Microscale Qualitative Organic Analysis
. . ~~~~~~. ~.~
Column Chromatography Separations Suggested for -.unaergraauare txperiments Mixture
Stationary Phase
Mobile Phase
Lycopene$-carotene
Alumina
Petroleum ether-acetone Petroleum ether
(R)- and (SF carvone
Alumina
Hexane
Ferrocene-acetyl Silica gel ferrocene
Petroleum ether- diethyl ether
CholesterolSilica gel cholesteryl acetate
Ligroin:ether (93)
Fluorenefluorenone
Alumina
Ligroin:ether (1 :1)
Caffeine
Silica gel
Ethyl acetate-methylene chloride
Leaf spinach pigments
Alumina
Hexane, toluene, ethyl acetate, methylene chloride
Syn- and antiazobenzene
Alumina
Petroleum ether
Chlorophyll and &- Alumina carotene
Petroleum ether, ethyl acetate
replaced by water and the second component-the yellow fluorescein-is collected in another test tube. If desired, the concentration of the recovered methylene blue can be measured quantitatively from absorbance readings using a W-visible spectrophotometer. In this case the amount of dye applied to the column (0.2-0.3 mL) must be measured accurately. The entire methylene blue eluate is diluted with 95% ethanol to 100 mL in a volumetric flask. One milliliter of this solution is further diluted with 95% ethanol to 50 mL in another volumetric flask. From the absorbance of this solution, the concentration of methvlene icalculated in millimams of methvlene blue oer milliliter of mixture) can be found by reference to a calibration curve made from suitably diluted samples of a stock solution containing 3.8 x 104 g of methylene blue in 1L of 95% ethanol. All absorbance readings are made a t Lnx= 663 nm. The quantitative determination of fluoresceinis not as accurate because commercially available fluorescein contains some impurities and the quantitative analysis of these samples is not as precise. Literature Cited 1. Lehman, J. W. Operational Organic Chemistry, 2nd ad.; AUyn and Bacon: Boston, 1988.
2. Fieser, L. F.;WiUiamson, K L.Organic Experiments, 8th ed.; D.C. Heath: Lexington, 1979. 3.Ault.A.Technkes and Experimentsin Organic Chemistry, 4th ed.;Allyn and Bacon: Boston. 1979. 4, Williaroaon,K L. M m m i f - and MicroscaleOrganic Exaerimerits;D. C. Heath:Lexington. 1989, 5. - f a k . D. F.; Hoemer, R. S, J. C k m . Educ. 1991,6873. 6. Rodig,O.R.;Bell,C. E.,Jr.;Clark,A. K OrganicchemistryLabom10ry:Stdrdand MicroscaleExperiments; Saundem: Philadelphia. 1990. 7. Roberts. R.M.;Gilbert, J, C.; Rodewald, L. B.; Wingrove, A . S. Modern Experimental Organic Chemistry, 5th ed; Philadelphia: Saunders, 1988. 8. Nilaitz, J. S. Experiments in Organic Chemistry:From M i c m x ~ kto Macrmde; En. dewood Cliffs:Prentice-Hall. 1991.
