LSC in Practice: Radio-Carbon Dioxide Trapping and Counting

Introduction
N O T E
The PerkinElmer Sample Oxidizer is perfectly designed
to trap 14CO2 originating from the combustion of large
amounts of samples containing 14C. However discrete
gaseous 14CO2 samples not originating from combustion
require a completely different approach for the trapping
of the gas and subsequent liquid scintillation counting
(LSC). 14CO2 gas samples originate from a variety of
sources, including:
There are a large number of potentially useful
reagents available for trapping carbon dioxide,
including the following:
1. Sodium hydroxide (0.1 - 1.0 M)
2. Potassium hydroxide (0.1 - 1.0 M)
3. Hyamine Hydroxide® in methanol (1.0 M)
4. Ethanolamine®
5. Carbo-Sorb®
Table 1 lists some basic information which will be
helpful in selecting the best trapping reagent for a
particular application.
1. 14CO2 in expired breath
2.
14CO
2
expired by plants
3.
14CO
2
expulsion from blood
4.
14CO
2
released in enzymatic studies
Table 1. Trapping capacity of suitable reagents for carbon dioxide.
mM CO2
per mL
mL required
for 10 mM CO2
0.1M Sodium/Potassium hydroxide
0.05
20.00
1.0M Sodium/Potassium hydroxide
0.50
2.00
®
mL required
for 5 mM CO2
mL required
for 1 mM CO2
Flash-Point (°C)
—
—
—
10.00
—
—
1.23
93 °C
1.0M Hyamine Hydroxide in methanol
0.50
2.00
10.00
Ethanolamine
8.10
0.12
0.62
11 °C
Carbo-Sorb
5.80
0.17
0.86
1.72
7 °C
Carbo-Sorb E
4.80
0.21
1.04
2.08
27 °C
Having selected a suitable carbon dioxide trapping
reagent for the application, the next step is to ensure
that a compatible liquid scintillation cocktail is
chosen. Therefore, each reagent will be considered
separately.
1-2. Sodium/Potassium hydroxide
Sodium hydroxide absorbs/traps CO2 by a reaction
which produces a sodium carbonate solution.
Potassium hydroxide performs in a similar way
by forming potassium carbonate.
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Recommended LSC Cocktails
1. 10 mL of Emulsifier-Safe™* will accept up to 2 mL
of 0.1 M sodium hydroxide/CO2.
2. 10 mL of Opti-Fluor®* will accept up to 5 mL of
0.1 M sodium hydroxide/CO2.
3. 10 mL of Hionic-Fluor™ will accept up to 5 mL of
1.0 M sodium hydroxide fully saturated with CO2.
4. 10 mL of ULTIMA-Flo™ AF* will accept 10 mL of
0.5 M NaOH/CO2 or 5 mL of 1.0 M NaOH/CO2.
* High flash-point cocktails.
A P P L I C A T I O N
L S C C O C K TA I L S
LSC in Practice
Radio-Carbon Dioxide (14CO2)
Trapping and Counting
Notes:
Recommended LSC Cocktails
1. Hionic-Fluor is suitable for use with hydroxide/carbonate solutions due to its sample capacity for concentrated solutions and alkaline pH. Ideally the final pH
should be above 9 to avoid liberation of trapped CO2.
1. 10 mL of Insta-Fluor™ will accept up to 7.5 mL of
Hyamine Hydroxide® saturated with carbon dioxide.
2. Cocktails containing mixed surfactant systems, such
as ULTIMA Gold™ or ULTIMA Gold XR can be used,
however counting should be performed the same day
as these cocktails have the potential for slow release of
CO2 on prolonged storage (i.e. characterized by
dropping CPM levels).
2. 10 mL of Emulsifier-Safe™ will accept up to 3 mL of
Hyamine Hydroxide® saturated with carbon dioxide
providing a safer system due to the high flash-point of
this LSC cocktail.
Note:
3. Hyamine Hydroxide® in methanol
Foaming of Hyamine Hydroxide® used to absorb carbon
dioxide expelled in rat breath has been reported. This
can be overcome by the addition of one drop of silicone
antifoam per 10 mL of Hyamine Hydroxide®.
Chemically, Hyamine Hydroxide® performs similarly to
sodium and potassium hydroxide in that it forms
hyamine carbonate on reaction with CO2.
The chemiluminescence resistance of both these cocktails is shown in Table 2, which clearly demonstrates
their suitabilities for use with this reagent system.
Table 2. Chemiluminescence resistance of LSC cocktails to Hyamine Hydroxide®/CO2.
Up to 3,0 mL Hyamine Hydroxide®/CO2
in 10 mL Emulsifier-Safe
Counting window
0 - 156
2 - 156
4 - 156
Up to 7.5 mL Hyamine Hydroxide®/CO2
in 10 mL Insta-Flour
Counting window
0 - 156
2 - 156
4 - 156
CPM after one minute
53
32
24
CPM after one minute
35
28
22
CPM after five minutes
39
27
22
CPM after five minutes
34
30
27
CPM after one hour
36
31
25
CPM after one hour
24
23
18
1) All counting in a PerkinElmer Tri-Carb 1900@ 20 °C.
2) All samples fully saturated with carbon dioxide.
3) All counting in high performance glass vials.
4. Ethanolamine
Recommended LSC Cocktails
Ethanolamine reacts with CO2 in a rather different way
than previously discussed reagents in that a carbamate is
formed as opposed to a carbonate. The main difference
between these two species is that a carbamate is more
stable under slightly acidic conditions whereas a carbonate reacts rapidly with acids to release carbon dioxide.
The main system known which is capable of accepting
this unusual reagent is shown in Table 3. The recommended LSC cocktail requires the use of a co-solvent e.g.
methyl cellosolve to facilitate the take up of the
ethanolamine/CO2.
