Introduction

The liquid scintillation process is the conversion of the energy of a radioactive decay event into photons of light. Photomultiplier tubes (PMT’s) are used to

detect and convert the photons into electrical pulses. Both the sample and the scintillator are dissolved in an aromatic solvent, which allows energy to be transferred. Any factor, which reduces the efficiency of the energy transfer or causes the absorption of photons (light), results in quenching in the sample. There are two main types of quench: chemical quench1,2 and color quench1,3,4.

Chemical QuenchChemical quench occurs during the transfer of energy from the solvent to the scintillator. Any chemical species that is electronegative (electron capturing) will affect the energy transfer process by capturing or stealing the π electrons associated with the aromatic solvent and thus reduce the availability of π electrons necessary for efficient energy transfer.

Use and Preparation of Quench Curves in Liquid Scintillation Counting

A P P L I C A T I O N N O T E

Authors:

Jock Thomson

PerkinElmer, Inc. Waltham, MA

Liquid Scintillation

2

Color QuenchColor quench is an attenuation of the photons of light. The photons produced are absorbed or scattered by the color in the solution, resulting in reduced light output available for measurement by the PMT’s. The steps in the energy transfer process affected by chemical and color quenching are indicated in Figure 1.

The collective effect of quench is a reduction in the number of photons produced and, therefore, detected CPM (counts per minute). Counting efficiency is affected by the degree of quenching in the sample.

To determine absolute sample activity in DPM (disintegrations per minute or absolute activity), it is necessary to measure the level of quench of the samples first, then make the corrections for the measured reduction in counting efficiencies.

Measurement of QuenchIt is possible to measure quench accurately via high-resolution spectral analysis. Quenching manifests itself by a shifting of the energy spectrum toward lower energy channels in the Multichannel Analyzer (MCA). On PerkinElmer's Tri-Carb® series LSA’s, there are two methods of spectral analysis for measuring quench.

The first method is the Spectral Index of the Sample (SIS) which uses the sample isotope spectrum to monitor the quench of the solution. The SIS value decreases as quench increases, reflecting the shift of the spectrum to lower energy. The second method used to measure quench is the transformed Spectral Index of the External Standard (t-SIE) which is calculated from the Compton spectrum induced in the scintillation cocktail by an external 133Ba gamma source.

The source is positioned under the sample vial, causing a Compton spectrum to be produced in the cocktail solution. From a mathematical transformation of this spectrum, the t-SIE value is determined, and t-SIE is a relative value, on a scale from 0 (most quenched) to 1000 (unquenched). The calculated t-SIE value is adjusted to 1000 when the instrument is calibrated. Like SIS, t-SIE decreases as quench increases. Both SIS and t-SIE are used as Quench Indicating Parameters (QIP’s). t-SIE is independent of the sample isotope and of the activity in the vial and has a large dynamic range. This makes it a very reproducible means of tracking the quench of the cocktail. SIS uses the sample isotope spectrum to track quench; it is most accurate with high-count rate samples. The range of SIS values reflects the energy range of the isotope. Both can be used as QIP’s to create quench curves, although use of the external standard is preferred for samples containing low activity and is required for multilabeled samples.

Quench CurveA quench standard curve is a series of standards in which the absolute radioactivity (DPM) per vial is constant and the amount of quench increases from vial to vial. A quench curve uses the relationship between counting efficiency and QIP to correct the measured CPM to DPM. When a quench curve is made, the DPM value in each standard is known. Each standard is counted and the CPM is measured. The counting efficiency is calculated using the following relationship:

At the same time, the QIP is measured for each standard. A correlation is made using the QIP on one axis (X) and the % efficiency on the other axis (Y). A curve is fitted to the standard points. Figures 2 and 3 show quench curves for 3H and 14C using SIS as the QIP and quench curves for the same isotopes using t-SIE as the QIP. Once the quench curve is stored in the instrument computer, it can be used for automatic DPM calculations.

When unknowns are counted, the sample CPM and the QIP are measured. Using the QIP, the counting efficiency is determined from the quench curve. Sample DPM are then calculated by applying the appropriate efficiency to the CPM of the sample.

The standards and unknowns must be counted with the same energy regions. PerkinElmer's LSC’s with spectra based libraries (2500 series, 2700 series and the new 2900 and 3100 series) store the curve in a 0-Emax window and allow the curve to be recalculated for the windows used in the protocol. For other LSC’s (1600,1900, 2100, 2200 and 2300) the windows used to acquire the quench curve must be used in the actual DPM determination.

