US 3113103 A
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Dec. 3, 1963 J. A. LOWERY RAPID CENTRIFUGAL CHROMATOGRAPHY Filed Dec. 20, 1960 INVENTOR. vMAM/F5 JZ/Iffl 10/71 2) United States Patent 3,113,103 RAPID CENTRIFUGAL CHRQMATQGRAPHY I James Alfred lowery, New City, N.Y., assrguor to American Cyanamid Company, New York, N.Y., a corporation of Maine Filed Dec. 20, 196i), Ser. No. 77,097 1 Claim. (Cl. 219-198) This invention relates to an apparatus and method for the rapid centrifugal chromatographic development of small samples in which the sample is placed approxi mately centrally on a rapidly rotating chromatographic disk confined between plates with the developing liquid solvent being fed as a solid, continuous stream, approximately centrally to :the rotating disk, whereby the sample is centrifugally developed, in a much shorter time than would be possible with osmosis or gnavity as the sole driving forces to establish a concentration gradient.
Chromatography has become of increasing interest as a method for identifying and analyzing small quantities of material. In general, in chromatography a sample is placed at one end of an absorbent material used as a carrier and a developing fluid is used to move the sample along the carrier, with certain components being moved more napidly than others so that different materials can be, at least in part, identified by the rapidity with which they are moved along the chromatographic system. In general, the rates of motion are empirical and determined by experience. It is generally customary to establish the rates of movement along a chromatographic system using known standards, and having once established the rates of movement, these rates of movement apply to unknowns. Thus, a particular chromatographic system is calibrated, and after being calibrated becomes a very valuable research tool.
Whereas the carrier may be either a gas or a liquid, the present invention is concerned solely with liquid systems. Usually, the liquid, here called the solvent, moves along the column under the influence of gravity, or for instance where paper is used as the absorbing material, the solvent may move along the paper under the influence of both gravity and capillary attraction. Usually, gravity and capillarity result in a comparatively slow development.
It has now been found that the rate of development can be markedly accelerated by the use of centrifugal force as an additional gradient to increase the rate of migration of the components under consideration. In the past, the use of centrifugal force has been considered and discarded because the sample could not be evenly distributed, and more particularly the developing liquid was fed incrementally or by flooding, and as a result the motion of the developing fluid was not essentially uniform. For these reasons, centrifugal chromatographic development has not been recognized as a consistent and even type of developing.
In contradistinction thereto, it has now been found that if the sample is placed on a rapidly revolving disk and the developing liquid is added as an essentially axial continuous stream, the developing is uniform and is highly reproducible. Where the developing liquid is added drop-wise, each drop presents a separate problem as to concentration gradient, rates Otf diffusion, concentricity and directions of diffusion. Whereas single solid liquid stream is injected, all radii of a centrifugal disk receive the same treatment, and as a result highly repro ducible and consistent diffusion gradients are established.
Whereas a drum could be packed with an absorbent, and a comparatively large sample used, as for example, where a hollow drum would be packed with diatomaceous earth and both the absorbent sample and :the solvent or 3,1 13,103 Patented Dec. 3, 1963 developing liquid fed axially; it is preferred that for small scale operations a paper disk be used, which paper disk is clamped between non-corrosive plates so that the paper is uniformly md firmly held in position, the sample is placed essentially axially, and the solid stream of solvent causes the sample to move radially away from the center at reproducible and consistent relative speeds.
While it is preferred that the sample and the developing liquid both be fed exactly axially, it is found that over a reasonably wide range of concentrations, the rate of migration of the sample is a constant and, accordingly, if the sample is slightly off center or if the developing liquid solvent stream is slightly off center, still reproducible consistent results are obtained, because the solvent is being fed as a single solid stream. Discrete drops are not satisfactory because each drop then spreads differently, depending upon how it is impacted upon the paper disk. For that reason, with drop feeding the concentration gradient and rate of diffusion is not essentially and necessarily substantially reproducible.
Whereas the rate of diffusion varies with centrifugal speed, and whereas the thickness of the paper, the solvent system, and temperature all have an effect, a system can be calibrated using a standard paper, a standard rate of rotation, and standard conditions so that highly reproducible results are obtained, and when calibrated with knowns, unknowns can be easily fed into the system and their composition determined.
