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Modern Methods for the Separation and Continuous Measurement in the Gas Phase of Compounds Labelled with Radiocarbon. Radio and Reaction Gas Chromatography

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The same reaction process can be assumed for the fluorination by SF4 of cyanogen bromide and inorganic
cyanides, cyanates, and thiocyanates [76].
tacked by water and by alcohol, with cleavage of the
S-F and S-N bonds. In many reactions, C ~ H S - N = S C ~ ~
behaves chemically like thionyl chloride; for example, it
gives 4,4'-dimethoxydiphenyl sulfide with anisole in the
presence of zinc chloride [77]. The reaction of phenyliminosulfur difluoride with lithium aluminum hydride
to form aniline is interesting.
Thanks are due to Dr. L. fhfackerfor stimulating and informative discussions.
The organic iminosulfur difluorides have remarkable
thermal stability. Phenyliminosulfur difluoride is at-
Received. February 22nd, 1963 [A 301/103 IE]
[76] Additional iminosulfur difluorides are reported by W. C.
Smith et al. [74].
[77] See F. Loth and A . Michaelis, Ber. dtsch. chem. Ges. 27,
2540 (1894).
German version: Angew. Chem. 75,697 (1963)
Modern Methods for the Separation and Continuous Measurement in the Gas
Phase of Compounds Labelled with Radiocarbon
Radio and Reaction Gas Chromatography [ *]
In biochemical work with isotopic tracers, it is frequently advantageous to investigate
samples in the gas phase. In this way, the selectivity of gas-chromatographic separating
columns and the sensitivity of detectors can be utilized for both the detection of gas-chromatographic fractions and the determination of their radioactive portions. Conversion of
materials separated by gas chromatography into a single standard chemical compound
prior to measurement of the radioactivity is recommended. Methods suitable for 14Clabelled compounds can also be applied to those labelled with 3 H or 35s.
I. Introduction
Radiochemical detection of a compound is far more
sensitive than other methods, since the disintegration of
single atoms can be recorded. Under favorable conditions, activities of 10-7 to 10-9 mC [**I can be measured [1,2].
Good radiochemical detection methods and gas-chromatographic separation methods are of comparable
power. Both methods have been highly developed tech[*] Also Part VI of the series on Reaction Gas Chromatography.
Part V: F. Drawert and G . Kupfer, Hoppe-Seylers Z . physiol.
Chem. 329, 90 (1962).
[**I 1 mC = 3 . 7 ~10' disintegrationslsec.
[l] Cf. H. Gotte in Houben- Weyl: Methoden der organischen
Chemie. 4th Ed., Thieme, Stuttgart 1955, Vol. III/l, p. 751 et
seq.; G. Herrmann in Schwiegk and F.Turba: Kunstliche
radioaktive Isotope in Physiologie, Diagnostik und Therapie.
2nd Ed., Springer, Heidelberg 1961, pp. 121-304.
[2] H . Simon and F. Berthold, Atomwirtschaft 7,498 (1962).
nically, and the constructional elements of their respective detection systems can be readily combined. Gaschromatographic detectors can be linked to radiation
detectors and both worked continuously, so that both
the total amount of material and the radioactive portion
of fractions leaving a gas-chromatographic column can
be measured simultaneously. The conversion of nonvolatile compounds into volatile derivatives is, however,
a serious problem [3]. It is also possible to carry out reactions with radioactively labelled compounds in a reaction chamber connected to the column inlet and thus
to separate the reaction products immediately after they
left the reaction chamber [4].
In radio gas chromatography, liquid scintillation counters, ionization chambers, and Geiger-Muller or proportional counting tubes have been employed.
[3] Literature references: F. Drawert, R. Felgenhauer, and G .
Kupfer, Angew. Chem. 72, 555 (1960).
[4] F. Drawert: Gas Chromatography 1962. Butterworth, London 1963, p. 347, where further references are given.
