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Chemical composition and color attributes of Foch and deChaunac wines at various ages Scaman, Christine H. 1987

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CHEMICAL COMPOSITION AND COLOR ATTRIBUTES OF FOCH AND DECHAUNAC WINES AT VARIOUS ASES by CHRISTINE H. SCAMAN A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n FACULTY OF GRADUATE STUDIES Department of Food Science We accept t h i s t h e s i s as CDn-forminq to the required standard THE UNIVERSITY OF BRITISH COLUMBIA Feburary 1987 ©Christine H. Seaman, 1787 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Food Science The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date March 17, 1987 i i QBBIRACT P h e n o l i c and c o l o r parameters of Marechal Foch and deChaunac wines of 19B0 through 1983 v i n t a g e s were analysed t o determine v a r i e t y and aging e f f e c t s . C e n t r o i d Mapping O p t i m i z a t i o n together with the Simultaneous F a c t o r S h i f t a l g o r i t h m were used t o determine the HPLC o p e r a t i n g c o n d i t i o n s which r e s o l v e d the maximum number of p h e n o l i c components of whole red wine. A t e r n a r y g r a d i e n t system u s i n g 1 % a c e t i c a c i d : methanol i a c e t o n i t r i l e was changed from 100s0:0 t o 4.8:82.8:12.4 i n 130.6 minutes. A flow r a t e of 1.1 mL/min and a temperature of 32.9 °C were used. The HPLC system was used t o separate at l e a s t 50 components from each i n j e c t i o n of wine and of an e t h y l a c e t a t e e x t r a c t of wine. Foch wines were found t o have more c a t e c h i n and e p i c a t e c h i n than deChaunac wines. Peak areas f o r the e t h y l a c e t a t e e x t r a c t s common t o a l l wines, and areas of peaks i n the chromatograms of the whole wine, not present i n the n e u t r a l e x t r a c t , were used t o form a data s e t f o r m u l t i v a r i a t e a n a l y s e s . Strong l i n e a r c o r r e l a t i o n s were found between t r i s t i m u l u s and s p e c t r o p h o t o m e t r y measurements f o r each wine. The c o l o r of Foch wines was more s t a b l e and contained more brown and yellow hues than deChaunac wines, as determined by t r i s t i m u l u s measurements. T o t a l pigment l e v e l s of each wine and v a r i o u s f r a c t i o n s of the t o t a l , i n c l u d i n g i o n i z e d , u n - i o n i z e d , polymeric and I i i s u l f u r dioxide-bound anthocyanins, were determined s p e c t r o -p h o t o m e t r i c a l l y . T o t a l anthocyanin l e v e l s ( u n - i o n i z e d and i o n i z e d ) i n deChaunac wines decreased s i g n i f i c a n t l y with i n c r e a s i n g age but remained constant i n Foch wines. Tannin l e v e l s as determined by absorbance r e a d i n g s at 280 nm and by the F o l i n - C o i c a l t e u reagent method were h i g h l y c o r r e l a t e d . Foch had more f l a v o n o i d and l e s s n o n f l a v o n o i d s than deChaunac wines. The d i f f e r e n t c o l o r parameters, pigment and t a n n i n f r a c t i o n s , as well as t i t r a t a b l e a c i d i t y , pH and i n d i v i d u a l o r g a n i c a c i d s were used as a second data s e t of a n a l y t i c a l parameters f o r m u l t i v a r i a t e a n a l y s e s . A t h i r d data s e t composed of the combination of the a n a l y t i c a l data and the HPLC peak areas was a l s o used. D i f f e r e n c e s between the f o u r v i n t a g e s of wines, the two v a r i e t i e s , and between o l d and young wines w i t h i n v a r i e t i e s were found using stepwise d i s c r i m i n a n t a n a l y s i s (SDA). D i s c r i m i n a t i o n of v a r i e t y d i f f e r e n c e s was more s u c c e s s f u l (100 V. c o r r e c t c l a s s i f i c a t i o n by the j a c k k n i f e procedure) and r e q u i r e d fewer v a r i a b l e s than c l a s s i f i c a t i o n by age. C l u s t e r a n a l y s e s , performed with v a r i a b l e s chosen by SDA, gave s i m i l a r r e s u l t s t o the SDA. IT Table of Contents ABSTRACT - i i LIST DF TABLES v i LIST OF FIGURES v i i i ACKNOWLEDGEMENT i x I. INTRODUCTION 1 I I . LITERATURE REVIEW 3 A. Changes i n P h e n o l i c s and Co l o r i n Red Wine During Aging 3 B. O p t i m i z a t i o n 10 C. M u l t i v a r i a t e A n a l y s i s - 17 a. P r i n c i p a l Component A n a l y s i s .....18 b. Stepwise D i s c r i m i n a n t A n a l y s i s 18 c. C l u s t e r A n a l y s i s 20 I I I . METHODS AND MATERIALS 22 A. Wines 22 B. HPLC Chemicals and Instrumentation 23 C. C e n t r o i d Mapping O p t i m i z a t i o n 24 a. Procedure 24 b. O p t i m i z a t i o n F a c t o r s 25 D. Wine Analyses 26 a. T i t r a t a b l e A c i d i t y and pH 26 b. Organic A c i d Determination 27 i> Sample P r e p a r a t i o n 27 i i ) HPLC System 28 c. Spectrophotometric E v a l u a t i o n s ...28 V d. PVP ( P o l y v i n y l p y r r o l i d i n e ) Index *.29 e. T r i s t i m u l u s E v a l u a t i o n ..31 f . Paper Chromatography of Anthocyanins ....32 g. P h e n o l i c Content 32 h. Neutral P h e n o l i c E x t r a c t i o n 33 i . HPLC of the Neutral P h e n o l i c Sample and Wine .....34 E. S t a t i s t i c a l A n a l y s i s .....35 a. A n a l y s i s of Variance 36 b. P r i n c i p a l Component A n a l y s i s 36 c. Stepwise D i s c r i m i n a n t A n a l y s i s .36 d. C l u s t e r A n a l y s i s 37 IV. RESULTS AND DISCUSSION 39 A. A n a l y t i c a l Parameters 39 a. Mine C o l o r 39 b. Anthocyani ns 45 c. Other P h e n o l i c s ...50 B. C e n t r o i d Mapping O p t i m i z a t i o n f o r HPLC Se p a r a t i o n of P h e n o l i c Compounds of Red Mine .52 C. M u l t i v a r i a t e A n a l y s i s 62 a. P r i n c i p a l Component A n a l y s i s 62 b. Stepwise D i s c r i m i n a n t A n a l y s i s 65 c. C l u s t e r A n a l y s i s 71 V. CONCLUSIONS 77 VII. REFERENCES 78 APPENDIX I 84 APPENDIX II 93 vi L i s t of Tables Table Page 1. Color and composition of wine c a l c u l a t e d from absorbance readings ...30 2. Color and phenolic parameters and pH of Foch and deChaunac wines 40 3. Linear c o r r e l a t i o n between c o l o r and a n a l y t i c a l parameters of Foch (top value) and deChaunac (bottom value) wines 41 4. Anova of some c o l o r parameters of Foch and deChaunac wines: expressed as 100 R ('/.) 43 5. Anova of some pigment paramters of Foch and deChaunac wines: expressed as 100 R (7.) 44 6. Phenolic components of Foch and deChaunac wines ..47 7. Anova of some phenolic parameters of Foch and deChaunac wines: expressed as 100 R ('/.) ....48 8. Anova of some ph e n o l i c s and polymeric parameters of Foch and deChaunac wines: expressed as 100 R (7.) 49 9. I n i t i a l l i m i t s of f a c t o r s , f a c t o r l e v e l s of s t a r t i n g simplex ( v e r t i c e s 1 - 6) and f i r s t c e n t r o i d search (vertex 7) ... S3 10. Factor l i m i t s of second simplex search ( v e r t i c e s 8 - 13) and second c e n t r o i d search ( v e r t i c e s 14 - 15) 54 11. New l i m i t s of f a c t o r s a f t e r mapping, f a c t o r l i m i t s of t h i r d simplex ( v e r t i c e s 16 - 21) and t h i r d c e n t r o i d search ( v e r t i c e s 22 - 23) 56 12. Simultaneous f a c t o r s h i f t of f a c t o r l e v e l s 58 13. Eigenvalues (VP), sum of VP and cumulative p r o p o r t i o n i n t o t a l v ariance (7.) i n p r i n c i p a l component a n a l y s i s using a n a l y t i c a l v a r i a b l e s 63 14. Sorted, r o t a t e d f a c t o r loadings associated with the f i r s t s i x p r i n c i p a l components based on a n a l y t i c a l v a r i a b l e s .64 v i i 15. E i g e n v a l u e (VP), sum of VP and cumulative p r o p o r t i o n i n t o t a l v a r i a n c e (7.) i n p r i n c i p a l component a n a l y s i s u s i n g peak area v a r i a b l e s 66 16. C o o r d i n a t e s of i n d i v i d u a l wine samples f o r c a n o n i c a l p l o t of v a r i e t y x age d i s c r i m i n a t i o n based on combination data s e t 70 v l l l L i s t of F i g u r e s F i g u r e Page 1. Anthocyanin s t r u c t u r a l t r a n s f o r m a t i o n s with pH. M a l v i d i n 3 - g l u c o s i d e (25 C; 0.2 M i o n i c s t r e n g t h ) . (From: Timber l a k e , 1980) .4 2. D i s t r i b u t i o n of s t r u c t u r e s with pH ( M a l v i d i n 3 - g l u c o s i d e : 25 C). AH+ = red c a t i o n ; B = c o l o u r l e s s c a r b i n o l base; C = c o l o u r l e s s chalcone; A = blu e q u i n o i d a l base. (From: Timberlake, 1980) 5 3. I n i t i a l RP-HPLC s e p a r a t i o n of p h e n o l i c compounds df 1983 Foch wine. For c o n d i t i o n s see Table 9, vertex #3 59 4. Optimal RP-HPLC s e p a r a t i o n of p h e n o l i c compounds of 1983 Foch wine. For c o n d i t i o n s see Table 12, S h i f t Combination #2 60 5. Canonical p l o t of the group means of wine samples. V a r i e t y x age d i s c r i m i n a t i o n based on combination data s e t 69 6. H i e r a r c h i c a l c l u s t e r a n a l y s i s of wines u s i n g v a r i a b l e s chosen i n age stepwise d i s c r i m i n a n t a n a l y s i s . V a r i a b l e s were chosen from chromatographic peak areas and a n a l y t i c a l data 73 7* H i e r a r c h i c a l c l u s t e r a n a l y s i s of wines using v a r i a b l e s chosen i n v a r i e t a l stepwise d i s c r i m i n a n t a n a l y s i s . V a r i a b l e s were chosen from chromatographic peak areas and a n a l y t i c a l data .74 8. H i e r a r c h i c a l c l u s t e r a n a l y s i s of wines u s i n g v a r i a b l e s chosen i n age x v a r i e t y stepwise d i s c r i m i n a n t a n a l y s i s . V a r i a b l e s were chosen from a n a l y t i c a l data 75 Ix ACKNOWLEDGEMENT I would l i k e t o express my g r a t i t u d e t o Dr. W. Powrie f o r h i s a s s i s t a n c e , support and encouragement throughout the course of t h i s study. I a l s o Wish t o thank the members of my graduate committee, Drs. S. Nakai, B. Skura and P. Townsley f o r t h e i r c o n s t r u c t i v e suggestions and review of t h i s work, and Dr. 6 . Eaton f o r h i s a s s i s t a n c e . F i n a l l y , very s p e c i a l thanks goes t o David McArthur f o r h i s enthusiasm and h i s humour when i t was needed most. 1 I-. INTRODUCTION During the aging of red wines, the red hue g r a d u a l l y decreases, the a s t r i n g e n c y and b i t t e r n e s s i s decreased and the f l a v o u r and body are improved. P h e n o l i c compounds are r e s p o n s i b l e f o r the red c o l o r , a s t r i n g e n c y and b i t t e r n e s s of red wine. I t has been p o s t u l a t e d t h a t the p h e n o l i c compounds are a l t e r e d d u r i n g red wine aging and such a l t e r a t i o n l e a d s t o q u a l i t y changes ( S i n g l e t o n and Esau, 1969). An improved understanding of these changes and t h e i r e f f e c t on the sensory c h a r a c t e r i s t i c s of wine would be b e n e f i c i a l f o r the c o n t r o l of wine q u a l i t y . A t y p i c a l red wine c o n t a i n s approximately 1200 - 1500 mg/L p h e n o l i c m a t e r i a l ( S i n g l e t o n and Noble, 1976), c o n s i s t i n g of s e v e r a l hundred d i f f e r e n t types of p h e n o l i c compounds. Advancements i n the knowledge of p h e n o l i c s i n red wine have p a r a l l e l e d improvements i n chromatographic techniques. Somers (1966) used gel f i l t r a t i o n t o s e p a r a t e monomeric and polymeric c o l o r e d m a t e r i a l from red wines. As HPLC techniques were p e r f e c t e d , i n d i v i d u a l p h e n o l i c compounds c o u l d be separated. Now i t may be p o s s i b l e t o determine which p h e n o l i c compounds are a l t e r e d d u r i n g the aging of red wines made from d i f f e r e n t grape c u l t i v a r s , p a r t i c u l a r l y the h y b r i d grape v a r i e t i e s . With p o t e n t i a l l y l a r g e amounts of HPLC data on p h e n o l i c compounds i n red wines at v a r i o u s ages a v a i l a b l e , methods of d i s t i n g u i s h i n g the d i s c r i m i n a t o r y v a r i a b l e s r e l e v a n t t o wine aging c h a r a c t e r i s t i c s and q u a l i t y are necessary. P a t t e r n r e c o g n i t i o n techniques i n c l u d i n g p r i n c i p a l component, stepwise d i s c r i m i n a n t and c l u s t e r a n a l y s e s have been used t o c l a s s i f y wines by q u a l i t y , o r i g i n and v a r i e t y . The o b j e c t i v e s of t h i s work were t o examine the p h e n o l i c composition and c o l o r c h a r a c t e r i s t i c s of two v a r i e t a l red wines at v a r i o u s ages. The two v a r i e t i e s of grape examined were deChaunac ( S e i b e l 9549) and Marechal Foch (Kuhlmann 18B-2). Reverse phase HPLC was used i n t h i s study t o s e p a r a t e the p h e n o l i c compounds of the wines. Optimal chromatographic s e p a r a t i o n of p h e n o l i c s was achieved u s i n g C e n t r o i d Mapping O p t i m i z a t i o n . Groups of compounds, i n c l u d i n g o r g a n i c a c i d s , anthocyanins and other p h e n o l i c s were q u a n t i f i e d i n the wines. Several v i n t a g e s of each v a r i e t y were examined t o determine changes i n composition with time. 3 I I - . L I T E R A T U R E R E V I E W 8i CHANGES I N P H E N O L I C S AND COLOR I N RED WINE DURING A G I N G The c o l o r of young red wines i s due t o the f l a v o n o i d p h e n o l i c s , with a 4' - h y d r o x y - f l a v y l i u m s t r u c t u r e , known as anthocyanins. A mixture of anthocyanins have been found i n a s i n g l e grape v a r i e t y (Ribereau-Gayon, 1982). These anthocyanins d i f f e r i n the number and type of r e s i d u a l sugars, number and type of a c y l a t i n g a c i d s present on the sugar molecules, and the s u b s t i t u e n t s (methyl and hydroxyl groups) present on the B - r i n g (Hrazdina, 1981). At pH va l u e s below 6 anthocyanins can occur i n f o u r d i s t i n c t chemical s t r u c t u r e s ; the q u i n o i d a l base, f l a v y l i u m c a t i o n ( i o n i z e d ) , c a r b i n o l base and chalcone (Figure 1). The p r o p o r t i o n of each form i s h i g h l y dependent on pH. At low pH v a l u e s (pH < 0.05), the c o l o r e d f l a v y l i u m c a t i o n i s the only s p e c i e s present. As pH i n c r e a s e s , the m a j o r i t y of the c o l o r e d form i s converted i n t o the c o l o r l e s s c a r b i n o l base with minor amounts of the other two s p e c i e s . Between the pH va l u e s of approximately 4 t o 6, a small amount of the blu e q u i n o i d a l base i s the onl y c o l o r e d s p e c i e s present (Figure 2 ) . Wines, which have been aged f o r even s h o r t p e r i o d s of time, have been found t o c o n t a i n s i g n i f i c a n t l y lower c o n c e n t r a t i o n s of t o t a l anthocyanins. Ribereau-Gayon (1974) and Nagel and Wulf (1979) found approximately a 50 "A decrease i n f r e e anthocyanins i n wines a f t e r one year of OGI 1. Anthocyanin s t r u c t u r a l t r a n s f o r m a t i o n s with pH. M a l v i d i n 3 - d i g l u c o s i d e (23 Cj 0.2 M i o n i c strength) (Froms Timberlake, 19B0). 