sumably a reducible heteropoly complex is formed between molybdate and niobate. LITERATURE CITED
(1) “ASTM Methods for Chemical Analysis of Metals,” p. 151, hmerican Society
for Testing Materials,
Philadelphia, 1960. (2) Bailar, J. C., Jr., ed., “The Chemistry of the Coordination Comoounds.” D. 474, Reinhold, New Tork, i956. (3) Crouthamel, C. E., Hjelte, B. E., Johnson, C. E., ASAL. CHEM.27, 507 ( 1955). (4) Davydov, A. L., Vaisberg, Z. M., I
I
Burkser, L. E., Zauodslcaya Lab. 13, 1038 (1947). (5) Emeleus, H. J., Anderson, J. S., “Modern Aspects of Inorganic Chemistry,” p. 326, \‘an Nostrand, Princeton, 1960. (6) Hague, J. L., hlachlan, L. A., Jr., J. Research Natl. Bur. Standards 62, 11 ( 1959). (7) Kitson, R. E., Mellon, M. G., IND. ESG.CHEM. ANAL.ED. 16, 379 (1944). (8) Lundell, 6.E. F., Hoffman, J. I., Bright, H. A., “Chemical Analysis of Iron and Steel,” p. 365, Wiley, New
(11) Rosenheim, A,, in Abegg und Auer-
bach, “Handbuch der Anorganischen Chemie,” Vol. 4, Part I,i, p. 977, Hirzel, Leipzig, 1921. (12) Schoeller, W. R., “The Analytical Chemistry of Tantalum and Xiobiurn,” p. 122, Chapman and Hall, London, 1 Q.77
(IiY-Smith, E. F., Proc. Am. Phil. SOC. 44, 151 (1905). (14) Wallace, G. W., Jr., M.S. thesis, Purdue University. Lafayette, Ill., 1957.
York. 1931. I
G., Codell, M., ANAL. CHEM.26, 1230 (1954). (10) Rose, H., Ann. Physik u. Chem. 141 112, No. 4, 480 (1861).
(9)-Norwitz,
RECEIVEDfor review May 8, 1961. Resubmitted March 7, 1962. Accepted March 7, 1962. Work was supported by Eli Lilly and Co.
Determination of Radioactive Calcium by Liquid Scintillation Counting MARLENE SARNAT and HENRY JEFFAY Department of Biochemistry, College of Medicine, University of Illinois, Chicago, 111.
A liquid scintillation method for measuring calcium-45 is described. The method consists of precipitating calcium as the oxalate salt, converting this salt to the nitrate, and counting the nitrate in a solvent system of toluene-ethyl alcohol-ethylene glycolnitric acid containing an organic scintillator.
A
thercx iq gron ing literature on the usr of liquid scintillation counting techniques for carbon-14 and tritium, and to a lesser extent for inorganic radioactive nuclides, there is only one publishd liquid scintillation method for calcium45 Lutwak (4) has reported two procedures for calcium45) including the counting as calcium 2-cthylhexanoate in a solution of toluene, and as calcium chloride (or salt) in a tcutiary mixturc of absolute ethyl alcohol - hydrochloric acid - toluene. 1,utxik found the use of 2-ethj-lhexanoic acid somewhat limited in that it could not be used for urine, land that certain calcium salts required special pretreatment. He further statt,d that his other mctliod using the t d i a r y mixture should not be uscd for more than 50 mg. of calcium. Our laboratory, though obtaining essentially the same results as 1,utn ak, did find his tertiary mixture could not be u w d with much more than 25 mg. There have been other reports in which calcium-45 n a s counted in a liquid scintillation s j stem, but either insufficient details were given ( 1 . 7 ) , or the procedure is not ~ w l suitcd l for the routinc determination of this isotope (8). The presrnt n-ork dcscrihes a method LTHOUGH
in which calcium-45 nitrate is counted in an ethylene glycol-ethyl alcoholtoluene scintillation system. The method has the advantage of a relatively low background, high efficiency usefulness over a very 11-ide range of sample sizes, and direct applicability to calcium in any chemical form or from any biological source. Furthermore, the method is suitable for counting relatively large quantities (125 mg. of calcium) of low specific activity. EXPERIMENTAL
Apparatus. T h e liquid scintillation counter is a Tri-Carb liquid scintillation spectrometer, Model 314-A (Packard Instrument Co., LaGrange, Ill.). T h e freezer is maintained a t 00 c. Samples are counted in 5-dram cvlindrical Crvstallite vials fitted with 2%-mm. polirethylene snap caps (Wheaton Glass Co., Millville, N.J.). Materials. All chemicals used were ACS reagent grade. T h e scintillation system used was 1 liter of toluene containing 11.4 grams of 2,5-diphenyloxazole (DPO) (scintillation grade. Packard Instrument Co., Inc., LaGrange, Ill.), 1 liter of absolute ethyl alcohol, 250 ml. of ethylene glycol, and 12.4 ml. of nitric acid. T h e solution is stored in a n amber bottle in t h e dark. The standard used was a certified 10-pc. calcium-45 chloride solution (Nuclear-Chicago Corp., Chicago, Ill.). Procedure. PREPARATIONOF L I A C R O SAhSPLES (bone, milk, etc.). T h e sample is placed in a porcelain crucible in a muffle furnace 600-800’ C. for 8 or more hours. T h e ash is dissolved in concentrated HC1, transferred to a volumetric flask, a n d diluted t o a known volume. An
