THIN-LAYER the separation is accomplished on a thin layer of adsorbent material wetted with solvent. The technique is quite simple. The specimen is applied as a spot to the edge of the layer. That edge is then wet with the solvent, and the components of the mixture are separated as the solvent carries them across the surface.

The technique is within the reach of most amateurs in terms of cost, but it has a number of limitations. Roger Baker, Jr. (Box 7854, University Station, Austin, Tex. 78712), has therefore developed a new form of the technique, which he calls ultrathin-layer chromatography. The thinness of the layer allows the separation procedure to be reduced in scale so that smaller samples can be separated. Baker describes the technique and its uses as follows.

"The thin layers most commonly used in thin-layer chromatography are applied to a substrate such as glass in the form of a slurry of adsorbent and liquid (usually water) in much the same way that butter is spread on bread. For mechanical reasons, such as minute irregularities in the substrate, it is difficult to apply uniform layers less than about millimeter thick by spreading a slurry.

"The method of making thin layer described here is based on an old technique for dusting resin powder onto cop per plates for photomechanical printing The basis of the procedure is to shake o agitate a powder of the adsorbent material, a silica gel, inside a box so that some of the powder is left suspended in the air inside the box [see illustration at right]. A certain period of time is allowed for the larger particles of the powder to settle to the bottom of the box. Then the substrates are introduced through a previously sealed door near the bottom of the box. An additional period of time is allowed for the finest dust particles to settle uniformly onto the substrates, which are then removed. The dusted substrate is chilled so that a film of water condenses on the surface, drawing the particles into close contact with the substrate and causing the layer adhere when it is dry.

"The technique was developed with microscope slides (75 by 25 millimeter in size) as substrates. After a slide is coated the adsorbent layer of silica gel is wiped from the edges, leaving a central strip. This strip can be scribed into narrower strips, so that several dozen individual samples can be resolved on one slide simultaneously.

"After the samples are applied to the adsorbent layer near one end the slid is placed adsorbent side down over shallow cavity covered on the bottom with blotting paper that has been saturated with solvent. Solvent vapor is induced to condense as a liquid at the en of the adsorbent near the sample b cooling the top surface of the glass slightly. The chromatogram is developed as the solvent is drawn by capillary force toward the dry end.

"A little-known variation of thin-layer chromatography, called vapor-impregnation-gradient chromatography, is in some ways similar to the solvent gradient elution employed in column chromatography. This gradient effect is easily achieved with my apparatus. Th gradient technique tends to compress the sample components into narrow bands as development proceeds, so the longitudinal concentration of the sample before it is developed is not necessary.

"The gradient technique requires that the developing chamber be lined with two sections of blotting paper soaked with different solvents. The two sections should not touch. At one end of the chamber, below the sample, install a short double-layer section of blotting paper soaked with a relatively strong polar solvent such as acetone. The rest of the length of the chamber is lined on the bottom with a longer strip of blotting paper soaked with a relatively weak nonpolar solvent such as naphtha (lighter fluid). Various solvent combinations are satisfactory, but they should be miscible in all proportions and chosen so that the components to be resolved migrate rapidly in the first solvent and slowly in the second.

"The sample is carried down the length of the chamber by the stronger solvent, which passes over the area of the chamber lined with the blotting paper soaked with the weaker solvent. An exchange of solvent vapors occurs, causing the migration to slow with distance. Certain components of the sample are able to migrate farther than others under these conditions, so that the components are separated into bands along the length of the adsorbent strip. In addition to the sharp separations yielded by the gradient technique a rather wide variety of substances can be separated with a simple solvent combination such as acetone and naphtha. Thus the search for solvents that yield satisfactory resolution is simplified.

"Whether the sample is resolved in the ordinary way with a single solvent or with a gradient, it is useful to apply the warm zone to the far end of the adsorbent strip in order to continuously remove the solvent by evaporation as it reaches the end of the chamber. In this way the development can be continued as long as it is wished, so that even the more slowly moving components will be moved far enough to be well separated.

