GB2108654A - Continuous radioactivity flow monitor - Google Patents
Continuous radioactivity flow monitor Download PDFInfo
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- GB2108654A GB2108654A GB08224522A GB8224522A GB2108654A GB 2108654 A GB2108654 A GB 2108654A GB 08224522 A GB08224522 A GB 08224522A GB 8224522 A GB8224522 A GB 8224522A GB 2108654 A GB2108654 A GB 2108654A
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- flow cell
- tubing
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- photomultiplier tube
- planar spiral
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01T—MEASUREMENT OF NUCLEAR OR X-RADIATION
- G01T1/00—Measuring X-radiation, gamma radiation, corpuscular radiation, or cosmic radiation
- G01T1/16—Measuring radiation intensity
- G01T1/20—Measuring radiation intensity with scintillation detectors
- G01T1/204—Measuring radiation intensity with scintillation detectors the detector being a liquid
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01T—MEASUREMENT OF NUCLEAR OR X-RADIATION
- G01T1/00—Measuring X-radiation, gamma radiation, corpuscular radiation, or cosmic radiation
- G01T1/003—Scintillation (flow) cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N2030/77—Detectors specially adapted therefor detecting radioactive properties
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Abstract
In a chromatographic system for analyzing materials containing radioactive components, a sample eluted from a column is mixed with a scintillation cocktail and then passed through a flow cell positioned adjacent at least one photomultiplier tube preferably between two tubes, to detect scintillations in the mixture. The flow cell 20 comprises a planar spiral of transparent tubing 23 encased on each side by transparent plates 21, 21'. The photomultiplier tubes are placed on opposite sides of the cell in contact with the faces of the plates 21, 21'. This flow cell can also be used to analyze gel extruded from a gel electrophoresis column after admixture with a scintillation cocktail in a homogenizer. <IMAGE>
Description
SPECIFICATION
Continuous radioactivity flow monitor
This invention relates to the detection of radioactivity in flowing fluid streams, and is particularly advantageous in the detection and measurement of radioactivity in fluid streams such as those obtained from chromatographic columns or the like. More particularly, the present invention relates to apparatus and methods for detecting and measuring radioactively tagged or untagged material, such as chromatographically separated materials, contained in a flowing stream of material, e.g., the eluate of a chromatographic column. A particularly preferred embodiment of the present invention measures radioactively tagged or untagged materials in the eluate of a high pressure liquid chromatography system.
Another particularly preferred embodiment of the present invention measures radioactively tagged or untagged materials in a mixture, suspension, or slurry obtained by progressively suspending the gel resulting from electrophoresis in a carrier fluid, so that the radioactivity measured at a given time corresponds to the radio-labelled compounds contained in the various segments or strata of the electrophoresed gel after completion of gel electrophoresis.
Over the years, chromatography has become increasingly important as a means for separating chemical compositions into their constituent parts, thereby to permit analysis of those compositions.
For example, chromatographic methods and apparatus for resolution and analysis of chemical compounds, such as amino acids, are well known.
Because of different affinities for the adsorbing medium used, different compounds are eluted from a chromatographic column at different times.
Measurement of the amounts eluted of individual components can provide a measurement of the relative amounts of the compound in the original sample. By using such techniques, mixtures of a number of chemically similar compounds, such as amino acids, can be analyzed to determine which compounds are present in the sample, and in what amounts.
Another method utilized for separating and analyzing chemical compounds is electrophoresis.
In that technique, advantage is taken of the fact that different materials (particularly different large molecules, e.g., proteins) migrate at different rates through a supporting medium such as a gel under the influence of an electric field. Differences in rates depend on a variety of factors, e.g., differences in size, shape, or number of charged sites on the molecule. Thus, under the influence of an electric field, the fastest moving component moves out ahead of the other components, and the slower moving components iag behind. In a given time with a given current flow, each protein or other molecule travels a characteristic distance, resulting in a series of zones in the gel or other support medium.By analyzing the composition of the various sequential zones in a tube or column of gel, the identity and relative amounts of different compounds in a sample can be determined.
