A Standardized TLC Analysis of β-Orcinol Depsidones

Abstract

Many p-orcinol depsidones with low Rf values in the three stan- dardized TLC solvents commonly used in lichenology are better resolved by a solvent system of toluenelethyl acetate/formic acid. Standardized chromatographic data and examples of specific problems resolved by one- and two-dimensional TLC with this solvent system are given. Chemical analyses are reported for 25 lichen species from 17 genera. A vexing problem in the identification of lichen products by thin-layer chromatography (TLC) has been the separation of some 35 low-Rf, p-orcinol compounds many of which are still unknowns. In the three standardized solvent systems (Culberson & Kristinsson 1970; Culberson 1972, 1974; Culberson & Ammann 1979), most of these compounds run below the control substance norstictic acid, which has Rf values between 28 and 40. Even if the spots for these substances were evenly spaced, the Rf values would differ by only about one millimeter. In addition, we now know that these compounds occur in complicated mixtures and consequently accurate identifications will require more sophisticated chro- matographic techniques than those routinely used. Reports in the recent literature of low- Rf compounds (even common ones like salazinic, constictic and connorstictic acids) may be misidentifications based upon insufficient TLC evidence. The purpose of the present paper is to give standardized TLC data for a fourth solvent system (Solvent G) to assist the resolution of difficult mixtures of low-Rf, p-orcinol depsidones by one- and two-dimen- sional TLC. The chromatographic procedure followed the standardized method (Culberson 1972) except that toluene (dried over CaCl2 and redistilled) was used in place of benzene. Solvent G, toluene/ethyl acetate/formic acid (139:83:8, v/v/v; 230 ml), is modified from previously suggested systems (Stahl & Schorn 1961; Hirayama et al. 1976). Formic acid (95-97%) and ethyl acetate were not purified or redistilled; it is not necessary to purify a good grade of toluene for satisfactory results with Solvent G. As with Solvent B, however, a grade of formic acid with low water content is essential. TLC plates were Merck Silica Gel 60 F254 (0.25-mm layer; No. 5765). Chromatographic tanks (Desaga- Heidelberg) were insulated from external temperature fluctuations but were not lined with filter paper. Pre-equilibration of the plates in 60% formic acid (Culberson 1974) did not improve the quality of chromatograms in Solvent G. Unless noted otherwise, the standardized data in Table 1 were from shortened plates (12.5 x 20 cm) developed to 10 cm. Initial spots were 2 cm from the bottom of the plate and 0.5 cm above the solvent. Controls for standardization were included at three positions on every plate. The Rf data in Table 1 are standardized with respect to fumarprotocetraric acid in addition to the usual control substances, atranorin and norstictic acid. Two-dimensional chromatograms were prepared by the standardized method (Culberson & John- son 1976) except that full plates (20 x 20 cm) were used with seven one-dimensional comparisons (including two atranorin-norstictic acid controls) in each direction. In addition, for the second direc- tion, an atranorin-norstictic acid control was added just to the left of the origin on the two-dimensional portion of the plate. This control helps to align spots and to monitor effects of residual first-direction solvent on R, values in the second direction. (In the present report, first- and second-direction solvents