Supercritical Fluid Extraction and Chromatography - American

Chapter 12 Supercritical Fluid ChromatographyMass Spectrometry of Carotenoid Pigments 1

1

Nelson M. Frew , Carl G. Johnson , and Richard H.

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Bromund

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Department of Chemistry, Woods Hole Oceanographic Institution, Woods Hole, M A 02543 Department of Chemistry, College of Wooster, Wooster, OH 44691

Downloaded by CORNELL UNIV on July 20, 2016 | http://pubs.acs.org Publication Date: March 17, 1988 | doi: 10.1021/bk-1988-0366.ch012

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Combined supercritical fluid chromatography-mass spectrometry is shown to be a useful new tool for the separation and identification of carotenoids, relatively involatile, labile pigments which contain multiple functional groups spanning a range of polarities. The most promising stationary phases for capillary SFC of complex natural mixtures of carotenoids are the cyanopropylpolysiloxanes and polyethylene glycols. The extremely mild ionization conditions which prevail using supercritical CO as the mobile phase, produce superior quality mass spectra for fragile carotenoids such as fucoxanthin and its derivatives, as compared with earlier in-beam desorption CI techniques. The CI-CH fragmentation of many other carotenoids under these conditions is minimal; the simplicity of their spectra may be advantageous in determining low level distributions using molecular ion abundances. 2

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The separation and s t r u c t u r a l characterization of complex mixtures of natural products i s an important problem i n food and a g r i c u l t u r a l chemistry, pharmaceutical research and environmental and geochemical studies. Gas chromatography (GC) and high performance l i q u i d chromatography (HELC), now well-established methods, have largely supplanted e a r l i e r techniques, including thin-layer chromatography and open-column l i q u i d chromatography, f o r reasons of chromatographic e f f i c i e n c y and speed of analysis. The use of combined gas chromatography-mass spectrometry (GC-MS) has been p a r t i c u l a r l y valuable i n the analysis of complex mixtures, and more than a decade of research on combined HPLC-MS has resulted i n i n t e r f a c i n g techniques such as thermospray ionization and e l e c t r o spray i o n i z a t i o n which show s i m i l a r promise. The introduction of open-tubular columns and improved hardware f o r s u p e r c r i t i c a l f l u i d chromatography (SFC) has renewed i n t e r e s t 0097-6156/88/0366-0208$06.25/0 ©

1988

A m e r i c a n C h e m i c a l Society

Charpentier and Sevenants; Supercritical Fluid Extraction and Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1988.


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i n t h e use o f s u p e r c r i t i c a l f l u i d s as mobile phases f o r c h r o m a t o graphic separations ( 1 - 3 ) . T h i s new t e c h n i q u e supplements t h e s e p a r a t i o n c a p a b i l i t i e s o f b o t h GC and HPLC f o r two r e a s o n s : (1) the a b i l i t y t o d e a l w i t h l e s s v o l a t i l e , t h e r m o l a b i l e and h i g h m o l e c u l a r weight m a t e r i a l s n o t g e n e r a l l y amenable t o GC a n a l y s i s , but w i t h h i g h e r chromatographic e f f i c i e n c i e s than a r e a t t a i n a b l e i n HPLC and (2) t h e g r e a t e r f l e x i b i l i t y i n a v a i l a b l e d e t e c t i o n modes, i n c l u d i n g U V - v i s i b l e , f l u o r e s c e n c e , flame i o n i z a t i o n , t h e r m i o n i c , F o u r i e r t r a n s f o r m i n f r a r e d and mass s p e c t r o m e t r i c detection. The p o t e n t i a l f o r combining SFC w i t h t h e l a t t e r o f t h e s e d e t e c t o r s , t h e mass s p e c t r o m e t e r ( M S ) , i s an i m p o r t a n t advantage because o f t h e u n i v e r s