Ex: Elix, JA; Ernst-Russell, KD (1993): A Catalogue of Standardized Thin Layer Chromatographic Data and Biosynthetic Relationships for Lichen Substances, 2nd Edn, Australian National University: Canberra.

The TLC Method

The basic methodology has been described in detail by Culberson and Kristinsson (1970), Culberson (1972), Culberson and Ammann (1978), and White and James (1985) and was used with minor modifications (detailed below). In particular we have introduced the use of a new solvent system (25% ethyl acetate/hexane) and the measurement of relative Rf values (rather than absolute Rf values or Rf classes).

i The Plates
Merck silica gel 60 F254 precoated glass-backed TLC plates were used (layer thickness 0.25 mm) and were stored in a dry cupboard over selfindicating silica gel, but were not activated. In her exposition Culberson (1972) recommended the trimming of 20 cm × 20 cm plates to 20 cm × 12.5 cm and only eluting to a height of 10 cm (the level of the solvent front). We consistently find that better resolution of spots is obtained if the full 20 cm × 20 cm (or alternatively 10 cm × 20 cm) plate is used, and elution continued to the top of the plate (i.e. approximately 18 cm). For solvent E, elution to a height of 10 cm is quite adequate. In all solvent systems the spots were placed 1 cm apart, 2 cm above the base and beginning 2 cm from the edge of the plate (in oder to avoid edge effects). After elution the plate is air-dried for approximately 30 minutes in a well-drafted fume cupboard before viewing under ultraviolet light, spraying, etc.

ii. Extraction of the Lichen Material
Our normal procedure is to soak the lichen fragments in ca. 1 ml of acetone for 5 minutes in a small test tube and then #### bdi~ the solution to concentrate the original I m1 of acetone to approximately 0.1 ml. This concentrated solution is then used for spotting on the TLC plate using a capillary tube – several times if necessary. It is advisable to view the plate under short wavelength ultraviolet light before proceeding, in order to ascertain whether the spots are sufficiently intense – if not, further spotting may be necessary.

iii. Solvents
Culberson's improved standardized method (Culberson 1972) used the three solvents A, B and C (modified), constituted as detailed above, and these three solvents are still widely used in routine analyses. We find that solvent C provides the best discrimination of lichen substances, and is particularly stable and reliable. Solvent A also is very useful, but as dioxane is hygroscopic, it absorbs water over a period of time and Rf values obtained with 'aged' mixtures are unreliable and a secondary solvent front may develop. The standard solvent B is reliable provided the solvent is replaced at frequent intervals, but the actual frequency depends on the atmospheric conditions and the number of chromatograms run. In our experience the substitution of methyl t-butyl ether (solvent B') significantly improves the reliability and stability (lasting up to 4-5 days) of this comparable mixture and we now routinely use this in preference to standard solvent B (which rarely lasts beyond 6 hours). Solvent G (Culberson, Culberson & Johnson 1981) is a very stable and reliable eluant and is particularly useful in separating compounds with relatively low Rf values in solvents A, B, B', and C (b-orcinol depsidones, secalonic acids and hopane triterpenoids). Solvent E (25% ethyl acetate / 75% cyclohexane) and Solvent F (50% ethyl acetate / 50% cyclohexane) have been developed to discriminate non-polar derivatives and those compounds with very high Rf values in solvents A, B, B', and C (esters such as atranorin, chloroatranorin, pannarin, physciosporin, usnic acid, terpenes, xanthones and other pigments). These solvents are prepared fresh daily

iv. Equilibration and Elution
When using solvent A, B or B' a filter paper should be placed at the back of the tank and saturated with the solvent – the TLC plate is then placed in the tank with the silica-side faang the filter paper. This help achieve uniform vapour saturation of the solvent throughout the tank and assures an even running of the solvent front. If a lower secondart solvent front is observed to develop, this indicates that the solvent mixture is wet and should be discarded and replaced. When using solvents B, B' and C, it is important to 'pre-equilibrate' the plate with 60% formic acid vapour (for solvents B, B') or glacial acetic acid vapour (for solvent C) before proceeding with elution. Thus, after the plate has been spotted with the samples, it is placed in a tank saturated with acid vapour for the required time (5 minutes for B, B'; 10 minutes for C). The plate must not be wetted by the liquid acid. This is achieved by having a small quantity of liquid acid covering the base of a closed tank and supporting the dry plate to be pre-equilibrated on several glass islands (above the level of the liquid). Such pretreatment of the plates again ensures uniform travel of the solvent front and prevents the development of secondary solvent fronts.

