Counting Solutions: Counting Aqueous Samples by LSC - LSC Technical Tips

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CS-005
Counting Aqueous Samples by LSC
Introduction
The primary objective of all sample preparation
procedures for liquid scintillation counting (LSC) is
to obtain a homogeneous solution for efficient energy transfer from the sample to the LS cocktail.
Aqueous solutions are some of the simplest and most
commonly found in liquid scintillation analysis. In
general, they provide the environment necessary
for many assays and separations, and include the
most encountered solvent media for the numerous
radioisotopes used in LSC. The main methods for
producing aqueous samples are by dissolution,
extraction, and distillation. Dissolution simply involves dissolving the sample in water. Extraction
can be extraction of the sample from a solid matrix
by water (solid/liquid extraction), or extraction of
the sample from a liquid matrix by water (liquid/
liquid extraction). Distillation involves separation
of the aqueous component by evaporation. A variety
of LS cocktails have evolved over the years to
accommodate the diverse types, volumes, and
concentrations of aqueous samples presented for
analysis by LSC.1,2,3 Different types of anions
encountered in LSC include chlorides, nitrates,
phosphates, acetates, and formates, with sample
volumes ranging from less than 100 µL to greater
than 10 mL, and concentrations varying from less
than 10 mM to greater than 2M. For the purpose of
cocktail selection, these aqueous samples can be
roughly divided into the following categories:
1. Buffers (e.g., sodium chloride, PBS, potassium
phosphate, etc.)
Low ionic strength (less than 0.1M)
Medium ionic strength (0.1M to 0.5M)
High ionic strength (0.5M to greater than 1M)
2. Acids (e.g., hydrochloric acid, nitric acid, etc.)
3. Alkalis (e.g., sodium hydroxide, potassium
hydroxide, etc.)
4. Other types (e.g., urea, sucrose, imidazole, etc.)
By using this list of categories, it is now possible to
assign cocktails for each category and therefore
present a simpler and more comprehensive method
of cocktail selection than was previously possible.
For each category, cocktails will be recommended
based on sample acceptance, performance and safety.
Buffers – Low Ionic Strength
Buffers encountered in this group include 0.01M
PBS (phosphate buffered saline), 50 mM Tris-HCl
[Tris(hydroxymethyl)aminoethane hydrochloride],
0.1M NaCl (sodium chloride), 0.01M Na2SO4 (sodium sulphate), etc. Since these aqueous buffers are
relatively dilute, there are comparatively few problems. Both di- and tri-valent anions such as SO42- and
PO43- are potentially problematic, due in part to their
charge and in part to their relative size [e.g., chlorides (Cl-) are much smaller than sulphates (SO42-)].
These characteristics can impede the formation of a
stable microemulsion and can cause phase instability, especially with high concentrations and large
volumes. Surprisingly, similar problems can occur
with small volumes, particularly within the range of
0.1 mL to 0.5 mL sample in 10 mL cocktail (1 to 5%
sample load). The only other area of concern is color
quench problems when using certain metallic salts
which are intrinsically colored [e.g., FeCl3 (ferric
chloride)]. Any phase instability problem can usually be resolved by decreasing the sample volume or
by increasing the cocktail volume. If the problem
persists, then it may be necessary to change to a
cocktail which can accept higher strength ionic
samples. Color quench problems can be reduced
by either diluting the sample with water (if
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practicable), or by using a cocktail which is more
resistant to color quenching e.g., any of the
Ultima Gold™ cocktails.
will accept small volumes of certain sample types,
Pico-Fluor 40 is the recommended classical cocktail
for these samples.
Among the safer cocktails, the Ultima Gold family
[based on DIN (di-isopropylnaphthalene)] will give
higher 3H efficiency than Opti-Fluor® or Emulsifier-Safe™/Poly-Fluor™ [based on LAB (linear alkyl
benzene)]. Of the classical solvent-based cocktails,
Insta-Gel® Plus/XF (10 mL) can accommodate
greater than 2.5 mL of certain sample types and
forms a stable gel (usually at greater than 3 mL
sample volume), thus making large sample volumes
possible. Pico-Fluor™ 40 (10 mL) can accommodate
greater than 2.5 mL of certain samples and remains
in a single liquid phase. Cocktails suitable for these
samples are shown in Table 1.
Buffers – High Ionic Strength
Buffers – Medium Ionic Strength
Acids
Aqueous buffer concentrations encountered in this
group range from 0.1M up to 1.0M and typical
buffers are 0.1M to 0.5M PBS, 0.15M to 1.0M NaCl,
0.25M ammonium acetate, etc. The cocktails suitable for these sample types (shown in Table 2) are
designed to overcome any phase instability problems and therefore cocktail selection is limited by
volume and concentration factors.
