Automatic Paper Cksrolmatography RALPH H. MULLER A N D DORIS L. CLEGG, New York University, New York, N . Y . The preferential elution of a mixture of pigments is conduoted in a restricted channel on ordinary filter paper. The progress of the elution is followed automatically as each fraotion crosses a patch of monochromatic light. The variations in transmittaney are detected by a photomultiplier tube and recorded by a pen-and-ink recording potentiometer. The separation of microgram quantities is readily attained.
HE use of paper as a chromatographic medium is very old; are summarized by Rheinboldt (7). Its use has been revived, however, and some of the most striking accomplishments in modern chromatography involve its use, notably in the work on partition chromatography by Martin and his co-workers
the optimum value depends upon the ease with whioh the cornponents of a given system are separated and i t is best judged by a preliminarv trial. The passam of successive comuonents throueh
(4). As part of %generalprogram on this technique, the authors have developed a method for the rapid and automatic examination of paper chromatograms during the ~ ~ O W S Bof development (5). Great convenience attaches t o the use of a confined eone or channel which speeds up the diffusion process and enables one to work with smaller quantities. A typical specimen is shown in Figure 1, in which filter paper is impregnated with a paraffin barrier by a heated die. The process is essentially the one described by Ysgoda (8)for confined spot tests. The mixture to be separated is deposited in a s m l l drop a t A from an appropriate solution and rapidly dried in place by a micro air jet. When the matrix is mounted vertically and a suitable eluent is delivered at point B, it spreads rapidly throughout the circular &reaof this eone and soon begins to rise in the restricted rectangular channel. When the liquid reaches point A , i t begins to displace the various components of the sample mixture. At some point, C, beyond the site of the sample, optical examination of the displaced substances can begin. Some judgment and discretion me required in choosing the distance betureen A and C;
EQUIPMENT
The preparation of the confined barriers is a simple matter. An embossing tool was constructed na follows:
A length of wire of rectangular crom section (bus bar) was formed into the contour shown in Figure 1. After forming. it w m
bra& which had&
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heen tinned. For convenience in attaohine B
heatmn"the entire asssimblv with a Bunsen burn&. the temwri-
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technique is to machine the die out of a solid bl&k of brass, by routing out the chnnels on a milling machine, followed hy B surface cut to finish the top. I n preparing the matrices, a piece of filter paper was phced upon a flat layer of filter papers. A piece of paraffined paper was placed on top and the heated die wna pressed on the stack. Most commercial forms of wax paper were unsuitable, and it was found necessary to use very fine tissue which had been dipped in melted paraffin and hung up to drain and cool. The die was kept in the inner pot (dry) of a double hailer. If a large number of matrices
arrangement for bringin"g the die drawn upon thk paper, and a
other ikpregnants, iniludi'ng low melting point allovs, &ght be required. The general arrangement of the equipment is shown in Figure 2.
A concentrated filament lamp, operated from a constant~~~~
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tive was mounGd which focused ~~~
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tin" ikaae of the en&nce"slit
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technique to the ultraviolet. Otherwise a much cheaper instrument is possible; indeed, very useful results can be achieved with selectivefilters. The filter paper matrix was supported a t one edge between two microscope slides which in turn were mounted on the vertical oedestal of 8 Reichert mechanical stage. The latter was capable
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Figure 1. Setup for Analysisof Typical Specimen
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by an externally blackened glass rod to the en~losurehousing the 1123
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ANALYTICAL CHEMISTRY
photomultiplier tube. This simple light-collecting means could be improved upon by an elaborate optical system, but only if nonreflecting optical elements were used to minimize reflection losses. The cathode, dynodes, and anode of the photomultiplier tube n-ere fed from a stabilized 1000-volt direct current electronic pon-er supply. A tn-o-stage inverse feedback, battery-operated amplifier coupled the photomultiplier tube with a Br0n.n Electronik recorder. This amplifier was operated a t low gain and served primarily as an impedance-matching stage for the recorder. The microcapillary feed for the eluting agent consisted of a fluid rescrvoir, a stopcock, and bent capillary tube, all mounted on a stand housing vertical and horizontal feedscrew adjustments. By means of these adjustments, the capillary tip could be brought in gentle contact with the filter paper a t position B (Figure 1). At the desired time, the stopcock could be turned, admitting the eluting liquid to the paper strip. From this point on, recording of the phenomenon cvas automatic. TYPICAL RESULTS
The typical results shown in Figure 3 illustrate three cases, the last of which presents an alternative means of separation. For ease of reproduction, these curves have been traced from the original chart records. Pictures of the final samples are not shown here, because ordinary black and white photographs do not appear as striking and definite as the original colored patterns. For the authors’ records, each sample has been mounted on black cardboard and filed with the corresponding recorder chart. Greater permanence can be secured by dipping the entire sample in melted paraffin before mounting it. Presumably, any important case could be incorporated in a thermoplastic “sandwich,” similar to the practice of prepsling identification cards, etc. In some cases, notably C, examining the final specimen in ultraviolet light hm been useful. The well separated eosin fraction is strongly fluorescent.
