Sample Preparation Techniques for Gas-Liquid Chromatographic

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4 Sample Preparation Techniques for Gas-Liquid Chromatographic Analysis of Biologically Derived Aromas Thomas H . Parliment General Foods Technical Center, 555 South Broadway, Tarrytown, NY 10591 Sample preparation is a c r i t i c a l step in the analysis of bio-generated aromas. This paper will review techniques necessary to isolate and concentrate these volatile components. Particular emphasis will be placed on procedures which are relatively simple and which permit high sample throughput. The techniques to be discussed can be classified as follows: 1. 2. 3. 4. 5. 6.

Headspace Sampling Headspace Concentrating Distillation/Extraction Direct Analysis of Aqueous Samples Direct Adsorption of Aqueous Samples Direct Extraction of Aqueous Samples

The purpose o f t h i s p r e s e n t a t i o n is t o review procedures f o r t h e a n a l y s i s o f v o l a t i l e compounds generated through b i o l o g i c a l p r o cesses. Numerous t e c h n i q u e s have been proposed t o s e p a r a t e t h e v o l a t i l e c h e m i c a l s from the n o n v o l a t i l e m a t e r i a l s and water, and t o c o n c e n t r a t e them. A f t e r sample p r e p a r a t i o n , the complex aroma samp l e can be s e p a r a t e d i n t o i t s i n d i v i d u a l components by h i g h r e s o l u t i o n gas chromatography and t h e aroma c h e m i c a l s then s t r u c t u r a l l y i d e n t i f i e d by s p e c t r a l t e c h n i q u e s . Sample p r e p a r a t i o n o f b i o l o g i c a l l y generated aromas is c o m p l i c a t e d by a number o f f a c t o r s : 1.

Concentration Level The l e v e l o f these v o l a t i l e c h e m i c a l s is f r e q u e n t l y l o w — t y p i c a l l y , at t h e p a r t s per m i l l i o n (mg/kg) l e v e l o r l e s s . Thus, J. is f r e q u e n t l y n e c e s s a r y not o n l y t o i s o l a t e the v o l a t i l e mater i a l s , but a l s o t o c o n c e n t r a t e them by s e v e r a l o r d e r s o f magnitude. 0097-6156/ 86/ 0317-0034S06.00/ 0 © 1986 American Chemical Society

In Biogeneration of Aromas; Parliment, Thomas H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

PARLIMENT


2.

Gas-Liquid Chromatographic Analysis Matrix V o l a t i l e chemicals are f r e q u e n t l y i n t r a c e l l u l a r and c o m p a r t m e n t a l i z e d , thus r e q u i r i n g d i s r u p t i o n t o l i b e r a t e t h e s e aromas. In biological systems, water is g e n e r a l l y t h e component in g r e a t e s t amount. I n a d d i t i o n t o water, t h e r e a r e also lipids, c a r b o h y d r a t e s and proteinaceous m a t e r i a l s p r e s e n t which compound t h e i s o l a t i o n problem. The presence o f i n s o l u b l e m a t e r i a l s normally precludes d i r e c t gas chromatographic a n a l y s i s o f t h e aroma b e a r i n g b i o l o g i c a l m a t e r i als.

3.

Complexities

of Aromas

Biologically generated aromas a r e f r e q u e n t l y q u i t e complex and i n c l u d e a wide range o f p o l a r ities. F o r example, s t r a w b e r r i e s have been shown t o p o s s e s s more than 350 v o l a t i l e compounds (1.). I n a d d i t i o n , t h e c l a s s e s of compounds f r e q u e n t l y encountered in b i o l o g i c a l mat e r i a l s includes a l c o h o l s , aldehydes, ketones, esters, ethers, s u l f i d e s , mercaptans, amines, a r o m a t i c and h e t e r o c y c l i c compounds and hydrocarbons. Representative c l a s s e s o f compounds i d e n t i f i e d in s t r a w b e r r i e s a r e i n d i c a t e d in Table I . T a b l e I . C l a s s e s of Compounds in S t r a w b e r r i e s Class Hydrocarbons Alcohols Aldehydes Ketones Acids, aliphatic A c i d s , aromatic Esters Lactones

4.

Quantity 34 56 18 20 40 6 130 10

Class Bases Acetals Phenols Furans Mercaptans Sulfides Thio e s t e r s (Ep) O x i d e s Total

Quantity 1 20 3 8 2 3 2 5 358

V a r i a t i o n in V o l a t i l i t y Aroma c h e m i c a l s encompass a wide range o f v o l a tiles. F o r example, tomatoes contain both methyl mercaptan (bp 6°C) and v a n i l l i n (bp 285°C). Techniques a p p r o p r i a t e f o r t h e a n a l y s i s o f low b o i l i n g components a r e q u i t e d i f f e r ent from those f o r h i g h e r b o i l i n g c o n s t i t u e n t s .


36

BIOGENERATION OF AROMAS 5.

Instability


Many aroma compounds generated b i o l o g i c a l l y are unstable. Examples of t h i s a r e mercaptans which can be o x i d i z e d t o s u l f i d e s , and t e r p e n e s which can be t h e r m a l l y degraded. There is no u n i v e r s a l t e c h n i q u e which can be used f o r the p r e p a r a t i o n of a l l samples under a l l c o n d i t i o n s . One must be aware of the l i m i t a t i o n s of each sample p r e p a r a t i o n t e c h n i q u e , and d e s i g n the t e c h n i q u e t o the o b j e c t i v e of the s t u d y . A number of y e a r s ago, Weurman (2) reviewed t e c h n i q u e s of aroma i s o l a t i o n ; more r e c e n t l y , a number of a d d i t i o n a l r e v i e w s on aroma r e s e a r c h have appeared ( 3 - 1 1 ) . An i n t e r e s t i n g paper comparing s e v e r a l sample p r e p a r a t i o n t e c h n i q u e s was p u b l i s h e d by J e n n i n g s and F i l s o o f (12). In t h i s work, e q u a l amounts of t e n aroma c h e m i c a l s ( T a b l e I I ) were combined and then s u b j e c t e d t o v a r i o u s i s o l a t i o n t e c h n i q u e s . T a b l e I I . C o m p o s i t i o n of Model System Compound Bp,°C Ethanol 78 102 Pentan-2-one n-Heptane 98 138 Pentan-l-ol Hexan-l-ol 157 178 n-Hexyl formate Octan-2-one 174 176 d-Limonene n-Heptyl a c e t a t e 192 y-Heptalactone 84.8 (5 mmHg) (Reproduced in p a r t from Ref. American C h e m i c a l S o c i e t y . )

12, C o p y r i g h t

Wt.