a l i t y and s p e c i f i c i t y o f MS and i t s a b i l i t y t o supply e x p l i c i t s t r u c t u r a l i n f o r m a t i o n . S e v e r a l of the common MS i o n i z a t i o n modes, i n c l u d i n g e l e c t r o n i o n i z a t i o n ( £ 1 ) , c h e m i c a l i o n i z a t i o n ( C I ) and c h a r g e - e x c h a n g e (CE) have been shown t o be c o m p a t i b l e w i t h o n - l i n e SFC ( 4 - 1 2 ) . V a r y i n g degrees of hardware m o d i f i c a t i o n a r e r e q u i r e d , b u t a r e g e n e r a l l y much l e s s e x t e n s i v e t h a n r e q u i r e d i n HPLC-MS, where t h e mass s p e c t r o m e t e r must cope w i t h h i g h f l o w r a t e s of p o l a r l i q u i d s . The i n c r e a s i n g prominence o f SFC i s r e f l e c t e d i n t h e many recently-published applications, primarily involving industrial u s e s , i n c l u d i n g a n a l y s i s of s y n t h e t i c s u r f a c t a n t s , polymers, food and c o s m e t i c f o r m u l a t i o n s and p e t r o l e u m d i s t i l l a t e s ( 1 3 - 1 9 ) . Rela t i v e l y l i t t l e work has been p u b l i s h e d o n SFC of n a t u r a l p r o d u c t s , p a r t i c u l a r l y of p o l a r m a t e r i a l s w i t h molecular weights g r e a t e r than 400 d a l t o n s ( 2 0 - 2 3 ) . The c h o i c e o f p o l a r s u p e r c r i t i c a l f l u i d s i s r e l a t i v e l y l i m i t e d i n p r a c t i c e , s i n c e c r i t i c a l t e m p e r a t u r e s and chemical r e a c t i v i t y increase d r a m a t i c a l l y with p o l a r i t y . Some p r o g r e s s has been made w i t h t h e use o f p o l a r m o d i f i e r s , b u t most i n v e s t i g a t i o n s o f mixed f l u i d s have been c o n f i n e d t o s t u d i e s o f e l u t i o n o r d e r e f f e c t s w i t h r e l a t i v e l y n o n - p o l a r o r low m o l e c u l a r weight p o l a r s o l u t e s ( 2 4 - 2 9 ) . The e x t e n t t o w h i c h SFC w i l l be u s e f u l f o r r e l a t i v e l y p o l a r m a t e r i a l s (and thus a s t r o n g competitor w i t h HPLC) i s s t i l l an open q u e s t i o n and a m a t t e r f o r much f u t u r e research. We have s e l e c t e d a c l a s s o f l a b i l e p o l a r l i p i d s , t h e c a r o t e n o i d s , w i t h w h i c h t o e x p l o r e t h e p o t e n t i a l o f SFC-MS. We a r e s p e c i f i c a l l y i n t e r e s t e d i n t h e s e compounds as b i o l o g i c a l s o u r c e markers and as model i n d i c a t o r s o f t h e f a t e o f l a b i l e o r g a n i c m a t t e r w h i c h i s produced i n t h e s u r f a c e ocean and i s s u b j e c t t o v a r i o u s b i o l o g i c a l and c h e m i c a l d e g r a d a t i o n p r o c e s s e s . However, our f i n d i n g s s h o u l d be o f g e n e r a l i n t e r e s t t o o t h e r s w o r k i n g t o a p p l y SFC-MS i n o t h e r a r e a s o f n a t u r a l p r o d u c t c h e m i s t r y . The c a r o t e n o i d s ( c a r o t e n e s and x a n t h o p h y l l s ) a r e w i d e l y d i s t r i b u t e d i n b o t h p h o t o s y n t h e t i c and n o n - p h o t ο s y n t h e t i c o r g a n i s m s , f u n c t i o n i n g as a c c e s s o r y l i g h t r e c e p t o r s a s s i s t i n g i n t h e t r a n s f e r of e n e r g y t o t h e c h l o r o p h y l l s and a c t i n g as a n t i o x i d a n t s w h i c h p r o t e c t t h e c h l o r o p h y l l s from p h o t o o x i d a t i o n d u r i n g p h o t o s y n t h e s i s . The c a r o t e n o i d s a r e t e t r a t e r p e n o i d s ( F i g u r e 1 ) , each c o n s i s t i n g o f a c o n j u g a t e d p o l y e n e backbone o f t e n t e r m i n a t e d a t e a c h end w i t h six-membered r i n g s of v a r y i n g u n s a t u r a t i o n . The t e r m i n a l r i n g s (as w e l l as t h e backbone) a r e t y p i c a l l y s u b s t i t u t e d w i t h v a r i o u s p o l a r f u n c t i o n a l g r o u p s , i n c l u d i n g h y d r o x y l , methoxy, k e t o , a c e t y l