5 Figure 2. D i s t r i b u t i o n of structures with pH (Malvidin 3-glucoside: 25 C). AH+ = red cation; B = c o l o r l e s s carbinol base; C = c o l o r l e s s chalcone; A = blue quinoidal base. (From: Timberlake, 1980). 6 aging. McCloskey and Yengoyan (1981) a l s o noted a decrease i n anthocyanins but found 50 X present a f t e r f i v e years of aging. T h i s decrease corresponds t o a bathochromic s h i f t i n the wavelength of the c o l o r of the wine, from a p u r p l e - r e d t o an orange-brick hue. Tannin p o l y m e r i z a t i o n has been found t o i n c r e a s e as red wine ages (Peynaud, 1984). P h e n o l i c s which may p l a y an important r o l e i n the formation of complexes are c a t e c h i n s , f l a v a n - 3 , 4 - d i o l s and anthocyanins. The c a t e c h i n s and f l a v a n - 3 , 4 - d i o l s a re known t o form polymers y i e l d i n g condensed t a n n i n s , even i n the absence of anthocyanins. C a t e c h i n s as a group have a f l a v a n - 3 - o l s t r u c t u r e . ThB term c a t e c h i n i s a l s o used t o r e f e r t o a s p e c i f i c f l a v a n - 3 - o l with two hydroxy groups on the s i d e r i n g . Understanding the s t r u c t u r e of the polymers and f a c t o r s a f f e c t i n g t h e i r f o rmation has been the f o c u s of re c e n t r e s e a r c h . The ahthocyanin-phenolic l i n k a g e was suggested t o be one of two types known t o occur i n condensed t a n n i n polymers. D i r e c t l i n k a g e between anthocyanins and c a t e c h i n s or p r o c y a n i d i n s was thought t o occur (Jurd, 1967). Linkage i n the 4 p o s i t i o n of the anthocyanin t o the 8 or 6 of the f l a v o n o i d t o make a dimer or l a r g e r u n i t was co n s i d e r e d most l i k e l y . T h i s was supported by evidence t h a t s y n t h e t i c 4-aryl s u b s t i t u t e d f l a v y l i u m s a l t s a re almost u n a f f e c t e d by s u l f u r d i o x i d e (Timberlake and B r i d l e , 1968) j u s t as anthocyanin-tannin polymers are very r e s i s t a n t t o the d e c o l o r i z i n g e f f e c t s of s u l f u r d i o x i d e . A second type of polymer may be formed by an i n d i r e c t 7 l i n k a g e b B t w e e n phenol i c e and anthocyanins. Acetaldehyde produced through coupled a u t o - o x i d a t i o n of ethanol and p h e n o l i c s (Wildenradt and S i n g l e t o n , 1974), was shown by Timberlake and B r i d l e (1976) t o i n c r e a s e the amount of p o l y m e r i z a t i o n . Baranowski and Nagel (1983) showed t h a t the compound acted as a b r i d g e between the anthocyanin and c a t e c h i n molecules through the Baeyer condensation r e a c t i o n . In the absence of acetaldehyde, p o l y m e r i z a t i o n seemed t o be dependent upon the presence of f l a v y l i u m i o n i n s o l u t i o n . They found evidence t h a t p o l y m e r i z a t i o n c o u l d occur through both acetaldehyde b r i d g i n g and d i r e c t condensation between anthocyanins and t a n n i n s i n wine. Ribereau-Gayon e t a l t , (1983) concluded t h a t o x i d a t i o n r e a c t i o n s d u r i n g aging of red wines were important i n c a u sing a decrease i n f r e e anthocyanin content. Although s e v e r a l r e a c t i o n s may occur, the most important was the forma t i o n of acetaldehyde. Work done by Somers and Evans (1986) confirmed t h a t although two types of p h e n o l i c condensation r e a c t i o n s occur i n r e d wine, acetaldehyde-induced p o l y m e r i z a t i o n d i d not seem t o be as important as once b e l i e v e d . They found t h a t s i m i l a r aging r e a c t i o n s , i n c l u d i n g c o l o r a l t e r a t i o n , took p l a c e i n e i t h e r the presence of oxygen or under a n i t r o g e n atmosphere. The p h e n o l i c composition of the wine, as i n f l u e n c e d by v a r i e t y and v i n t n i n g technique were important i n determining the aging r e a c t i o n s . The d i g l y c o s i d i c anthocyanins d i f f e r from t h e i r monoglycosidic d e r i v a t i v e s i n s e v e r a l ways. Due t o the 8 lower pK of the d i g l y c o s i d e s , t h e i r c o n t r i b u t i o n t o the c o l o r of wine at pH v a l u e s of 3.0 t o 4.0 was found t o be l e s s than t h a t of the monoglycosides (Hrazdina, 1981). F u r t h e r , t h e r e i s some evidence t h a t anthocyanins d i f f e r i n s t a b i l i t y depending on t h e i r form (Jurd, 1972). S t u d i e s have shown t h a t the r a t e of anthocyanin d e s t r u c t i o n was pH dependent, and seemingly p r o p o r t i o n a l t o the amount of pigment i n the c o l o r l e s s c a r b i n o l base form. The two types of anthocyanins d i f f e r i n the r a t e t h a t they are degraded as wine ages, and i n t h e i r r e a c t i v i t y with s u l f u r d i o x i d e . The d i g l y c o s i d e s are not as l i k e l y t o form the chromen-4-d e r i v a t i v e due t o s t e r i c hinderance (Hrazdina, 1974). G a r o g l i o (1968) was a b l e t o d i s t i n g u i s h between d i g l u c o s i d e s and monoglucosides as the l a t t e r d i d not r a p i d l y p r e c i p i t a t e with acetaldehyde. A l s o , Van Buren e t a h (1974) found t h a t the second g l y c o s i d e moiety i n c r e a s e d the blueness of the anthocyanin, as determined by the t r i s t i m u l u s method. G e n e r a l l y , the 5 - g l y c o s i d i c group has an e l e c t r o n withdrawing e f f e c t on the r i n g s t r u c t u r e (Timberlake and B r i d l e , 1977). The changes i n c o l o r and i n p h e n o l i c composition of wines have important i m p l i c a t i o n s f o r wine q u a l i t y . In a study of young red wines, Somers and Evans (1974) found s i g n i f i c a n t c o r r e l a t i o n s between o v e r a l l wine q u a l i t y and c o l o r d e n s i t y , and the percent i o n i z e d anthocyanins and q u a l i t y . The i n f l u e n c e of s u l f u r d i o x i d e and pH on anthocyanin e q u i l i b r i a was found t o be r e s p o n s i b l e f o r these o b s e r v a t i o n s . F u r t h e r work (Somers e t al... , 1983) 9 i n d i c a t e d t h a t sul-fur d i o x i d e , i n i t s e f f e c t on i o n i z e d anthocyanins, was most important i n i n f l u e n c i n g c o l o r . Jackson et al._ , (1978) examined anthocyanin e q u i l i b r i u m s t a t e s and t h e i r r e l a t i o n s h i p with q u a l i t y parameters. They found t h a t simple c o l o r measurements of wine provided a u s e f u l i n d i c a t i o n of young red wine q u a l i t y . 10 i i QPIIMIZAHQN O p t i m i z a t i o n has been d e f i n e d as "the c o l l e c t i v e p r o c e s s of f i n d i n g the s e t of c o n d i t i o n s r e q u i r e d t o achieve the best r e s u l t from a g i v e n s i t u a t i o n " (Beveridge and Schechter, 1970). Informal o p t i m i z a t i o n i s o f t e n guided by a broad base of experience and i n depth knowledge of t h B area. However i n the absence of such e x p e r t i s e , or i n cases where f a c t o r s may i n t e r a c t , formal or d e f i n e d methods of o p t i m i z a t i o n should be used. The development of chromatographic methods f o r high p r e s s u r e l i q u i d chromatographic (HPLC) systems through the use of formal o p t i m i z a t i o n techniques has become a well accepted procedure (Costanzo, 19B6). The goal of these o p t i m i z a t i o n s has been t o o b t a i n the best p o s s i b l e s e p a r a t i o n or r e s o l u t i o n of components of a mixture i n a minimum of time through the m o d i f i c a t i o n of the chromatographic system. R e s o l u t i o n of compounds i n HPLC can be a f f e c t e d by t h r e e independent f a c t o r s , as shown i n equation 1 (Snyder and K i r k l a n d , 1979). Rs • 0.25 * (<* - 1) /<* • (N °-=) * (K' / K' + I) (1) Rs = r e s o l u t i o n f a c t o r N = column p l a t e number, or e f f i c i e n c y f a c t o r 11 K' => s o l u t e c a p a c i t y f a c t o r oi = s e l e c t i v i t y f a c t o r < k ' 2 / k ' i ) f o r two peaks. To o b t a i n the best r e s o l u t i o n between peak p a i r s , a l l t h r e e f a c t o r s should be optimized t o g e t h e r . However, m o d i f i c a t i o n t o the s o l v e n t s t r e n g t h has been found t o p r o v i d e a l a r g e change i n r e s o l u t i o n f o r a small amount of work, due t o i t s e f f e c t on K' (Snyder and K i r k l a n d , 1979). T h i s w i l l o f f e r e f f e c t i v e change i n R s i n the range 1 < K' < 10, s i n c e R a v a r i e s d i r e c t l y with the r a t i o K' / K' + 1 . The s e l e c t i v i t y f a c t o r , o< has a l s o been used widely t o improve R a. T h i s has been accomplished most o f t e n by mobile phase m o d i f i c a t i o n , i n c l u d i n g the use of g r a d i e n t e l u t i o n . Wallace (1983) showed t h a t small changes i n mobile phase composition r e s u l t e d i n r e l a t i v e l y l a r g e improvements i n r e s o l u t i o n of v a n i l l i n and r e l a t e d phenol compounds. Snyder (1978) c l a s s i f i e d l i q u i d chromatographic s o l v e n t s i n t o groups based on p o l a r i t y and s e l e c t i v i t y (the a b i l i t y of a g i v e n s o l v e n t t o s e l e c t i v e l y d i s s o l v e one compound as opposed t o a n o t h e r ) . In g r a d i e n t e l u t i o n s , s o l v e n t s t r e n g t h i s c o n t i n u a l l y i n c r e a s e d t o improve r e s o l u t i o n , peak shape and a n a l y s i s time with complex samples. G l a j c h gt a l . . (1980) used a l t e r a t i o n s i n s o l v e n t s t r e n g t h and s e l e c t i v i t y t o o p t i m i z e HPLC s e p a r a t i o n s . The most widely used HPLC methods have been r e v e r s e phase (RP) column s e p a r a t i o n s . These methods are based on passage of compounds through a nonpolar hydrocarbon bonded phase column with an aqueous mobile phase c o n t a i n i n g m i s c i b l e o r g a n i c m o d i f i e r s (Poole and Schuette, 19B4). Reverse phase HPLC (RP-HPLC) has been the most s u c c e s s f u l method of s e p a r a t i n g complex groups of p h e n o l i c compounds. Because they are p o l a r compounds, some p h e n o l i c s may adhere i r r e v e r s i b l y t o h y d r o p h i l i c p o l a r phase columns. Andersen and Pedersen (19B3) separated 11 common aglycone p h e n o l i c compounds i n l e s s than 25 minutes u s i n g RP-HPLC. Vande C a s t e e l e et a L (1983) separated a mixture of 41 p h e n o l i c s and r e l a t e d compounds i n approximately t h i r t y minutes u s i n g RP-HPLC. Ret e n t i o n of s o l u t e s d u r i n g RP-HPLC i s a f u n c t i o n of sample h y d r o p h o b i c i t y while the s e l e c t i v i t y of the s e p a r a t i o n r e s u l t s almost e x c l u s i v e l y from i n t e r a c t i o n s between the mobile phase and the column. T h i s s e l e c t i v i t y can e a s i l y be changed by the use of o r g a n i c m o d i f i e r s . Methanol and a c e t o n i t r i l e are the most commonly used o r g a n i c m o d i f i e r s (Hardin and S t u t t e , 1984). S o l v e n t s used f o r RP-HPLC s e p a r a t i o n s are a c i d i f i e d t o supress i o n i z a t i o n of the a c i d groups (Wallace, 19B3). Numerous procedures are a v a i l a b l e f o r the s y s t e m a t i c o p t i m i z a t i o n of RP s e p a r a t i o n s . These have been based on t h e o r e t i c a l models, which p r e d i c t r e t e n t i o n as a f u n c t i o n of chromatographic f a c t o r s ( Q l a j c h et a L , 1980) or on s t a t i s t i c a l or s e q u e n t i a l search t e c h n i q u e s (Sachok et a l . . , 1980). In some cases the former method has r e q u i r e d the 13 development o-f e x t e n s i v e data bases (Jinno and Kawasaki , 1984). Spendley et a l . . (1962) developed a v e r s a t i l e m u l t i d i m e n s i o n a l s e q u e n t i a l search a l g o r i t h m known as Simplex O p t i m i z a t i o n . The a l g o r i t h m d i r e c t s the experimental c o n d i t i o n s away from those which g i v e poor r e s u l t s , towards more f a v o r a b l e c o n d i t i o n s . A simplex i s a geometric f i g u r e d e f i n e d by n + 1 e q u i d i s t a n t p o i n t s or v e r t i c e s , where n i s equal t o the number of f a c t o r s i n v o l v e d i n o p t i m i z a t i o n . T h e r e f o r e an e q u i l a t e r a l t r i a n g l e i s the simplex i n an o p t i m i z a t i o n i n v o l v i n g two f a c t o r s . T h i s concept can r e a d i l y be extended t o t h r e e or more dimensions although i t soon becomes d i f f i c u l t t o v i s u a l i z e such f i g u r e s . The f a c t o r s t h a t are i n c l u d e d i n the o p t i m i z a t i o n can be determined by p r e l i m i n a r y f a c t o r i a l experimentation or by the r e s e a r c h e r s judgement. Both upper and lower l i m i t s of the f a c t o r s are then s e t as determined by p h y s i c a l , economic or p r a c t i c a l c o n s t r a i n t s . The v e r t i c e s , c h a r a c t e r i z e d by l e v e l s of the f a c t o r s chosen w i t h i n upper and lower l i m i t s , are then ev a l u a t e d by a pre-determined c r i t e r i o n . Based on " r u l e s " , the experimentation c o n t i n u e s u n t i l the optimum i s reached. A new procedure, C e n t r o i d Mapping O p t i m i z a t i o n (Aishima and Nakai, 1986) o f f e r s improved e f f i c i e n c y i n r e a c h i n g the g l o b a l optimum of a response s u r f a c e . The o p t i m i z a t i o n begins with a Spendley simplex. A f t e r the i n i t i a l e v a l u a t i o n of the v e r t i c e s , the worst vertex i s d i s c a r d e d and a c e n t r o i d i s c a l c u l a t e d as an average of the remaining v e r t i c e s . T h i s means t h a t the c e n t r o i d moves i n the space d e f i n e d by the best and next best responses. A f t e r e v a l u a t i o n of the c e n t r o i d , a l l data are mapped on two dimensional response scattergrams t o a i d , by v i s u a l i z a t i o n , i n the l o c a t i o n of the optimum. P o i n t s are l i n k e d and new ranges f o r the f a c t o r s are chosen a c c o r d i n g t o the method of Nakai et a l t (1984). The mapping a l g o r i t h m d i v i d e s f a c t o r l e v e l s i n t o small and l a r g e l i m i t s based on responses, with small l i m i t s being r e s t r i c t e d t o good responses. L e v e l s i n s i m i l a r groups are l i n k e d and w i l l tend t o converge on the l o c a t i o n of the optimum. T