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Separation of Plant Pigments of Komote Tops byThin Layer ChromatographyAdapted from: Quach, H. T.; Steeper, R L.; Griffin, G. w. J. Chem. Educ.2004,g1. 3g5-7 andDepartment of Chemistry, Troy University

Introduction

Chromatography is widely used for the identification of components as well as itsseparation into molecular components of a mixture. It was discovered by M. Tswett in 1906,where he dissolved a mixture of plant pigments in petroleum ether ani passed the solutionthrough a column of calcium carbonate. He was able to demonstrate that chromatography wasapplied only to coloured compounds, and the results of separation could be followed by visualobservation.

Chromatography is an analytical method permitting the separation of a mixture into itsmolecular components. The basic principle upon which it works is that a mixture first adheres tothe dry chromatography plate. A developer or solvent is then passed through the coating on theplate in a fixed direction moving the pigment molecules of the mixture along at different rates.The greater the attraction between the molecules and the absorbing medium (coated on theplate), the slower the molecules ascend the coating. The greater the soiubility of the componentsin the developer, the greater the distance the molecules move.

The developer commonly used contains a mixture of nonpolar.or polar solvents. (Ensureth,at there are no open flames in the laboratory because solvents are extremely flammable!)The solvent molecules contain non-polar covalent bonds and any net charges are equallydistributed within the molecules.

Chromatography has been the very suitable method for separation of pigments of plantextracts, especially when a small amount of material is available. In higher forms of planti, theprincipal pigment is chlorophyll a. Chlorophyll b, carotenes and xanthophylls play a secondaryrole by transferring the energy they absorb to chlorophyll a for use in photosynthesii.Chlorophyll a is colored dark green to blue green, whereas, chlorophyll b is yillow green. B-carotene is colored light yellow

ln the following experiment, the pigments found in the leaves of kamote tops, Ipomoeabatotas, are separated by means of thin layer chromatography.

Materials Needed

*Fresh Kamote tops (violet colored,PER SECTION)

+*Lab Kit (shoebox with section andnames of GROUP members)- 2 rags, paper towels or toilet paper,

hand soap, liquid detergent, dishsponge, test tube brush, pair ofscissors, masking tape, fine-tipmarker, wooden pencil, ruler (w/

Science and Technologt Research I

mm), lab gown, surgical masks,pairs of gloves

(to be provided by the PSHS LAB)(l) TLC plate (3.0 x 6.0 cm) and(l) TLC chamber(l) set mortar and pestle (small)(l) 4.0 mL test tube(l) 1.5 mL eppendorftube(l) capillary tube

PSHS-MC Sy 2010-20t 1

(l) 2.0 mL measuring pipetParafilm (as needed)(l) spatula( l) pair of forceps

Experimental Procedure

AcetonePetroleum etherIsopropanolDistilled water

On a balance weigh out 1.0 gram of kamote tops. Manually tear/cut the leaves into smallpieces and transfer to a mortar and using a pestle, grind the leaves very well. Transfer thecrushed leaves to a small test tube and add 2.0 mL of acetone. Cover the test tube with aparafilm and shake vigorously for approximately one minute between hands.

Allow this mixture to stand for about l0 minutes, then using a pipet carefully transfer L0mL of the solvent above the solid into a small 1.5 mL eppendorf tube. Use care not totransfer any of the solid material. The solvent extract should be green. Cap the tube tominimize solvent evaporation .lDispose remaining extract to an orgonic waste jar!l

Obtain a TLC chamber with developing solvent: a mixture of 25.0 mL petroleum ether,2.5 mL and 5 drops distilled water (to be prepared by the instructor).The solvent shouldcompletely cover the bottom of the chamber to a depth of approximately 0.3 cm (-2.0mL). Keep the chamber covered so that evaporation doesn't change the composition ofthe solvent

Obtain a TLC plate (a silica gel coated plastic sheet) and draw a line measuring 0.5 cmfrom the bottom as well as from the top. Place two small dots of equal distance apart witha lead pencil on the coated side approximately 0.5 cm from the bottom of the strip. Thedots should be parallel with the bottom of the strip. Label the first two dots with thenumbers."" l and 2" to indicate two trials

