Pharmaceutical Analysis

• It involves a series of process for identification, determination, quantification, purification of

asubstance.
• Separation of components from a mixture solution
• Determination of structure of compound
Example: The substance may be simple, single compound, mixture of compounds and may be in of
the dosage form. The substance used as pharmaceuticals are animal, plants, micro-organisms,
minerals andvarious synthetic products
The sample to be analyzed is called “Analyte”.
Trace analysis:
The development of pharmaceutical brought a revolution in human health. There pharmaceutical
would serve their if they are free from impurities and administered in appropriate amount.
To make drugs serve their purpose, various chemicals and instrumental methods were developed at
regular intervals which are involved in estimation of drugs
Types of chemical analysis:
Qualitative analysis: In this, which analyte is present in the sample is to be determined or to
identify a compound / mixture.
Quantitative Analysis: In this how much amount of analyte is present in the sample is to be
determined.
Types of quantitative Analysis:
1. Chemical methods
• volumetric/titrimetric method
• gravimetric method
• gasometric method
2. Electrical method
3. Instrumental method
4. Biological and microbiological
Chemical method:
a. Volumetric / titrimetric method: It is a method of analysis in which a solution of the substance
of unknown concentration is treated with a solution of a suitable reagent of known concentration.
The reagent is added to the substance until the amount is equivalent to the amount of substance to be
determined
various types of volumetric methods acid base titration, non-aqueous titration oxidation-reduction
complexometric titration precipitation titration

b) Gravimetric method: In this a substance to be determined is converted into an insoluble

precipitate in the purest form, then it is collected and weighed. It is a time-consuming process. In
electrogravimetry, electrolysis of the sample is carried out in the electrodes is weighed afterdrying
Electrogravimetry: Analyte solution is electrolyzed. Electrochemical reduction causes the analyte
to be deposited on the cathode. The cathode is weighed before and after the experiment and the
weight difference is used to calculate the amount of analyte in the original solution
Thermogravimetry: Records the change in weight. It continuously measures mass while the
temperature of a sample is changed overtime
Differential thermal analysis: Records the difference in temperature between test and reference
material
Differential scanning calorimetry: Energy needed to establish a zero-temperature difference
between a test and reference material.
c) Gasometric Analysis: It involves measurement of volume of gas evolved or absorbed in a
chemical reaction.
Examples of some gases which are determined by gasometry is NO2, cyclopropane etc..

Electrical method:
It involves the measurement of electrical current, voltage or resistance in relation to the
concentration of some species in solution
Electrical method of analysis includes:
1. Potentiometry
2. Conductometry
3. Polarography
4. Voltametry
5. Amperometry
Potentiometry measures electrical potential of an electrode in equilibrium with an ion to be
determined
Conductometry measures electrical conductivity of an electrode with a reference electrode
Polarography, voltametry, and amperometry measures electrical current at micro electrode
Instrumental method of analysis:
It measures physical properties of the compound and employed for the determination of trace
concentration of element in the sample. Instrumental methods are preferred due to their selectivity,
high speed, accuracy and simplicity of analysis. Any change in properties of system is detected by
measurements of absorbance, specific rotation, refractive index, change in mass Spectroscopic
method:
This analysis involves measurement and interpretation of electromagnetic radiation
(absorbed/emitted)
Methods which include absorption of radiation are uv, visible, IR, atomic absorption, NMR etc.
Emission methods involve heating or electrical treatment of the sample so that the atoms are raised
to excited state to emit the energy and the intensity of the emitted energy is measured.
Example: Emission spectroscopy, flame photometry
Chromatography technique and electrophoretic method are separation of mixture of components but
also utilize for identification of compounds

Microbiological methods:
They are based upon a comparison of the inhibition of growth of bacteria by a measured
concentration of antibiotics to be examined with that produced by known concentration of standard
preparation of the antibiotics having a known activity
Biological method: biological standardization on bioassay are the procedure by which the potency
or the nature of the substance is estimated by studying its effect on living matter Bioassays are
generally done using animal tissue or organ as in case of use of guinea pig ileumfor the estimation of
histamine or using the intact animal as in case of insulin using mouse
The precision, reliability and reproductivity as a bioassay depends on proper selection of the tissue or
method with highest selectivity and sensitivity for the drug
Bioassays are designed to measure the relative potency of two preparations, usually a standardand an
unknown solution
The observed response of the unknown would be always relative to the effect that produced by a
standard substance
The standard substance is pure substance and in official bio assays it refers to pharmaceutical
standards

