Channel ratio

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). 

OF THE RADIOACTIVITY CONCENTRATION Radioactivity measurements are carried out by the liquid scintillation counting procedure in -spectrometers using an external standard device which permitted the counting efficiency to be determined by the channel ratio method (explained for instance by Dyer (1980)). 

The channels ratio method makes use of existing counts within the sample vial. This method is suitable when large numbers of counts are present, but it becomes very time consuming with samples containing few counts, because a long time is required to accumulate sufficient counts for statistical accuracy. Most modern scintillation counters therefore employ an automatic external standardization system of quench analysis to avoid the time required for the internal channels ratio method. This method utilizes a specially selected external y radiation source carried in a lead-shielded chamber that is buried in the instrument. Before the regular counting of the sample, the external standard is... 

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.

This experiment demonstrates two methods for analysis of quenching within samples, namely, the channels ratio method and the automatic external standardization method. Either or both of the methods may be demonstrated with the quench series of bottles described in the following protocol, depending on the capabilities of your particular scintillation counter. 

If the channels ratio method is used, proceed with Steps 3 to 8. If the automatic external standardization method is used, proceed with Steps 9 to 13. 

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. 

The channel 1/channel 2 ratio is a value that will be provided by the instructor. It is a correction factor that takes into account the fact that some 14C counts per minute were counted in channel 1. The instructor determined this ratio by scintillation counting of a sample containing a known number of 14C disintegrations per minute in channel 1 and channel 2 under conditions identical to your experiment. Refer to Section I, Experiment 3 for further discussion of the channel ratio method of quantifying radioactivity. 

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 3-16. Channels ratio counting of quenched samples. Quenching increases sequentially from sample 1 to sample 4. Arrows above the figure indicate the range of the A and B channel discriminators. (Courtesy of Beckman Instruments, Inc.)...

Example. A sample of C was counted using the channels ratio technique and the following data were obtained ... 

What was the efficiency of counting and the absolute radioactivity (dpm) in the sample These data give a channels ratio of... 

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 ... 

Dividing equation 3-22 by 3-21 gives the channels ratio for the external standard ... 

With a statistically significant channels ratio in hand, it is only necessary to relate this ratio to a specific efficiency. In the channels ratio technique this was done by determining the ratios and efficiency of counting for a set... 

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.

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. 

Since we have already calculated dpm C above, equation 3-26 has only one unknown (the efficiencies are obtained from Figure 3-20 at the appropriate channels ratio values), the dpm of in the sample. If the amount of radioactivity in the sample is sufficiently low that the counter... 

Why is the channels ratio not affected by the nature of the isotope contained in the vial ... 

Counting Quenched Samples Using the Channels Ratio Technique... 

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

Plot the efficiency (in channel A) of each quenched standard as a function of the external standard channels ratio value it yielded. This type of plot is shown in Figure 3-20. 

E. T. Bush, Anal. Chem., 35 1024 (1963). General Applicability of the Channels Ratio Method of Measuring Liquid Counting Efficiencies. 

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