Non-aqueous titration

1. COURSE: PHARMACEUTICAL ANALYSIS-I (BP102T)
PROF. K.N.RAJINI KANTH, DEPARTMENT OF PHARMACEUTICAL ANALYSIS
CHALAPATHI INSTITUTE OF PHARMACEUTICAL SCIENCES, GUNTUR, A.P

2. CONTENT
Introduction to non- aqueous titration
Need of non-aqueous titrations
Principle and technique of non-aqueous titration
Solvents used in non-aqueous titrations
Levelling solvents
Titrants for non-aqueous titration
Indicators for non-aqueous titration
Drugs analysed by non-aqueous titration
Advantages of non-aqueous titration

3. NON-AQUEOUS TITRATION
• Non aqueous titration is the titration of substances
dissolved in solvents other than water.
• It is one of the most common titrimetric procedure used
in pharmacopoeial assays
• It is suitable for the titration of very weak acids and very
weak bases
• It provides a solvent in which organic compounds are
soluble.
• The most commonly used procedure is the titration of
organic bases with perchloric acid in anhydrous acetic
acid.
• These assays judge the endpoint more precisely.
• The Karl Fischer titration for water content is another
nonaqueous titration, usually done in methanol.

4. NON-AQUEOUS TITRATION (NAT)
Titration performed in solvent medium which does not
contain water. Substance is dissolved in a solvent and
titrated using acid or base as titrant.
• Theory is same as Acid-Base titration
• Reaction carried out in non-aqueous medium
• Extensively used for organic acids and bases

5. NEED OF NON-AQUEOUS TITRATIONS
Often times we need to perform an acid-base titration in non-
aqueous solvent due to:
• The analyte is too weak acid or a base to be titrated in H2O
• Reactants or products are poorly soluble in H2O
• Reactants or products weak reactivity with H2O
• Titration in H2O doesn’t allow a sharp end point
• In a non-aqueous solvent, it is possible to get sharp end point

6. The principle and technique of non-aqueous
titrimetric method is very simple. It is based on
• The acid-base theory proposed by lowry-bronsted
• Nature and influence of levelling effect of non-aqueous
solvents on substances

8. CONJUGATE ACID CONJUGATE BASE
Strongest HClO4 ClO4
- Weakest
H2SO4 HSO4
-
HCl Cl-
H3O+ H2O
CH3COOH CH3COO-
NH4
+ NH3
HCO3
- CO3
2-
Weakest H2O OH- Strongest
CONJUGATE ACIDS AND BASES

9. IT FOLLOWS FROM THESE DEFINITIONS THAT
An acid may be either:
• An electrically neutral molecule, e.g. HCl, or
• A positively charged cation, e.g. C6H5NH3+, or
• A negatively charged anion, e.g. HSO4
-
A base may be either:
• An electrically neutral molecule, e.g. C6H5NH2, or an anion,
e.g. Cl-
• Substances which are potentially acidic can function as acids
only in the presence of a base to which they can donate a
proton. Conversely basic properties do not become apparent
unless an acid also is present.
• The apparent strength of an acid or base is determined by the
extent of its reaction with a solvent.

10. • In aqueous solution all strong acids appear equally strong
because they react with the solvent to undergo almost complete
conversion to hydronium ion (H3O+) and the acid anion.
• In a weakly protophilic solvent such as acetic acid, the extent
of formation of the acetonium ion (CH3COOH2
+) due to the
addition of a proton provides a more sensitive differentiation of
the strength of acids.
• Acetic acid reacts incompletely with water to form hydronium
ion and is, therefore, a weak acid.
• In contrast, it reacts with strong acid like perchloric acid and
behaves as a strong acid. This so-called leveling effect.

