GTP in Pharma

Good Titration Practice
in the Pharmaceutical Industry

Agenda
The strength of acids and bases
How to titrate a weak acid or base?
Advantages of non-aqueous titrations
Non-aqueous base titrations
- Titration of bases – levelling/differentiating
- Solvents for titration of bases
- Titrants for titration of bases
Non-aqueous acid titrationstitration)
- Solvents for titration of acids
- Titrants for analysis of acids
- Tips for non-aqueous acid titrations
Standardization of non-aqueous titrants
Sensors for non-aqueous titrations
Blank determination
Effect of temperature
Application examples
1
Examples of acids Examples of bases
Acid pKa Base pKb
Perchloric acid -9 Guanidine 0.5

Formic acid 3.75 Methylamine 3.36
Acetic acid 4.73 Ammonia 4.75
Lactic acid 3.86 Diethanolamine 5.12
Benzoic acid 4.20 Picoline 7.52
Phenol 9.95 Quinoline 8.94
Hydroquinone 10.0 Pyrazol 11.51
Caffeine 13.39
The weaker an acid (base) the higher the pKa (pkb) value
Note: the pK values are defined in aqueous systems

2
Agenda
Blank determination
3
In aqueous media the H3O+ or the OH- ions are the strongest
acids or bases
The solvent water shows a levelling effect on the acid or
base strength
Very strong acids or bases [pKa(b) <0] or very weak acids or
bases [pKa(b)>9] cannot be titrated in water
In non-aqueous media there is no competition from the auto
dissociation of water
H2O + H2O H3O+ + OH-
In non-aqueous solvents the dissociation of an acid or a
base is totally different compared to water
Example: A solution of picrinic acid in acetone is more
acidic than a solution of HCl in water
4
Agenda
Blank determination
5
Expansion of the solubility range: many substances that are
insoluble in water can be titrated in non aqueous solvents.
Expansion of the application range: weak bases and weak acids

that cannot be analyzed in water can be easily titrated in non-
aqueous solvents. Example: Pyrazole (pKb = 11.51), 2-
Aminobenzoicacidmethylester (pKb = 11.82), Phenol (pKa = 9.95)
Titration of substance mixtures that cannot be separated in

aqueous media. Example: Isopropylamine (pKb = 3.37) and
Aniline (pKb = 9.42) mixture
Undesirable side reactions of titrant and sample with water are

avoided
6
Agenda
Blank determination
7
Non-aqueous base titration
Determination of amines
R-NH2 + HClO4 R-NH3+ + ClO4-
Examples for direct titration of amines

- Benzylnicotinate 1 (Rheumatism) 1
- Dequalinium chloride 2 (Anti-bacterial)
Examples of protonated amine salts titrations
2
- Diphenylpyraline Hydrochloride 3 (Anti-Histamine)
- Tramadol Hydrochloride 4 (Analgesic, “pain killer”)
Note:
Mercuric acetate is used to facilitate titrations by 3
Releasing free amine from the protonated amine salts:
2R-NH3+Cl- + Hg(OAc)2 2R-NH2 + HgCl2 + 2HOAc

4
8
Titration of bases – levelling/differentiating
In aqueous systems only weak bases with a pKb value of 7 can be

titrated, e.g. aliphatic amines
Bases with a pKb value from 7 – 12 (aromatic amines or

heterocycles) can be titrated in organic solvents, mostly glacial
acetic acid.
Perchloric acid in glacial acetic acid is used as the titrant
9
Solvents for titration of bases
Crucial for the success of a titration

Must have a high dielectric constant in order to
solubilize the analyte and to enable a sharper
potential jump
The nature of the solvent determines the maximum
permissible base strength
Must be available in sufficient purity and dryness
10
Solvents for titration of bases
Acetic acid O
- Very good solvent
- Enables titration of weak bases up to pKb values of 12 H3C OH
- Analysis of bases mixtures is not possible – levelling effect
- Rarely acetylation is a side reaction
Acetic acid anhydride O O
- Very good solvent, similar to acetic acid
- Added as auxiliary reagent to maintain anhydrous conditions
H3C O CH3
- Preferred solvent for titration of very weak bases up to pKb
values of 12
Methanol
- Very good solvent, similar to water H3 C OH
- Enables titration of bases up to a pKb value of 9
2- Propanol (Isopropanol)
- Good differentiation of base mixtures H3C CH CH3
- Enables titration of bases up to a pKb value of 10 OH
Acetone
- Excellent differentiation of base mixtures O
- Very good solvent

H3C CH3
11
Titrants for the titration of bases
Perchloric acid can be purchased as a standard stock solution (0.1M in
70% aqueous solution) for dilution with the appropriate solvent
Perchloric acid in glacial acetic acid