%

9.6 9.5 8.2 9.7 9.8 10.5 9.8 11.3 10.3 11.5 1977,

The samples were s e p a r a t e d by c a p i l l a r y gas chromatography and peak areas compared. The r e s u l t s of t h a t study which are shown in F i g u r e 1 demonstrate t h a t no s i n g l e i s o l a t i o n and c o n c e n t r a t i o n t e c h n i q u e is u n i f o r m l y s a t i s f a c t o r y . R a t h e r , the c h o i c e of t e c h nique is determined by the i n f o r m a t i o n d e s i r e d . They d i d c o n c l u d e , however, t h a t d i s t i l l a t i o n - e x t r a c t i o n gave r e s u l t s which most n e a r l y agreed w i t h d i r e c t i n j e c t i o n s of the neat m i x t u r e . The g o a l of t h i s a r t i c l e is t o r e v i e w c u r r e n t sample i s o l a t i o n and c o n c e n t r a t i o n t e c h n i q u e s which have v a l u e in the a n a l y s i s of b i o l o g i c a l l y g e n e r a t e d aromas. R e l a t i v e l y s i m p l e and s t r a i g h t f o r ward t e c h n i q u e s w i l l be emphasized s i n c e the r e s e a r c h e r f r e q u e n t l y wishes t o a n a l y z e a number of samples, e.g., c e l l c u l t u r e s , ferment a t i o n b r o t h s and p l a n t m a t e r i a l s , in a s h o r t p e r i o d of t i m e . Headspace

Sampling

Manual Procedures D i r e c t a n a l y s i s of v o l a t i l e s above an e q u i l i b r a t e d sample c o n t a i n e d in a s e a l e d system is a t e c h n i q u e which has



PARLIMENT

Gas-Liquid Chromatographic Analysis

nil,

LL neat

headspace,

headspace,

headspace,

solution

neat solution

1 0 0 ppm a q .

100 ppm a q . , t'd.

T e n a x GC "essence"

Porapak Q sol vent e x t r a c t ion

Porapak Q "essence"

4

0

%

N a C 1

s a

T e n a x GC sol vent extract!on

LU J J J headspace, 1 0 0 ppm a q . 80% NaCl s a t '

di s t i 1 1 a t i one x t r a c t i on

F i g u r e 1. R e l a t i v e i n t e g r a t o r response ( a r b i t r a r y u n i t s ) f o r s e v e r a l methods of sample p r e p a r a t i o n , (Reproduced from Ref, 12, C o p y r i g h t 1977, American Chemical S o c i e t y ) ,



38

BIOGENERATION OF AROMAS

g r e a t a p p e a l . Arduous sample p r e p a r a t i o n is e l i m i n a t e d ; one s i m p l y homogenizes the sample, t r a n s f e r s J. t o a v i a l , s e a l s t h e v i a l w i t h a septum and e q u i l i b r a t e s J. f o r a p e r i o d of time at a c o n s t a n t temperature. H y p o v i a l s ( P i e r c e Chemical Company, R o c k f o r d , I L ) a r e q u i t e s a t i s f a c t o r y f o r t h i s t y p e of a n a l y s i s . Gas-tight glass s y r i n g e s s h o u l d be used f o r s a m p l i n g t h e v a p o r s , s i n c e d i s p o s a b l e s y r i n g e s have a d s o r p t i v e p r o p e r t i e s . S y r i n g e needles w i t h Huber p o i n t s a r e u s e f u l s i n c e they do not cause c o r i n g of t h e septum. O c c a s i o n a l a n a l y s i s of water vapor s h o u l d be made as a blank t o ens u r e no r e s i d u a l components a r e c o n t a m i n a t i n g t h e s y r i n g e or t h e i n j e c t o r of the gas chromatograph. S a l t may be added t o the aqueous samples which w i l l i n c r e a s e the amount of a r o m a t i c m a t e r i a l s in t h e headspace; however, t h i s may cause d i s p r o p o r t i o n a t i o n of the c h e m i c a l s in the headspace. For example, a d d i t i o n of s a l t t o a p p l e j u i c e i n c r e a s e d the concent r a t i o n s of a l c o h o l s more than a l d e h y d e s , and the l a t t e r more than esters (13). R e p r o d u c i b i l i t y can be a s i g n i f i c a n t problem w i t h manual headspace t e c h n i q u e s ; t h e r e f o r e , r i g o r o u s e f f o r t s must be made t o s t a n d a r d i z e a l l t i m e s , t e m p e r a t u r e s , and p r o c e d u r e s . An improvement on the manual s y r i n g e t e c h n i q u e is shown in F i g u r e 2. I n t h i s c a s e , two s y r i n g e needles a r e permanently a f f i x e d t o two p o r t s of a three-wave v a l v e ( 3 ) . A g a s - t i g h t s y r i n g e (1) is a t t a c h e d t o the t h i r d p o r t . The needle at (4) is p l a c e d i n t o the i n j e c t o r of t h e gas chromatograph and t h e a p p a r a t u s is clamped i n t o p l a c e . The b i o l o g i c a l sample is p l a c e d in a v i a l (2) and h e l d f o r a p r e s c r i b e d p e r i o d of time at a c o n s t a n t temperature to permit e q u i l i b r a t i o n to occur. The v i a l is then punctured by the n e e d l e ( 2 ) . When ready t o i n j e c t , t h e three-way v a l v e is t u r n e d so t h a t vapors in the sample can be t r a n s f e r r e d back and f o r t h between v i a l (2) and s y r i n g e ( 1 ) . T h i s s h o u l d be done seve r a l t i m e s t o ensure e q u i l i b r a t i o n of the sample. F i n a l l y , the v a l v e is r o t a t e d so t h a t a l l of the vapors can be i n j e c t e d d i r e c t l y from the s y r i n g e i n t o the gas chromatographic i n j e c t i o n p o r t which is l o c a t e d at p o s i t i o n ( 4 ) . A f t e r i n j e c t i o n , the v a l v e is c l o s e d . The apparatus is l e f t in p l a c e between gas chromatographic runs so t h a t c o r i n g of the septum and c o n c u r r e n t leakage is a v o i d e d . I t is our e x p e r i e n c e t h a t t h e s e t e c h n i q u e s a r e a p p l i c a b l e t o components a p p r o a c h i n g 200°C in b o i l i n g p o i n t such as ethyl heptanoate (bp 187°C). Compounds w i t h h i g h e r b o i l i n g p o i n t s do not have s u f f i c i e n t vapor p r e s s u r e t o be sampled and d e t e c t e d r e p r o d u c a b l y by t h i s t e c h n i q u e u n l e s s the sample temperature is raised. Another d i f f i c u l t y a r i s e s from t h e f a c t t h a t t h e l a r g e s t component n o r m a l l y i n j e c t e d u s i n g t h i s t e c h n i q u e is water vapor. Water can cause d e t e r i o r a t i o n of p o l a r gas chromatographic l i q u i d phases and p r e c l u d e s c h i l l i n g the head of the column t o f o c u s the o r g a n i c sample. The presence of water a l s o p r e v e n t s the use of h i g h r e s o l u t i o n gas chromatographic columns u n l e s s s p e c i a l p r e c a u t i o n s are taken. These t e c h n i q u e s a r e s i m p l e t o p e r f o r m , and they a r e f r e q u e n t l y a b l e t o a n a l y z e the l o w e r - b o i l i n g components in a s a t i s f a c t o r y manner. A d i s t i n c t advantage they possess is t h a t i n s o l u b l e and c e l l u l a r m a t e r i a l do not cause i n t e r f e r e n c e . Another advantage