Methods of expressing concentration:

Different ways of expressing concentration
1. Normality
2. Molarity
3. Molality
 4. Formal concentration
5. Percent solution
6. Parts per million(ppm)

Normality: Number of gram equivalents of solute in one liter of solution. Indicated by N Equivalent
weight = gram equivalent weight of solute / No of replaceable H+ and OH-
Example:
Preparation of 1N NaOH Molecular weight of NaOHAtomic weight of Na = 23 Atomic weight of O
= 16 Atomic weight of H = 1
1N = 40 gm of NaOH in 1 lit/1000 ml of water
0.1N = 4 gm of NaOH in 1 lit/1000 ml of water
0.01N = 0.04 gm in NaOH in 1 lit/1000 ml of water

Molarity:
Number of molecular weight of the substance in one liter of solution. Indicated by M
M = Gram molecular weight of solute / 1000 ml of solution Example: 1M NaOH Molecular weight =
23+16+1 = 40 gm
1M = 40 gm in 1 lit of solution
0.1M = 4 gm 1 lit of solution
HCl = 1 + 35.5 = 36.5 gm of HCl in 1 lit of solution

Molality: gram molecular weight of the substance in 1 kg of solution. Denoted by m M = number of

molecular weight of substance

1000 gm of solution NaOH = 23+16+1 = 40 gm of NaOH in 1000 gm of water

Formal concentration:
Some substances do not exist in molecular form, they remain in ionic form in solid state as well as in
solution. In such cases grams of molecular weight ; formula weight is used in preparation of solution
and its concentration is expressed in terms of formality
F = weight of solute in grams formula weight / Volume of solution in liter

Percent solution:
Sometimes concentration is expressed in percent (parts per hundred) composition of a solution can
expressed as
 percent W/W = weight of the solute / 100 Weight of the solution
percent V/V = volume of the solute / 100 Volume of solution
percent W/V = weight of the solute / 100 Weight of the solution
a) Percent W/W frequently employed to express the concentration of commercial aqueous reagent
b) Percent V/V used to specify the concentration of a solution prepared by diluting a pure liquid with
another liquid
c) percent W/V employed to indicate the composition of dilute aqueous solution of solid reagents.

parts per million and parts per billion (ppm and ppb)
Parts per million is frequently employed to express the concentration of very dilute solution and is
expressed as PPM
Concentration in PPM = Mass of solute / 106 Mass of solvent
Ppm and ppb are used to express the concentration of impurities in pharmaceuticals

Error: It is defined as the difference between true value and observed mean value
Absolute error = true value - observed mean value
Types of errors:
It is of two types.
1. Determinate error:
As the name indicates these errors are determinable and can be either avoided or corrected
2. Indeterminate error:
These errors are random and cannot be avoided or corrected by the use of high purity reagents or
calibrated equipment
Types of determinate errors:
1. Operational or personal errors: These are associated with individual analyst and not associated
with the procedure or method
Ex: wrong observation, wrong transferring
2. Instrumental errors:
These errors occur due to improper functioning of the instruments or due to the use of uncalibrated
equipment or instruments
3. Reagent error:
These errors occur due to the use of reagent supplied in impure form or substandard form.
 4. Additive or constant error:
These errors are constant throughout the analysis
5. Proportional error:
These errors will be proportional throughout the analysis
6. Errors in method:
These errors occur due to the wrong selection of procedure or method
Indeterminate errors:
They cannot be pin-pointed to any specific well-defined resources
They are random in nature and take place in several successive measurements performed by the
same analyst under the same conditions and identical experimental parameters.
Example: some persons are unable to judge color changes sharply in visual titrations, which may
result in a slight overtopping of the end point.
• An analyst may use the instrument incorrectly.
• Under washing or over washing of precipitate.

