A
PROJECT REPORT
FOR
ELECTIVE SUBJECT
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LIMIT TEST
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SUBMITTED
TO
HEMCHANDRACHARYA NORTH GUJARAT UNIVERSITY,
PATAN.
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In Partial Fulfillment of the requirement For the Degree of bachelor of
Pharmacy
Year: 2006-07.
SUBMITTED BY,
Mr. NIRAV G. PRAJAPATI
SHREE S. K. PATEL COLLEGE OF PHARMACEUTICAL
EDUCATION AND RESEARCH
GANPAT VIDYANAGAR
KHERVA-382711.
Certificate
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This is to certify that the project work for elective subject entitled “Limit
test” represent the bonafide work of NIRAV.G.PRAJAPATI carried out
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under my guidance and supervision at the department of Pharmaceutical
Chemistry, Shree S.K. Patel College of Pharmaceutical Education and
Research, Ganpat Vidyanagar during academic year 2006-2007. He has
collected the literature very sincerely and methodically. This work is up to
my satisfaction.
GUIDE
Head of the Department
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Dr. S. A. PATEL
M. Pharm., Ph. D.
Assistant Professor,
Department of Pharmaceutical
Chemistry,
Shree S.K. Patel College of.
Pharmaceutical Education &
Research,
Ganpat Vidyanagar.
Date:
Place:
Dr. P. U. PATEL
M. Pharm., Ph. D.
Assistant Professor,
Department of Pharmaceutical
Chemistry,
Shree S.K. Patel College of
Pharmaceutical Education &
Research,
Ganpat Vidyanagar.
Principal
Dr. M. M. Patel
M. Pharm., Ph. D., L.L.B., F.I.C.
Shree S.K. Patel College of
Pharmaceutical Education & Research,
Ganpat Vidyanagar.
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ACKNOWLEDGEMENT
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I take this opportunity to express my deep sense of gratitude to those
who have helped me in this work by their help, guidance, suggestion and
encouragement. This project report has been prepared to give a brief
introduction and the project “Limit test” that was under taken for the
fulfillment of degree course in pharmacy. The project work has to be
undertaken and completed as per the direction of the syllabus.
I express my sincere thanks and gratitude to Dr. S. A. Patel, who
become my friend, philosopher, & a guide in true sense. Dr S. A. Patel
constantly helped me by suggestion, encouragement, and giving his valuable
time to discuss the progress of work at every stage.
I am grateful to our principal Dr. M. M. Patel who gave me this
golden opportunity for this work and also gave constant co-operation and
valuable guidance.
My sincere thanks to Dr. P. U. Patel, Mr. B. H. Patel, Mrs. S. K.
Patel, Mrs. H. J. Panchal, Mrs. D. B. Patel and Dr. B. G. Chaudhari of
department of Pharmaceutical Chemistry to help me for reference work.
I am thankful to Mr. P. I. Patel, librarian, Mahadevbhai and
Mukeshbhai who helped me in easy finding of my topic related books &
provides books to me as and when required.
I am also thankful to my friends who gave me constant
encouragement & strength to complete my work.
I am sincerely thankful to all other staff member of the college, who
gave their valuable suggestion.
Last but not least thanks to my mom, dad, brother and all my family
member who are not directly interact with this work but they are always with
me at my all critical stages of this work.
Ganpat VidhyaNagar
2006-07
Mr. Nirav G. Prajapati
Final B. Pharm.
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Dedicated to
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My Family,
My Teachers,
And
My Friends.
SR NO.
CONTENTS
PAGE NO.
INTRODUCTION OF LIMIT TEST
1
2
LIMIT TEST
5
2.1
LIMIT TEST OF CHLORIDE
6
2.2
LIMIT TEST OF SULPHATE
10
2.3
LIMIT TEST OF IRON
14
2.4
LIMIT TEST OF HEAVY METAL
17
LIMIT TEST OF LEAD
20
LIMIT TEST OF ARSENIC
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1
2.5
REFERENCE
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INDEX
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CHAPTER - 1
OF
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INTRODUCTION
LIMIT TESTS
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INTRODUCTION
Limit tests are quantitative or semi-quantitative tests designed to identify and
control small quantities of impurity, which are likely to be present in the substance. The
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quantity of any one impurity in an official substance is often small, and consequently the
visible reaction response to any test for that impurity is also small. The design of
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individual tests is therefore important if errors are to be avoided in the hands of different
operators. This is accomplished by giving attention to a number of factors, which are
discussed below.
SPECIFICITY OF THE TESTS
Any test used as a limit test must, of necessity, give some form of selective reaction
with the trace impurity. Many tests used for the detection of inorganic impurities in
official inorganic chemicals are based upon the separations involved in inorganic
qualitative analysis. A test may be demanded which will exclude one specific impurity,
but highly specific tests are not always the best; a less specific test, which limits several
likely impurities, at once, is obviously advantageous, and in fact can often be
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accomplished. An example of such a test is the heavy metals test applied to alum, which
not only limits contamination by lead, but also other heavy metal contaminants
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precipitated by thioacetamide as sulphide at pH 3.5.
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SENSITIVITY
The degree of sensitivity required in a limit test varies enormously according to the
standard of purity demanded by the monograph. The sensitivity of most tests is dependent
upon a number of variable factors all capable of strict definition, and all favorable
towards the production of reproducible results. Thus the precipitation of an insoluble
substance from solution is governed by such factors as concentration of the solute and of
the precipitating reagent, duration of the reaction, reaction temperature, and the nature
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and concentration of other substances unavoidably present in solution. As a general rule,
cold dilute solutions give light precipitates, whereas more granular ones are obtained
from hot concentrated solutions. Many of the limit tests, however, are concerned with
very dilute solutions, which are often slow to react, and here sensitivity of the reaction
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can often be increased by extending the duration of the reaction or by raising the reaction
temperature. Similar considerations apply in the design of colour and other tests
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employed as limit tests. With suitable control of the factors described the same degree of
reproductivity can be guaranteeed in all cases.
CONTROL OF PERSONAL ERRORS
It is essential to exclude all possible sources of ambiguity in the description of a
test. Vague terms such as 'slight precipitate,' should be avoided as far as possible. The
extent of the visible reaction to be expected under the specified test conditions should be
clearly and precisely defined. This is usually accomplished in one of three ways.
(a) Tests in which there is no visible reaction: A definite statement is incorporated
in the wording of the test, which states that there shall be no colour, opalescence or
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precipitate, whichever is appropriate to the particular test. One example of this type of
requirement is the test for barium, and calcium in Dilute Hypophosphorus. Acid (BP
Appendix I), where the additions of dilute sulphuric acid under precisely controlled
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condition shall produce 'no turbidity, or precipitate' within one hour. The time factor is
used here as a means of increasing the sensitivity of the test.
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Tests such as these which give negative results do not necessarily imply the
complete absence of the impurity, the test as laid down merely indicating the absence of
an undesirably large amount of the impurity
(b) Comparison methods: Tests of this type require a standard containing a
definite amount of impurity, to be set up at the same time and under the same conditions
as the test experiment. In this way the extent of the reaction is readily determined by
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direct comparison of the test solution with a standard of known concentration. The
official limit tests for chlorides, sulphate iron and heavy metals are based on this
principle. The limit tests for lead and arsenic are, in practice, also comparison methods.
They are, however, so designed that they can be readily applied as quantitative
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determinations.