4.

PAR LI MENT


39


of headspace sampling is t h a t t h e sample r e p r e s e n t s , most accur a t e l y , t h e c o m p o s i t i o n which we p e r c e i v e by odor, s i n c e no d i s p r o p o r t i o n a t i o n due t o a d s o r p t i o n o r s e l e c t i v e s o l v e n t e x t r a c t i o n o c curs. An American C h e m i c a l S o c i e t y Symposium was devoted t o headspace t e c h n i q u e s , and t h e p r o c e e d i n g s have been p u b l i s h e d (]_). Automated P r o c e d u r e s Some of t h e d i f f i c u l t i e s a s s o c i a t e d w i t h manual p r o c e d u r e s c a n be e l i m i n a t e d w i t h an automated headspace sampler. Such a d e v i c e has been d e s c r i b e d in t h e l i t e r a t u r e ( 14), and is c o m m e r c i a l l y a v a i l a b l e . The s c h e m a t i c diagram of such a semi-automatic headspace a n a l y z e r is shown in F i g u r e 3. P r e c i s e c o n t r o l o f times and t e m p e r a t u r e s , as w e l l as t h e c a p a b i l i t y t o h o l d samples at h i g h t e m p e r a t u r e s , produces b e t t e r chromatographic reproducibility. We have been a b l e t o a n a l y z e ppm l e v e l s o f e t h y l dodecanoate in aqueous s o l u t i o n w i t h t h i s system. Significant amounts of water vapor a r e i n t r o d u c e d i n t o t h e gas c h r o m a t o g r a p h i c column under these c o n d i t i o n s . F o r t h i s r e a s o n , columns w i t h non-polar l i q u i d phases o r bonded Carbowax-type l i q u i d phases s h o u l d be used. Two v e r s i o n s of these automated headspace samplers are commercially a v a i l a b l e (Perkin-Elmer Corp., Norwalk, CT; H e w l e t t - P a c k a r d Corp., P a l o A l t o , CA). Both can be i n t e r f a c e d w i t h f u s e d s i l i c a c a p i l l a r y gas chromatographic columns. The

advantages of automated headspace a n a l y s i s a r e : 1. 2. 3. 4. 5.

6.

Sample p r e p a r a t i o n is m i n i m i z e d . Various types o f samples (liquids, solids, s l u d g e s ) can be a n a l y z e d . A significant concentration factor may be achieved. S o l v e n t peaks a s s o c i a t e d w i t h e x t r a c t i o n s a r e eliminated. GC columns l a s t l o n g e r s i n c e o n l y v o l a t i l e s a r e i n t r o d u c e d onto t h e column and e x c e s s i v e column temperatures a r e not r e q u i r e d . R e p r o d u c i b i l i t y is good.

I f i n c r e a s e d s e n s i t i v i t y is r e q u i r e d : 1. 2. 3.

I n c r e a s e t h e sample t e m p e r a t u r e . Use a l a r g e r sample. Add an i n o r g a n i c s a l t (Na2S0 4) t o an aqueous sample t o i n c r e a s e t h e amount o f o r g a n i c compound in the headspace.

Headspace c o n c e n t r a t i n g by C o n d e n s a t i o n In cases where t h e p r e c e d i n g t e c h n i q u e s do not possess s u f f i c i e n t s e n s i t i v i t y , c o n c e n t r a t i n g t e c h n i q u e s may be employed. T r a p p i n g o f v o l a t i l e o r g a n i c compounds may be a c c o m p l i s h e d d i r e c t l y on t h e gas chromatographic column i f t h e b o i l i n g p o i n t o f t h e c h e m i c a l s is s u f f i c i e n t l y h i g h , o r i f t h e gas chromatographic column is c o o l e d . Such t e c h n i q u e s have been d e s c r i b e d by J e n n i n g s {15).



