Toxicology Manual Series XIII

SCREENING/SPOT TEST OF ACIDS & ALKALIS (BASES)

A. K. Jaiswal*, Kamna Sharma**, S. Lukose**, T. Millo*, O.P Murthy* * Department of Forensic Medicine and Toxicology, All India Institute of Medical Sciences, Ansari Nagar, Sciences, New Delhi- 110029 **AIFS, Amity University Campus, Sector 125, Noida-201303

ABSTRACT Mineral acids and strong alkalis are corrosive poisons. In dilute solutions, they act as irritants. In forensic autopsy case, the forensic pathologist may require a complete toxicological investigation for different poisons including antibiotics. In India, Forensic Science Laboratories run by Government under the Home ministry usually carry out this. The samples have to be analyzed by the forensic toxicologist/chemists/scientist. This article deals with the screening/spot test for acids/alkalis. It attempts to simplify the standard procedures in a stepwise manner, which can be of handy reference for the forensic toxicologist. It is in continuation of Toxicology Manual Series- XII, screening/spot test for antibiotics Vol 12, No 5, July-Sept 2010. Keywords: Acid, Alkalis, spot test, colour test etc.

INTRODUCTION An acid is defined as a substance which when dissolved in water, undergoes dissociation with the formation of hydrogen ions as the only positive ions. Degree of dissociation differs from acid to acid. Strong acids dissociate almost completely at medium dilutions whereas weak acids dissociate only slightly at medium or low concentrations. A base is defined as a substance which when dissolved in water, undergoes dissociation with formation of hydroxyl ions as the negative ions. We have tried to set out standard procedures for screening/spot test for acids and alkalis that are easily available and useful for the forensic science laboratory. This article covers the screening test of acids & alkalis of Hydroiodic acid, hydrobromic acid, hydrochloric acid, nitric acid, sulphuric acid Acetic acid, formic acid, hydrocyanic acid, oxalic acid, nitrous acid and in alkalis sodium hydroxide, potassium hydroxide, ammonia and pyridine etc.

Acids

y y y y y y y y y

Strong Perchloric acid Hydroiodic acid Hydrobromic acid Hydrochloric acid Nitric acid Sulphuric acid Sodium hydroxide Potassium hydroxide Barium hydroxide

y y y y y y y y y

Weak Acetic acid Formic acid Hydrocyanic acid Oxalic acid Nitrous acid Hydrofluoric acid Ammonia Methylamine Pyridine

Bases

ACIDS Litmus paper test 1. One drop of extract is taken on a blue litmus paper. 2. Blue litmus turned red which shows the presence of acids. Congo red paper test 1. One drop of extract is taken on a congo red paper. 2. Blue colour is observed which shows the presence of acids. TEST FOR SOME INDIVIUAL ACIDS PERCHLORIC ACID (HClO4) Methylene blue test 1. A drop of extract is taken on filter paper impregnated with solution of zinc sulphate and potassium nitrate. 2. Few drops of methylene blue are added to it. 3. Violet colour is observed which shows the presence of perchloric caid. Zinc uranyl acetate test 1. Two ml of extract is taken in a test tube. 2. Few drops of zinc uranyl acetate reagent is added to it. 3. Lemon- yellow precipitate is observed which shows the presence of perchloric acid. Diphenylamine test 1. A drop of extract is taken on a spot tile. 2. A drop of 1% diphenylamine solution is added to it. 3. Black colour is observed which shows the presence of perchloric acid.

