Principles and Applications of Liquid Scintillation Counting
Principles and Applications of Liquid Scintillation Counting
Principles and Applications of Liquid Scintillation Counting
A PRIMER
FOR
ORIENTATION
Principles 1.1 RADIOACTIVE EMISSIONS 1.2 MEASUREMENT OF RADIATION AND ISOTOPE QUANTITATION 1.3 MECHANISM OF LIQUID SCINTILLATION COUNTING 1.4 LIQUID SCINTILLATION SIGNAL INTERPRETATION 1.5 THE COMPLETE SCINTILLATION COCKTAIL 1.6 CHEMILUMINESCENCE AND STATIC ELECTRICITY 1.7 WASTE DISPOSAL ISSUES
Applications 2.1 COUNTING DISCRETE SAMPLES 2.2 SPECIAL SAMPLE PREPARATION PROTOCOLS 2.3 FLOW LIQUID SCINTILLATION 2.4 LIQUID SCINTILLATION AND RADIATION SAFETY
1
1.1 1.2 1.3
RADIOACTIVE EMISSIONS
Types of Radioactive Emission / Characteristics of Useful Isotopes / Use of Isotopes in Research
1.5
THE COMPLETE SCINTILLATION COCKTAIL CHEMILUMINESCENCE AND STATIC ELECTRICITY WASTE DISPOSAL ISSUES
1.6
1.7
he chemical properties of an element are determined by its atomic number - the number of protons in the nucleus (and electrons within neutral atoms of that element). Uncharged neutrons, within the nucleus along with protons, do not contribute to the atomic number, but will alter the atomic mass. This makes possible the existence of isotopes, which are atoms of the same element with different atomic weight. Most isotopes are stable, and do not undergo any spontaneous nuclear changes. A subset of isotopes possess too few or too many neutrons to be stable. These are radioactive. Radioactive atoms spontaneously rearrange their nuclei, emitting energy or particles in the process.
Radioactive isotopes of common elements are extremely useful in life science disciplines, among others, because radioactive atoms can be substituted for their nonradioactive counterparts in chemical formulations. The resulting radioactive compound is easily detectable but still chemically identical to the original material. Two detection methods predominate for assaying such incorporated radioactivity. In autoradiography, labeled material is allowed to expose a photographic emulsion. Development of the emulsion reveals the distribution of labeled material. In the second detection method, the amount of radioactivity in labeled samples is directly measured, either by a Geiger counter or by a scintillation counter. In scintillation counting, the sample is mixed with a material that will fluoresce upon interaction with a particle emitted by radioactive decay. The scintillation counter quantifies the resulting flashes of light.
Emission
Half Life (t1/2) 12.3 yrs 5730 yrs 87.2 days 14.3 days 25.3 days 59.9 days
Glycine [2 - 3H]
ATP [-35S]
ATP [-32P]
Figure 1.1.3a Examples of radiolabeled biological molecules including tritiated glycine and ATP labeled with either 35S or 32P.
1.2
To determine the number of radioactive atoms present within a sample at a given time, use the equations above.
Most research applications of radioisotopes, at some stage, require quantitation of the isotope, which is done by measuring the intensity of radiation emitted. Common nomenclature expresses this intensity as disintegrations per minute (DPM). The SI unit for radiation, the Becquerel (Bq), corresponds to 60 DPM (one disintegration per second). The curie, an earlier and still prevalent measure, is equal to 3.7 x 1010 Bq. Truly accurate measurement of DPM would require that every emission event be detected and counted, which is not possible in most situations. Additionally, naturally occurring isotopes and cosmic radiation contribute significant background radiation. Corrections for efficiency and background are needed to convert CPM, the counts per minute measured, into DPM, the number of decay events which actually occurred. Techniques have been developed for applying these corrections, and a great deal of research has been carried out to improve the efficiency of counting, using various detection systems.
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Liquid scintillation (LSC), detailed in the next section, was evolved to provide a usable method for counting organic isotopic compounds. These materials are most often water soluble -emitters. LSC addresses the need for convenience, reproducibility, and high sensitivity in these assays. It also offers solutions to the problem of counting aqueous (i.e. biological) samples in a nonaqueous environment.
1.3
Figure 1.2.1a The stylized representation of an ionization chamber above shows a beta particle colliding and ionizing a neutral particle.
