Liquid Scintillation Counting

6/21/2011
General

9/8/2008
Introduction

A scintillating liquid, referred to as the “cocktail,” sethe detector.The cocktail (perhaps 10 ml) is inside a plastic or gthat is transparent to the light emitted by the cocktaIdeally the sample (e g 1 ml) is dissolved in the cIdeally, the sample (e.g., 1 ml) is dissolved in the c

Failing that, the sample might be suspended as an or suspended in a gel. In some cases, a large solid (e.g., a smear) is simply placed into the cocktail wiattempt at dissolving it.
Introduction

Liquid scintillation counting is primarily used to quabeta emitters, e.g., H-3

Ni-63C-14S-35P-32

It is also used to quantify alpha emitters and nucliddecay by electron capture (e.g., Fe-55, Cr-51, I-12LSC incorporates elements of spectroscopy, but it used to identify radioactive material. As a rule, LSC to quantify the activity of a known nuclide.
Important Characteristics of L

9/8/2008
Important Characteristics of LSC

Three Important Characteristics of LSC:

1. High counting efficiency: 100% for many nu

Efficiencies as high as 70% for H-3

2. No backscatter

3. Low background
1. High Counting Efficiency

There are two reasons for the high counting efficie• There is no window through which the radiation 

pass in order to reach the sensitive part of the d

Important Characteristics of LSC• The system employs a “four pi” geometry. No m

which direction the radiation is emitted in, it will with the cocktail (assuming we are talking abouparticle radiation).
2. No Backscatter

When proportional counters or GM detectors count particles, backscatter directs some betas towards tthat otherwise would have gone in a different direc

DETECTOR

Important Characteristics of LSC

SOURCE S

SOURCE

While this increases the detector counting efficienour estimate of the counting efficiency less certainLSC involves no source support in which backscaoccur.
3. Low Background

The LSC vials (glass or plastic) are made of matervery low levels of naturally occurring radionuclidesWhen positioned inside the counting chamber, the is shielded from background gamma rays OtherwiImportant Characteristics of LSCis shielded from background gamma rays. Otherwi

gamma rays could interact with the vial wall or the (primarily via Compton scattering) and transfer theto electrons which could produce a signal indistingfrom that produced by the betas being counted.The shield might be passive (e.g., lead) or active (eanticoincident BGO guard detector)
3. Low Background

LSC systems use coincidence counting to reduce tnumber of spurious counts produced by electronic 

PMT

Important Characteristics of LSC

VialPMT

Coincidence 

Circuit

Summation 

Circuit
3. Low Background

When a beta particle is emitted in the cocktail, a siscintillation event (flash) occurs in which light photoemitted in all directions.The two photomultiplier tubes react to the scintillat

t i ti (i i id ) d h P

Important Characteristics of LSCsame moment in time (in coincidence) and each P

generates a pulse that travels to the coincidence asummation circuits.The coincidence circuit “recognizes” that this reprelegitimate decay event and it sends a logic pulse to summation circuit telling it to sum the two pulses aits electronic gate to allow the resulting pulse throu
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial Light Photons
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial
PMT

LogicPulse

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Open)

Vial
PMT Su

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Vial Circuit
Electronic noise from the PMTs (random fluctuation

voltage) is a potentially significant source of backgcounts. Although such noise “pulses” consists of smvoltage fluctuations, they still might be mistaken folegitimate pulses of the sort produced by tritiumImportant Characteristics of LSC3. Low Backgroundlegitimate pulses of the sort produced by tritium.

Only infrequently are “noise pulses” produced by thPMTs in coincidence. As such, in the absence of lepulses, the coincidence circuit rarely produces the pulses required to open the summation circuit’s galatter is kept closed, spurious noise pulses will not the summation circuit for analysis.
PMT

“Noise pulse”

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Circuit

(Gate Closed)

Vial
PMT

Important Characteristics of LSC3. Low Background

PMT

Coincidence 

Circuit

Summation 

Vial Circuit
Maximizing Light Collection

A given scintillation event (flash) consists of many light photons. But even a high energy beta emitter 32 emits some low energy betas that produce weascintillations.With the low energy beta emitter tritium (H-3), the bscintillations produced by its 18 6 keV betas consisscintillations produced by its 18.6 keV betas consis250 photons. The average energy betas of H-3 onlscintillations involving 70 – 100 photons.Because these scintillations can be so weak, the Lis designed to maximize the transmission of light frsample vial to the PMTs. It does so via reflectors paround the vial and PMTs. There might even be a rfoil inside the cap of the sample vial itself!
Typical LSC System

Courtesy of PerkinElmer, Inc. 940 Winter Street, Waltham, MA. 
The Liquid Scintillation Vial

The most common size of LSC vial is 20 ml (intend10 mls of cocktail). Smaller sizes include 6, 7, 8 ml

• Glass (borosilicate). Low 

potassium versions areavailable. Transparency 

allows visual inspectionallows visual inspection

of cocktail for color, inhomogeneity, etc.