h i s sequence of simplex and c e n t r o i d searches, f o l l o w e d by mapping, i s continued u n t i l the f a c t o r range appears t o narrowly c o n t a i n the optimum. At t h i s p o i n t , a l l f a c t o r l e v e l s are s i m u l t a n e o u s l y s h i f t e d w i t h i n the narrow range toward the optimum and e v a l u a t e d . S h i f t i n g c o n t i n u e s u n t i l t he response begins t o d e c l i n e . In work done with mathematical models, Aishima and Nakai (1986) have demonstrated the C e n t r o i d Mapping O p t i m i z a t i o n was more e f f i c i e n t i n r e a c h i n g a g l o b a l optimum i n complex models c o n t a i n i n g m u l t i p l e l o c a l optima than the Mapping Simplex O p t i m i z a t i o n of Nakai gt a L (1984). With a s i m p l e r model, no s i g n i f i c a n t d i f f e r e n c e i n e f f i c i e n c y between the two methods was found. The s e l e c t i o n of a range f o r the c e n t r o i d search a v o i d s the r e f l e c t i o n and c o n t r a c t i o n a l g o r i t h m s of r e g u l a r simplex o p t i m i z a t i o n . There-fore, time i s not wasted on experiments which may lead t o a l o c a l i z e d optimum. O p t i m i z a t i o n of a chromatographic system i s o n l y p o s s i b l e i f the d i f f e r e n c e between a chromatogram with good r e s o l u t i o n of peaks and one with poor r e s o l u t i o n can be determined. The problem of e v a l u a t i n g a s e p a r a t i o n of compounds i n c r e a s e s as the complexity of the sample t o be analysed i n c r e a s e s , e s p e c i a l l y i f a l l peaks are c o n s i d e r e d t o be e q u a l l y important. Morgan and Deming (1975) used a chromatographic response f u n c t i o n (CRF) t o judge the q u a l i t y of gas chromatographic s e p a r a t i o n s as k CRF = ^ In (P*) (2) where P± i s a measure of peak s e p a r a t i o n of the i t h p a i r of peaks with k t o t a l p a i r s . The measure of peak s e p a r a t i o n used was most u s e f u l when adjacent peaks were s i m i l a r i n h e i g h t ( G l a j c h gt a l . . , 1980). The degree of s e p a r a t i o n between two peaks may be d e f i n e d by t h e i r r e s o l u t i o n . B e r r i d g e (1982) m o d i f i e d the CRF t o i n c l u d e a r a p i d method of e s t i m a t i n g r e s o l u t i o n between adjacent peak as R = 2 ( t 2 - t i ) / ( M i + w2) (3) where t i s the r e t e n t i o n time and w i s the peak width at b a s e l i n e . Column p l a t e number can be determined from Gaussian peaks by (4) 16 (t/w) 2 or 2 (ht/A) = where h i s peak h e i g h t and A i s area. Combining these equations, peak width can be estimated as w • <4A / 2 ) °- = * h. (5) However, s i n c e chromatographic peaks are r a r e l y symmetrical, peak base width w i l l be underestimated by the equation. To account f o r peak asymmetry, width was c a l c u l a t e d as w - 2A/h. (6) 17 C . MULTIVARIATE ANALYSIS M u l t i v a r i a t e s t a t i s t i c a l t echniques are those which are a p p l i e d when one or more independent and one or more dependent v a r i a b l e s are being c o n s i d e r e d s i m u l t a n e o u s l y (Massart e t a l t , 1978). Each measured v a r i a b l e d e f i n e s i t s own dimension, which prevents man (with h i s i n a b i l i t y t o deal with g r e a t e r than t h r e e dimensions) from r e c o g n i z i n g groupings i n the data or e l i m i n a t i n g s u p e r f l u o u s i n f o r m a t i o n . Techniques which reduce the number of dimensions while r e t a i n i n g the i n f o r m a t i o n i n a l l the data, or are capable of f i n d i n g p a t t e r n s i n m u l t i p l e dimensions are extremely u s e f u l . A n a l y t i c a l chemists have been u s i n g p a t t e r n r e c o g n i t i o n t e c h n i q u e s f o r m u l t i v a r i a t e a n a l y s e s s i n c e the l a t e 1960's to f a c i l i t a t e the i n t e r p r e t a t i o n of complex data s e t s (Kryger, 1981). Recently these techniques have been used s u c c e s s f u l l y f o r c l a s s i f i c a t i o n of v a r i o u s f o o d s t u f f s a c c o r d i n g t o q u a l i t y , o r i g i n and f l a v o u r c h a r a c t e r i s i t i c s ( E l l i s et a L , 19B5; Smeyers-Verbeke et a L , 1977). The development of chromatographic equipment capable of high r e s o l u t i o n and s e p a r a t i o n , and computers t o a i d i n the a c q u i s i t i o n of data, have enabled s c i e n t i s t s t o o b t a i n huge i n f o r m a t i o n s e t s . T h i s i n t u r n has c r e a t e d demands on the techni q u e s a v a i l a b l e f o r d e f i n i n g s t r u c t u r e or f i n d i n g IB order i n such l a r g e data s e t s , s i P r i n c i p a l . Cgmrjonent A n a l y s i s P r i n c i p a l component a n a l y s i s (PCA) i s a technique which condenses mul t i d i m e n s i o n a l d a t a , d e s c r i b i n g n s u b j e c t s , i n t o a s m a l l e r number of important combinations or p r i n c i p a l components (PC). L i n e a r combinations of the o r i g i n a l v a r i a b l e s are formed, such t h a t the v a r i a n c e between the s u b j e c t s (and a t t r i b u t e d t o the f i r s t PC) i s as l a r g e as p o s s i b l e . The next PC chosen i s again a l i n e a r combination of the remaining v a r i a b l e s which accounts f o r the maximum amount of v a r i a n c e w i t h i n the data, but i n a d d i t i o n , i t must be u n c o r r e l a t e d with the f i r s t PC. T h i s process c o n t i n u e s on such t h a t each s u c c e s s i v e PC accounts f o r l e s s v a r i a n c e than the p r e v i o u s one and together t h e i r sum i s equal t o the sum of v a r i a n c e s of the o r i g i n a l data. A small number of PC w i l l o f t e n c o n t a i n a l a r g e p r o p o r t i o n of the v a r i a n c e , or the in h e r e n t i n f o r m a t i o n , of a l l the v a r i a b l e s . The p r i n c i p a l components can be used t o g r a p h i c a l l y i l l u s t r a t e the s e p a r a t i o n of the s u b j e c t s i n t o groups. Kwan and Kowalski (1980) used PCA t o i d e n t i f y compounds which were found t o be r e l a t e d t o o v e r a l l q u a l i t y of wines. Clapperton and P i g g o t t (1979) d i f f e r e n t i a t e d between a l e s and l a g e r s on the b a s i s of d e s c r i p t i v e f l a v o u r a n a l y s i s . Peppard (1985) d e s c r i b e d the use of PCA i n making between and w i t h i n brand comparisons, b). Stepwise D i s c r i m i n a n t A n a l y s i s Stepwise d i s c r i m i n a n t a n a l y s i s (SDA) i s a s u p e r v i s e d c l a s s i f i c a t i o n technique i n which parameters are s e l e c t e d , one step at a time. A v a r i a b l e i s chosen which o p t i m a l l y s e p a r a t e s the samples i n t o t h e i r pre-determined c a t e g o r i e s , or , has the best d i s c r i m i n a t i n g power. The next v a r i a b l e chosen, when taken i n t o c o n s i d e r a t i o n with the f i r s t , w i l l improve the d i s c r i m i n a t i o n the most, and so on. The sequence i s continued u n t i l a predetermined l e v e l of s i g n i f i c a n c e r e g a r d i n g the s e p a r a t i o n has been achieved, or a maximum number Of s t e p s have been taken. SDA was a p p l i e d by Powers and K e i t h (196B) t o c l a s s i f y c o f f e e i n t o f o u r f l a v o u r c a t e g o r i e s u s i n g r a t i o s of gas chromatographic peak h e i g h t s as v a r i a b l e s . In the same manner, potato c h i p s were c l a s s i f i e d a c c o r d i n g t o headspace v o l a t i l e s . Pham and Nakai (1984) used SDA t o c l a s s i f y cheeses i n t o m i l d , medium, o l d and e x t r a o l d ages u s i n g s e l e c t e d peaks from a chromatogram of water e x t r a c t s . They noted t h a t SDA allowed c l a s s i f i c a t i o n of an unknown by c a l c u l a t i n g a s c o r e from the d i s c r i m i n a n t f u n c t i o n . A l s o , through the c a n o n i c a l two dimensional p l o t , a v i s u a l i z a t i o n of how c l o s e l y an unknown sample f i t i n t o the-group c o u l d be determined. B e r t u c c i o l i e t a l . . (1982) were ab l e t o c l a s s i f y wines i n t o q u a l i t y groups u s i n g SDA. It was f e l t t h a t some r o u t i n e components chosen by SDA were s u i t a b l e f o r determining the sensory q u a l i t y of the wines. Several data s e t s of gas chomatographic v a r i a b l e s , a n a l y t i c a l v a r i a b l e s and v a r i o u s combinations of the two were used i n the d i s c r i m i n a t i o n . Many v a r i a b l e s from both s e t s were r e q u i r e d •for the c l a s s i f i c a t i o n , i n accordance with the complex sensory response evoked by the wines. Cabezudo et al.. (1983) used SDA t o c a t e g o r i z e e i g h t types of Spanish wine. From a l a r g e number of v a r i a b l e s d e r i v e d from r o u t i n e gas and l i q u i d chromatographic a n a l y s e s , a few were s e l e c t e d which s u c c e s s f u l l y separated the wines. I t was f e l t t h a t t h i s was f i r s t step i n s t r e a m l i n i n g a n a l y t i c a l procedures f o r examination of other wines. Moret et a h (1984) use SDA t o d i s c r i m i n a t e between t h r e e Venetian wines of s e v e r a l v i n t a g e s . Attempts were made t o c l a s s i f y the wines on the b a s i s of v a r i e t y without regard f o r age d i f f e r e n c e s . Parameters used i n c l u d e d v a r i o u s metals , pH, a c i d i t y , ash, and a l k a l i n i t y . Two of the v a r i e t i e s were not completely separated, i n d i c a t i n g t h a t the parameters examined, d i d not have s u f f i c i e n t d i s c r i m i n a t i n g power. £l C l u s t e r Analyses Another approach t o deal with m u l t i v a r i a t e data s e t s i s t o look f o r u n d e r l y i n g s t r u c t u r e or order i n the data by c l u s t e r i n g them. C l u s t e r i n g i s a numerical taxonometric technique which c l a s s i f i e s o b j e c t s i n t o groups by c h a r a c t e r i z a t i o n of t h e i r q u a l i t a t i v e or q u a n t i t a t i v e p r o p e r t i e s . I t i s done " b l i n d " , or with no p r i o r knowledge of which o b j e c t s belong t o which groups. Because no assumptions are made about the s t r u c t u r e of the groups, i t can p r o v i d e an in f o r m a l method of a s s e s s i n g d i m e n s i o n a l i t y , i d e n t i f y i n g o u t l i e r s and suggesting r e l a t i o n s h i p s i n the data (Aishima, i n p r e s s ) . There are two methods by which c l u s t e r s may be obtained. A l l o b j e c t s may be con s i d e r e d as a s i n g l e c l u s t e r , and may then be d i v i d e d up i n t o small groups on the b a s i s of d i s s i m i l a r i t i e s , o r , each o b j e c t may be c o n s i d e r e d as an i n d i v i d u a l c l u s t e r and they may be combined i n a s e q u e n t i a l manner. Measures of s i m i l a r i t y between two p a t t e r n v e c t o r s a re made on the b a s i s of E u c l i d e a n d i s t a n c e . When two p a t t e r n s are very s i m i l a r or d i f f e r e n t , the d i s t a n c e w i l l be small or l a r g e , r e s p e c t i v e l y . There are no s t a t i s t i c a l t e s t s of the "goodness" of the c l u s t e r a n a l y s i s , but i t s a p p r o p r i a t e n e s s must be judged by the r e s e a r c h e r . It has been recommended t h a t p a t t e r n r e c o g n i t i o n problems be approached from more than one d i r e c t i o n ( i . e . u s i n g more than one m u l t i v a r i a t e technique) (Massart and Kaufman, 19B3). Aishima (19B2) u s i n g m u l t i p l e r e g r e s s i o n a n a l y s i s , found ten peaks which d i s t i n g u i s h e d between genuine and semi-fermented soy sauces. Using the ten peaks i n c l u s t e r a n a l y s i s r e s u l t e d i n the grouping of s i m i l a r samples. Moll e t a l . . (1981) used PCA, c l u s t e r a n a l y s i s , and SDA f o r p r e d i c t i n g the o r g a n o l e p t i c s t a b i l i t y of beers from chemical a n a l y s e s . Van der Voet et a K (19B4) used c l u s t e r a n a l y s i s and SDA t o chose chemical f e a t u r e s which d i f f e r e n t i a t e d between Bordeaux and Bourgogne wines. 22 111-. METHODS AND MATERIALS B i WINES Two types of v a r i e t a l h y b r i d red wines, made by the s t a f f of the Food P r o c e s s i n g S e c t i o n , A g r i c u l t u r e Canada Research S t a t i o n (Summerland, B.C.), were chosen f o r t h i s work. The f i r s t was Marechal Foch (Kuhlmann 188 - 2) and the second was deChaunac ( S e i b e l 9549). Wines from the v i n t a g e s 1980 through 1983 were used. For Foch, two b o t t l e s from the 1980, two from the 19B1, f i v e from the 1982 and th r e e from the 1983 v i n t a g e were used. One b o t t l e from the 1980, two from the 1981, one from the 1982 and two from the 1983 v i n t a g e of deChaunac were used. These were a l l produced by a standard method of destemming and c r u s h i n g , f o l l o w e d by the a d d i t i o n of 35 ppm s u l f u r d i o x i d e and an inoculum of Saccharomyctts cerevisaa Y5. Skin c o n t a c t was maintained u n t i l adequate c o l o r i n the must was achieved. T h i s v a r i e d from two t o f o u r days f o r Foch wines and f i v e t o e i g h t days f o r deChaunac wines, at which p o i n t the must was pressed. Sucrose was added, i f necessary, t o i n c r e a s e the sugar content of the o r i g i n a l j u i c e t o 20 '/.. Fermentation was allowed t o con t i n u e u n t i l the r e s i d u a l sugar l e v e l s were l e s s than 0.5 '/.. A f t e r r a c k i n g , f i n i n g and c o l d s t a b i l i z a t i o n , the wines were adju s t e d t o 50 ppm f r e e s u l f u r d i o x i d e , s t e r i l e f i l t e r e d i n t o b o t t l e s and s t o r e d i n the dark at 13 °C. The wines, upon r e c e i p t i n the l a b o r a t o r i e s at the U n i v e r s i t y of B r i t i s h Columbia, were t r a n s f e r r e d i n t o 8 or 13 mL crimped cap v i a l s , which were then s e a l e d with t e f l o n coated rubber caps a f t e r the a i r s p a c e was f l u s h e d with n i t r o g e n gas f o r approximately 5 seconds. The v i a l s were encased i n aluminum f o i l t o l i m i t exposure t o l i g h t and kept at 4 °C u n t i l needed. i i HPLC CHEMICALS AND INSTRUMENTATION G l a s s - d i s t i l l e d HPLC grade methanol (BDH Chemicals Ltd.) and a c e t o n i t r i l e ( M a l l i n c k r o d t Inc.) were used f o r a l l chromatographic runs d u r i n g the o p t i m i z a t i o n procedure. D i s t i l l e d water and HPLC grade a c e t i c a c i d (Baker Chemical Co.) were used t o make up the IV. a c e t i c a c i d s o l u t i o n (pH= 2.8). A l l s o l v e n t s were f i l t e r e