SEPARATUM 30 - X I • 1964 No. 2-4

Qu a l i t a s p l a n t a r u mE T

ma t e r i a e v e ge t a b i l e sHereditas, Biochimia, Physiologia, Oecologia, Cultura et Praeparatlo plantarum

cdulium et artibus u tili um

^ rgan of the Confoederatio Internationalis ad Qualitates Plantarum Edulium >erquirendas (CIQ) and of the International Commission for Plant Raw

Materials

EDITORS:

A- Th. Cz a j a , Aachen - L. G e n e v o is , Talence - C. R e g e l , Izmir BornovaW . Schuphan, Geisenheim

Ga s c h r o m a t o g r a p h i c a n a l y s i s o f

C U R I N G SMOKES(A Progress Rep6rt)

Z d zislaw E. Siko rsk i

technical University, Dept, of Animal Products Technology, Politechnika Gdanska. Head: Prof. Dr. D. J . T il g n e r )

(with 3 figs.)

VOLUMEN X I

G I T G E V E R I J D R . W. J U N K - D E N H A A G - 1 9 6 4

NOTICE TO CONTRIBUTORS

’ ’Qualitas Plantarum et Materiae Vegetabiles” publishes original contribu­tions on genetics, biochemistry, physiology, ecology, culture and preparation of edible plants and of those used as pharmaceutical and raw materials, further notes, reviews and personalia. Contributions must be typewritten in duplo double-spaced on one side of the paper in English, French, German, Italian, or Spanish. Long historical introductions are not accepted. Protocols should be limited. Names of plants and animals must be given according to the laws of binominal nomenclature adopted at the recent International Congresses of Botany and of Zoology, and are to include the author’s name in full. Measures and weights should be given in the decimal system. Every paper should be accompanied by short summaries in english, french and german. Summaries in other languages (portuguese, russian, Spanish, inter­lingua) will also be accepted. Text figures will be reproduced by line engraving and hence should not include any shading, although figures which cannot be reproduced in this manner will be accepted if necessary. All drawings are to be made in indian ink on separate sheets of white paper, the reduction desired should be clearly indicated on the margin. The approximate position . of text figures should be indicated in the manuscript. A condensed titl® f should be cited as follows — in the text: T o b l e b , F. (1947); in the biblio­graphy: M à c k e l , H. G., (1948). Mikroskopie einiger weniger bekannten Lavendelblattdrogen; Bot. Oecon. 1, 129— 141. or in case of a book: T ren d e ­l e n b u r g , R. (1955.) Das Holz als Rohstoff, Carl Hanser Verlag, München- Names of perodicals should be abbreviated according to the rules given in the ’World List of Scientific Periodicals’.

The various types of printing should be indicated by underlining the words in the following way:

straight blue line: small capitals, e.g. all names of persons, both in the text and in the bibliography.straight red line: italics, e.g. all Latin names of plants and animals.

spaced type: for words to be stressed.

Fifty reprints of the paper with covers will be supplied free of charge W the publishers. Additional copies may be obtained at rates which will be quoted to the author with the proof.

Manuscriptson medicinally and technically on edible plantsused plants

to be sent to

P R O F . D R . A . T H . C Z A J A , B O T A N IS C H E S IN S T IT U T , T E C H N IS C H E H O C H S C H U L E , A A C H E N , G E R M A N Y (W E S T )

orP R O F . D R . C . R E G E L D E P T . O F B O T A N Y E G E U N IV E R S IT Y IZ M IR -B O R N O V A , T U R K E Y

Printed in

P R O F . D R . L . G E N E V O IS F A C U L T É D E S S C IE N C E S 3 5 1 , C O U R S D E L A L IB É R A T IO N T A L E N C E (G IR O N D E ), F R A N C E

orP R O F . D R . H A B IL . W . S C H U P H A N B U N D E S A N S T A L T F Ü R Q U A L IT Ä T S F O »^

S C H U N G P F L A N Z L IC H E R E R Z E U G N IS » G E IS E N H E IM /R H ., G E R M A N Y (W E S T /

the Netherlands

'

GAS CH RO M AT OGR APH IC ANA LY SIS OF C U R IN G SMOKES

(A Progress Report)

Zdzislaw E. Sikorski

(Technical University, Dept, of Animal Products Technology, Politechnika Gdanska. Head: Prof. Dr. D. J. Tilgner)

(with 3 figs.)

Introduction

During the VHth Conference of Meat Research Institutes (1961) the results of preliminary experiments in gas chromatographic analysis of curing smokes were presented (1).

