Exercise 4 (Chromatography)

Chem 31.1 EXPERIMENT 4 Chromatography Introduction Chromatography includes a variety of techniques for separating individual compounds or compound types from a mixture. Separation is affected by the distribution of the components of the mixtures between a stationary phase and a mobile phase. Various types of chromatography are possible depending on the physical states of the stationary phase and mobile phases involved. Adsorption chromatography uses a solid stationary phase and a liquid mobile phase. Separation using adsorption chromatography is governed by surface adsorption phenomena. On the other hand, partition chromatography uses a liquid stationary phase supported on the surface of a solid and a liquid or gas mobile phase which is insoluble in the stationary phase. Partition chromatographic separations may be due to differences in the solubility of the sample in the stationary and mobile phases. In adsorption chromatography, the mixture to be separated is adsorbed on the solid stationary phase over which the liquid mobile phase is allowed to flow. The transfer of the adsorbed compound between the stationary phase and the mobile phase is an equilibrium process. K compound in stationary phase

compound in mobile phase

The extent of adsorption of a single component depends on the polarity of the molecule, the activity of the adsorbent, and the polarity of the liquid mobile phase. In general, the more polar a functional group in the compound is, the more strongly it will be adsorbed on the surface of the polar stationary phase. The actual separation of the components in a mixture is dependent on the relative values of the adsorption-desorption equilibrium constant, K, for each of the components. The individual components will move with the mobile phase at different rate, resulting in their separation into different regions (as bands or spots) in the stationary phase. Thin layer chromatography (TLC) is a form of solid-liquid adsorption chromatography. It uses a thin layer of adsorbent (usually alumina or silica gel) supported on a flat surface (usually glass). TLC is very useful in monitoring the progress of the reactions, detecting intermediates in reactions, analyzing crude products or unknown mixtures, determining the number of components in a mixture and evaluating the efficiency of purification processes. Paper chromatography bears a resemblance to TLC but is slightly different in principle. Filter paper, which is made of highly purified cellulose, absorbs and retains water molecules strongly. This is because cellulose is a polyhydroxy compound. The paper (cellulose) and the bound water, which forms part of its structure, constitute the stationary phase in paper chromatography. Small spots of the mixture to be separated are placed near the bottom of a strip of filter paper and a solvent (mobile phase) is allowed to travel up the paper by capillary action. Separation takes place due to different affinities of the components of the mixture for the polar stationary phase and the mobile phase, which is relatively nonpolar solvent or solvent system. There is a continuous back-and-forth exchange of solutes between water and the solvent, but those, which are more soluble in the mobile phase, spend more time in it and are carried up the paper faster.

Chem 31.1 Paper chromatography is an example of liquid-liquid partition chromatography. Like TLC, paper chromatography is used in the rapid analysis of the components of reaction mixtures and as tentative means of identification. A compound will move up to the TLC plate or paper strip at a rate relative to that of the solvent front. The relative mobility is known as the Rf value of the compound and is defined by the equation: Rf =

distance travelled by compound from the origin distance travelled by solvent from the origin

where the origin is the midpoint of the original spot. The distance traveled by a compound is obtained by measuring the distance from the origin to the point of greatest density (center of mass) of the spot corresponding to the compound. Under a defined set of conditions (adsorbent, solvent, temperature and humidity) the Rf value is a characteristic property which can be used for the identification of a compound. I.

Objectives 1. To learn the techniques of paper chromatography and thin layer chromatography. 2. To apply chromatographic methods in the separation of the components of a mixture. 3. To identify an unknown by comparing Rf value and other characteristics with those of a standard.

II.

Procedure A. Separation of plant pigments by paper chromatography 1. Collect about 1 g of leaves from any one kind of plant in the locality. Take note of the plant’s scientific name and the place where it was collected. 2. Cut the leaves into small pieces. 3. Extract the leaf pigments with about 20 mL acetone using a mortar and pestle. Filter the extract through a piece of cotton. 4. Fill a capillary tube with a clear acetone extract and use this extract to make a spot on a strip of chromatographic paper about one centimeter from the bottom. (To save time and materials, practice your spotting technique on a piece of scratch paper before attempting to use the chromatography strip). Make sure that the spot does not exceed two millimeters in diameter. 5. Repeat step 4 five times, allowing the spot to dry each time. 6. Take another strip and do steps 4 and 5. 7. Pour 5 mL of 9:1:1 (v/v/v) petroleum ether-diethylether-acetone and 5 mL of 9:1 (v/v) petroleum ether-acetone into two separate 50 mL test tubes. 8. Mount the test tubes in 250-mL Erlenmeyer flasks as shown in Figure 4.1. 9. Attach the paper with the spotted side at the far end to a hook mounted on a stopper and carefully insert the spotted end of the strip into the test tube. Make sure that the paper does not touch the sides of the tube. See to it that the spot is above the level of the solvent. 10. Stopper the tube tightly and watch the solvent rise with the paper. 11. When the solvent front is about one inch from the top of the strip, remove the paper from the test tube and mark the solvent front with a pencil. 12. Allow the strip to dry.

