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ApPLE STORAGE VOLAfiLESu EVALUA'HON BY 6“
CHRGMATOGRAPW AND MASS SPECTRQMETK‘I

'E'hosig for the Degree» {:5 RED.
MICHIGAN STATE UNIVERSE?
Pia Angelini
1963

THESIS

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thesis entitled

Controlled Atmosphere Apple Storage Volatiles -
Evaluation by Gas Chromatography
and Mass Spectrometry

presented by
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has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Food Science

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ABSTRACT
CONTROLLED ATMOSPHERE APPLE STORAGE VOLATILES -
EVALUATION BY GAS CHROMATOGRAPHY
AND MASS SPECTROMETRY

by Pio Angelini

Chemical compounds that diffuse out of apple fruits during storage
under controlled atmosphere conditions, described generally as volatiles,
were studied using the techniques of low-temperature high-vacuum distil-
lation, gas chromatography, and mass spectrometry. Identification of
- apple storage volatiles was attempted from storage atmospheres, molecular
sieves, cold trap condensates, and activated carbon extracts.

Gas chromatography employing a hydrogen flame ionization detector
proved to be a simple, direct, and rapid method for ethylene concentra-
tion studies in controlled atmosphere apple storage rooms. The type of
detector used was an important feature in this procedure; insensitivity
to normal amounts of air gases, carbon dioxide, and water was required
plus a high sensitivity to organic compounds. Ethylene concentrations
were followed in eleven controlled atmosphere apple storages. In all
cases the ethylene concentration increased after sealing the storages
until it reached a maximum and then it gradually leveled off at this
maximum concentration. The ethylene concentrations then remained con-
stant, with few slight fluctuations until the storage rooms were openedc
The air changes of conventional controlled atmosphere storages are
directly proportional to the respiration rate of the fruit. From this
knowledge and the equilibrium ethylene concentration found in the
storages, it was deducted that ethylene production by apples is propor-

tional to reapiration rate of the apples. The equilibrium ethylene

concentrations assumed different values in some Storages, ranging from
concentrations of 1250 ppm to 250 ppm. Storages containing the same
variety of apples assumed approximately equal equilibrium ethylene
concentrations. These results in conjunction with the knowledge of
controlled atmosphere storages suggest a difference in the amount of
ethylene produced per unit amount of oxygen used for respiration among
apple varieties.

Qualitative analyses of cold trap condensates from storage atmos-
pheres by gas chromatography and mass spectrometry led to the identifi-
cation of only three compounds: ethylene, ethanol, and acetaldehyde.
Extracting activated carbon that was in contact with a controlled atmos-
phere containing McIntosh apples for one season produced a multitude of
components. Gas chromatograms of isopentane extracts of the activated
carbon show more than thirty peaks. The high complexity of the compounds
in the extract and lack of adequate reference mass spectra prevented the
definite identification of the extracted compounds. However, tentative
identification of saturated and unsaturated aldehydes containing from
four to ten carbon atoms was made from mass spectra of isopentane
extracts of the activated carbon.

Gas chromatographic analyses of cold trap condensates of storage
atmosphere were used in an attempt to determine the concentrations of
other storage volatiles. Ethanol was the only volatile found by this
method and was estimated at a concentration between 100 ppb and 2 ppm
in one storage room. Further development of techniques using gas
chromatography and mass Spectrometry appears very promising in apple

storage volatile studies.

CONTROLLED ATI'DSPHERE APPLE STORAGE VOLATHES -
EVALUATION BY GAS CHROMATOGRAPHY AND MASS SPECTROI'ETRI

By
Pic Angelini

A THESIS

Submitted to the College of Agriculture,
Michigan State University of Agriculture and Applied Science
for the degree of

DOCTOR ' OF PHILOSOPHY

Department of Food Science

1963

63% +1
9/e/c. 7‘

§CKNOWLEDGMENTS

Due acknowledgments are hereby expressed by the author to all
who knowingly'or unknowingly affected the doctoral program undertaken,
of which this thesis is a part. The members of the guidance committee -
Chairman, Dr. I. J. Pflug, Fbod Science Department; Dr. D. H. Dewey,
Horticulture Department; Dr. J. L. Fairly, Biochemistry Department;
Dr. J. B. Brunner, Food Science Department; Dr. B. S. Schweigert,
Chairman, Fbod Science Department; and Dr. R. J. Evans, Biochemistry
Department - were most essential to the doctoral program and the
arrangement of the thesis in its final form. The experimental con-
trolled atmosphere generator used in part of this study was provided
and installed by the Whirlpool Corporation. The Analytical Chemistry
Section, Pioneering Research Division, at the Quartermaster Research
and Engineering Command provided the facilities for the analyses

carried out by mass spectrometry.

TABLE OF CONTENTS

INTRODUCTION ..................................................
REVIEW OF LITERATURE ..........................................
Discussion of Literature Review .........................
EXPERIMENTAL ..................................................
Apparatus and Methods ...................................
LowATemperature High-vacuum.System ................

Mess Spectrometer .................................

Gas Chromatograph .................................

Procedures and Results ..................................
Exploratory Studies ...............................

Molecular Sieve Adsorption of Velatiles ...........

Activated Carbon Extraction .......................

Gas Chromatography of Atmosphere Samples ..........

Conditions for Direct Analysis ...............

Column Selection .............................

Identification of Second Peaks in
Chromatograms of Rooms 10 and 11 Atmospheres ..

Standard Curve for Ethylene Concentrations

inAir OOOOOOOOOOOOOOOOOOOOOO0......0.00.0.0.
Gas Sa-Ulpling Proced‘lres .COOOOOOOOOOOOOOOOOOOO

Results CCIOOOOOOOOOOOOOOOOOOO...0.0.0.0000...

Attempt to Determine Concentration of Other
volatiles in Controlled Atmosphere Storages ..

DISCUSSION .0OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOO

Apparatus OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOO...

Page
1
3

12
15
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l6
l6
l6
l7
17
2O
21
25
25
26

27

29
30

32
35
35

Page
Mass Spectra of Apple Storage Atmospheres ............... 36
Analysis of Extract from Activated Carbon ............... 37

Ethylene Concentration in Controlled Atmosphere
Storages 0.0.0....OOOOOOOOOOOOOOOOOOOOOOO00.0.0000...00. LO

Concentration of Other volatiles ........................ 43
Solubility and volatility’ ............................... 47
Source of volatiles ..................................... 50
Limiting Factors and Suggestions ........................ 51
SUMMARY AND CONCLUSIONS ....................................... 53

BIBLIOGRAPHY 0.0.0.0000...0.0.0.0000...0.0...OOOOOOOOOOOOOO0.0. 56

INTRODUCTION

The volatile chemical compounds produced by plant tissues are a
matter of interest theoretically and practically. All of these volatile
compounds are believed to be produced by cell metabolism and there is
much.evidence showing that at least some are products of the energy
producing metabolic process of cell respiration. Identification of
these metabolic products would aid in determining their route of bio-
synthesis and how they affect tissue metabolism.

Identification studies of plant tissue volatiles have been con-
ducted for decades. The more abundant volatiles, ethylene, ethyl alcohol,
and acetaldehyde, have been positively identified. Although these com-
pounds account for the majority of the total quantity of volatiles
produced by plant tissues, there is sufficient evidence to conclude that
the volatiles already identified are only a few of the total number of
volatile products produced by plant tissues.

Apple fruit volatiles are important in the storage of apples ‘
because the fruit is exposed to the volatiles for long periods of time.
Some researchers believe that the storage disorders of scald and pre-
mature fruit ripening are due to apple volatiles. Attempts to control
these problems by storage practices, such as the use of shredded oiled
paper, air purifiers and chemicals have been partially successful in
some instances. A higher concentration of volatiles is present in
fruit generated controlled atmosphere storages (0.02 air changes per
day) than in regular storages ( 1 to 2 air changes per day). The

higher volatile concentration in controlled atmosphere storages may have

a greater effect on apples held in these storages than apples held in
regular storages since the volatiles are in contact with‘susceptible
tissues for a longer period of time. A better understanding of apple
storage volatiles, particularly their chemical nature and concentrations,
is important from both its academic and practical standpoints, and
ultimately may be of great utility to the apple storage industry.

The purpose of this study was to increase the knowledge of
volatiles in controlled atmosphere apple storages by: (l) qualitative
and quantitative identification of apple storage volatiles; (2) estab-
lishing a feasible method for following the concentration of one or more
apple storage volatiles; and (3) Comparing the effects of apple variety,
type of carbon dioxide scrubber used, and activated carbon units on the
concentration of one or more of these volatiles in controlled atmosphere

storages.

W

The effect of storage volatiles on apples, especially with respect
to the physiological disorder of storage scald, has been a controver-
sial subject and still is not resolved. Brooks and Cooley (1919),
Brooks (1924), and Berl (1951) believe scald is caused by accumulations
of certain gases produced by apples in their life processes. On the
other hand Gerhardt (1953) presented evidence that these apple volatiles
do not cause scald. In reviewing the literature, Smock and Neubert
(1950) report that orchard fertilization, spray program, and seasonal
factors such as rainfall and temperature all seem to affect the suscep-
tibility of apples to scald. Greater emphasis, however, has been placed
on removing the apple volatiles from apple storages and prestorage
treatment of the fruit itself.

Gene (1934) identified ethylene (CZHZ) as the gas in the emana-
tions from ripe apples which has striking physiological effects on plant
tissues. Smock (1942) showed that emanations of one lot of apples
produced a ripening effect as evidenced by stimulated respiration of a
second lot of apples. He believed the ripening effect was largely due
to ethylene. Smock and Neubert (1950) point out that Rhode Island
Greening and Cortland apples stored in the presence of emanations of
McIntosh apples scalded much faster than when stored alone. They also.
point out that off-flavors and off-odors in storage apples may be
absorbed from.volatiles produced by surface molds or bacteria, from
volatiles from certain types of wood like yellow pine, or from other

\
vegetables or varieties of apples in the storage rooms.

Basically two approaches have been followed toward reducing the
problem of apple storage volatiles (Phillips, 1953). The more immediate
approach has been to investigate various methods of storage management
and fruit.handling to reduce storage volatiles. The long-range approach
has been to identify the volatile chemical compounds produced by apples
in storage.

Brooks and Cooley (1919, 1919 A) utilized paper impregnated with
mineral oil either as wrappers for apples or shredded and mixed with
the apples for removal of volatiles from apple storages. This method
has proved rather effective for controlling scald in many cases;
however, Smock and Neubert (1950) cite a number of objections to this
method: (1) it is costly and fails to control scald in severe cases;
(2) the mineral oil also absorbs foul storage room odors; and (3) fruit
buyers object to oily paper in packages of apples.

