A More Affordable Undergraduate Experiment on the Reduction of

Mar 1, 1994 - A More Affordable Undergraduate Experiment on the Reduction of Acetophenone by Yeast. Moses Lee and Martha Huntington. J. Chem. Educ. , ...
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the microscale laboratory concentrated pigment solution in 3 mL of 95% ethanol. The yellow pigment in the first fraction may be analyzed in a similar manner. Discussion This procedure is performed easily in a 2.75-h laboratory period with the possible exception of obtaining the IR and UVNis spectra. Care must be taken to prevent the uiament m i t u r e or the fractions wllected frbm going to dr;ness while concentrating them because they may oxidize in air. If storage of the is required, tb;! dissolved pigments may be sealed in a flask with Parafilm. The only difficulty encountered was a tendency for the column to separate if allowed to stand attached to the vacuum filter flask. Application of a gentle vacuum reunites the column without adverse results. This procedure has been well received by the students. It gives results comparable to the macroscale method while requiring less time and smaller amounts of chemicals. Literature Cited 1. Miller, J,A; Neuril, E. F Madern E x p r i m n f o l O w e Chemlatn: D.C. Heath: laxingtan, MA, 1982; pp 270-274.

Salt Bridge Using Soil Moist David W. Brooks and Helen B. Brooks Center for Curriculum & Instruction University of Nebraska-Lincoln Lincoln, NE 68588

Small scale electrochemical experiments are becoming more common. While 24-well and even 48-well plates are hand for setting up most half cells (e.g., Cu/Cu2+; PtiFe me3+)and modern electronics makes high impedance multimeters readily available to teachers, salt hridges remain a problem. Strips of paper or pieces of wettable thread moistened with electrolyte often are inadequate. Recently we attem ted to do a microscale potentiometric titration. The CdCu1;: couple was used as a reference cell. A titration of Fez+with Mn04- with a platinum wire electrode was the reaction cell. (Nichrome is usable over the range of the experiment where Fez+is available.) Under these conditions, salt bridges of paper and thread failed. However, the following procedure worked. A small piece of Soil Moist, a dehydrated polymer (25% hydroxyethyl methacrylate, acrylamide copolymer, crosslinked acrylic homopolymer) that swells in water, was inserted ahout 3 cm into the stem of a plastic transfer pipet. (An unwound paper clip works well.) The pipet was squeezed slowly to expel air, then inserted into a test tube containing 0.5 M Na2S04, to draw the electrolyte solution slowly into the bulb. The pipet was allowed to stand for about 3 min. Changes in the solid were noticeable a t first but, before long, the solid seemed to disappear. The pipet was removed from the solution, and the bulb cut off where it joins the stem. The resulting tube remains filled with electrolyte, conducts well, and is flexible. It readily can be bent to conn e d adjacent or nearby wells. For the purposes of the potentiometric titration, this tube served as a n excellent salt

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'Soil Moist is a product of JRM Chemical Division, 13900 BroadThe product is way Avenue. Cleveland. OH 44125.800-962-4010. available at garden stores. A62

Journal of Chemical Education

bridge. In addition, it was a practical laboratory use of the Soil Moist product.' The same approach can be used to construct a one-piece bridge-reference cell-stirrer. The tip of a standard plastic transfer pipet is cut to 4 cm. The polymer piece is placed about 2 cm into the stem. The pipet is then filled as above with 1M Na2S04After the gel swells, a small hole is made a t the end of the pipet bulb. A plastic transfer pipet is used to remove excess Na2S04 through the hole. Next 1 M CuS04 is added through the hole, a copper wire is inserted, and the entire piece is used a s a reference cell, salt bridge, and stirrer. When this device is used in the potentiometric titration, readings are remarkably stable aRer reaction in the working cell is complete.

