Notes to accompany lecture and early experimental design.
The basis of liquid scintillation counting is the emission of light
(fluorescence) by special organic molecules (called fluor molecules)
in response to emitted beta particles arising from radioisotopes
(such as Tritium, 14C, 35S, or 32P). This process occurs in a special
mixture called a Liquid Scintillation Cocktail. The components of it
include at a minimum an organic solvent and a fluor.
Several aspects of the ultimate detection of radioactive decay are
critical. First, the energy of the partical must be conveyed to the
fluor molecule. Solvents, such as toluene or xylene, perform this
function well. Other solvents, such as chloroform or water, perform
the function very poorly. Even tiny quantities of chloroform or water
in a liquid scintillation sample can inhibit the transfer of energy
from beta particles to fluors. The effect of inhibiting the transfer
of beta particle energy to fluors by a solvent is referred to as
chemical quenching. In this case, the fluor does not emit light
because it never receives the energy from the beta particle.
Another type of quench occurs after the fluor molecule emits photons.
In this type of quenching, the emitted photons are absorbed after
being emitted by the fluor. One example of this is in a strongly
colored solution. Such a type of quenching is called Color Quenching.
From these examples, one can see that the general term Quenching
refers to any inhibition of the process below:
beta particle energy --->solvent energy ----> fluor excitement
---> photon ----> exit from vial
Quenching can reduce the Counts Per Minute (CPM) detected for a
particular sample. At this point, it is easy to see the distinction
between the number of CPMs and the number of Disintegrations Per
Minute (DPM). Because of quenching (as well as other effects), not
every radioactive decay results in a detectable "count". In other
words, the liquid scintillation counter does not "see" every
radioactive decay. Fortunately, for a given sample in a given vial,
the rate with which the machine misses counting decays is constant.
Thus we can define a Counting Efficiency (C.E.) which takes this
constant into account. The C.E. for a sample is defined as
follows:
C.E. = (CPM - background counts)/DPM
The background counts in the above equation is necessary because
random events, such as cosmic rays, electronic noise, etc. contribute
to the number of CPMs. They are easily determined by counting a vial
containing only scintillation fluid and no added radioactivity.
We make further assumptions that all other things being equal, the
C.E. in two identical environments is the same. That is, if I take a
sample of 14C Toluene and I divide it equally into two vials
containing the same scintillation fluid, the same fluor, the same
volume, and no other changes, the two samples will yield the same
number of counts (within statistical expectations). This assumption
allows us to use the C.E. determined for a known sample and use that
efficiency on an unknown sample in an identical environment to
determine DPM by rearranging the above equation as follows:
DPM = (CPM - background counts)/C.E.
Thus, once I determine the C.E. for a given environment, I know the
C.E. for all samples placed in it. Normally in an experiment one
determines CPMs by counting decay of radioactive compounds produced
in a system. For example, one can easily follow the metabolic
pathways of a compound by starting with a radioactive precursor, such
as glucose, adding it to a biological system (for example, a mouse),
allowing the compound to be metabolized for a fixed period of time,
and then collecting samples from the organism and analyzing them.
Doing this, one can isolate radioactively labelled
glucose-6-phosphate, the first product of glycolysis. By putting the
sample in a liquid scintillation counter, one can determine the
number of CPMs of G-6-P. If one knows the counting efficiency under
those conditions, the number of DPMs can be determined from
DPM = (CPM - background counts)/C.E.
It is essential in almost all work with radiochemicals to express the
radioactivity in DPMs. CPMs have virtually no meaning, since the
number of CPMs varies considerably under different counting
conditions. There is a more important reason to work with DPMs,
however. DPMs can be easily interconverted to moles of material by
knowing the Specific Activity of a compound. There are many ways of
expressing Specific Activity, but all are based on the following
general idea:
Specific Activity = DPM/quantity
Thus one can have
Specific Activity = DPM/mole
or
Specific Activity = DPM/gram
or
Specific Activity = Ci/mole (One Curie -Ci - is the same as
2.2x10e12)
or other combinations.