(c) Quantitative determinations: Quantitative determination of impurities is only
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applied in special circumstances, usually in those cases where the limit is not readily
susceptible to simple and more direct chemical determination. The method is used in the
following different types of tests:
(i) Limits of insoluble matter
(ii) Limits of soluble matter
(iii) Limits of moisture, volatile matter, and residual solvents
(iv) Limits of non-volatile matter
(v) Limits of residue on ignition
(vi) Loss on ignition
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(vii) Ash values
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(viii) Precipitation methods.
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CHAPTER - 2
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LIMIT TEST
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(1) LIMIT TEST FOR CHLORIDE
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Principle
The limit test for Chloride has been based on the simple reaction between silver
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nitrate and soluble chlorides to obtain silver chloride which is insoluble in dilute nitric
acid. The silver chloride produced in the presence of dilute nitric acid makes the test
solution turbid, the extent of turbidity depending upon the amount of chloride present in
the substance is compared with a standard opalescence produced by addition of silver
nitrate to a standard solution having a known amount of chloride and the same volume of
dilute nitric acid as used in the test solution. If the turbidity from the sample has been less
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than the standard turbidity, the sample will pass the limit test and vice versa.
Nessler cylinder
Method for the limiting test for chlorides I.P. 85
Specified weight of the substance is dissolved in water or the solution is prepared
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by special treatment as directed in the pharmacopoeia and transferred to a Nessler
cylinder. To the solution 1 ml of nitric acid is added and the volume made up to 50 ml
AgNO3 + Cl-
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HNO3
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with water. Then 1 ml of silver nitrate solution is added and the solution stirred
AgCl + NO3-
and kept aside for 5 minutes. Simultaneously, for standard opalescence, place 1 ml of
0.05845 per cent w/v solution of sodium chloride in Nessler cylinder B and add 1 ml of
dilute nitric acid, make up the volume to 50 ml with water, add 1 ml of silver nitrate
solution, stir with glass rod and set aside for 5 minutes. The opalescence produced by the
sample (in cylinder A) should not be greater than standard opalescence.
If the opalescence from the sample has been less than the standard opalescence,
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the sample will pass the limit test.
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NaCl + AgNO3
HNO3
AgCl + NaNO3
(White turbidity)
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Standard
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Test
Specified substance (1 g) + 10 1 ml of 0.05845 % w/v solution
ml of water + l ml of HN03
of sodium chloride + l ml of
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HNO3
Diluted to 50 ml in Nessler
glass +l ml of AgN03 Sol.
Diluted to 50 ml in Nessler
glass +l ml of AgN03 Sol.
Opalescence/turbidity
Opalescence/turbidity
Note: Sometimes the solution, to be tested, has to be prepared by special
method and
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instruction to this effect, if given, must be followed for preparing test solution.
The opalescences in the sample and standard solution is compared by keeping the
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Nesslcr's cylinder against proper background and observing side by side.
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Preparation of the Solutions for the Limiting Tests of Chlorides
A specified amount of the substance is dissolved in distilled water, and the volume
made up to 50 ml in a Nessler cylinder. Depending upon the nature of the substance,
some modifications have to be adopted for the preparation of the solution.
(a) Alkaline substances have to be dissolved in acid so that effervescence ceases
and much of the free acid is left in the solution as is prescribed in the test.
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(b) Insoluble substances are generally extracted with water and then filtered, and
the filtrate is used for the test, because the presence of insoluble substance modifies the
opalescence and colour.
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(c) Salts of organic acids like sodium benzoate, sodium salicylate, etc. liberate
free water insoluble organic acid during acidification which is filtered off and the filtrate
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is employed for the test.
(d) Coloured substances like crystal violet, malachite green, etc. are carbonised
and the ash so produced is extracted in water.
(e) Deeply coloured substances have to be decolourised before test e.g., potassium
permanganate is reduced by boiling with alcohol and the filtrate is used.
(f) Reducing substances like hypophosphorus acid, which react with silver nitrate
in the limit test for chlorides should be oxidized with nitric acid or some other oxidizing
agents before carrying out the test.
Final Remark on Limiting Test for Chlorides
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For comparison of turbidity for different substances with varying amount of
impurity, the amount of substance to be used is varied, and not the standard turbidity.
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Pharmacopoeias do not give a numerical value to the limits, as is not practicable as its
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content will be influenced to great extent, by large quantities of other substances present.
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(2) LIMIT TEST FOR SULPHATE
Principle
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The Limit Test for Sulphate is carried out on the basis of the reaction between
barium chloride and soluble sulphates in presence of dilute hydrochloric acid. Then, the
comparison of the turbidity produced by a given amount of the substance is done with a
standard turbidity obtained from a known amount of sulphate, same volume of dilute
hydrochloric acid having been added to both the solutions. The barium chloride test
solution in the IP has been replaced by Barium Sulphate Reagent which is having barium
chloride, sulphate-free alcohol, and a solution of potassium sulphate. Potassium sulphate
has been added to increase the sensitivity of the test. The ionic concentrations in the
reagent has been so adjusted that the solubility product of barium sulphate gets exceeded,
and the very small amount of barium sulphate present in the reagent acts as a seeding
agent for precipitation of barium sulphate, if sulphate be present in the substance under
test. Alcohol helps to prevent super-saturation and thus produces a more uniform
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opalescence.
The substance passes the limit test if it produces a turbidity that is less than the
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standard.
In performing these tests, it becomes essential to follow the directions which are
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indicated by the Indian Pharmacopoeia.
Method for Limiting Test for Sulphate
A solution of specified quantity of substance is prepared in water or prepared as
directed in the pharmacopoeia in Nessler's cylinder and 2 ml dilute hydrochloric acid is
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added except where hydrochloric acid is used in the preparation of solution. Dilute to 45
ml with water, add 5 ml of barium sulphate reagent, stir immediately with the glass rod
and set aside for 5 minutes. In order to produce standard turbidity place 1 ml of 0.1089
per cent w/v solution of potassium sulphate and 2 ml of dilute hydrochloric acid in
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another Nessler's cylinder, dilute to 45 ml with water, add 5 ml of barium sulphate
reagent, stir immediately, and set aside for 5 minutes. The turbidity produced by the
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sample solution should not be greater than the standard turbidity.
If the turbidity produced by the test solution has been less than standard turbidity the
sample would pass the limit test for sulphate.
SO42- + BaCl2
HCl
BaSO4 + 2Cl-
B.P. makes use of barium sulphate reagent, which is having barium chloride,
alcohol and small amount of potassium sulphate. Alcohol does not allow super saturation,
and potassium sulphate is known to increase the sensitivity of the test, by giving the ionic
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concentration in the reagent, which just exceeds the solubility product of barium'sulphate.
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Test
Standard
Specified substance (1 g) + 2 ml 1 ml of 0.01089 % w/v sol. of
HC1 diluted to 45 ml+ 5 ml Sol K2SO4 + 2 ml HC1 diluted to 45
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of BaSO4
ml + 5 ml Sol of BaSO4
Turbidity
Turbidity
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Preparation of the Solutions for the Limiting Tests of Sulphates
A specified amount of the substance is dissolved in distilled water, and the volume
made up to 50 ml in a Nessler cylinder. Depending upon the nature of the substance,
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some modifications have to be adopted for the preparation of the solution.
(a) Alkaline substances have to be dissolved in acid so that effervescence ceases
and much of the free acid is left in the solution as is prescribed in the test.
(b) Insoluble substances are generally extracted with water and then filtered, and
the filtrate is used for the test, because the presence of insoluble substance modifies the
opalescence and colour.