By eliminating the combustion steps needed for gas phase analysis, the introduction of liquid scintillation counting (LSC) reduced the time required to analyze radioactive samples from hours to minutes. For low energy (soft) emitters, LSC offers unmatched convenience and sensitivity. LSC detects radioactivity via the same type of light emission events which are used in solid scintillation. The key difference is that in LSC the scintillation takes place in a solution of scintillator, rather than in a solid crystal. This allows close contact between the isotope atoms and the scintillator, which is not possible with solid scintillation. With LSC the short path length of soft emissions is not an obstacle to detection. Liquid scintillation cocktails absorb the energy emitted by radioisotopes and re-emit it as flashes of light. To accomplish these two actions, absorption and re-emission, cocktails contain two basic components, the solvent and the phosphor(s). The solvent carries out the bulk of the energy absorption. Dissolved in the solvent, molecules of phosphor convert the absorbed energy into light. Many cocktails contain additional materials to extend their range of use to different sample compositions, but the solvent and the phosphor provide the scintillation of the mixture.
Toluene
Figure 1.2.2a In a solid scintillator, beta and alpha particles cannot penetrate the barrier between the sample well and the NaI crystal, but gamma rays pass through easily.
Figure 1.3.1a Solvents employed in liquid scintillation cocktails, such as toluene, pseudocumene, or PXE, possess aromatic rings to absorb the energy of incident radiation.
Pseudocumene
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particle Toluene
p-Terphenyl
Figure 1.3.1b The solvent molecules in a scintillation cocktail absorb a portion of an alpha or beta particles energy. The energy passes between solvent molecules until the energy reaches a phosphor, which absorbs the energy and re-emits it as light.
1.4
Primary Scintillators
Scintillator Butyl PBD
2-[4-biphenylyl]-5-[4-tert-butylphenyl]-1,3,4-oxadiazole) Order No. SFC-20
Structure
Naphthalene
Order No. SFC-40
322nm
PPO
2,5-diphenyloxazole Order No. SFC-10
357nm
A particle, passing through a scintillation cocktail, leaves a trail of energized solvent molecules. These excited solvent molecules transfer their energy to scintillator molecules, which give off light. Each scintillator molecule gives off only one photon on activation, (and the wavelength of that photon is characteristic of the scintillator, not the -particle), but multiple scintillators are activated by the energized molecules generated by one -particle. The path of a -particle in a cocktail is generally less than 0.1 cm; and the half life is correspondingly short, which means that the burst of photons from an emission event derives from a small space, and reaches the PMT with sufficient simultaneity to be read as one pulse of light. The number of photons generated is directly proportional to the path length of the particle, which is in turn determined by its emission energy (the particle rebounds from solvent molecule to solvent molecule, until its incident energy is exhausted). The intensity of each light pulse corresponds to the emission energy and the number of pulses per second corresponds to the number of radioactive emissions.
p-Terphenyl
Order No. SFC-50
340nm
Secondary Scintillators
BBQ
(7H-benzimidazo[2,1-a]benz [de]isoquinoline-7-one) Order No. SFC-13
477nm
Bis-MSB
(1,4-bis[2-methylstyryl]benzene) Order No. SFC-90
420nm
Figure 1.4.1a Passing through scintillation fluid, a single beta particle gives rise to multiple, nearly simultaneous emissions of light. These photons are registered by the photomultiplier tube as one pulse of energy. The magnitude of this light pulse corresponds to the number of photons.
POPOP
(1,4-bis[5-phenyloxazol-2yl]benzene) Order No. SFC-60
410nm
TPB
(1,1,4,4-tetraphenyl-1,3-butadiene) Order No. SFC-15
455nm
Table 1.3.2a
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1.4.3 Quenching
Quenching is the loss of counts due to sample or cocktail characteristics and may result from a variety of components in a sample. Quenchers are customarily divided into the categories of chemical quenchers or color quenchers. Chemical quenchers absorb radioactive energy before it is converted to light. Therefore, chemical quenchers reduce the number of photons generated by each -particle. Color quenchers absorb light in the range of the wavelength emitted by the scintillator. In this case the number of photons emitted is not changed, but the number reaching the photomultiplier tube is reduced.
Figure 1.4.3a Strong quenching can shift the majority of pulses below the threshold of detection (marked by the dashed red line).