For further work on the same subject the Griffin M2B VPC Apparatus was additionally equipped in our Department for programmed temperature operation. Nevertheless the analytical results could not be significantly improved. It became clear that the great number of chemical compounds present in the curing smoke cannot be directly separated (without any chemical pre­treatment) and detected with this type of chromatograph. The idea of direct introduction of the smoke samples onto the top of the analytical column could not be realized with good results using this type of apparatus, too.

In further experiments in which only ether extracts of the curing smoke samples were analysed the more sensitive Pye Argon Chromatograph was used. With the help of this instrument much better results could be achieved.

Experimental

The curing smoke obtained from an amount of 30 to 60 g of beech sawdust in a laboratory smouldering-type all-glass smoke generator in constant conditions was trapped in ethyl ether. The ether extract of smoke components was divided into equal parts which were treated with different group reagents in order to separate the respective groups of compounds. The prepared samples were dried with anhydrous Na2S 04 and the solvent was removed at room temperature under vacuum in the presence of nitrogen. The

t tliu extracts condensed to constant volume were analysed on the ye chromatograph.

he group reagents and processing regime applied in this experi- ments are listed in Table I.

I he samples were introduced on the top of the column with the , e P of a 0.1 microliter micropipette. The columns were held under sot ermal conditions at 100°, 150° and 190°. Also other tempera­

tures were tested (125, 175°). When programmed temperature operation was applied the column was held initially at 100°; after

the temperature was raised up to 192° C. The Pye argon ta r0mat°graPh was not equipped for PTGC. This caused an simul-

neous rise of the base line during temperature programming, urthermore there was only one heating rate possible.

T a b l e I

Sample code T r e a t m e n t

or the current letter giving the number of the sample.

Untreated, dried original ethyl ether smoke solution

A-iOriginal sample extracted with distilled water

A-l.i Ether extract of the combined water portions used for thepreparation of sample No. A-l

A-2 Original sample extracted with NaHS03 solution

A-2.1 Ether extract of the combined fractions of NaHS03 so . used above, after washing of the solution with ether and

______ alkalization with Na2C03

A-3 Original sample extracted with Na2C 03 sol.

A-3.i Ether extract of the combined fractions of Na2COs sol., after washing with ether and acidification with H 3P 04

A-4 Original sample extracted with NaOH sol.

A 4 Ether extract of the combined fractions of NaOH sol.after washing with ether and acidification

A-5 Original sample after reaction with dry NH,

A-5.i Ether extract of the ether washed and acidificated precipitate obtained at A-5.

374 Z. SIK O B SK I

Different column filling materials and different temperatures were tested in order to find optimum conditions for the separation of the smoke components of the untreated sample.

R esults and discussion

Untreated samples of curing smoke contain a great number of compounds having different boiling points, from low boiling car­bonyl compounds to very high boiling phenols and hydrocarbons. At low column temperatures only few peaks could be separated.

Using columns filled with solvents specially recommended for the analysis of phenols only very unadequate results were obtained at considerably low temperatures. Columns filled with xylenyl phosphate at 115° and 70 ml/Ar min gave in 30 minutes only 4 peaks. On columns with Apiezon L working at 225 only 13 peaks could beseparated. 0

Columns with Carbowax MW 100 Polyethylene Glycol at 1-5gave better results.

The best stationary phase for direct separation of the components present in wood smoke seems to be polyethylene glycol adipate, described in the litterature as a very efficient solvent for the sepa­ration of unesterified acids (2).

Detailed data obtained at different temperatures on columns filled with PEGA on celite are given in Table II. Some of the typical chromatograms are presented in the fig. 1, 2, and 3.

The great number of smoke components having different boiling points made it difficult to choose the optimal processing parameters for the separation of maximal number of smoke components present in the original untreated mixture. The amount of peaks separated a different temperatus from 100 to 192° ranged in all cases from abou 30 up to 40. The greatest number of compounds was separated with programmed heating of the column during analysis (ovei 60) Detailed data are given in Table II and in the chromatograms.

Group identification of the peaks gave some preliminary results.Among the 33 peaks separated at 150° on PEGA-column 17 have

a good watersolubility. The largest part of the compounds represented by this peaks (13) seem to be free from carbonyl, acid or phenolic functional groups. Results of chromatographic analysis of the A-G, A-2, A-3 and A-4 samples indicate that among the 33 peaks 6 are formed by compounds having phenolic functional groups, 6 by aci compounds, 2 by aldehydes and ketones and 19 peaks represenneutral substances. ,,

Chromatographic analysis of samples of original smoke as we

' hA N A L Y S IS O F C U B IN G SM O K E S 375

T a b l e I I

Relative retentions of the components of the ether extract of the smouldering-typecuring smoke.

