WO2003062350A1

WO2003062350A1 - Liquid scintillation counting

Info

Publication number: WO2003062350A1
Application number: PCT/NL2003/000002
Authority: WO
Inventors: Jan Ter Wiel; Saake Kleinman; Swaantje Elisabeth Beukema
Original assignee: Packard Bioscience BV
Current assignee: Packard Bioscience BV
Priority date: 2002-01-07
Filing date: 2003-01-06
Publication date: 2003-07-31
Anticipated expiration: 2004-07-07
Also published as: NL1019704C2

Abstract

The present invention relates to a medium for scintillation counting comprising at least one fluor and a specific solvent. The invention also relates to a method for analyzing a sample by scintillation counting, wherein this medium is used. An important advantage of a medium according to the invention is that a more reliable measuring result is obtained within a shorter time than with known scintillation media.

Description

Title: Liquid scintillation counting

The invention relates to a composition for use in liquid scintillation counting, in particular to a solvent therefor. The invention further relates to a method for detecting β radiation in which this composition is used.

Liquid scintillation counting is a well-known method for detecting and measuring a concentration of radioactive elements or compounds in a sample. It is a particularly useful method for measuring elements emitting low-energetic β radiation, such as ³H, ¹⁴C, ³⁵S, ⁵⁵Fe, and ³⁶C1.

In a method for liquid scintillation counting, a radioactive sample to be analyzed is brought into a medium (or cocktail) for liquid scintillation. Such a medium generally comprises a solvent in the form of an aromatic hydrocarbon, one or more scintillators or fluors and optionally a surfactant (surface active substance) or emulsifier. Energy released during energetic decay in the sample excites the aromatic hydrocarbon. An energy transfer process takes place with the result that a fluor releases the energy in the form of light. This light can be quantified, or counted, with for instance photomultipliers and associated apparatus in a scintillation counter.

Surfactants are mainly used in liquid scintillation counting when aqueous samples are analyzed. The presence of a surfactant in a cocktail leads to the formation of a microemulsion in which the aqueous phase is kept close to the organic phase. Consequently, radioactive particles present can enter into the desired interaction with the aromatic hydrocarbon.

The primary scintillator or fluor used in liquid scintillation counting is a substance capable of emitting light through fluorescence if sufficient energy is received and the substance reaches an excited condition. Energy from a radioactive decay can be transferred by a solvent to the primary scintillator. The solvent thus functions as a link in a cascade of energy transfer processes.

When a β particle is emitted in the liquid scintillation medium by a radioactive isotope, it comes into contact with molecules of the solvent. A number of these molecules will be excited by the energy which the β particle transfers. Because the β particle then loses its energy, the number of excited solvent molecules will be directly proportional to the number of β particles. High-energy β particles will travel a longer path in a scintillation medium and excite many solvent molecules. Because a solvent for Hquid scintillation counting must be able to be efficiently excited by a β particle, it is generally aromatic by nature. Through the presence of double bonds in an aromatic solvent, energy can be efficiently collected and transferred after a specific time to a fluor molecule. The fluor molecule then releases, usually monochromatically, light that can be recorded with a Hquid scintiUation counter.

Examples of known and commercially used solvents for liquid scintillation are toluene, xylenes, ethyϊbenzenes, cumenes, pseudocumene, phenylcyclohexane, anisole, and dioxane. In case of dioxane a small amount of naphthalene can be added. These known solvents have many disadvantages, such as a high vapor pressure, toxicity, and a relative low flash point, so that they can be inconvenient and dangerous to use. Another disadvantage of these solvents is that they can diffuse through the (often polyethylene or polypropylene) walls of frequently used holders for scintillation counting, which results in measuring errors.

More recently, other solvents for liquid scintillation counting have been developed. US-A-4,657,696 describes the use of diisopropylnaphthalenes. US-A-5, 135,679 describes the use of 1,2- dicumylethane, and US-A-4,867,905 proposes to use 1-phenyl-l-xylylethane. These more modern solvents largely remove the disadvantages of the above-mentioned solvents. They all have a low vapor pressure, a low toxicity, and a high flash point. Also, they do not penetrate the materials of which the walls of holders for scintillation counting are often made. Nevertheless, a number of other disadvantages are connected with these solvents.