This group includes commonly encountered mineral
acids such as hydrochloric acid, nitric acid, sulphuric
acid, perchloric acid, orthophosphoric acid, and hydrofluoric acid as well as some aqueous miscible
organic acids such as acetic acid, formic acid, and
trichloroacetic acid (TCA). Acids are commonly
used as extractants,4 pH modifiers, and solubilizers.5
There are a number of potential problems associated
with this particular sample group and these include
quenching, reaction with cocktail components, and
chemiluminescence. Strong mineral acids can also
cause marked quenching effects, due primarily to
interaction with the scintillators.
With high ionic strength buffers, the choice of
cocktails is among Ultima Gold XR, Hionic-Fluor™,
and Pico-Fluor 40. Certain high ionic strength samples
can be accommodated in other cocktails, however
the capacity is usually very low (less than 0.5 mL).
The only other method of overcoming the problem
of low sample acceptance of high ionic strength
samples is to dilute the buffer sample with water and
convert it into a medium strength buffer which
simplifies cocktail selection. The sample capacity of
selected cocktails for increasing ionic strength solutions is shown in Table 3.
The Ultima Gold family will, in general, give higher
quench resistance than the classical cocktails i.e.,
higher efficiency at the same sample load. Ultima
Gold works well with low sample volumes of aqueous buffers up to 0.5M. Although Insta-Gel Plus/XF
Low sample volume
(0 to 2.5 mL)
High sample volume
(>2.5 mL)
Safer Cocktail
Ultima Gold
Ultima Gold MV
Ultima Gold AB
Ultima Gold LLT
Opti-Fluor
Emulsifier-Safe/
Poly-Fluor**
Ultima Gold
Ultima Gold XR
Ultima Gold AB
Ultima Gold LLT
Opti-Fluor
Classical Cocktail
Insta-Gel Plus/XF*
Pico-Fluor 15
Pico-Fluor 40
Insta-Gel Plus/XF
Pico-Fluor 40
Table 1.
Cocktail selection for low ionic strength samples (based on the use
of 10 mL of cocktail). * Sold as Insta-Gel XF in the USA. ** Sold as
Poly-Fluor in the USA.
2
Low sample volume
(0 to 2.5 mL)
High sample volume
(>2.5 mL)
Safer Cocktail
Ultima Gold
Ultima Gold XR
Ultima Gold AB
Ultima Gold LLT
Ultima Gold XR
Classical Cocktail
Insta-Gel Plus/XF
Pico-Fluor 40
Hionic-Fluor
Pico-Fluor 40
Hionic-Fluor
Table 2.
Cocktail selection for medium ionic strength samples (based on the
use of 10 mL of cocktail).
This can be overcome by using a cocktail which is
known to be compatible with mineral acids or,
preferably, by diluting the acid with water prior to
the addition of the cocktail. Certain strong mineral
acids will react with cocktail components causing
both color development and changes in surfactant
characteristics. For example, adding even small
amounts of concentrated sulphuric acid to a cocktail
will result in almost immediate color formation and
eventual sulphonation of the surfactants (emulsifiers). This alteration to the surfactants will result in a
change or loss of emulsifying properties and lead to
phase instability. The color formation is due to
sulphonation of minor impurities in the solvent and
in addition to color, significant amounts of chemiluminescence may be produced. Another example
involves adding small amounts of concentrated nitric acid which results in a yellow/brown color
forming due to the dissociation of nitric acid and
release of NO2. This problem can be overcome by
diluting the acid with water prior to adding it to the
cocktail. With some cocktails, the addition of TCA
can produce chemiluminescence. Although a rare
occurrence, acid-induced chemiluminescence can
be avoided by using a cocktail which is resistant to
it such as Ultima Gold LLT. An overview of suitable
cocktails for acids is shown in Table 4.
applications involving pH modification, cell lysis,
CO2 trapping,6 and solubilization. The major problem normally encountered is chemiluminescence
and in general the amount of chemiluminescence is
influenced by both the volume and concentration of
alkali added. The standard method of avoiding this
problem is to use a cocktail which is known to be
resistant to chemiluminescence. Other methods of
overcoming the problem include diluting the base
with water to reduce the effect, allowing the chemiluminescence to decay in the dark before counting,
and neutralizing the base with acid. Prolonged storage of cocktails with alkalis present is not recommended due to the potential for color formation.
Where possible, counting should be performed within
one or two days.