Figure 1. Quenching in the Energy Transfer Process

= % Counting EfficiencyCPM x 100

DPM

DPM =CPM

Efficiency (expressed as a decimal)

Figure 2. Quench Curves for 3H and 14C Using SIS as the QIP

Figure 3. Quench Curves for 3H and 14C Using t-SIE as the QIP

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Preparation of Quench Curves

Method 1As mentioned, a quench standard curve is a series of standards in which the absolute radioactivity (DPM) per vial is constant and the amount of quench increases from vial to vial. The quench is increased from vial to vial by the addition of a quenching agent. A quenching agent is any chemical or color material added to the vial which causes a shift in the standard spectrum to a lower energy and a subsequent decrease in the counting efficiency of the radioactive standard. Usually a series of six to ten quench standards are prepared per radionuclide. This series is sometimes referred to as the quench set.

PerkinElmer, Inc. provides factory stored quench curves in the instrument for 3H and 14C. On occasion, it is necessary for the investigator to prepare a quench curve, for isotopes other than 3H or 14C, (i.e., 35S). There are some basic considerations to note before preparing a quench curve:

It is necessary to obtain a calibrated source of radioactivity to use as the source of the activity (DPM). It is essential that a known amount of activity be added per vial. Also, the standard material must be compatible with the cocktail chosen.

A suitable quenching agent must be chosen. It is desirable to closely approximate the chemical environment in the samples. If samples contain water with various other constituents, add the same material in increasing amounts to the standards. Additional quenching agents that are most often used and available in the laboratory are carbon tetrachloride (CCl4), acetone (CH3CH3CO), chloroform (CHCl3) and nitromethane (CH3NO2). To prepare the quench standards, perform the following steps:

1. Make a batch of radioactive solution in the chosen cocktail so the desired DPM are transferred to each individual vial when dispensing the cocktail. Prepare the standards with a sufficient level of activity, typically 50,000 to 200,000 DPM per vial, in order to be able to count the standards with good statistics in a short time. If ten standards are to be made with 10 mL of cocktail per vial, then 100+ mL of radioactive cocktail solution are required. If 15 mL of cocktail is to be used then 150+ mL of radioactive cocktail is required. Note: If the unknowns to be counted contain two radioisotopes (i.e., 3H and 14C), then individual standard curves must be prepared for each isotope.

2. Count the individual standards for at least five minutes to check for constant activity (CPM). Any sample that deviates more than 2% from the mean should be discarded.

3. Add incremental amounts of the quenching agent to vials 2 ... n (quenching agent is not added to vial 1) to obtain the desired quench range. It may be necessary to predetermine the amounts to add per vial by testing various volumes of quenching agent added to the cocktail only (no radioactivity), and monitor the amount of cocktail quench using t-SIE. Otherwise, add the suggested amounts of nitromethane based on the information given in Table 1.

4. Count the complete set under the conditions described in the instrument operation manual for storing a quenchcurve. Practically we suggest that the standards are counted to a pre-selected level of statistical accuracy (generally 0.5%2s), and this is usually achieved within five minutes per sample with the sealed standards which we provide.

5. Once the quench curve(s) are counted and stored, count unknown samples using the stored quench curve(s) to determine the DPM value for each sample.

Method 2The preparation of a quench curve with any LSC cocktail is relatively straightforward and the following procedure is given as a guideline. Many researchers use their own methods and equipment and the procedure is, therefore, open to modification.

1. Dispense 10.0 mL or 15.0 mL of LSC cocktail into ten high performance glass vials.

2. Add activity to each vial (200,000 DPM for 3H or 100,000 DPM for 14C).

3. Count all ten vials to ensure that the same amount of activity is in each vial. A count time of about five minutes per vial will be sufficient. Any sample that deviates more than 2% from the mean should be discarded.

4. Number the vials 1 to 10 or A to J and add the suggested amounts of nitromethane based on the information given in Table 1.

5. Count the complete set under the conditions described in the instrument operation manual for storing a quench curve. PerkinElmer recommends counting each standard for 30 minutes or until a pre-selected level of statistical accuracy (using %2S terminator, generally 0.5%) is reached.

Notes1. For dispensing the activity, use a glass barreled microliter syringe

fitted with a Chaney adapter. Such an adapter ensures reproducible dispensing of activity.

2. After preparation the standards should be stored in the dark preferably at 5 °C to 10 °C for best stability.

Quench LevelToluene Standards

(15 mL)Ultima Gold Standards

(15 mL)

A (1) 0 μL 0 μL

B (2) 1 μL 5 μL

C (3) 5 μL 10 μL

D (4) 11 μL 15 μL

E (5) 17 μL 26 μL

F (6) 25 μL 45 μL

G (7) 35 μL 70 μL

H (8) 45 μL 110 μL

I (9) 55 μL 150 μL

J (10) 66 μL 230 μL

Table 1. Volume of Nitromethane Needed for Quench Curve

4

Notes on Using the Quench Curves1. t-SIE is independent of the sample isotope and of the activity in

the vial, and has a large dynamic range. This makes it a very reproducible means of tracking quench in the cocktail.