Addition of a sample to a scintillation medium may cause chemical quenching, color quenching or sample quenching. Chemical quenching occurs when an electronegative or electron sucking compound is present in the sample, or a sample consists thereof (for instance a halogenated hydrocarbon) and this sample is added to a medium for liquid scintillation counting. Such a sample can absorb the excitation energy of the aromatic solvent in the scintillation medium. Color quenching, also referred to as photon quenching, occurs when a colored sample is analyzed. The photons emitted by such a sample can be absorbed by a colored substance. Sample quenching, or dilution quenching, wherein the average distance between the molecules of the solvent in the scintillation medium increases as a result of the dilution occurring when a sample to be analyzed is added to the medium. Consequently, the risk of interaction between a radioactive particle and the solvent is reduced. All three forms of quenching result in a disturbance of the measurement/analysis.

Another disadvantage of the above-mentioned more modern solvents for liquid scintillation counting is their relative high viscosity. Because of a high viscosity of the solvent, the scintillation medium will have a high viscosity as weU. This has the result that a sample to be analyzed mixes relatively slowly with the scintiUation medium. IdeaUy, the sample is directly homogeneously distributed over the medium before molecules of the solvent are excited so that the final measurement proceeds as accurately as possible. Furthermore, a high viscosity makes it difficult to accurately dose a scintillation medium from a so-caUed 'bottle-top dispenser' generally used for this purpose. Moreover, a high viscosity may give rise to inaccuracy when dosing small amounts of scintillation medium, for instance in the wells of a microtiter plate. It is an object of the invention to provide a liquid scintillation medium which does not have the above disadvantages. More in particular, it is an object of the invention to provide a Hquid scintillation medium having a low vapor pressure, a low toxicity, a high flash point, and a low viscosity. Furthermore, it is an object of the invention to provide a medium that is as much insensitive to quenching as possible, that is to say has a high quench resistance so that a high counting efficiency is achieved in a scintillation measurement.

Surprisingly, it has been found that the above and other objects of the invention are achieved if a monoisopropylnaphthalene is used as solvent in a scintillation medium. The invention therefore relates to a medium for liquid scintiUation counting comprising a fluor dissolved in a solvent, wherein the solvent comprises a monoisopropylnaphthalene.

A medium according to the invention has a low vapor pressure, a low toxicity, and a high flash point. Furthermore, a medium according to the invention has a relative low viscosity. With the aid of a medium according to the invention, a liquid scintillation counting can be carried out with great accuracy because the risk that quenching occurs is very low. Furthermore, a medium according to the invention can be easily and accurately dosed from a manual or automized dosing system. A medium according to the invention can be used for analyzing of both aqueous and organic samples. Other advantages will become apparent from the following description of the invention.

As stated, a medium for scintillation counting comprises a solvent comprising a monoisopropylnaphthalene. EHgible therefor are both 1- monoisopropylnaphthalene and 2-monoisopropylnaphthalene. Monoisopropylnaphthalene s may be prepared by propylation of naphthalene with the aid of a suitable catalyst. They are also commercially sold by Rutgers Kureha Solvents GmbH, Duisburg, Germany. 1-Monoisopropylnaphthalene is preferably present in an amount of 0.5 to 50 wt.%, more preferably 25 to 45 wt.%, based on the weight of the solvent. 2-Monoisopropylnaphthalene is preferably present in an amount of 0.5 to 75 wt.%, more preferably 50 to 70 wt.%, based on the weight of the solvent. Although these substances can be used individually or in the form of a mixture, they are preferably used in combination. Their mutual ratio is preferably selected such that between 25 and 45 wt.% 1- monoisopropylnaphthalene and between 55 and 75 wt.% 2- monoisopropylnaphthalene is present in the mixture. Besides a monoisopropylnaphthalene, a medium according to the invention may comprise one or more other solvents. Eligible therefor are both the above-mentioned solvents for scintillation counting and other solvents. The latter can be used as co-solvent or diluent, to adjust the viscosity or to increase the compatibility with specific plastics (for instance polystyrene). Examples of other solvents to be used are toluene, xylenes, ethylbenzenes, cumenes, pseudocumene, mesitylene, phenylcyclohexane, anisole, dioxane, wherein in the latter a small amount of naphthalene is dissolved, diisopropylnaphthalenes, 1,2-dicumylethane, 1-phenyl-l- xylylethane, alcohols, cellosolves, and esters. If besides a monoisopropylnaphthalene one or more other solvents are used, the monoisopropylnaphthalene or the mixture of 1- and 2- monoisopropylnaphthalene is preferably present in an amount of 2 to 75 wt.%, more preferably of 15 to 65 wt.%, based on the weight of the solvent. Most preferably, however, monoisopropylnaphthalene, in the form of one of both or in the form of a combination of both isomers, is used as the only solvent in a medium according to the invention.

