Introduction to limit tests, the limit test is a semiquantitative test, limit test for chlorides, limit test for sulfates, limit test for iron, limit test for heavy metals, limit test for arsenic, Gutzeit apparatus

1. LIMIT TEST
Authored By: Dr. Alex Martin
Ph.D., M.Pharm, FAGE
‘’The one that is rotten, fails to get accepted’’

2. LIMIT TEST
• Most usual contaminants found in pharmaceuticals: CHLORINE, SULPHATE, IRON, HEAVY METALS AND ARSENIC.
• The quantity of these impurities in traces is not a problem. However, the quantity of these impurities should
not go beyond a certain level.
• If they increase beyond a certain level, they can cause adverse effects.
• Therefore, pharmacopeia has prescribed a limit for impurities that can be found in pharmaceuticals.
• They can be present in trace amounts but they cannot be present beyond a certain limit.
Eg 1: CHLORINE
Respiratory problems: Wheezing, sneezing, cough,
sore throat, airway irritation and chest tightness.
Eye (Blurred vision)and Skin Irritation (Rash)
Eg 2: IRON
GIT Symptoms: Nausea, Vomiting (may contain blood),
Stomach ache (sharp), Constipation or Diarrhea
If they are present within the permissible
amount, they pass the limit test and are accepted
as medicines after passing other tests.
If impurities are present beyond prescribed
pharmacopeia limit, they fail the limit tests and
are rejected as medicines.

3. Whether a pharmaceutical drug has passed or
failed the limit test…?
How to find out….?????
This is simply found out by comparison. There are two
cylindrical tubes in the test:
1. STANDARD TUBE
Contains impurity within prescribed higher limit
2. TEST OR SAMPLE TUBE
Contains impurity, the quantity of which we don’t
know….
Limit test involves comparison of opalescence,
turbidity and color of the test with that of the
standard.

4. • VISIBLE REACTION OUTCOMES ARE COMPARED:
• 1. OPALESCENSE
• 2. TURBIDITY
• 3. COLOR
• CASE 1: If any of these visible signs is more in standard, then test passes the limit test.
• If any of these visible signs is more in test than that in the standard, the test fails the limit test.
• LIMIT TEST IS A QUANTITATIVE OR SEMI-QUANTITATIVE
TEST
STD.
TEST
STD TEST
• Concentration of standard is known and is fixed by pharmacopeia.
•Concentration of test unknown.
We are comparing unknown with the known. Therefore, we are
performing semi-quantitative test.

5. • It is like comparing two apple, the one that is rotten fails to be accepted
LIMIT TEST FOR CHLORIDES
PROCEDURE:
Apparatus required:
1. Nessler’s Cylinder
2. Glass rod
3. Stand
Chemicals required:
1. Dilute Nitric acid
2. Silver nitrate
3. Sodium chloride
TEST STANDARD
1. Dissolve the specified quantity of distilled
water and tranfer it to Nessler’s cylinder.
2. Add 10 ml Dil. HNO3
3. Dilute to 50 ml distilled water
4. Add 1 ml AgNO3 solution
5. Stir immediately with glass rod and allow to
stand for 5 minutes.
1. Place 1ml of 0.05845% of NaCl and transfer to a
Nessler’s cylinder
2. Add 10 ml of dilute HNO3
3. Dilute to 50 ml distilled water
4. Add 1 ml AgNO3 solution
5. Stir immediately with glass rod and allow to
stand for 5 minutes.