HYDROIODIC ACID (HI) Silver nitrate test 1. Two ml of extract is taken in a test tube. 2. Few drops of silver nitrate solution are added to it. 3. Yellow colour is observed which shows the presence of hydroiodic acid. Mercuric nitrate test 1. A Strip of filter paper No.1is first dipped into mercuric nitrate solution. 2. Few drops of extract are added to it. 3. Orange or vermilion colour is observed on the test paper within a 10-min heating shows the presence of hydroiodic acid. HYDROBROMIC ACID (HBr) Silver nitrate test 1. Two ml of extract is taken in a test tube. 2. Few drops of silver nitrate solution are added to it. 3. Cream colour is observed which shows the presence of hydrobromic acid. Magnesium peroxide test 1. 2. 3. 4. 5. 6. Two ml of extract is taken in a test tube. Pinch of magnesium peroxide is added to it. Few drops of sulphuric acid are added to it Yellowish-red vapours are evolved. Vapours evolved brought in contact with starch paste. Orange-yellow colour is observed which shows the presence of hydrobromic acid.

HYDROCHLORIC ACID (HCl) Gunzbergs test 1. 2. 3. 4. Few drops of Gunzberg reagent is taken in a porcelain basin. Solution in basin was evaporated to dryness and cooled. Few drops of extract are added to dried reagent in porcelain basin. Purplish red colour is observed which shows the presence of hydrochloric acid.

Silver nitrate test 1. Two ml of extract is taken in a test tube. 2. Few drops of silver nitrate solution are added to it. 3. Curdy white precipitate is observed and is soluble in ammonia and insoluble in nitric acid which shows the presence of hydrochloric acid.

NITRIC ACID (HNO3) Caustic soda test 1. Two ml of extract is taken in a test tube. 2. Few drops of 1% caustic soda solution are added to it. 3. Orange colour is observed which shows the presence of nitric acid. Diphenylamine test 1. Two drop of extract is taken on a spot tile. 2. One drop of 1% diphenylamine solution is added to it. 3. Blue colour is obtained which shows the presence of nitric acid. Brucine test 1. Two drop of extract is taken on a spot tile. 2. One drop of 0.1 % brucine solution is added to it. 3. Red colour is obtained which shows the presence of nitric acid. Antistin test 1. 2. 3. 4. Two drop of extract is taken on a spot tile. One drop of 1% antazoline hydrochloride solution is added to it. One drop of concentrated sulphuric acid is added to it. Red colour is obtained which shows the presence of nitric acid.

Ferrous-sulphate test 1. 2. 3. 4. Two ml of extract is taken in a test tube. One ml of ferrous sulphate solution is added to it. One ml of concentrated sulphuric acid is slowly added by the side of the test tube. Formation of brown ring is formed at the junction of liquids shows the presence of nitric acid.

SULPHURIC ACID (H2SO4) Barium chloride test 1. 2. 3. 4. Two ml of extract is taken in a test tube. Few drops of nitric acid are added. One ml of barium chloride solution is added to it. White precipitate is observed which shows the presence of sulphuric acid.

Methyl violet test 1. Two ml of extract is taken in attest tube. 2. Few drops of aqueous solution of 0.01% methyl violet is added to it. 3. A greenish blue colour is observed which shows the presence of sulphuric acid.

Copper turning test 1. 2. 3. 4. 5. Two ml of extract is taken in a test tube. Solution is concentrated by heating it. Few pieces of copper turnings are added to it. Solution in the test tube is again boiled. Pink colour of the solution is observed which slowly fades out shows the presence of sulphuric acid.

ACETIC ACID (CH3 COOH) Ferric chloride test 1. Two ml of extract is taken in a test tube. 2. Few drops of neutral ferric chloride solution are added to it. 3. Red colour is observed which disappears on addition of few drops of hydrochloric acid shows the presence of acetic acid. Lanthanum nitrate test 1. One drop of extract is taken on a spot tile. 2. One drop of 5% solution of lanthanum nitrate is added to it. 3. One drop of 0.01N iodine solution is added followed by a drop of 1N ammonia solution. 4. Formation of blue or brown ring develops around the drop of ammonia in a few minutes shows the presence of acetic acid. Calcium carbonate test 1. 2. 3. 4. Two ml of extract is taken in a test tube. Few drops of neutral calcium carbonate solution are added to it. One drop of ferric chloride solution is added to it. Red-brown colour is observed which shows the presence of acetic acid.

