Assurance of the purity of the essential oil is of primary importance in an investigation of its chemical constituents. If there is the slightest doubt as to whether or not the oil may have been contaminated or adulterated, then such an oil is worthless for the examination, because the results obtained after much labor will be open to question. Therefore, it is best for the investigator to distill the oil from the botanical, or to supervise the distillation in the producing region or factory. Such distillations should be carried out on a commercial scale in the manner in which the oil of commerce is produced; otherwise, misleading results may be obtained. If this is impossible, the oil should be obtained directly from a prime source of unquestionable repute.
A representative sample of the oil to be investigated should be analyzed carefully. All physical and chemical properties should be determined, including specific gravity, optical rotation, refractive index, solubility and the percentages of esters, aldehydes, ketones, phenols, acids and alcohols. These physicochemical properties should be compared with values given in the literature for normal pure oils. Further examination should not be attempted if these properties show any suspicious deviation from normal values. Such deviation might indicate accidental contamination, adulteration, or the production of an abnormal oil.
Although an oil may have been distilled from the proper botanical material, nevertheless, it may not represent the normal article of commerce. Such factors as the degree of maturity of the botanical frequently exert an important influence on the composition of the oil. Consider, for example, oil of coriander. If an oil is distilled from the immature and green coriander seed it will show a high decyl aldehyde content, sometimes attaining a value as high as 70 per cent. As the seed matures, the aldehyde content of the oil decreases and the linalool content increases, until finally an oil is obtained from mature seed which shows an aldehyde content of about 1 per cent. Needless to say, the oil having this low aldehyde content is the oil accepted in commerce as normal oil of coriander.
A further difficulty exists in the proper selection of the botanical. Sometimes there are many species within a plant family but only one or more yields the desired oil or oils ; the eucalypts are a good example. Occasionally there are found several varieties of the same species which may yield different oils upon distillation. The production of juniper berry oil from Juniperus communis L. growing in America gives rise to an oil which differs from the normal commercial product formerly obtained from Juniperus communis L. grown in Central Europe. This has been explained by the fact that the American oil is distilled from a variety of the true Juniperus communis L.; viz., Juniperus communis L. var. depressa Pursh. Physiological varieties of the same species of certain plants are also known (e.g., Eucalyptus dives).
The geographical location of the growing section may exert an effect upon the composition and quality of the oil. This probably results from the nature of the soil, the altitude at which the plant grows, as well as factors such as intensity of sunlight, rainfall and temperature.
Consideration should be given to the methods of distillation and production of the commercial oil and to the handling of the botanical before distillation. Some plants should be distilled as soon as cut, some after sun drying for a day, some after thorough drying in the shade, some after drying and storage for several years. For details, the reader is referred to the section in Chapter III on "Practice of Distillation”
All of the above factors should be carefully considered, and as much information as possible concerning the botany, geographical source, maturity, preliminary treatment of the plant material and method of production of the oil should be included in the report on the chemical constituent of the oil.
The amount of oil used for the examination is a limiting factor. The availability and the cost of the oil enter in most commercial and academic investigations. For oils that are available in relatively unlimited quantity, the difficulty of handling large amounts in a research laboratory must be considered. Such difficulty may be overcome if the manufacturing plant or factory cooperates in the investigation. It then becomes possible to fractionate large quantities of the oil, even hundreds of pounds, and to investigate the individual fractions or aliquot parts of such fractions. Constituents occurring in minute amounts have been identified by such a procedure. Without benefit of this preliminary fractionation, it is difficult to handle much more than 15 liters of an oil in the laboratory.
For an oil which has not been investigated previously, the first step is a general examination, followed by an investigation which endeavors to discover as many of the constituents as possible. This usually reveals those constituents which occur in substantial amounts. Frequently, indications of the occurrence of other constituents are thereby obtained, whose presence, however, cannot be established conclusively. A subsequent investigation directed solely to the isolation and identification of such individual constituents often will prove successful.
