A LABORATORY COURSE ON QUALITATIVE ORGANIC ANALYSIS

Course designed by Dr. Archana Jain, DSc Professor Krishna K. Verma, DSc, CChem, FRSC

Department of Chemistry Rani Durgavati University, Jabalpur, Madhya Pradesh

A.Jain and K.K. Verma: A LABORATORY COURSE ON QUANTITATIVE ORGANIC ANALYSIS

General Scheme for the Separation of Organic Compounds

At no stage should hot solvents or reagents be used for separation. Members of different groups can be mixed to exercise mixtures. No two members of the same group should be mixed. After stirring the powdered mixture with water or a reagent (filled blocks in the flow sheet), separate the insoluble mass by filtration or preferably by centrifugation. Wash the residue with water before stirring with the subsequent reagent for separation. powdered organic mixture, about 0.5 g

stir with 3-5 ml of distilled water filtrate/supernatant: Group A evaporate a portion to recover compound; see notes (1) and (2)

residue stir with 3-5 ml of 2% NaHCO3 residue

filtrate/supernatant: Group B acidify by dropwise addition of conc. HCl to recover compound

stir with 3-5 ml ml of 2% NaOH filtrate/supernatant: Group C acidify by dropwise addition of conc. HCl to recover compound

residue

stir with 0.5-1 ml of conc. HCl, add 5 ml of water and stir again filtrate/supernatant: Group D make alkaline by dropwise addition of 5% NaOH to recover compound

residue: Group E

A.Jain and K.K. Verma: A LABORATORY COURSE ON QUANTITATIVE ORGANIC ANALYSIS

Identification of Organic Compounds

GROUP A Apply the following tests to the filtrate/supernatant for Group A to confirm the class of compound. (i) Molisch test. Mix the aqueous solution with 2-5 drops of alcoholic solution of 1-naphthol and allow 2 drops of concentrated H2SO4 to tumbledown the wall of test tube. A violet ring at the junction is a positive test. Carbohydrates. glucose, fructose, sucrose, maltose Note (1): To isolate a carbohydrate, stir the powdered mixture with 5 ml of dry acetone, filter the residue which is a carbohydrate, and wash it with 1-2 ml of acetone. The filtrate can be used to recover other component(s) of mixture after evaporation of acetone and washing the residue with cold water. (ii) Test for acid. To about 0.2 ml of aqueous extract, add 1 ml of water and 20-30 mg of sodium carbonate. Brisk effervescence of CO2 indicates acid nature of the compound. Alternatively, add in turn in a test tube, 1 ml of 1% KIO3 (potassium iodate), 2 ml of 10% KI, 1 ml of 1% starch solution (no blue colour should appear at this time), and few drops of aqueous extract. Immediate formation of a blue colour indicates the presence of acid group. Carboxylic acids. oxalic acid, succinic acid, tartaric acid and citric acid (sulphanilic acid and anthranilic acid also give a positive test) Note (2): Aromatic carboxylic acid, members of Group B, are partially soluble in cold water. Thus, slight acidity in the filtrate is due to them. On evaporation of aqueous extract too small residue is obtained. Absence of above acids indicates the presence of aromatic acids. They should be separated and confirmed as in Group B. (iii) Test with nitrous acid. A portion of aqueous solution is treated with 0.5 ml of 2% NaNO2 and 1 ml of 4M HCl. Rapid effervescence of N2 is observed in case the following compounds of diverse classes are present. Urea, thiourea, glycine (iv) Diazotization and coupling. A portion of aqueous solution is treated with 2-3 drops of 2% NaNO2 and 1 ml of 4M HCl and shaken for about 30 second. It is mixed with 2-3 drops of NED [N-(1-naphthyl) ethylene diamine dihydrochloride] when a pink to red colour is formed. Sulphanilic acid, anthranilic acid (v) Test with NaOH. Treat a small portion of aqueous solution with 0.5 ml of 5% NaOH and allow the solution to keep for some time when the solution turns to yellow, brown and finally dark brown. Hydroquinone, pyrogallol and resorcinol Identification of the Compounds of Group A Glucose. C6H12O6, m.p. 146o See box no. 1 for the reagents used in detection. Fehling test. Mix 0.5 ml each of Fehling solutions A and B. An intense blue solution should result; this solution is called Fehling reagent. Add to this, a small portion of the aqueous extract and boil gently. Formation of a precipitate (Cu2O) that is initially yellow but turns to red on standing is a positive test. This test indicates that the carbohydrate is reducing in nature. Tollen test. Add to this reagent solution about 0.5 ml of the aqueous extract and place the test tube in a water-bath at 50-60oC. A silver mirror is produced in 1-2 min. This test indicates that the carbohydrate is reducing in nature. Test for monosaccharide. To about 0.5 ml of aqueous extract, add 1 ml of Barfoed reagent or Benedict reagent. Place the tube in boiling water-bath for 3-4 min. Formation of a yellow-orange or orange-red precipitate (Cu2O) is a positive test. Note the influence the reaction medium!

A.Jain and K.K. Verma: A LABORATORY COURSE ON QUANTITATIVE ORGANIC ANALYSIS

Rapid Furfural test. To about 0.5 ml of aqueous extract, add 2-5 drops of alcoholic solution of 1-naphthol and 2 ml of concentrated HCl. Boil the solution gently. In case of glucose either no violet colour is formed or the colouration appears after 1 min of boiling. In this way glucose can be distinguished from fructose. Osazone formation. To 1 ml of aqueous extract, add about 0.2 g of phenylhydrazine hydrochloride, about 0.3 g of sodium acetate, 1 or 2 drops of glacial acetic acid and about 10 ml of water. Shake to dissolve the solids and place the tube in a boiling water-bath. Yellow crystals of osazone appear after about 10 min. Filter the osazone, wash with water and dry. m.p., 204o.

Box No. 1. Tollen reagent. To 0.5 ml of 1% silver nitrate solution, add a drop of 4M NaOH, and just dissolve the precipitate by dropwise addition of ammonium hydroxide. This solution is prepared only at the time of requirement since it has limited shelf-life. Barfoed reagent. Dissolve 15 g of copper(II) acetate in 250 ml of water and add 2.5 ml of glacial acetic acid. Benedict reagent. Dissolve 4.5 g of copper(II) sulphate (pentahydrate) in 40 ml of water. Dissolve separately 43 g of sodium citrate and 30 g of sodium carbonate in 150 ml of water. Mix the two solutions and dilute to 250 ml with water. Fehling solution A. Dissolve 15 g of copper(II) sulphate (pentahydrate) in about 150 ml of water and dilute to 250 ml. Fehling solution B. Dissolve 80 g of sodium potassium tartrate (Rochelle salt) in about 150 ml of water. Dissolve separately 30 g of sodium hydroxide in about 50 ml of water. Mix the two solutions, cool and dilute to 250 ml with water. When needed, equal volumes of Fehling solutions A and B are mixed together; the resulting solution is bright blue in colour. Copper(II) forms a complex with tartrate ion in alkaline medium.

Fructose. C6H12O6, m.p. 102o Fructose rapidly gives positive tests with Fehling solution and Tollen reagent as given under glucose. Test for monosaccharide is also positive. Rapid Furfural test is answered by fructose, i.e., the violet colouration appears rapidly and within 1 min. Seliwanoff test. To a small portion of aqueous extract, add 1-2 mg of resorcinol and 1-2 ml of concentrated HCl, and heat. A red colour is formed. This test indicates the presence of a ketose. Place in a test tube, 2 drops of concentrated H2SO4, 2-3 drops of SnCl2 solution, about 10 mg of urea and 23 drops of fructose solution. Heat gently for about 1 min. On cooling a blue colour appears indicating the sugar is ketohexose. Heat a portion of aqueous extract with a small volume of ammonium molybdate, a blue colour is formed. Fructose forms osazone by the same method as given for glucose and the product obtained is also the same. ( The reaction with phenylhydrazone is confined only at C1 and C2 and the structure of rest of two molecules, C3 to C6, is the same.) Maltose, C12H22O11, m.p. 102o (monohydrate). Maltose reduces Fehling solution ( it is a reducing sugar) but not Barfoed reagent ( it is a disaccharide). Thus, it is a reducing disaccharide; different from sucrose. Delayed formation of violet colour in Rapid Furfural test, as with glucose. Maltose forms osazone after 1 hour of heating, m.p. 206o. Sucrose. C12H22O11, m.p. 169o. Both tests with Fehling solution and Tollen reagent are not positive with sucrose. This confirms the nonreducing nature of sucrose. Test for monosaccharide is negative. No osazone is formed by sucrose. Thus different from glucose and fructose. Rapid Furfural test is positive, as with fructose. Sucrose is hydrolyzed to a mixture of glucose and fructose (responsible for rapid colouration) under the condition of test. Forms blue solution with copper(II) sulphate and NaOH.

