CHROMATOGRAPHY

By Dr. Stullken

Chromatography is a technique that is ordinarily used to separate a series of solutes mixed within a common solvent by utilization of one or more of the following phenomena: differential adsorption, liquid-liquid partition, or ion exchange. Generally the variations employed to achieve this end are column chromatography, paper chromatography and ion-exchange chromatography. In the first two types, it is not necessary that the solute molecules be charged in order to effect separation, whereas charged molecules are the only kind separable by ion-exchange chromatography. The name chromatography resulted from the fact that the first substances to be separated by this technique were colored, although such a property was not, a prerequisite to being separable by chromatography. This technique has been involved in some of the most outstanding work performed in the field of molecular biology.

Column Chromatography

The earliest version of chromatography is the column variety, in which a finely divided adsorbing material is carefully packed in a long glass tube. The solution containing the solute mixture is poured into the top of the column and allowed to "develop." Development is the name given to the separation process. As the different molecules come in contact with the packing material, each type of molecule will have a greater or lesser affinity for the adsorbent and will therefore soon show a tendency to migrate through the column at some characteristic rate. Those molecules that are more firmly bound to the adsorbent will ultimately move through the column more slowly than those molecules with a slight affinity for the adsorbent (or high affinity for the solvent). After the initial solution is poured into the column, solvent alone is continually added to maintain a constant rate of flow. When the combination of all factors involved reaches a sort of, equilibrium or balance, the overall effect becomes noticeable as a series of separate bands (fractions) moving toward the bottom of the column. If the fractions are colored, direct visualization is possible; if not, indirect means may be employed to accomplish the same thing. Measured volumes can be taken from the bottom of the column by a fraction collector, and each sample of effluent can be analyzed for its content (or possible content) of each of the solutes introduced originally. A band or fraction will tend to be located in one or two samples on the fraction collector.

Various substances are employed as adsorbents, i.e., charcoal, sugar, starch, cellulose and aluminum oxide. An often used example of column chromatography is the separation of plant pigments extracted from leaves with acetone and ether and poured through a column of sugar or starch. Four rather distinct fractions become visible with a good separation. One difficulty with this technique is the necessity for repeated practice before satisfactory results can be achieved. The most critical part of this procedure involves an even packing of the adsorbent to proper degree of firmness so that the separation will proceed smoothly.

Paper Chromatography

Paper chromatography is a much simpler technique and is more practical in our present situation. Both differential adsorption and liquid-liquid partition are operative in this version of the chromatographic process. Liquid-liquid partition refers to the fact that a substance (solute) may exhibit different degrees of solubility in two or more solvents.

A piece of filter paper is usually rolled into a tube and a small spot of the solvent-solute mixture is applied near the edge of one of the two free margins. Next the cylinder is placed in a jar saturated with the vapor of the second solvent (an organic one) with the "spot" side down. The bottom of the jar contains a small amount of the organic solvent, which begins to migrate up into the paper by capillary action as soon as contact is made. The lid is immediately replaced to make an air-tight seal, and development of the chromatogram proceeds until the solvent reaches the top of the paper. As the organic solvent passes over the sample spot, liquid-liquid partition comes into play. Of the several solutes dissolved in the sample spot (aqueous phase), some will dissolve more readily in the organic solvent (nonaqueous phase) than others, and these will begin to move up the paper as they are carried along by the nonaqueous phase. If the relative solubilities of A, B, and C (solutes in the nonaqueous phase are in the order C, A, B, then C will migrate the furthest up the paper and B will migrate the least. In this way separation of the three solutes will be achieved.

R_f Determination

Molecules may be characterized by measuring the distance traveled by the solvent and the distance traveled by a given fraction. If a ratio of such distances is computed, solute distance/solvent distance, a value known as the R_f value for that substance becomes known. If all conditions are kept carefully constant, this value is quite reproducible and therefore can be used to characterize a molecule, just as a molecule can be characterized by its boiling point, freezing point, molecular weight, or other similar criteria. It should be emphasized that the R_f will vary according to the solvent(s) employed, so these should always be stated when recording an R_f value. Also, other factors are involved, including temperature and nature of the paper used. It should also be pointed out that it is quite possible for two different substances to exhibit the same R_f value under closely controlled conditions, so it is not usable as an absolute method of identification.

