Gas ChromatographyâMatrix Isolation Infrared SpectroscopyâMass

Chapter 7

Gas Chromatography—Matrix Isolation Infrared Spectroscopy—Mass Spectrometry for Analysis of Thermally Generated Aroma Compounds William R. Croasmun and Robert J. McGorrin

Downloaded by UNIV LAVAL on April 29, 2016 | http://pubs.acs.org Publication Date: October 3, 1989 | doi: 10.1021/bk-1989-0409.ch007

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An integrated GC/IR/MS instrument is a powerful tool for rapid identification of thermally generated aroma compounds. Fourier transform infrared spectroscopy (GC/IR) provides a complementary technique to mass spectrometry (MS) for the characterization of volatile flavor components in complex mixtures. Recent improvements in GC/IR instruments have made it possible to construct an integrated GC/IR/MS system in which the sensitivity of the two spectroscopic detectors is roughly equal. The combined system offers direct correlation of IR and MS chromatograms, functional group analysis, substantial time savings, and the potential for an expert systems approach to identification of flavor components. Performance of the technique is illustrated with applications to the analysis of volatile flavor components in charbroiled chicken.

F l a v o r c h e m i s t s have t r a d i t i o n a l l y r e l i e d on mass s p e c t r o m e t r y i n conjunction w i t h gas chromatography (GC/MS) to i d e n t i f y the structures of v o l a t i l e f l a v o r components i n heated food systems. Mass s p e c t r o m e t r y p r o v i d e s the m o l e c u l a r w e i g h t s o f fragment i o n s , which a r e u s e f u l f o r d e d u c i n g m o l e c u l a r s t r u c t u r e . The MS d e t e c t i o n limit i s on the o r d e r o f 10" g, however d e t e c t i o n l i m i t s f o r t a r g e t compound a n a l y s i s o r c h e m i c a l c l a s s d e t e c t i o n v i a s e l e c t e d i o n m o n i t o r i n g can be much l o w e r . E x t e n s i v e l i b r a r i e s o f mass s p e c t r a a r e a v a i l a b l e ; even so, many new f l a v o r compounds can o f t e n not be i d e n t i f i e d from MS d a t a a l o n e . Complementary s t r u c t u r a l i n f o r m a t i o n i s a v a i l a b l e from gas chromatography/infrared spectroscopy (GC/IR), which provides f u n c t i o n a l group o r s u b s t r u c t u r e a n a l y s i s . Recent improvements i n light p i p e GC/IR (1) and the advent o f m a t r i x i s o l a t i o n GC/IR (2-4) have pushed d e t e c t i o n l i m i t s i n t o the 10" t o 10" g range. GC/IR a l l o w s the g e n e r a t i o n o f a f u n c t i o n a l group IR chromatogram, which d i s p l a y s i n f r a r e d absorbance a c r o s s a s e l e c t e d wavelength r e g i o n as a f u n c t i o n o f time. F o r example, i f a 1780-1680 cm" range i s chosen, t h e r e s u l t a n t chromatogram d i s p l a y s c a r b o n y l - c o n t a i n i n g q

0097-6156/89/0409-0061S06.00/0 © 1989 American Chemical Society Parliment et al.; Thermal Generation of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