Phillips (1953) reviewed Smock's modification of antanel's idea
to use charcoal to remove apple storage volatiles. By impregnating
activated charcoal with bromine, Smock attempted to remove, along with
other volatiles, the metabolic stimulant ethylene. He found the high
corrosiveness of bromine made it impractical for use in storages.
Nevertheless, activated carbon has been reported by Gross and Smock
(1945) to be an effective means of solving the pine wood volatiles
problem; by Uota and Smock (1948) as the most effective means tried for
removing food odors and volatiles; and by Southwick and Smock (1948) to
control scald more satisfactorily than did shredded oiled paper.
van Doren and Bullock (1950) report significant scald reduction, in-

crease in storage life, and control of off-flavors and storage odors

with highpgrade activated coconut shell carbon. Gerhardt (1950) showed
that, in commercial apple storages,'activated carbon failed to remove
ethylene and did not appreciably reduce other volatiles in the storages.
However, Gerhardt reports that activated carbon reduced the volatiles
to one third in small experimental apple storages as compared to the
control storages. Gerhardt, Sainsbury, and Siegelman (1953) report
that air purification with activated carbon failed to reduce signifi-
cantly storage scald of apples and that their study cast serious doubt
on the value of air purification of fruit storages. Despite conflicting
results concerning the control of volatiles, activated carbon has been
'widely employed by apple storage operators.

Recently, the treatment of apples with two chemicals, diphenylamine
(DPA) and 1, 2-dihydro-6-ethoxy-2, 2, A-trimethquuinoline (ethoxyquin
or Stop-Scald), has been extensively investigated by Mattus (1962) and
Hardenburg and Anderson (1962). Both DPA and Stop-Scald dips proved to
be more effective as scald inhibitors than oil paper wraps for all apple
varieties tested. Tests by Hardenburg and Anderson (1962) showed that
EPA was appreciably better for scald control than Stop-Scald. Several
tests by Hardenburg and Anderson (1962) using DPA dips have produced
100 per cent scald control. These authors indicate that dip treatments
of apples with either DPA or Stop-Scald are feasible methods for con-
trolling scald in commercial apple storages.

Power and Chesnut (1920 and 1922) found that apple fruits con-
tained several organic compounds which are believed to cause the charac-
teristic apple aromas. They used steam distillation to remove and

concentrate these compounds from the cortex and epidermal tissues

separately. They identified acetaldehyde and ethyl alcohol and show
some evidence for the presence of some methyl alcohol in the cortex.

A neutral ester fraction was obtained which upon hydrolysis yielded amyl
alcohol and formic, acetic, caproic, and a small amount of caprylic
acids. The apple peels contained, in addition, formic and acetic acids
in the distillate and from.McIntosh apple peels geraniol was identified.
They did not determine whether geraniol was esterified. Furfural was
also found but believed to be an artifact.

White (1950), modifying the method of Milleville and Eskew (191.4),
proceeded to concentrate apple juice by 150 fold. This concentrate was
fractionated by distillation both at atmospheric pressure and reduced
pressures (375 mm Hg absolute). Alcohols composed 92 per cent of the
essence which included: methanol, ethanol, propanol, 2-propanol,
butanol, isobutanol, d-2-methyl-l-butanol, and hexanol. Carbonyl com-
pounds (6 per cent) included: acetaldehyde, acetone, caproaldehyde, and
2-hexenal. Esters (2 per cent) included ethyl bugmate and ethyl caproate.
Components of other esters were identified as methanol, ethanol,
2-propanol, butanol, and formic, acetic, propionic, butyric, and caproic
acids. The concentration of these compounds in the original apple juice
was approximately 50 ppm.

Attempts to correlate acetaldehyde and ethyl alcohol with storage
scald and other storage disorders have been, for the most part, non-
conclusive (Smock and Neubert, 1950). Gerhardt (19%2) found large
accumulations of acetaldehyde in apples showing severe scald. It has
not yet been determined whether the scald is a result of the acetaldehyde
or vice versa. Off-flavors and physiological disorders may be due to the

presence of ethyl alcohol or acetaldehyde or both (Gerhardt, 1942).

Acetaldehyde and ethyl alcohol accumulations have been noted during
senescence (Smock and Neubert, 1950) or in low-oxygen atmospheres
(Thomas, 1925). Ethyl alcohol seems to act as a poison and depresses
the respiration rate when it accumulates in apple tissue.

Halls (1942) achieved some success in both qualitative and
quantitative analysis of apple volatiles. Sulphuric acid activated
with silver sulphate was used for total volatile adsorption. Quanti-
tative measurements of volatiles were expressed as amounts of carbon
dioxide produced by combustion of the absorbed volatiles. Ethylene was
shown to form a high proportion of apple volatiles absorbed by silver-
activated sulphuric acid. walls then experimented with three adsorbents
for collecting the odorous components of apple volatiles: calcium
chloride, silica gel, and adsorbent carbon (Desorex III). Calcium
chloride was the best of these three adsorbents, and from it two frac-
tions were obtained by extracting the volatiles by steam distillation.

A highly odorous oil, fraction A, composed largely of aliphatic esters,
was obtained from all apple varieties studied. A minute amount of
small crystals, fraction B, which was less volatile than fraction A,
was obtained in an appreciable amount, but only from apple varieties
susceptible to scald. Hydrolysis of fraction A yielded formic and
acetic acids and'an oil having the odor of amyl alcohol. An insuffi-
cient quantity of B was obtained to permit further examination.

Thompson (1951) found cold trapping to be very useful for collect-
ing volatiles given off by Granny Smith apples. The volatiles were con-
densed by passing purified air over the fruit and through a spiral

condenser cooled in liquid oxygen. The volatiles were hydrolyzed with

0.2N sodium hydroxide and the alcohols were distilled off while the
solution was alkaline; the acids were distilled off after acidifying
the solution. Sodium salts of these acids were obtained by bringing
the acid distillate to pH 8 with sodium hydroxide and evaporating to
dryness. By first cenverting the sodium.salts of the acids to
hydroxamic acids, followed by chromatographic separation on paper, the
acids were identified as formic, acetic, propionic, butyric (probably
normal), valeric, and caproic acids. Fbrmic and acetic acids were shown,
to be both in the free and esterified form. All other acids were found
in the esterified form. Ethanol and n-propanol were identified by paper
chromatography after converting them to the respective hydroxamic acids
and methanol was identified by a specific color test. The acids ob-
tained were virtually free from carbonyl, hydroxy, or unsaturated groups
and the alcohols were predominantly primary and saturated.

. Thompson and Huelin (1951) studied the production of esters by
Granny Smith apples stored at 0°C and 20°C. The esters were collected
by passing purified air over the fruit samples and through three cold
traps in series. The first trap consisted of a U-tube cooled in an ice-
calcium chloride mixture. ‘The second and third traps each consisted of
two spiral absorbers containing 30 ml. of ether and cooled in a dry ice-
alcohol mixture. The aqueous portion from the first trap was extracted
with ether. The ether extracts from all three traps were dried over
calcium chloride and made up to volume in a volumetric flask. Apple
ester concentrations were determined colorimetrically by conversion of
the esters to hydroxamic acids and then forming ferric hydroxamate

complexes. Thompson and Huelin (1951) also show that apples stored at

0°C appeared to increase steadily in ester production while those stored
at 20°C increased to a maximum of ester production and then decreased.
Early picking reduced whereas higher air flow rates increased ester pro-
duction. Apples stored in atmospheres of 6 per cent oxygen first
increased and then decreased in ester production. Ester production by
apples stored in air remained constant or increased during the storage
period.

Huelin (1952) continued the study of Thompson and Huelin (1951)
and identified the volatile aldehydes and ketones produced by Granny
Smith apples. Collection was carried out by passing purified air over
the fruit sample and through a spiral absorption trap which was cooled
in an ice bath and contained 30 ml. of concentrated metabisulfite solup
tion (25 g. K28205 per 100 ml.). The aldehydes were removed by oxida-
tion. Then dinitrophenylhydrazones were prepared, first of the total
carbonyl compounds and then of ketones alone. Identification of the
dinitrophenylhydrazones was carried out by paper chromatography.
Acetaldehyde was found to be the predominating carbonyl compound.
Smaller amounts of propionaldehyde and acetone were also found. The
presence of acetaldehyde and prepionaldehyde was confirmed by conversion
to hydroxamic acids which were identified by paper chromatography.

Turk and Smock (1951) used cold traps and activated carbon to
collect the volatiles from apples. The volatiles were driven off the
activated carbon by heat. Low-temperature high-vacuum distillation
procedures were used to fractionate the samples and mass spectrometry
and infrared spectroscopy were employed in the analysis of the samples.
From the mass spectra data ethanol and acetaldehyde were identified,

but most of the mass spectra data could not be resolved to show

 

10

specific compounds. Prominent peaks of the mass spectra indicate
aldehydes, acetates or methyl ketones, and secondary alcohols. The
infrared spectra of the samples is typical for a mixture of esters,
aldehydes, and perhaps alcohols, confirming the mass spectra data.

Henze et. a1. (1953) used a different approach to recover apple
storage volatiles. A quantity of activated carbon was acquired from an
air purification unit in a commercial refrigerated apple storage at the
close of the storage season. At least twenty extractions of the carbon
were made with other as the solvent. The ether was evaporated off over
a steam bath and the residue was analyzed. The neutral portion of the
extract, 98 per cent, consisted mainly of esters. By hydrolysis of this
fraction and subsequent chromatography on silicic acid columns, methyl,
ethyl, isopropyl, n-butyl, and n-hexy alcohols and proprionic, n-butyric,
n-caproic, and n-caprylic acids were identified. An impure amyl alcohol
and formic, acetic, and impure valeric acids were indicated present.

The free acid and free alcohol fractions were found to contain the same
alcohols and acids as did the ester fraction with the exception of
n-caprylic acid.

Meigh (1956, 1957) conducted a rather extensive study of volatile
compounds produced by three varieties of apples: Edward VII, Laxton
Superb, and Cox's Orange Pippin. The volatiles were collected by first
passing air through air-tight cabinets containing the apples and then
through a series of three cold traps where the volatiles were condensed.
The first trap was a U-tube emersed in an ice-salt mixture. The other
two traps were cooled to dry ice temperature and contained a pure solvent

through which the air samples were passed.

The aldehydes and ketones (Meigh, 1956) were converted to
2:4 dinitrophenylhydrazones and were analyzed by paper chromatography,
ultraviolet spectroscopy, and melting point determinations. Of the
aldehydes and ketones, acetone was in most abundance and smaller amounts
of acetaldehyde, n-butanal, propanal, ethyl methyl ketone, and isobutanal
were identified. The alcohols were converted to 3:5 dinitrobenzoates,
which were separated by paper chromatography and the fractions were
estimated with an ultraviolet spectrophotometer (Unicam SP500). The
esters were identified by converting them to hydroxamic acids which
were estimated colorimetrically as a group, according to the method used
by Thompson (1951) and identified by paper chromatography. The alcohols
identified were ethanol, D-2dmethylbutan-1-ol, 2—methylpropanel-ol,
methanol, isopropanol, and a 06 alcohol. Esterified acids from Cl to
06 inclusive were found.

Quantitative determinations were carried out by Meigh (1956, 1957)
for the volatile compounds and their production was followed throughout
a storage season. No correlation was found between high rate of evolup
tion of volatile substances and a heavy incidence of scald. Apples
stored in normal air produced all the volatile compounds at a greater
rate than those stored in controlled atmosphere storage. The air
storage rates of volatile production tended to increase, while in gas
storage rates of volatile production tended to remain constant or‘
decrease through the storage season. The percentage recovery of these
volatiles using this method varied considerably. Recovery tests for
acetaldehyde varied from near 100 per cent recovery to as low as 31

per cent.

