Column and Thin Layer Chromatography

Jan Michael V. Salcedo, Bea Marie B. Samar, Maria Ysabel O. San Juan, and Gabriel Jerome M. Sandimas Group 8 2G Pharmacy Organic Chemistry Laboratory

ABSTRACT
Chromatography is the physical separation of a mixture into its individual components. There are different types of chromatography and each has its own advantages and disadvantages. Column and Thin Layer Chromatography was performed by the group in this experiment. Malunggay leaves were used as the sample and its pigments were extracted using Hexane:Acetone (7:3). The extract was introduced in the column and eluate was collected, this process is the column chromatography. The purity of the components was determined by using thin layer chromatography. Rf values were also calculated on each pigment.

INTRODUCTION
Chromatography, firstly introduced by the Russian botanist Micharl Iswett, is a separation method that relies on differences in partitioning behavior between a flowing mobile phase, which is a liquid solvent (or mixture of solvents) that use to carry the sample solutes under analysis along the paper, and a stationary phase, which is the adsorbent, to separate the components in a mixture. Chromatography is a very special separation process for a multitude of reasons: (a) separates complex mixtures with great precision; (b) can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are employed; (c) separates delicate products since the conditions under which it is performed are not typically severe; and (d) used to separate the colored pigments in plants.[1] There are different techniques in Chromatography but this experiment will just focus on Column Chromatography and Thin Layer Chromatography. In Column Chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column. The mobile phase, a liquid, is added to the top and flows down through the column by either gravity or external pressure. Column chromatography is generally used as a purification technique: it isolates desired compounds from a mixture.[3] Thin Layer Chromatography is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the R f of a compound is compared with the Rf of a known compound (preferably both run on the same TLC plate). A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina).[4] In order to make the technique more scientific rather than a mere interpretation by sight, Retention Value (Rf value) was applied in chromatography. A particular compound will

travel the same distance along the stationary phase by a specific solvent (or solvent mixture) given that other experimental conditions are kept constant. In other words, every compound (dye, pigment, organic substance etc) have a specific Rf value for every specific solvent and solvent concentration. Rf values come very handy for identification because one can compare Rf values of the unknown sample (or its consituents) with Rf values of known compounds.[2] In this experiment, the group should be able to attain the following objectives: (1) to be able to separate the colored components of malunggay leaves using column chromatography, (2) to determine the purity of the components using think layer chromatography (TLC), and (3) to measure the Rf values of the colored components in TLC.

EXPERIMENTAL
A. Sample used Malunggay leaves were the sample used for both column and thin layer chromatography. B. Procedure To start the experiment, column chromatography was performed first. The group extracted the pigments of the malunggay leaves using Hexane:Acetone (7:3). The column of Pasteur pipette was plugged with cotton and uniformly packed with silica gel up to the indented part. A 0.5mL of the extract was placed on top of the column using a Pasteur pipette. The pigment mixture was eluted using 10mL of Hexane:Acetone (7:3). The solvent system was introduced in portions to prevent the column to run dry. The colored eluates were collected in separate test tubes. The number of drops of eluate collected in each test tube was recorded.

RESULTS AND DISCUSSION

The collection of eluate stopped when the solvent system that is introduced in portion ran out and all the colors of components were collected. Table 1 will indicate the summary of the different colors of component and the volume of eluate (drops). Table 1. Column Chromatography
Color of Component 1 2 3 4 Yellow Dark Green Yellow Green Light Yellow Volume of eluate (drops) 34 drops 54 drops 100 drops 104 drops

The table shows that there are 4 different colors collected namely: Yellow with 34 drops, Dark Green with 54 drops, Yellow Green with 100 drops, and Light Yellow with 104 drops. The Yellow eluate has the least volume while Light Yellow has the most volume. The collected eluates were spotted in a TLC plate then placed in a chamber with solvent system below the origin. Table 2 will indicate the summary of distance of component from origin and Rf value of each eluate. Table 2. Thin Layer Chromatography
Color of Component Distance of component from origin (x) in cm 6.8 cm 4.8 cm 4.7 cm 4.3 cm Rf Value

In thin layer chromatography, the eluates were applied on a 5cm x 8cm precoated TLC plate by spotting 10 times. The spot was allowed to dry before applying the next. The spots were required to be as small as possible. The developing chamber was prepared by placing an approximate amount of the solvent system: Hexane:Acetone (7:3). The inner wall of the chamber was lined with filter paper, covered with watch glass, and allowed to equilibrate. The plate was placed carefully inside the developing chamber. The solvent system was allowed to rise up to 1cm from the upper end. The plate was removed from the chamber and the group immediately marked the solvent front, and airdry. Rf values were calculated using this formula:

1 2 3 4

Yellow Dark Green Yellow Green Light Yellow

0.96 0.68 0.66 0.61

The table shows that Yellow eluate travelled the farthest and has the highest Rf value while Light Yellow travelled nearest to origin and has the lowest Rf value among the 3. The distance and Rf value of Dark Green and Yellow Green is close to each other.

Calculations: 1. Yellow

Elchem.kaist.ac.kr (n.d.) Retrieve August 9, 2012 from http://elchem.kaist.ac.kr/vt/chemed/sep/chromato.htm Phys.sinica.edu.tw (n.d.) Retrieve August 9, 2012 from http://www.phys.sinica.edu.tw/TIGPNANO/Course/2007_Spring/Class%20Notes/AC_c hapter%203%20Chromatography%200411.pdf

2. Dark Green

3. Yellow Green

4. Light Yellow

Rf values are calculated to determine the affinity of the solute to the solvent. And it is also used in identification purposes. The greater the Rf, the greater the affinity of the solute to the solvent.

REFERENCES
WEBSITES: [1] Cemca.org (n.d.) Retrieve August 9, 2012 from http://cemca.org/andcollege/andcwebsite/subject 01/CHEtext.htm [2] Marz-kreations.com (n.d.) Retrieve August 9, 2012 from http://www.marzkreations.com/Chemistry/Chromatography/Dyes/ RF-Values.html [3] Orgchem.colorado.edu (n.d.) Retrieve August 9, 2012 from http://orgchem.colorado.edu/Technique/Procedur es/Columnchrom/Columnchrom.html [4] Orgchem.colorado.edu (n.d.) Retrieve August 9, 2012 from http://orgchem.colorado.edu/Technique/Procedur es/TLC/TLC.html Chemguide.co.uk (n.d.) Retrieve August 9, 2012 from http://www.chemguide.co.uk/analysis/chromatog raphy/thinlayer.html#top