\s> Volatiles in Controlled-Atmosphere^ Apple Storage: Evaluation by Gas^ Chromatography and Mass Spectrometry

^- Pio Angelini" and I. J. Pflug1'

Fuel Science Department, Michigan Slate I'niversity, K.-isI f.:iiixiiiu. Michigan l-SSlM

SUMMARY

fins chromatographic, and massspeclrometric analyses methods wereused for ethyleneconcentration studies niiil (he identification of othervolatile* in cuul rolled atmosphere(('A) stomal! atmospheres. Directrhromatographing of ('A storage atmospheres provided a rapid, simplemethod lor following the ethylene concentrations in ]| (\\ apple storages. Comparisons of the elTeets ofapple variety, two types id' earhoiidioxide removal eipiipnielil, and thepresence or ahseiiee of aetivaled earhoii on ethylene concentration weremade. Kxtrncts of activated carhonfrom an air pnrilication unit whichwas used in a CA .Mcintosh applestorage lor one season were analyzed.Identification of many ol' these compounds was achieved by last scanning mass spectral analysis of diluentsfrom a temperature programmed gaschromatographic coluinn. Saturatedand unsaturated hydrocarbons predominated both in number and quantities. Several esters, primary andsecondary alcohols, and a I'vw aldehydes were also identified.

INTRODUCTION

The identity of volatile compoundsproduced by apple fruits has receivedconsiderable attention in past decades(Thompson, M)">1 ; Thompson rt al.,Mlfil; ilueliii, 1!)"j2; and ]len/.e el al.,I!)"»:i, .1S15-I). The literature on thecited of volatiles on product qualityis also ipiite voluminous and has beenrecently reviewed by Smock (liMil).Meigh (|!)JS(i, ji)57), and Phillips(!!)">!)). In general, the conclusionsare that no relationship between volatiles and scald has been establishedami that the number of volatiles analyzed, although lengthy, has been lacking especially in the quantitative analysis of the different components.

.Most of the methods and techniquesthat have been used for the earlier

" JVcsciil address: Analytical Cl.ein-istiy l.:il»ii;iloiy, I'iiinccriii^ Researchl>ivisimi, V. S. Army Xalick Lahnralo-ncs, Nalick, Massachusetts OI7<i<>.

''Present address: College itf MedicalScience, School of Public Health, University of Minnesota, Minneapolis, Minnesota.

studies of apple volatiles althoughsensitive are cumbersome for complete!analysis of such a complex mixture,of compounds. The methods and re-sulls of studies on apple storage volatiles and similar problems suggest thatthe usf of advanced physical-chemicalmethods of sampling and analysisshould add significant information tothe apple volatile picture as shown byTurk n al. (Ii)->l), MaeUregnr el al.{!!)<; I) and Meigh (HUM, HUM).

This study stresses the applicationof low-temperature high-vacuum distillation, mass spectrometry and gaschrouialography for qualitative andquantitative analysis of apple storagevolatiles. The purpose of this studywas the qualitative and quantitativeanalysis of some controlled atmosphereapple storage volatiles and how theyare affected by various storage conditions.

EXPERIMENTAL PROCEDUREDirect ethylene concentration anal

yses. Kthylene concentration in controlled atmosphere storages was analyzed directly with an Aerograph modelA-(i(MI My-Ki chroniatogrnph. A Vs-in.X 10-ft. stainless-steel column packedwith |II'A', Castorwax on ('hrnmosorh\V, an oven temperature of faTC amia gas sample size of JO /*| were usedfor all gas analyses of storage roomatmospheres. A management summaryof the II storage rooms used in thestudy is shown in Table I.

The desired levels of oxygen andcarbon dioxide were obtained purelyby (he respiration of the fruit withscrubbers to remove excess carbon dioxide in all storages except room II.This storage had an experimental controlled aim..sphere generator and acarbon dioxide scrubber to produceand maintain the desired concentrations of oxy-en and carbon dioxide.The oxygen .->ml carhon dioxide, onceobtained, were maintained at optimumconcentrations for the varielies ofapples | reseiiI in each storage chamberaccording (o the State of Michiganspecifications for CA storage of apples. In all cases the fruit was in very

STABY - OSUgood to excellent condition with minimal spoilage; when it was removedfrom storage.

