Lab #3: Components of Matter: Separation of Mixtures by Chromatography
Introduction:
Michael Tswett in 1906 described a simple method to separate chlorophyll into many different colored compounds.
He called his method the “chromatographic” method. In 1931 Kuhn and Lederer separated carotene, and orange substance
extracted from carrots, into two different compounds by chromatography. That marked the turning point for chromatography
to be accepted and used extensively for separating mixtures and for preparing pure compounds. Today chromatography has
become a very valuable technique very extensively used in both qualitative and quantitative analysis of a wide variety of
chemical compounds.
Separation of mixtures by chromatography is based on the property of adsorption. Adsorption may be described as
the adhesion of a substance to a solid surface. Different substances are adsorbed on solid surfaces with varying degrees of
tenacity. Therefore if a solvent is passed over a mixture of substances adsorbed on a solid surface, the solvent will carry the
different components of the mixture to different extents. The extent to which each component is carried will depend upon the
degree of the adsorption of that component on the solid surface and the solubility of that component in the moving liquid. If
the solid is packed in a column and the liquid is passed through the column, then the process is known as column
chromatography. The solid adsorbent in the column constitutes the stationary phase and the liquid that is passed through the
column constitutes the moving phase.
To separate a mixture using column chromatography, the mixture is dissolved in a suitable solvent and applied to the
top of the column. The components of the mixture will then be adsorbed on the stationary phase. Then the appropriate liquid
which serves as the moving phase is added to the top of the column. As the liquid moves down, the component which is very
soluble in the moving phase and which has love affinity to the stationary phase will move down rapidly, but not quite as fast
as the moving phase itself. On the other hand, the component which has low solubility in the moving phase and high affinity
to the stationary phase will move down very slowly. The components between these two extremes will move down as rates
determined by their relative affinities to the stationary phase and their relative solubility in the moving phase. Polarities of
the stationary phase and the moving phase play important roles in chromatography. If a compound is very polar it is
adsorbed strongly on a polar stationary phase; similarly less polar compounds are adsorbed less strongly on polar stationary
phases.
There are many variations of chromatography. The adsorbent can be paper or a thin layer of solid adsorbent coated
on a glass plate or plastic sheet. If the adsorbent is paper it is called paper chromatography; if it is a coating on a class or
plastic sheet it is thin layer chromatography (TLC). In some special cases, the stationary phase is a liquid coated on solid
support and the moving phase is a gas. Then it is called gas liquid chromatography (GLC).
In paper chromatography and TLC, the mixture is applied about 1” away from one edge of the paper or the thin
layer plate (see figure). When the applied spot is dry, the paper or the plate is placed vertically in a covered container
containing the liquid mobile phase, which is also known as the “developing solvent” (see figure). The liquid will move up
the paper or the thin layer plate by capillary action. As the solvent moves over the solid stationary phase, it will carry the
components of the mixture with it, the different components moving up the paper or plate at different rates. When the
advancing solvent front as reached within about 1” of the top edge of the paper or plate, the paper or plate is taken out of the
container and the solvent front is marked with a pencil. If the components of the mixture are colored, the location of each
component on the chromatogram can be noticed without further treatment. But, if the components are colorless, the
chromatogram is treated with a reagent which will react with the components to give colored products. This will give rise to
colored spots corresponding to the location of the components.
The “mobility” of a compound chromatographed is described in relation to the mobility of the developing solvent by
a term known as Rf value. The Rf value is defined as:
Rf =
distance moved by the compound
distance moved by the solvent
The distance moved by the compound is the distance between the applied spot and the spot at the end of the
chromatography, center to center; the distance the solvent moved is the distance between the center of the applied spot (of the
compound) and the solvent front. In the figure shown, Rf of the compound = dx
dy
Two factors which will very much affect the Rf value of a compound are (1) the nature of the stationary phase and
(2) the nature of the developing solvent. When the same stationary phase is used, the nature of the solvent is the predominant
factor which determines the Rf value of a compound. Sometimes two compounds may have the same Rf values when
developed in a solvent system. Then these two compounds will not separate when a mixture of these two is developed with
that solvent system. In such cases, the other solvents have to be tried until a solvent capable of separating the two
compounds is found.
In this experiment paper chromatography will be used to separate (1) the components of four food colors and (2) a
mixture of two amino acids.
Problem: In part I, which color will move fastest and why? In part II, what will determine the movement of the ions and
which will move the fastest?
Materials:
Developing solvent
Funnel
100 ml beaker
Staining solution
4 test tubes
chromatography paper
food coloring
pencil
0.1 M Ni(NO3)2 0.1 M Co(NO3)2
stoppers
0.1 M Cu(NO3)2
unknown solution
Eluting solvent
0.1 M Fe(NO3)3
capillary tubes
Procedure:
I.
