105Supplementary references
4.3 ALKANES AND RELATED HYDROCARBONS .1 Chemistry and distribution
Long-chain hydrocarbons are best known as constituents of petroleum, which is at least partly derived from fossilized plant matter. In living plants, hydrocarbons are universally distributed in the waxy coatings on leaves and other plant organs. The alkane fraction is commonly a mixture of hydrocarbons of similar properties. The qualitative pattern is relatively similar from plant to plant, but there are considerable quantitative varia- tions. Alkanes also occur in fungi and other lower plant groups, and the pattern is generally like that found in higher plants (Weete, 1972).
Biosynthetically, these hydrocarbons are related to the fatty acids (see p.
162)and, in the simplest instances, are formed from them by chain elon- gation and decarboxylation. The function of alkanes in the cuticle waxes of plants is a protective one, the water-repellent properties providing a means of controlling water balance in the leaf and stem. Their universal presence in leaf coatings may also be to provide a measure of disease resistance to the plant.
Alkanes are saturated long-chain hydrocarbons with the general for- mula CH3(CH2)/ICH3. They are usually present in the range of C2S to C3S carbon atoms, Le. general formula with n
=
23 to 33. Odd numbered members of the series, C2S' C27, etc., predominate over the even numbered members of the series, often to the extent of 10:1.The major constituents of waxes are thus C27-C31 alkanes. Examples are n-nonacosane, C29H60and n-hentriacontane, C31H64I the major constituent of candelilla wax from Euphorbia species.There are also a considerable number of alkane derivatives in waxes, formed by the introduction of unsaturation, branching of the chain, or oxidation to alcohol, aldehyde or ketone. Branching most commonly occurs near the end of the carbon chain. Two types may be mentioned;
isoalkanes, with general formula (CH3)2CH(CH2)II-CH3 and ante- isoalkanes, general formula (CH3)(C2Hs)CH(CH2)n-CH3' Branched alkanes are by no means universally present and rarely occur in any quantity.
Olefinic alkanes, or alkenes, have a similar distribution in that they occur fairly frequently but in relatively low amount. Exceptionally high amounts of alkenes have been detected in rye pollen, rose petals and sugar cane.
Alkanes and related hydrocarbons 171 Hydrocarbon alcohols are fairly common in plants, ceryl alcohol CH3- (CH2)24CH20H being a regular consituent in many cuticular waxes.
By contrast, aldehydes and ketones are infrequent. Two taxonomically interesting~-diketonesmight be mentioned: the compound CH3(CH2)1O- COCH2CO(CH2)14CH3 found in waxes of certain Eucalyptus species and the related hentriacontan-14,16-dione present as a major wax constituent of cereals and grasses.
Detailed knowledge on the plant hydrocarbons has only accumulated significantly in the last thirty years and this has been entirely due to the application of one particular technique, GLC, to their separation and estimation. A key paper on leaf waxes is that of Eglintonet al.(1962), who first applied this technique to a survey of alkanes in the Crassulaceae. Of the many more recent papers that have been published, those of Herbin and Robins (1968; 1969) onAloe,of Nagy and Nordby (1972) on citrus fruit hydrocarbons and of Evanset al. (1980) on Rhododendronleaf waxes may be mentioned. General reviews of plant wax constituents include those of Douglas and Eglinton (1966), Martin and Juniper (1970) and Baker (1982).
4.3.2 Recommended techniques (a) Extraction
As a general precaution against contamination of plant samples, it is essential to employ clean glassware and redistilled solvents and also to avoid contact with stopcock grease or plastic tubing. Another source of possible contamination is contact with 'Parafilm', a thermoplastic sealing material widely employed in phytochemical laboratories. Indeed, this material, when washed with benzene or hexane is very useful since it will give a solution containing the standard range of n-alkanes, which can then be used for GLC comparison with alkanes from a plant extract (Gaskin et al., 1971).
Extraction of plant waxes is simply carried out by dipping unbroken leaves or stems into ether or chloroform for very short periods of time (e.g.
30 s). This removes the surface alkanes without attacking cytoplasmic constituents. A filtration at this stage may be desirable to remove any dirt.
An alternative procedure is to Soxhlet-extract dried powdered leaf for several hours in hexane. Such an extract will be considerably contami- nated with leaf lipids and some fractionation will be necessary before the hydrocarbons are obtained pure. For example, steam-distillation may be desirable, in order to remove any essential oils from such an extract.
(b) Purification
Itis common practice to fractionate the direct wax extract either in order to remove undesirable components (such as lipids) or to separate the
172 Organic acids, lipids and related compounds
hydrocarbon classes according to polarity. In many cases, all that is neces- sary is to pass the crude extract through an alumina column (e.g. Alcoa F- 20 grade AI20J ) and elute with light petroleum. The first fraction contains the alkanes, the later fractions containing the alcohols, aldehydes or ketones. The purity of the alkane fraction can be checked by IR spectroscopy; oxygenated impurities if present will be apparent by IR absorption between 600 and 3500 cm-1.A more thorough approach to the purification of alkanes is to saponify the crude wax with methanolic KOH and then remove ketonic materials by reacting them with 2,4- dinitrophenylhydrazine in aqueous 2M HCl.
