Rod O'Connor
Montano State College Bozeman
I Natural Product Chemistry
I
laboratory research approach for elementary organic courses
T h e laboratory. . programs for students of beginning organic chemistry courses have often been less than inspiring, usually containing such procedures as thepreparation of soap, the synthesis of aspirin from salicylic acid, and similar experiments which are often better suited to science fair projects at the junior high school level. The experiments are typically unrelated to each other and often do not teach the chemical techniques most commonly used in modern laboratories. Observations and data are usually treated by the "fill-in-the-blank" method, which presents a far from real picture of true laboratory study. Many of the students in these courses eventually participate in some type of pure or applied research, yet colleges frequently treat these courses much more casually than either the needs or the abilities of the students warrant. Our program, begun in 1964, was designed to introduce macro, semi-micro, and micro techniques of organic chemistry of the types commonly employed in research in natural-product chemistry or in biologicallyrelated fields; to present a brief, but typical, approach to the chemical study of a physiologically-active natural product, from isolation to synthesis; and to teach the proper use of the chemical literature, the research notebook, and the formal report in systematic laboratory study. The program was designed for a one-quarter course of nine three-hour laboratory periods, using inexpensive and readily available equipment. Access to spectrophotometers and other special apparatus is desirable, but not essential. Student response to the course was uniforn~ly enthusiastic. The introduction of such techniques as microslide thin-layer chromatography, by discussions of practical applications in the students' own fields of study, provided added incentive; and the idea of carrying out a systematic study of a single compound, caffeine, proved quite appealing. Labors, tory examinations showed that the students gained at least as good an understanding of such techniques as distillation, melting point determination, etc., as had been obtained by earlier groups using conventional laboratory manuals. I n addition the students became reasonably proficient in several chromatographic procedures and in the interpretation of simple spectra. The laboratorv notebooks were used as the basis for final formal reports summarizing the entire laboratory investigation of caffeine. The series of experiments, which could easily be supplemented by additional studies for programs with more laboratory time, is summarized in the following sections. Each describes the work for One threehour laboratory period. 492
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Journal of Chemical Educofion
Extraction of Crude Coffeine from Coffee Beans1
Approximately 200 g of coffee beans are ground in a coffeemill or other grinder to a coarse powder which is tied in a bag made from a 10-inch square of clean cotton cloth. The bag is suspended in a 1000-ml beaker containing 600 ml of distilled water and the water is boiled gently for about 15 min, after which the bag is removed. allowed to drain into the beaker, and discarded. To the coffeesolution is added 100 ml of 10% lead acetate; the resulting mixture is boiled 5 min and then allowed to stand 15 min on an asbestos square. The supernatant liquid is decanted through a coarse filter and then the residue is filtered through the same systenl. The filtrate is boiled until the volume is reduced to 100 rnl. (A second filtration is necessary if much precipitate appears.) The solution is allowed to cool to room temperature and is then extracted twice in a separatory funnel (ungreased stopcock) with 50-ml portions of chloroform, using a few drops of glacial acetic acid to break up emulsions. The combined chloroform extracts are washed with 25 ml of 2 M sodium hydroxide followed by 25 ml of distilled water, using portions of saturated salt water to break up emulsions. The chloroform solution is placed in a stoppered flask ovel. anhydrous sodium sulfate to dry until the next laboratory period. Isolation of Crude Caffeine and Purification of Chloroform for Chromotogrophy
The chloroform solution of crude cafieine is evaporated to dryness in a 200-ml round-bottom flask under v a c u ~ mwith , ~ the vacuum trap set in an ice-salt bath to collect most of the chloroform. The crude caffeine is scraped out of the flask with a curved spatula and is weighed and stored in a sealed vial for future use. Usually 100-200 mg are obtained. The collected chloroform in the cold trap is washed by extraction with 5% sodium carbonate (one-half the volume of the chloroform used), followed by distilled water, and is then dried over anhydrous sodium sulfate about 30 min while a simple distillation apparatus is being assembled. The dried chloroform is decanted and distilled, collecting the fraction boiling around 6162'/1 atm. If time permits this is redistilled. One 'Adapted from the method described by JonNsoN, L. H., GOERING, K. J., AND BARER, G. L., "L~bomtOryExperiments in Fundamental Organic and Biological Chemistry," Montana State 1951. =Adapted from CASON,J., AND RAPOPORT, H., "Laboratory Text in Organic Chemistry," 2nd ed., Prentice-Hall, Inc., Englewood Cliffs, New Jersey, 1962, p. 328.
drop of absolute ethanol is added to the distillate as a preservative and the chloroform is stored in a labeled flask in a cool dark place until the next period.