A More Affordable Undergraduate Experiment on the Reduction of Acetophenone by Yeast Moses ~ e eand ' Martha Huntington Furman University Greenville, SC 29613

In a n earlier publication we described an experiment for the synthesis of (-1-(R)-a-deuteriovanillylalcohol by reduction of vanillin with yeast in D20 (11. While the ex~eriment was surcessful in i ~ i u s t r a t i n ~ t enantiomeric he &lecti\ity of the reaction, the expense of D20 may preclude some departments from adopting it. a he current contribution describes the reduction of acetophenone, 1,by yeast, which is more cost-effective in demonstrating the stereochemical control of such reactions. In the described experiment yeast is used to reduce 1to (Continuedon page A64) $C

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3(S,S)andior 2(S,R)

Synthesis of I-phenyiethanol 2 give the optically active (-)-1-phenylethanol2 in 5 %yield, and NaBH4 is used to form the racemic alcohol 2 in 73% neld (see reaction below). Althouah the vield of the optically active 2 is not as high as the;eported value (2)sifficient auantities of it are easilv obtained to comolete this exerciie. Also in this study thestructure of 2 can i e readily deduced by IR, 'H-NMR (31, GC-MS, and polarimetric analyses. The chiral and racemic 2 are then reacted with (S)-(-)-methoxytrifluorophenylacetic acid (MTPA), 'Author to whom correspondence should be addressed.

the microscale laboratory

Figdre 2. hewman Prolecf ons of tne d astereomers of ester 3. The lener a represents a aownf eld s gnal relawe to its counterpan b.

F,g~re1. NMR Ana yses of tne Mosher's esters 3 that are der~ved from tne opuca .y anwe an0 racemc a conols 2. Parts A and B snow the 300-Mdz d-hMR spectra of 3 oola nea from the racemlc and cnra 2. Parts C ano D oepct tne 282 Mhz 'F-NMR of 3 preparea from the respective alcohols

dieyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) to produce the corresponding Mosher's esters 3 (4,5). While the 'H-NMR spectrum of ester 3, derived form the racemic alcohol 2, shows two chemically nonequivalent signals for the a-methoxy group (3.57 ppm and 3.48 ppm, Fig. lA), the ester obtained from the optically active alwhol shows a n intense signal for the methoxy group a t 3.48 ppm (Fig. lB)? This signal corresponds to the (S,S) diastereomer of 3 because the methoxy group is shielded by the phenyl group whereas that of the (S,R) diastereomer is not (4, 5) (see Fig. 2). I n the 'SF-NMR studies, the 'racemic' ester 3 gives two signals of almost equal intensities a t 55.01 and 54.82 ppm (see Fig. 1C). However, the spectrum of the "chiral" ester depicted in Figure 1D gives mainly the lower field peak. Since there is less steric hindrance between the phenyl and methyl groups in the S,S diastereomer than the two phenyl moieties of the S,R isomer, the CF3 signal of the former isomer is more deshielded by the magnetic anisotropy of the carbonyl moiety (6). The S absolute configuration of the chiral alcohol agrees with the addition of a hydride to the re face of the prochiral ketone catalyzed by alcohol dehydrogenase (Za, 7). The enantiomeric excess (e.e.) of the chiral and racemic alcohols 2 can be measured by integrating either the diastereomeric aOCH3 signals in the 'H-NMR spectrum or the a-CF3 peaks in the "F-NMR spectrum. The values of e.e. obtained from the 'H- and 19F-NMR studies are 82 f 4 and 4 i 4 %, and 86?4% and 4i4%, respectively (4,6). 2 0 ~ ana r yses were parformed on a 300-Mhz as we1 as a 60-MHz hMR specfrometer In tne aner spennm the s gnals of the aiastereomers are sufficientlyresolved but not enough for accurate integration. It can nevertheless be useful for illustrating the sterwselectivity of yeast mediated reductions.

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Journal of Chemical Education

Reduction of Acetophenone with Baker's Yeast I n a 500-mL round-bottom flask suspend 56 g of Fleishmann's active dry yeast in 300 mL of water. Add 250 mg (2.08 mmol) of acetophenone 1 to the yeast solution, loosely plug the flask with cotton, and stir a t 30 'C for 72 h. Centrifuge the broth for 15 min and extract the supernatant three times (100 mL each) with CHzClz. Dry them with NazS04then gravity filter. Concentrate the filtrate in a tared flask on a rotary evaporator. Purify the oily residue using preparative TLC (20% ethyl acetate: 80% hexane, Rf = 0.21). View the TLC plate under UVlight then scrape the Silica Gel containing t h e product i n t o a 125-mL Erlenmeyer flask, add 40 mL CH2CI2,and sonicate to dissolve the product. Vacuum filter and concentrate with a rotary evaporator to produce 11.4 mg (5% yield) of 2 as a clear oil.

