Developers:

Cecelia Oberholzer
Little Flower High School
Philadelphia, PA

Dr. Peter Cartier
Sheryl Deley
Rohm and Haas Company
Spring House, PA

Grade
Levels:

High School

Discipline:

Chemistry

Goals:

• To acquaint students with the principles of liquid chromatography.
• To construct a liquid chromatography column.
• To investigate the effects of different solvents and solvent concentrations in the separation process.

Specific
Objectives:

To obtain separations of both synthetic dyes and natural plant pigments.

Background:

Chromatography is a method of separation mixture of two or more substances by distribution between two phases, one of which is stationary and one of which is moving.

The type of chromatography used depends on the nature of the two phases. Column chromatography, also called liquid chromatography or LC is a solid-liquid phase partitioning method. Small columns are packed with stationary phase material called adsorbent. This material may be any solid that does not dissolve in the liquid phase. Silica gel, SiO₂ X H₂O, is commonly used as the stationary phase.

The adsorbent (stationary phase) is continuously washed by a solvent (liquid phase) passing through the column. The mixture to be separated is introduced into the top of the column. The components of the mixture initially adsorb onto the silica particles and are then washed down by the solvent at differing rates, depending on their attraction, first to the adsorbent and then to the solvent. The components of the mixture form moving bands, and each band usually contains a single component.

Good separation of mixture components (solutes) depends on the kind of adsorbent and the solvent system. In general, nonpolar compounds pass through the column faster than polar compounds. If the adsorbent has equal affinity for the solute materials, the components will not move down the column. If the solvent is too polar, all the solutes may wash down the column with no separation.

Materials:

Clear drinking straws	Glass wool
Silica gel (70-230 mesh)	AmberchromÆ Ion Exchange Resin (Rohm and Haas)
Ethanol	Water
Acetic acid, 0.1M	Wash bottle
Ring stand	Clamp holder
Extension clamp micro jaws	Dropper pipet
Microtip dropper pipet	Red food color (Durkee)
Washable black ink	Green and blue food colors (McCormick)
Beet juice	100-ml beakers

A. Silica Gel Column

Wet a small piece of glass wool and plug the bottom of the straw. Clamp the straw vertically to the ring stand. Place a 100-ml beaker under the straw. In another beaker, add 20ml of water to a small amount of silica gel to form a slurry. Draw some slurry into the dropper pipet. With a finger on the bottom of the straw, holding the glass wool in place, add silica gel slurry to the top of the straw from the dropper pipet. Fill the straw with slurry to about three centimeters from the top of the straw. Add water to the top of the straw column using a wash bottle. Observe the water draining through the slurry. Using a microtip dropper, add a drop of green food color to the top of the silica gel in the column. Let the color go into the gel. Add water to the top of the column, continuing until the separation is complete. Collect the effluent from each band of color in a separate beaker.

B. Acid Silica Gel Column

 
1. Pack column using Procedure A, steps 1 though 9 (above).
2. Rinse with 0.1M acetic acid (CH₃COOH).
3. Rinse with one volume of water.
4. Using a microtip dropper, add one drop of beet juice.
5. Add water to the top of the column, continuing until the separation is complete.
6. Collect the effluent from each band of color in a separate beaker.

C. Amberchrom Resin Column

 
1. Wet a small piece of glass wool and plug the bottom of the straw.
2. Clamp the straw vertically to the ring stand.
3. Place a 100-ml beaker under the straw.
4. Pack the straw column with AmberchromÆ resin.
5. Rinse the column with two volume of 30% ethanol.
6. Add one drop of Durkee red food color to the top of the resin.
7. Add 30% ethanol until the color separation is complete.

Exercise:

AmberchromÆ Column Experiment

1. Pack column using Procedure C, steps 1 through 4 (above).
2. Rinse the column with two volumes of 10% ethanol.
3. Add a drop of Durkee red food color.
4. Continue the separation.
   a. What happens to the color bands?
   b. What is the effect of solvent concentration on the separation?
5. Repeat the procedure to separate washable black ink using 10% ethanol as the solvent.
   a. Is the separation of color complete?
   b. If color remains on the column, what should be done to remove the color?

Questions:

1. What is chromatography?
2. What two factors are important for good separation of mixtures?
3. Name one polar solvent and one nonpolar solvent.
4. Name two adsorbent materials used in chromatography.
5. What are the advantages of liquid chromatography techniques?
6. Investigate these other types of chromatography:
     Paper chromatography
     Thin layer chromatography
     Gas chromatography
     How do these types differ from liquid chromatography?
7. Investigate commercial uses of chromatography.

References:

1. Krstulovic, A.M., Reversed-Phase High-Performance Liquid Chromatography, John Wiley and Sons, New York, 1982.
2. Thompson, S., Chemtrek, Allyn and Bacon, Boston, 1990.