"After the sample has been resolved chromatographically it is possible to concentrate the bands of components sideways with a procedure resembling the one used in longitudinal concentration. Hence the components are deposited along a line running the length of the adsorbent strip. The procedure is called lateral concentration; it yields a greatly increased sensitivity of detection at the expense of a loss in resolution.

"An interesting method of detecting colorless materials that are not soluble in water is to stain them with a dye as they remain adsorbed on the silica gel. After the silica gel has been treated with a strong dye solution the excess dye is washed off, leaving the separated components visible as more highly stained areas on a light background. The method is sensitive, nondestructive and fairly general. It also gives a permanent record of the position and the relative amounts of sample components.

"The details of construction of the dusting box can be varied considerably without much affecting the results. I did my first experiments with a tin can. The setting periods involved should be increased in proportion to the height of the box.

"Satisfactory results can be obtained with a plywood box about 50 centimeters in height. The amount of silica-gel dust retained in suspension in the air, and accordingly the thickness of the layer produced during one coating operation, depend on factors such as the height of the box and the particle size of the powder. About a gram of adsorbent per 10 square centimeters of settling area is a suitable amount. The powder can be agitated by inverting the box end over end about a dozen times. The box should be tapped immediately after agitation to knock down any loose particles.

"After the initial settling period the cleaned microscope slides are introduced into the box on an aluminum plate. My plate holds 12 slides. The opening through which they are introduced should be sealed during agitation with a strip of foam rubber on a hinged door. Some kind of standoff, such as nails driven through the bottom of the box, is needed to support the aluminum plate above the bulk of the powder. With a dusting box 50 centimeters high, suitable results are obtained if the dust is allowed to settle for about four minutes before the substrates are introduced. The substrates should then remain inside the box for about 10 minutes to collect the adsorbent dust.

"The best procedure for dusting on the adsorbent layers has not been determined with certainty. On the basis of work that has been done with pure silica gel prepared for thin-layer chromatography, however, it seems best to apply the desired layer over several stages. Presumably other finely powdered adsorbents could be applied in much the same way.

"After an initial layer of adsorbent dusted onto the slides the aluminum plate on which they rest is chilled by placing it on a layer of crushed ice. The temperature of the glass is thereby lowered below the dew point of the air, causing a film of water to condense on the adsorbent layer. When the layer is wet and shiny, the excess water is wiped off the sides and bottom of the plate and the plate is dusted and wet by condensation once or twice more to thicken the final layer so that it is free of pinholes.

"The greatest uniformity is achieved by shifting the positions of the substrates between dusting steps. It appears that the layers should have a certain minimum thickness in relation to the size of the particles in order to minimize the effect of point-to-point variations in the capillary channels between the particles. The silica-gel layers that appeared to be the most useful far chromatographic purposes were from 15 to 50 micrometers thick. The thickness was measured with a micrometer both before and after wiping the adsorbent from a portion of the glass. A densitometer can be employed to compare the relative thicknesses of several layers.

"Since the slides are covered with adsorbent over their entire top surface at the time they are prepared, the adsorbent is wiped off each border for about five millimeters inward, leaving a central strip of adsorbent measuring about 65 by 15 millimeters. That can be done by sliding the end of a matchstick along the border, using your thumbnail as a guide. I made a tool for scribing lines about three millimeters apart along the length of the adsorbent strip by soldering a row of ordinary pins to a brass strip with the points extending like the teeth of a comb. A pin at the edge extends a little farther so that it rides along the edge of the slide and guides the rest of the pins. Any excess dust is blown off. The slides are stored in a microscopist's slide holder.