'H-NMR(CDCL,) G 7.36-7.35 (m, 5H), 4.89 (q, 6.4, lH), 2.00 br, lH), 1.49 (d, 6.9, 3H); IR (neat) 3358, 3040, 2971, 1604, 1491,1075,899,700em-'; GC-MS [HP4890A, 12 m x 0.2 mm Llp-1 cross-lrnkrd methyl sdiconcifilmthickncss0.33 $n,cnpillnry GC column1 retention time 7.35 mln; MStK1, nz z lrcl mtensitv 122 rM'.. 39 .: la^.^-,^^ CHCI? = -39.5' 10 = 0 0228 g/mL) [it. value is reported to be 43.5'for 589 nm (811 (S

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Reduction of Aceto~henonewith NaBH4 The prncedure is similar to that used in the reduction of vanillin (11. Starting with 500 mg of 1, the yield is 371 rng (73'il a s a clear colorless oil.

[als7s22 (CHC13)= O'(c= 0.0512 gimL). TLC, IR, GC-MSdata are identical to those of the chiral product. Preparation of the Mosher's Esters, 3 Dissolve MTPA (22 mg, 0.095 mmol) in 3 mL of dry CHzClzin a dry 15-mL round-bottom flask. To this solution add DCC (92.9 mg, 0.45 mmol), three crystals of DMAP, and the racemic alcohol 2 (50 mg, 0.41 mmol) dissolved in 1mL of dry CH2Cl2.Reflux gently under a drying tube for 12 h. Filter out the urea, add 40 mL of CHzCll to the filtrate, and wash the organic extract with 5 mL of NaHC03 (1.0 M). Dry with Na2SO4and remove the solvent on a rotary evaporator. Purify the residue with preparative TLC (25% ethyl acetatemexanes, Rf = 0.7). The yield is 46.9 mg (34%) a s a colorless oil.

'H-NMR (CDC13)S 7.44-7.26 [m, 10 HI, 6.14 and 6.10 (both q, 6.5, 1H for S,S and S,R), 3.56 (8, -OCH3, 3H, rel. intensity 48%. for S.Rl. . -.3.48 (s. . . -0CH..",3H., 52% for SS). . .. 1.65 Id. . . 6.6.. 3H, -CH3 for S,R), 1.59 (d,.6.6,3H,-CH3 for SS); e.e. = 4f4%, (Continued on page A661

the microscale laboratory 19F-NMR(trifluarotaluene internal standard 63.13 ppm) 6 55.01(s. 3F.-CF,. 52%forSS).54.82 (s.3F. -CFs48%f0rS.R): e.e. = 4490: i~ 1C%Cloeast) 3430.2948. i747.166"16.1496.1452: GC-MS retention tim; 1259, 1169, 1122, 10"18,762,69'8 14.02 min; MS(E1) 338 (M*,4. The Mosher's ester of the optically active alcohol (8.2 mg, 0.024 mmol) is prepared in 25% yield. 'H-NMH (CDCI?)6 35fi Is. 9'71, 3.47 Is, 9141, ex. = 8234'; 1YF-lWRS55.021s,Y31 1,54.821s,7Lbl,e.e.=86?4'1;TLC,IR, GC-MS are identical to thore of the rneemic pmduct. Literature Cited 1. Lee,M.L.J. Chem. Educ l993,70,A155-A158. 2. la1 Nakamura, K,Ushlo,K:Oka,S.; 0hno.A.TktrohdmnIatt. 1984,25,39795982. lbl Bucdarelli, M.; Forni, A ; Moretti. I.; lbm, G.Synthesis 1983, 897-899. 1 4 Elie1,E.L. J hChem. . Sac.I949,71,397W3972. 3. Pouch&, C.J. TheAldrichLibroryofNMR Spaclm, 2nd ed.;Ald?~chChemicalCompany,he.: Milwaukee, 1988;Vol.1, pp 921C. 922A. 4. Yamaschi, S. In Asymmtric Syntheses, Morrison, J.D., Ed.; Aesdemie Resa, he.: New York. 1983: Vol. 1. DO 125-152.

