US3027458A - Recording scanner for chromatograms having radio activity - Google Patents

Description

March 27, 1962 E. B. FOWLER ETAL 3,027,458

RECORDING SCANNER FOR CHROMATOGRAMS HAVING RADIO ACTIVITY Filed Aug. 19, 1955 2 Sheets-Sheet 1 INVENTORS ATTORNEY RECORDING SCANNER FOR CHROMATOGRAMS HAVING RADIO ACTIVITY Filed Aug. 19, 1955 March 27, 1962 E. B. FOWLER ETAL 2 Sheets-Sheet 2 f5 I I I 4%; ATTORIVFY 3,d27,d58 REQORDKNG SQANNLER FOR CHRQIVEATGGRAh/ih HAVNG RADEO ACTEVHTY Eric B. Fowler and Wayne A. Rhiuehart, Amos, Iowa, assignors to town State Cottage Research Foundation, Inc, Ar'njes, Iowa, a corporation of Iowa Filed Aug. 19, W55, Ser. No. 529,415 Ciaims. (C 250--83.6)

This invention relates to apparatus for surveying chromatograms having radio activity associated therewith, and more particularly to automatic recording scanner apparatus useful in scanning chromatogramized strips, detecting any radio activity associated therewith and recording the activity as to extent and location.

Chromatographic techniques have been developed in recent years for the separation of components of heterogeneous mixtures. Separation is accomplished generally by placing such a mixture on a supporting medium, such as a paper strip, and immersing one end of the medium in a multi-phasic solution so that the solution ascends or descends along the medium. Components of the unknown mixture will travel along the medium strip at difierent rates and thus be separated. The components may be identified later by spraying the strip with an indicator solution. Since micro amounts of each component may be detected in this Way, the technique is valuable for the identification of products in an enzymatic reaction mixture wherein the concentration of the components is unusually low.

Radio tracer techniques have also been recently developed and applied to the field of biology. The radio tracer method is now used extensively to demonstrate the up-take or incorporation of many compounds by biological systems. Tracer techniques may also be used to prove or disprove certain hypothesized chemical reac tions. The uptake of C 0 and its incorporation into amino acids, and hence protein of cellular systems, may be cited as an instance.

The combination of chromatographic and radio tracer techniques places in the hands of the research worker a valuable tool for the detection of products of reactions and their relationship to know precursors. By these two methods, micro amounts of a product can be separated at a high level of radioactivity or a high degree of purity, and the incorporation of labeled fractions demonstrated. However, the two techniques have been limited in application in that the element used extensively as a tracer (C is difiicult to detect by conventional means because of the low energy of its radiations. The flow type detector tubes available which were capable of detecting low activity were of such construction that the chromatographs had to be cut into small sectionsand the sections surveyed individually. Such a process is wasteful of both time and material. Isolated biological systems in general do not incorporate high concentrations of reactants, and :since the activity associated with any one zone might appear in several sections, a dependable total count could not be obtained.

It is, accordingly, an object of this invention to provide :a means for overcoming the limitations set fonh above, and to thereby extend the applicability of radio-tracer and chromatographic techniques. Another object of the invention is to provide apparatus designed to solve many of the limitations heretofore present in the art and to provide a research Worker with a dependable tool operative to increase the accuracy of his results, decrease the time required for any one determination, and to be of such an automatic nature as to require no attention after its being set in motion, thereby freeing the worker for .other endeavors.

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Still another object of the invention is in providing apparatus for scanning and recording indicia provided by chromatograms having radio activity in which the chromatograms are enclosed in a gas-tight housing which is an integral part of the counting chamber, whereby no windows or other barriers to the passage of the low energy radiations of the tracer element into the counting mixture are present. Yet another object is in the provision of scanning means for chromatograms comprising a defining slot interposed between the chromatogram strip and the actual counting portion of the tube, whereby the area of the strip being surveyed at any instant is determined by the size of the defining slotthe apparatus being equipped with means for readily altering the size of the defining slot so that the resolution of the apparatus may be varied.

Another object of the invention is in the provision of a drum rotatable and movable axially within a chamber provided by a casing thereabout, the drum being adapted to carry chromatogram strips wound spirally along the cylindrical surface thereof, whereby the strip is progressively scanned by a radio activity detector positioned in proximity with the drum and a strip carried thereon, and preferably separated therefrom by a plate having a resolution slot therein aligned with the detector element. Still a further object is in the provision of scanning and recording apparatus of the character described whereby the movement of the chart of the recorder is synchronized with the movement of the chromatogram strip in its scanning relation with the detector element so that following a scanning operation the recording chart and the chromatogram strip can be placed side by side and the areas of radio activity accurately located. in such an arrangement the relative amounts of activity in the various areas can be determined by integrating the area of each locatirig peak on the recorder chart.

