Miss Preeti Verma

Assistant Professor
Faculty of Pharmaceutical Sciences,
Rama University,
Kanpur, U.P.

UNIT-1

Pharmaceutical analysis
Pharmaceutical analysis is a branch of practical chemistry that involves a series of process for
identification, determination, quantification and purification of a substance, separation of the
components of a solution or mixture, or determination of structure of chemical compounds.

The substance may be a single compound or a mixture of compounds and it may be in any of
the dosage form. The substance used as pharmaceuticals are animals, plants, microorganisms,
minerals and various synthetic products.

The different pharmaceutical agents are as follows:

1. Plants
2. Microorganisms
3. Minerals
4. Synthetic compounds

Pharmaceutical analysis is traditionally defined as analytical chemistry dealing with drugs

both as bulk drug substances and as pharmaceutical products (formulations). However, in
academia, as well as in the pharmaceutical industry, other branches of analytical chemistry
are also involved, viz. bioanalytical chemistry, drug metabolism studies and analytical
biotechnology. The development of drugs in the pharmaceutical industry is a long-term
process, often taking more than a decade from the start of a research project to the appearance
of a drug on the market. That process involves several decision points, such as the choice of
the candidate drug after the preclinical screening phase, the investigational new drug (IND)
application before testing the compound for the first time in man, and finally the new drug
application (NDA) which summarizes the data obtained from all the studies needed for
marketing approval of the drug as a medicine. In all these steps, especially the IND and
NDA, the amount of data generated is enormous. Analytical chemists take part in many of the
studies that constitute this documentation. Substance quality and its specifications are based
on substance analysis, and that knowledge is later used for quality control during full-scale
production. Product analysis involves dealing with the various formulations and starts after
the IND has been approved. The results from such work lead to specifications that form the
basis for the quality control of the product. For both substances and formulations there is an
increasing interest in the introduction of process analytical chemistry.

The sample to be analysed is called as analyse.

 Quality control and quality assurance

 Chromatographic techniques
 Quantitative and qualitative analysis
 Validation methods
 Stoichiometry between reactants & products

Scope of Pharmaceutical Analysis

 Pharmaceutical Analysis is one of the most sort after specializations in masters of

pharmacy. People specialised in pharmaceutical analysis are indispensable to the
manufacturing, quality control and analytical manifestations of the industry.
 They can work in quality control department which oversees the purity, qualitative
aspects and the matching of the stringent regulatory limits required by a finished
product.
 Research and development has huge implications on the results of the analysis and
detection of new compounds. More and more companies are stressing on a separate
analytical R&D department.
 Pharmaceutical analysis students also find takers in the medical devices companies,
equipment companies, regulatory agencies etc.
 Always remember, no matter what compounds you discover or formulation you make
nothing is valid until it is evaluated, analysed and validated.
 Based upon the determination type, there are mainly two types of analytical methods.
They are as follows:

Qualitative analysis:
Quantitative analysis:

1. Qualitative analysis
This method is used for the identification of the chemical compounds. Qualitative analysis is
performed to establish composition of natural/synthetic substances. These tests are performed
to indicate whether the substance or compound is present in the sample or not.

2. Quantitative analysis

This method is used for the determination of the amount of the sample. Quantitative
analytical techniques are mainly used to quantify any compound or substance in the sample.
There are various methods to find out the quantity of a substance in a product.

Various types of Qualitative analysis:

1.Chemical methods
a) volumetric or titrimetric methods
b) gravimetric methods
c) gasometric analysis
2.Electrical methods
3.Instrumental methods
4.Biological and microbiological

Methods of Expressing Concentration of Solution

Concentration of solution is the amount of solute dissolved in a known amount of the solvent
or solution. The concentration of solution can be expressed in various ways as discussed
below,

(1) Percentage: It refers to the amount of the solute per 100 parts of the solution. It can also
be called as parts per hundred (pph). It can be expressed by any of following four methods,

(i)Weight to weight percent

% w/w = Wt of solute x 100

Wt of solution

(ii) Weight to volume percent

% w/v = Wt of solute x 100

Volume of solution

(iii) Volume to volume percent

% v/v= Volume of solute x 100

 Volume of solution

(iv)Volume to weight percent

% v/w = Volume of solute x 100

Wt of solution

(2) Parts per million (ppm) and parts per billion (ppb):

When a solute is present in trace quantities, it is convenient to express the concentration in

parts per million and parts per billion. It is the number of parts of solute per million (101) or
per billion (109) parts of the solution. It is independent of the temperature.

