Simultaneous Detection of Nitrogen and Sulfur Containing Flavor

5 Simultaneous Detection of N i t r o g e n a n d Sulfur Containing Flavor Volatiles DONALD A. WITHYCOMBE, JOHN P. WALRADT, and ANNE HRUZA

Downloaded by UNIV LAVAL on April 24, 2016 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0026.ch005

International Flavors and Fragrances, Inc., 1515 Highway 36, Union Beach, N. J. 07735

Element s e l e c t i v e gas chromatography detectors are used e x t e n s i v e l y i n the f i e l d s of p e s t i c i d e and environmental chemistry. Their relative merits have been compared i n recent reviews ( 1 , 2 , 3 ) . A very r e cent paper by McLeod et al. (4) described the s i m u l t a neous operation of 5 detectors for p e s t i c i d e residue a n a l y s i s . The detectors which they used were: flame i o n i z a t i o n (FID), flame photometric (FPD) at 526 nm for phosphorous, FPD at 394 nm for s u l f u r , Coulson electrolytic c o n d u c t i v i t y for n i t r o g e n , and e l e c t r o n capture for halogen. Our own system, which utilizes flame photometric (FPD) and alkali flame i o n i z a t i o n (AFID) detectors f o r the a n a l y s i s of s u l f u r and nitrogen c o n t a i n i n g compounds i n a d d i t i o n to the FID,has been the subject of a short a p p l i c a t i o n s note (5) and will be described i n detail here. While there are s e v e r a l a l t e r n a t i v e detector types which are s e n s i t i v e to nitrogen and/or s u l f u r c o n t a i n ing compounds t h i s d i s c u s s i o n will be l i m i t e d to those we consider to be the most u s e f u l . For s u l f u r selective response, the flame photometric detector (FPD) i s by far the e a s i e s t and most reliable to use. Organics in the column e f f l u e n t stream are burned i n a hydrogen-rich flame i n which they are fragmented and e x c i t e d to higher energy s t a t e s . The e x c i t e d S species formed from the combustion of s u l f u r c o n t a i n i n g compounds emits photons at a wavelength of 394 nm. The emitted light is passed through an appropriate interference filter and detected by a p h o t o m u l t i p l i e r tube. The response to s u l f u r c o n t a i n i n g compounds i s virtually free from interference by other compounds normally encountered i n f l a v o r research (selectivity i s about 10, 000:1). The s u l f u r response curve i s p e c u l i a r l y n o n - l i n e a r . Recently, e l e c t r o n i c linearizing circuits 2

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Charalambous and Katz; Phenolic, Sulfur, and Nitrogen Compounds in Food Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1976.


