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á921ñ WATER DETERMINATION

Many Pharmacopeial articles either are hydrates or contain water in adsorbed form. As a result, the determination of the
water content is important in demonstrating compliance with the Pharmacopeial standards. Generally one of the methods
given below is called for in the individual monograph, depending upon the nature of the article. In rare cases, a choice is allowed
between two methods. When the article contains water of hydration, Method I (Titrimetric), Method II (Azeotropic), or Method
III (Gravimetric) is employed, as directed in the individual monograph, and the requirement is given under the heading Water.
The heading Loss on Drying (see Loss on Drying á731ñ) is used in those cases where the loss sustained on heating may be not
entirely water.

METHOD I (TITRIMETRIC)

Determine the water by Method Ia, unless otherwise specified in the individual monograph.

Method Ia (Direct Titration)

PRINCIPLE
The titrimetric determination of water is based upon the quantitative reaction of water with an anhydrous solution of sulfur
dioxide and iodine in the presence of a buffer that reacts with hydrogen ions.

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In the original titrimetric solution, known as Karl Fischer Reagent, the sulfur dioxide and iodine are dissolved in pyridine and
methanol. The test specimen may be titrated with the Reagent directly, or the analysis may be carried out by a residual titration
procedure. The stoichiometry of the reaction is not exact, and the reproducibility of a determination depends upon such factors
as the relative concentrations of the Reagent ingredients, the nature of the inert solvent used to dissolve the test specimen, and
the technique used in the particular determination. Therefore, an empirically standardized technique is used in order to achieve
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the desired accuracy. Precision in the method is governed largely by the extent to which atmospheric moisture is excluded
from the system. The titration of water is usually carried out with the use of anhydrous methanol as the solvent for the test
specimen. In some cases, other suitable solvents may be used for special or unusual test specimens. In these cases, the addition
of at least 20% of methanol or other primary alcohol is recommended.
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APPARATUS
Any apparatus may be used that provides for adequate exclusion of atmospheric moisture and determination of the endpoint.
In the case of a colorless solution that is titrated directly, the endpoint may be observed visually as a change in color from canary
yellow to amber. The reverse is observed in the case of a test specimen that is titrated residually. More commonly, however,
the endpoint is determined electrometrically with an apparatus employing a simple electrical circuit that serves to impress about
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200 mV of applied potential between a pair of platinum electrodes immersed in the solution to be titrated. At the endpoint of
the titration a slight excess of the reagent increases the flow of current to between 50 and 150 microamperes for 30 s to 30 min,
depending upon the solution being titrated. The time is shortest for substances that dissolve in the reagent. With some
automatic titrators, the abrupt change in current or potential at the endpoint serves to close a solenoid-operated valve that
controls the buret delivering the titrant. Commercially available apparatus generally comprises a closed system consisting of
one or two automatic burets and a tightly covered titration vessel fitted with the necessary electrodes and a magnetic stirrer.
The air in the system is kept dry with a suitable desiccant, and the titration vessel may be purged by means of a stream of dry
nitrogen or current of dry air.

REAGENT
Prepare the Karl Fischer Reagent as follows. Add 125 g of iodine to a solution containing 670 mL of methanol and 170 mL
of pyridine, and cool. Place 100 mL of pyridine in a 250-mL graduated cylinder, and, keeping the pyridine cold in an ice bath,
pass in dry sulfur dioxide until the volume reaches 200 mL. Slowly add this solution, with shaking, to the cooled iodine mixture.
Shake to dissolve the iodine, transfer the solution to the apparatus, and allow the solution to stand overnight before
standardizing. One mL of this solution when freshly prepared is equivalent to approximately 5 mg of water, but it deteriorates
gradually; therefore, standardize it within 1 h before use, or daily if in continuous use. Protect from light while in use. Store any
bulk stock of the reagent in a suitably sealed, glass-stoppered container, fully protected from light, and under refrigeration. For
determination of trace amounts of water (less than 1%), it is preferable to use a Reagent with a water equivalency factor of not
more than 2.0, which will lead to the consumption of a more significant volume of titrant.
A commercially available, stabilized solution of Karl Fischer type reagent may be used. Commercially available reagents
containing solvents or bases other than pyridine or alcohols other than methanol may be used also. These may be single solutions
or reagents formed in situ by combining the components of the reagents present in two discrete solutions. The diluted Reagent
called for in some monographs should be diluted as directed by the manufacturer. Either methanol or other suitable solvent,
such as ethylene glycol monomethyl ether, may be used as the diluent.