d u s i n g M i l l i p o r e f i l t r a t i o n apparatus. Methanol and a c e t o n i t r i l e s o l v e n t s were f i l t e r e d through 0.45um polycarbonate f i l t e r s and the IX a c e t i c a c i d was f i l t e r e d through Mi 11ipore 0.45 urn HA type f i l t e r s . During chromatographic runs, a f i n e stream of helium was bubbled through the s o l v e n t s t o degas them. A S p e c t r a - P h y s i c s 8100 HPLC and 8400 v a r i a b l e wavelength d e t e c t o r (Spectra - P h y s i c s , Santa C l a r a , CA.) were used. Wavelengths of 254, 280 and 320 nm were used f o r an a l y s e s . A S p e c t r a - P h y s i c s 4100 computing i n t e g r a t o r was used t o c a l c u l a t e peak area and h e i g h t . A r e v e r s e phase column (250 x 4.6 mm, I.D.) packed with S u p e l c o s i l L C i s (5um) (Supelco, Inc., Pennsylvania) was used f o r a l l chromatographic runs. The column was p l a c e d i n s i d e a LC-23 Column Heater, r e g u l a t e d by a LC-22 24 Temperature C o n t r o l l e r ( B i o a h a l y t i c a l Systems). A 20 AJL sample loop was used -for p r e c i s e volume i n j e c t i o n . Q i QENIRQID MAPPING? OPTIMIZATION e i Procedure The C e n t r o i d Mapping O p t i m i z a t i o n (CMO) program of Aishima and Nakai (1986), w r i t t e n i n b a s i c f o r a Sharp PC-1500 Pocket Computer (Sharp Corp., Osaka, Japan) was used t o f i n d the HPLC c o n d i t i o n s which o f f e r e d the best r e s o l u t i o n of wine p h e n o l i c compounds. The CMO program i n c l u d e d the Mapping and Simultaneous S h i f t (Nakai et a l . . , 1984) a l g o r i t h m s . Chromatograms obtained f o r each vertex were ev a l u a t e d on the b a s i s of the two c r i t e r i a of r e s o l u t i o n between adjacent peaks and the t o t a l number of peaks r e s o l v e d . A r a p i d method used f o r e v a l u a t i n g r e s o l u t i o n (Rs) was Rs = 2 (t=> - tx) / (Wx + w2) (7) where t i s r e t e n t i o n time and w i s peak width at the b a s e l i n e . The S p e c t r a - P h y s i c s Sp 4100 i n t e g r a t o r was used t o c a l c u l a t e h e i g h t and area of each peak on the chromatogram. These were used t o c a l c u l a t e the width v a l u e of peaks. R e t e n t i o n time was determined i n 0.01 second i n t e r v a l s . The chromatographic response f a c t o r (CRF) used was L CRF = ,Z. R s * L (8) where L i s the t o t a l number of peaks d e t e c t e d . The CRF r e q u i r e d no assumptions about the number of peaks expected, the importance of any p a r t i c u l a r peak or the importance of r e s o l u t i o n r e l a t i v e t o the number of peaks r e s o l v e d . l t was modified from the CRF used by B e r r i d g e (1982) by the d e l e t i o n of the terms which allowed the t o t a l a n a l y s i s time t o i n f l u e n c e the CRF. In t h i s work, time was i n c l u d e d as a f a c t o r t o be v a r i e d with each v e r t e x . The product of the two c r i t e r i a was c a l u l a t e d s i n c e an i n c r e a s e i n both was the a b j e c t i v e of the o p t i m i z a t i o n . M u l t i p l i c a t i o n of c r i t e r i a should be used when the g o a l s of these c r i t e r i a are n o n c o n f 1 i c t i n g ( i . e . both are t o be maximized or both minimized). The wine samples used f o r the HPLC o p t i m i z a t i o n were obtained from a s i n g l e b o t t l e of a Marechal Foch of the 1983 v i n t a g e which had aged f o r approximately 1.5 years. For each chromatographic run, a wine sample was withdrawn from a s e a l e d v i a l u s i n g a s y r i n g e . The samples were then f i l t e r e d through a M i l l i p o r e Swinny f i t t e d with a 0.45 urn f i l t e r . At l e a s t two and i n some cases t h r e e chromatograms were obtained f o r each v e r t e x . O p t i m i z a t i o n was completed w i t h i n t h r e e months, duri n g which time changes i n the wine sample were minimized due t o the p r e c a u t i o n s taken t o l i m i t l i g h t and oxygen exposure and by s t o r a g e at 4 °C. b)_ QBtimizat^on F a c t o r s The f a c t o r s chosen f o r o p t i m i z a t i o n and l i m i t s f o r these f a c t o r s were s e t as f o l l o w s : 1. the c o n c e n t r a t i o n of a c e t o n i t r i l e i n the l e s s p o l a r mobile phase v a r i e d between 0.0 and 25.0 X; 2. the f i n a l c o n c e n t r a t i o n of the l e s s p o l a r mobile phase v a r i e d between 90.0 and 100.0 */. of the t o t a l ; 3. the flow r a t e of the mobile phase v a r i e d between 0.5 and 1.5 mL per minute; 4. the time of the run v a r i e d between 45.0 and 150.0 minutes; 5. the column temperature v a r i e d between 25.0 and 40.0 °C. The l i m i t s f o r flow r a t e and column temperature were d e f i n e d by p r a c t i c a l l i m i t s of p r e s s u r e and c o n t r o l , r e s p e c t i v e l y . The time l i m i t , or r a t e of change of s o l v e n t s t r e n g t h was s e t t o a l l o w s u f f i c i e n t s e p a r a t i o n of the numerous p h e n o l i c compounds i n the wine. From p r e v i o u s e x p e r i e n c e and l i t e r a t u r e , i t was known t h a t the s o l v e n t s t r e n g t h of the mobile phase would have t o encompass a wide range, from very p o l a r (100 V. a c i d i f i e d water) t o l e s s p o l a r (10-0 7. a c i d i f i e d water). A percentage of a c e t o n i t r i l e i n the s t r o n g s o l v e n t was i n c l u d e d t o decrease the high back p r e s s u r e caused by methanol. In a d d i t i o n , the s o l v e n t p o l a r i t y , and r e l a t i v e a b i l i t y t o i n t e r a c t as a proton - donor, proton - acceptor or d i p o l e d i f f e r s f o r methanol and a c e t o n i t r i l e ( G l a j c h gt a l . . , 1980) and c o u l d i n f l u e n c e compound r e t e n t i o n on the column. WINE ANALYSES a l l i t r a t a b l e A c i d i t y and p_H The t i t r a t a b l e a c i d i t y (TA) of the wines was determined a c c o r d i n g t o the method recommended by Amerine and Qugh (1974). F i v e mL of wine were d i l u t e d with 75 mL of degassed d i s t i l l e d water and t i t r a t e d with 0.10 N NaOH t o an e l e c t r o c h e m i c a l endpoint of pH 8.20 (+/- 0.02). T r i p l i c a t e d e t e r m i n a t i o n s were done f o r each wine. The r e s u l t s were averaged and converted t o grams t a r t a r i c a c i d per 100 mL wine by the f o l l o w i n g equation T a r t a r i c a c i d = * N * 100 * 75 / 1000 x v w l „ . (9) = the volume i n mL of NaOH t i t r a t e d N - n o r m a l i t y of NaOH used V N t n . = the volume i n mL of wine used The pH of each wine at 25 °C was obtained i n t r i p l i c a t e . fell Organic A c i d Determination Q u a n t i t a t i o n of or g a n i c a c i d s of wine were determined u s i n g i o n su p p r e s s i o n r e v e r s e phase HPLC. M a l i c , l a c t i c , t a r t a r i c , a c e t i c , c i t r i c , o x a l i c , s u c c i n i c and p r o p i o n i c a c i d s were determined. 1L2. Sample P r e p a r a t i o n Sample p r e p a r a t i o n was c a r r i e d out by using a Waters Sep-Pak® C i s c a r t r i d g e . The c a r t r i d g e was prepared by f l u s h i n g with 3 mL of HPLC grade methanol, f o l l o w e d by 10 mL of d i s t i l l e d water. Two mL of wine were d i l u t e d with 3 mL of a 0.01 M potassium phosphate b u f f e r pH B.0. The b u f f e r was made us i n g 0.01 M potassium phosphate monobasic^and d i b a s i c . One mL of the d i l u t e d sample was a p p l i e d t o the c a r t r i d g e and the e l u a n t c o l l e c t e d . The c a r t r i d g e was washed with one mL of b u f f e r and the e l u a n t was again c o l l e c t e d , combined with the p r e v i o u s e l u a t e , vortexed f o r 30 seconds and analysed immediately. D u p l i c a t e i s o l a t i o n s were done f o r each wine. l i . i l HPLC System The mobile phase used was 27. NH*H2P0* ad j u s t e d t o pH 2.4 with phosphoric a c i d (Shaw and Wilson, 1981) and f i l t e r e d through 0.45 jam f i l t e r s . During chromatographic runs, a f i n e stream of helium gas was bubbled through the mobile phase t o degas i t . Each run took 16 minutes. A flow r a t e of 1.00 mL/minute and a temperature of 25 °C was used. D u p l i c a t e d e t e r m i n a t i o n s were done f o r each e x t r a c t i o n . A f t e r every ten runs, the column was thoroughly washed with 807. methanol and B q u l i b r a t e d f o r the next run. The S p e c t r a - P h y s i c s HPLC equipment and r e v e r s e phase column p r e v i o u s l y d e s c r i b e d were used. A c i d s were d e t e c t e d at 216 nm. A g e n t l e stream of n i t r o g e n gas was flowed through the d e t e c t o r p r e v e n t i n g ozone accumulation i n the immediate v i c i n i t y of the d e t e c t i o n flow c e l l . A l i n e a r equation d e r i v e d from a standard curve of each a c i d was used t o q u a n t i f y t h a t a c i d i n the wine sample. E l §B§ctrop_hotometric E v a l u a t i o n s A s p e c t r o p h o t o m e t r i c examination of the wines was c a r r i e d out, a c c o r d i n g t o methods developed by Somers and Evans (1977). A Unicam SP 800 spectrophotometer was used t o o b t a i n a l l measurements. A l l chemicals used were reagent grade. The f o l l o w i n g procedures were used f o r a l l wines. Procedure 1. A p o r t i o n of wine was f i l t e r e d through a 0.45 iim M i l l i p o r e f i l t e r and 0.33 mL was t r a n s f e r r e d t o a 1.0 mm p a t h l e n g t h c u v e t t e u s i n g a 1.0 mL p i p e t e and absorbance at 420 and 520 nm was measured. Then 0.5 mL of sodium m e t a b i s u l f i t e (207. w/v) was added t o the c u v e t t e . F o l l o w i n g thorough mixing f o r one minute, the absorbance at 520 nm was determined. Procedure 2. To a new 2.0 mL sample of wine, 20 mL of a 10 "/. acetaldehyde was added and the mixture was vortexed f o r approximately 15 seconds. The mixture was h e l d a t about 25 °C f a r 45 minutes, and then absorbance at 520 nm was determined with a 1.0 mm pathlength c u v e t t e . Procedure 3. To 5 mL of a 1.0 M HCl s o l u t i o n , 0.1 mL of wine was added, and the mixture was vortexed f o r approximately 15 seconds. A f t e r t h r e e hours at room temperature, the absorbances a t 520 and 280 nm were determined with a 10 mm pathlength quartz c u v e t t e . D u p l i c a t e samples f o r each wine were used. These s i x absorbance r e a d i n g s , a f t e r c o r r e c t i o n t o a 10 mm pathlength and f o r d i l u t i o n where necessary, were used t o determine 11 a n a l y t i c a l c h a r a c t e r i s t i c s of each wine, as shown i n Table 1. d_2 FVP APgiyyi.nyl g y r r o l i d i n e l Index The PVP Index, or the percentage of polymeric anthocyanins were determined (Ribereau-Bayon et , Table 1. C o l o r and composition of wine c a l c u l a t e d from absorbance r e a d i n g s . " C o l o r d e n s i t y C o l o r hue T o t a l anthocyanins <mg/ L) Ionized anthocyanins (mg/L) 7. i o n i z a t i o n of anthocyanins (o< ) e* + SQ 2 bound anthocyanins (o( ' ) T o t a l p h e n o l i c s (Abs. u n i t s ) Chemical age A 420 + A 520 A 420 / A 520 20(A 520,HCl - 5/3 (A 520,SO2)> 20(A 520 - A 520, S0 2) 100 (Ionized antho. / T o t a l antho.> 100 ( A 520 A CH3CHO - A 52Q A SO2 > T o t a l anthocyanins A 2B0, HCl - 4 A 520, SOs- / A 520,CH3CH0 Free S0=» (mg/L) (SO 2 + S O 3 H ) Molecular S 0 2 (ug/L) ( S O 2 only) 3.84 ( - <X ) / Free S 0 2 * pH f a c t o r From : Somers, T. C. and Evans, M. E., 1977. at pH 4.0 - the pH f a c t o r = 6; at pH 3.4 - the pH f a c t o r • 23. 31 1983). Seven mL of an aqueous s l u r r y of PVP (SO V.) was f i l t e r e d i n a 15.0 mL g l a s s f i l t e r f u n n e l . Approximately 15 mL of water were used t o wash the PVP r e s i d u e . F i v e mL of wine was then a p p l i e d t o the PVP. Sugars and a c i d s were washed from the PVP r e s i d u e with 10 mL of water. Free anthocyanins were s e l e c t i v e l y e l u t e d with 30 mL of 70:30:1 ( w a t e r : e t h a n o l : h y d r o c h l o r i c a c i d ) and c o l l e c t e d . The anthocyanin s o l u t i o n was evaporated t o dryness with a r o t o r y evaporator at 35 °C. A 12 V. ethanol s o l u t i o n was added to the anthocyanin s o l u t i o n t o b r i n g the volume back t o 5 mL. The pH of the sample was ad j u s t e d back t o the pH of the o r i g i n a l wine (+/- 0.02) with 0.1 N NaOH. Absorbance at 520 nm of the f r e e anthocyanin (FA) s o l u t i o n and the o r i g i n a l wine were taken u s i n g a 1.0 mm pathle n g t h . The PVP index was c a l c u l a t e d as PVP Index = ( A = 2 D H s 2 o r=f± )/A= (10) * 100 e). Tristimul.us E v a l u a t i o n An e v a l u a t i o n of wine c o l o r was c a r r i e d out by us i n g a D25 Hunterlab C o l o r D i f f e r e n c e Meter (Hunterlab A s s o c i a t e L a b o r a t o r i e s , F a i r f a x , VA.) with a tungsten f i l a m e n t l i g h t source. A c l e a n white standard t i l e was used t o s t a n d a r d i z e the equipment. Measurements, made u s i n g a 10 mm l i g h t path i n the t r a n s m i s s i o n head, were obtained as "L" ( b r i g h t n e s s ) , "a" (red t o blue-green hues) and "B" (yellow t o p u r p l e - b l u e hues). T r i p l i c a t e d e t e r m i n a t i o n s were made f o r each wine, with the equipment being s t a n d a r d i z e d between each d e t e r m i n a t i o n with the white t i l e . The "a" and "B" rea d i n g s were used t o c a l c u l a t e the angle hue (6 = c o t a n - 1 a/B) and s a t u r a t i o n ( a 3 +B= a)°-=. f 1 Pager Chromatography of Anthocyanins S k i n s of approximately 50 grams of f r o z e n grapes were b l o t t e d dry and macerated i n a Waring blender f o r 1 minute with 50 mL of 2 7. f o r m i c a c i d i n methanol. T h i s e x t r a c t , along with r i n s i n g s of the macerated s k i n s , was f i l t e r e d under vacuum through Whatman #4 f i l t e r paper. The e x t r a c t was t r a n s f e r r e d t o a s e p a r a t o r y funnel and washed with 20 mL of hexane which was then d i s c a r d e d . The e x t r a c t was conce n t r a t e d i n a r o t o r y evaporator at 35 °C t o approximately two mL. Approximately 1.5 mL of t h i s e x t r a c t was a p p l i e d r e p e a t e d l y t o Whatman #1 f i l t e r paper t o form a spot. The paper was developed, by descending chromatography f o r 5.5 hours with 1 7. f o r m i c a c i d i n a n-pentanol s a t u r a t e d chamber (Fong gt a L , 1974). A v i s u a l comparison of the i n t e n s i t y of bands of high ( d i g l y c o s i d e s ) and low (monoglycosides) m o b i l i t y was made a f t e r d r y i n g . 