VII. A PROCEDURE FOR THE INVESTIGATION OF THE CHEMICAL CONSTITUENTS OF AN ESSENTIAL OIL
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."