12

DISCUSSION OF LITERATURE REVIEW

Control of apple scald and storage volatiles has been.attempted
in many ways. The use of treated wraps with mineral oil or chemicals,
although effective, has serious drawbacks, such as unsightliness,
excessive handling, and absorption of foul storage room odors. The use
of activated carbon in a so-called air purifier in storages gives
inconsistent and questionable scald control when used. The recent
experimental results of Mattus (1962) and Hardenburg and Anderson (1962)
on prestorage treatment of apples with StOp-Scald or DPA sprays or dips
for scald control have been very satisfactory and these chemicals seem
feasible for scald control in commercial installations.

Although scald control appears possible through the use of chemi-
cal dips or sprays, the cause of scald still remains obscure. Growing
seasons and orchard management apparently affect the susceptibility of
apples to scald. The results in attempts made by Gerhardt et. a1. (1953)
support the argument that scald is not caused by apple volatiles. Never-
theless, it has not been shown that apple volatiles or any one apple
volatile are not responsible for scald.

The mechanism of scald control by Stop-Scald and DPA is still
unknown. In the case of activated carbon and mineral oil impregnated
paper, scald control is attributed to the removal of volatiles.

Although Phillips (1953) doubts the control of scald by activated carbon
air filters, he does state that charcoal dust on the surface of the
apple does control scald. This leads to the assumption that removal of

volatiles by adsorption or absorption is more effective if the volatile

13

trapping agent is closer to the apple surface. Meigh (1957) suggests
that the less volatile compounds evolved by apples are possibly more
directly associated with scald than more volatile compounds. The fact
that DPA and Stop-Scald are most effective when applied to the surface
of the apple than when applied to paper wraps indicates that the scald-
control mechanism of these two chemicals is similar to that of mineral
oil or activated carbon next to the apple surface; namely, removal of
volatiles from the apple surface. DPA and Stop—Scald are antioxidants.
The mechanism by which these antioxidants prevent scald is not known
yet. It may be possible that certain metabolic reactions of the fruit
are affected by these chemicals. They may either cause or prevent
oxidative changes of compounds on the surface of the apple and prevent
scald in this way. On the other hand, these are organic chemicals and
may be good absorbents for volatiles, thus removing them from the sur-
face of the apple.

Many organic compounds given off by apples have already been
identified, as seen in the literature review. Several compounds, such
as ethylene, methanol, ethanol, acetaldehyde, and esters as a group,
have been found by many investigators. Nevertheless, most of these
investigators have identified compounds which are not common with the
general findings. Experimental procedures and apple differences (variety,
storage conditions, growing conditions, and seasons)are believed to be
responsible for the differences. The differences in odor and taste of
different apples indicate a difference in volatile composition. Arti-
facts or destruction of compounds may occur with different methods of

analysis. It is also quite possible that some methods of analysis do

not detect some of the volatiles due to the low concentration of most
of the volatiles. Furthermore, the ester fractions separated have been
hydrolyzed and the component alcohols and acids identified but little
has been done to identify the esters as such. It is evident, therefore,
that more qualitative and quantitative information would be very useful
in order to determine the role of apple volatiles in storage scald.

A great deal of work has gone into obtaining the available infor-
mation on apple storage volatiles. The acquiring of more information
by most of the methods and techniques already used is hampered by the
lack of sensitivity, lack of specificity, and/hr great laboriousness.
The determination of ethylene by absorption in a mercuric perchloric
solution followed by reduction of cerate sulfate provides a specific
and sensitive analysis for ethylene in apple storages but is rather
cumbersome for frequent analysis. The sulfuric acid ceric sulfate
method of analysis for the non-ethylenic volatiles has provided useful
information but it is not specific and is cumbersome. More work with
mass spectrometry would appear to be a good way to further resolve the
work started by Turk and Smock (1950). Meigh (1957) also points out
that gas chromatography, with properly developed techniques, provides a

potential method to further the work on apple storage volatiles.

 

15

EXPERIMENTAL

The Review of Literature cites a rather extensive effort and
considerable success in the identification of apple storage volatiles.
Most of the methods and techniques used in previous studies give evi- -
dence for the low concentration of most of these volatiles in storage
room atmospheres. The sensitivity of most of the above-cited methods
is great but the limitations of these methods make them difficult to
use. The many different kinds of compounds make the specificity of most
of these methods a hindrance to complete identification of all the com-
pounds in a sample by any one method. In forming derivatives of certain
types of compounds, reactions with other types of compounds present may
destroy existing volatiles and/hr produce artifacts. Most of the methods
used thus far are also so cumbersome that a great deal of work is neces-
sary for any complete analysis of apple storage volatiles.

The methods and results of studies on apple storage volatiles and
similar problems indicate that the use of advanced physical-chemical
methods of sampling and analysis will solve many of the problems
encountered by most previously used procedures. Lowatemperature high-
vacuum distillation used in handling the volatiles will keep to a mini-
mum the destruction of the compounds and/hr formation of artifacts.

Mass spectrometry eliminates the formation of derivatives in identifi-
cation and lends itself well to analyzing samples prepared by low-
temperature high-vacuum distillation. Gas chromatography can be a
rapid method for concentration studies of previously identified

volatiles.

 

l6

APPARATUS AND METHODS

nghTemperature High-vacuum Distillation

A lowbtemperature high-vacuum distillation apparatus was designed
and constructed using a Cenco Byvac 2 vacuum pump and a single stage
mercury diffusion pump. All Joints in the apparatus were ground glass
of standard taper; glass vacuumptype stopcocks were used throughout.
Apiezon N stopcock grease was used on all stopcocks and small ground
glass joints. The pressure in the system was measured with a Pirani
gauge, type GP-llO, manufactured by Consolidated Electrodynamics Corpora-
tion. Pressures of less than 1 f1 Hg absolute were obtainable in this
system.. At pressures of 0-5f7 Hg absolute, the system gained 4/7! Hg
pressure per day over a period of 4 days with all stopcocks closed and

vacuum.pump off.

Mass Spectrometgy
A Model 21-1030 Mass Spectrometer, having a vibrating reed ampli-

fier, manufactured by the Consolidated Electrodynamics Corporation was
used in this study. The vibrating reed amplifier increases the sensi—
tivity of the instrument by ten-fold over the amplifier normally in-

stalled in the instrument.

Gas Chromatogggphy
An Aerograph Model A9600 HthI gas chromatograph with the hydrogen

flame ionization detector was used in this study. A one-millivolt,

Leeds and Northrup Speedomax H, Model S recorder was used to record the

17

output potential of the chromatograph amplifier. An Aerograph hydrogen
generator, Mbdel Ap650, was used to generate and meter both the hydro- '
gen and the air used for combustion by the hydrogen flame detection
assembly.

A 1/3"-5' stainless steel column packed with SE-BO on acid-washed
chromosorb W was purchased with the chromatograph. Two l/B"-lO' stain-
less steel columns were obtained from Wilkins Instrument and Research,
Incorporated. One of these was packed with 15 per cent CarbowaxelS-AO
on Chromosorb W; the other, with.40 per cent Castorwax on Chromosorb'w.
Column selection was based on the nature of compounds anticipated to be
present in apple storage volatiles, that is, ethylene and oxygenated
organic compounds. The operating conditions for the desired analyses

were determined by trial and error.

PROCEDURES AND RESULTS

Exploratory Studies

Two bushels of yellow transparent apples were placed in an air-
tight metal container and held at refrigerated temperatures, 34 to 38°F.
The 002 in the container was maintained constant by controlled ventila-
tion to prevent 002 injury to the fruit. The mass Spectrum of the
atmosphere within the container was obtained and is shown in Figure l.
Subtracting the mass spectra due to air gas from the mass spectrum of
the total sample, Figure 1, results in the mass spectrum shown in
Figure 2. From this resulting spectrum no positive identifications were

made. The concentrations of the compounds giving the spectrum in

Relative Intensity

Relative Intensity

1000 y

800-
700‘

600-

500‘ .

400‘

300‘

200*

100‘

 

50d

20 ~

10 i

 

/‘./

4470

 

 

I
I

I
I

 

 

 

 

Fig.

10

Fig.

16

fa r

2.

Iii a I I I1; . i“ f t L i 1 . “r
20 30 40 50 60 '70 8O 90 100
. 111/6

1. thee spectrum of CA storage atmosphere

T {III {JIfiIIIjIJI III I I I

7'" T T 1 Vfi' f! f

203040506070809010

m/e

Mass spectrum of CA storage minus mass spectra
of air gases

 

18

Figure 2 were too small to give sufficient spectra for identification,
but short chained alcohols and aldehydes are indicated by peaks at
m/e 45, 43, 31, and 29. Peak m/e 57'may indicate the possibility of
esters of propionic acid and is an interesting peak to observe.

Low-temperature highpvacuum (LTHV) distillation was used to remove
the highly volatile air gases that interfered with analysis of the apple
volatiles. Further fractionation of the samples was carried out by LTHV
distillation after the removal of air in an attempt to separate the
apple volatiles according to their volatility.

One sample was taken by filling an evacuated bottle with the
atmosphere from the container. The sample was held at -l96°C and all
gases volatile at this temperature were removed by pulling a high
vacuum on the sample. The compounds remaining were fractionated into
three parts. This was accomplished by holding the remaining compounds
at -155°C, -lOO°C, and at room temperature, respectively. Collection
of the volatile compounds at these three temperatures was accomplished
by allowing them to condense in a liquid nitrogen trap. Compounds
exerting vapor pressures greater than 5 4/ Hg between the temperatures
-l96°0 and -l55°C, -l55°C and -lOO°C, and -lOO°C and room temperature
were collected in the three fractions prepared from this sample. Mass
spectra of these three fractions are shown in Figures 3, 4, and 5.

After allowing for CO2 in the ~155°C to ~lOO°C fraction and water
and some 002 in the -lOO°C to room temperature fraction, no identifica-
tions can be made since only a few peaks with very low intensities
remain. Ethylene is indicated as present in the -155°C to -l96°C

fraction by m/e's 26, 27, and 28. Peaks seen at m/e's greater than 44

(D
O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3 80 1
I --| I 44.5800
’ " 45: 229.5
70 ‘ 813 165 7° * 393 2910
1 ‘
60 « 60 I I
I? 5’
a a
3 50 . a 50 4
3 B
x: c:
m o
5 a.
+3 30 « *3 30~
3 32' I
20 a» 20 4
10 if | 10 4
0L I I .I‘IIIIITIILIII] 'J‘ 0 I VI fr Irv
O 10 20 I) 1.0 50 60 O 10 Z) 30 40 50 60
m/e m/e
Fig. 3. Mass spectrum of CA Fig. 4. Mass spectrum of CA
storage atmosphere storage atmosphere
-155°C to -l96°C -lOO°C to ~155°C
fraction fraction
:1 I I I
a , off ,
.5” I ‘
«4 50 " I
m I
§ 1,0 I
.5 .
o
.5 3° ‘
+3
.3 20.
32
1° " I II I
0 ,j, IIIIIIIIIILfir' II [I‘llfi

 

01020 3040 5060776 80 90
. m/e

Fig. 5. Mass spectrum of CA storage atmosphere
room temperature to -lOO°C fraction

 

19

would not be expected in the fractions collected at temperatures lower
than -lOO°C but are present. This is believed to be due to contamina-
tion from previous samples by absorption of compounds by the stopcock
grease used in the high vacuum system. Disregarding the peaks contri-
buted by air, 002, and H20, m/e's 57, 43, and 41 are of greatest sig-
nificance. These peaks indicate the same families of compounds,
aliphatic alcohols, aldehydes, and ketones, indicated by the scan in
Figure 2.