Other Types
This final group covers other aqueous samples/
mixtures which are occasionally used in LSC methods and assays. These aqueous mixtures are usually
fairly specific for certain types of assays, e.g., sucrose gradients in DNA and RNA separation, urea as
a denaturing and reducing buffer, and imidazole as
a biological buffer. Ultima Gold is the cocktail of
choice for these sample types.
Summary
Alkalis
This group of samples includes bases such as sodium
hydroxide, potassium hydroxide, and ammonium
hydroxide. Alkaline samples are produced from
Ionic Strength
0.5M NaCl
0.75M NaCl
1.0M NaCl
Ultima Gold
XR
9.0 mL
6.5 mL
5.5 mL
Hionic-Fluor
1.4 mL
2.25 mL
8.5 mL
A slightly expanded compilation of recommended
cocktails for different sample types is shown in the
Appendix in Table A (Safer Cocktails), Table B
Pico-Fluor
40
3.0 mL
2.75 mL
2.3 mL
Ultima Gold
1.5 mL
0.75 mL
0.5 mL
Ultima Gold Opti-Fluor Pico-Fluor
MV
15
1.25 mL
1.1 mL
1.2 mL
0.75 mL
0.75 mL
0.5 mL
0.5 mL
0.5 mL
0.25 mL
Table 3.
Sample capacity of selected cocktails for various ionic strength buffers (sample capacities are for 10 mL cocktail at 20 °C).
3
Acid
Mineral Acids
Concentration
0 to 2M
Safer Cocktail
Ultima Gold AB
Ultima Gold LLT
Ultima Gold
Ultima Gold AB
Ultima Gold LLT
Ultima Gold LLT
>2M
Trichloroacetic Acid
0 to 20%
Classical Cocktail
Insta-Gel XF
Pico-Fluor 40
Hionic-Fluor
Hionic-Fluor
Table 4.
Cocktail selection for acids (based on the use of 10 mL of cocktail).
(Classical Cocktails), and Table C (Safer Cocktails
for Acids). This may prove useful in providing a
basic guide to cocktail selection. To further help
with cocktail selection, sample capacities are presented to help with both the selection and suitability
of a cocktail.
Appendix
Selection and Suitability of a Cocktail
Based on Ionic Strength
1. Determine the approximate ionic strength using
the sample molarity:
Conclusion
Mixture Molarity = [(Va x Ma) + (Vb x Mb) + .....]
(Va + Vb + .....)
There are a variety of LSC cocktails, of
both safer, high flash-point and of classical solvent
types, which are suitable for use with the wide
diversity of aqueous samples commonly encountered in LSC. If problems with sample/cocktail
selection persist or help is needed with a particular
sample type not mentioned in this publication,
please call your local PerkinElmer representative
for further applications support.
Where:
Va = Volume of solution A
Ma = Molarity of solution A
Vb = Volume of solution B
Mb = Molarity of solution B
Example: 10 mL of 0.2M NaCl (solution A) added
to 2 mL of 0.5M KH2PO4 (solution B)
References
Mixture Molarity = [(10 x 0.2) + (2 x 0.5)]
(10 + 2)
= 0.25M (A medium molarity sample)
1. Kobayashi, Y. and Maudsley, D.V. (1974) Biological Applications of Liquid Scintillation Counting. Academic Press,
pages 58-68.
2. ter Wiel, J. and Hegge, Th.C.J.M. (1991) Advances in
Scintillation Cocktails. Liquid Scintillation Counting and
Organic Scintillators. Edited by Harley Ross, John E.
Noakes and Jim D. Spalding, Lewis Publishers, pages
51-67.
2. Select appropriate cocktail based on ionic strength.
3. Test for sample/cocktail compatibility.
3.1 Dispense 10.0 mL cocktail into a 20 mL
glass vial. The use of a glass vial allows a
clear view of the mixture.
3. Peng, C.T. (1983) Sample Preparation in Liquid Scintillation Counting. Advances in Scintillation Counting. Edited
by S.A. McQuarrie, C. Ediss and L.I. Wiebe, pages
279-306.
3.2 Add the desired sample volume, cap, and
shake thoroughly.
4. LSC Counting Solutions (1996) Environmental Sample
Preparation for LSC. PerkinElmer.
CS-004.
3.3 If the mixture is clear, proceed with the
stability test.
5. LSC Counting Solutions (1996) LSC Sample Preparation
by Solubilization. PerkinElmer. CS-003.
6. LSC Counting Solutions (1994) Radio-Carbon Dioxide
(14CO2) Trapping and Counting, PerkinElmer CS-001.