62

THERMAL GENERATION OF AROMAS

components. Because IR s p e c t r a a r e d e t e c t e d by a F o u r i e r t r a n s f o r m t e c h n i q u e , d e t e c t i o n l i m i t s a r e not improved i n f u n c t i o n a l group ( v e r s u s f u l l - s p e c t r u m ) chromatograms, as they a r e w i t h GC/MS. The IR spectrum l i b r a r i e s u s e f u l f o r GC/IR, which c o n t a i n about 3,000 t o 10,000 s p e c t r a , a r e s m a l l e r than MS libraries. However, an advantage f o r IR i s t h a t computer l i b r a r y s e a r c h i n g o f the spectrum o f an unknown tends t o produce b e s t matches which c o n t a i n s i m i l a r f u n c t i o n a l g r o u p s , even though the spectrum o f the s p e c i f i c compound i s not c o n t a i n e d i n the l i b r a r y . The complementary n a t u r e o f IR and MS d a t a i s i l l u s t r a t e d i n F i g u r e 1, w h i c h compares the IR and MS s p e c t r a o f f o u r i s o m e r i c dimethylphenols. The isomers a r e e a s i l y d i s t i n g u i s h e d i n the IR, a l t h o u g h t h e i r mass s p e c t r a a r e s i m i l a r . C o n v e r s e l y , MS i s b e t t e r suited than IR f o r d i s t i n g u i s h i n g a homologous s e r i e s o f compounds; whereas IR s p e c t r a appear s i m i l a r f o r a d j a c e n t members o f the s e r i e s , MS can p r o v i d e a c h a r a c t e r i z i n g m o l e c u l a r i o n . GC/IR has p r e v i o u s l y been u t i l i z e d i n f l a v o r r e s e a r c h to a n a l y z e aroma components i n t r o p i c a l f r u i t s ( 5 ) and a p p l e s ( 6 ) . W h i l e a l i n k e d GC/IR/MS system was proposed e a r l i e r ( 5 ) , the p r i n c i p a l l i m i t a t i o n a t t h a t time r e s u l t e d from the i n h e r e n t l a c k o f IR s e n s i t i v i t y i n o l d e r l i g h t p i p e systems. It i s p o s s i b l e to o b t a i n GC/MS and GC/IR d a t a i n s e p a r a t e e x p e r i m e n t s ( 7 - 1 1 ) , but t h e r e a r e p o w e r f u l reasons f o r i n t e g r a t i n g the measurements i n t o a s i n g l e i n s t r u m e n t (1,12-17). Data from two s e p a r a t e e x p e r i m e n t s can be d i f f i c u l t t o c o r r e l a t e , owing t o s l i g h t r e t e n t i o n time s h i f t s and d i f f e r e n c e s i n GC-spectrometer interfacing. Separate experiments are u n n e c e s s a r i l y time consuming. O f t e n , a f l a v o r sample may be l i m i t e d i n amount o r may d e t e r i o r a t e between e x p e r i m e n t s . Finally, i t i s n e c e s s a r y t o assemble the e n t i r e d a t a s e t i n a s i n g l e d a t a system i n o r d e r t o t a k e advantage o f the p o t e n t i a l f o r automated d a t a i n t e r p r e t a t i o n o f the combined data. Experimental Methods Commercial GC/IR/MS instruments are a v a i l a b l e from M a t t s o n I n s t r u m e n t s ( u s i n g a m a t r i x i s o l a t i o n GC/IR i n t e r f a c e ) and from H e w l e t t P a c k a r d ( u s i n g a h i g h l y o p t i m i z e d l i g h t p i p e GC/IR d e s i g n ) . Each uses a H e w l e t t P a c k a r d Mass S e l e c t i v e D e t e c t o r to o b t a i n e l e c t r o n impact MS d a t a . The i n s t r u m e n t i n our l a b o r a t o r y i s a p r o t o t y p e v e r s i o n o f the M a t t s o n i n s t r u m e n t , b u i l t i n c o l l a b o r a t i o n w i t h Mattson Instruments. Three key i s s u e s must be f a c e d i n c o n s t r u c t i n g an integrated instrument. F i r s t , the s e n s i t i v i t y o f the IR and MS d e t e c t o r s must be matched. We have chosen t o use a M a t t s o n I n s t r u m e n t s C r y o l e c t m a t r i x i s o l a t i o n GC/IR i n t e r f a c e , which has IR d e t e c t i o n l i m i t s comparable t o f u l l s c a n e l e c t r o n impact MS. I n t h i s system, the effluent s t r e a m from a c a p i l l a r y gas chromatograph i s doped w i t h IX argon and d e p o s i t e d on a g o l d - c o a t e d s u r f a c e o f a s l o w l y r o t a t i n g d i s k c o o l e d t o 12°K. Helium i n the e f f l u e n t s t r e a m i s removed by vacuum, w h i l e the argon forms a s o l i d m a t r i x on the c o l d s u r f a c e , t r a p p i n g the c h r o m a t o g r a p h i c e l u e n t s i n t o a s o l i d t r a c k . IR s p e c t r a o f i n d i v i d u a l f l a v o r components o r o f the e n t i r e chromatogram a r e o b t a i n e d by r e f l e c t i n g an IR beam o f f the d i s k s u r f a c e . I n our

Parliment et al.; Thermal Generation of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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Figure 1. Mass spectra (left) and matrix isolation infrared spectra (right) of isomeric dimethylphenols.