Angew. Chem. internat. Edit. Vol. 2 (1963) I No. 9
11. Radiation Detectors
1. Liquid Scintillation Counters [5]
If radioactive gas-chromatographic fractions are collected in a solution of a scintillator (diphenyloxazole in
toluene), the combination provides an important method
for the continuous measurement of 1%. The carrier gas
drives the scintillation fluid in a circulatory system past
a photomultiplier and increases in radioactivity are integrally indicated [6]. In another technique, the fractions
are collected in silicone oil in a fraction collector and are
measured discontinuously, using anthracene as scintillator [7]. Plastic helices with imbedded scintillator have
also been used for the continuous flow measurement of
1%-labelled compounds [8].
obtained if the fractions separated by gas chromatography are either ignited to CO2 at 650°C [I71 or converted into methane and hydrogen by treatment with
zinc and nickel oxide at 650 "C [18] before passing
through the ionization chamber.
Continuous-flow ionization chambers are predominantly
provided with oscillating capacitor type amplifiers whose
sensitivity is very dependent on the input resistance. Sensitivities of from ca. 10-2 p C are reported for 14C and 3H.
[15,16]. The background effects of ionization chambers are
somewhat high and cannot be reduced efficiently [2]. Heating the chamber alters the background unfavorably and
often causes base-line drift. One advantage of the ionization
chamber is that relatively high activities (0.1 t o 1.0 mC) can
be measured without difficulty.
3. Geiger-Muller and Proportional Counting Tubes
Evans and Willard [9] lead the gas-chromatographic fractions
past a crystal scintillator (detection of 10-13 g of CH382Br
and 10-15 g of CH380Br). Herr et al. [lo] put ampoules of
radiation products into a n ampoule-breaker (gas-chromatographic insertion accessory) and led the fractions through
a glass helix to a solid scintillator (an Nal(T1) crystal). Radiation products of benzene [ l l ] and other p-emitters [12] have
also been measured by the scintillation method after separation by gas chromatography.
Transference from the gaseous to the liquid phase is not
absolutely necessary, since numerous very sensitive
methods for measuring 14Cin the gas phase are available.
2. Ionization Chambers
Various types of continuous-flow ionization chambers
for use with gas chromatography have been described.
Cacace [13] described a method in which the effluent gas
is mixed with additional carrier gas after passing through
the katharometer cell (gas-chromatographic detector)
and before entering the ionization chamber. In this arrangement, it is possible to maintain a constant flux
through the ionization chamber (10 l/hr; chamber volume: 100 ml) independent of column conditions. A high
rate of gas flow prevents condensation of compounds
with boiling points up to 155 "C. The minimum detectable activity is about 2 x 10-4 pC. Various heatable
chambers have been described for measuring the radionuclides 3H, 14C, and 35s [23-16]. Improved results are
[5] E. Rapkin, Atomwirtschaft 7 , 508 (1962).
[6] G. Popjak, A . E. Lowe, D. Moore, L . Brown, and F. A . Smith,
J. Lipid. Res. 1, 29 (1959); Chem. Abstr. 54, 10409 (1960); cf.
A . E. Lowe and D . Moore, Nature (London) 182, 133 (1958).
[7] A . Karmen and H . R.Tritch, Nature (London) 186, 150 (1960).
[8] B. L . Funt and A . Hetherington, Science (Washington) 129,
1429 (1959).
[9] J . B. Evans and I . E.Willard, J. Amer. chem. SOC.78, 2908
[lo] W. Herr, F. Schmidt, and G. Stocklin, Z. analyt. Chem. 170,
301 (1959).
[ l l ] R. M . Lemmon, F. Mazzetti, F. L . Reynolds, and M . Calvin,
J. Amer. chem. SOC.78, 6414 (1956).
[12] A . A . Cordus and I . E. WiZZard,J. Amer. chem. SOC.79,4609
[13] F. Cacace and 1. UI Hag, Science (Washington) 131, 732
[14] L. H. Mason, H. J. Dutton, and L . R. Bair, J. Chromatogr. 2,
322 (1959).
