Preparation of Buffers, Indicators and Reagents Solutions

The preparation of buffers, indicators and reagents to the intended users or concerned personnel. XYZ Pharmaceuticals Limited's policy is that a written procedure shall be followed to prepare buffers, indicators and reagents to the intended users or concerned personnel for awareness and implementation. 

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Preparation of Buffers

Reference:            IP / In-house

Frequency:

Preparation:       1 Month

Storage conditions: - Store the solution in a well-stoppered glass bottle at ambient temperature                  

Precautions (if any):- If the buffer solutions become turbid then discard the Solution

Acetate Buffer pH 4.6 

Dissolve 5.4 g of sodium acetate in 50 ml of distilled water, add 2.4 ml of glacial acetic acid and dilute with distilled water to 100 ml. Adjust the pH, if necessary.

Acetate Buffer pH   5.0 

Dissolve 13.6 g of sodium acetate and 6 ml of glacial acetic acid insufficient water to produce 1000 ml. Adjust the pH, if necessary.

Acetic Acid-Ammonium Acetate Buffer 

Dissolve 77.1 g of ammonium acetate in distilled water, add 57 ml of glacial acetic acid and dilute with distilled water to 1000 ml.

Ammonia –Ammonium Chloride Buffer 

Dissolve 67.5 g of ammonium chloride in about 200 ml of distilled water, add 570 ml of strong ammonia solution and dilute with distilled water to 1000 ml. 

Benzalkonium Chloride Buffer pH 10.0 

Weigh accurately about 3.0 gm of boric acid, 3.725 gm of potassium chloride and 1.75 gm of sodium hydroxide in a 1000ml volumetric flask, add a sufficient amount of distilled water to dissolve and dilute it up to the mark with distilled water

Buffer solution pH 6.0 + 0.1 

Dissolve 2.0 gm of dipotassium hydrogen phosphate (K₂HPO₄) and 8 gm of potassium dihydrogen phosphate in sufficient distilled water to produce 1000 ml after sterilisation, adjusting the pH with 8 M phosphoric acid or 10 M potassium hydroxide.

Buffer solution pH 8.0 + 0.1 

Dissolve 16.73 gm of dipotassium hydrogen phosphate (K₂HPO₄) and 0.523 gm of potassium dihydrogen phosphate in sufficient distilled water to produce 1000 ml after sterilisation, adjusting the pH with 8 M phosphoric acid or 10 M potassium hydroxide.

Buffer solution pH 4.5 + 0.1 

Dissolve 13.61 gm of potassium dihydrogen phosphate in sufficient distilled water to produce 1000 ml after sterilisation, adjusting the pH with 8 M phosphoric acid or 10 M potassium hydroxide.

Buffer solution pH 10.5 + 0.1*

Dissolve 35.0 gm of dipotassium hydrogen phosphate (K₂HPO₄) in sufficient distilled water to produce 1000 ml after sterilisation, adjusting the pH with 8 M phosphoric acid or 10 M potassium hydroxide.

* After addition of 2 ml of 10 M potassium hydroxide

Buffer solution pH 7.0 + 0.2 

Dissolve 13.6 gm of dipotassium hydrogen phosphate (K₂HPO₄) and 4.0 gm of potassium dihydrogen phosphate in sufficient distilled water to produce 1000 ml after sterilisation, adjusting the pH with 8 M phosphoric acid or 10 M potassium hydroxide.

Buffer solution pH 3.0 + 0.1 

Dissolve 9.8 gm of potassium dihydrogen phosphate (KH2PO4) in sufficient distilled water to produce 1000 ml after sterilisation, adjusting the pH with 8M phosphoric acid or 10 M potassium hydroxide.

Fluoride Buffer 

Weigh about 58 gm of sodium chloride and 4 gm of 1, 2- cyclohexylenediamine tetra acetic acid in a 1000 ml beaker; add 57 ml of acetic acid and 500 ml of distilled water to dissolve. Add 125 ml of 6N sodium hydroxide adjust the pH 5.3 to 5.5 and dilute the volume to produce 1000ml.            

Phosphate Buffer pH 4.0, Mixed 

Dissolve 5.04 g of disodium hydrogen phosphate and 3.01 g of potassium dihydrogen phosphate in sufficient distilled water to produce 1000 ml. Adjust the pH with glacial acetic acid.

