APPARATUS AND METHOD FOR RADIOACTIVITY MEASUREMENT IN LIQUID CHROMATOGRAPHY
BACKGROUND OF THE INVENTION
This invention generally relates to an apparatus and method for measurement of radioactivity in liquid chromatography.
It is well known in the prior art that for accurate quantitation of radioactivity in a liquid sample, one has to measure or count the sample in a liquid scintillation counter for a period of time. The lower the radioactivity, the longer the measurement or counting time in order to achieve certain counting accuracy. This is due to the nature of radiation, which statistically follows the Poisson distribution. Due to the fact that emission from radioactive isotopes is a random process, the accuracy in detection of those isotopes based on the detection of emission depends greatly on the measurement time for the samples. The longer the samples are measured, the more accurate results will be obtained. The percent error based on the 95% confidence interval can be calculated as follows :
Percent error (or %2sigma) =200/ (square root of total counts accumulated)
Therefore, counting for a longer period of time is an effective way and in many cases is the only way to achieve the lower percent error or more accurate results, even though higher counting efficiency and lower counting background also contribute to higher counting accuracy or sensitivity. It is also known that in radio-chro atography a radioactivity flow-through detector with either a solid or a liquid cell is used to detect the radioactivity in an eluate. Since this is a dynamic process, the eluate flows through the flow-through detector constantly. Therefore the residence time of the radioactivity being measured inside the measuring chamber of the radioactivity detector is limited, and the accuracy and sensitivity of the measurement are also limited, especially when the level of radioactivity is low. One way of increasing residence time is to increase the cell volume. However, this approach will result in poor chromatographic resolution and also increase the cost.
In the prior art, one patent (US patent 5,166,526 by Dietzel) tried to improve the counting accuracy by counting the radioactive peaks in a multiple flow-through radioactivity detector arrangement. However, the proposed arrangement is not practical due to many disadvantages such as only being able to quantitate a limited number of peaks with a relatively high level of radioactivity, the higher cost associated with the implementation, and no radioactivity recovery data available using this method. In a practical analysis, since the positions or retention times, radioactivity level, and shapes of the peaks are unknown in most cases, every portion of the chromatogram needs to be counted accurately in order to obtain a true radio-chromatogram.
Another attempt to improve the measurement of radioactivity in an eluate was made in a patent in the prior art (US patent 5559324 by Rapkin) by using an external standard placed adjacent to the measuring cell. The low sensitivity problems associated with the flow-through detector was not solved since the residence time of the eluate being measured inside the cell was limited under the dynamic flow conditions. Therefore, the best approach to solve this problem in the prior art is to first collect fractions of the eluate into scintillation vials using a fraction collector, then mix each eluate fraction with a volume of scintillation cocktail, and finally measure the radioactivity of those fractions in a stationary manner in a liquid scintillation counter (LSC) . Since each fraction can be measured in a stationary manner for a longer period of time, the required counting accuracy can be achieved for each eluate fraction. The results from those fractions then are used to reconstruct the radio-chromatogram. The disadvantages associated with this method are obvious:
(a) Involving intensive manual operations and increasing the cost per analysis which results in poor productivity. (b) Generating a great amount of radioactive solid and liquid wastes including scintillation vials, contaminated scintillation cocktail and solvents. (c) Increasing the potential of creating unsafe working environments .
Eliminating this fraction collection/LSC operation in modern laboratories would improve not only the productivity but also working environments related to workers and to the public in general due to the benefit of waste reduction. Consequently, a need exists for new techniques in radioactivity measurement in liquid chromatography, which not only would have greater accuracy and sensitivity of radioactivity measurement for all desired portions of chromatogram, but also are operated in an automated and environment friendly manner.