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l a b o r a t o r y a N i c o l e t 7199 FTIR s p e c t r o m e t e r i s used t o a c q u i r e and p r o c e s s the IR d a t a . The second i s s u e i s t o i n t e r f a c e two d e t e c t o r s t o a s i n g l e GC. S i n c e b o t h the MS and the m a t r i x i s o l a t i o n i n t e r f a c e a r e d e s t r u c t i v e detectors, the sample i s s p l i t i n a 1:1 e f f l u e n t s p l i t t e r and h a l f the sample i s r o u t e d t o each d e t e c t o r v i a a s p e c i a l l y - d e s i g n e d open s p l i t i n t e r f a c e (18). The t h i r d key i s s u e i n v o l v e s data handling. To take full advantage o f the complementary n a t u r e o f the d a t a , i t i s d e s i r a b l e to h a n d l e a l l the s p e c t r a l i n f o r m a t i o n i n a s i n g l e system. I n our l a b the IR and MS d a t a a r e h a n d l e d s e p a r a t e l y by the N i c o l e t and H e w l e t t P a c k a r d computer systems, r e s p e c t i v e l y . C u r r e n t commercial instruments a l s o h a n d l e MS and IR d a t a on s e p a r a t e systems, with only l i m i t e d communications between t h e two computers. On our instrument, t h e f o l l o w i n g computer-based l i b r a r i e s were used t o search unknown s p e c t r a : I R - EPA Vapor Phase; MS - N a t i o n a l Bureau of S t a n d a r d s , R e v i s i o n F. A n a l y s i s o f t h e r m a l l y g e n e r a t e d aromas by GC/IR/MS w i l l be i l l u s t r a t e d w i t h d a t a from a c h a r b r o i l e d c h i c k e n d i s t i l l a t e . The chicken was g r i l l e d a t 400°F f o r a t o t a l o f 36 minutes (18 minutes on each s i d e ) . The f l a v o r i s o l a t e was o b t a i n e d from seven L i k e n s N i c k e r s o n d i s t i l l a t i o n / e x t r a c t i o n s u s i n g methylene c h l o r i d e , and was c o n c e n t r a t e d t o 2 ml o f s o l v e n t . A 2 - u l a l i q u o t o f the i s o l a t e (~2g chicken) was i n j e c t e d s p l i t l e s s onto a 50m by 0.32 mm 0V-1 c r o s s l i n k e d m e t h y l s i l i c o n e gum GC c a p i l l a r y column (0.52 urn f i l m t h i c k n e s s ) i n a H e w l e t t Packard 5790A GC. The column was h e l d a t 10°C f o r 1 minute, programmed a t 15°C/minute t o 50°C, h e l d a t 50°C f o r 3 m i n u t e s , and heated a t 2°C/minute t o 220°C. Mass s p e c t r a were acquired a t the r a t e o f ~2/second, s c a n n i n g from m/z 35 t o 250. I n f r a r e d s p e c t r a used t o compute^the IR chromatograms were a c q u i r e d a t 1-second i n t e r v a l s w i t h 8 cm" r e s o l u t i o n ; each spectrum was t h e average o f f o u r s c a n s . The IR s p e c t r a i n F i g u r e s 1,^ 4 ( t o p two spectra) and 6 (bottom spectrum) were a c q u i r e d a t 4 cm" r e s o l u t i o n and a r e the average o f 32 s c a n s . A n a l y s i s o f Charbroiled Chicken IR and MS chromatograms o f the d i s t i l l a t e a r e d i s p l a y e d i n F i g u r e 2. The IR chromatogram i s computed o v e r an absorbance window corresponding to saturated C-H s t r e t c h and t h e r e f o r e s e r v e s as a nearly universal detector. (Gram-Schmidt chromatograms o f m a t r i x i s o l a t i o n IR d a t a y i e l d poor r e s u l t s i n our hands, p r o b a b l y due t o background water i n the m a t r i x . ) Note t h a t the chromatograms match w e l l enough t h a t i t i s s t r a i g h t f o r w a r d t o i d e n t i f y c o r r e s p o n d i n g peaks. Observe a l s o t h a t the s i g n a l - t o - n o i s e i n the two chromatograms i s s i m i l a r . F i g u r e 3 i l l u s t r a t e s t h a t f u n c t i o n a l group chromatograms can be p a r t i c u l a r l y u s e f u l i n f l a v o r a n a l y s i s . The " u n i v e r s a l " C-H window (top) i s compared t o t h r e e d i f f e r e n t windows w h i c h d e t e c t c a r b o n y l compounds. The 1780-1680 cm~^ window p i c k s up most c a r b o n y l s , w h i l e the n a r r o w e r 1710-1690 cm" window d e t e c t s m o s t l y unsaturated a l d e h y d e s i n t h i s h i g h l y o x i d i z e d sample. The 1810-1790 cm" window shows m a i n l y a homologous s e r i e s o f gamma-lactones. I t s h o u l d be pointed o u t t h a t IR i s n o t a b l e t o produce f u n c t i o n a l group chromatograms o f h e t e r o c y c l e s , s i n c e they l a c k u s e f u l c h a r a c t e r i s t i c



