LIMIT TESTS

By,
CHAITHRA R SHETTY
Department of Ph.chemistry

Limit tests:
 Tests being used to identify the impurity.
 Tests being used to control the impurity.


Definition: Limit tests are quantitative or semi quantitative

test designed to identify and control small quantities of

impurities which are likely to be present in the substances.

Types:
 Tests in which there is no visible reaction
 Comparison methods
 Quantitative determinations
1.Tests in which there is no visible reaction :

This means there shall be no colour, opalescence or precipitate under the experimental
conditions.


Eg: Test for barium and calcium in dilute hypophosphorus acid where the addition of
dil.H2SO4 under precisely controlled condition shall produce “no turbidity’ or ‘precipitate”
within one hour.


If they give negative results, donot necessarily imply the complete absence of the impurity.


The test indicates the absence of an undesirably large amount of impurity.
2. Comparison methods:

These types of tests require a standard containing a definite amt. of impurity.


At the same time and same experimental conditions, test sample is prepared and experiment is
performed by direct comparison of test solution with std of known concentration.

Eg: limit tests for chlorides, sulphates and heavy metals.

3. Quantitative determination

These methods are applied only in special circumstances.


Usually in those cases, where the limit of impurities is not readily susceptible to simple and more
direct chemical determination.


Different types tests are,

Limit tests of insoluble matter

Limit tests of soluble matter

Limit tests of moisture, volatile matter etc.,

 Limit test for Chloride

The test is used to limit the amount of Chloride as an impurity in inorganic substances.


Chloride is one of the main impurity present in the pharmaceutical substances.


The main sources- tap water, HCl, NaCl etc- used during the process of pharmaceutical preparations.


Excessive chloride ions- may cause incompatibility in pharmaceutical preparations and may produce
toxic effects.


So it is essential to keep control over the limits of chlorides, and I.P prescribes chloride limit test for
almost all pharmaceuticals.
Principle:

Dil.HNO3

Std. solution – 1ml contains 0.05845%w/v of NaCl.

If the opalescence produced from the sample is less than std, and it passes the limit test (pure) and vice versa.

PROCEDURE
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 as Dilute HNO3 is insoluble in AgCl.

It also prevents the precipitation of other acid radicals with AgNO3.


Pharmacopoeia does not prescribe any numerical value of limit test for chlorides, sulphate and iron
because limit test is based on the simple comparison of opalescence or colour between the test and
standard solution prescribed according to pharmacopoeia.


In this type of limit test, the extent of turbidity or opalescence or colour produced in influenced by the
presence of other impurities present in the substance and also by variation in time and method of
performance of test.


Thus the pharmacopoeia does not prescribe any numerical value of the limit test.
 Limit test for Sulphates
The Sulfate Limit Test is designed to determine the allowable limit of sulfate contained in a sample.

Extent of turbidity depends on the concentration of sulphate ion in the sample.


Std. solution – 1ml contains 0.1089%w/v of K2SO4


If the turbidity produced from the sample is less than std, and it passes the limit test (pure) and vice
versa.

Uses of HCl:

Makes the solution acidic, added to prevent ppt of other acid radicals in BaCl2 solution in presence of
HCl, only sulphate precipitates.
The barium chloride test solution in the I.P has been replaced by Barium sulphate reagent,

Barium sulphate reagent: It consists of BaCl2, small amt of K2SO4 and sulphate free alcohol.

1. BaCl2: acts as seeding agent for the ppt of sulphates as BaSO4.

2. K2SO4: presence of very small proportion of K2SO4 increases sensitivity of the test by giving ionic
conc. in the reagent. (Its ionic conc. is adjusted in such a way that it also increases the solubility of
BaSO4)

3. Alcohol: It prevents the supersaturation and thus produces uniform ppt of BaSO4 in the form of
turbidity.

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.
LIMIT TEST FOR IRON

Thioglycolic acid performs following 2 functions:
I. Iron impurities may be present in either trivalent ferric (Fe 3+) or divalent ferrous
(Fe2+) form. If it is present in Fe3+ form, then thioglycolic acid reduces it to ferrous
form.