Edwin J. Goller and John H. ~ i l l e r ' Virginia Military Institute Lexington, VA 24450 In spite of the fact that most undergraduate chemistry programs have dropped qualitative organic analysis as a separate course, the chemistry faculty a t VMI believe this course remains a useful educational experience for our undergraduate majors. While the primary objective of the course has not changed over the years, the heavy reliance on the modern instrumental techniques of Fourier-transform infrared spectroscopy (FTIR), contmuous-wave 60MHz nuclear magnetic resonance spectroscopy (NMR), and gas chromatographylmass spectrometry (GCiMS), make the course a relevant capstone experience? Recently some chemists ( 1 ) have argued that the advent of sophisticated laboratory instruments equipped with computer-searchable spectral data bases may render traditional chemical classification tests and individual student interpretation of spectra obsolete. We, however, do not allow students to use computer-searchable spectral data bases to identify their "unknowns". We prefer that students gain experience from microscale experimentation and that they gain critical thinking skills by selecting their own chemical tests and by interpreting their own spectra. Students need to appreciate the fact that complex spectra are more than a jumble of bnes to be sorted by computer. The Course There are a number of standard texts for microscale (24) and spectroscopic techniques (5, 6)on the market, and we maintain copies of each on the laboratory reference shelf. A suitable microscale qualitative organic analysis text is not available so we have adopted references 2 and 5 as course texts and prepared a 15-pagelaboratory handout containing information about the course a s well as several specific procedures, a number of which are described below. Students also are provided cross references for the chemical classification tests found in reference 2 and the traditional Shriner text (7)available on the laboratory reference shelf. The laboratory portion of the course meets twice weekly for two hours and the students are expected to identify the structures of five "unknown&"-three (separate samples of 750 my each and a two-component mixture contain in^ 1.00 g of each compound-by the end of the fall semester. While the identification of these unknowns is based primarily on the interpretation of FTIR, NMR, and GCiMS (liquids only) spectral data, the determination of physical constants, the purification of solids and liquids, the performance of selected chemical classification tests and the preparation ofone derivative out of five unknowns are carried out using microscale techniques and equipment." The chemical classification tests help students reriolve uncertainties created by unexpected or ambigmous spectral dam. The 15-nagelaboratory handout contains a microscale ~ r o cedure for the separation of a two-component mixture
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'3910 Timber Ridge Place, Midlothain, VA 23113 %lost, but not all, chemistry majors perform senior thesis research or summer research beforegraduating from VMI = ~ cGlass, e lnc microscale laboratory equipment. (Continued on next page)
Volume 70 Number 6 June 1993
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the microscale laboratory adapted from an earlier report in this Journal by O'HaraMays, et al. (8). Procedure
tive test is the formation of a silver mirror or a black precipitate of finely divided silver. Heating in a sand bath a t 40 'C may help the appearance of the siluer mirror. Some suggested test compounds are nitrobenzene and p-nitrophenol.
Classification Tests
The Nitrous Acid Test for Amines Primary Amines. In a 3411. test tube dissolve five drops of a primary m i n e in 1.5 mL of a 6 M hydrochloric acid. Stir and cool to 0 T. In a separate 3-in. test tube dissolve 150 mg of sodium nitrite in 1.0 mL of distilled water. Add the nitrite solution slowly with stirring to the cold amine hydrochloride solution. Continue the addition until the mixture (diazonium solution) gives a positive test for nitrous acid with starch iodide paper (blue color). Remove 12 drops of this diazonium solution to another test tube, warm gently (sand bath), and note whether or not gas bubbles are pmduced within 5 min. Primary aromatic amines evolve N9 eas under these conditions. Primaw aliohatic amines lfbzrate Nz gas during the addition of sodi;m nitrite to the amine hydmchloride at 0 'C. Some suggested test compounds are aniline and sec-butylamine. P r e ~ a r ea solution containine 15 me of 8-na~hthol(carcinog&c) in 10 drops of 10%:odium-hydroxi'de. ~ d 1.0 d mL of distilled water followed bv the remainine cold diazonium solution and note if a re"d-orange dye produced. Primarv aromatic amines form azo dves via a coupline . reaction; whereas, primary aliphatic amines do not.
is
-
Secondary Amines. Dissolve 10 drops of a secondary amine in 2.0 mLof 6 M hydrochloric acidin a3-in. test tube and cool to 0 'C in an ice bath. Add slowlv. with stirrine. a solution of 0.25 g of sodium nitrite in 1.0-LL of water &d note whether or not a vellow oil is ~roduced.If the amine is highly colored to be& with the &lor of the oil produced will be brown or red-brown. Some suggested test compounds are N-methylaniline and diethylamine. Caution The N-nitrosoarnines produced by this reaction are carcinogenic. Tertiary Amines. Repeat the secondary amine procedure using a tertiary amine. Add 1.0 mL of the reaction product to 1.0 mL of 6 M sodium hydroxide to obtain a greenp-nitroso derivative with aromatic amines that have a free para position. Tertiary aliphatic amines simply dissolve in the acidic solution. Some suggested test compounds are N,N-dimethylaniline and triethylamine.