In both types of quenching, the energy of all light pulses is reduced, and the total CPM is reduced by the number of pulses quenched to below detectable levels. This leads to an underestimate of the total counts, and thus of the isotope present. It also leads to an apparent shift in the energy spectrum of the sample.
Figure 1.4.2a A scintillation counter collating the energy spectrum of emissions into three channels would read the majority of 3H emissions in the low energy channel, 14C in the intermediate channel, and 32P in the high energy channel.
Chemical quenching
Color quenching
Detection
Figure 1.4.3b Chemical quenching and color quenching can keep energy from a radioactive event from making its way through the scintillation mechanism to the photomultiplier tube. The stylized diagram above presents chemical quenching by water and color quenching by an organic nitrate as two possible obstacles to efficient counting.
Quench Correction
Various methods are available for quench correction. The most straightforward, but most laborious, is the use of an internal standard. A known amount of radioactivity, added to an unknown sample, will increase the DPM by a predictable amount. The difference between the increase in DPM observed and that expected is due to quenching, and allows the determination of counting efficiency for that sample. The drawback to the use of internal standards is that each sample must be counted twice. It is also inconvenient to add an internal standard to many vials. Many scintillation counters offer the use of an external standard to correct for quenching. After initial counting, a strong emission source is placed next to the vial and the sample is counted again. The rays cause secondary emission of Compton electrons, which scintillate in the cocktail like particles. The counts due to sample radioactivity are subtracted, leaving only the Compton electron counts. The theoretical energy distribution of the Compton electrons is compared with the measured energy spectrum to determine the extent of quenching. The samples must still be counted twice, but nothing need be added to the vials, and the process may be carried out automatically by the counter. The analysis of the energy spectrum is commonly done by computing a channels ratio. The detected counts are divided into channels based on their relative energies, and the number of high energy counts (with two channels, the B channel) is compared to the number of low energy counts (channel A). The ratio, calculated as B/A or B/B+A, will change if the sample is quenched. Quenching reduces the intensity of each light pulse, so counts will appear to be of lower energy. This will shift counts from high to low energy channels, and decrease the channels ratio. In practice, a set of quenched standards is created by adding a quenching agent to reduce the CPM of an internal standard. The channels ratio of the external standard is then determined, and a correlation is established between quench and channels ratio. The channels ratio analysis may also be applied to the sample itself to determine quenching. Again, a set of quenched standards is assembled, and a known amount of radioactivity is added to each. A curve is constructed, relating CPM/DPM to B/B+A. Once this curve has been generated, the quench of any subsequent sample can be determined from its channels ratio. This quenching factor is then used to correct CPM to DPM.
must escape from the micelle before beginning the scintillation process. Energy is lost while the emitted particle traverses the micelle, resulting in fewer photons per particle reaching the counter. The result is an effective quench, which can be corrected by the means given in the previous section. This quenching is dependent upon the size of the micelles, which in turn depends upon the ratio of sample to cocktail. It is important to use a correction curve which accounts for this volume dependence.
Figure 1.5a Micellar structure in a scintillation cocktail. Hydrophilic proteins (green) and water (blue) are emulsified by Triton X-100 (black). Radioactive emissions from the labeled protein must pass through the micelle to encounter the toluene solvent (brown) before energy can be passed to the primary and secondary phosphors (red) and be reemitted as light.
Ecoscint A LS-273 An excellent all around scintillation fluid, Ecoscint A is readily biodegradable with high flash point, low odor and low toxicity. It has exceptional sample holding capability (40% water) and high efficiency.
Ecoscint LS-271 The first biodegradable scintillation fluid introduced, the original Ecoscint is an excellent all around performer at an affordable price. Provides good counting efficiency and sample hold.
Ecoscint H LS-275 Ecoscint H is National Diagnostics highest efficiency scintillation fluid for aqueous samples. Ecoscint H delivers up to 62% 3H counting efficiency. It can hold up to 10% of its own volume of aqueous sample.
Monoflow 5 LS-285 Economical, biodegradable flow scintillator for HPLC effluents counted in flow detectors at ratios of up to 3:1 scintillator to sample. Monoflow 5 is nonhazardous and can be disposed of as normal liquid waste.
Ecoscint O LS-274 Ecoscint O is a biodegradable scintillation fluid designed to count non-aqueous (organic soluble) samples. Ecoscint O delivers ultra-high efficiency and extremely low background.