Primary and secondary standard solutions:

Standard solution: In pharmaceutical analysis, the word standard means a material containing a
substance with known concentration
Functions of standard solution:
1. To provide a reference for determining the concentration of unknown concentration
2. Standardization of volumetric solution
3. To calibrate an instrument
4. Preparation of secondary standard
Primary standard:
It is a reagent which is very pure generally representative of the number of moles of the substance
containing and easily weighed. It should satisfy the following requirements.
1. Easy to obtain, to purify, to dry, and to preserve in a pure state.
2. The substance must be unaltered in air during weighing (i.e. non-hygroscopic non-oxidized).
3. The total amount of impurities should not exceed 0.01-0.02%.
4. It should have a high equivalent so that the weighing errors may be negligible.
5. The primary standard should be readily soluble under the condition in which it is employed.
6. The titration error should be negligible or easy to demonstrate accurately by experiment.
7. The substances commonly employed as primary standards examples of acid-base titration,
oxidation-reduction titration.
Secondary standard:
It is a chemical which is standardized against a primary standard for use in a specific analysis.
It follows that a secondary standard solution is a solution in which the concentration of dissolved
solute has not been determined from the weight of the compound dissolved but by reaction of a
volume of the solution against a measured volume of primary standard solution.
Example: most of the alkali hydroxides NaOH, KOH.

STANDARD SOLUTION:
The solution of accurately known strength is called standard solution.
EQUIVALENCE POINT:
The point in a titration where the reaction is just completed /Theoretical end point/stoichiometric end
point
INDICATOR:
The regent employed in titration to get locate the end point
ACCURACY:
It is defined has the closeness of the measured value to the true value. It depends on the
minimization of the errors
PRECISION:
It refers to the agreement among a group of measured values
The errors in measurement can be broadly classified as systematic errors, random errors and gross
errors

PREPARATION AND STANDARDISATION OF 0.1M SODIUM HYDROXIDE SOLUTION

AIM: To prepare and standardize 0.1M sodium hydroxide solution.
Requirements:
Apparatus: Conical flask, Burette, Pipette, Water Bath, Burner, Measuring cylinder, Beaker,
Dropper, Glass rod, etc.
Chemicals: Sodium hydroxide pellets, Potassium hydrogen phthalate, Phenolphthalein, Distilled
Water.
Principle: Potassium hydrogen phthalate, (KHC8H4O4) is a non-hygroscopic, crystalline, solid that
behaves as a monoprotic acid. It is a water soluble and available in high purity. Because of its high
purity, we can determine the number of moles of KHP directly from its mass and is referred as
primary standard. Primary standard is used to determine the concentration of sodium hydroxide
solution.
Neutralization reaction takes between sodium hydroxide and known amount of acid in the flask. End
point is determined when all the acids has been neutralized and solution composition changes from
excess acid to excess base. Phenolphthalein acts as an indicator to determine the end point and gives
colour change.

KHC8H4O4 + NaOH → KNaC8H4O4 (aq) + H2O2

Titration: Acid base titration

Titrant: 0.1 M Sodium hydroxide
Indicator: Phenolphthalein
Endpoint: Appearance of pale pink
Primary Standard: Potassium hydrogen Phthalate.

Procedure:
Preparation of 0.1M sodium hydroxide:
Weigh accurately 4.0 gm of sodium hydroxide and transfer it to 100 ml distilled water in a beaker
with continuous stirring. Make up the volume to 1000 ml with distilled water. Keep the solution for
at least an hour and then carry out standardisation
Standardization of 0.1M NaOH:
* Weigh accurately 0.5 gm of KHP and transfer into a conical flask.
* Add total volume of 50-75 ml of water. Dissolve the sample properly.
* Add 2-3 drops of phenolphthalein indicator to the flask and titrate it against 0.1M NaOH until it
gives pale pink colour as end point.
Each 1ml of 0.1M NaOH = 0.0204 gm of KHP

PREPARATION AND STANDARDISATION OF 0.1 N SODIUM THIOSULFATE

AIM: To prepare and standardise 0.1N sodium thiosulfate.
Requirements: Apparatus: Conical flask, Burette, Pipette, Water Bath, Burner, Measuring cylinder,
Beaker, Dropper, Glass rod, etc.
Chemicals: Sodium thiosulfate, Potassium dichromate, Potassium iodide, sulphuric acid, Distilled
Water.
Principle:
Iodometry (Iodometric titration) is a method of volumetric chemical analysis. A redox titration
where appearance of elementary iodine indicates end point. Sodium thiosulphate a reducing agent is
standardised by a primary standard potassium dichromate. A known amount of excess potassium
iodide is added which oxidises to iodine in acidic medium. The liberated iodine is titrated with
sodium thiosulphate using starch as an indicator.
K2Cr2O7 + 6KI +7H2SO4 → 4K2SO4 + 7H2O +3I2

Titration: Redox titration

Titrant: 0.1N Sodium thiosulphate
Indicator: Starch solution
Endpoint: Appearance of pale blue
Primary Standard: Potassium dichromate.

