Demonstration pubs.acs.org/jchemeduc
Normal- and Reverse-Phase Paper Chromatography of Leaf Extracts of Dandelions Maria H. Du Toit,† Per-Odd Eggen,‡ Lise Kvittingen,*,‡ Vassilia Partali,‡ and Rudolf Schmid‡ †
School of Physical and Chemical Sciences, North West University, Potchefstroom 2522, South Africa Department of Chemistry, Norwegian University of Science and Technology, 7491 Trondheim, Norway
‡
S Supporting Information *
ABSTRACT: This demonstration describes how normal and reverse phase chromatography can be illustrated using only chromatography paper for the separation of extracts of dandelions.
KEYWORDS: High School/Introductory Chemistry, First-Year Undergraduate/General, Demonstrations, Physical Chemistry, Chromatography, Natural Products, Plant Chemistry, Separation Science
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EXPERIMENTAL OVERVIEW Fresh dandelion leaves soaked in acetone were crushed using a mortar and pestle and then extracted with hexane, filtered, and concentrated in a rotary evaporator. If no rotary evaporator is available, the hexane extract can be left in a container with a wide opening at room temperature in a fume hood. The chromatography chamber used was approximately 20 cm tall, and the inside walls were lined with filter paper. For the normal-phase chromatography, the solvent system was composed of n-hexane/acetone, 9:1, and the chamber was equilibrated for at least 30 min. The leaf extract was applied with a capillary tube onto the 20 cm long chromatography paper and developed until the eluent reached the top of the chromatography paper after approximately 20 min. Thereafter, the system was “overdeveloped” for another 20 min to obtain the required separation. Scotch tape was applied to the paper immediately after the solvent had evaporated, and the result documented by photography. To perform reverse-phase paper chromatography, chromatography paper was impregnated with a solution of plant oil (olive oil−sunflower oil)/n-hexane, 1:16, in an adaption of previous procedures,2,11 and left hanging to dry for at least 15 min in a fume hood before application of the sample. The eluent was in this case acetone/water, 9:1. Sample application and time required for sufficient separation were otherwise as for the normal-phase system described above.
hromatography is a central topic in chemistry curricula from high school through university in most countries. Introductory experiments of chromatography are often conducted using filter paper, chalk, or thin-layer chromatography (TLC) plates with various solvent systems to separate colored samples, such as inks, dyes, and plant extracts. Many simple experiments have been reported.1−6 Liquid chromatography techniques can be classified as either normal or reversed phase. In reversed-phase chromatography, the stationary phase is less polar than the mobile phase. The relation between normal- and reverse-phase chromatography is often presented as a fact, but seldom illustrated with experiments. Where such experiments exist, they often utilize column chromatography7 and, particularly, high-performance liquid chromatography,8,9 although TLC systems10 and paper chromatography11 have also been reported. Normal- and reverse-phase chromatography can be illustrated by simply using paper chromatography of extracts of dandelion leaves (Taraxacum off icinale). As is well-known, green leaves contain chlorophyll a and b. The main carotenoids in leaves are typically carotene and xanthophylls (lutein, violaxanthin, and neoxanthin).12 Compared to previous reports,11,13 the extraction process and solvent systems are simplified in the method reported here, making the procedure more suitable within constrained time limits. The solvent systems also comply with current safety regulations. The use of an overdevelopment technique improves the otherwise unsatisfactory separation. Various green leaves can be used, but dandelion leaves were deliberately chosen as these contain a substantial quantity of pheophytin. This compound is visible as a singular grayish spot in the separation, making pheophytin a particularly valuable reference when the sequence of the migration is to be compared between separation systems. © 2012 American Chemical Society and Division of Chemical Education, Inc.
