DeWiH B. Stone The Univers~tyof Tennessee a t Martm, 38237
Diazonium Salts as Spot Test and Synthetic Reagents
The synthetic utility of diazonium salts is emphasized in every elementary organic text. Of perhaps greater interest to many students are the a p plications of diazonium salts as spot test reagents in the identification of phenols, aromatic amines, and certain natural products. Two reports of current interest involve the detection of marijuana, Cannabis sativa L., and the analysis of its three phenolic constituents, tetrahydrocannabinol (THC), canuabinol (CBN), and cannabidiol (CBD)
The fragmented marijuana plant material reacts with p-nitrobenzenediazonium chloride to give a red-brown color (in alkaline solution), presumably due to azo dyes formed by coupling ortho and/or para to the phenolic hydroxyl groups. This is a useful forensic test as tobacco does not give a color reaction with this reagent.' Other workers report that p-nitrobenzeuediazonium chloride can be used to locate the colorless THC, CBN, and CBD spots when an extract of marijuana is analyzed by paper chromatography. THC gives red-violet, CBN gives brown-violet, and CBD gives brown spots when the chromatogram is sprayed with the diazouium salt solution and then with base.2 The laboratory experiment outlined below includes the paper chromatography of phenols, the use of p nitrobenzenediazonium chloride to locate and identify the phenols on the chromatogram, and the use of this IRUDAVASAMT, A., A N D NATARAJAN, A. R., Indian J . Chcm., 3,327 (1965). KORTE,F., AND SIEPER,H., Telrahedron, 10,153 (1960). 'BRAY, H. G., THORPE,W. V., A N D WHITE, K., Rioehemical J.,46,271 (1950).
diazonium salt in syntheses involving coupling or replacement of the diazonium functional group. Six phenols and an unknown mixture are chromatographed on Whatman #l paper using a chloroform/acetic acid/water solvent s y s t e m . T h e resulting chromatogram is then wetted with a solution of p-nitrobenzeuediazonium hydrogen sulfate to give the following results
com-
pound 1 2 3 4 5 6
Phenol 1,s-dihydroxyhenzene 1,3,~trihydroxyhenzene l,4-d~hydraxybenzene 2-hydroxynaphthdene 1,2-dihydroxybensene 1,2,3-trihydroxyhenzene
R, 0.63 0.10 0.61 0.98 0.86 0.41
Am Dye Color red-orange red-brown gray-green red-violet orange-brown orange-yellow
These R,values are averages taken from the data of 34 students. Suitable student unknowns can be prepared by mixing the following phenols: 1 and 2; 1 and 4; 2 and 3; 2 and 4; 2 and 5; 4 and 6; and 2, 3, and 4. The diazouium salt solution remaining is divided into halves and used to synthesize any two of the following compounds: pnitrophenol, p-nitrochlorobenzene, g nitrobromobenzene, pnitroiodobenzene, 4(p-nitropheny1azo)-1-naphthol, 4(p-nitropheny1azo)-N,Ndimethylaniline. The procedure can be altered to change the number of syntheses if desired. The complete experiment (chromatography and two syntheses) requires only 5 or 6 hr of laboratory time, subject to the usual restriction that the diazonium salt solution must be used on the day it is prepared. This experiment presents several opportunities for open-ended assignments for the interested student. The slightly more difficult syntheses of p-nitrofluorobenzene, p-nitrobenzonitrile, and nitrobenzene may be assigned as library/laboratory projects. The paper chromatography of other phenols, the use of other diazouium salts as color-test reagents, and the use of other chromatographic solvent systems may be studied. Also, similar chromatographic and color test procedures may be devised for aromatic amines. Experimental Paper Chromatography Mix 40 ml of chloroform, 40 ml of acetic acid, and 20 ml of water in s sepmtrstory funnel. Draw off the organic (lower) layer into a, clean, dry, 1000-ml heaker and cover the beaker with a sheet of paper. Discard the aqueous (upper) layer. Secure an 1 8 ' / h m circle of Whatman #I filter paper and trim handle the paper by the top it as shown in the figure (CAIJTION:
Q m
a
Mat'ng a chromatogram from a filter paper. First cut the paper as rhown i n A and then trim upvertically from the first edge as in B.