Yet a further object is that of providing scanning apparatus as described wherein chromatogram strips are carried on a drum mounted within a substantially closed chamber, means'being provided in the system for evacuating and flushing the chamber and for filling the same with a counting gas (.a commercially available counting gas known as Q gas may be used), and the pressure in the chamber accurately maintained at the same predetermined value. Additional objects and advantages will become apparent as the specification develops.

An embodiment of the invention is illustrated in the accompany drawing, in Which- FIGURE 1 is a front view in elevation of the scanning and recording apparatus; FIGURE 2 is a side' view in elevation of the apparatus; FIGURE 3 is a longitudinal sectional view or the sample box taken on the line 3 3 of FIGURE 2; FIGURE 4 is a transverse sectional view of the sample box taken on the line 4-4 of FIGURE 1; and FEGURE 5' is a top plan view of a resolution plate used in the apparatus, and which is seen most clearly in FIGURE 3.

The advance provided by the instant invention may be better appreciated when viewed with the background of the prior art in mind. Prior to the development of the instant invention, several methods were attempted to circumvent the limitations set out hereinbefore concerning the chromatographic and radio tracer techniques. One method entailed placing the chromatogram on sensitive film and obtaining a radio autograph which located the positions of the radio active areas. However, high concentrations of activity were required for successful use of this technique, and the method lacks accuracy when total activity associated with an active spot is required. Further, the time required for film exposure is relatively long, and the results of an experiment are not immediately available.

A second technique involves the use of replicate chromatogrammed strips. One strip is sectioned, and each section counted or detected separately. If the sections are counted in a flow type counter, low activities are not a primary problem since in the flow counter the sample is placed in the tube, and there is no loss of counts due. to window absorption as in the end window tube. Disadvantages of this technique lie in the fact that a large number of small sections must be surveyed which requires continued supervision of the counting mechanism over long periods and the use of large volumes of counting gas for the flow tube. For example, a 2" by 20" chromatogram strip will yield 160 half inch circular discs. Each disc must be mounted separately which means that the flow tube must be opened, samples changed, the tube out-gassed or flushed, and the sample counted. Assuming typical counting rates, each operation would require about thirty minutes, or a total of about 80 hours per 20 strip. Even so, the worker has only a poor estimate of the total activity at any one position of the strip, and the strip is useless for further study.

With the technique and apparatus herein being considered, the operation is simple and the results are highly accurate. Several strips may be surveyed or scanned with one loading, and constant supervision of the instrument is not necessary once selection of the type of survey has been made and the apparatus set in operation. A minimum amount of counting gas is needed for the survey since the system is sealed and gas does not flow through the tube during operation of the apparatus. survey may be made in about seven and one-half hours automatically, as compared to over 400 hours that would be required for a comparable survey in the disc-flow counter method. In addition, the total activity associated with the strip spot is read directly from the recorder chart, and the strip remains intact for further study. The position of activity is indicated on the recorder chart as well as total activity; low activities are detected, and a permanent record of the results is obtained.

In referring to the drawings for a detailed description of the apparatus, reference will first be made to FIGURES 1 and 2 showing the scanning and recording apparatus in its entirety. The apparatus is designated generally with the numeral 10, and for portability is carried on a base or platform 11 equipped with castors 12 and a vertically extending frame 13 having a top wall 14 and intermediate shelves 15 and 16. Carried on the shelf 15 is a rate meter assembly 17 comprised of electronic circuits and components and, as is seen in FIGURE 1, is provided with a front panel 18 having a volt meter 19 and micro ammeter 20. Mounted on the shelf 16 is a recorder 21 that may be a Brown recorder and is equipped with a front panel 22 (FIGURE 1) having a central window 23 therein that provides visibility for a recording chart advanced by the apparatus of the recorder.