aliquot is removed, and the calcium is precipitated as the oxalate salt (5). The calcium oxalate is collected on a sintered glass filter crucible and washed with water. The crucible is then placed in a 111’2inch diameter short stem glass funnel on a rack designed to support 40 funnels. Using a counting vial as the receiving vessel, a small portion of warm concentrated nitric acid is poured into the crucible. Additional warin acid is added until all the calcium oxalate is dissolved. The crucible and funnel are washed with acid. The vial is placed on a hot plate (approximately 60” to 70” C.), in a fume hood, and the acid viashings are evaporated. The resulting Iyhite salt res; idue is heated for 15 minutes at 120 C. Twenty milliliters of the scintillation solution is added; the vial is capped and shaken until all the salt dissolves. The sample is placed in the freezer unit of the spectrometer and counted after 1 hour. PREPBRATION O F LfICRO SAMPLES (serum. urine, dilute solutions, etc.). The sample is placed in a 12-ml. glass centrifuge tube, and the calcium is precipitated as the oxalate ( 2 ) . The sample is centrifuged, the supernatant is discarded, and the precipitate is washed 1%-ithwater. The n nshed precipitate is dissolved in warm nitric acid, and the solution is transferred to a counting vial by repeated washings of the centrifuge tube n-ith small volumes of nitric acid. The nitric acid solution is treated as described above. RESULTS
Operating Voltage. T h e operating voltage was determined in t h e usual manner (6). The final operating conditions chosen m-ere T a p 4.65 (1070 volts) with window settings of 10 to 90, VOL. 34, NO. 6, MAY 1962
643
Table 1.
Reproducibility of Method
Observed Counting Trial S o . Ratea 1 45,966 2 45,996 3 45,883 4 45,874 5 45,956 Average 45,935 a Each count represents t h e average of three 30-minute counts.
90 to 100. Background was usually 65 c.p.m. Selection of Solvent. It rr-as necessary to find a solvent capable of dissolving a readily obtainable calcium salt. The salts n hich are easily prepared include the oxide, carbonate, sulfate, oxalate, and nitrate, to name a feir-. All b a t the nitrate are insoluble in most polar and nonpolar solrentc. After considerable experimentation. i t n-as found t h a t calcium nitrate was soluble in ethylene glycol. Howerer, ethylene glycol is a poor solvent for most phosphors and a quencher. Ethyl alcohol is required to bring ethylene glycol and toluene (a good phosphor solvent) into a one phase system. The ratio of solvents chosen yielded a clear stable solution a t 0' C., with minimum quenching. The solubility of calcium nitrate is greatly increased by the addition of small quantities of nitric acid. However, our results, as well as those of Von Erdtmann and Herrmann (9), indicate nitric acid causes quenching. There is a shift to higher tap settings and a decrease in observed counting rate with increasing concentrations of nitric acid. This effect is similar to that reported by Packard ( 5 ) . Calcium nitrate, though soluble in the scintillation medium without nitric acid up to 100 mg. at room temperatures usually precipitates a t 0" C. if more than 40 mg. are present. I n the presence of 0.5501, nitric acid, approximately 500 mg. of calcium nitrate nil1 remain in (20 ml.) solution a t 0' C. Concentration of Scintillators. T o determine the optimum concentration of the scintillator, identical radioactive samples containing variable amounts of scintillator were prepared and counted. The results indicate a DPO concentration of 0.5y0 would be desirable. 1,4-Bis-2-(5-phenyloxazoyl) benzene (POPOP) did not appreciably increase the counting rate. Linearity of Counting. A stock solution of calcium-45 nitrate in dilute nitric acid was prepared. Varying quantities of this solution were placed in counting vials, and the liquid was evaporated. Each sample was dissolved in 20 ml. of the scintillator and 644
ANALYTICAL CHEMISTRY
28
2
4
6
8
IO
12
14
16
18
20
22
24
26
28
30
THEORETICAL COUNTING R A T E (c.P.M. a 10-3)
Figure 1.
