limit test.pdf..........................

limit test

1. Unit-v
Limit tests
1

2. Introduction
Definition and importance:
Limit test
is a quantitative or semi quantitative test.
It is designed to identifyand control
small quantities of impurity
which is
likely to be present in the pharmaceutical substanc e.
Impurityis undesirable foreign material that is normally pr esent in the
pharmaceutical substances.
Analytically 100% pure substances are not available .
So, drug regulatory authorities provide permissible limit of a impurity and
designate the pharmaceutical substance as standard provided it complies the
tests given under individual monographs.
2

3. Sources of impurities
•Impurities are imparted into the pharmaceutical sub stance through raw
materials, intermediates, reagents, catalysts, solv ents, reaction vessels,
improper storage, cross-contamination, microbial co ntamination, chemical
instability, storage containers etc.
•Wateris a rich source for chlorides, sulphates, carbonat es etc.
•Reactor materialsused for manufacturing are rich source of steel, co pper,
iron, zinc, lead.
•Reagents & catalysts are rich sources of arsenic, antimony, heavy metals ,
lead, cadmium, mercury, which are potent nerve pois ons on cumulative
accumulation.
3

4. Effects of impurities in pharmaceutical
•Some have toxic effects on body when present beyond certain limits. e.gLead
and Arsenic salts.
•May cause cumulative toxic effect,
•Make substance incompatible with other substances
•Affect the storage property of the pharmaceuticals if present beyond the limit.
•Decrease in shelf-life.
•Lower the active strength of the medicinal compound. E.gNa-salt in K -salt.
•May bring about technical difficulties in the use o f the substance. Eg.Alter test,
change in odor, color, taste and appearance
4

5. Importance of Limit tests:
To identify
the impurities present in the substance and
compare it
with standard.
To find out the harmful amount of impurities
Thequantityofany impurityinanofficialsubstanceisoftensmallandconsequently,
thevisibleresponse toanytestforthatimpurityisalsosma ll.
Therefore,designofindividualtestsisimportanttoavoid errors.
Thisisaccomplishedbygivingattentionforthefollowingfactors:
•Factors affecting limit tests:
Specificity of the tests
Sensitivity
Control of personal errors (Analyst errors)
5

6. Specificity of the tests:
Any test used as limit test
must
give
some
form of
selective
reaction with the
trace impurity.
N.B. Highly specific tests are not always the best , a less specific (selective)
test which limit several likely impurities at once is obviously advantageous.
•Example: Contamination of Pb
2+
and other heavy metal impurities in Alum is
precipitated by thioacetamide as their respective s ulphidesat pH 3.5.
•High specificity is readily achieved with limit tes t based on TLC, GC and
HPLC (Separated impurities identified by their ch aracter)
6

7. Sensitivity
The degree of sensitivity required in a limit test varies enormously according
to standard of purity demanded by the monograph.
Sensitivity of a tests depends up on a number of fa ctors.
Example a sensitivity of Gravimetric Analysis depen ds on
Concentration of the solute and of the precipitatin g reagent,
Duration of reaction,
Reaction temperature,
Nature and concentration of another substance prese nt etc.
7

8. Control of personal errors
•It is essential to exclude all possible source of ambiguity in the description
of a test.
•Vague term, like ‘slight precipitate’ should be avo id as far as possible.
•The extent of visible reaction expected under the s pecified test condition
should be clearly and precisely defined.

9. Test for purity:
•Pharmacopoeia prescribes the “Test for purity” for pharmaceutical substances
to check their freedom from undesirable impurities.
•Pharmacopoeia will decide and fix the limit of tolerance for these impurities.
•For certain common impurities for which pharmacopoeia prescribes the test of
purity are:
•Ash value
•Loss on drying (moisture content)
•Heavy metal like Arsenic, lead, etc
•Non-metals like Chloride, Sulphate
9