9 l P h e n o l i c Content The t o t a l p h e n o l i c content, as well as n o n - f l a v o n o i d and f l a v o n o i d f r a c t i o n s of the t o t a l of each wine were determined as g a l l i c a c i d e q u i v a l e n t s (GAE), a c c o r d i n g t o the method gi v e n by Amerine and Ough (1974), with minor m o d i f i c a t i o n s . For the t o t a l p h e n o l i c d e t e r m i n a t i o n , 0.5 mL of a l s l d i l u t i o n of wine (or 0.5 mL of d i s t i l l e d water f o r the blank) was p i p e t t e d i n t o a 25 mL v o l u m e t r i c f l a s k . Then 15 mL of d i s t i l l e d water and the 1.5 mL of F o l i n - C i o c a l t e u reagent were added and mixed. Between 30 seconds and B minutes a f t e r adding the reagent, 3.75 mL of 20 V. sodium carbonate was added and mixed. The s o l u t i o n was brought up t o volume with d i s t i l l e d water, mixed and p l a c e d i n a hot water bath at 50 °C f o r 5 minutes. The f l a s k s were then c o o l e d i n c o l d water f o r 5 minutes t o allow the pigment t o s t a b i l i z e . The absorbance of the c o l o r e d s o l u t i o n was read at 765 nm a g a i n s t the reagent blank. D u p l i c a t e r e a d i n g s D f each sample were obtained. P h e n o l i c content was determined by comparing the absorbance t o t h a t on a standard curve f o r g a l l i c a c i d . For n o n - f l a v o n o i d d e t e r m i n a t i o n , 2.5 mL of f i l t e r e d wine was p i p e t t e d i n t o each of two screw cap t e s t tubes f a l l o w e d by 2.5 mL of l s 4 H C l i d i s t i 1 l e d water and 1.25 mL of formaldehyde <B mg/mL). The mixture was vortexed f o r approximately 5 seconds, and the tube was capped and l e f t f o r 24 hours at 25 °C. The s o l u t i o n was then f i l t e r e d through a 0.45 Aim f i l t e r . A 0.5 mL volume of the s o l u t i o n was used t o determine the n o n - f l a v o n o i d p h e n o l i c s as noted above f o r t o t a l p h e n o l i c s . D u p l i c a t e d e t e r m i n a t i o n s were c a r r i e d out f o r each of the two t e s t tubes. F l a v o n o i d p h e n o l i c s were c a l c u l a t e d by s u b t r a c t i o n of the amount of the n o n - f l a v o n o i d f r a c t i o n from the t o t a l p h e n o l i c content f o r each wine, t i l Neutral. Phenol i c E x t r a c t i o n The c o n c e n t r a t i o n of n e u t r a l p h e n o l i c components of the wines, i n c l u d i n g c a t e c h i n s , p r o c y a n i d i n s and f l a v o n o l s was determined u s i n g the method of Sa l a g o i t y - A u g u s t e and Bertrand (19B4). A 20-mL sample of wine and 20 mL of d i s t i l l e d water were combined i n a small round bottomed f l a s k and concentrated t o l e s s than 20 mL u s i n g a r o t a r y evaporator at 35 °C. The volume was made up t o approximately 20 mL with d i s t i l l e d water r i n s i n g s of the f l a s k . The sample was a d j u s t e d t o pH 7.0 with 2 N NaOH and e x t r a c t e d t h r e e times with e t h y l a c e t a t e using 20, 10 and 10 mL of s o l v e n t . The e x t r a c t i o n s were performed by m a g n e t i c a l l y s t i r r i n g the sample and s o l v e n t i n a f l a s k f o r ten minutes. A f t e r t h i s time, the mixture was t r a n s f e r r e d t o capped tubes and c e n t r i f u g e d at approximately 1500 x g f o r 5 minutes. The top s o l v e n t l a y e r was then removed by Pasteur p i p e t t e . The 3 e x t r a c t s were combined i n a small round bottomed f l a s k along with an i n t e r n a l standard (4-OH m a n d e l l i c a c i d ) and evaporated t o dryness with a r o t o r y evaporator at 35 °C. The dry r e s i d u e was then d i s s o l v e d i n 2 mL of HPLC grade methanol and s e a l e d i n a v i a l . The samples were s u b j e c t e d t o RP-HPLC w i t h i n 24 hours. i i HPLC of the Neutral P h e n o l i c Sample and Wine The HPLC methodology was p r e v i o u s l y d e s c r i b e d . The HPLC o p e r a t i n g c o n d i t i o n s used were those which achieved the g r e a t e s t CRF i n the C e n t r o i d Mapping O p t i m i z a t i o n . Commercially-obtained standards of c a t e c h i n , and e p i c a t e c h i n (Sigma Chemical Co.) were used f o r the q u a n t i f i c a t i o n of these compounds i n the n e u t r a l e x t r a c t . G a l l i c a c i d standard was used f o r the q u a n t i f i c a t i o n of t h i s compound i n wine. The t h r e e p h e n o l i c s were determined by comparing r e t e n t i o n times of the sample peaks and peaks of standards, by c o e l u t i o n e v a l u a t i o n of peaks f o r spiked standards and f o r samples peaks and r a t i o s (A 2S4/A 280 and A 280/A 320). Ca t e c h i n and e p i c a t e c h i n were expressed as c a t e c h i n . Neutral p h e n o l i c peaks which were i d e n t i f i e d as common between a l l wines and a l l years were numbered and the peak areas were used i n f u r t h e r s t a t i s t i c a l a n a l y s e s . Common peaks were determined by t h e i r r e l a t i v e r e t e n t i o n times, based on d i s t i n c t i v e peaks i n the chromatogram. S i m i l a r l y , peaks on the chromatograms of the wine were i d e n t i f i e d and numbered. Those which were i d e n t i f i e d as not being present on the n e u t r a l p h e n o l i c chromatograms (by comparing r e t e n t i o n times) were a l s o used i n f u r t h e r s t a t i s t i c a l a n a l y s i s . SIAIISIICAL ANALYSIS Computer a n a l y s e s were performed on an Amdahl 470 vY8 and a Vax 730 computer. The data obtained from the v a r i o u s wine a n a l y s e s was d i v i d e d i n t o two groups f o r s t a t i s t i c a l treatment. The f i r s t group was composed of HPLC peak areas from chromatograms of both the n e u t r a l p h e n o l i c e x t r a c t and the wine samples and was r e f e r r e d t o as the "peak data". The second group of data was r e f e r r e d t o as the " a n a l y t i c a l data" and i n c l u d e s the or g a n i c a c i d p r o f i l e , pH, t i t r a t a b l e a c i d i t y , c o l o r parameters and v a r i o u s p h e n o l i c f r a c t i o n s , as 36 well as c a t e c h i n , e p i c a t e c h i n , and g a l l i c a c i d l e v e l s . The peak data and a n a l y t i c a l data were a l s o used together as a s i n g l e data s e t . Analyses were done u s i n g BMD programs (BMDP S t a t i s t i c a l Software Inc., 1983, Westwood B l v d . , S u i t e 202, U n i v e r s i t y of C a l i f o r n i a P r e s s L t d . , Los Angeles) and Genstat (Lawes A g r i c . T r u s t , Rothamsted Exp. S t a t i o n , 1982). i i i A n a l y s i s of Variance Two way a n a l y s i s of v a r i a n c e was used t o determine the c o n t r i b u t o r y e f f e c t s of age and v a r i e t y on the a n a l y t i c a l data. The program used was Genstat, A n a l y s i s of V a r i a n c e and Covariance. Two v a l u e s f o r each wine were used. E s t i m a t e s of missing v a l u e s f o r two deChaunac wines were made. Trend a n a l y s i s was conducted by using orthogonal c o n t r a s t s . bI P r i n c i e a l Component A n a l y s i s P r i n c i p a l component a n a l y s i s (PCA) i s a b l i n d c l a s s i f i c a t i o n technique which was used t o reduce the l a r g e number of v a r i a b l e s t o a fewer number of p r i n c i p a l components (PC). The program BMD s 4M was a p p l i e d t o each the a n a l y t i c a l data and the peak data. Varimax r o t a t i o n was used t o r o t a t e f a c t o r s so t h a t the v a r i a n c e of the squared f a c t o r l o a d i n g s f o r any f a c t o r was maximized. Graphic r e p r e s e n t a t i o n of the r e s u l t s i n two dimensional space was done u s i n g combinations of the f i r s t t h r e e PC as the x and y axes. &1 S-tep.wi.se D i s c r i m i n a n t A n a l y s i s Stepwise d i s c r i m i n a n t a n a l y s i s (SDA) i s a technique which seeks t o maximize the d i f f e r e n c e s between known groups of o b j e c t s , and t o c a t e g o r i z e new o b j e c t s i n t o a p r e v i o u s l y d e f i n e d category. Using the BMD : 7M program, wines were grouped a c c o r d i n g t o age, v a r i e t y , and age x v a r i e t y . In t h i s l a s t c l a s s i f i c a t i o n , young (19B2 and 1983 vintage) and o l d (1981 and 1980 vintage) wines were grouped w i t h i n v a r i e t i e s , forming f o u r groups. L i n e a r d i s c r i m i n a n t f u n c t i o n s were c a l c u l a t e d i n order t o make the r a t i o of between group t o w i t h i n group v a r i a t i o n maximum. Only forward s e l e c t i o n of v a r i a b l e s was used, so t h a t at each step the new v a r i a b l e which c o n t r i b u t e d the most t o the s e p a r a t i o n of the groups was added t o the d i s c r i m i n a n t f u n c t i o n . A j a c k k n i f e v a l i d a t i o n procedure was used t o help remove b i a s i n the groupings. Ob j e c t s were removed, one at a time, from the computation of the group means and c r o s s p r o d u c t s . Each objBct was then c l a s s i f i e d by the d i s c r i m i n a n t f u n c t i o n at each s t e p , and the number of m i s c i a s s i f i c a t i o n s was recorded. In some cases c r o s s v a l i d a t i o n was used, i n which 20 X of each group of wines was not used i n determining the d i s c r i m i n a n t f u n c t i o n . These wines were then assigned t o groups, a c c o r d i n g t o the f u n c t i o n . The number of c o r r e c t placements was used as an i n d i c a t i o n of the s u c c e s s of the d i s c r i m i n a t i o n . C l u s t e r A n a l y s i s A second b l i n d c l a s s i f i c a t i o n technique, c l u s t e r a n a l y s i s (CA) was done, u s i n g the BMD : 2M program. A n a l y t i c a l d ata, peak data and v a r i a b l e s chosen by both SDA and PCA were used t o -form c l u s t e r s . C l u s t e r s were based on E u c l i d e a n d i s t a n c e s between v a r i a b l e s . In a h e i r a r c h i c a l p r o c e s s , each wine was c o n s i d e r e d a c l u s t e r and these were then l i n k e d u n t i l a l l wines were combined i n t o one c l u s t e r . The d i s t a n c e between c e n t r o i d c l u s t e r s was used as the c r i t e r i o n f o r l i n k a g e . In t h i s l i n k a g e , a new weighted average l o c a t i o n of a c l u s t e r i s c a l c u l a t e d a f t e r any j o i n i n g of s m a l l e r c l u s t e r s . A t r e e diagram and amalgamation d i s t a n c e s were used t o determine the e f f e c t i v e n e s s of the a n a l y s i s . 39 IY.i RESULTS AND DISCUSSION B i ANALYTICAL PARAMETERS a)_ Wine Color Values -for c o l o r and p h e n o l i c parameters of Foch and deChaunac wines f o r the v i n t a g e s 19S0 through 19S3 are shown i n Table 2. There were c e r t a i n c o l o r parameters obtained s p e c t r o p h o t o m e t r i c a l l y and by the Hunterlab C o l o r D i f f e r e n c e Meter t h a t were c o r r e l a t e d . The t r i s t i m u l u s "L" or b r i g h t n e s s v a l u e s f o r red wines were s i g n i f i c a n t l y and n e g a t i v e l y c o r r e l a t e d with the c o l o r d e n s i t y v a l u e s obtained s p e c t r o p h o t o m e t r i c a l l y (Table 3.). Hue angle by the t r i s t i m u l u s method and c o l o r hue by the spectrophotometric method were a l s o c o r r e l a t e d , e s p e c i a l l y f o r Foch wines. There was a s i g n i f i c a n t c o r r e l a t i o n between "a" and the amount of i o n i z e d anthocyanins f o r both v a r i e t i e s (Table 3). It can be noted t h a t t r i s t i m u l u s parameters of s a t u r a t i o n and "a" were c o r r e l a t e d . The v a l u e s f o r "a" dominated the equation used f o r c a l c u l a t i o n of s a t u r a t i o n as they were l a r g e r than those f o r "B". E f f e c t s d i s p l a y e d by the s a t u r a t i o n term are t h e r e f o r e e x p l a i n e d by the "a" v a l u e s . T r i s t i m u l u s v a l u e s of L, a, B, hue, and s a t u r a t i o n have been r e p o r t e d t o d e s c r i b e c o l o r i n terms r e l e v a n t t o the human v i s u a l response (Ough et § L , 1962) but g i v e no 40 Table 2. C o l o r and p h e n o l i c parameters and pH o-f Foch and deChaunac wines. Parameter V a r i e t y 1983 1982 1981 19B0 Mean a |^  II Foch deChaunac 3.54 6.88 4.99 5.67 1.48 3.8B 2.27 4.29 3.07 5. 18 »a" Foch deChaunac 4.07 6.66 4.25 5.97 2. 15 2.70 3.4B 3.73 3.49 4.77 "B" Foch deChaunac 0.70 0. 10 0.55 0. 10 1. 10 0. 14 0.48 0.80 0.71 0.29 Hue Foch deChaunac 0.98 0.56 0.84 0.72 1.45 0.98 1.05 1.09 1.08 0.84 Color Densi t y Foch deChaunac 8.90 13.85 7.45 11.50 5.50 3.20 10.60 7.30 8. 11 8.96 T o t a l Anthocyanins <mg/L) Foch deChaunac 395.0 914.0 219.0 790.0 438.0 289.0 467.0 324.0 380.0 579.0 Ionized Anthocyanins (mg/L) Foch deChaunac 42.0 133.2 27.4 88.0 13.8 18.8 33.0 32.0 29.0 6B.0 Un-ionized Anthocyanins (mg/L) Foch deChaunac 353.0 7B1.0 191.0 702.0 425.0 270.0 434.0 292.0 351.0 511.0 Alpha (7. i o n i z e d / t o t a l anth.) Foch deChaunac 11.05 14.55 13.70 11. 10 3. 15 6.50 6.85 9.90 8.69 10.51 T o t a l Pi gment (mg/L) Foch deChaunac 448.0 961.0 275.0 843.0 475.0 316.0 516.0 373.0 400.0 623.0 PH Foch deChaunac 3.7 3. 1 3.6 3.3 4.0 3.0 4.0 3.4 3.8 3.2 Refer t o Tables 4 and 5 f o r Anova. 41 Table 3. L i n e a r c o r r e l a t i o n s between c o l o r and a n a l y t i c a l parameters of Foch (top value) and deChaunac (bottom value) wines. Age C o l o r C o l o r Satu- Ionized Free Molec-Dens i t y Hue r a t i o n Antho. SQ^ u l a r SQ^ L -0.93** -0.99** a -0.39 1.00** 0.83** -0.86** -0.85** -0.85* 1.00** 0.95* -0.93** -0.90* 0.69 Hue 0.93** Angle 0.70 Co l o r 0.04 Density-0.81* Free 0.89** -0.77** S 0 2 0.77 -0.89* Mol e c - 0.87** -0.76** u l a r S O 2 0.69 -0.84* pH 0.66* 0.59* -0.25 — 0.27 0.14 0.10 A s t e r i s k s i n d i c a t e l e v e l of s i g n i f i c a n c e : 0.05 (*),.01 (**) . i n f o r m a t i o n about the composition of the wine c o l o r . Absorbance r e a d i n g s can be r e l a t e d t o both types and q u a n t i t i e s of pigment pres e n t . The two methods, t h e r e f o r e , complement each other. The c o r r e l a t i o n s obtained between both instruments i n d i c a t e d t h a t some parameters gave s i m i l a r i n f o r m a t i o n , and l i k e Hunterlab v a l u e s , s p e c t r o p h o t o m e t r y v a l u e s might be r e l a t e d t o human c o l o r p e r c e p t i o n . Foch wines had l e s s red c o l o r than deChaunac wines, as i n d i c a t e d by lower "a" and high hue v a l u e s (Table 2 ) . The l a r g e r hue v a l u e s f o r Foch were a r e s u l t of a s m a l l e r A s 2 o r e l a t i v e t o deChaunac. T h i s was e s p e c i a l l y t r u e f o r young wines. Hue i n c r e a s e d with age and appeared t o s t a b i l i z e at a r a t i o of approximately one. The "a" v a l u e s decreased l i n e a r l y with age f o r both Foch and deChaunac wines (Table 4). T h i s i n d i c a t e d a decrease i n red c o l o r with age f o r both v a r i e t i e s of wine, and i s a common ob s e r v a t i o n f o r red wines. T h i s decrease i n "redness" as wines aged, shown by the t r i s t i m u l u s "a" v a l u e s , was p a r t l y a s s o c i a t e d with a b l e a c h i n g e f f e c t of s u l f u r d i o x i d e on the i o n i z e d anthocyanins. The "a" v a l u e s had n e g a t i v e l i n e a r c o r r e l a t i o n s with f r e e s u l f u r d i o x i d e l e v e l s and molecular s u l f u r d i o x i d e l e v e l s . T h i s r e s u l t e d i n the subsequent enhancement of the brown hue from t h B polymeric pigments which are minimally a f f e c t e d by s u l f u r d i o x i d e . In a d d i t i o n , i t has been shown t h a t as pH i n c r e a s e s , browning and c o l o r hue a l s o i n c r e a s e (Sims and M o r r i s , 1985). Foch wines were found t o have higher pH v a l u e s and g r e a t e r c o l o r Table 4. Anova of some c o l o r parameters of jrach and deChaunac wines: expressed as 100 R (*yi). Source D. F. Co l o r Hue a B V a r i e t y <V) 1 20.9* 16.5* 29.0* Age (A) 3 52.0* 57.0* 12.9 L i n e a r 1 31.7** 31.9* 8.2 Quadrati c 1 2.3 5.2 2.7 Cubic 1 18.0* 19.9* 2.0 V x A 3 16.3 8.9 27. 