Phosphate Buffer pH 7.2 for BOD

Weigh accurately about 2.125 gm of potassium dihydrogen phosphate (KH₂PO₄), 5.4375 gm of dipotassium hydrogen phosphate (K₂HPO₄), 4.42 gm of disodium hydrogen phosphate (Na₂HPO₄) or 8.35 gm of disodium hydrogen phosphate heptahydrate (Na₂HPO₄.7H₂O) and 0.425 gm of ammonium chloride (NH₄Cl) in 250 ml volumetric flask, add sufficient distilled water to dissolve and then dilute it up to the mark with distilled water.

Pyridoxine Hydrochloride Buffer 

Dissolve 16 gm of ammonium chloride 16 ml of ammonia solution and sufficient distilled water to produce 100 ml.           

Phosphate Buffer pH 7.4

Dissolve 2.38 g of disodium hydrogen phosphate, 0.19 g of potassium dihydrogen phosphate and 8.0 g of sodium chloride in sufficient distilled water to produce 1000 ml. Adjust the pH if necessary.

Phosphate Buffer pH 7.0 Mixed 

Dissolve 0.50 g of anhydrous disodium hydrogen phosphate and 0.301 g of potassium dihydrogen phosphate in sufficient water to produce 1000 ml.

Riboflavin Buffer 

Dissolve 33.0 gm of potassium hydroxide, 38 ml of phosphoric acid and 16 gm of sodium hydroxide in sufficient distilled water to produce 500 ml.

Sulphanilic Acid Buffer pH 4.5 

Dissolve 5.0 gm of sulfanilic acid in 5 ml of ammonia solution and add sufficient distilled water to produce 250 ml.

Zinc Buffer 

Mix 4.6 gm citric acid, 7.5 ml distilled water and 12 ml ammonia.

Indicators Preparation 

Reference: IP / In-house

Frequency

Preparation: 1 Month

Storage conditions: Store the solution in a well-stoppered glass bottle at ambient temperature 

Precautions (if any): None specific

Bromocresol Purple Solution 

Dissolve 50 mg of bromocresol purple in 0.92 ml of 0.1M Sodium Hydroxide and 20 ml of ethanol (95%).  If a solution is affected, add sufficient water to produce 100 ml.

Complies with the following requirements.

SENSITIVITY – A mixture of 0.2 ml of the solution and 100 ml of carbon dioxide-free water to which 0.05 ml of 0.02 M Sodium Hydroxide has been added is bluish violet.  No more than 0.2 ml of 0.02 M hydrochloric acid is required to change the colour to yellow.

Bromophenol Blue Solution

Strong Bromophenol Blue Solution; Ethanolic Bromophenol Blue Solution; Dissolve 0.1 g of bromophenol blue with gentle heating in 1.5 ml of 0.1 M Sodium Hydroxide and 20 ml of ethanol (95%) and add sufficient distilled water to produce 100 ml.

Complies with the following requirements.

SENSITIVITY mixture of 0.05 ml of the solution and 20 ml of carbon dioxide-free water to which 0.05 ml of 0.1 M hydrochloric acid has been added is yellow.  No more than 0.1 ml of 0.1M sodium hydroxide is required to change the colour to bluish violet.

Bromothymol Blue Solution 

Aqueous Bromothymol Blue solution: Dissolve 50 mg of Bromothymol blue in 4 ml of 0.02M Sodium Hydroxide and 20 ml of ethanol (95%).  After a solution is effected, add sufficient distilled water to produce 100 ml.  Complies with the following requirements.

SENSITIVITY – A mixture of 0.3 ml of the solution and 100 ml of carbon dioxide-free water is yellow.  No more than 0.1 ml of 0.02M sodium hydroxide is required to change the colour to blue.

Crystal Violet Solution 

A 0.5% w/v solution of crystal violet in anhydrous glacial acetic acid.

Complies with the following requirements.

SENSITIVITY – A mixture of 0.1 ml of the solution and 50 ml of anhydrous glacial acetic acid is bluish-purple.  Add 0.1 ml of 0.1 M perchloric acid; the solution turns blue-green.

Dimethyl Yellow – Oracet Blue B Solution 

Dimethyl Yellow – Solvent Blue 19 Solution.  Dissolve 15 mg of Dimethyl yellow and 15 mg of racet blue B in chloroform and dilute to 500 ml with chloroform.

Eosin Solution

A 0.5% w/v solution of eosin in distilled water.