s

to




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and epoxy g r o u p s . The h i g h l y r e a c t i v e n a t u r e o f t h e s e compounds w h i c h makes them a t t a c t i v e as i n d i c a t o r s of s h o r t - t e r m d é g r a d a t i v e pathways a l s o makes t h e i r i s o l a t i o n and mass s p e c t r a l c h a r a c t e r i z a t i o n d i f f i c u l t , p a r t i c u l a r l y a t low l e v e l s (< 100 n g ) . Carotenoids a r e n o t g e n e r a l l y amenable t o gas c h r o m a t o g r a p h i c a n a l y s i s due t o t h e i r t h e r m o l a b i l i t y and low v o l a t i l i t y (35 , 4 2 ) . The a n a l y s i s o f c a r o t e n o i d s c u r r e n t l y i n v o l v e s s e p a r a t i o n by HPLC and i d e n t i f i c a t i o n o f t h e p u r i f i e d compounds by a b s o r p t i o n s p e c t r o s c o p y and d i r e c t i n s e r t i o n probe mass s p e c t r o m e t r y ( 3 0 ) . The e l e c t r o n i o n i z a t i o n , c h e m i c a l i o n i z a t i o n and f i e l d d e s o r p t i o n f r a g m e n t a t i o n o f c a r o t e n o i d pigments have been s t u d i e d by a number o f workers (31-33). V e t t e r e t a l . (31) have d i s c u s s e d i n d e t a i l t h e problem o f t h e r m a l d e g r a d a t i o n o f c a r o t e n o i d s and t h e o b s e r v a t i o n t h a t carotenoid EI fragmentation patterns, t o a large extent, a c t u a l l y r e p r e s e n t t h e r m a l l y degraded and i s o m e r i z e d c a r o t e n o i d s . Combined o n - l i n e chromatography-mass s p e c t r o m e t r y (HPLC-MS o r SFC-MS) h a s not been p r e v i o u s l y demonstrated f o r t h e c a r o t e n o i d s . We have i n v e s t i g a t e d t h e p o s s i b i l i t y o f i d e n t i f y i n g c a r o t e n o i d pigments u s i n g c a p i l l a r y s u p e r c r i t i c a l f l u i d chromatography-mass spectrometry. I n v i e w o f t h e i r l a b i l i t y , l o w - v o l a t i l i t y , and p o l a r i t y r a n g e , t h e s e compounds p r e s e n t an i n t e r e s t i n g t e s t o f t h e c u r r e n t c a p a b i l i t i e s o f c a p i l l a r y SFC-MS. We demonstrate t h a t SFC-MS can be implemented w i t h r e l a t i v e l y few m o d i f i c a t i o n s t o a t y p i c a l q u a d r u p o l e GC-MS system and t h a t u s e f u l chromatography and s t r u c t u r a l i n f o r m a t i o n may be o b t a i n e d f o r t h e s e compounds u s i n g SFC. I n s t r u m e n t a t i o n and E x p e r i m e n t a l Methods Two s u p e r c r i t i c a l f l u i d chromatographs were used i n t h i s work. The f i r s t s y s t e m , used f o r SFC a l o n e , was c o n s t r u c t e d u s i n g a c o m p u t e r - c o n t r o l l e d ( A p p l e H e ) p r e s s u r e / d e n s i t y programmable Lee S c i e n t i f i c Model 250 s y r i n g e pump and a H e w l e t t - P a c k a r d 5710 chromatograph m o d i f i e d t o a c c e p t a Rheodyne 7520 i n j e c t o r ( r o t o r i n t e r n a l l o o p volume, 0 . 2 y l ) . Columns were c o n n e c t e d t o t h e Rheodyne i n j e c t o r t h r o u g h a s p l i t t e r t e e e i t h e r d i r e c t l y o r i n d i r e c t l y u s i n g a s h o r t r e t e n t i o n gap o f u n c o a t e d c a p i l l a r y . J e t - t y p e r e s t r i c t o r s were f a b r i c a t e d d i r e c t l y on t h e columns by flame s e a l i n g t h e column end t o form a s h a r p i n t e r n a l t a p e r , t h e n o p e n i n g t h e s e a l by g r i n d i n g t o t h e d e s i r e d o r i f i c e d i a m e t e r o f approximately 1 t o 3 microns ( 3 4 ) . When flame i o n i z a t i o n d e t e c t i o n (FID) was u s e d , some flame i n s t a b i l i t y was o b s e r v e d a t h i g h p r e s s u r e s u n l e s s t h e r e s t r i c t o r was p o s i t i o n e d s l i g h t l y below t h e t i p o f t h e FID j e t . T h i s n e c e s s i t a t e d h e a t i n g t h e FID t o t e m p e r a t u r e s o f 3 0 0 - 3 5 0 ° C t o a v o i d peak t a i l i n g . A second system used f o r SFC-MS c o n s i s t e d o f a Brownlee Model G Micropump, a Rheodyne 7520 i n j e c t o r ( 0 . 2 μ 1 r o t o r ; s p l i t t e r t e e ) and a C a r l o E r b a 4160 gas chromatograph. The i n j e c t o r was c h i l l e d to 18°C using a r e f r i g e r a t e d c i r c u l a t i n g bath. The chromatograph was d i r e c t l y c o u p l e d t o a F i n n i g a n 4500 q u a d r u p o l e mass s p e c t r o m e t e r ( e s s e n t i a l l y w i t h o u t m o d i f i c a t i o n u s i n g t h e same e l e c t r i c a l l y h e a t e d i n t e r f a c e oven used f o r GC-MS o p e r a t i o n ) s u c h t h a t t h e column e f f l u e n t e n t e r e d t h e i o n volume n o r m a l t o t h e q u a d r u p o l e


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SUPERCRITICAL FLUID EXTRACTION AND C H R O M A T O G R A P H Y

axis. The column r e s t r i c t o r was p o s i t i o n e d 0 . 5 mm back from the sample e n t r a n c e h o l e . While e l e c t r i c a l heating maintained the i n t e r f a c e r e g i o n a t t h e same t e m p e r a t u r e as t h e chromatograph o v e n , no a d d i t i o n a l heat was s u p p l i e d t o t h e r e s t r i c t o r o t h e r t h a n t h a t from t h e s o u r c e b l o c k , w h i c h was m a i n t a i n e d a t 1 0 0 - 1 2 0 ° C . Methane c h e m i c a l i o n i z a t i o n was a c h i e v e d u s i n g a methane p r e s s u r e of 0 . 5 t o r r ( u n c o r r e c t e d ) . A n a l y z e r p r e s s u r e s ranged from 3-6 χ 10" torr. The e l e c t r o n energy was 130 e V . The e l e c t r o n m u l t i p l i e r was o p e r a t e d a t 1.3 kV w i t h t h e c o n v e r s i o n dynodes a t 3 k V . P r e a m p l i f i e r g a i n was 10~8 A / V . The s p e c t r o m e t e r was tuned f o r u n i t r e s o l u t i o n and c a l i b r a t e d i n t h e C H 4 - C I mode u s i n g p e r fluorotributylamine. D a t a a c q u i s i t i o n was c a r r i e d out w i t h an INCOS 2300 d a t a s y s t e m . D a t a were a c q u i r e d o v e r t h e mass range o f 400-800 d a l t o n s u s i n g t w o - s e c o n d scans t o enhance t h e s i g n a l - t o noise r a t i o .


