Column Liquid Chromatography Experiment Adapted for Use in Secondary Schools

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Scot Henry Underwood
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1 Column Liquid Chromatography Experiment Adapted for Use in Secondary Schools Mark Langella WISTA The most modern and sophisticated methods of separating mixtures that the organic chemist has available all involve chromatography. Chromatography is the separation of a mixture of two or more different compounds by distribution between two phases, one of which is stationary and one of which is moving. All chromatography works on much the same principle as solvent extraction. The methods depend on differential solubilities (or absorptivities) of the substances to be separated relative to the two phases between which they are to be partitioned. In this lab, column chromatography, a solid-liquid method, is considered. Column chromatography is a technique based on both absorptivity and solubility. It is a solid-liquid phase-partitioning technique. The solid may be almost any material that does not dissolve in the associated liquid phase; those solids most commonly used are silica gel and alumina. These compounds are used in their powdered or finely ground (usually 200 to 400- mesh) forms. If powdered or finely ground silica (or silica gel) is added to a solution containing an organic compound, some of the organic compound will absorb onto to the fine particles of silica. Many kinds of intermolecular forces cause organic molecules to bind to silica. These forces vary in strength according to their type. Non-polar compounds bind to the silica using only Van der Waals forces. These are weak forces, and non-polar molecules do not bind strongly unless they have extremely high molecular weights. The most important interactions are those typical of polar organic compounds. Either these forces are of the dipole-dipole type, or they involve some direct interaction (coordination, hydrogen bonding, or salt formation). Similar interactions occur with Alumina. The strengths of such interactions vary in the approximate order: Salt formation > coordination > hydrogen bonding > dipole-dipole > Van der Waals Strength of interaction varies among compounds. The more polar the functional group on the molecule the stronger the bond to Silica. Polar solvents dissolve polar compounds more effectively than non-polar solvents; nonpolar compounds are dissolved best by non-polar solvents. Thus, the extent to which any given solvent can wash an adsorbed compound from silica depends almost directly on the solvent's relative polarity. If any adsorbed material, a kind of distribution equilibrium can be envisioned between the adsorbent material and the solvent. The distribution equilibrium is dynamic with molecules constantly adsorbing from the solution and desorbing into it. The average number of molecules remaining adsorbed on the particle at equilibrium depends both on the particular molecule involved and the dissolving power of the solvent with which the adsorbent must compete. Dynamic equilibrium mentioned above, and the variations in the extent to which different compounds adsorb on silica, underlie a versatile and ingenious method for separating mixtures of organic compounds. In this method, the mixture of compounds to be separated is introduced onto the top of a column pre-packed, or filled with fine silica particles (stationary solid phase). The adsorbent is then continuously washed by a flow of solvent (moving phase) passing through the column. 1

2 Initially, the components of the mixture adsorb onto the silica particles at the top of the column. The continuous flow of solvent through the column elutes, or washes, the solutes off the silica and sweeps them down the column. The solutes (or materials to be separated) are called eluates or elutants; and the solvents, eluents. As the solutes pass down the column to fresh silica, new equilibria are established between the adsorbent, the solutes, and the solvent. The constant equilibration means that different compounds will move down the column at differing rates depending on their relative affinity for the adsorbent on one hand and for the solvent on the other. Since the number of silica particles is large, the number of equilibrations between adsorbent and solvent that the solutes experience is enormous. As the components of the mixture are separated, they begin to form moving bands (or zones), each band containing a single component. If the column is long enough and the various other parameters (column diameter, adsorbent, solvent, and rate of flow) are correctly chosen, the bands separate from one another, leaving gaps of pure solvent in between. As each band (solvent and solute) passes out the bottom of the column, it can be collected completely before the next band arrives. If the parameters mentioned are poorly chosen, the various bands either overlap or coincide, therefore, you have a poor separation or no separation at all is the result. There are many different types of chromatography experiments. This column chromatography separation takes about 15 minutes to complete. You can use this activity as an introduction to column chromatography and intermolecular attractions. In this lab you will make two types of columns that can separate food dyes in different elution orders. The two dyes used are common dyes found in purple colored foods. The two types of columns constructed is Normal Phase and a Reverse phase columns. You will use a syringe to increase elution pressure and rate. In normal phase chromatography, the mobile phase is less polar than the column packing. Therefore, the non-polar components elute earlier than the more polar components, which are strongly retained by the column. 2

In reverse-phase chromatography, the mobile phase is more polar than the column packing. The more polar samples elute easier than the non-polar samples.

3 In reverse-phase chromatography, the mobile phase is more polar than the column packing. The more polar samples elute easier than the non-polar samples. Materials: Pre-Packed Column $ Silica $ Octyldecyl Silane A mixture of Blue #1 (brilliant Blue FCF) and Red # 3 Erythrosine Tert-butyl alcohol 50% v/v Vinegar/Methanol Solution Vacuum Pump Procedure: 1. Obtain Prepacked Silica and Octylecyl silane columns. 3

Turn on the vacuum pump and allow eluent

4 2. Place rubber tubing on end of packed column. 3. Place columns into suction flasks. 3. Connect Suction Flask to Vacuum pump. 4. Turn on the vacuum pump and allow eluent used for column to wash the packing in the column. The eluent for the octyldecyl silane packed column is the vinegar-methanol solution and the eluent for the 4

silica packed column is tert-butyl alcohol.

Place few drops of the solution of dye to be

Try to place dye into center of packing. 6.

Continue to add eluent until the separation

5 silica packed column is tert-butyl alcohol. Shut off vacuum pump. 5. Place few drops of the solution of dye to be separated onto packing layer in column. Try to place dye into center of packing. 6. Turn on pump so that the dye will be adsorbed by the packing 7. Add more eluent to top of column. 8. Turn on vacuum pump and allow eluent to enter column. Continue to add eluent until the separation of components is complete. 9. This is the result using the octyldecyl silane column. 20. Shut off vacuum pump and observe results. 5

21. Try the same procedure using the silica

6 21. Try the same procedure using the silica column but use the tert-butyl alcohol as the eluent. 22. Note the reverse order of elution. The final results are quite impressive. Literature Cited 1. Reynolds, R. C.; O'Dell, C. A., J. Chem. Ed. 1992, 69,

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Experiment 1: Thin Layer Chromatography

Experiment 1: Thin Layer Chromatography Part A: understanding R f values Part B: R f values & solvent polarity Part C: R f values & compound functionality Part D: identification of commercial food dye