4
3.4 If the mixture is cloudy or hazy, try increasing the cocktail volume and/or decreasing
the sample volume. If the mixture does not
clear, select a cocktail which can accept
higher ionic strength samples such as Ultima
Gold XR.
Use a mixture which has passed the sample/
cocktail compatibility test. Allow it to stand at the
LSC counting temperature for a minimum of two
hours or the proposed count time for the sample, if
this is greater than two hours. If the mixture remains
stable, successful counting will be possible.
3.5 If the mixture separates into two distinct
phases (like oil and water), or is milky, select
a cocktail which can accept higher ionic
strength samples.
5. Count.
Note: Once the proper proportions and stability of
the sample/cocktail mixture have been established,
plastic vials can be considered for routine counting.
4. Test for stability.
Sample
Ultima
Gold
Ultima
Ultima
Ultima
Ultima
OptiEmulsifierGold
Gold
Gold
Gold
Flour
Safe/
XR
AB
LLT
MV
Poly-Fluor
Deionized Water
3.2 mL >10.0 mL
10.0 mL
>10.0 mL
2.0 mL
1.8 mL
3.2 mL
0.01M PBS (pH 7.4)
6.5 mL
10.0 mL
8.5 mL
8.0 mL
4.0 mL
3.0 mL
3.0 mL
0.1M PBS (pH 7.4)
4.0 mL
8.5 mL
<0.25 mL
<0.25 mL 3.0 mL
2.0 mL 0.6-1.2 mL
0.5M PBS (pH 7.4)
0.5mL
1.25 mL
<0.5 mL
0.05M Tris-HCl (pH 7.4) 4.5 mL
10.0 mL
10.0 mL
9.0 mL
2.75 mL
2.7 mL
3.2 mL
0.15M Sodium Chloride
6.5 mL
10.0 mL
7.5 mL
6.5 mL
5.0 mL
3.5 mL
3.1 mL
0.5M Sodium Chloride
1.5 mL
9.0 mL
8.0 mL
6.0 mL
1.25 mL
1.1 mL
2.2 mL
1.0M Sodium Chloride
0.5 mL
5.5 mL
4.5 mL
3.5 mL
0.5 mL
0.5 mL
1.4 mL
0.04M NaH2PO4 (pH 7.4) 8.0 mL
10.0 mL 0.75-8.0 mL 0.75-7.0 mL
2.25 mL
4.25 mL 0.6-2.5 mL
0.1M NaH2PO4 (pH 4.9) 10.0 mL
10.0 mL
8.0 mL
8.0 mL
7.0 mL
7.5 mL
2.0 mL
0.2M NaH2PO4 (pH 4.9)
3.5 mL
10.0 mL
1.0-6.5 mL
1.0-5.0 mL
2.75 mL
3.0 mL
2.0 mL
0.25M Amm. Acetate
3.25 mL
8.0 mL
5.0 mL
5.5 mL
3.0 mL
1.5 mL
2.0 mL
0.1M Amm. Sulphate
3.25 mL 10.0 mL
1.0-7.0 mL
1.0-5.5 mL
2.25 mL
3.0 mL
2.0 mL
0.1M Sodium Sulphate
4.25 mL 10.0 mL
3.25 mL
4.0 mL
1.75 mL
0.1M HCl
6.5 mL
7.0 mL
10.0 mL
10.0 mL
4.5 mL
4.0 mL
2.7 mL
10% TCA
3.0 mL
7.0 mL
4.5 mL
4.0 mL
1.5 mL
2.5 mL
2.3 mL
20% TCA
2.0 mL
5.0 mL
3.0 mL
3.0 mL
0.5 mL
2.0 mL
1.5 mL
0.1M NaOH
2.5 mL
10.0 mL
10.0 mL
7.5 mL
1.5 mL
5.0 mL
3.0 mL
1.0M NaOH
3.0 mL
1.0 mL
1.75 mL <0.25 mL
0.75 mL
0.1M Imidazole (pH 7.4) 10.0 mL
10.0 mL
10.0 mL
10.0 mL
2.0 mL
4.5 mL
2.5 mL
8M Urea
1.0 mL
2.5 mL
2.0 mL
3.5 mL
0.5 mL
0.5 mL
1.0 mL
Table A.
Performance of safer cocktails with various aqueous buffers at 20 °C (based on the use of 10 mL of cocktail). Shaded area
indicates no or very limited sample capacity.
