Introduction
UV-Vis spectroscopy is a widely used quantitative analysis method in modern laboratories. It is a simple, accurate, rapid, versatile, and cost-effective method for different analyzing tasks.[1]
UV-Vis occupies only in a narrow frequency. The energy states of a molecule can be summarized in an energy-level diagram (figure 1) which can be used to explain the primary photophysical processes. Electronic states depend on the number of electrons and geometry or structure and symmetry of the molecule.[2]
An electronic spectrum can be used for structure determination, and absorption maxima correspond to the discrete molecular states. They are dependent on molecular symmetry, structure, and geometry. Also, the extinction coefficient is essential when interpreting the spectrum. Extinction coefficient gives the magnitude of the transition dipole moment and is also dependent on the geometry and structure. Structure in the absorption band gives information about normal vibrations coupled to the electronic excitation. Anisotropy, on the other hand, gives information about the electronic transition orientation. Electronic excitation spectra can give information on molecular structure.[2]
UV-Vis spectrometer can be used for example in quantitative analysis of light-absorbing molecules, for disrupting DNA double helix, for analyzing food, drug dissolution, enzyme kinetics, and monitoring ozone depletion.[3] In this page is going to be more about principle, apparatus, limitations, and interpretation of the UV-Vis spectrum.
Principle
The principle of the UV-Vis is quite simple.[4] Different molecules absorb light in different wavelengths. If this happens, the wavelengths that the molecule absorbs can be seen by using a UV-Vis spectrometer.[5] A UV and/or visible light source beam are separated into its component wavelengths by a prism or diffraction grating. Each of the monochromatic beams is split into two separate equal beams by a half-mirrored device. The sample beam passes through a small transparent cuvette which contains the sample to be examined in a transparent solvent. The second beam passes the cuvette that contains only the transparent solvent. Electronic detectors then measure these light beams and compared the light intensities with each other. The reference beam should not have suffered from light absorption and is defined as I0. Respectively, the sample beam intensity is I. The spectrometer scans all the component wavelengths in the described manner (UV region is 200-400 nm, the visible region is 400-800 nm).[4]
Absorption of the light happens only if the molecule can accommodate the additional energy by raising electrons to higher energy levels. This absorbed energy of light needs to be as large as the energy needed to raise the electron, and that is why not all wavelengths are absorbed equally by a sample. The absorbance spectrum chooses the light wavelengths the sample absorbs.[3]
The results from the spectrometer are presented as wavelength (λ, x-axis) versus absorption (absorbance (A = log(I0/I)) or transmittance (T = I/I0) from the Lambert-Beer law A = - log(T) = -log(I/I0) = εcd where T is the transmission, I0 is the intensity before passing through the sample, I is the intensity after passing through the sample, ε is molar absorption coefficient, c is concentration, and d is the pathlength of the measuring beam in the sample,[6] y-axis). If the sample does not absorb light of a particular wavelength, I = I0. On the other hand, when the sample absorbs light sample intensity is smaller than the reference one. This difference is then plotted in the graph. λmax is a unique value for each compound which tells is the maximum absorbance wavelength and every compound can have different absorption maxima and absorbances.[4]
Compounds that absorb intensively must be examined in completely transparent solutions so that the detector receives the vital light energies. Also, the absorbance of the sample is proportional to its molar concentration, and that is why is used molar absorptivity ε = Absorbance/(sample concentration • length of light path through cuvette) when comparing different compound spectra.[4]
Apparatus
There are two kinds of spectrometers, single- and double-beam instruments. The single-beam ones generally operate by substitution principle where the reference and measurement cuvettes are placed successively in the light path. The result is then automatically displayed in digital form. On the other hand, in the double-beam instrument, the primary light is split and directed along two paths. Other travels to reference cuvette and the other to the measurement cuvette. After both beams have been refocused, the varying intensities of light go to the detector generating an alternating-voltage signal.[2]
