ALINAO, Corina B. BASA, Giannah Nicole B.

April 27, 2019

ALVES, Lovely Clariza D. BAUTISTA, Daniel Adrian A. PHARMACEUTICAL ANALYSIS

ANURAN, Sharon Angela Marie BELENO, Jeevel Christine B.

AUM, Rayziel Vien D. BETE, Cheriza D.

BALAKI, Brooklyn Kate T.

WATER CONTENT DETERMINATION

A. DEFINITION

Water Content Determination is a process of determining the water content within a certain substance or any
pharmacological determination of water within drugs etc.

B. DISCUSSION

The determination of water content is an important aspect of the pharmaceutical industry. It is of great significance in
pharmaceutical development, process, production and quality control. Knowing the water content and understanding the
hygroscopic nature of a drug substance, as well as the final product in which it is contained is essential. Currently, the preferred
and most widely used method for water determination is KFT.

It has been a well-established standard method for water analysis for over 70 years, and is specified in the leading
pharmacopeias such as the USP and European Pharmacopoeia (EP).

Methods of Water Content Determination

B.1 Water Content Determination with Karl Fischer Titration

Background

KFT THEORY

The KF reaction is a specific quantitative reaction of water with an anhydrous solution of sulfur dioxide and iodine in the
presence of a buffer that reacts with hydrogen ions. The KF reaction is based on the well-known bunsen reaction in aqueous
media

The reagent is buffered with pyridine (Py) in order to neutralize liberated protons and ensure complete reaction

Fischer proposed the above equation for water determination, which requires a 2:1 molar ratio of H 2O to iodine. This
reaction led to the establishment of the classical KF reagent, which is a solution of iodine and sulfur dioxide in a mixture of pyridine
and methanol.

Later on, Smith et al. showed that methanol plays an important role in the KF reaction.They showed that water forms a
pyridine sulfur trioxide complex

in the first partial reaction that preferably

reacts with methanol, or in lack of alcohol, with a second molecule of water.

From the reaction scheme, it is clear that the stoichiometry between water and iodine is 1:1 in this solvent. Hence, the
amount of water can be determined by measuring consumption of iodine.

Principle:
 The Karl Fischer method is used for many substances as a reference method. It is a chemical analysis procedure which is
based on the oxidation of sulfur dioxide by iodine in a methanolic hydroxide solution. In principle, the following chemical reaction
takes place:

H2O + I2 + SO2 + CH3OH + 3RN -> [RNH]SO4CH3 + 2[RNH]I

The titration can be performed volumetrically or coulometrically.

a. Volumetric method

A solution with an exactly known concentration of iodine is added to sample by means of an electric burette. The
amount of iodine added to the sample is calculated from the volume of iodine solution used.

b. Coulometric method

The iodine participating in the reaction is generated directly in the titration cell by electrochemical oxidation of iodide until
again a trace of unreacted iodine is detected.

Faraday's law can be used to calculate the amount of iodine generated from the quantity of electricity required.

END POINT DETERMINATION

When reacting with water, the brown iodine is reduced to the colourless iodide. At the endpoint of the titration when all
the water is consumed the colour of the solution turns increasingly from yellow to brown. As there is no sharp colour change and
the coloration differs in nonpolar solvents (such as DMF) and polar solvents (as e.g. methanol) , it is not easy to determine the
endpoint of the titration visually. For this reason, the endpoint of the titration is usually determined electrometrically with a double
platinum wire electrode.

INDICATION METHODS

There are two types of indication methods commonly used, namely biamperometric and bivoltametric.

Biamperometric indication

A constant voltage of approximately 500 mV is applied to the wires of the electrode and the resulting current is
measured. As long as there is water in the sample, no free iodine is present in the solution. When the endpoint of the titration has
been reached, the following reactions occur at the wires of the electrode:

Cathode: I2 + 2e- → 2I- Anode: 2I-- 2e- → I2

When the endpoint has been reached the current thus increases from almost nil to a few μA.

Bivoltametric indication

A small current (normally in the range of 1 … 50 μA) is applied between the electrodes and the voltage required to
maintain this current is measured. Normally alternating current is used (AC) as it yields a higher sensitivity of the electrode than
direct current (DC). The voltage required to maintain the current is in the range of several 100 mV as long as an excess of water is
present in the sample. When the endpoint of the titration is reached, free iodine is available in the solution and the voltage drops
to 100 mV or less. Normally, the endpoint potential level (switch off voltage) must be selected according to the type of solvent
being used and/or the type of sample being titrated. The ideal switch off voltage depends on the type of sample and solvent
used. With normal Karl Fischer Titrators it must be determined experimentally:

• If the switch off voltage is too low, too much iodine is added before the endpoint is detected, the water contents yielded are
too high.