SUMMARY OF THE INVENTION
The present invention provides a stop-flow radioactivity measurement apparatus and method designed to satisfy the aforementioned needs. The stop-flow radioactivity measurement is carried out by a stop-flow system that stops and resumes the flow of eluate repeatedly in order to measure the radioactivity in each desired fraction or portion of an eluate in a stationary manner. Since the measurement time devoted for any fractions, peaks, or regions of a chromatogram is controllable, the accuracy and sensitivity of the measurement will increase dramatically. Furthermore, no regions in the chromatogram will be missed for accurate stationary measurement, thus providing a true on-line radioactivity detection method with high accuracy and sensitivity for liquid chromatography. With this invention, the fraction collection/LSC method used in the prior art can be completely eliminated, thus reducing the radioactive wastes and improving the working environments and productivity. Also, the cost associated with the implementation of current invention is much less than that of the prior art procedure.
Accordingly, the present invention relates to apparatus and method for measurement of radioactivity in liquid chromatography, which include the operative steps of (a) stopping the flow of the eluate and (b) measuring the radioactivity of the eluate present in the radioactivity detector in a stationary manner while the flow of the eluate stops, whereby the radioactivity in the eluate will be measurable for a longer period of time. The invention may also include the step of resuming the flow of the eluate from liquid chromatography to introduce the following fraction into the radioactivity detector for stationary measurement. The stop-flow operation can be activated by a timed sequence, radioactivity level detected by the radioactivity detector or an external signal. The flow cell can be cleaned, if necessary, by flushing the cell with a solvent or liquid scintillation cocktail prior to the introduction of the following fraction.
The current invention also provides a method which includes the operative steps of (a) collecting fractions of an eluate from liquid chromatography in a storage device and (b) feeding the fractions individually back into the radioactivity detector for counting in a stationary manner. Again, the operation as to when and where to collect fractions can be activated by either a timed sequence or other signals such as radioactivity signal observed in the radioactivity detector.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the first embodiment of the stop-flow radioactivity counting apparatus pursuant to the present invention; FIG. 2 shows the second embodiment of the stop-flow radioactivity counting apparatus of the present invention;
FIG. 3 shows the detailed sequences for a typical stop-flow radioactivity counting process of the present invention;
FIG. 4 shows the detailed sequences for another mode of operation for counting only the peaks in the present invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Referring to the drawings, and more particularly, FIG. 1, there is schematically shown the stop-flow radioactivity counting apparatus for detecting radioactivity from an eluate in liquid chromatograph. The apparatus basically comprises of pump 1 for delivering mobile phase (s), column 10 for fractionation of components of a sample, radioactivity detector 17 for measurement or counting of radioactivity, and control arrangement 35 for data collection and communication with various devices involved in the present invention.
Pump 1 can include one or more pumps for delivering a isocratic (one composition of solvent throughout the entire analysis or run) or a gradient of mobile phase. In the preferred embodiment, pump 1 is being controlled by control arrangement 35 for the stop-flow operation. Column 10 is usually a liquid chromatography column which comprises of a tubing and a packed stationary phase for fractionation of components of samples. Column 10 can be a analytical, narrow- bore, micro-bore, or semi-preparative LC column (LC equals to liquid chromatography) .
Radioactivity detector 17 can be a radioactivity flow-through detector suitable for high pressure liquid chromatography. Radioactivity detector 17 can also be a normal liquid scintillation counter as long as the flow cell is placed between two photomultiplier tubes. Radioactivity detector 17 can have a liquid cell or a solid cell or the combination thereof. The counting data from radioactivity detector 17 can be either digital format or a analog format which will be digitized in the control arrangement.
Control arrangement 35 communicates with pump 1 through signal line 34, with radioactivity detector 17 through signal line 14, with sample injector 3 through signal line 4, valve 47 through signal line 6, with pump 26 through signal line 48, with detector 12 through signal line 13, valve 18 through signal line 46, and fraction collector 22 through signal line 16.