(c) Salts of organic acids like sodium benzoate, sodium salicylate, etc. liberate
free water insoluble organic acid during acidification which is filtered off and the filtrate
is employed for the test.
(d) Coloured substances like crystal violet, malachite green, etc. are carbonised
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and the ash so produced is extracted in water.
(e) Deeply coloured substances have to be decolourised before test e.g., boiling
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with alcohol reduces potassium permanganate and the filtrate is used.
(f) Reducing substances like hypophosphorus acid, which react with silver nitrate
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in the limit test for chlorides should be oxidized with nitric acid or some other oxidizing
agents before carrying out the test.
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Final Remark on Limiting Test for Sulphates
For comparison of turbidity for different substances with varying amount of
impurity, the amount of substance to be used is varied, and not the standard turbidity.
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Pharmacopoeias do not give a numerical value to the limits, as is not practicable as its
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content will be influenced to great extent, by large quantities of other substances present.
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(3) LIMIT TEST FOR IRON
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Principle.
The Limit Test for iron is based on the reaction of iron in ammoniacal solution in
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presence of citric acid, with thioglycollic acid when a pale pink to deep reddish purple
colour is formed due to ferrous compounds. The colour produced from a specified
amount of substance from the test is compared by viewing vertically with a standard
(ferric ammonium sulphate). If the colour from test solution is less dark than the standard,
then the sample passes the test. Citric acid docs not allow the precipitation of iron by
ammonia by forming a complex with it. The colour due to ferrous compound gets
destroyed by oxidising agent’s anil alkalies. The state of oxidation of iron has been
immaterial as iron (III) gels rei.luced to iron (II) by thioglycollic acid.
Thioglycollic acid, (HS) CH2COOH, is a useful analogue of glycollic acid, CH2
(OH) COOH. This is prepared by the action of potassium hydrogen sulphide on
monochloroacetic acid:
CH2 (SH) COOH + KCl
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CH2ClCOOH + KSH
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It is a colourless liquid having unpleasant odour. The thioglycollic acid test for iron
has largely replaced the former test which was based on the formation of (lie highly
coloured ferric thiocyanate by reaction between ammonium thiocyanate and iron (III)
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salts. It is believed that the thioglycollic acid test has been more sensitive than the
ammonium thiocyanate test.
The colour produced from a specified amount of the substance is compared with
the standard colour obtained from a known amount of iron under exactly similar
conditions. Nessler cylinders of specified dimensions and uniform quality being used for
the standard and the test solution.
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This test is very sensitive. Interference of other metal cations is eliminated, by
Preparation of Standard Solution of Iron:
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It is prepared by adding 0.173 g of ferric ammonium sulphate
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making use of 20% citric acid, which forms a complex with other metal cations.
(NH4Fe (SO4)2 .12 H2O) to 1.5 ml of HCl and adding sufficient water to produce 1000
ml. Each ml of solution contains 0.02 mg of iron.
Method: A solution is first of all obtained from a specified amount of substance.
It is then taken in a Nessler cylinder and to it 2 ml of 20 per cent citric acid solution T.S.
and 2 drops of thioglycollic acid are added, the solutions are mixed, and made alkaline
with a solution of iron-free ammonia and diluted to the 50 ml mark with water. The
colour obtained is compared with a standard prepared from 2 ml of standard solution of
iron, with 40 ml water in a Nessler cylinder following the same procedure as for the test
solution.
Ferrous thioglycollate is colourless in acid or neutral solutions. In acidic media
2+
is oxidized to Fe3+ in presence of thioglycollic acid. Hence the solution becomes
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colourless. The purple colour is developed only in the presence of alkali.
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If the colour produced by test solution has been less than that of standard, me
sample passes the limit test for iron.
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Earlier, ammonium thiocyanate reagent was used for the limit test of iron. Since
thioglycolic acid is a more sensitive reagent for iron, it has replaced ammonium
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thiocyanate in the test.
Test Solution
Standard Solution
Sanple +40 ml of water + 2 ml of 2 ml of standerd solution of iron +
20% w/v (iron free) citric acid + 2 40 ml water + 2 ml of 20% citric
drop of thioglycolic acid ; solution acid + 2 drop of thioglycollic acid ;
mixed
,
made
alkaline
with solution made alkaline and adjusted
ammonia volume adjusted to 50 ml to 50 ml ; allowed to stand and
; allowed to stand and color color developed viewed vertically
developed viewed vertically and and compared with test solution .
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compared with standered solution .
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Note: All the reagents used in the limit test for iron should themselves be iron-free.
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Hence they themselves should conform to the limit tests for iron.
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(4) LIMIT TEST FOR HEAVY METALS
The Indian Pharmacopoeia and the U.S.P. have adopted a limit test for heavy
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metals for many official substances. The test has been designed to demonstrate that the
content of metallic impurities that are coloured by hydrogen sulphide (sulphide ion)
under the conditions of the test should not .exceed the heavy metals limit given under the
individual monographs. In substances the proportion of any such impurity (Heavy
Metals) has been expressed as the quantity of lead required to produce a colour of equal
depth as in a standard comparison solution having a definite quantity of lead nitrate. The
quantity is stated as the heavy metals limit and is expressed as parts of lead (by weight)
per million parts of the test substance.
Principle
The limit test for heavy metals has been based upon the reaction of the metal ion
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with hydrogen sulphide, under the prescribed conditions of the test causing the formation
of metal sulphides. These remain distributed in acolloidal state, and give rise to a
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brownish colouration. The test solution is compared with a standard prepared using a lead
solution (as the heavy metal). The metallic impurities in substances are expressed as parts
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of lead per million parts of the substance. The usual limit as per l.P. has been 20 ppm.
Methods for limiting test of Heavy Metals
The Indian Pharmacopoeia has adopted three methods for the limit tests for heavy
metals. The 'Method I' is used for the substance which gives a clear colourless solution
under specified conditions. 'Method II' is used for those substances which do not give
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clear colourless solution under the test conditions specified for method I. 'Method III' is
used for substances that give clear colourless solution in sodium hydroxide medium. The
reagents like acetic acid, ammonia, hydrochloric acid, nitric acid, potassium cyanide and
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sulphuric acid should be lead free and are designated as'Sp. Reagents.'
Method I:
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This method is applicable for the samples which give clear colourless solutions
under specified conditions of test.
Standard Solution:
Take 2 mi of standard lead solution by pipette in Nessler’s cylinder
and dilute
it with water to produce 25 ml. Adjust the pH 1between 3 -4 with dilute acetic acid "Sp'
or dilute ammonia solution 'Sp'. Dilutewith water to about 35 ml. Mix.
Test Solution:
Take 25 ml of the solution which is prepared as per the procedure given under
respective monograph from l.P. in Nessler cylinder.
Adjust the pH between 3 to 4 by adding dilute acetic acid 'Sp' or dilute ammonia
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solution 'Sp.' Dilute with water to about 35 ml. Mix.
To the above cylinders A and B each containing standard solution and test
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solution respectively, add 10 ml of freshly prepared hydrogen sulphide solution. Mix
well. Dilute with water to 50 ml. Allow to stand for five minutes and view downwards
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over a white surface.
The colour produced in the test solution is not darker than that produced in the
standard solution.