A.Jain and K.K. Verma: A LABORATORY COURSE ON QUANTITATIVE ORGANIC ANALYSIS

Forms violet colour on warming with cobalt nitrate and NaOH. Oxidation with nitric acid forms oxalic acid, m.p. 101o. To about 0.2 g of compound, add 2 ml of concentrated HNO3 and heat the tube in a boiling water-bath. Care, toxic fumes of nitrogen oxides are evolved. The reaction subsides in about 10 min. Continue heating until almost dry residue appears. Cool in ice, add about 2 ml of rectified spirit (95% ethanol) and filter the crystals of oxalic acid, m.p. 101o. Oxalic acid. C2H2O4, m.p. 101o. To a portion of aqueous extract, add dilute NH4OH until the solution gives smell of ammonia and then dropwise add dilute acetic acid until solution smells as vinegar. Add 2-3 ml of calcium chloride solution and briefly warm the solution. A white precipitate (calcium oxalate) appears. Filter the precipitate, wash with little water, and dissolve the precipitate in dilute H2SO4. Add 1 or 2 drops of dilute KMnO4 solution and warm. Quick disappearance of purple colour indicates oxalic acid. Reduction of MnO4- by oxalic acid in acid medium. S-Benzyl isothiouronium salt (SBT), m.p. 195o. Follow instructions for preparation as given in the box no. 2. Neutralization equivalent, 63 (oxalic acid dihydrate). Follow instructions given in box no. 3.

Box No. 2. Organic acids (as anions) react with S-benzyl isothiouronium chloride in aqueous medium to give corresponding salts (SBT), which usually have sharp m.p. Polyacids and amino acids do not give satisfactory results. Caution! S-Benzyl isothiouronium chloride undergoes hydrolysis (more rapidly in alkaline medium) to give off benzyl mercaptan which is obnoxious. Wash hands with rectified spirit in case of contact. Preparation of S-Benzyl isothiouronium salt (SBT) Dissolve or suspend about 0.1 g of compound in 2 ml of water, add dilute NH4OH dropwise with shaking until smell of ammonia persists. Heat to expel off excess ammonia. In another tube dissolve 0.1 g of S- benzyl isothiouronium chloride in 2-3 ml of water. Mix the two solutions and cool in ice-water. Filter off the S-benzyl isothiouronium salt that has separated, and recrystallize from ethanol-water (90:10, v/v).

Box No. 3. Mass of organic acid that neutralizes 1 mol of NaOH (1 litre of 1M NaOH) is its neutralization equivalent. Determination of Neutralization Equivalent Dissolve an accurately weighed amount (50 mg) of acid in about 20 ml of water in a 100 ml Erlenmeyer flask. Add 2 or 3 drops of phenolphthalein indicator and titrate with 0.05M NaOH till the appearance of a pink colour. If w = weight (mg) of organic acid; V = titre (ml) of NaOH solution; and M = concentration of NaOH solution, the Neutralization Equivalent = w/VM.

Succinic acid. COOH(CH2)2COOH, m.p. 185o. Gives buff (colour of dull yellow leather; colour of MnS) precipitate with neutral FeCl3 that is soluble in dilute hydrochloric acid. Follow instructions in box no. 4 for the preparation of neutral FeCl3. Fluorescein test. In a dry test tube, mix a small amount of succinic acid with roughly double the amount of resorcinol and about 2 to 3 drops of concentrated H2SO4. Gently fuse on a small flame. Pick up the fused mass at the tip of a glass rod and dip into dilute NaOH solution. A red dye with strong green fluorescence appears. S-Benzyl isothiouronium salt (SBT), m.p. 154o. Neutralization equivalent, 59.

Box No. 4. Stored solution of ferric chloride undergoes hydrolysis giving iron(III) hydroxide, brown-red sediment, and hydrochloric acid. Iron(III) complexes of many organic acids are soluble in acid medium. This makes necessary use of neutral ferric chloride chloride. Neutral Ferric Chloride solution. To about 1 ml of ferric chloride solution, add dropwise with shaking dilute solution of NH4OH until the redbrown precipitate that is initially formed disappears giving a faint red-brown coloured solution.

A.Jain and K.K. Verma: A LABORATORY COURSE ON QUANTITATIVE ORGANIC ANALYSIS

Tartaric acid. COOH(CHOH)2COOH, m.p. 170o (+); 203o (monohydrate). Tollen test. To about 0.5 ml of aqueous extract, add dropwise dilute NH4OH till the solutions smells of ammonia. Add 1-2 ml of Tollen reagent and place the tube in warm water. A silver mirror is formed in a few min. Fenton test. To about 0.5 ml of aqueous extract, add in sequence 1 or 2 drops of freshly prepared FeSO4 solution, 1 drop of H2O2 and 3 to 5 drops of NaOH solution. An intense violet colour is formed. Neutralization equivalent, 75 (anhydrous); 84 (monohydrate). Citric acid. COOHC(OH)(CH2COOH)2, m.p. 100o (monohydrate) Iodoform test. To about 0.5 ml of aqueous extract, add 1 ml of 2% KI3 (iodine solution in potassium iodide) and 2 ml of NaOH solution. Warm in a water-bath. Characteristic smell of iodoform appears with precipitation of yellow iodoform depending on the amount of citric acid taken for analysis. Evaporate a small portion of aqueous extract. Mix the residue with 0.5 ml of acetic anhydride and about 0.1 g of sodium acetate, and heat on a small flame. A blood red colour appears. Neutralization equivalent, 70 (monohydrate). Urea. NH2CONH2, m.p. 132o Lassaigne test. Test for nitrogen is positive. Follow instructions given in the box no. 5. Evaporate a small portion of aqueous extract. Heat the residue in a dry test tube above its melting point. Note the smell of ammonia. The liquid mass solidifies on cooling. Dissolve the solid in 1-2 ml of NaOH solution and add a drop of very dilute solution of CuSO4. A purple colouration is obtained. Urea nitrate, m.p. 163o. Add a few drops of concentrated HNO3 to a concentrated solution of urea. A white precipitate is formed. Urea oxalate, m.p. 171o. To a concentrated solution of urea, add concentrated solution of oxalic acid. Warm the tube and scratch the tube with a glass rod. White crystals of urea oxalate separate. Thiourea. NH2CSNH2, m.p. 182o Lassainge test. Tests for both nitrogen and sulphur are positive. Follow instructions given in the box 5. To a small portion of aqueous extract, add 1 ml of dilute HCl and 1 ml of 2% NaNO3. Shake the solution. Add 1 or 2 drops of FeCl3 and note the formation of blood red colour (ferric thiocyanate). Heat a small amount of solid thiourea in a dry test tube above the melting point, cool and dissolve the solid in 1 ml of water. Add 1 or 2 drops of FeCl3 and note the formation of blood red colour (ferric thiocyanate). To a small portion of aqueous extract, add 0.5 ml of 2% lead acetate (or 1% AgNO3) and 2 ml of NaOH solution. Heat the solution. A black precipitate of PbS (or Ag2S) is formed. To a small portion of aqueous extract, add 1 ml of K4[Fe(CN)6] and 1 ml of acetic acid. A green colour that soon changes to blue is formed. S-Benzyl isothiouronium chloride, m.p. 175o. Evaporate a small volume of aqueous extract. Shake the residue with 2 ml of ethanol and 2 drops of benzyl chloride (C6H5CH2Cl), and briefly heat in a water-bath. A white precipitate appears (the product is soluble in water). Glycine. NH2CH2COOH, m.p. 232o (why this simple compound has such a high m.p.?) Lassainge test. Test for nitrogen is positive. Follow instructions given in the box no. 5. To a portion of aqueous extract, add 2 or 3 drops of FeCl3 solution. A red colour is formed.