2. SIS should only be used when there is at least 500 CPM activity in the sample. Remember that SIS uses the sample isotope spectrum to track quench; it is most accurate with high-count rate samples. For an accurate SIS, a good sample spectrum needs to be acquired.

3. SIS should not be used for low activity samples since an accurate sample spectrum cannot be acquired.

4. Most customers prefer to purchase quench standards. For cocktails based on toluene, xylene, pseudocumene or LAB (linear alkyl benzene) as the solvent, toluene quench standards should be used. For cocktails based on DIN (di-isopropylnaphthalene) or PXE (phenylxylylethane) as the solvent, Ultima-Gold™ quench standards should be used. If the wrong quench standard is used there can be an error in DPM. This error is most pronounced with low energy isotopes such as tritium (see Tables 2-5).

5. Be sure that your prepared quench curve covers a wide t-SIE range (i.e., 800-300) in order to provide accurate DPM results.

Color QuenchWhen a small amount of color is present in a sample there is virtually no difference between chemical and color quenching and the standard chemical quench curves are suitable. This applies to

samples where the t-SIE is in the range 100 to 400. However, if a significant amount of color is present in the sample (t-SIE is <100) it may be necessary to consider preparing a color quench curve. When preparing a color quench curve, the selection of a suitable color quench agent is important. Aqueous soluble food dyes are usually a good choice as they provide both a stable color and a wide range of colors. The color of the sample must match the color used in the quench curve. Do not use a pH indicator since some cocktails contain acidic components and these will alter the color. Preparing a color quench curve is very similar to the method used for a chemical quench curve and the only difference is the quench agent. If considerable color is present in the sample it may be wise to modify or change the sample preparation method to either remove or reduce the level of color. Some techniques that have proved useful are shown in Table 2. The easiest way to reduce color quench is to either decrease the sample size or increase the cocktail volume or both.

Nature of Sample Suggested Remedy

Color from sample solubilizationTreat with hydrogen peroxide to bleach out the color

Plant material Consider sample oxidation

Inorganic matrix Change to alternate colorless anion

Table 2. Techniques for Reducing Color in Certain Samples

Quench Curve ErrorsThe errors that can be present when the wrong quench curve is used with an LSC cocktail are shown in the following tables:

Tritium: Ultima Gold Toluene Opti-Fluor Insta-Gel Plus Pico-Fluor 15

No quench -0.12% -1.04% 6.00% 2.70% 4.89%

Low quench -0.46% 4.24% 7.06% 5.14% 6.45%

Medium quench 0.04% 5.87% 8.43% 5.82% 6.91%

High quench -0.14% 10.10% 14.41% 10.02% 11.89%

Highest quench -0.20% 13.42% 18.01% 13.36% 13.43%

Tritium: Ultima Gold Toluene Opti-Fluor Insta-Gel Plus Pico-Fluor 15

No quench -4.10% -0.49% -0.57% -1.58% -0.32%

Low quench -5.33% -0.27% -0.13% -1.22% 0.23%

Medium quench -6.51% 0.01% 0.45% -0.19% 0.60%

High quench -10.39% -0.01% 1.21% -0.79% -0.49%

Highest quench -16.16% -0.70% 0.11% 0.56% -0.21%

Table 3. Various Cocktails vs. Ultima Gold Quench Curve (Tritium)

Table 4. Various Cocktails vs. Toluene Quench Curve (Tritium)

Table 3 shows that only Ultima Gold cocktails should be used for Tritium DPM measurements with the Ultima Gold Tritium quench curve.

Table 4 shows that the Ultima Gold cocktails should not be used for Tritium DPM measurements with the Toluene Tritium quench curve.

5

Using a Quench CurveOnce the quench curve is stored in the instrument computer, it can be used for automatic DPM calculations. When unknowns are counted, the sample CPM and the QIP are measured. Using the QIP (SIS or t-SIE) the counting efficiency is determined from the appropriate quench curve.