Ethanolamine/CO2 is notoriously difficult to incorporate
into LSC cocktails and consequently the recommended
LSC solutions may seem a little unusual.
Table 3. Suitable LSC solutions and capacity for ethanolamine/CO2.
Cocktail Volume (mL)
Methyl Cellosolve Volume (mL)
Ethanolamine Volume (mL)
Hionic-Fluor
10.0
4.0
1.0
8.1
Hionic-Fluor
10.0
6.5
2.0
16.2
Hionic-Fluor
10.0
8.5
3.0
24.3
COCKTAIL
2
mM CO2 Trapping Capacity
be phase separation of the formed carbamate. If however
this capacity is exceeded, then either of the following
two methods will produce a clear homogeneous mixture:
5-6. Carbo-Sorb/Carbo-Sorb E
These amines react with CO2 to form a carbamate as
well. Both reagents were developed to work in the
PerkinElmer Sample Oxidizer. It is however, possible to
use these reagents as carbon dioxide trapping agents
outside of the Oxidizer. As with the Oxidizer, the
recommended cocktail is Permafluor® E+. When using
Carbo-Sorb or Carbo-Sorb E with Permafluor E+ , the
following conditions are recommended:
i) Add methanol to the mixture until a clear solution
forms (usually 2-4 mL).
ii)Add extra Carbo-Sorb to the mixture until a clear
solution forms. This method simply dilutes the
absorption capacity below the critical 80% level.
Note:
1. Carbo-Sorb E with Permafluor E+
The use of Carbo-Sorb or Carbo-Sorb E is not recommended in enzymatic, plant or human studies due to the
corrosive nature of the volatile amine present.
For ratios of Carbo-Sorb E to Permafluor E+ from 1:10 up
to 1:1, maximum saturation of carbon dioxide is possible
with no phase separation of the resulting carbamate.
Summary
2. Carbo-Sorb with Permafluor E+
The information presented in the previous sections (1-6)
of this publication are condensed into a quick reference
table (Table 4). This may prove particularly useful when
the total trapping capacity per standard 20 mL LSC vial
is required.
When using Carbo-Sorb with Permafluor E+ at any ratio
up to 1:1 the recommendation is that 80% carbon dioxide
capacity is not exceeded. Above this capacity there may
Table 4. Reference table for CO2 trapping and LS counting.
CO2 Absorber
mM CO2
per mL
mL for
1 mM CO2
5mM CO2
mL for
10 mM CO2
mL for
LSC Cocktail
Cocktail
Volume
mL of Absorber
Max. CO2
Capacity (mM)
0.1 M Sodium &
Potassium Hydroxide
0.05
20.00
—
—
Emulsifier-Safe
15.0 mL
14.0 mL
3.00
7.00
0.15
0.35
0.5 M Sodium &
Potassium Hydroxide
0.25
4.00
—
—
ULTIMA-Flo AF
10.0 mL
10.00
2.50
1.0 M Sodium &
Potassium Hydroxide
0.50
0.50
2.00
2.00
10.00
10.00
—
Hionic-Fluor
ULTIMA-Flo AF
14.0 mL
14.0 mL
7.00
7.00
3.50
3.50
1.0 M Hyamine Hydroxide®
in Methanol
0.50
2.00
10.00
—
Emulsifier-Safe
Insta-Fluor
15.0 mL
12.0 mL
4.50
9.00
2.25
4.50
Ethanolamine
8.10
0.12
0.62
1.23
Hionic-Fluor +
Methyl Cellosolve
10.0 mL
8.5 mL
3.00
24.30
Carbo-Sorb
5.80
0.17
0.86
1.72
Permafluor E+
10.0 mL
8.00
46.40
Carbo-Sorb E
4.80
0.21
1.04
2.08
Permafluor E+
10.0 mL
10.00
48.00
Conclusion
Recommended Literature
There are a variety of PerkinElmer LSC cocktails, both
high flash-point and classical types, which are suitable
for 14CO2 absorption work regardless of the trapping agent
used. If problems with trapping and counting persist, or
an alternative trapping agent not covered in this publication
is used, please call your local PerkinElmer representative
for further applications support.
1. Accurate Determination of 14CO2 by Expulsion from
Blood. Kaczmar, B.U. and Manet R. Appl. Radiat. Isot.
Vol 38 No. 7, pp 577-578, 1987.
2. The Use of CO2 Absorbers for the Determination of
Specific 14C Activities. R.M. Qureshi, Peter Fritz and
R.J. Dsimmie, Int. J. Appl. Radiat. Isot. vol 36 No.2, pp
165-170, 1985.
w w w. p e r k i n e l m e r. c o m
3
3. 14C Dating of Hydrological Samples Using Simple
Procedures. Riffat M. Qureshi and Peter Fritz. Int. J.
Appl. Radiat. Isot. vol 36 No. 10 p 825,1985.
4. A Device for the Liberation and Determination of
14CO . Schadewaldt et. al. Analytical Biochemistry Vol
2
132, pp 400-404, 1983.
5. A Method of Counting 14C as CaCO3 in a Liquid
Scintillator with Improved Precision. Pfeiffer K.,
Rank, D. and Tschurlovits. Int. J. of App. Radiat. Isot.
vol 32, pp 665-667, 1981.
6. Improved Technique for Accurate and Convenient
Assay of Biological Reactions Liberating 14CO2. Sissons,
C.H. Analytical Biochemistry Vol 70, pp 454-462, 1976.
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©2008 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. Emulsifier-Safe, Hionic-Fluor, Insta-Fluor, ULTIMA-Flo and
ULTIMA Gold are trademarks and Carbo-Sorb, Ethanolamine, Opti-Fluor and Permafluor are registered trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other
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