1
Ppm= mass of solute component x 10

Total mass of solution

9
Ppb= mass of solute component x 10

Total mass of solution

(3) Normality (N)

It is defined as the number of gram equivalents (equivalent weight in grams) of a solute

present per litre of the solution. Unit of normality is gram equivalents litre–1. Normality
changes with temperature since it involves volume. When a solution is diluted times, its
normality also decreases by times. Solutions in term of normality generally expressed as,

N= Normal solution; 5N= Penta normal,

10N= Deca normal;N/2= semi normal

N/10= Deci normal; N/5= Penti normal

N/100 or 0.01N= centinormal,

N/1000 or 0.001= millinormal

Mathematically normality can be calculated by following formula

Normality (N) = Number of gm eq. of solute

Volume of solution (l)

 (* 1 equivalent = 1000 mill equivalent or meq.)

(4) Molarity

The number of moles of solute per litter of solution OR the molar concentration of a solution
usually expressed as the number ofmoles of solute per liter of solution.

It is also known as molar concentration, is the number of moles of a substance per litter of
solution. Solutions libelled with the molar concentration are denoted with a capital M. A 1.0
M solution contains 1 mole of solute per litter of solution.

Molarity (M) = Mole of solute

Litres of solution

Molarity - M → moles per litter solution

(5) Molality

The number of moles of solute per kilogram of solvent. It is important the mass of solvent is
used and not the mass of the solution. Solutions labelled with molal concentration are
denoted with a lower case m. A 1.0 m solution contains 1 mole of solute per kilogram of
solvent.

Molality (m) = Mole of solute

Kg of solvent

Molality - m → moles per kilogram solvent

Preparation and standardization of solutions of various molar and normal solutions

Oxalic acid (COOH)2
Oxalic acid is available in pure state and its standard solutions can, therefore, be prepared by
the direct method. Eq. wt. of hydrated oxalic acid (C2H2O4.2H2O), being 63 its N/10 solution
would contain 6.3 gm/litre, and N/20 solution would contain 3.15 gm/litre.
These standard solutions are employed to find the strength of solutions of alkalies (NaOH and
KOH) whose standard solutions cannot be prepared by the direct method.
Preparation of oxalic acid solution
Standard solutions are prepared by using standard substances. Here a known quantity of
standard substances depending upon the requirement is dissolved in a known amount of water
and desired volume is made. Since, these substances have a constant weight, high purity, non-
hygroscopic property, so the solution obtained is of known and definite concentration. The
examples of such solutions are as follows.

Standardization N/10 oxalic acid solution (Primary standard)

To prepare N/10 solution of oxalic acid, weigh 6.3 gm of oxalic acid & dissolve in distilled
water & finally make up the volume to one liter in a volumetric flask. The standard solution
of oxalic acid (Primary standard) is used to find the strength of solutions of alkalies like
NaOH, KOH (Secondary standards) whose standard solutions cannot be made by direct
weighing. Secondary standard substances. Those substances or reagents which cannot be
obtained in a sufficient pure state, e.g. NaOH, KOH, HCl, H2SO4 are called secondary
standard substances.

Preparation of Sodium Hydroxide (NaOH) solutions

N/10 NaOH

Prepare concentrated stock solution (say 50%) of NaOH by dissolving equal parts of NaOH
pellets (50 gm) & water (50 gm) in a flask. Keep it tightly stoppered for 3-4 days. Use the
clean, supernatant liquid for preparing N/10 solution. Approximately 8 ml of this stock
solution (50%) is required per litre of distilled water. This will give approximate solution.
Now take 10 ml of standard N/10 oxalic acid (primary standard) solution in conical flask and
add 2- 3 drops of phenolphthalein indicator. Take unknown solution i.e. approximate N/10
NaOH solution in burette and add to the conical flask containing standard oxalic acid solution
by continuous mixing by swirling the flask till the appearance of pink color. NaOH is taken
in burette and standard oxalic acid in conical flask. Note down the volume of approximate
N/10 NaOH solution used in the titration of 10 ml of standard oxalic acid. Calculate the
normality of the unknown sodium hydroxide solution by using following equation.