v. Examination of the Developed Plates
The dried plates are examined initially in visible light (daylight) for pigments which appear as coloured spots. Their colour and position are recorded. Some pigments, such as the secalonic acids and related compounds (which are relatively pale and streak along the plate), are most easily seen if the plate is examined whilst being held in front of a strong (visible) light. Next the plates are examined under short wavelength (### h 254 nm) ultraviolet light, where all aromatic lichen substances are indicated by dark spots on a fluorescent background (fatty acids and terpenes are not observed under these conditions unless they are extremely concentrated). These spots should be marked for future reference. Several substances, such as alectoronic acid and a-collatolic acid, fluoresce bright blue under short wavelength ultraviolet light before spraying – Subsequently the plates are sprayed with water. When the wet plate is illuminated from above and viewed against a dark background, fatty acid spots show up as opaque white spots against a relatively dull background. After brief drying the plates are sprayed with 10% sulfuric acid until wet (but with no run-off) and then heated at 110° in an oven or on a hotplate for 10 minutes to develop the spots. The various diagnostic colours of each lichen substance are well developed by this time and the Rf values and colour (acid spray) should be recorded. Note that colours should be recorded as soon as the plate has cooled, rather than later on, as they often alter with time. Extra purple or blue spots may also appear (which did not show up under ultraviolet light) – these are the lichen triterpenes and steroids. Additional useful information may also be obtained by noting the colour of fluorescence of the developed spots under long wavelength (h. 350 nm) ultraviolet light immediately after acid and heat treatment. Such colours are less prominent if the plates are stored. The colours which develop after spraying and charring are concentration dependent. Thus a strong or intense spot (especially for depsides and depsidones) will appear as a spot of one colour with a ring or halo of a different colour. If the spot is weak, that is, the compound is present in small quantities, it will appear as a uniform spot of the same colour as the halo of the corresponding intense spot. This phenomenon is observed in both visible light and under long wavelength ultraviolet light. Thus the colours available in the colour palette of the data base have all been cross-referenced (see Figure 9) to allow for differences due to concentration effects, as well as individual interpretations of colour. Other spray reagents may also be used. Recently the use of 3-methyl-2-benzothiazolone hydrazone hydrochloride (MTBH or Archers Solution) has been developed by Archer (Archer 1978) as a complementary spray reagent to sulfuric acid/heat. This reagent develops characteristic colours with a number of depsides, depsidones and dibenzofurans.

vi Relative rather than Absolute Rf Values
The standardized method (Culberson 1972; White and James 1985) utilized Rf classes determined on each plate by a control mixture (atranorin and norstictic acid), so that accurate reproducibility of the Rf values was not required. This method is very useful in limiting the number of possibilities for an 'unknown' spot, but often some ambiguity remains. Certainly better resolution is usually observed, but the measured or absolute Rf value may fluctuate depending on the atmospheric conditions, the age of the solvent, etc. In practice we find that the most satisfactory method is to use relative Rf values, where one achieves maximum resolution as well as reproducibility. The key to this method is to use more than two compounds in the control mixture (a choice of those listed in Table 1). Although the absolute Rf values of the 'unknowns' may fluctuate, the Rf values of the controls will fluctuate in a parallel manner. Hence the controls are assigned invariant Rf values, and all other spots are measured relative to them.

vii Confirmation of Identity
The identity of an 'unknown' substance can only be confirmed by comparative TLC in at least three of the solvent systems, that is, running the lichen extract adjacent to an extract containing this particular substance or preferably, using a pure sample of the particular lichen compound. Then the Rf values as well as the fluorescent properties can be compared under identical conditions. Even so, it is preferable to have independent confirmation of identity – via mass spectrometry or comparative HPLC.

Table 1
Standard Rf values of Control Compounds

Compound Rf in Solvent
A B B' C E G
Atranorin 75 78 73 79 57
Chloroatranorin 74 79 73 81 30
Usnic Acid 70 70 65 71 23
40-Methylhypoprotocetraric Acid 39 58 51 45 61
Notatic Acid 34 49 44 38 55
Norstictic Acid 40 29 32 30 57
Physodalic Acid 10 30 33 19 46
Stictic Acid 32 9 9 18 34
Salazinic Acid 10 7 7 4 26
Protocetraric Acid 19

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