10. I. The Ash Limit Tests
The ash values are designed to measure the
amount
of the
residual substances
when a sample is ignited under the conditions speci fied in the individual
monograph.
Ash values are helpful in determining the qualityand purityof the crude
drugs in powder form.
These values are categorized into fourheads, namely :
Ash Value (Total Ash).
Acid-Insoluble Ash.
Water-Soluble Ash.
Sulphated Ash.
10

11. 11
Crucible
Furnace
Desiccator
A.
Total
Ash
•The total ash is the residue remaining after incine ration.
•It usually represent the inorganic residue present in official herbal drugs and
pharmaceutical substances.
Procedure:
Ignite an empty cruciblein furnaceat 500 to 550
0
C for 30 min,
Allow to cool in a desiccatorfor 30minutes, and weigh it accurately.
Unless otherwise specified, place 2 to 3 g of the s ample in the crucible and
weigh it accurately.
Heat the crucible gently first, then raise the temp erature gradually,

12. 12
Weight of the
powder taken (g)
Weight of ash obtained (gm) % w/w total ash
Mean value ±
SD
3.0
0.32
3.0
0.339
3.0
0.292
3.0
0.284
3.0
0.294
3.0
0.266
10.67
11.30
9.73
9.47
9.80
8.87
Ignite at 500 to 550
0
C until it is white ash
Cool in a desiccator for 30min, and weigh accuratel y.
Then calculate the following formula and compare wi th the standard.
Unitn-i-vLnvm e
s1aq -nitnvm niu-vao1rn
s1aq -nitnmv.IL1n-vd1on
ngnwhh
Total ash represents inorganic salts (Ca-oxalate) o ccurring naturally in the
drug and inorganic matter from external sources.
So it is used to ensure the absence of an undue pro portion of extraneous
mineral or matter introduced accidentally.
9.97±0.36

13. Applications of total ash
•To ensure the absence of an undue proportion of extraneous minera l
matter introduced accidentally or at time of collection or in subsequent
treatment.
•To ensure the absence of other parts of the plant .
•To detect adulterationwith exhausted drug E.g. Ginger.
•To detect the adulteration with material containing cells or starch which
modify the ash values.

14. Weight of the
powder taken
(g)
Weight of total ash
(g)
Weight of acid
insoluble Ash
(gm)
Percentage w/w
AI ash
Mean value ±
SD
3.0 0.32 0.052
3.0 0.339 0.059
3.0 0.292 0.061
1.91±0.16
1.73
1.97
2.03
B.
Acid-insoluble ash
Is designed to measure the amount of ash insoluble in diluted HCl.
It consists primarily of sand andother siliceous matte .
Procedure:
Boil the
total ash
with 25 ml of diluted
HCl for 5 minutes.
•Filteredon an ash-less filter-paper
•Wash with hot water.
•Then
ignite
, cool in a desiccator, and weigh accurately.
•Then it was
calculated
as follows.
14
%
it
vcar
aomiLluL1
vm
=
k1aq -
it
y1marl1

s1aq -
it
mv.IL1
-vd1o

g
100

15. Weight of the
powder taken
(g)
Weight of
total ash (g)
Weight of
residue (gm)
Percentage w/w
WS ash
Mean value ±
SD
3.0 0.284 0.125
3.0 0.294 0.128
3.0 0.266 0.134
5.30
5.53
4.40
C.
Water soluble ash
5.07 ±0.49
The part of the total ash dissolved by water.
Applied only in specified case of an official drug( ginger)
Procedure Dissolve the total ash with 25 ml of
water
and boiled for 5 minutes.
Separate the insoluble residue by filteringon an ash-less filter-paper.
Subtract the weight of insoluble from the weight of total ash;
The difference weight represents the weight of wate r soluble ash
Calculate the % of water-soluble ash with reference to the air dried drug
%
it
kv-1y
miLluL1
vm
=
k1aq -nitnvm bk1aq -nitny1marl1
s1aq -
it
mv.IL1
-vd1o

g
100
15

16. D.
Sulfated ash
This test utilizes a procedure to measure the amoun t of residual substance
not volatilizedfrom a sample when the sample is ignited in the pre sence of
sulfuric acid.
The test is applied very widely to control the exte nt of contamination by
inorganic non volatile impurities in organic substances.
Ex. Aspirin, ligocain HCl
Used to control traces of alkali metals in chlorbut olresulting from method of
preparation (heating of acetone and chloroform in p resence of KOH).
16