Valve 47 is preferred in order to stop the flow of mobile phase completely to achieve the best performance in separation under stop-flow operation. Valve 47 can be a on/off valve or switching valve which enables the flow at one position and disables or block the flow of mobile phase (s) at another position, which is preferably disposed immediately after column 10. However, valve 47 can also be disposed immediately after radioactivity detector 17 if radioactivity detector 17 and other devices between radioactivity detector 17 and column 10 can tolerate the pressure during the stop-flow operation.
Pump 26 is used for pumping either scintillation fluid or non- radioactive solvent (s) or a solvent mixture in container 24 through line 25 and to radioactivity detector 17 through line 27 for either liquid scintillation counting purpose or flushing out the residual radioactivity residing inside the flow cell of radioactivity detector 17. No scintillation fluid is needed for a solid cell.
Sample injector 3, disposed between pump 1 and column 10 via lines 50 and 37, is either a manual injector (such as a loop on a multiple port switching valve) or automatic injector (auto-sampler) . Detector 12, such as a U (ultraviolet) detector, can be connected on-line between valve 47 and radioactivity detector 17 through lines 42 and 43. The connection lines between the column 10 and valve 47 is 41. Fraction collector 22 and waste container 20 are connected through lines 21 and 19 from valve 18 which is connected to the outlet of radioactivity detector 17 through line 28.
The stop-flow radioactivity counting method can be used in several different modes using the embodiment of FIG. 1. The first mode is to measure radioactivity by counting every fraction eluting from an entire LC run. The second mode is to count only the peaks which exceed predefined criteria (either by a threshold or other algorithms) . The third mode is to count only the regions of interest of the LC run. Any combination of these three modes can be used as well. Here are shown only the details of those common modes of operation. However, many variations of this stop-flow counting method are possible without departing from the spirit of the present invention.
The stop-flow radioactivity counting method for counting every fraction of an entire LC run using embodiment of FIG. 1 is shown in FIG. 3. FIG. 3 shows the beginning part of the operation sequences for a typical stop-flow radioactivity counting method with a predefined time interval for fractions. FIG. 3A shows the change of the mobile phase composition in pump 1 under a linear gradient. If a isocratic mobile phase is used, the line in FIG. 3A becomes a horizontal line parallel to the time (horizontal) axis. FIG. 3B shows the flowrate of mobile phase delivered by pump 1. FIG. 3C shows the flowrate of pump 26. FIG. 3D shows the radioactivity counting operation in radioactivity detector 17. FIG. 3E shows the results of the stop-flow radioactivity counting representing for the radioactivity in fractions FI through F5.
Before the sample is injected, valve 47 is at the open position and the pump 1 is at the initial conditions of the liquid chromatography. The gradient is divided into equal or different time intervals or fractions for counting radioactivity by radioactivity detector 17 in a stationary manner. As an example, the first five fractions (FI through F5) are shown in FIG. 3B. Fraction FI contains the eluate collected in the flow cell (either a solid or a liquid cell) of radioactivity detector 17 between time points TO and Tl . Fraction F2 is the eluate collected from time points Tl through T2, etc.
