CHEM 31.

1: Elementary Organic Chemistry Laboratory

Experiment 4: Chromatography

A. Separation of Plant Pigments by Paper Chromatography

CAUTION! Keep the chromatography paper clean and dry at all times to avoid contamination. Use gloves or tweezers
when handling the paper.

Materials Needed: Reagents needed:

(1) Mortar and Pestle Acetone (with 10-ml graduated cylinder)
(1) Cotton (whole class) Hexane (with 10-ml graduated cylinder)
(1) Funnel Ethanol (with 10-ml graduated cylinder)
(2) Capillary tubes Acetone (with 10-ml graduated cylinder)
(1) Chromatography paper
(1) Test tube with rubber stopper Sample needed:
(2) 250-ml Erlenmeyer flask 1g of leaves from any plant
(3) 50-ml beaker
(1) 10-ml graduated cylinder
(1) cutter
(1) scissors
(1) ruler
(1) pencil

Procedure
1. Collect about 1 g of leaves from any one kind of plant. Take note of the plant’s scientific name and the place where it was
collected.
2. Prepare two (2) chromatography paper strips with dimensions 15 cm width and 150 cm length. Using a pencil draw a horizontal
line that is 1 cm from the bottom of the paper then set aside.
3. Using a pair of scissors cut the leaf sample into small pieces.
4. Place the cut leaves in the mortar and add a small amount of acetone just enough to submerge the leaf samples and extract
using a pestle.
5. Filter the extract using a funnel with a cotton plug into a 50-ml beaker.
6. Using the capillary tube, practice your spotting technique on a piece of scratch paper with water. Make sure that the spot does
not exceed 2 mm in diameter.
7. When ready, draw a clear acetone extract into a capillary tube and use this extract to make spot on the line drawn your
chromatographic paper strip then allow to dry.
8. Repeat step 7 five times.
9. Take another strip and do steps 7 and 8
10. Prepare the following solvent systems: 9:1:1 (v/v/v) hexane:ethanol:acetone and 9:1 (v/v) hexane:acetone in separate
beakers.
11. Pour 5 mL each solvent system into two separate test tubes and label each test tube according to the solvent system.
12. Attach the top of the strip to the push pin on the rubber stopper using a masking tape.
13. Carefully insert the spotted end of the strip into the test tube. Make sure that the paper does not touch the side of the test
tube. See to it that the spot is above the level of the solvent and stopper the tube tightly
14. Place the test tubes in separate 250-ml Erlenmeyer flasks and watch the solvent rise on the paper.
15. When the solvent front is about 1 in from the top of the strip, remove the paper from the test tube and mark the solvent front
with a pencil.
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 16. Allow the strip to dry.
17. Mark the outline of the individual spots with a pencil. Draw the pattern produced on your data sheets and label each spot
according to its color and shape.
18. Compare the chromatograms produced by the two solvent systems as to the extent or degree of separation of the spots.
19. Determine the Rf values of the spots in the two chromatograms.

Figure 4.1 Setup for paper chromatography.

(http://www.tanlam.com/science/biology/eoi/eoi.htm)

B. Identification of Amino Acids by Paper Chromatography

Materials Needed: Reagents needed:
(1) Whatman chromatography paper Phenylalanine
(1) Ruler Tyrosine
(1) pencil Aspartic Acid
(1) stapler n-butanol
(1) cling wrap and aluminum foil (for the whole class) Glacial acetic acid
(1) tweezers Acetone
(1) Watch glass 2% ninhydrin solution in acetone

Procedure:
1. Obtain a clean sheet of Whatman chromatography paper, about and place it on a clean and dry surface. Cut the
chromatography paper with dimension width 8 cm and length 23 cm.
2. Using a pencil and a ruler, lightly draw a thin line parallel to one side 1.5 cm from the edge of the paper.
3. Lightly write 8 X’s along the line at 2 cm intervals.
4. Under each X, place an identifying mark, two for each standard (P=phenylalanine, T=tyrosine and A=aspartic acid) and two
for the unknown substance (U).
5. Spot a small amount of each standard and unknown solution on its designated position on the paper. Do these five times
allowing the spot to dry each time.
6. Roll the paper into a cylinder and staple the ends. Make sure that the edges of the paper do not touch each other.
7. Fill the developing chamber up to about 0.75 cm deep with the solvent system 4:1:1 (v/v/v) n-butanol: glacial acetic acid:
water.
8. Place the cylindrical paper in the chamber, observing the usual precautions.
9. Cover the chamber tightly using a plastic wrap (cling wrap) and allow the chromatogram to develop (about 1 and ½ hours).
DO NOT DISTURB the chamber.

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 10. After the chromatogram has been developed, remove the paper carefully and open it. Mark the solvent front lightly with a
pencil.
11. Allow the paper to dry.
CAUTION! Ninhydrin is a neurotoxin (this can change the function and structure of your nervous system),
Avoid direct skin contact!
12. In the fume hood, spray the chromatography paper with 2 % ninhydrin solution in acetone.
13. Dry the paper under the fume hood. Spots will appear after completely drying the paper.
14. Encircle each spot with a pencil and calculate Rf values.
15. Compare the spots in terms of shape and color.
16. Draw the chromatogram.

Figure 4.2 Setup for paper chromatography of amino acids

(http://employees.oneonta.edu/helsertl/AAChrom.html)

C. Analysis of Isolated Caffeine by TLC

Materials Needed: Reagents needed:
(1) TLC Plate 5 % acetic acid in ethyl acetate
(1) TLC developing chamber
(1) Capillary tube Sample needed:
(1) Ruler Isolated crude caffeine
(1) Pencil
(1) Aluminum foil or plastic wrap (whole class)

Procedure:
1. Prepare the TLC plate having the size of 3 cm by 5 cm.

2. Draw an aliquot of the caffeine solution into a capillary tube and spot it on one TLC plate approx. 1 cm from the bottom. Make
another spot for the standard dissolved in same solvent given by the instructor. The second spot should be approx. 1 cm from the
1st spot.

3. In a 250 mL beaker, pour about 10 mL of the solvent system (5 % acetic acid in ethyl acetate). Line the sides of the chamber
with a piece of filter paper, cover the beaker with a foil or plastic wrap and allow the system to equilibrate.

4. After about 3-5 minutes, place the TLC plate in the developing chamber, making sure that the spots are above the solvent
system. Cover the beaker with the aluminum foil or plastic wrap.

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5. Allow the solvent to migrate up the TLC plate until it is about 1 cm from the top. Make sure the solvent line will not reach the top
of the plate. Remove the TLC from the chamber and let it dry under the hood.

6. View the spots under UV.

7. Gently mark the spots using pencil. Analyze and compare the chromatograms of standard and the isolated caffeine.

8. Determine the Rf values of the spots.