EXPERIMENT 5 COLUMN AND THIN LAYER CHROMATOGRAPHY Michael Edward D. Real, Kriselle Dianne M. Rosario, James Urbano M. Santos, Angelyn M. Si, and Nicole Therese L. Siao Group 9 2E MedTech Organic Chemistry Laboratory
ABSTRACT Chromatography is a set of laboratory techniques for the separation of mixtures which involves passing a mixture dissolved in a mobile phase through a stationary phase.This experiment aims to separate the colored components of red Siling Labuyo and Malun Malungga ggay y leaves leaves using using col colum umn n chroma chromatog tograp raphy hy;; to determ determine ine the purit purity y of the the compon component ents s using using thin thin layer layer chroma chromatog tograp raphy hy;; and measu measure re the Rf values values of the col colore ored d compo compone nents nts in thin thin layer layer chroma chromatog tograp raphy. hy. Colum Column n Chromatography and Thin Layer Chromatography was used to separate the different colored pigments in Malunnggay leaves and these pigments found to be Xanthophylls, the yellow eluate; Chlorophyll A, the dark green eluate; and Chlorophyll B, the light green eluate . Colored eluate was collected through Column Chromatography and the purity of these eluates was tested tested through through Thin Layer Chromatog Chromatograph raphy. y. UV lamp was used as the visualizatio visualization n method method for the determina determination tion of colorless substrates and the Retention or Retardation Factor was measured.
INTRODUCTION Column Column chroma chromatog tograp raphy hy is the most most general general method for the purification of organic compounds. It involv involves es the separa separatio tion n of indivi individua duall compou compounds nds from from a mixt mixture ure by pass passin ing g it throu through gh a colu column mn packed with silica gel. After the mixture is applied to the the colu column mn,, an appr approp opri riat ate e solv solven entt is pass passed ed throug through h the col column umn.. Silica Silica gel was used used becaus because e comp compou ound nds s will will usua usuall lly y adhe adhere re to the the sili silica ca to differ different ent extent extents, s, they they travel travel at differ different ent speeds speeds down down the the colu column mn.. Each Each chrom chromat atog ogra raph phic ic zone zone represe represents nts a natura naturall consti constitue tuent nt of the organi organism sm from which which the mixture mixture was extracted. extracted. Thin layer layer chromatography is very useful for rapid analysis of organic mixtures. Plastic, metal, or glass plates are prepared with a thin layer of silica gel. The organic compound compound that was noticeable noticeable was the chlorophyll. chlorophyll. Thes These e are are the the gree green, n, magn magnes esiu ium m cont contai aini ning ng,, tetrapyrroli tetrapyrrolic c pigments pigments of autotrophic autotrophic plants. They are easily convertible to other greens, yellow-green, or gray gray pigm pigmen ents ts that that are are readi readily ly sepa separa rabl ble e by conven conventio tional nal chroma chromatog tograp raphic hic method methods. s. Throug Through h these processes the following objectives were met, mainly: 1. Separate the colored components of siling labuy abuyo o and malu malung ngga gay y leave eaves s usin using g colum olumn n chromatography. 2. Determine the purity of the components using thin layer chromatography (TLC). 3. Me Meas asur ure e the the Rf valu values es of the the color olored ed components in TLC.
The silica gel got up to the intended part acted as the stationary phase in the set-up. Using sing a Past Pastue uerr pipe pipett tte, e, 0.5 0.5 mL of the the extr extrac actt was was plac placed ed on top top of the the colu column mn.. The The pigments were then eluted with the use of 10 mL of hexane:acetone having a ratio of 7 is to3(7:3). The column was properly observed upon every now and then to avoid it to run dry. The solvent system system was then introduced into portions whilst discarding the colorless eluate. The collected colored eluates were placed in separate test tubes. Note taking of the number of drops drops of elua eluate te that that are are coll collec ecte ted d in each each tube tube became an important task and was done cautiously. Prepare a 5 x 8 TLC plate was prepared. With this TLC plate, eluates were applied by spotting five times. Spots were made sure to be a small one as possible.Each spot were dried first before applying the next eluate.
A developing chamber was then prepared by adding 10 mL of the hexane hexane:a :acet cetone one(7: (7:3) 3) sol soluti ution on and lined the inner wall with filter paper, covered with a watch glass and allowed to equilibrate. e quilibrate.
EXPERIMENTAL A. Compounds Compounds tested tested (or samples samples used) 10-15 leaves of malunggay, Hexane:Acetone(7:3 ratio), Acetone, Acetone Methanol. B. Proc Proce edure dure For the first procedure of this experiment, 10-15 malunggay leaves were selected and grinded using a mortar and pestle. Using hexane:acetone(7:3), pigments were extracted and later on, a portion of the extracts were set aside for TLC. After that, the column was plugged with cotton at the bottom and uniformly packed with silica gel.
TLC plate was placed carefully in the developing chamber to allow the solvent system to rise up to 1 cm from the upper end before the plate was removed from the chamber. Then, the solvent front and air dry were marked immediately. After many procedures had had done, components were visualized using UV lamp in order to measure RF values and to document the chromatoplate.
RESULTS AND DISCUSSION Table 1. Leaf Pigments and # of drops obtained to get the pigment in Column Chromatography
Leaf Pigment
Color
# of drops obtained before getting the pigment
Chlorophyll A Chlorophyll B Xantophyll
Dark Green Yellowish Green Yellow
75th drop 121st drop 135th drop
The mobile phase slowly flows down through the silica gel column by gravity leaving behind zones of color - the chromatogram. The theory of column chromatography is analogous to that of thin-layer chromatography. The different components in the sample mixture pass through the column at different rates due to differences in their partioning behaviour between the mobile liquid phase and the stationary phase.
In addition, to qualitative results, TLC can also provide a chromatographic parameter known as R f Value. The Rf value is the retardation factor or the ratio-to-front value expressed as a decimal fraction. The Rf value is the ratio of the distance travelled by the spot from the point of origin to the distance the solvent travels. The general formula for computing for the Rf value is
Computations: Chlorophyll A:
Chlorophyll B:
Xantopohyll:
The Rf values obtained will help us determine the identity of a substance. If two substances on the same TLC plate give spots in identical location, they may be the same. But still, comparisons of Rf values to be valid, TLC plates must be run under the exact same conditions for stationary phase, mobile phase, and temperature.
REFERENCE/S: Books: Pavia, D., Lampman, G., Kriz, G., & Engel, R. (1998). Introduction to organic laboratory techniques: a microscale approach.3rd Ed. USA: Saunders Publishing Comapany. Figure 1. TLC plate Pigments on the TLC plate after placing it on a solvent system Table 2. Thin Layer Chromatography: Leaf Pigments and Distance travelled by the spotted pigment from the origin
Leaf Pigment
Color
Distance (cm)
Chlorophyll A Chlorophyll B Xanthophyll
Dark Green Yellowish Green Yellow
6.0 cm 5.9 cm 5.0 cm
The distance travelled by the solvent of the “solvent front”. In the experiment, we obtained 7.5 cm as the solvent front.
Williamson, K; Minard, R; Masters K. (2007). Macroscale and microscale organic experiments. 5th Ed. Boston, USA: Houghton Mifflin Company. Garner, CM. (1997). Techniques & experiments for advanced organic laboratory. USA: John Wiley & Sons. Inc. MACAULAY, DB. (2007). General, organic & biological chemistry an integrated approach. USA: John Wiley & Sons. Inc.
