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United States Department of Agriculture

Agricultural Research Service

Survey of Phenolic Compounds Produced in Citrus
Experimental Methods

General Phenolic Analysis

The chromatographic peaks present in each sample were evaluated spectrophometrically to determine the class of phenolic compounds—flavonoid, coumarin/cinnamic acid, or psoralin—to which the peak belonged. This information was used to construct table 2, which summarizes the overall phenolic composition for each species examined.

Data Collection. For each discernible phenolic peak on the HPLC run trace at 285 nm examined, a spectrum and a relative retention time was obtained, and then it was classified as either one of the three classes of phenolics or an unknown. During the HPLC run, diode array absorption spectra were taken of each detectable peak at three points as it passed through the detector: up-slope, apex, and down-slope. This information was retrieved from the computer, and the peak apex spectrum was printed out on the plotter for each peak. The up- and down-slope spectra were examined if there was any ambiguity in assigning peaks to classes.

Purified standards, either isolated in this laboratory or purchased from chemical supply companies, were used to determine both a characteristic absorbance spectrum for each of the four classes of phenolics examined and their extinction coefficients at 285 nm. The characteristic spectra of standards of these compounds are shown in figure 2. Peaks from sample runs were then assigned a classification based on the following spectral criteria:

Flavanones. Flavanones are identifiable by a characteristic absorbance maximum at 280–288 nm and a broad shoulder at about 330 nm (figure 2). These features, as well as the large number of known spectra, were used to identify this class of compounds.

Flavones and Flavonols. Flavones and flavonols exhibit high absorbances in both the 240–270 nm region and the 320–380 nm region. The flavonones and flavonols have very similar spectra and it is difficult to differentiate between the two types of compounds based on spectra alone, so they are classified together here.

Coumarins and Cinnamic Acids. As a class, coumarins are identified on the basis of their characteristically large absorbance peak occurring in the region of 310–330 nm (figure 2). Cinnamic acid derivatives (including p–coumaric acid, caffeic acid, and ferulic acid), although they are distinct compounds and somewhat distant biosynthetically, are included in this class because of the similarity of their absorbance spectra to that of the coumarins. Cinnamic acid derivatives are present in relatively high concentrations in mature grapefruit (Maier and Metzler 1967).

Psoralens. Psoralens (furocoumarins) have been isolated from many citrus species. They are identifiable by absorbance maxima in the region of 240–265 nm, followed by a precipitous dip in the region of 270–290 nm, and a broader, smaller peak in the region of 320–350 nm (figure 2).

Unknown. Unknowns comprised those compounds that absorbed light at 285 nm but were either obvious mixtures of compounds (as determined by the examination of the up-slope and down-slope spectra of these peaks) or had a spectrum that did not match that of the four general classes outlined above.

After the peaks in a given sample were identified, the relative concentrations of each class in the sample were determined. The concentrations were ascertained from peak areas printed out by the computer in milliabsorbance units, using the extinction coefficients determined from the standards.

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United States Department of Agriculture
Agricultural Research Service

The material on this page is in the public domain.

Original posting: April 1, 1999.

Last Modified: 8/13/2016
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