Factors Affecting UV-Vis Spectroscopy

UV-Vis spectroscopy is a quantitative laboratory technique used for the measurement of the absorbance of light by a chemical compound. It is performed through measurement of the intensity of light, which passes through a sample with respect to the intensity of light through a reference. It is used particularly in ultraviolet and visible regions. The obtained light intensity versus wavelength is called the spectrum.

Sometimes, the spectrum obtained from the UV-Vis spectrophotometer need not be the same every time. The result may be erroneous. Reproducibility is missing if any parameters like temperature, pH, concentration are changed. Hence, it is essential to study the factors influencing UV-Vis spectroscopy, so that a clear picture will be obtained for careful measurement of the spectrum. This blog takes you to the study of variables, which affect the spectrum obtained from UV-Vis spectroscopy.

List of factors affecting UV-Vis Spectroscopy

For measurement of absorbance and studies, there are numerous factors, which impact the results in ultraviolet visible spectroscopy. The factors are as follows:

• Effect of sample temperature
• Effect of sample concentration
• Effect of sample pH
• Effect of solvent
• Effect of steric hindrance
• Effect of conjugation

Effect of Sample Temperature

The change in temperature of the sample plays a role in the spectrum.

• With the decrease in temperature, the sharpness of absorption bands increases.
• With the decrease in temperature, the position of the peak (absorption maximum) moves very little towards the longer wavelength side.
• However, total absorption intensity is independent of the temperature.
• Simple thermal expansion of the solution can change the intensity of the absorption band.
• Rotational and vibrational energy states depend on the temperature.
• When the temperature is decreased, rotational and vibrational energy states of the molecules also get lowered. 
• Fine absorption bands will be produced when absorption occurs at lower temperatures due to the smaller distribution of excited states..
• The position of the band maximum does not shift much with a decrease in temperature.
• Hence, for obtaining more accurate results, the spectrum needs to be taken at a constant or particular temperature.

Effect of Sample Concentration

The concentration of sample present is directly proportional to the intensity of light absorption, thus influencing the spectrum.

• At a high concentration of solvent, molecular interactions occur, which causes changes in the shape and position of absorption bands. For qualitative work, this effect needs to be identified and taken into consideration. 
• The type of solvent used also affects the fineness of the absorption band in the UV spectra. Polar solvents provide broader bands, but non-polar solvents give better resolution. Removing the solvent gives the best resolution. These effects are all due to solvent-solute interactions.
• There are stronger solvent-solute interactions if the dielectric constant of the solvent is high.
• Water and ethanol, which are polar solvents, exhibit a stronger binding to solute through induced dipole-dipole interactions or hydrogen bonding.
• Through London interactions in non-polar solvents, ground state and excited state will change and the frequency of absorbed photons can be changed. This leads to overlap of different transition energies in spectra, which broaden the absorption band.

Effect of Sample pH

The change of pH of the solution and the aquatic environment has an influence on the spectrum obtained from UV-Vis spectroscopy.

• If the pH of the solution is changed, absorption spectra of aromatic compounds like amines and phenols also change. Upon addition of a base, acidic compounds like phenols and substituted phenols undergo a change in absorption spectra. On removal of the phenolic proton, phenoxide ion is obtained, which increases the conjugation. This leads to a decrease in energy difference between LUMO and HOMO orbitals, resulting in a shift to a longer wavelength along with an increase in absorption intensity.
• If an aromatic amine gets protonated in an acidic medium, there is a disturbance of the conjugation system. The shift of peak towards shorter wavelength happens and a decrease in intensity also occurs.
• The acid-base indicators have an application due to their absorptions in the visible region of the UV-Vis spectrum.
• A minute change in the chemical structure of the indicator causes a change in the chromophore, which absorbs wavelength maximum at different values resulting in color change at different pH.
• An example is a phenolphthalein. It is a weak acid, dissociating in water to give anions, which adds a negative charge to the oxygen atom, contributing a shift of absorption maximum to a longer wavelength. The anion of phenolphthalein is orange, while non-ionized phenolphthalein is colorless. At neutral and acidic pH, the equilibrium shifts towards the left and there will be a lesser concentration of anions and pink color is not observed. But at basic pH, equilibrium shifts towards the right, leading to a higher concentration of anions, and pink color is observed.
• Hence, to maintain pH at a constant value, the UV-Vis spectrum should be measured in an appropriate buffer solution. Over the wavelength range of measurement, the buffer requires it to be transparent. The absorbance value is higher if the buffer solution also absorbs the light.

Effect of Solvent

To an extent, the choice of solvent also affects the spectrum.

• The absorption spectrum is also dependent on the solvent in which the absorption molecule gets dissolved. The option of choosing a solvent can shift the absorption peak to longer or shorter wavelengths. It is based on the interaction of solvent with the chromophore of the desired molecule.
• When compared to hexane solution, ethanol gives absorption maximum at longer wavelengths. 
• Alcohols and water can form hydrogen bonding with the substance, which shifts the absorption bands of polar molecules. As the polarities of ground and excited states of chromophore are different, a change in solvent polarity causes a change in the energy gap between the two states.
• Highly pure and non-polar solvents do not interact with the solute molecules either in the ground state or excited state. However, polar solvents impact the molecular orbitals at the ground state of the excited state. 
• Hence, the spectrum recorded in non-polar solvent differs from that one recorded in the polar solvent.

Effect of Steric Hindrance

The configuration of molecules also has a say in the spectrum.

• When a molecule is planar in conjugation, electronic conjugation works well. The position of the absorption peak is dependent on the effectiveness and length of the conjugative system. If autochrome is there, it prevents the molecule to exist in a planar configuration and shift towards longer or shorter wavelengths depending on the distortion. Distortion of chromophore causes the absorption peak to shift, due to loss of conjugation. 
• Steric hindrance can also be seen in geometric isomerism. Trans isomers exhibit absorption peaks at longer wavelengths and molar absorptivity is higher than the cis counterpart. Due to the steric effect, trans-stilbene absorbs with greater intensity at longer wavelengths.

Effect of Conjugation

Molecular conjugation plays a role in determination of spectrum.

• The absorption peak is shifted to a shorter frequency or longer wavelength when two or more chromophores are conjugated. Conjugation enhances the energy of the highest occupied molecular orbital and mitigates the energy of the lowest unoccupied molecular orbital. Hence, lesser energy is needed for an electronic transition to take place in a conjugated system. If the number of conjugated bands increases, the value of the absorption peak also increases. An increase in the double bonds of a conjugation leads to lesser energy needed for electronic transition. Conjugation of two chromophores also leads to an increase in molar absorptivity and intensity.
• An increase in the number of conjugated bonds leads to absorption of visible light and compounds will be colored. An example is beta carotene, which is a precursor compound of vitamin A. It has 11 conjugated bonds and the absorption peak is shifted from the UV region to the visible region (blue), giving it an orange color.

Conclusion

There are so many factors that impact the absorbance studies in UV-Visible spectroscopy. Choosing a proper solvent and buffer will make the reading accurate. At the same time, optimal temperature and concentration also provide the absorbance value with precision. Similarly, solvent pH should be optimized before taking the reading.