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Automatic Activity Measurements during the Separation of Radioactive Compounds by Thin-Layer Chromatography.

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Automatic Activity Measurements during the Separation of Radioactive
Compounds by Thin-Layer Chromatography
A method was developed for continuous automatic measurement of the radioactivity distribution of weak @-emitterson open columns,for example, thin-layerchromatograms.It may be
used for analytical separations, as well as for preparative separations on Q semi-micro scale.
During the measurements the substances are not contaminatedby addition ojother materials.
Since its rediscovery by Stahl [l-41, thin-layer chromatography has been used principally for the solution of
analytical problems [4a]. This paper will demonstrate
the utility of this method for checking the purity of
labelled compounds and for the preparative separation
of substances labelled with tritium by the gas exchange
procedure of Wilzbach [5,6]. In addition, chemically
synthetized compounds labelled with radioactive elements that emit soft p-rays, for example 14C-labelled
steroids or 35S-labelled sulfonamides, can be measured
directly and separated from impurities on the thin-layer
The aperture has no projecting parts, hence, the chromatogram can be guided along on a carriage situated
directly below the aperture slit (distance from the surface
of the layer to the interior of the counter tube: 1-3 mm).
Formerly, paper chromatography, gas chromatography, and
partition processes were used to check the purity of such compounds. In the latter two cases,however, accurate measurement
of the radioactivity requires complicated apparatus. In some
cases, paper chromatography has also been used for the
preparative separation of labelled compounds; however, this
procedure is not well adapted for this purpose owing to the
low capacity of the paper sheets, especially for lipophilic
substances [61.
Fig. 1. Gas flow counter with an extremely flat aperture plate (B) for
measurement of weak @-emitterson thin-layer chromatograms. The
aperture plate is attached to the counter tube without projecting parts.
a) Analytical Methods
Since thin-layer chromatography is essentially a separation on an open column, it offers the possibility of
direct measurement of radioactive substances. For this
purpose, a proportional gas-flow counter [*I tube with
an extremely flat aperture plate was developed (Fig. 1).
[ l ] E. Stahl, Chemiker-Ztg. 82, 323 (1958).
[2] E. Stahl, Arch. Pharrnaz. Ber. dtsch. pharrnaz. Ges. 292, 41 1
[3] E. Stahl, Angew. Chem. 73, 646 (1961).
[4] Diinnschicht-Chromatographie. Brochure of E. Merck,
Darmstadt (Germany) 1960.
[4 a] Compare also K.Randeruth : Diinnschicht-Chromatographie.
Verlag Chemie, Weinheim/Bergstr. 1962.
[5] K. E. Wilrbuch, J. Amer. chem. SOC.79, 1013 (1957).
[6] M . Wenrel and E. Schulze: Tritium-Markierung. W. de
Gruyter, Berlin 1962.
[*I We wish to thank Mr. Bust, precision mechanic at the Phyaiologisches Institut der Freien Universitat, for the manufacture
of the counter tube (patent applied for on December 7th, 1961).
5 80
Z = counter wire (30 p diameter). The measurements are given in mm.
With this arrangement, soft p-ray emitters - including
tritium - can be measured with high radiation yields.
The distribution of the radioactivity is recorded via a
cathode follower (connected to the counter tube) and a
scaling unit with a synchronized recorder [**I.
Calibration curves for 14C and 3H were prepared using
thin-layer plates with a layer 0.25 mm thick of Silica
Gel G (E. Merck) as layer material. Testosterone labelled with 14C in the 4-position (specific activity 0.016
mc/mg), and estradiol labelled with 3H in the 6- and 7positions (specific activity 1.3 mc/mg> were used as
standard substances. Fig. 2 shows the calibration curve
for 3H. With the lowest activity measured, the error is
naturally at a maximum; e.g. for 0.0oO1 pc 14CCM = f
8 % and for 0.1 pc 3H cM= f 9.3 %. For activities
[* *] Manufactured by Laboratorium Prof. Berthold, Wildbad/
Schwarzwald (Germany).
Angew. Chem. internat. Edit. Vol. 1 (1962) No. 11
greater than 0.1 pc for 3H, and 0.001 pc for
error is less than 5 3 %.
b) Preparative Separations
In labelling by gas exchange according to the method of
Wilzbach, sometimes radioactive impurities appear,
which are negligible in weight, although their specific
activities are greater than that of the main product.
