The document discusses four key factors that can affect UV-Vis absorption by organic compounds: solvent, concentration, pH, and temperature of the sample. The solvent, sample concentration, pH, and temperature can all influence the position and intensity of absorption peaks. It is important for analysts to control these parameters to obtain accurate, precise, and valid quantitative results by using standardized sample preparation and measurement conditions.

4. What Affects UV-Vis Absorption by Organic Compounds
There are a range of factors that can affect the ultraviolet
and visible absorption characteristics of an organic
compound. These are attributed to:
• solvent
• concentration of the sample
• pH of the sample
• temperature of the sample.
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.

5. 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.

6. 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.

7. Effect of Sample Temperature
Temperature impacts absorption measurements by various means:
 Expansion or contraction of the solvent - leading to lower/higher concentrations and absorbances, a particular
issue with some organic solvents.
 Shifts in equilibria between the chemical forms of an analyte – the nature of the absorbing species may be
changed.
 Changes to reaction rates - enzymatic reactions are particularly sensitive to temperature.
 Variation in refractive index within the sample solution due to convection currents – refractive index affects
absorbance particularly for some organic solvents.
For reliable results the analyst needs to ensure that samples/standards are being measured at a specified or constant
temperature.