Table II.
Properties of N-Substituted Tetrachlorophthalamides
Analyses M.P., "C.
Substituent
1 135-6'3' 135-6 * 130a 167d 201d 163-4'
Form u 1a CieHniClrXOt CisH2rCIaXOz C2oH2,CLS02 CzzHzqC14N02 C22H29Br4?i02 C22H291aX92 C.,H,,RrA O1
Calrulated H
C 50 51 53 55 40
9 8 0 0 2
42 0
4 5 5 6 4
9 2 7 0 4
4 9
c1 33 32 31 29 48 60 46
4 3 4 6 5. 0' 5"
Found Yc H
C 51 52 52 55 40
0 0 7 3 2
5 5 5 6 4
41 7
0 5 4 2 4
CT 33 32 31 29 48 59 46
4 7
7 4 2 6 Se
9, 3e
. 1 1 50 9 34 5 11 50 9 -CH;C€LCl CioHaCl5XOz 34 6 203c 38 4 2 2 47 3 38 2 -CH,CH2CHzCH,Cl 2 3 47 0 CI2HsCIaN02 153-4' h 27 2 1 1 60 46 27 1 -CHzCH,CH2CN 1 3 60 1" C,,HeBrrN202 230-20,' ' 19 8 0 8 71 Of 0 8 70 7 1 -CHzCH2CH2CN C12HJaS292 20 1 264-7 -CH&ONH2 35 2 1 2 41 4 35 4 300-2' 1 CtnHLCLh 2 0 9 1 2 41 7 3.0 35 7 41 9 -cH;coK(GH,)~ C;;H;,ci,s2o, 42.2 3.0 3F,. 5 240-lk -CH( CIBHBB)COOCIHO 609 C?~H,&lrN04 56.5 6 6 24.9 56.3 6.5 24 6 -CH( Ci6H3r)COOC2Hs 68-71',' C2sH,,Bra?iOc 4 1 . 4e 41.8" 40.4 2.3 34 1 -CH( COCH,)COOC2H, 40.7 2.2 34.4 CiaHsClrN05 173-4 * ' From chloroform-methanol. c Kork of bV. E. Yoerger. From dimethglformamide. Bromine. Iodine. a From acetone. K, calcd., 3.7%; found, 3.55;. C1 deriv., m.p. 194 ( I ) . 1 N, calcd., 8.2Yc; found, 7.9%. From ethanol. From acetic acid. K,ralcd. l.8Yc; found, l . 8 7 c . -
1
L "
"
'
Q
unused reagent. If the imide did not crystallize well, other solvents were employed; these are included as footnotes in Table TI, which lists properties of the neFv imides. Decolorizing carbon was used where needed. The volume of diniethylforniamide was increaied to 20 ml. for the bromo imide, and to 25 ml. for the iodo analog. IK THE STEAMBATH. The potassium salt, 1 gram, and 10 nil. of pure dimethyl sulfoxide were thoroughly mixed, 2.5 ml. of the halide were added, and the mixture heated was for 1 hour under a reflux condenser. After the mixture cooled, 10 ml. of water were added to the paste and the product was collected on a filter and rinsed (if colored) with 10 nil. of saturated aqueous sodium bisulfite. This product was then recryitallized as usual. (The smallest amount of halogen compound that was converted to a satisfactory derivative is 0.9 gram.) The use of the new solvents did not result in the formation of derivatives that were unattainable by the earlier
procedure. Other solvents that did not react or gave noncrystalline products were ethyl a-bromomyristate, ethyl dichloroacetate, bromochloromethane, and dichloroniethane. Chloroacetaldehyde diethylacetal gave a black solution ACKNOWLEDGMENT
We thank W.E. Yoerger for much of the work with the alkanesulfonates, and for some of the reactions a t 95' C.; and Donald Ketchum and the Analytical Department of the Research Laboratory of the Eastman Kodak Co. for the microanalyses. We also thank Rohm & Haas for a specimen of dodecenyl chloride.
