PHARMACOPOEIAL METHODS OF ANALYSIS

Water content

Osmolality & Osmolarity

Refractive index. Siva

Chaitanya

PA/2015/102

Contents

Water contentWater content determination

Direct titration

(1a)

Method 2(Azeotropic

Toulene distillation)Method 3

(Gravimetric)

Volumetric titration

 

Principle :

This reaction consumes water and iodine in a 1:1 ratio.

The choice of working medium and pH range are the most critical considerations.

CH3 OH + SO2 + RN → [RNH]SO3CH3 

H2O + I2 + [RNH]SO3CH3 + 2 RN → [RNH]SO4 CH3 + 2 [RNH]I 

(RN = Base)

Working medium

1. It assures the stoichiometry of the Karl Fischer reaction

2. Must be able to dissolve the sample

3. Allow confident end point determination

Examples: Methanol ( if other solvents are

added ,methanol content should never fall below 25 %)

1-Propanol, ethanol are better solubilizing agents for lipophilic molecules.Chloroform is a good solvent for solubilizing fats.Formamide improves the solubility of polar substances and can be mixed with methanol for the determination of water in proteins.

pH Value

In the ideal pH range 5-7, the Karl Fischer reaction runs quickly and stoichiometrically.

At higher pH values a side reaction occurs which consumes iodine and leads to vanishing end points.

In strongly acidic conditions the reaction constant of the Karl Fischer reaction decreases and the course of the titration is slower

Volumetric titration(method 1a &1b )

Method 1a (Direct titration): Quantitative reaction of water with Sulphar dioxide and Iodine .

End Point:If colourless solution ; canary yellow to amber ORSlight excess reagent increases flow of current to b/w 50 & 150 microamperes

121 g of iodine to 670 ml of methanol & 170 ml of Pyridine and cool

Allow this solution to stand overnight .

1 ml of this solution is equilent to 5 mg of water.

Preparation of KFR reagent:

Standardization of the reagent :

Sodium tartarate dihydrate

Add equivalent of b/w 2 & 250 mg water

Calculate ‘F’ in mg of water per ml of reagent

1• Transfer

enough methanol (30-40 ml ) & titrate with reagent to consume any moisture.

2•Quickly add test preparation mix , again titrate with reagent to electrometric or visual end point

TEST:

Accurately weigh test specimen specimen to contain 2 – 250 mg of water

C is b/w 30 – 100 % for manual titration and b/w 10 – 100 % for instruments

Method 1b (Residual titration) :Excess reagent is added to test specimen, sufficient time is allowed for the reaction toReach completion. Unconsumed reagent is titrated with a standard solution of water in a solvent such as methanol

Use:For substances where bound water is released slowly.

2ml water +methanol + other suitable solvent to 1000 ml

Standardize it with standardized reagent(25 ml)

Standardization of water solution:

X/- vol.of reagent after introducing specimenX-vol.in ml of water solution required to neutralize unconsumed reagentR - ratio

Transfer enough methanol or suitable solvent to the

titration vessel ; to cover electrodes (30-40 ml) . Titrate

with reagent till end point.

Add test mix and accurately measure

excess of the reagent

Procedure:

Coulometric titration(method 1c)Iodine is not added in form of vol. Solution ; but is produced in an iodide containing solution by anodic oxidation.

The Pt anode generates I2 when current is provided through the electric circuit, one mole of I2 is consumed for each mole of H2O. In other words, 2 moles of electrons are consumed per mole of water.

When all water consumed excess iodine occurs detected electrometrically,indicating end point.

B·I2 + B·SO2 + B + H2O → 2BH+I− + BSO3

BSO3 + ROH → BH+ROSO3−

Specimen Test preparationsoluble solid appropriate quantity, accurately

weighed, may be dissolved in anhydrous methanol or other suitable solvents.

insoluble solid an appropriate quantity, accurately weighed, may be extracted using a suitable anhydrous solvent, and may be injected into the analyte 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.

used directly without dissolving in a suitable anhydrous solvent

an appropriate quantity, accurately weighed, may be introduced into the chamber directly.