Method II:
The standard solution can be prepared as directed under Method I. Test solution
may be prepared by weighing a specified quantity of substance as per monograph in
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crucible. Moisten the substance with sulphuric acid; ignite on a low flame till completely
charred. Add few drops of nitric acid and heat to 500°. Allow to cool, add 4 ml of
hydrochloric acid and evaporate to dryness. Moisten the residue with 10 ml hydrochloric
acid and digest for two minutes. Neutralize with ammonia solution and make just acidic
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with acetic acid. Adjust the pH between 3.0 and 4.0, filter if necessary. Adjust the volume
of filtrate to 35 ml in Nessler's cylinder, add 10 ml of hydrogen sulphide solution, dilute
Method III:
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to 50 ml with water and compare the colour with the standard solution.
The standard solution could be prepared by using 2 ml of standard lead solution;
adding 5 ml dilute sodium hydroxide solution and making the volume to 50 ml with
water. For the test solution take either 25 ml solution prepared as directed in the
monograph or takes specified quantity of substance, dissolve in 20 ml water, add 5 ml of
dilute sodium hydroxide solution and make up the volume to 50 ml.
To each of the above solution in Nessler’s cylinder add 5 drops of sodium sulphide
solution, mix and set aside for 5 minutes. The colour produced by test solution is not
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darker than the standard solution.
Limit Test for Heavy Metals in Volatile Oils:
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In 25 ml glass stoppered test tubes, 10 ml of the oil are shaken with an equal
volume of water having a drop of hydrochloric acid. Hydrogen sulphide is passed through
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the mixture until it gets saturated. No darkening in colour should get produced either in
the oil, or in the water layer, for the sample to pass the test.
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Introduction:
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(5) LIMIT TEST FOR LEAD
Lead has been one of the most undesirable impurities in medicinal substances.
The chief sources of this impurity have been the sulphuric acid and the lead-lined
apparatus. Besides, glass bottles used for storage of chemicals have been dangerous
sources of contamination with lead.
Analyst Richmond reported that the lead content of potassium carbonate stored
under the above conditions increases from less than 5 parts per million to 150 p.p.m. in
course of nine weeks. Moreover, in the case of substances prepared by precipitation, the
lead content of all the chemicals used in manufacture may also get concentrated in the
precipitate and increase the lead content to dangerous limits. Hence, the lead content of
ferrous sulphate varies between 20 to 50 p.p.m. but that of iron and ammonium citrate
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was found by Powell and Hall to be as high as 600 p.p.m. because of the fact that all of
the lead present in chemicals used in the preparation of ferric hydroxide got absorbed by
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the precipitate.
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Special Procedures for Preparing Primary and Auxiliary Solutions for
carrying out Limiting Test for Lead
(1) Carbonates and Oxides: Carbonates are treated with acetic acid and boiled to
set off CO2 completely. So, colour interferes and contaminations are avoided.
(2) Sparingly Soluble Organic acids and their salts: Substances like aspirin, benzoic
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acid, salicylic acid, etc. and sodium and potassium tartrate liberate free acids when
treated with acetic acid and hence form precipitates. As precipitates have been
undesirable in the test, such substances have been dissolved in ammonia instead of acetic
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acid.
(3) Antimony Compounds and Sulpha Drugs: Antimony compounds and Sulpha
drugs give rise to the formation of precipitate on addition of ammonia. Therefore, they
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are made to treat with sodium hydroxide solution to avoid precipitation of antimony
sulphide when sodium sulphide is added to develop the colour.
(4) Kaolin: Kaolins are insoluble in ordinary solvents. Therefore, a hydrochloric
acid extract is prepared, filtered, the filtrate is treated with nitric acid and evaporated to a
low bulk. The concentrated filtrate is diluted, ammonium chloride and ammonium
thiocyanate added and the solution extracted with equal parts of amyl alcohol and solvent
ether. The aqueous solution so obtained and having lead freed from other impurities is
treated with citric acid. By the method described above lead has been freed from iron and
other metals which would otherwise interfere with the lead test.
(5) Iron Compounds. Iron compounds are oxidised with nitric acid and the ferric
chloride so produced is extracted with ether before carrying out the test. Iron interferes in
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the test by reacting with sodium sulphide.
(6) Magnesium Trisilicate: Magnesium trisilicate is first of all decomposed with
in
hydrochloric acid and then evaporated to dryness, followed by extraction of the residue
with hydrochloric acid and filtration of the insoluble silica. The lead in the filtrate has
been freed from other impurities by treating with ammonium chloride and ammonium
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thiocyanate and extracted as in the case of Kaolin. Magnesium trisilicate is insoluble and
hence there is need to extract lead from it.
(7) Methylene Blue, Brilliant Green, Crystal Violet, Phenolphthalein, Suramin
etc.: These substances are decomposed with sulphuric acid and nitric acid before carrying
out the limit test. Their decomposition becomes necessary because of the colour they
impart to the solutions and because of the interference of organic matter with the test.
- 21 -
Organic matter is destroyed by treating with the above acids. The sulphuric acid residue
retaining all the lead gets separated from impurities by extracting it in the form of its
complex with dipheny1 thiocarbazone by using chloroform. The lead complex so
obtained is extracted with hydrochloric acid and then the test is completed in the usual
in
way.
(8) Calcium Hydroxide Solution: Calcium hydroxide solution is having a very
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small limit and hence it should be concentrated by boiling to a define volume before it is
tested. Concentration should be done after acidifying it with,acetic acid.
Lead sulphide is soluble in dilute acids. Consequently, if the lead is present in
traces, no colour will get produced by addition of sodium sulphide to acidic solutions. On
the contrary, lead sulphide is insoluble in ammoniacal solutions and hence a deeper
colour will get developed by the same amount of lead in an ammoniacal solution than in
the acid one.
The substance under test may have traces of copper and iron salts which interfere
in the test. Copper yields a brown and iron a dark green colour on treated with sodium
sulphide because of the formation of their sulphides and may modify the colour produced
by reaction between lead salts and sodium sulphide. Copper and iron salts form complex
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compounds with potassium cyanide and hence the interference of copper and iron salts
can be eliminated by adding potassium cyanide before the addition of sodium sulphide.
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The reactions may be put as follows:
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2Cu2+ + 4CN -  2Cu (CN) 2
2Cu (CN) 2  Cu2 (CN) 2 + (CN) 2
Cu2 (CN) 2 + 6CN- 
FeSO4 + 2KCN 
Fe (CN) 2 + 4KCN 
K4Fe (CN) 6 
2[Cu (CN) 4]2Fe (CN) 2 + K2SO4
K4 [Fe (CN) 6]
4K+ + [Fe (CN) 6]4 –
- 22 -
These complexes do not react with sodium sulphide and hence produce no
Principle:
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(i) Limit test of lead as per IP& USP
in
colours.
The limit test for lead as per I.P. and U.S.P. has been based upon the reaction
between lead and diphenyl thiocarbazone (dithizone).
Dithizone in chloroform is able to extract lead from alkaline aqueousstions as a
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lead dithizone complex (red in colour).
in
‫׀‬
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‫׀‬The original dithizone is having a green colour in chloroform while the lead-
dithizone is having a violet colour. The intensity of the colour of complex is dependent
upon the amount of lead in the solution. The colour of the lead-dithizone complex in
chloroform has been compared with a standard volume of lead solution, treated in the
same manner.
In this method the lead present as an impunity in the substances, gets separated by
extracting an alkaline solution with a dithizone extraction solution. The interference and
- 23 -
influence of oilier metal ions etc. have been eliminated by adjusting the optimum pH for
the extraction by employing ammonium citrate, potassium cyanide, hydroxylamine
in
hydrochloride reagents, etc.