• Plastic(polyethylene). 

Less expensive, lower background, but permeable to toluene, xylene and benzene
The LSC Cocktail

and the 

Energy Transfer Process

9/8/2008
The Liquid Scintillation Cocktail

The two major components of the LSC cocktail are t

• Solvent - dissolves sample and fluor• Fluor - emits light
Solvent

The solvent makes up 60-99% of the cocktail by voSolvents are almost always aromatic organics.Properties of an ideal solvent:

• good solubility for sample• high flash point

g p

• low vapor pressure• low toxicity• biodegradable• good solubility for fluor• low photo and/or chemoluminescence• high counting efficiency (tritium)• chemical and color quench resistant
Solvent

Solvents can be characterized as:

• Aqueous solvent – for samples soluble in w• Non-aqueous solvent – for samples not solu

water

M lti l t f l th t 

• Multipurpose solvent – for samples that are 

or insoluble in water

Secondary Solvents - these might be added to thprimary solvent to improve the solubility of the sam(sometimes referred to as a solubilizer).
Older Solvents

• Toluene• Benzene• Dioxane• Xylene• Pseudodocumene

Modern Biodegradable/Disposable Solve

• Linear alkyl benzene (LAB), e.g., Packard’s 

Fluor, RPI’s Bio-Safe

• Di-isopropylnaphthalene (DIN), e.g., Packa

Ultima Gold, Wallac’s Optiphase Hi-Safe

• Phenylxylylethane (PXE), e.g., Beckman’s 

ReadySafe

g p
Fluor

The fluor (aka, scintillator) might make up 0.3 to 1.0cocktail by volume.Its purpose is to absorb excitation energy from the and emit some fraction of this energy as light.There are two categories of fluors:There are two categories of fluors:

• Primary Fluor – most primary fluors emit ligh

UV range with wavelengths shorter than 400 

• Secondary Fluor (wave shifter) – the presenc

secondary fluor increases the wavelength of emitted light to one more efficiently absorbed photomultiplier tubes.
Primary Fluor

The most common primary fluor is 2,5 diphenyloxazole (PPO) whose peak emission wavelength is at 357 nm.

Secondary FluorThe most common secondary fluors:

O

N

PPO

CH CH

1,4 bis[2-methylstyryl] benzene (Bis-MSB) 

1,4-bis[5-phenyloxazol-2yl] benzene (POPO

O

N

O

N

CH CH CH CH

CH3 CH3
Energy Transfer Process in the Cocktail

Radioactive decay in the cocktail results in the emicharged particle (e.g., beta particle) that travels (a most) through the cocktail.Pi electrons in the aromatic rings of the solvent moclose to the charged particle tract are excited.These excited solvent molecules collide with other These excited solvent molecules collide with other 

molecules and transfer the excitation energy to theEventually a primary fluor molecule picks up the exenergy from an excited solvent molecule – this dorequire a physical collision but can occur at some dBy some mechanism, not completely understood, tcan be transferred to a secondary fluor molecule wexcites and emits a light photon.
Energy Transfer Process in the Cocktail