Mounted on the platform 11, as is shown in FIGURE 2, is a tank 24 adapted to contain under pressure a volume of an inert counting gas. The tank is equipped with a manually operable valve 25 that leads into a conduit 26 that carries the gas into the chamber of a sample box 27 that will be described in detail hereinafter. That platform 11 also supports a motor driven pump 28 that is a high vacuum pump and, if desired, may be a high vacuum Cenco pump. The pump is connected to a manifold 29 that opens into a manifold extension 30 equipped with a Sylphon high vacuum shut-off valve 31. The extension 30 communicates with the sample box at four spaced apart points through branch conduits designated by the numerals 32a through 32d. The frame 13 also carries a mercury manometer 33 having a meter stick 34. The manometer is connected to the manifold branch 30 through a valve 35 and is employed to measure the pressure of the counting gas within the sample box 27. The sample box 27, referring now to FIGURES 3 and 4, comprises a generally cylindrical casing36 providing a chamber 37 therein and a drum 38 mounted within A double the chamber 37. The casing 36 at one end thereof is equipped with an end closure wall 39 that may be squareshaped, as seen in FIGURE 2, and dimensionally is larger than the casing 36 so that it provides outwardly extending flanges that may be employed in mounting the casing on the top wall 14 of the frame. At its opposite end the casing 36 is equipped with an outwardly extending perimetric flange 4t) equipped with a plurality of spaced apart threaded openings therein that are adapted to receive threaded screws 41 that may be provided with wing nut heads 42, which are employed in securing the end wall 43 to the flange 40. As is shown in FIGURE 3, the end wall 53 is provided at a spaced distance inwardly from the edge thereof and along its inner face with an annular channel 44 having seated therein a sealing gasket 45. The end walls 39 and 43 in combination with the casing 36 provide a substantially fluid-tight chamber 37.

The drum 3% comprises a cylindrical shell or tube 46 that is hollow and that is provided with spaced apart wall members 47 and 48, each of which is equipped with a plurality of central openings 49 therethrough and peripherally located openings 50. The walls 47 and 43 have aligned central openings provided with keyways therein and seat upon a shaft 51 equipped with a key 52 that is journalled for rotation adjacent the end wall 39 in a bearing 53 secured to the end wall by cap screws 54-, and in a bearing 55 carried exteriorly of the end wall 43 and secured thereto by screws 56.

Referring now to FIGURES l and 3, it is seen that the shaft 51 extends outwardly from the end Wall 43 and is connected through a gear reducer 57 to a synchronous motor 58. When the motor is energized, rotation of the shaft 51 rotates the drum 33 which is locked thereon to prevent relative rotational movement therebetween but is free to move axially along the shaft. By means of appropriate gear ratios in the gear reducer 57, the rate of travel of the periphery of drum 38 may be made the same as the rate of travel of the recording chart on the Brown recorder, so that the movement of the chart of the recorder is synchronized with the movement of the drum 38 within the chamber 3'7.

The tube or shell 46 of the drum 38 is provided about the circumferential outer surface thereof with a spiral channel or groove 59 which extends from end to end thereof. Adapted to ride in the spiral groove 59 is a guide pin 60 that extends through a boss 61 with which the casing 36 is provided. The boss 61 is threaded at its outer end and threadedly receives a cap 62 that bears downwardly on a flange 63 carried by the guide pin to force the same into tight engagement with a resilient seal member 64, whereby the mounting for the guide pin provides a substantially fluid-tight seal preventing the escape of gases from the chamber 37. It is clear from FIGURE 4 that the portion of the pin 60 that rides in the groove 59 may be reduced in cross section. It will be appreciated that the rotation of the drum 38 will cause the drum to be moved axially or bodily along the shaft 51 because of the engagement between the guide pin and the spiral groove.

As is shown most clearly in FIGURE 3, the casing 36 is provided along the wall thereof intermediate its ends with an opening 65 and with a short tubular conduit 66 that extends outwardly from the casing thereabout. The tube 66 is provided with a laterally extending flange 67 that has an annular channel in the upper face thereof that receives a gasket 68. Seated upon the flange in covering relation with the gasket or seal member is a short tubular section 69 provided with a seal member 70 that has seated thereon a laterally extending flange 71 of an outer closure member 72 provided with a central opening 73 therein. Elongated bolts 74 secure the flanges 67 and 71 and the member 69 together. Extending outwardly from the closure member 72 about the opening 73 therethrough is a collar member 75 that bears against a seal 76 carried by the closure member 72 to establish a fluidtight relation therewith. Collar member 75 has provided an internal shoulder at its lower opening onto which a Kovar glass seal 75a may be soldered to provide a fluidtight relation thereby.

Secured to the collar 75 is a fitting 77 of a coaxial cable 78 that, as is shown in FIGURE 2, is connected to the rate member assembly 17. The center conduit of the cable 78 is electrically connected to a detector element or anode 79 that may be a ten mil tungsten Wire provided at its end with a glass head 80. The detector 79 is aligned with a resolution slot 81 provided in a plate 82 that seats on an annular flange section 83 provided by the casing 36 about the opening 65. The plate 82, as is shown best in FIGURE 5, is provided with a notch 84 that receives therein a key 85 provided along the inner wall of the member 66. The resolution slot 81 has a predetermined area, and it will be appreciated that the plate 82 is readily removable and may be replaced with another having a resolution slot of greater or smaller area depending upon the degree of resolution that may be desired in a scanning operation.