A.Jain and K.K. Verma: A LABORATORY COURSE ON QUANTITATIVE ORGANIC ANALYSIS

To a portion of aqueous extract, add 1 or 2 drops of 1% CuSO4 solution. An intense blue colour is formed. N-Benzoyl glycine, m.p. 187o. Follow the instructions given in the box no. 6.

Box No. 5. Presence of certain elements such as nitrogen, sulphur, chlorine, bromine and iodine provides a valuable information in identification of an organic compounds. These elements are best detected by mineralization of organic compound by fusion with sodium and conducting test for anions that are formed. Caution! The compound should be free from traces of moisture. Wearing eye protection glasses recommended. Lassaigne test Preparation of sodium fusion extract. Place a small piece of Na metal in an ignition tube and cover with 30-40 mg of organic compound. Gently heat over a small Bunsen flame until the bottom of tube is red hot. Drop the hot tube in about 10 ml of distilled water taken in a hard glass boiling tube. The tube breaks liberating the fused mass. Boil for about 1 min and filter. Test for nitrogen. To about 2 ml of sodium fusion extract, add a few crystals of FeSO4 and 1 or 2 drops of NaOH, and boil for 1 min. Cool the solution, add 1 drop of FeCl3 solution. Formation of a Prussian blue colour is a positive test. Test for sulphur. To 2 drops of sodium fusion extract, add 1 or 2 drops of 5% sodium nitroprusside and shake. A violet colour is immediately formed. When both sulphur and nitrogen are present, sodium fusion can also give CNS- which can be tested by treating 2 or 3 drops of sodium fusion extract with 2 drops of dilute HCl and 2 drops of FeCl3. A blood red colour is formed. Test for chlorine. Acidify 0.5 ml of sodium fusion extract with dilute HNO3 and add 3 to 5 drops of AgNO3 solution. A white precipitate soluble in dilute NH4OH indicates chlorine. Test for bromine/iodine. Acidify 0.5 ml of sodium fusion extract with dilute HNO3, add 2 to 3 drops of CHCl3 or CCl4 and dropwise add with shaking chlorine solution in water. Formation of violet colour in organic layer indicates iodine. Formation of brown colour in organic layer indicates bromine. If the organic layer has violet colour, add excess of chlorine water and shake. Disappearance of violet colour leaving (i) a brown colour in organic layer indicates bromine, (ii) colourless organic layer indicates absence of bromine.

Box No. 6. Benzoates (benzoyl derivatives) of amines and phenols have higher m.p. than the corresponding acetyl derivatives. Owing to lower solubility, they can be most readily recrystallized. Caution! The reagent has a pungent odour and is lachrymatory. Schotten-Baumann reaction for benzoylation Dissolve (or suspend) about 0.1 g of compound in 4 ml of 5% NaOH in a boiling test tube provided with a rubber stopper, add 2 to 3 drops of benzoyl chloride (C6H5COCl), stopper the tube and shake vigorously for about 3-4 min. Usually a solid compound separates out. The product remains in solution if the a carboxyl or sulphonic group is also present in the substrate. In this situation, acidify the mixture by dropwise addition of concentrated HCl, filter the product and wash it with little warm water (to remove benzoic acid formed by hydrolysis of excess benzoyl chloride). Recrystallize the product from ethanol-water.

Sulphanilic acid. H2N-C6C4-SO3H (1,4), m.p. >300o (decomposition) Lassaigne test. Tests for both nitrogen and sulphur are positive. Follow instructions given in the box no. 5. Test for aromatic amino group positive. Follow the instructions given in the box no. 7. Test for -SO3H group para to -NH2 group. Acidify 3 drops of aqueous extract with 0.5 ml of dilute HCl and add dropwise with shaking a saturated aqueous bromine solution till the appearance of yellow colour of excess bromine. Keep for 1-2 min. A white precipitate of 2,4,6-tribromoaniline is formed (keep as derivative). Dilute with 1 ml of distilled water and filter (or centrifuge). To the clear filtrate, add 3 drops of BaCl2 solution. Formation of a white precipitate of BaSO4 is a positive test. Why is -SO3H group knocked off! 2,4,6-Tribromoaniline, m.p. 119o

A.Jain and K.K. Verma: A LABORATORY COURSE ON QUANTITATIVE ORGANIC ANALYSIS

Box No. 7. Test for aromatic amino group Diazotization and coupling reaction. Add to 2 drops of the aqueous extract or to about 5 mg of compound, 0.5 ml of dilute HCl and 1 drop of 2% NaNO2 solution. Add 2 drops of NED, [N-(1-naphthyl) ethylenediamine 2HCl], reagent solution. Immediate formation of a pink to red colour is a positive test. NED, [N-(1-naphthyl) ethylenediamine 2HCl], reagent solution. Dissolving 0.2 g of the reagent in 100 ml of water pre-mixed with 1 ml of concentrated HCl. This solution keeps well for about 15 days in amber bottle. Reaction with lead tetraacetate. Place 1 mg (or less) of compound in a depression on a spot plate or a watch glass, and dissolve it in 1 or 2 drops of glacial acetic acid. Add 1 drop of lead tetraacetate reagent solution to it. Red-brown-orange colouration usually develops. Lead letraacetate reagent solution. Heating in a water-bath 25 ml of glacial acetic acid and add to it in small increments 0.1 g of Pb3O4. Continue heating with occasional stirring until the whole red oxide dissolves. Cool and store in amber bottle. This solution keeps well for about 15 days. Reaction with 4-dimethylaminobenzaldehyde. Place 1 mg (or less) of compound in a depression on a spot plate or a watch glass, and dissolve it in 1 or 2 drops of methanol. Add 1 drop of reagent solution to it. A yellow colour appears. 4-Dimethylaminobenzaldehyde reagent solution. Dissolve 100 mg of reagent in 50 ml of methanol.

Hydroquinone (quinol). C6H4(OH)2 (1,4), m.p. 169o To a portion of aqueous extract, add a few drops of Tollen reagent. A black precipitate (Ag) is formed immediately. Mix 0.5 ml each of Fehling solutions A and B, add a portion of aqueous extract. A red-brown precipitate (Cu2O) is formed immediately in cold. Both these tests indicate the reducing nature of the compound. To a portion of aqueous extract, add a drop of neutral FeCl3 solution (use dropper); note the transient blue colour. Dibenzoate, m.p. 205o Pyrogallol. C6H3(OH)3 (1,2,3), m.p. 133o To a portion of aqueous extract, add a few drops of Tollen reagent. A black precipitate (Ag) is formed immediately. Mix 0.5 ml each of Fehling solutions A and B, add a portion of aqueous extract. A red-brown precipitate (Cu2O) is formed immediately in cold. Both these tests indicate the reducing nature of the compound. To a portion of aqueous extract, add FeSO4 solution; a blue colour/precipitate appears. Tribenzoate,, m.p. 90o Resorcinol. C6H4(OH)2 (1,3), m.p. 110o To a portion of aqueous extract, add neutral FeCl3 solution; a blue-violet colour is formed. Fluorescein test. Mix in a dry test tube about 20 mg of compound with 10 mg of phthalic anhydride and 1 ml of concentrated H2SO4. Heat on a small Bunsen flame for about 1 min. Pick up the fused mass at the tip of a glass rod and dip it into dilute NaOH solution. A red dye with strong green fluorescence appears. Dibenzoate, m.p. 117o