Linearity of counting
Line A represents results obtained with tracer quantities of calcium-45 nitrate. Line B represents results obtained using varying quantities ( 5 0 500 mg.) of calcium nitrate containing 35 c.p.m. per mg.
counted. The results (Figure 1, line A ) indicate a linear relationship between the true and observed counting rate over a wide range of activities when tracer quantities of calcium-45 nitrate are present. If larger quantities of calcium nitrate are present (Figure 1, line B ) , then only approximate linearity is obtained. Above a theoretical counting rate of 17,500 c.p.m. (more than 500 mg. of calcium nitrate), a precipitate was seen. When comparing samples containing more than a 200-mg. difference in calcium nitrate content, a correction factor should be used. Effect of Calcium-40 Nitrate on Counting Rate. Because i t may be desirable in many experiments t o compare the radioactivity in variable quantities of calcium with widely differing specific activities, i t was necessary to determine the effect stable calcium nitrate mould have on the counting rate of a known amount of radioactivity. A series of vials was prepared] each containing exactly the same quantity of calcium-45 nitrate but varying amounts of calcium-40 nitrate. These results are seen as Figure 2. A quenching effect was noted. If the assumption is made that the true counting rate would be observed at the trace levels of calcium nitrate (0 mg. added), then there was a loss of approximately 9.5% of the counts when 500 mg. of calcium nitrate was present. Since the slope of the line in Figure 2 is very gradual, the counts lost between two samples of similar total calcium content,
due to quenching, would be very small, 0.019yo lost per mg. of calcium nitrate. This quenching effect of calcium nitrate is probably responsible for the deviations from linearity (Figure 1) observed with nontracer quantities of calcium. Reproducibility. Aliquots of a radioactive bone ash solution were analyzed using this method. T h e results (Table I ) clearly show the method yields reproducible results. Efficiency. A certified standard solution of calcium-45 chloride was diluted so t h a t 5 ml. of the final solution contained 110.000 d.p.m. with a trace quantity of calcium. Five 5 m l . aliquots were placed in counting Yials, and t h e solution vias evaporated. One milliliter of nitric acid was added and evaporated. T h e samples were dissolved in the scintillating solution and counted. The average measured counting rate (each counted three times for 30 minutes) was 58,640 c.p.m., or 53.370 of the disintegrations were detected. If amounts of calcium comparable to what might be expected from an animal bone sample-e.g., 200 mg. of calcium nitrate-were present, the efficiency was somewhat lower, 51.1%. Stability of Samples. 9 series of identical samples was prepared and stored in the dark at room temperatures or in the freezer unit of the counter for varying periods of time u p t o 4 days. T h e results (Table 11) demonstrated t h a t t h e solution was stable for more t h a n 4 days. If the final solution was not kept in the dark,
i t became yellow after 2 days and the counting rate changed appreciably.
Table II.
Storage Time, Days
Storage Place Dark
DISCUSSION
This method is relatively simple and accurate for the determination of the radioactive calcium content of a wide variety of substances and can be used for the routine handling of a large number of samples. Forty samples have been simultaneously analyzed without difficulty. Furthermore, the analytical procedure may bp interrupted a t any point and continued when more convenient without a loss of accuracy. The exact conditions for ashing a sample before precipitating the calcium as the oxalate are not critical for this method. Recovery of calcium-45 nitrate from ashed unlabeled bone samples was essentially lOOs%, indicating no loss of calcium-45 was observed during the overnight ashing at 800' C. Undoubtedly, lower ashing temperatures (600' C.) are possible and in many circumstances desirable. It was necessary to store the scintillation solution in a n amber bottle in the dark at all times; otherwise, there is photodecomposition yielding yellow colored products. The solution is stable in the dark for a t least 2 to 3 months, Presumably this reaction involves nitric acid since the scintillation medium without nitric acid is quite stable (it must, hov-ever, be "dark adapted" before use). The D P O scintillator should be dissolved in the toluene before the other reagents are added. Care should be taken not to heat the dry calcium nitrate much above 120' C. since this usually results in a loss of activity. The reason for this loss is not clear. If desired, the dry calcium nitrate may be stored either in the light or dark, until convrnient to disbolve and count.