The detector 79 through the slot 81 is adapted to detect the radio activity of chromatogram strips 86 that are carried by the drum 38 about the outer surface of the tubular shell 46 thereof. The chromatogram strips are spirally wound about the shell 46 and may extend from end to end thereof. They may be secured in place by a plurality of Phosphor bronze clips 87. In an embodiment of the invention that has been successfully employed, three strips each 2" in width by 20 long may be secured to the shell 46 in end to end relation to provide a continuous chromatogram extending from end to end of the drum. Preferably, the drum 38 is formed of brass.

The member 66 is provided with a flow port 88 communicating with the conduit 26 that connects through the valve 25 to the countings gas container 24.

A micro switch 89 is carried by the end wall 43 and is provided with a pair of leads 90 and 91 that extend outwardly through the wall 43 and have, respectively, glass seals 92 and 93 thereabout. The end wall 39 carries a micro switch 94 having leads 95 and 96 extending therefrom outwardly through the end wall and through glass seals 97 and 98. All of the seal members function to provide a fluid-tight closure about the lead wires for the micro switches, and it has been found that Kovar seals may be employed to bond the glass to the metal end walls.

Referring back to FIGURE 1, it is seen that the front panel 101 of the apparatus is equipped with a plurality of switch members designated with the numerals 102, 103, 104, 105 and 106. Also, pilot lights 107, 108 and 109 are provided. The switches respectively are the pump switch for controlling the motor of the pump 28, the master switch for controlling the power connection to the apparatus generally, a momentary switch, a selector switch, and a relay reset switch.

Operation In use of the apparatus, the end wall 43 is released from its position of sealing engagement with the flange 40, and the drum 38 is moved bodily out of the casing 36. The chromatogram strip 86 is positioned about the shell 46 of the drum and is secured thereto by clips 87. If a plurality of short strips are placed in end to end relation about the drum, the end portions thereof may be secured together by tape or other appropriate means. The drum is then moved into the casing and is preferably positioned so that the starting end of the strip 86 is substantially adjacent the resolution slot 81 in the plate 82. The end wall 43 is then secured as is shown in FIG- URE 3.

In removing the drum 38 from the casing or returning it to position therein, the guide pin 60 may be withdrawn from engagement with the spiral channel 59, or alternatively may be rotated until the guide pin is released from the channel.

When samples or chromatogram strips are changed, the

counting chamber 37 must be flushed and a fresh supply of counting gas under pressure admitted thereto. In the system provided, it is not necessary to flush the chamber 37 several times with counting gas prior to starting the run of a new sample. Such a procedure is undesirable for it involves the use of large quantities of gas which is expensive. Instead, the pump 28 is actuated and the chamber 37 is evacuated since the pump is connected thereto through the manifold 29, manifold extension 30 and branch conduits 32a through 32d. After evacuation, a supply of counting gas is admitted to the chamber 37 through the valve 25. Preferably, that volume of gas is then evacuated from the chamber and a fresh quantity of counting gas admitted thereto. Such a flushing procedure substantially eliminates contaminating gas which would interfere with counting tube operation.

To actuate the apparatus, the various switches are moved to the on posit-ion, and the motor 58 through the shaft 51 rotates the drum 38. As the drum rotates, the coaction of the guide pin 60 and spiral channel 59 causes the drum to move bodily axially within the chamber 37, or toward the right as viewed in FIGURE 3. The chromatogram strip 86 then advances progressively beneath the resolution slot 81 and is scanned by the detector element or anode 79 of the scanning tube.

At the same time, the recorder 21 is functioning, and it is operatively coupled to the rate meter assembly 17 which in turn receives the counting impulses from the detector 79, and the radio activity of the strip at the location beneath the resolution slot 81 is recorded directly on the chart of the recorder. Thus, both the amount or extent of activity and the precise location thereof on the strip 86 are directly recorded. This procedure is valuable, for after a test has been run the strip 86 may be removed from the drum and placed beside the chart of the recorder so that the extent and exact location of the recorded activity can be transposed to the strip. The extent of activity may be determined by integrating the areas under the curves or pips on the recording graph.

The apparatus may be set up so that when the drum 38 engages the switch arm of the micro switch 94, the apparatus will be de-energized. Thus, one pass or one complete scanning cycle of the strip 86 will have taken place. On the other hand, if it is desired to provide a double-scanning cycle, the selector switch is so positioned, and upon the drum striking the switch arm 100, the motor 58 is reversed and the drum will then move axially to the left within the chamber 37 until it engages the arm 99 of the micro switch 89 which functions to deenergize the apparatus. By a suitable means, the recorder chart is marked at the end of the forward scan. In such a scanning cycle, strip 86 will be scanned first in one direction and then in the other.