• If the switch off voltage is too high, the titration does not start automatically as no free iodine is required for this voltage to be
achieved.

Which method should be used, coulometric or volumetric?

In general it can be said that the method has to be chosen depending on the water content of the samples to be
measured:

• the coulometric method is suitable for samples with a low water content (10 μg … 100 mg)

• the volumetric method is suitable for samples with a higher water content (0.1 … 500 mg).

APPLICATION
 The Karl Fischer titration method is specific for water and is suitable for samples with a high moisture content (titrimetry)
and also for those with water contents in the ppm range (coulometry). It was originally developed for nonaqueous liquids, but is
also suitable for solids if these are soluble or if the water they contain can be removed by heating in a stream of gas or by
extraction. The KFT method is a selective, quantitative, preferred and long-standing standard method in pharmacopeias for the
determination of water for both drug substances and drug products.
According to USP <921>,6 the KFT is specified as Method I (Titrimetric) for the determination of water and emphasizes Method
Ia (Direct Titration), Method Ib (Residual Titration) and Method Ic (Coulometric Titration), Method II (Azeotropic Distillation) and
Method III (Gravimetry) unless otherwise specified in the individual monograph.

Method I (Titrimetric)

Test specimen may be titrated with the KF reagent directly, or the analysis may be carried out by a residual titration
procedure which is specified in Method Ib.

Method Ia (Direct Titration)

Governed largely by the extent to which atmospheric moisture is excluded from the system.

Method Ib (Residual Titration)

Generally applicable and avoids the difficulties that may be encountered in the direct titration of substances from which
the bound water is released slowly. In this method, excess reagent is added to the test sample, sufficient time is allowed for the
reaction to reach completion, and the unconsumed reagent is titrated with a standard solution of water in a solvent such as
methanol.

Method Ic (Coulometric Titration)

Both the EP and the USP describe sample size/sample weight, titration method
(95% direct volumetric titration), the range of water content permitted, and the reagents to be used. The use of pyridine-
containing reagents for KF volumetry is described in both standards. The USP has fewer restrictions, which permits the use of
commercially available reagents containing other solvents than pyridine. Finally, KF titrations are effective at quantifying even
tightly bound water and often are considered a better method than LOD. Also, KF is a standard method that is used for
calibration of other indirect methods for water determination.

Method II Azeotropic
Principle: Moisture is removed from the sample by distillation as an azeotrope with toluene. The water is collected in a
suitable trap and its volume is measured at a known temperature.
Application: This method is applicable to all unmodified starches, most modified starches and many starch products
Toluene: Reagent Grade

Method III Gravimetric Analysis

Thermogravimetric analysis
Principle: A sample is heated, its mass changes. This change can be used to determine the composition of a material or
its thermal stability, up to 1000oC. Usually, a sample loses weight as it is heated up due to decomposition, reduction, or
evaporation.Release of water appears as a weight-loss step at a certain temperature that is easily evaluated and ideally
quantifies the amount of water present in a sample. Varying the heating rate for TGA provides the data for kinetic evaluations.
The technique allows kinetic parameters to be calculated and predictions about long-term behavior of the samples to be made.
Care must be taken with the applied temperature. The sample may decompose before the water is expelled
completely. Special standard methods have been established for this purpose. A vacuum is applied in order to shift the water
release to lower temperatures.

SIGNIFICANCE
The determination of water content is an important aspect of the pharmaceutical industry. It is of great significance in
pharmaceutical development, process, production and quality control. Knowing the water content and understanding the
hygroscopic nature of a drug substance, as well as the final product in which it is contained is essential. Water can affect the
physical and chemical stability of active pharmaceutical ingredients (APIs) and drug formulations,1 the microbial sustainability
and activity, potency, efficacy and shelf life.2 Therefore, it is very important to set proper specifications and have accurate
methods for the determination of water in pharmaceuticals while they are still in developmental stages.

Advantages: Accurate reference method, coulometry also suitable for trace analysis and water detection.

Restriction: The working method must be adapted to the specific sample.

OTHER PROCESSES OF WATER CONTENT DETERMINATION :

B.2 Dew point Determination of Humidity

A dew point hygrometer consists of a small mirror, a narrow light beam and a light detector. The light beam is reflected off
the mirror into the detector, which measures the brightness of the reflected light. When the mirror surface cools to the dew point
temperature, dew forms on the mirror.

The water droplets on the chilled mirror block light from reaching the detector, causing the reflected light beam to look less
bright. The reduction in the measured reflected light indicates the mirror is at the dew point temperature.

