Group Title: Flavor research investigations, 1960
Title: Flavor research investigations
Full Citation
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 Material Information
Title: Flavor research investigations
Series Title: Citrus Station mimeo series
Alternate Title: Recovery and analytical separation of volatile flavor components of Valencia orange gas chromatography
Identification of chromatographically separated flavor components
Physical Description: 3, 3 leaves : ; 28 cm.
Language: English
Creator: Wolford, R. W
Attaway, John A., 1930-
Citrus Experiment Station (Lake Alfred, Fla.)
Florida Citrus Commission
Publisher: Florida Citrus Experiment Station :
Florida Citrus Commission
Place of Publication: Lake Alfred FL
Publication Date: 1960
Subject: Orange juice -- Flavor and odor -- Florida   ( lcsh )
Orange juice -- Varieties -- Florida   ( lcsh )
Orange juice -- Processing -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
non-fiction   ( marcgt )
Statement of Responsibility: Richard W. Wolford and John A. Attaway.
General Note: Caption title.
General Note: "September 21, 1960."
Funding: Citrus Station mimeo report ;
 Record Information
Bibliographic ID: UF00072345
Volume ID: VID00002
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 75260988

Full Text

Flavor Research Investigations

II. Identification of Chromatographically Separated Flavor Components
John A. Attaway and Richard W. Wolford

Before going into the techniques used for identification of the various
flavor components separated by gas chromatography, it would be well to consider
some of what is already known about the chemical make-up of orange flavor.

It has been demonstrated by many investigators that most of the flavor and
aroma of orange juice is associated with the volatile fraction. Although some
of this volatile flavor is found in the water-soluble material, the bulk of it
is generally considered to be located in an oil fraction, which is slightly
soluble in water.

Recently, Kirchner and Miller (J. Agr. and Food Chem. 5, 283, 1957) identi-
fied many of the important volatile constituents of fresh, canned, and stored
canned Valencia orange juice. They used 1500-3000 gallon quantities of juice
as starting material, and employed the standard techniques of evaporation, ex-
traction, and fractionation. The components which they identified from fresh
juice may be broken down into classes as follows:

Carbonyl Components
(Aldehydes and Ketones)


n-2-dodecanal (?)
C15 carbonyls




ethyl isovalerate
ethyl C6H802
citronellyl acetate
terpinyl acetate



Terpene Hydrocarbons


C15H24 (I)
Cl5H24 (II)



3 unidentified

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
992 c 9/21/60-JAA



- ---e. --


Of these four classes of materials, the terpene hydrocarbons are by far
the major components as far as quantity is concerned. However, in terms of
contribution to flavor and aroma the oxygenated components are believed by
most investigators to be more important. This is particularly true of the
carbonyl components which are thought to be responsible for the greater part
of the characteristic orange flavor. Consequently, the carbonyls seem to
offer the most promise for fundamental research on orange flavor.

Now returning to the immediate problem. Gas chromatographic methods showed
the presence of approximately 40 components in the organic extract obtained from
aqueous orange essence, and it became necessary to identify these components so
that the number, sizes, retention times and other characteristics of the peaks
on the gas chromatograms would have some meaning. This was, and is, no routine
job as chromatograms using equipment initially available were prepared from
only 3-50 microliters of the organic extract. A microliter is one millionth of
a liter or approximately one millionth of a quart. Consequently, definite peaks
are regularly produced by quantities which are only small fractions of a micro-
liter, thus making it most difficult to obtain enough material for accurate

This problem was partially solved in the following manner. The carbonyl
components, the importance of which have already been emphasized, react readily
with 2,4-dinitrophenylhydrazine to give characteristic solid derivatives called
2,4-dinitrophenylhydrazones. This reaction is shown as follows using acetalde-
hyde as an example:
CH3CHO + HH2N9HC6H3(N02)2 -- CH3CH = NN!IC6H3(N02)2 + H20
44 198 224 18