Attempts at increasing the usefulness of chromatographic systems by providing a method for measuring the radioactivity of tagged compounds in the various segments of the chromatographic eluate have been made for many years. Scintillation counting techniques have been used, in which a material was employed which converted the energy resulting from radioactive decay of tagged molecules to electromagnetic radiation, which in turn is detected by photomultiplier tubes or other light sensitive devices. In recent years, liquid scintillation counting has become a very popular method for the measurement of low energy beta emitters such as 14c, 35, and 3H (tritium). Liquid scintillation counting (LSC) is commonly accomplished by combining, dissolving, dispersing or solubilizing a radioactive sample to be analyzed in a liquid scintillator so as to form a counting sample.Each radioactive disintegration may lead to the production of a light pulse, and these are counted by suitable equipment, as mentioned above. One measure of success of the analysis is the ability to obtain the same number of counts per unit time from a given sample over a period of time, allowing, of course, for the natural radioactive decay of the material. A measure of sensitivity and utility of the analysis is called the "counting efficiency", which is commonly defined as:
observed counts per unit time x 100
actual disintegrations per unit time
When applied to flowing systems, the efficiency can be calculated by the equation:
Observed output (cpm) flow rate x Sample activity (dpm) cell value
See Mackey et al., "High Efficiency Solid Scintillation Radioactivity Detection * * *," J.
Chrom.208: 1(1981).
The common constituents of a liquid scintillator are the solvent and one or more fluors. The solvent is usually an aromatic liquid such as an alkylbenzene. Its purpose is to absorb the energy of excitation due to radioactive disintegrations, and to transfer it to the fluor. The fluor (sometimes called a scintillator solute) converts the transferred energy to electromagnetic radiation. When a fluor does not produce this radiation, or fluorescence light, at the wavelengths of greatest sensitivity of the light measuring device available, a second fluor (sometimes called a wavelength shifter or secondary scintillator solute) may be added. It absorbs the light from the first or primary fluor, and re-emits at longer wavelengths, hopefully matching the sensitivity of the light measuring device, and leading to higher counting efficiency.
The solvent and fluors are commonly referred to as the "scintillation cocktail."
At first, the radioactivity measurements of the various samples or fractions, e.g. of the eluate from a chromatography column, were made by physically dividing the eluate into sequential segments, which were individually collected in vials, mixed with a scintillation cocktail, and each vial was then counted in a scintillation counter, to provide a measurement of the radioactivity of the sample, and thus the concentration of the tagged compound in the sample. This approach was highly expensive, time consuming and laborious, since it required the careful measuring and mixing of many individual samples, e.g., often about 30 or 40 to 100 samples for a typical amino acid measurement.This approach was also not practical for some types of chromatography, e.g., those which produce very small volumes of samples. See B. Bakay, "A Novel Method of
Sample Transport and its Application for
Continuous Detection of Radioactivity in the
Effluent of the High Speed Amino Acid Analyzer",
Analytical Biochemistry 63:87 (1975), and Reeve et al., "Radioactivity Monitor for High Performance
Liquid Chromatography," J. Chromatography 135:271-82(1977).
Attempts have been made to provide a means for measuring radioactivity in chromatographic eluates on a continuous basis. These attempts have historically been of two types. One approach has been to use solid scintillators, such as anthracene or certain crystals or glasses, or plastic scintillators such as scintillator-coated polyethylene tubing. The sample to be analyzed would continuously flow over crystals of anthracene scintillator or beads of glass scintillator in a clear tube. When plastic scintillators have been used, either the tube itself has been used as the scintillator, or plastic scintillator beads or filaments were used. See E.
Schram, "Flow-Monitoring of Aqueous Solutions
Containing Weak Beta-Emitters," in E. D.
Bransome (Ed.), Liquid Scintillation Counting (Grune S Stratton, 1970), at 95 et seq. The other approach that has been used is to mix a part of the eluate stream from the chromatograph with a continuous supply of liquid scintillator composition, and pass the mixture through a cell to measure radioactivity.
However, each of these approaches has been plagued by its own set of problems. The solid scintillator systems suffered from a tendency of the solid scintillators to absorb materials from the eluate stream. See Hunt, "Continuous Flow Monitor System * * *," Analytical Biochemistry 23, 280 (1968). Such detectors also suffered from seif quenching, i.e., the light impulses were quenched within the system before they could be detected by the photomultiplier tube or other detection apparatus. Alsom scintillators such as anthracene and various salts react with, or are dissolved in, certain compounds commonly found in chromatographic eluates. Further, heavy metals such as chromium, mercury and their salts are sequestered by anthracene.See McGuinness et al., "Continuous Flow Measurement of Beta
Radiation Using Suspended Scintillators",
J. Chem. Ed. 47:A9 (1970). Other problems experienced with solid scintillation systems included high background readings and very low efficiencies, especially for tritium (in the order of 0.1%).
