INTRODUCTION
Chromatography in all its variations is one of the most widely used and most important of all the laboratory purification methods. First demonstrated by Michael Tswett, a Russian botanist who reported the separation of plant pigments by this method in 1903, chromatography has since been applied to every conceivable type of compound in a wide variety of uses. The police and other investigators use chromatography to identify clues at a crime scene like blood, ink, or drugs. More accurate chromatography in combination with expensive equipment is used to make sure a food company's processes are working correctly and they are creating the right product. This type of chromatography works the same way as regular chromatography, but a scanner system in conjunction with a computer can be used to identify the different chemicals and their amounts. Chemists use chromatography in labs to track the progress of a reaction. By looking at the sample spots on the chromatography plate, they can easily find out when the products start to form and when the reactants have been used up ( i.e
., when the reaction is complete). Chemists and biologists also use chromatography to identify the compounds present in a sample, such as pigments used to identify plant and lichen species.
All types of chromatography have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. Numerous forms of chromatography have been developed:
1.
Column chromatography – The stationary phase is often crushed mineral (silica gel, for instance) and the mobile phase a solvent (acetone, benzene, etc.).
2.
Gas chromatography – (variously known as GC, GLC – gas liquid chromatography or VPC – vapor phase chromatography) – The stationary phase is an oily liquid coated thinly on a crushed mineral and the mobile phase is a gas (He, N
2
).
3.
Thin layer chromatography
– (TLC) – both stationary and mobile phases are as in column chromatography. However, the adsorbent is coated on a glass plate in a thin layer, the mixture is deposited near one edge of the layer as a spot or a streak and the plate is then dipped in a shallow bath of a solvent.
4.
Paper chromatography
– This is a variation of TLC where the stationary phase is a strip of very uniform absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents.
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This diagram shows what a paper chromatogram might look like after the solvent has moved almost to the top.
This is the frontline, the height reached by the solvent.
FIGURE 3-1
In this example, four ink mixtures (M, 1,
2, and 3) were applied to the baseline. the solvent wicks up the paper in this direction solvent
If “M” represents an unknown ink mixture and samples 1, 2, and 3 represent known ink mixtures, it is fairly easy to see from the final chromatogram that the unknown ink mixture contained the same dyes as pen #2. You can also see that pen #1 contains a mixture of two different blue dyes - one of which might be the same as the single dye in pen #3.
Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the base line. The distance travelled relative to the solvent is a constant for a particular compound as long as you keep everything else constant - the type of paper and the exact composition of the solvent, for example.
The distance travelled relative to the solvent gives the retention factor ( R f
value ). For each compound it can be worked out using the formula:
R f
=
For example, if one component of a mixture travelled 9.6 cm from the base line while the solvent had travelled 12.0 cm, then the R f value for that component is:
R f
=
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Although paper chromatography is simple to do, it is quite difficult to explain. The explanation depends to some extent on what sort of solvent you are using, and many sources gloss over the problem completely. Paper is made of cellulose fibers, and cellulose is a polymer of the simple sugar, glucose.
You can explain paper chromatography in terms of the way that different compounds are absorbed to different extents onto the paper surface. In other words, it uses a very similar mechanism as that for thin layer chromatography. Unfortunately, it is a bit more complicated than that.
The complication arises because the cellulose fibers attract water vapor from the atmosphere as well as any water that was present when the paper was made. You can therefore think of paper as being cellulose fibers with a very thin layer of water molecules bound to the surface. It is the interaction with this water which has the most important effect during paper chromatography.
Non-polar (hydrophobic) molecules in the mixture that you are trying to separate will have little attraction for the water molecules attached to the cellulose, and so will spend most of their time dissolved in the moving solvent. Molecules like this will therefore travel a long way up the paper carried by the solvent. They will have relatively high R f values presuming that the solvent is non-polar.
On the other hand, polar molecules will have a high attraction for the water molecules and much less for the non-polar solvent. They will therefore tend to dissolve in the thin layer of water around the cellulose fibers much more than in the moving solvent. Because they spend more time dissolved in the stationary phase and less time in the mobile phase, they will not travel very fast up the paper.
Water can serve as a polar solvent. The attraction of the water to the paper (adhesion force) is larger than the attraction of the water to itself (cohesion force), hence the water moves up the paper. The ink and plant pigments will also be attracted to the paper, to themselves, and to the water differently, and thus a different ink component or plant pigment will move a different distance depending upon the strength of attraction to each of these objects.
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MATERIALS chromatography papers petroleum ether/ethanol (strong non-polar) acetone (slightly non-polar) ethanol (borderline polar/non-polar) isopropyl alcohol (borderline polar/non-polar) methanol/water (polar) ruler pencils chromatography chambers with lids spinach leaves
Coleus leaves mortar and pestle capillary tube beakers (50 ml) gloves glass Pasteur pipettes for transferring solvents to chambers graduated cylinders (5-10 ml) fume hood
PROCEDURE
Separation of Ink Mixtures
1.
You should wear goggles and gloves during this experiment.
2.
Obtain five chromatography chambers and add to each, one of the five solvents (Acetone,
Petroleum ether/ethanol, Ethanol, Isopropyl Alcohol, and Methanol/water). Add just enough solvent to the bottom of each chamber to bring the solvent level roughly 5 mm from the base of the chamber. (This will be approximately 2-2.5 ml of each solvent.)
Petroleum ether/ethanol should only be poured under the fume hoods. Be sure to keep the lids on your chromatography chambers once they are filled with solvent.
Do not leave the chambers open.
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3.
Take five chromatography papers and draw a baseline with a pencil roughly 15 mm from the bottom edge of each paper. Limit handling of the chromatogram papers with your fingers.