Method
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A solution of the sample is prepared as directed earlier. A known quantity of the
sample solution is transferred to a separating funnel. To it 6 ml of ammonium citrate, 2
ml of potassium cyanide and 2 ml of hydroxylamine hydrochioride are added, following
by 2 drops of phenol red, and the solution' is made alkaline by adding an ammonia
solution. This is then extracted with 5 mi of portions of dithizone solution until it
becomes green. The combined dithizone extracts are shaken for 30 seconds, with 30 ml
of 1 nitric acid, and the chloroform layer is discarded. To the acid solution 5 ml of
standard dithizone solution is added along with 4 ml of ammonium cyanide and solution
shaken for 30 seconds.
A control is similarly prepared with a volume of diluted standard lead solution
equivalent to the amount of lead permitted in the sample under examination and
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containing all other reagents in the same quantity as in the test solution. The colour of the
chloroform layer in case of sample being tested should not be of deeper shade of violet
than that of control which is made with a volume of lead solution equivalent to the
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amount of lead permitted in the sample under examination.
During the preparation of a sample solution, an approp-iate preliminary treatment
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is given, so as to get lead in the solution, without any interfering substance or ion. All
reagents used under the test (except for standard lead solution), must be free from lead,
and have been designated as PbT reagents in pharmacopoeias.
- 24 -
Important Points:
in
(i) All reagents and solutions used in the test must be free from lead.
(ii) The reagents are used in the test to buffer the solution of the sample to the
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optimum pH for extraction of lead dithizonate and disallow interference with other metals
which produce similar colours with dithizone.'
(ii) Limit Test for Lead as per B.P.
Principle:
The test described in B.P. is dependent upon the formation of brownish
6colouration when sodium sulphide is added to dilute solution of lead salts, the intensity
of the coloration varying with the quantity of lead present. If the lead has been present in
more than traces, a colloidal brownish black precipitate of lead sulphide has been formed
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instead of the colour. The colour so obtained is matched against standard colour produced
from a known amount, of lead and thereby, the exact quantity of lead present in the
in
sample determined. The comparison is done in two similar 50 ml Nessler cylinder made
of thin lead free glass.
In order to carry out this test, two solutions called primary and auxiliary see
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';
prepared from the sample.
- 25 -
Method:
For the test, two standard solutions of the substance have to be prepared, a
greater amount of the substance than the auxiliary.
in
primary solution and an auxiliary solution, the primary solution containing a definite but
Two solutions of the substance under test are prepared; with hot water and acetic
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acid. The primary solution, containing a definite but greater amount of substance, is
placed in a 50 ml. Nessler cylinder. The auxiliary solution, containing a known amount of
the test substance is taken in another 50 ml Nessler cylinder. To this auxiliary solution, a
definite amount of a dilute solution of lead nitrate is added. Ammonia and potassium
cyanide solutions are added to the both solutions in the Nessler cylinders. If they are
coloured, then small amount of burnt sugar solution is added to both solutions, to correct
any difference of colour and the volume is made up to 50 ml. If the solutions appear '
turbid, then they are filtered, and the volume made up to 50 ml. Both solutions are treated
with sodium sulphide solution, and a colour is developed. If the colour in the auxiliary
solution becomes darker than that in the primary, then the substance is having lead within
limits.
The aim of using primary and auxiliary solutions of substances is to do a
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comparison under identical conditions. Interference by any unknown entity
present in
the solution gets eliminated by this technique.
in
The method may be used for determining approximate amount of lead in the
substance by preparing a number of auxiliary solutions and adding varying amounts of
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dilute lead solutions to each. The one nearest in colour to the primary will give the
required value. If more than 15 ml of dilute lead solution PbT is needed, a smaller
quantity of substance is to be taken.
The following tabulated statement will be able to make the Lead Limit Test more
easily understood. Suppose the B.P. prescribes the weight is to be taken as 4 g.
- 26 -
Primary
Auxiliary
4 g of substance dissolved in 1 g substance dissolved in water
water having 10 ml of Acetic having 7 ml of Acetic Acid PbT
is added.
in
Acid PbT.
2 ml of dilute solution of lead
The
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PbT is added
solution
alkaline
with
is
made
solution
just The
solution
of alkaline,
with
is
made
solution
just
of
Ammonia PbT and then 1 ml of Ammonia PbT and then 1 ml
solutionof potassium Cyanide solution of Potassium Cyanide
PbTis added.
PbT is added.
Filter both the solution if they get turbid. And insert colouring matter if colours are
different.
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Dilute the solution to 50 ml
Dilute the solution to 50 ml
with water, add 2 drops of with water, add 2 drops of
in
Solution of Sodium Sulfide Solution of Sodium Sulfide PbT,
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PbT, and stir.
and stir.
Compare the two colours now.
- 27 -
If the two solutions are having the same tint, it implies that 2 ml of dilute solution
of lead PbT is having the same amount of lead as is contained in the difference of weights
of substance in the two solutions, primary and the secondary. In the above case, the
difference has been 2 g in the weights of the substance in the auxiliary and the primary
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solutions. 2 ml of dilute solution of lead PbT is having 10 p.p.m. of lead.
Important Points:
(A) The colour in lead limit test has been due to colloidal particles of lead
sulphide and has been governed by the degree of fineness and degree of dispersion of
these particles. The dispersion of particles could be controlled by the magnitude of
electric charges which are carried by them. Addition of other substances modified the
charges on the particles and their dispersion, thereby altering intensity of the original
colour. That is why it is usual to use two solutions in the Lead Limit Test, both having
original substance in solution.
C.A. Hill showed that the modifying effect of other substances on the colour of
lead sulphide reached a maximum when the substances have been present to the extent of
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40 per cent. Therefore, the amount of the substance in auxiliary solution is generally 2 g
unless other considerations merit any alteration. The difference in colours of the primary
and auxiliary solutions has been then taken to be due to difference in amount of lead
in
contained in the two solutions. For calculations, the quantity of substance present in the
auxiliary solution has to be subtracted from that of the primary and the difference has
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been used as the basis
(B) As acetic acid is a good solvent for lead, it helps to take into solution any
insoluble lead. Hot water increases the solubility of lead and hence lowers the risk of its
absorption by filter papers. That is why substances are directed to get dissolved in hot
water having a specified amount of acetic acid.
- 28 -
(6) LIMIT TEST FOR ARSENIC
in
Arsenic is a well-known undesirable and harmful impurity which is present in
medicinal Substances. All pharmacopoeias prescribe a limit test for it. Many qualitative
and quantitative tests for arsenic are known. However, the pharmacopoeia method is
Principle:
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based on the Gutzeit test.
The pharmacopoeia test is based on the fact that arsenic in the arsenious state can
be easily reduced to arsine gas (AsH3). When this gas is passed over mercuric bromide
paper, it produces a stain, which ranges in colour from yellow to brown, the intensity and
length of which are proportional to the amount of arsenic. B.P. suggests the use of a
mercuric chloride paper instead of mercuric bromide paper. A standard stain prepared
from a definite quantity of arsenic, is used for comparison, and provides the limit which
the sample under test must not exceed.
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Reduction of the arsenic to arsine, both in the standard and the sample may be
achieved by the combined actions of zinc, acid, stannous chloride, and potassium iodide.
in
Analyst Monier Williams has devised an electrolytic apparatus which is used to generate
hydrogen for the reduction of arsenic to arsine. The arsine is carried over by hydrogen to
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the mercuric bromide or mercuric chloride papers which are supported in test apparatus.