The use of liquid scintillators did not provide a ready solution to the problem, particularly since common liquid scintillator systems have been unable to adequately incorporate and hold in solution sufficient quantities of the typical chromatographic eluates. Certain components in the chromatographic eluate further decreased the solubility of the samples and/or increased selfquenching of the scintillations. Further, liquid scintillator systems required large amounts of scintillators to be used with small amounts of sample, which made such systems more economically impractical.
Liquid scintillator systems also suffered from low efficiencies, especially for tritium counting.
Both systems required highly expensive and complex sample chambers or flow cells for flowing the eluate past the radiation detectors. In addition to being expensive and complex, sample chambers were low in efficiency and resolution. In order to improve counting efficiency, it had been necessary to increase sample size, which adversely affected resolution.
Another analytical technique which produces radio-labelled compounds which are difficult to analyze on a continuous basis is electrophoresis.
Typically in gel electrophoresis a sample containing a number of compounds is inserted into a column of gel, which is then subjected to an electric field for a period of time. The compounds contained in the sample migrate through the gel at different rates and therefore separate along the length of the gel column in accordance with their rate of migration. The end product of this technique is usually a column or tube or slab containing the gel, or other support medium, with the various compounds in the sample being separated into different strata, depending on their speed of migration. A convenient way to run a plurality of samples at the same time is to deposit a plurality of samples adjacent to one another at one end of a slab of gel or other support medium, and apply the electrical current across the slab.
After current flow is stopped, the slab can be cut into rows of electrophoresed gel corresponding to each of the adjacent samples, and each of the rows can be continuously analyzed in accordance with the present invention.
While there have been attempts to combine gel electrophoresis with scintillation counting techniques, see 0. G. Maizel, "Acrylamide Gel
Electrophoretograms by Mechanical Fractionation:
Radioactive Adenovirus Proteins," Science 151 988 (1966), prior to the present invention, practical methods conducting such analysis of gel electrophoresis samples on a continuous basis have been lacking. As a result, the art has generally been relegated to collection and analysis of myriads of individual electrophoresed gel samples. See Maizel, supra.
The present invention permits the accurate detection and measuring of radioactivity contained in a flowing fluid stream and provides for high counting efficiency and low background even for weak beta-emitters by flowing the sample past a photomultiplier in a flow cell which comprises a planar spiral of tubing which transmits light encased on two sides by members which also transmit light.
The present invention also provides a practical method of analysis for radio-labelled compounds in samples obtained from gel electrophoresis on a continuous basis, so that the identity and amount of radio-labelled compounds can be determined in relation to other compounds stratified in the gel sample as described hereinafter.
Exemplary embodiments of the invention are described hereinafter with reference to the accompanying drawings in which:
Figure 1 is a schematic representation of a detection apparatus useful in accordance with the present invention.
Figure 2 is a side view of a preferred flow cell for use in connection with the present invention.
Figure 3 is a cross-section frontal view of the flow cell as depicted in Figure 2, taken along section lines A-A' in Figure 2.
Figure 4 is a top view of a preferred photomultiplier tube system for use in the present invention.
Figure 5 is a frontal view, partially in crosssection of the preferred photomultiplier tube system taken along section lines B-B' of
Figure 4.
Figure 6 is a schematic representation of a detection apparatus useful in conjunction with gel electrophoresis in accordance with the present invention.
Figure 7 is a graph depicting the results of
Example 1 hereof as further described below.
Figure 8 is a graph depicting the results obtained in accordance with the present invention.
Figure 9 is a graph depicting the results of
Example 2 hereof, obtained by cutting, separating and counting each individual slice of electrophoresed gel.
As shown in Figure 1 of the accompanying drawings, one suitable fluid chromatographic system employing an embodiment of the present invention comprises a chromatographic fractionating column 2 fed by inlet conduit 1, and containing a suitable chromatographic medium for separating the components of the fluid stream. A wide variety of such materials suitable for chromatographic separations are known, including silica gel, alumina, glass beads, ion exchange resins, other natural or synthetic resins, polymers such as cellulose, polystyrene, clays such as bentonite, kaolin, etc., or other materials which have the property of selectively or preferentially absorbing or adsorbing one or more of the compounds or components in the liquid or other fluid to be analyzed.The chromatographic medium is supported in chromatographic column 2 by means well known in the art, e.g., screens mounted in the column proximate to each of its ends. While the invention is being exemplified by a description of a system for liquid chromatography, the invention can also be used with other types of separation or analysis, e.g., gas chromatography, gas/liquid chromatography, or in other situations in which it is desired to analyze the radioactivity of a moving stream or stratified column of material.