4.
With the black marker, mark a line of black ink over the pencil baseline on each of the five papers.
FIGURE 3-2 pencil base line with ink line marked on top of baseline
(15 mm) solvent level (5 mm)
5.
Put each of the five chromatography papers in one of the five solvent chambers and wait a few minutes for each to “develop”. BE SURE TO KEEP THE LIDS ON THE
SOLVENTS AT ALL TIMES TO PREVENT EVAPORATION AND EXPOSURE
TO FUMES.
The other reason for covering the container is to make sure that the atmosphere in the chamber is saturated with solvent vapor. Saturating the atmosphere in the beaker with vapor stops the solvent from evaporating as it rises up the paper.
6.
Allow the solvent to move within 5 mm of the top of the chromatography paper. Remove the papers and let them dry.
7.
Complete the Results table for the black pen by calculating R f
values for each separated pigment in the black ink. When measuring the distance the sample traveled, you should measure from the baseline (where the middle of the spot originally was) and then to the center of the spot in its new location.
To calculate the Rf value, we use the equation:
R f
= distance traveled by the sample component distance traveled by the solvent
Note that an R f
value has no units because the units of distance cancel.
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8.
Repeat steps 1-7 for the green pen and the brown pen.
9.
Thoroughly dispose of all solvents in appropriate waste containers and allow your chambers to air dry. DO NOT wash chambers out with water .
Separation of Plant Pigments
Chlorophyll absorbs red and blue light from the sun, but it does not absorb green light, it reflects it. This is why tree leaves are normally green. In the fall, the tree shuts down chlorophyll production to save energy, and recycles it for later use. Thus the green-reflecting chlorophyll disappears, and we are left with the orange and red carotenoids (pigments that capture light energy and transfer it to chlorophyll), and so the leaves turn red and orange. Paper chromatography will enable you to see these ‘hidden’ plant pigments.
1. Your instructor will grind up five spinach leaves and five Coleus leaves in 5 ml of acetone each using the mortar and pestle.
2. Take five chromatography papers and draw a baseline with a pencil roughly 15 mm from the bottom edge of each paper.
3. Using a capillary tube, mark a line of spinach extract on each of the five papers. Allow the line to dry for a minute and then repeat ten times in order to transfer a significant amount of pigment mixture to the baseline.
4. Put each of the five chromatography papers in one of the five solvent chambers and wait a few minutes for each to “develop”. Remove the papers to dry.
5. Complete the Results table for the spinach leaf by calculating R f
values for each separated pigment in the spinach leaf extract. Repeat steps 4-7 for the Coleus leaf extract.
6. Properly discard the used chromatography solvent in the appropriately labeled waste containers in the fume hoods.
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RESULTS
Mobile Phase
(Solvent)
Petroleum
Ether/Ethanol
Ethanol
Acetone
Isopropyl Alcohol
Methanol/Water
Distance
Traveled By
Solvent (mm)
BLACK PEN
Pigments
Separated
(Name the Color)
Distance
Traveled by
Pigment (mm)
TABLE 3-1
R f
Value for each
Pigment
Separated
Mobile Phase
(Solvent)
Petroleum
Ether/Ethanol
Distance
Traveled By
Solvent (mm)
Ethanol
Acetone
Isopropyl Alcohol
Methanol/Water
BROWN PEN
Pigments
Separated
(Name the Color)
Distance
Traveled by
Pigment (mm)
TABLE 3-2
R f
Value for each
Pigment
Separated
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GREEN PEN
Mobile Phase
(Solvent)
Petroleum
Ether/Ethanol
Distance
Traveled By
Solvent (mm)
Ethanol
Acetone
Isopropyl Alcohol
Methanol/Water
Isopropyl Alcohol
Methanol/Water
SPINACH LEAF
Mobile Phase
(Solvent)
Distance
Traveled By
Solvent (mm)
Petroleum
Ether/Ethanol
Ethanol
Acetone
Pigments
Separated
(Name the Color)
Distance
Traveled by
Pigment (mm)
TABLE 3-3
R f
Value for each
Pigment
Separated
Pigments
Separated
(Name the Color)
Distance
Traveled by
Pigment (mm)
TABLE 3-4
R f
Value for each
Pigment
Separated
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Coleus LEAF
Mobile Phase
(Solvent)
Petroleum
Ether/Ethanol
Ethanol
Acetone
Isopropyl Alcohol
Methanol/Water
Distance
Traveled By
Solvent (mm)
Pigments
Separated
(Name the Color)
Distance
Traveled by
Pigment (mm)
DISCUSSION QUESTIONS
1) Why do trees leaves turn different colors in the fall?
TABLE 3-5
R f
Value for each
Pigment
Separated
2) Why does a plant use more than one pigment?
3) Why do different plants have different amounts and different types of pigments?
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4) What makes a molecule polar or nonpolar? How does its polarity affect its interactions with other molecules?
5) How do the polarities of the solid phase and liquid phase affect how far chemicals travel on the paper?
6) What types of pigments do you think are present in each type of leaf? How can you tell?
(consider color and R f
value)
7) Were the R f
values similar or different for each pigment that occurred in both the spinach and the Coleus leaves? Why might you expect them to be similar? (Make sure to consider molecular structure and polarity.)
8) Why must you use lead pencil, instead of a pen, to mark the baseline on your chromatography paper?
9) Give two or three practical examples where chromatography would be valuable analytical tool.
ADAPTED FROM: Natural Sciences/Chemistry – Walter Scharf & Charles Malerich; Baruch College New York, NY 10010
Chemguide© Jim Clark 2009
Science Buddies – Author: Amber Hess Editor: Andrew Olson
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