40


A l t e r n a t i v e l y , headspace vapors may be c o n c e n t r a t e d by an app a r a t u s s i m i l a r t o t h a t designed by Chang £t al, (16) and shown in F i g u r e 4. P u r i f i e d n i t r o g e n is swept through t h e sample, which is c o n t a i n e d in v e s s e l (B)· The n i t r o g e n gas is d i s b u r s e d through a sparger ( D ) , and aroma and water a r e condensed in t h e f l a s k ( G ) . In a d d i t i o n , t h e system i n c l u d e s subsequent t r a p s c o o l e d at p r o g r e s s i v e l y lower t e m p e r a t u r e s . The v a r i o u s condensates a r e then e x t r a c t e d w i t h a l o w - b o i l i n g s o l v e n t such as methylene c h l o r i d e , which is c o n c e n t r a t e d and i n j e c t e d in t h e gas chromatograph. S e v e r a l pounds of m a t e r i a l may be t r e a t e d at one time u s i n g a l a r g e - s c a l e apparatus of t h i s type. T h i s t e c h n i q u e is n o n - d e s t r u c t i v e , s i n c e temperatures a r e kept low. Extended p e r i o d s of time are r e q u i r e d f o r t h e sweeping, thus t h i s t e c h n i q u e is not a p p r o p r i ate f o r r o u t i n e a n a l y s i s . Headspace C o n c e n t r a t i n g

by Contact

with

Solvents

Another t e c h n i q u e f o r c o n c e n t r a t i n g v o l a t i l e aroma compounds inv o l v e s sweeping t h e vapors at a low r a t e i n t o a l o w - b o i l i n g r e f l u x i n g s o l v e n t (17) as shown in F i g u r e 5. In t h i s way, t h e v o l a t i l e s a r e r e t a i n e d in a w a t e r - i m m i s c i b l e s o l v e n t which can be subseq u e n t l y a n a l y z e d by gas chromatography. A c o n t i n u o u s v a r i a t i o n o f the n i t r o g e n e n t r a i n m e n t / F r e o n e x t r a c t i o n t e c h n i q u e has r e c e n t l y been d e s c r i b e d (18) and is shown in F i g u r e 6. The sample is p l a c e d in v e s s e l (7) and p u r i f i e d n i t r o g e n is passed through J. at a r a t e of 50 ml per minute. The v o l a t i l e m a t e r i a l s a r e t r a n s f e r r e d t o an e x t r a c t o r ( 9 ) . The upper phase in e x t r a c t o r (9) is 10% a l c o h o l in w a t e r , and t h i s is c o n t i n u o u s l y e x t r a c t e d w i t h Freon 11. T h i s system has been r e f i n e d and e v a l u a t e d (_19) u s i n g a s e r i e s of c h e m i c a l s r e p r e s e n t a t i v e of f r u i t s o r fermented beverages. These r e s e a r c h e r s found t h e t e c h n i q u e t o be q u i t e r e p r o d u c i b l e and e f f e c t i v e f o r terpene h y d r o c a r b o n s . I t is not q u a n t i t a t i v e f o r p o l a r , w a t e r - s o l u b l e compounds. E x t r a c t i o n time f o r t h i s apparatus may be as l o n g as 24 h o u r s , hence J. cannot be used f o r r a p i d s c r e e n i n g of aromas generated from b i o l o g i c a l p r o c e s s e s . These headspace c o n c e n t r a t i n g t e c h n i q u e s have t h e advantage t h a t t h e v o l a t i l e c h e m i c a l s a r e removed and c o n c e n t r a t e d under very g e n t l e c o n d i t i o n s which reduces a r t i f a c t f o r m a t i o n . Headspace C o n c e n t r a t i n g

by A d s o r p t i o n

Over t h e l a s t s e v e r a l y e a r s , much r e s e a r c h has i n v o l v e d t h e use o f porous polymers and carbon f o r c o n c e n t r a t i n g aroma chemicals. M a t e r i a l s such as Tenax GC, Porapack and Chromosorbs .possess t h e v a l u a b l e c h a r a c t e r i s t i c of r e t a i n i n g a wide v a r i e t y of v o l a t i l e aroma compounds w h i l e p e r m i t t i n g water and low m o l e c u l a r weight alcohols to elute rapidly. Manual P r o c e d u r e s P r i o r t o use, these p a c k i n g s s h o u l d be c o n d i t i o n e d by p l a c i n g t h e polymer in a t r a p at an e l e v a t e d temperature f o r an extended p e r i o d of time under a f l o w of oxygen-free n i t r o gen. I f t h i s is not done, d i f f i c u l t i e s w i t h i m p u r i t i e s w i l l be encountered. W i l l i a m s eît a l (2£) recommended c o n d i t i o n i n g t r a p s at 180°C f o r 15 hours at 30 ml/min n i t r o g e n f l o w .



4.

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Figure

2.

Manual headspace sampling device.

Figure 3. Semiautomatic headspace sampling device. (Reproduced with permission from Ref. 14. Copyright 1977, Dr. A. Huethig Publishers).

Figure 4. Apparatus for the i s o l a t i o n of trace v o l a t i l e constituents in headspace gas of foods. (Reproduced from Ref. 16. Copyright 1977, American Chemical Society).




Γ\ SAMPLE ' STREAM OUT

SAMPLE S 'TREAM IN

Figure 5. A Freon co-condensation unit, (Reproduced with permission from Ref. 17. Copyright 1979, Dr. A. Huethig Publishers).

Figure 6. Apparatus for the enrichment of headspace components by nitrogen entrainment Freon/extraction. (Reproduced with permission from Ref. 18. Copyright 1980, F r i e d r . Vieweg & Sohn).



4.

