In Dashek, William V., ed.

Methods in plant biochemistry and molecular

biology. Boca Raton, FL: CRC Press: pp. 107-113. Chapter 9.1997.

Isolation, Separation, and

Characterization of
Organic Acids*
William V. Dashek and Jessie A. Micales

Contents

9.1 Overview — Chemistry, Occurrence, and Function

Many plants contain a variety of free acids which are colorless compounds soluble in water, ethanol,
and ether. but insoluble in the nonpolar solvents benzene or petroleum ether. These acids are weakly
acidic and can accumulate in the cellular vacuoles of certain plants. Some of the acids are compo-
nents of the citric acid cycle, while others are intermediates in the pathway leading from carbohy-
1
drates to either aromatic compounds or isoprenoid derivatives . Figure 9.1 presents the chemistry
of certain of these organic acids. Table 9.1 summarizes their possible functions. Oxalic acid appears
to be of marked significance in fungal-induced wood decay. i.e.. the acid could serve as a proton
donor for oxidative (Fenton's reaction) cleavages of wood cellulose.2,3

* The Forest Products Laboratory is maintained in cooperation with the University of Wisconsin. The chapter was co-
authored by J. A. Micales. a U.S. government employee, on official time and is, therefore, in the public domain and not
subject to copyright.
Isolation, Separation, and Characterization of Organic Acids

9.2 Protocols
9.2.1 Isolation
This chapter is concerned with the isolation, purification, and assay of organic acids. Whereas
Protocol 9.1 presents an acidified, ether extraction procedure for isolating organic acids from either
heat-dried or freeze-dried plant material, Protocol 9.2 summarizes an ion exchange chromatographic
protocol for extracting the acids.

9.2.2 Separation and Preparation Techniques and Characterization

Subsequent to extraction, organic acids can be separated and detected via a variety of techniques.
Table 9.2 lists the references for procedures that separate the acids in ether extracts. A gas
chromatographic separation of organic acids is depicted in Figure 9.2 and an HPLC elution profile
of seven organic acids can be seen in Figure 9.3 For rigorous identification of organic acids, gas
chromatography can be coupled to mass spectrometry.
In addition to the above techniques, paper and thin-layer chromatographies have been employed
to separate certain organic acids. Table 9.3 summarizes the conditions for paper and thin-layer
chromatographies. If Whatman® 3MM chromatography paper is employed, paper chromatography
can serve as a preparative procedure, partially purifying as much as 100 mg of organic acids.
 L

Methods in Plant Biochemistry and Molecular Biology

Isolation, Separation, and Characterization of Organic Acids
 Methods in Plant Biochemistry and Molecular Biology

Heat-dried or freeze-fried plant material.

Make strongly acidic (pH 1.0) with H 2SO4and extract up to several days with peroxide-free ether.

Ether extract contains free acids.

Transfer from ether to water by shaking with sodium hydroxide.

Remove sodium sulfate by adjusting aqueous solution to pH 1 H2SO4 two volumes of ethanol maintaining overnight in the cold.

Separate organic acids by filtation from precipitated sodium sulfate.

Note: (1) drying with heat can remove volatile acids, destroy keto acids, and cause ester formation: (2) volatile acids or esters
may be lost during freeze drying. Neutralizing the plant marerial before drying yields acids as nonvolatile salts.

Protocol 9.1
Isolation of organic acids by acidification and subsequent partitioning of plant extracts with ether. (Adapted from Robinson,
T., The Organic Constituents of Higher Plants, Their Chemistry and Interrelationships. Cordus Press. North Amherst, MA.
1980, 43-49. With permission.)

Prepare an aqueous extract of plant acids

Pass through a column of weakly basic anion exchange in the hydroxide form (cast separate several hundred milligrams of mixed
organic acids with 10 g resin).

Wash column.

Elute free acids with 0.1 N HCl (order of elution — mono, di, and tricarboxylic acids).
Note if a strongly basic anion exchange resin is utilized in the hydroxide form, sugars may form sugar acids.

Protocol 9.2
Anion exchange chromatography protocol for extracting organic acids. (Adapted from Robinson, T., The Organic Constit-
uents of Higher Plants, Their Chemistry and Interrelationships. Cordus Press, ,North Amherst, MA. 1980, 43-49. With
permission.)

References
Isolation, Separation, and Characterization of Organic Acids