A.Jain and K.K. Verma: A LABORATORY COURSE ON QUANTITATIVE ORGANIC ANALYSIS

GROUP B

Benzoic acid. C6H5COOH, m.p. 122o Suspend 10-20 mg of compound in 1 ml of water, add 2 or 3 drops of neutral FeCl3 solution. A buff precipitate appears. S-Benzyl isothiouronium salt (SBT), m.p. 167o. Neutralization equivalent, 122. Phenylacetic acid. C6H5CH2COOH, m.p. 76o S-Benzyl isothiouronium salt (SBT), m.p. 165o. Neutralization equivalent, 136. Salicylic acid, HOC6H4COOH (1,2), m.p. 158o Suspend 10 mg of compound in 1 ml of distilled water and add 2 or 3 drops of neutral FeCl3. Note the appearance of purple colour. FeCl3. shelf reagent, without neutralization, also gives the colour reaction. Place in a dry test-tube about 20 mg of compound, 1 ml of CH3OH and carefully add 0.5 ml of concentrated H2SO4. Heat in boiling water-bath for 1-2 min. Pour the reaction mixture in 5 ml of water, taken in boiling test tube. Note the smell of oil of wintergreen due to the formation of methyl salicylate. S-Benzyl isothiouronium salt (SBT), m.p. 146o. 2-Methoxybenzoic acid, m.p. 100o. Follow the instructions for methylation given in the box no. 8. Acetylsalicylic acid. CH3COOC6H4COOH (1,2), m.p. 135o Suspend 10 mg of compound in 1 ml of distilled water and add 2 or 3 drops of neutral FeCl3. Note a very faint or no purple colour. Faint colour is due to contamination with salicylic acid. Keep the tube in warm water, the purple colour deepens. S-Benzyl isothiouronium salt (SBT), m.p. 144o

Box No. 8. Methylation is a common reaction for hydroxyl compounds. Procedure for methylation with dimethyl sulphate To about 100 mg of hydroxyl compound, add 2 ml of 5% NaOH and 3 to 5 drops of dimethyl sulphate (Caution! Corrosive liquid). Stopper the tube and shake vigorously until the oily drops of the reagent disappear. A crystalline product separates at this stage. If the hydroxyl compound has a carboxyl group (as in salicylic acid), the product remains in solution as its sodium salt. Acidify the reaction mixture by dropwise addition of concentrated HCl and cool in ice-water. Filter the product. Recrystallize the product from ethanol-water.

Phthalic acid. C6H4(COOH)2 (1,2), m.p. 195o (decomposition) Fluorescein test. Place in a dry test about 10 mg of the compound, 20 mg of resorcinol and 1 ml of concentrated H2SO4. Heat on a small Bunsen flame for about 1 min. Pick up the fused mass at the tip of a glass rod and dip it into dilute NaOH solution. A red dye with strong green fluorescence appears. Place in a dry test about 10 mg of the compound, 20 mg of phenol and 1 ml of concentrated H2SO4. Heat on a small Bunsen flame for about 1 min. Pick up the fused mass at the tip of a glass rod and dip it into dilute NaOH solution. A pink dye appears. Add dilute HCl, the solution turns colourless.

A.Jain and K.K. Verma: A LABORATORY COURSE ON QUANTITATIVE ORGANIC ANALYSIS

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S-Benzyl isothiouronium salt (SBT), m.p. 157o. Neutralization equivalent, 83. Cinnamic acid. C6H5CH=CHCOOH, m.p. 133o To about 20 mg of compound add 50 mg of Na2CO3 and 2 ml of distilled water. Add dropwise with shaking 1% KMnO4 solution. Note the immediate decolourization of purple colour that denotes the presence of unsaturation. Also note the smell of benzaldehyde. To about 20 mg of compound add 50 mg of Na2CO3 and 2 ml of distilled water. Add bromine-water dropwise till a slight excess of bromine persists. Note the formation of an oily product (bromostyrene, C6H5CH=CHBr) with characteristic odour. S-Benzyl isothiouronium salt (SBT), m.p. 175o. Neutralization equivalent, 148. Benzilic acid. (C6H5)2C(OH)COOH, m.p. 170o In a dry test tube mix 5 mg of compound with 2 or 3 drops of concentrated H2SO4. A red colour is formed. The compound gives positive Molisch test (explain the chemistry; it is not a carbohydrate). Dissolve 0.2 g of K2Cr2O7 in about 1 ml of water, add about 1 ml of concentrated H2SO4 and 20 mg of the compound. Warm in a water bath. Dilute with 5 ml of water and cool in ice-water, filter and wash with ice-water. Colourless crystals of benzophenone, m.p. 48o, with characteristic odour are formed. S-Benzyl isothiouronium salt (SBT), m.p. 125o. Neutralization equivalent, 228. 4-Nitrobenzoic acid. O2NC6H4COOH (1,4), m.p. 241o Lassaigne test for nitrogen positive. Follow instructions given in the box no. 5. Tests for nitro group positive. Follow the instructions given in the box no. 9 for (i) reduction and dye test, and (ii) Mlliken test. Neutralization equivalent, 167.

Box No. 9. Tests for aromatic nitro group Reduction and dye test. Dissolve about 10 mg of compound in 1 ml of rectified spirit, add a piece of zinc metal, 1 ml of water and 1 ml of concentrated HCl. Allow to keep for 2-3 min with occasional shaking. Add 1 ml of water and filter. To the clear filtrate, add 1 drop of 2% NaNO2 solution and 2 drops of NED, [N-(1-naphthyl) ethylenediamine 2HCl], reagent solution. Immediate formation of a pink to red colour is a positive test. Mlliken test. Dissolve about 10 mg of compound in 1 ml of rectified spirit, add 1 ml of water, 20 mg of NH4Cl and about 20 mg of zinc dust. Shake the mixture and allow to keep for 2-3 min with occasional shaking. Add 2 ml of water and filter. Spot a drop of clear filtrate on filter paper and place over it a drop of Tollen reagent. Turning the spot to black is a positive test. (Chemistry of the test?) The Mlliken test has a special value in detecting nitro group in the presence of amino group, e.g., in nitroanilines. (Why is it so?)

3,5-Dinitrobenzoic acid. (O2N)2C6H3COOH (1,3,5), m.p. 205o Lassaigne test for nitrogen positive. Follow instructions given in the box no. 5. Tests for nitro group positive. Follow the instructions given in the box no. 9 for (i) reduction and dye test, and (ii) Mlliken test. To about 10 mg of compound, add 1 ml of acetone and 1 ml of 5% NaOH. A purple colour is formed. This test indicates the presence of two nitro groups that are 1,3 (meta) to each other. Neutralization equivalent, 212.

A.Jain and K.K. Verma: A LABORATORY COURSE ON QUANTITATIVE ORGANIC ANALYSIS

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2-Chlorobenzoic acid. ClC6H4COOH (1,2), m.p. 140o Lassaigne test for chlorine positive. Follow instructions given in the box no. 5. Neutralization equivalent, 156.5.

GROUP C

1-Naphthol. C10H7OH (1), m.p. 94o Dissolve about 10 mg of compound in excess of NaOH solution. Add iodine solution in KI (2% KI3). A violet colour appears. Diazotize 0.5 ml of 0.1% solution of 4-nitroaniline in 2M HCl by dropwise addition of 1% NaNO2 (the yellow colour of nitroaniline is decolourized). Dissolve 2-5 mg of 1-naphthol in 1 ml of 4M NaOH solution, add 2 or 3 drops of diazotized 4-nitroaniline solution to it and shake well. A red dye is formed. Divide in two parts. Part I. Add excess of NaOH. The red dye turns violet. Part II. Add 3 or 4 drops of 2% MgSO4 solution, excess of NaOH solution and shake to mix. A blue lake [the azo dye adsorbed on to Mg(OH)2] is formed. Warm 10 mg of compound with 1 ml of dilute NaOH, 25 mg of Cu powder and 0.5 ml of CCl4. A blue colour is formed. 2-Naphthol does not give any colour in above tests. Benzoate, m.p. 56o 2-Naphthol. C10H7OH (1), m.p. 122o Diazotize 0.5 ml of 0.1% solution of 4-nitroaniline in 2M HCl by dropwise addition of 1% NaNO2 (the yellow colour of nitroaniline is decolourized). Dissolve 2-5 mg of 2-naphthol in 1 ml of 4M NaOH solution, add 2 or 3 drops of diazotized 4-nitroaniline solution to it and shake well. A red dye is formed. Add excess of NaOH. The dye remains red-orange (different from 1-naphthol). Warm 10 mg of compound with 1 ml of concentrated NaOH and 0.5 ml of CHCl3. A blue colour is formed. Benzoate, m.p. 107o. Methyl 2-naphthyl ether (2-methoxynaphthalene, narolin), m.p. 107 Thymol. (CH3)2CHC6H3(CH3)OH, m.p. 50o Gently warm 10 mg of compound with 1 drop of 5% KOH solution and 2 drops of iodine solution in KI (2% KI3). A red colour results. Benzoate, m.p. 33o. Salicylamide. HOC6H4CONH2 (1,2), m.p. 139o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Boil 20 mg of compound with 1 ml of 10% NaOH. Note the odour of ammonia. Suspend 5 mg of compound in 2 ml of water. Add 2 drops of FeCl3. A violet colour results. Benzoate, m.p. 143o. Phthalimide. C6H4(CO)2NH, m.p. 233o