A shows the pattern obtained with approximately 1 microgram each of aniline orange and malachite green. From a methanol solution of the mixture, one small drop was deposited a t position A (Figure 1). The eluting liquid was methanol containing 5% water. In these records, elapsed time is indicated from right to left. Within 18 seconds after the eluting fluid was admitted, a wave of the fluid had advanced to the optical scanning zone, and the transmittancy increased sharply. An increase in transmittancy arises from the fact that the clear solvent increases the translucency of the paper. Shortly thereafter, the transmittancy decreased rapidly as the malachite green began to diffuse into the scanning zone. It reached a constant value within a few seconds and maintained this value for about 10 seconds. Thereafter a second sharp decrease set in, a t which the more strongly light-absorbing aniline orange began to appear. This attained a steady level which persisted for 14 seconds, after which a third unidentified fraction appeared. The aniline orange was known to be impure, as shown later by conventional chromatographic examination, and this record establishes the fact and also that it follow, rather than leads, the aniline orange portion. In this and the remaining examples, the monochromator was set a t 550 mp, Beyond the last decrease in transmittancy, the curve rises sloxly, and eventually rises to its original value as every trace of dye is wmhed out. B illustrates a somewhat less clear-cut case in the separation of malachite green and Congo red. The second plateau, corresponding to Congo red, is not as well defined, although visual examination of the paper chromatogram would lead one to believe that the separation was just as effective as in the previous c a ~ e . Cismarkedly different. Here a much more dilute solution was employed, a t higfier recorder sensitivity, and the light scanning zone wa4 located more remotely from the sample site. The methylene blue fraction appeared rapidly, followed by a relatively clear zone which increased the transmittancy almost to ita original value. The eosin fraction then appeared. The process required about four times as long as the preceding cases, as indicated by the time axis. In cases of this sort where the degree of sepa-
ration is fairly complete, it has been found possible to interrupt the process, remove the paper, and after drying it, cut it into two pieces and dissolve away the separate portions. This is not original, but has been employed with success in other investigations ( 1 , 8 ) . The automatically recorded chromatogram gves advance information about the completeness of separation, and it can be verified by spectrophotometric examination of the individual solutions. The choice of a single wave length is a disadvantage because this cannot be the optimum choice for each constituent. However, because it is monochromatic light, the exact value for the extinction coefficient can be calculated from measurements on large amounts of individual constituents. In some caes, it is actually an advantage, especially if one is examining a material for traces of impurities. If a wave length can be found for which the impurity shows high absorption and the main constituent loa absorption, the conditions are most favorable for the detection of very small contamination. These measurements are semiquantitative and there is good reason to believe that the advantage of the technique lies not merely in its quantitative possibilities, but in the information of other sorts which it can supply. The principal advantage residep in the enormous economy of material. \J7ith very little material it is possible to run dozens of tests with various eluting mixtures in order to establish the best eluent. Separation can then be effected in larger amount and the mixture analyzed by more conventional optical methods.
HONOCBRO3dTOR
Figure 2.
1
Diagram of Equipment
Figure 3.
A. B. C.
C
Typical Results
Aniline orange and malachite green Conso red and malachite green Eoain and methylene blue
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V O L U M E 21, N O . 9, S E P T E M B E R 1 9 4 9 Some advantage would accrue from the use of a logarithmic amplifier ( 6 ) , because the recorded wave heights would then be proportional to the concentration for those substances obeying Beer’s law. A modified form of apparatus would permit the measurement of light reflected from the sample to be recorded and the possibility of using the fluorescence of the material, although of restricted application, cannot be ignored any more than i t is in conventional chromatography. The extension to the ultraviolet, by either transmittance or reflectance, is even more important because then one is not restricted to colored substances. These, and other possibilities, are under investigation and will be reported later. The advantages of paper chromatography are not restricted to optical methods. I t has been found in this laboratory that conductance measurements are very useful in folloa~ingthe diffusion of ionic species through a paper strip and such measurements are even more easily recorded automatically. Although the use of paper as a chromatographic medium may be considered to have very limited use compared with other adsorbents, the possibility of impregnating i t lyith appropriate soluble or difficultly soluble reagents offers many opportunities to extend its usefulness (3). This instrument, and the several modifications that are under construction, are intended primarily for orientation studies on paper chromatography. Little or nothing lias been said here
about the type aiid tlilckness of the paper, its puiity a d structure. The instrument has proved very useful in evaluating these factors, the choice of eluting liquids, and almost every factor involved in a proposed separation. By a minor modification, in which the entire strip could be moved with uniform velocity across the scanning zone, one could obtain a conventional “densitome‘ter” record of a finished chromatogram. Whatex er advantages this mlght possess for quantitative work, it aould not provide as complete a picture of the actual dynamics of the process as the present arrangement. LITERATURE CITED
Consden, Gordon, Martin, and Synge, Biochem. J., 41, 690 (1947). Ibid.,41, 596 (1947). Flood, 2. anal. Chem., 120,327 (1940). Martin, Consden, and Gordon, Biochem. J . , 38, 224 (1944). hltlller. R. H., and Clegg, D. L.,ANAL.CHEM.,21, 192 (1949). Muller, R. H., and Kinney, G. F., J . Optical SOC.Am., 25, 342 (1936). Rheinboldt, H., in J. Houben’s “Die Methoden der organischen Chemie,” 3rd ed., 1‘01. I, p. 291, Leipzig. Gerge Thieme, 1926. Yagoda, H., IXD.EXQ.CHEM.,ANAL.ED., 9, 79 (1937). RECEIVEDDecember 29, 1948. A portion of 8 thesis submitted t o the Graduate School of New York University by Doris L. Clegg, in partial fulfillment of t h r requirrmrnts for the degree of doctor of philosophy.