For example:

A Tritium sample is analyzed and has: Count rate = 10,000 CPM} t-SIE = 400

From the quench curve shown in Figure 4. The instrument uses the t-SIE of 400 to determine that the counting efficiency is 42%. Since we now know the CPM and the counting efficiency it is possible to calculate the DPM:

CocktailRecommended Quench Curve

(3H AND 14C)

Ultima Gold™ Ultima Gold

Ultima Gold AB Ultima Gold

Ultima Gold LLT Ultima Gold

Ultima Gold MV Ultima Gold

Ultima Gold XR Ultima Gold

Ultima Gold F Ultima Gold

StarScint Ultima Gold

Opti-Fluor®/Opti-Fluor O Toluene

Emulsifier-Safe™ Toluene

Insta-Gel® Plus Toluene

Pico-Fluor™ 15 Toluene

Pico-Fluor 40 Toluene

Insta-Fluor™ Toluene

Hionic-Fluor™ Toluene

Filter-Count™ Toluene

Carbo-Sorb® E / Permafluor® E+ Toluene

Monophase® S Toluene

Formula 989 Toluene

AQUASOL™/AQUASOL II Toluene

AQUASSURE®/BIOFLUOR® Toluene

ATOMLIGHT® Toluene

ECONOFLUOR®-2 Toluene

Table 7. A Simple Guide to Quench Curve Selection

Carbon-14: Ultima Gold Toluene Opti-Fluor Insta-Gel Plus Pico-Fluor 15

No quench 0.06% -1.37% 2.51% 2.25% 1.96%

Low quench 0.03% 0.12% 2.04% 0.78% 0.80%

Medium quench -0.13% 0.84% 1.72% 1.15% 1.20%

High quench 0.02% 1.30% 2.51% 3.11% 1.71%

Highest quench -0.63% 4.52% 3.81% 3.59% 2.77%

Table 5. Various Cocktails vs. Ultima Gold Quench Curve (Carbon-14)

Table 5 shows that only Ultima Gold cocktails should be used for Carbon-14 DPM measurements with the Ultima Gold Carbon-14 quench curve.

Carbon-14: Ultima Gold Toluene Opti-Fluor Insta-Gel Plus Pico-Fluor 15

No quench 0.45% 0.42% 1.71% 1.90% 1.96%

Low quench 0.27% -0.03% 0.89% 1.20% 0.43%

Medium quench -0.54% -0.37% 1.28% 0.31% 0.83%

High quench -0.81% 0.01% 1.86% 0.77% 0.81%

Highest quench -16.51% 0.33% 1.04% 0.49% 0.88%

Table 6. Various Cocktails vs. Toluene Quench Curve (Carbon-14)

Table 6 shows that the Ultima Gold cocktails should not be used for Carbon-14 DPM measurements with the Toluene Carbon-14 quench curve.

DPM = CPM

10,000

Efficiency (expressed as a decimal)

0.42 =

= 23,809 DPM

Figure 4. How the Instrument Uses t-SIE to Determine Efficiency

Summary

A compilation of recommended quench curves for different LSC cocktails are shown in Table 7. This will provide a basic guide to correct quench curve selection.

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Conclusion

For accurate DPM recovery, it is imperative that the quench curve selected is appropriate for the LSC cocktail being used. PerkinElmer manufactures quench curves using either a safer, high flash point (Ultima Gold) cocktail or a classical solvent (Toluene) that are suitable for use with all PerkinElmer's LSC cocktails. If problems with quench curve selection or preparation persist, or help is needed with a particular isotope not mentioned in this publication, please call your local PerkinElmer representative for further applications support.

References

1. Gibson, J.A.B. 1980. Modern techniques for measuring the quench correction in a liquid scintillation counter. In Peng, C.T., Horrocks, D.L. and Alpen, E.L., eds., Liquid Scintillation Counting, Recent Applications and Developments. New York, Academic Press: 153-172.

2. Birks, J.B. 1971. Liquid scintillator solvents. In Peng, C.T., Horrocks, D.L. and Alpen, E.L., eds., Organic Scintillators and Liquid Scintillation Counting. New York, Academic Press: 3-23.

3. Ross, H.H. 1965. Color quench correction in liquid scintillator systems. Analytical Chemistry, 37(4): 621-623.

4. Ten Haaf, F.E.L. 1975. Color quenching in liquid scintillation counters. In Crook, M.A. and Johnson, P., eds., Liquid Scintillation Counting, Vol. 3. London, Heyden & Son, Ltd.: 41-43.

The application note information enclosed is used to illustrate the technique and may not represent the latest instrument, reagents and cocktails. Customers should validate the technique in their laboratory.

Contact Customer Care at www.perkinelmer.com/contact to find the current PerkinElmer instruments, reagents and cocktails.

