N;tration
The moist 4bmmo-2-nitmacetanilide is in a 20x 150-mm test tube with 2.0 mL of water and 3.0 mL of concentrated hydrochloric acid. Boiling chips are added and mixture i s ~ e n l y refluxed for 10 min. At the end ofthe hydrolysis, thekaction mixture is poured with mixinginto a mixture of 30 mL of ice water and 5 mL of concentrated ammonium hydroxide. If the mixture is not basic, it is made basic by the addition of more concentrated ammonium hydroxide. After being cooled 5 min in an ice bath, the precipitate is collected in a Hirsch funnel, washed with a small amount of ice water, and recrystallized in a minimal amount of 50% ethanol.
flask was filled to the mark with distilled water and set aside. Seven pairs of students prepared three serial dilutions from the stock solution using 10-mL volumetric flasks. The concentration range for the class was 6.758 x lo3 to 6.05798 x lo-' moles&. The experiment was conducted at room temperature. The baseline was determined using distilled water in both cells. The solution cell was drained and rinsed with each solution prior to recording the spectrum. The cell was drained again, and exactly 2.50 mL of the first solution was added to the cell using a pipet graduated in 0.1 mL units. The ceU was stoppered, and the spectrum was recorded. (Cyclohexanone bas an absorption maximum at 277 mu.) (The 2.50-mL volume was the minimum volume required to give the same absorbance obtained with the cell fdled. Smaller volumes gave absorbance values that were too high. Each instructor should fmd this minimum volume prior to the experiment.) After recording the spectrum, the cell was removed and exactlv 1.50 mL of cvclohexane was added to the cell from a p i p i graduated in 0.1 -mL units. The small free volume (0.05 mLl mmainine in the s t o ~ w r e dcell facilitated mixing of the two phages and w"usefu1 for removing any small bubbles that may adhere to the surface of the cell. The cell was shaken for two minutes and allowed to stand for 1min prior to being returned to the spectrophotometer. The spectrum of the extracted aqueous layer wasrecorded. Additional shaking was done as necessaq until the ahsorbance value remained constant. Most samples required 2 min of shaking. The solution cell was washed with acetone and soapy water and rinsed with distilled water after each run and a new baseline was obtained prior to each solution. Student pairs averaged 2 h to complete the experiment.
Literature Cited
Discussion
The dry crystals are weighed. The following directions are f o e mmol(428 mg) ofp-bmmoacetanilide.Pmportionate quantities should he used for different quantities of starting material. In a 25- x 150-mm test tube, thep-bmmoacetanilide is dissolved in 6 mL of concentrated sulfuric acid (with slight heating if necessary). A stirring bar is added and the solution is stirred in an ice bath. A mixture of 12 drops of concentrated nitric add and 20 dmps of concentrated sulfuric acid is prepared and woled in the ice bath. This nitrating mixtule is added to the bromoacetanilide solution at the rate of one drop everv 10 s while the mixture is stirred in the ice bath. ~ & the-addition r is complete the mixture is stirred another 15 min in the ice ba& and poured into 40 mL of ice water with mixing. After 5 min of frequent swirling in an ice bath, the precipitate is collected in a Hirsch funnel, washed with ice water, and partially dried by pulling air through it and pressing it between filter paper between paper towels. This moist pmduct is used directly in the hydrolysis step.
I .a Mayo. 0 W . nkc. R M..Buuhar, Y S Mumrob OwmcLobnmlw, 2nd d.. Wlly New Yark. 1989: pp J57-399. $bl W#llumam.K L Mnrnanb ond Mar r m b O r # a n r r h n w n b Hsalh I m n g n r . 198% pp 52R574
The Distribution of Cyclohexanone between Cyclohexane and Water John D. Worky St. Norbert College DePere, WI 54115
Although microscale experiments have been incorporated into general and organic lab manuals, analytical chemistry and physical chemistry generally have not adopted microscale techniques. This article describes a micmscale emriment that mav be used to demonstrate extraction, s&Arophotometric"analysis, and the determination of a distribution constant in an analvtical or . ~hvsical . laboratory Experimental
The experiment requires reagent grade cyclohexanone, cyclohexane, and distilled water. Measurements were made with a W spectrophotometer (e.g., a double beam Shimadzu Model 160) using matched rectangular 1-cm cells with tighbfitting Teflon stoppers. Astock solution was prepared by weighing to the nearest 0.1 mg a glass-stoppered 100-mLvolumetric flask containing about 15 mL of distilled water. Thirty drops of cyclohexanone were added, and the flask was weighed. The
The system studied is comparatively simple. Cyclohexanone has a large nonpolar moiety and should prefer the organic layer. In the absence of wmpeting equhbria, it can be shown that the distribution wnstant can be determined from the measured absorbance data according to Kd = ((AilA& 1)*(2.5/1.5)
(1)