It is obvious that no comprehensive procedure can be given which will prove applicable to all essential oils. The following notes are intended merely as an aid to the chemist embarked upon such an investigation. From a study of the physicochemical properties of the oil, a general plan for the investigation is formulated.
If the oil shows A large percentage of free acids, phenols or carbonyl compounds it is usually advisable to remove these components before fractionation. Any free acids should always be removed before further treatment of small amounts, it may be better to fractionate the oil and then separate these components from the enriched fraction or fractions.
Occasionally solid constituents (such as camphor, menthol, safrole, or anethole) may be separated from the whole oil by freezing, followed by filtration or centrifuging. Since such separations are never quantitative it may be advisable to freeze out these components from the enriched fraction rather than from the whole oil. If the solid constituents occur in large amounts, one may resort to a preliminary freezing, followed by fractionation of the filtrate so obtained. The enriched fractions should then be frozen and the material thus further separated added to that obtained from the original oil. The difficulty of maintaining sufficiently low temperatures during the filtration, especially for large amounts of oil, may make a separation from the whole oil impractical. In general, for the isolation and purification of the various constituents it is necessary to resort to chemical methods in addition to purely physical means.
After such preliminary treatment as indicated above, the oil or residual oil should be fractionated. This will result in a separation of the oil into a low boiling terpene fraction, and intermediate fraction, a fraction rich in oxygenated constituents, a second intermediate fraction, a fraction containing the sesquitcrpene constituents, and a distillation residue. The residue usually contains polymerization products and high boiling constituents, such as azulenic compounds, and the naturally occurring waxes in the case of citrus oils obtained by expression. These waxes show a tendency to "fix" part of the volatile components. If present to any appreciable extent these waxes should be freed from the more volatile components by steam distillation or by the addition of a water-soluble glycol (e.g., diethylene glycol), followed by vacuum distillation.206 The latter procedure will remove most of the volatile material from the waxes, leaving a relatively inodorous residue. The glycol may then be removed from the natural constituents by washing out with water or sodium chloride solution.
Should the original analysis show a high ester content it is usually best to fractionate the oil before saponification so that the ester may be obtained in a state of relative purity for a determination of physical properties. Its components may then be identified after saponification. Since the corresponding free alcohol usually is present with the ester, saponification of the whole oil (followed by fractionation) may be preferable, especially if only small amounts of ester are present.
The treatment of an oil or fraction with reagents for the purpose of separating and purifying various constituents may cause drastic changes to occur. This may give rise to new chemical compounds not originallypresent as such in the oil. Intra- and intermolecular rearrangements mayoccur as well as degradations and dehydrations. Such possibilities must beconsidered in the evaluation of the final results of the investigation.
For the identification of individual constituents which have beenseparated and purified from the oil, two general procedures are employed: (1) The determination of physical properties including melting point (or congealing point), boiling point, specific gravity, optical rotation, refractive index and solubility in alcohol of varying strengths. (2) The preparation of suitable derivatives, preferably solid compounds of definite melting point capable of purification by recrystallization. In general, the identification may be considered established if no depression is observed in the melting point when a derivative of the constituent is mixed with the corresponding derivative of a sample of known purity and constitution. The reader is referred to Volume II on the " Constituents of Essential Oils" for the properties of the individual compounds and for data on the melting points of certain frequently employed derivatives. In many cases compounds obtained by oxidation, reduction, and condensations may be used for identification.
Other methods are often employed in establishing the identity of a constituent or derivative : combustion to determine the percentage of carbon and hydrogen and to establish the empirical formula; molecular weight determinations, especially by cryoscopic methods ; molecular refraction; ignition of metallic salts, especially the silver salts of organic acids; determinations of the percentage of halogen in chlorides and bromides; and other procedures.
Detailed procedures for the separation of chemical groups and for the isolation and purification of individual constituents are given in Volume II dealing with the "Constituents of Essential Oils."

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