Introduction

Basically, the liquid scintillation process is the conversion ofthe energy of a radioactive decay event into photons of light.Photomultiplier tubes (PMT’s) are used to detect and con-vert the photons into electrical pulses. Both the sample andthe scintillator are dissolved in an aromatic solvent, whichallows energy to be transferred. Any factor, which reducesthe efficiency of the energy transfer or causes the absorp-tion of photons (light), results in quenching in the sample.There are two main types of quench: chemical quench1,2 andcolor quench1,3,4.

Chemical QuenchChemical quench occurs during the transfer of energy fromthe solvent to the scintillator. Any chemical species that iselectronegative (electron capturing) will affect the energytransfer process by capturing or stealing the π electronsassociated with the aromatic solvent and thus reduce theavailability of π electrons necessary for efficient energytransfer.

Color QuenchColor quench is an attenuation of the photons of light. Thephotons produced are absorbed or scattered by the color inthe solution, resulting in reduced light output available formeasurement by the PMT’s. The steps in the energy trans-fer process affected by chemical and color quenching areindicated in Figure 1.

Use and Preparation of Quench Curves inLiquid Scintillation Counting

By Jock Thomson, Packard BioScience Company, Meriden, Connecticut

800 Research Parkway Meriden, CT 06450 Tel: 203-639-2598 1-800-323-1891 Fax: 203-639-2172Web site: http://www.packardinstrument.com E-mail: webmaster@packardinstrument.com

Liquid Scintillation CountingApplication Note LSC-007

Figure 1.Quenching in the Energy Transfer Process

The collective effect of quench is a reduction in the numberof photons produced and, therefore, detected CPM (countsper minute). Counting efficiency is affected by the degree ofquenching in the sample.

To determine absolute sample activity in DPM (disintegra-tions per minute or absolute activity), it is necessary tomeasure the level of quench of the samples first, then makethe corrections for the measured reduction in countingefficiencies.

Measurement of QuenchIt is possible to measure quench accurately via high-resolu-tion spectral analysis. Quenching manifests itself by a shift-ing of the energy spectrum toward lower energy channels inthe Multichannel Analyzer (MCA). On Packard’s Tri-Carb®

series LSA’s, there are two methods of spectral analysis formeasuring quench.

The first method is the Spectral Index of the Sample (SIS)which uses the sample isotope spectrum to monitor thequench of the solution. The SIS value decreases as quenchincreases, reflecting the shift of the spectrum to lower en-ergy. The second method used to measure quench is thetransformed Spectral Index of the External Standard (t-SIE)which is calculated from the Compton spectrum induced inthe scintillation cocktail by an external 133Ba gamma source.

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The source is positioned under the sample vial, causing aCompton spectrum to be produced in the cocktail solution.From a mathematical transformation of this spectrum, thet-SIE value is determined, and t-SIE is a relative value, on ascale from 0 (most quenched) to 1000 (unquenched). Thecalculated t-SIE value is adjusted to 1000 when the instru-ment is calibrated. Like SIS, t-SIE decreases as quenchincreases. Both SIS and t-SIE are used as Quench Indicat-ing Parameters (QIP’s). t-SIE is independent of the sampleisotope and of the activity in the vial and has a large dy-namic range. This makes it a very reproducible means oftracking the quench of the cocktail. SIS uses the sampleisotope spectrum to track quench; it is most accurate withhigh-count rate samples. The range of SIS values reflectsthe energy range of the isotope. Both can be used as QIP’sto create quench curves, although use of the externalstandard is preferred for samples containing low activity andis required for multilabeled samples.

Quench CurveA quench standard curve is a series of standards in whichthe absolute radioactivity (DPM) per vial is constant and theamount of quench increases from vial to vial. A quench curveuses the relationship between counting efficiency and QIPto correct the measured CPM to DPM. When a quench curveis made, the DPM value in each standard is known. Eachstandard is counted and the CPM is measured. The count-ing efficiency is calculated using the following relationship:

CPM x 100 = % Counting EfficiencyDPM

At the same time, the QIP is measured for each standard. Acorrelation is made using the QIP on one axis (X) and the %efficiency on the other axis (Y). A curve is fitted to thestandard points. Figures 2 and 3 show quench curves for 3Hand 14C using SIS as the QIP and quench curves for thesame isotopes using t-SIE as the QIP. Once the quenchcurve is stored in the instrument computer, it can be usedfor automatic DPM calculations.

When unknowns are counted, the sample CPM and the QIPare measured. Using the QIP, the counting efficiency isdetermined from the quench curve. Sample DPM are thencalculated by applying the appropriate efficiency to the CPMof the sample.

DPM = CPMEfficiency (expressed as a decimal)

The standards and unknowns must be counted with thesame energy regions. Packard LSC’s with spectra basedlibraries (2500 series, 2700 series and the new 2900 and3100 series) store the curve in a 0-Emax window and allowthe curve to be recalculated for the windows used in theprotocol. For other LSC’s (1600,1900, 2100, 2200 and 2300)the windows used to acquire the quench curve must be usedin the actual DPM determination.

