University Southern of Mississippi Hattiesburg, 39401
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Conical chrornatoara~hv
Chromatography methods can he classified as adsorption or partition, liquid or gas eluent, and solid or liquid suhstrate. Another tvve of classification are the categories of ascending, descending, or hodzonal chromatography, Yet another classification not commonly thought of is the geometry involved, i.e., cylindrical or planar as in column and in paper or thin-layer chromatography. However, the geometrical types do not have to he limited to cylindrical or planar forms. This paper reports investigations into conical geometry and the advantages-disadvantages of conical geometry. Literature gives only two references to the effect of geometry on separations. One of the references2 is to paper chromatography where the paper is folded into a cone. The base of the cone is immersed in a liquid contained in a Petri dish with enclosure of the whole assembly. The other reference3 is to thin-layer conical separations. There is no reference to conical paper chromatography with the apex immersed in the eluent or to a study of the effects of a packed funnel. Both of these types of conical chromatography have been investigated in this study and are reported here. I agree with Zweig, Moore, and Skerma4 that chromatography should he performed with the equipment as simple as possible. For this reason, the experiments are devised to use common glassware such as might he found in any teaching and in any research laboratory. The experiments in paper chromatography were performed using a spotter (fountain pen), an eluent consisting of a mixture of hutanol, ethanol, and 2 M ammonia5, filter paper, inks, a paper clip, a ruler, and a heaker. The paper was folded into a cone and then folded such that it would rest either on the rim of the heaker or slip down the beaker with the apex on the bottom of the heaker (see Fig. 1). The effects of enclosure were tested by lowering a large inverted heaker over the assembly and resting the whole assembly on a piece of filter paper saturated with the eluent. The only experimental difference noted was in speed of development, rather than RF values. The fastest development was when a large, inverted heaker was lowered over the beaker holding the paper and eluent. The next fastest development is that of arrangement A in Figure 1. The development rate was also a function of the size of the heaker in the arrangement shown in part B in Figure 1. The smaller the beaker, and the more the filter paper projected ahove the rim of the beaker, the slower was the development rate. In every case the eluent was allowed to rise on the paper ahove the rim of the heaker. In no case were the R, values different, within experimental error. 'Present address: Chemistry Department, Clarkson College of Technology, Potsdam, New York 13676. ZOsawa, Y., Nature, 180,705 (1957). ZMusya, S., and Oehi, H., Kogoku No Ryoiki, 1956 19(12) 915; CA, 69R,R4336q. 'Zweia, G., Moore, R., and Skerms, J., Anal. Chem., 43, 349R (1970). JAbott, D., and Andrews, R., "An Introduction to Chromatography," Houghton-MifflinCo., Bostonp. 46, 1969. $Referencein Footnote 4, p. 59.
Figure 1.
A B Arrangement of paper canes in large and small beakers.
Typical Experimental RF Values
Red (Ship) Royal Blue (Parker) Blue (Skrip) Green 1Skrio)
0.08 0.18 0.09 0.37
0.25 0.33 0.19 0.45
0.54 0.52 0.39 0.66
0.64 0.74 0.73
I'ena were ured ru hold the m k and tu spot rhc paper S o currelatron between HS'S ior d~lfrrenr~ n kisi i n t m n r d
One difference between the A and B arrangements of Figure 1 is that in A, the paper flap is squeezed tight and presents a capillary structure to the eluent. The eluent races up the fold, pulling eluent up the sides of the cone close to the flap, effectively decreasing the area of the cone usable for separations. In method B, the flap can he left partially open. Method A , involves only a ml or so of eluent, whereas in B, the method involves filling the beaker about twothirds full. Occasionally the filter paper would fall over in method A, ruining the whole experiment. An experiment was performed in which the eluent was dripped on the apex of the cone (apex up). The eluent ran down the cone, ruining the experiment. Another experiment involved placing the cone inside a heaker, with the base down, with the beaker containing a few ml of the eluent. Tailing and crowding of the spots became extremely bad when the eluent reached the cone's apex. Filter paper of three sizes, 6, 9, and 11 em were used. As normal in chromatography, the further the eluent moved (the larger the filter paper) the longer the development time but the more reproducible were the RF'S and the separations were visibly better. The geometry of adding the ink to the paper was not important as long as the spot was not submerged in the eluent. The two geometries of spotting used were as a distinct spot or as a circle -2 cm in diameter when the cone was unfolded. Typical experimental RF values of the inks are given in the table. A "packed" funnel was made by plugging the stem of a 60" funnel with a piece of filter paper and adding a slurry of alumina, 80 to 200 mesh. The sample of malachite green and methylene bluea was added to the top of the packing and water used to elute the methylene blue. The pressure head was too low to cause a separation in less than a day. Suction applied to the bottom of the funnel Volume 50, Number 2, February 1973 / 133
Figure 2. Pressure system for ascending, conical chromatography. The pressure head is built up in the flask on the right by adding water (b) to the standpipe. The pressure forces the eluent (a) up through the packed
cone.
either was too weak to speed u p the elution or too strong and thus caused channeling. Ascending chromatography was tried using a vacuum flask as shown in Figure 2. The rate of development could
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Journal 01 Chemical Education
be increased to 5 to 10 min or faster, depending on the height of the pressure head. When the methylene blue reached the surface of the packing, it was removed with a pipet, the eluent changed to ethanol, and the malachite green was eluted. The only odd part of the experiment was adding the sample to the apex of the funnel by means of a long-needled syringe. An advantage of the ascending technique, besides the rapid development, was the change in hand width. The hand width visibly decreased as the band moved up the column. Thus, ascending, conical packed chromatography should aid in separating close hands by decreasing tailing and decreasing the band thickness. Another advantage of the ascending technique is the automatic closing of any channel that forms-unless the eluent flow is too great of course. In conclusion, the advantages of conical chromatography over other methods are simplicity of equipment for paper chromatography, a decrease in tailing effects for both paper and packed conical chromatography, automatic closing of channels in the packed funnel, and a decrease in the hand width.