* Excitation energy

Charged Particle

Solvent

Solvent

*
Energy Transfer Process in the Cocktail

* Excitation energy

Charged Particle

Solvent

Solvent

*

Solvent

Solvent 

*
Charged Particle

Solvent *

Energy Transfer Process in the Cocktail

* Excitation energy

Solvent

Solvent *

Solvent

Solvent *

Solvent
Solvent * Solvent *

Energy Transfer Process in the Cocktail

* Excitation energy

Solvent

Solvent *

Solvent

Solvent *

Solvent

Solvent *

Solvent

Solvent *

Solvent Solvent
Solvent * 10 Fluor *

Energy Transfer Process in the Cocktail

* Excitation energy

Solvent

Solvent *

10 Fluor

10 Fluor *

Solvent

Solvent *

10 Fluor

10 Fluor*

Solvent 10 Fluor
10 Fluor * 20 Fluor *

Energy Transfer Process in the Cocktail

Short wavelength light photon

* Excitation energy

10 Fluor

10 Fluor *

20 Fluor

20 Fluor *

10 Fluor

10 Fluor *

20 Fluor

20 Fluor *

10 Fluor 20 Fluor
20 Fluor *

Energy Transfer Process in the Cocktail

* Excitation energy Long wavelength light photon

20 Fluor

20 Fluor *

20 Fluor

20 Fluor *

20 Fluor
Charged Particle

Solvent * Solvent * 10 Fluor * 20 Fluor *

Short wavelength light photonLong wavelength light photon

Energy Transfer Process in the Cocktail

* Excitation energy

Solvent

Solvent *

Solvent

Solvent *

10 Fluor

10 Fluor *

20 Fluor

20 Fluor *

Solvent

Solvent *

Solvent

Solvent *

10 Fluor

10 Fluor 

20 Fluor

20 Fluor *

Solvent Solvent 10 Fluor 20 Fluor
Energy Transfer Process in the Cocktail

The greater the energy of the charged particle, the 

the number of excited solvent moleculesThe greater the number of excited solvent moleculgreater the number of light photons emitted by the 

The more light photons emitted by the cocktail, the The more light photons emitted by the cocktail, the 

the size of the pulse produced by the PMT.A given beta emitting radionuclide emits beta particrange of energies up to some maximum value.
Beta Spectrum

Number of Pulses

The beta spectrum looks something like this:

Pulse Size

(Beta Energy)

(Number of Betas)

Average 

Beta Energy

M

B
Problems to be 

Addressed

9/8/2008
Problems to be Addressed

There are several potential problems that must be addressed in LSC:

• Getting the sample into solution• Static Electricity

• Photoluminescence• Chemiluminescence• Quenching - Optical

- Color- Chemical
Getting the Sample in Solution

The goal is to get the sample completely dissolved cocktail or uniformly dispersed throughout the cocemulsion or gel.In addition, the solution should be clear, colorless neutral pH.If the radioactive component of the sample is not icontact with the cocktail, the emitted betas may loor all of their energy before the energy can be tranthe solvent molecules. As such, phase separation cocktail components would be a major problem.
Getting the Sample in Solution

If the sample is not completely dissolved in the cocounts may go up over time as the radioactive magradually “taken up” by the cocktail.If this might be a problem, a small amount (e.g., 5microliters) of a solution that the sample is known soluble in can be added to the sample before the soluble in can be added to the sample before the 

into the cocktail. Ideally this solution should produquenching. Water, ethanol, acetonitrile, ethyl acetahexane are relatively good in this regard.A more thorough mixing of the sample in the cocktvia ultrasonication) might help in some cases.
Getting the Sample in Solution

Organic samples are fairly easy to get into solution the cocktail solvents are organic molecules. With ssamples, the best efficiencies are obtained with coare only intended for organics.Aqueous samples require cocktails employing emThese cocktails can also be used for organic samThese cocktails can also be used for organic sam

for convenience they might be used for all types oWith aqueous samples, the trade off is between coefficiency and the sample capacity, i.e., the amounthe cocktail can hold.
Getting the Sample in Solution

Prior to counting, biological material might be hommacerated or combusted and then treated with a sSolubilizers might be:

• alkaline (e.g., Soluene-350). These solubiliz

be used with blood, urine, muscle. Solubiliz

accomplished by hydrolysis of the sample.

• acidic (e.g., perchloric acid, nitric acid). The

be used with bone, cartilage. Solubilization achieved via oxidation of the sample. 

• some other type (e.g., sodium hypochlorite)

material is often treated with sodium hypocSodium hypochlorite works by oxidative ble
Static Electricity

Static charges on the surface of the scintillation viaresult in the emission of random single photons. Thmaximum pulse size due to such static charge eveequate to 10 keV betas (i.e., H-3).Plastic vials, especially the small vials in adapters, prone to developing static charges than glass vialsprone to developing static charges than glass vialsHandling the vials in low humidity conditions with cgloves can exacerbate the problem.Static eliminators are optional equipment for most systems.
Photoluminescence

Excitation of the vial or cocktail by ultra-violet light spurious single photon emissions that can produce similar to those of H-3.The maximum height of photoluminescence inducecorrespond to 6 keV betas. The vast majority of the in the 0 – 2 keV rangein the 0 2 keV range.