(3) Billman, J. H., Cash, K. V.,Zbid., 75, 2499 (1953). ( 4 ) Zbid., 76, 1944 (1954). ( 5 ) Huebner, C. F., U. S. Patent 3,025,300 (19621: C. A . 58.3398 11963). ( 6 ) Hunsberger, I: 11.) Tien, J. l l . , Chem. Ind. 1959, p. 88. ( 7 ) Marvel, C. S., Sekera, V.C., ,'Organic Syntheses," E. C. Homing, ed., Coll. Vol. 3, p. 366, Wiley, New York, 1955. 18) Xefkens. G. H. L.. Tesser. G. I . Nivard, R . F. J., Rec.'Trav. Chem. 79; 688 (1960). ( 9 ) Pratt, D. S., Perkins, G. A,, J . d m . Chem. SOC.40, 205 (1918). (10) Pratt, D. S., Young, C. O., Zbid., p. 1417. (11) Ranky, W. O., Selson, 11. C., "Organic Sulfur Compounds," N. Kharasch, ed., Vol. 1, Chap. 17, Pergamon Press, New York, 1961. (12) Sheehan, J. C., Bolhofer, W.A , , J . Am. Chem. SOC.72, 2786 (1950). ~I
~
C. F. H. ALLEX W. R. ADAMS C. L. MYERS
LITERATURE CITED
(1) Allen, C. F. H., Laakso, T. T. .M., U. S. Patent 2,816,125 (1957). ( 2 ) Allen, C. F . H., Nicholls, R. V. V., J . Am. Chem. SOC.56, 1409 (1934).
Rochester Institute of Technology Rochester, N.Y .
Liquid Scintillation Counting of H3-Nucleic Acids SIR: Liquid scintillation counting of biological compounds such as sugars, amino acids, and proteins has been described by many workers ( I , 2, 4, 6, I O ) . The methods usually employed toluenehyamine or dioxane-water solvent systems to keep the compounds in solution. .i system including hyamine in the dioxane-a ater solvent which can be used for a number of low molecular organic compounds, including nucleic acid bases, nucleosides, and nucleotides, has been reported from this laboratory ( 9 ) . Vnfortunately, however, thls syhtem could hold only a minute amount of nucleic acids and was not suitable as a general method for the radioassay of H3-nucleic acids.
The present method, which employs digestion of the nucleic acid samples by hydrochloric acid prior to the addition of hyamine-dioxane solvent system appears to be a convenient method as a routine radioassay of nucleic acids. EXPERIMENTAL
The liquid scintillation spectrometer used in this experiment was a Packard Tri-Carb model 314.1X equipped with an automatic sample changer (Packard Instrument Co., Inc., La Grange, Ill.). Dioxane scintillator solvent consisted of 100 grams of naphthalene, 10 grams of PPO and 250 mg. of POPOP in 1 liter of dioxane ( 1 1 ) . Toluene scintillator solvent contained 4 grams of
PPO and 100 mg. of POPOP in 1 liter of toluene (8). Hydroxide of hyamine lox, 1 molar solution in methanol was purchased from the Packard Co. (this material will be called hyamine for convenience). H3-nucleic acids were prepared from Pseudomonas cells (adenine requiring mutant) which were grown in a medium containing H3-adenine. The nucleic acid fraction was purified by the sodium dodecyl sulfate-phenol method ( 5 ) . Vnless otherwise stated, this material with or without cold nucleic acid fraction prepared from Pseudomonss cells was used as a radioactive sample without further purification. Chromatographic analysis of this preparation with a methylated albuminkieselguhr column ( 7 ) revealed that this VOL. 37, NO. 1 , JANUARY 1965
159
Table 1.
H3-nucleir acid, mg 0 1 0 3 0 6 1 0 3 0 0 1 0 8 0 6
I 0 3 0 Table
II.
Counting of H'-Nucleic Acid b y Digestion Method
In Water Water Water Water Water 2M SaCl 2M XaC1 2M NaCl 2M KaC1 2Jf NaCl
cPm 3,846 11,507 23,194 36,457 104,698 3,863 10,461 20,875 35,683 99,201
Counting efficiency,
72
6 6 6 6 6 6 6 5 6 5
37 61 84 47 49 25 27 92 12 46
dpm 60,400 174 000 339,000 563,000 1,610,000 61,800 167,000 353,000 583,000 1,820,000 ~
d p m per mg -nucleic acid 604,000 580,000 565,000 563,000 537,000 618,000 557 000 588,000 583,000 607,000 ~
35
i Y
10.
