Group Title: Citrus Station mimeo report - University of Florida Citrus Experiment Station ; CES 67-8
Title: Techniques for estimating the relative concentrations of flavor constituents in orange essences
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 Material Information
Title: Techniques for estimating the relative concentrations of flavor constituents in orange essences
Series Title: Citrus Station mimeo report
Physical Description: 5 leaves : ; 28 cm.
Language: English
Creator: Attaway, John A., 1930-
Citrus Experiment Station (Lake Alfred, Fla.)
Florida Citrus Commission
Publisher: Florida Citrus Commission :
Citrus Experiment Station
Place of Publication: Lake Alfred FL
Publication Date: 1966
 Subjects
Subject: Orange products -- Quality -- Florida   ( lcsh )
Frozen concentrated orange juice -- Quality -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Includes bibliographical references (leaves 4-5).
Statement of Responsibility: John A Attaway ... et al..
General Note: Caption title.
General Note: "400-10/4/66-JAA."
 Record Information
Bibliographic ID: UF00072473
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 76752078

Full Text

, Q



Florida Citrus Commission and
Citrus Experiment Station CES 67-8
Lake Alfred, Florida 400-10/4/66-JAA


Techniques for Estimating the Relative Concentrations of Flavor
Constituents in Orange Essences

John A. Attaway, Marshall H. Dougherty and Richard W. Wolford
Florida Citrus Commission
and
George J. Edwards
University of Florida Citrus Experiment Station
Lake Alfred, Florida


The satisfactory blending of concentrate from the evaporator with cutback
juice, essence, and coldpressed oil to produce a more flavorful frozen concen-
trated orange juice requires a knowledge of the concentration of the volatile
constituents in the essence. In the past the Chemical Oxygen Demand (COD)
method described by Dougherty (3) has been used to obtain this information with
some measure of success. However, this method is very general in its appli-
cability to all types of oxidizable material, and consequently it does not
furnish any information with regard to the relative amounts of the various
classes of organic components present. This paper describes three totally new
procedures which permit the analyst to estimate the concentration of oxygenated
terpenes as C10H180, the concentration of saturated aliphatic aldehydes as
octanal, and the concentration of alpha, beta- unsaturated aldehydes as citral.
In addition, the adaptation of a known procedure for estimating esters as ethyl
butyrate, and a modification of the COD method are described.


EXPERIMENTAL
Method for Oxygenated Terpenes as ClOH180. The procedure for determining
oxygenated terpenes relies on their reaction with a strongly acid solution of
vanillin to produce colored reaction products as previously described for thin
layer chromatographic analysis (1), (2). Extensive experimentation was carried
out using vanillin solutions of different strengths and varying degrees of
acidity to determine the optimum conditions for studying solutions. Heating in
a water bath for various lengths of time to drive the color reaction to com-
pletion was also tried. However, it was found that the use of a solution of
2 g. vanillin.in 100 ml. cone. H2S04 solved the problem entirely. The intensity
of the blue-green color produced was ideal for colorimetric measurement, and the
heat of mixing between the H2S04 and the aqueous essence forced the reaction to
completion within 5 minutes without the use of supplementary heating. The
visible spectra of the essence-vanillin reaction product was determined over
the 400-900 mj range using the Beckman DK-2 Recording Ultraviolet-Visible
Spectrophotometer. An excellent analytical peak was found in the region of
580-610 mp. This same peak was obtained when using an aqueous solution contain-
ing equal parts of citronellal, linalool, terpinen-4-ol, and alpha-terpineol,
all compounds of empirical formula CO1H180. Consequently, this solution was
selected as the reference mixture. One gram was dissolved in a liter of 80%
ethanol to give a stock solution of 1000 ppm C10H180, which was diluted to the
25, 50, 100, 150, and 200 ppm levels for preparation of the standard curve.

In the analysis all quantities were measured using volumetric pipets as
follows: 1 ml. essence or standard solution was added to 10 ml. water in a











25 ml. Erlenmeyer flask. Then, 5 ml. of vanillin/H2S04 solution was added with
swirling. The hot solution was allowed to stand for 5 min., following which the
optical density (O.D.) was read using the 585 mn filter in a Fisher Electro-
photometer II, or at 600 mA in a Bausch and Lomb Spectronic 20. A blank, in
which 1 ml. of water was substituted for the essence or standard solution, was
used to set the colorimeter to 0.000 O.D. Readings were expressed as ppm
C10H180.

Method for Saturated Aliphatic Aldehydes as Octanal. This procedure was
based on the ability of aldehydes to catalyze the reaction between p-
phenylenediamine and hydrogen peroxide to form a black compound known as
Bandrowski's base (5). When saturated aliphatic aldehyde solutions or orange
essences, reacted according to the procedure, and orange color was produced
which was visibly darker than the orange color obtained from the blank or
from solutions of non-aldehydic components. The intensity of this color varied
with the concentration of saturated aliphatic aldehydes. It gave a broad
absorption maximum in the region of 425-475 m4. Unsaturated aldehydes and
aromatic aldehydes gave colors which did not appreciably increase the O.D. at
this frequency. The ketones, carvone and methyl heptenone, which are known to
occur in citrus oils also did not interfere. In Table I are shown relative
O.D. values of 500 ppm solutions of various saturated and unsaturated aldehydes,
These values were obtained with the Fisher Electrophotometer II.


