Internal Stalndardwith Identical System Properties for Determination of Liquid Scintillation Counting Efficiency A. A. Moghissi and M. W. Carter Southeastern Radiological Health Laboratory, P.O. Box 61, Montgomery, Ala. 36101 LIQUIDSCINTILLATION represents one of the most versatile counting systems for the determination of radionuclides which emit alpha and/or beta radiations. The major problem associated with its use is quenching, and the subsequent necessity of determining the counting efficiency for each individual sample. The earliest and a frequently applied method for efficiency determination is the internal standard. Statistical aspects of the internal standard technique have been discussed in detail by Herberg ( I ) . The results of his investigations indicate that the counted activity resulting from the addition of the standard activity should be significantly higher than that originating from the sample. The accuracy of the internal standard technique has been discussed and disputed by several authors (2-4). Davidson and Feigelson (2) indicate that the internal standard technique is not accurate when strong quenchers are present. Smith (3),as well as Butler (4), reached the same conclusion based on data using tritiated water as the internal standard. The internal standard technique has several limitations in its general application. Addition of a standard, even in small concentrations, changes the geometry of the system. As there is no ideal internal standard, the addition of any compound changes the counting efficiency. These changes are usually in the negative direction-e.g., by the addition of water-but occasionally in the positive direction-e.g., by the addition of naphthalene. To minimize this problem, efforts have been made to use small amounts of high specific activity standards. Measuring microliter amounts of a standard with high accuracy is, however, time-consuming and tedious. The elapsed time between sample counting and counting of the sample plus the standard may be appreciable. In this case, increased inaccuracy may be introduced by drift of the instrument. An additional limitation is the loss of the sample for a recount or any further study. Subsequent to addition of the standard, the sample characteristics are irreversibly altered. Moghissi and Hogrebe (5) describe a two-sample technique which partially eliminates or minimizes certain of the above limitations. These authors did not present any details of their technique. The present paper is a generalization of the two-sample technique. This method, the internal standard with identical system properties (ISISP), uses two sample aliquots which are iden(1) R. J. Herberg, ANAL.CHEM., 35, 786 (1963). (2) J. D. Davidson and P. Feigelson, Intern. J. Appl. Rad. Isotopes. 2, 1 (1958). (3) A. H. Smith and G . W. Reed, “Radioisotopes Sample Measure-
ment Techniques in Medicine and Biology,” International Atomic Energy Agency, Vienna, 1965, p. 427. (4) F. E. Butler, “Assessment of Tritium in Production Workers, Assessment of Radioactivity in Man,” International Atomic Energy Agency, Vienna, 1964, Vol. 11, p. 431. ( 5 ) A. Moghissi and K. Hogrebe, Intern. J . Appl. Rad. Isotopes, 15, 165 (1 964).
812
ANALYTICAL CHEMISTRY
tical in their chemical and quenching properties. In one aliquot a radionuclide is replaced by its stable isotope. The samples are then counted consecutively to minimize the effect of any instrumental drift. PROCEDURES
A Beckman “liquid scintillation system” operated at room temperature was used in this investigation. The scintillation solutions were prepared with reagent grade toluene or dioxane. The primary solute was diphenyloxazole (PPO) at a concentration of 4 grams per liter and the secondary solute was a P-bis-[2-(4-methyl-5-phenyloxazolyl)]-benzene (dimethyl POPOP) at a concentration of 0.7 gram per liter. To the solutions containing dioxane, 100 grams of naphthalene per liter were added. Naphthalene-T (NT), tritiated water (HOT), hexadecane-T (HDT), naphthalene- 4C (NC), hexadecane-14C (HDC), and benzoic a c i d - l c (BC) were used as standard activities. Tritium standards were prepared by the internal standard method with tritiated water which was received from the National Bureau of Standards. Benzoic acid-IC and naphthalene-l4C were calibrated by using a hexadecane-lC standard which was obtained from the Radiochemical Centre, Amersham, England. The following three types of counting vials were used: low potassium, 22-ml glass vials (LKG) and 25-ml polyethylene vials (PEP), which are widely used in liquid scintillation counting (Packard Instrument Co., Downers Grove, Ill.); and 20-ml polyethylene vials with snap tops (PST) (Nuclear Equipment Chemical Corp., Farmingdale, N.Y.). The procedure for the internal standard with identical system properties, using naphthalene as the standard, was as follows: The sample to be counted was placed into a volumetric flask and a sufficient amount of liquid scintillator was added and the