Without being limited to the details of a particular construction, here shown, but in fact being limited by the appended claim, one modification of the apparatus of the present invention is set forth in the accompanying drawings in which:
FIGURE 1 is a top view of the centrifugal chromatograph.
FIGURE 2 is a front View, in partial section, of the centrifugal chromatograph.
FlGURE 3 is an illustration of a typical development pattern as obtained from the centrifugal chromatognaph.
A circular plate 11 is mounted for rotation about a central axis perpendicular to the face of the plate on the end of the shaft of a motor 12 mounted on a motor support 13, which in turn is mounted on a frame 14.
Conveniently, but not necessarily, the circular plate is surrounded by a splatter shield 15. Conveniently, but not necessarily, the motor, the motor support, the frame and the splatter shield are parts of a conventional laboratory centrifuge. The splatter shield is not essential to the operating of the device except that it catches liquid which may be thrown from the rotating circular plate and prevents splashes about the room. Other vertical supports and vertically mounted motors may be used.
Removably mounted on the circular plate 11 is an annular circular plate 16. A small dowel l7 and a large dowel 18 are used between the plates, being fastened in the lower plate and extending into matching apertures in the upper plate, to insure that the removable annular circular plate is placed in exactly the same position each time. The two plates are held together by screws 19 Conveniently, but not necessarily, these screws are cap screws with an Allen wrench opening, that is, a hexagonal hole for a hexagonal wrench in the center, and fit into counterbores 25 in the annular circular plate. By having counter-bored holes, the screws do not stick up above the annular circular plate, and the risk of the operator catching his fingers in the screw-heads is minimized.
These screws are used to fasten the two annular circular plates together during rotation so that they do not move with respect to each other. The entire rotating assembly consisting of the circular plate, the annular circular plate, the do els and the screws is preferably dynamically balanced.
Above the plates is mounted a solvent feeder 21, in a support bracket 21A. At the lower end of the solvent feeder is a removable cap 22 which holds in position a feed disk :23. Inside of the solvent feeder is a filter disk 24. The filter disk is preferably of stainless steel or other resistant fritted material, and serves as a filter to prevent any particles which may inadvertently be introduced with the solvent from clogging the orifice in the feed disk. Above the filter disk is the solvent feeder cap 25 which has a removable feed cap 26 and a T-air connection 27.
The feed cap 26 is removed to permit the introduction of the solvent being used to develop a particular chromatograph. The feed cap is replaced, and air, under pressure, is introduced through the T-air connection to force the solvent 31 through the filter disk into the lower part of the solvent feeder. The feed disk 23 has a fine central orifice 28. This orifice is of such size that when the developing liquid is fed through the orifice, under the effect of gas pressure, a single solid stream is formed which does break into drops as it is fed to the chromatographic paper disk 29. It is important that the feed disk have a very small hole so that a small amount of solvent liquid, in the neighborhood of from 2 to 15 cubic centimeters, may be fed as the solid stream over a time of l to 4 minutes, more or less. An orifice having a diameter of from 0.001- to 0.003-inch generally gives good results.
The total amount of liquid fed is usually not greater than the paper disk will absorb. Thinner papers are usually preferred, hence for thinner papers, the amount of solvent used is less, and the relationship of gas pressure and orifice size is such that the desired quantity of liquid is fed in a solid stream in the time used for development. A time of less than a \minute gives poorer resolution. A time of over four minutes may be used, but slows down the rapidity of results.
Inasmuch as it is desirable to prevent contamination of the chromatogram with impurities or dissolved materials, it is desirable that the entire solvent feeder system be of a resistant material. Stainless steel gives good results. The plates themselves are conveniently of duralurnin, as durallumin is both corrosion-resistant and very light, which thus reduces the moment of inertia of the rotating mass.
Any suitable means may be used for drilling the hole in the feed disk. An electronic drill usually gives good results. These are devices in which a beam of electrons operating in a vacuum is used, rather than a steel drill, to form the hole; holes of smaller than 0.00 l-inch are comparatively readily formed by such electronic drills. The hole need not be circular, but a circular hole gives good. results, and is usually easier to drill so as to have a selected size than a hole of star shape or other configuration.