1 L i n e a r 1 5.0 8.5 12.5 Quadratic 1 1.0 0.2 11.3 Cubic 1 10.3 0.2 3.3 E r r o r a 6(2) 10.9 17.6 31.0 T o t a l 13 100.0 100.0 100.0 T o t a l Sum of Squares 13 1.1 39.8 2.5 A s t e r i s k s i n d i c a t e the l e v e l of s i g n i f i c a n c e : 0.05 (*), 0.01 (**) l e v e l . 8 2 missing v a l u e s estimated. 44 Table 5. Anova of some pigment parameters of Foch and deChaunac wines: expressed as 100 R* (•/.). Source D. F. T o t a l Antho-cyani Alpha T o t a l Pigment Ionized Antho-cyanin Un-i o n i z e d Antho-cyanin V a r i e t y (V) 1 18.2*** 5.4 15.0** 29.4*** 16.2** Age (A) 3 23.7** 68. 3* 23.6** 47.6*** 19. 1* L i n e a r 1 19.3*** 35. 0* 17.0** 35.0*** 15.9** Quadr a t i c 1 3. 8* 6.3 5.2* 8. 7** 2.9 Cubic 1 0.6 27.0** 1.4 3.9* 0.3 V x A 3 54.7*** 10.6 56.7*** 24.3** 60.6*** L i n e a r 1 41.B*** 0.4 44.4*** 22.6*** 44.4*** Quadratic 1 0. 1 3.4 0.2 0.6 0.2 Cubic 1 12.8 6.7 12.1** 1. 1 16.0** E r r o r * 6(2) 3.4 15.8 4.7 3.2 4.0 T o t a l 13 100.0 100.0 100.0 100.0 100.0 T o t a l Sum of Squares 13 875595.0 248.3 902395.0 24400.0 634706.0 A s t e r i s k s i n d i c a t e l e v e l of s i g n i f i c a n c e : 0.05 (*), 0.01 (**), 0.001 (***). a 2 mi s s i n g v a l u e s estimated. 45 hue v a l u e s than deChaunac wines. However, a low c o r r e l a t i o n between pH and c o l o r hue was found (Table 3 ) . The higher pH can o n l y be seen as one of s e v e r a l f a c t o r s c o n t r i b u t i n g t o the c o l o r d i f f e r e n c e s between the v a r i e t i e s of wine, b l Anthocyanin S u b s t a n t i a l amounts of anthocyanin, determined s p e c t r o p h o t o m e t r i c a l l y , were found t o be present i n the wines even a f t e r s e v e r a l years of b o t t l e aging. Using the same method of d e t e r m i n a t i o n , anthocyanin l e v e l s of 250 t o 630 mg/L (Somers and Evans, 1974) and 371 t o 486 mg/L ( Somers and Evans, 1977) were found i n young Cabernet Sauvignon and S h i r a z wines. These v a l u e s may be g r e a t e r than the a c t u a l amounts of anthocyanin i n the wines s i n c e the s p e c t r o p h o t o m e t r i c method has been found t o overestimate anthocyanin l e v e l s compared t o d e t e r m i n a t i o n by HPLC. Bakker et a K (1986) found t h a t the r a t i o of anthocyanins determined s p e c t r o p h o t o m e t r i c a l l y t o those determined by HPLC was g r e a t e s t f o r very young wines but decreased so t h a t at 46 weeks the r a t i o was o n l y 1.2. A s i m i l a r o v e r e s t i m a t i o n o c c u r s i n wines more than f i v e years o l d (McClosky and Yengoyan, 1981). I t would appear t h a t f o r the range of wine ages examined i n t h i s work (one t o f o u r y e a r s ) , the s p e c t r o p h o t o m e t r i c a l l y determined anthocyanin c o n c e n t r a t i o n may be somewhat g r e a t e r than a c t u a l v a l u e s but s t i l l p r o v i d e an i n t e r e s t i n g comparison of the two v a r i e t i e s . T o t a l anthocyanin and t o t a l pigment l e v e l s i n Foch wines showed l i t t l e change with age (Table 2 ) . It was expected 46 t h a t the l e v e l s of f r e e anthocyanins would decrease with i n c r e a s i n g age as they were i n c o r p o r a t e d i n t o polymeric pigments. However, t h e r e appeared t o be no i n c r e a s e i n pigment p o l y m e r i z a t i o n with i n c r e a s e d aging, as determined by the chemical age parameter and by the amount of polymeric pigment measured i n the wine (Table 6 ) . P o l y m e r i z a t i o n , as determined by the PVP Index was v a r i a b l e with age. P o l y m e r i z a t i o n may have been l i m i t e d by a f a c t o r such as acetaldehyde c o n c e n t r a t i o n or the c o n c e n t r a t i o n D f a r e a c t i v e t a n n i n component. DeChaunac wines c o n t r a s t e d s t r o n g l y with Foch wines i n r e g a r d s t o pigment changes with age. T o t a l anthocyanin of deChaunac wines decreased with i n c r e a s i n g age. The same tre n d was found f o r i o n i z e d and u n - i o n i z e d anthocyanins. T h i s change was u n l i k e l y a pH e f f e c t as t h e r e was no c o r r e l a t i o n of i o n i z e d anthocyanins with pH over the range of wines sampled (Table 3 ) . I t would be expected t h a t these d i s a p p e a r i n g monomeric forms would then be transformed i n t o polymeric pigments. However p o l y m e r i z a t i o n , as determined by the amount of polymeric pigment, d i d not i n c r e a s e with age (Table 6 ) . A l s o , as with Foch, v a r i a b l e amounts of p o l y m e r i z a t i o n were r e v e a l e d by the PVP Index. T o t a l pigment l e v e l s showed a decrease with i n c r e a s i n g age f o r deChaunac wines (Table 2 ) , i n d i c a t i n g i n s t a b i l i t y and p r e c i p i t a t i o n of the polymeric forms. Foch wines had more yellow hues, as i n d i c a t e d by the l a r g e r "B" v a l u e s (Table 2 ) . Yellow tones may be due t o the l a r g e amount of monoglycosides p r e s e n t , s i n c e the e x t r a 47 Table 6. P h e n o l i c components of Foch and deChaunac wines. Parameter V a r i e t y 1983 19B2 1981 1980 Mean E p i c a t e c h i n Foch 6.22 6.90 5.16 14.54 8.21 (mg/L) deChaunac 4.77 5.10 4.52 3.58 4.50 Ca t e c h i n Foch 4.54 4.58 6.39 22.67 9.54 (mg/L) deChaunac 1.42 1.65 2.80 2.78 2.16 Ca t e c h i n per Foch 1.19 2.25 1.46 4.80 2.42 Anthocyanins deChaunac 0.15 0.21 0.98 0.86 0.55 T o t a l Foch 1360.0 1157.0 1447.0 1792.0 1439.0 P h e n o l i c s deChaunac 1650.0 1722.0 1139.0 1498.0 1502.0 (mg/L) F l a v o n o i d s Foch 305.0 250.0 403.0 39B.0 339.0 (mg/L) deChaunac 343.0 159.0 100.0 30.0 158.0 Polymeric Foch 48.0 49.7 26.3 71.4 4B.8 pigment deChaunac 45.5 46.5 13.9 38.8 36.2 (mg/L) Chemical Age Foch 0.51 0.60 0.50 0.56 0.54 deChaunac 0.25 0.33 0.30 0.48 0.34 PVP Index Foch 66.6 60.0 21.3 46.6 48.6 deChaunac 49.1 56.0 0.0 53.0 39.5 Refer t o Tables 7 and 8 f o r Anova. 48 Table 7. Anova of some p h e n o l i c parameters of Foch and deChaunac wines: expressed as 100 R ( */. ). Source D. F. Cat e c h i n E p i -c a t e c h i n Catech/ E p i c a t e c C a t e c h i n / Anthocyanin V a r i e t y <V) 1 29.0** 2B.7* 45.0*** 42.2*** Age (A) 3 34.4* 21.7 48.4** 31.4** L i n e a r 1 25.1** 9.4 42.2*** 25.1** Quadrati c 1 8.5 7.2 2.4* 3.4 , Cubic 1 0.7 5. 1 3.7* 2.9 V x A 3 27.8* 36.8* 4.3 20.9* L i n e a r 1 17.3* 19.5* 3.2* 7.7* Quadr a t i c 1 9. 1* 12.9 0.5 4.5 Cubic 1 1.5 4.4 0.7 8.6* a E r r o r 6(2) 8.9 12.8 2.2 5.6 T o t a l 13 100.0 100.0 100.0 100.0 T o t a l Sum of Squares 13 751.7 192. 1 2.6 33.3 A s t e r i s k s i n d i c a t e l e v e l of s i g n i f i c a n c e : 0.05 (*), 0.01 (**) , 0.001 (***). 2 missing v a l u e s estimated. 49 Table 8. Anova of some p h e n o l i c and polymeric parameters of Foch and deChaunac wines: expressed as 100 R (*/.) Source D. F. T o t a l P h e n o l i c s F l a v o n o i d P h e n o l i c s Polymeric Pigment Chemical Age PVP Index V a r i e t y (V) 1 1.7 3B.4* 9.8 67.0*** 4.0 Age <A> 3 26.4 10.3 43.4 19.0** 74.7** L i n e a r 1 l . S 4.7 0.0 10.5** 12.4 Quadratic 1 18.0* 2.0 17.4 0.4 18.5* Cubic 1 6.9 3.7 26.0 9.9** 43.B** V x A 3 58.7* 31.0 9.0 10.0* 5.9 L i n e a r 1 35.6** 29.9* 7.5 8. 0** l.B Q uadratic 1 1.8 0.3 1.5 1.9 0.6 Cubic 1 21.4 0.8 0.0 0.0 3.5 E r r o r a 6(2) 13.2 20.3 37.7 2. 1 15.3 T o t a l 13 100.0 100.0 100.0 100.0 100.0 T o t a l Sum of Squares 13 969014.0 341453.0 6536.7 0.2 8214.0 A s t e r i s k s i n d i c a t e l e v e l of s i g n i f i c a n c e : 0.05 (*), 0.01 (**>, 0.001 (***). a 2 m i s s i n g v a l u e s estimated. 50 sugar of d i g l y c o s i d e s has been shown t o i n c r e a s e the blueness of the pigment (Van Buren e t a h , 1974). The "B" v a l u e s d i d not change with time f o r e i t h e r v a r i e t y . E l Qther P h e n o l i c s C a t e c h i n c o n c e n t r a t i o n s i n deChaunac wines i n c r e a s e d with i n c r e a s i n g age while e p i c a t e c h i n l e v e l s decreased (Table 6). T h i s suggested a p r e f e r r e d i n c o r p o r a t i o n of e p i c a t e c h i n over c a t e c h i n i n t o polymeric pigment. D i f f e r e n c e s i n grape m a t u r i t y at h a r v e s t and i n time of f e r m e n t a t i o n an the s k i n s do not appear t o be r e l a t e d t o the change i n the r a t i o of the two compounds over time (Czochanska e t a l ; . , 1979). In Foch wines, where p o l y m e r i z a t i o n appeared s t a t i c , both c a t e c h i n and e p i c a t e c h i n l e v e l s i n c r e a s e d with age. The r e s u l t s from the F o l i n - C i o c a l t e u and s p e c t r o p h o t o m e t r i c method f o r determining the t o t a l p h e n o l i c content of wine were c o r r e l a t e d (r = 0.84, p < 0.01). D i s c r e p a n c i e s between the two may be caused by the i n h e r e n t e r r o r s of each; namely, the use of an a r b i t r a r y value of 4 f o r the c o r r e c t i o n of absorbance of non-phenolic m a t e r i a l s i n the s p e c t r o p h o t o m e t r i c method and the a d d i t i v e e f f e c t s of s u l f u r d i o x i d e on the F o l i n - C i o c a l t e u reagent. Foch had more f l a v o n o i d but l e s s n o n - f l a v o n o i d m a t e r i a l than deChaunac. Somers and Evans (1986) found, i n a comparison of the r a t e s of aging of two red wines, the wine with the lower p h e n o l i c content aged r e l a t i v e l y s l o w l y . In t h i s work, deChaunac wines appeared t o age c o n t i n u o u s l y over the f o u r year p e r i o d examined but had higher l e v e l s of t o t a l 51 p h e n o l i c s i n young wines. They d i d , however, have lower o v e r a l l l e v e l s of both c a t e c h i n and e p i c a t e c h i n , and a lower c a t e c h i n / e p i c a t e c h i n r a t i o i n the wine as well as i n the grapes. The r a t i o of c a t e c h i n / e p i c a t e c h i n was 3.8 f o r Foch grapes while i n deChaunac grapes i t was 0.71. As w e l l , Foch wines had a higher r a t i o of c a t e c h i n : anthocyanin than deChaunac (Table 6). Timberlake and B r i d l e (1977) found t h a t the extent of p o l y m e r i z a t i o n between c a t e c h i n and anthocyanin i n a model system depended on the r a t i o of the two. The r e a c t i o n was f a s t e r and l e d t o more pigment p r e c i p i t a t i o n at a high r a t i o of c a t e c h i n : anthocyanin. Nagel and Wulf (1979) found s i g n i f i c a n t l y g r e a t e r amounts of e p i c a t e c h i n i n a f a s t aging wine v a r i e t y than a slow aging one, as was found with these wines. The impact of these s p e c i f i c types of f l a v o n o i d m a t e r i a l s would seem t o be more important than a measure of more general p h e n o l i c content. 52 CENTROID MAPPING OPTIMIZATION FOR HPLC SEPARATION OF PHENOLIC COMPOUNDS OF RED WINE The s t a r t i n g Spendley simplex and the chromatographic response f a c t o r s (CRF) obtained are shown i n Table 9. The c o n d i t i o n s of the t h i r d vertex were c l e a r l y s u p e r i o r t o the other f i v e . The f a c t o r which seemed t o have the g r e a t e s t e f f e c t on the high response was the run time, which was s i g n i f i c a n t l y longer than the other f i v e v e r t i c e s . The f i r s t c e n t r o i d search was attempted a f t e r t h i s but f a i l e d t o show any improvement a f t e r the f i r s t vertex was ev a l u a t e d . T h e r e f o r e a second Spendley matrix was generated t o d e f i n e a new s e a r c h range. T h i s new range was c a l c u l a t e d based on the r e l a t i o n s h i p between the best and the next best v e r t i c e s on the response s u r f a c e . I t was a narrower zone, centered on the area where the optimum may be l o c a t e d . A f t e r e v a l u a t i n g the second Spendley simplex, f o u r v e r t i c e s showed an improvement i n the CRF (Table 10). The c l o s e v a l u e s obtained f o r t h r e e of these v e r t i c e s ( v e r t i c e s 8, 9 and 12), i n d i c a t e d t h a t a l o c a l optimum may have been at or near the f a c t o r l e v e l s of these v e r t i c e s . The poor response from the t e n t h vertex seemed t o be due t o the s h o r t run time. It was noted t h a t the combination of a long run time and a high flow r a t e p r o v i d e d the best response. A second c e n t r o i d was generated, and although the f i r s t vertex 53 Table 9. I n i t i a l s t a r t i n g c e n t r o i d l i m i t s of f a c t o r s , f a c t o r l e v e l s of simplex ( v e r t i c e s 1 - 6 ) and f i r s t s earch (vertex 7). === === ==s= a s - s s s s s ==== F a c t o r L i m i t s A B C D E Lower 0.0 45.0 0.5 25.0 90.0 Upper 25.0 150.0 1.5 40.0 100.0 Vertex Response 1. 0.0 45.0 0.5 25.0 90.0 1303 2. 22.8 66.5 0.7 28. 1 92.0 2343 3. 5. 1 140.8 0.7 28. 1 92.0 6545 4. 5. 1 66.5 1.4 28. 1 92.0 2596 5. 5. 1 66.5 0.7 38.7 92.0 2414 6. 5. 1 66.5 0.7 28. 1 99. 1 2142 7. 8.7 81.4 0.9 30.2 93.5 3692 A = c o n c e n t r a t i o n CH^CN i n l e s s p o l a r mobile phase (*/.) B = run time (minutes) C = flow r a t e of mobile (mL/minute) 0 = column temperature ( ° C ) E = f i n a l c o n c e n t r a t i o n of l e s s p o l a r mobile phase 54 Table 10. F a c t o r l i m i t s of second simplex search ( v e r t i c e s 8-13) and second c e n t r o i d search ( v e r t i c e s 14-15). F a c t o r Vertex A B e D E Response 8. 3.9 140.8 0.7 27.3 91.5 7042 9. 6.2 125.6 0.8 27.6 91.8 6999 10. 4.4 73. 1 0.8 27.6 91.B 3193 11. 4.4 125.6 1.3 27.6 91. B 7206 12. 4.4 125.6 0.8 28.7 91.8 6695 13. 4.4 125.6 0.8 27.6 92.5 5032 14. 4.6 128.6 0.9 27.8 91.9 5833 15. 