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43

F i g u r e 7 shows a r e l a t i v e l y s i m p l e apparatus which was used f o r a study on c i t r u s a r o m a t i c s (21.)· The amounts o f p a c k i n g mater i a l s which have been employed range from 30 mg (21) t o g r e a t e r than 1 gm (22). D e s o r p t i o n o f t h e d e s i r e d aromas is g e n e r a l l y a c c o m p l i s h e d by h e a t i n g and may be performed d i r e c t l y i n t o t h e gas chromatography A l t e r n a t i v e l y , t h e sample may be desorbed i n t o an i n t e r m e d i a t e c o o l e d t r a p and sampled v i a a m i c r o s y r i n g e o r e v a l u ated by o r g a n o l e p t i c t e c h n i q u e s (^22). The most commonly used p o l y mers a r e Tenax GC and Porapack Q. S o l v e n t s may a l s o be used f o r d e s o r p t i o n of porous polymer t r a p s as d i s c u s s e d by S c h a e f e r (23). R e c e n t l y P a r l i m e n t (24) des c r i b e d a micro solvent e x t r a c t i o n apparatus f o r desorbing v o l a t i l e o r g a n i c s from C-18 o r Tenax t r a p s . L e s s than a m i l l i l i t e r of s o l vent is used t o desorb t h e v o l a t i l e s from t h e t r a p , and t h e s o l v e n t is r e c y c l e d t o a c h i e v e complete d e s o r p t i o n . The apparatus is shown in F i g u r e 8. The tube c o n t a i n i n g t h e adsorbent (3) is p l a c e d in a d a p t o r ( 1 ) , and m i c r o f u n n e l (2) is used t o d i r e c t s o l v e n t i n t o t h e adsorbent tube. A 5 ml pear-shaped f l a s k is p l a c e d at t h e lower end of a d a p t o r ( 1 ) , w h i l e a m i c r o s p i r a l condenser w i t h d r i p t i p is p l a c e d at t h e upper j o i n t . S o l v e n t is a l l o w e d t o p e r c o l a t e t h r o u g h t h e t r a p u n t i l about 0.5 ml c o l l e c t s in t h e pear-shaped f l a s k . Then t h i s s o l v e n t is brought t o r e f l u x and t h e r e s i n t r a p is c o n t i n u o u s l y e x t r a c t e d f o r 10-15 minutes. F i n a l l y , t h e a p p a r a t u s is d i s c o n n e c t e d and d i s t i l l a t i o n used t o reduce t h e volume t o about 75 m i c r o l i t e r s . Automated P r o c e d u r e s A t l e a s t f o u r commercial automated instruments e x i s t which w i l l c o n c e n t r a t e headspace v o l a t i l e s on a d s o r b e n t s and t h e r m a l l y desorb them i n t o a gas chromatograph. Times, t e m p e r a t u r e s , and gas f l o w r a t e s a r e a c c u r a t e l y c o n t r o l l e d . Manual h a n d l i n g s t e p s which can i n t r o d u c e v a r i a b i l i t y a r e e l i m i n a t e d . S i n c e t h e s e i n s t r u m e n t s a r e automated, sample throughput is e n hanced; they can be i n t e r f a c e d t o h i g h r e s o l u t i o n gas chromatographs. A major d i s a d v a n t a g e of t h e s e i n s t r u m e n t s is t h e i r c o s t , which is s e v e r a l thousand d o l l a r s . D i s t i l l a t ion/Concent r a t i o n Techniques I f t h e v o l a t i l e s can be removed from t h e sample v i a steam d i s t i l l a t i o n w i t h o u t d e c o m p o s i t i o n , then s e v e r a l p r o c e d u r e s i n v o l v i n g d i s t i l l a t i o n / e x t r a c t i o n are available. Manual Steam D i s t i l l a t i o n / E x t r a c t i o n Samples can be p r e p a r e d u s i n g the a p p a r a t u s shown in F i g u r e 9. The sample, c o n t a i n i n g an i n t e r n a l s t a n d a r d , is p l a c e d in pear-shaped f l a s k ( 2 ) . Steam is genera t e d e x t e r n a l l y and i n t r o d u c e d through i n l e t assembly (1) i n t o t h e bottom of f l a s k ( 2 ) . S t e a m - d i s t i l l a b l e o r g a n i c compounds a r e condensed in t h e s p i r a l condenser (3) and c o l l e c t e d in a r e c e i v e r . The aqueous condensate is e x t r a c t e d w i t h a few hundred m i c r o l i t e r s of a s o l v e n t (such as methylene c h l o r i d e ) and t h e o r g a n i c phase a n a l y z e d by gas chromatography. P r e p a r a t i o n times a r e q u i t e s h o r t , e.g., f i v e t o s i x minutes f o r d i s t i l l a t i o n , one minute f o r e x t r a c tion. U s i n g a system such as t h i s , methyl a n t h r a n i l a t e can be




Figure 7. Porous polymer trap for stripped v o l a t i l e s . (Reproduced with permission from Ref. 21. Copyright 1978, Academic Press).

Figure 8. Micro solvent extraction apparatus for desorbing aromatics from porous polymers. (Reproduced with permission from Ref. 24. Copyright 1985, Walter de Gruyter & Co.).


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q u a n t i t a t i v e l y d i s t i l l e d and c o n c e n t r a t e d from grape j u i c e in as l i t t l e as t e n minutes. I f t h e sample is heat s e n s i t i v e , t h e aqueous r e c e i v e r can be connected t o a vacuum s o u r c e , and t h e d i s t i l l a t i o n performed under vacuum. S i m u l t a n e o u s Steam D i s t i l l a t i o n / E x t r a c t i o n An e l e g a n t apparatus was d e s c r i b e d by N i c k e r s o n and L i k e n s (25) f o r t h e s i m u l t a n e o u s steam d i s t i l l a t i o n and e x t r a c t i o n (SDE) of v o l a t i l e components. T h i s d e v i c e has become one of t h e m a i n s t a y s in t h e f l a v o r f i e l d . In t h i s a p p a r a t u s , both t h e aqueous sample and w a t e r - i m m i s c i b l e solvent are simultaneously d i s t i l l e d . The steam which c o n t a i n s t h e aroma c h e m i c a l s and t h e o r g a n i c s o l v e n t a r e condensed t o g e t h e r , and the aroma compounds a r e t r a n s f e r r e d from t h e aqueous phase t o t h e o r g a n i c phase. T y p i c a l s o l v e n t s used a r e d i e t h y l e t h e r , pentane o r a m i x t u r e t h e r e o f ; normal e x t r a c t i o n times a r e one t o two hours. Advantages of t h e SDE Apparatus a r e as f o l l o w s : 1. 2. 3.

A s i n g l e o p e r a t i o n removes t h e o r g a n i c s from an aqueous m a t e r i a l and c o n c e n t r a t e s them. R e c o v e r i e s of v o l a t i l e , w a t e r - i m m i s c i b l e o r g a n i c compounds a r e g e n e r a l l y q u i t e h i g h . Only a s m a l l amount of o r g a n i c s o l v e n t is r e q u i r e d , thus one doesn't have t o c o n c e n t r a t e l a r g e volumes of s o l v e n t s .