contents were thoroughly mixed. Two aliquots (usually 20 ml each) were pipetted into counting vials. To the sample, a preweighed amount (usually 1 gram) of naphthalene (stable equivalent of standard activity) was added. The same amount of tritiated naphthalene (standard activity) was added to the standard. These aliquots were then counted in an appropriate manner. Care was taken to use counting vials of the same type and from the same batch for each pair of aliquots. In the following experiments, each sample was counted to achieve 0.5 counting error at the 2-sigma confidence level. A sufficiently high activity level was selected to assure a negligible effect on the accuracy owing to any change in background caused by quenching. Comparison of Counting Vials. According to the definition of the internal standard with identical systems properties, the counting vials for the standard and the sample must be identical. Differences in results could be caused by variation in the type material, the wall thickness, and configuration. To investigate certain of these possible differences, a number of experiments were conducted using three types of vials. Naphthalene-T was used as the source of activity in these experiments. The data derived are presented in Table I
Table I. Comparison between Various Types of Counting Vials Type and capacity, ml PEP 25 PST 20 LKG 22
Relative counting efficiency, 100 87.5 78.5
Range in counting efficiency, 0.9 1.1 0.9
and show that ranges in counting efficiency are within the pipetting and counting errors. Also, use of the glass vials produces the lowest counting efficiency for tritium, whereas the highest counting efficiency is achieved with the PEP counting vials. An intermediate counting efficiency was obtained with the PST vial. No attempt was made to compare one batch of vials with another obtained from the same supplier because the results of such a comparison would not necessarily be applicable to future batches. The results of the numerous experiments conducted routinely at this laboratory indicate that differences, if any, between batches of a particular vial are small. Comparison of Standard Activity and Its Stable Equivalent. One of the requirements of the internal standard with identical system properties is that the standard activity and its stable equivalent have identical quenching properties. Because small amounts of impurities may change the counting efficiency considerably, each radioactive standard and its stable equivalent should be compared for their purity or at least their quenching properties. This comparison may be conducted-e.g., using naphthalene-by preparing the scintillation solution in the usual manner and adding an appropriate mixture of naphthalene (NH) and tritiated naphthalene (NT). The results of such an investigation are presented in Figure 1. In all cases the total amount of naphthalene (NH NT) is kept constant, and the amount of tritiated naphthalene varies from 50 to 1000 mg. The straight line NH-NT indicates the linearity. A standard activity and its stable equivalent can be applied to the ISISP only when the measurements result in such a line, To observe the effect of impurities in the standard or its stable equivalent, benzoic acid (B) and methyl red (M) were added to the standard activity and its stable equivalent, respectively. Results are plotted in Figure 1 for cases when the standard activity (NT) was contaminated by mild (NTB) or strong (NTM) quenchers. The resulting curves deviate markedly from the NH-NT system. Similarly, curves are presented for systems where the stable equivalent of the standard activity (NH) is contaminated with mild (NHB) or strong (NHM) quenchers. These latter curves have different and distinguishable forms and deviate appreciably from the NH-NT curve. Errors arising from the incompatibility of the standard activity and its stable equivalent depend upon the nature and the amounts of impurities present. Accuracy of the ISISP. Table I1 contains comparison data for the ISISP and the internal standard using various scintillation solutions and standards. Attempts were made to include color and chemical quenchers as well as complex compounds and biological samples. The degree of quenching caused by the various amounts of quencher is presented as the ratio of the per cent counting efficiency of quenched (E,) to unquenched (E,) samples. Although the internal standard technique often does not use the large amounts of standard activity described in this paper, a comparison between the two methods shows the order of magnitude of errors introduced when compounds are added to a liquid scintillation solution. Results are presented in terms of
+
Measured Activity ( A r b It r a r y U n i t s )
0 L
1000
250
750
mg Naphthalene
- $H
500 rng Naphthalene
- 'H
2 50
0
Figure 1. Comparison between the standard activity and its stable equivalent = standard activity, NJ3 = stable equivalent of the standard activity, B = mild quencher, M = strong quencher.