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have been o f f e r e d by instrument m a n u f a c t u r e r s f o r the flame p h o t o m e t r i c s u l f u r d e t e c t o r . The two d e t e c t o r s most o f t e n used f o r n i t r o g e n a r e the a l k a l i flame i o n i z a t i o n d e t e c t o r (AFID o r t h e r m i o n i c ) and the e l e c t r o l y t i c c o n d u c t i v i t y d e t e c t o r . The AFID i s the s i m p l e s t i n d e s i g n , r e q u i r i n g o n l y a s o u r c e o f a l k a l i m e t a l as an a d d i t i o n t o the u s u a l flame i o n ization detector. The mechanism of enhanced r e s p o n s e t o n i t r o g e n and phosphorous compounds i s s t i l l l a r g e l y unknown. In the most f a v o r a b l e c a s e s , s e l e c t i v i t y f o r n i t r o g e n c o n t a i n i n g components may approach 1 0 , 0 0 0 : 1 . However, i n p r a c t i c e , s e l e c t i v i t i e s on the o r d e r of 100 t o 500:1 a r e more common. The AFID i s v e r y s e n s i t i v e t o changes i n the gas m i x t u r e and p o s i t i o n o f the a l k a l i m e t a l s a l t s o u r c e . A l t e r i n g the s i z e o r l e a n n e s s o f the flame changes the temperature o f the a l k a l i metal s o u r c e and can dramati c a l l y change the mode o f r e s p o n s e , s e l e c t i v i t y , and noise l e v e l . For example, the degree o f r e s p o n s e t o s u l f u r and h a l o g e n c o n t a i n i n g compounds when o p e r a t i n g as a n i t r o g e n d e t e c t o r a r e c o n t r o l l e d t o a c o n s i d e r a b l e e x t e n t by the flame c o n d i t i o n s . Recently, Perkin-Elmer has i n t r o d u c e d an improved AFID which they c l a i m t o be v i r t u a l l y f r e e of these e f f e c t s . The e l e c t r o l y t i c c o n d u c t i v i t y d e t e c t o r (ECD) (6, 7 ) , w h i l e a more complex d e v i c e , i s p r o b a b l y a more r e l i a b l e q u a n t i t a t i v e d e t e c t o r f o r n i t r o g e n compounds than the A F I D . The GC e f f l u e n t i s p y r o l y z e d i n a h y drogen atmosphere to produce N H 3 which i s absorbed i n a water o r e t h a n o l s t r e a m and passed through an e l e c t r o l y t i c c o n d u c t i v i t y c e l l f o r measurement. Halogen r e s p o n s e may be e l i m i n a t e d by u s i n g a b a s i c a b s o r b e r a f t e r the p y r o l y s i s u n i t . In the o x i d a t i v e mode t h i s d e t e c t o r i s s e n s i t i v e t o halogens and s u l f u r . F o r the p u r p o s e s o f the l a r g e l y q u a l i t a t i v e s u r vey type a n a l y s e s which we p e r f o r m , t h e improved v e r s i o n s o f the AFID would be p r e f e r r e d over the ECD f o r n i t r o g e n d e t e c t i o n from the s t a n d p o i n t o f s i m p l i c i t y . The flame p h o t o m e t r i c d e t e c t o r f o r s u l f u r compounds has been e x t r e m e l y r e l i a b l e i n our hands and we see no o t h e r d e t e c t o r comparable to i t . Instrument

D e s i g n and M o d i f i c a t i o n s

The p r i m a r y o b j e c t i v e i n the d e s i g n of our c h r o matographic system was the achievement o f a t o t a l l y i n e r t system i n which even the most u n s t a b l e compounds c o u l d be d e l i v e r e d t o the p o i n t o f d e t e c t i o n and/or e v a l u a t i o n without decomposition. Many c o m m e r c i a l l y a v a i l a b l e i n s t r u m e n t s and systems r e p o r t e d i n the



5.

WITHYCOMBE ETA L .