"The developing chamber can be machined from a solid metal such as aluminum. My chamber was constructed from two pieces of aluminum an eighth of an inch thick measuring 79 by 28 millimeters. A rectangular hole 70 by 18 millimeters was cut in the center of one of them with a jeweler's saw. The two pieces were cleaned well and cemented in close contact with epoxy glue to form the chamber [see illustration at left]. The top surface of the chamber, on which the slide rests, was ground flat with fine abrasive paper and finished by rubbing it against a flat piece of glass covered with a paste of kitchen cleanser and water.

"The sample solution should be applied to the adsorbent layer about 15 millimeters from one end. I make the application with a fine glass capillary drawn over a flame from larger tubing and having a square-cut or broken end. It can be difficult to apply the sample to the adsorbent layer from such a handheld capillary without disturbing the layer, particularly when the sample is to be applied to the narrow scribed strips. A small plywood jig makes the job easy. The jig consists of a microscope slide cemented to a piece of plywood [see illustration at right]. Another slide prepared with adsorbent can be made to adhere to the top of this first one with a small drop of naphtha.

"A small plywood bridge is made to straddle the slides and extend upward at a right angle to the surfaces. A glass or metal tube is fastened to the bridge with a rubber band. The tube acts as a guide for the capillary pipette, which is slid through until it touches the adsorbent surface at the desired place. By holding the jig so that the slide is nearly vertical and the capillary nearly horizontal and then tilting the two slowly, you can delicately adjust the height of the liquid column in the capillary, and therefore the rate of flow of the sample into the adsorbent, to match the rate of evaporation of the solvent from the sample spot.

"The glass is cooled to initiate condensation of the solvent by placing the adsorbent strip face down on the developing chamber and covering the glass above the end of the adsorbent strip (just beyond the sample spot) with a small, flat piece of aluminum about 15 by 10 millimeters. This piece of aluminum is cooled slightly by covering it with a strip of filter paper about a centimeter wide that extends over to a bottle cap filled with water alongside the chamber. The strip acts as a wick, drawing fresh water from the bottle cap and cooling the aluminum by evaporation.

"The time required for development will vary according to how rapidly the components migrate, how quickly the solvent is condensed and whether or not the experimenter is working with the gradient technique. It may be sufficient to simply let the solvent travel down the length of the strip, which might take 15 minutes. Often it is desirable to apply the warmth at the end of the strip, so that the solvent is continuously removed as it reaches the end and development is continued for a longer time. Since the migration rate of the components is restrained by the conditions of gradient development, an hour or more is often necessary for this type of development.

"It is convenient to mount the warmed aluminum strip at the end of a hinge so that it is free to swing down and rest gently on the glass. The strip is about 70 millimeters long, 20 millimeters wide and 1/16-inch thick. It carries six.5-watt, 10-ohm resistors attached with silicone rubber, parallel to one another and evenly spaced [see illustration above left]. They are wired together in three groups of two. The warmth of the strip is controlled by wiring all three of the groups in series with a one-ampere, 50-ohm rheostat (Ohmite). Everything is then connected to the low-voltage output a 6.3-volt filament transformer (Radio Shack).

"My method of detecting the location of colorless components of the sample is to stain them with a water-soluble dye such as rhodamine B. The stain can be applied by dipping the slide into a strong water solution of dye. The stain is allowed to dry on the slide. Then the slide is rinsed with a dilute solution of acetic acid in water, which will remove most of the dye from the background areas of silica gel and cause the areas that have adsorbed material from the sample to appear darker.

"Ordinary thin-film chromatography of black ink from a ball-point pen, with benzene as the solvent, is capable of resolving about 14 component pigments. The gradient technique, with acetone and naphtha as the solvents, can resolve the same ink into more than 20 pigments The same solvent gradient will I serve to separate acetone extracts of plant leaves into various yellow and gray pigments and as many as 10 distinct blue or green pigments."

Bibliography

THIN-LAYER CHROMATOGRAPHY. Kurt Randerath, translated by D. D. Libman. Academic Press, Inc., 1966.

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