A standard envvo for ethylene concentrations in air was prepared by obtaining gas chromatogratns of concentrations of ethylene in air ranging fromI to -1000 ppm. peak heights wereplotted against ethylene, concentration;the resulting curve (Fig. 1) is withinthe limits of experimental error astraight line. This curve was used todetermine the ethylene concentrationsof all storage atmosphere samples. Acheck on the calibration of the ehro-matograph was made with known concentrations of ethylene in air eachlime storage samples were analyzed forethylene concentration.

Samples of the storage room atmospheres were taken in 4-L polyethylenebags from the storage room gas sampling tubes. The gas sampling tubeswere connected to the storage atmosphere recirculating lines between theblowers and the adsorption towers ofthe respective atmosphere scrubbingsystems. The polyethylene bags werefilled with the storage room atmospherebeing collected and collapsed completely three times to flush out anyresidual gas. Tins open end of thepolyethylene bag was gathered tightlyaround a rubber-vaccine-bottle-stopperand held in place by a rubber band.The gas was taken from the polyethylene sample bag with a syringeand injected directly into the gaschroma!ograph injection port.

No change in ethylene concentrationwas detected over a period of 0 hrin samples collected and analyzed inthe manner described previously. All

IICOIDM llirONSI - OIVIUONS

Fig. ]. Standard curve for ethyleneconcentration in air.

Vol. 21. 1643 II Fnnn TtruMni Arv n ^r

r"

-A

Tablo I. Storage room management summary.

'l'<-iii per •1 l.'S reel i:as cone nlriilions

Typa Aclivalei

Room Dnla i.l n ni Date RC.ruhliiiiK carhein Dateno, Variety scaled •F % Os % CDs uhtnincel unit uscel tlfCfll opened

1 Mel nl..si. 10/2/01 :i7 :i 5 10/2K/01 Water Yes 1/15/622 Mcliiteixh 10/10/01 :i7 3 ij 11/H/0I Water Yes 3/4/623 ltd Delicious 10/21/01 :i2 3 3 11/21/01 Water Yes 3/4/62•1 Jonathan 10/10/01 :t2 3 5 1 1/22/01 Water Yi's 2/11/82

5 Northern Spy 11/15/01 :i7 5 8 12/1/01 Water Yes After 5/1/026 Mcintosh 10/10/01 37 8 5 11/5/01 Water Yes 4/27/627 Jonathan ami

U olden amiRed Delicious

10/28/61 32 3 3 11/28/01 Water Yes 3/22/62

8 Jonathan amiCol.Icn and

]o/:io/gi 32 8 12/0/61 Water Yes •1/21/62

0 Mcintosh 10/8/01 37 8 11/10/01 Caustic Soil a Yes 4/28/621(1 10/12/01 37 3 5 11/29/01 Caustic Soda No 5/18/6211 Mixture ' 10/15/01 32 3 3 10/20/01 Water No 5/J/62

1 Mclnlosli, Jonathan, Rome licauty, ami Golden and Red Delicious

TIME-DAYS

Fig. 2. lOthylenc concent rat ion in atmosphere of ("A storage rooms \\, .r>, ami Gversus time.

1000 -

250 -

210 240

11M E - 0 A Y S

Fig. :?. ethylene concent ration in atmospheres of <'A storage roonia !>, 1", andvs. time.

100—DECEMBER 1967 II FOOD TECHNOLOGY || Vol. 21. 16-1-1

samples used for ethylene concentration determinations were analyzedwithin 4 hi" after collection.

