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Rapid determination of balance point in multichannel liquid scintillation counters
ANALYTICAL
Rapid
49, 511-516
BIOCHEMISTRY
Determination Liquid
of Balance Scintillation
K. D. NEAME Department
(1972)
AND
Point in Multichannel Counters
C. A. HOMEWOOD’
of Physiology, University of Liverpool, Liverpool School of Tropical Medicine,
and Department Liverpool,
of Parasitology,
England
ReceivedMarch 10, 1972; acceptedApril 21, 1972 A method is describedfor the rapid determination of balancepoint in multichannel
liquid
scintillation
counters. The random
element
associated
with the rate of radioactivity decay is eliminatedby calculatingthe ratio of the countsin the analyzing channelwhosebalancepoint is required to the counts in detectable number of accurately
a second channel, which is set to count all the scintillations by the instrument. If the instrument is set to stop at a preset counts in the second channel, balance point may be obtained as from counts in the one channel only.
It is common practice in the liquid scintillation counting of P-emitting radioisotopes to measure the detected radioact’ivity at what is usually referred to as “balance point” (1). This represents the photomultiplier gain required to obtain maximum counts within a particular energy range or ‘iwindow width.” It may take considerable time to find the correct instrument setting by the method commonly used (in which the gain or attenuator control is turned to various positions until maximum daunts are obtained), since radioactive decay is a random process, and this affects the reproducibility of the counts obtained at each trial setting (see Channel A counts in lower portions of Figs. 1-3, open circles). Balance point is affected by the constitutents of the sample to be measured, by the energy of p-emission, and by the properties of the instrument components; it may therefore need to be checked frequently, so that a great deal of time may be spent in determining this single parameter. A method of determining balance point is described here in which the random element mentioned above is eliminated, so that it t#akes considerably less time than the orthodox method. Two analyzing channels are required in which the same disintegrations can be counted at different settings. One channel (A) is set to count all the /3-particles which can possibly be detected by the instrument; for this, 1 MRC Research Group Resistance.
on the Chemotherapy
511 Copyright @ 1972 by Academic Press, Inc. A11 rights of reproduction in any form reserved.
of Protozoa1
Diseases and Drug
512
NEAME
AND
HOMEWOOD
the gain is set at maximum, i.e., zero attenuation, the lower discriminator (which determines the lower limit of the energy range detected) is set at its lowest voltage setting, and the upper discriminator made inoperative so that the window examined has no upper limit (i.e., infinity setting). The other channel (B), in which balance point is required, is set wit,h the a z E 2 Y m E
l.OO-
l
o.gg
. l ee l
2 2
0.98
-
l
l l
6e
l
i l
c
’
l l
ci
0.97
z 9 0 5
0.96-
l
-
l
l
.0 ‘0 @z
l
0
0 0
0
0
0
0
0
l 0
oee
l
0
0
0 0
0
l l
l 0
0 0
l
0
3
00
l e
E l
l l
3
l ee l
l
I 500
I 600
l
2.8001
' 400
Channel
I 700
I 800
B, fine ottenuotion
I 900
I loo0
setting
FIQ. 1. Determination of balance point of carbon-14. Lower diagram, counts in Channel A (open circles) and Channel B (closed circles), vertical pairs being measured together. Channel A: window 0.1 V-W, attenuation zero (i.e., maximum gain). Channel B: window 0.1-9.9 V, coarse attenuation set at factor of 8 (instrument setting D). Upper diagram, ratio of counts in Channel B/counts in Channel A. Balance point indicated by maximum value of ratio. Fine attenuation by 500 ohm ten-turn helipot, full scale O-1,000. Counting time, 0.1 min. Radioactive source1 Amersham/Searle sealed toluene C-14 standard (31,000 dpm), measured in NuclearChicago Unilux II liquid scintillation counter.
DETERMINATION
OF
BALANCE
,513
POINT
particular window width desired, and the gain or attenuator setting is then determined as follows: Instead of measuring only the counts obtained in Channel B at various gain or attenuator settings, as would be the usual practice, t,he ratio of the counts in Channel B to those in Channel A ia calculated; the setting on Channel B which then produces the maximum ratio represents the balance point. The reason for this is that the ratio is the fraction of the maximum counts obtainable (counts in Channel A) which are measured in Channel B at, any pa~i~ular setting. Thus the setting which gives maximum counts in Channel B, i.e., balance point, also gives the maximum ratio. At the same time the random element in the rate of disintegration affects both channels equally (this is particularly obvious in Fig. 3, lower portion) and so is eliminated in the resulting ratio. Examples of the use of this method are shown in Figs. X-3, which may
l
* l
0 0 EVI 2
30.m
0
-
0
*oO
O
o 0
0 0
0
0
0 m
l
b &
o” 0 o”
0
l OI 0
l
*
0
*.e*
*
0
b 29,000-
I 400
I 500 Channel
FIG. 2. Determination fig.