17. Method
Take the amount of test sample specified in the ind ividual monograph in the
crucible and weigh the crucible accurately.
Moisten the sample with a small amount (usually 1 m L) of sulfuric acid, heat
gently at until the sample is thoroughly charred.
After cooling moisten the residue with a small amou nt (usually 1 mL) of
sulfuric acid TS, heat gently until white fumes are no longer evolved
Then ignite at 550 °C to 650 °C until the residue i s completely incinerated.
17

18. 18
Ensure that flames are not produced at any time dur ing the procedure.
Cool the crucible in a desiccator (silica gel or ot her suitable desiccant), weigh
accurately and calculate the percentage of residue.
UnitnplLtv-1rnvm e
k1aq -nitnvm
s1aq -nitnmv.IL1n-vd1on
ng
100

19. II. Limit on Moisture content
The presence of water plays an important role in th e physicaland chemical
stability of the AI, and pharmaceutical preparation s.
•It may lead to their degradation.
•It ma provides a favorable environment for microbialgrowth.
•Therefore, the presence of water in the pharmaceuti cal substances affects:
»Qualityof the product
»Storageof the product
»Efficacyof the product
There are different methods which used for determin ations of moisture
Los on Drying Method, Karl fisher titration, Azeotr opic Distillation
19

20. A. Loss on drying method
The loss on Drying test is a method to measure the loss in mass of the sample,
when dried under the conditions specified in the mo nograph.
This method is applied to determine the amount of water and volatile mater in
the sample, which is removed during the drying.
The amount of heatto which the substance is subjected varies considerably
according to the
nature of the substance
i.e -Sufficient to produce required effect in read on ti me
-Not so high as to cause decomposition
If the substance is thermo stable the test is usually applied by drying to
constant weight at 105
o
C
20

21. Example of monograph description
•“not more than 1% (1 g, 105
0
C, 4 hours)”
Indicate that mass is not more than 10 mg per 1 g o f the substance in the
test in which about 1 g of the substance is accurat ely weighed and dried at
105
0
c for 4 hour.
•“not more than 0.5% (1 g, in vacuum, phosphorus (v) oxide, 1 hours)”
Indicate that mass is not more than 5 mg per 1 g of the substance in the
test in which about 1 g of the substance is accurat ely weighed and
transferred in to a desiccator (phosphorus (v) oxid e ) and dried in vacuum
4 for hour.

22. Procedure
•Takespecified amount of the drug in to a
crucible
.
•Dry in an
oven at 105 °C
until two successive weights get constant.
22
•Calculateusing the following formula. •Eg.Three samples of 5g of a drug that is powdered w as dried in oven.
•After drying 4.300, 4.253 and 4.284 g were obtaine d.
•Calculate mean moisture content of this drug.
•If a limitfor this drug is 14% as per pharmacopoeia , can we accept the drug?
Ans= 14.42%

23. B. Karl fisher titration method
Karl Fischer Titration Method is designed to determ ine water content in a
substance, utilizing the quantitative reaction of waterwith Sulphur dioxide
and iodinein the presence of alcohol.
It is specific method in which only water will be determined.
The method is rapid(a few minutes) and determine both surface water (water
on crystals) and the water contained insidethem.
It works over a wide concentration range from ppm up to 100% and supplies
reproducible and correct results.
The titration can be performed volumetricallyor coulometrically.
23