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l i t e r a t u r e i n c o r p o r a t e an e s s e n t i a l l y i n e r t i n j e c t i o n system and column, o n l y to d e l i v e r the sample to the d e t e c t o r through a h i g h l y r e a c t i v e hot m e t a l t r a n s f e r line. When c h e m i c a l s a r e t o be e v a l u a t e d f o r use i n f l a v o r s and f r a g r a n c e s , even the s l i g h t e s t d e c o m p o s i t i o n o f the c h e m i c a l may l e a d t o erroneous c o n c l u s i o n s c o n c e r n i n g i t s t r u e aroma. A l t h o u g h the c h r o m a t o g r a p h i c system which w i l l be d i s c u s s e d i s c o m p a t i b l e w i t h a l l t y p e s o f columns, i t has been s p e c i f i c a l l y m o d i f i e d and o p t i m i z e d f o r use w i t h l a r g e bore w a l l - c o a t e d g l a s s c a p i l l a r y c o l u m n s . We have found i n our l a b o r a t o r y t h a t many n a t u r a l l y o c c u r r i n g s u l f u r c o n t a i n i n g compounds a r e l e s s s u b j e c t t o d e c o m p o s i t i o n on a l a r g e bore g l a s s c a p i l l a r y column than on a packed g l a s s c o l u m n . The instrument which has been used i n our l a b o r a t o r y i s a T r a c o r MT-220 gas chromatograph equipped w i t h a l i n e a r temperature programmer, t h r e e e l e c t r o m e t e r s , d u a l flame i o n i z a t i o n d e t e c t o r s ( F I D ) , and a Melpar flame p h o t o m e t r i c d e t e c t o r (FPD) (8) ( F i g . 1 ) . The Melpar flame p h o t o m e t r i c d e t e c t o r i n c o r p o r a t e s a flame i o n i z a t i o n e l e c t r o d e which p e r m i t s the s i m u l t a n e o u s output o f a flame p h o t o m e t r i c response and a flame i o n i z a t i o n response. The FPD has been r e p o s i t i o n e d on the i n j e c t o r and d e t e c t o r m a n i f o l d t o accommodate the a d m i s s i o n o f the column e f f l u e n t d i r e c t l y i n t o the f l a m e , as i n d i c a t e d by the g l a s s - l i n e d s t e e l l i n e s , t h e r e b y e l i m i n a t i n g the p o s s i b i l i t y o f c o n t a c t between the sample gas and m e t a l d e t e c t o r components p r i o r to com bustion. One o f the s t a n d a r d flame i o n i z a t i o n d e t e c t o r s has been c o n v e r t e d to an a l k a l i flame i o n i z a t i o n d e t e c t o r ( A F I D ) ( F i g . 2)by c a r e f u l l y p o s i t i o n i n g a s m a l l wedgeshaped segment o f a 1.5 χ 10 mm r u b i d i u m s u l f a t e - p o t a s sium bromide p e l l e t on the a n n u l a r g l a s s s l e e v e which s u r r o u n d s the b u r n e r t i p . The p e l l e t i s h y d r a u l i c a l l y p r e s s e d from a homogeneous 1:1 m i x t u r e o f r u b i d i u m s u l f a t e (99% Matheson Coleman & B e l l ) and i n f r a r e d q u a l i t y p o t a s s i u m bromide (Harshaw C h e m i c a l C o . ) (9). The sample i s combusted i n a h y d r o g e n - p o o r f l a m e . When the flame has been p r o p e r l y o p t i m i z e d , the AFID p r o v i d e s an i o n i z a t i o n c u r r e n t which i s a p p r o x i m a t e l y 100 f o l d enhanced i n response t o n i t r o g e n c o n t a i n i n g com pounds . A diagram i l l u s t r a t i n g the r e l a t i o n s h i p o f the v a r i o u s components o f the system i s shown i n F i g u r e 3 . The i n j e c t i o n p o r t i n s e r t i s c o n s t r u c t e d from a 15 cm segment o f 6 mm o . d . χ 1 mm i . d . c a p i l l a r y bore g l a s s tubing. One end i s s l i g h t l y f l a r e d t o f a c i l i t a t e d i r e c t s y r i n g e i n j e c t i o n and the o t h e r end i s drawn t o



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C O L U M N EFFLUENT INLET

Figure 1. Schematic of Melpar flame photometric detector designed for simultan flame ionization and flame photometric (sulfur) operation with glass capillary c

Figure 2. Schematic of a Tracorflameionization detector modified for operation as an alkali flame ionization detector (nitrogen)


5.

WITHYCOMBE ET AL.

