Accelerated Microparticulate Bed Liauid Chromatoaraahy Elmar V. Piel, Flo1
o i paper C chromatograms and of thin layer chromatograms has enabled considerable ENTRIFUGAL DEVELOPMENT
increase in speed for paper and thin Vacuum layer chromatography ( I ) . and pressure differentialsapproaching an atmospherehave,in thepast,heenused to accelerate column chromat,ography (2). Xevertheless, in order to maintain reasonahle development times, present column chromatography practice involves use of bed particles of at least about 25 microns average diameter. Two different methods for forcing liquids through microparticulate beds have been found to increase speed for column chromatography, and to produce advantages in resolution, adsorbent capacity, and handling ability for micro or macro chromatographic problems. Centrifugal forces which are higher than those used for uauer or thin laver chromatographic methods are used in one technique; the other technique involves the use of high liquid pressure differentials. Best results are achieved with beds in which particles are not greater than ahout 1.5 microns in size and in which there is uniform particle size. The centrifugal fields required vary widely in centrifugal microparticulate bed column chromatography, depending upon specific particle size and upon . . .. . . . how firmly compacted the beds become. Preferably force fields of a t least 800 times gravity are used. Pressure differentials n'ecessary to drive the devel'.ng liquid through beds in high pres1.1 .L..-~.L.~~ 'e micropal WUAW ueu crlrumawg,hy generally amount to at least 250 .i.g. per centimeter of length of chromatographic bed. The two methods are not equivalent in all respects. In general, lower pressure and more uniform conditions of pressure and bed compaction characterize the centrifugal method. Thus, the centrifugal method is adaptable to a greater variety of adsorbents and developing liquids than the high pressure differential technique. In the latter type, compaction of the bed is greater a t the bed exit than at its entrance. Similarly, liquid pressures at the entrance are high, while at the exit they are low. Pressure affects R, value, so that there can he appreciable R, changes as the liquid progresses down the column. In addition, high pressure causes many developing liquids to freeze at temperatures higher than normal operating temperature. Thus, it may not he possible to use some solvents for a given separation with this
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ANALYTICAL CHEMISTRY
tecnnique. Un the other hand, high pressure apparatus is more simple than centrifugal apparatus; particularly, consider the ease of collecting and monitoring chromatographic eluates from micro chromatographic apparatus. Centrifugal force followed by pressure provides an advantageous means For rapidly establishing a more uniformly compacted bed. CENTRIFUGAL MICROPARTICULATE BED CHROMATOGRAPHY
Apparatus requirements for centrifugal microparticulate bed chromatography are very simple. A high speed centrifuge equipped with a horizontal swinging head (such as a clinical centrifuge) or with a fixed horizontal head (such as in a hematocrit) is suitable. Simple capillary tubes with tapered tips may he used in a hematocrit. More practically, tubes with a relatively large solvent reservoir may he used in a swinging-horizontal head equipped centrifuge. Figure 1 shows a micro column. It wm fmhioned by blowing a
Figure 1. Separation of Butter Yellow, Sudan Red, and Indophenol Blue on alumina
bubble to serve as solvent reservoir in the thickened wall of Zmm. i.d. borosilicate glass tubing of 1-mm. wall thickness. The column is supported hy having the shoulder of the solvent reservoir bear on a short section of polyethylene tubing which rests on the open end of a heavy walled test tube of 7-mm. 0.d. The latter, which collects eluate and supports the column, is in turn held within the centrifuge bucket by cork supports. The fine particles of the chromatographic bed are contained within the tube by a layer of raw diatomaceous earth and a layer of 80-mesh crushed firebrick which becomes lodged in the tapered exit of t,he column. Larger size columns were similarly constructed from regular weight 10- and 14-mm. i.d. borosilicate glass tubes. The shoulder of the solvent reservoir was supported by a polyethylene seat cut from the neck of a detergent bottle and inserted in an aluminum sheet punched for the plastic insert and cupped to fit over the top of the centrifuge bucket. In these tubes the ehromatographic bed IV&$ confined in the tube at its exit by a layer of raw diatomaceous earth and a thin layer of glass wool covering a polyethylene or wooden plug. The latter fitted relatively loosrly into the tube and was supported by the bottom of t,he centrifuge bucket during centrifugation. Thus, these thin-wallrd and large tubes easily withstand centrifugal fields of at least 1600 times gravity when doubly supported. Eluate was collected directly in the centrifuge bucket with this arrangement. The separation shown in F i y r e 1 was achieved in 4 minutes with a centrifugal field varying from ahout 900 to 1800 times gravity on a mixture of the test dyes ]
