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Michael Dominique B. Allag, Eryll Joy H. Agojo, Camille A. Baetiong, Greanne P. Danica Ballesta, Rachel Anne S.

Barlao Group 1 2C Medical Technology Organic Chemistry Laboratory


Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and quantify the mixture or components. There are different types of chromatography and each has its own advantages and disadvantages. In this experiment, DCM-hexane was used to extract the different pigments of the siling labuyo. Extract was introduced into the column and eluate was collected, this process is the column chromatography (CC) method. The purity of the components was determined by using thin later chromatography (TLC). UV lamp was used to visualize the developed TLC plate and the Retention or Retardation Factor was measured.


surface. All types of chromatography are useful for analytical purposes. Under appropriate c o n d itio n s all , types of chromatography can be used for preparative s c a l e s e p a r a t i o n s . In e v e r y type of chromatography there are three elements to be considered: the size of the sample (Load), relative se par atio n o f co mpo ne nts (Resolution), and the Speed.

Chromatography can be defined as a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary medium through which they pass. The underlying principle of chromatography is that different substances have different partition coefficients between the stationary and mobile phases. A compound that interacts weakly with the stationary phase will spend most of its time in the mobile phase and move rapidly through the chromatographic system. Compounds that interact strongly with the stationary phase will move slowly. All forms of chromatography work on the same principle. Diverse types o f Chromatography are possible, depending on the physical states of the phases. Employing a gas the mobile phase is termed gas chromatography (gc) or vapor phase chromatography (vpc). Separations using gas chromatography involve vapor phase versus adsorption and/or equilibria. Liquid Chromatography (lc) refers to any chromatographic process that employs a mobile liquid phase. Chromatographic separations can also be carried out using thin layer chromatography (tlc) and column chromatography which a variety of supports, including immobilized silica on glass plates.

It would be ideal if all three elements could be maximized so that complete separation of samples of any desired size could be quickly achieved. In practice, generally two of these elements can be maximized at the expense of the third. For routine analytical work, resolution and speed are maximized at the expense of the load. In preparative scale separations, load, and speed can be maximized but then separations are usually incomplete. Complete separations of large samples can be achieved but the overall operation is likely to be slow and tedious, and may involve the use of large quantities of solvent that must be distilled for reuse, or discarded.

Chromatography separates a substance into its component parts, which is very useful, as substances are often unique in their composition. It can identify a substance and show how it differs from others that may look alike on the

In the experiment, Chromatography and Chromatography were used.

Column Thin

TLC has a number of advantages: It is simple, fast, efficient to use and it requires only small amounts of sample. TLC is generally used a qualitative analytic technique, such as checking the purity of a compound or determining the number of components in a mixture or column chromatographic function. In addition, TLC is useful for determining the best solvents for a column chromatographic separation. It can be used for an initial check on the identity of an unknown sample. Preparative plates can be carried out with special thick-layered TLC plates.

Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analytical and preparative applications. Not only can column chromatography be used to determine the number of components of a mixture, but it can also be used to separate and purify substantial quantities of those components for subsequent analysis. This is in contrast to paper chromatography, which is solely an analytical method. The disadvantage of a column chromatography is that it is time-consuming and tedious, especially for large samples. If it is unnecessary to preparative separate large quantities of sample, analytical methods such as paper chromatography may be more suitable and easier to perform. Thin-Layer Chromatography (TLC) involves the same principles as column chromatography; it is also a form of solid liquid adsorption chromatography. In this case, however, the solid adsorbent is spread as a thin layer on a plate of glass or rigid plastic. The solvent travels up by plate through capillary action. A drop of the solution to be separated is placed near one edge of the plate, and the plate is placed in a container, called a developing chamber, with enough of the eluting solvent to come to a level just below the point of origin. The solvent migrates up the plate, carrying with it the components of the mixture at different rates. The result then, is a series of spots on the plate, falling on a line perpendicular to the solvent level in the container.

DCM hexane or Dichloromethane hexane is the solvent system used to elute through a chromatography column. This means that the mobile phase (solvent system) consists of 1:1 (ratio of volume) mixture of dichloromethane (DCM; CH2Cl2), and hexane (C6H14). The solid phase (silica gel) is eluted with this solvent system until fully solvated, the compound to be purified is then loaded onto the solvated solid phase, and the column is eluted with the same solvent system until your desired compound has come off the column T he Retention or Retardation Factor (R value) is the ratio of the distance that the spot travelled relative to the distance moved by the solvent which in this case is the DCMhexane.