No. of Peak 100°

Tern p e r a t u r e o f a n a 150°

l y s i s192°

0.009 0.03 0.060.030 0.06 0.100.055 0.09 0.190.074 0,11 0.330.080 0.13 0.530.086 0.16 0.650.101 0.21 0.700.127 0.23 0.820.145 0.28 1.00 phenol0.192 0.35 1.120.224 0.42 1.360.236 0.52 1.720.261 0.59 2.150.282 0.68 2.400.336 0.76 2.810.392 0.87 3.400.430 1.00 phenol 4.000.478 1.14 4.500.575 1.24 4.800.649 1.48 5.300.775 1.62 5.900.835 1.84 6.800.930 2.06 8.001.00 guaiacol 2.13 9.801.15 2.54 10.001.33 2.87 13.11.43 phenol 3.20 14.11.58 3.55 15.51.68 4.06 17.92.00 4.45 18.2

5.105.506.307.808.25

^farneters: 4 ft x 4 mm i.D. of 10 % polyethyleneglycol adipate on 100— 120 esh celite, argon flow rate 60 ml/min, chart speed 12 in/hr, sample size 0.1

lo0IOliter’ attenuatiorl x !> x 3 and x 10; voltage 1750 V at 100°, 1500 Vat and 192°; argon inlet pressure 0.7 atm.; outlet pressure atmospheric.

T

V

378 Z. S 1K O R S K I

as smoke treated with group reagents as given in Table I, on PEGA column at 192°, gave better results as compared with the a >o mentioned. Thanks to the variety of different treated samples m u d more individuals could be distinguished. On the B O chromatogra 36 peaks were separated (without the first one - solvent and vola­tiles). Among this number 16 are composite peaks, gathered mam y in the first half of the chart; 7 peaks seem to have carbonyl functio al groups, 11 react as acids and 17 as phenolic compounds, compounds corresponding to 7 peaks, at the end of the c OIf® , gram, did not show the presence of any of the above men functional groups and could not be detected on the chromatogram B-2.1. B-3.1, B 4.1, B-4. Furthermore, 13 compounds do not s any of the functional groups but could be detected on the chroma o grams B-2, B-3, and B-4. thus they are called neutral compounds

the smoke.Detailed data are given in Table III.

Conclusions

ofThe results indicate that direct gas chromatographic

the ether extract of curing smoke can lx* successfully usee separation of the compounds present in the curing smoke. jn

The greatest number of jx*aks (above 60) can be sep about 4 hours using the programmed temperature gas c ¿g) graphv method. Up to 36 peaks. representing at least con ¡0„ can be separated at 192° on the PEGA column, using argon

detector. .,,-tionsc»11For group identification of the peaks simple group n . furthcr

be used. Among the corresponding groups of compound identification can lx* made on the basis of retention < afor pure standards. nonent*

Much lx*tter results in the separation of the con | , „graphoriginal smoke samples should be achieved using a < ^ (.„Him11equipped with ionizing detector and programmed « »

oven- .. ~kw*matogrftl,,,|CFor practical utilization of this direct gas « . furtber

analysis of untreated curing-smoke samples (ether « xtra< ^ \ftt*r work on the identification of the ¡x»aks should be■ car'Mj ■ ^ fora complete identification of all jx*aks this meth<s <oU proes*®6* control purposes in smoke generation and smoke cu as well as in research work on smoke-curing phenotnen

A N A L Y S IS O F C U B IN G SM O K E S 379

T a b l e III

Results of gas chromatographic analysis of original caring smoke ether extract and samples treated with group reagents.

Sampledumber Sample code (see Table I)

Preliminary*)identification

! B-0 B-2.1 B-2 B-3.1 B-3 B-4.1 B-4B-5. 1

*

1 + + + + + + + +2 + + + + + + +3 + + + +4 + + + + +5 + + + + + + +6 + + + 1 + + +7 + + + + + +8 + + + —9 + + + + +

10 + + + + + +11 + + + _L112 —L + + +13 + + + + +14 + + + + + +15 ~r + + + + +16 + 4_r + + + + +17 + + + + + + +18 + + + + _1_T +19 + + + + + + +20 + + + _L1 + + + +21 + +22 + + + + +23 + + + + +24 + + +25 + + +26 + +27 + + + + +28 -j_ +29 + +30 + + + +31 +32 + +33 + +34 + _|_ + +35 + + + +36 + +37 + + + +

) c.a.P-n.?.

Compounds with carbonyl functional group compounds with acid functional group compounds with phenol functional group neutral compoundsunidentified with the help of the group reactions

solvent and valatilesc acc a

n an an p

a pp

n pp

n pn pn pn p

c a pn a

c an

c ap

?PP

cP?PP

?