11. SOLVENTS USED IN NON-AQUEOUS TITRATIONS
Solvent which are used in non-aqueous titration are
called non-aqueous solvent.
Classified as four types:
1. Aprotic solvents: Chemically neutral
2. Protogenic solvents: Acidic nature readily donate
protons
3. Amphiprotic solvent: Which slowly ionize and donate
and accept protons
4. Protophilc solvents: Posses high affinity for protons

12. APROTIC SOLVENTS:
• solvents are chemically neutral
• un-reactive under the titration condition
• do not undergo reactions with acids and bases
• they possess low dielectric constants
• do not cause ionization in solutes and
• aprotic solvents are frequently used to dilute reaction
mixture
Example: Toluene, Carbon tetrachloride,
Acetonitrile and Benzene

13. PROTOGENIC SOLVENTS:
(PROTOGENIC–PROTON PRODUCING)
• also called as acidic solvents
• acidic in nature and readily donate protons
• solvents which are more acidic than water
• strength and ability to donate protons
• they enhance the strength of weak bases
• these solvents have a more acidic character
• tend to have a levelling effect on the bases they come in
contact with
Example: Hydrogen fluoride, Sulphuric acid,
Formic acid and Acetic acid

14. PROTOPHILIC SOLVENTS: (PHILIC-AFFINITY)
• solvents which are more basic than water
• they will react with an acidic solute with the formation of a
solvated proton
• the conjugate base of the acid possess a high affinity for
protons
Example: Liquid ammonia, Pyridine, Acetone
Weak acids are normally used in the presence of strongly
protophilic solvents as their acidic strengths are then
enhanced and then it behave like strong acids, this is known
as the levelling effect
• Strong bases are levelling solvents for acids
• Weak bases are differentiating solvents for acids

15. AMPHIPROTIC SOLVENTS
• Solvents have properties which are protophilic as well as
protogenic
• Similar to water possesses both acidic and basic
properties
• donate and accept of protons
• which are slightly ionisable and donate or accept
protons
Example: Methanol, Ethanol, Acetic acid

16. ACETIC ACID
Acetic acid slightly ionise and combine both protogenic and
protophilic properties and able to donate and to accept
protons
Acetic acid ionize and dissociate to produce protons
CH3COOH ↔ CH3COO- + H+
But in the presence of perchloric acid, a far stronger acid, it
will accept a proton:
CH3COOH + HClO4 ↔ CH3COOH2
+ + ClO4
–
The CH3COOH2
+ ion can very readily give up its proton to
react with a base, so basic properties of a base is enhanced,
so titrations between weak base and perchloric acid can often
be accurately carried out using acetic acid

17. LEVELING SOLVENTS
In general, strongly protophilic solvents are important to force
equilibrium equation to the right.
CH3COOH + HClO4 ↔ CH3COOH2+ + ClO4 –
This effect is so powerful that, in strongly protophilic solvents,
all acids act as of similar strength.
The converse occurs with strongly protogenic solvents, which
cause all bases to act as they were of similar strength.
Solvents, which act in this way, are known as levelling solvents.

18. NON-AQUEOUS TITRIMETRY PERFORMED FOR
Acids, acid halides, acid anhydrides, carboxylic acids, amino
acids, enols such as barbiturates and xanthines, imides,
phenols, pyroles, and sulfonamides.
Bases, amines, nitrogen-containing heterocyclic compounds,
oxazolines, quaternary ammonium compounds, alkali salts
of organic acids, alkali salts of weak inorganic acids, and
some salts of amines.