NT
per cent deviations as (1 - measured activity/added activity) X 100. From these data (for the ISISP), it is apparent that the deviations in counting efficiency are small and caused by normal pipetting and weighing errors. It is obvious, under the described experimental conditions, that in the presence of quenchers, appreciable errors in counting efficiency are introduced if the internal standard technique is applied. These errors may be either positive or negative depending upon the quencher, degree of quenching, scintillation solution, and the standard used. When cadmium nitrate was used to produce 6 % quenching, a deviation in counting efficiency of about 10% was introduced. When Congo red was used to produce 99.8 % quenching, the deviation in counting efficiency was 980 %. In each case, as shown by data in Table 11, the deviation in counting efficiency using the ISISP is small. Consequently, accurate measurements can be made even in the presence of strong quenchers, whereas the conventional internal standard technique is unreliable. A comparison of the two systems using various standards containing 4c for different scintillators, quenchers, and degrees of quenching is given in Table 111. In general, the deviations in counting efficiency using the internal standard technique are not so large for l4C as for tritium. This is primarily due to the lower beta energy of tritium. Nevertheless, the presence of quenchers adversely affects the accuracy of counting efficiency using the internal standard technique, whereas it has no appreciable effect using the ISISP method. DISCUSSION
Because of the energy dependency of quenching effect, the most severe problems are observed for low energies. Therefore, primary emphasis was given to tritium counting. The improvements in liquid scintillation counters have resulted in lower backgrounds and higher counting efficiencies. Thus, the levels of activity necessary for a particular VOL 40, NO. 4, APRIL 1968
e
813
Table 11. Comparison of ISISP and IS Methods for 3H and Various Degrees of Quenching Scintillation soh.. T T T T D D D D D
Quencher CCla CC1,
cc1; Fe(N0d3 F"03)3 Urine Urine Milk Milk Diethanolamine Diethanolamine Tributyl phosphate Tributyl phosphate EDTA EDTA Pyridine Pyridine Instant coffee Instant coffee Cd(N03h Congo red
D
T T T T D D T T D D D D
Standardb
100 EqIEo 10.8 10.8 99.3 99.3 49.3
NT HDT NT HDT NT HDT HOT NT HOT NT HDT NT NT HDT HOT NT HDT NT NT HOT HOT NT
49.3
41.8 41.8
45.1 45.1 31.8 31.8 30.9 30.9 47.5 47.5 40.0 40.0 37.2 37.2
6.3 99.8
a
T = toluene-based liquid scintillator, D = dioxane-based liquid scintillator.
b
NT
=
naphthalene-T, HDT
=
hexadecane-T,HOT
=
+O. 3
-0.2 +O. 5 +O. 5 +O. 3 +O. 3 -0.5
+1.2
+o. 8 +13.2 +3.8 -25.1 -3.2
-20.8
$0.4
-2.3
-0.8
-18.0 +11.0 -6.5 +3.1 -0.8
-0.7 f1.0 -0.3 -0.5 -0.5
-0.7
+o. 1
-0.8
+l.l
+0.7 +0.7 -1.1 -1.0
-1.2
-14.9 +11.0 -3.1 1-12.4 +11.1
-12.8 -10.3 t980
tritiated water.
Table 111. Comparison of ISISP and IS Methods for 14Cand Various Degrees of Quenching Scintillation so1n.a D T D D D D D T D D D
Quencher Methyl red Triethanolamine Rhodamin B Rhodamin B Congo red Congo red CCl4
cc14
Glutaric acid Tartaric acid Methylene blue
100 EJEo 88.0 53.2
24.2 4.0 97.4 67.6 80.6 30.8 10.8 15.0 31.8
Standardb HDC HDC BC NC NC NC HDC HDC BC NC BC
-0.8 +0.7
+6.3 -2.2
+0.9
-7.6 -1.1 +358 +180
-0.3
+9.5 -0.9
+O. 5 +O. 3 -0.5
+o. 1 -0.5
- 16
+0.8 -0.9
-24.6
-0.5
= T = toluene-based liquid scintillator, D = dioxane-based liquid scintillator. bHDC = hexadecane l4C, BC = benzoic acid 14C, NC = naphthalene 14C.
determination have tended to decrease. The required division of the sample in the ISISP is, therefore, generally no problem. On the other hand, this technique can be used to preserve one half of a given sample in its original form for the isolation of a labeled compound or a recount. The statistical considerations described by Herberg ( I ) for the IS technique are applicable and valid for the ISISP as well. If, after an unknown sample is counted, it is found that the ratio between the sample plus the standard activity and the sample is too small, more standard activity can be added to satisfy statistical requirements in ISISP with no sacrifice in accuracy of counting. The nature of quenching materials and the degree of quenching have no effect on the accurate determination of counting efficiency in the ISISP method, the only limitations being statistical considerations. Also, the new procedure permits consecutive counting of its two parts, standard and sample, which tends to minimize possible effects of instrumental drift. 8 14
ANALYTICAL CHEMISTRY
The selection of a standard for the ISISP method is much less of a problem than for the internal standard technique. Many compounds may be used in the ISISP method with little or no disadvantage. The ISISP represents an excellent method for the preparation of secondary standards of labeled organic and inorganic compounds for a variety of alpha- and beta-emitting radionuclides. ACKNOWLEDGMENT
The authors express their appreciation for the technical assistance of Mae W. Williams in performing the laboratory experiments. RECEIVED for review October 16, 1967. Accepted January 22, 1968. Presented at the 153rd meeting, Miami Beach, Fla., April 9-14, 1967. Mention of commercial products used in connection with work reported in this article does not constitute an endorsement by the Public Health Service.