TEST PREPARATION
Unless otherwise specified in the individual monograph, use an accurately weighed or measured amount of the specimen
under test estimated to contain 2–250 mg of water. The amount of water depends on the water equivalency factor of the

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Reagent and on the method of endpoint determination. In most cases, the minimum amount of specimen, in mg, can be
estimated using the formula:

FCV/KF

in which F is the water equivalency factor of the Reagent, in mg per mL; C is the used volume, in percent, of the capacity of
the buret; V is the buret volume, in mL; and KF is the limit or reasonable expected water content in the sample, in percent. C
is generally between 30% and 100% for manual titration, and between 10% and 100% for the instrumental method endpoint
determination.
[NOTE—It is recommended that the product of FCV be greater than or equal to 200 for the calculation to ensure that the
minimum amount of water titrated is greater than or equal to 2 mg.]
Where the specimen under test is an aerosol with propellant, store it in a freezer for not less than 2 h, open the container,
and test 10.0 mL of the well-mixed specimen. In titrating the specimen, determine the endpoint at a temperature of 10° or
higher.
Where the specimen under test is capsules, use a portion of the mixed contents of not fewer than four capsules.
Where the specimen under test is tablets, use powder from not fewer than four tablets ground to a fine powder in an
atmosphere of temperature and relative humidity known not to influence the results.
Where the monograph specifies that the specimen under test is hygroscopic, take an accurately weighed portion of the solid
into the titration vessel, proceeding as soon as possible and taking care to avoid moisture uptake from the atmosphere. If the
sample is constituted by a finite amount of solid as a lyophilized product or a powder inside a vial, use a dry syringe to inject
an appropriate volume of methanol, or other suitable solvent, accurately measured, into a tared container, and shake to dissolve
the specimen. Using the same syringe, remove the solution from the container and transfer it to a titration vessel prepared as
directed for Procedure. Repeat the procedure with a second portion of methanol, or other suitable solvent, accurately measured,

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add this washing to the titration vessel, and immediately titrate. Determine the water content, in mg, of a portion of solvent
of the same total volume as that used to dissolve the specimen and to wash the container and syringe, as directed for
Standardization of Water Solution for Residual Titration, and subtract this value from the water content, in mg, obtained in the
titration of the specimen under test. Dry the container and its closure at 100° for 3 h, allow to cool in a desiccator, and weigh.
Determine the weight of specimen tested from the difference in weight from the initial weight of the container.
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When appropriate, the water may be desorbed or released from the sample by heat in an external oven connected with the
vessel, to where it is transferred with the aid of an inert and dried gas such as pure nitrogen. Any drift due to the transport gas
should be considered and corrected. Care should be taken in the selection of the heating conditions to avoid the formation of
water coming from dehydration due to decomposition of the sample constituents, which may invalidate this approach.
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STANDARDIZATION OF THE REAGENT
Place enough methanol or other suitable solvent in the titration vessel to cover the electrodes, and add sufficient Reagent to
give the characteristic endpoint color, or 100 ± 50 microamperes of direct current at about 200 mV of applied potential.
Purified Water, sodium tartrate dihydrate, a USP Reference Standard, or commercial standards with a certificate of analysis
traceable to a national standard may be used to standardize the Reagent. The reagent equivalency factor, the recommended
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titration volume, buret size, and amount of standard to measure are factors to consider when deciding which standard and
how much to use.1 For Purified Water or water standards, quickly add the equivalent of between 2 and 250 mg of water. Calculate
the water equivalency factor, F, in mg of water per mL of reagent:

W/V

in which W is the weight, in mg, of the water contained in the aliquot of standard used; and V is the volume, in mL, of the
Reagent used in the titration. For sodium tartrate dihydrate, quickly add 20–125 mg of sodium tartrate dihydrate (C4H4Na2O6
· 2H2O), accurately weighed by difference, and titrate to the endpoint. The water equivalence factor F, in mg of water per mL
of reagent, is given by the formula:

W/V (36.04/230.08)

in which 36.04 is two times the molecular weight of water and 230.08 is the molecular weight of sodium tartrate dihydrate;
W is the weight, in mg, of sodium tartrate dihydrate; and V is the volume, in mL, of the Reagent consumed in the second
titration. Note that the solubility of sodium tartrate dihydrate in methanol is such that fresh methanol may be needed for
additional titrations of the sodium tartrate dihydrate standard.