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edge only). The following marks must be made lightly, with Draw a line parallel to, and about in. from the bottom of the paper. Equally space seven x-mxks along this line. Label these x-marks by writing below the line es follows: 1, 2, 3, 4, 5, 6, M. Roll the paper into a cylinder and use two staples to hold the two edges about in. apart. Use s. round toothpick to place 3 drops of a 1% solution of compound 11in ether on the #1 x-mark. The wet spat should be no larger than I/, in. in diameter. Spot compounds 2 through 6 and the mixture by the same procedure. After the spots have dried, carefully stand the pbper cylinder in the beaker of solvent and again cover the beaker. Allow the chromatogra.phy to proceed undisturbed for 45 mi?. During this time prepme the p-nitrohenzenediazonium hydrogen sulfate solution and any two of the solutions in A through F below. After 45 min have elapsed, remove the paper cylinder from the beaker, mark the position of the solvent, and allow the paper to dry (about 15 min). Then carefully remove the staples and place the paper on three thicknesses of paper towel. With a medicine dropper wet the entire chromatogram with the diazonium salt solut,ion, using as little of the solution as possible. After the towels have soaked up the excess water, carefully move the chromatogram to a. dry paper towel. Record the R, value for each phenol and describe the color of each a m dye formed. The colors will fade and should he described without undue delay. Divide the remaining diazonium salt solution into halves and carry out two of thesyntheses below. Diazotizalion qf p-Nitroaniline. Prepare a solution of 0.108 mole (6 ml) of concentrated sulfuric acid in 25 ml of water in s n Erlenmeyer flesk. Add 0.03 m& of p-nitroaniline (weigh i t in a beaker) .and heat on the stesm bath to effect solution. (Caution: p-nitromilineis toxic and is absorbed through the skin. Do not breathe the dust or dlow this compound or its solutions to contact your skin.) Cool the solution to below 10'C in an ice-water bath. Slowly add 25 ml of aqueous 1.5M sodium nitrite solution (0.0375 mole), keeping the solution temperature below 10°C. Stir the cooled solution for 5 min and then filter it into mother cold Erlenmeyer flask to remove any remaining solid. Keep this solution of pnitrobenzenediazonium hydrogen sulfate in en iee-water bath until needed. A. p-Nilrophaol. Prepare a solution of 25 ml of coneentrbted sulfuric acid in 150 ml of water. Add one-hdf of the remaining diaeonium salt solution to the acid solution and carefully heat the mixture to boiling with a burner for 15 mi". Notice the evolution of nitrogen gas when the temperature reaches about 70°C. Cool the mixture in an ice-water bath and extract the p-nitrophenol with two 15-ml portions of ether. Distill off or evaporate the ether in an area free from flames. Recrystallisao pan'l.
414 / Journal o f Chemical Education
tion of the crude product from toluene yields ahout 1.39 g (67%) of yellow-brown needles, mp 1134'C. B. p-Nit~ochlorobazae. Prepare a solution of 0.01 mole of ouprous chloride and 7%/rml of concentrated hydrochloric acid in 10 ml of water. Stir this solution a t room temperature while adding one-half of the remaining diazonium salt solution dropwise over s. period of 10 min. Allow the reaction mixture to stand with occasional stirring for 10 mi", and then add 100 ml of water. Filter the crude product with suction and weshit with wster. Recrystdlimtion of the crude product from ethanol/ water yields about 1.60g (68%) of tan product, mp 78-80°C. C. p-Nitrobromobazae. Prepare a solution of 0.01 mole of cuprous bromide and 10 ml of 48% hydrobromic mid in 10 ml of water. Heat this solution on the steam bath while adding anehalf of the remaining diszonium salt solution dropwise over a. period of 10 min. Allow the reaction mixture to stand with occasional stirring for 10 min, and then add 100 ml of wster. Filter the crude product with suction and wash i t with water. Recrystallization of the crude product from ethanol/water yields about 1.93 g (64%) of pink-brown product, mp 12.5-6'C. D. p-Nitroiodobenzae. Prepare a solution of 0.03 mole of potassium iodide in 50 ml of water. Stir this solution a t room tempereture while adding one-half of the remaining diisanium salt solution dropwise over a, period of 10 min. Allow the reaction mixture to stand with occasional stirring for 10 min, and then add 100 ml of water. Filter the crude product with suction and wash i t with water. Reerystdlization of the crude product from ethanol/water yields about 2.60 g (70y0) of pinkyellow product, mp 171-3% E. 4-(p-Nitrophay1aro)-l-naphthol. Prepare a solution of 0.015 mole of l-naphthol in 30 ml of 5% aqueous sodium hydroxide. Stir this solution a t room temperature while rapidly adding ane-half of the remaining diasonium salt solution. Allow the reaction mixture to stand for 10 min then add 100 ml of wster. Filter the product with suction, warh i t twice with cold ethanol, and allow i t to dry. The yield is about 3.35 g (76%) of briek-red product, mp 278-9 d. F. 4-(p-Nilrophaylazo)-N,N-dimethylanilinePrepere a solution of 0.015 mole of N,N-dimethylaniline and 3 ml of concentrated hydrochloric acid in 10 ml of water. Stir this solution a t room temperature while rapidly adding one-half of the diazonium salt solution. Allow the reaction mixture to stand for 10 min with occasional stirring, m d then add 10% aqueous sodium hydroxide dropwise until the reaction mixture is alkaline to litmus. Filter the product with suction, wash it twice with cold ethanol, and d o w it to dry. The yield is about 2.97 g (70%) of deep red-purple product, mp 150-60 d.