The plate 82 is removably mounted within the member 66 which permits a plurality of plates 82 to be interchanged with each other. The various plates will have resolution slots 81 of different size, whereby the degree of resolution of the scanning operation may be selected by the operator. Ordinarily, the narrower the slot 81, the greater will be the degree of resolution. It is also desirable to reduce the operational speed of the apparatus where greater resolution is being provided by a smaller resolution slot 81.

The amount of counting gas consumed in operation of the apparatus is maintained at a minimum because no gas flows through the counting chamber 37 during the testing or scanning of a strip. The only gas movement through the chamber comes during the flushing operation prior to the scanning of a new test sample. The operation is substantially automatic, and once the apparatus is set in motion and the mode of operation selected, attention from an operator is unnecessary for the apparatus scans and records automatically and terminates its own operation upon the completion of a scanning cycle, whether the cycle be a single or double-scanning cycle.

While in the foregoing specification an embodiment of the invention hasbeen ,set forth in substantial detail for purposes of making a full and complete disclosure of the invention, it will be apparent to those skilled in the art that numerous changes may be made in those details without departing from the spirit and principles of the invention.

We' claim:

1. In apparatus for scanning chromatogram strips to detect the radio activity thereof, a casing providing a counting chamber therein, a counting tube carried by said casing and having a. stationary detector element in facing relation with the interior of said counting chamber, a drum having a cylindrical surface for spirally supporting an elongated and discontinuous chromatogram strip thereon and being mounted within said chamber for rotational and axial movement, and guide means cooperating with said drum for progressively advancing said drum axially as the same is rotated and thereby orienting said spirally wound chromatogram strip, in scanning relation with said stationary detector element as said drum is rotated.

2. The apparatus of claim lin which said drum is substantially hollow and is provided with spaced apart partitioned walls each having a plurality of apertures therethrough, and in which a rotatable shaft extending axially through said chamber extends through said walls and provides a mounting for said drum..

3., In a chromatogram scanning apparatus of the character described, a casing providing a substantially fluid-tight counting chamber therein, a rotatable shaft disposed within said chamber and fixed against axial movement with reference thereto, a. drum carried by said shaft for axial movement therealong but being constrained on said shaft to prevent relative rotational movement therebetween, said drum having a cylindrical outer surface and being provided along said surface with a spiral groove, a stationary guide provided by said casing and adapted to ride along 'said groove for progressively and gradually advancing said drum axially as the same is rotated, said drum being adapted to support an elongated spirally-wound and dis continuous chromatogram strip upon said cylindrical surface and between the spirals of said groove, a counting tube carried by said casing in open communication with tector element and said guide being positioned and arranged so that as said drum is axially advanced upon rotation thereof said spirally-Wound chromatogram strip is carried in progressive scanning relation with reference to said detector.

. 4. In an apparatus for scanning chromatogram strips to detect the radioactivity thereof both as to extent and location along said strips, a casing providing a substantially fluid-tight counting chamber, a cylindrical drum adapted to carry an elongated discontinuous chromatogram strip in spirally wound fashion along the outer surface thereof, means for supporting said drum for rotational and axial movement Within said chamber, a guide element provided by said casing and cooperating with said drum for progressively moving the same along its rotational axis as said drum is rotated, and a counting tube communicating with said chamber and having a stationary detector element in facing relation with the outer surface of said drum, said detector and guide elements and said drum being related with respect to each other so that upon rotational and axial movement of said drum a chromatogram spirally wound thereon passes longitudinally in scanning relation with reference to said detector element, whereby, said spirally-wound strip is scanned by said detector as said drum is rotated and axially advanced to detect both the extent and location of radioactivity therealong.

5. The structure of claim 4 in which said drum is hollow and is provided with spaced partion Walls each having a plurality of apertures therein.

References Qited in the file of this patent UNITED STATES PATENTS 1,806,375 'ifieclernan May 19, 1931 2,490,298 Ghiorso et al. Dec. 6, 1949 2,751,505 Anderson June 19, 1956 OTHER REFERENCES Use of Beta-Ray Densitometry in Paper Chromatogra- .phy, from Analytical Chemistry, vol. 23, No. 1, January 1951, pages 207 to 208.

Apparatus for Automatically Scanning Two-Dimensional Paper Chromatograms for Radioactivity, by W. J. Wingo, from Analytical Chemistry, vol. 26, July-December 1954, pp. 1527, 1528.

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