Cold trap samples. Cold trap samples wen; collected by passing 10 ft'of storage chamber atmosphere IhroiHia liquid nitrogen cold trap. The samples collected in this manner wereheld tit. —196°C and evacuated toremove all gases at this temperaturewith a low tentpernturc-high vacuum(T7TIIV) apparatus similar to thatdescribed by Merrill ct «/. (1050).The compounds remaining were selectively fractionated into five parts bycollecting compounds that were volatileat -155, -125, -100, -78°C, andat room temperature. The volatilecompounds wen; collected in a liquidnitrogen trap. The five fractions contained compounds exerting vapor pressures greater than 5 /a Hg betweenthe temperatures —100 and —155°C-155 and -125°C, -125 and'-100°C, —100 and -7S°C, -7S°Cand room temperature and were analyzed with a consolidated model 21-10.'}(J mass spectrometer.

Activated carbon extracts. Concentrates of apple storage volatiles wereobtained from activated carbon thathad been in a controlled atmosphereMcintosh apple storage during oneseason oi' approximately 200 days,Three methods for removing volatilesfrom activated carbon were tried andcompared. The first method consistedof placing n \D-x carbon sample in «gas bottle and cooling it to -196°C.All materials volatile tit —li>G0G werepumped off using the LTIIV system.Subsequently, the carbon was heatedto 1S0°C and the remaining compound;driven off and condensed in a —10G°Ccold Irap under high vacuum.

Compounds absorbed on the activatedcarbon wen; also removed by [Kl)solvent-extraction techniques usin" di-ethyl ether and methyl butane (jsa.penlane). Identical procedures werecarried out tor extraction by each sol.

'

^.v«'titrr"Tcn g of carbon were extractedwith three 10-ml portions of solvent.The solvent extract was placed in agas hullU' and the solvent was distilh'doff at —7S°C iimler high vacuum,leaving behind the apple storage volatiles that were not volatile at —7S°(\

Tin* isopi'iitane extract e»f activatedcarbon was analyzed by wide rangeIcmperature progr;tmined gas chroma-lography combined with fast seanuiiigmass spectrometry. A Vs-in. X 10-ftstainless-steed column packed with 5%Carbowax 20M on (ire-brick was tem

perature programmed from —50 tei|50°C at 2°C per min. The effluentof the column was directed into the

ionization source of modified liendixmodel 1-1 titne-of-tlight mass spectrometer as described by Merrill rt al.(I0(il). Mass spectra of the samplecompounds separated on the columnwe're oblaineel as the compounds wereelitled from th«> column.

RESULTS AND DISCUSSION

The ethylene e-otu-ent ration of elevenCA storage- rooms was cheeked at intervals throughout the liHil 02 storageseason by sampling and analyzing Ihestorage- atmospheres as eleseribeelabove. The results oblaineel from six

nf the storage's are- shown in Kigs. 2ami '1. Ilootns 1 through S we're e-om-nicrcial storages reunite tei the laboratory and we-re sampled less frequently.

In all case's the' ethylene' eoueentration iucrenseel steaelily after sealing therooms. When the room remained seah'dfor a long e'lieiugh jie'rieiil of time', aswas the ease; for rooms '.i, 5, 0, 7, S,ami 0. the ethylene eoueentratiouIcveh'il ofT and varied about an eepii-lihriuin concentration until the roomwas opened. Kootn .1 was opened before the ethylene' coite-e'titration reaehe-elcipiilibrium and rooms 2 and A were-opened before snllicient data cotilel heeollecte'd to establish equilibrium ethylene ceine-eiitrations. The- ethylene e-on-ccnlrations in rooms JO and .11 showmore llucluatieitts than in other rooms.The irregularities are due to the- factthat other relatively large atmosphere'samples were reuioveel from room 10for analysis and that the- experimentalrout rolled atmosphere ge-nerateir use-don room II was very erratic in itsoperation.

The results suggest that the' equilibrium ethylene' coue-eutration levelreached in a storage1 room depends on(he- variety of apples stored in the-room if oxygen concentration is maintained at a cms!.-nil level. Koom 5

contained Northern Spy apple's andrcaclii'd an equilibrium ethylene eon-rcnlralion id'approximately 1250 ppm.h'ooins with Mcintosh apples ivaeheel

an equilibrium ethylene concent rationol' 1000 ppm, while- (lie* room with ItcdIh'licioiis apples achieved an equilibrium in ethylene! e-oiicentrnlion of 500ppm. It'ooiiis e-oiilaiuiiig mixtures ofapple- varieties produced equilibriumethylene' eoneeulrsitioii between thosefound for 1he* rooms containing Ilieri-spi-clive single- varietie-s of apples.

