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Water content
Osmolality & Osmolarity
Refractive index.
Siva Chaitanya
PA/2015/102
Water content
determination
Method 1
(Titremetric)
Karl fisher titration
Direct titration (1a)
Coulometric titration (1c)
Residual titration(1b)
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
Method 1a (Direct titration):
 Quantitative reaction of water with Sulphar dioxide
and Iodine .
 End Point:
If colourless solution ; canary yellow to amber OR
Slight 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
In 250 ml beaker take
100 ml pyridine and
pass in dry so2 volume
reaches 200 ml under
cooling
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 to
Reach 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 specimen
X-vol.in ml of water solution required to neutralize unconsumed reagent
R - 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:
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 preparation
soluble 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 flask
B: Reflux condenser
C: ground glass joints
D: 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.
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 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 solution
ϕ – molal osmotic coefficient
units : 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/kg
Kf – 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 solution
2. Determine the zero of the apparatus using water
3. Calibrate the apparatus using reference solution
4. Test sample
5. 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
Abbe refractometer
Refractive index
 Refractive index of a medium with reference
is 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
 INDIAN PHARMACOPOEIA,
VOLUME 1
 UNITED STATES
PHARMACOPOEIA ,
VOLUME 1
 BRITISH PHARMACOPOEIA,
VOLUME 4
pharmacoepial methods-physical tests

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pharmacoepial methods-physical tests

  • 1.
  • 2. Water content Osmolality & Osmolarity Refractive index. Siva Chaitanya PA/2015/102
  • 3. Water content determination Method 1 (Titremetric) Karl fisher titration Direct titration (1a) Coulometric titration (1c) Residual titration(1b) Method 2 (Azeotropic Toulene distillation) Method 3 (Gravimetric) Volumetric titration
  • 4. 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)
  • 5. 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
  • 6. Method 1a (Direct titration):  Quantitative reaction of water with Sulphar dioxide and Iodine .  End Point: If colourless solution ; canary yellow to amber OR Slight excess reagent increases flow of current to b/w 50 & 150 microamperes
  • 7. 121 g of iodine to 670 ml of methanol & 170 ml of Pyridine and cool In 250 ml beaker take 100 ml pyridine and pass in dry so2 volume reaches 200 ml under cooling Allow this solution to stand overnight . 1 ml of this solution is equilent to 5 mg of water. Preparation of KFR reagent:
  • 8. 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
  • 9. 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
  • 10. Method 1b (Residual titration) : Excess reagent is added to test specimen, sufficient time is allowed for the reaction to Reach 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:
  • 11. X/- vol.of reagent after introducing specimen X-vol.in ml of water solution required to neutralize unconsumed reagent R - 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:
  • 12. 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 −
  • 13. Specimen Test preparation soluble 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.
  • 14. 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
  • 15. A: Glass flask B: Reflux condenser C: ground glass joints D: 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.
  • 16. 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
  • 17.
  • 18. 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 solution ϕ – molal osmotic coefficient units : Osm/kg or mOsm/kg Osmolality
  • 19. 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.
  • 20. units : Osm/kg Kf – 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
  • 21. 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.
  • 22. Method: 1. Prepare the required reference solution 2. Determine the zero of the apparatus using water 3. Calibrate the apparatus using reference solution 4. Test sample 5. Calculate the osmolality Note:The test is not valid unless the value found is within two values of the calibration scale
  • 23. 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.
  • 24. 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
  • 26. Refractive index  Refractive index of a medium with reference is 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( )
  • 27. 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
  • 28.  INDIAN PHARMACOPOEIA, VOLUME 1  UNITED STATES PHARMACOPOEIA , VOLUME 1  BRITISH PHARMACOPOEIA, VOLUME 4