Hydriodic Acid Test IZeisel'.. Alkoxyl Method This test is reliable for ethers and esters containing the methoxy, ethoxy, and pmpoxy groups. Place about one drop (20 mg) of the compound to be tested in a 5-in. test tube. Carefully add, by means of an automatic pipet, 200 WLof glacial acetic acid and 200 pL of 57% hydriodic acid (sp. gr. 1.7). Add a small porcelain boiline stone and insert into the mouth of the test tube a eauze pl;g saturated with sodium plumbite solution (see b&ow). The gauze plug is folded to make a tight fit with the walls of the test tube and pushed down so that it is 4 cmfrom the mouth of the tube. (Blot off any excess liauid settine on top of the plug.) A small piece of ionabsorbent cotton isgentG tamped on top of the d u e bv means of a glass rod so as to make a disk okmtton :Im i ;hick. Apiece of filter paper cut 2 cm x 10 cm is fidded lonpitudinallv, moistened with a soon the cotton disk. lution of mercuric nitrate; and The test tube is placed at a depth of 0.5 in. into a sand bath maintained a t i30 'C. When the reaction mixture boils, vapors rise through the porous gauze plug. The volatile alkyl iodide, rising through the plug, reacts with the mercuric nitrate to produce a light orange or vermilion color due to the formation of mercuric iodide within 10 min. Ayellow color constitutes a negative test. Some suggested test compounds are anisole and methyl benzoate. Gauze Plugs
About 3.0 or 4.0 mL of sodium plumbite solution (3)is transferred to a gauze plug and the gauze plug is folded to fit a 5-in. test tube. The sodium plumbite solution should be stored in a refrigerator. Acknowledgment The authors wish to acknowledee the s u ~ ~ oofrthe t VMI Research Committee, the ~ h ~ho u n & t i o n and the Gwathmcv Research Fund for their sumort of this work. We also want to thank Stanley wetmb;e for his helpful suggestions during the preparation of this manuscript. Literature Cited
ZinclAmmonium Chloride Reduction Place 20 mg of the nitro compound to be tested in a 1.0mL conical vial. Add 200 pL of 50% ethanol, 20 mg of ammonium chloride. and 20 me of zinc dust. Shake the mixture then connect to an air-&led condenser and reflux for 30 s using a sand bath or aluminum block (6, 7).Let the mixture stand for 5 min then transfer two drops of this solution with a Pasteur filter d ~ etot a freshly made solution of Tollen's reagent (3-in. te& tube) and shake. A posi-
A160
Journal of Chemical Education
1. Zub"~k, J. W.J. C h m . Educ. 1981.69.389, 2. Mayo. D. w.: Pike, R. M.; Butcher, S. S. Miemseole OgonieLobommry:John w s e y &
Sons. 1989. 3. Williamson, K L Mocmsmk a d M h m a k Organic Exprimis; D . C. Heath and Company: New York, 1989. 4. Rodig, 0. R: Bell. C. E.;Clark. A. K Oganie Chemialry hbomtory: Slondani and M ~ m s m d ~ E r p e ~Savnderj ~ i i i ~ , College Publishing: New Yark, 1990. 5. K-p, W. Organic Spedmsmpy; W. H. Freeman and Company: NewYork, 1991. 6. silver stein,^. ~ . ; ~ s s s l e rorrill . ~ . rill,^ ~ . s ~ c ~ r o ~ r r i e r & n r i f ~ o t l a l a o f o g o n l e Compounds, 5th ed. John Wiley & Sons: New Ymk, 1991. 7. Shriner,R. L . : F u s o n , R . C . ; C u ~ ~ , D . Y , : M o ~ lC. l , TT.h S y s r o m o t i c I & n t i ~ t i o n of 0rg.nieCompounds: John Wiley & Sons: New York. 1980. 8. O'HsmeMays, E. P.:Yuen,G. U.J C h m . Edue. 1989.66. 961.