A great advantage of a medium according to the invention is that the employed solvent has a low viscosity. Preferably, the solvent has a viscosity of less than 3.5 centiStokes (cSt) at 40°C. More preferably, the viscosity is lower than 3.0 cSt at 40°C, and still more preferably, the viscosity is lower than 2.5 cSt at 40°C. The lower limit of the viscosity is not very critical, but wiU usually be 1.0 cSt at 40°C. Due to a low viscosity of the employed solvent, a sample to be analyzed mixes very quickly and homogeneously with a medium according to the invention. Consequently, not only time is gained during a scintillation counting, but also the reliability increases considerably.

The solvent present in a medium according to the invention is preferably Hquid at a temperature of 10°C. More preferably, the solvent is liquid at a temperature of 5°C, more preferably also at -5°C and still more preferably also at -10°C.

Besides the described solvent, a medium for scintillation counting according to the invention comprises at least one fluor. EHgible therefor are all the usual fluors. A fluor frequently used in scintiUation counting is 2,5- diphenyloxazole (PPO), which has a fluorescence peak at 365 nm. Other known fluors are p-terphenyl, 2,5-bis(5-tert-butyl-benzoxaloyl)-thiophene, 2- phenyl-5-(4'-biphenyi)-l,3,4-oxadiazole (PBD), and 2-(4'-tert-butylphenyl)-5- (4'-biphenyl)-l,3,4-oxadiazole (butyl-PBD). The last is a very efficient fluor and has a fluorescence peak at 366 nm. Other known fluors may be used as well. For a discussion thereof, reference may be made to J.B. Birks, "The Theory and Practice of Scintillation Counting", Pergamon Press, 1964. A fluor is generally present in a medium according to the invention in low concentrations, preferably 1 to 15, more preferably 4 to 10 g/1.

In many cases, a secondary fluor, or wavelength shifter, will also be used to shift the wavelength of the scintillation light to a wavelength which the photomultiplier can record more sensitively. By the use of a secondary scintillator, the wavelength of the light is typically shifted to a value above 400 nm. Some typical examples of secondary fluors are l,4-di(2- methylstyryl) -benzene (Bis-MSB), l,6-diphenyl-l,3,5-hexatriene, 9,10- dimethylanthracene (DMA), 9,10-diphenylanthracene, 9,10- ditolylanthracene, l,4-bis-[4-methylphenyl-2- oxazolyljbenzene, and 2,5- di(4-biphenylyl)oxazole. Secondary fluors are generally used in small amounts relative to the (primary) fluor. Preferably, the secondary fluor is present in the medium in an amount between 0.2 and 1 g/1. In a preferred embodiment, particularly when it is (also) intended for use of analysis of aqueous samples, a medium according to the invention may comprise a surfactant. Eligible therefor is, in principle, any surfactant generally used in scintiUation media. The surfactant may be a so-called non- ionic surfactant, for instance a polyethoxylated alkylphenol (for instance nonylphenol), but also anionic, for instance dialkyl sulfosuccinate, or cationic or amphoteric. Other suitable surfactants are polyethoxylated alcohols, dodecylbenzenesulfonates, sulfonates, ether sulfates, and dialkyl sulfosuccinates. The amount of surfactant in the medium will depend on the specific use for which the medium is intended and can be selected within the limits conventional for cocktails for liquid scintillation counting.

The invention further relates to a method for analyzing a sample by means of scintillation counting in which a medium as described above is used. In the conventional manner, according to this method, a sample is brought into the medium, and a light signal is measured with the aid of a scintillation counter.

The invention wUl now be explained in more detail with reference to the following examples, not to be construed as limitative.

Example 1

The viscosity of different solvents was determined with a Gardner EZ viscosity Cup according to ASTM D 4212. Analyzed were monoisopropylnaphthalene (66% 2-isopropylnaphthalene and 34% 1- isopropylnaphthalene) (IPN), 1-phenyl-l-xylylethane (PXE), diisoprop lnaphthalene (DIN), and pseudocumene (PC). The results are listed in Table 1.