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Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Boil 20 mg of compound with 1 ml of 10% NaOH. Note the odour of ammonia. Place in a dry test about 10 mg of the compound, 20 mg of resorcinol and 1 ml of concentrated H2SO4. Heat on a small Bunsen flame for about 1 min. Pick up the fused mass at the tip of a glass rod and dip it into dilute NaOH solution. A red dye with strong green fluorescence appears. Place in a dry test about 10 mg of the compound, 20 mg of phenol and 1 ml of concentrated H2SO4. Heat on a small Bunsen flame for about 1 min. Pick up the fused mass at the tip of a glass rod and dip it into dilute NaOH solution. A pink dye appears. Add dilute HCl, the solution turns colourless. N-Benzylphthalimide, m.p. 116o. Suspend 50 mg of phthalimide in 4 ml of water. Add dropwise with shaking 5% NaOH till the compound dissolves. Add 2 or 3 drops of benzyl chloride (C6H5CH2Cl) and warm in a boiling water-bath. Add 10 ml of water and filter. Wash the solid product with 1:1 ethanol-water. Recrystallize from glacial acetic acid. Uric acid. C5H4O3N4, m.p. the compound decomposes Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Dissolve 25 mg of compound in NaOH solution. Add Fehling solution (equal volumes of Fehling solution A and B) drop by drop. Note the formation of a white precipitate (copper ureate). Add an excess of Fehling solution and boil. A red precipitate (Cu2O) is formed. Dissolve 10 mg of compound in NaOH solution. Combine 1 or 2 drops of this solution with 1 drop of 2% AgNO3 over a filter paper. A black stain (Ag) is formed. Murexide test. Place 50 mg of uric acid and 2 or 3 drops of concentrated HNO3 on a porcelain dish and heat it very gently to dryness. Cool and add 1 drop of NH4OH. A purple colour is formed (ammonium purpurate or murexide). Now add 1 drop of NaOH solution, the colour changes to blue.

GROUP D

4-Toluidine. CH3C6H4NH2 (1,4), m.p. 43o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Test for aromatic amino group is positive. Follow instructions given in the box no. 7. Benzoate, m.p. 144o. 4-Anisidine. CH3OC6H4NH2 (1,4), m.p. 43o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Test for aromatic amino group is positive. Follow instructions given in the box no. 7. Dissolve the compound in dilute HCl. Add 2-3 drops of FeCl3. A violet colour is formed (different from 4toluidine). Benzoate, m.p. 154o. 2-Nitroaniline. O2NC6H4NH2 (1,2), m.p. 71o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Test for aromatic amino group is positive. Follow instructions given in the box no. 7. Mlliken test for nitro group is positive. Follow instruction given in the box no. 9.

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Dissolve 2-3 mg of compound in dilute HCl. Add 3 to 4 drops of 1% NaNO2 solution. Add 2 or 3 drops of 1-naphthol solution in 1% NaOH. An orange-red dye is formed. Add excess of NaOH, the colour deepens to red. Benzoate, m.p. 98o. 3-Nitroaniline. O2NC6H4NH2 (1,3), m.p. 114o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Test for aromatic amino group is positive. Follow instructions given in the box no. 7. Mlliken test for nitro group is positive. Follow instruction given in the box no. 9. Dissolve 2-3 mg of compound in dilute HCl. Add 3 to 4 drops of 1% NaNO2 solution. Add 2 or 3 drops of 1-naphthol solution in 1% NaOH. An orange-red dye is formed. Add excess of NaOH, the colour changes to brown. Benzoate, m.p. 155o. 4-Nitroaniline. O2NC6H4NH2 (1,4), m.p. 147o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Test for aromatic amino group is positive. Follow instructions given in the box no. 7. Mlliken test for nitro group is positive. Follow instruction given in the box no. 9. Dissolve 2-3 mg of compound in dilute HCl. Add 3 to 4 drops of 1% NaNO2 solution. Add 2 or 3 drops of 1-naphthol solution in 1% NaOH. An orange-red dye is formed. Add excess of NaOH, the colour changes to violet. Benzoate, m.p. 199o.

GROUP E

1,3-Dinitrobenzene. C6H4(NO2)2 (1,3; meta), m.p. 90o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Mlliken test for nitro group is positive. Follow instruction given in the box no. 9. Dissolve 2-4 mg of compound in acetone and add 2 or 3 drops of NaOH. A deep violet colour is formed. Add 0.5 ml of dilute acetic acid, the colour turns into red. This test confirms the presence of two nitro group in the compound which are 1,3 or meta to each other. Boil about 5 mg of compound with 2 ml of water and 1 drop of dilute NaOH solution. Add a very dilute solution of glucose. A purple colour appears. Adduct with naphthalene (charge-transfer complex). A 50 mg portion of compound and an equal amount of naphthalene are separately dissolved in 2 ml each of benzene (Caution! Benzene vapours are highly toxic), warming the tubes in hot water-bath. Next, the two solutions are mixed together and heated in water-bath for further 1 min. The mixture is poured on to watch glass and the solvent allowed to evaporate slowly under hood. The adduct of 1,3dinitrobenzene with naphthalene is left as yellow residue, m.p. 52o. 1-Nitronaphthalene. C10H7NO2 (1 or ), m.p. 60o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Mlliken test for nitro group is positive. Follow instruction given in the box no. 9. Place in a dry test tube about 5 mg of 1-nitronaphthalene, pour 2-4 drops of concentrated H2SO4. A blood red colouration appears.

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Warm in a water-bath 50 mg of compound with 1 ml of concentrated HNO3 and 1 ml of concentrated H2SO4. 1,3,8-Trinitronaphthalene, m.p. 218o forms. Add 5-6 ml of water and filter the product. 4-Nitrotoluene. CH3C6H4NO2 (1,4; para), m.p. 52o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Mlliken test for nitro group is positive. Follow instruction given in the box no. 9. Warm in a water-bath 50 mg of compound with 1 ml of concentrated HNO3 and 1 ml of concentrated H2SO4. 2,4-Dinitrotoluene, m.p. 70o forms. Add 5-6 ml of water and filter the product. 2,4-Dinitroaniline. NH2C6H3(NO2)2 (1,2,4), m.p. 180o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Test for aromatic amino group, by diazotization and coupling, is positive. Follow instructions given in the box no. 7. Mlliken test for nitro group is positive. Follow instruction given in the box no. 9. The compound does not dissolve in acids (Why?). Mix 10 mg of compound with 1 ml of acetone and dilute NaOH. A red colour appears. This confirms the presence of two nitro groups (1,3) meta to each other. Boil 20 mg of compound with 5 ml of concentrated NaOH. Note the smell of ammonia (Why is NH3 evolved? What does it suggest about the position of amino group with respect to nitro groups?). The solution turns intense red due to the formation of 2,4-dinitrophenol (Na salt). Acidify the solution with concentrated HCl. Collect the yellow product, wash with little ice-water, m.p. 114o. Benzoate, m.p. 202o. Diphenylamine. C6H5NHC6H5, m.p. 53o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Dissolve 2 mg of compound in 2 or 3 drops of concentrated H2SO4 and add 1 drop of dilute HNO3. A purple-blue colour is obtained. Reaction with lead tetraacetate (follow instructions given in box no. 7). A red-brown colour changing to permanent green is obtained. Benzoate, m.p. 180o. Benzamide. C6H5CONH2, m.p. 128o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Boil 10 mg of compound with 2 ml of concentrated NaOH solution. Note the smell of ammonia. Acidify with concentrated HCl and cool in ice-water. White crystals of benzoic acid, m.p. 121o, are formed. Acetanilide. C6H5NHCOCH3, m.p. 114o Lassaigne test for nitrogen is positive. Follow instructions given in the box no. 5. Boil 10 mg of compound with 2 ml of concentrated HCl and 1 ml of water. Dilute with 2 ml of water and cool in ice-water. Apply test for aromatic amino group. Follow instructions given in box no. 7.