6. Procedure (Block Diagram)
STEP I: PLACING OF STANDARD/TEST SOLUTION IN TEST TUBE
STEP II: ADDITION OF 10 ML OF HNO3
STEP III: ADDITION OF 50 ML OF DISTILLED WATER
ADDITION OF 1 ML OF AgNO3
STEP V: STIR IMMEDIATELY AND OBSERVE AFTER 5 MINUTES

7. • OBSERVATION
The Nessler’s cylinders containing the test and the
standard are observed against a dark background.
• Observation:
• Case I: STANDARD IS MORE OPALESCENT THAN TEST
• In cases where the standard is more opalescent than
test; the test passes the limit test for chlorides.
• It means that test has chloride ions within the
prescribed limits as set by pharmacopeia. The test
complies with the limit.
• Case II: TEST IS MORE OPALESCENT THAN STANDARD
• In such cases, the test contains chloride ions more than
that prescribed by pharmacopeia. In other words, the
test does not comply within the limits.
• Therefore, the test fails the limit test for sulphate.

8. REACTION INVOLVED (REASON FOR OPALESCENSE)

10. PROCEDURE
TEST STANDARD
1. Specific weight of compound is dissolved in 1ml
of water as directed in pharmacopeia (in
nessler’s clinder labeled test)
1. Take 1 ml of 0.1089% w/v solution of potassium
sulphate in Nessler’s cylinder.
1. Add 2ml Dil. HCl 1. Add 2ml Dil. HCl
1. Dilute to 45 ml with distilled water 1. Dilute to 45 ml with distilled water
1. Add 5 ml BaSO4 reagent 1. Add 5ml BaSO4 reagent
1. Stir with a glass rod and keep aside for 5
minutes.
1. Stir with a glass rod and keep aside for 5
minutes.
1. Observe the turbidity against a black
background
1. Observe the turbidity against a black
background
COMPARE
TEST
STD.

11. PROCEDURE(BLOCK DIAGRAM)
STEP I: PLACING OF STANDARD/TEST SOLUTION IN NESSLER’S CYLINDER
STEP II: ADDITION OF 2 ML OF DIL. HCl
STEP III: ADDITION OF 45 ML OF DISTILLED WATER
STEP IV: ADDITON OF 5 ML OF BaSO4
STEP V: STIR IMMEDIATELY AND OBSERVE TURBIDITY AFTER 5 MINUTES

12. CASE I: STANDARD IS MORE TURBID THAN DENSE
OUTCOME: TEST (OUR SAMPLE) HAS PASSED THE LIMIT TEST FOR SULPHATE
CASE II: TEST IS MORE TURBID THAN STANDARD
OUTCOME: TEST (OUR SAMPLE) HAS FAILED THE LIMIT TEST FOR SULPHATE
STD IS MORE TURBID TEST IS LESS TURBID
STD IS LESS TURBID TEST IS MORE TURBID

13. PRINCIPLE (REASON FOR TURBIDITY)
• SULPHATE reacts with BaCl2 to form Barium Sulphate which are white particles that make the solution
turbid.
(White ppt; appear as turbidity)
Dil. HCl
Dil.HCl
• Reaction takes place in acidic medium
BaSO4 reagent: Contains BaCl2 + suphate free alcohol + pottasium sulphate
•1. BaCl2 is the reactant
•Alcohol prevents supersaturation
•Pottasium sulphate increases ionic concentration, thereby increasing ionic concentration;
ultimately increasing the sensitivity of reaction.

15. PROCEDURE
TAKE TWO NESSLER’S CYLINDER AND LABEL THEM AS TEST AND STANDARD
TEST STANDARD
1. Sample is dissolved in specific amount of
water and then volume is made upto 40
ml water.
1. 2ml of Standard iron solution is diluted with
water to upto 40 ml water.
1. Add 2 ml of 20 % w/v of citric acid 1. Add 2 ml of 20% w/v of citric acid
1. Add 2 drops of thioglycollic acid 1. Add 2 drops of thioglycollic acid
1. Add ammonia till the solution is alkaline
and then adjust the volume to upto
50ml.
1. Add ammonia till the solution is alkaline
and then adjust the volume to upto
50ml.
1. Stir and keep aside for 5 Mins 1. Stir and keep aside for 5 Mins
COMPARE THE TUBES BY VIEWING VERTICALLY DOWNWARDS