Eriochrome Black T Solution 

Mordant Black 11 Solution: Dissolve 0.2 g of ferrochrome black T and 2 g of hydroxylamine hydrochloride in sufficient methanol to produce 50 ml.

Prepare immediately before use.

Ferroin Solution; Ferroin Sulphate Solution 

Dissolve 0.7 g of ferrous sulphate and 1.5 g of 1, 10-phenanthroline hydrochloride in 70 ml of distilled water and add sufficient distilled water to produce 100 ml.

Complies with the following requirements.

SENSITIVITY – Add 0.1 ml of the solution and 0.15 ml of osmic acid solution to 50 ml of 1M sulphuric acid.  Add 0.1 ml of 0.1M ceric ammonium nitrate; the colour changes from red to light blue.

Methyl Orange Solution

Dissolve 0.1 g of methyl orange in 80 ml of distilled water and add sufficient ethanol (95%) to produce 100 ml.

Complies with the following requirements.

SENSITIVITY – A mixture of 0.1 ml of the solution and 100 ml of carbon dioxide-free water is yellow.  Note more than 0.1 ml of 0.1 M hydrochloric acid is required to change the colour to red.

Methyl red indicator preparation 

Dissolve 50 mg of methyl red in a mixture of 1.86 ml of 0.1 M sodium hydroxide and 50 ml of ethanol (95%).  After a solution is effected, add sufficient distilled water to produce 100 ml.

Complies with the following requirements.

SENSITIVITY – A mixture of 0.1 ml of the solution, 100 ml of carbon dioxide-free water and 0.05 ml of 0.02 M hydrochloric acid is red.  No more than 0.1 ml of 0.02M sodium hydroxide is required to change the colour to yellow.

Methylene Blue Solution 

Dissolve 150 g of methylene blue in 100 ml of ethanol (95%) and dilute with ethanol (95%) to produce 250 ml.

Mixed bromocresol green-methyl red indicator solution (Code No. IND12)

Use either the aqueous or the alcoholic solution

Dissolve 100 mg bromocresol green sodium salt and 20 mg of methyl red sodium salt in 100 ml of distilled water.

Dissolve 100 mg of bromocresol green and 20 mg methyl red in 100 ml ethyl alcohol (95%) or isopropyl alcohol

Neutral Red Solution 

A 0.1% w/v solution of neutral red in ethanol (50%)

Ninhydrin Solution 

A 0.2 % w/v solution of ninhydrin in a mixture of 95 volumes of 1-butanol and 5 volumes of 2 M acetic acid.

Ninhydrin Solution 0.1%w/v

A 0.1 % w/v solution of ninhydrin in a mixture of 95 volumes of 1-butanol and 5 volumes of 2 M acetic acid.

Phenol Red Solution 

Dissolve 0.1 g of phenol red in 2.82 ml of 0.1 M sodium hydroxide and 20 ml of ethanol (95%).  After a solution is effected, add sufficient water to produce 100 ml.

Complies with the following requirements.

SENSITIVITY – A mixture of 0.1 ml of the solution and 100 ml of carbon dioxide-free water is yellow.  No more than 0.1 ml of 0.02 M sodium hydroxide is required to change the colour of the solution to reddish-violet.

Phenolphthalein Solution 

A 1.0 % w/v solution of phenolphthalein in ethanol (95%).

Potassium chromate 

Weigh 5 gm of potassium chromate (K₂CrO₄) in 100 ml volumetric dissolve in a sufficient amount of distilled water and then add silver nitrate solution till red precipitation occurs, wait for 12 hours. Filter the solution and dilute it up to the mark with distilled water.

Thymol Blue Solution 

Dissolve 0.1 g of thymol blue in 2.15 ml of 0.1M sodium hydroxide and 20 ml of ethanol (95%).  After a solution is effected, add sufficient water to produce 100 ml.

Complies with the following requirements.

SENSITIVITY-A mixture of 0.1 ml of the solution and 100 ml of carbon dioxide-free water to which 0.2 ml of 0.02 M sodium hydroxide has been added is blue.  No more than 0.1 ml of 0.02 M hydrochloric acid is required to change the colour to yellow.

Starch Solution 

Dissolve 25 mg of starch in 10 ml of distilled water; warm on a water bath for 15-20 min till transparent liquid occurs.