1. Counting
time,
*
.
of 1 min.
balance
i 600 Et, fine
point
I 700
I
800
attenuation
of
carbon-14.
I
900
I 1000
setting
Details
as in
legend
to
514
NEAME
ANll
HOMEWOOD
l l
#n E 5
300,000-
0
0
O
O
o”oooooo
l
l
ooooo @emem
: b
l mee
0
l em l
l 290,000-
0
l
l l
I
I
I
I
I
400
500
600
700
SKI
Channel
FIG. 3. Determination of balance Fig. 1. Counting time, 10 min.
6, fine
point
attenuation
I
900
I 1cxxl
setting
of carbon-14.
Details
as in legend
to
be compared directly one with another, since the proportions of the “counts” (and ‘katio”) scales are the same in each. The ratios are shown in the upper part of each figure, while in the lower part are shown the separate counts in each channel from which the ratios have been derived. By the orthodox method of analysis, balance point would be determined solely from an examination of the counts in Channel B (closed circles). It can be seen that randomness of the radioactive decay, as shown by the counts in Channel A (open circles), affects the counts in a similar way in both channels. With a gross count as low as 3,000 no balance point is obtainable from the counts in Channel B alone (Fig. 1) ; on the other hand, the ratio of the counts in the two channels shows clearly that the balance point lies between the fine attenuation settings of 600 and 800. Figure 2 shows a gross count of ten times that in Fig. 1, and Fig. 3 ten t,imes that in Fig. 2. In Fig. 2, the gross counts in Channel B still show no definite balance point, but the ratio is a more reliable indicator
DETERMINATION
OF
BALANCE
515
POINT
than in Fig. 1. In Fig. 3, with gross counts of about 300,000, the counts in Channel B suggest the position of a balance point which is barely more accurate than that determined by the plot of ratios shown in Fig. 1, yet the total actual counting time for the measurements in Fig. 3 is about a hundred times that for the measurements in Fig. 1. It is thus clear that for the same total actual counting time the acbx $ cl 2:
0.9:
“6 I= eY g;
0.91 c
2s g 5
0.9
,
l
e
-a
40,oot
-0
0
0
0
000000000000000
0
39,001 L-7 ; 2 v
e
2 ;
4,ooc
,o
0 0 0
00000000000000000
3,9oc e l
I 400
I 500 Channel
I 600 B, fine
I 700 attenuation
I 800
I 900
I to00
seiting
FIQ. 4. lIetermination of balance point of carbon-14. Instrument and source details as in legend to Fig. 1. Lower ~~r~~, inst~ment set to stop at preset counts of 4,000 in Channel A (open circles); balance point shown by maximum counts in Channel B (closed circles). Middle diqnwn, details as in lower diagram, but instrument set to stop at preset counts of 40,000 in Channel A. Upper diagram, closed circles, ratio of oounts Channel B/Channel A in middle diagram; crosses, ratio of 10 min counts Channel B/Channel A measured on same day.
516
NDAME
AND
HOMEWOOD
curacy of determination of balance point by the ratio method is much greater than by counting in a single channel only. (The time required for the whole procedure will of course include the time taken to print out data and to calculate the ratios.) Balance point can be determined in even shorter time by this method if the liquid scintillation counter can be stopped when a predetermined number of counts in any particular channel has been reached (“preset count,” “precount stop”). In this case, the instrument is set to stop when the counts in Channel A have reached a fixed number, the counts chosen depending on the accuracy required. The counts measured in Channel B at the same time will then be some proportion of that number depending on the gain or attenuator setting, as described above. In the ratio (counts in Channel B/counts in Channel A) the denominator is now a constant, so that only the counts in Channel B need to be recorded, the balance point being obtained directly from these. This is shown in Fig. 4, where the circles have the same meaning as in Figs. 1-3. The lower diagram shows data obtained using a preset count in Channel A of 4,000, while the circles in the middle diagram show data using a preset count of 40,000. Balance point, as shown by the peak value of counts in Channel B, can now clearly be determined as effectively by counting in that Channel alone as by the use of a ratio. As a direct comparison, the closed circles in the upper diagram of Fig. 4 show the ratio (Channel B/ Channel A) of the counts in the middle diagram ; a comparable analysis by measuring separately counts for a 10 min period (as in Fig. 3) gave almost identical ratio values, which are shown as superimposed crosses. Although the data given are derived only from measurement of carbon14 radioactivity, the method has been found to be equally effective and rapid when measuring the balance point of tritium. REFEREKCE 1. WANQ, C. H., AND WILLIS, D. L., “Radiotracer Methodology p. 126. Prentice-Hall, Englewood Cliffs, N. J., 1965.