Uniscint BD LS-276 Specially formulated to accommodate high salt and buffer samples while still delivering efficiency. Accepts NH4HPO3 gradients up to 2M in concentration. Suitable for both flow or vial counting.
Sample Capacity
Increasing the amount of water dispersed in an emulsion cocktail will increase micellar size, decreasing the energy in any given particle when it finally escapes the micelle and begins to generate light. At some point, the amount of water added causes a micellar inversion, in which the organic solvent is surrounded by the surfactant, while the water makes up the bulk solution. Efficiency will decrease drastically at this point. The inversion process generally does not yield a clear solution. Above the cocktail sample holding capacity, the mixture is cloudy or opaque, and photons emitted within such a solution are lost to internal reflectance. Sample holding capacity is dependant upon sample composition and upon temperature. In planning scintillation counting experiments, it is crucial to ensure that the sample volume not be too close to the capacity of the cocktail. Such samples may turn opaque with a 1-2 change in temperature, and give falsely low readings. With the use of translucent plastic scintillation vials this type of artifact can be very difficult to detect. Sample Holding Capacities of National Diagnostics Ecoscint A
Sample Capacity (ml sample /10ml cocktail)
Many scintillation counters use coincidence counting to eliminate counts due to chemiluminescence. This system uses two photomultiplier tubes, generally mounted opposite each other. Because chemiluminescence only generates one photon at a time, only one photomultiplier tube will be activated. In contrast, the burst of photons from a genuine decay event will activate both photomultiplier tubes. Coincidence counting eliminates those emission events which do not appear at both photomultiplier tubes, thus eliminating chemiluminescence counts. However, coincidence counting will also cause some low energy emission events to be missed. A further source of spurious counts is static electricity. The energy from a static electric buildup can be released as a burst of light from the cocktail. In dry environments, with plastic vials and latex gloves, high levels of static can build up, sufficient to give 104 cpm or higher from an affected sample. Static is the likely cause if counts from an individual sample vary unpredictably from one measurement to the next. Static can be minimized by wiping the vials with a wet paper towel (water dissipates the static) or by wiping with an antistatic laundry dryer sheet.
Water (20C) Water (25C) Water (15C) 0.05M Tris-HCl 0.15M NaCl 10% Sucrose 8M Urea
Table 1.5a
Figure 1.6a Spurious counts due to chemiluminescence (green) dissipate over the course of hours, while the true count stays nearly constant. With the common isotopes used in life sciences research, the rate of radioactive decay is much slower than the decay of chemiluminescent reactions.
2
2.1 2.2
Cocktails have been developed for discrete samples and for flow applications, answering the specific needs of each type of experiment. In addition, cocktails are available for a variety of specific applications for the counting of discrete samples with unique characteristics. Most of these cocktails are designed to simplify sample preparation in such applications as counting samples in electrophoresis gels, samples on filters, whole tissue samples or combusted materials.
hours, or in some cases, overnight, before counting. This allows time for the chemiluminescent reaction to run its course and die out. If chemiluminescence is suspected, samples should be counted repeatedly at intervals of greater than 1 hour until a stable reading is obtained.
2.1.2 Decolorizing
Achieving a colorless solution of sample in cocktail is generally not problematic. Many samples are colorless, or contain so little color that dilution into the scintillation cocktail gives an essentially colorless solution. In those cases where samples are deeply colored, particularly when the sample absorbs in the region of 300-400 nm, where scintillation phosphors emit, several decolorizing protocols are available. As visible color often depends upon long conjugated polyene systems, strong oxidants are used to bleach the samples. Samples can be treated quite harshly prior to counting, because chemical changes to the labeled compounds will not alter the number of DPM emitted.
Protocol 2.1.2a Decolorizing LSC Samples with Ultraviolet Light Ultraviolet irradiation is often effective in bleaching visibly colored samples. The optimal wavelength, intensity and time must of course be determined for each sample. In many cases, exposing samples to sunlight for 1-2 hours is sufficient. Bleaching by UV has a great advantage over other methods nothing is added to the sample, avoiding the potential quenching or chemiluminescent effects of other bleaching agents. Protocol 2.1.2b
H2O2 is a strong oxidant and a very effective bleaching agent. It is inexpensive, easy to work with, and miscible with aqueous samples. The only disadvantage to using H2O2 is that it decomposes to produce molecular oxygen, which is an effective quenching agent. Samples must be heated to drive off the O2 following H2O2 bleaching, to ensure reproducible results. 1. Mix: 0.1 - 0.3 ml of 30% H2O2 with 1ml sample 2. Incubate 1 hour at 50C, shake occasionally. 3. Cool to room temperature, add scintillation cocktail and count.
