Measuring and testing – Instrument proving or calibrating – Gas or liquid analyzer
Reexamination Certificate
1999-09-07
2003-01-07
Williams, Hezron (Department: 2856)
Measuring and testing
Instrument proving or calibrating
Gas or liquid analyzer
C073S023410, C073S023420, C073S864830, C073S863730, C210S198200, C250S252100, C422S089000, C436S180000
Reexamination Certificate
active
06502448
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to detecting emissions in eluate from a chromatography column, and in particular, to devices for incorporating test samples directly into the flow of the eluate.
2. Description of Related Art
Known emissions detectors can detect activity in the eluate from a chromatography column. These detectors allow eluate to flow through a sample cell located between a pair of photomultipliers. The eluate produces scintillations due to the interaction of radioactive components and solid scintillator located in the sample cell, or because a liquid scintillator is mixed with the eluate upstream of the sample cell. Counts thus detected can be analyzed by a computer-based system that can graphically present the counts as a function of time or as a function of energy. These known analyzers can perform various calculations with the data, and can correct the data based on various criteria.
Injector valves are commonly used in high-performance liquid chromatography (HPLC) as well as many GC systems and other analytical instruments. In HPLC, the injector valves are placed ahead of the chromatography column and serve as the means for placing a measured amount of sample into the column at a precise time. Such valves typically have a single or dual external loops. Loop volumes from perhaps 5 to 100 &mgr;L are common, but larger ones can be had. For smaller volumes a groove of suitable size is machined into the valve rotor and there is no external loop.
Multiport valves with or without sample loops have other uses. They have been used as switching valves to interchange chromatography columns while using a single pumping system. They have been used to direct the eluate exiting a column from one detector to another. They have been used to load two columns simultaneously, and to backflush one column while a second is actively used. They have been used to switch from one mobile phase to another.
Conventionally, means are provided to fill a loop while still maintaining mobile phase flow through the column. At the start of a run, a rotor in the valve is moved, which changes the porting so that mobile phase flow is now directed through the loop thereby washing its contents into the column. The valves are made to have minimum dead volumes so that sample is not lost in them and is expressed into the HPLC column without being spread out. Typically they withstand pressures of several thousand psi.
In the past, to check the efficiency of the radioactive detection process, the typical user collects eluate in a vial from a chromatography column at a peak by watching for the peak on a monitor screen, which is providing measurement data from a flow-through detector, a continuous detector of radioactivity connected to the output of the column. The user then takes this vial to a liquid scintillation sample counter and measures it. The peak may or may not be well-defined depending upon the chromatography. Collecting a sample in this fashion will only produce small volumes: just the peak volume of the mobile phase if using solid scintillator, or the mobile phase plus 3-4 additional volumes with liquid scintillator. Most static sample counters require much larger volumes, so the user must dilute the sample with scintillator solution thereby changing the performance from what it was in the flow-through detector. Then, since the user is counting an unknown, that sample is calibrated by addition of an internal standard of known activity and recounting. The overall result is not very accurate. Finally the calibration must be brought back to the original system and entered into the associated software.
Double isotope counting is avoided by many who do not appreciate the mathematics of correcting for spectral overlap in HPLC. One simplification many people make is to spend a great deal of time adjusting their counting windows so that they only look at that portion of the more energetic isotope that lies completely above the most energetic events of the less energetic isotope. Doing that can lose significant counting efficiency, e.g. for
3
H/
14
C dual-label counting, to eliminate the last 1% of
3
H in the
14
C channel might result in a 10% reduction in
14
C counts. If users had a simple way to obtain measurements of the spectral distribution of the different isotopes, they might be more inclined to use such measurement data to calculate corrections for such overlap or spillover.
Variable quench correction is not widely practiced, and then only with liquid scintillator, not with solid (though there is some belief that it should be). First, the sample concentration in an HPLC eluate stream is extremely low; quenching, if any, primarily comes from the composition of the mobile phase itself and variable quenching occurs because the mobile phase is deliberately changed during a run to push different compounds off the column. The normal terminology is “gradient elution” and the gradients often are of different salts and buffers, or water and miscible organic solvents.
Quench correction is not normally practiced for several reasons. Users hope that they will not need the correction, and, because the method seems complex, they do not try to learn whether they really do need this correction. Further, the correction when practiced in the conventional manner, may offer the promise of accuracy, but will require that substantial isotope be consumed, which also discourages frequent repetition.
The method commonly recommended by manufacturers of flow-through detectors—which is only applicable to liquid scintillation counting—is to make a dummy run with no sample, but otherwise identical in every aspect to the anticipated sample runs. Radioactive standard is added to the scintillator solution prior to the dummy run. The run is made and the activity is counted throughout. Since activity level of the scintillator is known, as well as its flow rate, one can create a table—from data collected minute by minute—of performance vs. time. When the sample runs are later made, measurements are corrected minute-by-minute using the above performance table.
The problem with this technique is that very large quantities of standard are required. If the chromatography run lasts one-hour with scintillator flowing at 3 ml/min through a 1 ml cell, and one needs to count at 10,000 counts per minute to obtain good statistics and the counting efficiency is about 50% (if it was much higher no one would worry about quenching), the total activity needed is about 3,600,000 disintegrations per minute. That activity is fairly high for a calibrated standard; it does not pre-suppose that this would be done often, yet it is necessary after any change in the analytical procedure: run length, gradient composition, gradient change rate, counting windows, counting cell size, etc.
Percent recovery is also of interest to many users (that is, determining how much of the activity that went into the HPLC column was actually measured in the flow-through detector.) Sometimes activity stays on the HPLC resin, and sometimes it sticks to the stainless steel or plastic tubing. A convenient measurement technique does not currently exist. The operator might pipette a known volume of the original sample mixture into a liquid scintillation vial and count it in a static sample counter. Then the operator must apply all the corrections mentioned above to translate that result to what the flow-through detector gives. It would be better if the raw sample prior to chromatographic separation could be counted in the flow-through detector and each peak subsequently separated is reported as a percent of that sample.
In FIG. 1 of U.S. Pat. No. 4,775,476 eluent passes through 10-port valve 20 to pickup sample through tubular membrane 35. Thereafter, the sample and eluent are directed by the valve through sample loop 27 before arriving at detector 34. In FIG. 2 valve 20 is switched so that eluent is pumped through sample loop 27 to the chromatography column 30, whose eluate is supplied to the detector 34. In this system
Adams Thomas L.
Cygan Michael
Williams Hezron
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