in Biological
Science,”
Rapid
49, 511-516
BIOCHEMISTRY
Determination Liquid
of Balance Scintillation
K. D. NEAME Department
(1972)
AND
Point in Multichannel Counters
C. A. HOMEWOOD’
of Physiology, University of Liverpool, Liverpool School of Tropical Medicine,
and Department Liverpool,
of Parasitology,
England
ReceivedMarch 10, 1972; acceptedApril 21, 1972 A method is describedfor the rapid determination of balancepoint in multichannel
liquid
scintillation
counters. The random
element
associated
with the rate of radioactivity decay is eliminatedby calculatingthe ratio of the countsin the analyzing channelwhosebalancepoint is required to the counts in detectable number of accurately
a second channel, which is set to count all the scintillations by the instrument. If the instrument is set to stop at a preset counts in the second channel, balance point may be obtained as from counts in the one channel only.
It is common practice in the liquid scintillation counting of P-emitting radioisotopes to measure the detected radioact’ivity at what is usually referred to as “balance point” (1). This represents the photomultiplier gain required to obtain maximum counts within a particular energy range or ‘iwindow width.” It may take considerable time to find the correct instrument setting by the method commonly used (in which the gain or attenuator control is turned to various positions until maximum daunts are obtained), since radioactive decay is a random process, and this affects the reproducibility of the counts obtained at each trial setting (see Channel A counts in lower portions of Figs. 1-3, open circles). Balance point is affected by the constitutents of the sample to be measured, by the energy of p-emission, and by the properties of the instrument components; it may therefore need to be checked frequently, so that a great deal of time may be spent in determining this single parameter. A method of determining balance point is described here in which the random element mentioned above is eliminated, so that it t#akes considerably less time than the orthodox method. Two analyzing channels are required in which the same disintegrations can be counted at different settings. One channel (A) is set to count all the /3-particles which can possibly be detected by the instrument; for this, 1 MRC Research Group Resistance.
on the Chemotherapy
511 Copyright @ 1972 by Academic Press, Inc. A11 rights of reproduction in any form reserved.
of Protozoa1
Diseases and Drug
512
NEAME
AND
HOMEWOOD
the gain is set at maximum, i.e., zero attenuation, the lower discriminator (which determines the lower limit of the energy range detected) is set at its lowest voltage setting, and the upper discriminator made inoperative so that the window examined has no upper limit (i.e., infinity setting). The other channel (B), in which balance point is required, is set wit,h the a z E 2 Y m E
l.OO-
l
o.gg
. l ee l
2 2
0.98
-
l
l l
6e
l
i l
c
’
l l
ci
0.97
z 9 0 5
0.96-
l
-
l
l
.0 ‘0 @z
l
0
0 0
0
0
0
0
0
l 0
oee
l
0
0
0 0
0
l l
l 0
0 0
l
0
3
00
l e
E l
l l
3
l ee l
l
I 500
I 600
l
2.8001
' 400
Channel
I 700
I 800
B, fine ottenuotion
I 900
I loo0
setting
FIQ. 1. Determination of balance point of carbon-14. Lower diagram, counts in Channel A (open circles) and Channel B (closed circles), vertical pairs being measured together. Channel A: window 0.1 V-W, attenuation zero (i.e., maximum gain). Channel B: window 0.1-9.9 V, coarse attenuation set at factor of 8 (instrument setting D). Upper diagram, ratio of counts in Channel B/counts in Channel A. Balance point indicated by maximum value of ratio. Fine attenuation by 500 ohm ten-turn helipot, full scale O-1,000. Counting time, 0.1 min. Radioactive source1 Amersham/Searle sealed toluene C-14 standard (31,000 dpm), measured in NuclearChicago Unilux II liquid scintillation counter.
DETERMINATION
OF
BALANCE
,513
POINT
particular window width desired, and the gain or attenuator setting is then determined as follows: Instead of measuring only the counts obtained in Channel B at various gain or attenuator settings, as would be the usual practice, t,he ratio of the counts in Channel B to those in Channel A ia calculated; the setting on Channel B which then produces the maximum ratio represents the balance point. The reason for this is that the ratio is the fraction of the maximum counts obtainable (counts in Channel A) which are measured in Channel B at, any pa~i~ular setting. Thus the setting which gives maximum counts in Channel B, i.e., balance point, also gives the maximum ratio. At the same time the random element in the rate of disintegration affects both channels equally (this is particularly obvious in Fig. 3, lower portion) and so is eliminated in the resulting ratio. Examples of the use of this method are shown in Figs. X-3, which may
l
* l
0 0 EVI 2
30.m
0
-
0
*oO
O
o 0
0 0
0
0
0 m
l
b &
o” 0 o”
0
l OI 0
l
*
0
*.e*
*
0
b 29,000-
I 400
I 500 Channel
FIG. 2. Determination fig.