1 2 3 4 1 2 3 4
Light
a
Ma.
c. c. 0" c. 0" c. 0" 0"
Counting Rate4 (C.P.M.) 41,497 41,775 40,487 41,108 42.229 41 I377 40,022 41 ,076 40,009 26,215 31,421 24,684
Room temperature Room temperature Room temperature Room temperature Immediately after preparation, the average counting rate as 41,253 c.p.m.
However, once dissolved, the solution should be stored in the dark. When trace amounts of calcium-45 nitrate were counted in the presence of equimolar quantities of nitric acid or calcium-40 nitrate, the results indicated that the nitric acid causes a majority of the quenching seen in Figures 1 and 2. It is not known whether the quenching is due to the presence of nitric acid molecules, a lower pH, or the water content of concentrated nitric acid. Our results show that calcium nitrate, in the absence of nitric acid, will also cause some quenching. This difficulty may be easily overcome in either of two ways. Since the quenching effect of calcium nitrate is rather small, 0.019yo per mg., counting samples of similar calcium contents eliminates the necessity of using a correction factor. Thus, a 100-mg. difference in calcium nitrate content between samples would have only 1.970 error if not corrected. This is usually less than the statistical counting error. For most studies where calcium-45 is used, bone, blood, urine, or milk is
300
200
Storage Temperature Room temperature Room temperature Room temperature Room temperature
1 2 3 4
I I00
Stability of Samples
400
500
603
C O ~ O I N O ~ ) ~
Figure 2. Effect of unlabeled calcium nitrate on counting rate
analyzed. If interest is focused on comparing different samples of the same type of material-e.g., all blood samples -then no correction is necessary, since all samplrs n ould have a similar calcium content. If interest is focused on comparing samples of 1Tidely differing calcium content, dilution of the more concentrated samples to approximate the concentration of the other samples would also eliminate any quenching error. The reported efficiency of counting, 53%, is with trace amounts of calcium nitrate. Decreasing efficiencies mould result with increasing calcium nitrate concentrations. Improved efficiency yields are possible b y eliminating nitric acid from the scintillation solution. This procedure is recommended only when small quantities of calcium are to be counted-e.g., serum samples. The function of the nitric acid in the scintillator is to increase the solubility of the calcium nitrate. With the recommended quantity of nitric acid, 0.550jo, 500 mg. of calcium nitrate can be solubilized. This method should be very useful for the determination of very low specific activity calcium samples. It is estimated that the lower range of detection is 10 mp per m J 1 calcium, or 0.5 d.p.m. per mg. of calcium. The method is probably even more useful for the accurate determination of very high specific activity calcium. It is assumed that this method would be equally applicable to the beta emissions of the other calcium isotope, calcium-47. The only modification would be a change in operating voltages of the spectrometer. Of course, the counting efficiency of the gamma ray activity of calcium-47 by this technique would have to be determined. Originally i t mas hoped that other radionuclide nitrates, especially of the alkaline earth series, would be soluble in this scintillation solution. The solubility of strontium, barium, manganese, magnesium, iron, copper, sodium, poVOL. 34, NO. 6 , MAY 1962
645
tassium, and cesium was tested. Onlv iron, COPPCr, and manganese nitrates were soluble to any appreciable extent, and these all yielded brightly colored solutions. ACKNOWLEDGMENT
The authors acknowledge the help of Julian Alvarez in preparing the drawings, and H. R. Bayne for checking the method.