Operation In operation, the chromatographic paper disk 29 is placed between the circular plate and the annular circular plate. Any of the papers normally used for chromatographic work may he used. The paper disk is preferably selected so that it is centered by the holes for the screws 19. The paper is placed on the circular plate, and clamped bet-ween the annular circular plate and the circular plate by the screws '19. High pressure is not required, but it is desirable that the plates be held together with sutficient force so that they cannot shift during rotation. The access hole 30 in the annular circular plate is preferably coaxial with the axis of rotation of the plates. The plates are rotated by hand, and a pencil is used to describe a small circle in the center of the paper, thus circumscribing the center which is the point at which the sample is to be placed. The sample to be developed is then placed in the center of the paper, the orifice in the feed disk is aligned with the center of the paper, the developing liquid is placed in the solvent feeder, and the rotating assembly is brought up to speed. After the assembly is rotating at a substantially constant speed, air pressure is applied through the T-air connection 27 which l blows the liquid down through the orifice 2 8, and onto the chromatographic paper disk. The rate of feed is such that the access hole 30 is not flooded, but instead the paper absorbs the liquid as fast as it is fed.
While it is preferred that everything be completely axial, if the sample is not completely centered, the quantity of sample is not uniform with respect to the chromatographic paper disk, but the relative rate of migration of the various components is not adversely affected by an unsymmetrical feeding. Similarly, if the paper disk is not exactly centered, the center of rotation is established and the solvent firont may reach one portion of the edge of the disk ahead of the time it reaches another portion, but this is not serious. Even if the stream of liquid from the orifice in the feed disk is not exactly centered, the high rate of rotation of the plate assembly is such that the liquid is uniformly, radially fed and that there is no asymmetric component introduced. If drop-wise feed is used, each drop as it hits, sets up a different center of diffusion and these various centers of diffusion usually result in an uneven distribution of the liquid. By using a solid stream of liquid, an even feed is assured.
Example It is to be understood that any of the papers normally used for chromatographic work may be used in the present system. The only problem is getting a sheet of the paper of the proper size. Similarly, any of the solvent systems normally used in either ascending or descending paper chromatography are perfectly satisfactory. The time required for development is markedly reduced because of the high forces of centrifugal force operating which tend to increase the rate at which the solvent migrates along the paper.
It might be noted that the solvent is under a much higher feed gradient as it diffuses away from the center, as the force tending to feed the solvent along the paper is that defined by the centripetal equation, where the force is equal to the mass, multiplied by the radius, multiplied by the rate of rotation squared.
One particular system which gives good results, is an 8-inch circle of Schleicher and Schuell paper No. 470. Amino acids in water containing approximately 10 to 20 milligrams of acids per milliliter were placed in the center of the paper. About 20 lambda were used. A developing solvent used consisting of 50 parts by weight of butanol, 12 parts by weight of acetic acid, and 50 parts by weight of water. Six and one-half milliliters of developer was used under a pressure of 4 lbs. per square inch. From 2 /2 to 3 minutes were required to feed this amount of developing liquid through the orifice.
After the developing liquid was fed, the spinning plates stopped, the screws loosened, and the disk of paper removed. A ninhydrin solution was used to spray the paper after which it was heated for a few minutes at C. to develop the colors in accordance with conventional practice. For the individual amino acids, the Rf (ratio of flow, that is, the ratio of the distancewhich the particular material moves to the distance which the solvent front moves) was found to be for leucine 0.93, and 0.94. For proline it was found to be 0.61 and 0.64. For histidine it was found to be 0.42 and 0.45. When a mixture of these were developed together, the ratios were found to be 0.95, 0.62 and 0.47. These values are consistent within the accuracy of a paper chromatographic system.
FIGURE 3 shows the results obtained chromatographically separating thneedyes. A mixture of PD. and C. Red No. 4, RD. and C. Green No. l, and FD. and C. Yellow No. 8 was used as a sample on the same paper treated with a phosphate buifer to a pH of 3.0. With normal butyl alcohol, pre-equilibrated with water as a developing solvent, the respective R values were found to be 0.43, 0.62 and 0.99.
