Measuring and testing – Liquid analysis or analysis of the suspension of solids in a... – Content or effect of a constituent of a liquid mixture
Reexamination Certificate
2001-03-28
2003-04-15
Williams, Hezron (Department: 2856)
Measuring and testing
Liquid analysis or analysis of the suspension of solids in a...
Content or effect of a constituent of a liquid mixture
C250S328000
Reexamination Certificate
active
06546786
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for detecting and measuring radioactivity in liquid samples and devices for detecting and measuring radioactivity in liquid samples. Particularly, the invention relates to methods of detecting and statically counting concentrations of radioisotopes, and devices for detecting and statically counting concentrations of radioisotopes.
BACKGROUND
Radioisotopes are an important tool in many industries and various types of research, including medical research. For example, radioisotopes are used to label developmental compounds to understand their pharmacokinetics and how they are metabolized in animals and humans. In agrochemical research, radioisotopes are sometimes an important tool for complete understanding of the chemical behavior in the environment. Common radioisotopes used in research include
14
C and
3
H.
High performance liquid chromatography (HPLC) is widely used for detection and quantifying of radioisotope compounds. Radioactivity flow-through detectors are used to detect radioactive components in an HPLC eluate containing radioisotopes and radioisotope-labeled compounds. There is constant need for improved devices and methods for detecting and quantifying radioisotopes and radioisotope-labeled compounds.
Radioactive decay of a radioisotope is a random process following a Poisson distribution where the standard deviation, &Sgr;, equals the square root of the total number of times a radioactive decay was observed, or counted, during a counting experiment:
&Sgr;={square root over (Total Number of Decays)}
This formula is the basis for calculating the practical lower radioactivity detection limit of a sample containing radioisotopes when combined with information about background levels of radiation. The standard deviation, A, of background radiation produced by a reference sample is expressed in the number of times a radiative decays is measured in the reference sample, i.e. disintegrations per min, “DPM.” The scope of radioactivity detection is then expressed using the following equation:
Ld
∝
B
E
*
T
wherein Ld is the lower limit of detection measured in DPM, B is the background radiation level measured in DPM, E is the counting efficiency measured as a percentage, and T is the length of time spent measuring radioactive decay, i.e. counting time, measured in minutes.
As shown by the formula, the limits of radioactivity detection can be improved by reducing the background level of radioactivity, increasing the counting efficiency of the system, and/or increasing the counting period. Improvement of any one of these parameters would lower the limit of radioactivity detection and thereby enable the measurement of lower concentrations of radioisotopes in a sample.
Several attempts have been made to increase detection sensitivity by reducing background radioactivity levels, increasing counting efficiency, and/or increasing the counting time. For example, Packard Instruments attempted to reduce the background level of radioactivity and thereby lower the limit of radioactivity detection by using time-resolved background reduction technology. Similarly, liquid scintillants can be used to replace solid scintillants for liquid cells in order to increase counting efficiency.
Conventional methods of improving detection sensitivity also involve taking fractions from a continuously running chromatography column and analyzing them in batches, i.e. taking them “off-line,” in order to increase counting time. Off-line methods are undesirable because they can not detect and quantitate volatile radioactive components with a very high degree of accuracy because the volatile components tend to evaporate during the fraction collection process.
Conventional methods of increasing counting time are accomplished by collecting eluate in fractions in individual vials using a fraction collector or well fraction collector. Scintillate is then added “off-line” so that the number of radioactive decays can be measured. This manual process is not desirable in modem laboratories because it is labor intensive, inefficient, and costly. An exemplary “off-line” counting process is accomplished by manually collecting fractions in a large number of well plates, such as 96 well plates. The eluate is evaporated and manually counted statically using a multi-well counter.
A stop flow apparatus for determining radioactivity of chromatographic samples is disclosed by Lee, D. Y., PCT/US98/20324 filed Sep. 25, 1998, incorporated herein by reference in its entirety.
Conventional methods have also attempted to lengthen counting time without using a fraction collector. However, these methods fail to accurately detect all radioactive components in samples that contain a number of radioactive components and where the radioactivity of each component is different. Examples of typical methods are provided by A. C. Berick (U.S. Pat. No. 4,137,451. 1981) herein incorporated by reference in its entirety, Berthold (U.S. Pat. No. 4,704,531. 1987) herein incorporated by reference in its entirety, and Dietzel (U.S. Pat. No. 5,166,513. 1992) herein incorporated by reference in its entirety.
A. C. Berick (U.S. Pat. No. 4,137,451. 1981) collects fractions in rotating devices with many tubes that contain scintillant. The tubes are then measured sequentially. Berthold (U.S. Pat. No. 4,704,531. 1987) detects radioactive peaks using a mass detector such as an ultravoilet (UV) detector and then diverts radioisotope containing fractions as they appear as peaks on the UV detector to a radioactivity detector for static counting. In addition, Diezel (U.S. Pat. No. 5,166,513. 1992) detects radioactive peaks using a monitor radioactive detector instead of using a ultraviolet detector, and then diverts radioisotope containing fractions into a secondary radioactivity detector for static counting. In Diezel's attempt, when multiple radioisotope containing fractions elute, the second and subsequent fractions are diverted to a third radioactive detector and subsequent radioactive detectors for static counting. Diezel's method also has the undesirable problem of increased difficulty of recognizing peaks of radioisotope containing fractions on the monitor as radioactivity levels get closer to background levels of radioactivity. Therefore, methods and devices that address these needs have long been sought.
SUMMARY OF THE INVENTION
An apparatus and method for accurate radioisotope counting in radio-LC is described. An chromatogram or a region of chromatogram is divided into multiple fractions. Each fraction is fed/delivered precisively into the transparent and effective section of the flow cell before conducting static counting. When a radioactive fraction is detected, the content of the flow cell is flushed out using either scintillant, solvent, or a gas. Thus, in some embodiments, the current invention includes following steps: a) flow, b) stop-flow, c) precise positioning of the fraction, d) static counting, and e) memory effect removal. In some embodiments Step c) can optionally be eliminated if a fine tube is positioned right at the beginning of the effective section of the flow cell to flush the flow cell with either a scintillant, solvent or gas. The sensitivity and accuracy are improved significantly.
In some embodiments of the invention, methods are provided for measuring radioactivity in an eluate from a chromatography column comprising the steps of:
a) providing a liquid chromatograph comprising:
(i) a chromatography column;
(ii) a radioactivity detector having a flow cell, said flow cell having a radiation detection area;
(iii) a conduit for flowing eluate from said chromatography column into said flow cell of said radioactivity detector;
(iv) means for introducing a gas disposed in either:
1) said conduit; or
2) said flow cell; and
(v) a controlable source of said gas;
b) flowing said eluate from said chromatography column through said conduit and into said flow cell;
c) stopping said flow of said eluate;
d) counting the radioactivity of said eluate in sa
Politzer Jay L
Straher Michael P.
Williams Hezron
LandOfFree
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