Reagents Preparation 

Reference:     IP / In-house

Frequency

Preparation:    1 Month

Storage conditions:  Store the solution in a well-stoppered glass bottle at ambient temperature                  

Precautions (if any): None specific

Acetic Acid 2M 

Dilute 114 ml (120 g) of glacial acetic acid to 1000 ml with distilled water.

Acetic Acid 5M 

Dilute 285 ml (300 g) of glacial acetic acid to 1000 ml with distilled water.

Acidify water 

Dilute 65 ml of 1 N HCL to 1000 ml with distilled water.

Ammonia Solution, Dilute 

Contains approximately 10% w/w of NH_3. Dilute 425 ml of strong ammonia solution to 1000 ml.

Store in well-closed containers in a cool dry place.

Ammonium Hydroxide Solution 0.5 N 

Dilute 35 ml of fresh conc. Ammonia hydroxide in sufficient distilled water to produce 1000 ml

4- Aminoantipyrine solution 

Dissolve 2 gm of amino antipyrine in sufficient distilled water to produce 100 ml.

Ammonium Molybdate reagent for phosphate 

Weigh accurately about 25 gm of ammonium molybdate in a 1000 ml volumetric flask dissolve it in 175 ml of distilled water and add 280 ml of conc. Sulphuric acid (H₂SO₄) and dilute the volume up to the mark with distilled water.

Ammonium Molybdate reagent for silica 

Weigh accurately about 10 gm of ammonium molybdate in a 100 ml volumetric flask, add sufficient water to dissolve, warm gently and dilute the volume up to the mark with distilled water. Adjust the pH of the solution to 7-8 with sodium hydroxide solution.

Ammonium Sulphamate Solution (5% w/v) 

Dissolve 5 gm of ammonium sulphamate solution in 100 ml distilled water.

Ammonium Sulphamate Solution (15% w/v) 

Dissolve 15 gm of ammonium sulphamate solution in 100 ml distilled water.

Arsenic Standard Solution 10 ppm As 

Dissolve 0.330 g of arsenic trioxide in 5 ml of 2M sodium hydroxide and dilute to 250.0 ml with distilled water.  Dilute 1 volume of this solution to 100 volumes with distilled water.

Borax solution 1% w/v 

Dissolve 1 g of borax in sufficient distilled water to produce 100 ml

Boric acid solution 4% w/v 

Dissolve 4 g of boric acid in sufficient distilled water to produce 100 ml.

Borax solution 5% w/v

Dissolve 5 g of boric acid in sufficient distilled water to produce 100 ml.

Barium Chloride solution 10%w/v 

Dissolve 10 g of barium chloride in sufficient distilled water to produce 100 ml.

2, 2- Bipyridyl Solution 0.5% w/v 

Dissolve 500 mg of 2, 2 - Bipyridyl in sufficient methanol to produce 100 ml.

Calcium Chloride Solution 

Dissolve 6.875 gm of calcium chloride in sufficient distilled water to produce 250 ml.

Citric acid solution 20% w/v 

Dissolve 20 g of citric acid in sufficient distilled water to produce 100 ml

Chloride Standard Solution 25 ppm Cl 

Dilute 5 volumes of a 0.0824% w/v solution of sodium chloride to 100 volumes with distilled water.

Chlorpheniramine Maleate buffer 

2.5 gm sulphanillic acid add 15 ml water and 2 ml dilute ammonia solution. Mix, add with stirring more dilute ammonia solution if necessary to dissolve the substance, and adjust the pH of the solution to 4.5 with dilute ammonia to 100 ml with water.

Cobalt Thiocyanate Solution 

Dissolve 6.8 g of cobalt chloride and 4.3 g of ammonium thiocyanate in sufficient distilled water to produce 100 ml.

2, 6-Dichloroquinone-4-chloromide 0.05%w/v

Dissolve 50 mg of 2, 6-Dichloroquinone -4-chloromide in sufficient isopropyl alcohol to produce 100 ml.

Disodium Edetate 5%w/v 

Dissolve 5 mg of disodium edetate in sufficient distilled water to produce 100 ml.

Dipotassium hydrogen phosphate 7.5%w/v 

Dissolve 7.5 gm of dipotassium hydrogen phosphate (K₂HPO₄) in sufficient distilled water to produce 100 ml

Disodium Edetate 0.5 M 

Dissolve 186 gm of disodium edetate in sufficient distilled water to produce 1000ml.