Figure 2.Quench Curves for 3H and 14C Using SIS as the QIP

Figure 3.Quench Curves for 3H and 14C Using t-SIE as the QIP

Preparation of Quench Curves

Method 1As mentioned, a quench standard curve is a series of stan-dards in which the absolute radioactivity (DPM) per vial isconstant and the amount of quench increases from vial tovial. The quench is increased from vial to vial by the additionof a quenching agent. A quenching agent is any chemical orcolor material added to the vial which causes a shift in thestandard spectrum to a lower energy and a subsequentdecrease in the counting efficiency of the radioactive stan-dard. Usually a series of six to ten quench standards areprepared per radionuclide. This series is sometimes referredto as the quench set.

Packard BioScience provides factory stored quench curvesin the instrument for 3H and 14C. On occasion, it is necessaryfor the investigator to prepare a quench curve, for isotopesother than 3H or 14C, (i.e., 35S). There are some basic con-siderations to note before preparing a quench curve:

It is necessary to obtain a calibrated source of radioactivityto use as the source of the activity (DPM). It is essential thata known amount of activity be added per vial. Also, the stan-dard material must be compatible with the cocktail chosen.

Page 3

A suitable quenching agent must be chosen. It is desirableto closely approximate the chemical environment in thesamples. If samples contain water with various other con-stituents, add the same material in increasing amounts tothe standards. Additional quenching agents that are mostoften used and available in the laboratory are carbontetrachloride (CCl4), acetone (CH3CH3CO), chloroform(CHCl3) and nitromethane (CH3NO2). To prepare the quenchstandards, perform the following steps:

1. Make a batch of radioactive solution in the chosen cock-tail so the desired DPM are transferred to each individualvial when dispensing the cocktail. Prepare the standardswith a sufficient level of activity, typically 50,000 to200,000 DPM per vial, in order to be able to count thestandards with good statistics in a short time. If ten stan-dards are to be made with 10 mL of cocktail per vial,then 100+ mL of radioactive cocktail solution are re-quired. If 15 mL of cocktail is to be used then 150+ mL ofradioactive cocktail is required. Note: If the unknowns tobe counted contain two radioisotopes (i.e., 3H and 14C),then individual standard curves must be prepared foreach isotope.

2. Count the individual standards for at least five minutesto check for constant activity (CPM). Any sample thatdeviates more than 2% from the mean should bediscarded.

3. Add incremental amounts of the quenching agent tovials 2 ... n (quenching agent is not added to vial 1) toobtain the desired quench range. It may be necessary topredetermine the amounts to add per vial by testing vari-ous volumes of quenching agent added to the cocktailonly (no radioactivity), and monitor the amount of cock-tail quench using tSIE. Otherwise, add the suggestedamounts of nitromethane based on the information givenin Table 1.

4. Count the complete set under the conditions describedin the instrument operation manual for storing a quenchcurve. Practically we suggest that the standards arecounted to a pre-selected level of statistical accuracy(generally 0.5%2s), and this is usually achieved withinfive minutes per sample with the sealed standards whichwe provide.

5. Once the quench curve(s) are counted and stored, countunknown samples using the stored quench curve(s) todetermine the DPM value for each sample.

Quench Level Toluene Ultima Goldstandards standards

(15 mL) (15 mL)

A (1) 0 µL 0 µL

B (2) 1 µL 5 µL

C (3) 5 µL 10 µL

D (4) 11 µL 15 µL

E (5) 17 µL 26 µL

F (6) 25 µL 45 µL

G (7) 35 µL 70 µL

H (8) 45 µL 110 µL

I (9) 55 µL 150 µL

J (10) 66 µL 230 µL

Table 1.Volume of Nitromethane Needed for Quench Curve

Method 2Basically the preparation of a quench curve with any LSCcocktail is relatively straightforward and the following proce-dure is given as a guideline. Many researchers use theirown methods and equipment and the procedure is, there-fore, open to modification.

1. Dispense 10.0 mL or 15.0 mL of LSC cocktail into tenhigh performance glass vials.

2. Add activity to each vial (200,000 DPM for 3H or 100,000DPM for 14C).

3. Count all ten vials to ensure that the same amount ofactivity is in each vial. A count time of about five minutesper vial will be sufficient. Any sample that deviates morethan 2% from the mean should be discarded.

4. Number the vials 1 to 10 or A to J and add the suggestedamounts of nitromethane based on the information givenin Table 1.