The present invention is particularly advantageously used in connection with high pressure liquid chromatography (HPLC), other types of chromatography, and electrophoresis.
Upon leaving column 2, the eluate typically is passed through standard analysis equipment, such as a spectrophotometric, coiormetric, conductometric, or fluorometric analyzer, a flame ionization detector, or other device, indicated schematically at 3, which measures the concentration of one or more of the sample components as they are sequentially eluted from the chromatographic material. From the spectrophotometer or other device 3, the eluate passes through conduit 4. At least a portion of the column eluate is taken off through conduit 5 for radioactivity analysis. The remaining eluate can be further analyzed, tested, or collected and saved, as indicated schematically at station 6 in Figure 1, or simply discarded.
The portion of the eluate which is taken off by conduit 5 may be passed through pump 7 before being mixed with the scintillation cocktail.
Preferably pump 7 is of the type which provides a constant volume of flow of the eluate so that it can be mixed accurately in a predetermined volume ratio with the scintillation cocktail.
However, it should be noted that in some circumstances, e.g., HPLC, wherein the eluate flow and pressure are fairly constant, a pump in eluate line 5 will not be necessary.
As indicated in Figure 1, the liquid scintillation cocktail flows from its supply 8 through pump 10, preferably also of the constant volume variety, through conduit 11 and into conduit 12, which carries the portion of eluate from the chromatographic column. By varying the rate of pump 10 and/or pump 7, the ratio of liquid scintillation cocktail to eluate can be accurately controlled. Other means of controlling that ratio, e.g., a valve in conduit 11 and/or conduit 12, can also be used.
When the stream of scintillation cocktail in conduit 11 joins the stream of eluate in conduit 12, a certain amount of mixing of those two streams will occur. If desired, means can be provided for enhancing the mixing of the cocktail and the eluate to provide a maximally dispersed solution to be counted in the scintillation counter.
A wide variety of mixing means (designated schematically at 13 in Figure 1) can be employed.
For example, a magnetic stirrer bar can be employed in a small mixing chamber at or near the junction of conduits 11 and 12. See, e.g., Hunt, supra. Other known mixing devices can be used, e.g., a mixing coil, see, e.g., D. R. Eyre, "An
Automated Method for Continuous-Flow Analysis of Radioactivity ** *," Analytical Biochemistry 54:619 (1973).
The eluate/cocktail admixture is then passed through flow cell 14 situated between a pair of photomultiplier tubes (16 and 16') all described at greater length below. The eluate/cocktail mixture leaves the flow cell through conduit 15, and may be subjected to further analysis, or may be discarded.
The output signals from the photomultiplier tubes are monitored by a ratemeter or other counting device 17. Preferably, the counting device is connected to a chart recorder 18, so that the radioactivity can be charted over the time period of the chromatographic analysis. The chart produced gives a substantial amount of information, particularly when utilized in combination with the results given by the spectrophotometer or other measuring device indicated at 3 in Figure 1. For example, analyzer 3 may give a measurement of the total concentration of a particular component in the mixture, whereas the results from chart recorder 1 8 wouid indicate what percentage of that component is radio-labelled.Alternatively, the radioactivity detector 1 7 may provide information on components in the sample which the analyzer shown at 3 is not capable of measuring. Other methods and means for advantageously handling the information provided by the systems of the present invention, such as digital or analog data processors are known to the art.
Turning to Figures 2 and 3, the special flow cell 20 utilized in accordance with the present invention has two sidewalls in the form of flat, circular disks, 21 and 21', which are preferably made of optically clear material. Disks 21 and 21' support and contain sandwiched between them the tubing which carries the sample to be counted.
The disks are held together by a plurality of spacers, 24, 25, 26, 27, and 28. The thickness of the spacers Is preferably about the same as or slightly smaller than the outer diameter of the tube 23 being used, so that the inside faces of disks 21 and 21' are in close contact with the outside of the tube, so as to reduce the amount of scintillations which are undetected due to surface effects. The tubing 23 as shown is turned a number of times around the supports 27 and 28, proximate the center of the flow cell, so as to increase the amount of time the flowing sample is held exposed to the photomultiplier tubes.The material used for the tubing is preferably clear to light in the wavelengths of the scintillations and impervious to the samples, solvent, and other materials used, and which also does not readily absorb or adsorb any of the components of the sample. Polytetrafluoroethylene is the preferred material, although other materials, e.g., polyethylene, polypropylene, and silicones, etc., are also available. The diameter of the tubing used is chosen in consideration of the radioactivity of the sample and the flow rate of the sample, so as to provide an adequate number of counts to obtain an accurate reading on the sample content. Higher volumes of the flow cell lead to higher counting efficiencies, but the distinctiveness of separation of the components (i.e., the resolution) suffers.