19. TITRANTS FOR NON-AQUEOUS TITRATION AND
PHARMACEUTICAL SUBSTANCES ANALYSED
• Perchloric acid in acetic acid
Amines, amine salts, amino acids, salts of acids
• Potassium methoxide in Toluene-Methanol
Weak organic acid
• Quaternary ammonium hydroxide in acetonitrile-
pyridine
Acids, enols, imides and sulphonamides

20. TITRANTS USED IN NON-AQUEOUS TITRATION
Acidic titrants:
• Perchloric acid
• p- Toluene sulfonic acid,
• 2,4-Dinitrobenzene sulfonic acid
Basic titrants
• Tetrabutyl ammonium hydroxide
• Sodium acetate
• Potassium methoxide
• Sodium amino ethoxide

21. INDICATORS FOR NON AQUEOUS TITRATION
1. Crystal violet is used as a 0.5% (w/v) solution in glacial acetic
acid. Its colour change is from violet through blue, followed by
green, then to greenish-yellow,
2. Methyl red is used as a 0.2% (w/v) solution in dioxane with a
yellow to red colour change.
3. 1-Naphthol benzein 0.2% (w/v) solution in acetic acid gives a
yellow to green colour. It gives sharp end points in nitromethane
containing acetic anhydride for titrations of weak bases against
perchloric acid.

22. INDICATORS FOR NON AQUEOUS TITRATION
4. Oracet blue B is used as a 0.5 % (w/v) solution in acetic
acid and is considered to be superior to crystal violet for
titrations of bases in acetic acid with standard perchloric
acid. The end point is from blue to pink.
5. Thymol blue 0.5 % (w/v) in methanol is used for titrations
of substances acting as acids in dimethylformamide solution.
The end point: change from yellow to blue.
6. Methyl violet 0.2% (w/v) in chlorobenzene, violet to blue.

23. CLASSES OF NON-AQUEOUS TITRATION
• ACIDIMETRIC TITRATION (Basic substances)
• ALKALIMETRIC TITRATION (Acidic substances)

24. ACIDIMETRIC TITRATION
• The titrant should be a very strong acid, eg., perchloric acid
in acetic acid/dioxane
• The solvent should not possess any basic properties
• Aprotic solvents, such as benzene, chloroform, carbon
tetrachloride, chlorobenzene, either alone or mixed with
glacial acetic acid may sometimes be used for titration with
acetous perchloric acid
• To determine primary, secondary, tertiary amines and
heterocyclic amines

25. ALKALIMETRIC TITRATION
• The titrant should be a solution of a strong base
• Solutions of quaternary ammonium hydroxides in organic
solvents,
• e.g. tetra-butylammonium hydroxide in benzene-
methanol or n-butylammonium hydroxide in benzene-
ethanol
• Solution of sodium or potassium methoxide in benzene-
methanol
• Solvent(s) include a mixture of benzene and methanol
• Very weak acids (e.g., many phenols) usually require a
more strongly basic solvent, such as DMF, anhydrous
ethylene diamine or butylamine
• To determine weak organic acids

26. ENDPOINT DETECTION
End point detection is critical for titration; it is to know the
completion of reaction and accurate determination.
Visual indicators:
Observe a colour change or precipitation at the endpoint.
Reaction progress checked by addition of external or self-
indicator
Electrochemistry:
Potentiometry -measure voltage change (pH electrode)
Amperometry-measure change in current between
electrodes in solution
Conductance – measure conductivity changes of solution

27. POTENTIOMETRIC TITRATION
The end-point of the titration is determined by variation of
the potential difference between two electrodes in the
solution to be examined as a function of the quantity of
titrant added.
Determine the volume consumed between the two points of
inflexion.

28. ELECTRODE
Selection of appropriate electrode is important to determine
the accurate determination
Preferred electrode for acid-base titrations is glass-calomel or
glass- silver-silver chloride electrode, unless otherwise
specified in Pharmacopoeia

29. ESTIMATION OF SODIUM BENZOATE
Molecular Formula: C7H5NaO2
Molecular Weight: 144.1
Category: Pharmaceutical aid
Sodium Benzoate contains not less than 99.0 per cent and
not more than 100.5 per cent of C7H5NaO2 calculated on
the dried basis.