N1V1 = N2V2
(Base) = (Acid)

N1 = Normality of NaOH solution. (ml)

V1 = Volume of NaOH solution used (ml)
N2 = Normality of standard oxalic acid solution (0.1 N)
V2 = Volume of standard oxalic acid solution (10 ml)

If the volume of approximate NaOH used in the titration is less than 10 ml, means the
solution is strong and its normality is not N/10, so dilute the basic solution and again
standardize with standard oxalic acid solution till normality of approximate solution is same
as that of standard solution.

Preparation of Sodium Hydroxide Concentrated acids (HCl)

Preparation of Concentrated acids

Prepare approximately 0.1 N solutions on the basis of strength diluting it 120 times with
distilled water. Then standardize it against standard N/10 Na2CO3 using methyl orange as an
indicator.

Standardization of Concentrated acids (HCl)

Prepare approximetly 0.1N solution on the basis of the strength given on the label by diluting
it 120 times with distilled water. Then standardize it against standard N/10 NaOH which is
already
Standardized against N/10 oxalic acid using phenolphthalein indicators.

Preparation of sulphuric acid (H2SO4)

Concentrated H2So4 is very corrosive in nature; therefore, it should be handled carefully.
And always remember add acid to water under cold condition this is done to avoid pumping
due to the heat generated.

Standardization of Sulphuric Acid H2SO4

For the preparation of N/10 H2SO4, take 10 ml of concentrated H2SO4 (usually about 36 N),
dilute 36 times by adding acid in small quantity to distilled water in a cold water bath, to
make it 1N and then dilute this 1N solution further 10 times to make it N/10. Then
standardize against standard N/10 NaOH or N/10 KOH using phenolphthalein indicator.

Preparation of nitric acid ( HNO3)

Take 10 ml of concentrated nitric acid HNO3 ( about 16N), and dilute 16 times by adding
acid to distilled water to make it 1N and then dilute this 1N solution futher 10 tiimes to make
it N/10 then standerize agaianst standard N/10 KOH using phenolphthalein indicator.

Preparation of 0.1N sodium thiosulphate solution ( Na2SO3.5H2O)

Dissolve approximately 24.8 gm of sodium thiosulphate crystals in previously boiled and

cooled distilled water and make the volume to 1000 ml. Store the solution in a cool place in a
dark colored bottle. After storing the solution for about two weeks, filter if necessary and
standardize as follows.Weigh accurately about 5.0 gm of finely ground potassium dichromate
which has been previously dried to a constant weight at 105 ± 2° in to a clean 1.0 litre
volumetric flask. Dissolve in water make up to the mark; shake thoroughly and keep the
solution in dark place. Pipette 25.0 ml of this solution into a clean glass stoppered 250 ml
conical flask. Add 5.0 ml of concentrated hydrochloric acid and 15.0 ml of 10% potassium
iodide solution. Allow to stand in dark for 5 minutes and titrate the mixture with the solution
of sodium thiosulphate using starch solution as an indicator towards the end. The end point is
taken when blue color changes to green. Calculate the normality (N) of the sodium
thiosulphate as follows:

N = 25W
49.03 V
W= Weight in g of the potassium dichromate
V= Volume in ml of solution thiosulphate solution required for the titration

Preparation of 0.1N ceric ammonium sulphate (NH₄)₄Ce(SO₄)₄2 H₂O

66gm of ceric ammonium sulphate was dissolved with gentle heat in a mixture of 30 ml of
sulphuric acid and 500 ml of water. The mixture was cooled and filtered. The resulting
solution was diluted to 1000ml with water

Standardisation of 0.1 N Ceric Ammonium Sulphate

1. About 0.2 gm of Arsenic trioxide which was previously dried for about an hour was
accurately weighed and transferred into a 500 ml conical flask.
2. The inner walls of the flask were washed with 100 ml of water and mixed thoroughly
3. Then 300 ml of dil. sulphuric acid , 0.15 ml of osmic acid, 0.1 ml of ferroinsulphate
indicator were added
 4. Titration was carried out until pink colour of solution changed to pale blue or
yellowish green colour
5. Each ml of 0.1 N ceric ammonium sulphate ~ 0.6326 gm of ceric ammonium sulphate
~ 4.946 grams of arsenic trioxide