FORMIC ACID (HCOOH) Sulphuric acid test 1. 2. 3. 4. 5. Two ml of extract is taken in a test tube. One ml of sulphuric acid is added to it. Solution is warmed for few minutes. Carbon monoxide is evolved at the mouth of test tube. Blue flame is observed at the mouth of the test tube which shows the presence of formic acid.

Ferric chloride test 1. Two ml of extract is taken in a test tube.

2. One ml of sodium hydroxide solution is added to it. 3. One ml of ferric chloride solution is added to it. 4. Reddish orange colour is observed which shows the presence of formic acid. HYDROCYANIC ACID (HCN) Guaiacum Copper sulphate test 1. A Strip of filter paper No.1is first dipped into 0.1% aqueous solution of copper sulphate. 2. After that the filter paper is dipped in a tincture of 10% guaiacum solution. 3. Filter paper turns to blue colour after 30 minutes shows the presence of hydrocyanic acid. Picric acid test 1. A Strip of filter paper No.1is first dipped into solution containing 1 gm of picric acid in 10% sodium carbonate solution. 2. Play of colour is observed on filter paper from yellow to tan or brown within 5 minutes shows the presence of hydrocyanic acid. Prussian blue Test 1. Two ml of concentrated alkaline distillate is taken in a porcelain basin. 2. Distillate is concentrated by heating it on a water bath. 3. Few drops of freshly prepared 5% solution of ferrous sulphate followed by a drop of ferric chloride solution are added to it. 4. Mixture is heated till boiling. 5. Few drops of sulphuric acid are added to it to make the solution acidic. 6. A Prussian blue colour is observed shows the presence of hydrocyanic acid. Vortmans Test 1. Two ml of concentrated alkaline distillate is taken in a porcelain basin. 2. Distillate is concentrated by heating it on a water bath. 3. Few drops of freshly prepared potassium nitrite solution followed by few drops of ferric chloride solution. 4. Few drops of sulphuric acid is added to it and a bright yellow colour is observed 5. Solution is boiled. 6. One ml of ammonium hydroxide solution is added after the solution is cooled. 7. Filter the solution to remove excess of iron. 8. To the filtrate few drops of ammonium sulphide are added. 9. Violet colour is obtained which gradually changes to blue and finally to yellow shows the presence of hydrocyanic acid. Sulphocyanate Test 1. Two ml of concentrated alkaline distillate is taken in a porcelain basin. 2. Distillate is concentrated by heating it on a water bath. 3. One ml of ammonium sulphide is added in a porcelain basin

4. Solution is evaporated to dryness on a boiling water bath. 5. Residue obtained is dissolved in 1 ml. of acidulated water (containing 2 drops of hydrochloric acid). 6. Solution is warmed and filtered. 7. To the filtrate, a few drops of very dilute ferric chloride solution are added. 8. A blood red colour is obtained. 9. On adding a few drops of mercuric chloride solution, the red colour disappears which shows the presence of hydrocyanic acid.

OXALIC ACID (C2 O4H2) Lime water test Two ml of extract is taken in a test tube. Few drops of lime water are added to it. Formation of white precipitate is observed. Few drops of acetic acid and ammonia solution are added to it which remains insoluble in white precipitate. 5. Few drops of dilute hydrochloric acid or nitric acid are added to it. 6. White precipitate is soluble shows the presence of oxalic acid. Calcium chloride test 1. 2. 3. 4. Two ml of extract is taken in a test tube. One ml of ammonia solution is added to neutralize the solution. Few drops of calcium chloride solution are added to it. White precipitate is formed which shows the presence of oxalic acid 1. 2. 3. 4.