[15] H . E. Dobbs, J. Chromatogr. 5, 32 (1961).
[16] H.W. Schurpenseel, Angew. Chem. 73,615 (1961).
Angew. Chem. internat. Edit. 1 Vol. 2 (1963) No. 9
In recent years, Geiger-Muller and particularly proportional counting tubes have been introduced for the
measurement of weak P-rays. Both types are applied as
continuous-flowcounting tubes in combination with gas
Emmett et al. [19] studied the behavior of [14C]-ethylene and
unlabelled propylene toward catalysts (micro-reaction
chamber connected to the column) by separating the products
by gas chromatography and mcdsuring their radioactivity
with a continuous-flow Geiger-Muller counting tube [20].
Cacace et al. [21] separated the acids produced by irradiation
of mixtures of pentane and W O z by gas chromktography
and oxidized the fractions to COz, which was then converted
into barium carbonate for counting with a stationary Geiger
counter. Yanovskii et al. [22] employed a counter provided
with a thin mica window. James used continuous-flow tubes
[23] or a continuous-flow proportional counting tube [24]
made of copper and brass with polyethylene insulators and
with a tungsten counting wire as anode and determined the
radioactivity of gas-chromatographic fractions after ignition
Extensive investigationsof continuous-flowproportional
counters have been published by Wolfgang and Rowland
[25]; metal counting tubes gave good results using
helium/methane mixtures. Precise working conditions
(plateau; counting and carrier gas ; resolving power;
interferences) are reported for internally silvered glass
counting tubes (volumes : 10,20, and 85 ml) fitted with a
tungsten counting wire [26]. For y-rays, Herr et al. [lo]
[17] F. Cacace, A. Guerino, and I. UI Hag, Ann. Chimica 50,915
(1960); Chem. Abstr. 55, 3269 (1961).
[I81 F. Cacace, Ann. Chimica 50, 931 (1960); Chem. Abstr. 55,
4338 (1961); F. Cacace and G . Cironni, Atti Accad. naz. Lincei,
Rend., C1. Sci. fisiche, mat. natur. 28, 865 (1960); Chem. Abstr.
55, 1342 (1961).
[19] R. I . Kokes, H,Tobin, and P . H . Emmett, J. Amer. chem. SOC.
77, 5860 (1955).
[20] I.T. Kummer, Nucleonics 3, 27 (1948).
[21] B. Aliprandiand F. Cacace, Gazz. chim. ital. 89,2268 (1959);
Chem. Abstr. 55, 6364i (1961).
[22] M . I. Yanovskii, D . S. Kapustin, and V . A . Nogotkov-Ryutin,
Dokl. Akad. Nauk USSR 9,391 (1956); Chem. Abstr. 53,7719b
[23] A.T. James in D. Click: Methods of Biochemical Analysis.
Interscience, New York 1960, Vol. X, p. 1.
[24] A.T. James and E . A . Piper, J. Chromatogr. 5 , 265 (1961).
[25] R. Wolfgang and F. S . Rowland, Analytic. Chem. 30, 903
[26] I. K . Lee, E. K . C. Lee, B. Musgrave, Yi-No0 Tang, I. W.
Root, and F. S. Rowland, Analytic. Chem. 34, 741 (1962).
used a continuous-flow counting tube provided with a
capillary flow tube in its counting chamber, or a continuous-flow proportional counting tube made of stainless
steel with teflon insulators and a tungsten counting
wire of 0.05 mm diameter; the latter tube showed a back
ground effect of 40 impulses/min at 210 "C [27]. Lieser et
al. [28] fitted a continuous-flow proportional counting
tube (glass tube with fused-in stainless-steel cathode and
a tungsten counting wire of 26 diameter) into the
thermostat of a gas chromatograph.