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Dissolve 25 mg of acetanilide in 2 ml of glacial acetic acid. Add 1 ml of concentrated HCl and a solution of 100 mg of KBr in 2 ml of water. Add dropwise with shaking 5% KBrO3 solution until a yellow colour of excess bromine appears. Add 5 ml of water. Filter the precipitated 4-bromoacetanilide, m.p. 167o, and wash with water. 1,4-Dichlorobenzene. C6H4Cl2 (1,4; para), m.p. 53o Lassaigne test for chlorine is positive. Follow instructions given in the box no. 5. 1,4-Dibromobenzene. C6H4Br2 (1,4; para), m.p. 89o Lassaigne test for bromine is positive. Follow instructions given in the box no. 5. Naphthalene. C10H8, m.p. 80o Place 10 mg of the compound in a dry test tube. Add 2 or 3 drops of concentrated HNO3, mix by rotating the tube. Carefully add 1 or 2 drops of concentrated H2SO4. A blood red colour is formed. Picrate (yellow long needles), m.p. 150o. Follow instructions given in the box no. 10. Biphenyl (diphenyl). C6H5C6H5, m.p. 70o No specific direct colour test. Dissolve 0.1 g of compound in 2 ml of glacial acetic acid. Add 1 ml of concentrated HNO3 and heat the tube in a boiling water-bath. Pour the mixture into 10 ml of water. 4,4-Dinitrobiphenyl, m.p. 235o, separates out which is filtered and washed with water.

Box No. 10. Picrates are charge-transfer complexes. They have characteristic m.p. and are valuable aid in the identification of a number of compounds which behave as Lewis bases. Picrates also have value in purification of compounds which can form picrates. The latter are decomposed by hot water, picric acid being soluble in large excess of water, the Lewis base is liberated in pure state. Procedure for picrate formation. A 50 mg portion of compound and an equal amount of picric acid (dried by pressing between folds of filterpaper) are separately dissolved in 2 ml each of benzene (Caution! Benzene vapours are highly toxic), warming the tubes in hot water-bath. Next, the two solutions are mixed together and heated in water-bath for further 1 min. The mixture is poured on to watch glass and the solvent allowed to evaporate slowly under hood. This leaves picrate in characteristic shape and colour.

Anthracene. C14H10, m.p. 217o Mix 10 mg of anthracene with 1 ml of concentrated HNO3 and heat the tube in boiling water-bath. Add 4 ml of water and filter the product. Boil the product with 2 ml of NaOH and 20 mg of Zn dust. A red colour is formed which changes to colourless on cooling and becomes red on reheating. Picrate (bright red mass), m.p. 138o. Follow instructions given in box no. 10. Acenaphthene. C12H10, m.p. 95o No specific direct colour test. Picrate (yellow), m.p. 161o. Follow instructions given in box no. 10.

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Phenanthrene. C14H10, m.p. 100o No specific direct colour test. Picrate (yellow), m.p. 143o. Follow instructions given in box no. 10. Benzophenone. C6H5COC6H5, m.p. 48o Place 5 mg of compound in an ignition tube, add a piece of Na metal, about 40 mg of powdered naphthalene and briefly heat over a small Bunsen flame. Note a blue colour around Na metal. The colour persists on cooling the mass. (What is chemistry of this test?) Formation of an orange product on reaction with 2,4-dinitrophenylhydrazine confirms the presence of carbonyl group. Dissolve 50 mg of compound in about 2 ml of rectified spirit. Add 1 ml of 2,4dinitrophenylhydrazine reagent solution (see box no. 11 for preparing the reagent) and warm in a water-bath for about 2 min. Add 5 ml of water. An benzophenone-2,4-dinitrophenylhydrazone, m.p. 238o, separates out. Filter the product and wash with water.

Box No. 11. Formation of an orange product with 2,4-dinitrophenylhydrazine is a positive test for the carbonyl compounds. The products are also used as derivatives for carbonyl compounds. Preparation of the reagent solution. Suspend 0.5 g of powdered 2,4-dinitrophenylhydrazine in about 30 ml of methanol, carefully add in small portions 3 ml of concentrated H2SO4. Cool the solution and dilute to 50 ml with methanol.

Anthraquinone. C14H8O2, m.p. 284o (approximately) Boil the compound with 2 ml of NaOH and 20 mg of Zn dust. A red colour is formed which changes to colourless on cooling and becomes red on reheating. Reductive acetylation. Heat under reflux 0.1 g of anthraquinone, 0.1 g of Zn dust, 1 ml of acetic anhydride and 0.5 ml of pyridine for 15 min. While still hot, decant the liquid into 20 ml of water. Anthrahydroquinone diacetate, m.p. 260o, separates out. Filter the product and wash with water.

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COURSE ON ORGANIC SYNTHESIS

9,10-Anthraquinone
Place 0.1 g of anthracene in a 20 ml boiling test tube, add 1.5 ml of glacial acetic acid (Caution! Glacial acetic acid is pungent. Do not inhale the vapours) and warm in a water bath to dissolve the solid. Mix with 2 ml of concentrated nitric acid and heat in a boiling water bath for 15 min. Pour the contents into 30 ml of water taken in a 100 ml beaker and stir well. Filter at the pump, wash with water and drain well. Dry the product in an air oven. Yield, 0.1 g; m.p. 273o.

Methyl 2-naphthyl ether (2-methoxynaphthalene; nerolin)

Place 1 g of sodium hydroxide and 5 ml of water in a 20 ml boiling test tube and shake to dissolve the solid. Add 0.5 g of finely powdered 2-naphthol and again shake the mixture until a clear solution results. Add 0.5 ml of dimethyl sulphate (Caution!) and shake vigorously. Warm (70-80o) in a water bath for 30 min with occasional shaking. Allow to cool the contents. Filter off the methyl 2-naphthyl ether at the pump and wash first with 1% sodium hydroxide solution and then with water. Drain the product well. Yield, 0.5 g. Recrystallise about 0.1 g of product from ethanol-water; m.p. 72o.

Caution! Dimethyl sulphate must be handled with great care is it causes severe burns on the skin. In case of accident, wash the infected area with dilute ammonia and then liberally with water. Consult a doctor

Methyl orange
In a 20 ml boiling test tube place 0.5 g of sulphanilic acid (dihydrate), 0.2 g of sodium carbonate and 10 ml of water. Stir the mixture (warming slightly if necessary) until a clear solution of sodium sulphanilate results. Dissolve 0.2 g of sodium nitrite in 2 ml of water and add it to the sodium sulphanilate solution. Shake the mixed solution and cool in ice. Pour this solution slowly and with stirring into a 100 ml beaker containing 1 ml of concentrated hydrochloric acid and about 10 g of crushed ice. Stir for 5 min more and test for the presence of nitrous acid.

Test for nitrous acid: Place a drop of stirred solution in a depression in a spot plate already charged with a drop each of 10% potassium iodide and 1% starch; immediate formation of a blue colour indicates free nitrous acid.

Dissolve separately 0.5 ml of N,N-dimethylaniline in 2 ml of glacial acetic acid in a test tube and add it with vigorous stirring to the above diazotized sulphanilic acid solution. Allow the mixture to stand for 10 min. Add to it slowly and with stirring a solution of 4 g of sodium hydroxide in 10 ml of water. Then add 2-3 g of sodium chloride and warm the solution in a water bath pre-heated to 80-90o. Allow the mixture to cool undisturbed to room temperature and then in ice bath. Filter off the methyl orange under gentle suction and drain well. Yield, 0.6 g. Recrystallise 0.1 g from hot water; reddish-orange crystals of methyl orange separate as the solution cools. Methyl orange, being a salt, has no well-defined m.p. Note the indicator properties of methyl orange.

1,2,3,4-Tetrahydrocarbazole
In a 100 ml round-bottomed flask place 0.5 ml of cyclohexanone and 5 ml of glacial acetic acid. Attach a reflux water condenser to the flask and a separatory funnel to the top of the condenser. Use a T-joint with an open

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side arm placed between condenser and separatory funnel to avoid build up of pressure in the assembly. Charge the separatory funnel with 0.5 ml of phenylhydrazine (Caution! Phenylhydrazine is toxic. Do not inhale the vapours) and 5 ml of glacial acetic acid. Heat the mixture in the flask to gentle boiling and slowly run in phenylhydrazine solution during a period of 30 min. Reflux for further 5 min. Pour the reaction mixture into 50 ml of water (taken in a 100 ml beaker), stir vigorously while the product solidifies. Cool in ice-bath for 10 min and filter at the pump. Wash the product with water and dry. Yield, 0.7 g. Recrystallise from ethanol-water (shinning colourless plates); m.p. 116117o.

