LIMIT TEST ❖ Test being used to identify the impurity.
❖ It is quantitative or semi-quantitative test design to identify and
control small quantity of impurities (which is likely to be present in the substance).
❖ Limit test Generally carried out to determine the inorganic
impurities present in compound . DEPTH OF BIOLOGY LIMIT TEST ➢ Lead and Heavy Metal ➢ Sulphate ➢ Chloride ➢ Arsenic ➢ Iron ➢ Lead ➢ Modified Limit test for Sulphate ➢ And modified Limit test for chloride and sulphate LIMIT TEST FOR HEAVY METALS Heavy Metal like Co, Sn, Mn, Pb, Ag, Sb
PRINCIPLE:- Limit test for heavy metal is based on the reaction of metallic impurities with Hydrogen, Sulphide or Sodium sulphide in acidic medium to produce metal sulphides which gives Brown colour. [LEAD IS USED AS STAND] Heavy Metals + H2S/ Na2S Heavy metal sulphides (brown colour ppt.)
Method A Method C
Method B Method D DEPTH OF BIOLOGY Method A = For the substance give clear colourless solution under specific condition give in monograph (pharmacopeia).
Method B = For the substance not give clear colourless solution.
Method C = For the substance who give clear colourless solution
in NaOH solution.
Method D = For the substance not give clear colourless solution.
DEPTH OF BIOLOGY
Means, here we check impurities of sulphate in sample with the help of Limit Test. Here we use 2 Nessler cylinder ❑ Test solution ❑ Standard solution Chemical required :- Potassium Sulphate , Distilled water, Barium sulphate reagent, Dilute HCl DEPTH OF BIOLOGY
1. Take 1 ml of a 25 % w/v solution of barium chloride in a Nessler’s cylinder
2. Add 1.5 ml of ethanolic sulphate standard solution(10 ppm SO4) Mix and allow to stand for 1min.
3. Take 15ml of specific amount of test substance dissolve in distilled water or
25ml of test solution prepared as directed in monograph in IP.
4. Add 0.15ml of 5M acetic acid
5. Make up to 50ml with distilled water.
6. Stir with glass rod and allow to stand for 5min
7. Observe the turbidity and compare with standard solution.
DEPTH OF BIOLOGY STANDARD SOLUTION (HCl) 1. Take 1.0ml of a 25% w/v solution of barium chloride in a Nessler’s cylinder
2. Add 1.5ml of ethanolic sulphate standard solution (10ppm SO4) Mix and allow to stand for 1min.
3. Take 15ml of sulphate standard solution (10 ppm SO4)
4. Add 0.15ml of 5M acetic acid
5. Make up to 50ml with distilled water
6. Stir with glass rod and allow to stand for 5min.
7. Compare with sample solution
DEPTH OF BIOLOGY
PRINCIPLE . BaSO4 reagent react with sulphate . In the presence of dilute HCl . And form BaSO4 . Lead to formation of white ppt. . Lead to turibidity in solution Means, here we check impurities of chloride in sample with the help of Limit test. For Limit Test, we use Nesseler cylinder [2] Test solution Standard solution Chemical required :- Standard NaCl solution – 0.05845%, distilled H2O, Dilute HNO3, AgNO3 solution- 5%
TEST OR SAMPLE SOLUTION
1. Specific amount of test substance dissolve in distilled water or solution is prepared as directed in monograph and take it into Nessler’s cylinder. 2. Add 10ml dilute Nitric acid 3. Dilute upto 50ml with distilled water 4. Add 1ml of AgNO3 solution 5. Keep aside for 5mins 6. Observe the opalescence and compare with stardand solution STANDARD SOLUTION(25 ppm) 1. Take 10ml standard chloride solution + 5ml distilled water in Nessler’s cylinder 2. Add 10ml dilute Nitric acid 3. Dilute upto 50ml with distilled water 4. Add 1ml AgNO3 solution 5. Keep aside for 5mins 6. Compare with sample solution 7. Now, compare both test and standard solution 8. If, test solution less turbidity as compared to standard then test pass 9. [test solution > standard solution (turbidity) test fail
PRINCIPLE – Limit Test of chloride Here we add AgNO3 → this react with chloride in the presence of dilute HNO 3 form AgCl This leads to turbidity DEPTH OF BIOLOGY LIMIT TEST FOR ARSENIC Arsenic easily reduce into Arsine gas If we use Nessler cylinder then gas release out , so we use Gutziet apparatus. As may be present as As+3 (Trivalent) As+5 (Pentavalent) As+3 HCl H3AsO3 Arsanious Acid HCl As+5 H3AsO4 Arsenic Acid
SnCl Arsenic 2 Arsanious Acid reduction Standard Solution Test Solution
Standard solution of Arsenic. Test solution of Arsenic.
Add 10gm KI Add 10gm KI
10gm granulated Zn 10gm granulated Zn
5ml of SnCl2 5ml of SnCl2
Kept solution for 40 min. Kept solution for 40 min.