Potassium permanganate test 1. 2. 3. 4. 5. Two ml of extract is taken in a test tube. Few drops of (0.1%, w/v) dilute potassium permanganate solution are added to it. Few drops of dilute sulphuric acid are added to acidify the solution. Slightly warmed the solution. The pink colour of the solution is discharged shows the presence of oxalic acid.

Silver nitrate test 1. Two ml of extract is taken in a test tube. 2. Few drops of silver nitrate solution are added to it. 3. White precipitate is formed which is soluble in ammonia shows the presence of oxalic acid. NITROUS ACID (HNO2) Starch-iodide test 1. One drop of extract is taken on starch iodide paper.

2. Blue colour is observed which shows the presence of nitrous acid Sulphanilic acid test 1. 2. 3. 4. Two ml of extract is mixed with few drops of sulphanilic acid. Solution is warmed on water bath. One ml of the 1% naphthalamine solution is added to it. Red colour is formed which shows the presence of nitrous acid

AKALIS Litmus paper test 1. One drop of extract is taken on a red litmus paper. 2. Red litmus turned blue which shows the presence of alkalis. Chloroplatinic acid test 1. 2. 3. 4. Two ml of extract is taken in a test tube. Few drops of hydrochloric acid are added to neutralize the solution. Few drops of chloroplatinic acid are added to it. Yellow crystalline precipitate is formed which is soluble in alcohol shows the presence of alkalis.

TEST FOR SOME INDIVIUAL ALKALIS SODIUM HYDROXIDE (NaOH) Flame test 1. Apply a small amount of extract on a glass rod. 2. Bring the glass rod on flame. 3. Golden yellow flame is observed shows the presence of sodium. Potassium antimonate test 1. Two ml of extract is taken in a test tube. 2. Few drops of potassium antimonate solution are added to it. 3. White crystalline precipitate is formed which is soluble in ammonia shows the presence of sodium hydroxide. POTASSIUM HYDROXIDE (KOH) Flame test 1. Apply a small amount of extract on a glass rod. 2. Bring the glass rod on flame. 3. Violet colored flame is observed shows the presence of potassium hydroxide.

Perchloric acid test 1. 2. 3. 4. Two ml of extract is taken in a test tube. Few drops of hydrochloric acid are added to neutralize the solution. Few drops of perchloric acid are added to it. White crystalline precipitate is formed which is soluble in alcohol shows the presence of potassium hydroxide.

Zinc uranyl acetate test 1. Two drops of extract is taken in a test tube. 2. Three to four drops of zinc uranyl acetate are added to it. 3. Yellow precipitate is observed which shows the presence of potassium hydroxide. Sodium hydrogen tartrate test 1. Two ml of extract is taken in a test tube. 2. Few drops of Sodium hydrogen tartrate solution are added to it. 3. White crystalline precipitate is formed which is soluble in ammonia shows the presence of potassium hydroxide. BARIUM HYDROXIDE [Ba(OH)2 ] Flame test 1. Apply a small amount of extract on a glass rod. 2. Bring the glass rod on flame. 3. Green colored flame is observed shows the presence of barium hydroxide AMMONIA or AMMONIUM HYDROXIDE (NH3 or NH4OH) Nesslers reagent test 1. Two ml of extract is taken in a test tube. 2. One ml of nesslers reagent is added to it. 3. Yellow to brown colour is observed which shows the presence of ammonia. Mercurous Nitrate test 1. Two ml of extract is taken in a test tube. 2. One ml of mercurous nitrate reagent is added to it. 3. Black precipitate is observed which shows the presence of ammonia. CALCIUM HYDROXIDE Ca(OH)2 Flame test 1. Apply a small amount of extract on a glass rod. 2. Bring the glass rod on flame. 3. Orange red colored flame is observed shows the presence of calcium hydroxide.

Sodium hydroxide test 1. Two ml of extract is taken in a test tube. 2. One ml of sodium hydroxide is added to it. 3. White precipitate is observed which shows the presence of calcium hydroxide. Ammonium oxalate test 1. Two ml of extract is taken in a test tube. 2. One ml of ammonium oxalate is added to it. 3. White precipitate is observed which shows the presence of calcium hydroxide.

