Liquid Scintillation Counting of Biological Compounds in Aqueous Solution SIR: The finding of Langham, Eversole, Hayes, and Trujillo ( 6 ) , that a solvent system composed of dioxane, naphthalene, 2,5-diphenyloxazole (PPO) 1,4-bis-2-(5-phenyloxazolyl)-benand zene (POPOP) is suitable for counting radioactivity in biological fluids with a liquid scintillation spectrometer, led many workers (1, 2, 5, 10) to explore a modified dioxane system for counting tritiated water as well as aqueous solutions of radioactive materials. For the purpose of assaying aqueous solutions such as column eluates containing (314and H3-amino acids and -nucleotides, the diosane system described by N'erbin, Chaikoff, and Imada (10) has been used in this laboratory. The system is quite satisfactory for counting of tritiated water but has the disadvantage that it can hold only small quantities of these organic compounds in solution. For example, I ml. of water plus 10 ml. of the solvent can hold only 1 mg. of leucine. The recent report by Hash (4) indicates that small amounts of H3bacterial fractions suspended in water can be solubilized with the aid of trypsin, lysozyme, and hyamine hydroyide.
Table 1.
The counting had been done with the dioxane-anisole-dimethoxyethane system ( 3 ) . Hash's finding points to a new approach for the counting of biological materials in aqueous solution using a hyamine hydroxide-dioxane system. Here we describe an extension of Hash's observation utilizing hyamine hydroxide in a diosane-naphthalenewater system n-hich was found to hold relatively large amounts of biological materials and to give a high counting efficiency. EXPERIMENTAL
The liquid scintillation spectrometer used in this experiment was a Packard Tri-Carb model 314 AX equipped with an automatic sample changer. All chemicals and radioactive materials were commercial preparations. The dioxane solvent system contained 100 grams of naphthalene, 10 grams of PPO, and 250 mg. of POPOP in 1 liter of dioxane (20). The toluene solvent system consisted of 4 grams of PPO and 100 mg. of POPOP in 1 liter of toluene
(8).
Relatively large amounts of leucine could be held in solution when hyamine hydroxide (one molar solution of hy-
Counting Efficiencies of H3-Toluene in the Presence of Various Biological Compounds
Compound Adenosine LAspartic acid L-Dihydroorotic Glucose Hydroxyproline I,-Phenylalanine L-Serine Sucrose Thymine Uracil Uridylic acid
Amount Counting Amount tested, tested, efficiency, nig. c /C Compound nig . Solvent system which contained 0.1 nil. of liyarnine 8.2 11. 8 rlscorbic acid 20.0 11.5 Citric acid 13.8 11.4 acid 13.6 11.7 Fructose 12.2 11 . 8 L-Glutamic acid 11.0 9.8 9.8 L-Methionine 9.6 9.6 11.6 14.4 8.7 D-Ribose 11 , 4 proli line 11.2 8.2 10.0 12.3 DL-Threonine 6.2 10.2 11.8 L-Tryptophan 8.2 12 . 0 DL-Ureidosuccinic 7.4 7.6 acid 11 , 6 Xanthosine 9.0 9.0
L-Alanine Cytidylic acid Glycine Hippuric acid L-Leucine Thymidine Uridine
Solvent system which contained 0.2 nil. of hyamine 9.2 ;Idenosine 5'-uhos3.4 11.2 pliate 9.0 10.3 Cytidine 22 Galactose 40 3.0 11.3 10.6 11.0 L-Histidine-HC1 9.8 22.0 11. 0 DL-Isoleucine 11 . 8 11.8 11.0 L-Ornithine-HC1 9,4 3.0 11.3 Urea 11.2 4.6 11.6 DL-T-aline 11.6
~-Arginine-HCl Cytosine Guanosine L-Tyrosine Xanthine
Solvent system which contained 0.4 nil. of hyarnine 5.2 8.3 L-Cystine 3.0 7.4 9.0 Guanine-HC1 11.2 2.6 8.9 L-Lysine-HC1 10.2 6.0 8.5 Urir acid 15.4 9.2 8.9
Sdenine
1982
ANALYTICAL CHEMISTRY
Counting eficien(,>.j C'
4.S
12.1 11.9 11.6 11.2 11.4 7.4 11.8 9.0 13.7 11.2
