This document discusses various methods for analyzing complexes, including continuous variation (Job's) method, distribution method, solubility method, pH titration method, and spectroscopy. The continuous variation method analyzes changes in physical properties like dielectric constant when complexes form to determine stoichiometric ratios. The distribution method examines how the distribution of a solute between immiscible liquids changes with complexation to estimate stability constants. The solubility method observes whether solubility increases or decreases with the addition of a complexing agent. pH titration is reliable for complexes that affect pH upon formation. Spectroscopy techniques like UV and NMR are also used to determine rate constants and equilibrium constants.

1. IIIrd Semesester B. pharmacy
Physical Pharmaceutics-I
Unit-IV
Complexation and Protein Binding [Part-2]
(Method of analysis, Complexation and drug action )
Miss. Pooja D. Bhandare
(Assistant professor)
Kandhar college of pharmacy

2. Method of Analysis
• The analysis of complexes involves the
estimation of two parameter.
1. The stoichiometric ratio of ligand – to
metal or donor-to acceptor.
2. Stability constant of the complex.
Study of rate of complex degradation is
very important in the determination of
complex application.

3. Method of Analysis
1. Methods of continuous variation / JOB’S
method of continuous variation.
2. pH titration method.
3. Distribution method.
4. Solubility method.
5. Spectroscopy and charge transfer
complexation.
6. Miscellaneous method

4. 1. Continuous variation method
• The physical properties, such as dielectric constant, refractive index
and spectrophotometric extinction coefficient, are characteristics of
particular species.
• When there is no complexation between these species ( A and B), the
value of property is additive.
• This particular behaviour is represent in figure as dotted line. In case
of complexation, these properties change, i.e. additives phenomena do
not hold good.

6. Continuous variation
1. Due to complexation physical
properties result may be
maximum or minimum.
2. At maximum/minimum point
note concentration of individual
species.
3. Calculate stoichiometric ratio of
species.

7. 2. Distribution method
• The distribution behaviour of a solute between two immiscible liquid
is expressed by distribution coefficient or partition coefficient.
• When a solute complexes with an added substance, the solute
distribution pattern changes depending on the nature of complex.
• A set of such experiment provides data for the estimation of complex
equilibrium stability.

8. • Example: The complexation of
iodine (𝐼2) with potassium iodide
(𝐼−
) can be represented by the
following equilibrium.
𝐼2 + 𝐼− = 𝐼3
−
𝐾𝑜/𝑤 =
[𝐼2] 𝑜
[𝐼2] 𝑤
K=
[𝐼3] 𝑤
[𝐼−] 𝑤 𝑋 [𝐼2] 𝑤

10. 3. Solubility method:
• When mixture form complexes solubility
may increase/ decrease.
• Experiment are conducted to estimate
parameters
• Experiment:
1. Caffine ( Complexing agent) taken in
different concentrations.
2. Add PABA, Agitate, Filter and analyse
drug content.

12. 4. pH Titration method :
• Most reliable method
Complexation should be affected
by change in pH.
• E.g. : Glycine with copper

13. 4. pH titration method:
This method if complexation produce change
in pH.
Experiment:
1. Glycine solution (75ml) titrated with
NaOH, pH is recorded.
2. Glycine solution (75ml) + Cu+2 Complex
titrated with NaOH.
3. pH is recorded. (complexation releases
Proton and pH decreases)
4. Quantity of alkali = Concentration of
ligand bound.

14. 5. Spectroscopy method
• The UV spectroscopy is used as extensively in determining rate
constant , equilibrium constant, acid-base dissociation constant etc for
chemical reaction

15. 6. Miscellaneous method
• Several other method are available for the analysis of complexes like
NMR and IR spectroscopy, polarography, circular dicromism, kinetics,
X- ray diffraction and electron diffraction.

17. Thank you!