Preparation and standardization of EDTA solutions

1. Preparation of 0.01 M EDTA solution: Dissolve 3.8 g of disodium ethylene diamine
dihydrogen tetraacetate (EDTA, M.Wt. 372.25) in distilled water and volume is made
to 1 litre. Mix it well, store in polyethylene reagent bottle. It is standardized against
0.01 M CaCO3 or CaCl2.
2. Preparation of 0.01 M CaCl2 solution: Prepare standard Ca solution (1 ml = 1 mg
CaCO3, M.wt. 100) by weighing 1 g CaCO3 into 500 ml conical flask or beaker and
adding dilute HCl through funnel until CaCO3 is dissolved. Add 20 ml water, boil to
expel CO2 and cool. Add few drops of methyl red indicator and adjust colour
intermediate orange (brownish red) with dilute NH4OH or HCl as required. Transfer
quantitatively to 1 L volumetric flask and make up volume to the mark. Shake it well
and store it well and store in air-tight reagent bottle.
3. Erichrome Black T indicator: Dissolve 0.5 g of Erichrome black T in 100 ml of
triethanolamine. Or 0.4 g in 100 ml methanol.
4. Buffer solution: Dissolve 16.9 g NH4Cl in 143 ml NH4OH, and dilute to 250 ml with
water. Store in tightly stoppered Pyrex of plastic bottle. Dispense from bulb-operated
pipette. Discard after 1 month or when 1-2 ml added to sample fails to produce pH
10.0±0.1 at end point titration.

Standardization of EDTA solution

Rinse and then fill burette with prepared EDTA solution. Pipette 25 ml of standard
CaCO3 solution into 250 ml Erlenmeyer flask, add 1 ml ammonia buffer (to raise the pH as
reaction takes place at high pH) and 3-4 drops of Erichrome black T indicator. Titrate the
EDTA solution until colour changes from wine red to dark blue with no reddish tinge
remaining. Calculate the molarity of EDTA (M1V1 = M2V2), if excess follows the procedure
for the standardization, recheck the molarity and it should be 0.01 M.

Preparation of Potassium Permanganate

Potassium Permanganate 0.1 N: Dissolve 3.3 g of reagent grade potassium permanganate
(KmnO4 ) in 1 L of purified water and heat on a steam bath for two hrs. Cover and allow to
stand for 24 hrs. Filter through a fine porosity sintered glass crucible, discarding the first 25
mL. Store in a glass-stoppered, amber-colored bottle. Avoid exposure to direct sunlight;
cover the neck of the bottle with a small beaker as a protection against dust. If manganese
dioxide precipitates on standing, refilter and restandardize before use.

Standardization of Potassium Permanganate

Potassium Permanganate 0.1 N: Weigh accurately 0.2-0.3 g sodium oxalate (Na2 C2 O4 )
(dried 2 hrs., 105-110 °C) National Institutes of Science and Technology, U. S. Department
of Commerce. Cool in a desiccator and transfer quantitatively to a 600 mL beaker. Add 250
mL of purified water (freshly boiled and cooled) and 10 mL sulfuric acid (96% H2 SO4 , sp g
1.84). Add rapidly from a buret about 95% of the theoretical quantity of potassium
permanganate solution needed; stir until the solution is clear. Heat the solution to 55-60 °C
(Maintain temperature range during titration.) and complete the titration by slow dropwise
addition until the appearance of a pink color which persists for 30 secs. Determine and
subtract a blank titration run at 55-60 °C on a mixture of 250 mL of purified water (freshly
boiled and cooled) and 10 mL of concentrated sulfuric acid.

