In the investigation of 10-hydroxy chlorophylls a and b novel techniques included modification of chromatography and the use of fully-deuterated compounds isolated from fully-deuterated autotropic algae to determine the molecular structure of the chlorophylls

Katz, J. J.

Pennington, F. C.

Strain, H. H.

Svec, W. A.

English

NASA Technical Reports Server

December 1968
	 Brief 68-10409 
• j:	 AEC-NASA TECH BRIEF 
AEC-NASA Tech Briefs describe innovations resulting from the research and development program 
of the U.S. AEC or from AEC-NASA interagency efforts. They are issued to encourage commercial 
application. Tech Briefs are published by NASA and may be purchased, at 15 cents each, from 
the Clearinghouse for Federal Scientific and Technical Information, Springfield, Virginia 22151. 
The Preparation, Identification and Properties of Chlorophyll Derivatives 
A thorough investigation into the identity of chloro-
phyll derivatives, entitled "Preparation and Proper-
ties of 10-Hydroxy Chlorophylls a and b," has been 
conducted by F. C. Pennington, H. H. Strain, W. A. 
Svec, and J. J. Katz of Argonne National Laboratory, 
Argonne, Illinois. This investigation has been based 
upon several novel techniques that are of wide applica-
tion not only to pigment investigation but also to the 
study of many other substances. These methods 
include modifications of chromatography, and the use 
of fully-deuterated compounds isolated from fully-
deuterated autotropic algae: 
The enzymatic oxidation of chlorophyll a and b 
produced various green substances. The principal 
oxidation products, which were isolated by chroma-
tography, have been identified as 10-hydroxy chloro-
phylls. Identification techniques included chemical 
analysis, chromatography, nuclear magnetic reson-
ance, and infrared spectroscopy. These 10-hydroxy 
chlorophylls are also shown to be major products of 
the spontaneous oxidation of the chlorophylls in 
methanol, the so-called allomerization reaction. 
Similar allomerization of the full-deuterated 
chlorophylls, with H substituted at C-10 by normal 
exchange, provided the corresponding, fully-deuter-
ated oxidation products, with the —H at C-b 
replaced by —OH. These results, verified by n. m. r., 
provided critical evidence for the molecular structure 
of the oxidized chlorophylls and strong support for 
the structure of the methoxy lactones also formed in 
the allomerization reactions. 
In exploratory enzymatic hydroxylation experi-
ments, the formation of the oxidized chlorophylls in 
leaf material was followed by chromatography of the 
reaction products in columns of powdered sugar with
petroleum ether plus 0.5% n-propanol as the wash 
liquid. The extent of the reactions was indicated by 
the diminution of the chlorophyll zones and by the 
appearance of more-sorbed green substances. 
All allomerization experiments were conducted at a 
chlorophyll concentration of about 0.0067M. Chloro-
phyll a was dissolved with stirring in 20ml of spectro-
grade methanol and allowed to stand at room tem-
perature in the dark for three days. The solvent was 
then evaporated away. The solid residue was chro-
matographed on powdered sugar using petroleum 
ether with propanol as a developing solution. Two 
major fractions were obtained. The one obtained in 
smallest yield was identical to the enzymatically 
produced product, which was identified by n. m. r. 
and infrared spectra as 10-hydroxy chlorophyll a. The 
second major product had an n. m. r. spectrum con-
sistent with a 10-methoxy lactone structure. 
Allomerization of chlorophyll b also yielded two 
major products. One was the same as the enzymati-
cally produced 10-hydroxy chlorophyll b, the other 
was consistent with the 10-methoxy lactone formula-
tion. 
N. m. r. spectra were recorded on a Varian HA-100 
spectrometer. Infrared spectra were recorded on a 
Beckman lR-7. Absorption spectra in the visible and 
ultraviolet were recorded on aCary 14 spectrophotom-
eter. Molecular 'weights were measured on a Michro-
lab vapor phase osmometer. 
Notes: 
I. The report includes a complete discussion of the 
procedures and results, including sample spectra. 
2. Reference: Additional details may be found in 
Amer. Chem. Soc., 89, 3875, (1967)
(conti)ued overteal) 
This document was prepared under the sponsorship of the Atomic	 liability resulting from the use of the information contained in this 
Energy Commission and/or the National Aeronautics and Space document, or warrants that the use of any information, apparatus, 
Administration. Neither the United States Government nor any per- 	 method, or process disclosed in this document may not infringe pri-
son acting on behalf of the United States Government assumes any vately owned rights.
https://ntrs.nasa.gov/search.jsp?R=19680000409 2020-03-12T05:55:31+00:00Z
Inquiries concerning this innovation may be di-
rected to:
Office of Industrial Cooperation 
Argonne National Laboratory 
9700 South Cass Avenue 
Argonne, Illinois 60439 
Reference: B68-10409 
Source: F. C. Pennington, H. H. Strain, 
W. A. Svec, J. J. Katz, 
of the Chemistry Division, 
(ARG-10205)
Patent status: 
Inquiries about obtaining 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 68-10409	 Category 03


The preparation, identification and properties of chlorophyll derivatives

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