P
P
2
2?
Y
i
Counting of H3-Nucleic Acids by Digestion and Combustion Methods"
Digestion method Combustion method Counting Counting Sample efficiency, Sample efficiency, ?io. c.p.m. 72 d.p.m. No. c.p.m. 7% d.p.m. a 7646 6.68 114,000 e 20,750 20.04 103,500 b 7927 7.36 108,000 f 19,616 18.45 106,300 r 7402 7.00 106,000 20,033 18.87 106,200 d 7905 7.41 107,000 19,918 19.11 104,200 hIean 109,000 d.p.m. Mean 105,100 d.p.m. a H3-nucleic acid (49 pg.) was assayed by the digestion method. The combustion n'as done with H3-nucleic acid (49 p g . ) and approximately 7 mg. of yeast RXA.
E
material contained approximately 66% ribosomal-, 8% soluble-RXXs, and 26% DX.L That the label was located in the adenylic and the guanylic acid residues in each nucleic acid was determined by paper chromatography after hydrolysis (1%'). H3-DKA\ preparation used was the fraction isolated by the methylated albumin-kieselguhr column as described above which was precipitated by two volumes of ethanol after addition of commercial DKA sample as a carrier. The combustion of the H3-nucleic acid was done with an automatic combustion furnace for the determination of carbon and hydrogen (hlitamura Riken Kogyo, Tokyo) equipped with a dry ice cold trap. After the combustion of the samples in an atmosphere of oxygen, the resulting water in the trap was transferred quantitatively to a vial with 0.5 ml. of water and 10 ml. of the dioxane solvent. Direct counting of the nucleic acid on the membrane filter was carried out according to the method described by Hall and Spiegelman (3). The filters (type GM-7, 2-inch diameter) were purchased from the Gelman Instrument Co. (.inn hrbor, Mich.). All the countings were done at 5-6' C. with standard 20-ml. glass vials a t the 10-100 window a t 5.76 high voltage taps (ca. 1115 volts) which gave the highest counts in this window with a standard H3-toluene vial. The counting efficiencies were determined with H3toluene internal standard. Counting of Nucleic Acid Preparation by Digestion Method. Various amounts of H3-nucleic acid, 0.1 to 3 mg., were placed in the vials. To each vial, 0.5 ml. of water or 0.5 ml. of 251 KaC1 solution and 0.04 ml. of concentrated HC1 were added (final concentration of HC1 was 0.9h'). Sam160
15
i
ANALYTICAL CHEMISTRY
ples were incubated a t 50' C. for 20 to 24 hours in silicone rubber stoppered vials. After the incubation, to the vial was added 0.1 ml. of hyamine followed by 10 ml. of the dioxane scintillator. Samples which contained NaCl were treated with 0.5 ml. of methanol in order to give homogeneous solution. RESULTS
The observed counts and the corrected radioactivity of each sample are shown in Table I. A proportional relationship existed betaeen the amounts of H3-nucleic acid added and the corrected radioactivities. Kone of the vials showed visible precipitates except the vials containing NaC1. The presence of YaC1 in the sample somewhat affected the corrected activities. The error due to the presence of S a C l , however, was approximately 4%) which is almost negligible in the routine assay of nucleic acid fractions. Therefore, the samples which contained high concentrations of salts such as the eluates from column chromatography, could be applied directly to this method without troublesome desalting procedures. Table I1 summarizes the counting data of the H3-nucleic acids by the combustion and the digestion methods. As shown, the data coincided very closely, indicating the digestion method is useful. To determine how much nucleic acid can be assayed with this method, the following experiment was set up. Appropriate amounts of the Ha-nucleic acids (30 fig. each) with various amounts of carrier yeast R K h , 1 to 25 mg., were
Figure 1 . Counting of H3-nucleic acid on membrane filters
added to vials. The digestion by HCl, addition of hyamine, and the dioxane solvent were done as before. The counting data and the corrected radioactivities are shown in Table 111. The counting was reliable up to 10 mg. of RS.l per vial. -1slight decrease in the corrected radioactivity was observed with increasing amounts of yeast RKA. The vials which contained more than 12 mg. of RK-\, showed appreciable precipitation. Therefore, the amount of RS.\ which apparently can be assayed per vial is approximately 10 mg. Counting of DNA by Digestion Method. A known amount of H3DKA in aqueous solution was placed in each vial and was counted as described above. Table IV summarizes the counting results. There existed a good linear relationship between the corrected radioactivities and the DNA added up to 2.54 mg. per vial. Again, the corrected radioactivities by the digestion method coincided with those obtained by the combustion method. S o precipitate was observed within this range of DK.1. Amorphous precipitates appeared in vials which contained more than 5 mg.