5

S e p a r a t i o n s were c a r r i e d o u t u s i n g 50 o r 100 μ m ID D B - 5 , D B - 1 7 , DB-225 and DB-WAX c o a t e d f u s e d s i l i c a c a p i l l a r y columns (J&W S c i e n t i f i c , Rancho C o r d o v a , CA) o f 2 t o 20 meters i n l e n g t h . SFC Grade c a r b o n d i o x i d e ( S c o t t S p e c i a l t y G a s e s , P l u m b s t e a d v i l l e , PA) was used as t h e c a r r i e r f l u i d a t 6 0 - 1 0 0 ° C w i t h c a r r i e r l i n e a r f l o w v e l o c i t i e s o f 2-5 c m / s e c . U n d e r i v a t i z e d carotenoid standards ( i s o l a t e d from n a t u r a l s o u r c e s by D . Répéta o r o b t a i n e d from Sigma C h e m i c a l C o . , S t . L o u i s , MO) were d i s s o l v e d i n methylene c h l o r i d e t o a c o n c e n t r a t i o n o f 400-4000 n g / μ ΐ and i n j e c t e d u s i n g a 0 . 2 u l r o t o r and a s p l i t r a t i o i n t h e range o f 2 - 1 0 : 1 . R e s u l t s and D i s c u s s i o n Chromatography o f C a r o t e n o i d s . One o f t h e e a r l i e s t l i t e r a t u r e r e p o r t s on SFC demonstrated t h e m i g r a t i o n and s e p a r a t i o n o f a l p h a and b e t a - c a r o t e n e i n s u p e r c r i t i c a l CO2 ( 3 5 ) . Enhanced s o l u b i l i t y of t h e s e compounds i n CO2 was a s c r i b e d t o f o r m a t i o n o f a c c e p t o r donor complexes w i t h t h e p o l a r i z a b l e p i - e l e c t r o n s i n t h e h i g h l y c o n j u g a t e d backbone. I n t h e p r e s e n t work, a l l o f t h e c a r o t e n o i d s shown i n F i g u r e 1, as w e l l as s e v e r a l r e l a t e d i s o m e r s , a r e f o u n d t o be s u f f i c i e n t l y s o l u b l e i n s u p e r c r i t i c a l CO2 t o a l l o w chroma t o g r a p h i c e l u t i o n from t h e s t a t i o n a r y phases t e s t e d . We examined t h e c h r o m a t o g r a p h i c b e h a v i o r o f c a r o t e n o i d s on s e v e r a l t y p e s o f s t a t i o n a r y phases u s i n g s i m p l e m i x t u r e s s p a n n i n g a range o f p o l a r i ties. I n g e n e r a l , s e l e c t i v i t i e s f o r t h e c a r o t e n o i d s on p o l y s i l o x a n e phases a r e l i m i t e d . On D B - 5 (5% p h e n y l , m e t h y l - p o l y s i l o x a n e ) , r e l a t i v e l y s h a r p c h r o m a t o g r a p h i c peaks a r e o b t a i n e d , b u t t h e s e l e c t i v i t y i s i n a d e q u a t e t o s e p a r a t e even s i m p l e m i x tures. U s e f u l s e p a r a t i o n s a r e o b t a i n e d o n l y on m o d e r a t e l y p o l a r to p o l a r s t a t i o n a r y phases. S e p a r a t i o n o f s e v e r a l c a r o t e n o i d s on a 20 m χ 100 y m ID DB-17 (50% p h e n y l , m e t h y l - p o l y s i l o x a n e ) column i s i l l u s t r a t e d i n F i g u r e 2A. The u n u s u a l e l u t i o n b e h a v i o r o f f u c o x a n t h i n (V) i s e x e m p l i f i e d by i t s e l u t i o n from DB-17 w e l l before other carotenoids. T h i s behavior i s temperature s e n s i t i v e and n o t e x h i b i t e d by o t h e r p o l a r c a r o t e n o i d s . DB-225 (50% c y a n o p r o p y l m e t h y l , 50% m e t h y l p h e n y l - p o l y s i l o x a n e ) e x h i b i t s s i m i l a r moderate s e l e c t i v i t y ( F i g u r e 2 B ) , b u t s t i l l does n o t p r o v i d e s u f f i c i e n t r e s o l u t i o n o f t h e more p o l a r c a r o t e n o i d s . The h i g h e s t


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A

Fluid: C 0 at 70°C 2


Column: DB-17 20m χ 100/* ID Inject: 80 barsj fast ramp to 200 bars Program-. 200 to 320 bars at 4 bar/min.

ι 80

«—ι

1

1

80 200

230

260

PRESSURE

1

290

1

320

1 320

(Bars)

F i g u r e 2A. SFC-FID chromatograms o f c a r o t e n o i d s s e p a r a t e d on DB-17 s t a t i o n a r y phase. See F i g u r e 1 f o r key t o compound s t r u c t u r e s .


214


Β Fluid :C0 at 60°C 2


Column: DB-225 10mx50/i.lD Inject: 80 bars; fast ramp to 200 bars Program: 200 to 320 bars at 4 bar/min.

Π

m

—ι 80

80 200

230

260

PRESSURE

290

1

320 320

(Bars)

F i g u r e 2B. SFC-FID chromatograms o f c a r o t e n o i d s s e p a r a t e d on DB-225 s t a t i o n a r y phase. See F i g u r e 1 f o r key t o compound s t r u c t u r e s .


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C

Fluid: C 0 at 80°C


2

Column: DB-WAX 1.8mx50/xlD Inject: 80 bars; fast ramp to 120 bars Program: 120 to 300 bars at 6 bar/min.

I 80

, 120

1

1 180

1

PRESSURE

1 240

1

1— 300

(Bars)

F i g u r e 2C. SFC-FID chromatograms o f c a r o t e n o i d s s e p a r a t e d on DB-WAX s t a t i o n a r y phase. See F i g u r e 1 f o r key t o compound s t r u c t u r e s .


216


selectivity

is

o b t a i n e d w i t h DB-WAX ( a c r o s s - l i n k e d Carbowax

20M).


