The document discusses factors that can affect UV-Vis absorption characteristics of organic compounds, including: - Solvent choice can shift peaks to shorter or longer wavelengths depending on solvent-analyte interactions. For example, ethanol often gives longer wavelengths than hexane. - Sample concentration is proportional to absorption intensity but high concentrations can cause non-linearity due to molecular interactions. - Sample pH can significantly impact spectra; maintaining a stable pH buffer is recommended but the buffer must be transparent in the measurement range. - Temperature impacts absorption through solvent expansion/contraction, shifts in analyte equilibria, and changes to reaction rates. Heating a sample can result in higher or lower absorbances.

1. M A D E B Y: L A L I T S I N G H
S T U D E N T O F M - P H A R M
( P H A R M A C E U T I C A L A N A LY S I S )
I S F C O L L E G E O F P H A R M A C Y, M O G A
Factor affecting UV-Vis
Absorption

2.  There are a range of factors that can affect the ultraviolet
and visible absorption characteristics of an organic
compound.
Concentration
of the sample
Temperature
of the sample
pH of the
sample
Solvent
Factor

3. 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.

4. 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).

5. Conti…
 Sample concentration ∞ Absorbance
 Higher the concentration of the analyte show higher abs.
they don’t follow Lambert beer law.
 Optimum concentration of analyte will be use , the abs.
will below then 0.8 .

6. Effect of Sample pH
 The pH of the sample solution can have a significant
impact on absorption spectra.
 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. Conti…
 If the optimum pH
buffer solution is
suitable with
analyte
 The abs. spectra
will show clear
peak of the analyte
• If the optimum pH
buffer solution is
not suitable with
analyte
• The abs. spectra
will not show
clear peak of the
analyte, they
merge with buffer
peak.

8. 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.

9. Conti…
 Sample solution
Heat
 Expansion or contraction of the solvent
 lower/higher concentrations
 Absorbance will alter(May be higher abs. or lower abs.)