PROCEDURE
Unless otherwise specified, transfer enough methanol or other suitable solvent to the titration vessel, ensuring that the volume
is sufficient to cover the electrodes (approximately 30–40 mL), and titrate with the Reagent to the electrometric or visual
endpoint to consume any moisture that may be present. (Disregard the volume consumed, because it does not enter into the
calculations.) Quickly add the Test Preparation, mix, and again titrate with the Reagent to the electrometric or visual endpoint.
Calculate the water content of the specimen taken, in mg:

1 Consider a setup in which the reagent equivalency factor is 5 mg/mL, and the buret volume is 5 mL and an instrumental endpoint. Standard amounts
equivalent to between 2.5 mg and 22.5 mg of water (10%–90% of buret capacity) could be used based on the buret and the reagent equivalency factor.
The upper end of this range would involve an excessive amount of sodium tartrate dihydrate. If Purified Water or a standard is weighed, an analytical
balance appropriate to the amount weighed is required.

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SF

in which S is the volume, in mL, of the Reagent consumed in the second titration; and F is the water equivalence factor of the
Reagent.

Method Ib (Residual Titration)

PRINCIPLE
See the information given in the section Principle under Method Ia. In the residual titration, excess Reagent is added to the
test specimen, 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. The residual titration procedure is applicable generally and avoids the
difficulties that may be encountered in the direct titration of substances from which the bound water is released slowly.

APPARATUS, REAGENT, AND TEST PREPARATION

Use Method Ia.

STANDARDIZATION OF WATER SOLUTION FOR RESIDUAL TITRATION

Prepare a Water Solution by diluting 2 mL of water with methanol or other suitable solvent to 1000 mL. Standardize this
solution by titrating 25.0 mL with the Reagent, previously standardized as directed under Standardization of the Reagent.

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Calculate the water content, in mg per mL, of the Water Solution taken:

V′F/25

in which V′ is the volume of the Reagent consumed, and F is the water equivalence factor of the Reagent. Determine the water
content of the Water Solution weekly, and standardize the Reagent against it periodically as needed.
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Where the individual monograph specifies that the water content is to be determined by Method Ib, transfer enough methanol
or other suitable solvent to the titration vessel, ensuring that the volume is sufficient to cover the electrodes (approximately
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30–40 mL), and titrate with the Reagent to the electrometric or visual endpoint. Quickly add the Test Preparation, mix, and add
an accurately measured excess of the Reagent. Allow sufficient time for the reaction to reach completion, and titrate the
unconsumed Reagent with standardized Water Solution to the electrometric or visual endpoint. Calculate the water content of
the specimen, in mg, taken:

F(X′ − XR)
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in which F is the water equivalence factor of the Reagent; X′ is the volume, in mL, of the Reagent added after introduction of
the specimen; X is the volume, in mL, of standardized Water Solution required to neutralize the unconsumed Reagent; and R is
the ratio, V′/25 (mL Reagent/mL Water Solution), determined from the Standardization of Water Solution for Residual Titration.

Method Ic (Coulometric Titration)

PRINCIPLE
The Karl Fischer reaction is used in the coulometric determination of water. Iodine, however, is not added in the form of a
volumetric solution but is produced in an iodide-containing solution by anodic oxidation. The reaction cell usually consists of a
large anode compartment and a small cathode compartment that are separated by a diaphragm. Other suitable types of reaction
cells (e.g., without diaphragms) may also be used. Each compartment has a platinum electrode that conducts current through
the cell. Iodine, which is produced at the anode electrode, immediately reacts with water present in the compartment. When
all the water has been consumed, an excess of iodine occurs, which usually is detected electrometrically, thus indicating the
endpoint. Moisture is eliminated from the system by pre-electrolysis. Changing the Karl Fischer solution after each determination
is not necessary because individual determinations can be carried out in succession in the same reagent solution. A requirement
for this method is that each component of the test specimen is compatible with the other components, and no side reactions
take place. Samples are usually transferred into the vessel as solutions by means of injection through a septum. Gases can be
introduced into the cell by means of a suitable gas inlet tube. Precision in the method is predominantly governed by the extent
to which atmospheric moisture is excluded from the system; thus, the introduction of solids into the cell may require
precautions, such as working in a glove-box in an atmosphere of dry inert gas. Control of the system may be monitored by
measuring the amount of baseline drift, which does not preclude the need of any blank correction when used as a vehicle for
sample introduction. This method is particularly suited to chemically inert substances like hydrocarbons, alcohols, and ethers.
In comparison with the volumetric Karl Fischer titration, coulometry is a micro-method.
When appropriate, the water may be desorbed or released from the sample by heat in an external oven connected with the
vessel, to where it is transferred with the aid of an inert and dried gas such as pure nitrogen. Any drift due to the transport gas
should be considered and corrected. Care should be taken in the selection of the heating conditions to avoid the formation of
water coming from dehydration due to decomposition of the sample constituents, which may invalidate this approach.