F i g u r e 2C i s a chromatogram o b t a i n e d on a s h o r t ( 1 . 8 m χ 50 μ m ID) DB-WAX column u s i n g a v e r y h i g h l i n e a r f l o w v e l o c i t y . These n o n i d e a l c o n d i t i o n s r e s u l t i n lower c h r o m a t o g r a p h i c e f f i c i e n c y , b u t are necessary f o r e l u t i o n of the s t r o n g l y r e t a i n e d p o l a r c a r o tenoids. The c a r o t e n o i d s I - V e l u t e from DB-WAX w i t h the e x p e c t e d o r d e r based on p o l a r i t y . We have a l s o used l o n g e r (10 and 20 meter) DB-WAX columns u s i n g more o p t i m a l ( l o w e r ) l i n e a r f l o w v e l o c i t i e s , w i t h a t t e n d e n t improvement i n c h r o m a t o g r a p h i c e f f i c i e n c y . However, v e r y h i g h f l u i d d e n s i t i e s (> 0 . 9 g / m l ) and t h e use o f p o l a r m o d i f i e r s ( e . g . 2-5% MeOH) a r e r e q u i r e d f o r e l u t i o n o f the more p o l a r c a r o t e n o i d s from l o n g e r DB-WAX columns. Under t h e s e c o n d i t i o n s , the s t a t i o n a r y phase i s n o t s t a b l e and c o n t i n u o u s d i s s o l u t i o n o f t h e column c o a t i n g l e a d s t o r e p e a t e d p l u g g i n g o f the c a p i l l a r y r e s t r i c t o r . We c o n c l u d e t h a t f u r t h e r improvements i n s t a t i o n a r y phase c r o s s - l i n k i n g t e c h n o l o g y w i l l be r e q u i r e d f o r s u b s t a n t i a l p r o g r e s s i n t h e use o f p o l a r m o d i f i e r s f o r s t r o n g l y p o l a r s o l u t e s . The s t r o n g r e t e n t i o n and h i g h s e l e c t i v i t y of DB-WAX make t h i s phase t h e most p r o m i s i n g one f o r t h e SFC s e p a r a t i o n of c a r o t e n o i d s and a f f o r d the p o s s i b i l i t y o f u s i n g p o l a r m o d i f i e r g r a d i e n t s t o enhance the q u a l i t y of the chromatography. I t i s i m p o r t a n t t o emphasize t h a t s t a t i o n a r y phase s e l e c t i v i t y i s t h e dominant f a c t o r i n t h e s e p a r a t i o n o f t h e s e compounds, w h i c h d i f f e r p r i m a r i l y i n end-group s u b s t i t u t i o n . O t h e r f a c t o r s s u c h as column d i a m e t e r appear t o be l e s s i m p o r t a n t . The advantage of i n c r e a s e d column e f f i c i e n c y o b t a i n e d u s i n g s m a l l e r d i a m e t e r columns ( i . e . 50 μ m) tends t o be outweighed by t h e lower l o a d i n g c a p a c i t y , p a r t i c u l a r l y f o r SFC-MS, because the s i g n a l / n o i s e r a t i o i s l e s s f a v o r a b l e t h a n f o r S F C - F I D and h i g h e r amounts of a n a l y t e a r e required. Due t o t h e i r p o l a r i t y and m o l e c u l a r w e i g h t , the s u c c e s s f u l e l u t i o n o f t h e s e compounds i s c r u c i a l l y dependent on t h e d e s i g n of the flow r e s t r i c t o r . We were n o t a b l e t o use s m a l l d i a m e t e r ( e . g . 5 m i c r o n ) s t r a i g h t c a p i l l a r y r e s t r i c t o r s because o f pronounced s o l u t e p r e c i p i t a t i o n and p l u g g i n g p r o b l e m s . I n s t e a d , we f a b r i c a t e d j e t - t y p e r e s t r i c t o r s d i r e c t l y on t h e columns u s i n g t h e method o f Guthrie ( 3 4 ) · The r e s t r i c t o r s thus formed a r e m e c h a n i c a l l y s t u r d y and t a p e r from t h e column i n t e r n a l d i a m e t e r t o a p p r o x i m a t e l y 1-3 m i c r o n s o v e r a l e n g t h o f 1 mm, p r o v i d i n g n e a r l y i d e a l decompres s i o n of the f l u i d . S i g n i f i c a n t advantages of t h e use o f r e s t r i c t o r s i n t e g r a l w i t h the column a r e the e a s e o f i n t e r f a c i n g t o the mass s p e c t r o m e t e r , t h e absence of c o n n e c t i n g dead volumes and a c t i v e s u r f a c e s and the f a c t t h a t the s t a t i o n a r y phase extends v i r t u a l l y to the p o i n t of d e t e c t i o n . Mass S p e c t r o m e t r y . The use o f s u p e r c r i t i c a l CO2 as a m o b i l e phase p r o v i d e s an e f f i c i e n t means o f t r a n s f e r r i n g t h e s e l a b i l e compounds t o t h e mass s p e c t r o m e t e r i o n s o u r c e . M o d i f i c a t i o n s t o the F i n n i gan 4500 a r e m i n i m a l and f u l l y c o m p a t i b l e w i t h n o r m a l GC-MS o p e r ation. C o n v e r s i o n between GC-MS and SFC-MS modes i n v o l v e s o n l y an i n j e c t o r s u b s t i t u t i o n and a column change. Figure 3 i l l u s t r a t e s t h e r e c o n s t r u c t e d i o n chromatogram o b t a i n e d f o r the s e p a r a t i o n of


12.

FREW ET AL.

SFC-MS

of Carotenoid

Pigments

217

100 Downloaded by CORNELL UNIV on July 20, 2016 | http://pubs.acs.org Publication Date: March 17, 1988 | doi: 10.1021/bk-1988-0366.ch012

fucoxanthin ce- carotene echinenone

1

50

β - carotene

200 10=00

400 20=00

canthaxanthin

ft

600 30=00

800 Scan Number 40=00 Time (min)

F i g u r e 3. SFC-MS t o t a l i o n chromatogram o b t a i n e d f o r s e p a r a t i o n of a c a r o t e n o i d t e s t m i x t u r e on 0V-17 ( c o n d i t i o n s as i n F i g u r e

2A).



