COLUMN AND THIN LAYER CHROMATOGRAPHY
RAMOS, Sharina Joy; REYES Aina Marie; REYES Jallisa Maan;
RUBIO, John Michael; SABINO Patricia Anne; SANTOS, Carlos Rafael
To separate the colored components of siling labuyo and to determine the purity of the components, the students performed a column and thin or solid-liquid chromatography procedure. The solid may be almost any material that does not dissolve in liquid phase. But for this experiment, the solid used by the students was silica gel. Chromatography is a method of separating mixtures and at the same time useful in the identification of compounds. It is based on both solubility and absorptivity. The students were able to extract, more or less, six (6) colors and was further broken down into three (3) colors specifically yellow, red and orange. Using a precoated TLC plate, the students then was able to observe and measure the Rf values of the different components of the siling labuyo through the use of the TLC plates with eluates applied and was then placed in the developing chamber. At the end of the experiment, the students were able to discover the different components of the siling labuyo, its purity and the Rf values of each components of the red siling labuyo.
The experiment was done to separate the colored components of siling labuyo using column chromatography; to determine the purity of the components using the thin layer chromatography; and to measure the Rf values of the colored components in thin layer chromatography. Chromatography is the most modern and sophisticated method of separating mixtures and identifications of compounds in a solution. The procedures rely on the adsorptivities of the components to be separated. There are two involved phases in chromatography namely, the stationary phase which is the substance fixed in place and the mobile phase which is the phase that moves in definite direction. The experiment focused on the column and thin layer chromatgraphy which is based on both the solubility and adsorptivity of the compounds to be separated. EXPERIMENTAL
A. Sample used
1. Siling Labuyo
A short pipette will be used as the column. Place small paper plug loosely in bottom of column. A small piece of Kim-wipe works well for this. Do not jam it down into tapered portion. Add approximately 1/2 cm of sand. Slowly, with constant tapping, add approximately 6 cm of dried Alumina to column. Be sure to have alumina seat evenly. Do not waste time getting exactly 0.50 cm of sand and 6.00 cm of Alumina. Add approximately 1/2 cm of sand to top. Tap gently so bed seats evenly. Attach to ring stand using small three pronged clamp. The column is now ready to use. Label and weigh two watch glasses. Place one under pipette column. The solvents are volatile. When pipetting, be sure to keep pipette in strict vertical position (up and down, not sideways). Tilting pipette will cause liquid to drip or spray out. Have 4 mL methyl t-butyl ether (MTBE), 4 mL hexane, and the F:AF solution labeled and within reach at your workspace. Have a pipette with a rubber bulb ready for each solution. Once you begin the next step you may not stop until complete. DO NOT allow your column to run dry. Additions must be made quickly to avoid the column running dry and cracking. Be fully prepared before beginning the next step. Running Column Add approximate 2cm depth of hexane to top of column. When liquid level drains down and reaches top of sand, IMMEDIATELY add the entire F:AF solution to top of column. Try to add directly to top of column and not to drip down inside of glass pipette. When the F:AF solution drops into sand, carefully add hexane to top of column. Continue adding hexane to prevent solvent level dipping below top of column. DO NOT allow column to run dry. Collect effluent in watch glass. You will notice color bands separating. Collect the yellow band in the first watch glass. When all...
References: From books:
 Bayquen, A.V., Cruz, C.T., de Guia, R.M., Lampa, F.F., Pena, G.T., Sarile, A.S., and Torres, P.C. (2009) Organic Chemistry. 839 EDSA, South Triangle, Quezon City: C & E Publishing, Inc.
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