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APPARATUS
Any commercially available apparatus consisting of an absolutely tight system fitted with the necessary electrodes and a
magnetic stirrer is appropriate. The instrument’s microprocessor controls the analytical procedure and displays the results.
Calibration of the instrument is not necessary, as the current consumed can be measured absolutely.

REAGENT
See the manufacturer’s recommendations.

TEST PREPARATION
Where the specimen is a soluble solid, an appropriate quantity, accurately weighed, may be dissolved in anhydrous methanol
or other suitable solvents.
Where the specimen is an insoluble solid, an appropriate quantity, accurately weighed, may be extracted using a suitable
anhydrous solvent, and may be injected into the anolyte solution. Alternatively, an evaporation technique may be used in which
water is released and evaporated by heating the specimen in a tube in a stream of dry inert gas. The gas is then passed into
the cell.
Where the specimen is to be used directly without dissolving in a suitable anhydrous solvent, an appropriate quantity,
accurately weighed, may be introduced into the chamber directly.
Where the specimen is a liquid, and is miscible with anhydrous methanol or other suitable solvents, an appropriate quantity,
accurately weighed, may be added to anhydrous methanol or other suitable solvents.

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PROCEDURE
Using a dry device, inject or add directly an accurately measured amount of the sample or sample preparation estimated to
contain between 0.5 and 5 mg of water, or an amount recommended by the instrument manufacturer into the anolyte, mix,
and perform the coulometric titration to the electrometric endpoint. Read the water content of the liquid Test Preparation
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directly from the instrument’s display, and calculate the percentage that is present in the substance. Perform a blank
determination, as needed, and make any necessary corrections.

METHOD II (AZEOTROPIC—TOLUENE DISTILLATION)

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Apparatus
Use a 500-mL glass flask A connected by means of a trap B to a reflux condenser C by ground glass joints (see Figure 1).
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Figure 1. Toluene moisture apparatus.

The critical dimensions of the parts of the apparatus are as follows. The connecting tube D is 9–11 mm in internal diameter.
The trap is 235–240 mm in length. The condenser, if of the straight-tube type, is approximately 400 mm in length and not less
than 8 mm in bore diameter. The receiving tube E has a 5-mL capacity, and its cylindrical portion, 146–156 mm in length, is
graduated in 0.1-mL subdivisions, so that the error of reading is not greater than 0.05 mL for any indicated volume. The source
of heat is preferably an electric heater with rheostat control or an oil bath. The upper portion of the flask and the connecting
tube may be insulated.
Clean the receiving tube and the condenser with a suitable cleanser, thoroughly rinse with water, and dry in an oven. Prepare
the toluene to be used by first shaking with a small quantity of water, separating the excess water, and distilling the toluene.

Procedure
Place in the dry flask a quantity of the substance, weighed accurately to the nearest centigram, which is expected to yield
2–4 mL of water. If the substance is of a pasty character, weigh it in a boat of metal foil of a size that will just pass through the
neck of the flask. If the substance is likely to cause bumping, add enough dry, washed sand to cover the bottom of the flask,
or a number of capillary melting-point tubes, about 100 mm in length, sealed at the upper end. Place about 200 mL of toluene

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in the flask, connect the apparatus, and fill the receiving tube E with toluene poured through the top of the condenser. Heat
the flask gently for 15 min and, when the toluene begins to boil, distill at the rate of about two drops per s until most of the
water has passed over, then increase the rate of distillation to about four drops per s. When the water has apparently all distilled
over, rinse the inside of the condenser tube with toluene while brushing down the tube with a tube brush attached to a copper
wire and saturated with toluene. Continue the distillation for five min, then remove the heat, and allow the receiving tube to
cool to room temperature. If any droplets of water adhere to the walls of the receiving tube, scrub them down with a brush
consisting of a rubber band wrapped around a copper wire and wetted with toluene. When the water and toluene have
separated completely, read the volume of water, and calculate the percentage that was present in the substance.

METHOD III (GRAVIMETRIC)

Procedure for Chemicals

Proceed as directed in the individual monograph preparing the chemical as directed under Loss on Drying á731ñ.

Procedure for Biologics

Proceed as directed in the individual monograph.

Procedure for Articles of Botanical Origin

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Place about 10 g of the drug, prepared as directed (see Methods of Analysis under Articles of Botanical Origin á561ñ) and
accurately weighed, in a tared evaporating dish. Dry at 105° for 5 h, and weigh. Continue the drying and weighing at 1-h
intervals until the difference between two successive weighings corresponds to not more than 0.25%.
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