The main parts of UV-Vis spectrometer are a light source, monochromator (most crucial component, usually grating), sample cuvette, detector, and an amplifier with an indicating device. There are two light bulbs, one for ultraviolet (deuterium lamp) and the other for visible light (tungsten-halogen) wavelength region. The radiation sent by the light source is directed through the monochromator to the cuvette. The lamps and the monochromator make it possible to go through the whole spectral region. Depending on the instrument there can be one or two monochromators. With two monochromators there is an advantage that there is less probability of getting stray light. It is the light from other spectral regions, and it can distort the measurement considerably.[2]
After the monochromator, there is only left the wavelength of light that is crucial for the measurement. This light absorbs to the atoms or molecules, and this absorbed light is registered in the detector, and from there to the computer. As a result, we get the light absorbance is in proportion to the sample concentration. The radiation from the lamps can be transformed with different lens-, gap-, prism-, and catcher-systems.[2][7]
Interpretation of UV-Vis spectrum
The detector measures a transmittance which is interpreted with a computer program. From the UV-Vis spectrum can find out how many double bonds is in the compound. The number of double bonds and conjugation causes both increase of absorption and the movement of it to longer wavelengths. Also, the UV-Vis spectrum can be used for tracking how the reaction is progressing. It requires that the spectrum for sources and products are different. From the UV-Vis spectrum can be quite reliably determined the degree of purity of the compound and the presence of different compounds.[2] In the figure 2 is shown an example result of UV-Vis spectrometer whiskey samples.[8]
Figure 2. UV-Vis spectra for whiskey samples in different temperatures [8]. (License: CC BY 4.0)
Limitations
In real life, the measurements are not ideal. Usually for UV-region the light source is typically a tungsten-iodine light and for visible a deuterium discharge lamp. For lamp, switching is used a flipmirror, and if this would be perfect the absorbance values before and after flipping mirror would be identical. To obtain the monochromatic light, most instruments use a grating monochromator which is moved by a stepper motor. Problems with a monochromator come from the diffraction order, and this can be corrected via order filters in the beam after the monochromator that let the desired light pass and block or at least reduces the higher order light intensity. The unwanted measuring beam light limits the use of the Lambert-Beer law, which is called as stray light. Also, in the monochromator, the stray light causes false in the spectrum. A small fraction of the incoming white light will also appear at the output. Spectral false can also occur from a strongly fluorescent sample. The best way would be to correct this with a second detector between the sample and the detector.[6]
To minimize the problems occurring some precautions need to be considered. First, the instrument should be checked before starting analyzing. In the apparatus check the measuring beam and how it is placed concerning cuvette. Run the machine at the wanted wavelength range without the sample or reference cuvette. Then magnify the measured baseline to see the noise. After that block the light path and measure a dark spectrum. Check the measuring beam with two identical cuvettes with the same solvent. Run the spectrum with sample and reference cuvette. If it is possible to see the scattering background, it can be reduced by changing geometry.[6]
Each of the components produces photon shot noise, dark current noise, and Johnson noise, and they are never constant varying between samples. The noise can be expected to follow Gaussian distribution and has a significant effect on the resulting quantitative spectra. This is why the noise is difficult to get rid of. Few methods have been proposed for removing the noise: Savitzky-Golay, Fourier transform, and Wavelet transform. The first mentioned method is to fit low-frequency components of the signal and smooth the high-frequency components of noise to not effectively denoise low-frequency noise signal. In the Fourier transform, the signal spectrum is analyzed and then eliminate the unwanted spectrum directly according to the filter requirements. Wavelet transforms, on the other hand, is a time-frequency analysis method by threshold denoising method. In this method the size of the threshold is important, and it can improve signal-to-noise ratio and decrease root mean square error.[1]
References
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I. Joshi, V.K. Truong, A. Elbourne, J. Chapman, D. Cozzolino, Influence of the scanning temperature on the classification of whisky samples analysed by UV-VIS spectroscopy, Applied Sciences (Switzerland), 2019, 9, 3254-1 - 3254-9 (https://doi.org/10.3390/app9163254). |