218

SUPERCRITICAL FLUID EXTRACTION AND CHROMATOGRAPHY

a simple carotenoid test mixture on a 20 meter χ 100 y m ID OV-17 column under c o n d i t i o n s s i m i l a r t o F i g u r e 2A. Adequate s e n s i t i v i t i e s (S/N - 5-10 for 50-100 ng injected on the column) are obtained using column effluent introduction normal to the quadrupole a x i s . However, we have not yet explored other ion source geometries with respect to optimizing s e n s i t i v i t y and better performance may be possible using a x i a l effluent introduction. Ion source and a n alyzer pressure changes are substantial, increasing from 0.5 to 1.0 t o r r and from 3 to 6 χ 10" ^ t o r r , respectively, during pres sure programming from 80 to 350 bars. However, these pressures are within the normal l i m i t s for chemical i o n i z a t i o n . Background noise does not increase s i g n i f i c a n t l y . Observed CO2 cluster ions (protonated) are not s i g n i f i c a n t above the tetramer. We do not observe a s i g n i f i c a n t effect on s e n s i t i v i t y due to increasing pressure, although Pinkston et a l . (13) have reported lowered sen s i t i v i t i e s f o r t h e i r SFC-MS system at analyzer pressures i n the 10~4 t o r r range. In our experience, SFC pressure programming up to 400 bars using a properly dimensioned r e s t r i c t o r i s compatible with normal mass spectrometer operating pressures. Several workers have reported that a d d i t i o n a l heating of SFC-MS r e s t r i c t o r s i s required as with FID detection (8, 36, 37). In the present work, mass chromâtograms f o r the most polar carotenoids are observed to give smooth e l u t i o n envelopes ( e . g . fucoxanthin; c f . Figures 3 and 6), which suggests that p a r t i c l e formation during decompression i s minimal without additional heating of the r e s t r i c t o r . I t i s poss i b l e , however, that we are detecting only that small percentage of the carotenoid analyte a c t u a l l y i n the gas phase, the remainder forming p a r t i c l e s to which the spectrometer i s i n s e n s i t i v e . Smith et a l . (38) have studied r e s t r i c t o r performance i n d e t a i l with respect to p a r t i c l e nucleation as a function of r e s t r i c t o r geomet r y , temperature, f l u i d flow rates and analyte v o l a t i l i t y . However, effects of r e s t r i c t o r heating on thermolabile compounds have not been extensively evaluated. Pinkston et a l . (13) have reported degradation of benzoyl peroxide used as a test compound when high r e s t r i c t o r temperatures were used f o r SFC-MS. Our attempts to use elevated r e s t r i c t o r temperatures with carotenoids resulted i n reduced s e n s i t i v i t y , due to either analyte p r e c i p i t a t i o n or to decomposition. Thus, although we generally operate our FID detector at 300-350°C, we are not convinced that t h i s additional heating i s required or b e n e f i c i a l i n the case of MS detection, part i c u l a r l y when dealing with thermolabile components. Carotenes, Keto-carotenoids and Carotenoid D i o l s . The CH4-CI mass spectra of beta-carotene ( I ) , echinenone ( I I ) , canthaxanthin (III) and astacene (IV) are shown i n Figure 4. Under the soft i o n i z a t i o n conditions used, these compounds exhibit extremely simple spectra consisting primarily of [M+l] . The usual [M+29] and [M+41] adduct ions are present but are of very low i n t e n s i t y . A small [M+H+(X>2] peak i s also observed f o r some of the compounds. The spectrum of the carotenoid d i o l , zeaxanthin (VIII. Figure 5), i s also very simple, exhibiting a base peak, [M+l]+, along with a prominent [M+l-H20] i o n . The presence +

+

+

+


+

FREW E T AL.

SFC-MS

of Carotenoxd

219

Pigments

(M + H) 537 β-CAROTENE

Ί'

I"'

I ' I

1

I

* I

'*l ' I

1

400

I

1

I

'ι '

I '

,i.

,1 .,

(I)

h, .,

500

600


• ι (M + H) 551

ECHINENONE

1

11

400

(H)

,1'1• 1•1'1 ί

500

ι 700

+

1

700

600

(M+H) 565 CANTHAXANTHIN

(HI)

• I' 1

400

500

ι '

I ' I

I ' I ' ι

600

700

(M+H)+ 593 ASTACENE

(JSL)

'p"

400

500

600

| S

•r-'V'

I· 4

700

m/z F i g u r e 4 · Methane c h e m i c a l i o n i z a t i o n mass s p e c t r a o f s e v e r a l carotenoids. ( S t r u c t u r e s g i v e n i n F i g u r e 1.)


220

SUPERCRITICAL FLUID EXTRACTION AND

Z E A X A N T H I N

CTH)

CHROMATOGRAPHY

(M + H)

569

(M + 1-H 0) 551


2

400

500

ZEAXANTHIN

600

ACETATE

(IX)

(M + 1-CH C00H) 593 3

533 ^ 400

500

700

(M+H) 653 +

I

600

J,

681 700

m/z Figure 5. Methane chemical i o n i z a t i o n mass spectra of the carotenoid d i o l , zeaxanthin and i t s acetylated derivative, zeaxanthin acetate.



12.

FREW E T A L .