4.7 129.2 0.9 27.8 91.7 5671 A = c o n c e n t r a t i o n CH^CN i n l e s s p o l a r mobile phase (V.) B = run time (minutes) C = flow r a t e of mobile (mL/minute) o D = column temperature( C) E = f i n a l c o n c e n t r a t i o n of l e s s p o l a r mobile phase (X) 55 f a i l e d , the second was e v a l u a t e d i n order t o i n c r e a s e the number of responses a v a i l a b l e f o r mapping. The advantages of i n c o r p o r a t i n g a v i s u a l e v a l u a t i o n such as mapping i n t o an i t e r a t i v e o p t i m i z a t i o n process and the technique of mapping have been presented by Nakai et a l i . (1984). Mapping pr o v i d e d a convenient method f o r e s t i m a t i n g the e f f e c t s of l e v e l changes t o a s i n g l e f a c t o r and thereby aided i n s e t t i n g new f a c t o r l i m i t s . Decreasing the range of v a l u e s of f a c t o r s may improve the e f f i c i e n c y with which the o p t i m i z a t i o n i s reached, e s p e c i a l l y i n the f i n a l s t a g e s of the o p t i m i z a t i o n . The t r e n d of an i n c r e a s i n g CRF with i n c r e a s i n g run time was very c l e a r from the mapping. I t was a l s o apparent t h a t i n c r e a s i n g the flow r a t e improved the CRF. The remaining t h r e e f a c t o r s showed no c l e a r t r e n d s . The upper and lower l i m i t s f o r the f i v e f a c t o r s were narrowed t o those shown i n Table 11 and used t o e s t a b l i s h the t h i r d simplex. During the e v a l u a t i o n of t h i s simplex, i t was noted t h a t as the column p r e s s u r e i n c r e a s e d , peak r e s o l u t i o n was lower than expected. The c o n d i t i o n of the column was e v a l u a t e d by r e p e a t i n g a p r e v i o u s vertex (vertex 11). The CRF was found t o have decreased 37 "/.. At t h i s p o i n t the o l d column was r e p l a c e d with an i d e n t i c a l one from the same s u p p l i e r ( S u p e l c o s i l LC 18, 5 mm, 250 x 4 mm ID, Supelco L t d , Penn.). The performance of the new column was e v a l u a t e d by r e p e a t i n g vertex 11. The r e s u l t obtained was very s i m i l a r t o the o r i g i n a l e v a l u a t i o n and the o p t i m i z a t i o n was continued. The t h i r d Spendley matrix was e v a l u a t e d with 56 Table 11. New l i m i t s D-f f a c t o r s a f t e r mapping, f a c t o r l i m i t s of t h i r d simplex ( v e r t i c e s 16-21) and t h i r d c e n t r o i d search ( v e r t i c e s 22-23). F a c t o r L i m i t s A B C D E Lower 3.9 125.6 0.7 27.3 91.5 Upper 22.8 150.0 1.3 38.7 99. 1 Vertex Response 16. 3.9 125.6 0.7 27.3 91.5 6467 17. 21. 1 130.6 0.8 29.7 93. 1 6581 18. 7.8 147.9 0.8 29.7 93. 1 7274 19. 7.8 130.6 1.3 29.7 93. 1 B700 20. 7.8 130.6 0.8 37.7 93. 1 8138 21. 7.8 130.6 0.8 29.7 98.5 7475 22. 9.7 133.0 0.8 30.8 93.9 6478 23. 10.0 133.5 O.B 29.4 94.0 6777 A = c o n c e n t r a t i o n CHjCN i n l e s s | p o l a r mobili e phase (7.) B = run time (minutes) C = flow r a t e of mobile (mL/minute) D = column temperature (C) E = f i n a l c o n c e n t r a t i o n of l e s s p o l a r mobile phase (7.) 5 7 improved responses being achieved. Two v e r t i c e s -from a c e n t r o i d s e a r c h , conducted a f t e r the t h i r d simplex, f a i l e d . Mapping was attempted again with the a d d i t i o n a l e i g h t data, f o r a t o t a l of 23 p o i n t s . A f t e r mapping, run time was f i x e d at 130.6 minutes, and l i m i t s f o r the remaining f a c t o r s were narrowed and a p p l i e d t o the simultaneous f a c t o r s h i f t program. T h i s program allowed the examination of experimental c o n d i t i o n s near the optimum by s h i f t i n g a l l the f a c t o r l e v e l s between t h e i r present best and t a r g e t v a l u e s . In a d d i t i o n , t h r e e other s h i f t v a l u e s are provided which examine the r e g i o n beyond the t a r g e t l e v e l s , t o ensure t h a t the optimum i s not beyond the t a r g e t . Three combinations were ev a l u a t e d , with the second p r o v i d i n g the best response (Table 12). A f o u r t h s h i f t combination was attempted but f a i l e d t o improve the response, i n d i c a t i n g t h a t the g l o b a l optimum had been reached with the second combination. The average percent standard d e v i a t i o n of the CRF f o r r e p l i c a t e v e r t i c e s was 13, and ranged from 0.1 "/. t o 35 '/.. A 70 7. improvement i n the CRF was o b t a i n e d , c a l c u l a t e d from the best vertex of the s t a r t i n g simplex ( F i g . 3.) and the vertex which gave the f i n a l best response ( F i g . 4.). The number of peaks separated i n c r e a s e d 16 7. and the average r e s o l u t i o n of the peak p a i r s i n c r e a s e d 26 7.. Chromatograms t y p i c a l of Foch and deChaunac wines and t h e i r n e u t r a l p h e n o l i c e x t r a c t s run under optimal RP-HPLC c o n d i t i o n s are shown i n Appendix I. It should be noted t h a t even an i n s i g n i f i c a n t f a c t o r 58 Table 12. Simultaneous f a c t o r s h i f t of f a c t o r l e v e l s . F a c t o r Combination A B C D E Response 1. 15.8 130.6 1.0 34.5 96.3 8555 2. 13. 1 130.6 1.1 32.9 95.2 11136 3. 11.8 130.6 1.2 32. 1 94.7 B578 4. 10.4 130.6 1.2 31.2 94. 1 9621 A = c o n c e n t r a t i o n CH3CN i n l e s s p o l a r mobile phase (X) B = run time (minutes) C = flow r a t e of mobile (mL/minute) o D = column temperature ( C) E = f i n a l c o n c e n t r a t i o n of l e s s p o l a r mobile phase (X) F i g u r e 3. I n i t i a l RP-HPLC s e p a r a t i o n b-f p h e n o l i c compounds o-f 1983 Foch wine. For c o n d i t i o n s see Table 9, Vertex #3. F i g u r e 4. Optimal RP-HPLC s e p a r a t i o n o-f p h e n o l i c compounds of 1983 Foch wine. For c o n d i t i o n s see Table, 12, S h i f t Combination #2. 61 may seem t o converge on the optimum (Parker e t a l ^ , 1975); t h e r e f o r e the c o n t r i b u t o r y e f f e c t of a f a c t o r t o an o p t i m i z a t i o n must be c a r e f u l l y c o n s i d e r e d . As expected, the long run time, a l l o w i n g f o r a slow r a t e of change of s o l v e n t s , was found t o be d e s i r a b l e . With the l a r g e number of compounds being e l u t e d , adequate time was r e q u i r e d f o r t h e i r r e s o l u t i o n . The f i n a l flow r a t e was s e t at a moderate l e v e l . Higher flow r a t e s caused column back pr e s s u r e t o g r e a t l y i n c r e a s e w hile slower flow d i d not allow adequate peak r e s o l u t i o n . Few p u b l i s h e d methods on p h e n o l i c a n a l y s i s make r e f e r e n c e t o the temperature at which the s e p a r a t i o n was done with the i m p l i c a t i o n of ambient temperature. Temperatures r e p o r t e d t o have been used are 40 °C (Andersen and Pedersen, 1983; Wallace, 1983) and 35 °C (Vande C a s t e e l e et , 1983). The temperature obtained i n the o p t i m i z a t i o n of 32.9 °C tended t o decrease the r e t e n t i o n time of compounds on the column compared t o lower temperatures. A s t r o n g s o l v e n t of 13 7. a c e t o n i t r i l e i n methanol was found t o g i v e the best r e s o l u t i o n . The l i m i t s f o r the f i n a l percent of the l e s s p o l a r mobile phase co u l d probably have been s e t at 100 "/., and not i n c l u d e d i n the o p t i m i z a t i o n . However, i t i s p r e f e r r a b l e t o i n c l u d e as many f a c t o r s as p o s s i b l e i n the simplex, so as t o be l e s s l i k e l y t o miss a s i g n i f i c a n t f a c t o r (Yarbro and Deming, 1974). T h i s reasoning may be extended t o CMO because of the s i m i l a r i t y of the technique t o Simplex O p t i m i z a t i o n . 62 Q i . MULTIVARIATE ANALYSIS §2. Princip_al Comrjonent A n a l y s i s P r i n c i p a l component a n a l y s i s (PCA) was a p p l i e d t o the i n f o r m a t i o n i n each of the a n a l y t i c a l and peak area data s e t s . T h i r t y one v a r i a b l e s were entered from the a n a l y t i c a l data s e t . Six p r i n c i p a l components (PC) accounted f o r 91.1 7. of the v a r i a n c e of the o r i g i n a l data (Table 13). V a r i a b l e s with the c l o s e s t a s s o c i a t i o n t o the f i r s t PC were i o n i z e d and t o t a l anthocyanin l e v e l s , c o l o r d e n s i t y , L, F-C p h e n o l i c s , s a t u r a t i o n , t o t a l p h e n o l i c s determined s p e c t r a l l y , t r i s t i m u l u s s a t u r a t i o n and "a", and non-f1avonoid p h e n o l i c s (Table 14). For the second PC, hue angle, c o l o r hue, molecular and f r e e s u l f u r d i o x i d e l e v e l s , and t r i s t i m u l u s "a" and "B" v a l u e s had the l a r g e s t f a c t o r l o a d i n g s . Poor d i s c r i m i n a t i o n of the wines by e i t h e r age and/or v a r i e t y was found, when the wines were graphed a g a i n s t the f i r s t and second, f i r s t and t h i r d , and second and t h i r d f a c t o r s c o r e s . S i m i l a r l y , Amantea (19B4) found t h a t two dimensional p l o t s u s i n g f a c t o r l o a d i n g s as axes, d i d not r e s u l t i n d i s t i n c t age c l a s s i f i c a t i o n s f o r cheese samples of v a r y i n g ages. I t was suggested t h a t PC were c l a s s i f y i n g o b j e c t s on an unknown c r i t e r i o n . F a c t o r l o a d i n g s f o r each PC may l a c k d i s c i m i n a t i n g i n f o r m a t i o n (Aishima, 1979). 63 Table 13. Eigenvalue (VP), sum of VP and cumulative p r o p o r t i o n i n t o t a l v a r i a n c e (7.) i n p r i n c i p a l component a n a l y s i s using a n a l y t i c a l v a r i a b l e s . PC Eigenva l u e Sum VP Cumulative P r o p o r t i o n 1 10.44 10.44 33.7 2 6.32 16.76 54.1 3 5.26 22.02 71.0 4 2.77 24.79 80.0 5 2.02 26. B l 86.5 6 1.43 28.24 91.1 64 Table 14. Sor t e d , r o t a t e d -factor* l o a d i n g s a s s o c i a t e d with the f i r s t s i x p r i n c i p a l components based on a n a l y t i c a l v a r i a b l e s . FACTOR PRINCIPAL COMPONENTS 1 2 3 4 5 6 Ionized Anthocyanin 0.B9 -0.25 -0.31 0.00 0.00 0.00 T o t a l Anthocyanin 0.89 0.00 0.00 -0.38 0.00 0.00 Co l o r D e n s i t y 0.86 0.00 0.00 0.34 0.00 0.00 "L" -6.83 0.40 0.00 0.00 0.00 0.00 F.C. P h e n o l i c s 0.80 0.00 0.50 0.00 0.00 0.00 S a t u r a t i o n 0.79 -0.43 0.00 0.00 0.00 0.00 "a" 0.76 -0.51 0.00 0.00 0.00 0.00 P h e n o l i c s (A280) 0.71 0.00 0.38 -0.40 0.00 0.00 Non-Flavonoid P h e n o l i c s 0.67 0.00 0.37 0.00 0.00 0.51 Hue angle -0.26 0.92 0.00 0.00 0.00 0.00 Free S u l f u r D i o x i d e 0.00 0.87 0.00 0.00 0.00 0.00 Molecular S u l f u r D i o x i d e 0.00 0.80 0.00 0.00 -0.36 0.00 Co l o r Hue -0.48 0.76 0.27 0.00 0.00 0.00 "B" -0.36 0.66 0.00 0.00 0.53 0.00 Catechin 0.00 0.00 0.96 0.00 0.00 0.00 E p i c a t e c h i n 0.00 0.00 0.85 0.00 0.00 -0.30 S a l l i c 0.42 0.00 0.78 0.00 0.26 0.00 C a t e c h i n / E p i c a t e c h i n 0.00 0.38 0.76 -0.27 0.00 0.00 Alpha <•/. Ionized Antho.) 0.00 -0.30 -0.26 0.84 0.00 0.00 Chemical Age -0.40 0.00 0.42 0.54 0.52 0.00 PVP Index 0.33 -0.33 0.00 0.36 0.76 0.00 pH 0.00 0.43 0.50 0.00 0.64 0.00 T a r t a r i c A c i d 0.25 0.00 0.00 0.00 -0.29 0.82 A c e t i c A c i d 0.00 0.00 0.00 0.00 0.29 0.81 F l a v o n o i d P h e n o l i c s 0.00 0.34 0.00 0.00 0.30 -0.64 M a l i c A c i d / L a c t i c A c i d 0.00 0.44 0.00 0.43 0.26 -0.39 T i t r a t a b l e A c i d i t y 0.38 0.00 -0.47 0.00 -0.41 0.37 * F a c t o r l o a d i n g s <0.25 are r e p l a c e d by 0.00. 65 S i m i l a r r e s u l t s were achieved i n the PCA using HPLC peak area data. Twelve PC e x p l a i n e d 95.8 V. of the v a r i a n c e c o n t a i n e d i n 59 peaks (Table 15). The two dimensional p l o t of the f a c t o r s c o r e s f o r the f i r s t and second, second and t h i r d and f i r s t and t h i r d PC r e s u l t e d i n poor d i s c r i m i n a t i o n on an age or v a r i e t y b a s i s . To determine i f some s t r u c t u r e c o u l d be found u s i n g the r e s u l t s of e i t h e r of the PCA, the f a c t o r s were a p p l i e d t o c l u s t e r a n a l y s i s . I n c o n s i s t e n t c l u s t e r i n g of ages and/or v a r i e t i e s of the wines r e s u l t e d from both attempts. In the case of the s i x f a c t o r s o b tained from PCA based on the a n a l y t i c a l data, young deChaunac wines (1982 and 1983 v i n t a g e s ) c l u s t e r e d , as d i d the 1981 deChaunac wines. Some 1982 and 1983 Foch wines a l s o c l u s t e r e d . PCA was not done on a combined data s e t of HPLC peak areas and a n a l y t i c a l data. With only poor v a r i e t y and age d i s c r i m i n a t i o n o c c u r r i n g with each separate s e t of data, i t was thought t o be h i g h l y improbable t h a t b e t t e r r e s u l t s would be found i n a l a r g e r , more complex, data s e t . b)_ Stepwise D i s c r i m i n a n t Analyses One of the most widely used m u l t i v a r i a t e techniques i s stepwise d i s c r i m i n a n t a n a l y s i s (SDA). The d i s c r i m i n a n t f u n c t i o n s f o r the age, v a r i e t y and v a r i e t y x age groups based on each of the t h r e e data s e t s were c a l c u l a t e d . The data s e t s used were the HPLC peak area data, a n a l y t i c a l data and the combination of the two. 66 Table 15. Eigenva l u e (VP), sum of VP and cumulative p r o p o r t i o n i n t o t a l v a r i a n c e (7.) i n p r i n c i p a l component a n a l y s i s u sing peak area v a r i a b l e s . * Cumulative PC Ei g e n v a l u e Sum VP P r o p o r t i o n 1 18.97 18.97 32. 1 2 9.25 28.22 47.8 3 5.78 34.00 57.6 4 4.94 3B.94 66.0 5 4.07 43.01 72.9 6 3. 19 46.20 78.3 7 2.74 48.94 83.0 8 2. 11 51.05 86.5 9 1.63 52.68 89.3 10 1.43 54. 11 91.7 11 1.28 55.39 93.9 12 1.09 56.48 95.8 * F a c t o r l o a d i n g matrix i s not presented as RP-HPLC peaks were not i d e n t i f i e d . 