Radioactive-Tracer Techniques 
in Paper Chromatography 
Associating one or more radioisotopes with one or more substances separated 
on a paper chromatogram offers the possibility of quantitative, 
as well as qualitative, component analysis. 
A simple automatic device is used to 
scan the paper radiometrically and locate the labeled substance 
By F. P. W. WINTERINGHAM,* A. HARRISON, and R. G. BRIDGES 
Department of Scientific and Industrial Research 
Pest Infestation Laboratory, Slough, England 
THE USE of paper chromatography as 
a micro-analytical tool is well estab- 
lished. In many instances, paper 
chromatography is capable of separat- 
ing substances in amounts below the 
limits of chemical detection. In other 
cases, the resolving powers of pape1 
chromatography may be undiscovered 
or unexploited because of the lack of 
suitable chemical methods of detection. 
The application of radiotracer tech- 
niques to paper chromatography may 
permit the separated components not 
only to be located and characterized 
but to be estimated quantitatively. 
In the combined technique, the prin- 
ciple is to associate one or more suitable 
radioisotopes with one or more of the 
components of the mixture, either 
before or after chromatography. The 
components separated on the chro- 
matogram are then located and esti- 
mated by their associated radioactivity. 
In some cases, the radioactivity may 
be used for characterizing or identify- 
ing a particular component when its 
association may be made to depend 
upon a specific chemical reaction. 
Locating and estimating the radio- 
* Fellow of the Commonwealth Fund of 
New York. Present Appress: Agricultural 
Experiment Station, University of Califor- 
nia, Berkeley, Calif. 
176 
activity is done by systematically 
scanning the paper chromatogram with 
a Geiger-Miiller counter, for example, 
or by clamping whole or part of the 
paper chromatogram to a photographic 
plate in the dark. In the photographic 
method, the radioactive zones, if suffi- 
ciently intense, will have autoradio- 
graphed themselves. 
The scanning method is capable of 
great sensitivity and is quantitative, 
while the photographic method is some- 
times more convenient in certain 
qualitative work. 
Preparing the Chromatograms 
The association of a suitable radio- 
isotope with one or more bands of the 
resolved components of the paper 
chromatogram may be brought about 
in any one of three ways. 
1. Labeling of mixture before pa- 
per chromatography. The mixture 
for chromatography may already con- 
tain one or more labeled components as 
in tracer experiments. For example, 
Benson et al. (1) studied the path of 
radiocarbon in the photosynthetic 
incorporation of this element by respir- 
ing plant cells. Labeled intermediates 
were located and estimated on paper 
chromatograms by autoradiographic 
and counting techniques. 
The metabolism of a radioactive 
bromine analog of DDT absorbed by 
susceptible and DDT-resistant house- 
flies was studied (2, 3) by the combined 
techniques in which paper chromato- 
grams were scanned by the methods 
to be described. 
Keston, Udenfriend, and Levy (4) 
analyzed an unlabeled mixture of 
amino acids by treating the mixture 
with a labeled reagent before applying 
paper chromatography. The paper 
chromatograms were cut up into small 
sections which were mounted in turn 
below a Geiger-Miiller tube and the 
measured rate of count plotted against 
distance along the strip. In this way 
they were able to determine quantita- 
tively glutamic acid, serine, glycine, 
and alanine in a mixture of amino acids. 
Another example in which the mix- 
ture already contained labeled com- 
ponents is illustrated by radiochro- 
matogram A in the chart opposite. A 
paper chromatogram was run on a pro- 
tein hydrolysate prepared from wheat 
grown on a solution containing S*°. 
It was scanned radiometrically and the 
radiochromatogram shown was ob- 
tained by plotting the S** activity 
against the R; value.t The two peaks 
correspond to the cystine and methio- 
nine biosynthesized in the plant. 