Figure 2. Chromatograms for matrix isolation capillary GC/IR (top) and GC/MS (bottom) of charbroiled chicken obtained by Likens-Nickerson distillation/extraction.



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30

T 1 me

(min.)

40

50

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Figure 3. Selected matrix isolation GC/IR absorbance windows for charbroiled chicken isolate (cf. Figure 2).

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1810-1790 cm-1

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7.

CROASMUN AND McGORRIN

GC-IR-MS Analysis ofAromas

67

group f r e q u e n c i e s . However, these compounds a r e e a s i l y d e t e c t e d i n selected i o n MS chromatograms, s i n c e they produce intense, c h a r a c t e r i s t i c molecular ions. Consistent w i t h thermal p r o c e s s i n g of chicken, pyrazine compounds are generated v i a M a i l l a r d browning r e a c t i o n s , and c o n t r i b u t e browned, r o a s t e d aromas to c h a r b r o i l e d c h i c k e n flavor. The u t i l i t y o f IR i n r e s o l v i n g p y r a z i n e isomers i s i l l u s t r a t e d i n F i g u r e 4. The peak a t 19.685 minutes i s e a s i l y i d e n t i f i e d as 2,5or 2,6-dimethylpyrazine by MS (not shown), but the MS cannot distinguish these c o e l u t i n g i s o m e r s . The IR shows t h a t both a r e p r e s e n t and i n d i c a t e s the r e l a t i v e p r o p o r t i o n s ; i t a l s o ^ d e t e c t s a c o e l u t i n g c a r b o n y l compound (bands a t 1720 and 1080 cm ). Only a s i n g l e i o n i n the mass spectrum i n d i c a t e s the presence of a t h i r d component. To i l l u s t r a t e the complementary n a t u r e o f IR and MS d a t a we will f o c u s on t h r e e a d j a c e n t peaks near 29 minutes ( F i g u r e 5, boxed r e g i o n ) . Note t h a t the c e n t e r peak shows a s h o u l d e r i n the MS. The c o r r e s p o n d i n g i n f r a r e d and mass s p e c t r a a r e p r e s e n t e d i n F i g u r e 6. T h e - p e a k on the l e f t (28.821 m i n u t e s ) i s a n - e n a l by IR (C=0 a t 1700 cm and aldehyde C-H s t r e t c h a t 2700 cm ). The molecular weight i s 124, y i e l d i n g a m o l e c u l a r f o r m u l a o f CgH^O and r e q u i r i n g a second double bond o r a r i n g i n the hydrocarbon p o r t i o n . There i s no e v i d e n c e f o r a r i n g ; the p o s i t i o n o f the C=0 s t r e t c h i m p l i e s t h a t a second double bond i s not c o n j u g a t e d w i t h the f i r s t . The r a t i o o f CH to CH s t r e t c h i n g bands (2900-3000 cm ) s u g g e s t s a branched structure. The combined d a t a i n d i c a t e a branched e i g h t carbon d o u b l y - u n s a t u r a t e d a l d e h y d e , w i t h o n l y one double bond c o n j u g a t e d to the c a r b o n y l . T h i s c o n c l u s i o n i s c o n s i s t e n t w i t h an observed