These by-products (e.g. compounds where double
bonds have been hydrogenated with tritium) can often
be removed only with difficulty by reprecipitation or
recrystallization. Purification is best carried out by
recrystallization and chromatography. The separation
of impurities from a nucleoside labelled with 3H according to Wilzbach by thin-layer chromatography [8] has
proved satisfactory.
In combination with the present method of rneasurement, preparative thin - layer chromatography offers the
possibility of combining easy measurement with sharp
separations. A slight increase in the thickness of the
layer does not alter the excellent separating properties
of normal thin-layer plates and permits separations on a
preparative scale [9]. Examples are given in Table 1 and
Fig. 3. The fact that the counting efficiency decreases
with increasing layer thickness is not important, since,
in the separation of mg quantities, large activities are to
be expected anyway.
0 0714
Fig. 2. Dependence of the radioactivity measured (average of
10 measurements) on the amount of radioactivity applied. Layer
material: Silica Gel G (E.Merck). Layer thickness: 0.25 mm.
Developing agent: cyclohexane/ethyl acetate (90:60, by volume).
Increasing amounts of a methanolic solution of estra-6.7-diol-3Hz were
initially applied as spots at intervals of approximately 15 mm. Ordinate:
countslmin. at the activity maximum. Abscissa: Upper numbers:
radioactivity applied [gcl; lower numbers: amount of substance
applied C g 1 .
If several substances of different Rfvalues are present
on a thin-layer chromatogram, the determination of
their relative radioactivities is even more accurate, since
Table 1. Compounds separated by thin-layer chromatography on a preparative scale. Layer material: Kieselgel 0, Merck (Germany)
Substance applied
N-Methylthiourea-JSS [++I
Substance isolated
[‘I Measurement of the radioactivity of an infinitely thin layer (I
Androstan-17 P-ol-3-one-sH
1-Methyl-A1-androsten- 17P-01-3-one-3H
1 ~-Methyl-androstan-l7~-ol-3-one-3H
y/sq. cm.) of pure substances
lwlme.1 [*I
3 “*I
2.7 I**]
1.1 “*I
5.7 [*‘I
on a plaochet, using a methane flow counter.
(FH 407) with a 44 %efficiency and an average error of f 6 % [6,10].
In the elution, small amounts of carrier substance were added.
[+I Labelled by the Wilzbach procedure.
[++IHighly impure synthesis residues.
Ial Cyclohexane/ethyl acetate (3: 2 v/v).
tbl Butanol saturated with conc. NH3.
changes in the distance between plate and counter tube
aperture are more readily obviated in a single measurement using a single plate than when different plates are
inserted in sequence. With tritium, a small change in the
plate-counter distance causes an especially large error,
because of the low average range of its P-particles in air
(approximately 4.5 mm). With layer thicknesses of 0.25
mm, the efficiency of the measurement is approximately
40 % for 1 4 C and approximately 0.3 % for tritium. For
tritium this is less than the efficiency attainable by the
use of liquid scintillation spectrometry described by
Scharpenseel[7], but the simplicity of the present
method compensates for this disadvantage, especially
since the sample is not contaminated in the measurement and can be recovered.
‘71 H. W. Schurpenseel et al., 2. analyt. Chem. I80, 81 (1961);
i82, 1 (1961); Angew. Chem. 73,615 (1961).
4ngew. Chem. internat. Edit.
Vol. I (1962)1 No. II
Fig. 3. Chromatography of crude Al-androsten-17P-ol-3-one-3H
Silica Gel G (E.Merck). Layer thickness: 0.9 mm. Developing agent:
cyclohexanelethyl acetate (90: 60, vtv).
O n scratching off zones A and B no mixtures of A and B were obtained,
as was shown by test-chromatograms. That the radioactivity between
A and B does not return to zero is partly due to the eeometry of the flow
S = start
F =front
A = androstan-17P-ol-3-one-3H
B = A1-androsten-17P-ol-3-onc3H
Ordinate: countslmin.
[8] M . Wenzeland H.P.Kortge,Naturwissenschaften48,431(1961).
[9] P. Schrniuleck succeeded in separating 250mg. of a lipoid
ester mixture on a silicagel layer 20 cm2 in size and cu. 1 mrn.
thick (personal communication).