5
Sample
Deionized Water
0.01M PBS
0.1M PBS
0.5M PBS
0.05M Tris-HCl
0.15M Sodium Chloride
0.5M Sodium Chloride
1.0M Sodium Chloride
0.04M NaH2PO4
0.1M NaH2PO4 (pH 4.9)
0.2M NaH2PO4 (pH 4.9)
0.25M Amm. Acetate
0.1M Amm. Sulphate
0.1M Sodium Sulphate
0.1M HCl
Pico-Fluor 15
1.6 mL
Pico-Fluor 40
2.3 mL
Hionic-Fluor
1.2 mL
2.9 mL
10.0 mL
1.4 mL
<0.25 mL
0-1.8 mL
3.0-10.0 mL
0-1.8 mL
4.9-10.0 mL
0-2.1 mL
3.0-10.0 mL
0.4-7.0 mL
0.3-1.9 mL
3.0-10.0 mL
0.6-2.0 mL
1.0-2.0 mL
4.0-10.0 mL
0-1.75 mL
3.5-10.0 mL
0.5-1.75 mL
3.5-10.0 mL
2.0 mL
6.4 mL
2.0 mL
3.0 mL
1.6 mL
7.0 mL
3.0 mL
4.0 mL
10.0 mL
1.1 mL
1.2 mL
3.0 mL
1.4 mL
<0.3 mL
2.0 mL
2.3 mL
4.0 mL
8.5 mL
1.75 mL
6.0 mL
2.0 mL
10.0 mL
7.1 mL
1.75 mL
1.75 mL
1.5 mL
5.0 mL
1.75 mL
2.5 mL
8.5 mL
1.75 mL
2.5 mL
1.8 mL
8.5 mL
7.25 mL
1.75 mL
1.3 mL
1.5 mL
4.0 mL
1.5 mL
1.25 mL
3.3 mL
3.25 mL
2.5 mL
4.5 mL
1.2 mL
0.5 mL
3.0 mL
4.5 mL
5.5 mL
1.2 mL
1.75 mL
1.0 mL*
2.5 mL
1.0 mL
0-1.5 mL
3.0-10.0 mL
2.1 mL
(No gel phase)
3.75 mL
(No gel phase)
0-2.0 mL
3.0-10.0 mL
10% TCA
20% TCA
0.1 M NaOH
1.0 M NaOH
0.1M Imidazole (pH 7.4)
8M Urea
Insta-Gel Plus/XF
0-1.7 mL
2.9-10.0 mL
0.2-1.6 mL
3.1-10.0 mL
1.0-2.0 mL
0-1.75 mL
3.0-10.0 mL
0-1.5 mL*
Table B.
Performance of classical cocktails with various aqueous buffers at 20 °C (based on the use of 10 mL of
cocktail). Shaded area indicates no or very limited sample capacity. * Clears only after extended agitation.
Sample
0.1M HCl
1.0M HCl
2.0M HCl
5.0M HCl
1.0M HNO3
2.0M HNO3
3.0M HNO3
1.0M H2SO4
2.0M H2SO4
1.0M HClO4
2.0M HClO4
1.0M H3PO4
2.0M H3PO4
Ultima Gold
6.5 mL
0.5 mL
0.5 mL
2.0 mL
1.5 mL
Ultima Gold
XR
7.0 mL
2.5 mL
1.0 mL
<0.5 mL
2.5 mL
2.0 mL
1.0 mL
0.25 mL
2.0 mL
1.5 mL
1.5 mL
0.5 mL
Ultima Gold
AB
10.0 mL
5.5 mL
2.25 mL
2.0 mL
3.25 mL
2.25 mL
2.0 mL
6.5 mL
4.0 mL
2.25 mL
2.0 mL
0.5-10.0 mL
0.5-4.0 mL
Ultima Gold
LLT
10.0 mL
5.0 mL
3.0 mL
1.5 mL
3.5 mL
2.5 mL
2.25 mL
7.0 mL
4.0 mL
2.25 mL
2.5 mL
0.5-10.0 mL
0.5-6.0 mL
Opti-Fluor
Emulsifier-Safe/
Poly-Fluor
4.0 mL
2.7 mL
0.5 mL
3.0 mL
4.5 mL
0.5 mL
0.75 mL
3.5 mL
0.75 mL
3.5 mL
0.5 mL
1.0 mL
2.0 mL
2.75 mL
1.5 mL
1.0 mL
1.0 mL
0.75 mL
0.5-1.5 mL
3.0 mL
0.5-1.0 mL
3.0 mL
Table C.
Recommended safer cocktails for mineral acids (based on the use of 10 mL of cocktail). Shaded area indicates no or
very limited sample capacity.
6
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