Iflipids are present, these may be separated by TLC of the wax extract on silica gel in chloroform-benzene (1:1) followed by detection with a Rhodamine B fluorescein spray. The hydrocarbons haveRrca.90 and the fatty acid esters have Rrca.50. Alternatively, separation from lipids may be achieved by argentative TLC with light petroleum as solvent.
Argentative TLC (see p. 169) may also be employed for fractionating alkane types; in 2% ether in petroleum (b.p. 30-60°C) saturated, mono- unsaturated and polyunsaturated hydrocarbons separate in order of decreasing RF•
Ifthe leaf waxes contain 13-diketones as happens in many grass species, these can be separated by column chromatography on copper acetate- silica gel, previously prepared by mixing 80 g silica gel (200-400 mesh) with a solution of 25g cupric acetate in 100ml H20 and then drying at 120°C. Other wax constituents are first eluted off with hexane-ether (9: 1) and then the 13-diketone-Cu complex formed on the column is eluted with chloroform-ethanol (17: 3) at 50°C. The f3-diketones are recovered by de- composing the complex with acid and are then identified by GLC-MS of the trimethylsilyl enol ethers (Tulloch, 1983).
(c) Gasliquid chromatography
The earlier procedures described by Eglintonet aI.(1962) for the GLC ofn- alkanes have been employed, with relatively little modification, up to the present time. These authors used a 120 x O.5cm column of 80-100 Celite coated with 0.5% Apiezon L grease, and obtained separations as indicated in Fig. 4.6. They found also that there was a linear relationship between the logarithm of the relative retention times and the alkane carbon number, in both then-and isoalkane series. Identification was based in the first instance on the use of this linear relationship and on the intensifica- tion of appropriate peaks when genuine n-alkanes of known carbon number were added to the plant extract.
More recently, other types of column packing have been used. Two useful additional systems are 3% 5E-30 on 100-120 Varaport 30 and 10%
polyethylene glycol adipate. When a wide range of alkanes are being
Alkanes and related hydrocarbons 173
80
Species 60 237°C
Species 33 235°C
'lq
It)c: ._I I Species 73
225°C
M
1
c:I
Q) Ihc:
oa.
Ih~
u
o
Q)oQ)
Retention time (min)
Fig. 4.6 Gas liquid chromatography of plant alkanes. Key: Species 60 isMonanthes polyphylla,species 33 isAeonium saundersii-Bolleand species 73 isEuphorbia aphylla.
Column0.50/0Apiezon L on 80-100 celite (from Eglintonet al., 1962).
separated, a programmed temperature operation is desirable, such as one based on raising the temperature of the column from 70°C to 300°C at 6°C min-I.
Alkenes can be tested for, using preparative GLC, by their reaction with bromine in carbon tetrachloride. Also, they have shorter relative retention times(RRTs)than the corresponding alkanes on an Apiezon L column, but longerRRySon a polyethylene glycol adipate column. Alkene peaks can also be made to disappear by catalytically hydrogenating the plant extract before GLC; on the subsequent GLC trace, the peaks of those alkanes related to the alkenes reduced will be intensified.
Confirmation of identification is most frequently done by mass spectral studies, and in many recent studies of plant alkanes, combined GC-MS is used. IR spectroscopy may also be employed for identification, but it is not
174 Organic acids, lipids and related compounds
very sensitive to impurities. The advantage of mass spectral studies is that they may well reveal trace amounts of isomers in what otherwise appear to be pure GLC fractions (Misra and Ghosh, 1991).
(d) Ethylene
The identification of this simple hydrocarbon (CH2=CH2) is of especial interest to plant physiologists since it has been recognized to be an impor- tant natural growth regulator. Ethylene produced by plants is measured using a GLC apparatus set up for gas analysis. Since only very small amounts of ethylene are produced by plant tissues, it is essential that the GLC recorder used is operating at the maximum level of sensitivity.
Before analysis, ethylene can be condensed in a liquid oxygen trap, or by passing it on to a column of silica gel impregnated with mercuric perchlorate (Phan, 1965).
Originally, GLC was carried out on a column of 30% silicone oil 550 coated on Firebrick (60-80 mesh) (Meighet al., 1960). Galliard et al. (968) have employed a stainless steel column 050cmX 3mm) packed with 10%
Triton x-305 on NAW Chromosorb G (80-100 mesh), operating isother- mally at room temperature with a flame ionization detector. The same authors removed polar volatile compounds from the gas samples by employing a pre-column 07cm X 4mm) packed with 20% diglycerol on celite and fitted with a back-flushing device. Using this procedure, 0.03 p.p.m. ethylene can be detected.
Muir and Richter (1972) recommended GLC on a column of Porapak, at a temperature of 80°C. In a typical run on this support, the authors found oxygen emerging after 1.5 min, methane at 2 min and ethylene at 4 min.
Although ethylene clearly separates from related hydrocarbons (e.g.
methane, ethane, propylene) on most column stationary phases, it is as well to confirm its identification in natural plant vapours by GLC on at least two different types of column, e.g. on Porapak together with silicic acid (80-100 mesh) or alumina Fj (80-100 mesh) as the contrasting phase.
Conclusive identification of ethylene in a new plant source really requires GLC-MS analysis as well (Wardet al., 1978).