SFC-MS

of Carotenoid

Pigments

221

o f two hydroxy1 groups i s n o t a p p a r e n t . The mass s p e c t r u m o f t h e a c e t y l a t e d p r o d u c t , z e a x a n t h i n d i a c e t a t e ( I X ) , however, e x h i b i t s an 84 d a l t o n s h i f t f o r t h e m o l e c u l a r i o n and s e q u e n t i a l l o s s e s of a c e t i c a c i d , c l e a r l y i n d i c a t i n g t h e s t r u c t u r e t o be a d i o l . The s i m p l i c i t y o f t h e c a r o t e n o i d s p e c t r a under t h e s e c o n d i t i o n s l i m i t s the s t r u c t u r a l i n f o r m a t i o n a v a i l a b l e t o the a n a l y s t . As noted e a r l i e r , s t r u c t u r a l i n f o r m a t i o n regarding the carotenoid p o l y e n e c h a i n and e n d - g r o u p s p r o v i d e d by e l e c t r o n i o n i z a t i o n f r a g m e n t a t i o n , may a r i s e l a r g e l y from t h e r m a l d e g r a d a t i o n p r o c e s s e s (31). C a r n e v a l e e t a l . ( 3 2 ) , have demonstrated t h a t s i m i l a r s t r u c t u r a l i n f o r m a t i o n c a n be o b t a i n e d u s i n g c h e m i c a l i o n i z a t i o n , a l t h o u g h heated d i r e c t i n s e r t i o n p r o b e s were a l s o used i n t h e i r study. F u r t h e r r e s e a r c h i s needed t o determine t h e i m p o r t a n c e of t h e r m a l d e g r a d a t i o n p r o c e s s e s ( a p p a r e n t l y m i n i m i z e d i n SFC-MS) i n p r o v i d i n g s t r u c t u r a l I n f o r m a t i o n as opposed t o p r o c e s s e s i n i t i a t e d by i o n i z a t i o n a l o n e . There a r e s e v e r a l p o s s i b i l i t i e s f o r c r e a t i n g more e n e r g e t i c i o n i z a t i o n c o n d i t i o n s , i n c l u d i n g h i g h e r s o u r c e t e m p e r a t u r e s , t h e use o f h y d r o g e n ( l o w e r p r o t o n a f f i n i t y ) as a r e agent gas o r t h e use o f mixed c h e m i c a l i o n i z a t i o n - c h a r g e exchange ionization. I n r e c e n t work, we have noted i n c r e a s e d f r a g m e n t a t i o n o f t h e p o l y e n e backbone f o r some c a r o t e n o i d s ( e . g . e l i m i n a t i o n o f t o l u e n e ) a t h i g h CO2 p r e s s u r e s , a p p a r e n t l y due t o charge e x change. T h i s s u g g e s t s c h a r g e - e x c h a n g e i o n i z a t i o n as a means o f p r o d u c i n g s i g n i f i c a n t l y more f r a g m e n t a t i o n and we a r e a c t i v e l y exploring this area. I t s h o u l d be p o i n t e d o u t , however, t h a t t h e l a c k o f f r a g m e n t a t i o n c a n be used t o advantage i n t h e a n a l y s i s o f complex m i x t u r e s t o o b t a i n m o l e c u l a r i o n abundances w i t h o u t t h e p r o b l e m of i n t e r f e r i n g f r a g m e n t s . M i n i m a l f r a g m e n t a t i o n would a l s o be advantageous f o r tandem mass s p e c t r o m e t r y o f complex c a r o t e n o i d m i x t u r e s , s i n c e s i m p l e m o l e c u l a r i o n s c o u l d be c o l l i s i o n a l l y d i s s o c i a t e d p r i o r t o t h e second a n a l y z e r t o p r o v i d e t h e necessary s t r u c t u r a l information. F u c o x a n t h i n and R e l a t e d P i g m e n t s . F o r some t y p e s o f p i g m e n t s , n o t a b l y t h e f u c o p i g m e n t s , we observe c o n s i d e r a b l e f r a g m e n t a t i o n e v e n under t h e s e m i l d i o n i z a t i o n c o n d i t i o n s . Fucoxanthin ( s t r u c t u r e V ) , an abundant pigment i n marine d i a t o m s , i s e x t r e m e l y f r a g i l e and s u s c e p t i b l e t o r a p i d d e g r a d a t i o n i n t h e o c e a n i c water column by v a r i o u s pathways i n c l u d i n g h y d r o l y s i s , d e h y d r a t i o n and epoxide rearrangement. We have p r e v i o u s l y i d e n t i f i e d f u c o x a n t h i n and i t s d e g r a d a t i o n p r o d u c t s u s i n g a c o m b i n a t i o n of o f f - l i n e HPLC and i n - b e a m d e s o r p t i o n c h e m i c a l i o n i z a t i o n ( 3 9 ) . The q u a l i t y o f t h e s p e c t r a and d e t e c t i o n l i m i t s o b t a i n e d by t h i s method v a r y c o n s i d e r a b l y and a r e s t r o n g l y dependent o n f a c t o r s s u c h as probe h e a t i n g r a t e , c o n d i t i o n o f t h e probe t i p and d i s t r i b u t i o n o f sample on t h e t i p . Here we compare r e s u l t s o f t h e d e s o r p t i o n CI probe method w i t h some o f o u r SFC-MS r e s u l t s . The SFC-MS a n a l y s i s o f a 100 ng sample o f f u c o x a n t h i n i s shown i n F i g u r e 6. F u c o x a n t h i n e l u t e s as a s h a r p c h r o m a t o g r a p h i c peak and produces a v e r y h i g h q u a l i t y s p e c trum e x h i b i t i n g a n i n t e n s e p r o t o n a t e d m o l e c u l a r i o n a t m/z 659 (Figure 6). O t h e r k e y d i a g n o s t i c fragments ( c f . F i g u r e 7) i n c l u d e [ M + l - n l 8 ] + a t m/z 641 and 623, [ M + l - 6 0 ] a t m/z 599, [ M + l - 6 0 - n l 8 ]+ a t m/z 581 and 563, [M+l-170]+ a t m/z 4 8 9 , [M+l-170-60]+ a t m/z 429 +