Chromatography of Food Colors
1. Record a hypothesis to answer the problem/objective.
2.
Create a data sheet in your lab notebook.
3.
Take four 8” test tubes and pour about 10 ml of the developing solvent into each of them using a funnel. Make
sure the sides of the test tubes do not become wet with the solvent. Stopper the test tubes tightly and set them
aside.
4.
Obtain 4 strips of chromatography paper from the cart. On each strip, draw a pencil line across the strip 4 cm
away from one of the ends (see figure above). In the center of each line, make a mark. This will be the starting
line where the dye will be spotted for chromatography. Label the top of each strip red, blue, yellow, and green.
5.
Take a few drops of one of the food colors, say red, in a small test tube (or a microvial). Dip the end of the
capillary tube into the food coloring. When you take the capillary out of the tube, there will be a very small
amount of dye solution in it, drawn up by capillary action.
6.
Touch the tip of the capillary tube containing the dye on to the pencil mark on the red-labeled chromatography
strip. The dye in the capillary tube will flow onto the paper and make a spot.
7.
Spot the other three strips using the same technique and their matching colors.
8.
Allow the strips to dry completely.
9.
Uncork each of the tubes and insert one strip into each, with the spotted end toward the bottom of the test tube
and replace the stopper immediately. Leave the test tubes aside undisturbed.
10. While the strips are running, you can move onto part 2, but keep an eye on them.
11. After the solvent front has reached almost all the way up the strip, remove the corks, take the wet strips out of
the test tubes carefully, and place them flat on clean paper towels.
12. Mark the solvent front immediately, before the solvent evaporates off, with a pencil.
13. When the strips are dry, outline the spots on each strip with a pencil and mark the center of each spot.
14. Measure on each chromatogram the distance of the solvent front and the center of each spot from the starting
line. Record these values for each color in your lab book.
15. Complete the data analysis by calculating the Rf for each chromatogram.
II.
Chromatography of Selected Ions
1. Take five 8” test tubes and pour about 10 ml of the eluting solvent into each of them using a funnel. Make sure
the sides of the test tubes do not become wet with the solvent. Stopper the test tubes tightly and set them aside.
2.
Obtain 5 strips of chromatography paper from the cart. On each strip, draw a pencil line across the strip 4 cm
away from one of the ends (see figure above). In the center of each line, make a mark. This will be the starting
line where the dye will be spotted for chromatography. Label the top of each strip Cu(NO3)2, Fe(NO3)3,
Ni(NO3)2, Co(NO3)2 and unknown.
3.
Take a few drops of one of the cations in a small test tube (or a microvial). Dip the end of the capillary tube
into the cation. When you take the capillary out of the tube, there will be a very small amount of cation solution
in it, drawn up by capillary action.
4.
Touch the tip of the capillary tube containing the solution on to the pencil mark on the matching
chromatography strip. The solution in the capillary tube will flow onto the paper and make a spot.
5.
Spot the other three strips using the same technique and their matching cations.
6.
Allow the strips to dry completely.
7.
Uncork each of the tubes and insert one strip into each, with the spotted end toward the bottom of the test tube
and replace the stopper immediately. Leave the test tubes aside undisturbed.
8.
After the solvent front has reached almost all the way up the strip, remove the corks, take the wet strips out of
the test tubes carefully, and place them flat on clean paper towels.
9.
Mark the solvent front immediately, before the solvent evaporates off, with a pencil.
10. When the strips are dry, spray the strips with the staining solution.
11. When the strips are dry, outline the spots on each strip with a pencil and mark the center of each spot. If the
spots are difficult to see, rinse the strips in tap water to remove the staining solution.
12. Pour the eluting solution into the waste container provided by the teacher.
13. Measure on each chromatogram the distance of the solvent front and the center of each spot from the starting
line. Record these values for each color in your lab book.
14. Complete the data analysis by calculating the Rf for each chromatogram.
15. Answer the questions.
Questions:
1.
A student chromatographs a mixture, and after developing the spots with a suitable reagent, he observes the
following:
What are the Rf values?
2.
Explain, in your own words, why samples can often be separated into their components by chromatography.
3.
The solvent moves 3 cm in about 5 minutes. Why shouldn’t the experiment be stopped at that time instead of
waiting 20 minutes for the solvent to move 10 cm?
4.
In this experiment it takes about 1- microliters of solution to produce a spot 1 cm in diameter. If the Cu(NO 3)2
solution contains about 6 g of Cu2+ per liter, how many micrograms of Cu2+ ions are in the spot?
Conclusion:
 What did you learn? Be specific.
 Was your hypothesis correct? Why or why not?
 What went well? Why?
 What didn’t go well? Why?
 What were your sources of error? What could be done to correct them?