24. Basic ingredients of KF reagents
Iodine
-------------------- I
2
Base
(
Buffer)
--------Imidazole
Sulphur dioxide
--------SO
2
Solvent
----------------Methanol
The reaction of KFR
SO
2
+ CH
3
OH + RN ⇌[RNH]SO
3
CH
3
RN-base
alkylsulfite salt
•This intermediate alkyl sulfite salt is then oxidized by
iodine
to an alkyl
sulfate salt.
•The reaction consumes H
2
O.
H
2
O+I
2
+ [RNH]SO
3
CH
3
+2RN ⇌[RNH]SO
4
CH
3
+ 2[RNH]I
alkylsulfite salt
alkylsulfate salt hydroiodic acid salt
24

25. An endpoint is reached when all the water is consum ed.
The reaction rate depends on pH value for KF.
Its ideal pH range is 5-7.5
Above 8
the
reagent decompose
so the titration end point may not be found.
At low pH the reaction is very slow and even the rxnis not take place at pH=1
Note: Both pyridine and methanol should be anhydrous.
25

26. The following precautions must be observed rigidly using the Karl Fischer
reagent, namely:
Always prepare the reagent a day or two before it i s to be used,
Great care must be taken to prevent any possible co ntamination of the
reagent or the sample by atmospheric moisture,
All glassware(s) must be thoroughly driedbefore use,
Standard solution should be stored out of contact w ith air,
Essential to minimize contact between the atmospher e and the solution
during the course of titration.
Stability of the Reagent:

27. Volumetric titration Method
Iodine required for reaction with water is previous ly dissolved in water
determination TSand form Karl Fischer reagent.
Karl Fischer reagent consists of known iodine conce ntration, excess sulfur
dioxide, base and solvent, such as alcohol.
A Karl Fischer reagent is added from a burette.
Water content is determined based on volume of Karl Fischer reagent or by
measuring the amount of iodine consumed as a result of reaction with water
in a sample
27

28. How does a Volumetric Titrator work?
•The volumetric titrator performs the following thre e key functions:
1. It dispenses KF titrating reagent containing iodine into the cell using the
burette
2. It detects the endpoint of the titration using the double platinum pin
indicator electrode.
•During titration when excess H
2
O is available there is High voltage b/n
the Pt wire.
•At the end of titration voltage decrease sharply as all water consume.
3. It calculates the end result based on the volume of KF reagent dispensed
using the onboard microprocessor.
28

29. 29
Method for Karl Fischer titration
•It involve two steps
1. Standardization of the reagent (titer determinati on)
2. Sample water determination
Step 1: Standardization
•Fill methanol in the titration flask to cover the e lectrodes
•Quickly add a water standard, that is accurately we ighed and containing 20 to
25 mg of water in to the titration flask
•Stir and titrate until the end point
•Then calculate a water equivalency factor (F) in mg of water per ml of reagent,
by the formula: F=w/v

30. F=W/V
•W= weight in mg of water contained in aliquot of st andard used.
•V= the volume in ml of reagent used in the titratio n.
•A KFR may state water equivalency factor like “Hydr nalcomposite 5”
•This means 1ml KF solution is equvalentto approx. 5 mg water.
•But because the reagent deteriorate gradually, titr ant standardization is
necessary before use.
Step 2: Sample water determination
•After determining F, quickly transfer the test prep aration in to the titration
flask and stir.
•Then titrate the solution with KFR to the end point .
30

31. •Calculate a water content in the sample in mg by mu ltiplying volume (ml) of
KFR consumed with water equivalency factor (F).
W=VF
•Then water content in percent is calculated
Unkv-1yncio-1o-nitnmv.IL1 e
k1aq -nitnkv-1ynA.q0
k1aq -nitnmv.IL1n
.q
gwhh
•Eg.If 0.0244g of standard water consume 5.249ml of KFR on titration and a
sample of lidocaine HCl that is 0.1564g consume 2.0 94ml KFR. What is the
percentage concentration of water in this product? If the limit test for
lidocaine HCl is between 5 to 7% can this product p ass the test?
31

32. •Sln: F=w/v24.4mg/5.249ml= 4.6485mg/ml
•Wtof water in lidocaine= VxF2.094mlx4.6485mg/ml= 9.73mg
•Unitnkv-1ynen.vmmnit
!"#$%
&'()*"'+$
X100
,../0
123.40
x100 =6.22%
•Yes it can pass the test because it is within the r ange.
32