useful information. Typical data obtained are shown in the table. Melting Range Comparisons
Alumina Column Chromatography
A small column is prepared by partially flame-sealing (to about a 4-mm diameter) one end of a 15-cm length of 8-mm soft glass tubing. To this tube, cleaned and dried, a 1-inch tip cut from an eyedropper is connected by a 1-inch length of 6 mm ID Tygon tubing. The column is clamped in a vertical position and the Tygon tubing is closed with a screw clamp. Light petroleum ether is placed in the column and a small wad of soft glass wool is tamped gently down into the bottom of the 8-mm tube. While the column is tapped gently, washed sea sand is added to give a layer about in. deep, 1.5 g of "neutral" alumina is added in small portions, and a h a l '/l-in. layer of washed sea sand is added. The screw clamp is opened to drain the petroleum ether down to a level just above the upper sand layer. The column is washed successively with 3-ml portions of 3: 1 petroleum ether-benzene, 1:1 petroleum ether-benzene, benzene, and 3 : 1 benzene-chloroform (using the distilled chlorofornl), allowing each wash to run down to just above the upper sand layer. About of the sample of crude caffeine (but no more than 40 mg) is dissolved in 5 1 0 drops of 3:l benzene-chloroform and this solution is carefully pipetted onto the column and is then chromatographed using 3 : l benzene-chloroform for elution. Six fractions of 40 drops each are collected in 2dram vials, previously numbered and weighed. Each vial contains a boiling stone and the vials are handled only with folded filter paper. The vials are set in a clean dry 150-ml beaker on an asbestos-covered hotplate in the fume hood to evaporate the chloroform, and the column is examined for evidence of retention of colored impurities. The vials are allowed to cool and are again weighed. A graph is prepared, plotting weight of purified caffeine versus fraction number, and the color of the purified caffeine is compared with that of the crude sample. Each vial is sealed and saved for future use. Recovery of 8090% of the caffeine is usual. Purification by Sublimation and Determination of Melting Range
A simple apparatus is prepared for sublimation as shown in Figure 1. A 20-30 mg sample of the crude caffeine is placed in the bottom of the side-arm tube and this is lowered into a heating-oil bath to a depth of about 3 / r in. Vacuum is obtained from a water aspirator and the oil is heated slowly to about 16&180°, carefully watching for the appearance of sublimed caffeine on the cold finger. When sublimation is complete, the heating bath is carefully lowered, the tube is allowed to cool, the vacuum is carefully vented, and the cold finger is removed and scraped over a small square of aluminum foil. The sublimed sample is saved for further study. The melting ranges of sublimed caffeine, stock "pul.e" caffeine, crude caffeine, and all the fractions of chromatographed cdeine are compared, using samples sealed in evacuated capillaries. Mixed melting points may also be determined if the student feels this will provide
Student
Stock ceffeine
Crude caffeme
234-236'
229" dec.
Sublimed caffeine
Chramrttographed caffeine
235-236"
235-237"
der.
D
Two-Dimensional Paper Chromatography
Three-inch squares of Whatman #1 chromatography paper are provided for comparison chromatograms of stock "pure" caffeine, a stock mixture of caffeine and theobromine, crude caffeine, and caffeine purified by sublimation and/or colunln chromatography. The chromatograms are developed first xith water-saturated 1-butanol and then with 1-propanol-ammonia (3: 2), using a 600-ml beaker for the chromatography chamber. The papers are suspended in the chamber by paper clips from a horizontal wire and the chamber is sealed with Saran wrap. Detertion of the chromatographed alkaloids is done by spraying the developed papers with Dragendorff's R e a g e n t h n d allowing them to stand for about 30 min. Microslide Thin-Layer Chromatography and Calculation of the Molecular Formula
Two ultra-clean microslides are placed together and are dipped to about 5/6 of their length into a freshly shaken slurry of silica gel G in chl~roform.~ The dipped slides are separated and placed, silica gel up, on a paper towel to dry. When the slides are dry, excess
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HAI IS, I. M., AND MACEII,K., "Paper Chromatography," Academic Press, New York, 1963, p. 814. 4 The slurry is made by mixing 1 part silica gel with 3 parts (hy weight) of chloroform. The technique for preparation of these slides is in common use now, but was first suggested by Mr. Kile Baker, of Bozeman, Montana, while he was a high school student working on a science project.
f=l7 RUBBER SMPPER
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10 MM 0. D. PYREX TUBE, SEALED ON ONE AND - END VACUUM TESTED. (FILL WITH CRlSHED ICE AND GOLD W A m ~
Y
1.5 GM I.D. SIDE ARM TEST TUBE
u Figure 1.
Simple sublimation apparatus.