8. Downer, E.; Kenyan,J. J. Chom. Soc. 1939, 1156.

Temperature Control for a Small-Scale Kinetics Experiment Patrick lash' Kent State University-Ashtabula Campus Ashtabula, OH 44004 This note describes a microscale version of the classic iodine clock kinetics experiment with iodide and peroxydisulfate under temperature-controlled conditions that also permits calculating the activation energy. The iodine clock experiments available in current microscale general chemistry lab books ( I , 2) do not offer a way to control temperature using microwell plates. Thompson (3)uses a styrene coffee cup to provide temperature control for a methylene blue decolorization study. Construction of the Bath One-inch, blue polystyrene insulation foam is cut into pieces 6.5 x 8 in. (for bases) and 1 in. x 1 in. strips (for edges). Sauare edges are essential. The l-in.-sauare strips are cut tdthe length and fastened to the base and each other with bathtub caulk, which seals all the seams to provide a shallow water bath that fits a 24-well plate perfectly. Four to SIX 3 8-in. holes are dnlled in the base of the 24-well olate between the wells and each edee of the d a t e is notchLd with a paper punch to allow wat& circulacon. Use of the Bath A 60-drop volume of stock ~eroxvdisulfatesolution is placed in two wells of the 24-well and a third well is filled with a stock mixture of iodide. thiosulfate. and starch indicator. The bath is preheated/~ooledby rinsing with water above or below the temperature ultimately desired.

About 200 mL of water slightly above or below the desired temperature is poured in, depending on whether the kinetic run will be above or below room temperature. The plate is submerged carefully, making sure the bath water does not rise above the rims of the wells. The plate is held submerged by placing a thermometer in well B3 and clamping it so that the thermometer forces the base of the plate down. A photograph of the apparatus is shown below. When the appropriate temperature has been reached as indicated by the thermometer in well B3, 40 drops of the iodide solution is transferred rapidly to a sample of the peroxydisulfate solution via a calibrated Pasteur pipet. The solution is stirred with a capillary melting point tube until the blue color appears. Use of a room temperature pipet to transfer the solution does not seem to affect the calculated activation energy significantly. Time versus temperature &dies with thermometers in water-filled wells shows that the maximum time for solutions to reach thermal eauilibrium is about 10 min for a bath temperature of 35 '6 and 15 min for a bath temperat u r e of 5 ~ C These . times a r e for a b a t h not precooledheated as described above. Unclamping the thermometer and .. wntlv. ~ . u s h i n ethe plate down a few times every few mlnutes encourages water c~rculat~on and faster thermal eau~libnum.The thermometer ul well H.3 seems to give a good indication of the overall bath temperature. Duplicate kinetic runs are recommended for each set of conditions. If the two times deviate significantly from the average (+/-lo%), a third trial is required. Even with some repetition of individual trials, students can finish the experiment in a three-hour period. Results The order of the reaction with respect to peroxydisulfate is determined using five room-temperature runs of variable peroxydisulfate composition. (The order with respect to iodide is given.) Initial reaction rates are calculated from the known change in concentration of iodine with time. Rate constants &r four temperatures (5 OC, 15 T, room temperature. and 35 "C) are then calculated and a graph of the log ofthe rate constant versus the reciprocal of the Kelvin temperature is plotted. The slope of the straight line yields Emt from the equation

slope = -E,&.303R The calculated activation energy of 48.5 k J (average of three classes) agrees quite well with the reported value of 51.8 k J ( 4 ) and is near the minimum error expected for drop wise measurements (5). At the suggestion of a reviewer, the experiment was re~ e a t e du s i n ~10- bv 60-mm test tubes. a 100-mLbeaker as water hat(, and total reaction volime of 2.5 mL. Duplicate runs a t five temperatures from 20 'C to 55 'C pave an activation energy 2 48.7 k J (R = 0.9816), in excellent agreement with the microwell determined value.

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Literature Cited 1. Mills, J. L.: Hampton, M. D.Miemsmle Labomtory Manual fw Ceneml Chemistry; ~~

'Presented at the 199th National Meeting of the American Chemical Society, Boston, MA, April 1990;paper CHED 246. Copies of the experimental handout given to the students can be obtained by sending a SASE to the author. A66

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Random House:New York, 1988. 2. Russo, T Micm Chemistry; Kemtec Educational: Korsington, MD. 1985. 3. Thompson, S. Chomtmk: Micmscois E ~ p p n m n t afor Gmernl Chomishy: Allyn and Bacon:Needham Heights, MA, 1989. 4.

Moews,P.C.:Petmi.R.H.J

Cham. E d u r 1%,41,549.

5. E d 6 James: Fickdng, Milea. J. Chem. Educ. 1991,68,A120-122.