Plant Physiol.

(1971) 48, 228

Short Communication

A Simple Method for Synthesis of Ferric

Ethylenediaminetetraacetate in a Pure and Stable Form1
Received for publication February 11,1971

SAMUEL I. BEALE2
Departmentt of Botanical Sciences, Uniiversity of California, Los Anigeles, Californwia 90024
Following the introduction of EDTA as a chelator of metals
in the water culture of plants (5), it has been used extensively
as a carrier for iron. A commonly used method for the preparation of FeEDTA is based on the air-oxidation of a solution
of FeSO4, KOH, and EDTA (6). More recently, the method
has been modified so as to substitute chloride for sulfate and
ammonium for potassium, for use in physiological experiments
where potassium or sulfate would interfere. This method is
still based on air-oxidation of the ferrous salt (7). I have been
using a method which I believe is simpler and more direct,
and which has the added advantages that no extraneous ions
are introduced, and the product is obtained in an indefinitely
stable form.
The basic method is as follows: EDTA is placed in a flask
with an equimolar amount of Fe(OH)3 or a half-equimolar
amount of Fe2O,, and one liter of deionized water is added for
each mole of EDTA. The mixture is autoclaved overnight,
cooled to room temperature, filtered, and lyophilized. The
green powder product is stored in a brown glass jar.
In a typical run, 0.25 mole of FeEDTA was prepared as
follows: 19.96 g of Fe,03 (Baker and Adamson reagent grade,
code 1741), 73.06 g of EDTA (Fisher Certified Reagent grade,
catalog No. E-478) and 250 ml of deionized water was placed
in a one-liter Erlenmeyer flask, autoclaved for 16 hr at 122 C,
cooled to room temperature, and filtered through Whatman
No. 1 paper on a Buchner funnel. A small amount of orange
colored residue (364 mg) could have resulted from incomplete
reaction or from a slight excess of Fe2O, in the reaction mix'Supported in part by a National Institutes of Health Predoctoral Fellowship No. GM-28419.
'Present address: Department of Biology, Brookhaven National
Laboratory, Upton, N. Y. 11973.

ture. The pH of the coffee-brown filtrate was 3.5. The filtrate

was transferred to a 1200-ml Virtis lyophilizing flask, shellfrozen, and lyophilized for 48 hr. The 86.09 g of green powder
collected represented a 98% recovery of FeEDTA, or a 93%
recovery as the monohydrate (4).
A batch of FeEDTA prepared in the above way has been
stored in a brown jar for over 5 years, during which portions
have been used successfully in growth media for Chlorella
vulgaris (1, 2), Acetobacter peroxidans, Euglena gracilis, Anabena variabilis, Anacystis nidutlans, and Prototheca zopfii (unpublished).
The FeEDTA is readily soluble in water. An 85 mm (5 g/l
Fe) solution was stable for at least a year when stored at room
temperature in a brown bottle. Solutions did not support the
growth of mold, as did solutions containing other ions. Also,
there was much less tendency to decompose upon exposure to
direct sunlight (3). Perhaps this was due to a difference in pH,
or the absence of extraneous ions.
LITERATURE CITED
1. BEALE, S. I. 1970. The biosyntlhesis of 5-amino-levulinic acid in Chllorella.
Plant Physiol. 45: 504-506.
2. BEALE, S. I. AND D. APPLEMAN. 1971. Chlorophyll syntlhesis in Chlorella. Regulation by degree of light limitation of growth. Plant Physiol. 47: 230-235.
3. HILL-COTTINGHAM, D. G. 1935. Photosensitivity of iron chelates. Nature 175:
347-348.
4. HOARD, J. L., M1. LIND, AN-D J. V. SILVERTON. 1961. The stereochemistry of the
ethy-lenedliaminetetraacetatoaquoferrate (III) ion. J. Amer. Chem. Soc. 83:

2770-2771.
5. HUTNER, S. H., L. PROVASOLI, A. SCHATZ, AN-D C. P. HASKINS. 1950. Some approaches to the study of the role of metals in the metabolism of mircoorganisms. Proc. Amer. Phil. Soc. 94: 152-170.
6. JACOBSON, L. 1951. Maintenance of iron supply in nutrient solutions by a single
addition of ferric potassium ethylenediamine tetraacetate. Plant Physiol. 26:
411-413.
7. STEINER, A. A. AND H. VAN WINDEN. 1970. Recipe for ferric salts of ethylene(lalaminetetraacetic acid. Plant Physiol. 46: 862-863.

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