Pharmacopoeia

Pharmacopoeia the word derives from the ancient Greek pharmakopoiia from (pharmako-)
″drug″, followed by the verb-stem (poi-) ″make″ and finally the abstract noun ending -ια (-
ia). These three elements together can be rendered as ″drug-mak-ing″ or ″to make a drug″. A
pharmacopoeia, pharmacopeia, or pharmacopoeia, in its modern sense, is a legally binding
collection, prepared by a national or regional authority, of standards and quality
specifications for medicines used in that country or region. A quality specification is
composed of a set of appropriate tests that will confirm the identity and purity of the product,
ascertain the strength (or amount) of the active substance and, when needed, its performance
characteristics. Reference substances, i.e. highly-characterized, physical specimens, are used
in testing to help ensure the quality, such as identity, strength and purity, of medicines. The
texts cover pharmaceutical starting materials, excipients, intermediates and finished
pharmaceutical products (FPPs). General requirements may also be given in the
pharmacopoeia on important subjects related to medicines quality, such as analytical
methods, microbiological purity, dissolution testing, stability, etc. (1). The role of a modern
pharmacopoeia is to furnish quality specifications for active pharmaceutical ingredients
(APIs), FPPs and general requirements, e.g. for dosage forms. The existence of such
specifications and requirements is necessary for the proper functioning or regulatory control
of medicines. Pharmacopoeial requirements form a base for establishing quality requirements
for individual pharmaceutical preparations in their final form. According to the information
available to the World Health Organization (WHO), 140 independent countries are at present
employing some 30 national as well as the African, European and International
Pharmacopoeias (2). Compared to national and regional pharmacopoeias, The International
Pharmacopoeia (Ph. Int.) is issued by WHO as a recommendation with the aim to provide
international standards – including less technically demanding alternatives where needed - for
adoption by Member States and to help achieve a potentially global uniformity of quality
specifications for selected pharmaceutical products, excipients and dosage forms. After
discussion with many representatives of world pharmacopoeias and in response to feedback,
WHO has initiated steps based on WHO's first attempts during various meetings of the
International Conference of Drug Regulatory Authorities (ICDRA), especially the 10th
ICDRA meeting held in Hong Kong in 2002 and a further discussion among regulators
during the 11th ICDRA meeting held in Madrid in 2004, to organize an official meeting
entitled International meeting of world pharmacopoeias for participation of all WHO Member
States' pharmacopoeias worldwide, be they national, regional or international. The aim was to
discuss topics of common interest and challenges. The meeting dates were 29 February–2
March 2012. In order to prepare for the meeting in a timely manner, WHO sent a preliminary
agenda and Questions to pharmacopoeias in advance to receive feedback and enable
comprehensive input to the agenda. The questions, participant’s presentations and the
meeting report are shown on the meeting web site (3). This document presents a summary of
the answers to the Questions to pharmacopoeias provided by representatives of world
pharmacopoeias participating in the international meeting, and of other related information
received from those that were unable to actively participate in this meeting. History and
background Overwhelming empirical knowledge of mankind gained during cent

Indian Pharmacopoeia Commission (IPC) is an autonomous institution of the Ministry of

Health and Family Welfare which sets standards for all drugs that are manufactured, sold and
consumed in India. The set of standards are published under the title Indian
Pharmacopoeia (IP) which has been modelled over and historically follows from the British
Pharmacopoeia. The standards that are in effect since 1 December 2010 is the Indian
Pharmacopoeia 2010 (IP 2010). The Pharmacopoeia 2014 was released by Health
Minister Ghulam Nabi Azad on 4 November 2013.

I.P., the abbreviation of 'Indian Pharmacopoeia' is familiar to the consumers in the Indian
sub-continent as a mandatory drug name suffix. Drugs manufactured in India have to be
labelled with the mandatory non-proprietary drug name with the suffix I.P. This is similar to
the B.P. suffix for British Pharmacopoeia and the U.S.P. suffix for the United States
Pharmacopeia.

The IPC was formed according to the Indian Drugs and Cosmetics Act of 1940 and
established by executive orders of the Government of India in 1945.

History of Pharmacopoeia

The actual process of publishing the first Pharmacopoeia started in the year 1944 under the
chairmanship of Col. the I. P. list was first published in the year 1946 and was put forth for
approval. The titles are suffixed with the respective years of publication, e.g. IP 1996. The
following table describes the publication history of the Indian Pharmacopoeia.

