the microscale laboratory
vanillyl-OH).GC-MS: 7.79 min; no M+ ion was observed under
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Downloaded via UNIV DEL VALLE DE GUATEMALA on March 4, 2019 at 17:12:43 (UTC).
Acknowledgment
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,
...,
. .
.. .
...
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.
The Microscale Separation of Lycopene
and p-Carotene from Tomato Paste
James Goodrich. Chris Parker. and Ruff Phelos
Lou~s~anaState ~ 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 instruc-
tional 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 chromatog-
raphy (for separation) and UV-VIS spectrometry (for anal-
ysis).
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 op-
tional 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. Pos-
itive 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
Support from NSF-ILIP and NSF-Young Scholars grants
(USE-8851427 and RCD-9055029) are gratefully acknowl-
edged.
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: h-
ington, 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
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. How-
ever, many of the procedures-found in laborkary manuals
are both tedious and comdicated for students in a beein-
ning 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 non-
toxic dves. The exneriment is suitable for laree
" laboratom
sections because it requires inexpensive and easily dispos-
able eaui~mentand cheniicals. In addition. the results can
be qu&&ied by measuring the collected &ethylene blue
spectrophotometrically.
Procedure .
Asmall uniform plug of glass wool is placed into the bot-
tom 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% eth-
anol 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 alu-
mina 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 experi-
ment 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 col-
umn, 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
. . ~~~~~~.
Column Chromatography ~.~
Separations Suggested for
-.-
unaergraauare txperiments Edwin J. Goller and John H. ~ i l l e r '
Virginia Military Institute
Lexington, VA 24450
Mixture Stationary Mobile Phase
Phase
In spite of the fact that most undergraduate chemistry
Lycopene- Alumina Petroleum ether-acetone programs have dropped qualitative organic analysis as a
$-carotene Petroleum ether separate course, the chemistry faculty a t VMI believe this
(R)- and (SF Alumina Hexane course remains a useful educational experience for our un-
carvone dergraduate majors. While the primary objective of the
course has not changed over the years, the heavy reliance
Ferrocene-acetyl Silica gel Petroleum ether- diethyl on the modern instrumental techniques of Fourier-trans-
ferrocene ether form infrared spectroscopy (FTIR), contmuous-wave 60-
Cholesterol- Silica gel Ligroin:ether (93) MHz nuclear magnetic resonance spectroscopy (NMR),
cholesteryl acetate and gas chromatographylmass spectrometry (GCiMS),
make the course a relevant capstone experience?
Fluorene- Alumina Ligroin:ether (1 :1) Recently some chemists ( 1 ) have argued that the advent
fluorenone of sophisticated laboratory instruments equipped with
Caffeine Silica gel Ethyl acetate-methylene computer-searchable spectral data bases may render tra-
chloride ditional chemical classification tests and individual stu-
dent interpretation of spectra obsolete. We, however, do
Leaf spinach Alumina Hexane, toluene, ethyl not allow students to use computer-searchable spectral
pigments acetate, methylene
chloride data bases to identify their "unknowns". We prefer that
students gain experience from microscale experimentation
Syn- and anti- Alumina Petroleum ether and that they gain critical thinking skills by selecting their
azobenzene own chemical tests and by interpreting their own spectra.
Chlorophyll and &- Alumina Petroleum ether, ethyl Students need to appreciate the fact that complex spectra
carotene acetate are more than a jumble of bnes to be sorted by computer.

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 volumet-
ric 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 calibra-
tion 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:Lex-
ington. 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.
The Course
There are a number of standard texts for microscale (2-
4) 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 ref-
erence 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 con-
stants, the purification of solids and liquids, the perfor-
mance of selected chemical classification tests and the
preparation ofone derivative out of five unknowns are car-
ried out using microscale techniques and equipment." The
chemical classification tests help students reriolve uncer-
tainties 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
'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 A159