33. Coulometric titration methods
•I
2
is produced by electrolysis of I
-
ions and the quantity of electricity used in
electrolysis is measured.
•i.e. The amount of iodine added to the sample is de termined by measuring the current
needed for the electrochemical generation of the io dine.
•Water is quantified on the basis of the total charg e passed (Q), as measured by
current (amperes) and time (seconds), according to the following relationship:
Q = 1C (coulomb) = 1A x 1S
Where 1mg H
2O = 10.72 C
•When reacting with water, the brown iodine is reduc ed to the colorless iodide.
•This method measures water levels much lower than the volumetric method.
33

34. How does a Coulometric Titrator work?
•The Coulometric titrator performs the following thr ee key functions:
1. It generates iodine at the anode of the titration cell, instead of dispensing KF
reagent as in volumetric titration.
2. It detects the endpoint of the titration using the double platinum pin indi cator
electrode.
3. It calculates the end result based on the total charge passed (Q), in
Coulombs, using the onboard microprocessor.
34

35. Which method should beused, coulometricorvolumetric?
Ingeneral,itcanbesaidthatthemethodhastobechosendependingonthe
watercontentofthesamplestobemeasured.
Thecoulometricmethodissuitableforsampleswithalowwatercontent(1
ppmand5%).
Thevolumetricmethod is suitable for samples with a higher water content
(100ppmand100%).

36. C. Azeotropic Distillation
Procedure:
Add a water immiscible solvent to the material containing mo isture and co-distill
anywater present.
Condensationof the vaporresultsin separationof water fro mimmiscible solvent.
Benzene,Tolueneandxyleneareused as immiscible solvents .
The method is successful for determination of plant parts an d aqueous solutions
such as medicinal soapsolutions.
Disadvantage:Largesamples arerequired.

37. The schematic graph of Azeotropic distillation apparatus

38. •The official compendia lay a great deal of emphasis on the control of
physiologically dangerous, cumulative poisonous and harmful impurities, such
as
lead, arsenic
and
iron
present in a host of pharmaceutical chemicals.
•These impurities very often creep into the final product through a number of
means:
»Through atmospheric pollution.
»Most frequently derived from the raw materials.
»From materialsused in the process of manufacture.
»Due to solventaction on the metal of the plant in which the subst ance is
prepared
Pharmacopoeia usually fix certain limits of toleran ce.
38
III. Limit Test For Heavy Metals

39. •Limit test for heavy metal are semi quantitative analytical test.
•It is performed to identify the trace of heavy meta l impurities which are likely
to be present in a substance.
•It is based on the reaction of metallic impurities and standard lead metal with
hydrogen sulfide in acidic medium to form brownish colour solution.
•Depth of color formed represent a concentration of metals.
•The test solution color is then compared with a col or of standard prepared
using a lead solution (as the heavy metal).
39

40. •In substances the proportion of any such impurity ( Heavy metals) has been
expressed as the quantity of lead required to produ ce a color of equal depth as
in a standard comparison solution having a definite quantity of lead nitrate.
•The test colour should not exceed the heavy metal l imits given under the
individual monograph.
•The quantity is stated as the heavy metal limit and expressed as parts of lead
per million parts of the substance.
•Metals that response to this test are lead, mercury , bismuth, arsenic, antimony,
tin, cadmium, silver, copper, and molybdenum.
•Indian Pharmacopoeia limit for heavy metals is 20 p pm
40

41. •Procedure: •The IP has adopted three methods for the limit test of heavy metals:
•Method I: Used for the samples which give
clear colourless solutions
under
specified conditions of test.
•Method II: The method is applicable for the samples which
DO NOT give
clear colourless solutions
under specified conditions of test.
•Method III: Used for substances which give
clear colourless solutions
in
sodium hydroxide medium
.
41