Ecoscint A LS-273 An excellent all around scintillation fluid, Ecoscint A is readily biodegradable with high flash point, low odor and low toxicity. It has exceptional sample holding capability (40% water) and high efficiency.
Ecoscint LS-271 The first biodegradable scintillation fluid introduced, the original Ecoscint is an excellent all around performer at an affordable price. Provides good counting efficiency and sample hold.
Ecoscint H LS-275 Ecoscint H is National Diagnostics highest efficiency scintillation fluid for aqueous samples. Ecoscint H delivers up to 62% 3H counting efficiency. It can hold up to 10% of its own volume of aqueous sample.
Monoflow 5 LS-285 Economical, biodegradable flow scintillator for HPLC effluents counted in flow detectors at ratios of up to 3:1 scintillator to sample. Monoflow 5 is nonhazardous and can be disposed of as normal liquid waste.
Samples which are not soluble in water or tissues which have been dissolved in organic solubilizers, can be bleached with Benzoyl Peroxide. 1. Dissolve 1g Benzoyl Peroxide in 5 ml Toluene - heating to 60C may be required. Filter solution if cloudy. (Caution: Toluene has a flash point of 7C. Heating must be carried out in a spark-free fume hood to avoid an explosion hazard.) 2. Add 2 ml Benzoyl Peroxide/Toluene solution to 1 ml sample. 3. Incubate for 30 minutes at 50C. 4. Cool to room temperature, add scintillation cocktail and count.
Ecoscint O LS-274 Ecoscint O is a biodegradable scintillation fluid designed to count non-aqueous (organic soluble) samples. Ecoscint O delivers ultra-high efficiency and extremely low background.
Uniscint BD LS-276 Specially formulated to accommodate high salt and buffer samples while still delivering efficiency. Accepts NH4HPO3 gradients up to 2M in concentration. Suitable for both flow or vial counting.
2.1.1
The neutralization of strongly alkaline samples is necessary to avoid chemiluminescence (Section 1.6). Neutralization can be accomplished by the addition of acetic acid. If the sample contains a high concentration of alkali, the addition of acetic acid may increase the overall salt content beyond the capacity of the scintillation cocktail. In such situations, the sample will need to be diluted prior to the addition of cocktail. If neutralization is not practical, samples may be left to stand 1-3
10
Protocol 2.2.2a Counting Samples on Cellulose-Ester Filters Do not dry the filters, because this will slow the dispersion process. If a filter has dried, dampen it with 1-2 drops of distilled water. 1. Place damp filter with sample into 10ml of National Diagnostics Filtron X.
2.2.1
TLC Plates
2. Allow to stand at room temperature for 15 minutes. Shake well and count.
In a typical TLC experiment, the radioactivity is detected at two points: after TLC it is analyzed by autoradiography (Electrophoresis Theory, Section 4.1.3), to locate radioactive spots. These spots are then scraped off of the plate and counted to provide quantitative information. Each of these steps can be enhanced using the following protocols.
Protocol 2.2.1a Autoradiography and LSC with TLC Plates A. Autoradiography (Fluorography) After the plate has been developed, spray twice with National Diagnostics Autofluor and allow the plate to dry. This impregnates the plate with phosphors, which will convert the emissions to more readily detectable photons. Autofluor enhances the speed and sensitivity of detection when the plate is placed on film. For details on autoradiography procedures, see Electrophoresis Theory, Section 4.1.3. B. Liquid scintillation counting of scraped TLC silica Option 1: Suspend the silica powder in 10ml of a cocktail which has a gel phase with water such as National Diagnostics Hydrofluor. Shake well, and add 3ml H2O. Shake until gel forms, and count.
Option 2: Suspend the silica in 10ml of an organic based (non-emulsion) cocktail such as National Diagnostics Ecoscint O. Add 0.5-1g finely divided silica thixotrophic agent to form a clear gel in the solution, keeping the TLC particles suspended. Count as usual.