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.

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 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 directly from the instrument’s display, and calculate the percentage that present in the substance.

Perform a blank determination, as needed, and make any necessary corrections.

Procedure

A: Glass flaskB: Reflux condenserC: ground glass jointsD: connecting tube E: Receiving tube

An azeotrope or a constant boiling mixture is a mixture of two or more liquids whose proportions cannot be altered by simple distillation This happens because, when an azeotrope is boiled, the vapour has the same proportions of constituents as the unboiled mixture.

Method II- Azeotropic distillation

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 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 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 second until most of the water has passed over, then increase the rate of distillation to about four drops per second.

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

Procedure

Osmolality & Osmolarity

Osmolality is a practical means of giving an overall measure of the combination of the various soutes present in a solution to the osmotic pressure of the solution.

Osmolality is a measure of the osmotic pressure excerted by the real solution across a Semi-permeable membrane.

ν - total no of ions already formed by solvolysis from one molecule of solute

m – molality of the solutionϕ – molal osmotic coefficientunits : Osm/kg or mOsm/kg

Osmolality

ϕ – molal osmotic coefficient

It takes into account the deviation of solution from from ideal behaviour.

Its value depend upon conc. Of solute in solution,its chemical properties & ionic charactersticis.

Its vaue can be determined by measuring freezing point depression at different molal concentrations.

units : Osm/kgKf – molal cryoscopic constant(for water it is 1.86 per osmol)ΔTf - freezing point depression

Directly related to collegative properties

It is determined most accurately and conveniently by measuring freezing point depression.

Osmole is the number of moles of solute that contribute to the osmotic pressure of a solution

Osmometer:

It consist of :1. a system of cooling the container

used for the measurement for mixing the sample

2. a system for measuring the temperature by means of device for determining the current or potential difference, graduated in temperature depression or directly in osmolality.

Method:

1. Prepare the required reference solution2. Determine the zero of the apparatus using water3. Calibrate the apparatus using reference solution4. Test sample5. Calculate the osmolality

Note:The test is not valid unless the value found is within two values of the calibration scale

Calibration:

1. Introduce 50µl to 250µl of the sample into the measurement cell and start the cooling.

2. Operate the cooling device at a temperature below that expected through cryoscopic depression to prevent super cooling.

3. When equilibrium is attained record the freezing point.

4. Before each measurement, rinse the measurement cell with the solution to be examined.

5. The instrument is calibrated by using two standards of NaCl that span the expected range.

osmolarity, is the measure of solute concentration defined as the number of osmoles (Osm) of solute per litre (L) of solution (osmol/L or Osm/L). The osmolarity of a solution is usually expressed as Osm/L

Widely used in clinical practice because it expresses osmoles as a function of volume

Can’t be measured experimentally but is calculated theoritically from osmolality

Osmolarity

Refractive index

Abbe refractometer

Refractive index Refractive index of a medium with referenceis equal to the ratio of sine of the angle of incidence of a beam of light in air to the sine of the angle of refraction of refracted beam in the given medium.

Measured at with reference to the Wavelength of the D-line of sodium( )

To achieve the theoretical accuracy of ± 0.0001,it is necessary to calibrate the instrument against a standard provided by the manufacturer and to check frequently the temperature control and cleanliness of the instrument by determining the refractive index of distilled water, which is 1.3325 at 250 or against the reference liquids given in the table

Reference liquid Dn20 Temperature coefficient

Carbon tetrachloride 1.4603 -0.00057

Toluene 1.4969 -0.00056

α-Methylnaphthalene 1.6176 -0.00048

REFERENCES

INDIAN PHARMACOPOEIA, VOLUME 1

UNITED STATES PHARMACOPOEIA , VOLUME 1

BRITISH PHARMACOPOEIA, VOLUME 4