A number of m o d i f i c a t i o n s of t h i s apparatus have been proposed. One such improvement is d e s c r i b e d by S c h u l t z e t a l , (26) and is shown in F i g u r e 10. I n t h i s paper, t h e authors d e s c r i b e t h e e f f e c t s of pH, t i m e , p r e s s u r e and e x t r a c t i n g s o l v e n t on t h e r e c o v e r y of t y p i c a l aroma compounds. R e s u l t s showing p e r c e n t r e c o v e r y at v a r i o u s i n i t i a l c o n c e n t r a t i o n s of aromas a r e shown in T a b l e I I I . Table I I I . Recovery of Components by SDE from t h e Model M i x t u r e at V a r i o u s Degrees o f D i l u t i o n . (Recovery as P e r c e n t of I n i t i a l Amount) 3

Cone. of each component , ppm 210 21 2.1 0.21 Ethyl butyrate E t h y l hexanoate E t h y l octanoate Ethanol 1-Hexanol Linalool Carvone Limonene

95 100 101 0 99 99 99 99

a

A t pH 5.0 and atmospheric hexane; SDE time 1 h r . (Reproduced in p a r t from Ref. Chemical S o c i e t y . )

98 104 98

95 100 90

98 101 97 93

91 97 90 80

pressure,

93 96 89 «*0.01 86 95 83 85

with

26, C o p y r i g h t

125

1977,


ml o f

American



Figure

9.

M i c r o steam d i s t i l l a t i o n a p p a r a t u s .

F i g u r e 10. Simultaneous s t e a m - d i s t i l l a t i o n e x t r a c t i o n (SDE) head. (Reproduced from R e f . 26. C o p y r i g h t 1977, American Chemical S o c i e t y ) .



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I t is apparent t h a t compounds which a r e water i m m i s c i b l e and steam d i s t i l l a b l e are recovered q u i t e e f f e c t i v e l y . A m i c r o v e r s i o n of the d i s t i l l a t i o n e x t r a c t i o n apparatus has been d e s c r i b e d by G o d e f r o o t et a l , (27). T h i s a p p a r a t u s uses h e a v i e r than water s o l v e n t s , e.g., methylene c h l o r i d e or carbon d i s u l f i d e as the e x t r a c t a n t . Because o n l y one m i l l i l i t e r of s o l vent is used, no f u r t h e r c o n c e n t r a t i o n of s o l v e n t is r e q u i r e d . The a u t h o r s found 15 minutes d i s t i l l a t i o n / e x t r a c t i o n time s u f f i c i e n t f o r r e c o v e r y of n o n p o l a r compounds, e.g., mono and s e s q u i t e r p e n e s , w h i l e one hour was r e q u i r e d f o r oxygenated and h i g h e r b o i l i n g compounds. T h i s a p p a r a t u s was e v a l u a t e d by Nunez and Bemelmans (28) f o r low l e v e l s of aroma compounds in water. They r e p o r t e d t h a t r e s u l t s were s a t i s f a c t o r y f o r v o l a t i l e l e v e l s g r e a t e r than 1 ppm. Both h e a v i e r and l i g h t e r than water v e r s i o n s of the steam d i s t i l l a t i o n apparatus a r e c o m m e r c i a l l y a v a i l a b l e ( A l l t e c h , D e e r f i e l d , I L ; Chrompack, B r i d g e w a t e r , N J ) . Diagrams of t h e two v e r s i o n s a r e shown in F i g u r e 11. S i m u l t a n e o u s D i s t i l l a t i o n and A d s o r p t i o n J e n n i n g s and N u r s t e n (29) suggested steam d i s t i l l i n g d i l u t e aqueous s o l u t i o n s , f o l l o w e d by p a s s i n g the condensate over a c t i v a t e d carbon. The v o l a t i l e a r o m a t i c compounds were desorbed w i t h carbon d i s u l f i d e . A simultaneous d i s t i l l a t i o n and a d s o r p t i o n (SDA) v e r s i o n of t h i s procedure has r e c e n t l y been p u b l i s h e d ( 30_), and the a p p a r a t u s is shown in F i g u r e 12. Ten mg of a c t i v a t e d carbon (60-80 mesh) a r e used in the t r a p p i n g tube and t y p i c a l d i s t i l l a t i o n times a r e 40 minutes. The r e s e a r c h e r s found methylene c h l o r i d e and carbon d i s u l f i d e t o be the most e f f e c t i v e d e s o r p t i o n s o l v e n t s . The procedure was a p p l i e d t o both a model system as w e l l as P e r i l l a Leaves. The chromatograms o b t a i n e d compared f a v o r a b l y w i t h those g e n e r a t e d by an SDE apparatus. D i r e c t A n a l y s i s of Aqueous Essences The most s i m p l e sample p r e p a r a t i o n t e c h n i q u e is d i r e c t gas c h r o matographic i n j e c t i o n of an aqueous essence on a bonded f u s e d s i l i c a column. T h i s t e c h n i q u e may be employed when aqueous d i s t i l l a t e s are a v a i l a b l e . For example, Moshonas and Shaw (31.) d e s c r i b e d a method f o r the a n a l y s i s of aroma c o n s t i t u e n t s of n a t u r a l f r u i t essences. The essences were c o l l e c t e d from the f i r s t s t a g e of an e v a p o r a t o r and the essence i n j e c t e d d i r e c t l y i n t o a c a p i l l a r y gas chromâtograph. They s t r e s s e d t h a t the a v a i l a b i l i t y of f u s e d s i l i c a c a p i l l a r y columns c o a t e d w i t h c r o s s - l i n k e d n o n - p o l a r l i q u i d phases p e r m i t t e d development of t h i s t e c h n i q u e . Such columns r e s i s t the d e t e r i o r a t i o n which was p r e v i o u s l y encountered w i t h aqueous samples. These a u t h o r s a p p l i e d t h i s t e c h n i q u e t o s e v e r a l c i t r u s essences as w e l l as t o f r u i t essences such as grape, a p p l e and s t r a w b e r r y . A d s o r p t i o n of O r g a n i c s from Aqueous S o l u t i o n s I f p a r t i c u l a t e s can be removed from an aqueous sample, J. is p o s s i b l e t o c o n c e n t r a t e the v o l a t i l e a r o m a t i c compounds d i r e c t l y on

American Chemical Society Library 1155 18th St., N.W. Washington, D.C. 20036



BIOGENERATION OF A R O M A S

A

Β

Figure 11. Micro steam d i s t i l l a t i o n - e x t r a c t i o n apparatus. A: For solvents l i g h t e r than water. B: For solvents heavier than water. (Reproduced with permission from Chrompack, I n c . , Bridgewater, N.J.) To S p i r a l

Condenser

Condenser

-Water

Trapping

To

Tube

Sample

Flask

Figure 12. Simultaneous s t e a m - d i s t i l l a t i o n adsorption (SDA) head. (Reproduced with permission from Ref. 30. Copyright 1981, Walter de Gruyter & Co.).