Preferably, smaller sizes of tubing are utilized, e.g., tubing having an outer diameter of from about 0.04 to 0.2 inches, more preferably from about 0.06 to about 0.10 inches, since their smaller, thinner tube walls decrease the amount of quenching which occurs in the tube wall, and their smaller volume permits less chance of backmixing between components. Preferably, the flow cell will have a total sample volume of about 0.05 to 10 ml, more preferably from about 0.2 to 1.0 ml. Preferably, the inner diameter of the tubing is about 0.02 to 0.15 inches, more preferably about 0.03 to about 0.07 inches. It has surprisingly been found that this simple, straightforward flow cell, which costs less than one-tenth as much as standard commercially available flow cells, can give higher counting efficiencies and more accurate results than the more costly flow cells.The low cost and high efficiency of this type of flow cell permits the researcher to optimize his work by having a large number of different flow cells, e.g., with different volumes or different types of tubes, for use in different situations. Preferably all working elements of the flow cell pass light very readily, and preferably at least the disks 21 and 21' are optically clear, to maximize the light reaching the photomultiplier tubes. Plexiglass (polywarbonate) is a suitable material for the disk-cell walls 21 and 21', as well as for the spacers 24-28. Other optically clear plastics, e.g., acrylic and methacrylic resins, are well known. Glass or other optically clear materials may also be used. It is preferred but not critical that the spacers be optically clear as well as the faceplates of the flow cell.
The details of one preferred assembly for the photomultiplier/flow cell unit are shown in Figures 4 and 5. The unit is mounted on a base 30, and comprises two identical photomultiplier tube housings 16 and 16'. Within each housing is a photomultiplier tube 31 (and 31') with its associated circuitry 32 (32'). Such photomultiplier tubes and their circuitry are well known and commercially available. Since each side of the photomultiplier tube assembly is identical with the other side, the components of the housing on the left of Figures 4 and 5 will be particularly discussed, it being understood that the right half of the assembly has corresponding components for each component of the left hand side.
Unlike previous systems, the photomultiplier tubes in each of the housings 16 and 16' are mounted in the housing with a means for longitudinal adjustment of the tube. The adjustment means shown in the embodiment depicted in the drawings is an adjusting screw 33, mounted in end cap 34, and connected to the photomultiplier tube via network support plate 35 which in turn is connected with tube 31 through a plurality of posts 36 and plug 37, which also serves to connect photomultiplier tube 31 to its circuitry 32. Photomultiplier tube 31 is also slidably mounted at the other end in coupler block 38, which permits the photomultiplier tube to be adjusted longitudinally within the tube housing by means of the adjustment screw 33.
Each of Figures 4 and 5 depict the assembly as containing a flow cell 20 mounted between the photomultiplier tubes 31 and 31'. Note that photomultiplier tubes 31 and 31' are directly in contact with the faces 21 and 21 ' of the flow cell 20. In order to achieve that end, the position adjustment means, 33, 33', are adjusted after the flow cell is inserted into the tube assembly, so that the front face of each photomultiplier tube is in direct contact with the face of the sample holder.
This factor, together with the unique design of the flow cell, is believed partially responsible for the improved counting efficiencies and reproducibility obtained with the present invention. Other means can also be used for mounting the photomultiplier tube in the housing to insure direct contact between the face of the tube and the sample flow cell, such as a piston or plunger assembly. Fittings 39 and 40 are light-tight connections to the sample tube 23 in flow cell 20. Fluid to be analyzed from conduit 12 in Figure 1 enters flow cell 20 through one of the light-tight connectors 39 or 40 in Figure 4, flows through the flow cell 20, and thence through the other of the connectors into exit line 1 5 shown in Figure 1.As noted, these connections must be light-tight, i.e., they must not admit any light into the interior of the flow cell or the photomultiplier tube assembly, lest the photomultiplier tubes be excited in false indications of scintillation. To that end, it is preferred that lines 12 and 1 5 in Figure 1 be opaque to light, e.g., black teflon, stainless steel, or other opaque substances can be used as the material for such tubing.
