=====Lab Exercise - Double Labeling in Liquid Scintillation Counting ===== **Developed by**\\ Department of Chemistry\\ Radiochemistry\\ University of Helsinki\\ ====Learning Goals==== 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. ====Explanation and Exercise Guide==== ===Theory=== Three commonly used beta emitters 3H (Emax 18 keV), 14C (Emax 180 keV), and 32P (Emax 1700 keV) have significantly different maximum beta energies (see the next figure). {{ :laboratory_exercises:three_beta_emitters_in_the_same_lsc_spectrum.png?600 |}} //Separately measured LSC spectra of 3H (Emax 18 keV), 14C (Emax 180 keV), and 32P (Emax 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. {{ :laboratory_exercises:quenching_of_3h_and_14c_in_lsc.png?600 |}} //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. // ===Experimental Procedure=== __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. \\ {{ :laboratory_exercises:fluorescein.png?600 |}} \\ 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: {{ :laboratory_exercises:table_quench_series_preparation.png?600 |}} 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 {{ :laboratory_exercises:selection_of_windows_in_lsc_3h_and_14c.png?600 |}} //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: {{ :laboratory_exercises:equations_double_labelling_exercise.png?400 |}} 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 = N2C\\ There are pulses from both 3H and 14C in window 1, therefore, N1 = N1H + N1C \\ ====Questions for Students==== * 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. \\ ====Work report==== 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 \\ ====Safety Aspects==== * 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 \\ ====Preparation for the Lab Supervisor==== ==Equipment== * Liquid scintillation counter * 100-1000 µl autopipette ==Consumables== * MilliQ water * quenching agent Fluorescein or some alternative * LSC cocktail * polythene LSC vials * 100-1000 µl pipette tips * permanent marker ==Radioactive sources== * Standard capsules of 14C and 3H dissolved to 5 ml MilliQ water per capsule (3H and 14C are dissolved in separate vials) \\ ====Feedback from Users and Supervisors==== Leave feedback in comments