The stoichiometric advantages of this reaction are apparent. The dinitrophenyl-
hydrazone has a weight more than 5 times greater than the aldehyde. This is an
important advantage when dealing with such minute traces as are encountered in
gas chromatographic separations. Furthermore, the physical characteristics of
the dinitrophenylhydrazones are more favorable in many ways than those of the
free aldenyde. Whereas the latter is a volatile liquid with storage and handling
problems, the derivative is a crystalline solid without these problems. It may
be easily purified in small quantities by recrystallization. It has a character-
istic melting point, infrared spectrum, and Rf value when paper chromatographed.
Derivative color also gives useful information since saturated aldehydes always
yield yellow precipitates, unsaturated aldehydes orange to orange-red precipi-
tates, and ketones orange to red precipitates. Through the use of this reaction
it was possible to identify the carbonyl components responsible for a number of
peaks obtained on a gas chromatogram of the oil extracted from aqueous essence.
This was accomplished by bubbling the effluent gas from the column through an
ethanol solution of 2,4-dinitrophenylhydrazine sulfate. The arrival of the
component in the effluent gas is almost simultaneous with the recording on the
chromatogram of the peak produced by this particular component. When the com-
ponent is a carbonyl, a cloudiness is produced in the solution by the precipi-
tation of the dinitrophenylhydrazone. Repeated chromatographic runs make it
possible to accumulate sufficient quantities of the derivative for use in its
identification. However, when small quantities are present, 20 or more large
scale runs require ing about 1 hour each must be made to obtain the 2-5 milligrams
of precipitate required for study.
Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
992 d 9/21/60 JAA


A gas chromatogram of oil extracted from aqueous orange essence showed 11
peaks which were either definitely or tentatively identified as carbonyl
components. The steps in the identification of these carbonyl components using
their dinitrophenylhydrazone precipitates were as follows: (1) comparison of
gas chromatographic retention time of peak, (2) filtration and recrystallization
of derivative, (3) melting point determination of derivative, (4) paper chro-
matographic analysis of derivative, and (5) determination of infrared spectrum
of derivative. Steps 2 and 3 are fairly elementary as the precipitates re-
crystallize nicely from ethanol. However, care must be taken because of the
small quantities being handled.

Paper chromatographic analysis is best done using Whatman No. 3MM filter
paper. The choice of 2 solvent systems by Ellis, Gaddis, and Currie (Anal.
Chem. 30, 475, 1958) is based on the chain length of the parent carbonyl com-
pound. When there are 6 or fewer carbon atoms in the aldehyde or ketone, the
chromatogram should be developed d on paper impregnated with propylene glycol
using heptane-methanol as the developing solvent. When there are 6 or more
carbons, the paper should be impregnated with petrolatum and developed with
89% methanol. Since the derivatives are highly colored, the spots show up
without a spray reagery although a 10% NaOH spray can be used to intensify the
color of the spots.

The infrared spectra were determined by G. J. Edwards of this Station using
a rectangular KBr pellet, a modification of Jones, Holmes, and Seligman (Anal.
Chem. 28, 191, 1956), whereby approximately 1 mg of the derivative is weighed
into 200 mg KBr and the mixture converted into a pellet under high pressure. In
many instances it is possible to distinguish between an aldehyde and a ketone
derivative by the location of the N-H stretching frequency in the range of 3.01
to 3.09 microns.

Elemental analysis and molecular weight determination are other aids which
can also be used in identifying the dinitrophenylhydrazones.

On the gas chromatogram the following definite or tentative identifications
have been assigned to nine of the eleven carbonyl produced peaks: acetaldehyde,
hexanal, hexenal (?), cis-2-hexen-al-l, octanal, furfural (?), citral A, citral
B and carvone. To our knowledge this is the first time that hexenal has been
reported as a constituent of orange juice.

In addition to the carbonyl components, the two alcohols present in largest
volume have lhso been identified; these were identified as ethanol and linalool.
As they were present in large quantities, they could be identified directly by
condensation followed by infrared analysis and formation of the 3,5-dinitro-
benzoate derivative.

Florida Citrun -xperiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
992 e 9/21/60 JAA

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