16. PROCEDURE (BLOCK DIAGRAM)
STEP I: PLACING OF STANDARD/TEST SOLUTION IN NESSLER’S CYLINDER
STEP II: ADDITION OF 2 ML 20% w/v CITRIC ACID
STEP III: ADDITION OF 2DROPS OF THIOGLYCOLLIC ACID
STEP IV: ADDIITION OF NH3 TILL ALKALINE AND MAKE UP THE VOLUME
STEP V: STIR IMMEDIATELY AND OBSERVE COLOR AFTER 5 MINUTES

17. CASE I: TEST IS MORE COLORED THAN STANDARD
OUTCOME: THE TEST (OUR SAMPLE) FAILS THE LIMIT TEST FOR IRON
CASE II: TEST IS LESS COLORED THAN STANDARD
OUTCOME: THE TEST (OUR SAMPLE) PASSES THE LIMIT TEST FOR IRON
STD. IS LESS COLORED TEST IS MORE COLORED
LIMIT TEST
FAILS
STD. IS MORE COLORED TEST IS LESS COLORED
LIMIT TEST
PASSES

18. PRINCIPLE
REACTION:
REASONS FOR ADDITION OF EACHN REAGENT
IRON IN AMMONICAL SOLUTION REACTS WITH THIOGLYCOLLIC ACID IN THE PRESENCE OF
CITRIC ACID TO FORM FERROUS THIOGLYCOLATE COMPLEX WHICH IS PALE PINK TO DEEP
REDDISH PURPLE IN COLOR.
-HELPS TO CONVERT IRON(II) TO IRON (III). FORMS THE FINAL COMPLEX
-REACTION TAKES PLACE IN ALKALINE SOLUTION
-CITRIC ACID FORMS COMPLEX WITH OTHER METAL CATIONS AND
THEREFORE PREVENT PERCIPITATION OF OTHER METAL IONS WITH
THIOGLYCOLLIC
Dil. NH3

19. LIMIT TEST FOR LEAD

20. SOURCES OF LEAD

21. Intellectual disability Irritability
Memory problems
Inability to have children
Abdominal Pain
Tingling in hands and
feet

22. PRINCIPLE
• Pb + diphenylthiocarbazone [Pg-dithizonecomplex]
REACTION
•
Red color
Alkali solution
• To prevent interference of other metal ions.
• To provide optimum pH for the product to be formed
Ammonium citrate, potassium
cyanide, hydroxylamine chloride
• To produce color at alkalin pH(pH above 8.2)
Phenol red

23. PROCEDURE
TEST STANDARD
1. A known quantity of solution of drug is
transferred into a separating funnel.
2. Add 6ml of ammonium citrate
3. Add 2 ml of potassium cyanide and 2ml
of hydroxylamine hydrochloride.
4. Add 2 drops of phenol red
5. Make solution alkali by adding ammonia
6. Extract with 5ml of dithizone until it
becomes green.
7. Extract with 5ml dithizone extraction
solution until it becomes green.
8. Add 30 ml of 1% HNO3. Shake well and
keep aside until chloroform layer is
formed. Discard the chloroform layer.
9. Add 5 ml std dithizone solution
10. Add 4ml of ammonium cyanide
11. Shake for 30 mins
1. A known quantity of solution of drug is
transferred into a separating funnel.
2. Add 6ml of ammonium citrate
3. Add 2 ml of potassium cyanide and 2ml
of hydroxylamine hydrochloride.
4. Add 2 drops of phenol red
5. Make solution alkali by adding ammonia
6. Extract with 5ml of dithizone until it
becomes green
7. Extract with 5ml dithizone extraction
solution until it becomes green.
8. Add 30 ml of 1% HNO3. Shake well and
keep aside until chloroform layer is
formed. Discard the chloroform layer.
9. Add 5 ml std dithizone solution
10. Add 4ml of ammonium cyanide
11. Shake for 30 mins
COMPARE