Fortunately, photoluminescence decays away very can be completely gone within 5 minutes or so.Photoluminescence is independent of quenching areduced somewhat by cooling the sample.
Chemiluminescence

Chemiluminescence is the spurious production of lchemical reactions that involve various componentsample.Unfortunately this light emission can last for up to a more!Chemiluminescence is primarily a problem when:

• the sample is alkaline (high pH)• the sample contains oxidizers• the cocktail contains solubilizers
Chemiluminescence

To a large extend chemiluminescence, which consmultiple single photon events, is minimized by the counter’s coincidence circuitry.Chemiluminescence can also be reduced by ensurthe cocktail has a neutral pH. This is often accompadding acetic acid (alkaline cocktails are the mostadding acetic acid (alkaline cocktails are the most

problematic). This can compromise the sample holcapacity however.A simpler approach is to wait several hours to a dasample has been added to the cocktail to count.Some folks heat the cocktail in order to drive the chreactions to completion, let it cool down, and then 
Quenching

While quenching is a “good” thing in GM detectorsundesirable in liquid scintillation counting.In essence, it is an interference in the energy transprocess that reduces the number of photons reachPMTs.This handout will consider three types:

• optical quenching• color quenching• chemical quenching
Optical Quenching

This term is usually treated as a synonym for color quenching.As used here, the term applies to the absorption of the emitted light by something other than the cocktail.For example putting a paper label on theFor example, putting a paper label on the

wall of the vial or labeling the side of the vial (instead of the cap) with a marker.

Other examples of what we call optical quenching fingerprints on the vial wall or condensation.Vials should be handled by the cap when possibleare worn or the vial is cleaned prior to counting, it sdone so as to minimize the generation of static.
Color Quenching

As used here, the term refers to the absorption of emitted light by color in the cocktail itself. This often a problem with biological samples, e.g., blood, urine.A simple and effective approach is to decolor with ultraviolet radiation (UV).decolor with ultraviolet radiation (UV).

Exposing the samples to sunlight for a few hours might be all that is necessary. 

Aqueous samples are often bleached by mixing 0.30% hydrogen peroxide in 1 ml of sample. The O2 

produced is a strong quenching agent and must be by heating to 50 oC for and shaking occasionally.
Color Quenching

Non-aqueous samples and samples digested with organic solubilizers can be bleached with benzoyl peroxide. Two mls of a benzoyl peroxide/toluene solution is used per ml of sample. The solution is prepared by dissolving onesolution is prepared by dissolving one

gram of benzoyl peroxide in five mls of toluene.

The sample is then incubated at for half an hour, acool to room temperature, and added to the cockta
Chemical Quenching

Chemical quenchers are components of the samplthat absorb excitation energy from solvent moleculdirectly from the charged particle) but fail to transfeenergy to the fluor molecules. Common examples of chemical quenchers include and chloroformand chloroform.
Consequences of Quenching

Quenching reduces the number of photons reachinPMT in a given scintillation. This reduces the size opulse.The result is that the spectrum shifts to the left.
No Quenching

LSC Vial

PMT

Spec

Pulse Siz

Pulse

Counts

Quenching

LSC Vial

PMT

Spe

Pulse

Pulse Si

Counts
Consequences of Quenching

unts

1. Quenching reducpulse sizes and shifspectrum to the left.2. Quenching reduccounting efficiency bthe very smallest pu

Pulse Size (channel number)

Comade so small that t

not counted.
More quenching

unts

Consequences of Quenching

Less quenc

Pulse Size (channel number)

Co
Quench Correction

9/8/2008
Quench Correction

Since the amount of quenching affects the counting efficiency (counts per disintegration) and because amount of quenching varies from sample to samplecounting efficiency can vary from sample to sampleDetermining the counting efficiency for a sample is quench correction There are several quench correquench correction. There are several quench corre

techniques that can be used, e.g.,

1. Internal Standard method2. Channels Ratio method (or variant)3. External Standard method (or variant)
Quench Correction

1. Internal Standard MethodThis involves the use of a “spike.”

i. The sample is counted and the resulting count 

(cpm or cps).

ii A d d ( h ik ) i i k i ii. A standard (the spike) containing a known activ

the radionuclide of interest is added to the cocktailthat the volume of the spike must be small in order minimize quench effects means that skilled personrequired. This method is particularly inconvenient isamples are being analyzed.

iii. The sample is counted again. The resulting cou

R2.
Quench Correction

1. Internal Standard Method

iv. The efficiency is calculated as follows:v. The activity in the sample is then calculated as 
This technique employs a standard containing a k

activity of the radionuclide of interest to determine relationship between the counting efficiency and thof the beta spectrum.Quench Correction2. Channels Ratio MethodOnce we know this relationship, we then use the sbeta spectrum to determine a samples counting efThe shape of the spectrum is described via the “chratio” (CR). Similar correction methods describe the spectrum different parameters, e.g., the spectral index of the 
Channel B

In the Channels Ratio method we divide the spectwo “channels”: A and B.

Channel A

Quench Correction2. Channels Ratio Method

Pulse Size 

Counts
i. A set of standards is counted. Each of these con

radionuclide of interest (e.g., H-3) at the same kactivity (Q

k).