No great difference in equilibriumethylene concentration was observe-elhetwe-e-n rooms using water or causticsoela absorption systems and/or thepresene-e or ahsenee of activated carbon. Iiooms (i, !) ami 10 all contained

Me'lnlosh apples and all reached anequilibrium elliyh-ne e-onccntration ofapproximately 1000 ppm. Room (i hada wate-r absorption system, and rooms0 and 10 had e-austie soda absorbers.

Rooms (> and 0 contained a diva led

carbon air purifiers while room 10 didnot. Then-lore, these two factors didlied appear |o affect. Hie equilibriumethylene concent rat ion in colli rolledatmosphere storage rooms.

Mass spectra of the cold trap condensate frnclions enabled the' ide-ulifi-

e-alioti of ethylene from the —l!Hi to—I55"C I'raclioii and accl.-ildchydc amie-lhvl alcohol from the -125 to - 100"

C fraction. No either positive ielenli-fic.-ilions could he made from the1 colel

trap e-otideiisate's. Nevertheless, massspectra of the- —7S°C te) room temperature fraction shows ni/e's charae-te-rislic of organic compouuds alongwith a large1 amount of water. TheLTI1V distillation technique's, therefore-, are effective in separating these

compounds from the more volatile airgases but not from water since watere-xerls a significant vapor pressureabove —7S°C Water vapor was present in far greater amounts in applestorage- atmospheres than were theseoilier compounds. This fad was quitee-videnl when the mass spectrum of—7S"C to room tempe-rature fractionwas considered.

(las chromatogrnms of the he-adspacefrom each of the activated carbon

extracts are shown in Fig. A. Thechroniatograins are correlated with theobservations made by smelling the samples. Both solvent extracts had a sweetapple-like odor combined with a mustystorage room odor. Kxce-pt for size ofthe solvent peak, the first large peakin each cbroniatograni as comparedwith the other peaks; both chroniatograins from solvent extracted sample'sare- quite similar. A radically differente-hroniatograni anil odor were producedby samples removed from activatedcarbon by heat. It is believed thatthese differences are due to heat degradation of the* components during removal from the carbon. Isopentaneproved a more satisfactory solvent forthis extraction than diethyl ether because it is more volatile and thus easierlo dislall off at low temperatures.

Compounds identified from isopentane extracts of activated carbon bycombined gas chromatography and fast.scanning mass spectrometry are listedin Table 2 according to class. Thesecompounds were identified by comparing their respective retention times and

TIMC-MIN.

rig. I. (ins clironi.-itograins of apple .storage volatiles removed from activatedc.-irl.on liy A, heal ; I?, diethyl e-llier extraction; C, isopentane extraction. AerographModel A-lJtm M.v-I-'I gas cliroinnlngrnpli; Column: XA" X •>' Stainless steel columnpacked with fi'/e SK-ItO; on acid-wa.xlicel cliromosorl) W; Carrier fias: Helium atL't) ml/miti; Oven Temperature: Isothermal at 50°C.

Vol. 21. (645 || FOOD TECHNOLOGY || DECEMBER 1967—101

EVALUATING VOLATILES IN CONTROLLED ATMOSPHERE STORAGE concluded

Table 2. Compounds identified

lyilrnrnrlimiM Alrnlinlx

from activated carbon extract;

Ksl.'is

ii I.nl fiuiiiali.

sci- Imt formulainii n.-atiitcml iii-i-tiili-

.•I liMlyrululull Inilyi-ali'li.'X lii.lyial.'ln-x pi-nlannali-

Alil.'liyil.'S

•J in.- pl-lipi-iilllisi.vali-ral.li'liyilnln-ptaiialni'liinnl

IKIII.M11 111

.l.-.-anal

of dett! rminlug the validity of thistheory.

