Thus, there is no simple guide to modern methods of plant analysis and the purpose of this book is to fill this gap. Since the preparation of the first edition, several important developments have taken place in the field of phytochemical techniques.
INTRODUCTION
Because the number of known substances is so large, special introductions have been written in each chapter of the book, indicating the structural variations that exist within each class of compounds, describing those compounds that occur frequently, and illustrating the chemical variations from a representative formula. Tables are included showing the RF values, color reactions, and spectral properties of most of the more common plant constituents.
METHODS OF EXTRACTION AND ISOLATION .1 The plant material
In the case of phytochemical analysis, the botanical identity of the plants examined must be authenticated by a recognized authority at some point in the study. The identity of the material should either be beyond doubt (eg a common species collected in the expected habitat by a field botanist) or it should be possible to establish the identity by a taxonomic expert.
METHODS OF SEPARATION .1 General
The temperature of the column inlet is controlled separately so that the sample can be vaporized rapidly as it is transferred to the column. Separation of the compounds on the column depends on passing this gas at a controlled rate.
METHODS OF IDENTIFICATION .1 General
The phytochemist therefore hands over his sample for analysis and receives the results back in the form of the graph shown in Figure. Its structure as 6-(4-hydroxymethyl-trans-2-butenylamino)purine was determined by Shannon and Letham. (1966); the results of the MS (Fig. 1.6) significantly assisted in this identification.
ANALYSIS OF RESULTS .1 Qualitative analysis
Just as important as qualitative measurements of a plant extract are determinations of the amounts of the components present. In the simplest approach, quantitative data can be obtained by weighing the amount of plant material initially used (assuming dried tissue) and the amount of product obtained.
APPLICATIONS .1 General
Phytochemistry plays an important role in plant tissue culture by detecting and analyzing the products of secondary metabolism that can be formed in such tissues. Phenolic compounds are all aromatic, so they all exhibit intense absorption in the UV region of the spectrum.
PHENOLS AND PHENOLIC ACIDS .1 Chemistry and distribution
However, most phenolic compounds (mainly flavonoids) can be detected on chromatograms by color or fluorescence in UV light, with the colors intensifying or changing if the papers are steamed with ammonia vapor. An introduction to the analysis of phenolic plant metabolites was given by Waterman and Mole (1994).
OH Hydroquinone
Y R R=H, resorcinol
PHENYLPROPANOIOS .1 Chemistry and distribution
Isomerization of the allyl to the propenyl form can be achieved in the laboratory, but only under drastic conditions (eg with strong alkali). Compounds and 7 can be detected as various shades of blue under UV light and fuming with ammonia. Separation of the glycosides from hydroxycoumarins can be carried out with similar procedures as for the free coumarins (Table 2.4).
The synthesis of scopolin can be observed in partially burned potatoes collected in autumn, immediately after harvest.
- ANTHOCYANINS
- FLAVONOLS AND FLAVONES .1 Chemistry and distribution
- MINOR FLAVONOIDS, XANTHONES AND STILBENES .1 Chemistry and distribution
- TANNINS
Detailed references to the natural distribution of the flavonoids can be found in Harborne (1967a), Harborneet al. Flavones occur as glycosides, but the range of different glycosides is smaller than for the flavonols. A more detailed account of the phytochemistry of these flavonoids can be found in Harborneet al.
The Rr values of the isomers formed from orientin and vitexin are shown in the table.
Tannins can be detected as dark purple spots under short UV light, which react positively to any of the standard phenolic reagents (see Section 2.2) (Haslam, 1989). One or more of the following procedures may be used, depending on the types of tannin present. This procedure, giving the relative astringency of the plant extract, is a direct measure of total soluble tannin (Bate-Smith, 1973).
Alternatively, the affinity of tannin for methylene blue solution (7.0 X 10-5M) can be measured (Okuda et al., 1985).
Other chemical tests for functional groups have been used in the past
- QUINONE PIGMENTS .1 Chemistry and distribution
A very complete account of the natural occurrences and properties of all vegetable quinones may be found in Thomson. Again, shanorellin, the orange-yellow pigment of the ascomycete Shanorella spirotricha, can be separated from its methyl and acetyl derivatives in benzene-acetic acid (9: 1) or chloroform-acetic acid (9: 1) (Wat and Towers, 1971). . Finally, mention should be made of the excellent separations of anthraquinone that can be achieved on silica gel plates treated with tartaric acid in chloroform-methanol (99: 1).
Spectrally, anthraquinones can be distinguished from other classes of quinone by the fact that they have four or five absorption bands in the UV and visible regions.
105Supplementary references
INTRODUCTION
Thus, terpenoids are all based on the isoprene molecule CH2=C(CH3)-eH=CH2, and their carbon skeletons are built up from the union of two or more of these Cunits. Although terpenoids are derived biogenetically from the molecule isoprene, which occurs as a natural product, this substance is not the in vivo precursor. Chemically, terpenoids are generally lipid soluble and are located in the cytoplasm of the plant cell.
Reviews of many aspects of plant terpenoids are included in Part 4 of a comprehensive treatise on plant biochemistry (Stumpf, 1980).
ESSENTIAL OILS
Other iridoids, such as loganin, are of interest because they are intermediates in the biosynthesis of indole alkaloids (see Chapter 5). Simple monoterpenes are widespread and occur as constituents of most essential oils. Specific methods have been developed for the detection and evaluation of the sesquiterpene growth inhibitor abscisic acid in plants.