When a sample is injected onto sample injector 3, a signal is generated and sent through signal line 4 to control arrangement 35 which triggered pump 1. Alternatively, the injection signal can be sent directly from sample injector 3 to pump 1 through signal line 49 for triggering of the run. Once pump 1 is triggered, the solvent elution or gradient will start. The sample is pushed onto column 10 for fractionation and the eluate is collected in the flow cell (not shown) of radioactivity detector 17. If a liquid cell is used, pump 26 also starts pumping liquid scintillation fluid which is mixed with eluate from line 43 before the liquid cell. If a solid cell is used, pump 26 does not pump anything into radioactivity detector 17. When time point Tl is encountered, control arrangement 35 will send a stop-flow signal to pump 1 to stop the flow of the mobile phase through signal line 34, and both flow and gradient are stopped or paused. At the same time, if valve 47 is used, control arrangement 35 also sends a signal through line 6 to turn valve 47 to the close or off position in order to stop the flow of mobile phase completely. Once the flow is stopped, radioactivity detector 17 starts measuring (or counting) the radioactivity contained in the flow cell (from time points Tl to a) ) . The radioactivity is measured by detecting the flash of photons generated by the interaction of radiation particles (such as beta particles from carbon-14 isotopes, etc.) with either liquid or solid scintillator by using usually a pair of photomultiplier tubes inside radioactivity detector 17. This process is also called counting radioactivity because the measurement process is actually counting the radiation events associated with the decay of radioactive isotopes. Since the counting time can be controlled by the user, any desired levels of accuracy on the radioactivity counting or measurement can be achieved. The more counts are accumulated or the longer the fraction is counted, the more accurate results one will obtain from radioactivity detector 17 for the fraction. This is due to the nature of the radiation which is a random process and follows the distribution curve of the Poison distribution statistically. The counts accumulated from this entire counting period is calculated as the mean to represent the radioactivity of fraction FI . The percent error of the counting, together with other parameters, can be calculated as well to indicate the performance of the counting process .
After accumulating the counts for either a predefined period of time (e.g. 2 min) or based on predefined criteria for counting accuracy or counting errors, time point a) is encountered. In order to remove the residual radioactivity residing inside the flow cell and eliminate any possible cross contamination for the next incoming fraction, pump 26 starts pumping at the same or different flowrate. Pump 26 will pump liquid scintillation fluid if a liquid cell is used. If a solid cell is used, pump 26 will pump a non-radioactive solvent or solvent mixture. The pumping operation of pump 26 between points a) and b) can be eliminated if the volume of the fraction (for a solid cell) or the combined volume of the fraction and the liquid scintillation fluid (for a liquid cell) approximately equals to the cell volume of radioactivity detector 17. This pumping operation is designed to remove any residual radioactivity out of the either solid or liquid cells before the introduction of the next new fraction (e.g. fraction F2) . At time point b) , the flow and gradient (if any) , of pump 1 is resumed for another cycle. If a solid cell is used, pump 26 stops pumping any solvent. As we can see, this stop-flow radioactivity counting method cycle includes the following steps: collecting the eluate into the flow cell; stopping the flow and stopping (pausing) the gradient (if any) ; counting the radioactivity inside the flow cell in a stationary manner; removing the residual radioactivity from the flow cell; and finally resuming the flow and gradient (if any) of pump 1 and pump 26 (if a liquid cell is used) . When the gradient is resumed, it starts from the point where it was stopped previously. This will ensures the best performance in separation and resolution comparable to the prior art continuous run or analysis.
After finishing the stop-flow counting cycle for fraction FI, next cycle continues for next fraction F2, etc. In FIG. 3, we observed five counting cycles (from time points TO through n) ) , each of which counts the corresponding fraction (FI through F5) . The results of the counting cycles are shown in the radio-chromatogram in FIG. 3E, which has five data points, each of which representing the counting results from corresponding fractions (FI through F5) . In this example, a peak is accurately detected in fraction F4 at time point T4 of FIG. 3E . The time scale on FIG. 3A is the time scale for a continuous run. The time scales in FIG. 3B through FIG. 3D are the actual time scales during the stop-flow counting process. The time scale in FIG. 3E is the reconstructed time scale by eliminating the time spent during the stop-flow counting cycles. The reconstructed radio-chromatogram represents the radioactivity distribution of fractions in FIG. 3A. There was not changes in the chromatographic separation and retention times using the stop-flow counting method comparing to the normal continuous run in the prior art. One explanation is that the binding force of the sample components toward the bonded phase of column 10 is stronger than the diffusion force of the components inside column 10. Furthermore, the sensitivity and accuracy of radioactivity counting is dramatically improved due to the fact that the counting time for each individual fraction of the eluate can be lengthened as long as the user preferred.