111. Methods of Measurement
Precise measurement of weak P-rays demands that all
samples be in the same chemical form. If the radioactivity of gas-chromatographic fractions is measured in a
counting tube without preliminary conversion into a
common chemical compound, the count obtained depends on both the quantity and composition of the
material [26]. According to Simon [2], there is no counting method available by which any and every 14C-labelled compound can be determined as such with good sensitivity and reproducibility. We therefore converted gaschromatographic fractions either into C02 by ignition
according to the methods of James [24] and Cucace[17],
or into methane as previously described by Zlatkis [29]
before measurement of their radioactivity. In this way,
there is no need to correct the results obtained since the
measurement of a variety of radioactive compounds
with different counting efficiencies is not involved [26].
As shown in Fig. 5 (see below), the plateau length, and
hence the counting efficiency, is constant for mixtures
containing 20-40 % methane and 80-60 % hydrogen.
Conversion of gas-chromatographic fractions into a common
compound also makes it superfluous to heat the counting
tube. Even with heated tubes, a certain amount of condensation and hence of contamination of the counting tube is
unavoidable. Raising the operating temperature gives rise
to plateau shifts with simultaneous flattening of the plateau
and increase in the background effect. Moreover, counting
tubes are not indifferent to the passage of inactive compounds
which often give rise to pseudosignals and uncontrollable
side effects.
For several reasons hydrogen is used most frequently as
carrier gas. In combination with a continuous-flow proportional counting tube, the most frequently used carrier
and counting gases have been argon [24], helium [26,27],
rare gases mixed with methane or propane, and methane
[28] or propane alone. Lieser et al. [28] found no plateau when using argon or hydrogen as counting gas. We
have concentrated on measurements using hydrogen as
carrier gas with admixture of methane or propane as
counting gas. The various experimental arrangements
are shown in Fig. 1.
1. Experimental Arrangement I
For exploratory measurements, combination of a continuous-flow cuvette with a methane continuous-flow
counter (Fig. 2) proved useful. The cuvette is separated
from the counting chamber by a thin window, so that
as a result of the spatial separation of carrier and counting gas, the results measured are relatively independent
of the nature of the carrier gas. The cuvette (Fig. 2) is
constructed in such a way that its base can be moderately heated. In order to obtain a favorable counting geometry, two windows on opposite walls of the cuvette
may be used. The relation between the volume of a radioactive standard gas and the measured number of impulses is linear. Experimental arrangement I is particularly advantageous for respiration and assimilation
studies with 14C-labelled gases.
Fig. 1. Block diagram for radio-gaschromatographic arrangements.
G = Gas chromatograph; F = Bridge current unit (Siemens and Halske,
Karlsruhe); K = Katharometer (Siemens); S1 = Compensating linear
recorder (Kompensograph, Siemens); RT,R2 = Reaction chambers;
T = Drying tube.
Measuring arrangement:
I = End-window continuous-flow cuvette (see Fig. 2).
I1 = Absorption of W02after combustion of gas-chromatographic
111 = Continuous-flow proportional counting tube (see Fig. 3).
C, = Counting tube; M = Mixing cell; A = Pre-amplifier (FH
524); CZ= Counting apparatus (scaler FH 49); H = High voltage
source, 5 kV (FH 505); I = Impulse timer (FH 449); Sz = Potentiometric recorder (FH 585).
F H indicates apparatus mannfacturd by Frieseke and Hoepfner,
Erlangen-Brnck (Germany).
[27] H . I . Ache, A.Thiemann, and W . Herr, Z. analyt. Chem. 181,
551 (1961).
[28] K. H. Lieser, H . Elias, and F. Sorg, Z. analyt. Chem. 191, 104
[29] A . Zlatkis and J. A . Ridgwuy, Nature (London) 182, 130
Fig. 2. End-window continuous-flow cuvette and continuous-flow
methane counter (FH 407) in combination with scaler FH 49.
1 = Inlet tube connection (Errneto tubing, 8 mm external diameter,
1 mm wall thickness); 2 = Base plate; 3 = Flow guide; 4 = Endwindow (gilded Hostaphan f o i l , surface coverage ca. 0.8 mg/cmz);
5 = Cathode; 6 = Counting wire loop (anode); 7 = Gasket; 8 = Screw
closure (flow control).