Nitrogen and Sulfur Containing Flavor Volatiles 89

a t a p e r e d 1/16" o . d . t i p . The 4 0 0 χ 0.032" i . d . g l a s s c a p i l l a r y column (C) i s connected t o the i n j e c t i o n p o r t i n s e r t and the e x i t s p l i t t e r assembly by s m a l l segments of h e a t - s h r i n k a b l e T e f l o n tubing ( B ) . The column e f f l u e n t i s d i r e c t e d i n t o one p o r t o f a 5-port e f f l u e n t s p l i t t e r assembly. The s p l i t t e r i s d i v i d e d i n t o two s e c t i o n s w i t h a l l i n t e r c o n n e c t i n g l i n e s f a b r i c a t e d from 1/16" o . d . χ 0 . 5 mm i . d . g l a s s lined s t e e l tubing ( S c i e n t i f i c Glass Engineering P t y . L t d . ) ( A ) . The j u n c t i o n p o i n t s a r e f a b r i c a t e d from low dead volume Swagelok u n i o n s , s p e c i a l l y bored and f i t t e d t o m i n i m i z e sample c o n t a c t w i t h s t a i n l e s s s t e e l surfaces. To f u r t h e r reduce t h e p o s s i b i l i t y o f sample d e c o m p o s i t i o n , the i n t a c t s p l i t t e r assembly was d e a c t i vated by c o a t i n g w i t h a 10% SE-30 s o l u t i o n and h e a t i n g at 4 0 0 ° C a c c o r d i n g t o the p r o c e d u r e o f Thompson and Goode ( 2 2 ) · The f i r s t s e c t i o n o f the s p l i t t e r assembly m e r e l y p r o v i d e s f o r the a d d i t i o n o f h e l i u m make-up gas from one o f the a u x i l i a r y i n j e c t i o n p o r t f l o w l i n e s (D). The d i l u t e d e f f l u e n t i s then d i r e c t e d t o t h e 3 way s p l i t t e r o f the second s e c t i o n . At t h i s p o i n t , 20% o f the e f f l u e n t i s d i r e c t e d t o the FID/FPD, 30% t o the AFID, and 50% t o a heated e x i t p o r t . The s i g n a l s from the t h r e e d e t e c t o r s a r e f e d i n t o t h r e e e l e c t r o meter c h a n n e l s which a r e connected t o a S o l t e c / Rikadenki three-channel recorder. The FID s i g n a l i s connected i n p a r a l l e l t o a c e n t r a l GC computer system which p r o v i d e s a q u a n t i t a t i v e e s t i m a t i o n o f the sample components and c a l c u l a t e s t h e i r r e t e n t i o n index ( I E ) (11). The heated e x i t p o r t i s a m o d i f i c a t i o n o f a t h i r d i n j e c t i o n p o r t which p r o v i d e s two o p t i o n s : 1) sample c o l l e c t i o n f o r subsequent a n a l y t i c a l r o u t i n e s , o r 2) s i m u l t a n e o u s odor e v a l u a t i o n o f the e l u t i n g components. When used i n t h e l a t t e r manner, a d d i t i o n a l h e l i u m i s p r o v i d e d (E) c o n c e n t r i c t o the f l o w l i n e t o " l i f t " the components away from the hot s u r f a c e . The chromatogram d i s p l a y i n g t h e r e s p o n s e s from the t h r e e d e t e c t o r s i s s i m u l t a n e o u s l y a n n o t a t e d t o p r o v i d e an "aroma p r o f i l e " o f the c h r o m a t o g r a p h i c a l l y r e s o l v e d s a m p l e . D e t e c t o r s e l e c t i v i t y i s demonstrated i n F i g u r e 4 . Shown i s the s e p a r a t i o n o f a model system composed o f a number o f s u l f u r and/or n i t r o g e n c o n t a i n i n g com pounds . The top t r a c e i s the FID r e s p o n s e . As i n d i c a t e d i n the second t r a c e , the FPD responds e x c l u s i v e l y to components 2, m e t h y l e t h y l d i s u l f i d e ; 15, 2 - n - p e n t y l t h i o p h e n e ; and 8, t e t r a h y d r o t h i o p h e n e - 3 - o n e . The AFID responds t o components 3, 1 - n - p e n t y l p y r r o l e ; 6, 2 m e t h y 1 - 5 - v i n y l p y r i d i n e , and 10, n - p e n t y l p y r a z i n e . Dual r e s p o n s e s a r e o b s e r v e d on the FPD and AFID f o r


f



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PHENOLIC, SULFUR, AND NITROGEN COMPOUNDS I N FOOD FLAVORS

Figure 3. Schematic of the Tracor MT-220 gas chromatograph equipped with a 0.032" id glass capillary column and modified for simultaneous detection and aro tion of nitrogen and sulfur containing compounds