The o b j e c t i v e s o f t h e e x p e r i m e n t are the following: separate the colored components of red siling labuyo using column chromatography, t o predict the purity of components using column and thin layer chromatography (TLC) and lastly, to measure the Retention/Retardation Factor (R values) of colored components in TLC.


The e l u a t e s w e r e a p p l i e d o n t h e 5cm X 8cm pre-coated TLC plate by equidistantly spotting each spot 10 times. The spot was allowed to dry first before applying the succeeding spots. It was ensured that the spots made were small as possible so that when the plate develops, the colors would not be disarray. Developing Chamber was prepared by placing the approximate amount of DCM hexane. The inner wall of the chamber was lined with filter paper to allow the TLC plate to stand. The developing chamber was covered with watch glass and was allowed to equilibrate. The TLC plate was carefully introduced in the developing chamber. The solvent system was allowed to rise up until it reaches just 1cm from the upper end. The TLC plate was then removed carefully from the chamber. The solvent front was immediately marked and the plate was allowed to dry. The components were visualized using the ultraviolet lamp after the plate has developed after elution process and this causes substances to appear as colored spots. The R values were measured and chromatographic plates were documented.

Pigments of the siling labuyo were extracted by cutting it to pieces and by pouring DCMhexane and eventually triturating it by using a mortar and pestle with the ratio of 1:1. The extracted pigments were set aside for a while. Silica Gel Column was prepared by plugging the column with cotton followed by the silica gel which was uniformly packed and contained no holes or air bubbles until it reached the indented part of the Pasteur pipette. 0.5 ml of the extract was placed on top of the column using Pasteur pipette. The p i g m e n t m i x t u r e w a s e l u t e d u s i n g 10ml DCM-hexane. The s y s t e m s o l v e n t was introduced in portions. The column was not allowed to run dry and the colorless eluate collected was discarded. Test tubes were changed each time the color of the eluate varies. The number of drops for each color was noted. After collecting the eluates from the column, Thin Layer Chromatography was performed. The color of the eluate varies. The number of drops for each color was noted. After collecting the eluates from the column, Thin Layer Chromatography was performed.


Results and Discussion

Plant used: Siling Labuyo Solvent System used: DCM-Hexane

Column Chromatography: Two eluates were yielded from the extraction of the colored components of siling labuyo using column chromatography. Two different shades of colors were obtained: Light yellow and light orange. The volume of the light yellow eluate collected from the column was 35 drops while on the other hand, the volume of the light orange was 85 drops.

travelled 4.3 cm while the light orange eluate travelled 0.5 cm. The color of the developed plate was not visible by the naked eye. It was placed UV light for viewing. The general formula for computing the R value is shown below:

1 2

Color of Componen light yellow light orange

Volume of eluate (no. of drops) 35 85

Table 1 Column Chromatography (Table of Results)

After measuring the distance traveled for each spot, The R value (also known as Retardation or Retention Factor was computed) Retardation or Retention Factor is the ratio of time spent in the stationary phase relative to time spent in the mobile phase. Distance of solvent: 5cm

Thin Layer Chromatography

Table 2 Thin Layer Chromatography (Table of Results) Color of Component Light Yellow Light Orange Crude Distance of Component from origin (x) in cm 4.3 cm 0.5 cm 4.0 cm Rf Value 0.86 0.1 0.8

With reference to Figure 4, (From left to right) the first spot is the Crude Eluate; the second spot is the first eluate collected from the column and the third spot is the second eluate collected from the Column Chromatography. The crude eluate travelled 4.0 cm from the origin; the light yellow eluate

1 2 3

The developed plate wasnt able to show completely the separation of colors. The possible sources of error are from the spotting of the TLC plate. When the extracted pigments of siling labuyo were spotted on the plate, it was not left completely dry before placing the succeeding spots in

addition to that; the spots werent small enough which have caused color the color to disarray. Another source of error is not covering completely the developing chamber during the development of TLC plate.

V. References BOOKS:
Fedessenden, R.J., Fedessenden, J.S., & Feist P. (2001). Organic Laboratory Techniques. Canada: Brooks/ Cole. Pg. 119-140 Robards, K., Haddad,P.R., Jackson,P.E., (1994). Principles and Practice of Modern Chromatographic Methods. San Diego,CA: Academic Press Inc. Pg. 1-34, 36-225 Williams, T. I., (1947). An Introduction to Chromatography.New York: Chemical Publishing Co., Inc. Pg. 1-85


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