Filter Paper Chromatography of Penicillin Broths M. L. KARNOVSKY AND RI. J. JOHNSON University of Wisconsin, Madison 6 , Wis. The c o n d i t i o n s for satisfactory paper-chromatographic separation of penicillins h a v e been e x a m i n e d critically, and a s y s t e m of o p e r a t i o n giving good r e s o l u t i o n is described. T w o assay methods a r e indicated, one rapid a n d of l i m i t e d applicat i o n based on m a x i m u m d i a m e t e r s of i n h i b i t i o n zones, a n d the o t h e r less s i m p l e but of greater accuracy and applicability. I n the l a t t e r the paper tape i s cut, a f t e r d e v e l o p m e n t , into u n i f o r m squares, and the penicillin c o n t e n t of e a c h is determined. The t y p e s of penicillin produced b y Penicillium rhrysopenum Q176 on a s y n t h e t i c m e d i u m , and t h e i r proportions. are discussed.
T
111.: unalysis of mixtures of penicillins by paper chromatography was introduced by Goodall and Levi (,2, S), who dewribed an approximate determination of the different penicillins i n a mixture. A qualitative modification of this procedure, more rapid but possessing somewhat lower resolving power, has been reported by n’insten and Spark ( 7 ) . In the present work a number of factors involved in the paper-chromatographic separation of the penicillins have been studied, together with aspects of the assay of the different components. Procedures are outlined which, it is helieved, result in improved resolution, reproducihilitv, and accuracy. GENERAL PROCEDURE
A filter paper strip, impregnated with buffer solution aud then
dried, is suspended vertically over the lip of a trough which is later filled with ether. About 0.002 to 0.004 nil. of broth or other aqueous sample, containing 1 to 2 units of penicillin (optimal figures), is spotted at a point on the tape, below the lip, on the outside of the trough. Water-saturated ether, progressing down the tape, is the mobile phase. The stationary phase is water absorbed by the impregnated paper. The entire operation is carried out in a sealed ciiamber. The distance moved by a given penicillin is a function of the pH of the buffer on the tape, the water content of the system, the distribution coefficient of the penicillin component, and tlie volunie of the ether allowed to flow. After development, the positions and concentrations of the individual compoueuts are determined either by measurement of the zones of iiihihitio~iproduced when the entire strip is laid on a
large agar plate inoculated with Bocillus subtilis spores, or by cutting the strip into small uniform squares and measuring the circular inhibition zones produced on B. subtilis plates by the individual squares-Le., the equivalent of a filter-disk assay. Figure 1 illustrates results obtained hy the use of both these methods, and the general chromatographic procedure outlined above. It may ?.JP seen {,hat: The major penicillin coniponent,s are well separated. Plating out individual squares gives somewhat better reuolution, for there is 110 loss of resolution due to diffusion of one component into another-which does occur on plating the ent,ire t,ape. Rleasurement of such zones as that of the I< types is not easy when the entire strip is plated out. In addition, Some doubt was entertained as t,o the validity of the maximum diameter of a zone as a true criterion of the amount of penicillin present in that zone, irrespective of the shape. (This is the method of assay used by Goodall and Levi, 2 . ) In the “squares” method, a convenient measure of tlie amount of each penicillin is given by the a.rea under the relevant portior~of the curve.
A detailed desciiption of the chromatograpliic and analytical procedures of the method finally adopted is given below, as well aa several factors investigated before establishing this final method. The general procedures adopted during the examination of these factors were t.hose of the esperimental part, and only the particular conditions under invactigation in each section were varied.
