NAME: Shawn Shaw ID#: 2014080314

LAB TITLE: Paper & Thin Layer Chromatography DATE: 03rd/Nov/ 16

LAB PARTNER(S): Lsaedai Williams
ABSTRACT:
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HYPOTHESIS:
Chromatography is not used for the separation of a mixture of compounds into its
individual components hence the components plant pigments separating visibly will not occur.

INTRODUCTION:
Chromatography is a technique for the separation of a mixture by passing it in solution or
suspension through a medium in which the components move at different rates. It is also a
separation technique in which the mixture to be separated is dissolved in a solvent and the
solvent is passed through a column packed with an absorbent stationary phase. All forms of
chromatography have a stationary phase and a mobile phase. The mobile phase flows through the
stationary phase and carries the components of the mixture with it. Simple chromatography is
carried out on paper. A spot of the mixture is placed near the bottom of a piece of
chromatography paper and the paper is then placed upright in a suitable solvent, eg water. As the

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solvent soaks up the paper, it carries the mixtures with it. Different components of the mixture
will move at different rates. This separates the mixture out.

Paper chromatography is used to separate colored chemicals or substances. It is primarily

used as a teaching tool, having been replaced by other chromatography methods, such as thin-
layer chromatography. The paper exhibits weak ion exchange and adsorptive properties. Thin
layer chromatography uses a thin, uniform layer of silica gel or alumina coated onto a piece of
glass, metal or rigid plastic. The silica gel is the stationary phase. The stationary phase for thin
layer chromatography also often contains a substance which fluoresces in UV light. The mobile
phase is a suitable liquid solvent or mixture of solvents; it is usually an organic solvent or solvent
mixture. The mobile phase is drawn up the plate via capillary action. Thin layer chromatography
is normally done on a small glass or plastic plate coated with a thin layer of a solid and the most
common are silica or alumina.

This experiment is conducted to investigate the components Plant Pigments separating

visibly. The most important and abundant chemical pigment found in plants is chlorophyll. There
are two types of chlorophyll found in the plant; Chlorophyll A and Chlorophyll B. Chlorophyll A
is the main photosynthetic pigment. It absorbs most energy from wavelengths of violet-blue and
orange-red light. It also reflects green/yellow light, and as such contributes to the observed green
color of most plants. It converts light energy to chemical energy used by the plant itself.
Chlorophyll b absorbs light in a region of the spectrum apart from the dominant chlorophyll, and
transfers the energy it produces to chlorophyll A.

Different chromatograms and the separated components of the mixtures can be identified
by calculating the Rf value using the equation. The Rf value of a particular compound is always
the same - if the chromatography has been carried out in the same way.

Rf = distance moved by the compound ÷ distance moved by the solvent

Various substances used in this experiment is highly flammable so caution is advised to

protect each individual.

PROCEDURE
Refer to: CHEM 241 Lab Manual, Lab #5 Paper & Thin Layer Chromatography, pg. 2-5

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 FLOW CHART
A) Paper Chromatography B) Thin- Layer Chromatography

Make chromatogram strip, taper the Prepare a chamber for TLC with a 100mL
bottom. Draw a pencil line 3cm from end, beaker film. Fill with 0.1% NaCl solution
loading strip with chlorophyll. and at 1cm from edge of TLC strip, draw
pencil line, continuing at 1cm intervals.

Load a capillary tube with chlorophyll acetone

solution, and then mark the center of the strip. Dip toothpick in distilled water and touch to
marking pen tip and immediately spot on the line
of a TLC plate just once, repeating for each
color.
Load the chromatogram with the chlorophyll
solution and touch the dot on the paper with the
capillary tube.

Place in developing cooler and seal with Para

film. Identify the number of dyes, colors and
relative positions
Allow drop to dry and repeat the process until 15
drops have been added. Add 3ml of petroleum
ether chloroform to a test tube.

Mark the solvent front on the strip when the

solvent is 2 cm from top and allow drying.
Place paper strip end-down into solvent container
and run chromatogram upright at room temp with a
stopper on the tube.

Circle the position of the most intense part of

When solvent reaches 1cm from top, remove from separated colors. Measure distance from straight
tube and mark the colored bands. Allow the paper line to solvent front. Calculate Rf values
to dry.

Determine the most and least soluble. Calculate Repeat the procedure for each color spot.
the Rf value for each pigment.

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PRE-LAB QUESTIONS:
1. The two samples were not necessarily the same substances. To prove unambiguously that
they were identical using TLC the same Rf value would have to be determined under the
various conditions and/or double spotting can be done.

2. The solvent used for the TLC with 2-Phenylethanol and acetophenone is acetone. The solvent
used for the TLC with the bromobenzene and p-xylene is hexane. The solvent used for the
TLC with benzoic acid, 2, 4-dinitrobenzoic acid, and 2, 4, 6-trinitrobenzoic acid is methylene
chloride.

3. The corrections for the errors are:

a) A less polar mobile phase should be used instead.
b) A more polar mobile phase should be used instead
c) This will result in incorrect data as the distance traveled by the solvent will be
inaccurate. To correct this the experiment would have to be redone with a new plate.
4.

TABLE SHOWING DISTANCE TRAVELLED BY EACH MOLECULE ON A

CHROMATOGRAM.

Distance Travelled (mm)

Molecule Rf value
D =Rf * Sf

N,N-fictionol 0.41 22

Cis-2,4-pretendium 0.20 11

dl-made-upelene 0.72 39

D(+)-tetra-imaginase 0.91 49

Polysynthetic acid 0.78 42

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5.

TABLE SHOWING DATA FROM CHROMATOGRAPHY

Chlorophyll A Chlorophyll B Xanthophyll Carotene

Pigment Origin (cm) 1 1 1 1

Pigment Front (cm) 5.2 4.7 9.0 7.7

Solvent Front (cm) 12 12 12 12

Rf Value (cm) 0.43 0.40 0.75 0.64

RESULTS:

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 TABLE 1: RESULTS OF PIGMENTS IN PLANTS USING PAPER
CHROMATOGRAPHY
Migration Distance
Band # Pigment Color Rf Value
(mm)

Solvent - - -

TABLE 2: RESULTS OF THIN LAYER CHROMATOGRAPHY OF INK

Migration Distance
Band # Pigment Colour Rf Value
(mm)

Solvent - - -

OBSERVATIONS:

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CALCULATIONS:

DISCUSSION:

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CONCLUSION:

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REFERENCES:

Ault, A. (1998). Techniques and Experiments for Organic Chemistry (6th ed.). California:
University Science Books.
Bruice, P. (2011). Organic Chemistry (7th ed.). Santa Barbara: Pearson.
Jonathan Clayden, N. G. ((May 4, 2012)). Organic Chemistry (2nd ed.). New York: Oxford
University Press. Retrieved October 21, 2016

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