24. TAKE TWO SEPERATING FUNNELS. LABEL ONE AS TEST AND THE OTHER AS STANDARD
1. Place test/
standard solution
2. 6ml ammonium citrate
3. 2ml pottasium
cyanide
4. 2ml hydroxylamine
hydrochloride
5. 2 drops of phenol red
6. Add NH3 till alkaline
7. Extract with 5ml
dithizone extraction
solution until green

25. CHCl3
1. Add
30 ml
dil.
HNO3
2. Shake well
and keep aside
3. Discard
the
chloroform
layer
4. Add 5ml
std
dithizone
solution
5. Add 4 ml
ammonium
cyanide
solution
6. Shake for
30 mins

27. LIMIT TEST FOR HEAVY METALS

28. Brownish colored solution
Std.
Test
Brownish colored solution
NOT MORE THAN 20PPM
TEST STANDARD
1. Dissolve the test drug in 25ml of water. Adjust
to a pH b/w 3 and 4 by adding dil. Acetic acid or
ammonia and again dilute to about 35ml and
mix.
1. Place 0.1 ml of lead standard solution and dilute
it to 25ml with water. Adjust pH b/w 3 and 4 by
addition of acetic acid or ammonia and again
dilute with water to about 35ml and mix.
2. Add 10ml freshly prepared hydrogen sulphide
solution.
1. Add 10ml freshly prepared hydrogen sulphide
solution.
1. Mix and dilute it with water to about 50ml. 1. Mix and dilute it with water to about 50ml.
1. After 5 minutes view the cylinder vertically
downwards over a white background.
1. After 5 minutes view the cylinder vertically
downwards over a white background

29. Std. Test
1. Dissolve 0.1ml std
lead solution and dilute
it to 25ml water
1. Dissolve the test
drug in 25 ml water
2. Adjust the pH b/w 3 and 4 by addition of NH3 and
CH3COOH. Dilute to about 35ml with water
3. Add 10ml H2S solution and mix
4. Make up the volume upto 50ml with water
5. After 5 minutes view the cylinder vertically downwards

30. TEST STANDARD
1. Moisten sample with H2SO4 and ignite on a
low flame till completely charged.
Add few drops of nitric acid and heat it to
500° C.
Allow to cool. Add 4 ml of HCl and
evaporate to dryness.
Moisten the residue with 10ml HCl and
digest for two minutes.
Neutralize with ammonia and make acidic
with acetic acid.
1. Take 2ml of standard lead solution and
dilute to 25 ml of water
STEPS 2,3 AND 4 ARE SAME AS METHOD A
Std Test
Take 2ml
standard
lead
solution and
dissolve it in
25ml water.
a) Moisten sample with H2SO4
and ignite on low flame till
charred.
b) Add few drops of conc. HNO3
and heat it to 500°C.
c) Allow to cool and then add
4ml HCl. Evaporate to
dryness.
d) Moisten the residue with
10ml HCl and digest for two
mins.
e) Neutralize with ammonia and
then add acetic acid to make
it acidic.
STEPS 2,3 AND 4 ARE SAME AS METHOD A

31. METHOD C
TEST STANDARD
1. Dissolve specified amount of
substance in 20ml of water.
1. Place 2ml of std lead solution
2. Add 5ml of dilute sodium hydroxide solution.
3. Make up the volume up to 50ml with distilled water.
4. Add 5 drops of sodium sulfide solution.
5. Mix and set aside for 5 minutes.
6. View downwards over a white surface.
Std Test
1. Place
2ml std
lead
solution
1. Dissolve specified qty. of substance in 20ml water
2. Add 5ml dil. NaOH solution
3. Make up the volume to 50ml with distilled water
4. Add 5 drops of sodium sulfide solution
5. Mix and set aside for 5 minutes
6. View vertically downwards against a white
background