Volume 42, Number 9, September 1965
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ailiv:~g1.1 is w n ~ l x r lfrnm t11r nlgrs of rnc.11 slidr rind :III i l n t i f y i I I I I I I I ~ I P is ~ I : l on t11r I I I ~ I : I ~ N I top ~nrlirmof rnrh slid?. A s1mI nf . w l ~ ~ t i oton 11r rhmmntogr:q~hwlis applinl nt n p i n t in tlw rrntrr of n slirlc :it :I ~ l i r t m r rof ' in. fmm tl~rhottom. Solrrtio~~s to hr r n ~ n l ~ n r i~wlurlr c~l stork "1111rr"rnflrinr, slrwk t l ~ n ~ I ) m n h . . :Ls1oc.k mixturr of c d r i n c RIICI t h r n I ) r n n ~ i ~ ~ r . :1n11 snmplra of I I I ~ .stlalrnt's r r ~ a l rrnflrirw, s ~ ~ h l i m n l rnflrirrr. :rnd ~ M r i n rfrnm nlunli~~n I.II~II~~~o~I:IIIII~. Tlw ~.:~flc.ilwis clisu~lvcrrlin rl~lomformn ~ r lt11r thwh r n ~ n i ~I ~ Ic i l ~ t rI ~ I h yI c x i c l r . Tlw n~ixtrtrris I i ~ I l~ I I I : I I I ~ I I ~I I ~ I I I I II y l m x i l . Tlw sliclrs nrr ~lr*vc*lopcrl i l l :I 2.W-111l hr:~krr rontnining !) rill nf purilid vl1lomfrwn1 :lncl I nil of !Xw0 rthnnol. T h r r l i ~ h:irr si111pI.vpI:~rcvli n the hm~kcrnt :I slight mgle F l ~ u n2. NMR spechum of roffclne by Dr. Groem. Baker. M a t m a nnrl ilw In.:tk~*rir w:llcd with S:I~:IIIwrnp. Sliclrs :lrv Slots Unlvenlly. Sok.nt. CdCh; Rller bandrldth, 4 .psi w e e p time. rrmovnl \~II(.II t h r snlvrnt fmnl rrnrl~rsn l i ~ wI.1, in. 250 sr;w e e p rldlh, 500 cps. hrlow tlw to11 nlge of thr silir:~grl ronting. Sliclrs w c drinl : I I ~thrn q1~11ywIwith 2"; ~rntnswi~tn~ triidide infrnml s~wrtrn,to "rount" S-methyl hyclmgrns in nn I in W rthnnol. H, in I I I I I ~ fI n l l ~ v c lI I S M I 1 s p n ~ l n ~ mto, rnrnnlrnl i~~trlligrntly on lhr invnluvs of alwls nrc* III)~:I~IIIYIhy divicling tl~rclistnnce f l w ~ w of r pII on tlw ullrnviolrt s l w t r a of r n f l r i ~ ~nnrl r fmm slx~t.origin lo 1i11:ll spnt rrntrr 11y t h r disl:~nrc t h r n h m n ~ i ~and ~ c ~ to . notc diITrrr~~ws in ~ I I P1111 rnviolvt fmm slmt origiu t o 1i11:d wlvrnt fmnt. Tlw KI \ d u r sprrtni of r . n d ~and * lmrifinl rnlTc4nr. for rnlfrinr wit11 this pmt.nl~trr is nppmximnlrly 0.57 Trnt nt ivr st r11r111rw for raflril~rrl~o~tlrl he mtmrst rrl n11r1ll1:11 for ~ I I I Y ~ ~ I N I I I is ~ ~:~ppmxin~:itrIy IIP 022. and drfrncl~rl1)y 1 1 1 rt~tdrnts, ~ i~sruminpthat thv . ; ~ I I I ~ Typical Molecular Formula Prablem t r m of t l ~ r v ~ l ~ r ois~ kn ni ~ o \~vr~~~ . S l r a l r ~ ~ tnrr x r~~pplicrl with romhnation and molrrSynthesis of Caffeine from Theobromine 111w wight. dntn :IS shown hy the following tyl~ivnl ~\hnut0.1 gnl of thmhmmine is pl:wd in n I 4 r n m pmhlrm :~ndnrr rxpr(.lnl to rnlrwlntr the ~nolnwlnr a I v i n T o this nre nddnl 1 1111of 10C; runlium formul:~of c.nfl~.inr11y tlw rral of t h r Inhorntory pericd. hyrlmxiclr nucl .5 clmps of din~ethylsulfntr (Cnitl~orP~. " l h w n t : ~ r y :~n:~lysisof mflrine rrvrnls the n l r Tlw vinl is rovrrnl with Saran wrnp, r:lppnl wrurc4y, srnw nf 11:110grns:1c1c1 s111f11rrind