Chem 31.1 cork stopper

hook chromatographic paper

test tube sample spot (origin) Erlenmeyer flask

solvent

Figure 4.1. Setup for paper chromatography. 13. Mark the outline of the individual spots with a pencil. Copy the pattern produced on your data sheets and label each spot as to its color. 14. Compare the chromatograms produced by the two solvent systems as to the extent or degree of separation of the spots. B. Analysis of the component dyes of black ink by TLC NOTE: Avoid inhaling silica gel. 1. Mix 45 grams of silica gel G and 150 mL of dichloromethane in a wide-mouth screw cap bottle. 2. Wipe the surface of two microscope slides with cotton soaked in dichloromethane and then with cotton soaked in acetone. Allow the solvent to evaporate. 3. Put the slides together back to back handling them only at their edges. 4. Stir the slurry of silica gel and dip the slides ones, leaving about 0.5 cm from the top clean. 5. Slowly remove the plates from the slurry and allow the solvent to drain back into the bottle. 6. Check the coating of the gel. It must be uniform across the plate and without any break on the surface. 7. Remove the gel from the sides of the slides with a finger. 8. Separate the plates and put them on a clean sheet of paper with the silica gel facing upwards. 9. Allow the plates to dry for about 5 minutes. 10. As soon as the plates are dry, draw an aliquot of the ink sample into a capillary tube and spot it on one TLC plate, ~1 cm from the bottom. Spot the other plate using another ink brand. 11. In a wide-mouth screw –cap bottle, pour about 20 mL of the solvent system 6:2:2 (v/v/v) n-butanol-ethanol-NH3). Line the sides of the chamber with a piece of filter paper and allow the system to equilibrate. The setup for TLC is shown in Figure 4.2 12. After about two minutes, place the TLC plates in the developing chamber. Cover the chamber tightly. 13. Allow the chromatogram to develop (about 20 min). 14. Analyze and compare the chromatograms as in part A.

Chem 31.1

cover

developing chamber filter paper liner TLC plate sample spot

solvent

Figure 4.2. Setup for thin layer chromatography. C. Identification of amino acids by paper chromatography 1. Obtain a clean sheet of Whatman no. 1 filter paper, about 12 cm by 18 cm. Handle it only at its corners. 2. Using a pencil, lightly draw a thin line parallel to one side, ~1.5 cm from the edge of the paper. 3. Lightly place 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). See Figure 4.3 18 cm

12 cm 1.5 cm P

T

A

U

P

T

A

U

Figure 4.3. Preparation of paper for chromatography of amino acids. 5. Spot a small amount of each solution on its designated position on the paper. Do this five times allowing the spot to dry each time. 6. Roll the paper into a cylinder in such a way that the labels are facing out and staple the ends as shown below. Make sure that the edges of the paper do not touch each other. 7. Fill the developing chamber with the solvent system 1:2 (v/v) 2% ammonium hydroxide-isopropyl alcohol, up to about 0.75 cm deep. 8. Place the cylindrical paper in the chamber, observing the usual precautions. 9. Cover the chamber tightly and allow the chromatogram to develop (about 1 and ½ hours).

Chem 31.1 10. After the chromatogram has been developed, remove the paper and open it. Mark the solvent front lightly with a pencil. 11. Allow the paper to dry. 12. Dip the paper in a 2% ninhydrin solution in acetone. NOTE: Ninhydrin is a neurotoxin. Avoid direct skin contact. 13. Allow the solvent to evaporate. 14. Dry the paper for about 10 minutes using a blow dryer. 15. Encircle each spot with a pencil and calculate the Rf values. 16. Compare the spots in terms of shape and color. 17. Draw the chromatogram. III.

Questions 1. What would be the effect of the following errors in chromatographic work? a. The solvent level in the developing chamber is higher than the spotted sample. b. Too much sample is applied to the paper. c. The paper is allowed to remain in the chamber after the solvent front has reached the top of the plate. 2. Why is it necessary to cover the developing chamber tightly during the development of a chromatogram? 3. Identify your unknown. Explain clearly how you made this identification. 4. Can TLC or paper chromatography be used to separate and identify very volatile substances? Explain your answer. 5. Why were you required to handle the chromatographic paper only at its corners in part C?

Chem 31.1

IV.

Data and Results

A. Separation of Plant Pigments by Paper Chromatography Sample: ________________________________________ Solvent System Spot No.

9:1 (v/v) pet-ether-acetone 2

X

2

Y

Rf

9:1:1 (v/v/v) pet-ether-diethyl ether-acetone X2

Color

Y2

Rf

Color

NOTE: Include sketch of chromatogram B. Analysis of the Component Dyes of Black Ink by TLC Solvent System: __________________________________ Sample: (include brand name) _______________________ Spot No.

X2

Y2

Rf

Color

NOTE: Include sketch of chromatogram C. Identification of Amino Acids by Paper Chromatography Solvent System: __________________________________ Visualization method: ______________________________

X2 Y2 Rf Color Average Rf

Phenylalanine (P) Trial 1 Trial 2

Tyrosine (T) Trial 1

Trial 2

Aspartic acid (A) Trial 1

Unknown is: ______________________________________ NOTE: Include sketch of chromatogram

Trial 2

Unknown Trial 1

Trial 2