- Glacial acetic acid (99.8%) as the solvent
- This solution contains approx. 0.5% water
- If non aqueous conditions are required, acetic anhydride must be added. Tip:
0.5% water require 28 g acetic anhydride
Perchloric acid in acetic anhydride
- Dilution of the standard stock solution with acetic anhydride
Perchloric acid in 2-Propanol (Isopropanol)
- The standard stock solution is mixed with 2-Propanol into 500 mL of 2-
Propanol
- This titrant is stable
12
Agenda
Non-aqueous acid titrations
Blank determination
13
Non-aqueous acid titration
Determination of aromatic alcohols
R-OH + (R‘)4NOH R-O - + N(CH3)4+ + H2O

R‘ = Methyl, Butyl
OH
Examples for direct titration of phenol

and derivatives OH
- Phenol HO O
- Rutoside OH
OH
HO OH
O
O
O
OH O
H3C O
HO
HO
OH
14
Solvents for titration of acids
1,2 - Diaminoethane H2
- Determination of acids with pKa values up to 11 C NH2
H2N CH2
- Increasing levelling effect with increasing acid strength, at pKa<6 it is
not possible to distinguish between acids in a mixture
- Must be protected against CO2 contamination
Dimethylformamide O
- Excellent solvent, high dielectric constant
- Highly differentiating solvent, only strong acids with pKa<0 are levelled, H N(CH3)2
i.e. mixtures of strong acids cannot be determined
- Steep inflections at the equivalence point
- Must be protected against moisture to avoid hydrolysis
2-Propanol (Isopropanol)
- Good solvent H3C CH CH3
- Highly differentiating solvent, only strong acids with pKa<0 are levelled, OH
i.e mixtures of strong acids cannot be titrated
Acetone O
- Excellent solvent
- Even very strong acids can be differentiated from each other H3C CH3
15
Titrants for titration of acids
Almost exclusively Tetramethylammoniumhydroxide (TMAH) or
Tetrabutylammoniumhydroxide (TBAH)
TMAH solutions in 2-Propanol yield maximum specifity but are not
stable
TMAH solutions in methanol yield medium to low specificity but
higher stability
Mixtures of 2-propanol and methanol and a small portion of water
(1%) increase the stability: titrants may be stored for 1 year
TBAH has the same basicity as TMAH
The TBA salts generated during the titration are more soluble than
the TMA salts
Some TMA salts are poorly soluble and precipitate at the electrode
surface
16
Titrants for titration of acids
0.1 M TMAH or 0.1 M TBAH can be purchased as a standard stock

solution in 2-propanol/methanol
TMAH can be stored for 1 year, however slight decomposition to
TMA can be observed
TBAH is less stable, shall be consumed within 6 months
If other solvents are required the respective standard stock solution
is diluted with the required solvent.
Protect titrant from atmospheric CO2!

Recommendation: Use NaOH coated, activated Charcoal
17
Tips for non-aqueous acid titrations
TMAH and TBAH are susceptible to

- Decomposition to the free amines TMA and TBA
- CO2 contamination from ambient atmosphere
The free amine causes an additional inflection in the titration curve
before the actual equivalence point of the analyte
The CO2 causes an additional inflection in the titration curve after
the actual equivalence of the analyte
The CO2 inflection can be surpressed by saturation of the solution
with nitrogen
18
Agenda
Blank determination
19
Standardization of titrants
Standardization of perchloric acid

- Potassium hydrogenphtalate (KHP)
8Stable
8Non-hygroscopic
8Easy to dry (105 – 135°C)
8Must be dissolved in hot glacial acetic acid
8Disadvantage: precipitation of KClO4
- 2-Methylimidazole
8Stable
8Good solubility in organic solvents
Standardization of TMAH or TBAH

- Benzoic acid
- Perchloric acid in 2-Propanol (not in acetic acid!)
20
Agenda
Blank determination
21
Potentiometric indication – requirements for electrode
Large pH glass membrane to achieve fast signal

response and equilibration
Electrostatic shielding in order to minimize interferences
that contribute to noisy curves
Large (moveable) junction to provide sufficiently large
electrolyte flow for signal stabilization
LiCl in Ethanol recommended as electrolyte
22
Sensor maintenance
Cleaning
- Rinse sensor in pure solvent followed by a water rinse
Conditioning in water.
- After 5 - 10 samples condition electrode about 5 minutes.
- Before the next titration, rinse off the conditioning solution with
solvent
After daily use:

- Store electrode over night in diluted acid or KCl 3 mol/L for
regeneration and hydration.
The sensors must be calibrated after regeneration
23
Agenda
Blank determination
24
Blank determination
In non-aqueous media it is highly recommended to perform a blank determination
Application:
Solvent: DMA with 10 % water (DMA = N,N-Dimethylacetamid)
Titrant: HClO4 0.1 mol/L in ethanol; Potentiometric indication
Blank determination with 60 mL solvent.
The solvent is without ions!

