This document discusses principles of non-aqueous titration and its applications in pharmaceutical industries. It describes the limitations of aqueous titration and how non-aqueous titration overcomes them. Various types of solvents used in non-aqueous titration are defined including aprotic, protogenic, protophilic and amphiprotic solvents. Acid-base theories, choice of titrants and endpoints, indicators and potentiometric titration are also covered. The document emphasizes the importance of solvent selection and various techniques to improve accuracy in non-aqueous titration.

1. Principles of Non-Aqueous Titration &
Application in Pharma Industries
Dr. A. Amsavel, M.Sc., B.Ed., Ph.DDr. A. Amsavel, M.Sc., B.Ed., Ph.D

2. ContentContent
 Introduction – Volumetric analysis
 Non- Aqueous Titration
 Acid Base Theory
 Solvents for NAT
 End Point Detection
 Indicators Indicators
 Potentiometric End Point
 Acid & Base Titration
 Pharmacopeia USP <541>
 Potentiometric Analysis
 Precaution To Be Taken
 Estimation Of Errors And Elimination Of Errors
 References

3. IntroductionIntroduction
Tirtimetric / Volumetric analysis:
Volumetric analysis is performed for Quantitative determination of
assay / content. It is simple and commonly used technique in Chemical
Industries. Analysis conducted in Aqueous and non-aqueous medium.
◦◦ Simple and easy
◦ Fast and can be done on site
◦ Less expensive
◦ Estimation of content or Assay of chemical
◦ Precise and accurate - depends on method and specificity

4. Limitation in Aqueous TitrationLimitation in Aqueous Titration
Titration in water solutions has limitation:
 To titrate week acids or weak bases
 To titrate separately for a mix of acids (bases) with near
dissociation constants.
 To determine the substances which are insoluble in
water.
 The substances, which are either to weakly acidic or too
weakly basics to give sharp end point in water
 The above can be overcome by non-aqueous to
perform easily and with accuracy

5. NonNon--Aqueous Titration (NAT)Aqueous Titration (NAT)
Non aqueous titration: 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 carry out in non-aqueous medium
 Extensively used for organic acids and bases
Principle is based on Brønsted-Lowry Theory

6. Where to use NATWhere to use NAT
NAT is applied where;
◦ To titrate week acid or weak bases
◦ To titrate separately for a mix of acids (bases) with
near dissociation constants.near dissociation constants.
◦ To determine the substances which are insoluble in
water.
◦ The substances, which are not give sharp end point in
aqueous solutions, can be titrated non-aqueous solvent
( eg too weakly acidic or basic)

7. Advantages of NATAdvantages of NAT
 Organic acids and bases that are insoluble in water can be tested
using appropriate solvents
 Principle of NAT is same as aqueous titration.
 Very week acid and bases can be analysed in NAT
 Mixture of week acids can be tested in single or mixture of
solvents.The individual acid can give separate end point in differentsolvents.The individual acid can give separate end point in different
solvent.
 Biological ingredients of a substance whether acidic or basic can be
selectively titrated using proper solvents.eg Nitrogen containing
compounds
 Non aqueous titrations are simple and accurate, examples of non
aqueous titration are;
◦ Ephedrine , codeine phosphate, tetracycline, piprazine Anti- histamines etc

8. What are Acids & BasesWhat are Acids & Bases
Acids:
Arrhenius acid: Any substance that, when dissolved in
water, increases the concentration of hydronium ion
(H3O+)
Bronsted-Lowry acid: A proton donor; conjugate base
Lewis acid: An electron acceptorLewis acid: An electron acceptor
Bases:
Arrhenius base: Any substance that, when dissolved in
water, increases the concentration of hydroxide ion (OH-)
Bronsted-Lowery base: A proton acceptor ; conjugate
acid
Lewis acid: An electron donor

9. Brønsted-Lowry Theory
The conjugate acid of a base is the base plus the attached proton and
the conjugate base of an acid is the acid minus the proton