FIG. 4 shows the sequences for counting only the peaks which exceed the predefined criteria (either by a threshold or other algorithms) . As an example, the threshold is used to detect the peaks. FIG. 4A shows the radio-chromatogram of a sample containing two radioactive peaks if the sample were run under normal method in the prior art (i.e. continuous run with a on- line radioactivity flow-through detector) . The threshold is preset to recognize any peaks for stop-flow counting. FIG. 4B shows the flowrate of pump 1. FIG. 4C shows the flowrate of pump 26 for delivering liquid scintillation fluid. If a solid cell is used, pump 26 only delivers the non-radioactive flushing solvent (s) after counting of each fraction and before introduction of next new fraction. In the other words, pump 26 pumps solvent (s) only between time points p) and q) , s) and t) , and v) and w) , etc. FIG. 4D shows the counting operation in radioactivity detector 17. Basically, all the data from the entire run are being used to generate the radio-chromatogram as shown in FIG. 4E . The data acquired while the pump 1 stops is calculated as a single data point (as the mean) to represent the corresponding fraction inside the flow cell. FIG. 4E shows the reconstructed radio-chromatogram obtained from this counting method.
After the sample is injected onto sample injector 3, a signal is generated and sent to control arrangement 35 which in turn sends a signal to pump 1 to start the flow and gradient (if . any) . At the same time, pump 26 starts pumping liquid scintillation fluid to radioactivity detector 17 if a liquid cell is used. If a solid cell is used, pump 26 does not pump anything. When radioactivity detector 17 detects Peak 1 (based on either a higher radioactivity than the preset threshold or other algorithms such as changes in slope, etc.), a fraction of eluate, the size of which is determined either by the predefined time intervals or levels of radioactivity detected or detection of the end of the peak. After a fraction of eluate is collected, the control arrangement will stop the flow of pump 1 and the gradient (if any) and count the radioactivity inside the flow cell for a predefined period of time or to accumulate enough counts to reach certain levels of counting accuracy.
In this example, fraction F6, containing eluate from time points TO' through Tl' , is counted first (from time points Tl' to p) ) . The accumulated counting data are calculated as the mean which represents the radioactivity level of fraction F6, which is shown at time point x) in FIG. 4E . After the counting of fraction F6 is finished, the content of the flow cell is flushed out by solvent (s) delivered by pump 26 between time points p) and q) . At time point q) the flow and gradient (if any) of pump 1 is resumed until time point r) , where fraction F7 (eluate from time points Tl' to T2' ) is ready for counting.
Fraction F7 is subjected to a similar counting cycle as shown for fraction F6 from time points r) to t) , resulting the data at time point y) calculated as the mean. When no peak is detected, the flow of pump 1 continues until the next peak (Peak 2) is encountered. In this example, Peak 2 is narrow enough so that only one fraction (fraction F8) containing eluate from time points T3' through T4' is needed to count the entire peak. The entire Peak 2 is counted in a stationary manner in the counting and flushing cycle between time points of u) and w) . The obtained data is presented at time point z) in the radio-chromatogram of FIG. 4E .
The accumulated counts for each fraction is calculated as the mean to represent the radioactivity of that fraction and positioned in the radio-chromatogram at the center position of that fraction on the time axis. In other words, radioactivity data from fraction F6 is positioned at the center (time point x) ) in the fraction between time points TO' and Tl' on the time axis and data from fraction F7 is positioned at the center between time points T3' and T4', etc. The counting data which are obtained by radioactivity detector 17 during the regions where no peaks are detected and counted, will be treated as raw data in the normal manner as used in the prior art.
This counting cycle can be applied to all the peaks detected in a LC run. The accurate quantitation of radioactive peaks are thus possible by my stop-flow counting method. A broad peak can be counted in several consecutive fractions. A sharp or narrow peak can be counted in a single fraction. Since the counting time can be controlled, any desired levels of counting accuracy and sensitivity can be obtained. Furthermore, since the fraction size is controllable, the desired resolution of radio-chromatogram can be achieved for any peaks .