Sampling procedures used thus far were not suitable for collecting
sufficient volatiles for identification. In the next test, samples were
collected by passing ten cubic feet of the chamber atmosphere through a
liquid air cold trap. This sample was also fractionated using LTHV
distillation. vacuum pumping was not as complete at -l96°C as it was
for the previous sample. Fractions prepared from this sample were be-
tween the temperatures of ~196°C to ~15500, -155°C to ~125°C, ~125°C to
~lOO°C, -lOO°C to -78°C, and -78°C to room temperature. Mass spectra
of these fractions are illustrated in Figures 6-10.

Figure 6 shows the presence of ethylene. Acetaldehyde was identi-
fied from the -l25°C to -lOO°C fraction, Figure 8, and both acetaldehyde
and ethyl alcohol were identified from Figure 9. No other positive
identifications could be made from these spectra. The m/e's above 44
show up in greater intensities in the higher temperature fraction, -78°C
to room temperature, than in the other fractions, as is expected from
the types of compounds, aliphatic alcohols, aldehydes, and ketones,
believed present and indicated by these m/e values. The LTHV distilla-

tion techniques, therefore, are effective in separating these compounds

 

Relative Intensity

 

 

 

 

 

 

 

 

 

HI 26:4720
27:5170
1750 . 28—off scale
1500 ‘ .
I
I
1250 ‘
n
1000 ~ j
750 4
500 i
V
250 « I
I
I
I
z
0 ,I§I I II . j
0 10 20 30 40 50
m/e
Fig. 6. Mass spectrum of

condensed CA storage
atmOSphere
~155°c to ~196°C.fraction

1750

1500

1250

1000

750

500

250

Fig.

 

 

 

 

5810
.I
I
.1
I
I
gig ILTIlHIIIY r gj
O 10 20 30 40 50
m/e
7. Mass spectrum of

condensed CA storage
atmosphere
-125°C to -155°C fraction

 

Relative Intensity

 

 

 

 

 

 

 

 

 

 

1176
175d ”I
150“
125*
b.
.p
100‘ “a;
a
o
.p
a
H
0
.3
.p
75~ .93
; :3;
50-4
25~
0..,I IIIIIIII
0 10 20 30 40 50 60
m/e '
Fig. 8. mass spectrum of

condensed CA storage
atmosphere

-lOO C to -125 C
fraction

175 -

150 I

125 ‘

a

q
U'l

50

25 I

Fig.

‘1

A

 

 

 

_.—_.. .. ~--.—-—--o—v—<o—. o~—A.——..--

 

 

 

 

_.._....-—-.-.- -_. a..-__,_ _-... l
.—

 

 

 

 

 

 

O

V

9.

I
M II III

I
10 20 30 40 50
m/e

mass spectrum of
condensed CA storage
atmosphere

~78 C to -100 C
fraction

 

Relative Intensity

200

175

150

125

100

75

50

25

1830'

 

 

I
. gr . IIhIY IIIT h I , g h,

0 10 20 30 40 50 60 7O 80 90 100
m/e

 

 

 

 

p—c———-—_

 

Fig. 10. Mass spectrum of condensed CA storage
atmosphere
room temperature to ~78°C fraction

 

 

20

from the more volatile air gases but not from water since water exerts
a significant vapor pressure above -78°C. Water vapor is present in

far greater amounts in apple storage atmospheres than are these other
compounds. This fact is quite evident when the mass spectrum of -78°C

to room temperature fraction is considered.

Melecular Sieve Adsorption of Vblatiles
An attempt was made to separate the volatiles from water by em-

ploying molecular sieves. One-sixteenthpinch pellets of Linde Melecular
sieves, type 5A, were activated by heating to 180°C and evacuating under
high vacuum (1-10‘6/ Hg) each time before using. Atmosphere samples of
various sizes were taken from a 35-bushel controlled atmOSphere storage
chamber filled with.McIntosh apples. The atmosphere samples were held
at ~135°C and all materials volatile at this temperature were pumped
off. The remaining volatiles from each sample were adsorbed onto 15g.
of activated molecular sieves. The sieves were then heated slowly until
the materials were driven off. There was no increase in vapor pressure
above the sieves for any samples until the temperature of the sieves
approached 180°C. The materials given off at this temperature were
collected in a liquid nitrogen trap and its vapor pressure was observed
at various temperatures from 0°C to -78°C. The vapor pressures at

these temperatures coincided well with those of water.

Activated Carbon Extractions

As a result of the work with molecular sieves, an attempt was
made to remove apple storage volatiles from the activated carbon of air
purification units that had been in a controlled atmosphere McIntosh
apple storage during one season of approximately 200 days. The precise
procedure for activation of the carbon used was not obtainable due to
company policies. The Condensed Chemical Dictionary (1956) states that
activation of carbon is generally achieved by heating to 800°C to 900°C
in an atmosphere of steam or carbon dioxide. Procedures for activation
by commercial companies are expected to meet defined standards. After
activation, the carbon is placed in sealed containers until ready for
use.

The carbon was removed from two 6“x10" units from a controlled
atmosphere storage room. The carbon was held in a polyethylene bag
until ready to use. A 10 g. sample of the carbon was placed in a gas
bottle. The bottle was cooled to -l96°C and all materials volatile at
this temperature were pumped off on the LTHV system. The volatiles
were then driven off the carbon by heating it to 180°C. Condensation
of the volatiles took place in a cold trap at ~196°C under high vacuum.
Approximately one-fourth milliliter of clear, colorless liquid was ob-
tained which had a strong acrid odor not resembling apple odor in the
least.

Solvent-extraction of volatiles from carbon was also attempted.
Diethyl ether and isopentane were the two solvents used for extraction.
Identical procedures were carried out for extraction by each solvent.

Ten grams of carbon were extracted with three ten-milliliter portions

22

of solvent. The solvent extract was placed in a gas bottle and the
solvent was distilled off at ~78°C under high vacuum. The solvent was
condensed in a gas bottle at -l96°C, leaving behind the apple storage
volatiles. Extractions using both diethyl ether and isopentane yielded
approximately one-half milliliter of clear, colorless liquid. Unlike
the volatiles removed by heat, the volatiles removed by solvent extrac-
tion in both cases had a sweet, apple-like odor combined with a foul,
musty odor.

Gas chromatograms of the head space from each of these samples
were obtained and are presented in Figures 11, 12, and 13. Similar
chromatograms were obtained from.a11 three columns available. The
silicone column was chosen to be used in further investigations because
it gave the best resolution of the extracts. The chromatograms are
correlated with the observations made by smelling the samples. A
radically different chromatogram was obtained from the volatiles removed
from the carbon by heat as compared to the chromatograms from the vola-
tiles removed from carbon by solvent extraction. Little difference is
noticed from the chromatograms obtained from the solvent—extracted vola-
tiles. Degradation of the volatiles due to heat appears to have taken
place while attempting to drive them off the carbon by heating. Extrac-
tion by isopentane was the method used in obtaining more volatile samples
from carbon because isopentane is more volatile than diethyl ether and
easier to distill off at low temperatures. The extraction method rather
than heat was used in obtaining other samples because of the degradation
believed due to heat. An unused sample from.the same lot of activated

carbon used was not available for a control. waever, the defined

nonemo popm>flpom mo pomapxm papa mo Emamoemanno who .HH .mwm
means? I mag I.v

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IF); ... . ._... _ ..i .............. _

 

é

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_ M
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mmpdcdH II 059i

000m II .ofmp no>o

CHEVH: ON I 3cm .6: on
8,330 mcooaflnm

‘

ea

Om

ow

scheme

 

Ill

pmpu>HpOQ no pomawxm ocwpcodomfl mo Euawopcfioaco who .MH .tam

mopedflz II mafia.lmv

ON om oq

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ooom nu .asea eeao
aaa\aa om n meme seam em
mfidfloo mcooflaflm

o

m

23

activation treatment is severe enough to destroy all oxygenated hydro-
carbon compounds (Royals, 1958).

A sample of volatiles was obtained from 10 grams of carbon by
extraction with isopentane followed by subsequent distillation at 778°C
under high vacuum. LHTV distillation was used to fractionate the
sample. Two samples were collected at -78°C. The first reduced the
vapor pressure of the sample from.800 hf to 400L4 Hg. pressure; the
second, from 400,, to O’WI Hg. pressure. Other fractions were collected
of materials volatile between temperatures of -60°C to -78°C, .4000 to
.60°c, -30°c to 4.0%, -20°C'to -30°c, -10°C to -20°c, 0°C to -10°C, and
room temperature to 0°C. All samples were condensed at ~196°C under
high vacuum. Mass spectra of some of these fractions are shown in
Figures 14, 15, l6, l7, and 18.

A comparison of Figure 14, the mass spectrum of the fraction col-
lected at ~78°c between 800,5 and 400,4 pressure, with Figure 15, the
mass spectrum of iSOpentane, shows this fraction of the sample to be
composed almost entirely of iSOpentane, the solvent used for extraction.
Inspection of Figures 16, 17, and 18 confirms similarities in these
three sample fractions, indicating likeness in composition. The promi-
nent peaks in these spectra occur at the same m/e values in each case.
Some variations of proportions in intensity between these major m/e
values is noticed between different sample fraction spectra due to vary-
ing proportions of the compounds in the different samples. This is
expected when the techniques of fractionation are considered.

The groupings of m/e values which are prominent and of interest
in these spectra are 55, 56, 57; 69, 70, 71; 82, 83, 84, 85, 86; 95, 96,

4.8.30me gunman mm H.400.» op mm I cow «comb!

 

 

 

 

 

 

 

 

 

 

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m\a a}
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IF I? —~p P I“— Hu—q—W HP flud Pia“ —b P qbr: » b O L hIfl—ap h h — “Nu—.i—fl b— — h» 4* — *n b dP—< L, I» O
m a ON _ Y OOOH
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9
I
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1-
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T...
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Relative Intensity

4000 ‘

5280

3000 a

 

-

I
2000 .. ! I

 

10004

W‘- ~4-‘u- an" .—.—-.—-—

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O ‘ '1 .. I LT ‘IIIII'IIIII JthIIIII Il.§II1?‘I Lil] 1HILI # fig?

0 10 20 30 40 50 60/70 80 90 100110 120
me

Fig. 16. Mass spectrum of isopentane extract of activated carbon
-60°C to -78°C fraction

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I
I
«Aeeo ‘2313
I;
1500 a

«E: I

g 1000 i I

*3 a

“I I

2 z

:3 .

