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. w w w. p e r k i n e l m e r. c o m 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. PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA Phone: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/lasoffices ©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 countries. Hyamine Hydroxide is a registered trademark of Lonza. All other trademarks not owned by PerkinElmer, Inc. or its subsidiaries that are depicted herein are the property of their respective owners. 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