Angew. Chem. intomat. Edit.
Vol. 2 (1963) / No. 9
2. Experimental Arrangement I1
This arrangement permits discontinuous measurement
of W 0 2 .
A furnace, charged with copper oxide (R2 in Fig. l), is
attached to the outlet of the gas chromatograph [30]. The
ignition tube (quartz) is joined to the metallic outlet of the
gas chromatograph and to the water-absorption tube
(T in Fig. 1; magnesium perchlorate) by short connections of silicone rubber tubing. Each individual substance
revealed by the detector of the gas chromatograph can be
collected after combustion using an absorption tube filled
with soda-lime. The soda-lime tubes are easily interchangeable. The hold-up time of a substance between the detector
and the diversion cock depends on the volume of the tube
connections and on the rate of gas flow; this can be determined by means of a radiation detector. The soda-lime is
decomposed with 89 % phosphoric acid in a glass apparatus
[2,31,32] provided with a two-armed vessel [l], and the
14CO2 in the gas phase determined.
3. Experimental Arrangement I11
A continuous-flow proportional counting tube was tried
out for continuous measurements, especially with
hydrogen as carrier gas. Proportional counting tubes
have the advantage over Geiger-Muller tubes of much
shorter recovery times, with consequent reduction in
coincidence losses at high counting rates, and the ability
to detect primary electrons of differing ionizing power.
Furthermore, they generally have good, relatively long
plateaux [2,33-361 and are comparatively simple to set
up. This also applies to a counting tube we built ourselves following the constructional details given by
Curran [37].
passed centrally through an insulator plug of teflon by
means of a glass capillary (5) and fixed to a glass bead (6)
close to a second teflon insulator at the opposite end of the
tube. The glass bead prevents distortion of the electric field.
A thicker platinum wire (0.4mm in diameter) is fused into
the glass bead, passes centrally through the second teflon
insulator, and is attached to a tightening device (7). The
direction of gas flow is shown by arrows in Figure 3. Threeway stopcocks are provided at the inlet and outlet to allow
for both continuous or stationary measurements. The brass
pin soldered to the counting wire is connected with the preamplifier. In order to minimize the background effect, the
counting tube is maintained in the vertical position and is
covered by a lead shield (wall thickness: 3-10 mm). The
background effect depends on the carrier gas and the quantity of methane mixed with it. Characteristics and working
data are obtained using a standard radiation source [**I or
with a standard gas (14CO&O2).
Using hydrogen as carrier gas and (with admixture of
about 10% of methane) as counting gas, the background effect of the counting tube lies under 100 impulsesjmin and becomes less with increasing additions
of methane (3.6 kV; test source). Under the same conditions, the counting rate [38] is substantially constant for
methane contents of more than 30 %. The plateau curves
Fig. 3. Continuous-flow proportional counting tube.
A = Anode (counting wire): K = Cathode (counting tube wall);
1 = Plug for pre-amplifier; 2 = Screw thread for adaptor connection
for pre-amplifier; 3 = Counting wire anchor (soldered); 4 = End-cap;
5 = Passage for counting-wire through the Teflon insulator; 6 = Glass
bead; 7 = Counting-wire tension adjuster. All dimensions in mm.
The cathode is a brass cylinder with an internal diamater of
20 mm. The inner surface was finely ground, polished, and
occasionally gilded. As counting wire we used a platinumiridium wire [*] soldered onto a brass pin ( I ) which was
[30] F. Pregland H . Roth: Quantitative organische Mikroanalyse.
7th Ed., Springer, Vienna 1958.
[31] H. Simon, H . Daniel, and J. F. Klebe, Angew. Chem. 71,303
[32] F. Drawert and 0 . Bachmann, unpublished data.
[33] K. H. Schweer and E. Uhlmann, Atompraxis 7, 453 (1961).