BIOSOL/BIOSCINT 1. Place up to 200 mg of tissue, or 1 ml of blood, in a glass scintillation vial. Ground or minced tissue will dissolve more rapidly. Avoid adhesion of the sample to the bottom of the vial, as this will extend the digestion time. 2. Add 1 ml of Biosol. Agitate gently (do not vortex).
3. Incubate in shaking water bath at 50C for 1-4 hours, until clear. 4. If necessary (for blood or other pigmented samples) decolorize with 0.2 ml of 30% H2O2. Cap loosely and incubate at 50C 1 hour. 5. Cool to room temperature, add 10 ml of Bioscint and count.
SOLUSOL 1. Place up to 100 mg of tissue, or 0.5 ml of blood, in a glass scintillation vial. Ground or minced tissue will dissolve more rapidly. Avoid adhesion of the sample to the bottom of the vial, as this will extend the digestion time. 2. Add 0.2-0.4 ml of Solusol. Agitate gently (do not vortex). 3. Incubate at 50C for 1-2 hours, or at room temperature for 3-5 hours, until clear. 4. If necessary (for blood or other pigmented samples) decolorize with 2 volumes of 20% benzoyl peroxide in toluene. Cap loosely and incubate at 50C 30 minutes. Lightly colored samples may be bleached by UV or sunlight exposure. 5. Cool to room temperature, add 10 ml of Soluscint O and count.
2.2.2
A common radiotracer technique is to precipitate macromolecules (protein & DNA) with TCA or some other strong denaturant, collect the precipitate on a filter and count it. Often such procedures give variable results, depending upon the degree to which the sample disperses from the filter into the cocktail. A typical artifact is counts which rise over time as more material dissolves off of the filter. It is possible to avoid these artifacts by using a cocktail which dissolves the filter, reproducibly releasing all of the sample for counting.
Figure 2.2a The well-dispersed sample on the right achieves 4 counting geometry, while half of the counts with the sample on the right are lost due to absorption and attenuation of emissions by the filter paper.
11
4. Place gel directly onto filter paper and dry under heat (80C) and vacuum. 5. Expose at -80C. 24 hours is generally sufficient for 14C or 3H samples, although up to 72 hours may be required for maximum detection of 3H.
DISSOLUTION AND COUNTING OF GEL SLICES 1. Using the autoradiography film as a template, cut out the band(s) of interest. 2. To every 100mg of gel, add 0.5ml 30% H2O2. 3. Incubate in a LSC vial at 50C until digested (1-4 hours). 4. Heat at 37C for one additional hour to drive off residual O2.
Figure 2.2.4a Bubbling 14 CO 2 through carbamate traps the gas in the liquid phase, yielding a suitable sample for liquid scintillation counting.
5. Cool, add 10ml of a scintillation cocktail capable of holding 0.6ml of aqueous material (such as Ecoscint H) and count.
Protocol 2.2.4a Counting 14CO2 OXOSOL C14 This is a single solution containing both carbamate and scintillators. Gas containing 14CO2 is shaken with the cocktail or bubbled through with a sparger. It is advisable to extract the gas with a second volume of solution to ensure capture of >90% of the 14CO2 . Cap the vials and count. CARBAMATE-1 + OXOSOL 306 In this system, the carbamate is provided as a separate solution, which may enhance capture efficiencies. Shake or bubble the gas with 1ml of carbamate. Add 10ml of Oxosol 306 and count.
Scintillation fluid
Pump PMT 1
PMT 2
continued
Flow detectors operate by passing a mixture of column effluent and scintillation cocktail through a transparent or translucent channel, which is monitored by a photomultiplier tube. The flow rates of effluent and cocktail are metered to provide a constant ratio, and the channel (generally a plastic tube) is coiled to cover the entire window area of the PMT, (see figure 2.3a). Cocktails for flow LSC must provide for high efficiency, high sample capacity, and low viscosity. High efficiency allows for more sensitive detection, and minimizes any decrease in signal due to sample composition or detector geometry. The sample capacity is important because flow LSC tends to generate surprising amounts of waste materials. A typical HPLC flow rate is 1ml/min. Over an 8 hour day, continuous use of such a system will generate 1 X 60 X 8=480ml of spent solvent. If a low capacity cocktail is used (one which requires 10 volumes of cocktail per volume of sample), up to 5 liters of waste will be produced. Use of a high capacity cocktail (3:1 or 2:1) will reduce this amount by up to 70% and keep disposal costs under control. Because of the large amounts of cocktail used, switching of labeled peaks to a separate waste collection and using a biodegradable cocktail are recommended.