4-Nitrotoluene 4- nitrobenzylidene diacetate 4-nitrobenzaldehyde

4-Nitrobenzylidene diacetate Take 3 g of 4-nitrotoluene and 25 ml of acetic anhydride in a 250 ml round-bottomed flask, and place the flask in a bath of ice and salt (sodium chloride). Add slowly, with shaking, 6 ml of concentrated sulphuric acid (the addition is exothermic). Allow the flask to remain in ice bath for 10-15 min to fall the temperature below 10o. Dissolve 7 g of chromiun trioxide in 30 ml of acetic anhydride, and add this solution to above mixture slowly in small portions of about 1 ml at a time, stirring the mixture vigorously and do not allowing the temperature to rise above 10o. Keep the flask for 1 h when all chromium trioxide solution has been added. Pour the contents of flask on to 100 g of crushed ice being taken in a 250 ml beaker. Keep the flask until all ice has melted. Filter the product at the pump and wash with cold water until the washings are colourless. Dry in a vacuum desiccator. Yield 3.5 g; m.p. 106o. 4-Nitrobenzaldehyde Place 25 ml of water in a 100 ml round-bottomed flask and add slowly and carefully with good shaking 2 ml of concentrated sulphuric acid. Add 20 ml of ethanol and 3 g of powdered 4-nitrobenzylidene diacetate. Attach a reflux water condenser and boil the contents for 20 min. Filter any insoluble through a fluted filter paper, cool the filtrate in ice-bath for precipitation of the product. Filter at the pump, wash with cold water and dry in a vacuum desiccator. Yield, about 1.5 g; m.p. 106o.

Nitrobenzene 1,3-dinitrobenzene 3-nitroaniline

1,3-Dinitrobenzene Caution: This experiment should be carried out in a fume cupboard. Toxic fumes of nitrogen oxides (NO x) are evolved during this experiment. Place 2 ml of nitrobenzene in a 40 ml round-bottomed flask (or a boiling tube), cautiously add 8 ml of concentrated sulphuric acid and dropwise with shaking 6 ml of concentrated nitric acid. Heat in a boiling water bath for about 30 min with occasional shaking. Allow the mixture to cool somewhat and pour it cautiously with good stirring into 100 ml of cold water taken in a 250 ml beaker. The product soon solidifies. Filter at pump, wash the product thoroughly with cold water and allow to drain. Yield, 2.5 g. Recrystallise about 0.1 g of compound from ethanol-water mixture; m.p. 89-90o (colourless crystals). 3-Nitroaniline Dissolve 3 g of sodium sulphide (Na 2S.9H2O) in about 20 ml of water, add 1 g of finely powdered sulphur and warm with occasional stirring until a clear solution is formed (sodium polysulphide). Heat 2 g of 1,3-dinitrobenzene and 25 ml of water in a 250 ml beaker until water boils gently. Whilst stirring the boiling solution vigorously add sodium polysulphide solution in small portions of about 1 ml each during a period of 25-30 min. Boil gently for a further 15 min. Allow to cool in ice, filter the precipitate at pump and wash with little ice-water. Transfer the precipitate to a beaker, add 25 ml of water and 3 ml of concentrated hydrochloric acid, stir and warm in a water bath for 5 min. Filter the insoluble sulphur and any unchanged 1,3-dinitrobenzene. Cool the filtrate

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in ice bath and add about 2 ml of concentrated ammonium hydroxide. Keep in ice-bath to precipitate 3-nitroaniline completely. Filter the product at pump. Yield 1 g. Recrystallise about 0.1 g of compound from boiling water; m.p. 114o (bright yellow needles).

Acetanilide 4-nitroacetanilide 4-nitroaniline

4-Nitroacetanilide Place 3 g of powdered acetanilide in a 100 ml beaker, add 6 ml of glacial acetic acid and, whilst stirring the mixture, 5 ml of concentrated sulphuric acid. The mixture becomes warm and a clear solution results. Place the beaker in freezing mixture (ice-NaCl bath) and allow the temperature to fall to 5-10o. Add 2 ml of concentrated nitric acid in small portions by sliding over a glass rod and stirring well after each addition. Do not allow the temperature to rise above 10o. Remove the beaker from freezing mixture and allow it to stand on bench top at room temperature for 30 min. Then, pour the reaction mixture on to 50 g of crushed ice taken in a 250 ml beaker and allow to stand for 15 min. Filter the precipitated 4-nitroacetanilide at pump, wash it thoroughly with cold water to remove acids and drain well. Yield, 2.5 g. Recrystallise 0.1 g of compound from ethanol-water mixture; m.p. 214o (colourless crystals). 4-Nitroaniline Place 10 ml of water in a 50 ml round-bottomed flask and add to it 5 ml of concentrated sulphuric acid cautiously in small portions with good shaking. Add 2 g of powdered 4-nitroacetanilide to the acid solution and reflux in a boiling water bath for about 20 min. During this period all solid dissolves. Add about 20 ml of water and transfer the hot clear liquid to a 250 ml beaker. Cool it in an ice bath. Dissolve about 8 g of sodium hydroxide in 30 ml of water. Cool the solution in ice bath. Precipitate 4nitroaniline by adding sodium hydroxide solution to the above solution in small portions and with good stirring. Keep the beaker in ice bath for further 15 min. Filter the yellow crystalline precipitate at the pump, wash it with 3-4 ml of ice cold water and drain. Yield, 1.6 g. Recrystallise about 0.1 g of compound from hot water; m.p. 148o. TLC of crude 4-nitroaniline (contaminated with 2-nitroaniline).

Acetanilide 4-bromoacetanilide 4-bromoaniline

4-Bromoacetanilide
Potassium bromate-potassium bromide mixture: Dissolve 30 g of potassium bromate in about 200 ml of water (warm if necessary), add 180 g of potassium bromide and 200 ml of water, stir until a clear solution results, and dilute the solution to 1 litre with water.

Dissolve 1.5 g of finely powdered acetanilide in 5 ml of glacial acetic acid in a 100 ml conical flask, and add 20 ml of water and 2 ml of concentrated hydrochloric acid. Add bromate-bromide solution dropwise with constant shaking. A white product starts forming. Continue the addition of reagent until the solution acquires yellowbrown colour due to a slight excess of bromine. About 15 ml of reagent is required. Allow the reaction mixture to stand for 10 min with occasional shaking. The yellow-brown colour of excess bromine should persist at the end of this period. Pour the reaction product into 60 ml of water taken in a 250 ml beaker. Stir the mixture well and filter off the product at the pump and wash with cold water. Yield, 2 g. Recrystallise about 0.1 g of product from ethanolwater; m.p. 167o. 4-Bromoaniline Place in a 100 ml round-bottomed flask 1.8 g of 4-bromoacetanilide, 20 ml of ethanol and 3 ml of concentrated hydrochloric acid. Shake the contents and reflux gently over a small flame for 20 min. Cool the contents, remove the condenser and add 30 ml of water. Boil the contents (without condenser) for 10 min to remove

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ethanol as far as possible. Pour residual solution onto 20 g of crushed ice and stir well. Dissolve 3 g of sodium hydroxide in 6 ml of water, cool the solution, and add with vigorous stirring to the hydrolysed solution. Test for complete neutralisation (use red litmus paper). Cool in ice bath. The 4-bromoaniline separates as an oil which soon crystallises. Filter at the pump, wash with cold water and dry between pads of filter-paper. Yield, 1.4 g. Recrystallise from ethanol-water; m.p. 66o.

Caution! Bromine causes severe burns. As an antidote for it, dissolve 20 g of sodium thiosulphate in 500 ml of water in a 1 litre beaker. Keep ready the antidote solution before handling bromine. Wash the affected area with this solution liberally and then with water. See a doctor.