Comparative Effects


table I
of Aldehydes on Reagent


Aldehyde
formaldehyde
acetaldehyde*
n-butyraldehyde
n-valeraldehyde
alpha-ethylbutyraldehyde (C6)*
n-octanal*
n-nonanal*
n-decanal*
n-undecanal*
n-dodecanal*
2-hexenal*
2,4-hexadienal
citronellal*
citral*
nonen-2-al-l
furfural*
phenylacetaldehyde


Type
saturated
I
If

I"
I"
"I

"I


unsaturated
a I
11
"I
11

aromatic


Optical Density
0.392
.640
.552
.403
.349
.170
.120
.133
.078
.172
.000
.020
.082
.000
.000
.042
.050


Has been reported in orange essence.
Has been reported in orange essence.


It is seen that the highest values are given by the saturated
particularly those with short carbon chains, while citral and
zero readings. However, the data for essences were expressed

Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida.
400-10/4/66 JAA


aldehydes,
2-hexenal gave
as ppm octanal










because that aldehyde is always found in orange essences and oils in substantial
concentration. Since standard octanal solutions were used to prepare the
reference curve, the presence of significant quantities of acetaldehyde in a
particular essence will give it an inflated value.

This reaction was found to be very time dependent. Consequently, the
preparation of a standard curve with each set of samples is recommended. The
standard solutions were prepared by dissolving 1 g. of octanal in 1 liter of
80% ethanol to give a 1000 ppm solution which was diluted to the 150, 250,
350, 450, and 650 ppm levels. Three solutions with octanal values nearest
those of the essences to be tested were selected to prepare the standard curve
for a particular set of essence determinations.

All quantities were measured with volumetric pipets according to the
following procedure: 1 ml. essence or standard solution was added to 10 ml.
water in a 25 ml. Erlenmeyer flask, following which 2 ml. 3% H202 was added
with swirling. Finally, 1 ml. of 0.25% aqueous p-phenylenediamine solution
was added with swirling. In exactly 15 minutes the O.D. was read using the
425 mj. filter of the Fisher Electrophotometer II, or at 435 mp with the Bausch
and Lomb Spectronic 20. When several samples were run at the same time as a
set, the p-phenylenediamine was added to all of them in a predetermined order
without interruption. Then, the O.D.'s were read in that same order without
interruption so that the time of reaction for each sample was practically the
same. Values were expressed as ppm octanal.

Method for Unsaturated Aldehydes as Citral. The technique for unsaturated
aldehydes depends on their ability to combine with primary aromatic amines to
form colored Schiff bases (4). The particular aromatic amine used was
o-dianisidine. Under the conditions of the analysis the alpha, beta-unsaturated
aldehydes gave intense greenish-yellow to red colors. The DK-2 showed the ab-
sorption maximum to occur near 380 mi. A blue color was obtained with both the
blank and the saturated aliphatic aldehydes.

Standard reference solutions of 5, 10, 20, 30, 40, and 50 ppm were prepared
by dissolving 1 g. citral in a liter of 80% ethanol and diluting. The analysis
was carried out as follows using volumetric pipets: 1 ml. of 2.5% wt./vol.
o-dianisidine in glacial acetic acid was added to 10 ml. essence or standard
solution in a 25 ml. Erlenmeyer flask. The sample solutions turned light to
dark yellow depending on concentration, while the blank remained lavender.
Readings were taken at 385 mp, using the Bausch and Lomb Spectronic 20, and
expressed as ppm citral.

Method for Esters as Ethyl Butyrate. The method for esters, which is
based on hydroxamic acid formation, is now being used in the industry. However,
it was adapted to ethyl butyrate as standard, rather than ethyl acetate, because
the former has been found to be the major ester in orange essence extracts. The
standard solution was prepared by dissolving 1 g. of ethyl butyrate in a liter
of 80% ethanol and diluting to the 5, 10, 25, 50, and 100 ppm levels. The
analysis was carried out as follows using volumetric pipets for all liquid
measurements: 2 ml. of 13.9% aqueous hydroxylamine hydrochloride solution was
added to 5 ml. essence in a 25 ml. Erlenmeyer flask. Immediately, 2 ml. of
3N NaOH was added and the solution allowed to stand for 5 minutes, following
which 2 ml. of 4N HC1 was added. Finally, 2 ml. of FeC13 was added and the
O.D. read at 525 mjp. The blank was yellow and the ester solutions progressively

Florida Citrus Experiment Station
and Florida Citrus Commission,
Lake Alfred, Florida.
400-10/4/66-JAA









more orange as concentration increased. The results were calculated as ppm
ethyl butyrate.

