There are a range of factors that can affect the ultraviolet and visible absorption characteristics of an organic compound. These are attributed to:
Uncontrolled changes in these factors can introduce inaccuracy and imprecision by altering the position ( max) and intensity (max) of the absorption peaks of the chromophore in the compound. The analyst must control these parameters if comparison of spectra is to be meaningful and if quantitative results are to be valid.
With the job of the analyst in mind, sample and standard preparation will play a key role particularly in regard to using standardised conditions.
Effect of Solvent
The choice of solvent can shift peaks to shorter or longer wavelengths. This will depend on the nature of the interaction of the particular solvent with the environment of the chromophore in the excited state of the molecule.
Depending on the chromophore in the particular analyte, changes in the
polarity of the solvent can influence shifts to longer or shorter wavelengths.
For instance, it is usually seen that ethanol solutions give longer wavelength
maxima than hexane solutions.
Effect of Sample Concentration
As you might expect, sample concentration is proportional to the intensity of the absorption. At high concentrations however, molecular interactions (for example, polymerisation) can take place causing changes to the position and shape of absorption bands. Such an outcome can affect the linearity of the relationship between sample concentration and absorbance (remember Beer’s Law).
Such effects need to be identified and taken into consideration for quantitative work.
Effect of Sample pH
The pH of the sample solution can have a significant impact on absorption spectra. The mechanism for this is primarily a shift in the equilibrium between the different chemical forms of an analyte. To illustrate this, pH indicators used in acid/base titrations change colour at a particular pH because the chemical form of the indicator-compound undergoes a change at this point.
If pH is known to be a factor, a remedy is to prepare the sample in a suitable buffer solution so as to maintain the pH at a steady value. The buffer though needs to be transparent over the wavelength range of the measurements – if the buffer absorbs radiation, absorbance readings attributed to the analyte may be higher than they should because the buffer and analyte absorptions will add together at each wavelength.
Effect of Sample Temperature
Temperature impacts absorption measurements by various means:
For reliable results the analyst needs to ensure that samples/standards are being measured at a specified or constant temperature.