COLUMN AND THIN LAYER CHROMATOGRAPHY Ivy Rose C. Orozco, Arnold V. Paguirigan Jr., Sarrah Mae B. Punzalan, Jayvee S. Rivera and Rico Maria R. Rivera Group 8 2E Pharmacy Organic Chemistry Laboratory
ABSTRACT The pigments present in malunggay leaves and the process of chromatography was the main focus of this study. The results, both qualitative and quantitative, that was obtained for this experiment was interpreted. The qualitative results of the experiment were about the colour and dominance of different pigments in malunggay. Both yellow pigments and green pigments were both seen in both set-ups. Also, the column chromatography set-up was only able to extract green and yellow pigments with almost equal amount in drops. The quantitative results, in the other hand, were about the retention factor value of the different pigments especially the green and yellow pigments, and also the presence of different compounds. The retention factor of the yellow pigment was 0.98 since it travelled 59 cm from the origin. While the green pigment retention factor was 0.33 since it travelled 20 cm only from the origin. Since retention factor is inversely proportional to polarity [8], a conclusion that yellow pigment was less polar than the green pigment was deduced.
INTRODUCTION This experiment is all about the use of Chromatography in separation, identification, and determination of purity of different compounds present in the sample. Chromatography is a proven method for separating complex samples into their constituent parts, and it is undoubtedly the most important procedure for isolating and purifying chemicals [4]. Chromatography relies on the differential solubilities and adsorptive of the components to be separated with respect to two phases; stationary phase and mobile phase [1]. Stationary phase is the part of the chromatographic system though which the mobile phase flows where distribution of the solutes between the phases occurs [3]. Mobile phase, on the other hand, is the part of the chromatographic system which carries the solutes through the stationary phase [2]. The two types of chromatography that will be used in this experiment is Column Chromatography and Thin Layer Chromatography Column Chromatography is the most advantageous over most of the chromatographic techniques. It can be used to determine the number of components in a mixture but also to separate and purify substantial quantities of those components for further analysis [6].
determining their purity and following the progress of a reaction [7].
Figure 2. Thin Layer Chromatography
The sample we used is the Malunggay plant. It is rich in Vitamin A and C, iron, and High-Density Lipoprotein [5]. It is naturally green in colour. Its scientific name is Moringa oleifera. The objectives of this experiment are to (1) separate the coloured components of malunggay leaves using column chromatography, (2) determine the purity of the components using thin layer chromatography, and (3) measure the Rf values of the coloured components in Thin Layer Chromatography
EXPERIMENTAL A. Sample Used Malunggay (Moringa oleifera )
Figure 1. Column Chromatography
Thin Layer Chromatography, on the other hand, is a very commonly used technique in synthetic chemistry for identifying compounds,
Figure 3. Malunggay Leaves
B. PROCEDURE 1. COLUMN CHROMATOGRAPHY First, the malunggay leaves were triturated with the use of a mortar and pestle. Second, 5 mL of hexane: acetone (7:3) was poured to the triturated malunggay leaves. After that, the dropping pipette that will be used for the set up was plugged by cotton and was uniformly packed with silica gel up to the indented part of the dropping pipette. 3 mL of the extract was placed on the top of the column using a dropping pipette. Then, the pigment mixture was eluted using 2 mL of hexane: acetone (7:3), 2 mL of acetone, and 2 Ml of acetone: methanol (1:1). The solvent systems were introduced in portions and the column was not allowed to run dry. The colourless eluate gotten from the column was discarded while the coloured eluates were collected in different test tubes. Data, like the number of drops per eluate collected in each test tube, were collected and noted.
Dried silica gel Cotton Test tube
Figure 4. Column Chromatography Set-Up
2.
THIN LAYER CHROMATOGRAPHY First, the malunggay leaves were triturated with the use of a mortar and pestle. Second, 5 mL of hexane: acetone (7:3) was poured to the triturated malunggay leaves. After that, the eluates were applied by spotting ten times on a 5 cm x 8 cm pre-coated TLC plate. Each spot was allowed to dry before applying the next and each spot was assured to be as small as possible. Then, the developing chamber was prepared by placing an approximate amount of solvent system. The inner wall of the chamber was lined with the filter paper, was covered with a watch glass and was allowed to equilibrate. After that, the plates were carefully placed in the developing chamber. The solvent system was allowed to rice up to 1 cm from the upper end. Then, the plates were removed from the chamber and the solvent front was immediately marked, and air-dried. Finally, the components were
viewed under a UV lamp and the Retention Factor (Rf ) values of the components were calculated and noted. Watch Glass Thin Layer Plate Solvent Mixture
Figure 5. Thin Layer Chromatography Set-Up
Figure 6. Formula for Retention Factor (R ) f
RESULTS AND DISCUSSION Different observations were noted during the experiment. First of all, the presence of the yellow pigment and green pigment were seen in both set-ups. Yellow pigments and green pigments dominance was proven by this observation. This was also proven by the column chromatography set-up which only has extracted yellow and green pigments. If one focused on the data of the thin layer chromatography, the yellow pigments proved to have a higher retention factor (Rf ) value than the green pigments. The greater polarity of green pigments over yellow pigments was shown by this because R f value is indirectly proportional to polarity. Computations: Yellow pigment:
Gray pigment:
Green pigment:
Yellow pigment:
Dark green pigment:
Light yellow pigment:
Light yellow pigment:
Colourless (seen in UV) pigment:
Table 1. Compound and Solvent system used Plant Used Solvent System Used
Table
2.
Malunggay (Moringa oleifera) Hexane: acetone (7:3)
Column Chromatography
Colour of Component 1 Yellow 2 Light green 3 Green 4 Yellow green 5 Light yellow 6 Yellow
Volume of eluate 31 drops 31 drops 65 drops 96 drops 109 drops 44 drops
Table 3. Thin Layer Chromatography
Colour of Component
1 2 3 4 5 6 7 8
Yellow Gray Green Yellow Dark green Light yellow Light yellow Colourless (UV)
Distance of component from origin (X) in centimetres 59 cm 29 cm 20 cm 18 cm 16 cm 12 cm 6 cm 54 cm
Retention Factor (Rf ) values 0.98 0.48 0.33 0.30 0.27 0.20 0.10 0.90
REFERENCES
[1] Bayquen, A.V., Cruz, C.T., de Guia, R.M., Lampa, F.F., Peña, G.T., Sarile, A.S., Torres, P.C. (2010). Laboratory Manual in Organic Chemistry. Manila, Philippines. C& E Publishing Inc. [2] Chromatography-online.org. Mobile Phase. http://www.chromatographyonline.org/topics/mobile/phase.html 08/24/2010 [3] Chromatography-online.org. Stationary Phase. http://www.chromatographyonline.org/topics/stationary/phase.html 08/24/2010 [4] Miller, James (2005). Chromatography: Concepts and Contrast . New Jersey: John Wiley & Sons Inc. [5] Tacio, H.D. Malunggay: The miracle vegetable. http://www.sunstar.com.ph/static/dav/20 07/10/13/bus/malunggay.the.miracle.veg etable.html 08/24/2010 [6] University of British Columbia. Column Chromatography.
http://www.chem.ubc.ca/courseware/154/ tutorials/exp3A/columnchrom/ 08/24/2010 [7] University of California, Los Angeles. Thin layer Chromatography. http://www.chem.ucla.edu/~bacher/Gene ral/30BL/tips/TLC1.html 08/24/2010 [8] University of Colorado at Boulder. T LC: Retention Factor. http://orgchem.colorado.edu/hndbksuppo rt/TLC/TLCrf.html 08/24/2010