10. Equilibrium constant KEquilibrium constant Kaa and Kand Kbb
The equilibrium constant for a Brønsted acid is
represented by Ka, and base is represented by Kb.
CH3COOH(aq) + H2O(l) H3O+(aq) + CH3COO–(aq)
[H O+][CH COO–][H3O+][CH3COO–]
Ka = –––––––––––––––––
[CH3COOH]
[NH4
+][OH–]
Kb = –––––––––––––
[NH3]
NH3(aq) + H2O(l) NH4
+(aq) + OH–(aq)
Notice that H2O is not
included in either
equilibrium expression.
pH of 1M ACOH =2.4

11. Solvents used in NATSolvents used in NAT
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 donate2. Protogenic solvents: Acidic nature readily donate
protons,
3. Amphiprotic solvent: Which are sly ionize and
donate and accept protons,
4. Protophilc solvents: Posses high affinity for protons.

12. AproticAprotic Solvents:Solvents:
Aprotic Solvents:
◦ Solvents are chemically neutral,
◦ un-reactive under the titration conditions; do not
undergo reactions with acids and bases
◦ they possess low dielectric constants,◦ they possess low dielectric constants,
◦ do not cause ionization in solutes and
◦ Aprotic solvents are frequently used to dilute reaction
mixture.
 Eg. Toluene, carbon tetrachloride , acetonitrile,
benzene, and chlorinated hydrocarbons

13. ProtogenicProtogenic SolventsSolvents
 Protogenic Solvents: ( protogenic – proton producing)
 Also called as Acidic solvents are 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 and tend to
have a leveling effect on the bases they come in contact
with.
 Eg . Anhydrous Hydrogen fluoride , Sulphuric acid,
Formic acid , acetic acid etc.

14. ProtophilicProtophilic SolventsSolvents
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 and the conjugate base of the acid :
 which possess a high affinity for protons.
Eg. Liq ammonia, Amine , Ketones, ethers etc.
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.

15. AmphiproticAmphiprotic Solvents:Solvents:
Amphiprotic Solvents:
 Solvents have properties which are protophilic as well as
protogenic.
 Similar to water possesses both acidic and basicSimilar to water possesses both acidic and basic
properties (donating and accepting of protons)
 Which are slightly ionize and donate or accept protons,
◦ Eg Alcohols (Methanol, Ethanol, etc.) , weak
organic acids ( Acetic acid)
◦ Acetic acid makes weak acid into storing base

16. Acetic AcidAcetic Acid
Acetic acid slightly ionise and combine both protogenic and
protophilic propertiesamd able to donate and to accept protons
 Acetic acid is slightly 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 Acidic acid .

17. LevellingLevelling Solvents:Solvents:
Levelling 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 protophillic solvents, all
acids act as of similar strength.
HB B- + H+
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. Properties of a solventsProperties of a solvents
The choice and Properties of a solvent for the particular
titration is very critical.
◦ the solvent should dissolve the substance to be titrated,
◦ should not introduce interfering side reactions with either
the substance to be titrated or the titrant,
◦ should permit a large change in the solvated proton◦ should permit a large change in the solvated proton
concentration near the equivalence point,
◦ should be easily purified, and
◦ preferably should be less expensive.
If the solvent is to be used for a differentiating titration it
should be neither strongly acidic nor strongly basic to avoid
"LEVELING" effects.

19. Titration Of BasesTitration Of Bases
 The titrant should be a very strong acid. Ie Perchloric
acid in Dioxane
 The solvent should not be basic properties
 Aprotic solvents, such as benzene, chloroform, carbon 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 , tertrary amines,
heterocyclic amines

20. Titration Of AcidsTitration Of Acids
 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 IPA
or triethyl-n-butylammonium hydroxide in benzene – methanol.
◦ Solution of sodium or potassium methoxide in benzene - methanol
 Solvent (s):
◦ A mixture of benzene and methanol
◦ very weak acids (e.g., many phenols) usually require a more
strongly basic solvent, such as DMF, anhydrous ethylenediamine or
butylamine
 To determine week organic acids.
Precaution: Amine may absorb carbon dioxide from the atmosphere