A psychrometer is an alternative instrument for determining the dew point. A psychrometer consists of two thermometers,
one of which has a wet wick around its bulb and is called the wet-bulb thermometer.

Evaporation of water off the wick removes heat from the thermometer. The temperature of the wet-bulb thermometer drops
according to the rate of evaporation, which depends on the dew point.

The thermometers have to be ventilated by either whirling the instrument around or using a fan.

After a few minutes, the temperature of the wet-bulb stabilizes at a particular temperature.

The temperature difference between the two thermometers is converted into a dew point temperature using a chart.

Dew point temperature is noted on the temperature ‘X-axis’ and move vertically on the psychrometric chart. The
intersect point at saturated curve (100%) is identified. The coordinates of the point (temperature, humidity) are noted. The Y-axis
point is the humidity of air. These values are substituted in the equation:

% Relative humidity = Humidity of air / Humidity of saturated air × 100

B.3 Electrolytic Hygrometer

Faraday’s law of electrolysis forms the basis of the electrolytic hygrometer. A bifilar platinum winding on a quartz or Teflon
tube coated with phosphorous pentoxide provides the measuring cell . The moisture contained in a stream of gas is adsorbed by
the hygroscopic phosphorous pentoxide and continuously electrolysed into its constituent hydrogen and oxygen gases. The
electrolysing current supplied is directly proportional to the water electrolysed and is used to display in suitable units.
 Hygrometers are always used in pharmaceutical manufacturing because a lot of pharmaceutical ingredients are
hygroscopic and high humidity may cause problems in formulation in those compounds.

C. PROCEDURE (KFT)

Water Content Determination

 Experiment 1 – Oven Drying

Materials: 10 g of sample

Procedure:

1. Powder the sample in a mortar

2. Weigh 10 g of the sample in a tared evaporating dish.

3. Dry at 105 degrees Celsius for 5 hours and weigh.

4. Continue drying and weighing at 1 hour intervals until the lost is not more than 0.25% in 1 hour drying.

 In case of other unpowdered samples, prepare 10 g of sample by cutting/shedding, 3 mm in thickness. Seeds

and fruits should be smaller than 3mm.
 Experiment 2: Toluene Determination Method

Materials: 50 g of sample

200 ml of toluene

Toluene moisture Apparatus

(Clean condenser and Mtube with cleaning mixture, rinse with distilled water and then
with alcohol, dry in oven at 100 degree Celsius)

Procedure:

1. Place in a dry flask a quantity of the substance which is estimated to yield from 2-4 ml of water.

2. Place about 200 ml of toluene in the flask, connect the apparatus and fill the receiving tube E with
poured through top of condenser.

3. Heat the flask gently for 15 minutes.

4. When toluene begins to boil, distil to 2 drops/seconds until most of the water has passed, then increase rate
of distillation to about 4 drops/sec.

5. When water has apparently all distilled over, rinse the inside of condenser tube with toluene while brushing
down the tube with a tube brush attached.

6. Continue distillation for 5 minutes.

7. Remove Heat

Karl Fischer Method

Reagent: Iodine (45g), Sulphur Dioxide (20g), Pyridine (80mg), and Methanol (400 ml)

Preparation:

1. 400 ml of methanol in a stopper conical flask.

2. Dissolve 80 mg pyridine in the methanol.
3. Keep the solution in a freezing bath.
4. Supply sulphur dioxide in the cold solution till the weight increased by 20 g.
5. Add 45g of iodine and dissolve properly and kept for 24 hours.
6. Karl Fischer Reagent is ready to use (1ml = 3.5mg water).
Karl Fischer Procedure:

Solid Sample:

1. Put the desired sample in a weighing boat that is previously tared.

2. Weigh and tare the sample.
3. Press the start button on the volumetric karl fischer titrator.
4. Put the weighed sample.
5. Back weigh the empty weighing boat then enter sample weight in the titrator then press start.

Liquid Sample:

1. Use a syringe in obtaining your sample.

2. Obtain a small amount of sample using the syringe to wash off the syringe.
3. Discard the washing and obtain the amount of sample needed.
4. Weight the syringe filled with the sample then tare.
5. Add the sample by injecting the syringe in the titrator.
6. Remove the syringe and weigh what was left, the negative weight that appears is the weight that should be entered in
the titrator.

D. SAMPLE VIDEO OF TITRATION

E. COMPUTATION

𝑆 𝑥 𝐹 𝑥 100
Formula: % moisture =
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔)

Wherein:

F = water equivalence factor

S = Volume of reagent used to titrate the sample

Problem:
𝑚𝑔
If a percent moisture of Acetic Acid was computed as 3.767 and the water equivalence factor of 4.90 was consumed by a
𝑚𝑙
0.302g sample, what is the amount of KF reagent in ml?