[lo] J. Rydberg, Acta chem. scand. 12, 399 (1958).
Material labelled with tritium by the Wilzbach procedure [6] can be applied without preliminary purification. Only loosely attached tritium is previously
removed. After chromatography a n d measurement of
the radioactivity, the width of radioactive band given by
the recorder are transcribed onto the chromatogram,
and the silica gel of the different bands is scraped off;
the sample is eluted and worked up. Then an aliquot
portion is tested for radioactive homogeneity on a
further chromatogram and purified, if necessary, by
sublimation or recrystallization.
The distribution of radioactivity can also be determined by
autoradiography. For the determination of tritium by this
procedure, scintillators such as 1,4-diphenylbenzene or 1,4di-(5-phenyloxazol-2-yl)benzene are admixed with the developing liquid. Subsequent spraying of the chromatogram
with a solution of scintillator is also possible [ll]. The
darkening of superposed film plate is evaluated photometrically. This method of recording, however, requires considerable time.
drawn slowly and uniformly over the glass plate. The plates
are dried for one hour at 120 "C, and then stored in a desiccator over calcium chloride.
Fig. 4. Apparatus for coating glass plates for thin-layer
chromatography. The bottom plate (A) and the coating apparatus ( S )
are made of poly(viny1 chloride). The measurements are given in mm.
See text for details.
G = glass plate, 4 mm. thick
B = layer material. V = plastic ledge
Experimental Procedure
Commercial counting apparatus supplied by Frieseke and
HBpfner Co. (FH 49) was used as scaler and H.T.-unit,
together with cathode follower FH 484; the gas flow counter
was attached directly to the latter.
The counter tube potential was 2100 volts; methane was used
as counter gas. The flow velocity was one bubble per second;
the background counting rate attained in this standardization
not more than 30 counts/min. For the measurement of the
radioactivity distribution, the thin-layer plate was placed
on a small carriage moving on a rail. By means of a waterlevel and screw adjustment, the carriage with the plate is
adjusted to the horizontal. The carriage was drawn along by
means of a cord coiled around the conveyor cylinder of a
paper chromatogram scanning apparatus F H 552 (Frieseke
and HBpfner), and the activity recorded by a synchronized
recorder. The distance from the surface of the layer to the
counter tube aperture was not greater than 1 mm. Stepwise
measurement by means of a preset count device and a time
recorder is also possible.
In order to prepare uniformly coated glass plates, the coating
apparatus shown in Fig. 4 was used. By placing aluminum
foil on the poly(viny1 chloride) support to the right and left
of the glass plate, the thickness of the layer can be Varied.
To make the coating, the sliding bar is placed in front of the
glass plate, the slurry is poured on, and the sliding bar is
[l I] P. Sehmialeck, personal communication.
The substances to be chromatographed were dissolved in
enough methanol or benzene so that a strip of about 3 cm
broad could be applied across the whole width of the plate.
In order not to disturb the layer, the solution was applied
with a capillary in drops, at intervals of 3 mm. The capillary
opening should be at least 0.6 to 0.8 mm in diameter in order
to avoid clogging. Ten to forty mg of the substance were
After development, the chromatograms were dried at 60°C
(in some cases in a vacuum drying oven), and the radioactivity was measured as described. The radioactive zones
were marked on the basis of the distribution of radioactivity
given by the recorder, and were scraped off. Instead, the nonradioactive zones can also be scraped off, and the radioactive substance can be eluted with a solvent from the
remaining bands. It is frequently expedient to measure the
right and the left edge of the plate in order to determine
whether the bands have run uniformly.
For elution of steroids, ethyl acetate which had been washed
with water and dried over sodium sulfate was used. After
concentration of the solvent in vacuo, frequently more of the
crude material was obtained than had been added (calcium
sulfate from binder?).
This possibly can be avoided by pretreating the layer
material with the eluting solvent.
We wish to thank Miss D . Muchow and Miss G . Fischer
for their interested and diligent cooperation.
Received, April 30th, 1962
Angew. Chem. internat. Edit.
1 Vol. I
[A 230/50 IEl
(1962) No. 11
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measurements, automatic, separating, compounds, radioactive, thin, layer, activity, chromatography
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