Table 111. Counting of Same Amounts of H3-Nucleic Acid in Presence of Various Amounts of Yeast-RNA by Digeslion Method
Yeast-
RNA, mg. 0 1
3 6 8 10 12 15 20
25
c.p.m 4600 4750 4615 4632 4489 4503 4164 3590 3182 2492
Counting efficiency, % 7.12 7 15 7 16 7 30 7 14 7.29 7 67 7 62 8 11 8 49
d.p.m. 64 600 66,400 64,500 63,500 62,900 61,800 54,300 47,100 39,200 29,400 ~
of DS.1. Therefore, the maximum amounts of DKAAwhich can be counted with this method are approximately 3 mg. per vial. Counting of Nucleic Acid on M e m brane Filters. Various amounts of H3-nucleic acid and the carrier DXA\ (250 pg.) were precipitated by trichloroacetic acid (final concentration 10%). The resulting precipitate was filtered on the membrane filter and was washed with small amounts of 0.01N HCI. The air-dried filters were placed in the vial and 15 ml. of the toluene scintillator was added. .After the direct counting, the filters were washed with toluene and air-dried. The digestion method was then applied to the filters and recounting was done. In Figure 1, the counting data by the direct and the digestion methods are summarized. The direct counting method gave a linear relationship between the observed counts and the applied radioactivities up to 300 pg. of nucleic acid samples. Above this range, the observed counts were no longer proportional to the radioactivity of the sample. The digestion method, on the other hand, could be applied up to the maximum amount of nucleic acid tested (10 mg.). This was in accord with the previous observation.
Toble IV.
Counting of H3-DNA by Digestion Method”
Counting efficiency, d.p.m. per c.p.ni. % d.p.m. mg.-DNA 6.?9 19,100 22,500 i f294 3.429 6.23 55.000 21.700 20,900 6: 056 5 69 106,000 5.08 8 46 8,237 5.13 161,000 19,Ooo 5 29 210,000 16,500 12.69 11,124 Radioactivity obtained by the combustion method was 22,600 d.p.m. per mg. of DNA.
H3-DNA, mK. 0.85 2 54
a
ACKNOWLEDGMENT
The authors are grateful to Yao Tung Liu, Laboratory of Radiation Genetics, Faculty of Agriculture, University of Tokyo, for his kind supply of the adenine requiring mutant of Pseudomonas. LITERATURE CITED
(1) Bray, G. A , , Anal. Biochem. 1, 279 (1960). (2) Bruno, G. A , , Christian. J. E.. ANAL. CHEM.33. _ _ 1216 (1961) - (3) Hall, B. D., Spiegelman, S., Proc. ‘VatZ. Acad. Sci. CJ.S . 47, 137 (1961). (4) Hash, J. H., Anal. Biochem. 4, 257 (1962). (5) Kirby, K. S., Biochem. J. 64, 405 f 1906). (6) Langham, W. H., Eversole, W. J., Hayes, F. N., Trujillo, T. T., J . Lab. \
I
(7) Mandell,
Biochem. 1, 66 (1960).