t l ~ r~ I N W I I W of nitmI I : I on n : I o r : 1 1 t n i n Thrnhrnmin~ I . Whrn :I 15.07 mp n ~ m p l rof ci~flrinrwas sub(I) is r o 1 1 v ~ r t In ~ 1rnRrinr (11) in r x r ~ l l r n tyield :I* j w t d to c w n l w r t i o ~n~lnlysia, ~ Ii'2.i-l rng of CnC03 rind shown I)rlo\v. 7.01 nlg of II,O ~ v r r or l > t : ~ i ~ ~ tAnalysis d. of n m n r l anmplr al~o\v~rl 2R.8.iC~nilrngrn. IVhrn 21.8 mg of r n f l c h - w:In mixcrl with X 2 mgof rnnlphor, l h r mrlting nlncv of thr r n ~ n l ~ l ~\vns o r rnlurecl by nlmnt i.7". I n o l m r l ~ i n gp i n t drprrssion of cwnphor is ! l i o . ) ( ' i i l w l i ~ t c Il~rrmpiriri~lfnrmttln, the nppmximnlr niolriwlnr wrigl~t,:ind t11r moIcr111nrfnrni~~ln for rnflrinr." Spechu of Caffeine and Theobramine S ~ I I I I I II I I to t h r nprctmsropy Inhorntory with snnlplm of tlwir rntclr and purified 11ndurts. If timr n11d st~tdrnt nutnl)cn permit, rnrh at~alrnr mnv ~ l c ~ t c r n ~thc i n r infrnml or nuclenr mngnrtir rrwn:wrr s y r t n m of n pnrifinl sample and may nlrnntn? thc, r t l l n ~ \ d e ls1wtntm of nny of thrir snniplrs, ns wrll :IS thr influ~.l~c.r of pH on tlw ultrnviolrt aprc.lrn of r:ilTc,i~~r: I I I ~I l ~ c v h n n ~ i ~ ~ r . I f ~ I I R ~ ~ I I I I ~ :Ire I I I S not nv~ilnhlr,liternturr spwtrn niny he w ~ ~ ~ ~ l i ~ d , ~ 1I1v s111dr11Is:trr r x p r r l ~ ~tol n l ~ k er r n ~ ~ n n h inlc I r r ~ r r t : l i nr ~ I~ S 1 1 nncl rnrhonyl rrgiona of the
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PX(CIICIII.~~Y~.IIC-~,~~Bof 1.he1Rspectra of ~ ~ f l r i n ~related md r n s q ~ w n d si* givwt I { ~ r , n .1,:. It., . m n FIF.I.IN.>I., J. A m . A :\I>
494 : Journal of Chemical Mucotion
I h r i n g the rcnrtion timr, prrpn~ntionsnrr mnde for t \ \ . ~ l i ~ ~ r r ~ ~ t spnprr i o r ~ nr l ~ r n n ~ n t o g n ~ pnnd h y mirmsliclr thin-l:~yr,r~~l~mtnntogrnpl~y. At. 1111. fwd of l11r shttking inlrn.nl. 1 ml of rhlomfor~n is ndclrrl to t h r vial : I I ~i l is shnkrn tl~orn~tghIy will1 orrxsicmal rnrrfr~lvrnting. T h r rhlomforn~ln.vrr is rrmovrcl wit11 n rnpill:~ry piprt nnrl lhir wl~ltion is
clried nnd cnmpnml hv pnlrr n ~ ~ thin-Inyer d rhmmntogrnphy with known ~lunplwof rnffrine nnd thwhmminc nncl will1 n mixtrlrr of the two. Finn1 ro~~rlunions ns to thr stnwlure of rnfieine nrr mndr nnd clrfrnrlnl hy the -tudrnt*.
The nloclrnLq nm r r q o i d to prrpnrc pnpcm surveying whnt they rnnsirlrr importnnl infornmtion
about rnffeine rts found in C b ~ n i c oAbalracls l and nvnilnhlr primnry liternture. Thin survey in followed hy n clrtnilnl clisrn~~ion of the students' own lnlmrntory ntucly. Report.; nrr grndnl rompnrnl.ivrly nnd somr nrr srlrrtrd for ornl prrsrntntion to the gmup. Wr lrlirve thnt this n p p m ~ ~ rish both more intern t i ~ ~nnd g more useful to studrntn in n "wrvice" murw for hiologirnlly-orier~t~lprrwnrr thnn thr more rlaesirnl nppmarhrs of most rornmrrvinl lnlmrntor?. ~nnnunlsnt this lrrrl.
Vokme 42, Number 9, September 1965 ' 495