In order that the results are obtained as reproducible, all the variables should be
kept constant. It is very essential to maintain the rate of evolution of hydrogen which in
turn is dependent upon the amount and surface area of zinc, the concentrations of the acid
and the salt in the reaction medium, the temperature and the dimensions of the apparatus
etc. Rapid evolution gives rise to a long and diffuse stain while a slow evolution gives
- 29 -
rise to a short stain of intense colouration. Neither stain has been found to be satisfactory
for quantitative comparisons. The specifications in the pharmacopoeia have been given to
in
standardise the variations and must be strictly followed.
The chemical reactions
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The chemical reaction involved in the method are outlined as follows When__the
sample is dissolved in acid, the arsenic present in the sample gets coverted to arsenic acid
which gets reduced, by reducing agents (like_potasium iodide, stannous acid etc.) to
arsenious acid. The nascent hydrogen formed during the reaction, further reduces
arsenious acid to arsine (gas), which reacts with mrcuric chloride paper, giving a yellow
stain.
●
H3AsO3+ 3H2  AsH3 + 3H2O
Arsine
●
H3AsO4 + 3H2  H3AsO3
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Arsenic
Acid
Arsenious
acid
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● H3AsO3 + 3H2  AsH3 +3H2O
Arsenious
Arsine
asid
The depth of yellow stain on mercuric chloride paper will depend upon the
quantity of arsenic present in the sample.
In order to carry out the test, a specified apparatus (as described in
pharmacopoeias) is employed. In order to convert arsenic into arsine gas, various
reducing agents such as zinc, hydrochloric acid, stannous chloride, and potassium iodide
- 30 -
are used.
It becomes essential to maintain the rate of evolution of hydrogen gas by using a
particular size of zinc, and controlling the concentration of acids and other salts of the
2S),
in
reaction medium, besides temperature. If any impurity is coming along with the gas (as H
it is trapped by placing a lead acetate soaked cotton plug in the apparatus. All the
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pharmacopoeias.
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reagents used for the test should be arsenic free, and are designated as As T in
Fig. 4.2 Apparatus used for arsenic limit test: on the left is an alternative device for
securing mercuric chloride paper.
- 31 -
Apparatus:
It is having a wide mouthed glass bottle of 120 ml capacity having mouth of about
2.5 cm in diameter. This bottle is fitted with a rubber bung through which passes a glass
in
tube, 20 cm long having an external diameter of about 0.8 cm and internal diameter of
0.65 cm. The tube is constricted at its lower end extremity to about 1 mm diameter and
constricted part.
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there is blown a hole, not less than 2 mm in diameter, in the side of the tube near the
The upper end of the glass tube has been fitted with two rubber bungs (about 25
mm x 25 mm), each having a hole bored centrally and exactly 6.5 mm in diameter. One
of the bungs has been fitted to the upper end of the tube, while the second bung has to be
fitted upon the first bung in such a way that the mercuric chloride paper gets exactly
sandwiched between the central perforations of the two. The bungs are kept in close
contact by using rubber band or spring clip in such a manner that gas evolved from bottle
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must have to pass through the 0. 65 mm internal circle of mercuric chloride paper.
- 32 -
in
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in
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gn
Regular arsenic apparatus with alternate devices (a) and (b) for fixing mercuric chloride
paper.
- 33 -
During the test, the evolved gases have been passing through the side hole, the
lower hole serving as an exit for water which condenses in the constricted part of the
tube. An important feature of the apparatus has been the standardisation of the area of
in
mercuric chloride paper which is exposed to the action of arsine gas.
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Reagents:
All the special reagents used in the limit test for arsenic are marked and
distinguished by letter 'As T', which means that they all should be arsenic free and should
themselves conform to limit test for arsenic. However, dilute and strong arsenic solutions
which are used for standard stains have been exceptions.
Hydrochloric Acid 'As T.
HCLshould comply with the following additional tests:
(i)
10 ml hydrochloric acid is diluted with sufficient water to produce 50 ml.
To this, 5 ml of solution of ammonium thiocyanate is added and the
solution is stirred immediately. No colour is produced
To 50 ml, 0.2 ml of bromine solution 'As T' is added. This solution is
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(ii)
evaporated on a water bath until reduced to 16 ml followed by addition of
in
more bromine solution 'As T', if necessary, in order that an excess, as
indicated by the colour, may be present throughout the evaporation, and
50 ml of water and 5 drops of stannous chloride solution 'As T' and then
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the general test is applied. The stain produced is not deeper than a 0.05
part per million.
Mercuric Chloride Paper. It is a smooth white filter paper, not less than 25 mm in
width which is first of all soaked in a saturated solution of mercuric chloride and two
pressed to remove superfluous solution, and finally dried at about 60° in the dark. The
grade of the filter paper must be such that the weight in g per sq. m. shall lie between 65
- 34 -
and 120 g, the thickness in mm of 400 papers must be approximately equal, numerically,
to the weight in g per sq m.
Note:mahloride paper must be preserved in a stoppered bottle in the dark. Paper which
stain or stain at all when it is used in the quantitative test for arsenic.
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Potassium Iodide As T
in
has got exposed to sunlight or to the vapour of ammonia, gives rise to a lighter coloured
Potassium iodide should comply with the following additional test:
10 g of potassium iodide is dissolved in 25 ml of hydrochloric acid 'As T' and 25 ml
of water. To this five drops of stannous chloride solution 'As T' are added and the general
test is applied; no visible stain is produced.
Zinc'As T'
Granulated zinc must comply with the following additional test:
10 ml of stannated hydrochloric acid 'As T' is added to 50 ml of water, and now
the general test is applied using 10 g of the zinc, but allowing the action to continue for
one hour; no visible stain gets produced (limit of arsenic). The test is repeated with the
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addition of. 1 ml of dilute arsenic solution As T; a faint but distinct yellow stain gets
produced (test for sensitivity)
in
Nitric Acid As T
Nitric acid should comply with the following additional test:
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20 ml of nitric acid is heated in porcelain dish with 2 ml of sulphuric acid ‘As T'’
until white fumes are given off. This solution is now cooled. To this 2 ml of water is
added. Now this is again heated until the white fumes are given off. The solution is again
cooled. To this 50 ml of water and 10 ml of stannated hydrochloric acid As T are added.
Now with this solution general test is applied. No visible stain should be produced.
- 35 -
Potassium Chlorate As T
Potassium chlorate must comply with the following additional test:
5 g potassium chlorate is mixed in the cold with 20 ml water and 22 ml of
in
hydrochloric acid 'As T'; when the first reaction has subsided; it is heated gently to expel
chlorine. The last traces of chlorine are removed with a few drops of stannous chloride
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solution 'As T'. To this 20 ml of water are added and the general test is applied. No
visible stain is produced.
Sodium Carbonate Anhydrous As T
Anhydrous sodium carbonate must comply with the following additional test:
5 g of sodium carbonate anhydrous is dissolved in 50 ml of water. To this 20 ml
ofbrominated hydrochloric acid 'As T' is added. The excess of bromine is removed with a
few drops of stannous chloride 'As T' and the general test is applied; no visible stain is
produced.