By knowing the specific activity of the starting compound (glucose in
the above example), and assuming that the radioactively labeled
compound is metabolized at the same rate as the non-radioactive
compound in the organism (a reasonable assumption), one can easily
determine the quantity of metabolite produced (G-6-P in the example
above) by rearranging one of the above equations:
mole = DPM/Specific Activity
Since DPM = (CPM - background)/(Counting Efficiency), determining the
CPMs and the counting efficiency of a sample tells exactly how much
material (G-6-P) was produced.
The descriptions above are useful for all of the experiments we will
perform this quarter. There are two other important considerations in
liquid scintillation counting that we will be concerned with. One has
to do with the liquid scintillation counter itself. The liquid
scintillation counter is able, using a trick, to count not only the
number of decay events happening in a sample, but the energy of each
decay event as well. This is accomplished by the fact that the energy
of the decay is a function of the number of photons released by the
fluor (obviously this is related to quench). While it might seem that
a very "hot" sample might confuse the counter by emitting a lot of
photons, in practice it does not, because the counter counts photons
only in tiny 20-30 nanosecond "windows" of time that reduce
tremendously the likelihood of even hot samples having two decays
occurring in the same window. Thus, each count is individually
registered and the number of photons corresponding to it are
determined, allowing the counter to assign an energy to it as well.
The counter we use in the lab has three energy "Windows" installed in
it. They are called A, B, and C. Respectively they measure the energy
corresponding to 3H decay, 14C energy above the 3H window, and the
complete 3H and 14C energy spectrum. The energy of decay of the
various radioisotopes is spread across a range of energies, as
discussed in class, with 3H having the lowest peak of energy, 14C
having a higher peak of energy, and 32P having an even higher peak of
decay energy. The low end of the 14C energy spectrum and the very low
end of the 32P energy spectrum overlap with the 3H spectrum, but the
3H high energy spectrum does not overlap with the peak energies of
either 14C or 32P.
Thus, an appropriately set up scintillation counter can distinguish
counts as coming from either 14C or 3H fairly easily if one assumes
that for a given amount of quench, the amount of CPMs of 14C seen in
the 3H window (Window A) is a constant and that all of the counts in
the B window came from 14C. (Please note that in all of the
calculations involving CPMs, I am assuming you have subtracted the
background.)
C.E. in A Window = (CPM in A- background)/DPM in sample
C.E. in B Window = (CPM in B - background)/DPM in sample
As an example, I might add 1000 DPM of 14C toluene to a vial and
count it, observing that window A had 250 counts and window B had 650
counts and the background of each window was 50 counts. The
efficiency of counting in each window then is 20% in A and 60% in B.
However, from above I noted that for a given quench, the CPMs in A
for 14C is a constant fraction of the CPMs in B. In this case the
"spill" of 14C counts into the A window was 33.3% of the counts of B.
This will hold true for all samples with the same amount of quench.
Thus if I have 900 CPM in the B window (after subtracting
background), the "spill" into the A window will be 300 CPM. Using
this information, I can take an unknown sample that has both 3H and
14C and determine the number of DPMs of each in the mixture. Let us
assume that we have determined that the counting efficiency of 14C is
as shown above and further that the C.E. of 3H in the A window is
40%. Let's say that under these conditions I count an unknown sample
and obtain the following CPMs:
A = 1450 CPM
B = 1850 CPM
Background in A = 50 CPM
Background in B = 50 CPM
The number of DPMs of each sample then is as follows:
B CPM for 14C = 1800 (1850 - 50 background CPM)
Spill 14C into A = 600 (33.3%)
A CPM for 3H = 800 (1450 - 50 CPM background - 600 CPM 14C)
DPM 14C = 3000 (1800 cpm in B/0.6 C.E. in B)
DPM 3H = 2000 (800 CPM in A/0.4 C.E. in A)
One last consideration about radioactive decay is that it occurs not
as a linear process, but rather as an exponential decay. Each
radioisotope has associated with it a "half-life", which describes
the amount of time it takes for half of the sample to decay. For some
isotopes, such as 14C (half life about 5000 years), the decay that
happens over the span of a few days to even a few years is trivial.
For other samples, such as 32P (half life = 14 days), it is critical
to know how old the sample is to determine the number of dpms in it.
I will talk about how to take these factors into consideration in
class on 4/11/96.
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