Disodium Edetate 0.01M 

Dissolve 3.72 gm of disodium edetate in sufficient water to produce 1000 ml.

Ephedrine Reagent (Code No. RS27)

Dissolve 5 gm of copper sulphate in sufficient distilled water to produce 100 ml.

Ferric Chloride Solution 1% w/v 

Dissolve 1 gm of ferric chloride in sufficient distilled water to produce 100 ml.

Ferric chloride solution 

Dissolve 0.0625 hexahydrated ferric chlorides (FeCl₃.6H₂O) or 0.0375 gm of ferric chloride (FeCl₃) in a sufficient amount of distilled water to produce 250 ml.

Ferrous Ammonium Sulphate 

Weigh accurately about 19.6 gm of ferrous ammonium sulphate (Fe (NH₄)₂(SO₄)₂.6H2O) in a 500 ml volumetric flask, add a sufficient amount of distilled water to dissolve and 10 ml of concentrated sulphuric acid (H₂SO₄) and dilute the volume up to the mark with distilled water.

Formaldehyde 0.2% 

Dilute 0.2 ml of formaldehyde to 100 ml with distilled water.

Hydrochloric acid, Dilute 

Dilute 10 ml of Hydrochloric acid to 100 ml with distilled water.

Hydrochloric acid 0.2M 

Dilute 17 ml of hydrochloric acid to produce 1000 ml with distilled water.

Hydrochloric Acid 5M 

Dilute 425 ml of Hydrochloric acid to 1000 ml with distilled water.

Hydrochloric acid - concentrated (11 N) for Iron 

Containing less than 0.00005% iron.

Hydroxamine Solution 

Dissolve 10 gm of hydroxylamine in sufficient distilled water to produce 100 ml.

Iodine Monochloride solution 

Dissolve 8 g of iodine trichloride in about 200 ml of glacial acetic acid and separately dissolve 9 g of iodine in 300 ml of dichloromethane. Mix the two solutions and dilute to 1000 ml with glacial acetic acid. Store in stoppered, light-resistant glass containers at a temperature not exceeding 15^oC

Iodide Solution 

Weigh accurately 12 gm of potassium iodide and 1.6 gm of iodine in a 500 ml volumetric flask. Add sufficient distilled water to dissolve and dilute it up to the mark with distilled water.

Iodide, Alkali 

Dissolve 50 gm of Sodium hydroxide and 13.5 gm of potassium iodide or 70 gm of potassium hydroxide and 15 gm of potassium iodide in sufficient distilled water to produce 100 ml.

Iron Standard Solution (20ppm Fe)

Dilute 1 volume of a 0.1726% w/v solution of ferric ammonium sulphate in 0.05M sulphuric acid to 10 volumes with distilled water.  Contains iron in the ferric state.

Lead Standard Solution 0.1% Pb 

Dissolve 0.400g of lead nitrate in distilled water containing 2 ml of nitric acid and add sufficient distilled water to produce 250.0 ml.

Magnesium Sulphate Solution 

Dissolve 5.625 gm of Magnesium Sulphate (MgSO4.7H2O) in sufficient distilled water to produce 250 ml.

Magnous Sulphate

Dissolve 36.4 gm of Magnus sulphate monohydrate (MnSO₄.H₂O) or 40 gm of magnous sulphate dehydrate (MnSO₄.2H₂O) or 48 gm of magnous sulphate tetrahydrate (MnSO₄.4H₂O) in sufficient distilled water to produce 100ml.          

1-Napthyl- Ethylene diamine dihydrochloride 0.1%w/v 

Dissolve 100 mg of 1- Napthyl - Ethylene diamine dihydrochloride in sufficient distilled water to produce 100 ml.

Nitric Acid, dilute 

Dilute 10 ml of Nitric acid to produce 100 ml with distilled water.

Niacinamide Buffer

2.5 gm sulphanilic acid adds 15 ml water and 2 ml dilute ammonia solution. Mix, add with stirring more dilute ammonia solution if necessary to dissolve the substance, and adjust the pH of the solution to 4.5 with dilute ammonia to 100 ml with water.

Oxalic Acid

Dissolve 7.5 FM of oxalic acid in sufficient water to produce 100 ml

1, 10-Phenanthroline solution 

Dissolve 100 mg of 1, 10 - Phenanthroline in 100 ml distilled water, heat at 80˚ C to avoid the boiling of the solution. Discard the solution if it darkens and add 2 drops of hydrochloric acid. The solution is stable for 1 week.