Fill a capillary tube (TLC applicator) by placing it in the leaf extract (it will fill bycapillary action). Place one finger at the end of the capillary tube to control application of'extract to the TLC plate. Apply the extract to the center of the first dot on the TLC plate

by quickly touching the end of the TLC applicator to the plate. Allow to dry. Repeat

several times (-20 times) to make a concentrated dot of extract. Be sure to let drybetween applications. Try to make the spots as small as possible but dark enough to see

the color clearly. lDispose used capillary tube at designated waste beaker!l

Using a pair of forceps, careflully place the TLC plate in the TLC chamber. The TLCplate should sit on the bottom of the chamber and be in uniform contact with the solvent(solvent surface must be below the extract dots). Cover the TLC chamber.

7. Allow the TLC plate to develop (separation of pigments) for approximately 10-15

minutes. As the solvent moves up the TLC plate you should see the different coloredpigments separating.

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2.

a

4.

5.

6.

Science and Technologt Research I PSHS-MC SY 20 1O-201 1

8. Remove the TLC plate from the chamber when the solvent front reaches the 0.5 cm fromthe top of the TLC plate using a pair of forceps. With a*pencil, mark the level of thesolvent front (highest level the solvent moves up the TLC plate) as soon as vou removethe strip from the chamber (the solvent evaporates and disappears quickly). Using a

ruler, also measure the different pigment distances (in mm) quickly as some pigmentsmay fade over time.

9. Record the results of the separation on the data sheet.

Results

t. Tape your chromatography plate to the data sheet in space provided. Draw arrows to thelocations of the solvent front and the colored bands. Label each band as to its type ofpigment.

For the following calculations mark the center of the initial pigment dot; this will be thestarting point for all the following measurements. Also mark the middle point of each

pigment band and the solvent front.

The rate at which a pigment moves up the plate is reported as an Rs value which is

defined as the ratio of the distance moved by the spot to the distance moved by thesolvent. Determine the Rrvalues for each of the pigments you observe using the formulaprovided below.

distance moved by solute (pigment)

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distance moved by solvent

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Chromatography Apparatus

PSHS-MC SY 20IO.2O11

Separation of Plant Pigments by TLC Plate Data Sheet

Project Code: Names:

I. Objectives:State your own objective/s.

II. Materials and Methods:Construct a flowchart or a schematic diagram to summarizethe materials and

methods used in the experiment.

III. Data and Results

Distance (mm)Trial I Trial2

Rf ValueTrial 1 Trial2

solvent front .

B-carotene

chlorophyll a

chlorophyll b

Tape, Draw, and Label TLC Plate Here

Science and lechnologt Research I PSHS-MC SY 20]O'2OI I

t.

ry. Discussion and Analyses:Discuss the results of the experiment by answering the following questions

completely and concisely.

Based on the results of the experiment, what evidence, if any, do you see for the presence ofother pigments aside from chlorophyll a and b? Identify these pigments and label properly inthe data sheet.

Which among the pigment molecules, chlorophyll a, b and B-carotene was the most

nonpolar? . The most polar pigment?

Explain your answer briefly. I

Why shoutd you use pencil on the coating to mark your pigment movement?

In the TLC experiment, why must

a) the TLC plate be coated thinly with silica gel?

b) the spot to be applied to the TLC plate be placed above the level of the developingsolvent?

c) you wear a mask when handling solvents like petroleum ether?

5. Consider three compounds to be separated on a TLC plate:

benzoic acid naphthalene

Which compound corresponds to the:

Highest Rfl:Intermediate Rfl:Lowest Rfl:Explain your answer.

Conclusion/s:State your conclusion in answer to your objective/s.

References:List references used to complete this report. Observe corect APA format for the

bibl iographical entries listed.

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Science and Technolog,, Research I PSHS-MC Sv 2010-201 I