Channels ratio quench correction

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. 

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.

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

The following channels ratio quench correction curve was obtained for C... 

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). 

In practice a sample is counted normally as described for the channels ratio method and then counted a second time with a source of y-rays (usually Cs or Ba) positioned in the center of the counting chamber just below the sample vial (see Figure 3-19). Any quenching that occurs has the same effect on the efficiency and spectrum of the Compton electrons as it does on those of the sample j8 particles. Since this technique has incorporated the procedures of both internal standardization and channels ratio correction methods, it is no surprise that the data obtained must be treated as described above for both of these techniques. The first or normal count rate obtained in each channel arises only from the sample and may be represented as follows ... 

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. 

The radioactivity of samples was measured by liquid scintillation with toluene+POPOP or with Triton X-100 (Isocap-300).Counting efficiencies,determined via the channel ratio method and quench correction curve,were sufficiently constant (about 70-80%).Gas exchange measurements of respiration of leaf were conducted by manometric tech-nique(9).It was found that the chosen concentrations of exogenous substrates had no effect on dark respiration of leaves studied plants. 

Reactions were terminated by the addition of C M and either aqueous KCl or KCl in dilute phosphoric acid (4) so that C M aqueous = 1 1 1 v/v/v. Lipids were separated by TLC using either C Acet M AcH H20 50 20 10 10 5 by volume or pet ether-.diethyl ether formic acid 80 20 2. Lipids were quantified by GLC using an internal standard of heptadecanoic acid. Radioactivity was measured by liquid scintillation counting and counts were quench corrected when necessary using the channels ratio... 

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