(8) Packard Instrument Co., Inc., La
Grange, Ill., “Tri-Carb Liquid Scintillaiion Spectrometer Operation Manual, 1959. (9) Takahashi, H., Hattori, T., Maruo, B., ANAL.CHEM.35, 1982 (1963). (10) Vaughan, hl., Steinberg, D., Logan, J., Science 126, 446 (1957). (11) Werbin, H., Chaikoff, I. L., Imada, M. R., Proc. Sac. Exptl. Biol. M e d . 102, 8 (1959). (12) Wyatt, G. R., Biochem. J., 48, 584 (1951). TOSHIE HATTORI AOKI HIROKO ITSUKO MATSUZAKI BUNJIMARUO Institute of Applied Microbiology University of Tokyo Bunkyo-ku, Tokyo HAJIMETAKAHASHI Laboratory of Radiation Genetics Faculty of Agriculture University of Tokyo Bunkyo-ku, Tokyo, Japan
A High Sensitivity Detector for Gas Analysis SIR: An extremely sensitive gas detector based on the work described by Winefordner, Steinbrecher, and Lear ( I O ) is described. The detector cell is a modified capacitor of small internal volume through which the carrier gas and sample gas flow. The detector cell capacitor is a part of the tank circuit of a Clapp oscillator which oscillates at about 65 Mc. The output of the sample oscillator is beat against a similar Clapp oscillator containing a variable air capacitor for initial zeroing of the two oscillators to a zero beat frequency. The beat frequency is measured using a simple frequency meter and potentiometric recorder. The detector shows extreme sensitivity to such gases as O,, N2,Hz, CO,, CO, CH,, C2H2,Ar, NOz, NO, NzO, and SO, when present in trace concentrations in a helium carrier gas. The detector also appears to show great sensitivity to organic vapors of less volatile organic compounds as well as to permanent gases. The detector system is simple in design, inexpensive to construct, nearly insensitive to small variations of carrier gas flow rates, and reproducible over a long period of time.
EXPERIMENTAL
Apparatus. A block diagram of t h e experimental setup is given in Figure 1. T h e sample a n d reference oscillators are 65-Mc. Clapp oscillators ( I ) and are similar to the ones previously described ( I O ) . However, because of a tendency for the oscillators to “lock-in” near the zero beat-frequency, the outputs of each oscillator were fed into a cathode follower and then into a diode-mixer circuit. The Clapp oscillators and mixer circuits were modified considerably from those previously used (IO), and so the modified diagrams are given in Figures 2 and 3. The values of all components are listed at the bottom of each diagram. The Clapp oscillators and the mixer were built on aluminum chassis (3 X 6 X 4 inches). The coil Ls (or L R ) in the tank circuit was rigidly mounted on a porcelain plate on the top of the chassis and was shielded by means of an aluminum box (3 X 4 X 3 inches). The oscillator and cathode follower tubes were enclosed in tube shields. The B+ and filament connections for the oscillators were connected to 500-ppf. button-type feed-through condensers and R F chokes (Ohmite 2-500). The feed-through condensers and the chokes are not shown in Figure 2. h ,Heathkit
power supply (Model PS-3, Heath Co., Benton Harbor, Mich.) was used to supply the power to the filaments and B+ of the oscillators and mixer. Ai similar power supply was used for the frequency meter. I t was of great importance that the components of the tank circuits be mounted and wired as rigidly as possible. Square bus bar wire was used for all connections in the tank circuit. The coils were wound from silver coated copper wire (No. 10) and were mounted rigidly on polystyrene rods (”, inch 0.d.). Connections to the coils were made through the base of the porcelain plate. Condenser C S (see Figure 4) was a cylindrical condenser with inlet and outlet for gas flow and was made from a male coasial connector (Type UG-58/TJ Amphenol). The top of the coaxial connector was carefully cut off to give a hole about inch deep. About 5/16 and Ijg inch from the edge of the open end of the connector, two short sections of stainless steel tubing (1 inch long X inch 0.d.) were force fitted into two 7/M-in~h holes. These provided for gas flow into and out of the detector cell. Then a stainless steel tube (3/s inch long X ‘/4 inch 0.d.) with a small hole in the center wab polished until it would fit tightly over the central alignment pin of the coaxial connector. The outside VOL. 37, NO. 1, JANUARY 1965
161