67 E f f e c t i v e s e p a r a t i o n of the wines by age was d i f f i c u l t t o a c h i e v e , and r e q u i r e d a g r e a t e r number of v a r i a b l e s than d i d v a r i e t y d i s c r i m i n a t i o n . F i v e , t h r e e and f o u r v a r i a b l e s were r e q u i r e d f o r v a r i e t y d i s c r i m i n a t i o n using the peak area, a n a l y t i c a l and combination data s e t s r e s p e c t i v e l y . Seven, seven and nine v a r i a b l e s were r e q u i r e d from the same data s e t s f o r age d i s c r i m i n a t i o n . i i i s c i a s s i f i c a t i o n s of wines by age occurred when the HPLC peak area data and the a n a l y t i c a l data were used s e p a r a t e l y . In the case of the HPLC peak areas, one wine of the 1981 v i n t a g e was c l a s s i f i e d as 1982 i n the j a c k k n i f e v a l i d a t i o n procedure (94.4 "/. c o r r e c t c l a s s i f i c a t i o n ) . The HPLC peaks chosen i n c l u d e d peak 38, 40, 56, 61, 62, 68 and 69. The peaks 38 and 40 were from the e t h y l a c e t a t e e x t r a c t . Of the remaining peaks from the chromatogram of the whole wine, 68 and 69 were found t o absorb at 520 nm. With the a n a l y t i c a l d a t a , t h r e e 1982 wines were c l a s s i f i e d as 1983, and two 1983 wines were c l a s s i f i e d as 19B2 i n the j a c k k n i f e procedure (72.2 7. c o r r e c t c l a s s i f i c a t i o n ) . The seven v a r i a b l e s chosen were c o l o r hue, t a r t a r i c a c i d l e v e l s , c a t e c h i n l e v e l s , the r a t i o of c a t e c h i n / e p i c a t e c h i n , PVP index, t i t r a t a b l e a c i d i t y and the r a t i o of m a l i c / l a c t i c a c i d . The f i r s t f o u r v a r i a b l e s i n c r e a s e d with age, w h i l e the remaining v a r i a b l e s decreased. S i n c e aging i s a continuous phenomenon), i t i s not s u r p r i s i n g t h a t some m i s c i a s s i f i c a t i o n s o c c u r r e d between c o n s e c u t i v e years. From the c a n o n i c a l p l o t s , a l i n e a r t r e n d with i n c r e a s i n g age was e v i d e n t along one a x i s , when 1982 and 68 19B3 v i n t a g e wines were grouped t o g e t h e r . T h i s was t r u e -for a l l t h r e e age d i s c r i m i n a t i o n s done. The two v a r i e t i e s of wine were c l e a r l y d i s c r i m i n a t e d . In the SDA us i n g the HPLC peak areas, c a t e c h i n , peak 26, peak 36, peak 57 and peak 70 were used. C a t e c h i n , peak 36 and peak 57 l e v e l s were higher i n Foch wines and the other two peaks were found i n lower c o n c e n t r a t i o n s than i n deChaunac wines. Ca t e c h i n and peak 57 were a l s o chosen from the combination data s e t along with peak 64 and i o n i z e d anthocyanin l e v e l s . Both i o n i z e d anthocyanin and peak 64 l e v e l s were lower i n Foch wines than deChaunac wines. In the d i s c r i m i n a t i o n using o n l y the a n a l y t i c a l data, pH was s e l e c t e d f i r s t as the most e f f e c t i v e v a r i a b l e . T h i s v a r i a b l e has a s i g n i f i c a n t (p < 0.01) v a r i e t y e f f e c t by ANOVA, with Foch wines having a mean pH of 3.B4 while deChaunac wines had a mean pH of 3.19. Non-flavonoid p h e n o l i c s and g a l l i c a c i d l e v e l s were a l s o used i n the d i s c r i m i n a n t f u n c t i o n and were present at higher l e v e l s i n Foch wines. In the d i s c r i m i n a t i o n of o l d and young wines w i t h i n a . v a r i e t y , the f i r s t s i x v a r i a b l e s chosen u s i n g the peak and combination data s e t s were i d e n t i c a l . These s i x were peak 13, peak 56, peak 34, peak 67, peak 60, and e p i c a t e c h i n . These peaks, with the ex c e p t i o n of peak 60, were a l l e l u t e d i n t he chromatographic runs from 27 minutes t o 59 minutes. Peak 13, e p i c a t e c h i n and peak 34 were q u a n t i f i e d from the e t h y l a c e t a t e e x t r a c t of the wine while the ot h e r s were from the whole wine chromatograms. In a d d i t i o n t o these s i x 69 30 w 20' G1980/1981 deChaunac ui OQ < § iok o 5 1980/1981 Foch _ i < O z S o -10 • 1982/1983 Foch 20 O1982/1983 deChaunac -.40 -30 -20 -10 0 10 20 30 CANONICAL VARIABLE 1 F i g u r e 5. Canonical p l o t of group means of wine samples. V a r i e t y x age d i s c r i m i n a t i o n based on combination data s e t . * For c o o r d i n a t e s of i n d i v i d u a l wine samples see Table 16. Table 16. Coor d i n a t e s of i n d i v i d u a l wine samples f o r c a n o n i c a l p l o t of v a r i e t y x age d i s c r i m i n a t i o n based on combination data s e t . BROUP CANONICAL FIRST VARIABLE SECOND Old Foch 10.07 9.88 (1980 fc 1981) 10.12 11.20 9.76 11.74 11.29 10.14 Young Foch 17.55 -7.94 <1982 & 1983) 16.88 -6.48 17.70 -6. 18 17.37 -9. 19 17.25 -6.44 17.48 -6.55 18.39 -7.8B 15.87 -9.08 Old deChaunac -17.B7 18.84 (1980 fc 1981) -21.08 19.54 -18.23 20.22 Young deChaunac -42.18 -13.74 (1982 fc 1983) -39.93 -14.34 -40.42 -13.75 71 v a r i a b l e s , t h r e e other v a r i a b l e s chosen from the combination data s e t were used i n the age x v a r i e t y d i s c r i m i n a n t f u n c t i o n . These i n c l u d e d peak 47 from the e t h y l a c e t a t e e x t r a c t , alpha (X i o n i z e d anthocyanin) and the PVP index. DeChaunac wines had higher and lower amounts of peak 13 and e p i c a t e c h i n than Foch wines. S i m i l a r l y , the two o l d e r v i n t a g e s of wine of both v a r i e t i e s had higher q u a n t i t i e s of peak 56 and lower v a l u e s f o r alpha and PVP index than young wines. L i n e a r combinations of these n i n e v a r i a b l e s were a l s o used t o form the t h r e e c a n o n i c a l v a r i a b l e s . The mean value s of the wine groups f o r the f i r s t two c a n o n i c a l v a r i a b l e s are p l o t t e d i n F i g u r e 5 with the c o o r d i n a t e s f o r i n d i v i d u a l wine samples shown i n Table 16. I t can be noted t h a t the v a r i e t i e s were separated c o n s i s t e n t l y and s u c c e s s f u l l y by the f i r s t c a n o n i c a l v a r i a b l e while age groups were separated by the second. From the a n a l y t i c a l data s e t , s i x v a r i a b l e s were needed t o d i s c r i m i n a t e on the b a s i s of v a r i e t y x age. The v a r i a b l e s chosen i n c l u d e d pH, t o t a l anthocyanins, the r a t i o of c a t e c h i n / e p i c a t e c h i n , PVP index, a c e t i c a c i d l e v e l s and the chemical age parameter. Both chemical age and pH were higher f o r Foch wines than deChaunac. Older wines had a higher r a t i o of c a t e c h i n / e p i c a t e c h i n and a c e t i c a c i d l e v e l s and a lower PVP index than young wines. £l Cluster Analysis C l u s t e r a n a l y s i s (CA) can be a u s e f u l technique f o r determining s t r u c t u r e of o b j e c t s or when v e r i f y i n g r e s u l t s from other m u l t i v a r i a t e a n a l y s e s . However, i f too many v a r i a b l e s are used i n the a n a l y s i s , some of which may c o n t a i n meaningless data, t r u e d i s t a n c e s between c l u s t e r s can be obscured (Aishima, 1982). T h i s proved t o be t r u e when CA was attempted u s i n g the HPLC peak area data and the a n a l y t i c a l data. In both cases, wines were fused with o t h e r s o n l y a t l a r g e d i s t a n c e s , c r e a t i n g many small c l u s t e r s . B e t t e r r e s u l t s were obtained when wine were c l u s t e r e d u s i n g the v a r i a b l e s chosen i n the SDA. The r e s u l t s of the CA tended t o resemble those of the SDA. The best c l u s t e r i n g of wines by age occurred when using the combination data s e t v a r i a b l e s chosen by SDA (Figure 6 ) . A c l e a r " inner c o r e " of 19B2 and 1983 v i n t a g e wines was obvious. In a d d i t i o n , t h e r e was some secondary c l u s t e r i n g of v a r i e t i e s w i t h i n age. The c l u s t e r s formed by usi n g v a r i a b l e s chosen i n SDA based on o n l y HPLC peak areas or a n a l y t i c a l data showed some i n c o n s i s t e n c i e s , s i m i l a r t o those i n the o r i g i n a l SDA c l a s s i f i c a t i o n . The best c l u s t e r i n g of wines by v a r i e t y occurred u s i n g the SDA v a r i a b l e s from the HPLC peak area data set and the combination data s e t . The r e s u l t s of the two were s i m i l a r ; i n both cases the 1980 Foch wines formed a group, the remaining Foch wines were a l s o c l u s t e r e d and deChaunac wines formed a t h i r d group. Amalgamated d i s t a n c e s between the c l u s t e r s were s l i g h t l y g r e a t e r when the combination data v a r i a b l e s were used ( F i g u r e 7) as opposed t o the c l u s t e r s formed u s i n g the peak area data. b f f e c d c c c c h h g d d b a a 6. H i e r a r c h i c a l c l u s t e r a n a l y s i s of wines u s i n g v a r i a b l e s chosen i n age stepwise d i s c r i m i n a n t a n a l y s i s . V a r i a b l e s were chosen -from chromatographic peak areas and a n a l y t i c a l data. a - 1980 Foch wine e - 1980 deChaunac wine b - 1981 Foch wine f - 1981 deChaunac wine c - 1982 Foch wine g - 1982 deChaunac wine d - 1983 Foch wine h - 1983 deChaunac wine 74 Q) CO CO •o . c Si E 03 O) . 03 l o r f i b c c c c d d d c b h h g f f e a a F i g u r e 7. H i e r a r c h i c a l c l u s t e r a n a l y s i s of wines using v a r i a b l e s chosen i n v a r i e t a l stepwise d i s c r i m i n a n t a n a l y s i s . V a r i a b l e s were chosen from chromatographic peak areas and a n a l y t i c a l data. a - 1980 Foch wine b - 1981 Foch wine c - 1982 Foch wine d - 1983 Foch wine e - 1980 deChaunac wine f - 19B1 deChaunac wine g - 19B2 deChaunac wine h - 1983 deChaunac wine 75 O (0 £ 2 c o • M M (0 E o> (0 E < 1 n l-g h h f f c c d c c c d d e b b a a F i g u r e B. H i e r a r c h i c a l c l u s t e r a n a l y s i s of wines using v a r i a b l e s chosen i n age x v a r i e t y stepwise d i s c r i m i n a n t a n a l y s i s . V a r i a b l e s were chosen from a n a l y t i c a l data. a - 1980 Foch wine b - 19B1 Foch wine c - 1982 Foch wine d - 1983 Foch wine e - 1980 deChaunac wine f - 1981 deChaunac wine g - 1982 deChaunac wine h - 1983 deChaunac wine The most s u c c e s s f u l c l u s t e r i n g of o l d and young wines w i t h i n a v a r i e t y o c c u r r e d u s i n g the seven a n a l y t i c a l v a r i a b l e s chosen by the age x v a r i e t y SDA c l a s s i f i c a t i o n ( F i gure 8 ). Old Foch and deChaunac wines, and young Foch wines formed d i s t i c t c l u s t e r s . The 1980 deChaunac wine was not c l o s e l y a s s o c i a t e d with the 1981 deChaunac wines, but more c l o s e l y a s s o c i a t e d with the o l d Foch wines. C O N C L U S I O N In t h i s work, C e n t r o i d Mapping O p t i m i z a t i o n (CMO) improved the q u a l i t y of the s e p a r a t i o n of p h e n o l i c compounds of red wine. The optimized HPLC system was a l s o very u s e f u l i n s e p a r a t i n g the n e u t r a l p h e n o l i c compounds e x t r a c t e d from the wines. Manual manipulation of f a c t o r s i n v o l v e d i n the o p t i m i z a t i o n , with a reasonably a c c u r a t e i n t e r p r e t a t i o n of t h e i r i n t e r a c t i o n s would have been very d i f f i c u l t . CMO i s well s u i t e d f o r experimentation i n which a s e t of v e r t i c e s may be done s i m u l t a n e o u s l y and where the number of f a c t o r s i s s m a l l . N e i t h e r of these c r i t e r i a a p p l i e d t o the HPLC o p t i m i z a t i o n done i n t h i s work. If the response s u r f a c e of the HPLC o p t i m i z a t i o n i s assumed t o c o n t a i n one or more l o c a l optima, CMO l i k e l y o f f e r e d the most e f f i c i e n t method of e s t a b l i s h i n g the g l o b a l optimum HPLC o p e r a t i n g c o n d i t i o n s . There were s e v e r a l a n a l y t i c a l parameters examined which i n d i c a t e d t h a t Foch and deChaunac wines v a r i e d i n t h e i r r a t e s of aging. Foch wines had more brown tones and lower, more s t a b l e l e v e l s of t o t a l and i o n i z e d anthocyanins than deChaunac wines. DeChaunac wines had lower l e v e l s of c a t e c h i n and e p i c a t e c h i n and a lower c a t e c h i n : anthocyanin r a t i o than Foch wines. Age, v a r i e t y and v a r i e t y by age d i s c r i m i n a t i o n of the wines was s u c c e s s f u l u s i n g stepwise d i s c r i m i n a n t and c l u s t e r a n a l y s e s . Fewer v a r i a b l e s were r e q u i r e d and fewer m i s c i a s s i f i c a t i o n s of wines occurred d u r i n g the v a r i e t y d i s c r i m i n a t i o n compared t o the age d i s c r i m i n a t i o n . 78 REFERENCES Aishirna, T. 1979. 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Chromat. S c i . 21: 139. Wildenradt, H. and S i n g l e t o n , V. 1974. ThB p r o d u c t i o n of aldehydes as a r e s u l t of o x i d a t i o n of p o l y p h e n o l i c compounds and i t s r e l a t i o n t o wine aging. Amer. J . Enol V i t i c . 25: 119. Yarbro, L. A. and Deming, S. p r e p r o c e s s i n g of f a c t o r s f o r Acta. 73: 391. N. 1974. S e l e c t i o n and simplex o p t i m i z a t i o n . Anal. Chim. 84 APPENDIX I. T y p i c a l chromatograms of 1980 and 1983 Foch and deChaunac wines and e t h y l a c e t a t e e x t r a c t s of n e u t r a l p h e n o l i c compounds. Samples were run under optimal chromatographic c o n d i t i o n s as shown i n Table 12, Combination #2. Chromatogram o-f 19B0 Foch wine. Peak #1 i s g a l l i c a c i d . 86 Chromatogram o-f 1983 Foch wine. Peak #1 i e g a l l i c a c i d . Chromatogram of 1980 deChaunac Peak #1 i s g a l l i c a c i d . Mine. Chromatogram of 1983 deChaunac wine. Peak #1 i s g a l l i c a c i d . Chromatogram of n e u t r a l p h e n o l i c compounds of 1980 Foch wine (from e t h y l a c e t a t e e x t r a c t i o n ) . Peak #1 i s the i n t e r n a l standard (4-OH-mandel1ic a c i d ) ; peak #2 i s c a t e c h i n ; peak #3 i s e p i c a t e c h i n . Chromatogram of n e u t r a l p h e n o l i c compounds of 1983 Foch wine (from e t h y l a c e t a t e e x t r a c t i o n ) . Peak ttl i s the i n t e r n a l standard (4-OH-mandel1ic a c i d ) ; peak #2 i s c a t e c h i n ; peak #3 i s e p i c a t e c h i n . 91 Chromatogram of n e u t r a l p h e n o l i c compounds of 1980 deChaunac wine (from e t h y l a c e t a t e e x t r a c t i o n ) . Peak #1 i s the i n t e r n a l standard <4-OH-mandel1ic a c i d ) ; peak #2 i s c a t e c h i n ; peak #3 i s e p i c a t e c h i n . UJUQ8Z Chromatogram o-f n e u t r a l p h e n o l i c compounds of 1983 deChaunac wine (from e t h y l a c e t a t e e x t r a c t i o n ) . Peak #1 i s the i n t e r n a l standard (4-OH-mandel1ic a c i d ) ; peak #2 i s c a t e c h i n ; peak #3 i s e p i c a t e c h i n . 93 APPENDIX I I . A n a l y t i c a l parameters of Foch and deChaunac wines. 94 Parameter V a r i e t y 1983 19B2 19B1 1980 Mean T i t r a t a b l e A c i d i t y <gm/L) Foch deChaunac 8.09 11.44 8.75 9.03 9.56 10.43 7. 11 9.21 8.3B 10.03 A c e t i c A c i d (gm/lOOmL) Foch deChaunac 0.84 0.67 0.45 0. 19 0.86 0.82 0.75 1.54 0.72 0.80 M a l i c / L a c t i c Foch deChaunac 0.70 0.67 0.85 0.52 1.06 0.45 0.72 0.63 0.83 0.57 T a r t a r i c A c i d (gm/lOOmL) Foch deChaunac 1.25 1.79 1. 10 1.50 1.31 1.71 1.48 2.47 1.2B 1.B7 Hue Angle Foch deChaunac 10.29 12. 17 37.22 2.49 7.79 0.00 9.74 0.89 16.26 3.89 Molecular SO (ug/L) Foch deChaunac 7.40 0.95 15.90 6.80 269.65 95. 10 18.70 26.90 77.93 32.44 Free SQ (mg/L) Foch deChaunac 0.56 6.03 0.99 0.26 26.98 2.84 2.79 1.20 7.B3 1.08 T o t a l P h e n o l i c s (A 280) Foch deChaunac 28. 13 47.50 15.70 49.60 20.75 25.35 50.05 38.80 28.67 40.31 G a l l i c a c i d (mg/lOOmL) Foch deChaunac 2.77 2.61 2.41 2.29 1.84 1.33 4.97 2.25 3.00 2. 12 

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