P?j1

aa

?n

380 Z. SIK O R SK I

Summary

It is nowadays possible, by direct gas chromatography, to separate the components of smoke as their ether extracts. The use o a chromatograph equiped with an Argon ionization detector and a programmed temperature heated oven makes it possible to obtan results of a precision unknown up to now. But the problem remains still so the control of smoke curing operation will be made possib e only when all the peaks of the chromatogram have been identified.

R ésumé

Il est possible actuellement, à l’aide de la chromatographie ^ f l directe, en phase gazeuse, de séparer les composants de la fumée sous forme de leurs extraits à l’éther. L ’emploi de chromatographes munis de détecteurs d’ionisation à Argon et de fours colonne avec chauffage à température programmée permet d ’obtenir des résultats d une précision inconnue jusqu’ici. Le problème n est cependant pa épuisé et l’application de cette méthode au contrôle des opérations de fumage ne pourrait s’effectuer qu’après l’identification compte e de tous les pics du chromatogramme.

ZusammenfassungX

Mit direkter Gaschromatographie kann man heute die Bestand teile des Gazes in Form ihrer aetherischen Auszüge trennen. Die Verwendung des Argon-Ionisationsdetektors und eines tem peraturprogrammierten Säulenofens verleiht dem gaschronia graphischen Verfahren eine bisher unerreichte Genauigkeit. Die D« tersuchung auf diesem Gebiete sind jedoch nicht abgeschlossen. Anwendung dieses Verfahrens zur Kontrolle der Verdampfung Vorgänge kann erst vorgenommen werden, wenn sämtliche Chroma togrammspitzen identifiziert sind.

R e f e r e n c e s

1. S ik o r s k i , Z. E.: VII Conference of Meat Research Institutes, Warszawa’ 1961, Proceedings, in the press.

2. Pye Separation Techniques Xo. 8.

t i v e ana q u a n t i t a t i v e d . t a on the com posit ion o f th e

'ro u p s o f smote components,

jy p e r im e n ts .

/ 2/

c a *{• v ' j i' Tn' va e* ct-.i cm. b o--r i . . * o - c . b, In ore.ox to overcome both- d i f f i c u l t i e s

®au3 t o a p p ly th e ¿ a s chrom atographic method i . smeh’• a n a l y s i s .

In th e f i r s t approach pas chromatography ve.s a p p l ie u t o

the a n a l y s i s o f th e main ¿roups o f compounds, i s o l a t e d from

the or i , i n a l m ix tu re / s' oho cond on s a t e / .

In a ll experiments th e G riffin ¿.art 2f Apparatus '."as used

"" U e h j h - o t - f—a.-,-h; -CV«vli2Lriia f i l l i n g R t?^ e in t emuera-

t u r e s up t o 200°C.

d a t a rnj^ardinp th e c om eos it io n o f the carbonyl compounds

*?toup h r e been p r e s e n t e d a t t h e Gaansh's Se&sinn. ^ 3 j . , f f i v e -

fw .iif .— ——4*7 /G; acetone —

-e h, bonsuldehycn 11 f ■ v o l e r ic old ehycLo ii uhhneun.

fh jj?iuji.--ic»clatou from t h e o r i i n a l smote coon n s a t e

-re s *parat 2d on s±liooa.> j>aliuiia t j i c ± ’A£; tla > t.yp g-r —

02-if ioat io n. rnothoa p re p 0sea by J . m.Ralls / $ / . A sp e c i a l

£ - a c t o r kt . s mad: i n s t e a d o f th e c a p i l l a r y u s d by R a l l s ana

the r e a c t i o n ~'as c a r r i e d out i n th e c a r r i e r oas stream .

F iv e a c i d s v*:x- found, i , e. a c e t i c a c id / So,-/, form ic

Sold / 29?/, p r o p i o n i c a c i d /%*■■•/ and t^o 'mhneni s t i l l un iden ­

t i f i e d . 93xe l a s t t r o pres sat i n t r a c e s c o u la o.ot he d e t e c t e d

th t h e o r i g i n a l u n t r e a t e d nd-^ture o f «odium s ^ l t s and ’’n r 1 found

:>x 3n th e major p a r t o f t h e j f o r -‘Ooin^ a c i u s h-.-.a b e . n r n.-ovea

t o r . t he miutus. e.

T y p ic a l chroma t o ^ o b t a in ec. i n l o t h ano.l - sea 00: sho” n

1;- f i r u r ' do. 2.