1. Counting
time,
*
.
of 1 min.
balance
i 600 Et, fine
point
I 700
I
800
attenuation
of
carbon-14.
I
900
I 1000
setting
Details
as in
legend
to
514
NEAME
ANll
HOMEWOOD
l l
#n E 5
300,000-
0
0
O
O
o”oooooo
l
l
ooooo @emem
: b
l mee
0
l em l
l 290,000-
0
l
l l
I
I
I
I
I
400
500
600
700
SKI
Channel
FIG. 3. Determination of balance Fig. 1. Counting time, 10 min.
6, fine
point
attenuation
I
900
I 1cxxl
setting
of carbon-14.
Details
as in legend
to
be compared directly one with another, since the proportions of the “counts” (and ‘katio”) scales are the same in each. The ratios are shown in the upper part of each figure, while in the lower part are shown the separate counts in each channel from which the ratios have been derived. By the orthodox method of analysis, balance point would be determined solely from an examination of the counts in Channel B (closed circles). It can be seen that randomness of the radioactive decay, as shown by the counts in Channel A (open circles), affects the counts in a similar way in both channels. With a gross count as low as 3,000 no balance point is obtainable from the counts in Channel B alone (Fig. 1) ; on the other hand, the ratio of the counts in the two channels shows clearly that the balance point lies between the fine attenuation settings of 600 and 800. Figure 2 shows a gross count of ten times that in Fig. 1, and Fig. 3 ten t,imes that in Fig. 2. In Fig. 2, the gross counts in Channel B still show no definite balance point, but the ratio is a more reliable indicator
DETERMINATION
OF
BALANCE
515
POINT
than in Fig. 1. In Fig. 3, with gross counts of about 300,000, the counts in Channel B suggest the position of a balance point which is barely more accurate than that determined by the plot of ratios shown in Fig. 1, yet the total actual counting time for the measurements in Fig. 3 is about a hundred times that for the measurements in Fig. 1. It is thus clear that for the same total actual counting time the acbx $ cl 2:
0.9:
“6 I= eY g;
0.91 c
2s g 5
0.9
,
l
e
-a
40,oot
-0
0
0
0
000000000000000
0
39,001 L-7 ; 2 v
e
2 ;
4,ooc
,o
0 0 0
00000000000000000
3,9oc e l
I 400
I 500 Channel
I 600 B, fine
I 700 attenuation
I 800
I 900
I to00
seiting
FIQ. 4. lIetermination of balance point of carbon-14. Instrument and source details as in legend to Fig. 1. Lower ~~r~~, inst~ment set to stop at preset counts of 4,000 in Channel A (open circles); balance point shown by maximum counts in Channel B (closed circles). Middle diqnwn, details as in lower diagram, but instrument set to stop at preset counts of 40,000 in Channel A. Upper diagram, closed circles, ratio of oounts Channel B/Channel A in middle diagram; crosses, ratio of 10 min counts Channel B/Channel A measured on same day.
516
NDAME
AND
HOMEWOOD
curacy of determination of balance point by the ratio method is much greater than by counting in a single channel only. (The time required for the whole procedure will of course include the time taken to print out data and to calculate the ratios.) Balance point can be determined in even shorter time by this method if the liquid scintillation counter can be stopped when a predetermined number of counts in any particular channel has been reached (“preset count,” “precount stop”). In this case, the instrument is set to stop when the counts in Channel A have reached a fixed number, the counts chosen depending on the accuracy required. The counts measured in Channel B at the same time will then be some proportion of that number depending on the gain or attenuator setting, as described above. In the ratio (counts in Channel B/counts in Channel A) the denominator is now a constant, so that only the counts in Channel B need to be recorded, the balance point being obtained directly from these. This is shown in Fig. 4, where the circles have the same meaning as in Figs. 1-3. The lower diagram shows data obtained using a preset count in Channel A of 4,000, while the circles in the middle diagram show data using a preset count of 40,000. Balance point, as shown by the peak value of counts in Channel B, can now clearly be determined as effectively by counting in that Channel alone as by the use of a ratio. As a direct comparison, the closed circles in the upper diagram of Fig. 4 show the ratio (Channel B/ Channel A) of the counts in the middle diagram ; a comparable analysis by measuring separately counts for a 10 min period (as in Fig. 3) gave almost identical ratio values, which are shown as superimposed crosses. Although the data given are derived only from measurement of carbon14 radioactivity, the method has been found to be equally effective and rapid when measuring the balance point of tritium. REFEREKCE 1. WANQ, C. H., AND WILLIS, D. L., “Radiotracer Methodology p. 126. Prentice-Hall, Englewood Cliffs, N. J., 1965.
in Biological
Science,”