amine hydroxide in methanol; this material will be called hyamine in this test) was added to the dioxanewater system. T o examine whether or not this technique can be applied to various water soluble compounds, the following esperiment was carried out. Approximately 5 to 20 mg. of each compound was placed in a counting vial (standard 20-ml. low potassium glass), 0.4 nil. of water and 0.1 ml. of hyamine xere then added. Incubation vias carried out at 45' C. until the solution became clear. An additional 0.1 ml. of hyamine was added to vials which did not give a clear solution and incubation was continued. When needed, a further addition of 0.2 nil. of hyamine was made and incubation was continued for a longer time. To a clear hyaminewater solution, 15 nil. of the dioxane solvent was added and the vial was kept a t 6" C. which was the counting temperature for this solvent system. If a precipitate formed upon addition of the dioxane solvent or on standing at 6" C., further addition of hyamine and incubation a t room temperature \vere carried out. Finally, 0.02 ml. of the standard H3-toluene (217,600 d.p.m.) \vas added to each vial and the counting efficiency was determined with the 10100 viindow. iable I summarizes the compounds tehted, the amounts, and the efficiencies obtained. As can be seen, the hyaminetem seems t'o be a useful solvent system for the counting of various compounds. Hyamine was known as a solubilizing agent of amino acids and proteins with the toluene solvent system (9). Noht of the amino acid> were dissolved to an extent of approximately 20 mg. per nil. of hyamine ( 9 ) . shown in Table I, I .
Table It. Counting Efficiencies of H3Leucine b y Toluene-Hyamine and b y Hyamine-water-Dioxane Systems
Counting efficiency, Solvent A . 0.2 ml. of hyamine, 1.0 ml. of water, and 10 ml. of
11.0 11 1 11 4
9.3
10.8 12.1
10.8 10.9 8.9
9.0 8.8 8.2
5
the dioxane scintillator 6.27 B. 0.2 ml. of hvamine, 0.5 nil. of water,"and 10 mi. of the dioxane scintillator 9.29 C. 1.0 ml. of hyamine and 10 ml. of the toluene scintillator 6.58 D. 0.5 ml. of hyaniine and 10 ml. of the toluene scintillator 9.09 Seven milligrams of Ha-leucine (114,400 d.p.ni.) was dissolved in hvamine-Fater or hyamine. After completesolubilization the dioxane or toluene scintillators were added as described. They were counted with the 10-100 window.
oxane system for the correction of quenching in determinations of C14, but was not so satisfactory as the use of a n internal standard in tritium assays.
comparable amounts of various biological compounds can be counted with the hyamine-water-dioxane system. Therefore, these two solvent systems were compared using H3-leucine. As shown in Table 11, the counting efficiency with the hyamine-water-dioxane system was comparable to that with the hyamine-toluene systcmm. The solubility of this particular amino acid was almost equal in solvents -1 and C, B and D, respectively, under t i e conditions employed. Sample prepitration was much easier and faster with the hyaminewater-dioxane system than the hyamine-toluene system. The former did not require the prolmged drying and digestion needed for the hyamine-toluene system. The ratio method ( 2 , 7 )was applicable to the hyamine-water-di-
LITERATURE CITED
(1) Bray, G. A., Anal. Biochem. 1, 279 (1960). (2) Bruno, G. A., Christian, J. E., ANAL. CHEX 33,1216 (1961). (3) Davidson, J. D., Feigelson, P., Intern. J . Appl. Radiation Isotopes 2, 1 (1957). (4) Hash, J. H., Anal. Biochem. 4, 257 (19621. -(5) Kidard, F. E., Rev. Sci. Instr. 28, 293 (1957). (6) Langham, W. H., Eversole, W. J., Hayes, F. N., Trujillo, T. T., J . Lab. Clin. Xed. 47, 819 (1956). \ - -
(7) Takahashi, H., Hattori, T., Maruo, B., Anal. Biochem. 2, 447 (1961).
(8) "Tri-Garb Liquid Scintillation Spectrometer Operation Manual," Packard Instrument Co., Inc., La Grange, Ill.,
1959. (9) Vaughan, M., Steinberg, D., Logan, J., Science 126, 446 (1957). (10) Werbin, H., Chaikoff, I. L., Imada, M. R., Proc. SOC.Exp. Biol. Med. 102, 8 (19593.