CONCLUSION In any analysis of poison, screening/spot test is very useful in knowing the presence of the poison which can be confirmed by the more confirmatory tests. It saves time for the toxicologist in ruling out the poisons which can be confirmed by the more confirmatory tests. It saves time for the toxicologist in ruling out the poisons and gives a quick clue to the doctors for patient management in emergency poisoning cases. The result of the analytical methods depends on the amount and purity of the sample extracted. Screening/spot test has been developed after repeated trial and testing. The techniques are being improved everytime. It is important for the forensic toxicologists to know the best available method and help to detect the poison in the crime investigations.

PREPARATION OF SOLUTIONS 1. Antazoline hydrochloride solution: 2. Bromine solution: Saturated solution of bromine in water. 3. Calcium chloride: 55 g of the hydrated calcium chloride is dissolved in 100 ml of water 4. Chloroplatinic acid: It is prepared by dissolving 2.6 gms of hydrated chloroplatinic acid in 10 ml. of water. 5. Copper sulphate: 125 g of copper sulphate is dissolved in 100 ml of water containing 3ml of conc sulphuric acid. 6. Congo red: 0.4 g of congo red is dissolved in 20 ml of 50% ethanol and the final volume of the solution is made upto 100 ml with 50% ethanol. 7. Diphenylamine solution: 1 g of diphenylamine is dissolved in 100 ml of redistilled ethanol. 8. Ferric chloride solution: 10 g of ferric chloride is dissolved in 100 ml distilled water

9. Gunzbergs reagent: It is prepared by dissolving 2gms. of phloroglucinol and 1gm. of vanillin in 100 ml. of 95% alcohol. 10. Lanthanum nitrate solution: 4.33 g of lanthanum nitrate hexahydrate is dissolved in 100 ml of water. 11. Mercurous nitrate reagent: To a saturated solution of mercurous nitrate, add solid sodium bicarbonate until effervescence ceases and the precipitate formed becomes yellow. The precipitate then changes to a biscuit colour. This reagent should be freshly prepared, shaken immediately before use and should not be kept for more than an hour. 12. Methylene blue solution: 0.05 g methylene blue is dissolved in 100 ml of water. 13. Millons reagent: 23.5 g of mercury is dissolved in 34 ml of cold fuming nitric acid and the solution is diluted with water upto 100 ml. 14. Naphthalamine: 0.3 g in 70 ml water, filter or decant, and mix with 30 ml of glacial acetic acid. 15. Nesslers reagent: Solution (I) 50 g of mercuric chloride and 35 g of potassium iodide in 200 ml of water and cool. Solution (II) 50 g of sodium hydroxide in 250 ml of water and cool. Add the cold solution (II) to cold solution (I) and make up to 500 ml. Allow the mixture to stand and decant the clear supernatant for use. Store in dark brown bottles away from light. 16. Potassium antimonate solution: 10.5 g of potassium antimonate is dissolved in 100 ml water & dilute the solution to 200 ml. 17. Potassium nitrate solution: 10.1 g potassium nitrate in 100 ml of water. 18. Sodium hydrogen tartrate solution: 10 g of sodium hydrogen tartrate monohydrate in 100 ml of water. Allow to stand for 24 hrs and use the clear supernatant liquid for test. 19. Sodium hydroxide solution: 4 g of sodium hydroxide is dissolved in 100 ml of water. 20. Silver nitrate: 17g of silver nitrate is dissolved in 100ml of water. 21. Zinc sulphate solution: 28.8 g zinc sulphate hepta-hydrate is dissolved in 100 ml of water. 22. Zinc Uranyl Acetate: It is prepared by dissolving uranyl zinc acetate in appropriate volume of water or 2 M acetic acid.

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