History and background Overwhelming empirical knowledge of mankind gained during

centuries and constant effort to establish better health care possibilities have led to the
creation of a list of origin, preparation and healing properties of medicines. The term
Pharmacopoeia first appears as a distinct title in a work published in Basel, Switzerland in
1561 by Dr A. Foes, but does not appear to have come into general use until the beginning of
the 17th century. Today’s pharmacopoeias focus mainly on assurance of quality of products
by various tools of analytical sciences. The aim to achieve a wide global harmonization of
quality specifications for selected pharmaceutical products, excipients and dosage forms
came with increased globalization and reciprocal collaboration. History of these approaches
goes back to 1902–1925 when agreements established a ″Unified″ Pharmacopoeia. In 1929
the "Brussels Agreement" stipulated the League of Nations to carry out related administrative
functions. Eight years later, in 1937, the first meeting of the ″Technical Commission of
Pharmaceutical Experts″ was held. An important date in the history of quality assurance of
medicines is 1948, when the First World Health Assembly (WHA) approved the Expert
Committee on Unification of Pharmacopoeias to continue this work. One year later, the
WHA renamed it the Expert Committee on International Pharmacopoeia.

Current Issues

 Amendments to IP 2007 have been published in IP Addendum 2008. Further amendments

are to be taken care of in IP 2009 edition.
 Publication of IP 2009 by Dec. 2009 is the immediate priority. Work is in full swing.
 159 Drug molecules have been short listed for the IP 2009. The APIs and their
formulations monographs are being prepared at the IPC.
 Monograph inclusion/deletion criteria and the Monograph Inclusion Form have been
uploaded on the website of the Commission

Indian National Formulary

1st Edition 1960

2nd Edition 1966

3rd Edition 1979

It is a reliable reference book on drugs formulations for the practicing physicians/ clinicians,
pharmacists, clinical pharmacists,nurses and others engaged in healthcare profession

 PHAMACOPOEIA  OFFICIAL DRUGS

 PHARM.CODEX  OFFICIAL+UNOFFICIAL DRUGS
 EXTRAPHARACOPOEIADETAILED INFORMATION OF
OFFICIAL+UNOFFICIAL DRUGS
 FORMULARYREGISTERED DRUGS FOR MARKETING

Table: 2 TYPES OF PHARMACOPOEIAS

Edition Year Addendum/Supplement

1st
1955 Supplement 1960
Edition

2nd
1966 Supplement 1975
Edition

Addendum 1989
3rd
1985
Edition
Addendum 1991

1996 Addendum 2000

4th
 Edition
Vet Supplement 2000

Addendum 2002

Addendum 2005

5th
2007 Addendum 2008
Edition

6th
2010 Addendum 2012
Edition

Addendum 2015
7th
2014
Edition
Addendum 2016

8th
2018 Addendum 2019
Edition

Sources of Impurities in Medicinal agent

The origin of impurities in drugs is from various sources and phases of the synthetic process
and preparation of pharmaceutical dosage forms. Majority of the impurities are characteristics
of the synthetic route of the manufacturing process. There are several possibilities of
synthesizing a drug; it is possible that the same product of different sources may give rise to
different impurities. According to the ICH impurities are classified as organic impurities,
inorganic impurities and residual solvents. Organic impurities may arise from starting
materials, by products, synthetic intermediates and degradation products. Inorganic impurities
may be derived from the manufacturing process and
are normally known and identified as reagents, ligands, inorganic salts, heavy metals,
catalysts, filter aids and charcoal etc. The number of inorganic impurities and
residual solvents are limited. These are easily identified and their physiological effects and
toxicity are well known. For this reason the limits set by the pharmacopoeias and the ICH
guidelines can guarantee that the harmful effects of these impurities do not contribute to the
toxicity or the side effects of the drug substances. The situation is different with the organic
impurities. Drugs prepared by multi-step synthesis results in various impurities, their number
and the variety of their structures are almost unlimited and highly dependent on the route and
reaction conditions of the synthesis and several other factors such as the purity of the starting
material, method of isolation, purification, conditions of storage etc. In addition, toxicity is
unknown or not easily predictable. For this reason the ICH guidelines set threshold limit
above which the identification of the impurity is obligatory.