Stannated Hydrochloric Acid As T
It is prepared from solution -of stannous chloride by adding an equal volume of
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hydrochloric acid, boiling down to the original volume and filtering through a finegrained filter paper.
in
It should comply with the following test:
To 10 ml of the above solution 6 ml of water and 10 ml of hydrochloric acid As T
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are added. Now the solution is distilled to collect 16 ml. To the distillate 50 ml of water
and a few drops of stannous chloride solution "As T' are added and the general test is
carried out. The stain produced is not deeper than a 1 ml standard stain showing that the
proportion of arsenic present does not exceed 1 part per million.
- 36 -
Stannated Hydrochloric Acid As T
Stannous chloride solution 'As T'........... 1 ml.
Sulphuric Acid As T
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Sulphuric acid should comply with following additional test:
in
Hydrochloric Acid As T....................... 100 ml.
10 g of Sulphuric acid is diluted with 50 ml of water. To this 0.2 ml of stannous
chloride solution As T is added and the general test is applied. No visible stain is
produced.
Arsenic Solution Dilute As T
Strong arsenic solution As T ................... 1 ml.
Water, sufficient to produce................. 100 ml.
Dilute Arsenic solution As T must be freshly prepared. 1 ml contains 0.01 mg of arsenic,
As T.
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Arsenic Solution Strong As T
Arsenic trioxide....................................0.132 g.
in
Hydrochloric acid................................... 50 ml,
Water, sufficient to produce................. 100 ml.
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Brominated Hydrochloric Acid As T
Bromine solution As T............................. 1 ml.
Hydrochloric acid As T........................ 100 ml.
- 37 -
Bromine Solution As T
Bromine.................................................... 30 g.
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Water sufficient to produce................. 100ml.
in
Potassium bromide ....................................30 g;
General method of limiting test of arsenic
Preparation:
The solution of substances (simple organic compounds and many inorganic acids
and salts) which are soluble is prepared with water and stannated HCL As T.
If the substances to be examined have been insoluble (e.g., BaS04, bentonite or
kaolins), they are diffused in water.
The solution of substances, such as metallic carbonates, which effervesce with
acids, is obtained with brominated HCL As T. The I.P. provides the methods of preparing
the solution of most of the substances which are needed to be tested for arsenic impurity.
in
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Special reagents needed or making solutions are provided in I.P.
Procedure for test for Arsenic
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The glass tube is first of all lightly packed with cotton wool, which is previously
moistened with solution of lead acetate and dried (because if impuirity of H2S is present
it will be trapped by lead acetate present in cotton, which otherwise itself would stain the
mercuric chloride paper). The cotton has been so arranged in the tube that the upper
surface of the cotton should not be less than 2.5 cm below the top of the tube and has
been lightly packed to allow the gas quite efficiently.
The upper end of the tube has been then inserted into the narrow end of one of the
- 38 -
bungs and the two bungs secured by using the rubber band after keeping the mercuric
chloride paper in between them as described above.
The solution to be examined and prepared as specified usually 5 ml from 1 g of
in
the substance is kept in the wide mouthed bottle. To this 1 g of KI As T (5 ml of KI), 5
ml of stannous chloride acid solution and 10 g of zinc As T are added. The glass tube is
kept in position quickly. The action is allowed to continue for 40 minutes. A yellow stain
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which gets produced on the HgCl2 paper, if arsenic is present, has been compared by
daylight with the standard stains which are produced by operating in a similar manner
with known quantities of dilute arsenic solution As T. The most suitable temperature for
running the test is generally about 40°C. However, the action may be hastened by
keeping the glass bottle on warm surface.
The comparison of the stains is done immediately at the completion of the test and
the standard stains used for comparison are prepared freshly as they start fading on
keeping. As a matter of fact both these tests should be carried out simultaneously.
Standard Stains: Standard stains are produced by using dilute arsenic solution.
Arsenic solution strong As T is having 0.132 g of As2O3 per 100 ml of solution. One ml
of the above solution is diluted with water to make 100 ml of dilute arsenic solution, one
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ml of which would be having 0.0000lg of As. A Stain equivalent to I ml of the dilute
arsenic solution produced by operating on 10 g of the substance would, therefore, show
in
that the proportion of arsenic has been 1 part per million (0.00001 g/10 = 1 x 10-6). It is
suggested that only such an amount of the substance should be taken as will be having a
maximum of 0.00002 g.
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If the sample under investigation shows a stain of lesser intensity than that of the
standard, then it passes the test.
Instead of mercuric chloride paper other methods can be used provided gas passes
through the paper which has been protected from sunlight in the course of the test.
The stain produced on paper fades on keeping, and therefore comparison should
- 39 -
be done immediately. Stained papers can be preserved by dipping in hot melted paraffin,
and keeping away from light. In order to get reproducibility of the results, it is essential to
in
follow the directions given in pharmacopoeias.
Important Points:
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(1) Lead acetate pledger or papers are used to trap any hydrogen sulphide which
may be evolved along with arsine.
(2) Stannous chloride is essential for the complete evolution of arsine. In the
Arsenic test, preference is given to stannous salts because they reduce arsenic to
arsenious state and sometime to metallic state whereas cadmium salts in themselves are
not reducing agents.
(3) Care must be taken that the mercuric chloride paper remains quite dry during
the test.
(4) The most suitable temperature for runiimg the test is generally about 40°C.
(5) The tube must be washed with hydrochloric acid As T, rinsed with Water and
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dried between succeeding tests.
The British pharmacopoeial method has been more or less similar to I.P. method.
in
However, the apparatus and design have been some what different. The amounts of zinc,
hydrochloric acid and other reagents used are also different. Further B.P. adopts the use
of mercuric bromide test paper. The Gutzeit test for arsenic has been very sensitive and
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hence has been adopted by pharmacopoeias of various countries.
- 40 -
Modification of the general method of limit test of arsenic for the
different compounds
Modification of the general method of testing is done for certain substances. It is
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substances, ions etc. gets eliminated by preliminary treatment.
in
to have arsenic in the final solution, in a readily reducible form, the interference of other
It is illustrated by the following examples:
(i) For the carbonates, hydroxides and oxides. When carbonates are treated with
hydrochloric acid, effervescence takes place, heat is generated and some hydrochloric
acid gas is evolved along with carbon dioxide. Oxides and hydroxides react similarly on
treatment with hydrochloric acid though carbon dioxide is not evolved. Arsenious
chloride (AsCl3) so formed volatilises with hydrochloric acid and if samples of AsCl3 are
dissolved in HCL, a part of arsenic is likely to get lost. Therefore, carbonates, oxides and
hydroxides are first treated with excess of brominated hydrochloric acid; the bromine
oxidizes arsenious divalent arsenic to the pentavalent form which is not volatile with
hydrochloric acid. In order to complete the test the arsenic should again be converted tc
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arsenious state. This is carried out by removing excess of bromine with a few drops of
stannous chloride solution which is able to reduce arsenic to the arsenious state.
in
(ii) Organic compound. There are many organic compounds which are insoluble
in acid and in water. Consequently when gas is evolved in the liquid having such
substances in suspension, frothing takes place, therefore any interfering organic matter
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should be removed by igniting with anhydrous sodium carbonate or calcium hydroxide
before carrying out the test. The ignited mixture is treated in the same way as carbonates.
Carbon particles should not be filtered out otherwise some arsenic may get lost.
Salicylic acid, benzoic acid, bismuth sub-gallate etc. are prepared in stannated
hydrochloric acid.