21. Selection of SolventsSelection of Solvents for NATfor NAT
 Acetic acid used for titration of weak bases, Nitrogen
containing compounds
 Acetonitrile / with ACOH: Metal ethanoates
 Alcohols (IPA, nBA) : Soaps and salts of organic
acids,
 DMF: Benzoic acid, amides etc

22. TitrantsTitrants for NATfor NAT
 Perchloric acid in acetic acid
◦ Amines, amine salts, amino acids, salts of acids
 Potassium Methoxide in Toluene-Methanol
◦ Week organic acid
Quaternary ammonium hydroxide in Quaternary ammonium hydroxide in
acetonitrile- pyridine
◦ Acids, enols, imides & sulphonamides

23. Endpoint DetectionEndpoint Detection
End point detection is critical for titration, it is to
know the completion of reaction and accurate
determination.
1) Visual indicators:
• Observe a colour change or precipitation at the endpoint.
– Reaction progress checked by addition of external or self– Reaction progress checked by addition of external or self
indicator
2) Electrochemistry:
• Potentiometry - measure voltage change ( pH electrode)
• Amperometry - measure change in current between
electrodes in solution
• Conductance – measure conductivity changes of solution
Later two used for coloured, turbid & accurate end point

24. Improvement of end pointImprovement of end point
 The end points may often be improved by the addition of aprotic
solvents in order to depress the solvolysis of the neutralisation product.
 Potentiometric titrations are used for coloured solutions and
also for compounds which remain feebly acidic or basic
notwithstanding the levelling effect of the solvent.
Visual indicators may be employed for compounds which behave as Visual indicators may be employed for compounds which behave as
sufficiently strong acids or bases in appropriate non-aqueous solvents.
 The suitability of a visible indicator for a particular titration must be
determined by performing a potentiometric titration and observing the
colour change of the indicator simultaneously.
 Temperature of standardization and test shall be same since
coefficients of expansion and dissociation constant may vary

25. Indicators for NATIndicators for NAT
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. I-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.

26. Indicators for NATIndicators for NAT
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 : from blue to pink.point : 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.

27. USPUSP TitrimetryTitrimetry <541><541>
Non-Aqueous titration
 Non-Aqueous titrimetry performed for ,
◦ Acids, acid halides, acid anhydrides, carboxylic acids,
amino acids, enols such as barbiturates and xanthines,
imides, phenols, pyrroles, 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.

28. USPUSP TitrimetryTitrimetry <541><541>
Choice of Titrants:
Basic compound:
 A volumetric solution of perchloric acid in glacial acetic
acid is preferred, or perchloric acid in dioxane is used as
required.required.
Acidic compound:
 A volumetric solution of sodium methoxide or Lithium
methoxide in a mixture of methanol and toluene
 tetra-n-butylammonium hydroxide and trimethyl
hexadecyla mmonium hydroxide (in benzene-methanol
or isopropyl alcohol),

29. USPUSP TitrimetryTitrimetry <541><541>

30. USPUSP TitrimetryTitrimetry <541><541>

31. PontentiomerticPontentiomertic titrationtitration
Potentiometric titration :
 The end-point of the titration is determined by variation of the
potential difference between 2 electrodes in the solution to be
examined as a function of the quantity of titrant added.
 Determine the volume consumed between the 2 points of
inflexion.inflexion.
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 Pharmacopeia

32. Typical titration graphTypical titration graph

33. Preparation of 0.1NPreparation of 0.1N PerchloricPerchloric AcidAcid
 Preparation of 0.1N Perchloric Acid in Glacial Acetic
Acid
◦ Mix 8.5 ml 70% of perchloric acid with 500 ml of
glacial acetic acid and 21 ml of acetic anhydride, cool,
and add glacial acetic acid to make 1000 ml.and add glacial acetic acid to make 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% and 0.5% (if >
0.5%, add acetic anhydride).