where Ai and Ar are absorbances for the unextracted (initial) and extracted (final) solutions. resoectivelv. Volumes are incorporated in the constant fa&or at the riiht of eq 1. Cvclohexanone has no sienificant acid or base ~ r o ~ e r t i e s in aqueous solution. 1Cis reasonable to assume that tautomeric forms would be negligible under the conditions of this experiment. A typical student result and calculation follows. A solution with a cyclohexanone concentratim of 5.385 x lo-' M had an initial absorbance of 1.170 and a fmal absorbance of 0.498. The distribution constant as calculated from eq 1 is 2.25. Student values ranged from 1.83 to 2.48 with a mean of 2.22 and a relative standard deviation of 5%. The lowest concentrations of cyclohexanone gave low values (Ka= 1.83,1.91) for the distribution constant and were not included in the average. The standard Gibb's free energy accompanying the transfer of a mole of cyclohexanone from the aqueous phase to the organic phase was calculated using the class mean for the distribution constant. At 23 "C. AGO = -1.96 kJ demonstrating the preference by cyclohexknone for the nonpolar phase. The molar absomtivitv for cvclohexanone (Continued on next page)
Volume 71 Number 6 June 1994
A145
the microscale laboratory was calculated as an internal check on the precision of the work. The average value was E = 21.61 Llmol-cm. The relative standard deviation was 1.1%.Christian et. al. report a molar absorptivity for cyclopentanone in water of 21.1 Llmol-cm ( I ) . They also report a Kd = 2.64 for cyclopentanone distributed between carbon tetrachloride and water at 25 OC. The Gibb's free energy change for their system is -2.41 kJ. All of these values are in excellent agreement with the values reported in this study There are two observations that may be useful in carrying out this experiment. The lowest concentration of cyclohexanone may require extra shaking. Cyclohexanone appears to have some surfactant properties. The low concentrations of cyclohexanone do not allow as much surfise area between phases to form in the unstable emulsion as will form at the hieher concentrations. Eouilibriurn is attained more slowly &r the lower concentrat;ons. This effect may be responsible for the low values of Kd a t the low end of the concentration range. In addition, the measured absorbance values for the extracted low concentrations are small which makes these solutions sensitive to enor. The lower concentrations also may give some difficulty with air bubbles or bubbles of eyelohexane adhering to the cell wall and thereby interfering with the optical analysis. If this happens tip the cell over so that the bubble at the top of the cell passes over the transparent surface of the cell. If this fails, allow the cell to sit for a longer time. As variations, a class might be divided into groups each of which is assigned a different compound to explore the effect of various substituent groups on the distribution constant. Alternative15 the effect of added salts on the value of Kdcould be determined. The class used approximately 30 drops of cyclohexanone and 35 nL of cyclohexane. The entire class used the same set of cells, pipets, and volumetric flasks. The cost of the chemicals is estimated, using current prices, to be $15.23 for the cyclohexane and $0.62for the cyclohexanone. Most of the waste cyclohexane may be recovered by distillation. The cost of reagents works out to $1.13 per student.
of limonene have somewhat different odors, the students usually are able to determine (by comparison to authentic materials) which enantiomer is present in orange peel, illustrating the different properties of mirror images in biological systems. We have found this experiment to require about 2 hand to be rather popular with the students. Experimental Extraction
The rind of an orange is grated onto a piece of wax paper or aluminum foil (not plain paper; it will absorb the oil!) until approximately 2 g are obtained; the exact weight is recorded. The grated peel is transferred to a 10-mL Erlenmeyer flask and mixed with 2 mL of hexanes for a few minutes, after which the hexanes are pipetted into a filter pipet containing about 1 cm of granular sodium sulfate. The filtrate is collected in a tared 5-mL conical vial. The extraction is repeated a second time using another 2 mL of hexanes. Finally, the transfer pipet and filter are rinsed with a small amount of hexanes (approximately 0.5 mL). The solvent is removed by evaporation until the level of the orange liquid stops decreasing andlor the weight remains nearly constant for a few minutes. The weight and appearance of the crude oil is recorded, and the odor of the crude limonene is compared to that of authentic samples of (+)and (-)-limonene (available from Aldrich) provided by the
Acknowledgment
The author thanks the students of Quantitative Analysis for performing the experiment. Literature Cited I . L W ~ ,R.~.;christl.~.,s.~.:~ffap-g,~. E.J ~ h y cham e im,73,an3a78.
A Microscale Isolation of Limonene from Orange Peels Charles M. Gamer and Chad Garibaldi
Baylor University Waco, TX 76798 As recently observed (11,the teaching of laboratory techniques in the context of natural product isolationslmanipulations makes for especially interesting and relevant experiments. However, many natural products are found a t low concentrations in nature, complicating their isolation at the microscale. Orange peel is a rich and convenient source of the temene limonene. Macroscale isolations of limunene that rely on steam distillation have been reported (1, 2,. We have developed a rapid and reliable extmclive microscale isolation ofiimonene This experiment provides a context in which to teach extraction, preparative GC, IH spectroscopy, and capillary GC. Because the enantiomers A146
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Time (mid Preparative GC chmmatogram of orange peel extract (180 "C)