For those who have ordinary skills in this field, it is easy to understand that many variations of this method can be used. Based on the same principle, a region or regions of the interest during a LC chromatogram can be measured accurately for radioactivity. Similarly, when the beginning of the region (s) is encountered, the control arrangement will send signals to stop the flow and pause the gradient through signal lines 34 and 6 and radioactivity detector 17 starts the counting process in a stationary manner for a period of time. Depending on the width of the regions of interest, each region can be counted as more than one fraction.
As shown in FIG. 1, fraction collector 22 and waste container 20 can be used to collect useful radioactive components detected by radioactivity detector 17. Valve 18 is a three- way valve which is controlled by control arrangement 35 through signal line 46. Fraction collector 22 is controlled also by control arrangement 35 through signal line 16.
The components of interest in the eluate can be collected using fraction collector 22. The fraction collection can have several modes of operation. The first one is to collect peak(s). When a peak is detected in radioactivity detector 17 based on predefined criteria, the eluate is directed to the fraction collector through the valve after a delay time which compensates the time needed for eluate traveling from the outlet of radioactivity detector 17 to valve 18. When the end of the peak is detected, the eluate is directed to the waste container through valve 18 after the delay time. The second mode is collection of predefined regions of interest. Again, when the beginning of region (s) of interest is encountered, the control arrangement will send signals through line 46 which turns the valve toward the fraction collector. When the end of the regions of interest is encountered, the valve is turned back to the waste container by control arrangement 35 through signal line 46 for collection of waste. This is an excellent tool to collect the needed peaks or components for further analysis by other means such as mass spectrometry or NMR (nuclear magnetic resonance) . This method is also good for waste management where separation of high level of radioactive wastes from low level or non-radioactive wastes are desired, thus saving cost in waste disposal. Coupling of line 21 in FIG. 2 to a LC/MS (liquid chromatography-mass spectrometry) is also possible and will provide a unique way of analyzing peaks more efficiently. This method is especially useful for a LC/MS/MS experiment where several modes of operation are needed for a single peak which is usually eluting from a column too fast and several runs of the same sample are needed to obtain the desired results. With stop-flow counting method, the peak(s) can be stopped allowing MS to have enough time to conduct a series of experiments on a single peak detected by the method of present invention.
FIG. 2 shows another embodiment of the present invention. In addition to the devices shown in FIG. 1, valves 9, 2 , 44 and storage devices 31 and 33 are incorporated. Valves 2 and 44 are two position switching valves (such as the four-port switching valves from Valco Instruments Co. Inc., P.O. Box 55603, Houston, TX 77255) . Valve 9 is a multiple position valve (such as the one similar to Valco stream selection valve for selection of up to six LC columns, part number: DCST6UW) for column selection. Storage devices 31 and 33 are multiple position valves (such as the one similar to the multiposition stream selection valve from Valco Instruments Co. Inc., part number: DCST16MWEY) .
Storage device 31 or 33 comprises of multiple capillary tubing for storage of eluate fractions. Depending on the desired number of capillary tubing used, one such device can be sufficient. If more than one storage devices are used, they can be connected in series or parallel to each other.
Valve 2 is disposed through line 37 immediately before valve 9 where multiple LC columns can be connected. In FIG. 2, column 10 is connected through lines 41 and 40 and column 11 is connected through lines 38 and 39. Valve 44 is disposed between radioactivity detector 17 through line 28 and valve 18 through line 45. Valves 2 and 44 is connected through line 8. Valve 2 is connected with control arrangement 35 through signal line 5. Storage devices 31 and 33 are connected in series and connected to valve 2 and 44 through lines 23, 32 and 7.
The stop-flow radioactivity counting method using embodiment of FIG. 2 is shown as follows. Since storage devices 31 and 33 can store the fractions of the eluate into the capillary tubing, the eluate can be collected and stored in the storage devices and counted after the LC run is finished. The size and length of the capillary tubing on storage devices 31 and 33 can vary depending on the fraction size and total number of fractions per LC run.
