the microscale laboratory vanillyl-OH).GC-MS: 7.79 min; no M+ ion was observed under our conditions. Acknowledgment The Experimental Techniques 1Class of Winter 1992 is acknowledged. Literature Cited 1. Gmas, M A n n u o l Rep.Med Chem. 1990,25,32&231. 2. Remos T o m b G.M.: Bellus, D.Anpelu Chem. Inl. Ed. E n d 1993.30, 1193-1215. 5. Csuk,R.; Glanzer, B. I. Chem Re". 1991,31,41C97. 6 . Nimitz J. S. Er~rrimpnfsin O w n i c Chemistry : Rentice-Hall: Endewood Clifi,
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7 . Banesbv,A.R.:Stauton, J.;Wfitshire,H.R.J C S . Perkin 1 1 W h 11561162
11. Ward, D.; Rhee, C. K lbhohedmn Lett 1991,32,7165-7166. 12. Yamaguehi, S. hkymmtric Synthosla;Mamison, J. D., Ed.:Academic Reas, Ine.: New York. 1983: Val. 1,pp 125-152.
Acknowledgment Support from NSF-ILIP and NSF-Young Scholars grants (USE-8851427 and RCD-9055029) are gratefully acknowledged. Literature Cited 1. Lehman, J. W. Opmtioionol O~gonicChemlafry;A l l p andBsmn: Bostrm, 1981. 2. Ranman, P J Chem. Edrrc. 1985,62,640. 3. Wiltiaman, K L. Mocmsmlaond Microscale OgonicExprimntn, D.C. Heath: hington, MA, 1989. 4. Fieser, L.F.:Williamson, K L. organic Experiments, 6thed.;D.C.Heath: Lexingtrm, M A 1987 5 . McKone, H . T . J Chem.Edue. 1973,56,6'76.
6 . Tantillo, M.J . Chem. Edue. IS-, 65,254. 7 . p s n a , D. L.; L-pman, G. M.; and k e , 0 . S. Infmdudion to Organic Iabomtory Tmhniguss; Q.B. Saunders Ca.:Philadslphia,PA, 1976. 8 . Sadier, G.;Davis, J.; Derman, D.J. FoodSci. 1990, hi, 1460.
Separation of Methylene Blue and Fluorescein: A Microscale Undergraduate Experiment in Column Chromatography
The Microscale Separation of Lycopene and p-Carotene from Tomato Paste James Goodrich. Chris Parker. and Ruff Phelos Lou~s~ana State ~ n ~ ; e r sIn~~hreve~orl t~ Shrevepoll. LA 71 115
Experiments for the isolation and analysis of carotenoids from carrot andlor tomato pastes are popular in instructional chemistry laboratories (Id)but oRen require 3.5-5 h. We have developed an experiment for the isolation and separation of p-carotene and lycopene from tomato paste on the microscale level that affords the isolation of two compounds from a single common foodstuff and requires less than 3 h to complete. The compounds can be rapidly analyzed. It provides experience with column chromatography (for separation) and UV-VIS spectrometry (for analysis). The column is prepared with insertion of a glass plug into the tip of a 5.75 in. Pasteur pipet. Alayer of alumina (80-200 mesh) (dry-packed or slurried in 99:l petroleum ether-acetone) ( 6 ) is added to a height of -8 an.An optional layer of sand (3 mm) may be added to complete the column. The use of approximately 3 g of tomato paste provides enough lycopene extract for spectral analysis. The tomato paste is extracted with petroleum ethedacetone (50:50) (3 x 10 mL) and filtered. The combined extracts are washed with saturated aqueous sodium chloride (25 mL), 10% aqueous potassium carbonate (25 mL), water (25 mL), and dried with sodium sulfate. After concentrating the volume in vacuo to several milliliters, it is placed on the column for separation. The C)-carotene1s eluted with petroleum ether acetone (99:11andthe lycopene ~selutedwitha 9W10 mlxture. Positive oressure noolied with a ruhber bulh will soeedelution but can cause band spreading, particularly with the lycopene. Eluants were analyzed spectrophotometrically using (90:lO) petroleum ether-acetone as a reference. The characteristic absomtions of lvco~enewere observed near 504, 473, and 446 i m ; whereas, 'p-carotene occurs at 476 and 450 nm (7).Quantitative vields can be calculated using the molar absorptivity at 470 nm (1.85 x 105) (8). A158
Journal of Chemical Education
Paris ~voronos'and Edward Sarlo Queensborough College of the City University of New Yorh Bayside. NY 11364
Often column chromatography (CC) is used to separate mixtures and/ or purify compounds. The procedure is so commonly employed that the technique usually is included in the first semester of an oreanic chemistrv course. However, many of the procedures-found in laborkary manuals are both tedious and comdicated for students in a beeinning organic labllratory c k w ctable,. We offer a simple microscale CC experiment for separatin~and isolating the componentsofa 5Q50 methylme hlue-fluorescein mixture that uses a disp~~sahle pipet, two nontoxic solvents (water and ethanol.. and aluminn and that allows the student to see the clean separation of the blue and yellow colored nontoxic dves. The exneriment is suitable for laree " laboratom sections because it requires inexpensive and easily disposable eaui~mentand cheniicals. In addition. the results can be qu&&ied by measuring the collected ðylene blue spectrophotometrically.
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Procedure Asmall uniform plug of glass wool is placed into the bottom of a disposable pipet with the aid of a thin glass rod, wire or "unfolded" paper clip. Alumina is added until it forms a 3-in. high column. The pipet is filled with 95% ethanol and, with a finger placed on top, inverted and shaken vigorously to ensure that the alumina is evenly suspended in the solvent and that all air bubbles are eliminated. The pipet is turned right-side up and secured over a test tube using a clamped, one-hole rubber stopper. Traces of alumina adhering to the glass may be washed down with a few drops of ethanol. The solvent level must be kept above the top of the column of alumina throughout the experiment by continuously adding ethanol. Two to three drops of a green dye solution composed of 5%methylene blue and 5% fluorescein in 95% ethanol is added directly to the column, which is then eluted using 95% ethanol. The first component, methylene blue, is collected in a test tube placed under the tip of the pipet. When the eluate is no
'Author to whom correspondence shoud be addressed.
Microscale Qualitative Organic Analysis
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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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