34. Precaution forPrecaution for PerchloricPerchloric acid preparationacid preparation
 Perchloric acid (70 to 72%) addition in solvent contain water is
exothermic and may decompose, handle carefully,.
 Conversion of acetic anhydride to acetic acid requires 15-45 min
 Allow to cool to room temperature before adding glacial acetic acid
 Avoid adding an excess of acetic anhydride especially when
primary and secondary amines are to be assayed, because it may
react and convert acetylation .

35. Standardization ofStandardization of PerchloricPerchloric AcidAcid
Standardization of 0.1N perchloric acid
 Accurately weigh about 700 mg of potassium biphthalate,
previously crushed lightly and dried at 120° for 2 h, and dissolve it
in 50 mL of glacial acetic acid in a 250-mL flask. Add 2 drops of
crystal violet TS, and titrate with the perchloric acid solution untilcrystal violet TS, and titrate with the perchloric acid solution until
the violet color changes to blue-green.
 Standardization: perform potentiometrically as specified
 Sample shall be tested the same type of end point determination (
visual or potentometry) followed for standardization
Note: Use benzoic acid as primary standard where required as
specified in USP

36. Electrolytes forElectrolytes for EectrodeEectrode
Electrolyte for electrode
 Suitable electrolyte is important for accurate result :
 Electrolyte shall be filled appropriately in the electrode
example
◦ LiCl saturated in ethanol
◦◦ KCl saturated in ethanol
◦ TEA-Br on Ethylene glycol
 Filling of electrolyte:
◦ remove the aqueous KCl completely
◦ rinsing with water if any residual KCl
◦ rinsing with the nonaqueous solvent to remove water
◦ finally filling the electrode with the suitable electrolyte.

37. Assay Determination of API (USP)Assay Determination of API (USP)
Typical test method as per UPS of API
Assay: (98.0%– 100.5%)
Dissolve about 150 mg of Pseudoephedrine Sulfate,
accurately weighed, in 50 mL of glacial acetic acid.
Titrate with 0.1 N perchloric acid VS, determining theTitrate with 0.1 N perchloric acid VS, determining the
endpoint potentiometrically.
Perform a blank determination, and make any necessary
correction. Each mL of 0.1 N perchloric acid is
equivalent to 42.85 mg of (C10H15NO)2 . H2S04.

38. Precaution to be taken during TitrationPrecaution to be taken during Titration
 Stirring rate is adequate. As a general rule, it should be as high as
possible. However, try to avoid the formation of a vortex during
stirring.
 If endpoint recognition criteria (ERC) is too small can result in an
incorrect equivalence point
 use the titration parameters “signal drift” and “min./max. waiting use the titration parameters “signal drift” and “min./max. waiting
time” to adjust the titration rate.
 Sensor is still suitable for the titration. If sensors is old or dirty can
result in longer response times.
 Check that the anti-diffusion tip is functioning correctly.
 Check the stirring direction. The titrant should not be stirred directly
onto the electrode, but rather away from it.

39. Precaution to be taken during TitrationPrecaution to be taken during Titration
 Selection of appropriate Electrode
 Electrolyte filled shall be appropriate
 Diaphragm shall be free from clogging
 Titration vassal shall be closed while titration
 Avoid expose to atmospheric air, CO may interfere Avoid expose to atmospheric air, CO2 may interfere
 Titration vassal shall not contain water or previous residue
 Sample shall be completely transferred and dissolved
 Air bubbles in the dosing cylinder and/or in the tubing connections
 Air bubbles in the measuring vessel and/or on the sensor during
photometric titrations
 Burette tip for any signs of blockage.

