Developed by
Department of Chemistry
Radiochemistry
University of Helsinki

Students will get deeper understanding about the functions of a liquid scintillation counter (LSC) and the double labeling technique with two radioactive standards will be illustrated. The students will determine quench curves (counting efficiency curves) for radionuclides ³H and ¹⁴C by using external standard quenching parameter method. Based on the obtained quench curves, the activities of unknown samples will be calculated.

Three commonly used beta emitters ³H (E_max 18 keV), ¹⁴C (E_max 180 keV), and ³²P (E_max 1700 keV) have significantly different maximum beta energies (see the next figure).

Separately measured LSC spectra of ³H (E_max 18 keV), ¹⁴C (E_max 180 keV), and ³²P (E_max 1700 keV), presented in the same graph.

The effect of quenching on beta spectra of ³H and ¹⁴C is presented in the next figure. It can be seen that as quenching increases in the counting sample from an unquenched sample (solid line) to varying levels of quenching (dashed lines), the energy position of the peak as well as the count rate decrease. The effect on quenching on beta spectra of ³H and ¹⁴C in LSC. The solid line is a spectrum of an unquenched sample and dashed lines are spectra of the same sample with increasing quench level.

Sample preparation
Two sets of quench series samples are prepared. In one series, all six samples will have the same added activity of ³H. In another series of six samples, all samples have the same added activity of ¹⁴C. Quenching and resulting counting efficiencies are different among the samples, due to either different water volumes added to samples, or because of different volume of added quenching agent. In this exercise, we use fluorescent colour agent Fluorescein as a quenching agent, but alternative quenching agent can be used.

¹⁴C and ³H standards have been prepared before the exercise by dissolving standard capsules to 5 ml MilliQ water. The measurement samples (2 x 6 samples in total) are prepared to LSC vials of 20 ml volume by pipetting this active water, MilliQ water, quenching agent and LSC cocktail in varying volumes, according to the next table:

Therefore, each sample contains 5.5 ml of water and 14.5 ml of LSC cocktail. In addition to quench series, two background samples are prepared by mixing 14.5 ml of LSC cocktail and 5.5 ml of Milli-Q water. Unknown samples have been prepared by the Lab Supervisor before the exercise.

Selection of the window settings used in the measurements

• unquenched samples of both quench series (³H and ¹⁴C) are measured first with the background samples and the spectra is saved
• Figure of Merit (FOM) is determined for the both radionuclides, with the formula E²/B, where E is counting efficiency and B is background count rate (as cpm)
• based on the highest values of the FOM, the channel range (window) for both radionuclides are selected

Determination of two isotopes from the same sample by using external standard quenching parameter SQP(E)

• Channel ranges (windows) for ³H (window 1) and ¹⁴C (window 2) are selected. ³H doesn’t cause significant amount of pulses in ¹⁴C window, whereas ¹⁴C causes pulses in ³H window, which has to be taken into account in calculations. The spectral interference from ¹⁴C to ³H determination and optimal window selection has been described in the following figure

Selection of channel ranges, i.e. windows, for simultaneous determination of ³H and ¹⁴C.

• the quench series samples of ³H and ¹⁴C are measured and quench curves will be drawn: counting efficiency as a function of quenching parameter SQP(E) (LSC will give the SQP(E) values)
• two samples, containing unknown amounts of both ³H and ¹⁴C, will be measured
• the activities of ³H and ¹⁴C in these double labelled samples are calculated based on the quench curves as follows:

where
N₁ = total count rate in window 1 (³H + ¹⁴C)
N₂ = total count rate in window 2 (¹⁴C) a = counting efficiency for ³H in window 1 b = counting efficiency for ¹⁴C in window 1 c = counting efficiency for ¹⁴C in window 2

There are only pulses from ¹⁴C in window 2, so N₂ = N_2C
There are pulses from both ³H and ¹⁴C in window 1, therefore, N₁ = N_1H + N_1C

• What are the main principles of LSC measurement technique?
• Describe the external standard window ratio method.
• Why two quench series were prepared in this exercise?
• Consider possible uncertainty sources in this work, and how they affect to the final result, which is the activity of a radionuclide. A complete uncertainty analysis is not needed here but instead, give an estimation with justifications, how high could be the uncertainties of the isotopic activities in the unknown samples.

The work report should be a freely formed essay, containing

• concise description of the performed work, used equipment and radioactive standards
• answers to the fore mentioned questions.
• activities of ¹⁴C and ³H in the unknown samples, which you have calculated based on the fore mentioned formulas
• graphs and calculations

• Lab coat, gloves and safety spectacles must be worn.
• MSDS (Material Safety Data Sheet) for each of the used reagent gives necessary information about working safely with that particular chemical, and its disposal after the work. The MSDSs are easily available from the websites of the chemical manufacturers and authorities
• Lab Supervisor takes care of collecting and disposing radioactive waste solutions and samples after the exercise, so don’t throw chemicals or samples down sinks or in waste bins

• Liquid scintillation counter
• 100-1000 µl autopipette

• MilliQ water
• quenching agent Fluorescein or some alternative
• LSC cocktail
• polythene LSC vials
• 100-1000 µl pipette tips
• permanent marker

• Standard capsules of ¹⁴C and ³H dissolved to 5 ml MilliQ water per capsule (³H and ¹⁴C are dissolved in separate vials)

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