Phthalic anhydride phthalimide anthranilic acid 2-chlorobenzoic acid

Phthalimide Method 1. Ground together 4 g of phthalic anhydride and 0.8 g of urea into a fine powder and place the mixture in a 100 ml round-bottomed flask. Heat the flask in an oil bath at 130-135 o. The mixture melts giving effervescence that gradually increases, and finally the mixture froths up and becomes solid (10-20 min). Allow the flask to cool, add about 30 ml of water and stir to disintegrate the solid. Filter at pump, wash with little water and dry. Yield, 3.2 g. Recrystallise 0.1 g from ethanol-water mixture; m.p. 234o. Method 2. Ground together 4 g of phthalic anhydride and 0.8 g of urea into a fine powder and place the mixture in a 100 ml beaker. Heat in a microwave oven at medium power for 5 min. The mixture melts giving effervescence that gradually increases, and finally the mixture froths up and becomes solid. Allow the beaker to cool, add about 30 ml of water and stir to disintegrate the solid. Filter at pump, wash with little water and dry. Yield, 3.2 g. Recrystallise 0.1 g from ethanol-water mixture; m.p. 234o. Anthranilic acid Dissolve 2 g of sodium hydroxide in 10 ml of water in a 100 ml beaker, cool the solution to room temperature, and add 2.5 g of phthalimide. Stir to make a clear solution (sodium phthalimide solution). Dissolve 3 g of sodium hydroxide in 15 ml of water in a 100 ml conical flask and cool in ice bath to 5-10 o. Add 0.8 ml (about 2.6 g) of bromine in a single lot and stir the mixture until all bromine has completely reacted. Again cool in ice bath to about 5-10o (sodium hypobromite solution). Add rapidly sodium phthalimide solution to sodium hypobromite solution, remove from ice bath and shake vigorously. Heat the solution to 80o in a heated water bath for 2 min. Cool the solution in ice bath and neutralise it by dropwise addition of concentrated hydrochloric acid (about 6 ml of acid is required; test with litmus). Precipitate anthranilic acid by gradual addition of glacial acetic acid (2 to 3 ml of acid is required). Cool in ice bath for 10 min and filter the product and wash with little cold water. Yield, 1.8 g. Recrystallise 0.1 g of anthranilic acid from hot water; m.p. 145o. 2-Chlorobenzoic acid Dissolve 2.6 g of copper(II) sulphate (CuSO4.5H2O) and 1.5 g of sodium chloride in 10 ml of water in a 100 ml round-bottomed flask. Add 5 ml of concentrated hydrochlorid acid and heat to boiling. Add 1 .5 g of copper turnings and continue to boiling until the solution is almost colourless. Decant the hot clear solution into 150 ml conical flask, lightly stopper the flask and cool in ice bath. Copper(I) chloride crystallises out as a white precipitate on cooling. Do not leave it for long periods. Dissolve 1.5 g of anthranilic acid in a solution of 2 ml of concentrated hydrochloric acid and 10 ml of water, and cool in ice bath to below 10o. Diazotize by dropwise addition of solution of 0.7 g of sodium nitrite in 10 ml of water until starch-iodide solution (used as an external indicator) gives distinct blue colour when treated with a drop of diazonium solution. Add cold diazonium solution into copper(I) chloride solution slowly and with shaking. The reaction proceeds rapidly and with frothing. Allow the mixture to stand at room temperature for 1 h with occasional shaking.

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Filter the precipitated 2-chloribenzoic acid and wash with little cold water. Yield, 1.2 g. Recrystallise 0.2 g from hot water; m.p. 138-139o (use decolourising carbon).

Benzaldehyde benzoin benzil benzilic acid

Benzoin In a 100 ml round-bottomed flask place about 0.5 g of sodium cyanide (Caution! observe safety) and 10 ml of water, shake to dissolve the solid. Add 20 ml of rectified spirit and 5 ml of benzaldehyde. Attach a reflux water condenser and boil the mixture for 20 min over a small Bunsen flame. Cool to the room temperature and then place the flask in an ice bath. Add about 30 ml of water, break the limps with a glass rod, and filter at pump. Wash the product with cold water, drain well and dry. Yield, 5 g (white or pale yellow). Recrystallise about 0.1 g from ethanolwater; m.p. 137o (white crystalline).

Caution! Sodium cyanide is a deadly poison. Before disposal, treat the filtrate from product separation with about 0.5 g of iron(II) sulphate and 5 ml of dilute sodium hydroxide, shake and keep for 5 min (during this time a relatively less toxic ferrocyanide is formed).

Benzil Place 4.5 g of crude benzoin and 20 ml of concentrated nitric acid in a 100 ml round-bottomed flask. Heat on a boiling water bath (in the fume cupboard) with occasional shaking until the formation of nitrogen oxide ceases (about 1 h). Pour the reaction mixuture into 150 ml of water taken in a 250 ml beaker, stir well until the oily product crystallises completely as a yellow solid. Filter at the pump, and wash it thoroughly with water to remove nitric acid. Yield, 4.2 g. Recrystallise about 0.1 g from ethanol-water; m.p. 94-96o. Benzilic acid Place in a 100 ml round-bottomed flask 3.5 g of potassium hydroxide and 10 ml of water. Shake until the solid dissolves. Add 20 ml of rectified spirit and 3.5 g of benzil. Shake the mixture. Fit a reflux water condenser to the flask and heat the mixture on a boiling water bath for 10 min. Pour the hot mixture into a 100 ml beaker. Add about 2-3 ml of rectified spirit to the flask, rotate the flask to dissolve the viscous residue and transfer to the same beaker. Cool the contents by placing the beaker in an ice bath for 10-15 min. Repeatedly scratch the sides of beaker with a glass rod, above and below the meniscus of solution, until a thick precipitate of potassium benzilate appears. Filter off the crystals at the pump and wash it with 1-2 ml of ice-cold rectified spirit. Drain the product. Transfer the potassium benzilate into a 100 ml beaker, dissolve in about 50 ml of water and precipitate benzilic acid whilst stirring the solution by dropwise addition of about 1 ml of concentrated hydrochloric acid (test with litmus for complete neutralisation). Filter off the benzilic acid at the pump, wash with little ice-cold water and dry. Yield, 3 g. Recrystallise from hot water (use decolourising charcoal); m.p. 150o.

Aniline 2,4,6-tribromoaniline 1,3,5-tribromobenzene

2,4,6-Tribromoaniline Dissolve 1.5 ml of distilled aniline in 10 ml of glacial acetic acid in a 100 ml beaker and place in an ice bath. The temperature should not be more than 10o during bromination. Dissolve 3 ml of bromine in 15 ml of glacial acetic acid and place this solution in a burette or a dropping funnel. Add bromine solution slowly and in about 0.5 ml portions to the aniline solution, and stir vigorously after each addition. A white product separates during bromine addition. Stop the addition of bromine when the product is coloured slighly yellow owing to the excess of bromine. Pour the product into 100 ml of water taken in a 250 ml

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beaker and stir well. Filter the product, wash with water, drain and dry. Yield, 3.2 g. Recrystallise about 0.1 of product from ethanol-water; m.p. 120o. 1,3,5-Tribromobenzene Place 2 g of 2,4,6-tribromoaniline in a 100 ml round-bottomed flask, add 20 ml of rectified spirit (commerical 95% ethanol) and 5 ml of benzene. Attach a reflux water condenser and heat the flask in a warm (4050o) water bath until a clear solution results. Caution! There should not be any nacked flame in the vicinity as benzene is highly inflammable. Loose and lift the condenser slightly up, and add 1 ml of concentrated sulphuric acid (use a pipette) to the clear solution and then quickly replace the condenser. Gently swirl the liquid and allow the flask to keep in water bath (40-50o) for 10 min. Divide 0.7 g of sodium nitrite in two equal portions on paper. Slightly lift the water condenser up, add the first portion of sodium nitrite and quickly replace the condenser. Shake the flask. Brisk effervescence is seen. When the reaction subsides, add the second portion of sodium nitrite in the same manner. Heat the water in bath to boiling and reflux the solution as long as gas is evolved (about 10 min), shaking occasionally. Allow the solution to cool for 10 min and then immerse the flask in an ice bath. A mixture of tribromobenzene and sodium sulphite crystallises out. Filter at pump, drain well and wash the product with water to remove sodium sulphite. Yield, 1.2 g. Recrystallise about 0.1 g of compound from ethanol-water; m.p. 122o.