Electrical connections 42 and 43 are provided at each end of the photomultiplier tube assembly in order to provide power to the photomultiplier tubes and to convey the signals corresponding to radiation-induced scintillations which are detected by the photomultiplier tubes to suitable counting, recording, or other apparatus. Generally the photomultiplier tubes require a high voltage power supply. The electronic equipment which may be used to process the signals from the photomultiplier tubes may include a variety of items known to the skilled in the art. Amplifiers and discriminators can be utilized to amplify and refine the signals. Preferably a coincidence circuit is employed, which compares the signals from both photomultiplier tubes to eliminate detection of spurious signals, such as those caused by cosmic radiation and the like.
Figure 6 depicts an embodiment of the present invention as applied to the column of gel obtained from gel electrophoresis. A cylindrical column of electrophoresed gel is contained in cylindrical container 50 which is open at its ends to conduits 51 and 52. Advantageously, the same column and tube utilized for electrophoresis can be simply plugged into the present system.
In the embodiment depicted, a fluid under pressure is utilized to force the column of gel out of container 50, through conduit 52, and into a low volume homogenizer 55. The fluid may be water or any other fluid (liquid or gas) which does not react with, dissolve or otherwise adversely affect the electrophoresed gel, and which is operable to extrude or force the gel out of container 50 and into the homogenizer 55 at a constant rate. The fluid is supplied from reservoir 54 preferably via pump 53 to increase the supply pressure, and through conduit 51 into cylindrical container 50.Preferably the pressure and viscosity of the fluid should be such as to extrude the electrophoresed gel from container 50 at a rate of about 0.001 to 1.0 milliliters per minute, more preferably from about 0.05 to 0.5 milliliters per minute, and most preferably from about 0.08 to about 0.2 milliliters per minute.
The electrophoresed gel is mixed with the scintillation cocktail in the low volume homogenizer 55. the remainder of the system shown in Figure 6 may be basically the same as that depicted in Figure 1 for use with a chromatographic column. Thus the scintillation cocktail from reservoir 1 is pumped by pump 10 through conduit 11 to mix with the electrophoresed gel which is extruded or forced into low volume homogenizer 55. The cocktail containing the gel may then be passed through flow cell 14 in a photomultiplier tube assembly (16-16') as previously described. The special flow cell 20 depicted in Figures 2 and 3 and described above, is well suited and highly effective in handling the scintillation cocktail/gel admixture.
The low volume homogenizer is primarily a device designed to provide intimate mixing between the extruded electrophoresed gel from conduit 52 and the scintillation cocktail from conduit 11. Where the flow rate of scintillation cocktail from conduit 11 is large compared to the flow rate of electrophoresed gel from conduit 55, and particularly where the gel is relatively miscible with or dispersible in the scintillation cocktail, a simple homogenizing or mixing device such as a "T" pipe joint may well be all that is needed.
Dispersion of the gel in the cocktail can be enhanced by extrusion through dies having small holes or by use of a rotating cutting or impacting blade or similar device to break the gel. The means previously discussed as improving the mixing of a scintillation cocktail with the effluent from a chromatographic column, e.g., a mixer with a rotating stirring bar, can also be used with advantage.
The embodiment of the invention relating to the use of electrophoresis has many present and potential uses including analysis of many proteinaceous or other macromolecules, such as radioactively tagged or untagged enzymes, antibodies, molecules tagged with antibodies, hormones or hormone-like molecules, and other endogenous or synthetic molecules such as polypeptides.
The invention will be further clarified with reference to the following illustrative embodiments, which are purely exemplary and are not to be taken as limiting the invention.