1341 R. F. Glascock: Isotopic Gas Analysis for Biochemists.
Academic Press, New York 1954; Atomics 6,329 (1955).
[35] W. Bernstein and R. Ballentine, Rev. Sci. Instruments21, 158
[36] H . Kienitz and D . Riedel, 2.analyt. Chem. 179, 93 (1961).
[37] S. C. Curran in S. Fliigge: Handbuch der Physik. Springer,
Heidelberg 1958, Vol. 45, p. 174.
[*] Platinum-iridium (9O:lO) wire with a very constant diameter
of 0.08 mm from Heraeus, Hanau (Germany).
Angew. Chem. internat. Edit. / Vol. 2 (1963) 1 No. 9
Fig. 4. Plateau curve obtained for the continuous-flow proportional
counting tube shown in Figure 3 with a test source. The numbers on the
curves indicate the proportions of hydrogenlmethane in the mixture
Ordinates: Counting rate [countslminl
Abscissae: Counting wire potential IkVl
for selected hydrogenlmethane mixtures, measured
using a constant radiation source (standard source) are
shown in Figure 4. A plot of different hydrogen/methane ratios in the gas mixture against the plateau length
(Fig. 5 ) shows the optimum ratio. In the optimum range
(80-60 % H 2 ; 20-40 % CH4), there is a certain buffer
action against variation of the gas composition.
The geometry of the counting tube (internal volume,
62.8 ml) ensures a relatively long retention time for the
gases in the counting chamber. The flushing time is short
[**I 137Cs, 25 NC (5 mr/h).
[38] The counting rate is the number of impulses per minute.
Cf. Friedlrinder and Kennedy: Lehrbuch der Kern- und Radiochemie. Thiemig, Miinchen 1962, p. 218 et seq.
enough to ensure that sharp activity-maxima are recorded. The general detection limits for 14C are from
5x10-4 to 2x10-4 pC (using propane as counting gas:
2x10-5 pc).
2. In order to analyse fatty acids by gas chromatography,
these were converted into their methyl esters by treatment with boron trifluoride and methanol [43,44]. The
method was tested using [ICI-acetic acid to see how
much esterification already occurs at room temperature.
600 -
Fig. 5. Optimum operating conditions for the counting tube shown in
Figure 3.
Ordinate: Plateau length [V]
Abscissae: Composition of the counting gas
We used two procedures to convert the gas-chromatographic fractions into a common chemical compound:
a) Combustion of the organic material to COz and H2O
in a flow of helium over CuO at 700 OC (Pregl-Roth furnace as used for N analysis [30]; R2 in Fig. l). The water-absorption tube was packed with coarse Mg(C104)~
and was 10--20 cm long and 0.6 cm in diameter.
b) Cracking of the organic material at 420 "C in a flow
of hydrogen over Raney-nickel [39]. The metal crackingtube was 10 mm in external and 8 mm in internal diameter, and was 20 cm long (Rzin Fig. l).
IV. Applications of
Radio and Reaction Gas Chromatography
1. Using experimental arrangement I, preliminary investigations were made of the I4C-labelledalcohols arising from fermentations in the presence of added [14C]glutamic acid. The alcohols were extracted with ether/
pentane (70: 30), converted into olefins [3,40-421, and
then separated by gas chromatography. The distribution
of radioactivity among the individual alcohols produced
during the fermentation was then determined more accurately using experimental arrangement 11. The alcohols were concentrated by extraction with ethyl chloride
and separated by gas chromatography; the fractions
were ignited, and the C02 was absorbed in soda-lime.
Figure 6 shows the distribution of radioactivity.
[39] The catalyst was prepared from Raney-alloy (50% Al,
50 % Ni) from Messrs. Schuchardt, Munchen (Germany). Cf.
K . Wimmer in Houben- Wcyl: Methoden der Organischen Chemie.
4th Ed., Thieme, Stuttgart 1955, Vol. IV/2, p. 173.