Sources of organic impurities:

 Impurities originating from drug substance synthetic processes

 Starting materials and intermediates
 Impurities in the starting materials
 Reagents, ligands and catalysts
 By-products of the synthesis
 Products of over-reaction
 Products of side reactions
 Impurities originating from degradation of the drug substance.

Limit Test

Limit = a value or amount that is likely to be present in a substance

Test = to examine or to investigate
Impurities = a foreign matter present in a compound

Limit test is defined as quantitative or semi quantitative test designed to identify and control
small quantities of impurity which is likely to be present in the substance. Limit test is
generally carried out to determine the inorganic impurities present in compound. In short,
limit test is nothing but to identify the impurities present in the substance and compare it with
standard.

Importance of Limit tests

 To find out the harmful amount of impurities

 To find out the avoidable/unavoidable amount of impurities.

Limit test for Chlorides

Principle:
Limit test of chloride is based on the reaction of soluble chloride with silver nitrate in
presence of dilute nitric acid to form silver chloride, which appears as solid particles
(Opalescence) in the solution.
Procedure:

Test sample Standard compound

Specific weight of compound is dissolved Take 1ml of 0.05845 % W/V solution of sodium
in water or solution is prepared as directed chloride in Nessler cylinder
in the pharmacopoeia and transferred in
Nessler cylinder
Add 1ml of nitric acid Add 1ml of nitric acid
Dilute to 50ml in Nessler cylinder Dilute to 50ml in Nessler cylinder
Add 1ml of AgNO3 solution Add 1ml of AgNO3 solution
Keep aside for 5 min Keep aside for 5 min
Observe the Opalescence/Turbidity Observe the Opalescence/Turbidity

Observation:
The opalescence produce in sample solution should not be greater than standard solution. If
opalescence produces in sample solution is less than the standard solution, the sample will
pass the limit test of chloride and visa versa.
Reasons:
Nitric acid is added in the limit test of chloride to make solution acidic and helps silver
chloride precipitate to make solution turbid at the end of process.

Limit test for Sulphates

Principle:
Limit test of sulphate is based on the reaction of soluble sulphate with barium chloride in
presence of dilute hydrochloric acid to form barium sulphate which appears as solid particles
(turbidity) in the solution.

Procedure:

Test sample Standard compound

Specific weight of compound is dissolved Take 1ml of 0.1089 % W/V solution of potassium
in water or solution is prepared as directed sulphate in Nessler cylinder
in the pharmacopoeia and transferred in
Nessler cylinder
Add 2ml of dilute hydrochloric acid Add 2ml of dilute hydrochloric acid
Dilute to 45 ml in Nessler cylinder Dilute to 45 ml in Nessler cylinder
Add 5ml of barium sulphate reagent Add 5ml of barium sulphate reagent
Keep aside for 5 min Keep aside for 5 min
Observe the Turbidity Observe the Turbidity

Barium sulphate reagent contains barium chloride, sulphate free alcohol and small amount of
potassium sulphate.

Observation:
The turbidity produce in sample solution should not be greater than standard solution. If
turbidity produces in sample solution is less than the standard solution, the sample will pass
the limit test of sulphate and vice versa.

Reasons:
Hydrochloric acid helps to make solution acidic. Potassium sulphate is used to increase the
sensitivity of the test by giving ionic concentration in the reagent. Alcohol helps to prevent
super saturation

Limit test for Iron

Principle:
Limit test of Iron is based on the reaction of iron in ammonical solution with thioglycollic
acid in presence of citric acid to form iron thioglycolate which is pale pink to deep reddish
purple in colour.
Procedure:

Test sample Standard compound

Sample is dissolved in specific amount 2 ml of standard solution of iron diluted with water upto
of water and then volume is made up to 40ml
40 ml
Add 2 ml of 20 % w/v of citric acid Add 2 ml of 20 % w/v of citric acid (iron free)
(iron free)
Add 2 drops of thioglycollic acid Add 2 drops of thioglycollic acid
Add ammonia to make the solution Add ammonia to make the solution alkaline and adjust
alkaline and adjust the volume to 50 ml the volume to 50 ml
Keep aside for 5 min Keep aside for 5 min
Color developed is viewed vertically Color developed is viewed vertically and compared with
and compared with standard solution standard solution

Earlier aamoniumthiocyanate reagent was used for the limit test of iron. Since thioglycolic
acid is more sensitive reagent, it has replaced ammonium thiocyanate in the test.