EXAMPLE 1
A cross check of a continuous radiation analyzer in accordance with the present invention was run against analysis of the same composition by UV spectrometer. A solution of the tritium isotope of spiperone in 70% acetonitrile and 30% (.7%) diethylamine was eluted at 1 ml/min through a 30 cm chromatographic column padded with duPont Zorbax BP ODS chromatographic material. The elution was run at 4500 psig. The effluent stream was monitored by running it through a Waters Associates Model 450 UV
Absorbance detector set to monitor UV light absorbance at 254 mm at 0.1 absorbance units equals full scale. Thereafter the eluate was mixed with 3 ml/min of a scintillation cocktail sold and commercially available under the trademark
ATOMLITE from New England Nuclear and passed through a flow cell having the structure depicted in Figures 2 and 3.The flow cell was made out of 1/32nd inch Teflon (Registered Trade Mark) polytetrafluoroethylene tubing wound around three times and sandwiched between two 1/16th inch thick plates of clear Lucite (Registered Trade
Mark) acrylic plastic. The cell volume was about 400 microliters. The plates of the sample cell were coupled with the photomultiplier tubes by coating them with laboratory grade white mineral oil and utilizing the longitudinal adjustment screw to place the ends of the photomultiplier tubes in direct contact with the plates. The tubes were supplied with power from a Canberra Model 3102 high voltage power supply made by Canberra
Industries. The output signals from each photomultiplier tube were fed to a Canberra Model 814 preamplifier/amplifier discriminator, and the two signals were compared using a Canberra
Model 1446 coincidence circuit.A Canberra
Model 1480 Linear Rate Meter was used to provide the range selection for the chart recorder display, which was run at 5 mm/min.
The results for the UV mass measurement (dotted line) and the radioactivity detector (solid line) are portrayed in Figure 7. Note that there is a slight time delay between the mass measurement peak and the radioactivity measurement peak, caused by the fact that the eluate spends some time in conduits 4, 5, 12, and 13 before reaching the radiation detector at 14. As can be seen from
Figure 7, the continuing radioactivity flow monitor of the present invention was as effective as, if not more effective than, the UV spectrometer mass measurement technique in determining the amount of spiperone in the sample.By following the amount of radiation detected as compared with the total amount of radiation injected, e.g., by use of a radiation counter such as a dual counter/timer (e.g., Canberra Model 1776), which displays total counts and elapsed time, and/or a printing counter such as the Canberra Model 2089
Serial Scanner Printer, which provides a printed record of the number of counts per unit time, it was determined that the counting efficiency of this embodiment of the system was over about 50%, a substantial improvement in tritium counting efficiency over previous continuous scintillation counting systems.
EXAMPLE 2
In this example, an embodiment of the invention was utilized to analyze on a continuous basis an electrophoresis column containing the carbon 1 4 isotope of the protein macroglobulin.
The column analyzed was a 10 cm long tubular column of 5% acrylamide gel, having an inner diameter of 5 mm, and an outer diameter of 7 mm. The tube was extruded out of its container at a rate of 0.1 ml/mjn, using water as the extruding medium. The liquid scintillation cocktail was again NEN's Atomlight with the 10% Protosol to enhance dispersibility of the gel in the cocktail.
A single mixing "T" tube connection was utilized to homogeneously mix the extruded gel with the scintillation cocktail. Figure 8 shows the result utilizing the continuous radioactivity analyzer of the present invention. Figure 9 shows results for the same system obtained by the tedious method of sequential analysis of electrophoresed gel slices, which involves the laborious task of slicing the gel into thin slices, placing the slices into individual liquid scintillation vials, and individually counting the vials for scintillations. In this case the gel was sliced into forty-six individual pieces of 2 mm thickness each, placed in counting vials, and each was counted'in a scintillation counter in turn.
It is apparent from Figures 8 and 9 that the continuous scintillation system of the present invention, applied to the same electrophoresed gel analysis problem, is more accurate than the prior art gel slicing technique, and at a vastly reduced cost in time and manpower.
While a number of embodiments have been described with particularity, other embodiments and advantages will be apparent to the skilled in the art from the present disclosure and from practice of the invention described herein. It is intended that the present disclosure be merely exemplary and not limiting, but that the true scope of the invention is indicated by the following
Claims (16)
1. A radiation detector comprising at least one photomultiplier tube for detecting scintiliations in a flowing fluid, flow cell means for flowing a fluid sample past the photomultiplier tube, which flow cell means comprises a planar spiral of tubing which transmits light, encased on two sides by members which also transmit light, adjusting means for changing the position of the photomultiplier tube relative to the flow cell
means, and means for counting scintillations in the flowing fluid detected by said photomultiplier tube.
2. The radiation detector of Claim 1, wherein the adjusting means is operable to position the photomultiplier tube in direct contact with the flow cell means.
3. The radiation detector of Claim 2, wherein the light transmitting members comprise sheets of transparent material located adjacent to the planar spiral and in planes generally parallel to the planar spiral of tubing.
4. The radiation detection of Claim 3, wherein the flow cell further comprises spacer means located between the sheets of transparent material, for supporting said sheets of transparent material in contact with the planar spiral of tubing.