REFERENCES

XpH&Hfcc, R. E., Raker, C. R. and QuneV-.hush, !•'. W. 1953. The chcmScomposition of apple storage vol*.files. Purdue Univ. Agr. Rxnl siItull. No. 070. ' •

mJio, It.. I-;., linker, 0. R. nnd Quack*,bush, \>\ YV. l!»r,-t. fJarbonyl «j!l.nimils in apple, storage volnthYtPurdue Univ. Agr. Kxpt. sia. r„No. L'L'.

jfnelin, I-'. K. 1052. Volatile products ofapples. II i. identification of a|,u.Iiydes and ketones. Austral j'eRes., />' 5. .'{28.

Mficfireuor, I). ]i., Sugisawa, II •„, iMatthews, .1. S. 1004. Apple'jui'volatile*. ./. Food Sri. 29 -l-|.s

Vf-iKli, I). V. 105G. Volatile compoUm].produced by apples. T. Aldelivdr,ami ketones. J. Sci. Food Agr T'vte

y%h, !'. I''. 1057. Volatile compound.produced by apples. II. Alcoholi

y and esters. ./. Sri. Food Ai/r. 8 JOK?Meigh, I). I-'. 1050. Nafiiro of olefin,"

produced by apples. Nature IBi proMejtfh, I). |.\ mO. Use of tras oh'roni;

j logrnphy in niensurhif; the ethyleneproduction of stored apples j' <sr;Food Apr. 11, .'{.Si. ' '

fVMeilch, !>. P. I0G4. The natural skinA) coaling df the apple and its influence

on scald in storage. T. Patty acidaand hydrocarbons. J.Sri. Food Agr15, 435, ' ' '

/'•'•'ill. C. Uresnick. fi. R„ RatinelM. Ii., Walsh, J. T. and Angelini, P."1050. Determination of volatile com.I nls of foodstuffs. Techniquesand their application to studies ofirradiated beef. ./. Agr. Fond Chrnx7, 784.

M&rriff, ''., Walsh. .1. T.. Porss, D. A' Ani,'.'iiiii, p. am] Swift, s. .\i. Jg '̂"

Wide-range progranuncil tempera-lure gas clirmuntogrnpliy jn jneseparation of very complex mixture*1 Anal. Clirm. 36, 1502.

'..' mi' lillllllli'

Illll'XIIIIi'

'J inr pell 111 IIP(MtI'iIii-MIIIC

ll-licplillli1II lll'llllll!

ii milium'

in.' ...Ian.'

n lll'llllll

Mil' IIIIMilllll

II lllllll'.'lllll'I III- Ill'I ui'li'iin

I IIOIII'llll

1 .l.r.ii.'

I -iiiui.'ri'ii.'

liii'l.vm:1 II.ill \ lie

I ili'i'_\ noiiiC'lioir/.cncft li.'ii/.i'iii'

n prop lii'iixciici prop li.'ii/.'lull lull Iii'Ii/.imii-

l.il I.ut I..-II/.-M.

II |ll-llt lll'll/.l'IIC

- t»«.|«'_' llli' I Jp|-<• |i:i II«>l- i 2-prnpunnl2-|M-|11.'lllll|M pciiliin.il•J III.- I llllllllMll:: mm- i iMititnol•J in.- :: liiiiaiiol:i mi- -' Iiiiliiin.1

I licxilllfil:i inr I pcl.wml

mass spcefrn WJtll tllnsi' ni" klldWII compounds using llic same iii.-tniini'iitsystem am! iili'iilir.-il operating parameters. Severn I oilier compounds couldno) In- positively identified because ni'one in1 more of the following reasons:insufficient separation by gas chromatography; unavailable, matching mussspeetra; amounts of eonipoiinds toosmall In obtain identifying massspeel la.