30. Preparation of 0.1N Perchloric acid solution:
Dissolve 8.5 ml of perchloric acid (72%) in about 500 ml
glacial acetic acid with constant stirring, add about 21ml of
acetic anhydride, cool and add more glacial acetic acid and
make up the volume to 1000 ml.
Allow the prepared solution to stand for 1 day for the excess
acetic anhydride to be combined, and determine the water
content 0.02% to 0.5% (if > 0.5%, add acetic anhydride).
Standardise the solution as per I.P using potassium
hydrogen phthalate as primary standard.
Note: Acetic anhydride absorbs all the water from HClO4
and glacial acetic acid and renders the solution virtually
anhydrous. HClO4 must be well diluted with glacial acetic
acid before adding acetic anhydride because reaction
between HClO4 and acetic anhydride is explosive.

31. Standardisation of 0.1N Perchloric acid Solution:

32. In usual practice, potassium hydrogen phthalate (or
potassium biphthalate, KHC8H4O4) is employed as a
standardizing agent for acetous perchloric acid. The
reaction may be expressed as follows:

33. Procedure for standardisation: Weigh accurately about
0.5gm of potassium hydrogen phthalate and dissolve in 25 ml
of glacial acetic acid in a 150ml conical flask and add few
drops of 5% w/v crystal violet in glacial acetic acid as
indicator. Titrate the solution with 0.1N perchloric acid
taken in the burette until violet colour changes to emerald
green.
Each ml of 0.1N perchloric acid is equivalent to 0.02042gms
of potassium hydrogen phthalate

34. Procedure for assay of sodium benzoate:
Weigh accurately about 0.25 g of sodium benzoate, dissolve
in 20 ml of anhydrous glacial acetic acid, warming to 50º if
necessary, cool. Titrate with 0.1 N perchloric acid, using 0.05
ml of 1-naphthol benzein solution as indicator. Carry out a
blank titration.
Equivalent factor or I.P factor: Each ml of 0.1 N perchloric
acid solution is equivalent to 0.01441 g of sodium benzoate.

35. ESTIMATION OF EPHEDRINE HYDROCHLORIDE
Molecular Formula: C10H15NO, HCl
Molecular Weight: 201.7
Category: Bronchodilator
Ephedrine Hydrochloride contains not less than 99.0 per
cent and not more than 101.0 per cent of C10H15NO, HCl
calculated on the dried basis.

36. Procedure for assay of Ephedrine HCl:
Weigh accurately about 0.17 g of ephedrine hydrochloride,
dissolve in 10 ml of mercuric acetate solution, warming gently,
add 50 ml of acetone and mix. Titrate with 0.1 N perchloric
acid, using 1 ml of a saturated solution of methyl orange in
acetone as indicator until red colour is obtained. Carry out a
blank titration.
Equivalent factor or I.P factor: Each ml of 0.1 N perchloric
acid solution is equivalent to 0.02017 g of ephedrine
hydrochloride

37. DRUGS ANALYSED BY NON-AQUEOUS TITRATION
Adrenaline
Caffeine
Ephedrine HCl
Metronidazole
Sodium benzoate
Sodium citrate
Ethosuximide
Fluorouracil
Mercaptopurine
Phenobarbitone

38. ADVANTAGES OF NAT
• Organic acids and bases that are insoluble in water
can be analysed using appropriate solvents
• Principle of non-aqueous titration is same as aqueous
titration
• Very weak acid and bases can be analysed by non-
aqueous titration
• Mixture of weak acids can be tested in single or
mixture of solvents
• The individual acids can give separate end point in
different solvents
• Biological ingredients of a substance whether acidic or
basic can be selectively titrated
• Example: Nitrogen containing compounds
• Non-aqueous titrations are simple and accurate

39. ACKNOWLEDGEMENT
I acknowledge the valuable inputs of teachers and
research scholars who have shared their knowledge,
ideas and expertise in various platforms of pharmacy
and who have inspired me to prepare this presentation
for the benefit of students and faculty
- Prof. K.N.Rajini Kanth