12
Biodegradable
Applications
All purpose scintillation fluid for a wide range of sample types. Ultra-high sample hold. Organic effluents. Lipid and steroid separations.
Monoflow 1
Order # LS-281
N/A
is exposed by the emissions which affect the worker. A correlation between film exposure and worker exposure allows the detection of dangerous levels of radiation in the lab. Radiation safety departments also use film badges to ensure that no user exceeds their long term exposure limits in a given year. Short term monitoring of exposure to high-energy emissions can be done with a Geiger counter, which detects the ionization of a gas in a sealed tube. Only those emissions which are energetic enough to penetrate the tube can be detected: 32P and the highest energy emissions from 35S. Geiger counters can be invaluable for checking gloves for contamination during the course of an experiment.
Wipe Testing
Monoflow 2
Order # LS-282
Routine low salt aqueous effluents (<200mM salt) Routine low salt aqueous effluents. Higher sample holding capacity than Monoflow 2
Monoflow 3
Order # LS-283
Once an experiment is finished, a comprehensive and sensitive check of all work areas is required. This is accomplished by use of wipe tests. A 4 cm2 piece of paper or other absorbent material is rubbed vigorously over the work area, placed in scintillation cocktail and counted. If counts above background are detected, the contaminated area is subdivided and the divisions wipe tested. Contaminated areas are cleaned and retested until no contamination can be detected. The type of filter used in wipe testing has a marked effect on the reliability of the results obtained. Often standard filter paper discs are used. Such discs generally adhere to the side or the bottom of the scintillation vial. If the vial is placed in the counter such that the filter is on the side facing the photomultiplier tube, much of the light emitted by the cocktail will be absorbed by the filter. This will give artificially low numbers of counts, measuring contaminated areas as clean. This hazard is avoided by the use of a wipe which dissolves in the scintillation cocktail such as National Diagnostics Nuc-Wipes.
Monoflow 4
Order # LS-284
High salt aqueous samples. Can accommodate 2M salt gradients. Biodegradable cocktail for routine low salt aqueous samples (<200mM salt) Biodegradable cocktail for high salt aqueous samples. Can accommodate up to 2M salt gradients.
Monoflow 5
Order # LS-285
Uniscint BD
Order # LS-276
Table 2.3a
Figure 2.4b Nuc-Wipes eliminate the dependence of results on the direction of the filter paper and time. Because NucWipes dissolve in scintillation fluid, there is no intact filter to absorb or attenuate beta emissions. 4 counting efficiency is achieved, giving reproducible results.
Efficient and effective cleaning of spills requires some knowledge of the chemical nature of the labeled compound. Water soluble materials will come off of nonabsorbent surfaces with detergent solutions. Hydrophobic compounds require the use of higher detergent concentrations. Extremely hydrophobic compounds or absorbent surfaces may require the use of organic solvents. If an unknown sample is spilled (spent culture medium, cellular extracts, etc.), a general purpose radioactive decontamination agent should be tried, such as National Diagnostics Nuclean, followed by solvents if necessary.
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Sample Overload
Counting Geometry
Exceeding the sample holding capacity of a scintillation cocktail will yield unpredictable results. The mixture will become opaque, with most photons being lost to internal reflectance. Phase separation may occur, which can increase or decrease counts depending on how the sample partitions. 1) Sample composition: The salt content, pH, protein content, etc., of a sample will determine the amount of cocktail needed to provide a clear, countable emulsion. Samples above 0.5M salt, or with pH < 4 or pH > 10 will generally require more cocktail. 2) Temperature: Sample/cocktail emulsions have a range of temperature in which they remain clear and countable. This range narrows as the sample volume approaches the cocktail holding capacity. The heat of mixing associated with adding sample to the cocktail can cause sample/ cocktail mixtures which are initially clear to cloud on standing, leading to a drop in counting efficiency. Addition of more cocktail will restore a clear solution. 3) Phase separation: The opaque solution generated by sample overload may separate into two phases on standing. If the sample molecules partition into the organic phase, this can increase efficiency. However, because this is a slow process, the effect is unpredictable, and manifests as erratic counting from sample to sample. 4) Standard curves: It is important to plan standard curves so that the sample capacity is not approached, to ensure that the efficiency is the same for all points.
