Methods for isolation of low concentration pigments of the cocklebur species are described. The methods entail two step chromatography so that the different sorption properties of the various pigments in varying column parameters can be utilized. Columnar and thin layer methods are compared. Many conditions influence separability of the chloroplasts

Grandolfo, M.

Sherma, J.

Strain, H. H.

English

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September 1969	 Brief 69-10425 
,V co
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the Clearinghouse for Federal Scientific and Technical Information, Springfield, Virginia 22151. 
Comparative Chromatography of Chioroplast Pigment 
S
Methods for isolation of low-concentration pig-
ments of the cocklebur species are described (1). The 
methods entail two-step chromatography so that the 
different sorption properties of the various pigments, 
in varying column parameters, can be utilized. 
There are few systematic and reliable chromato-
graphic procedures for complete resolution of the leaf 
pigments. Usually the chromatographic separations 
must be carried out empirically with various chro-
matographic systems until no further resolution is 
possible. 
In order to provide more-comparable results from 
chromatography of the leaf pigments, separations 
with various adsorbents and wash liquids were stud-
ied. Columnar methods and thin-layer methods were 
employed under comparable conditions, and the 
separations were followed in relation to the loading 
of the systems with the pigment mixtures. The pur-
poses included direct comparison of thin-layer and 
columnar techniques, determination of whether or 
not the major pigments may be resolved further, 
and confirmation of identities of the minor pigments. 
For the present, the more-promising chromatographic 
systems were utilized for separation of the pigments 
of a single species (Xanthium, or cocklebur) grown 
under reproducible greenhouse conditions. 
Results indicate that many conditions influence 
separability of the chloroplast pigments by chroma-
tography. These conditions include the nature and 
treatment of the sorbent, the solvent or wash liquid, 
the form in which the sorbent is employed, and the 
loading of the sorbent with the mixture. In the form 
of columns or thin layers, a given adsorbent provides 
reproducible capacity and selectivity for resolution 
of chloroplast pigments only when the same wash 
liquid is employed.
Various adsorbents exhibit different capacities and 
selectivities for the pigments, and they often provide 
different sequences of the separated pigments. Some 
adsorbents such as magnesia show variation in 
capacity and selectivity when made into thin layers 
from aqueous slurries. Very finely divided adsorbents, 
such as activated magnesia, may be employed both in 
columns and in thin layers if mixed with a nonsorp-
tive filter aid. 
With all the chromatographic systems tested, the 
lower the initial loading with the mixture, the 
greater the resolution. Conversely, the higher the 
loading, the poorer the resolution. With either under-
loading or overloading, minor constituents may not 
be detectable. 
Readsorption of large fractions of the pigments, 
obtained from one chromatographic system, improves 
separation and detection of the minor leaf pigments. 
Readorption with a second chromatographic system 
permits the separation of pigments such as lutein 
and zeaxanthin that were inseparable in the first 
system. For these chromatographic and rechromat-
ographic procedures, columns are much more adapt-
able than thin layers and sheets of the adsorbent. 
For preparation of quantities of the leaf pigments, 
columnar methods are more convenient than thin-
layer and paper techniques. Separations are faster 
and more adaptable in columns than in thin layers 
or in paper. 
The major pigments of cocklebur proved to be 
chlorophylls a and b, neoxanthin, violaxanthin, 
lutein, and fl-carotene. The minor pigments are 
cryptoxanthin, zeaxanthin, and traces of a-carotene, 
lutein epoxide, and (possible minute traces) zeaxan-
thin monoepoxide. There was no evidence that any 
(continued overleaf) 
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of these pigments could be further resolved chro-
matographically. 
Reference: 
I. H. H. Strain, J. Sherma, and M. Grandolfo, Com-
parative Chromatography of the Chioroplast Pig-
ment (Argonne National Laboratory, July 1967). 
Notes: 
1. These methods may interest researchers in plant 
pigments, photosynthesis, or energy-transfer. 
2. Inquiries maybe directed to: 
Office of Industrial Cooperation 
Argonne National Laboratory 
9700 South Cass Avenue 
Argonne, Illinois 60439 
Reference: B69-10425 
Source: H. H. Strain, J. Sherma, 
and M. Grandolfo
Chemistry Division
('ARG- 104 15)
Patent status: 
Inquiries concerning rights for commercial use of 
this innovation may be made to: 
Mr. George H. Lee, Chief 
Chicago Patent Group 
U.S. Atomic Energy Commission 
Chicago Operations Office 
9800 South Cass Avenue 
Argonne, Illinois 60439
. 
Brief 69-10425	 Category 03, 04


Comparative chromatography of chloroplast pigment

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