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COLUMN AND THIN LAYER CHROMATOGRAPHY

Alava, P.J., Alcausin, D.A., Andal M.I., Bagon, N.E., Barretto, D., *Bautista, C. 2D-Pharmacy, Faculty of Pharmacy, University of Santo Tomas

Abstract Chromatography is a useful technique of separating mixtures and identifying of compounds. There are different types of Chromatography to be used based on the phases of the components of the mixture. It can either be solid-liquid, liquid-liquid and gas-liquid. In the conducted experiment, Column chromatography (solid-liquid) was used to extract the components of the Malunggay leaves with the aid of Hexane-Acetone as the solvent . Thin layer chromatography (TLC) was also used to determine the purity of the colored components. UV lamp was then used to visualize the components in the developed TLC plate. Retention factor was finally measured and computed. Introduction In Chemistry, there are different ways to separate mixtures. It can either be through physical or chemical means. An example of the most commonly used in separating mixtures is Chromatography. Chromatography is commonly described as an easy and efficient way of separation of mixtures of chemical compounds with the aid of Adsorption. It usually involves a sample or extracts being dissolved in a mobile phase that will be forced to move through an immiscible and nonmoving phase called stationary phase. Different types of Chromatography will be employed depending on the phases that will be used in the mixture. In this experiment, Column chromatography and thin layer chromatography were used to extract and purify the components of a mixture and Retention factor was then measured. Column chromatography separates and purifies the chemical compounds in the mixture. It is a technique in which a column of stationary phase is used based on the stationary phase nature. Usually, the vertical glass column used in this type of chromatography is packed with Adsorbents such as Alumina and Silica gel. Column chromatography can be also be used as an analytical and preparative applications. The disadvantage of this technique is that it is very time-consuming especially when used in large quantities of sample. Thin layer chromatography (TLC) is commonly used in separating and identifying compounds in a mixture. It can also determine the purity of the substance. TLC is helpful in determining the best solvent to be used in Column chromatographic separation. This analytical technique requires only small sample, thus, making it quick, simple and inexpensive. Retention factor (Rf), also known as Retardation factor, is the ratio of distance traveled by the compound over the distance travelled by the solvent. The long distance traveled by the compound will result to larger Rf value, which will exhibit less polarity. The objectives of this experiment are to separate the colored components of Malunggay leaves using column chromatography, to determine the purity of the components using thin layer chromatography (TLC), and to measure the Rf values of the colored components in TLC. Materials and Methods The materials used in the extraction of compounds are Hexane, Acetone, Mortar and Pestle and Malunggay leaves (Moringa oleifera). The Malunggay leaves were finely triturated with the use of Mortar and pestle.

1mL of Hexane-Acetone (7:3) was mixed with the crushed Malunggay leaves. The dark green solution was placed in the test tube and was set aside. The materials required for the Column chromatography setup are Iron stand, Iron clamp, Pasteur pipettes, Silica gel and cotton. The Pasteur pipette was filled with cotton and uniformly packed with silica gel up to its indented part. The prepared pipette was placed on the iron stand with the help of an iron clamp. 0.5mL of the extract was introduced to the column of the Pasteur pipette. 10mL of Hexane-Acetone (7:3) was instilled in portions for elution. The colored eluates were then collected separately into different vials according to their color while the colorless eluates were discarded. The number of drops of each color of the eluates was recorded. The collected colored eluates were applied on a 5cmx8cm precoated thin layer chromatography plate. Using a capillary tube, colored eluates were spotted 10 times on the 1 inch margin from the lower end. Each spot were allowed to dry before another layer was spotted. The distance among each color should be equal to each other. 400-ml beaker, watch glass, hexane, acetone and filter paper were needed for the set-up of the developing chamber for the thin layer chromatography. The beaker was filled with approximate amount of hexaneacetone (7:3) such that the height of the solvent will not exceed 1.5 cm. A filter paper was used in order to cover the inner wall of the beaker. The set-up was then covered using a watch glass and was allowed to equilibrate to prevent the evaporation of the volatile components inside the beaker. The prepared precoated TLC plate was placed in the TLC developing chamber and the solvent was permitted to spread in the plate. Once it reached the margin (1cm)

from the upper end, the TLC plate was then removed from the TLC developing chamber. The solvent front and the compound front were marked and air-dried. The TLC plates were placed under the UV light and the invisible spots were marked. The distance travelled by the components and the solvent were recorded for the computation of the Rf values. Results and Discussion Table 1 Column Chromatography
Color of Component Green Yellow Green-Yellow Yellow-Green Volume of Eluate (drops) 21 16 18 18

1 2 3 4

With the use of Hexane-Acetone as the solvent, 4 colors of eluates (Green, Yellow, Green-Yellow and Yellow-Green) were collected. As shown in the table above (Table 1), the first colored eluate (Green) had the most number of drops. The second colored eluate (Yellow) got the least number of drops while the third and fourth colored eluates (Green-Yellow, Yellow-Green) were equal with the same number of drops. Table 2 Thin Layer Chromatography
Color of Component Distance of Component from origin (X) in cm 2.9 2.2 1.9 6.2 2.0 3.3 2.3 2.1 Rf value

1 2 3 4

Green Yellow Green-Yellow Moss Green

0.414 0.314 0.271 0.886 0.286 0.471 0.324 0.286

The thin layer chromatography produced four different colors from the four colored eluates produced in the column chromatography. Malunggay leaves were then classified to be an impure substance. The distance of the spots of each color from the base line (1.5cm margin from the lower part of the plate) was recorded in Table 2. The Yellow component, which had the longest spot from the origin, traveled 6.2cm. The Green component had three distances which resulted to 2.9cm, 2.2cm and 1.9cm. Moss green component also had 3 distances that traveled 3.3cm, 2.3cm and 2.1cm. The last color which is GreenYellow had the shortest spot from the origin with the distance of 2.0cm from the origin. The Rf value for each colored components were then computed using the formula:

In this experiment, the Green-Yellow was the most polar while the yellow component was the least polar. References Chromatography Retrieved on August 12, 2013, from http://www.organo.co.jp/technology/hisepa/e n_hisepa/chromato/c1.html Column Chromatography Retrieved on August 12, 2013, from http://orgchem.colorado.edu/Technique/Pro cedures/Columnchrom/Columnchrom.html Thin layer Chromatography Retrieved on August 12, 2013, from http://orgchem.colorado.edu/Technique/Pro cedures/TLC/TLC.html Retardation Factor Retrieved on August 12, 2013, from http://www.wisegeek.com/what-is-aretardation-factor.htm#didyouknowout

The solvent traveled 7cm, thus, each distance of the components were substituted in the formula that produced different Rf values for each color. The Green had 0.414, 0.314 and 0.271 with respect to decreasing distance travelled by the component. The Yellow component, which had the longest spot traveled, also had the highest Rf value with 0.886. The Green-Yellow component, which had the shortest distance traveled, also had the lowest Rf value with 0.286. The last component which is the Moss Green had 0.471, 0.324 and 0.286 with respect to decreasing distance travelled by the component. Based on the experiment, it was noticeable that the larger the Rf value of a compound, the longer the distance the compound will travel on the TLC plate, making it less polar.