Procedure:
Preparation of 0.1 N Potassium dichromate:
Weigh accurately about 1.227 gm of Potassium dichromate in 250 ml Volumetric flask and makeup to
volume with distilled water. Preparation of 0.1 N Sodium thiosulphate (Na2S2O3.5H2O): Dissolved accurately
about 25 g of Sodium thiosulphate in 1000ml of freshly boiled distilled water. Improve its stability by adding
0.1 g of Sodium carbonate, stored the solution overnight and filter.

Standardization of 0.1 N Sodium thiosulphate with potassium dichromate: 10 ml of 0.1 N

Potassium dichromate solution is taken in iodine flask.
To this solution add 5 ml of potassium iodide solution (10%) and add 5 ml of 1 M H2SO4 Cover the
iodine flask with stopper and keep the solution in dark for 10 minutes.
The liberated iodine is titrated with 0.1 N Sodium thiosulphate until the solution becomes pale
yellow. Introduce 5 drops of starch indicator and titrate with constant stirring till the appearance of
blue colour
1ml of 0.1N Sodium thiosulphate ≈
S. No. Content of Burette reading Volume of 0.1 N Indicator End point
flask Initial Final Na2S2O3 consumed used

M = (Weight of K2Cr2O7) (Expected normality) / Volume of H2SO4

Report: 0.1N Sodium thiosulphate solution was prepared and standardised. Concentration of the
solution was found to be N.
PREPARATION AND STANDARDISATION OF 0.1 M SULPHURIC ACID SOLUTION

AIM: To prepare and standardise 0.1 M sulphuric acid solution.

Requirements: Apparatus: Conical flask, Burette, Pipette, Measuring cylinder, Beaker, Dropper,
Glass rod, etc.
Chemicals: Sulphuric acid, Sodium carbonate, Methyl red, Distilled Water.

Principle: It involves neutralisation reaction between sulphuric acid and sodium carbonate. (primary
standard) Sulphuric acid reacts with sodium carbonate and forms sodium sulphate with water
molecule. End point is seen when excess of methyl red indicator reacts with excess acid in the flask
giving faint pinkish red colour.

Na2SO3 + H2SO4 → Na2SO4 + H2O + CO2

Titration: Acid base titration

Titrant: 0.1M sulphuric acid
Indicator: methyl red
Endpoint: Appearance of faint pinkish red
Primary Standard: sodium carbonate

Procedure:
Preparation of 0.1M sulphuric acid:
Add 6 ml of concentrated sulphuric acid to about 800 ml distilled water. Make up to volume 1000
ml with distilled water and cool the solution to 25°C
Standardisation of 0.1M sulphuric acid:
Weigh accurately 0.2gm of anhydrous sodium carbonate previously heated at 270°C for 1hr
Dissolve it in 100 ml of water and add 0.1 ml of methyl red solution. Add acid slowly from burette
with continuous stirring, until the solution becomes faint pink. Heat the solution to boiling, cool and
continue the titration. Heat again to boiling and titrate further as necessary until faint pink colour is
no longer affected by boiling.
1 ml of 0.1M sulphuric acid = 0.0106 gm of Sodium carbonate
Standardization of 0.1 M Sulphuric acid :
S. No. Content of Burette reading Volume of 0.1 N Indicator End point
flask Initial Final H2SO4 formation used

M = (Weight of Na2SO3) (Expected molarity) / 0.0106 (volume of 0.1 M H2SO4)

Report:
0.1M sulphuric acid solution was prepared and standardised. Concentration of the solution was
found to be M.

PREPARATION AND STANDARDISATION OF 0.1 N POTASSIUM PERMANGANATE

AIM: To prepare and standardise 0.1N potassium permanganate
Requirements:
Apparatus: Conical flask, Burette, Pipette, Water Bath, Burner, Measuring cylinder, Beaker,
Dropper, Glass rod, etc.
Chemicals: Oxalic acid, sulphuric acid, Potassium permanganate.