42. Method I
Test sample Standard compound
Solution is prepared as per the monograph and
25 ml is transferred in Nessler’s cylinder
Take 2 ml of standard lead solution and dilute to 25 ml with water
Adjust the pH between 3 to 4 by adding dilute
acetic acid ‘Sp’ or dilute ammonia solution‘Sp’
Adjust the pH between 3 to 4 by adding dilute
acetic acid ‘Sp’ or dilute ammonia solution ‘Sp’
Dilute with water to 35 ml Dilute with water to 35 ml
Add freshly prepared 10 ml of hydrogen
sulphidesolution
Add freshly prepared 10 ml of hydrogen
sulphide solution
Dilute with water to 50 ml Dilute with water to 50 ml
Allow to stand for five minutes Allow to stand for five minutes
View downwards over a white surface View downwards over a white surface
42
Result
: The turbid produce in sample solution should not be greater than standard solution.

43. Method II
Test sample Standard compound
•Weigh specific quantity of test substance, moisten
with sulphuric acid and ignite on a low flame till
completely charred
•Add few drops of nitric acid and heat to 500 °C
•Allow to cool and add 4 ml of hydrochloric acid
and evaporate to dryness
•Moisten the residue with 10 ml of hydrochloric
acid and digest for two minutes
•Neutralize with ammonia solution and make just
acid with acetic acid
•Take 2 ml of standard lead solution
and dilute to 25 ml with water
•Adjust the pH between 3 to 4 and filter if
necessary
•Adjust the pH between 3 to 4 by
adding dilute acetic acid ‘Sp’ or dilute
ammonia solution ‘
Sp
’
43

44. •Dilute with water to 35 ml•Dilute with water to 35 ml
•Add freshly prepared 10 ml of hydrogen
sulphidesolution
•Add freshly prepared 10 ml of hydrogen
sulphidesolution
•Dilute with water to 50 ml•Dilute with water to 50 ml
•Allow to stand for five minutes•Allow to stand for five minutes
•View downwards over a white surface•View downwards over a white surface
44
•Observation
: The color produce in sample solution should not b e greater
than standard solution.
•If color produces in sample solution is less than t he standard solution, the
sample will pass the limit test of heavy metals and vice versa.

45. Method III.
Test sample Standard compound
Solution is prepared as per the monograph and 25 ml is transferred to Nessler’s cylinder or
weigh specific amount of substance and
dissolve in 20 ml of water and add 5 ml of
dilute sodium hydroxide solution
Take 2 ml of standard lead solution and
dissolve in 20 ml of water and add 5 ml of
dilute sodium hydroxide solution
Make up the volume to 50 ml with water Make up the volume to 50 ml with water
Add 5 drops of sodium sulphide solution Add 5 drops of sodium sulphide solution
Mix and set aside for 5 min Mix and set aside for 5 min
View downwards over a white surface View downwards over a white surface
45
Observation
:The color produce in sample solution should not be greater than standard
solution.

46. Limit Test for Lead
•Lead is a most undesirable impurity in medical comp ounds and comes
through use of sulphuric acid, lead lined apparatus and glass bottles use for
storage of chemicals.
•Limit test of lead is based on the reaction of leadand diphenyl thiocarbazone
(dithizone).
•Dithizone in chloroform, is able to extract lead fr om alkaline aqueous
solutions as a lead dithizone complex (Red in colou r)
•Dithizone is green in color in chloroform and lead- dithizone complex is violet
in color, so the resulting color at the end of proc ess is red.
•The intensity of the color of complex is dependent upon the amount of lead in
the solution.
46

47. Procedure
Test sample Standard compound
A known quantity of sample solution is
transferred in a separating funnel
A standard lead solution is prepared equivalent
to the amount of lead permitted in the sample
under examination
Add 6ml of ammonium citrate Add 6ml of ammonium citrate
Add 2 ml of potassium cyanide and 2 ml of
hydroxylamine hydrochloride
Add 2 ml of potassium cyanide and 2 ml of
hydroxylamine hydrochloride
Add 2 drops of phenol red Add 2 drops of phenol red
Make solution alkaline by adding ammonia
solution.
Make solution alkaline by adding ammonia
solution.
Extract with 5 ml of dithizone until it becomes green
Extract with 5 ml of dithizone until it becomes
green
47