[40] F. Drawert, Vitis, Ber. Rebenforsch. 2, 172 (1960).
[41] F. Drawert and K . H . Reuther, Chem. Ber. 93, 3066 (1960).
[42] F. Drawert and G . Kupfer, Hoppe-Seylers Z . physiol. Chem.
329,90 (1962).
50 %CHI
Fig. 6. Radio-gaschromatographicanalysis of 14C-labelledcompounds
from a yeast fermentation containing [W]-glutamic acid.
a) Gas chromatogram; b) Radiogram.
1 = Ethyl chloride; 2 = Low-boiling fraction (esters, aldehydes,
methanol); 3 = Ethanol/isopropanol; 4 = Isobutanol; 5 = Isoamyl
alcohol; 6 = n-Amy1 acetate; 7 High-boiling fraction.
The dotted lines indicate where the soda-lime COyabsorption tubes
were exchanged.
Separation column: 5.80 m Ermeto tube (8 mm external diameter,
1 mm wall thickness); Packing: dinonyl phthalate/sterchamol(20 100);
connected to an 80 cm tube packed with ethyl D-tartrate/kieselguhr
(65 mesh) (20
100); 80°C; 50 ml of Helmin; Radiation detector:
85 ml gas-filled counting tube (Bcrthold, Wildbad (Germany)) combined
with apparatus similar to Fig. 1.
Ordinatas: a) Recorder potential [mV]
h) Activity [ % of tolal activity]
Abscissae: Time [min]
For this aim, acetic acid was liberated from sodium
[14C]-acetate by treatment with 89 % phosphoric acid,
and an excess of boron trifluoride/methanolwas added.
The esterificationmixture was then either passed through
a heated reaction vessel (a brass tube 10 cm long and
8 mm in diameter) or passed directly into the separation
column. It was found that esterification was complete
only when the reaction chamber was heated at 180°C.
3. Saponification of K14CN to [14C]-formate [45]was
investigated to determine whether the reaction goes to
completion and whether there are volatile by-products.
Investigation of the formic acid liberated or of its methyl
ester (BF3/CH30H) by gas chromatography and radiochemistry showed that the formic acid is formed in
satisfactory purity.
4. In order to study transesterification, the following
experiment was carried out: 10 ml of methyl propionate,
10 ml of ethyl propionate, 0.2 ml of 98 % phosphoric
acid, and 0.94 mg of sodium [14C]-acetate [46] were refluxed for 30 min. Samples of the mixture were sub[43] F. Drawert, H. J. Kuhn, and A . Rapp, Hoppe-Seylers 2.physiol. Chem. 329, 84 (1962).
I441 L. D . Metcalfe and A. A. Schrnitz, Analytic. Chem. 33, 363
[45] D . G. Grant and H . S. Turner, Nature (London) 165, 153
(1950). We prepared K*4CN in 0.1% aqueous solution in a
sealed tube; cf. A . Murruy atid D. L. WiZZiam8:Organic Syntheses
with Isotopes. Interscience, New York 1958, Part I, p. 31.
1461 Sodium [W]-acetate (27.6 vC/mg) was supplied by the
Isotopenlaboratorium, Kern Torschungszentrum Karlsruhe (Germany).
Angcw. Cheni.iritrrnat. Edit.1 Vol. 2 (1963) 1 No. 9
mitted to gas chromatography, the separated fractions
were cracked to methane, and their radioactivity was
measured in a continuous-flow proportional counting
5. The 14CO2 assimilation or a vine was investigated
using experimental arrangement I11 without reaction
units. Gas samples were removed from the closed experimental chamber after equal time intervals and analysed by gas chromatography (Fig. 8).
6. The efficiency of hydrogenation catalysts at different
temperatures can be investigated by hydrogenating over
the catalyst in a reaction tube, separating the products
by gas chromatography, and determining their radioactivities after cracking to methane [4].
tube. Propionates were shown to transesterify with the
labelled acetic acid (Fig. 7).