'73 I

a: I.
I

500 I I
I
0 . v 1 I LJ II IrIIIIIInLIL @1pr I. VIIIIIIIIIIr I‘ L‘IIII 4L,

 

0102030405060/708090100110120130
me

Fig. 17. Mass spectrum of isopentane extract of activated carbon
-40°c to -6o°c fraction

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97, 98, 99, 100; 111; 112; 125, 126; 140; and 154. These m/e values
indicate a series of singly oxygenated aliphatic hydrocarbons. The
lack of any significant intensity of m/e 31 and 45 eliminates the possi-
bility of any significant amounts of alcohols in these fractions. Ketones
are very unlikely in these fractions. Ketones of 5 or less carbon atoms
may be possible, but the spectra show no possibility of any significant
amounts of ketones with more than 5 carbons. A comparison of figures
20, 21, and 22 emphasizes the differences in mass spectra between alde—
hydes and ketones. The parent peak (m/b indicating the molecular weight
of the compound) for ketones stands alone and the m/e for the parent
peak minus 1 is very small compared to the parent peak. For aldehydes,
on the other hand, the parent peak minus 1 is not insignificant in com-
parison with the parent peak. This is due to the fact that the aldehyde
hydrogen is much.more easily removed by electron bombardment than are
any of the hydrogens on the ketone molecule. Furthermore, the saturated
and unsaturated aldehydes show'large peaks at m/b 29 and 27‘respectively,
also present in the spectra of the sample fraction, whereas m/e 43 and
45 are expected to be of considerable size for ketones. Therefore, m/b
154, 140 and 139, 126 and 125, 112 and 111, 100 and 99, and 98 and 97
are very probably due to decenal, nonenal, octenal, heptenal, hexanal,
and hexenal respectively. In order to prove the presence of these and
other compounds present in these fractions, mass spectra of these com-
pounds are necessary but were not all available.

The series of peaks in these spectra at m/e 57, 89, and 117 indi-
cate the possibility of a small amount of esters in these fractions.

The inability to obtain mass spectra of the more prominently suspected

25

compounds in these fractions made it impossible to verify the presence
of esters. Water, of course, is the compound in greatest abundance in
all of these fractions, as indicated by the size of m/e 18.

The mass spectrum in Figure 19 shows the room temperature to 0°C
fraction is composed largely of air and isopentane. Peaks present in
the spectrum of this fraction other than those due to air and isopentane
are similar to the ones noted and discussed in the spectra of foregoing.

fractions.

Gas Chromatography of Atmosphere Samples '
Conditions for Direct Analysis

various operating conditions for gas chromatographic procedures
were tried and the following conditions appeared to be best suited for
direct analysis in controlled atmosphere storages. An oven temperature
of 50°C was selected for operation. At this temperature any thermal
destruction would be very unlikely and, since the compounds in question
are volatile at 0°C, this temperature would be high enough for volatili-
zation of the compounds in question. The hydrogen flow rate and carrier
gas flow rate were set at 20 ml. per minute as suggested by the operating
manual. The recorder chart rate of §’ per minute was very satisfactory
for this study. By trial and error a gas sample size of lO;/ 1 was
found most appropriate. These operating conditions were used for all
gas analyses of storage atmospheres. A summary of the storage rooms

used in this study is shown in Table I.

Relative Intensity

m/e 18:1350 m/e 45:1200
800 ..

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

600 e» I
zoo -

}
200 “I I

0 10 20 30 40 50 39 70 80 90 100 110 120
m c

Fig. 19 Mass spectrum of isopentane extract of activated carbon
room temperature to 0°C fraction

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26

Column Selection

Samples from.three of the storage rooms were analyzed on the three
different columns available. The three rooms from which these samples
were taken were on the Michigan State University campus and readily
accessible. These results are shown in Figure 23. The silicone column
was eliminated due to its inability to resolve ethylene from the other
peaks found in the atmospheres of rooms number 10 and 11. The castorwax
column was selected for routine analysis of ethylene in controlled at-
mosphere storages because it gave better resolution of the peaks in
Figure 23 than did the carbowax.column.

A small amount of ethylene was added to the atmosphere samples
from rooms number 10 and 11. Chromatograms were obtained from these
samples before and after the addition of ethylene using the castorwax
column. These results are shown in Figure 24. From the increased
height of the first of the two peaks from each of the samples from
rooms number 10 and 11 after ethylene addition, it was concluded that
the first of the two peaks in the chromatograms from.rooms number 10
and 11 are due to ethylene.

Chromatograms of samples from each of the eight storage rooms,
numbers 1 through 8, at Belding Fruit Sales, Balding, Michigan, using
the castorwax column are shown in Figure 25. A chromatogram of a sample
of the same size, 10,571, of air is shown in the same Figure, showing
that air makes no contribution to the ethylene peak or anything else
for samples of this size. All eight of the Belding storages show a
single peak, as did room number 9, Figure 23, when chromatograms were

obtained using the castorwax column.

Cas tor-wax Carbowax Sil icone

 

 

l O l 0
(——Time - Minutes

 

 

 

 

 

7‘— I
" “" z I
Rm ::—Z -‘~~.
11 3:7]? . ‘I :
_ 7tf,.- : “Tn
.- _. .4. -C
Z ._J-,:.i
E;- v.7
-1 ‘35-!

 

 

Fig. 23. Gas chrome.to;t-rrc 01‘ CA storage atmospheres

 

 

 

 

 

' {- Time - Minutes

Fig. 24. Gas chromatograms of CA storage atmospheres and
CA storage atmospheres plus ethylene

 

 

 

 

€—- Time — 21inutes
-——w v-. -'—‘~— -~
fl ,. 'h“ . .1,

Room 8

 

 

I , C
.;, ‘\
I
l
'. -94“: _ u

 

 

1 o
é-Eine - I—Linutes

 

.-_ 25. Gas chrcvmtogl‘azs of CA storage :ztg,‘:osr:lier<£ and air

27

The gas chromatographic technique was found to be an excellent
method for routine analysis of ethylene concentration in controlled
atmosphere apple storages. Two characteristics of the method as de-
scribed make it especially advantageous: first, a sharp peak is obtained
for ethylene; and,second, the retention time for ethylene is less than_
one minute, making possible the analysis of many samples in a relatively
short period of time. The sharpness of the ethylene peak makes it pos-
sible to use peak height as a measure of ethylene concentration rather

than resorting to the area measurement method.

Identification of Second Peaks in Chromatograms
of Rooms 10 and 11 Atmospheres

The chromatograms of atmospheres from rooms 10 and 11 both show a
second peak. This characteristic is peculiar to these two rooms aloner
In all the other nine rooms, ethylene alone is detected by the analysis
method described above. It would be helpful to identify the compound or
compounds responsible for the second peak in the atmosphere chromato-
grams of rooms 10 and 11.

Compounds possibly producing the second peak in the chromatograms
of atmosphere samples from.rooms 10 and 11 were considered. Reinforce-
ment of the unknown peaks was achieved by adding a small amount of known
compounds, one at a time, to the unknown sample. Ethylene was found to
reinforce the first of the two peaks found in the atmospheres from
storage rooms 10 and 11. Three compounds, propane, used in the con-
trolled atmosphere gas generator, propylene, a homolog of ethylene, and

Refrigerant l2 (dichlorodifluoromethane), the refrigerant used, were

28

found to enforce the second peak detected from the atmospheres of
storage rooms 10 and 11, Figures 26 and 27.

By bubbling the atmosphere from storage room 10 through a solup
tion of HgClO4 (mercuric perchlorate), also used for ethylene analysis
from apples, it was possible to obtain a sample of all unsaturated hydro-
carbons present in the shuosphere. The addition of LiCl (lithium
chloride) to the HgClQL solution causes the release of all absorbed
unsaturated hydrocarbons. The gas chromatogram.of the gases released
from the H'gClO4 solution shows a single peak. This gas reinforced the
first of the two peaks obtained by chromatographing the atmosphere of
room number 10, Figure 28. From.these observations it was concluded
that the single peak seen in the gas chromatograms of the atmospheres
from rooms 1 through.9 and the first of the two peaks found in rooms 10
and 11 are due to ethylene present in the atmosphere of the storage
rooms. The second peaks (the ones not due to ethylene) detected from
the atmospheres of rooms 10 and 11 consequently are not due to the pres-
ence of propylene in the storage room atmospheres.

An acetylene halide detection torch was used to determine the
presence of Refrigerant 12 in storage room 10. The positive halide
test lead to the conclusion that the second peak in the chromatogram
was due to Freon 12 in storage room 10. In the case of room 11, the
second peak in the chromatogram.is believed due to a compound from the
experimental CA storage atmosphere generator which was used with this
storage room. The gas fed into the storage room.from this generator

t

produced this second peak when chromatogramed.

      

(r—‘Time - Minutes

Gas chromatograms of propylene, CA storage atmospheres,
and CA storage atmospheres plus propylene

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Fig. 28. Gas chromatograms of gas absorbed by HgClO , CA storage
atmosphere, and CA storage atmosphere plus gas absorbed

b HPClO
y O 4

29

Standard Curve for Ethylene Concentrations in Air

A standard curve for ethylene concentrations in airewas deter-
mined. Gas chromatograms of concentrations ranging from.lOO ppm to
4000 ppm of ethylene in air were made at random. Peak heights were
plotted against ethylene concentration to establish the standard curve.
The resulting standard curve, Figure 29, is for all practical purposes
a straight line. This straight line standard curve was used to determine
the ethylene concentration of all storage atmosphere samples. A check
on the chromatograph was made each month with known concentrations of
ethylene in air for assurance that the sensitivity of the chromatograph

to ethylene was not changing with time.

Gas Sampling Procedures

The atmospheres of several controlled atmosphere storage rooms
-were analyzed at intervals throughout the 1961-1962 storage season.
Atmosphere samples were taken from the storage rooms in 4-1iter poly-
ethylene bags. Samples were taken from the storage gas-sampling tubes.
The polyethylene bags were filled with the sample atmosphere and col-
lapsed completely three times to flush out any residual air. The mouth
of the polyethylene bag was gathered tightly around a rubber vaccine-
bottle stepper and held in place by a stretched rubber band. A pre-
determined amount of gas was removed from the polyethylene sampling bag
with a syringe and injected directly onto the gas chromatographic
column for analysis.

'Figure 30 illustrates the stable ethylene concentration of an

atmosphere sample in a polyethylene bag obtained as described above

Ethylene Concentration in Air - ppm

 

 

 

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Scale Divisions
Fig. 29. Standard curve of ethylene concentration in air

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during a period of six hours. Ten H 1 portions were analyzed from this
sample 0, 1, 2, 1., and 6 hours after collection. The constant height of
the ethylene peak (first peak) can readily be seen from-the figure.

Since ethylene concentration is determined by peak height, no detectable
change in ethylene concentration-takes place in a sample collected in the
manner described and held for a period of six hours. All samples used
for ethylene concentration determinations were analyzed within four
hours after collection. It is, therefore, evident that the samples when

analyzed contained the same ethylene concentration as when collected.

Results

The ethylene concentration of eleven controlled atmosphere storage
rooms was followed throughout the 1961-1962 storage season by sampling
and analyzing the storage atmospheres as described above. The sampling
dates are indicated by the points on the graphs. Since rooms 9, 10, and
ll were on the Michigan State University campus, they were sampled more
frequently than rooms 1 through 8 which were located at Belding,‘Michi-
gen. The management conditions of all eleven storage rooms are summarized
in Table I.