The standard vials and, if possible, the cocktail, 

identical to those that ill be sed to co nt the s

Quench Correction2. Channels Ratio Method

identical to those that will be used to co

unt the s

Since each standard contains a different amounquenching agent, the spectra of these standards different shapes.The greater the amount of quenching agent, the counts (i.e., the lower the counting efficiency) anfurther to the left the spectrum is shifted.
untsMore Quench

A B

Quench Correction2. Channels Ratio Method

Pulse Size

Cou

Less que
ii. We integrate the counts in Channel A and Chan

each spectrum we calculate two values:

An efficiency (E)

Quench Correction2. Channels Ratio Method

A channels ratio (CR)

The efficiency and channels ratios could be definedifferently. The key is that we standardize the defi
In general, as quench is added:

• the count in Channel B decreases• the efficiency (E) decreases

Quench Correction2. Channels Ratio Method

• the channels ratio (CR) decreases
Efficiency

iii. We then plot efficiency (E) as a function of the c

ratio (CR). This is our quench correction curve. 

Quench Correction2. Channels Ratio Method

Channels Ratio (CR) 

Efficiency

(E)

The channels ratio (CR) describes the spectrum 
iv. We then acquire a spectrum for our sample, a

calculate the channels ratio:

A

B

Quench Correction2. Channels Ratio Method

Pulse Size 

Counts
v. On the quench correction curve, we find the eff

that corresponds to the channels ratio for the s

Efficiency for 

sample

Quench Correction2. Channels Ratio Method

Channels Ratio (CR) 

Efficiency

(E)

CR calculat

for sample
vi. Using the efficiency (E) obtained from the quen

correction curve, we determine the activity of tsample:

Quench Correction2. Channels Ratio Method
The Spectral Index of the Sample (SIS) techniqu

similar to the channels ratio method.The difference is that the shape of the spectrum via the “spectral index of the sample” rather than channels ratio As such the quench correction c

Quench Correction2. Channels Ratio Method

channels ratio. As such, the quench correction c

plot of the counting efficiency as a function of the Mathematically, the calculation of the SIS is somcomplicated than the calculation of the CR since the processing and weighting of each individual An advantage is that the SIS method is more sethe CR method. Nevertheless, neither method is low activity (e.g., < 500 dpm) samples.
Despite the name, this technique is unrelated to th

standard method discussed earlier.What it does is employ a clever “trick” to make accevaluations of the amount of quenching and the coefficiency even when the sample activities are low Quench Correction3. External Standard Methodefficiency even when the sample activities are low 

counting statistics are poor.

i. The sample is counted but no attempt is made 

evaluate the spectral shape.

ii. A gamma emitting source (e.g., Ba-133 or Cs

automatically brought into position underneath sample vial and the sample is counted again.
The low energy gamma rays/x-rays from the “e

standard” interact with the vial and cocktail via scattering. The resulting Compton scattered eletravel through the cocktail mimicking beta partic

Quench Correction3. External Standard Methodiii. The counting efficiency is then determined from 

shape of the spectrum generated by the Compscattered electrons via a quench correction cucurve was produced using the same type of staused in the CR and SIS methods to generate qcorrection curves. 
Quench Correction

3. External Standard Method

First Count 

of Sample

Second 

Count of 

Sample

Low Counts (A1 and B1)

CR = B1 /(A1 + B1 )

Large uncertainty in channels ratio means large uncertainty in efficiency (E1).

High counts (A2 and B2

CR = B2 /(A2 + B2 )

Little uncertainty in charatio means little uncerefficiency (E2).

Sample Activity = B1 /E2
Because of the high count rates associated with t

Compton scattered electrons, the spectral shape and efficiency determination can be highly accurairrespective of the sample activity.

Quench Correction3. External Standard Method

Some LSC systems use a “quench indicating paraknown as the “transformed Spectral Index of the EStandard” (t-SIE) to describe the spectral shape. systems, the quench correction curve plots countefficiency as a function of the t-SIE value which rafrom 0 – 1000. Like the CR and SIS values, the tvalues decrease with increasing quenching. 
Cerenkov Counting

9/8/2008
Cerenkov Counting

Cerenkov radiation, the blue light produced when travel faster than light in a transparent medium, cato assay high energy (> ca. 500 keV) beta emittersThe sample is simply dissolved in a transparent soas water or alcohol.Although Cerenkov counting can be used to count 

energy beta emitter, its primary application has beSr-90/Y-90.Advantages of Cerenkov counting:

• Low background• No chemiluminescence• No chemical quenching