Two-dimensional Paper Chromatography

Another variation of paper chromatography that adds considerable usefulness is the two-dimensional version. The sample is applied in one corner of a square piece of filter paper and development in a vertical fashion proceeds as discussed above. After drying, the paper is developed in, the horizontal plane by turning it 90E and using another nonaqueous solvent. In this way additional liquid-liquid partition separation is possible by virtue of adding a second nonaqueous solvent. The solubility characteristics of the various solutes will be different in this second system, and the end result is further separation that is not possible when using only one nonaqueous solvent.

Recovery and Quantitation of Fractions

It should also be noted that separation is not the only possible objective of paper chromatography. It is also possible to recover the separated fractions quantitatively by elution of each one separately. Usually two chromatograms are run simultaneously under identical conditions. One is used to locate the separated fractions by some suitable staining procedure. Which then makes it possible to locate the invisible (unstained) fractions on the second chromatogram. Staining usually renders a substance unusable if the material is desired in the pure, undenatured form. Once the fractions are located they can be separated by cutting out each section of filter paper. The separate fractions can then be eluted from the paper by appropriate solvents. Substances thus isolated are considered to be in a high state of purity and are often referred to as "chromatographically pure.@

EXTRACTION AND SEPARATION BY PAPER CHROMATOGRAPHY OF

PHOTOSYNTHETIC PIGMENTS FROM SPINACH LEAVES

Materials:

1. Frozen or fresh spinach, (about 1 package per class). This material should be oven or air dried before use.

2. Acetone

3. Acetone (5%), petroleum ether (95%) solution

4. Blender

5. 10 ml pipette

6. Buchner funnels, suction flasks and vacuum pump

7. Filter paper discs

8. Paper chromatography jar

9. Petri dish bottom (must be glass)

10. Spectrophotometer and cuvettes

11. Parafilm

12. Fine-tip paint brushes

13. Whatman #1 chromatography paper, 8" x 8" squares

14. Stapler

Procedure:

1. Prepare a soluble extract of photosynthetic pigments as follows:

a. Pour about 200 ml of acetone into the blender. (Remember that acetone is extremely flammable!)

b. Take about 2 lb. frozen and dried spinach leaves (or just dried, if fresh) and tear into small pieces, discarding the stems. Start the blender and toss in all of the small pieces, a few at a time, blend the mixture until the leaves are well homogenized.

2. Suction filter the homogenate using a Buchner funnel, filter paper disc, suction flask and vacuum pump.

3. Set up your chromatography apparatus as follows:

a. Take a Petri dish bottom and fill it about 2 full with the acetone-pet. ether solution. This is your solvent reservoir.

b. Quickly Invert, the chromatography jar over the solvent reservoir and let it stand at least 15 minutes to allow the air inside to become saturated with solvent.

c. Take a piece of chromatography paper, handling only the edges, and draw a straight line very lightly with a pencil 1 inch from the bottom edge of the paper all the way across. This will serve as a guide for pigment application.

4. Trace a straight line of pigment extract about 2 to 1 inch long parallel to your pencil line at four different spots along the line (one for each member of your group). Repeat the application of pigment to each line, 10-20 times. Allowing it to evaporate completely between each application.

5. Take the paper and roll it into a cylinder, so that the pigment line faces outward, making sure that, the edge nearest the pencil line is one of the free edges. Staple the two other edges together so that they do not quite touch.

6. After the atmosphere has been well saturated with acetone-ether in the chromatography jar, quickly insert your chromatography paper with applied pigments and allow the solvent to travel almost to the top of the filter paper before removal. Dry these chromatograms in a well-ventilated area, not in an oven.

7. Take your original sample of the photosynthetic pigments and prepare an absorption spectrum of it. Use acetone as your solvent, and blank. Since this substance evaporates rapidly, the cuvette should be sealed with Parafilm to prevent gradual changes: in concentration of the dissolved pigments. Begin by using 450 nm wavelength setting on your spectrophotometer and dilute your sample with acetone until an absorbance reading of a little less than 1.0 is obtained. Now begin a scan of the complete visible spectrum (750 nm-380 nm) using intervals of 15 nm.

8. Cut out the strips of the chromatogram showing the separate pigment bands. Make a drawing to represent the chromatogram and measure the distance from the line of origin and the leading front for each pigment. Then cut each pigment away from the others. Insert all 4 copies of each pigment into a spec tube 3/4 full of acetone. After the pigment has dissolved off the paper remove the paper. Then produce an Amax curve for each pigment. This is best done using a range of 400 nm - 700 nm at intervals of 20 nm.

9. How many fractions did you observe? Identify them and determine their R_f values. How does the absorption spectrum for each extract compare with the absorption spectrum for all the pigments?