At default, valve 2 is at the position connecting lines 36 and 37 and valve 44 connecting lines 28 and 23. In the other words, the eluate flows through the following lines and devices sequentially: 1, 50, 3, 36, 2, 37, 9, 40, 10, 41, 42, 12 (if any), 43, 17, 28, 23, 33, 32, 31, 7, 8, 45, 18, 19, and finally arrives waste container 20.
Once a sample is injected into the LC system through sample injector 3, a sample injection signal is generated and sent through line 4 to the control arrangement. Control arrangement 35 then sends a signal through line 34 to trigger pump 1 to start the run. The control arrangement 35 will turn storage device 33 to the first position to collect eluate which fills the first capillary tubing through signal line 29. After the predefined time interval for collection of the first fraction, the control arrangement 35 will turn the storage device 33 into its second position to collect the second fraction, etc. When all the capillary tubings on storage device 33 are filled with eluate fractions, the control arrangement 35 will turn storage device 31 through signal line 30 to its first position and so on until all the desired fractions are collected.
After the LC run is finished, each of the fractions collected and stored in storage devices 33 and 31 is individually pushed back to radioactivity detector 17 for radioactivity counting in a stationary manner. In order to accomplish this, the control arrangement 35 will first turn valves 2, 44 (through signal line 15) and 9 such way that the flow path of the solvent from pump 1 is going through the following lines and devices: 1, 50, 3, 36, 2, 7, 31, 32, 33, 23, 8, 2, 37, 9 (without going through any columns) , 42, 12, 43, 17, 28, 44, 45, 18, 19, and waste container 20. Then the control arrangement will trigger pump 1 to deliver mobile phase at certain flowrate. The control arrangement 35 will turn storage device 33 to its first position to feed the first fraction back to radioactivity detector 17. A delay time is calculated by the control arrangement based on the flowrate of pump 1 and volume of the flowpath from storage device 33 through lines or devices of 23, 44, 8, 2, 37, 9, 42, 12, and 43. If a liquid cell is used, the LSC pump will start pumping the liquid scintillation fluid preferably just before the fraction arrives the flow cell. After the delay time, the flow is stopped by control arrangement 35 and the fraction is counted in radioactivity detector 17 in a stationary manner for a period of time and the data is collected through line 14. The counting time is predefined or judged by the control arrangement based on, for example, predefined intervals, the counting accuracy or magnitudes of counting errors. After the counting step is finished, pump 26 will flush out any residual radioactivity inside the liquid cell at preferably a higher flowrate in order to save time. For a solid cell, pump 26 will be pumping a non-radioactive solvent or a solvent mixture instead of liquid scintillation fluid which is not usually comparable with a solid cell. If the volume of the flow cell equals to the volume of the fraction in case of a liquid cell or the combined volume of the fraction and the liquid scintillation fluid in case of a liquid cell, this flushing step can be eliminated since the following fraction will replace the previous fraction inside the flow cell. This is the counting cycle for counting one fraction stored in storage device 33.
Similarly, control arrangement 35 will send a signal to pump 1 to resume the flow of the mobile phase and repeat the cycle described above to count the radioactivity of the subsequent fractions one by one. After all the fractions are counted, a radio-chromatogram containing all the counting results, each of the data points representing the counting results of each fraction, can be obtained.
The desired radioactive peaks or regions of interest can be collected into fraction collector 22 which is controlled by control arrangement 35 through line 16. When peak(s) is detected in radioactivity detector 17, control arrangement 35 will turn valves 44 and 18 such way that the eluate will go through: 28, 44, 45, 18, 21 and finally to fraction collector 22. If more than one peaks are being collected, the control arrangement will advance the fraction collector to the next tube or vial by sending a signal through line 16. Same procedures can be used for collecting region (s) of interest. Moreover, more than one fraction can be collected from one region of interest.