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Gas-Liquid Chromatographic

Analysis

49

adsorbents. C l a r i f i c a t i o n can be a c h i e v e d by c e n t r i f u g a t i o n , u l t r a f i l t r a t i o n o r r e v e r s e osmosis. The c l a r i f i e d aqueous m a t e r i a l can be c o n c e n t r a t e d on m i n i columns of m a c r o r e t i c u l a r r e s i n s , e.g., XAD-4 o r on a c t i v a t e d carbon as d e s c r i b e d by Tateda and F r i t z (32.)· These i n v e s t i g a t o r s used d i s p o s a b l e p i p e t s as t h e i r s o r p t i o n columns, w i t h column dimensions of 1.2 mm by 2.5 cm. T y p i c a l aqueous sample volumes a r e about 100 ml and f l o w r a t e s a r e 1 ml/min. D e s o r p t i o n is a c h i e v e d w i t h l e s s than 100 m i c r o l i t e r s of an o r g a n i c solvent. They found acetone t o be an e f f e c t i v e s o l v e n t f o r XAD-4 d e s o r p t i o n , and carbon d i s u l f i d e t o be most s a t i s f a c t o r y f o r des o r p t i o n from a c t i v a t e d c a r b o n . The purpose of t h e i r study was t o i n v e s t i g a t e t y p i c a l o r g a n i c c o n t a m i n a n t s found in d r i n k i n g w a t e r , but they a l s o i n v e s t i g a t e d a l c o h o l s , e s t e r s and c a r b o n y l compounds, which a r e t y p i c a l aroma c h e m i c a l s . In a s i m i l a r v e i n , P a r l i m e n t (33_) used r e v e r s e phase C18 a d s o r b e n t s t o c o n c e n t r a t e and f r a c t i o n a t e low l e v e l s of v o l a t i l e o r g a n i c compounds from d i l u t e aqueous streams. The aqueous phase must be p a r t i c u l a t e f r e e t o p r e v e n t f o u l i n g o f t h e a d s o r b e n t , but s o l u b l e s o l i d s a r e not n e c e s s a r i l y a problem. F o r example, a comm e r c i a l c o l a beverage c o n t a i n i n g c a r a m e l c o l o r , c a f f e i n e , phosp h o r i c a c i d and sweetener was passed over a r e v e r s e phase column. D e s o r p t i o n w i t h acetone produced an aroma c o n c e n t r a t e which c o u l d be a n a l y z e d by gas chromatography. E x t r a c t i o n o f V o l a t i l e O r g a n i c s from Aqueous S o l u t i o n s C e r t a i n aqueous m a t e r i a l s can be e x t r a c t e d d i r e c t l y w i t h a s o l v e n t t o p r o d i c e an aroma c o n c e n t r a t e . T h i s t e c h n i q u e has found u t i l i t y in s t u d i e s on wine aroma ( 3 U ) . The i n v e s t i g a t o r s s t u d i e d model wine systems c o n t a i n i n g 12% e t h a n o l and 4.4% s u c r o s e . Extractions were performed m a n u a l l y , and t h e o r g a n i c s o l u t i o n s were concent r a t e d under vacuum. They c o n c l u d e d t h a t Freon 11 (bp 24°) was the s o l v e n t of c h o i c e ; i f t h e o r g a n i c essence is t o be s t o r e d f o r an extended p e r i o d of t i m e , they recommended methylene c h l o r i d e as the s o l v e n t . The p r e s e n c e of sugar d i d not c r e a t e any d i f f i c u l t i e s . More r e c e n t l y , a 2:1 m i x t u r e of pentanermethylene c h l o r i d e has been used t o e x t r a c t t h e a r o m a t i c s from w h i t e wines (35.). These i n v e s t i g a t o r s used a c o n t i n u o u s l i q u i d / l i q u i d e x t r a c t o r f o r e i g h t hours t o remove t h e a r o m a t i c s ; t h e o r g a n i c phase was then concent r a t e d t o 1 ml volume u s i n g a V i g r e u x column. Such a p r o c e d u r e s h o u l d have a p p l i c a t i o n in s t u d i e s on f e r m e n t a t i o n b r o t h s . We have found d i r e c t e x t r a c t i o n s o f b i o l o g i c a l l y - g e n e r a t e d aromas can be performed q u i t e r a p i d l y by a p r o c e d u r e which has r e c e n t l y been d e s c r i b e d (36). The e x t r a c t i o n d e v i c e is a M i x x o r ( L i d e x Tech., B e d f o r d , MA), and is shown in F i g u r e 13. A q u a n t i t y of aqueous sample ( e . g . , 10 m i s ) is p l a c e d in t h e lower v e s s e l , and a low d e n s i t y s o l v e n t is added. The sample is mixed by moving t h e p i s t o n up and down. Phases a r e a l l o w e d t o s e p a r a t e , and t h e p i s t o n is s l o w l y depressed f o r c i n g t h e s o l v e n t i n t o t h e upper c o l l e c t i n g chamber. We have found t h a t i f o n l y a few hundred m i c r o l i t e r s o f s o l v e n t a r e used J. is p o s s i b l e t o f o r c e t h e s o l v e n t i n t o t h e a x i a l chamber ( C ) . The o r g a n i c sample can be removed from (C) w i t h a s y r i n g e f o r gas c h r o m a t o g r a p h i c a n a l y s i s . This extraction techn i q u e is r a p i d and produces an e f f i c i e n t e x t r a c t i o n . The p r o c e d u r e




F i g u r e 13. Mixxor e x t r a c t o r , used t o e x t r a c t aqueous s o l u t i o n s w i t h minimal amount of s o l v e n t .