48. Combine dithizone extracts are shaken for
30 mins with 30 ml of nitric acid and the
chloroform layer is discarded
Combine dithizone extracts are shaken for
30 mins with 30 ml of nitric acid and the
chloroform layer is discarded
To the acid solution add 5 ml of standard
dithizone solution
To the acid solution add 5 ml of standard
dithizone solution
Add 4 ml of ammonium cyanide Add 4 ml of ammonium cyanide
Shake for 30 mins Shake for 30 mins
Observe the color Observe the color
48
•Observation:The intensity of the color of complex, is depends o n the
amount of lead in the solution.
•The color produce in sample solution should not be greater than
standard solution.

49. Indication of each reagent •Ammonium citrate, potassium cyanide, hydroxylamine hydrochloride is
used to make pH optimum so interference and influen ce of other impurities
have been eliminated.
•Phenol red is used as indicator to develop the color at the end of process.
•Leadpresent as an impurities in the substance, gets sep arated bye extracting
an alkaline solution with a dithizone extraction so lution.
49

50. Limit test for Iron (Fe)
•This test is based on the reaction of ironin ammonicalsolution with
thioglycolic acid in presence of citric acid to form iron thioglycolate (Ferrous
thioglycolate complex)
•This produces pale-pink to deep reddish-purple colo r in alkaline media.
•Thioglycolic acid is used as reducing agent.
•The color of the Ferrous thioglycolate complex fade s in the presence of air,
oxidizing agents and strong alkalis due to oxidatio n.
50

51. Procedure
Note: All the reagents used in the limit test for I ron should themselves be iron free.
51
Test sample Standard compound
Sample is dissolved in specific amount of
water and then volume is made up to 40ml
2ml of standard solution of iron diluted with
water up to 40ml
Add 2ml of 20%w/v of citric acid (iron free) Add 2ml of 20%w/v of citric acid (iron free)
Add 2 drops of thioglycolic acid Add 2 drops of thioglycolic acid
Add ammonia to make the solution alkaline
and adjust the volume to 50ml
Add ammonia to make the solution alkaline
and adjust the volume to 50ml
Keep aside for 5min Keep aside for 5min
Color developed is viewed vertically and
compared with standard solution
Color developed is viewed vertically and
compared with sample solution

52. Reason for each reagent:
Ammonia is added to makesolution alkaline because the purple color is
developed only in alkaline media.
But ammonia reacts with iron and forms precipitate of ferrous hydroxide.
Thus citric acid is used to prevent the precipitate of iron with amm onia,
The citric acid form a complex with iron as iron citrate.
Thioglycolic acid helps to oxidize iron(II) to iron (III).
Observation:
The purple color produced in sample solution should not be greater than
standard solution.
52

53. Limit test for Arsenic (Ar)
•Arsenic is a well known undesirable and harmful imp urity which is present in
medicinal substances.
•All pharmacopoeias prescribe a limit test for it.
•Pharmacopoeial method is based on the Gutzeittest.
•All the special reagents used in the limit test for Arsenic are marked and
distinguished by letter ‘As T’,
•It means they all should be Arsenic free and should themselves conform to the
test for Arsenic.
53

54. Principle:
•Limit test of Arsenic is based on the reaction of a rsenic gas with hydrogen ion to form
yellow stain
on mercuric chloride paper in presence of reducing agents like potassium
iodide.
•It is also called as
Gutzeittest
and requires special apparatus.
•When the sample is dissolved in
acid
, the Arsenic present in the sample gets converted
to
Arsenic acid
(H
3AsO
4)
.
•By action of reducing agents like Potassium iodide, stannous acid, zinc, hydrochloric
acid etc., Arsenic acid gets reduced to
arsenious acid
(H
3AsO
3) .
•The
nascent hydrogen
formed during the reaction, further reduces
Arsenious acid
to
Arsine gas
(AsH
3).
•The arsine gas reacts with mercuric chloride paper to give a yellow stain.
•The depth of yellow stain on mercuric chloride pape r will depend upon the quantity of
arsenic present in the sample.
54