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HAZARDS All procedures should be performed in a fume hood and suitable gloves and eye protection should be used. n-Hexane Published: August 17, 2012 1295
dx.doi.org/10.1021/ed200851w | J. Chem. Educ. 2012, 89, 1295−1296
Journal of Chemical Education
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and acetone are highly flammable liquids and vapors and should be kept away from sources of ignition. Hexane and acetone are harmful if ingested, inhaled, or in contact with the skin. Vapors may cause drowsiness and dizziness. Acetone causes serious eye irritation. There is a danger of serious damage to health by prolonged exposure through inhalation of hexane, including a possible risk of impaired fertility. Hexane may be fatal if swallowed and enters airways and may cause skin irritation. Magnesium sulfate may cause eye and skin irritation as well as irritation to the respiratory and digestive tract.
Demonstration
ASSOCIATED CONTENT
S Supporting Information *
Instructor notes with supplementary experimental details. This material is available via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
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The authors declare no competing financial interest.
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RESULTS AND DISCUSSION The effect of the normal- and reverse-phase systems is illustrated in Figure 1. In Figure 1A, dandelion leaf extract is
REFERENCES
(1) Braithwaite, A.; Smith, F. J. Chromatographic Methods; Chapman and Hall: New York, 1985; pp 359−362. (2) Stock, R.; Rice, C. B. F. Chromatographic Methods Chapman and Hall: New York, 1963; pp 334−335. (3) Drudling, L. F. J. Chem. Educ. 1963, 40, 536. (4) Gilbert, J. C.; Monti, A. J. Chem. Educ. 1973, 50, 369. (5) Wollrab, A. J. Chem. Educ. 1975, 52, 809. (6) Heumann, L. V.; Blanchard, D. E. J. Chem. Educ. 2008, 85, 524. (7) Sander, L. C. J. Chem. Educ. 1988, 65, 373−374. (8) Dean, J. R.; Jones, A. M.; Holmes, D.; Reed, R.; Jones, A.; Weyers, J. Practical Skills in Chemistry, 2nd ed.; Prentice Hall: Essex, U.K., 2011; p 379. (9) Skoog, D. A.; Holler, F. J.; Crouch, S. R. Principles of Instrumental Analysis, 6th ed.; Brooks/Cole: Belmont, CA, 2007; p 829. (10) Cooley, J. H.; Wong, A. L. J. Chem. Educ. 1986, 63, 353. (11) Strain, H. H.; Sherma, J. J. Chem. Educ. 1969, 46, 476−483. (12) Britton, G. In Carotenoids Vol. 1A: Isolation and Analysis; Britton, G., Liaaen-Jensen, S., Pfander, H., Eds.; Birkhäuser: Basel, Switzerland, 1995; pp 201−203. (13) Anwar, M. H. J. Chem. Educ. 1963, 40, 29−31. (14) Rollins, C. J. Chem. Educ. 1963, 40, 32.
Figure 1. (A) Normal-phase and (B) reverse-phase chromatography of dandelion leaf extracts on paper, developed with n-hexane/acetone 9:1 and water/acetone 1:9, respectively.
separated in a normal-phase system, and in Figure 1B, the same extract is separated in a reverse-phase system. As can be observed, the elution sequence is opposite in these two systems. In the normal-phase system the main spots appear, with increasing Rf values, as one yellow spot with tailing (partly hidden by the first green spot), two green spots, a grayish spot, and a yellow-orange spot. According to the polarity of the components and literature referring to normal-phase systems,11,13,14 these spots most likely correspond to xanthophylls (with some tailing), chlorophylls a and b (the two green spots), pheophytin (gray), and β-carotene (yellow-orange), respectively. In the reverse system, the sequence of the spots is reversed; one orange spot (nicely rounded), one gray spot, two green spots, and (with longest migration) at least three yellow spots. The first four spots correspond to carotene, pheophytin, and the two chlorophylls. The next three are expected to be xanthophylls, most likely lutein, violaxanthin, and neoxanthin,13 of which the order is based on their expected polarities, but difficult to reliably deduce here due to the possible interference of xanthophyll fatty ester derivatives.
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SUMMARY These two experiments can be demonstrated in the classroom. They provide a springboard to discussing polarity and migration rate, the characteristics of normal- and reversephase systems, and a comparison between them. In short, these experiments illustrate that polar mobile phases promote migration of polar compounds, whereas nonpolar mobile phases promote migration of nonpolar compounds.10 1296
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