40. Blank correctionBlank correction
 Blank correction is usually obtained by means of a
residual blank titration, wherein the required procedure
is repeated in every detail except that the substance
being assayed is omitted.
 An appropriate blank correction shall be included in
calculation
 difference between the volume consumed in the residual
blank titration and that consumed in the titration can be
considered for assay
 If blank is high or suspected, repeat the analysis

41. CalculationCalculation
o Normality: Eq.wt/1000ml or meq/mL
o Morality: Mole/1000ml
o V1 N1 = V2N2
o N1 = V2N2/V1
 Normality = Wt of sample x 1000 / Eq. Wt x V
 Wt of sample (mg) = V x N x Eq. wt
 Assay = Qty estimated in sample x 100/ wt of sample
 Assay = V x N x Eq. wt x 100/ wt of sample x 1000

42. Errors inErrors in TitrimetryTitrimetry
Error in methods:
The endpoint method may not show a change exactly
at the equivalence point due to the reactions involved
Titration Error = Volume at endpoint – Volume at
equivalence point
 Lower assay: Negative error due endpoint is early
/before equivalency point
 Higher Assay: Positive error due to later endpoint /
after equivalency point

43. Error EstimationError Estimation
Possible Error due to titrant volume or sample weight:
 10 ml titre volume = 100 %
◦ If difference is 0.1ml error is 1% or 0.2ml = 2%
 5ml titre volume = 100 % difference in 0.1ml = 2% error
 25 ml titre volume = 100 % ie difference 0.1ml = 0.4% error 25 ml titre volume = 100 % ie difference 0.1ml = 0.4% error
Error can be minimized :
 Ensure optimum titre volume is around 25ml for manual titration
 Use autotitrator to keep lower dispensing increment ie burettewith
0.001ml or 0.01ml least count is preferred to reduce the error
 Higher weight of sample

44. Operational & personal errorOperational & personal error
The variables influence the error lead to volume in
pipette/ burette
 drain time;
 possible beads on the inner surface
 temperature; temperature;
 bringing meniscus to the proper level;
 angle of drain;
 touching off last drop;
 rinsing of the pipet with the solution used;
 Pipet calibration; etc.

45. Possible Error in weighing.Possible Error in weighing.
• Misreading of the balance,
• Balance sprit level is not eccentric
• Not cleaning the surface of the balance first,
• Touching the weighed object with moist hands,
• Leaving the balance doors open during weighing,• Leaving the balance doors open during weighing,
• Error due to incorrect calibrated balance,
• Not cooling the sample down to near room temperature,
• Not removing a static charge from the sample,
• Excess vibration or air currents from people or nearby equipment,
• Prolonged time sample left on pan adds/loses moisture.

46. Possible ContaminationPossible Contamination
An analyst could
 contaminate a sample during weighing by placing a
contaminated spatula
 placing the sample on or into a contaminated holder
during weighing,during weighing,
 dropping some lint/hair/skin or sneeze into the sample
while weighing,
 opening up a bottle of chemicals near the sample being
weighed.
 When performing trace analysis, it is possible for just a
microgram even massive fingerprint!

47. Units of measurementUnits of measurement
Name Defining Units
Molarity
(e.g. 0.1200 M)
moles of solute/liter (solutions), or
millimoles/milliliter (solutions)
Percent
(e.g. 23.45 %)
(grams of substance/grams of sample) x
100%, or
(e.g. 23.45 %)
centigrams/gram (seldom used)
Parts per million
(e.g 2.34 ppm, 2.34
mg/L)
milligrams/liter (solutions), or
micrograms/milliliter (solutions)
milligrams/kilogram (solids), or
micrograms/gram (solids)
Parts per billion
(e.g. 0.45 ppb, 0.45
ug/L)
micrograms/liter (solutions), or
nanograms/gram (solids)

48. ReferenceReference
 USP
 European Pharmacopoeia
 Non-aqueous titration of acids and bases with potentiometric
endpoint indication- Peter Bruttel , Metrohm
 E. G. Wollish, C. W. Pifer, and Morton. Schmall. -Titration in
Nonaqueous Solvent to Pharmaceuticals- Analytical ChemistryNonaqueous Solvent to Pharmaceuticals- Analytical Chemistry
 Vogel's Text Book of Qualitative Inorganic Analysis 6th Edition

49. Thank You