HAJIMETAKA HAS HI^ TOSHIE HATTORI BUNJIM m u o Institute of Applied Microbiology University of Tokyo Bunkyo-ku, Tokyo, Japan Present address, Laboratory of Radiation Genetics, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Distribution of Ion Pairs between Immiscible Nonaqueous Solvents SIR: Few studies have been made of the suitability of nonE,queous immiscible solvent pairs for extIactive separations ( 1 , 3, 4 ) . This paLer summarizes in brief the estent to which different ion pairs distribute thems,elves in three such systems . EXPERIMENTAL
Materials and Scdutions.
X com-
mercial grade of formamide purified according t o T'erhocbk's procedure ( 5 ) w a b distilled under vacuum. Middle fractions t h a t had melting points greater t h a n 2.15" C. were obtained from numerous diqt llations and combined for use. Ethanolamine -cas repeatedly extracted with diethyl ether and then distilled. Only the fraction t h a t boiled between 169' and l i l O C. was collected. Adiponitrile, Eastinan White Label,
Table I.
Metal ion Fe(II1) RZo(V1) hIO(V)
Cu(I1) SnlII)
m.p. 1' to 3' C., n'," 1.4382, was used without further purification. Prior to use, all three solvents were equilibrated with diethyl ether. All other materials were reagent grade. Aqueous solutions of the metal ions were prepared by dissolving that weight of a metal salt necessary to give a solution 0.04ilI in metal ion. Where needed, a minimum amount of acid was added to prevent hydrolysis. Apparatus. The extractions were carried out i n 125-ml. separatory funnels t h a t had carefully shaped short stems t o prevent holdup. Procedure. hfetal stock solution, 25 ml., was treated with appropriate reagents so t h a t t h e metal would be quantitatively extracted into ethere.g., t h e Fe(II1) solution was made 6 F in HCl. T h e ether phase was withdrawn, dried over CaC12, a n d equilibrated with a second immiscible phase.
Extraction of ion Pairs from Diethyl Ether by Immiscible Nonaqueous Solvents
Associated anion
e1
Br SCN
e1 Br
scs Br c1 Br NO3 e1
Immiscible phase Ethanolamine Formamide Adiponitrile E
For the most part, the degree and direction of extraction could be satisfactorily estimated from the color of the phases. Where the ions were colorless, an appropriate analytical measurement was made. RESULTS AND DISCUSSION
The results of these experiments are shown in Table I. Only Sn(1V) showed a n y preference whatsoever for the ether phase. An attempt to neutralize the basicity of the amine was unsuccessful: the amine hydrochloride precipitated. The most striking characteristic of the extraction was the color of the ion pair in the nonether phase. I n adiponitrile for example, CuBr4-2 is dark olive green and C O ( S C N ) ~ -is~ light blue. Since the amine nitrogen is more basic than the ether oxygen, the lack of specificity is not unexpected. Adiponitrile, however, has proven to be a satisfactory solvent for extracting Fe (111) and Mo(V1) from aqueous HCl ( 2 ) and further investigations with it as one of the immiscible, nonaqueous phases may show more significant differences.
E'
LITERATURE CITED
E'
( 1 ) Larson, E. M., Trevorrow, L. E., J . Znorg. A'ucl. Chem. 2,254 (1956).
E
(2) Latimer, G. W., Furman, N. H., Zbid.,
...
... ... ...
Th(1V) Au(II1) ... Ce(1V) IT03 ... U(V1) NO3 ... Sn(IV) c1 ... co SCX ... M = phases miscible E = >90% extracted to the non-ether phase P = lo% extracted . . . = Experiment not performed
...
... ... ... 11
24, 729 (1962). (3) Lien, A. P., McCauley, D. A., Everiny, B. L., Ind. Eng. Chem. 41, 2689 (1949). (4) Tsai, K. R., Fu, Y., ANAL.CHEM.21, 818 (1949). (5) Verhoek, F. J., J . Am. Chem. SOC.58, 2577 (1936).
E'
GEORGE W. LATIMER JR.
Pittsburgh Plate Glass, Chemical Division P.O. Box 4026 Corpiis Christi, Texas VOL. 35, NO. 12, NOVEMBER 1963
0
1983