Rr e l im in ary a n a l y s i s o f th e p h e n o l ic f r a c t i o n o f t h "

chrin._ smote r e s u l t e d i n chromstoprams sho^uny o n l y on; main

°°mpon;ent. T h is i n d i c a t os t h a t s i w i l i a r l y as i .. th.; c-: s e o f

acide. ; en enrichment o f the t r ? c ' coupon ent r ir ?ec '^rpr^.In further erperiæents th p rob lm o f fcmoke t . e,plia._ vas

at te ckJd. According to th e opinion o f K. - i l >r / t / th ; ideal a; o.h î sampling proc edur > shoolu h"? the d ir et in tro miction of SEoh'! on top o f th? chromatographic column. This -v o it - : ' any

^lùJeirebla reactions between tiu components ¿nu any lo ss o f const itu mt s dur in,-; th *> pr • y t r eatfc ent.

Attempts r;r.? maae to re a lise th is ic.aa. 5ha ¿ nierai arran- Ô.ment o f the- apparatus used in th~ '•r'O ■'riment s i.e sho’m in ■ iv. • i o . 3 •

hurinw th.- periou of sa oho . in f la t io n th? chrom.-: to rap'iic

column rrn h?lcl rt amM nit t imp eretur The or *}anic ra o'-e con­stituents d issolved i the column iio u id <u?d fu r in ? the 1 to

5 minutes o f sampling did not mov ; far enough, to cause any .c-on- siaerabl e overlapping and peak “cJ.stoo;ti«ns»--f?ubs?qu m tly the Propre.' E?c t Jüiporaturv opnm tlon ^as appli .*4. TTnfortuaat *lv -our chromatograph is not provided with separate chambers for th?

detector and columns ana the b eso -lin e he,«, therefer^ 0reat ion.- term d r i f t . If everthel ess th > p eaks resu ltin ', dur inn elu-

dioii o f components coula bo cl earl/ a istin ru ish ¡d,P.onc 1 u s i o n s .

>

smoke on ¿as c hr ora. to -

. rairr •d terp erbtur *as¡on ent s of th v- o r ! ,in?.l

n'irtu V . n '»’ meeems to he ? valuahle method fo r the a 'termination ° f the inuiviaual main sroke conctitu n ts. The b'-st column ^illin .,. m aterials ana workin, parameters for- the n '-lv ci? o f Sl-ch complex mirtures should be found in ora a to improve­r s technique.

Glace reaction should bo used fo r the id e n t if ica t io n o fi* "*-- ■' Peaks reru ltinp in chrome top::,? ms instead of: v e in th is r?ae

t io hs to separate the main w roups o f compounds fee- the o r i^ i-

&

Á

- 4 -

dal cii^irr^prl-cnc^ i>o chro mato ara piiic ~nalye i ",.

1. Spanyar P. et a l l .s S e itsch rift fu ir LVo^nsöitt ¡1 TTnt ei suchunyuna Forschun¿, 112,5» 353»19¿0.

I2. .SiEpson E.H., Campbell : Advanc^s in fho 'Piyineerin;- o f th?

Smolce Curin¿ ?r*coss, I I .In t ernational Session,Oäansio, 19^0, Paper IV / 1.

3* Sikorski Z. 1.: 'I I Int ornational Session, Gaunslo 19ó0, Paper1/7 .

4. T il m x i .J .e t a l l . : II International Session, ? a p r IV/2.

% 5* Halle J*T". : Analytic.0 Ghemistry, 52,332,195®.■J* M i 't E .: Per son? 1 conmuni cat i ons, 1950.

AHsaiiijg enfas sunffOas chromatographiache Methoden •■nzra >n in a r 7:auch erraue’i -

•Attalys > ansevendet. ~'r verdón ¿.i~ rauptkoirponent e c r eina elneni’Upp en von vji binaun0 en / Sauren, nariDonyl-Veibinaun_ on / b

^ in cit. Auch ”iirc! en Ver such e durch •- ofuehrt, in c "•non P er Pauclier- ^Uch airekt auf ¿..en V-ei-t >ilun._ drolonn en abyesetzt una ansclili esc end

'GM fcontinui erlicher ’brrrarwun?; chromato; raphi ert rurdr.

% jsz csanio .Sait. o so vano motody chromat o. _ic-.fi i ;,rnoT:,ej do oro. essuia slslau-

:"qt- -1dvrvch -rvn sM orhov fry ocrebnionych s üymu v,7ods-'■mica er%,

^ z aproare, s on o takz e proby boapoár etinio.io ^pro^ac: %■? rin tfrnrou na *-°lumny tíiromatoyraficzne i roádzialu silaanilió". pray proyramovymo •.orzeuaniu v cae.si 2 analizy.

\