It is possible to divide one fraction into several portions with same or different sizes and count them individually. This will result in better resolution on the radio-chromatogram. In this case, only a fraction of the whole fraction content stored in each individual capillary tubing will be fed into radioactivity detector 17 for counting in a stationary manner. Between each portion of whole fraction, no delay time is needed since the whole fraction migrates together toward radioactivity detector 17. In order to minimize the diffusion of the radioactive components in the capillary tubing, one fraction in one tubing is preferred.
It is also possible to only collect the peaks detected by radioactivity detector 17 or the regions of interest into the storage devices and the rest of the eluate goes to waste container 20 or fraction collector 22. The use of a solid cell is preferred in this method due to the fact that most of the liquid scintillation fluids generates back pressure when flowing through a narrow tubing. In order to accomplish this, the control arrangement 35 will turn valves 44, 2, 9, storage devices 33 and 31 in such way that before the run the mobile phase will use the following flow path going sequentially through parts of 1, 50, 3, 36, 2, 37, 9, 40, 10, 41, 42, 12, 43, 17, 28, 44, 45, 18, 19, and 20. When the beginning of the radioactive peaks or regions of interest is encountered, control arrangement 35 turns storage devices 33 and 31 to their first positions and will turn valve 44 to connect lines 28 and 23, the peak or the region of interest is collected into the first capillary tubing of storage device 33. If the first capillary tubing is full and the peak or region of interest is not finished, control arrangement 35 will turn storage device 33 into the next position to continue the collection, etc. When the peak or the region of interest is finished, control arrangement 35 will turn valve 44 back to connect line 28 to line 45 until the next coming peak or region of the interest, if any. This process is repeated until all the peaks or regions of interest is collected and stored in the capillary tubings on storage device 33 and/or 31.
The collected peaks or regions of interest on storage devices 33 or 31 can be counted for radioactivity in a stationary manner in a similar way as described above for counting fractions stored on storage devices 33 or 31. The content of each capillary tubing can be counted once or divided into many portions and counted separately. After each counting process, the flow cell can be flushed with a organic solvent or solvent mixture delivered by pump 26. This will ensure the elimination of the possible cross contamination resulting from the residual radioactivity from the previously counted sample.
The detailed description of the present invention as shown above will enable anybody with ordinary skills in the art to make and use the present invention without any undue experiments .
It is thought that the stop-flow measurement apparatus and method of the present invention and many of its attendant advantages will be understood from the foregoing description and it will be apparent that various changes may be made in the form, construction and arrangement of the parts thereof without departing from the spirit and scope of the invention or sacrificing all of its material advantages, the form hereinbefore described being merely a preferred or exemplary embodiment thereof.
REFERENCE NUMERALS IN DRAWINGS (CONTINUED) No. Part Name No. Part Name
39 line F5 fraction
40 line F6 fraction
41 line F7 fraction
42 line F8 fraction
43 line TO time point
44 valve Tl time point
45 line T2 time point
46 signal line T3 time point
47 valve T4 time point
48 signal line T5 time point
49 signal line TO' time point
50 line Tl' time point
T2' time point
FI fraction T3' time point
F2 fraction T4' time point
F3 fraction a)-n) time points
F4 fraction p)-z) time points
REFERENCE NUMERALS IN DRAWINGS
No. Part Name No. Part Name
1 pump 20 waste container
2 valve 21 line
3 sample injector 22 fraction collector
4 signal line 23 line
5 signal line 24 container
6 signal line 25 line
7 line 26 pump
8 line 27 line
9 valve 28 line
10 column 29 signal line
11 column 30 signal line
12 detector 31 storage device
13 signal line 32 line
14 signal line 33 storage device
15 line 34 signal line
16 signal line 35 control arrangement
17 radioactivity detector 36 line
18 valve 37 line
19 line 38 line