The water soluble carbohydrates of Pinus radiata : identification and quantitation
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
A wide range of techniques have been used in the analysis of carbohydrates from plant sources. Many of these techniques are fraught with difficulties and are not at all suitable for routine analytical procedures. A joint project with N.Z.F.R.I. was undertaken to develop suitable rapid methods for the routine analysis of the water soluble carbohydrates (w.s.c.) present in the various tissues of Pinus radiata. Seasonal variations of carbohydrate levels were also of particular interest to the plant physiologists at the N.Z.F.R.I. The traditional extraction procedure uses 80% or 95% ethanol/water, (Holligan 1971 b, Ford 1974) and requires either a 'soak’ or a soxhlet extraction of the plant tissue for a predetermined period. Unfortunately not all starch is removed by ethanol or water (Smith 1971) so a separate analysis in which the tissue is directly analysed for starch is usually necessary. The techniques used for the analysis of carbohydrates range from paper chromatography to, more recently, carbon magnetic resonance (c.m.r.) spectroscopy. Paper chromatography is still used when a small number of samples are to be analysed, although it is more useful for identification than quantitation because the process becomes both complex and inaccurate when the chromatograms are required to be measured colorimetrically (Dubois 1956, Jeffery 1960). Thin layer chromatography (t.l.c.) using silica and cellulose absorbent phases and a wide range of solvent systems have been reported (Jeffery 1960, Hehl 1973), but the time involved in obtaining chromatograms is such that other methods are preferable. Paper electrophoresis with borate buffer systems has been used with some success (Pettersson 1973), but it has the same limitations as paper chromatography. Column chromatography has also been used to isolate certain w.s.c.'s. For example, a sugar-free mixture of the cyclitols was separated on a cellulose packing in conjunction with a Dowex ion-exchange column (borate form) (Dittrich 1971). Affinity chromatography, a more advanced form of column chromatography, enabled Kennedy (Kennedy 1973) to separate mono- and polysaccharides, but as with paper chromatography he had to rely on clumsy colorimetric methods for quantitation. Automation would be necessary for large numbers of samples. Gas-liquid chromatography (g.l.c.) is the best of the chromatographic techniques available - offering rapid qualitative and quantitative analysis on relatively small amounts of material. The trimethylsilyl (TMS) ether derivatives of carbohydrates are easy to prepare and produce good chromatograms with compounds of similar or widely varying molecular weights (Holligan 1971 a, b). Acetate derivatives are more stable than their TMS counterparts, but separation of the aldose and ketose acetates was not satisfactory (Holligan 1971 a). G.l.c. relies largely on 'spiking' for identification of the peaks of a chromatogram. Linked to a mass spectrometer, the combined gc/ms provides mass spectra for all resolvable peaks and they can be identified by comparison with authentic spectra. Proton n.m.r. spectroscopy has been attempted, but it was not possible to identify individual components due to the multiplicity and overlapping of peaks. In contrast, c.m.r. has been shown to have great possibilities for qualitative and, more importantly for this work, quantitative analysis of aqueous solutions of carbohydrates. Starch in plant tissue has been analysed by a number of methods (Bailey 1958, stoddart 1966, Ebell 1969 a, MacRae 1968, 71), nearly all based on the analysis of the glucose formed by acid or enzymic hydrolysis. MacRae (MacRae 1971, 74) has reviewed the more traditional methods of starch analysis. Identification and quantitation of the w.s.c. is, and probably will continue to be a problem as molecules of similar structure and molecular weight are involved. In P. Radiate there are some 8 to 9 components to be considered. C.m.r. has greatly simplified the identification, and g.l.c. enables a large number of samples to be analysed accurately.