Principle:
Potassium permanganate (KMnO4) is a strong oxidising agent. Permanganate is intense dark purple
colour. Reduction of purple permanganate ion to the colourless Mn+2 ion the solution will turn to
faint pink from dark purple colour at the equivalence point No additional indicator is needed for the
titration as KMnO4 acts as self-indicator. The reduction of permanganate requires strong acidic
medium.
In this permanganate is reduced by oxalate in acidic condition Oxalate reacts very slowly at room
temperature so to enhance it the solution is heated.

2KMnO4 + 3H2C2O4 + 5H2SO4 → 2MnSO4 + K2SO4 + 10CO2 + 8H2O

Titration: Redox titration

Titrant: 0.1N Potassium permanganate
Indicator: Self indicator
Endpoint: Appearance of pale pink
Primary Standard: Oxalic acid

Procedure:
Preparation of 0.1N Potassium permanganate:
Weigh accurately 3.2gm of Potassium permanganate transferred to 250ml beaker containing cold
water and stir thoroughly.
Decant and makeup the volume to 1000ml.

Standardization of 0.1N Potassium permanganate:

Weigh accurately 6.3 gm of Oxalic acid and transfer it a 1000 ml graduated flask and makeup the
volume with water. Pipette out 20 ml of this solution and add 10 ml of diluted sulphuric acid and
warm to about 70°C. Add KMnO4 solution from burette until pink colour appears. Wait until the
colour disappears and continue until it persists faint pink colour for about 30 seconds.

Standardization of 0.1N Potassium permanganate:

S. No. Content of Burette reading Volume of 0.1 N Indicator End point
flask Initial Final KMnO4 consumed used

Each 1ml of 0.1N KMnO4 = 0.00063 gm of Oxalic acid

M = (Weight of oxalic acid) (Expected molarity) / 0.0063 (volume of 0.1 M KMnO4)
Report:
0.1N Potassium permanganate solution was prepared and standardised. Concentration of the
solution was found to be N.

PREPARATION AND STANDARDISATION OF 0.1 M CERRIC AMMONIUM SULPHATE

AIM: To prepare and standardise 0.1 M cerric ammonium sulphate

Apparatus: Conical flask, Burette, Pipette, Water Bath, Burner, Measuring cylinder, Beaker,
Dropper, Glass rod, etc.
Chemicals: Ceric ammonium sulphate, Arsenic trioxide, sodium hydroxide and sulphuric acid.
Principle:
Ceric ammonium sulphate is a powerful oxidising agent in acidic solution. It is bright yellow in
 colour and corresponding cerium salt form by reduction is colourless strong solution are self-
indicating. However since dilute solutions are used so indicators are used for observation of end
point. Aresnic trioxide used as primary standard in presence of sulphuric acid and osmic acid using
ferroin sulphate as an indicator.

As2O3 +NaOH → NaAsO2

NaAsO2 + H2O →NaH2AsO4 +2H +4e
4 (Ce4+ + e → Ce3+)

PROCEDURE:
Preparation of ceric ammonium sulphate:
66 gm ceric ammonium sulphate was dissolved with gentle heat. Mix 30ml of sulphuric acid and
500ml of water. Cool and filter the solution if turbid and dilute to 100ml with water.
Standardisation of 0.1M ceric ammonium sulphate:
Weigh accurately 0.2gm of arsenic trioxide previously dried at 105°C for 1hr and transferred to a
500 ml conical flask.
Wash down the flask with 25ml of 8% w/v solution of sodium hydroxide, swirl to dissolve, add
100ml of water and mix.
Add 30ml of dilute sulphuric acid, 0.1ml of ferroin sulphate solution.
Titrate with ceric ammonium sulphate until pale blue colour appears from pink colour.

Standardisation of 0.1M ceric ammonium sulphate:

S. No. Content of Burette reading Volume of 0.1 M Indicator End point
flask Initial Final ceric ammonium used
sulphate consumed

Each 1ml of 0.1M Cerric ammonium sulphate = 49.46 gm of As2O

M = (Weight of As2O3) (Expected molarity)/ 0.0063 (volume of ceric ammonium sulphate consumed)
Report:
0.1M Ceric ammonium sulphate solution was prepared and standardised. Concentration of the solution
was found to be M.