Besides the main components indicated in Table 3.5, most fruits contain other compounds of higher or lower RF•The higher RF compounds are monoterpene hydrocarbons such as limonene (RrB0), a-phellandrene and a-pinene.
DITERPENOIDS AND GIBBERELLINS .1 Chemistry and distribution
He also details how to separate the components of clove oil and citrus oils by column chromatography on silica gel. Recently, TLC on silica gel-AgN03 00:1) with light petroleum as solvent has been utilized (Norin and Westfelt, 1963). At least thirty solvents have been used for the separation of gibberellins on layers of inactive or activated silica gel (Kaldewey, 1969).
The material was isolated by extraction with acetone and purified by bicarbonate fractionation and column chromatography on silica gel with chloroform as eluent.
TRITERPENOIDS AND STEROIDS .1 Chemistry and distribution
On the other hand, the separation of about twenty-eight cardenolides in foxglove, Digitalis purpure requires two-dimensional TLC in ethyl acetate-methanol-water and chloroform-pyridine (6: 1) (Sjoholm, 1962). For the separation of cardiac glycosides of Asclepia species and of butterflies that ingest them in the larval stage, multiple development on silica gel plates is recommended either in ethyl acetate-methanol (97: 3) twice or in chiro-form-methanol-formamide (90 :60:1) four times (Broweret al., 1982). TLC of the final extract of chloroform in hexane-acetone (1:4) separates sterols (RF50 and above) from sapogenins (RF 45 and below).
Sterols are widely distributed in seeds, and sitosterol may be present in sufficient quantity to be isolated in crystalline form (eg in seeds of chickpea, eicer arietinum). The general procedure can be used for isolation.
CAROTENOIDS
Stransky (1978) has described a simple isocratic elution from a Nucleosil 50-S column with isooctane-ethanol (9:1) for estimating chloroplast carotenoids in the picomoles range. This applies to capsanthin from pods of the red pepper or paprika, Capsicum annuum. The following procedure is taken from Ikan (1969). 1991) in Modern Methods of Plant Analysis NS, Volume 12, Essential Oils and Waxes, pp. ed.) (1976) The Chemistry and Biochemistry of Plant Pigments, 2nd edition, Academic Press, London.
Passage through a weakly basic ion exchange resin in the hydroxyl form will lead to the absorption of the organic acid anions.
FATTY ACIDS AND LIPIDS .1 Chemistry and distribution
The saturated cutic acids generally have a longer chain length than palmitic or stearic acids, the range is from C24 to C32. • The other main chemical feature of the cutinic acids is the presence of hydroxy groups in their structures. In the case of the fatty acids, this can be overcome by derivatizing them and separating them as their phenacyl or p-bromophenacyl esters. The lipid extract is weighed and the percentage of lipid in the original nuts can then be calculated.
After cooling by adding 15 ml of water, the methyl esters are taken up in petroleum (2 x 15 ml) and the extracts are washed, dried and evaporated to dryness. 7) The methyl esters are incorporated into a minimal amount of petroleum and an aliquot is injected into the inlet port of a GLC apparatus, which has a column of 10% diethylene glycol adipate on 100-120 mesh Celite and operates at temperatures between 180°C and 180 °C. and 200°C.
ALKANES AND RELATED HYDROCARBONS .1 Chemistry and distribution
Finally, the concentrations of the various acids in peanut oil can be determined by measuring peak areas (see Chapter 1, p. 14). Of the many recent papers that have been published, those of Herbin and Robins on Aloe, of Nagy and Nordby (1972) on the hydrocarbons of citrus fruits, and of Evans et al. 1980) can be mentioned on Rhododendron leaf waxes. If lipids are present, these can be separated by TLC of the wax extract on silica gel in chloroform-benzene (1:1) followed by detection with a fluorescein Rhodamine B spray.
They also found that there was a linear relationship between the logarithm of the relative retention times and the alkane carbon number, both in the isoalkane and isoalkane series.
POLYACETYLENES
Triton x-305 on NAW Chromosorb G (80-100 mesh), operating isoterically at room temperature with a flame ionization detector. The same authors removed volatile polar compounds from gas samples using a precolumn (07cm X 4mm) packed with 20% diglycerol on celite and equipped with a backwash device. Acetylenics are also found in higher fungi, in two families of basidiomycetes, the. The UV spectra of several pure polyacetylenes found in the Umbelliferae are given in Table 4.4, to further illustrate the range and number of spectral maxima.
Fresh roots may be collected, and after careful washing, extracted in the same manner as in the above carrot experiment.
SULPHUR COMPOUNDS .1 Chemistry and distribution
Two indirect methods are recommended for the quantification of ~ucoSinolatesin plant extracts. The first provides concentration of total glucosinolate and involves hydrolysis of the purified glucosinolate fraction with myrosinase and measurement of released glucose with a standard glucose test kit (see Chapter 6). . 1975) Determination of the structure of fatty acids in Recent Advances in the Chemistry and Biochemistry of Plant Lipids (eds. T. Galliard and E.I. Mercer), Academic Press, London, p. 1980). The distribution of sulfur compounds in Comparative Phytochemistry (ed. T. Swain), Academic Press, London, p. 1978) Crassulacean Acid Metabolism, Springer-Verlag, Berlin.
Mangold, H.K. (1969) Alifatski lipidi v tankoplastni kromatografiji (ed.E.Stahl), George Allen in Unwin, London, str. 1970) The CuticleofPlants, Edward Arnold, London.