4.

PARLIMENT


51

was a p p l i e d t o a model system o f e t h y l e s t e r s in water. D e t e c t i o n l i m i t s were l e s s than 0.2 ppm and r e c o v e r i e s were g e n e r a l l y over 90%. The procedure was a p p l i e d t o s e v e r a l p l a n t m a t e r i a l s such as f e n n e l , rosemary and c e l e r y seed w i t h s a t i s f a c t o r y r e s u l t s .


Conclusion There a r e many sample p r e p a r a t i o n t e c h n i q u e s which one can choose. C h o i c e w i l l be determined by t h e r e q u i r e m e n t s o f t h e s t u d y . Quest i o n s t o be c o n s i d e r e d i n c l u d e : I s h i g h sample throughput r e quired? I s h i g h p r e c i s i o n d e s i r e d ? How much time is a v a i l a b l e f o r the s t u d y ? Does t h e sample s u r v i v e 100°C? What i n s t r u m e n t a t i o n and a p p a r a t u s a r e a v a i l a b l e ? Each method d e s c r i b e d has advantages and d i s a d v a n t a g e s . No s i n g l e method is i d e a l , and t h e c h o i c e o f t e c h n i q u e s h o u l d be based on informed d e c i s i o n s . Acknowledgment The a u t h o r wishes t o thank Judy S c h i n k e l f o r s e c r e t a r i a l a s s i s t a n c e . Literature

Cited

1. van Straten, S.; Maarse, H. "Volatile Compounds in Food." Division for Nutrition and Food Research TNO, Zeist, The Netherlands, 1983. 2. Weurman, C. J. Agric. Food Chem. 1969, 17, 370. 3. Schreier, P. In "Chromatographic Studies of Biogenesis of Plant Volatiles"; Huthig: New York, 1984; pp. 1-32. 4. Maarse, H.; Betz, R. In "Isolation and Identification of Volatile Compounds in Aroma Research"; Akademie - Verlag: Berlin, 1981; pp. 1-59. 5. Teranishi, R.; Flath, R.; Sugisawa, H. In "Flavor Research Recent Advances"; Marcel Dekker, Inc.: New York, 1981, pp. 11-51. 6. Morton, I.; MacLeod, A. In "Food Flavors. Part A. Introduction"; Elsevier: New York, 1982; pp. 15-48. 7. Charalambous, G. "Analysis of Foods and Beverages. Headspace Techniques"; Academic Press: New York, 1978. 8. Jennings, W. In "Gas Chromatography with Glass Capillary Columns"; Second Edition. Academic Press: New York, 1980; pp. 183-200. 9. Reineccius, G.; Anandaraman, S. Food Sci. Technol., 1984, 11, 195. 10. Leahy, M.; Reineccius, G. In "Analysis of Volatiles. Methods. Applications"; Schreier, P., Ed.; de Gruyter: New York, 1984; pp. 18-47. 11. Schreier, P.; Idstein, H. Z. Lebensm. Unters. Forsch. 1985, 180, 1. 12. Jennings, W.; Filsoof, M. J. Agric. Food Chem. 1977, 25, 440. 13. Poll, L.; Flink, J. Food Chem. 1984, 13, 193. 14. Kolb, B.; Pospisil, P.; Borath, T.; Auer, M. J.H.R.C.&C.C. 1979, 2, 283.


52


15. Jennings, w. In "Gas Chromatography with Glass Capillary Col umns"; Second Edition. Academic Press: New York, 1980; pp. 54-56. 16. Chang, S.; Vallese, F.; Hurang, L . ; Hsieh, Α.; Min, D. J. Agric. Food Chem. 1977, 25, 450.

Jennings, W. J.H.R.C.&C.C. 1979, 2, 221. Rapp, Α.; Knipser, W. Chromatographia 1980, 13, 698. Guichard, E.; Ducruet, V. J. Agric. Food Chem. 1984, 32, 838. Williams, Α.; May, H.; Tucknott, O. J. Sci. Fd. Agric. 1978, 29, 1041. 21. Lund, E.; Dinsmore, H. In "Analysis of Foods and Beverages.


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Headspace Techniques.";

Charalambous, G., Ed.; Academic Press:

New York, 1978; pp. 135-186. 22. Withycombe, D.; Mookherjee, B.; Hruza, A. In "Analysis of Foods and Beverages. Headspace Techniques"; Charalambous, G., Ed.;

Academic Press: New York, 1978; pp. 81-94. 23. Schaefer, J. In "Flavor '81"; Schreier, P., Ed.; de Gruyter: New York, 1981; pp. 301-314. 24. Parliment, T. In "Semiochemistry: Flavors and Pheromones"; Acree, T.; Soderlund, D., Eds.; de Gruyter: New York, 1985; pp. 181-202. 25. Nickerson, G.; Likens, S. J. Chromatogr. 1966, 21, 1. 26. Schultz, T.; Flath, R.; Mon, R.; Eggling, S.; Teranishi, R. J. Agric. Food Chem. 1977, 25, 446. 27. Godefroot, M.; Sandra, P.; Verzele, M. J. Chromatogr. 1981, 203, 325. 28. Nunez, Α.; Bemelmans, J. J. Chromatogr. 1984, 294, 361. 29. Jennings, W.; Nursten, H. Anal. Chem. 1967, 39, 521. 30. Sugisawa, H.; Hirose, T. In "Flavor '81"; Schreier, P., Ed.; de Gruyter: New York, 1981; pp. 287-299. 31. Moshonas, M.; Shaw, P. J. Agric. Food Chem. 1984, 32, 526. 32. Tateda, Α.; Fritz, J. J. Chromatogr. 1978, 152, 329. 33. Parliment, T. J. Agric. Food Chem. 1981, 29, 836. 34. Cobb, C.; Bursey, M. J. Agric. Food Chem. 1978, 26, 197. 35. Moret, I.; Scarponi, G.; Cescon, P. J. Sci. Food Agric. 1984, 35, 1004. 36. Parliment, T. Perfum. Flavorist 1986, in press. Received January 15, 1986


Sample Preparation Techniques for Gas-Liquid Chromatographic

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