Solution:

Given:
3.767 % moisture
F = 4.90
0.302 g sample
Unknown: KF reagent in ml
Solution:
1000𝑚𝑔
0.302𝑔 × = 302𝑚𝑔
𝑔

Formula:
𝑆 𝑥 𝐹 𝑥 100
% 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒 =
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 𝑚𝑔

(% 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒)(𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 𝑚𝑔) 𝑆 𝑥 𝐹 𝑥 100

=
𝑆 × 100 𝐹 × 100

(% 𝑀𝑜𝑖𝑠𝑡𝑢𝑟𝑒)(𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑖𝑛 𝑚𝑔)

𝑆=
𝐹 × 100

(3.767%)(302𝑚𝑔)
𝑆= 𝑚𝑔
4.90 × 100
𝑚𝑙
 Answer: 2.322𝑚𝑙

F. THREE SAMPLE PROBLEMS

1. Calculate the water equivalence factor F of Karl Fischer reagent if a 180-mg sample of sodium tartrate dehydrate required
15.00 ml of Karl Fischer reagent.

Given: 180 mg sample of tartrate dehydrate

15.00 ml of Karl Fischer reagent
Unknown: Water equivalence factor
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑑𝑖𝑢𝑚 𝑡𝑎𝑟𝑡𝑟𝑎𝑡𝑒 𝑑𝑒ℎ𝑦𝑑𝑟𝑎𝑡𝑒 (𝑚𝑔)
Formula: 0.1566 x (𝑚𝐿)
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑟𝑒𝑎𝑔𝑒𝑛𝑡 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑
Solution:
*Substitute data given
180 𝑚𝑔
Water equivalence factor = 0.1566 x
15.00 𝑚𝑙
Answer: 1.8792 mg/ml
2. Calculate the percent moisture in aminosalicylic acid if 9.00 ml of Karl Fischer reagent, having a water equivalence factor of
4.10, was consumed by a 5.100 g sample

Given: 9.00 ml of Karl Fischer reagent

Water equivalence factor of 4.10
5.100 g sample
Unknown: Percent moisture in aminosalicylic acid
𝑆 𝑥 𝐹 𝑥 100
Formula: % moisture =
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔)

Where in: S = volume of reagent used to titrate

F = water equivalence factor
Solution:
*Convert given weight to mg
1000 𝑚𝑔
=5.100 𝑔 𝑥
1𝑔
= 5100 mg

*Substitute data
9.00 𝑚𝑙 𝑥 4.10 𝑥 100
% moisture =
5100 𝑚𝑔
Answer: 0.724 %
3. Find the water equivalence factor if the percent moisture for a given acid is 3%, 14 ml of Karl Fischer reagent was consumed
by a 6.5 g sample.

Given: 3% moisture content

14 ml of Karl Fischer reagent
6.5 g sample
Unknown: Water equivalence factor
𝑆 𝑥 𝐹 𝑥 100
Formula: % moisture =
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔)

*Derive formula to solve for F

% 𝑚𝑜𝑖𝑠𝑡𝑢𝑟𝑒 𝑥 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝑔)

F=
𝑆 𝑥 100

Solution:

*Convert given weight to mg

1000 𝑚𝑔
= 6.5 𝑔 𝑥 = 6500 mg
1𝑔
*Substitute data
3% 𝑥 6500 𝑚𝑔
F=
14 𝑚𝑙 𝑥 100

Answer: 13.929%
 G. REFERENCES

Hoffmann, H.; Felgner, A. Water Determination in Pharmaceutical Compounds. Analytix, Article 4, 9.

Fisher Scientific. (n.d.). Retrieved from https://www.fishersci.com/us/en/products/JFV8PEKF/coulometric-karl-fischer-titration.html

Karl-Fischer Titration – the method for determining water. (n.d.). Retrieved from http://www.si-
analytics.com/fileadmin/upload/Informationen/Titration/INT/Technical-Data_TitroLine-KF-trace_742-KB_PDF-English.pdf

Water determination by Karl Fischer titration : Theory and applications. (2019, February 08). Retrieved from
https://www.worldcat.org/title/water-determination-by-karl-fischer-titration-theory-and-applications/oclc/679975899

https://www.researchgate.net/publication/315676636_Water_Determination

Determination of Moisture Content. (n.d.). Retrieved from https://www.scribd.com/doc/24474810/Determination-of-Moisture-

Content?fbclid=IwAR1ilIGQ-ohuHfiQsqCPBvvFRDCaAyBBuPLR0i45fuWqMs3Sc1DQb8sRow4