Particulate samples, or samples bound to a solid support, will have some emission events which are absorbed by the solid before reaching the cocktail. This can reduce efficiency by as much as 50%.
1 Particulate samples: Insoluble powders must be suspended in a cocktail which forms a gel, to avoid changes in counts as the particles settle. 2) Samples on solid support: For TLC plate scrapings, see (A) particulate samples, above. Samples bound onto modified cellulose filters, use Filtron-X to dissolve the filter. For glass fiber filters, use a cocktail which will dissolve the sample components, and allow sufficient time and mixing for complete elution. Be sure filter is not in the path of the PMT.
Diagnosis
Sample not fully separated from solid support (B-2) or solid sample not fully dispersed/dissolved in cocktail (B-1). Chemiluminescence (C-1) or sample overload (A-2,3).
Static electricity (C-2), sample overload (A-2,3), or solid sample settling out of cocktail (B-1). Color Quenching (D-1), chemical quenching (D-2), or sample overload (A-1). Sample not fully dispersed or dissolved (B-1).
Sample not eluted from filter (B-2), filter blocking PMT window (B-2), or quenching (D-1,2). Sample overload (A-4).
Quenching
Light emission from cocktails can be stimulated by static electricity or chemically excited molecules. Such emissions are relatively short lived. 1) Chemiluminescence: Caused by reactions which generate chemically excited products, its lifetime is limited by the amount of substrate in the sample. Samples should be recounted repeatedly until a stable result is obtained. Alkaline samples are particularly susceptible; neutralization can minimize the problem. 2) Static electricity: Static buildup, particularly on plastic vials, can cause bursts of light emission, giving wildly erratic counts. Run water over the vial, or wipe with an antistatic dryer sheet.
Scintillator light emissions can be absorbed by colored sample components (color quenching). In addition, the energy from radioactive emissions can be trapped by sample components before it reaches the cocktail phosphors (chemical quenching). In both cases, total counts are reduced, and the ratio of high to low energy counts is decreased. 1) Color quenching: Samples which absorb light at 350-450nm will attenuate the light emitted from the phosphors. In general, samples which appear yellow or brown will be quenched to some extent. Quenching shifts counts from high to low energy- a shift in the ratio of high and low channels is diagnostic. Samples can be bleached with hydrogen peroxide, taking care to remove the residual oxygen, which is a chemical quencher (see (2), below). 2) Chemical Quenching: Many chemicals are able to intercept the energy from a radioactive emission event before it can be converted to light. The effect is the same as color quenching: lower counts, and proportionally more low energy counts. Water can be a chemical quencher- to ensure consistent efficiency, all samples should have the same amount of water added. Molecular Oxygen is another quenching agent, it can be removed by warming the sample to degas it. Organic compounds containing oxygen (i.e. aldehydes and alcohols), or halogens (e.g. chloroform) are generally strong chemical quenchers. Such samples should be counted in as dilute a solution as possible, to minimize the quenching effect.
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12.3 yrs 5730 yrs 87.2 days 14.3 days 25.3 days 59.9 days
0.6 24 26 800 50
Iodine (125I)
Units of Radioactivity
1 Becquerel(Bq) = 1 disintegration per second (dps) = 60 disintegrations per minute (dpm)
1 Curie(Ci)
= 3.7 X 1010 disintegrations per second = 2.2 X 1012 disintegrations per minute
(decay constant) = 0.693/t1/2 N = N0e-t where: N= number of atoms remaining at time t N0=number of atoms at start (t=0)
General Resources
Schimel, David S. (1993) Theory and Application of Tracers. Academic Press. Knoche, H.W. (1991) Radioisotopic Methods for Biological and Medical Research Oxford University Press, NY. Slater, R.J. (ed) (1990) Radioisotopes in Biology - A Practical Approach. Oxford University Press. Coleman, David C and Fry, Brian, (eds) (1991) Carbon Isotope Techniques. Academic Press. Ehmann, Wm. D. and Vance, Diane E. (1991) Radiochemistry and Nuclear Methods of Analysis. J. Wiley & Sons.
Press, Boca Raton. Slack, L. and Way, K. (1959) Radiations from Radioactive Atoms in Frequent Use. U.S. Atomic Energy Commission Report.
Ionization Detection
Wilkinson, D.H., (1950) Ionization Chambers and Counters. Cambridge University Press, Cambridge, UK.
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