Table 1: Viscosities in centiStokes (cSt)

0°C 20°C 40°C

PC 1.2 1.0 0.8

PXE 32.0 11.0 5.2

DIN 45.0 14.0 7.0

IPN 10.5 3.5 2.0

Example 2

Three scintillation media were prepared by mixing the following substances in the indicated amounts: - solvent 230 g

- nonylphenolethoxylate 170 g

- 2,5-diphenyloxazole (PPO) 2 g

- 9,10-dimethylanthracene (DMA) 0.3 g. As solvents were used monoisopropylnaphthalene (66% 2- isopropylnaphthalene and 34% 1-isopropylnaphthalene) (IPN), medium A, and diisopropylnaphthalene (DIN), medium B.

Of both media, the viscosity was determined with a Gardner EZ viscosity Cup according to ASTM D 4212 at a temperature of 20°C. During the preparation of the medium, the solutions of nonylphenolethoxylate were first placed in a thermostatic bath for half an hour to accurately adjust the temperature. The viscosity of medium A was 21.7 cSt; that of medium B

50.7 cSt.

With both media, tritium was analyzed in the form of tritiated valine of tritiated water. Each time 200 μl medium and 10 μl radioactive label were brought together. To this end, the indicated amount of sample was brought into a well of a microtiter plate. To this was added the indicated amount of scintiUation medium, after which the microtiter plate was covered with a seal and agitated for some time to mix the sample and the medium. Subsequently, a counting was carried out with the aid of a Top Count scintillation counter at 19°C, after which agitation and counting were carried out again. The results are listed in the following tables as averages of three -fold measurements.

The presence of water is assumed herein to be responsible for the formation of quench, because this reduces the counting efficiency. In the tables, the amount of water present is based on the sample as percentage. When 100 μl water is added to 1 ml medium, the percentage 100 μl / total volume is 100%. The total volume is 1100 μl, so that the percentage is 9%.

The indicated efficiency is determined by dividing the measured number of counts per minute (cpm) by the number of disintegrations per minute (dpm). The number of disintegrations per minute is a measure for the activity added to a well.

Table 2: scintiUation counting when adding 216000 dpm valine in microtiter plate with 24 wells

Quench Medium A Medium B water

% sample

Counts per Efficiency (%) Counts per Efficiency (%) minute minute

0 113258 52.4 110885 51.3

9 100996 46.7 99121 45.8

Table 2 shows that with medium A a more efficient and more reliable measuring result was obtained than with medium B. Table 3: scintiUation counting when adding 15299 dpm water after mixing for specific time t (minutes) in microtiter plate with 96 wells Quench Medium A Medium B water % sample t = 15 t = 65 t = 115 t = 15 t = 65 t = 115

0 5466 5414 5426 5406 5581 5488

9 5312 5334 5321 4129 4927 5170

16 4800 4854 4848 3217 4296 4730

From these results it appears that medium A shows a higher counting efficiency. This appears particularly after a mixing time of 115 minutes. It further appears that the measured values for medium A in the first measuring cycle are already at the required level, while this is the case for medium B only after 115 minutes. From this it appears that with medium B it is necessary to wait for a time and to agitate more than with medium A before an accurate measurement can be carried out.

Claims

1. A medium for liquid scintillation counting comprising a fluor dissolved in a solvent, wherein the solvent comprises a monoisopropylnaphthalene.

2. A medium according to claim 1, wherein the solvent comprises from 0.5 to 50 wt.%, preferably 25 to 40 wt.%, based on the weight of the solvent,

1 -monoisop ropylnaphthalene .

3. A medium according to claim 1 or 2, wherein the solvent comprises from 0.5 to 75 wt.%, preferably 50 to 70 wt.%, based on the weight of the solvent, 2-monoisopropylnaphthalene.

4. A medium according to any one of the preceding claims, wherein the solvent comprises a mixture of 1-monoisopropylnaphthalene and 2- monoisop ropylnaphthalene .

5. A medium according to any one of the preceding claims, wherein the solvent has a viscosity lower than 3.5 cSt, preferably between 1.0 and 2.5 cSt, at 40°C.

6. A medium according to any one of the preceding claims, wherein the solvent has a melting point lower than 10°C, preferably lower than -10°C.

7. A medium according to any one of the preceding claims, which also comprises a secondary fluor.

8. A medium according to any one of the preceding claims, which also comprises a surfactant.

9. A method for analyzing a sample by means of liquid scintillation counting, wherein the sample is brought into a medium according to any one of the preceding claims and a Hght signal is measured with the aid of a scintillation counter.

10. The use of a monoisopropylnaphthalene as solvent in a medium for liquid scintiUation counting.