5. Count the complete set under the conditions describedin the instrument operation manual for storing a quenchcurve. Packard recommends counting each standard for30 minutes or until a pre-selected level of statisticalaccuracy (using %2S terminator, generally 0.5%) isreached.

Notes1. For dispensing the activity, use a glass barreled microli-

ter syringe fitted with a Chaney adapter. Such an adapterensures reproducible dispensing of activity.

2. After preparation the standards should be stored in thedark preferably at 5 °C to 10 °C for best stability.

Page 4

Notes on using the Quench Curves1. t-SIE is independent of the sample isotope and of the

activity in the vial, and has a large dynamic range. Thismakes it a very reproducible means of tracking quenchin the cocktail.

2. SIS should only be used when there is at least 500 CPMactivity in the sample. Remember that SIS uses thesample isotope spectrum to track quench; it is mostaccurate with high-count rate samples. For an accurateSIS, a good sample spectrum needs to be acquired.

3. SIS should not be used for low activity samples since anaccurate sample spectrum cannot be acquired.

4. Most customers prefer to purchase quench standards.For cocktails based on toluene, xylene, pseudocumeneor LAB (linear alkyl benzene) as the solvent, toluenequench standards should be used. For cocktailsbased on DIN (di-isopropylnaphthalene) or PXE(phenylxylylethane) as the solvent, Ultima-Gold™quench standards should be used. If the wrong quenchstandard is used there can be an error in DPM. Thiserror is most pronounced with low energy isotopes suchas tritium (see Tables 2-5).

5. Be sure that your prepared quench curve covers a widetSIE range (i.e., 800-300) in order to provide accurateDPM results.

Color QuenchWhen a small amount of color is present in a sample there isvirtually no difference between chemical and color quench-ing and the standard chemical quench curves are suitable.This applies to samples where the tSIE is in the range 100to 400. However, if a significant amount of color is present in

Nature of Sample Suggested Remedy

Color from sample Treat with hydrogen peroxidesolubilization to bleach out the color

Plant material Consider sample oxidation

Inorganic matrix Change to alternatecolorless anion

Table 2.Techniques for Reducing Color in Certain Samples

the sample (tSIE is <100) it may be necessary to considerpreparing a color quench curve. When preparing a colorquench curve, the selection of a suitable color quench agentis important. Aqueous soluble food dyes are usually a goodchoice as they provide both a stable color and a wide rangeof colors. The color of the sample must match the color usedin the quench curve. Do not use a pH indicator since somecocktails contain acidic components and these will alter thecolor. Preparing a color quench curve is very similar to themethod used for a chemical quench curve and the only dif-ference is the quench agent. If considerable color is presentin the sample it may be wise to modify or change the samplepreparation method to either remove or reduce the level ofcolor. Some techniques that have proved useful are shownin Table 2. The easiest way to reduce color quench is toeither decrease the sample size or increase the cocktailvolume or both.

Quench Curve errorsThe errors that can be present when the wrong quenchcurve is used with an LSC cocktail are shown in the follow-ing tables:

Tritium: Ultima Gold Toluene Opti-Fluor Insta-Gel Plus Pico-Fluor 15

No quench -0.12% -1.04% +6.00% +2.70% +4.89%

Low quench -0.46% +4.24% +7.06% +5.14% +6.45%

Medium quench +0.04% +5.87% +8.43% +5.82% +6.91%

High quench -0.14% +10.10% +14.41% +10.02% +11.89%

Highest quench -0.20% +13.42% +18.01% +13.36% +13.43%

Table 3 shows that only Ultima Gold cocktails should be used for Tritium DPM measurements with theUltima Gold Tritium quench curve.

Table 3.Various Cocktails vs. Ultima Gold Quench Curve (Tritium)

Table 4 shows that the Ultima Gold cocktails should not be used for Tritium DPM measurements with theToluene Tritium quench curve.

Table 4.Various Cocktails vs. Toluene Quench Curve (Tritium)

Tritium: Ultima Gold Toluene Opti-Fluor Insta-Gel Plus Pico-Fluor 15

No quench -4.10% -0.49% -0.57% -1.58% -0.32%

Low quench -5.33% -0.27% -0.13% -1.22% +0.23%

Medium quench -6.51% +0.01% +0.45% -0.19% +0.60%

High quench -10.39% -0.01% +1.21% -0.79% -0.49%

Highest quench -16.16% -0.70% +0.11% +0.56% -0.21%

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Table 5 shows that only Ultima Gold cocktails should be used for Carbon-14 DPM measurements with theUltima Gold Carbon-14 quench curve.