Fig. 7. Transesterificationof esters of propionic acid with [14C]-acetic
a) Gas chromatogram; b) Radiogram; 1 = Methyl acetate; 2 = Ethyl
acetate; 3 = Methyl propionate; 4 = Propyl acetate; 5
propionate. Column: 5.20 m long, Packing: Polywax 2000/Kieselguhr
(65 mesh) (20
100); S O T ; 52 rnl of Hz/min. Radiation detector:
Arrangement 111; 17.4 ml of CHdmin; Hz:CHq = 3 : l ; 3.6 kV.
Ordinates: Recorder potential ImV]
Abscissae: Time [minl
Fig. 8. Course of assimilation of 14COz (initial concentration 0.913
yC/ml of COz) by a vine in a closed chamber..The gas mixture was
initially 13.7 1 of air and 20 ml of COz.
Ordinate: 14CO2 remaining in the reaction chamber [coun~s/min/ml]
Abscissa: Time [min]
Our investigations show that results of good reproducibility are obtained using hydrogen with methane or
helium with methane as carrier and as counting gases, if
the fractions separated by gas chromatography are converted into either methane or C02 prior to radiometry.
Increases in the sensitivity and precision of radiometry
in the gas phase effected corresponding reductions in
the amounts of labelled material required. Apart from
the often considerable savings in the costs of materials,
the radiation level in biological investigations is reduced.
Complete destruction of the xnalysed material can be
avoided by applying stream splitting and measurement
of the radioactivity in parallel.
The authors wish to thank Prof. Husfeld for his interest in
their worlc, which was carried out with the support of the
Bundesministerium fur Atomkcrnenergie and the Deutsche Forschurigsgemeinschaft.
Received, March Ilth, 1963
[A 306/108 IE]
German version: Angew. Chem. 75, 716 (1963)
Interaction of Triphenylmethyl Salts and
Quaternary Cycloimmomium Salts with Pyridine
and Aromatic and Aliphatic Amines
By Prof. Dr. G.Briegleb, Dr. W. Ruttiger, and Dr. W. Jung
Institut fur Physikalische Chemie der Universitat Wurzburg
Dedicated to ProJ Dr. F. Krohnke on the occasion of his
60. birthday
Compounds of the n-electron-donor type (D), e.g. pyridine,
add onto the triphenylmethyl cation (Tro), which is formed
from triphenylmethyl halides in solvents of high dielectric
constant without pronounced n-donor properties [I], and thus
bring about the disappearance of the characteristic visible
absorption bands (halochromic bands) at 435 and 415 mp
+D +
+D +
Angew. Chem. internat. Edit. / Vol. 2 (1963) / No.9
These processes are reversible equilibria that are dependent
on theconcentration and solvent, as has been shown by means
of spectroscopic and conductivity measurements with triphenylmethyl bromide [2]. The association constant K1 for
TrD@...XQ in 1,1,2,2-tetrachlorocthaneat 20 "C is between
1.1 x 105 and 6.6 x lo5 I/mole. Triphenylpyridinium bromide
can be isolated as a colorless salt [3].
In order to investigate the eleci ron-accepting capacity of
resonance-stabilized aromatic cations more fully, the behavior of the N-2,6-dichlorobenzylquinolinium cation
(2,6-DClBQN*) was studied. Optical measurements indicate
that aromatic amines, even N,N, N', N'-tetramethyl-p-phenylenediamine, exhibit electron donor-acceptor interaction only
at very high concentrations (revcrsible concentration-dependent red coloration, e.g. in CHCIj and CH3CN at Csalt
=3x 10-3 mole/l and carnine= 2x I 0 -1 --I .O mole/l). Aliphatic
amines (n-butylamine, ethanolamine, piperidine, triethylamine), on the other hand, yield completely reversible complexes very readily in CHC13 [3,4]. These are solvent- and
concentration-dependent equilibria which, on addition ofHC1,
CH30H, or HzO (i.e. on raising the dielectric constant) shift
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