Method for COD Determination. The new method for COD determination
differed from the old method in the distillation procedure. It was carried
out as follows: 50 ml. of sample was diluted to 100 ml. with distilled water
in a 500 ml. round bottom distilling flask. Boiling chips were added and the
flask was fitted into a distillation apparatus equipped with an oil trap.
Fifty ml. of oil free distillate was collected in a graduated cylinder. The
distillate, or an appropriate aliquot diluted to 50 ml., was transferred to a
50 ml. round bottom flask containing a few glass beads. Then, 25 ml. of 0.25N
K2Cr207 was added followed by 75 ml. of reagent grade H2SO4. The flask was
fitted to a water cooled condenser and refluxed for 10 min. The mixture was
then cooled in running water and the % transmission determined using the Fisher
Electrophotometer II with the 650 mj filter. The COD was read from a standard
curve based on sucrose (6).


RESULTS AND DISCUSSION

Table II shows the data obtained from 17 different essences. Values are
shown from ppm COD, ppm oxygenated terpenes as C10H180, ppm esters as ethyl
butyrate, ppm saturated aliphatic aldehydes as octanal, and ppm alpha, beta-
unsaturated aldehydes as citral. The increase in concentration of esters and
saturated aldehydes with maturity in Hamlin essence is particularly interesting.
In the 4 time-of-harvest experiments, samples 14-17, the esters increased from
only a trace on November 16, 1965 to 10 ppm on January 19, 1966, while the
saturated aldehydes from 0 to 195 ppm. Oxygenated terpenes and unsaturated
aldehydes also increased, but with less regularity.

Samples 10 and 11 show an interesting comparison between essences made
from a single lot of fruit at two different locations and two different essence
recovery systems. The fruit was Florida Valencias, half of which were processed
at the University of Florida Citrus Experiment Station, and the other half at
the USDA laboratory in Albany, California. It is seen that the essence prepared
here was stronger in COD and oxygenated terpenes, while that prepared in
California was higher in esters and aldehydes.

Samples 8 and 9 are also interesting. Although these essences are both
fairly dilute and equivalent in concentration as determined by COD, the new
tests show sample 8 to give much higher values in all tests. It actually gave
the highest ppm C10H180 recorded.


LITERATURE CITED

1. Attaway, J. A. and R. W. Wolford. 1965. Isolation and Identification
of Volatile Flavor Components in Recovered Orange Essences Using Gas
Chromatography. Proceedings of the Fifth International Symposium of Gas
Chromatography. The Institute of Petroleum, London. PP 170-179.


Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida.
400-10/4/66 JAA









2. Attaway, J. A., R. W. Wolford, and G. J. Edwards. 1965. Determination
of Esters by Thin Layer Chromatography. Anal. Chem. 37, 74-76.

3. Dougherty, M. H. 1964. Method for Measuring the Water-Soluble Volatile
Constituents of Citrus Juices and Products. Citrus Station Mimeo Report CES 65-7.

4. Feigl, Fritz. 1946. Qualitative Analysis by Spot Tests, Third Edition.
Elsevier Publishing Co., New York, N. Y. PP 340-341.

5. Ibid. PP 345-346.

6. McNary, R. R., M. H. Dougherty, and R. W. Wolford. 1957. Determination
of the Chemical Oxygen Demand of Citrus Waste Waters. Sewage and Industrial
Wastes 29(8), 894-900.


Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida.
400-10/4/66 JAA







Table II
Comparative Data from Analysis of 17 Essences Representing 5 Citrus Varieties


Sample
Number

1


COD
ppm

57,700


C10H180
ppm

167


Fruit variety used
as source of essence

Valencia-Pineapple
blend
Valencia
Pineapple
Pineapple
Hamlin
Valencia, 1965
Valencia, 1965 (#391)
Pineapple, 1965
Valencia, 1965
Florida Valencia, #451
California, #457
Temple
Grapefruit
Hamlin, picked 11/16
Hamlin, picked 11/30
Hamlin, picked 12/21
Hamlin, picked 1/19


Esters as
ethyl butyrate
ppm

22


22
20
13
32
14
13
19
9
19
30
7
6
trace
2
4
10


Aldehydes
Saturated aliphatic
as octanal
ppm

551


523
523
408
398
115
195
451
160
485
2255
115
0
0
95
130
195


Unsaturated
as citral
ppm

18


18
44.5
26.5
25
10
10
19.5
11.5
17
25
11
1.5
1.5
6
11.5
10


Florida Citrus Commission and
Florida Citrus Experiment Station,
Lake Alfred, Florida.
400-10/4/66 JAA


56,500
55,000
42,000
36,500
25,500
22,800
15,150
14,400
49,000
30,000
3,300
4,340
8,000
8,000
25,000
18,000




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