I lyilriir.'irbiiiis were found in muchgreater abundance, both in number .Miniin .•minimis Iha ii any of the otherclasses of compounds. Solvent andunexposed activated carbon blanksyielded Ilie presence of isopriilauc, u-hcxatic ami 2-metliyl pciitnuc only. Itwas concluded, therefore, thai the restof llie compounds listed iii 'ruble "_!were absorbed onto the activated car

bon from the apple storage atmosphere.The origin of the identified com

pounds could be from several sources.The combined apple-like ami mustyodor of the Solvent extracts of Un

exposed activated carbon suggests twoof the more obvious sources: namely,from (.lit* apples and from microorganisms growing within the storagechamber. Other sources, some uoli|iiile so obvious, must also be considered. Volalilcs may be contributedto storage atmospheres by solvents andimpurities in the building materialsused to construct the chamber. Tin-

wooden container used to hold fruitmay contain volatile resins whichwould add to storage atmosphere Voia-liles. The refrigeration equipmentwithin the storage chamber would beexpected to contribute volatile substances from lubricants and from

lllilllllc refrigerant leaks if present.Storage room ventilation ami or at-

V

mosplierie washing (C.\ storages)would lower the concentration of Vol

alilcs produced within the storagechamber but would in turn add Voht-Iiles from outside tin: chamber. If airfor ventilation is taken from a service

room, this operation could add considerable volatile materials to the storage room atmosphere.

Mosl ol Hit! alcohols, esters, andaldehydes identilied are produced byllie apple fruits (MacfJregar rl, nl..I.'HM ami Meigh, |J)5(j, |{):"»7). Somenl' the oxygenated Volnl.iles are likelyto In- products of iiiieroorgaiiismswithin the storage chamber. The hydrocarbons, however, could originatefrom any of these sources. Kllivleueand other short chained hydrocarbonsind other short chained hydrocarbons ,^ _-- ' Anal. Clirm. 36, 1502.ire known to be present in the euinna-^jl'hillips. VV. It. 1050. Scald control inion from apples (Meigh, 1050, 1000), commercial CA storage. Can. Dept.

i ,ii i'ii i AKr<> Coinm. on Fruit and V»-lid recent V loll"- chain hvi roea r inns ^ i> .:.... i>..... ,,„-,>> ' ' ' U-and recently long chain hydrocarbons

containing from 1(1 to 30 carbons havebeen isolated and identified from apples ( Meigh, 1!XM). In view of this.Ihe apples could be responsible formany of Ihe aliphatic hydrocarbonsidentified in Ibis study. The aromatichydrocarbons are more probably derived from building materials and/orlubricants used oil storage room equipment.

The theory that apple scald is produced by volatile emanations fromsome apple varieties has not ye! beenlulls established, even (hough there ismuch evidence supporting this theory.Meigh's ( 1004) results indicate thatneither the fatty acids nor the hydrocarbons found in his study are involved in producing scald hut furtherinvest igaIions would be necessary I"prove this. Nevertheless, analyticaltechniques presently available and inuse on Ihe problem show great promise

Preservation Kept. (1058): 4.Smock, It. M. 1001. Methods' 0l WaM

conl nil on the apple. Cornell UnitAjjr. Kxpt. Si. Itull. No. 970.

/lonipsou, A. It. 1051. Volatile products of apples. T. Identification ofacids and alcohols. Austral J <inilien., II 4. 283. ' ' n-

yininipsnn, A. II., Iluclin, P. g, ]f)5if Volatile products of apples. II. pro|

din-lion of esters by Rrnnny smithapple Austral. .1. Sci. Rci n ,

/ink, A., Smock, It. M. nnil Taylor T I' 1051. Mass anil infrared spectra of

apple vapors. Food Technol 5 isMs. ree'd 8/10/66. * ' '

.Journal Article No. .'{971 of the Mjfnj.gnn Agr. Kxpt. Sta., East Lansing, Michigan. Presented at the 23rd annual meeting of the Institute of Food TcchnoloRi»l«.

The authors are grateful to J KUriuiuer and I). II. Dewey „f Mid,'}-,,,Stale University for advice and assi>tance; and Charles Mcrritt and perion.in-] of flic Analytical Chemistry Labor*lory al the U. S. Army Xati.-k Lahoralolies for use of the mnss-spoctroinetrrfacilities. •

102—DECEMBER 1967 || FOOD TECHNOLOGY |] Vol. 21, 16-16