Table 5.Various Cocktails vs. Ultima Gold Quench Curve (Carbon-14)

Carbon-14: Ultima Gold Toluene Opti-Fluor Insta-Gel Plus Pico-Fluor 15

No quench +0.06% -1.37% +2.51% +2.25% +1.96%

Low quench +0.03% +0.12% +2.04% +0.78% +0.80%

Medium quench -0.13% +0.84% +1.72% +1.15% +1.20%

High quench +0.02% +1.30% +2.51% +3.11% +1.71%

Highest quench -0.63% +4.52% +3.81% +3.59% +2.77%

Table 6 shows that the Ultima Gold cocktails should not be used for Carbon-14 DPM measurements with theToluene Carbon-14 quench curve.

Table 6.Various Cocktails vs. Toluene Quench Curve (Carbon-14)

Carbon-14: Ultima Gold Toluene Opti-Fluor Insta-Gel Plus Pico-Fluor 15

No quench +0.45% +0.42% +1.71% +1.90% +1.96%

Low quench +0.27% -0.03% +0.89% +1.20% +0.43%

Medium quench -0.54% -0.37% +1.28% +0.31% +0.83%

High quench -0.81% +0.01% +1.86% +0.77% +0.81%

Highest quench -16.51% +0.33% +1.04% +0.49% +0.88%

Using a Quench CurveOnce the quench curve is stored in the instrument computer,it can be used for automatic DPM calculations. Whenunknowns are counted, the sample CPM and the QIP aremeasured. Using the QIP (SIS or t-SIE) the counting effi-ciency is determined from the appropriate quench curve.

For example:

A Tritium sample is analyzed and has:Count rate = 10,000 CPMt-SIE = 400

From the quench curve shown in Figure 4. The instrumentuses the t-SIE of 400 to determine that the countingefficiency is 42%. Since we now know the CPM and thecounting efficiency it is possible to calculate the DPM:

DPM = CPMEfficiency (expressed as a decimal)

= 10,0000.42

= 23,809 DPM

Figure 4.How the Instrument Uses t-SIE to Determine Efficiency

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Summary

A compilation of recommended quench curves for differentLSC cocktails are shown in Table 7. This will provide a basicguide to correct quench curve selection.

Cocktail RecommendedQuench Curve(3H AND 14C)

Ultima Gold™ Ultima GoldUltima Gold AB Ultima GoldUltima Gold LLT Ultima GoldUltima Gold MV Ultima GoldUltima Gold XR Ultima GoldUltima Gold F Ultima GoldStarScint Ultima GoldOpti-Fluor®/Opti-Fluor O TolueneEmulsifier-Safe™ TolueneInsta-Gel® Plus ToluenePico-Fluor™ 15 ToluenePico-Fluor 40 TolueneInsta-Fluor™ TolueneHionic-Fluor™ TolueneFilter-Count™ TolueneCarbo-Sorb® E / Permafluor® E+ TolueneMonophase® S TolueneFormula 989 TolueneAQUASOL™/AQUASOL II TolueneAQUASSURE®/BIOFLUOR® TolueneATOMLIGHT® TolueneECONOFLUOR®-2 Toluene

Conclusion

For accurate DPM recovery, it is imperative that the quenchcurve selected is appropriate for the LSC cocktail beingused. Packard manufactures quench curves using either asafer, high flash point (Ultima Gold) cocktail or a classicalsolvent (Toluene) that are suitable for use with all Packard’sLSC cocktails. If problems with quench curve selection orpreparation persist, or help is needed with a particular iso-tope not mentioned in this publication, please call your localPackard representative for further applications support.

References1. Gibson, J.A.B. 1980. Modern techniques for measuring the quench

correction in a liquid scintillation counter. In Peng, C.T., Horrocks,D.L. and Alpen, E.L., eds., Liquid Scintillation Counting, RecentApplications and Developments. New York, Academic Press:153-172.

2. Birks, J.B. 1971. Liquid scintillator solvents. In Peng, C.T., Horrocks,D.L. and Alpen, E.L., eds., Organic Scintillators and LiquidScintillation Counting. New York, Academic Press: 3-23.

3. Ross, H.H. 1965. Color quench correction in liquid scintillatorsystems. Analytical Chemistry, 37(4): 621-623.

4. Ten Haaf, F.E.L. 1975. Color quenching in liquid scintillationcounters. In Crook, M.A. and Johnson, P., eds., Liquid ScintillationCounting, Vol. 3. London, Heyden & Son, Ltd.: 41-43.

M4608 (M4608-00) 5/01 Printed in U.S.A. 2001 Packard BioScience Company. All rights reserved.

Table 7.A Simple Guide to Quench Curve Selection

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