II. TGA- then reacts with ferrous form of iron in the ammonical alkaline medium to
produce purple coloured ferrous thioglycolate.

Citric acid
Citric acid prevents the precipitation of iron with NH3 (as ferrous hydroxide). It keeps
iron in solution form even in presence of ammonia, by forming a soluble complex of
iron citrate or ferrous citrate.


Ammonia
Helps in producing purple colour, because ferrous thioglycolate complex gives purple
colour only in alkaline media.

The intensity of purple colour produced in test solution is compared with that of
standard.


If the intensity of purple colour produced in the test solution is less than that of
standard, then it passes the limit test and vice versa.
Limit tests for Heavy metals:

IP prescribes limit test for heavy metals in drugs. These heavy metals may be bismuth, tin,
cobalt, manganese, antimony, mercury, chromium, cadmium, selenium, lead and arsenic etc.,

Their quantity is expressed in terms of lead i.e parts per weight of lead in 1 million parts by
weight of the substance

(expressed as the quantity of lead required to produce a colour of equal depth as standard-
having definite quantity of lead nitrate)

I.P limit for heavy metals in 20 ppm.

The latest I.P prescribes 3 methods for the limit test of heavy metals:

Method A: It is for colourless substances/solutions,

Principle: It is based on the reaction between hydrogen sulphide and heavy metals in an
acidic medium to produce the corresponding heavy metal sulphides.

These remain distributed in a colloidal state and produce brownish or blackish colouration.

The test solution is compared with a standard prepared using a lead solution.

Acidic
medium
Heavy metals+ H2S Acetic Heavy metal sulphides(black/brown colour)
acid/NH3
Procedure
Standard Test
Ppt out 2ml of std. lead solution Weigh specific amount of substance
Add 30-35ml 0f D.W
Adjust pH 3-4 either by adding acetic
acid/ NH3 solution

Add 10ml of freshly prepared

saturated solution of H2S.

Make upto 50ml with D.W

Compare the brown colour produced

Method B: for coloured substances.


It is similar to method A except in this case the coloured substances is given special treatment.

Test solution is prepared by weighing specified quantity of substance in crucible.
 Moisten the substance with H2SO4

Ignite it on a low flame till completely charred.

Add few drops of nitric acid and heat to 5000C.

Allow to cool, add 4ml of HCl and evaporate to dryness.

Moisten with HCl and keep for 2 min, neutralize with ammonia solution.

Make just acidic with acetic acid (pH- 3-4).
 35ml DW and 10ml H2S

Make upto 50ml.
Method C: It is used for both substances which form clear and colourless solution
with NaOH.

The test by this method is based upon the reaction between Na2S and heavy metals in an
alkaline medium to produce corresponding heavy metal sulphides.

This results in production of colour----compared in Nessler cylinder.

Alkaline
Heavy metals + Na2S medium Heavy metal sulphides (brown/black)
Procedure

Standard Test
Ppt out 2ml of std. lead solution Weigh specific amount of substance
Add 30-35ml 0f D.W
5ml of dl.NaOH

Make upto 50ml with D.W

Add 5 drops of Na2S solution.

Compare
Limit tests for Lead

 Lead has been one of the most undesirable impurities in medicinal substances.

 The chief source of this impurity in pharmaceutical substance is due to its presence in
sulphuric acid and in the apparatus which are used for their manufacture.

 Glass bottles used for storage of chemicals increases the lead impurities in
pharmaceutical substance.

 There are two methods for the limit tests of lead,

1. Limit test for lead as per B.P

 It mainly depends on the reaction between lead salts with Na2S, which
gives a colloidal brownish black ppt due to the formation of PbS.
 Dilute solutions of Pb produce brown colouration with Na2S.

Intensity of colour depends upon the amount of lead present in the
sample.
Dil.lead + Na2S PbS+ sodium salt
Pb(NO3)2 + Na2S PbS+ 2NaNO3

Other metals such as copper, iron also produce black or brownish colouration
with Na2S, which would interfere with this test.

 This difficulty can overcome by the addition of NH3 and KCN, which forms
the complex with CN and thus prevent the ppt of copper and Fe as sulphides.