5. The radiation detector of Claim 2, wherein the tubing has an outer diameter of 0.04 to 0.2 inches.
6. The radiation detector of Claim 3, wherein the sheets of transparent material have a thickness of 0.05 to 0.3 inches.
7. The radiation detector of Claim 3, wherein the internal volume of the tubing with flow cell means is 0.05 to 10 ml.
8. A chromatographic analysis system for analyzing materials containing radioactive components, comprising a chromatographic column, means for eluting a sample of material to be analyzed through the chromatographic column, means for analyzing the composition of sample material eluted from said chromatographic column, means for mixing a scintillation cocktail with at least a portion of the sample eluted from the chromatographic column, a pair of multiplier tubes for detecting scintillations in the mixture of scintillation cocktails and the sample, flow cell means for flowing the mixture of sample and scintillation cocktail past the photomultiplier tubes, which flow cell means comprises a planar spiral of tubing which transmits light encased on two sides by members which also transmit light, means for adjusting the position of the photomultiplier tubes relative to the flow cell means, and means for counting the scintillations in the flowing mixture.
9. The system of Claim 8, wherein that adjusting means is operable to position the photomultiplier tube in direct contact with the flow cell means.
10. The system of Claim 9, wherein the light transmitting members comprise sheets of transparent material located adjacent to the planar spiral and in planes generally parallel to the planar spiral of tubing.
11. A system for analyzing electrophoresed gel for radioactive components comprising mixing means for mixing electrophoresed gel with a scintillation cocktail, means for forcing a length of gel into the mixing means, flow cell means for flowing the mixture of electrophoresed gel and scintillation cocktail past at least one photomultiplier tube, means for detecting scintillations occurring in the flow cell means, and means for counting the scintillation detected.
12. The system of Claim 11 wherein the flow cell means comprises a length of tubing which transmits light encased on two sides by members which are also transparent to light.
1 3. The system of Claim 12 wherein the length of light-transmitting tubing is a planar spiral of light-transmitting tubing, and further comprising adjusting means for adjusting the position of the photomultiplier tube means relative to the flow cell means.
14. The system of Claim 1 3 wherein the lighttransmitting members comprise sheets of transparent material located adjacent to the planar spiral and in planes generally parallel to the planar spiral, and wherein the adjusting means is operable to position the photomultiplier tube means in direct contact with the flow cell means.
1 5. The radiation detector of Claim 1 substantially as described herein with reference to
Figures 2 to 5 of the accompanying drawings, or in
Example 1.
16. The system of Claim 8 substantially as described herein with reference to Figure 1 of the accompanying drawings, or in Example 1.
1 7. The system of Claim 11 substantially as described herein with reference to Figure 6 of the accompanying drawings, or in Example 2.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US30229881A | 1981-09-14 | 1981-09-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
GB2108654A true GB2108654A (en) | 1983-05-18 |
Family
ID=23167143
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB08224522A Withdrawn GB2108654A (en) | 1981-09-14 | 1982-08-26 | Continuous radioactivity flow monitor |
Country Status (3)
Country | Link |
---|---|
JP (1) | JPS5882176A (en) |
DE (1) | DE3233187A1 (en) |
GB (1) | GB2108654A (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4841151A (en) * | 1987-02-13 | 1989-06-20 | Beckman Instruments, Inc. | Variable volume flow cell |
DE4017810A1 (en) * | 1990-06-01 | 1991-12-05 | Raytest Isotopenmessgeraete | ARRANGEMENT AND METHOD FOR MEASURING THE RADIOACTIVITY OF AN ELUATE |
JPH04184287A (en) * | 1990-11-19 | 1992-07-01 | Aloka Co Ltd | Monitor of underwater radioactive substance |
DE19613971C2 (en) * | 1996-04-09 | 1998-12-24 | Forschungszentrum Juelich Gmbh | Production of a flow scintillation cell tube |
DE19743862C1 (en) * | 1997-10-04 | 1998-12-10 | Forschungszentrum Juelich Gmbh | Method for producing a flow scintillation measuring cell |
-
1982
- 1982-08-26 GB GB08224522A patent/GB2108654A/en not_active Withdrawn
- 1982-09-07 DE DE19823233187 patent/DE3233187A1/en not_active Ceased
- 1982-09-14 JP JP57160712A patent/JPS5882176A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
JPS5882176A (en) | 1983-05-17 |
DE3233187A1 (en) | 1983-03-31 |
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