‘ Plots of ethylene concentration versus time were made for the
eleven storage rooms studied (Figures 31, 32, and 33). The ethylene
concentration increased steadily after sealing the room until it leveled
off at a constant maximum level. After reaching constant concentration,
no significant persisting change took place but remained the same until
the storage room was opened. Although equilibrium ethylene concentra-

tions were observed, not all the rooms leveled off at the same

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31

concentration. .The equilibrium ethylene concentration level reached in
a storage room-appears to depend on the variety of apples stored in the
room. The lower ethylene concentration, approximately 250 ppm, found

in room 11 is believed due to dilution of the atmosphere by the con-
tro11ed atmosphere generator. The high equilibrium concentration,
approximately 1250 ppm, was reached in room-5, containing Northern Spy'
apples, and the low, approximately 500 ppm, was in room 3, containing
Red Delicious apples. Rooms with McIntosh apples reached an equilibrium
concentration of approximately 1000 ppm and the room with Jonathan apples
had an ethylene concentration of approximately 750 ppm. Rooms 7 and 8,
containing mixtures of apple varieties, produced equilibrium-ethylene
concentrations between those found for the rooms containing the single
varieties of apples.

No great difference in equilibrium.concentration was observed
between rooms using water or caustic soda absorption systems and/hr the
presence or absence of activated carbon. Rooms 1, 2, 6, 9, and 10 all
contained Mhlntosh apples and all reached an equilibrium.concentration
of approximately 1000 ppm. Rooms 1, 2, and 6 had water absorption
systems and rooms 9 and 10 had caustic soda absorbers. Rooms 1, 2, 6,
and 9 contained activated carbon air purifiers while room 10 did not.
Therefore, these two factors did not appear to affect the equilibrium
ethylene concentration in controlled atmosphere storage rooms.

Room 11 was very irregular in its ethylene concentration. This is
believed due to the irregular operation of this storage room. The
storage room was opened from time to time to remove samples. The experi-

mental controlled atmosphere generator was very erratic in its operation

32

and at times was not in Operation. Although it was not determined, the
irregularities observed could all be due to the irregular operation of

this particular storage room.

Attempt to Determine Concentrations of Other volatiles
in Controlled Atmosphere Storage

The concentration of other storage volatiles during the storage
season holds a great deal of interest in this investigation. Atmosphere
samples were taken directly from storage room number 10 and injected
onto the castorwax column. Figure 34 shows a chromatogram of a 20 ml
gas sample from room number In. Other than the large peak including
both ethylene and Refrigerant 12, no other peak is detected. One
hundred ppm-of ethylene are easily detected from a 10 #11 sample.
Assumdng equal sensitivity for other compounds as for ethylene, any
other volatiles present must, therefore, be in concentrations of less
than 0.05 ppm.

Ry recirculating the storage room atmosphere through a liquid
nitrogen cold trap, the volatiles from various volumes of atmosphere
from room number 10 were concentrated. After collection, the concen-
trate was held at ~155°C and air gases, ethylene, and 002 were removed
by vacuum-pumping. Samples of the remaining water solution of volatiles
and equal-sized samples of water and a 0.01 per cent solution of ethanol
in water were chromatogramnai Figure 35. One/7’1 of the concentrate
would be equivalent to 295 ml of atmosphere. This is based on the
assumption that 100 per cent of the volatiles of interest was condensed

in the concentrate, since, at the temperature and relative humidity of

 

 

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Fig. 35. Gas cnrOII-Iatograms of 2H1 of water, 2H1 of 0.01 per cent
ethanol in water standard, and 2H1 of CA storage atmos-
phere

33

the storage room (100 per cent at 35°F), each 295 ml of atmosphere
would contain approximately'llj 1 of water.

A study of Figure 35 shows the 0.01 per cent standard ethanol
solution produced four times the response as an equal amount of the
storage room atmosphere concentrate. It was also noted that the single
sharp peak produced by the concentrate has the same retention time as
does ethanol and responds in direct proportion to the sample size. It
was,therefore, concluded that the atmosphere concentrate contained
ethanol in one-fourth the concentration of the 0.01 per cent standard,
which would be 25 ppm. Air saturated with water vapor at 35°F contains
0.43 per cent water by weight. This is equivalent to a concentration of
.107 ppm or 107 ppb of ethanol in the atmosphere of storage room 10.

Let it be assumed that it is possible to detect an ethanol peak
using a 1/1 1 sample of atmosphere concentrate. This would mean that
295 ml of atmosphere would be necessary for ethanol detection at a level
of approximately 100 ppb. Therefore, it can no longer be assumed that
the gas chromatograph used is as sensitive for ethanol as it is for
ethylene. One hundred ppm of ethylene in a lofy 1 air sample,which is
equal to 1.25x10-3I4 g of ethylene,is easily detected. On the other
hand, 25 ppm of ethanol in a lf4 1 water sample, which is equal to
5x10'2 H g of ethanol, is necessary for detection. This indicates that,
under the conditions employed, this chromatograph is more than 40 times
as sensitive to ethylene than it is to ethanol. Also, under these cone
ditions, a concentration of 2 ppm ethanol is necessary for detection in

a 20 ml atmosphere sample.

Extreme difficulty encountered in reproducing these results made
it infeasible to pursue this method for routine analysis of ethanol

concentration in storage room atmospheres.

35

DISCUSSION

APPARATUS

The low-temperature high-vacuum system was used to obtain initial
separations of complex mixtures of volatiles. Considering the complex
nature of apple volatiles, lowatemperature handling should minimize the
possibility of chemical reactions among the compounds during concentra-
tion and separation procedures. Fewer chemical reactions, if any, would
be expected from.1owetemperature handling as compared to handling the
volatiles at higher temperatures. Low-temperature high~vacuum.distilla-
tion also lends itself well to working with small quantities of materials
and to preparation for analysis by mass spectrometry.

Mass spectrometry is one of the methods used to identify volatiles
in this study. This method of identification is sensitive, rapid,
simple, and direct. Merritt et a1. (1958) identified ten compounds in
a sample preparation from irradiated beef from a total of approximately
lxlO'5 moles of material. The unknown volatiles are discharged directly
into the instrument for analysis without any previous chemical altera-
tions. This feature minimizes the possibility of chemical changes or
destruction of the unknown compounds. Mass spectrometry makes possible
the identification of the individual volatile compounds in a mixture
provided the mixture is not too complex,.in which case only a few of the
total compounds may be identified due to interfering mass spectra of the
compounds. In such a case, separation of the complex mixture into less
complex fractions would be necessary for complete identification. Posi-

tive identification by mass spectrometry requires the mass spectrum of

the compounds being identified. Tentative identifications of compounds
can be made from mass spectra from the physical and chemical knowledge
of compounds without the known spectra of the compounds. Specific
types of compounds, such as alcohols, sulfur, or nitrogen compounds,
can be deduced present from characteristic peaks of mass spectra.

Gas chromatrography was quite useful in obtaining a pattern of
compounds present in a sample using the three extraction methods.
Differences in patterns indicate different compositions of the samples,
Figures ll, 12, and 13. This technique was very useful for following
the ethylene concentration in storage atmospheres. The hydrogen flame
ionization detector was an important feature in this work due to its

insensitivity to normal amounts of air gases, 002, and water and high

sensitivity to organic compounds of interest in this study.

MASS SPECTRA 0F APPLE STORAGE ATMOSPHERE

Identification of apple volatiles from.mass spectra of storage
atmosphere without previous concentration was unsuccessful. This indi-
cates the concentration of any single volatile in apple storage atmos-
pheres below an estimated 0.1 per cent. Concentration of the volatiles,
followed by fractionation by LTHV distillation, was necessary in order
to identify ethylene, ethanol, and acetaldehyde by mass spectrometry.
The vapor pressure characteristics of these compounds made it possible
to separate them from the air gases, carbon dioxide, and water present
in the atmosphere by LTHV distillation and thus penmitted their identi-

fication by mass spectrometry.

37

A greater percentage of acetaldehyde was expected to be separated
from water by LTHV distillation than ethanol, due to the greater vola-
tility of acetaldehyde. The fact is also shown by the fractions in.
which these two compounds are most prevalent, acetaldehyde being in
greater concentration in the ~125°C to -100°C fraction and ethanol in a
greater amount in the -1oo°c to -78°C fraction. Any other volatiles
with similar vapor pressure characteristics are either not present or

present in lower concentrations than either of these two compounds.

ANALYSIS OF EXTRACT FROM ACTIVATED CARBON

Solvent extraction appears to be a better method of recovering
volatiles from activated carbon than removal by heating. The solvent
extract had a characteristic apple odor, while the compounds removed by
heat had a strong, unpleasant acrid odor not resembling apple odor at
all. It is probable that the heat necessary to drive the volatiles off
the activated carbon causes a degradation of the apple volatiles. This
theory is supported by the gas chromatograms obtained from the extracts.
The chromatograms of the solvent extracted volatiles show compounds
being eluted from the column as much as an hour after injection. For
exactly the same chromatographing conditions, all compounds were eluted
from-the column after 35 minutes for the volatiles removed by heat.
This suggests that the compounds of higher molecular weight obtained by
solvent extraction are not present in the extraction obtained hy-heating
the activated carbon. The chromatograms Show little similarity even
among the lower molecular weight compounds. This is a good indication

that much of the material shown in the lower molecular’weight region of

38

the chromatogram of the volatiles extracted by heating is due to degra-
dation products of higher molecular weight compounds.

Two differences were observed between the chromatograms of the.
volatiles extracted by isopentane and diethyl ether. The large peak
near the beginning of the chromatogram from the diethyl ether extracted
volatiles is due to diethyl ether. The higher volatility and greater
physical inertness of isopentane enabled better separation of this sol-
vent from the extracted volatiles than did diethyl ether. With the
exception of the peaks obscured by the diethyl ether peak, the peaks in
both chromatograms exhibit identical retention times, indicating identical
compounds. A second difference is that the relative ratios of amounts
of the different compounds differ between the two chromatograms. This
fact cannot be used as a criterion for concluding that one of the two
solvents is better in this extraction than the other because the relative
ratios of the compounds as they exist on the activated carbon were not
known. By the same token, it cannot be concluded that all of the com-
pounds are at least partially extracted from the activated carbon. To
arrive at such a conclusion would require extraction by many different
types of solvents, chromatographing by many different types of columns
and column systems, and identification of all the compounds obtained and
artifacts fomed from this long, involved investigation.

The validity of the assumption that the compounds adsorbed by
activated carbon are a valid representation of volatiles produced by
apples can be attacked from several standpoints. Firstly, storage room
odors produced by microorganisms in the storage room can be adsorbed on

activated carbon. Secondly, all compounds do not have the same affinity

for activated carbon, or any adsorbent. It is probable that the com-
pounds present in apple storages are not adsorbed in the same proportions
in which they are present in the storage room and possible that some com-
pounds are not adsorbed in any detectable amount. Thirdly, there is evi-
dence that reactions of some compounds can take place while adsorbed on
activated carbon. The actual determination of the effects of these pos-
sible sources of error could be determined by recovery experiments using
compounds both identified as apple volatiles and believed to be produced
by apples.

The combination of apple-like and foul, musty, or moldy odor of
the extract indicates the presence of compounds produced by both apples
and molds present in apple storages. The musty odor was probably re-
sponsible for some of the peaks of the chromatograms. Chemical reac-
tions are not likely to any great extent, considering the lowhtemperature
conditions used in the handling of the activated carbon and treatments
of the extract. Therefore, it is quite conceivable that most of the
peaks on the chromatograms of the solvent-extracted volatiles are due
to volatiles produced by apples.