The intensity of brown colour produced in the test is compared with the std.


The limit test is carried out in Nessler’s cylinder and all the reagents which
are used in this are must completely free from lead.

For this limit test following 2 solutions are prepared,

a. Primary solution (test)- it contains definite but greater amount of test sample than
the auxiliary.

b. Auxiliary solution (std)- it contains very small quantity of test sample and specified
volume of std lead solution, prepared from PbNO3.

The primary solution is taken in one Nessler's cylinder and the auxiliary solutions in another.

 Excess of NH3 and small quantity of KCN are then added to each cylinder.


At this stage solution in 2 nesseler’s cylinders are examined for any change or difference in
colour.


If there is any change/difference in colour is removed by the addition of Burnt sugar( to clear
colour variation).


Volume made upto 50ml– with D.W

 2 drops of Na2S solution is added to each and mixed.


The colour produced in the test is compared with standard.

For calculation, quantity of the test sample in the auxiliary solution is
subtracted from that in the primary solution


Eg: 12g of test sample taken in primary solution, 2g of substance is taken
in auxiliary solution, then test is regarded as 12-2=10g of the test sample.


The reason for having small qty of substance in auxiliary solution is to
enable the comparison to be made under, as comparable conditions as
possible.
 Standard Test
Procedure
Weight accurately 2g of NH4Cl Weight accurately 12g of
and transfer it to a Nessler’s NH4Cl and transfer it to a
cylinder labelled as ‘S’ Nessler’s cylinder labelled as
‘T’
Dissolve it in hot distilled
water containing 5ml acetic
acid
Add 5ml of dil.Pb solution -

Make alkaline with NH3

Add 1ml of KCN solution

 Compare the intensity of color in 2 Nessler’s cylinder.
 Incase there is any noticeable diff. in color, it should removed by adding a very dilute
solution of burnt sugar.
 Make up the solution up to 50ml with D.W

 Add 2 drops of Na2S solution.

 Stir with glass rod and compare intensity of colour produced in test with that of std.
 pH 3-4 is necessary for the precipitation of PbS. So aq. Ammonia / acetic acid is
added to maintain that pH.
2. IP or USP method:

Principle: Based on the reaction between lead impurities and dithizone (diphenyl
thiocarbazone) which result in the formation of lead dithizonate complex(violet colour).
 Dithizone is soluble in chloroform.

 Dithizone in chloroform, is able to extract lead from alkaline aqueous solutions as a lead dithizone
complex (Red in colour)

 The original dithizone is having a green colour in chloroform while the lead dithizone is having a red
color. However the miscible of these two appears as violet at the end of the reaction.

  To avoid interference by other metals and make the pH optimum, reagents like ammonium citrate,
KCN, and NH₂OH.HCI is employed. Phenol red is used as an indicator to develop colour at the end of
the process.

 This limit test is not carried out in Nessler’s cylinder, double extraction method is followed here with
the help of separating funnel, where Dithizone-chloroform solution is the extraction solution.
 The intensity of the colour of complex, is depends on the amount of lead
in the solution. The colour produced in sample solution should not be
greater than standard solution. If colour produces in sample solution is
less than the standard solution, the sample will pass the limit test of lead
and vice versa.
Limit test for Arsenic:

Arsenic is a well known undesirable and harmful impurity which is present in medicinal substances.


If it is present even in traces, it is not desirable because it is toxic and cumulative.


All pharmacopoeias prescribes a limit test for it, to detect the presence of arsenic as an impurity in
various pharmaceutical substances.


There are many qualitative and quantitative test for arsenic are known, However the pharmacopeial
method is based on Gutzeit test.


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 the test for
Arsenic.
PRINCIPLE:

Limit test of Arsenic is based on the reaction of arsine gas with hydrogen to form yellow stain on
mercuric chloride paper in presence of reducing agents like potassium iodide.


Arsenic impurity can be present in pharmaceutical substance in 2 valency states, as As5+ and As3+.


When the sample is dissolved in an acid, As impurities will get converted to Arsenic acid or Arsanious

acid depending upon the valency states of As present in pharmaceutical substance.