55. 55

56. Procedure
•Stain obtained on mercuric chloride paper is compar ed with standard solution.
•Standard stain must be freshly prepared as it fades on keeping.
•Inference
: If the stain produced by the test is not deeper t han the standard stain,
the sample complies with the limit test for Arsenic .
56
Test sample Standard compound The test solution is prepared by dissolving
specific amount in water and stannatedHCl
and kept in a wide mouthed bottle.
A known quantity of dilute arsenic solution
in water and stannatedHCl is kept in wide
mouthed bottle.
1 g of KI 1 g of KI
5 ml of stannous chloride acid solution 5 ml of stan nous chloride acid solution
•10 g of granulated zinc is added (all this
reagents must be arsenic free).
•Keep the solution aside for 40 min
•10 g of granulated zinc is added (all this
reagents must be arsenic free).
•Keep the solution aside for 40 min

57. IV. Limit Test For Non Metals
A. Limit Test For Chloride: •Limit test of chloride is based on the reaction of soluble chloride with silver
nitrate in presence of dilute nitric acid to form s ilver chloride, which appears
as solid particles (Opalescence) in the solution.
•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 HNO
3
is insoluble in AgCl.
57

58. Procedure
Test sample Standard compound
Specific weight of compound is dissolved in
water or solution is prepared as directed in the
pharmacopoeia and transferred in Nessler
cylinder
Take 1ml of 0.05845 % W/V solution of sodium
chloride in Nessler cylinder
Add 1ml of nitric acid Add 1ml of nitric acid
Dilute to 50ml in Nessler cylinder Dilute to 50ml in Nessler cylinder
Add 1ml of AgNO
3
solution Add 1ml of AgNO
3
solution
Keep aside for 5 min Keep aside for 5 min
Observe the Opalescence/Turbidity Observe the Opalescence/Turbidity
58
The opalescence produce in sample solution should n ot be greater than standard solution.

59. •Pharmacopoeia does not prescribe any numerical valu e of limit test for
chlorides, sulphate and iron.
•So, the limit test is based on the simple compariso n of opalescence or colour
between the test and standard solution prescribed a ccording to pharmacopoeia.
59

60. B. Limit Test For Sulphate
•The Sulfate Limit Test is designed to determine the allowable limit of sulfate
contained in a sample.
•Limit test of sulphate is based on the reaction of soluble sulphate with barium
chloride in presence of dilute hydrochloric acid to form barium sulphate
which appears as solid particles (turbidity) in the solution.
60

61. 61
Test sample Standard compound
Specific weight of compound is dissolved in
water or solution is prepared as directed in
the pharmacopoeia and transferred in Nessler cylinder
Take 1ml of 0.1089 % W/V solution of
potassium sulphate in Nessler cylinder
Add 2ml of dilute hydrochloric acid Add 2ml of dilute hydrochloric acid
Dilute to 45 ml in Nessler cylinder Dilute to 45 ml in Nessler cylinder
Add 5ml of barium sulphate reagent Add 5ml of barium sulphate reagent
Keep aside for 5 min Keep aside for 5 min
Observe the Turbidity Observe the Turbidity

62. •Barium sulphate reagent contains barium chloride, s ulphate free alcohol and
small amount of potassium sulphate.
Observation:
•The turbidity produce in sample solution should not be greater than standard
solution.
•If turbidity produces in sample solution is less th an the standard solution, the
sample will pass the limit test of sulphate and vic e versa.
Reasons:
•Hydrochloric acid helps to make solution acidic.
•Potassium sulphate is used to increase the sensitiv ity of the test by giving ionic
concentration in the reagent
•Alcohol helps to prevent super saturation.
62

63. 63