Observation:
The purple color produce in sample solution should not be greater than standard solution. If
purple color produces in sample solution is less than the standard solution, the sample will
pass the limit test of iron and vice versa.

Reasons:
Citric acid helps precipitation of iron by ammonia by forming a complex with it.

Limit test for Heavy Metals

Principle:
Limit test of heavy metals is based on the reaction of metallic impurities with hydrogen
sulfide in acidic medium to form brownish colour solution. Metals that response to this test
are lead, mercury, bismuth, arsenic, antimony, tin, cadmium, silver, copper, and
molybdenum. The metallic impurities in substances are expressed as parts of lead per million
parts of the substance. The usual limit as per Indian Pharmacopoeia is 20 ppm

Procedure:
The Indian Pharmacopoeia has adopted three methods for the limit test of heavy metals.

Method I: Use for the substance which gives clear colorless solution under the specific
condition.

Test sample Standard compound

Solution is prepared as per the Take 2 ml of standard lead solution and dilute to
monograph and 25 ml is transferred in 25 ml with water
Nessler’s cylinder
Adjust the pH between 3 to 4 by adding Adjust the pH between 3 to 4 by adding dilute
dilute acetic acid ‘Sp’ or dilute acetic acid ‘Sp’ or dilute ammonia solution ‘Sp’
ammonia solution ‘Sp’
Dilute with water to 35 ml Dilute with water to 35 ml
Add freshly prepared 10 ml of hydrogen Add freshly prepared 10 ml of hydrogen sulphide
sulphide solution solution
Dilute with water to 50 ml Dilute with water to 50 ml
Allow to stand for five minutes Allow to stand for five minutes
View downwards over a white surface View downwards over a white surface

Observation:
The color produce in sample solution should not be greater than standard solution. If color
produces in sample solution is less than the standard solution, the sample will pass the limit
test of heavy metals and vice versa. [7-9]

Limit test for Lead

Lead is a most undesirable impurity in medical compounds and comes through use of
sulphuric acid, lead lined apparatus and glass bottles use for storage of chemicals.
Principle:
Limit test of lead is based on the reaction of lead and diphenylthiocabazone (dithizone) in
alkaline solution to form lead dithizone complex which is read in color.

Dithizone is green in color in chloroform and lead-dithizone complex is violet in color, so the
resulting color at the end of process is red.

Procedure:

Test sample Standard compound

A known quantity of sample solution is A standard lead solution is prepared equivalent to the
transferred in a separating funnel amount of lead permitted in the sample under
examination
Add 6ml of ammonium citrate Add 6ml of ammonium citrate
Add 2 ml of potassium cyanide and 2 ml Add 2 ml of potassium cyanide and 2 ml of
of hydroxylamine hydrochloride hydroxylamine hydrochloride
Add 2 drops of phenol red Add 2 drops of phenol red
Make solution alkaline by adding Make solution alkaline by adding ammonia solution.
ammonia solution.
Extract with 5 ml of dithizone until it Extract with 5 ml of dithizone until it becomes green
becomes green
Combine dithizone extracts are shaken Combine dithizone extracts are shaken for 30 mins
for 30 mins with 30 ml of nitric acid and with 30 ml of nitric acid and the chloroform layer is
the chloroform layer is discarded discarded
To the acid solution add 5 ml of standard To the acid solution add 5 ml of standard dithizone
dithizone solution solution
Add 4 ml of ammonium cyanide Add 4 ml of ammonium cyanide
Shake for 30 mins Shake for 30 mins
Observe the color Observe the color

Observation:
The intensity of the color of complex, is depends on the amount of lead in the solution. The
color produce in sample solution should not be greater than standard solution. If color
produces in sample solution is less than the standard solution, the sample will pass the limit
test of lead and vice versa.

Reasons:
Ammonium citrate, potassium cyanide, hydroxylamine hydrochloride is used to make pH
optimum so interference and influence of other impurities have been eliminated