As shown in FIGURE 3, an elliptical solvent pattern is obtained, and with minor variations, rather than a circular one. This is due to the longitudinal orientation of the cellulose fibers resulting from the processing of the paper on the Fourdrinier wires. The long axis is known as the machine direction, and on most chromatographic papers, is indicated by an arrow. The R values were essentially the same whether measured along the long axis, the short axis, or other axis of the ellipse. Hence, the R; is a constant for a particular developing system, including the paper, the pH, the developing liquid, rotating speed, and size of paper. In a standardized system the R values can once be measured, and be used on unknowns after they have once been calibrated from known materials.
Other materials, such as the tetracyolines, may be separated, as may the steroids. For steroids, for example, it is convenient that colors be developed by a blue tetrazolium dye, which is a conventional method of treating steroids to show up on a blue spot.
The large number of different solvent systems, methods of developing papers for the purpose, etc. precludes a comprehensive survey of all of the systems that could be used. Suffice to say that the same solvent systems, papers, developing procedures, etc. as can be used for conventional ascending or descending paper chromatography gives good results with the centrifugal chromatograph.
It is found that in addition to the conventional systems, the extreme rapidity with which results can be obtained, that is, the fact that the complete chromatogram can be developed in less than five minutes, permits use of the present chromatograph to develop fugitive material. There are instances where a conventional paper chromatogram, which would take several hours to develop, would be completely unsatisfactory, because a mixture would attain a different equilibrium point in the time required for such development. In contradistinction tiereto, in view of the fact that a complete development can be made in about 3 minutes, and in view of the fact that it can be developed immediately thereafter, the entire present procedure, including the development, can occur in less than five minutes and, accordingly, materials which would not have a desired long term stability are easily developed with the device of the present invention.
The speed of the chromatograph can be from less than 500 to more than 1,000 revolutions per rninute. Conveniently, it is about half the speed used in an ordinary centrifuge for centrifuging. A higher rate of rotation permits a faster feed of a chromatographic liquid. A fixed speed should be used for a particular series to avoid the differential forces of acceleration acting on the solvent from changing the R values as will occur if an extremely low, or an extremely high rate of speed be used. It is desirable that the speed be the same for samples as for calibration with knowns, and if this precaution is observed the R values are perfectly satisfactory and consistent.
If a material has a particularly low R value, it is possible to run the centrifuge long enough so that the solvent 5 front comes off the paper completely, and is spun out onto the splatter shield. "For such a type of development,
this system needs to be more accurately calibrated as to time and quantities, as there is no solvent front with which to compare the front of the migrating material. Where a series of compounds are used which have a comparatively low R they can be spread out, and from the relative positions of one which is known, used as a calibration line, the positions of the others may be determined.
The intensity of the various lines gives a quantitative estimate of the amount of material present. Because the periphery of the diffusion patterns at the outside of the paper are much longer, the lines in general tend to be sharper. That portion of the paper containing particular materials may be cut out, with the sample being eluted for further study where desired.
Other examples of chromatographic systems will be apparent to those skilled in the art.
An apparatus for rapid centrifugal chromatography comprising: a flat corrosion-resistant circular plate, mounting means to position the circular plate for rotation about a central axis perpendicular to the face of the plate, drive means to rapidly rotate said circular plate, an annular circular plate having a small central hole, positioning means to position, axially and radially, said annular circular plate adjacent to and separated by a chromatographic paper disk from said circular plate, so that the said plates are in contact with and press against the surfaces of the said chromatographic paper disk, and are held apart by said disk, and means to feed a solid stream of solvent approximately centrally of said paper disk comprising a solvent feeder, a feed disk having therein an orifice of a diameter of about 0.001 to 0.003 inch, at the bottom of said feeder, a filter disk in said feeder positioned above the bottom thereof, a solvent feeder cap, to close the solvent feeder, and an air pressure connection above the filter disk, to, by air pressure, force solvent through said filter disk, and then through said orifice as a fine solid stream.
References Cited in the file of this patent UNITED STATES PATENTS 2,986,280 Magnuson et al. May 30, 1961 OTHER REFERENCES Caronna: La Chimica e LIndustria, vol. 37, No. 2, 1955, pages 113, 114. Copy in Sci. Lib., 210-31.
Block: Paper Chromatography and Paper Electrophoresis, second edition (1958), Academic Press, Inc. (New York), page 49 of interest. Copy in Div. 67.