LITERATURE CITED
(1) Flynn, K. F,, Glendenin, L. E., Phys. Rev. 116, 744 (1959). ( 3 ) Hawk, p. B., Oser, €3. L., Summerson,
R. H., “Practical Physiological Chemistry,” p. 644, Blakiston, New York, 1954. (3) Kolthoff, I. M., Sandell, E. B., “Textbook of Quantitative Inorganic Analysis,” p. 337, Macmillan, New York, 1952. (4)Lutwak, L., ANAL. CHEM. 31, 340 (1959). ( 5 ) .Packard, L. E., in “Liquid Scintillation Counting” C. G. Bell, F. N. Hayes,
eds., p. 63, Pergamon Press, London, 1958. (6) Packard Tri-Carb Liquid Scintillation Spectrometer Operation Manual, Packard Instrument Co., LaGrange, Ill. ( 7 ) Peng, C. T., . ~ K A L . CHEM.32, 1292 (1960). (8) Ronzio, A . R., Intern. J. A p p l . Radiation and Isotopes 4, 196 (1959). (9) Von Erdtmann, G., Hermann, G., 2. Elektrochem. 64, 1092 (1960). RECEIVED for review Kovember 24, 1961. Accepted March 9, 1962. Work waa partially supported by U. S. Public Health Service Grant D-877.
Effect of Preheater Contamination on Gas Chromatographic Analysis of Strongly Adsorbed Substances EDGAR
D. SMITH
and AUBREY B. GOSNELL
Graduate Institute of Technology, University of Arkansas, little Rock, Ark.
b
The presence of carbonaceous deposits in the preheater section of a gas chromatographic apparatus may lead to serious complications in the gas chromatographic analysis of polar organic compounds. The main effects of such a deposit are low detector responses and false peaks. Work with fatty acids and amines has shown that these effects may sometimes be of such a magnitude as to invalidate either qualitative or quantitative analyses. These effects can be eliminated by rigorous cleaning of the preheater section, but they gradually return as the carbon deposit again builds up through thermal decomposition of the samples injected.
the very first reported application of gas chromatography involved the analysis of organic acids ( 3 ) and bases (2, 4, there have been LTHOUGH
10-4
3 4
L 4
0 INJECTION
NUMBER
Figure 1. Typical plot of recorder response vs. injection number
0=
propionic acid injection numbers 1-8 and
21-31 0 = n-valeric acid injection numbers 9-20
646
ANALYTICAL CHEMISTRY
relatively few similar applications since these early papers. The major reasons for the lack of progress in these fields are discussed in recent articles by Hunter, Xg, and Pence (1) and by Smith and Radford ( 5 ) , and methods for overcoming these experimental difficulties are suggested. The present work is concerned with still another obstacle which must be surmounted before highly polar organic compounds can be analyzed satisfactorily by gas chromatography. It has been discovered that the preheater section of the chromatograph must be maintained free of carbon deposit when strongly adsorbed materials are to be analyzed. If this is not done, low and erratic detector responses, as \Tell as false responses, may be obtained. The false responses are apparently due to partial displacement of one or more components from previously injected samples which had been retained on the carbon deposit in the preheater. Numerous false responses may be obtained from a badly contaminated preheater, and accordingly this phenomenon has been called the repeater effect. The effects described are principally illustrated in this work using propionic and valeric acids, but similar effects have been observed with other polar organic compounds. These effects are particularly noticeable with acids and amines, but they have also been observed to a minor extent with alcohols and ketones. EXPERIMENTAL
-4 Perkin-Elmer Model 154-C Vapor Fractometer equipped with a thermistor detector was used. One meter stainApparatus and Reagents.
less steel columns were packed with t h e partition phases and substrates listed below, t h e partition phases being applied b y the conventional slurry a n d evaporation techniques using acetone as the solvent. -411 columns were conditioned overnight a t 180’ C. in a stream of helium gas, and the columns were reweighed after conditioning to determine the final per cent of partition phase.
A. 12.4% LAC-296 on firebrick (regular 60/80 mesh, Wilkens Instrument and Research Inc.. Cat. Eo. XA-165). B. 15.5% LAC-296 on unactivated Celite (Perkin-Elmer Corp., Cat. No. 154-0048). C. 19.5% L4C-296 on Chromosorb-W (30/60 mesh, Johns-Manville Corp., Cat. S o . -1-472). D. 10% LAC-296 S% Hap04 on acid washed Chromosorb-R (Johns-
+
“I
h,e
I
I
S S C O L U M N ISSCOLUMN S S C O L U M N
Ill
p
2
1
’
I
Ill
INJECTION NUMBER
Figure 2. Recorder response v5. injection number interchanging two identical columns of packing D in stainless. steel
0 = propionic acid 0 = n-valeric acid