K o v a t s r e t e n t i o n i n d e x o f about 1000. The c e n t e r peak (28.986 m i n u t e s ) i s c l e a r l y a m i x t u r e o f two compounds, as i n d i c a t e d by the s h o u l d e r i n the t o t a l ion chromatogram, by the s t r o n g 85 and 91 i o n s i n the mass spectrum, and the presence o f two c a r b o n y l bands i n the IR. The i n d i v i d u a l compounds a r e most e a s i l y i d e n t i f i e d by spectrum s u b t r a c t i o n i n the IR, y i e l d i n g r e s o l v e d s p e c t r a o f the o v e r l a p p i n g components. The f i r s t i s gamma-hexalactone and the second p h e n y l a c e t a l d e h y d e ( F i g u r e 7 ) . The t h i r d o f the t h r e e a d j a c e n t peaks (29.207 m i n u t e s ) i s readily i d e n t i f i e d as 2-hydroxybenzaldehyde ( s a l i c y l a l d e h y d e ) by comparison o f the MS o r IR spectrum w i t h l i b r a r y d a t a . I t s h o u l d be noted t h a t the automated s e a r c h r o u t i n e i n the MS s o f t w a r e p i c k e d 3and 4-hydroxybenzaldehyde as b e t t e r matches than the 2-hydroxy compound, even though the 76 i o n i n the spectrum o f the unknown i s p r e s e n t o n l y i n the mass spectrum o f the 2-hydroxy compound. However, the IR s e a r c h r o u t i n e c o r r e c t l y i d e n t i f i e d the 2-isomer. T h i s i l l u s t r a t e s t h a t c a s u a l o p e r a t o r s who r e l y on automated s e a r c h r o u t i n e s f o r compound i d e n t i f i c a t i o n a r e much l e s s l i k e l y t o make e r r o r s when they have a c c e s s to both IR and MS s e a r c h i n g . 2

3

Future D i r e c t i o n s From the p o i n t o f view o f aroma a n a l y s i s , the u l t i m a t e o b j e c t i v e o f d e v e l o p i n g s o - c a l l e d " m u l t i p l y hyphenated" i n s t r u m e n t s i s t o produce a d e v i c e which can a u t o m a t i c a l l y determine the i d e n t i t y o f a l l o f the c o n s t i t u e n t s o f a complex v o l a t i l e m i x t u r e . I n t e g r a t e d GC/IR/MS i s a s t e p a l o n g t h a t p a t h , but a h o s t o f c r u c i a l i s s u e s remain.


68



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2 . BEG 1 .BEG 1 .6E6 1.4C6 1.2EG 1.0EG 8.0E5 6.0E5 4.0E5 2.0E5 0

Figure 5. Expanded chromatogram region (26-32 min.) of charbroiled chicken isolate (cf. Figure 2). Top: GC/IR. Bottom: GC/MS.



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Figure 6. Matrix isolation IR spectra (left) and MS spectra (right) for selected unknowns in charbroiled chicken (cf. Figure 5). Top: Rt 28.821 min., C -dienal. Middle: Rt 28.986 min., mixture of 7-hexalactone and phenylacetaldehyde. Bottom: Rt 29.207 min., 2-hydroxybenzaldehyde.

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Gas ChromatographyâMatrix Isolation Infrared SpectroscopyâMass

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