ETHANOL

2-METHYL-5-VINYL PYRIDINE

HETHYLETHYLDISULFIDE

4 -METHYL-5-VINYL THIAZOLE

1- N-PENTYLPYRROLE

TETRAHYDR0THI0PHENE-3-0NE

Figure 4. Chromatogram of synthetic FURFURAL mixture using 3-detector chromato2 - H-PENTYLTHIOPHENE graphic system

5 - METHYL FURFURAL JL-PENTYLPYRAZINE



5.

W I T H Y C O M B E ET A L .

Nitrogen and Sulfur Containing Flavor Volatiles 9 1

component 7, 4 - m e t h y 1 - 5 - v i n y l t h i a z o l e , as would be expected. A l s o observed i s a r e l a t i v e l y s m a l l r e sponse o f the AFID t o the l a r g e q u a n t i t y o f s o l v e n t , ethanol. T h i s s y m p a t h e t i c response can r e a d i l y be d i s t i n g u i s h e d from a t r u e r e s p o n s e by comparing the FID and AFID r e c o r d e r t r a c e s and c o i n c i d e n c e t i m e s . Under normal o p e r a t i n g c o n d i t i o n s the d e t e c t o r s e n s i t i v i t y i s a d j u s t e d t o g i v e comparably s i z e d peaks f o r a l l three detectors. The u s e a b l e s e n s i t i v i t y ( c o n s i d e r i n g the S/N r a t i o ) o f t h i s AFID i s o f the same o r d e r o f magnitude as the F I D ; however, the FPD may a c h i e v e 10 to 100 times g r e a t e r s e n s i t i v i t y . The p r i n c i p l e f e a t u r e s and advantages o f t h i s m u l t i p l e s e l e c t i v e d e t e c t o r system may be summarized i n the f o l l o w i n g t h r e e p o i n t s : 1.

An I n e r t , H i g h C a p a c i t y , H i g h R e s o l u t i o n Chromatpgraphic System. The column systems developed p e r m i t the r e s o l u t i o n o f 1 t o 2 m i c r o l i t e r d i r e c t i n j e c t i o n s of s o l v e n t - f r e e n a t u r a l e x t r a c t s w h i l e m a i n t a i n i n g column e f f i c i e n c e s on the o r d e r of 0.5 t o 1.0 χ 1 0 t h e o r e t i c a l p l a t e s . The use o f wide bore c a p i l l a r y columns i s e s s e n t i a l s i n c e the sample c a p a c i t y of narrow bore columns w i t h i n j e c t i o n s p l i t t e r s i s not s u f f i c i e n t f o r use w i t h m u l t i p l e e f f l u e n t splitting. Our columns p r o v i d e s u f f i c i e n t c a p a c i t y to a l l o w p r e p a r a t i v e i s o l a t i o n o f microgram q u a n t i t i e s of material for further spectral characterization. 6

2.

Simultaneous N i t r o g e n , S u l f u r , Flame I o n i z a t i o n , and P h y s i o l o g i c a l D e t e c t i o n . The s e n s o r y e v a l u a t i o n o f GLC e f f l u e n t s has been r e c o g n i z e d as an a i d i n a s s e s s i n g the s i g n i f i c a n c e o f the v a r i o u s components t o the aroma o f the t o t a l s a m p l e . When s e n s o r y é v a l u â t i o n s a r e made i n p a r a l l e l w i t h a F I D , aromas and aroma changes a r e d e t e c t e d i n a r e a s which cannot be a s s i g n e d s p e c i f i c a l l y t o a change i n d e t e c t o r response . However, when m u l t i p l e s e l e c t i v e d e t e c t o r s a r e u s e d , t h e s e aromas may o f t e n be a s s i g n e d s p e c i f i c a l l y t o t r a c e o r u n r e s o l v e d n i t r o g e n and/or s u l f u r c o n t a i n i n g components, many of which e x h i b i t e x t r e m e l y low odor t h r e s h o l d s . T h i s c a p a b i l i t y p r o v i d e s the f l a v o r i s t , p e r f u m e r , or a n a l y s t w i t h the a b i l i t y to b e t t e r c h a r a c t e r i z e the sample and l o c a t e components o f spec i f i c i n t e r e s t . 3.