If, [test solution < standard solution ( yellow strain)] → Test Pass But, [standard solution < test solution ( yellow strain)] → Test Fail
Principle Arsine Gas react with HgCl2 paper Form Diarsino Mercury which leads to yellow strain. DEPTH OF BIOLOGY LIMIT TEST FOR IRON Small amount of Inorganic impurity are detect. Here, we check impurity of Fe so we perform limit test and called limit test for Iron. Use 2 Nessler cylinder Chemical required:- • Standard Ferric ammonium sulphate solution =20ml • Citric acid solution = 20% w/v • Ammonia solution • Thioglycolic acid Test sample Standard compound Sample is dissolved in specific amount of water 2ml of standard solution of iron diluted with and then volume is made up to 40ml water upto 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 Add ammonia to make the solution alkaline and adjust the volume to 50ml adjust the volume to 50ml
Keep aside for 5min Keep aside for 5min
Color developed is viewed vertically and Color developed is viewed vertically and compared with standard solution compared with standard solution DEPTH OF BIOLOGY Now, compare the color of Test solution with Standard solution. If , Test solution have less color as compared to standard then test pass [standard < test solution (color) Test Fail ]
Principle ➢ Citric acid forms complex with metal cation and helps precipitation of iron by ammonia by forming a complex with it. ➢ Thioglycolic acid helps to oxidize iron(II) to iron (III). ➢ Ammonia is added to make solution alkaline. The pale pink to reddish purple color is visible only in the alkaline media. The color is not visible in acidic media as ferrous thioglycolate complex decomposes in high acidic media. DEPTH OF BIOLOGY LIMIT TEST FOR LEAD Most undesirable impurity in medical compound and come through use of H2SO4 • Lead line apparatus • Glass bottles used for storage of chemicals Principle Based on the reaction of lead and diphenyl thiocarbazone (dithizone) in alkaline solution to form lead dithizone complex (Red color).
Dithizone → is green color in chloroform
and lead dithizone complex is violet in color. So, the resulting color at the end of the process is red. Test solution :- A known quantity of sample Standard solution :- A standard lead solution is solution is transferred in a separating funnel prepared equivalent to the amount of lead permitted in the sample under examination. Add 6ml of ammonium citrate Add 6ml of ammonium citrate
Add 2ml of potassium cyanide and 2ml of hydroxylamine Add 2ml of potassium cyanide and 2ml of hydroxylamine hydrochloride hydrochloride
Add 2 drops of phenol red and make solution alkaline by Add 2 drops of phenol red and make solution alkaline by adding ammonia solution adding ammonia solution
Extract with 5ml of dithizone until it becomes green Extract with 5ml of dithizone until it becomes green
Combine dithizone extracts are shaken for 30mins with Combine dithizone extracts are shaken for 30mins with 30ml of nitric acid and the chloroform layer is discarded 30ml of nitric acid and the chloroform layer is discarded
To the acid solution add 5ml of standard dithizone To the acid solution add 5ml of standard dithizone solution solution Add 4ml of ammonium cyanide Add 4ml of ammonium cyanide Shake for 30 mins and observe the color Shake for 30 mins and observe the color DEPTH OF BIOLOGY OBSERVATION The intensity of the color of complex, is depends on the amount of lead in the solution. [Standard > Test solution (Red color) Test Pass] [Test > Standard solution (Red color) Test Fail]
REASONS:- Ammonium citrate, potassium cyanide, hydroxylamine hydrochloride is used to make pH optimum so interference and influence of other impurities have been eliminated. Phenol red is used as indicator to develop the color at the end of process Lead present as an impurities in the substance, gets separated bye extracting an alkaline solution with a dithizone extraction solution. DEPTH OF BIOLOGY
Principle :- Used for those color compound who does not pass normal limit test. It carried out on the basis of reaction between BaCl 2 and soluble in the presence of Acetic acid . Leads to formation of BaSO 4 Here reagent used → BaCl2, sulphate free alcohol and a solution of Potassium sulphate ( is added to increase sensitivity of test) and here alcohol is helps in prevention from supersaturation and produce uniform turbidity. Here Acetic acid makes acidic solution → BaSO4 ppt. form ( insoluble) which give turbidity.
(Sample) (ethanol) Test solution Standard solution Take 1 ml 25% w/v barium chloride in Nessler’s Take 1 ml 25% w/v barium chloride in Nessler’s cylinder and add 1.5 ml of ethanolic sulphate cylinder and add 1.5 ml of ethanolic sulphate standard solution (10 ppm SO4-2). Mix and allow to standard solution (10 ppm SO4-2). Mix and allow to stand for 1 minutes stand for 1 minutes Transfer prepared test solution and add 0.15 ml of Add 15 ml of standard sulphate solution (10 ppm 5 M acetic acid. SO4-2) and 0.15 ml of 5M acetic acid.
Add sufficient distilled water to produced 50 ml. Add sufficient distilled water to produced 50ml. Stirred it immediately and allow standing for 5 Stirred it immediately and allow standing for 5 minutes. minutes.
[ Standard solution > Test solution (Turbidity) Test Pass ]
[ Standard solution < Test solution (Turbidity) Test Fail ] DEPTH OF BIOLOGY MODIFIED LIMIT TEST FOR CHLORIDE Used for color compound which cannot be tested with normal test. Principle → based upon the chemical reaction between silver nitrate and soluble chloride to obtain AgCl ( makes the test solution turbid) in the presence dilute HNO3 Procedure Test solution Standard solution 1.5gm of KMnO4 Take 40ml of the test solution in Nessler 10ml standard chloride solution cylinder 50ml distilled H2O and heat on a water Add 0.1ml dilute HNO3 Add 5ml H2O and add 1ml dilute HNO3 bath Now gradually add 6ml of Ethanol (95%) 50ml distilled H2O 50ml distilled H2O
Now, cool the solution and dilute to 60ml Now add 1ml 0.1M AgNO3 solution 0.1M AgNO3 solution with distilled water and then filter
Take two 50ml Nessler cylinder Stir immediately with glass rod Stir with glass rod
Labelled one test And placed it for 5min (protected from And placed it for 5min light) And other on standard
Reagent used:- Dilute HNO3 → makes solution Acidic which helps in formation of AgCl ppt. (Leads to turbuidity) This test is not used for H2O immiscible liquid.
