Quench correction

In column 110, it is also theoretically possible that glycine com-plexed with the added humic acid and that it was sequestered in the aqueous phase of the Teflon eluate and bound to the Teflon bed. To test this explanation, a 1/10-scale parfait column was constructed and 4 liCi of 14C-glycine, 40 fig total, was applied in 800 mL of synthetic hard water (column 123). In this experiment, the alcohol and solvent 1 conditioning washes were combined with the standard eluates of each bed before counting. These solutions were not concentrated before counting. Quench correction was by the channels ratio method. 

For general purpose tracer work, however, and particularly in polymer chemistry, the liquid scintillation counter surpasses all other instruments in its sensitivity and adaptability. There is no question on the author s mind that at the present time such an instrument would be the first choice, particularly where tritium, carbon-14 or sulphur-35 were involved. Samples for assay are dissolved in a phosphor whose major solvent usually consists of toluene, toluene-alcohol, or dioxan. Many polymers and low molecular weight compounds are readily soluble in these solvents. Prospective users should not be deterred by alleged complications due to "variable quench effects" as these effects are readily corrected for via internal or external standards or the channels ratio method (7, 46, 91). Dilution quench corrections, though valid, are tedious and unnecessary. Where samples are insoluble in phosphor they may be suspended (e.g. as gels or as paper cut from chromatograms, etc.) or they can be burnt and the combustion products absorbed in a suitable phosphor solution. A modification of the Schoniger flask combustion technique is particularly suitable for this purpose (43—45). 

Since quenching can occur during all experimental counting of radioisotopes, it is important to be able to determine the extent of the reduced counting efficiency. Two methods for quench correction are in common use. 

The internal standard ratio method for quench correction is tedious and time-consuming and it destroys the sample, so it is not an ideal method. Scintillation counters are equipped with a standard radiation source inside the instrument but outside the scintillation solution. The radiation source, usually a gamma emitter, is mechanically moved into a position next to the vial containing the sample, and the combined system of standard and sample is counted. Gamma rays from the standard excite solvent molecules in the sample, and the scintillation process occurs as previously described. However, the instrument is adjusted to register only scintillations due to y particle collisions with solvent molecules. This method for quench correction, called the external standard method, is fast and precise. 

If the available scintillation counter does not have an adequate quench correction function, it may be necessary to process a non-radioactive fecal sample as described above and add an equivalent amount of the hexane extract to the counting vials for the starting sterol mix (from Section 2.1 or 2.2 above) so it is quenched similarly to the fecal samples. 

The measurement of light in colored samples this can be overcome by color quench correction programs applied by the appropriate scintillation counter. 

Figure 3-9 Channels ratio quench correction plot for 14C. Quench correction data may curve slightly up or down depending on the make and model of counter used.

Using the data obtained from samples 1 to 4, the known number of 14C disintegrations per minute added to each of these samples, and considering the counts per minute data obtained from the widest window as a reflection of overall counting efficiency (the total possible number of counts per minute that can be detected in each sample), construct a channels ratio quench correction curve similar to that shown in Figure 3-9. 

Extraction and purification of the sample containing is not required as y-rays penetrate coloured solutions and soft tissue with negligible loss of energy. In the case of p-emitters, the scintillation solution must be colourless or quenching corrections must be applied. 

Such curves are necessary for quench correction when, for the example given, the counting efficiency changes during gradient elution. If standard sensors and conditions are used, appropriate quench corrections are usually made available with the instrument. If not, standards are usually provided, and specific calibration instructions are given for each type of instrument. 

Three methods have evolved to ascertain the degree of efficiency loss both within the instrument and as a result of quenching. These techniques are termed (1) internal standardization, (2) channels ratio quench correction, and (3) external standard channels ratio quench correction. Determination of counting efficiency by internal standardization may be performed in two steps. The sample is first accurately counted followed by the addition of a precisely known quantity of radioactivity to the vial (50,000-80,000 dpm C or 100,000-150,000 cpm H). It is important for the amount of added radioactivity to be considerably larger than that originally present in the vial. The sample is then counted a second time. The first count is the sample cpm and the second count is the sample cpm + (efficiency)(standard dpm). That is. 