Effective separation of the isopentane extract of apple storage
volatiles was not achieved by LTHV distillation. Mass spectra of the
fractions obtained by LTHV distillation, by and large, show the same
characteristic peaks. Much of this similarity in mass spectra is be-
lieved due to absorption of the volatiles by the stopcock grease used
in the LTHV system. This would tend to redistribute the volatiles into
all of the fractions after separation of the volatiles by LTHV distilla-

tion. The mass spectra from these fractions were, nevertheless, very

useful in the tentative identification of some volatiles. The lack of
mass spectral patterns of the more abundant, greater molecular weight
compounds believed present prevented definite mass spectrometric identi-
fication of these and other more abundant lower molecular weight com-
pounds present.

The identification of the less abundant volatiles extracted from
activated carbon by mass spectrometry requires effective separation of
these from the more abundant volatiles. This can probably be accomplished
by chromatographic separation of a large sample of extracted volatiles
and collection of the fractions as they are eluted from.the column.

Mass spectra of the less abundant volatiles could then be obtained since
there would be no interfering spectra from.the more abundant volatiles.
The use of a time-of-flight mass spectrometer would eliminate the neces-
sity of collection of the fractions after elution. Elution of the
column directly into a time—of-flight mass spectrometer would enable

the instantaneous acquisition of the mass spectrum of each fraction as

it was eluted from the column.

ETHYLENE CONCENTRATION IN CONTROLLED ATMOSPHERE STORAGES

1n the eleven controlled atmosphere storage rooms studied, the
ethylene concentration increased rapidly at first and then leveled off
to a relatively constant concentration. This relatively constant
ethylene concentration was called the equilibrium.ethylene concentra-
tion. At this equilibrium concentration, the amount of ethylene being
produced by the apples is equal to the amount being removed from the

storage room.

Removal of ethylene from the conventionally operated controlled
atmosphere storage room could have been done in only two ways. The
first way, and perhaps the most important, was by limited ventilation,
which is necessary to replace the oxygen used up in respiration. The
other possible way was by scrubbing, necessary in removing the excess
carbon dioxide produced by respiration. The rates of oxygen consumption
and carbon dioxide production in conventional controlled atmosphere
storages are a result of respiration. Therefore, the amount of venti-
lation and scrubbing necessary to keep the oxygen and carbon dioxide
concentrations constant in a storage room.are directly proportional to
the respiration rate of the apples. The possible ways of removing
ethylene from the storage and the constant concentration of ethylene
found in the storage room indicate that ethylene production must be
proportional to respiration rate. This is in agreement with post
climacteric studies on apples (Smock and Neubert, 1950).

The ethylene concentration did not assume the same concentration
in all of the storage rooms. Room 5 came to equilibrium at an ethylene
concentration of’about 1250 ppm; rooms 1, 2, 6, and 9, at a concentra-
tion of about 1000 ppm; rooms 4, 7, and 10, at a concentration of about
750 ppm; rooms 3 and 8, at a concentration of about 500 ppm; and room
11, at a concentration of about 250 ppm. These equilibrium concentra-
tions are correlated with the varieties of apples stored in the storage
rooms. Room 5 contained Northern Spy apples; rooms 1, 2, 6, and 9 con-
tained McIntosh apples; room.4 contained Jonathan apples; room 3 con-
tained Red Delicious apples; rooms 7 and 8 contained a mixture of Jona-

than, Red Delicious, and Golden Delicious apples.

In room 2, containing McIntosh apples, the equilibrium ethylene
concentration is slightly less than the other rooms which contained
McIntosh apples. This is believed to be due to the fact that the
scrubbing units on rooms 3 and 4.were not operating properly, thus
forcing the part-time use of the scrubbing unit on room 2 on these
rooms. As a result, some mixing of the atmospheres of these three
rooms took place. This would not only tend to lower the ethylene con-
centration in room.2 but also increase the ethylene concentration in
rooms 3 and A.

A greater proportion of Jonathan apples in room 7 thah in room 8
could account for the difference in the equilibrium ethylene concentra-
tions in these rooms. The sharp irregularities in ethylene concentra-
tion in rooms 1 through 10 is assumed to be the result of sampling
before or after limited ventilation of the storage room. No reasonable
explanation could be found for the lower equilibrium ethylene concentra-
tion in room.lO as compared with the other storage rooms containing
McIntosh apples. These observations strongly support studies (reported
by Smock and Neubert (1950)) showing that a variation of the amount of
ethylene produced per unit amount of oxygen used in respiration exists
among apple varieties. No difference in equilibrium ethylene con-
centration was observed between storage rooms employing water scrubbers
and those employing caustic soda scrubbers. Therefore, if these carbon
dioxide absorbers have any effect on ethylene concentration, the effect
is very similar for each system.

Storage room 11 exhibits the lowest equilibrium ethylene concenp

tration of the eleven rooms studied. This is due to the controlled

43

atmosphere gas generator used with this room causing a constant dilution
of the room.atmosphere. The two unusually high concentrations of ethylene
found in this room occurred when the generator was not in operation.

Since lowering of ethylene concentration is believed due to dilution of
the controlled atmosphere generator, the same is believed to hold true

for all of the volatiles produced by the apples in the storage room.

CONCENTRATION OF OTHER VOLATILES

Perhaps the most convincing argument that volatiles are present
is the odor that is perceived from the apple storage atmospheres. Al-
though proving the presence of volatiles, odor tells little about the
concentration of apple storage volatiles. volatile concentration studies
using sensory methods are based on odor thresholds. Odor thresholds
vary greatly for different substances. Some substances may be detected
by odor in concentrations of fractions of parts per billion, whereas
some are odorless at all concentrations. Nevertheless, most of the
types of compounds believed present in apple storage atmospheres (acids,
alcohols, esters, aldehydes, and ketones) would be expected to have
their individual thresholds of concentrations in parts per million or
parts per billion. Odor thresholds of combinations of volatiles bear
no relationship to odor thresholds of the individual components. For
this reasOn and the factthat the composition of apple storage volatiles
has not yet been resolved, it is still impossible to use odor thresholds
as a study of apple storage volatile concentrations.

A more meaningful indication of apple storage volatile concentra-

tions may be derived from a consideration of sampling and concentration

procedures and the results obtained from these procedures. By the reduc-
tion of ceric sulphate after proper absorption (Gerhardt, 1950), the
concentration of volatiles has been expressed as milligrams ceric sul-
phate reduced per cubic foot of storage atmosphere. By separative
absorption techniques, ethylene has been separated from other storage
volatiles in this type of analysis. In the case of ethylene, the ceric
sulphate reduction method furnishes data from which ethylene concentra-
tion may be calculated, since the reducing power of ethylene can be
determined for the system; but the non-ethylenic volatiles have not all
been identified, and the reducing power of this mixture is not known.
Consequently, the concentration of any one compound of the mixture or
of the whole mixture cannot be precisely determined from the eerie sul-
phate reduction method.

Concentration methods using absorbers, adsorbers, and cold trap-
ping have been used to collect apple storage volatiles. These methods
have been used chiefly for identification rather than concentration
determination because of the question of the quantitative recovery of
the volatiles.. Gas chromatography has been shown to be very useful in
ethylene concentration studies, but more appropriate techniques must be
developed before this method can, by itself, provide a complete study
of apple storage volatile concentrations.

By combining cold trapping and gas chromatography, the only vola-
tile found in this study was ethanol. This was the only compound deter-
mined by chromatographing the cold trap condensate of apple storage
atmospheres. The low concentration of ethanol in the storage room at—
mospheres is supported by the mass spectra of the storage room atmos-

pheres.

45

The actual value of 100 ppb of ethanol in the storage room atmos-
phere is subject to question. Inconsistent results were obtained when
duplicate determinations were made. Although recovery of the water vapor
from the atmosphere appears to be quantitative, the same does not neces-
sarily hold true for ethanol. Recovery experiments using these methods
yield results as low as 20 per cent recovery. Loss of volatiles could
also take place when the more volatile air gases and 002 were being
pumped out of the bottle.

Since 100 ppb of ethanol was detected in storage room 10 by
chromatographing cold trap concentrates of the atmosphere, at least this
concentration is reached. Since ethanol was not detected by direct
chromatographing of the atmosphere in storage room 10, it was concluded
that the ethanol concentration in room 10 was less than 2 ppm. It must
be borne in mind that storage room 10 contained no activated charcoal.
Due to the great affinity of activated charcoal for ethanol, lower con-
centrations of ethanol would be expected in storage rooms containing
sufficient activated charcoal. It would, therefore, appear that the
nonpethylenic volatiles exist in concentrations of a few ppm or in ppb
each.

Practices and equipment used on controlled atmosphere storages
will undoubtedly affect the concentrations and pattern of volatiles in
storages. The effect produced by the presence of activated carbon is
demonstrated by Gerhardt (1950). Although activated carbon caused no
significant change in ethylene concentration, it greatly lowered the
ceric sulphate reducing power of the storage atmosphere. This is a

good indication of the removal of volatiles from the storage atmosphere.

46

The differences in affinity of activated carbon, or of any other ad-
sorbent or absorbent, for the volatiles present in apple storage atmos-
pheres cannot be determined until the volatiles have been identified.
Recommendations for minimum amounts of adsorbents or absorbents used to
obtain desired effects in the storage rooms have been made. These mini-
‘mum recommendations indicate saturation of the adsorbents or absorbents
with storage volatiles or an equilibrium condition set up between the
"scavengers" (such as activated carbon and oiled paper) and the storage
volatiles when insufficient amounts of the scavengers are‘used.
volatiles may also be removed from controlled atmosphere storages
by equipment used for this type of apple storage. Any volatile com-
pound, such as an acid, that could form a non-volatile compound with
sodium hydroxide will be removed, at least in part, by a caustic soda
absorption system used for carbon dioxide control. The detectable apple
odor of the gas desorbed from the water carbon dioxide removal equip-
ment indicates at least partial removal of volatiles from controlled
atmosphere apple storages by water absorption systems. Where controlled
atmosphere generators or bottled nitrogen are used to acquire and main-
tain controlled atmosphere conditions, dilution of the storage volatiles
would be inevitable. The extent of dilution would depend upon the
amount of nitrogen or generator gas being fed into the storage room.
volatile differences due to apple variety have been shown to
exist by the effects of one lot of fruit on another. The different
aromas of apple varieties also indicate differences of volatiles.
Apple volatile analyses have shown good evidence for these differences.

The effect of storage practices and equipment on concentrations of

47

apple storage volatiles has not yet been fully shown. Failure of pre-
viously used methods to accomplish this feat emphasizes the need for
more appropriate methods of analysis. Mass spectrometry appears to be
the ideal method for qualitative analysis of apple storage volatiles.
Collection and preparation of the volatiles for mass spectral analysis
are critical steps in this procedure. The steps in this procedure must
exclude the presence of large quantities of water with respect to other
apple volatiles. Preliminary separations with gas chromatography would
be helpful in obtaining a more complete qualitative analysis by mass
spectrometry. Quantitative analysis of apple storage volatiles requires
more investigation into techniques. Multiple column gas chromatography
techniques with detectors sensitive to organic compounds in the range
of parts per million to fractions of parts per billion would prove very
useful. Quantitative analysis by less sensitive methods would require
quantitative concentration of volatiles before analysis. Recovery
experiments should be carried out on any of these concentration proce-
dures before using them. This would permit quantitative analysis with
less sensitive gas chromatographic detectors or mass spectra used in

conjunction with volume-pressure data.