 This solution when treated with a reducing agent ( KI/stannous chloride in IP or BP and H 2SO4 in USP)

to convert As5+ acid to trivalent Arsenious acid.


The arsenious acid is then converted to arsenious hydride (arsine gas) with the help of
nascent hydrogen which is produced by Zn and HCl.

 Arsine gas is carried through the tube by the steam of H2 and out through the mercuric
chloride paper.


Reaction between Arsine gas and mercuric chloride will result in formation of mercuric
arsenide which gives yellow or brown stain on mercuric chloride paper.


The intensity of the color is proportional to the quantity of Arsenic.
Apparatus:

It consists of a wide mouth glass bottle fitted with rubber bung, through which it passes
a glass tube (length 200mm, outer diameter-8mm, inner diameter-6.5mm)


This tube is open at its upper end- but at the lower end it is narrow out through a smaller
diameter(tip aperture 1mm).


A hole is blown at the side at its lower end where it starts getting narrower(d=2mm).


The purpose of this narrowing is to provide a small aperture at the lower end for smooth
and slow passage of arsine gas from bottle to the tube.


The purpose of side hole is to provide an alternate passage for arsine gas if some water
condenses at he narrow lower end.

The glass tube is tightly packed with cotton wool, which has been previously moistened
with lead acetate and dried.

 The purpose is to trap any H2S, which would otherwise interfere with this test as it also

given some stain HgCl2 paper.


The tube is fitted at its upper end with 2 rubber bungs as shown in the diagram.

 Piece of dry HgCl2 paper is placed flat on the top of the bung and other bung is placed
over it and secured by means of clips in a such a manner that borings of the 2 rubber
bungs meet to form a true tube of the same diameter(6.5mm) interrupted by a diaphragm
of mercuric chloride paper.
Method:

The solution of test sample is prepared in the acid or SnCl 2 as specified in IP or BP.

It is placed in wide mouth glass bottle.

To this 1g of KI and 10g of Zn are added.

The glass tube with its other fittings is placed quickly in position in the bottle and the
reaction is allowed to proceed for 40 min.

All the reagents used in this limit test should be free from arsenic impurities and they are
labelled as AsT.

The yellow stain is produced on HgCl2 paper if As impurities is present.


It is compared with the standard stain by doing the test separately in similar manner with known
quantity of As solution.


This solution contains 1.32mg of arsenic trioxide in 100ml i.e 1mg of As in 100ml.

Notes:

This test is the modification of Gutzeit test and therefore called as modified Gutzeit test – original gutzeit
test was quantitative one, liberated arsine gas is detected by covering the mouth of the flask with filter
paper on which small amount of silver nitrate crystals were placed.

The liberated arsine turns silver nitrate complex - black


KI is used because it helps in the reduction of As5+ (arsenic acid) into trivalent arsenious acid.


KI is first converted into HI acid which helps in this reduction process.

 Granulated zinc is used instead of ordinary Zn because evolution of nascent H2 is steady and
prolonged in granulated zinc.

 If the evolution of H2 gas is very slow- reaction is very slow.


The apparatus should be warmed to 400C by placing these in a warm place or in a water
bath at 400C.


The As impurities is expressed in ppm- 1 ppm is 1mg/kg or 1µg/g.
 std test
Procedure:
Pipette 1ml of std. solution of As. Weigh the specified quantity of
substance and transfer it to another
bottle labelled as Test
Add 50ml of distilled water

Add 10ml of stannated HCl

Add 1g of KI

Add 10g of granulated zinc.

Place a prepared glass tube quickly in position in both the bottles and allow the reaction
to proceed for 40 min. Compare the yellow stain produced in test with that of std.
Modifications/special procedure for limit test for chloride and sulphate:

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 filtrate is
used for the test, because the presence of insoluble substances 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 carbonized and so
produced ash is extracted in water.

e. Deeply coloued substances: have to be decolorized before test. Eg : KMnO4 is reduced

by boiling with alcohol and the filtrate is used.

f. Reducing substances: like hypo phosphorous 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.
THANK YOU