chromatography

Column and Thin layer Chromatography of Red Siling Labuyo
Marian Angelu Ramos, Rose Ann Refuela, Leomarie Duanne Sanchez, Paula Lynne Santos, Geraline Sarmiento, Jon Carlo Semana
Group number Seven, 2E- Pharmacy, Faculty of Pharmacy, University of Santo Tomas

ABSTRACT
Chromatography is the separation of mixtures into their constituents. It relies on the differential solubilities or absorptivities of the components to be separated with respect to two phases, one of which is stationary and the other, mobile. The group aims to separate the coloured components of siling labuyo using column chromatography then determine the purity of the components using thin layer chromatography and lastly, measure the RF values of the coloured components in thin layer chromatography. The students plugged the column with cotton and silica gel inside the pasture pipette which later introduce the extract and eluent inside. The colored eluate was then collected and applied in precoated thin layer chromatography plate by spotting it ten times. Afterwards, the precoated thin layer chromatography plate was placed inside the closed beaker with 15 milliliter of dichloromethane-hexane and filter paper. The chromatography plate was removed; it was visualize under ultraviolet light; then, retention was measured at each components. Therefore,....

INTRODUCTION Chromatography can be explained as the separation of mixture into different fractions by disseminating into different phases, one being stationary and the other mobile. The principle of chromatography is that substances differ in their partition coefficient between the stationary and the mobile phase. The compound that weakly merges with the stationary phase will consume most of the time in mobile phase and the compound that interacts strongly with the stationary phase will move slowly. There are a couple types of chromatography and each differ in their uses. There is a type for solid – liquid separation, which is column and thin layer chromatography, paper or high performance liquid chromatography for liquid – liquid separation and vapor-phase chromatography for gas – liquid chromatography. Column chromatography is used to separate the coloured particles. Column chromatography is relies on the solubility and absorptivity of the mixture. The solid that is used must not be soluble in the liquid phase. Silica gel and alumina are the most common solids. Thin layer chromatography is also used to test the purity of the components. Thin layer chromatography is performed using a sheet of glass, plastic or aluminium foil with a thin layer of adsorbent known as the stationary phase. Siling labuyo or chili pepper (CApsicum Frutescens) is common backyard plant in the Philippines especially in the province. It grows about 0.8-1.5 meters in height. Fruit is commonly red when ripe, oblong-lanceolate, 1.5 to 2.5 centimeters long.

The objective of this experiment is to separate the colored component of siling labuyo using column chromatography. Also, the group’s objective is to determine the purity of the components using thin layer chromatography. Lastly, the objective is measure th retention factor of the colored components in thin layer chromatography.
MATERIALS AND METHODS
MATERIALS
The apparatus used are the mortar and pestle – where siling labuyo is triturated; pasture pipette- where cotton and silica gel was placed and set-up for column chromatography; dichloromethane-hexane, dichloromethane and dichloromethane-methanol-used as the eluent in column chromatography; precoated thin layer chromatography plate- where the colored eluate was spotted ten times; capillary tube- used to spot the eluate to the precoated thin layer chromatography plate; beaker- used as chamber for thin layer chromatography; watch glass-use to cover the beaker; filter paper- helps in saturating the chamber.
METHODS
The group removed the seeds of five siling labuyo by cutting it on sagittal portion. Afterwards, the siling labuyo was triturated until orange liquid is visible. Upon extraction of the pigments, five milliliter of dichloromethane was used; then, extracts was set aside for thin layer chromatography.
To prepare the column chromatography, Pasteur pipette was plug with cotton and uniformly packed with silica gel up to the indented part. On top of the column, 0.5 milliliter of extract was placed. Then, the extract was added with eluent of 10 milliliters of dichloromethane -hexane(1:1). The solvent system for siling labuyo was 3 milliliter of dichloromethane-hexane and dichloromethane. The column was not allowed to dry. The eluate was collected and separated in terms of color. Colored eluate was given more importance while colorless was discarded. Also, the number of drops of colored eluate from the column was noted.
A 5 centimeter by 8 centimeter precoated thin layer chromatography plate was marked 1 centimeter at each top and bottom part. On the bottom part, 0.5 centimeter from the edge was measured towards the center to apply the spots of the eluate. Each spots were dried and as small as possible.
Fifteen milliliters of dichloromethane hexane was used in preparing the developing chamber for red siling labuyo. The inner wall of the chamber was lined with filter paper, covered with watch glass and allowed to equilibrate. Afterwards, plate was placed inside the developing chamber to allow the solvent system to rise up to 1 centimeter from the upper end. If the solvent system reached 1 centimeter from the upper end, the plate was removed and immediately marked the solvent front. Also, the components was visualized under ultraviolet lamp and measure the retention factor.
RESULTS
One can see in Table 1-1, the colored eluate collected in drops from the column.

Color of Component
Volume of Eluate(number of drops)
1
Light Yellow
43
2
Orange
36
Table 1 Results of Column Chromatography
As seen in Figure 1, the set-up of the chamber for thin layer chromatography

Figure 1Thin layer Chromatography
One can see in Table 2, the retention factor was calculated by getting the distance from the origin of each component.

Color Component
Distance of component from the origin (X) in centimeter
Retention factor value
1
Light yellow
7.8
6.6
5.5
0.975
0.825
0.6875
2
Orange
2.8
2.6
2.1
1.1
0.9
0.7
0.5
0.35
0.325
0.2625
0.1375
0.1125
0.0875
0.0625
Table 2 Results of Thin layer Chromatography
RF=
Light Yellow
Retention Factor=0.975
Retention Factor=0.825
Retention Factor=0.6875
Orange
Retention Factor=0.35
Retention Factor=0.325
Retention Factor=0.2625
Retention Factor=0.1375
Retention Factor=0.1125
Retention Factor=0.0875
Retention Factor=0.0625
As seen in Figure 2, this is the plate after placed in the chamber. Also, there are two spots fluoresce under ultraviolet light.

Figure 2 Precoated Thin layer Chromatography Plate

DISCUSSION As seen in Table 1-1, there are light yellow, orange and colorless eluate was collected. There are 43 drops of light yellow and 36 drops of orange. Colorless eluate is not included in the data gathering because it is not part of the components of extraction of red siling labuyo. Only dichloromethane-hexane and dichloromethane eluent was used because the components of siling labuyo already flowed out of the column before the dichloromethane-methanol was added. The eluent affects the flow of the compounds whether they will remain in the column or out of the column. Light yellow and orange pigment tend to move along with the solvent. Orange component is more polar than light yellow because orange have the tendency not to travel quickly and absorbs more strongly by silica. I Figure 1 shows the set-up of the chamber for the thin layer chromatography. The beaker is covered with watch glass so that the atmosphere remains saturated with solvent vapour. Saturation of atmosphere is important because it hinders the evaporation leading to the rising up to the plate. There is a separation of components of siling labuyo at different rates.
In Table 2, it shows the amount of distance travelled by each component. Orange component shows that it remains in the stationary phase while light yellow component moves closely to the mobile phase.
In Figure 2, it shows that two spots were floresce under ultraviolet light which means it is the final chromatogram. REFERENCE
Clark, J. (2007). Thin Layer Chromatography. Retrieve from http://www.chemguide.co.uk/analysis/chrom atography/thinlayer.html
Clark, J. (2007). Column Chromatography. Retrieve from http://www.chemguide.co.uk/analysis/chrom atography/thinlayer.html
Stuart, G. Siling Labuyo. Retrieve from http://www.stuartxchange.com/SilingLabuy o.html