution is another important parameter that impacts the performance of the spectrophotometer. Sufficient resolution in the instrument helps to distinguish different absorbance peaks in the UV/Vis spectrum of the sample. If the resolution is low then identification of absorbance peak is challenging and hence the absorbance value is lower than the true value.
Photometric noise emerges from the fluctuation caused by the light source and the other electronic components. This Photometric noise affects the precision of the absorbance measurements.
Low absorbance levels are affected by the photometric noise from the light source while high absorbance levels are affected by the photometric noise from the electronic components. Noise is measured with no sample in the light path at zero absorbance.
Noise decrease for longer measurement and increases for shorter measurement and it varies with wavelength as the characteristics of light source and detectors vary with wavelength.
Photometric drift or photometric stability determines how stable the photometric results extended over a period of time. Photometric stability is measured at a zero absorbance value at a single wavelength for an hour.
Baseline flatness defines the relative noise level at all wavelengths. The problems with the switching filters or light source exchanges are revealed with the measurement of baseline flatness
UV/Vis spectroscopy has been used in a lot of emerging applications in the research field. It is a must to choose a UV-Vis spectrophotometer with optimal parameters that are well suited for the application. It is important to consider the instrument performance and the factors such as temperature, pH, solvent, and more to get accurate results.