Titration curve: Add inert ions: KCl or LiCl
E [mV] E [mV]
0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.1 0.2 0.3 0.4 0.5
V [mL] V [mL]
25
Agenda
Blank determination
26
Problem
The thermal expansion coefficient of the non-aqueous solvents is 5
to 10 times higher than that of water
A temperature increase will significantly decrease the density of
the solvent and consequently reduce the titer of the titrant
A temperature difference of 1 causes an error of 0.3%
27
Solution
Maintain constant temperature conditions, i.e. titrant and sample are

at the same temperature
If a temperature change has occured between the standardization
and sample titration the standardization of the titrant must be
repeated
Measure the temperature during standardization and titration and
calculate a correction factor. In the case of perchloric acid the
correction factor is in the range from 0.9988 – 1.0020
28
Agenda
Blank determination
29
Assay Benzylnicotinate
Titrant: 0.1 M HCLO4

Solvent: Acetic anhydride, 50 mL
Sample: 0.17 – 0.19 g Benzylnicotinate
Sensor: pH glass electrode with 1M LiCl in ethanol
(DGi113-SC or DGi116-Solvent)
Blank determination on 50 mL solvent (triple determination)
Correction factor CF of perchloric acid solution: temperature measurement
during standardization and titration (0.999644)
Reaction
- R-C5H4N + HClO4 → R-C5H4NH++ClO4-
Result: % Benzylnicotinate = (Consumption [mmol] – Blank mean value
[mmol])x CF x Molar Mass Benzyl nicotinate x 10/Sample weight [g]
Result: 97.921%, RSD (6 samples): 0.047%
30
Assay Sodium Citrate, tribasic, anhydrous
Titrant: 0.1 M HCLO4

Solvent: Glacial acetic acid, 50 mL
Sample: 0.05 g Sodium citrate
(DGi113-SC or DGi116-Solvent)
Blank determination on 50 mL solvent (triple determination)
Correction factor (CF) of perchloric acid solution: temperature
measurement during standardization and titration (0.99734)
31
Assay Sodium Citrate, tribasic, anhydrous
Reaction
C6H5Na3O7 + 3HClO4 → C6H5O4(OH)3+NaClO4
Result: % Benzylnicotinate = (VEQ*c*t [mmol] – Blank
[mmol]) x CF x Molar Mass Benzyl nicotinate x
10/Sample weight [g]*3
VEQ: Volume to equivalence point [mL]
c: Nominal volume of titrant mol/L
t: Titer of titrant
3: Equivalent number, since 3 moles of perchloric
acid are required for the reaction
32
Assay Rutoside
Application description
Titrant: 0.1 M TBAH

Solvent: Dimethylformamide
Sample: 0.19 – 0.21 g Rutoside
Reaction
- R-(OH)2 + TBAH → R-O2TBA2+2H2O
Result: % Rutoside = Consumption [mmol] x 2 x Molar Mass
Rutoside x 10/Sample weight [g]
33

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GTP in Pharma

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Good Titration Practice

in the Pharmaceutical Industry

Examples of acids Examples of bases

Acid pKa Base pKb

Perchloric acid -9 Guanidine 0.5

Note: the pK values are defined in aqueous systems

Expansion of the application range: weak bases and weak acids

Titration of substance mixtures that cannot be separated in

Undesirable side reactions of titrant and sample with water are

R-NH2 + HClO4 R-NH3+ + ClO4-

Examples for direct titration of amines

2R-NH3+Cl- + Hg(OAc)2 2R-NH2 + HgCl2 + 2HOAc

In aqueous systems only weak bases with a pKb value of 7 can be

Bases with a pKb value from 7 – 12 (aromatic amines or

Perchloric acid in glacial acetic acid is used as the titrant

Crucial for the success of a titration

- Enables titration of bases up to a pKb value of 10 OH

- Very good solvent

Perchloric acid in glacial acetic acid

Determination of aromatic alcohols

R-OH + (R‘)4NOH R-O - + N(CH3)4+ + H2O

Examples for direct titration of phenol

0.1 M TMAH or 0.1 M TBAH can be purchased as a standard stock

Protect titrant from atmospheric CO2!

TMAH and TBAH are susceptible to

Standardization of perchloric acid

Standardization of TMAH or TBAH

Potentiometric indication – requirements for electrode

Large pH glass membrane to achieve fast signal

After daily use:

The sensors must be calibrated after regeneration

The solvent is without ions!

Maintain constant temperature conditions, i.e. titrant and sample are

Titrant: 0.1 M HCLO4

Titrant: 0.1 M HCLO4

Titrant: 0.1 M TBAH

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