Figure 3-17. Channels ratio quench correction curve. This curve was constructed using the sample and discriminator settings described in Figure 3-16. (Courtesy of Beckman Instruments, Inc.)...

Figure 3-20. External standard channels ratio quench correction curves. These data were obtained using the procedures outlined in steps 3-50 to 3-57 of the experimental section.

What is the efficiency of C counting in channel B and what is the absolute radioactivity (dpm) in the sample Use the quench correction curve in Figure 3-20. 

Once these standard curves have been prepared the multiply labeled sample may be counted in the presence and absence of the external standard. The counting rate in channel B (cpm C). and the channels ratio value may be used in conjunction with the quench correction cutve (Figure 3-20) to calculate the absolute dpm of C using equation 3-24. 

Count a set of unknown variably quenched samples using the procedures described in steps 3-44 to 3-47 and then, using the quench correction curve obtained in step 3-48, calculate dpm C in each sample. 

Fignre 3-39, Channels ratio quench correction curves for C and H. 

Count each of the vials using the counter parameters established in steps 3-50 to 3-55, in the presence and absence of the external standard. 3-64. Calculate the cpm of C and in each vial using the quench correction curve obtained from steps 3-57 and 3-58 and equations 3-24 to 3-26. 

The index of quenching with respect to sample is found from Compton spectrum created by y-ray irradiation to the sample. A set of quenched standards are employed for the construction of a quenching correction curve which represents the relationship between the index of quenching and the counting efficiency. The radioactivity of the sample to be measured can be determined by using the quenching correction curve. 

The index of quenching in this method is obtained from an areal ratio or a centre of gravity with respect to a 3-ray spectrum. The radioactivity is determined through a similar quenching correction curve to that of the external standard method. 

For liquid scintillation counting (LSC), a Packard Tri-Carb 460 CD microprocessor controlled liquid scintillation spectrometer, with automatic external standarization, quench correction, and conversion of counts per minute (cpm) into disintegrations per minute (dpm), was utilized. An appropriate scintillation cocktail was selected according to sample characteristics Insta-Gel II (Packard) was used for aqueous solutions and organic extracts, Dimilume-30 (Packard) for NaOH solutions and... 

The traditional method for quench correction is to add an internal standard to each sample to determine the counting efficiency for each sample matrix. This method continues to be one of the most accurate but is labor intensive and expensive. A set of samples is counted without the internal standard. A duplicate set, with a small volume (e.g., 0.1 ml or less), of known activity of the internal standard spiking solution is added to each sample, and is then counted. The counting efficiency for each sample is as follows ... 

An external standard spectrum to determine the QIP is popular with users and instrument vendors. The external gamma-ray source (e.g., Cs, or Eu) that is part of the detector system induces a Compton-electron spectrum in the scintillation cocktail. Each sample is automatically counted with and without the external standard. The Compton-electron spectrum produced in each sample vial is applied with mathematical techniques to derive a QIP for a quench correction curve. 

The tritium measurement protocol Is relatively simplistic and consists of neutralization of the sample, single plate distillation, removal of an appropriate size aliquot (usually eight mL.) via reproducible automatic pipets and the addition of 15 mL. of dark adapted scintillation cocktail under Incandescent lighting conditions. After shaking to ensure a uniform gel, the sample is allowed to settle and dark adapt for up to twenty but no more than sixty minutes. The liquid scintillation unit efficiency Is determined dally on a previously prepared standard and background measurements are determined at least dally. Quench corrections are not applied to the system due to the lack of an external standards ratio capability and an effort to minimize the amount of hazardous waste which would be generated if an Internal standards approach were adopted. 

The effect of the lack of a quench correction was evaluated by reviewing the same technique accomplished at a different laboratory within the Corporation but with the added protocol step of efficiency determination via the Internal standard method. A total of 66 data points comprise the distribution depicted In Figure 2. This data Is considered normally distributed with 2Sq 3 0.071. These two relative systematic uncertainty boundary conditions for the efficiency have been propagated in quadrature to yield a relative systematic uncertainty of 0.074. 

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