SOLUBILITY AND VOLATILITY
Two physical properties of the compounds which determine their
activity are solubility and volatility. Solubility is the ability of
the molecules of a substance to become dispersed in among the molecules
of another liquid or solid substance. volatility is the tendency of

the molecules of a substance to escape from the liquid or solid state

into the gaseous state. Generally, water solubility of an organic com-
pound is proportional to its volatility. The Handbook of Chemistry and
Physics (1953) shows that methanol, ethanol, acetaldehyde, propanol,
and acetone are all very soluble in water. Butanol, propanal, and amyl
acetate are slightly soluble in water, and amyl propionate, hexanal,
and geraniol are listed as insoluble in water. The vapor pressures
exerted by some of these compounds at 0°C are found to be:

vapor Pressure

Compound in mm of fig
acetaldehyde > 200
acetone 85
methanol 38
ethanol 13
propanol 5
butanol l
geraniol < l

Damage caused to the apples by the more volatile compounds could be.
controlled by reducing the partial pressure of the more volatile com-
pounds in the surrounding atmosphere, thus causing the escape of these
compounds from the aqueous intercellular solution of the apple. Damage
to apple tissues from.the more soluble of the apple volatiles,such as
acetaldehyde, acetone, and ethanol, would be expected to appear through-
out the apple flesh with the most damage to the flesh most remote to the
atmosphere. Such storage disorders as internal breakdown, mealy break-
down, and internal browning, if affected by volatiles, would be affected
to a greater extent by the more soluble volatiles than would the surface
disorders.

The less water-soluble compounds, such as amyl propionate, hexanol,
and geraniol, would have a tendency to leave the aqueous phase of the

intercellular spaces of the apple tissues. Since apple fruits respire,

49

heat is generated by them. This heat of respiration is removed from
the surface of the apple by cold air circulation. In order for heat to
move from within the apple to the apple surface where it is removed, a
temperature gradient is necessary. Therefore, the apple flesh must be
at a higher temperature than the apple surface. Generally, since vapor
pressure of a material increases with temperature, a concentration
gradient of these volatiles could exist between the warmer and cooler
portions of the apple fruit. The temperature gradient is indeed small '
but exists continuously in the same direction. Since mass transfer will
proceed with the heat energy transfer, this mechanism could concentrate
certain volatiles in the surface layers of the apple tissue.

On reaching the surface of the apple, the non-waterbsoluble com-
pounds would have to overcome three difficulties (low volatility, the
skin barrier, and the wax cuticle barrier) in order to escape from the
apple surface into the surrounding atmosphere. Because of their low
volatility at cold storage temperatures, these compounds may collect on
the apple surface. The apple skin may provide a barrier to slow down
the escape of the volatiles. The wax cuticle may be a more difficult
barrier to overcome than the skin. The apple cuticle is composed of
waxes which may act as an organic solvent for apple volatiles. The wax
cuticle may also act as an adsorbent for organic volatiles. Although
there is a lack of data for proof, it seems quite plausible from the
foregoing argument that a high enough concentration of apple volatiles
may be trapped on the apple surface to cause damage to the tissues in
that immediate area. Scald, Jonathan spot, and other spot disorders

could be effected by these entrapped apple volatiles.

SOURCE OF VOLATILES

Apple storage volatiles may come from any of several sources.
Molds or other microbial life are believed to contribute foul odors
to apple storages. Building materials, equipment, or containers may
also contribute to apple storage volatiles. Nevertheless, the volatiles
generated by the apple fruit itself are of greatest interest here.

Apple fruits produce organic acids, alcohols, aldehydes, ketones,
and esters. Ethanol and acetaldehyde are undoubtedly the products of
anaerobic respiration. The lack of sufficient oxygen would cause a .
shift in the energy producing reactions of the electron transport system
and the Krebs cycle, causing a decrease in the utilization of pyruvate
which is produced by glycolysis of carbohydrates. When the rates of the
Krebs cycle reactions have been decreased to such an extent that all of
the pyruvate produced by glycolysis cannot be utilized, the pyruvate is
irreversibly converted to acetaldehyde plus carbon dioxide in plant
cells. This conversion produces some energy. More energy may be ob-
tained by the plant cell by the reversible conversion of acetaldehyde
to ethanol. This theory is supported by the fact that apples produce
more ethanol and acetaldehyde when oxygen is lacking than when oxygen
is in abundance (Smock and Neubert, 1950).

Acids and hydrocarbons could be produced from amino acids by
deamination and/or decarboxylation. Deamination of amino acids could
directly form fatty acids which are observed as apple volatiles either
in the free or esterified state. Deamination and decarboxylation of
amino acids would produce an aliphatic hydrocarbon that, if oxidized to

various extents, could produce the respective alcohols, aldehydes, or

51

ketones. However, since no correlation has been observed in the initial
rapid drop of amino acids and the production of volatiles in grape juice
fermentation, some believe that, if amino acids contribute to apple
volatiles, they do so indirectly. On the other hand, Meigh (1957) shows
that apples produce larger quantities of volatiles other than acetalde-
hyde and ethanol when oxygen is in abundance than when oxygen is re-
stricted. This would tend to show that apple volatiles are produced

by an altogether different system than fermentation, one requiring
oxygen.

The pathways for production of volatiles from fatty acids is less
obscure. It is conceivable that the carbon chains of these volatiles
could be produced by the successive combination of acetyl-co-enzyme A
or acetyl-co-enzyme A and propionyl-co-enzyme A.molecules (Neigh, 1957).
There is little scientific evidence supporting these theories for apple
biochemistry but the formation of ketone bodies and many other compounds
similar to apple volatiles, such as esters and aldehydes, is part of

fatty acid metabolism.

LIMITING FACTORS AND SUGGESTIONS
The feat of showing the biochemical pathways by which volatiles
are formed is hampered by two very obvious factors. The direct approach
of finding the systems by biochemical studies of all metabolic pro-
cesses is complicated by the vast number of biochemical reactions al-
ready known about the cell and undoubtedly many more unknown biochemical
pathways and reactions. The indirect approach consists first of identi-

fying the end products, or volatiles, setting up model biochemical '

52

reactions that could produce these compounds, and then showing the
presence of these reactions in the apple fruit cell. The pathways of
production of acetaldehyde and ethanol have been successfully deter-
mined by the indirect approach. The indirect approach is hindered by
the limitations on identification of the products of the reactions.
The small amount of volatiles produced by apples, resulting in low cone
centrations of volatiles in storage atmospheres, make volatile identi-
fication difficult. However, advanced present-day equipment and techp
niques look very promising for overcoming the low volatile concentration
drawback. work is presently in progress with elaborate gas chromato-
graphic procedures which will overcome at least part of the problem.
Mass spectrometry has contributed toward the identification of vola-
tiles in this thesis and, in conjunction with gas chromatography, can
contribute a great deal more.

volatile collection methods leave much to be desired. Much of
the problem here is due to low volatile concentration. Cuticle removal
from apples might cause a greater concentration of volatiles in storage
chambers. Such studies could also be used as evidence for or against
the theory that the cuticle acts as a barrier for apple volatiles.
Placement of good organic absorbers and adsorbers on the surface of
apples for collection of volatiles might also be tried. Removal of
these collecting agents, along with the cuticle after storage may pro-
vide a more appropriate means of concentrating apple volatiles for
analysis. A great deal can be added to the present knowledge of apple
storage volatiles by keeping in mind what has already been found and by
applying new methods to the problems as both the problems and methods

develop.

SUMMARY AND CONCLUSIONS

A selected review of literature on apple storage volatiles is
presented.) Methods used for controlling storage volatiles and their”
effectiveness are discussed. Identified volatiles and the analysis
methods used are presented. The results obtained by previous methods
of isolation and identification indicate that apple storage volatiles
are organic acids, alcohols, aldehydes, ketones, esters, and hydro-
carbons in individual concentrations of a few ppm in the storage at-
mosphere. The application of gas chromatography and mass spectrometry
provided a promising outlook on gaining extensive knowledge of apple
storage volatiles.

Ethylene, ethanol, and acetaldehyde were identified in controlled
atmosphere apple storage atmospheres by LTHV distillation and mass spec-
trometry. The other volatiles were too low in concentrations and their
vapor pressure characteristics are too similar to those of water to be
separated from water by LTHV distillation alone. molecular sieve
adsorption techniques also failed to give satisfactoryirecovery and
separation of volatiles. Recovery of volatiles from activated carbon
air purifier units gave some results. The harsh acrid odor, not resem-
bling apple odor, of the heat eluted volatiles from activated carbon
gave evidence of degradation of the volatiles when compared with the
sweet apple-like odor of solvent extracts of activated carbon. Isopen-
tane extraction combined with LTHV distillation was found to be the
best of the methods tried for extracting volatiles from activated

carbon. Gas chromatograms indicate at least 30 compounds in the

isopentane extract from activated carbon. Saturated and unsaturated
aldehydes containing from 4 to 10 carbon atoms were tentatively identi-
fied from.mass spectra of the activated carbon extract.

The volatile ethylene was found to be in greater concentration by
far than any other apple volatile, except, of course, for carbon dioxide
and water vapor. Gas chromatography using the hydrogen flame ionization
detector provided a rapid, simple, and very satisfactory way to carry
out routine analysis for ethylene concentrations in controlled atmos-
pheres as is shown in this thesis.

The ethylene concentrations in conventional controlled atmosphere
storage rooms were studied. After sealing the room, the ethylene con-
centration builds up and then gradually levels off at a maximum equili—
brium concentration which holds rather constant until the storage room
is opened. Ethylene production appears to be proportional to the
amount of oxygen used for respiration for a particular variety of apples.
This is deduced from the constant equilibrium ethylene concentration.

No difference in equilibrium ethylene concentration was noticed in
storage rooms employing caustic soda scrubbers as compared with those
using water scrubbers. The presence of activated carbon had no effect
on equilibrium ethylene concentration. The equilibrium.ethylene concen-
tration values varied with apple variety. Approximate equilibrium
ethylene concentration values for the different varieties studied are:
1250 ppm for Northern Spy, 1000 ppm for McIntosh, 750 ppm for Jonathan,
and 500 ppm for Red Delicious. It would appear, therefore, that
ethylene production varies per unit amount of oxygen used for respira-

tion among different varieties of apples.

55

The lowest concentration of 250 ppm.was found in the storage room
which employed an experimental controlled atmosphere generator. The
inverse proportionality noticed between ethylene concentration in the
room and amount of generator gases metered into the room.shows the lower
ethylene concentration to be due to dilution of the storage room atmos-
phere with the controlled atmOSphere generator gases.

Chromatograms of direct sampling of storage room atmosphere and
of cold trap condensates of storage room atmosphere show only the pres-
ence of ethanol. Its concentration in the atmosphere of storage room 10
was calculated to between 100 ppb and 2 ppm. Therefore, ethanol was the
next most abundant volatile to ethylene in storage room 10, which con-
tained McIntosh apples but no activated carbon.

In view of the information obtained, mass spectrometry, with the
development of collection techniques after chromatographing and more
elaborate gas chromatographic equipment, would make possible more quali-

tative and quantitative information of apple storage volatiles.

10.

11.

12.

13.

15.

53

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