- 41 -
There are many other organic compounds and foods which get destroyed by wet
oxidation for estimation of arsenic in them. The substance should be treated with HNO3,
and then heated until vigorous reaction stops. The solution should then be cooled and
sulphuric acid added to it. The mixture should be again heated until it gets darkened. As
in
the mixture starts to darken more nitric acid should be added and the solution heated until
white fumes are evolved and a pale yellow or colourless liquid remains behind. The
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solution should then be cooled, diluted with water and evaporated until white fumes are
given off. The solution must then be distilled after treatment with stannated hydrochloric
acid and stannous chloride. The distillate must be treated with bromine, excess bromine
removed and the general test performed.
(iii) Nitric Acid and Nitrates. It is possible to remove nitrates and nitric acid by
heating the substance with cone. sulphuric acid. Arsenic remains behind in the sulphuric
acid. It is possible to remove the last traces of nitric acid by diluting residual sulphuric
acid with a little water and evaporating a second time. The second treatment decomposes
the nitrosyl sulphuric acid formed during first evaporation thereby freeing the sulphuric
acid entirely from nitric acid. Solution of ferric chloride is also having some nitric acid.
Hence it has to be treated similarly.
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(iv) Boric Acid and Borax. Boric acid is only sparingly soluble in hydrochloric
acid but it is known to form a visible compound with citric acid. Borax yields boric acid
on treatment with hydrochloric acid. Hence citric acid should be used for dissolving boric
in
acid and borax before addition of stannated hydrochloric acid.
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(v) Solutions containing free Ammonia. Solutions containing free ammonia are
evaporated on water bath till most of the ammonia gets evolved. Arsenic is left behind as
ammonium arsenite and arsenate because both of these salts are not volatile at the
temperature of water bath. Later the solutions have been acidified with brominated
hydrochloric acid, and stannous chloride added.
(vi) Liquid Glucose and Potassium Acid Tartrate. Liquid Glucose is;i having
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some sulphur dioxide and potassium tartrate is having traces of sulphites. These should
be treated with brominated hydrochloric acid to oxidize the sulphurous acid before
running the test. Excess bromine must be removed by adding a few drops of stannous
in
chloride solution.
(vii) Hypophosphorus Acid. When hypophosphorus acid is reduced it forms
phosphine which interacts with mercuric halide papers, thereby interfering with the test.
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Therefore, it gets oxidized to phosphoric acid by treatment with potassium chlorate and
hydrochloric acid. Excess chlorine must be expelled by boiling and last traces could be
removed by treatment with stannous chloride.
(viii) Powerful Oxidising Agents. Powerful oxidising agents like potassium
chlorate should be suitably reduced, otherwise, whole of the hydrogen will get used up in
reducing the substances under examination and arsine would not get liberated.
(ix) Compounds of Copper, Bismuth, Antimony and Iron. Cooper deposits on zinc
forming a zinc-copper couple which prevents steady evolution of hydrogen and all the
arsenic does not get converted into arsine. Bismuth deposits on zinc forming a sponge
like mass stopping the evolution of hydrogen. Antimony compounds are forming
antimony hydride in presence of nascent hydrogen which produces a dark stain on
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mercuric halide paper masking the stain due to arsine. Iron reduces the rate of evolution
of hydrogen and hence much of arsenic docs not get reduced to arsine.
in
Arsenic in the arsenious stale has been volatile with strong hydrochloric acid.
Antimonous chloride too has been volatile but the chlorides of copper, bismuth and iron
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are not. 20 per cent hydrochloric acid distills over unchanged in strength.
Therefore, copper, bismuth and iron salts are put in 20 per cent HCL (stannated)
and distilled. Whole of the arsenic present in the salts comes over in the first 75 per cent
of the distillate. In case of ferric salts, enough stannous chloride has been added to reduce
the salts to ferrous state.
A double distillation becomes necessary to run the test on antimony compounds
- 43 -
because antimony is slightly volatile in hydrochloric acid. In the second distillation,
amount of antimony trichloride passing over will be too small to interfere with the test.
If sulphates of the metals are subjected to distillation with hydrochloric acid and
in
stannous chloride, part of the sulphuric acid gets reduced to sulphurous acid which passes
over in the distillate, and part of it is likely to get further reduced to H 2S in the test.
Hence distillate of a solution having sulphate or sulphuric acid should be treated with
chloride.
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brominated hydrochloric acid and the excess bromine again reduced with stannous
In order to run the tests on metallic iron or bismuth, the metals are dissolved in a
mixture of potassium chlorate and hydrochloric acid causing the formation of chlorine
dioxide which dissolves the metals as chlorides. If iron and bismuth are dissolved in
hydrochloric acid as such, hydrogen gets evolved and part of arsenic may get lost.
Potassium chlorate oxidizes the hydrogen liberated preventing loss of arsenic. The
mixture should then be boiled to get rid of excess chlorine and the test is run.
(x) Sulfur. Arsenic present in sulfur has been in the form of arsenic sulfide.
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Arsenic sulphide is soluble in ammonium sulfide and ammonia. Sulfur should, therefore,
be digested with ammonia whereby ammonium polysulfides get produced in which
arsenic sulphide is soluble. Thereupon, the mixture must be filtered, the undissolved
in
sulfur separated and the filtrate evaporated to dryness. Dried mass having whole of
arsenic and little sulphur is treated with anhydrous sodium carbonate and water,
whereupon sodium polysulfide is formed in which arsenic sulfide is soluble. The solution
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is then boiled and bromine added to it to oxidize sulfide to sulfate and arsenic to arsenate.
The solution is acidified, boiled and treated with stannous chloride and then subjected to
the general limit test.
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in
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CHEPTER-3
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REFERENCE
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(3) REFERENCE
1. Chatwal G. R., Pharmaceutical inorganic chemistry volume-I, Third edition, Himalaya
in
Publication, Mumbai-35, 2006, pp 103-108.
2. Rajasekaran V.N., Textbook of pharmaceutical inorganic chemistry, Sixth edition, Sun
Publication, Chennai-31, pp 80-96.
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c.
3. Qadry J.S. & Qadry S.Z., Textbook of pharmaceutical & medicinal chemistry, Seventh
edition, B. S. Shah Prakashan, Ahemedabad-38007, 2005-2006, pp 3-14.
4. Pandeya Surendra N., Text book of inorganic & medicinal chemistry, Second edition,
SG Publisher, Varanasi, 2001, pp 423-426.
5. Bhandari Anil, Sing G. K., Pharmaceutical chemistry volume-I, First edition, CBS
Publisher, New Dilhi-110032, 1998, pp 6-11.
6. Prakash Satya, Tuli G.D., Basu S. K., Madan R. D., Advanced inorganic chemistry,
19th edition, S. Chand Publication, New Dilhi-110055, 2005-06, pp 504-510.
7. Siddiqui Anees A., Wani Sachin M., Pharmaceutical inorganic chemistry, Second
edition, Birla Publication, Dilhi-110032, 2005-06, pp 5-15.
8.Rao Gundu P., Inorganic pharmaceutical chemistry, Second edition, Vallabh prakashan,
New Dilhi-110034, 1993, pp 173-182.
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9. Siddiqui Anees A., Siddiqui Seemi, Advanced pharmaceutical chemistry, 1st edition,
CBS Publisher, New Dilhi-110032, 2005, pp 82-84.
10. Mahadik K.R. & Kuchekar B.S., Concise pharmaceutical inorganic chemistry, 2nd
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u.
in
edition, Nirali Prakashan, Pune-411002, 1995, pp164-183.