P r o v i d e Maximum I n f o r m a t i o n f o r the A c q u i s i t i o n and I n t e r p r e t a t i o n o f GC-MS D a t a . In the same manner as above, i t i s p o s s i b l e t o e f f e c t i v e l y l o c a t e t r a c e and u n r e s o l v e d components p r i o r to analysis by


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FLAVORS

gas chromatography - mass s p e c t r o m e t r y (GC-MS) a n d , through the a i d of c o m p u t e r i z e d MS d a t a h a n d l i n g f a c i l i t i e s , o b t a i n i d e n t i f i a b l e mass s p e c t r a o f components which would n o r m a l l y be o v e r l o o k e d . When t h i s t e c h nique i s used i n c o n j u n c t i o n w i t h s p e c i f i c or m u l t i p l e i o n m o n i t o r i n g o f the mass s p e c t r a l d a t a the a n a l y s t i s armed w i t h p o w e r f u l a n c i l l a r y t e c h n i q u e s f o r the l o c a t i o n and mass s p e c t r a l c h a r a c t e r i z a t i o n o f o r g a n i c f l a v o r and aroma components.


Application In our l a b o r a t o r y n a t u r a l e x t r a c t s a r e r o u t i n e l y s u r v e y e d w i t h the system j u s t d e s c r i b e d b e f o r e GC-MS analysis. An a n a l y s i s of t h e v o l a t i l e components o f p r e s s u r e cooked pork l i v e r , which has p r e v i o u s l y been r e p o r t e d i n p a r t by Mussinan and Walradt (12) w i l l p r o v i d e an example o f the a p p l i c a t i o n o f the t e c h n i q u e . The d e t a i l s o f the sample p r e p a r a t i o n a r e e s s e n t i a l l y the same as p r e v i o u s l y r e p o r t e d , the p r o d u c t b e i n g a c o n c e n t r a t e d e t h e r s o l u t i o n o f cooked pork l i v e r v o l a tiles. The t h r e e chromatograms shown i n F i g u r e 5 a r e those o f a 2 ^ul i n j e c t i o n o f the e x t r a c t on a 4 0 0 χ 0.032" i . d . g l a s s SE-30 c a p i l l a r y column, tem p e r a t u r e programmed from 5 0 - 1 9 0 ° C at l ° C / m i n u t e . The upper t r a c e i s the flame i o n i z a t i o n r e s p o n s e . The second t r a c e i s the AFID r e s p o n s e t o n i t r o g e n compo nents and the flame p h o t o m e t r i c response to s u l f u r com pounds i s the t h i r d t r a c e . Due t o the i n a b i l i t y t o s u i t a b l y a n n o t a t e the f i g u r e w i t h the odor d e s c r i p t o r s , they have been o m i t t e d . The major component o f the sample, 1_, i s f u r f u r y l alcohol. The r e s p o n s e o f the AFID to the p y r a z i n e s and p y r r o l e d e r i v a t i v e s compared to o t h e r c o n s t i t u e n t s o f s i m i l a r FID r e s p o n s e i l l u s t r a t e s t h e d e t e c t o r s e l e c t i v i t y as i n t h e c a s e o f components 2_ (methyl p y r a z i n e ) , 3 (2,5(6)-dimethyl pyrazine), 4 (2-ethyl-6-methyl p y r a z i n e ) and

Simultaneous Detection of Nitrogen and Sulfur Containing Flavor

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