32. COMPARE THE BROWN COLOUR
CASE I: TEST IS MORE COLORED THAN STANDARD
• CASE II : TEST IS LESS COLORED THAN STANDARD

33. SPECIAL DISCUSSION
HOLISTIC STUDY APPROACH
https://www.youtube.com/watch?v=VJbKXmujI
00

35. ADVERSE EFFECTS OF ARSENIC
SKIN DAMAGE:
HYPERKERATOSIS AND
PIGMENT CHANGES
CARCINOGENIC: CAUSES
CANCER IN LUNG,
BLADDE, KIDNEY AND
LIVER
NERVE DAMAGE
CIRCULATORY PROBLEMS

36. GUTZEIT APPARATUS
4. A disc or a piece of
mercuric chloride
paper is placed b/w
the two triangular
rubber stopper
which connects the
two tubes

37. PRINCIPLE
2. Arsenic acid is reduced by reducing
agents such as Zn and HCl, SnCl2 and KI
to arsenious acid.
3. Arsenious acid is further reduced
by nascent hydrogen produced by
the reaction of Zn and HCl to arsine
gas.
4. The evolved arsine gas ascends
up through the tube and reached
the juncture of the two tubes
where HgCl2 paper is placed.
5. Here, arsine gas reacts with
HgCl2 paper to form a yellow
stain.
6. The depth of yellow stain is depends
upon the quantity of arsine gas reacted
which in turn depends upon the quantity
of arsenic present in the sample.
1. Metal arsenic in the sample
dissolves in HCl as arsenic acid

38. PRINCIPLE WITH DIAGRAM
1. Metal arsenic in the sample
dissolves in HCl as arsenic acid
2. Arsenic acid is
reduced by
reducing agents
such as Zn and
HCl, SnCl2 and KI
to arsenious acid.
3. Arsenious acid is
further reduced by
nascent hydrogen
produced by the
reaction of Zn and
HCl to arsine gas.
4. The evolved arsine gas ascends up through the tube and reached the
juncture of the two tubes where HgCl2 paper is placed.
5. Here, arsine
gas reacts with
HgCl2 paper to
form a yellow
stain.
6. The depth of
yellow stain is
depends upon
the quantity of
arsine gas
reacted which in
turn depends
upon the
quantity of
arsenic present
in the sample.

39. Limit
test
for
arsenic
Limit
test
for
arsenic
Limit
test
for
arsenic

40. PROCEDURE
TEST STANDARD
1. Place the sample in 100ml conical
flask or a wide mouthed bottle.
Dissolve it in a mixture of water and
HCl.
1. A known quantity of dilute arsenic
solution (1ml arsenic solution diluted
to 50ml with water) is kept in the
conical flask.
2. To this solution add 1g of KI, 5ml SnCl2
and 10g of Zn.
2. To this solution add 1g of KI, 5ml SnCl2
and 10g of Zn.
3. Immediately assemble the apparatus
and immerse in a water bath provided
with a uniform temperature such that
uniform evolution of gas takes place.
3. Immediately assemble the apparatus
and immerse in a water bath provided
with a uniform temperature such that
uniform evolution of gas takes place.
4. After 40 mins., observe the HgCl2
paper that has been placed betwixt
the two tubes
4. After 40 mins., observe the HgCl2
paper that has been placed betwixt
the two tubes
COMPARE THE YELLOW STAIN

41. PROCEDURE
2. To this solution add 1g of KI, 5ml SnCl2
and 10g of Zn.
3. Immediately assemble the apparatus and
immerse in a water bath provided with a
uniform temperature such that uniform
evolution of gas takes place
4. After 40 mins., observe the HgCl2 paper
that has been placed betwixt the two tubes
5. COMPARE THE
YELLOW STAIN
1. TEST: Place the sample in 100ml
conical flask or a wide mouthed
bottle. Dissolve it in a mixture of
water and HCl.
1. STANDARD: A known quantity of dilute
arsenic solution (1ml arsenic solution
diluted to 50ml with water) is kept in the
conical flask.

42. TWO CASES
• CASE I: TEST IS MORE COLORED
• CASE II: TEST IS LESS COLORED
STD. TEST
STD. TEST

43. THANK YOU…..