Phosphate Buffer for phenol 

Weigh accurately about 104.5 gm of potassium hydrogen phosphate (K₂HPO₄) and 72.3 gm of potassium dihydrogen phosphate (KH₂PO₄) in a 1000 ml volumetric flask, add a sufficient amount of distilled water to dissolve and then dilute the volume up to the mark with distilled water.

Potassium Bromate Solution 

Dissolve 200 mg of potassium bromate (KBrO₃) in sufficient distilled water to produce 250 ml.    

Potassium Ferricyanide solution 1%w/v 

Dissolve 1 gm of potassium ferricyanide in sufficient distilled water to produce 100 ml.

Prepare immediately before use.

Potassium Hydroxide solution 50 %w/v 

Dissolve 50 gm of potassium hydroxide in sufficient distilled water to produce 100 ml.

Potassium Hydroxide, 0.5M Ethanolic 

Dissolve 2.8 g of potassium hydroxide in sufficient ethanol (95%) to produce 1000 ml.

Potassium Cupri -Tartrate Solution; Alkaline Cupric - Tartrate Solution; Fehling‘s Solution. (Code No. RS54)

Solution I (Copper Solution) - Dissolve 34.66 gm of carefully selected small crystals of cupric sulphate, showing no trace of efflorescence or of adhering moisture, in sufficient distilled water to produce 500 ml. Store this solution in small, well-stoppered bottles

Solution II (Alkaline Tartrate Solution) - Dissolve 176 gm of sodium potassium tartrate and 77 gm of sodium hydroxide in sufficient distilled water to produce 500 ml.

Mix equal volumes of the solutions (I) and (II) immediately before use.

Potassium Dichromate 0.01667 M for COD 

In a 200 ml volumetric flask containing 100 ml of distilled water accurately about 98.06 mg of potassium dichromate (K₂Cr₂O₇), add 33.4 ml of sulphuric acid and 6.6 gm of mercuric sulphate (HgSO₄) and then dissolve. Allow to cool to room temperature and dilute the volume up to the mark with distilled water.

Potassium Permanganate 0.2%w/v 

Dissolve 200 mg of potassium permanganate in sufficient distilled water to produce 100 ml.        

Nitric Acid 2M 

Dilute 126 ml of nitric acid to 1000 ml with distilled water.

Sodium Nitrite Solution 2%w/v

Dissolve 2gm of sodium nitrite in sufficient distilled water to produce 100 ml.

Sodium Nitrite Solution 10 %w/v 

Dissolve 10 gm of sodium nitrite in sufficient distilled water to produce 100 ml.

Sodium Arsenite Solution 0.1M

Dissolve 9.892 g of arsenic trioxide in 80 ml of 1 M sodium hydroxide, dilute to 800 ml with distilled water and add 2 M hydrochloric acid until the solution is neutral to litmus paper. Add 2 g of sodium bicarbonate dissolved by stirring and diluting with distilled water to produce 1000 ml.

Sodium Hydroxide 10 %w/v

Dissolve 10 gm of sodium hydroxide in sufficient distilled water to produce 100 ml.

Sodium Hydroxide 20 % w/v 

Dissolve 10 gm of sodium hydroxide in sufficient distilled water to produce 100 ml.

Sodium Hydroxide 1N 

Dissolve 40 gm of sodium hydroxide in sufficient distilled water to produce 1000 ml.

Sulphuric Acid 1M 

Dilute 60 ml of sulphuric acid to 1000 ml with distilled water.

Sulphuric Acid, Dilute 

Dilute 10 ml of sulphuric acid to produce 100 ml with distilled water.

Sulphate Standard Solution 10 ppm SO_4. 

Dilute 1 volume of a 0.181% w/v solution of potassium sulphate in distilled water to 100 volumes with the same solvent.

Sulphuric acid for COD

In a 1000 ml volumetric flask dissolve 543 ml of conc. Sulphuric acid and 5.5 gm of silver sulphate and kept solution overnight.

Stannous Chloride solution 

Dissolve 2.5 gm of fresh stannous chloride in 1000 ml glycerol. Heat on the water bath and stir. This reagent does not require preservatives or special storage.

Thioglycolic Acid 10% Solution 

Dilute 10 ml of thioglycolic acid to 100 ml with distilled water

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