222

SUPERCRITICAL FLUID EXTRACTION AND CHROMATOGRAPHY 100

TOTAL ION CHROMATOGRAM


50 J

0

20=00 290

1320 260

640 230

0 200 100 η

26=40 320

TIME (min) PRESSURE (bars)

581

CH -CI 4

659 641

50 489 599 429 411 11 ι

563

50(Γ

400

I

623

687

700

600

m/z

F i g u r e 6 . SFC-MS t o t a l i o n c u r r e n t chromatogram and methane c h e m i c a l i o n i z a t i o n spectrum o f f u c o x a n t h i n .

m/z 659

623-

-641-

-HOAc -H0 2

•599 F i g u r e 7.

-H 0

—581

2

—563

Schematic f r a g m e n t a t i o n o f f u c o x a n t h i n .


12.

FREW E T A L .

SFC-MS

of Carotenoid

Pigments

223

and [M+l-170-60-18]+ a t m/z 4 1 1 . The e f f e c t s o f t h e r m a l d e g r a d a t i o n a r e a b s e n t and n o i s e due t o background i s n e g l i g i b l e . In c o n t r a s t , F i g u r e 8 shows r e s u l t s w h i c h were o b t a i n e d by i n - b e a m d e s o r p t i o n CH4-CI from a s i m i l a r amount (100 ng) o f f u c o x a n t h i n . The mass chromatograms f o r t h e [ M + l ] i o n (m/z 659) and [M+l-18]+ i o n ( m / z 641) i n d i c a t e t h e p r e s e n c e o f b o t h f u c o x a n t h i n and f u c o x a n t h i n d e h y d r a t e , r a i s i n g t h e q u e s t i o n o f whether t h e dehydrate i s p r e s e n t i n t h e o r i g i n a l sample o r i s produced by t h e r m a l d e h y d r a t i o n on t h e p r o b e . F u r t h e r m o r e , t h e mass s p e c t r a t a k e n o v e r d i f f e r e n t i n t e r v a l s e x h i b i t s i g n i f i c a n t v a r i a t i o n s and d i s p l a y e x t r a n e o u s fragments due t o t h e r m a l d e g r a d a t i o n and reduced i n t e n s i t i e s f o r molecular i o n s . T h u s , SFC-MS appears t o be a more r e l i a b l e means f o r i n t e r p r e t i n g fucopigment s t r u c t u r e s from r e l a t i v e intensity information. F r a g m e n t a t i o n p a t t e r n s o b t a i n e d by SFC-MS f o r t h e r e l a t e d f u c o p i g m e n t s , i s o f u c o x a n t h i n ( s t r u c t u r e V I ) and f u c o x a n t h i n - 3 - a c e t a t e ( s t r u c t u r e V I I ) , a r e i l l u s t r a t e d i n F i g u r e 9. The h i g h q u a l i t y o f these spectra again r e f l e c t the gentle i o n i z a t i o n conditions a c h i e v a b l e w i t h CO2 SFC/CH4-CI MS. A s expected f o r i s o f u c o x a n t h i n , no i o n i s p r e s e n t a t m/z 4 8 9 , showing c l e a r l y t h a t t h e 5 , 6 - e p o x y group i s a b s e n t . L o s s o f ketene ([M+I-CH2CO] ) i s a l s o a p p a r e n t from t h e i o n a t m/z 6 1 7 . F r a g m e n t a t i o n o f f u c o x a n t h i n - 3 - a c e t a t e produces r e l a t i v e i n t e n s i t i e s f o r t h e major i o n s ( e x c e p t f o r [M+1-2H20]*) w h i c h a r e v e r y s i m i l a r t o t h o s e o b t a i n e d f o r f u c o xanthin.


+

+

Summary and C o n c l u s i o n s The c a r o t e n o i d s , l a b i l e pigments w h i c h c o n t a i n m u l t i p l e f u n c t i o n a l groups s p a n n i n g a range o f p o l a r i t i e s a r e n o r m a l l y c o n s i d e r e d t o f a l l w i t h i n t h e sphere o f HPLC a n a l y s i s . The p r e l i m i n a r y r e s u l t s r e p o r t e d here show t h a t s e p a r a t i o n s o f t h e s e compounds a r e a l s o p o s s i b l e u s i n g s u p e r c r i t i c a l f l u i d chromatography w i t h CO2 as t h e mobile phase and t h a t t h i s t e c h n i q u e e v e n t u a l l y may be com p e t i t i v e w i t h HPLC. U s i n g n o n - p o l a r s t a t i o n a r y p h a s e s , even r e l a t i v e l y p o l a r c a r o t e n o i d s c a n be e l u t e d w i t h pure s u p e r c r i t i c a l carbon dioxide alone. However, t h e p o t e n t i a l advantage o f SFC i n terms o f column e f f i c i e n c y has n o t y e t been a t t a i n e d e x p e r i m e n t a l l y i n t h e case o f t h e c a r o t e n o i d s . The p r a c t i c a l r e s o l u t i o n and s e l e c t i v i t i e s o b s e r v e d f o r t h e s e compounds on t h e s t a t i o n a r y phases s t u d i e d a r e as y e t r a t h e r l i m i t e d compared w i t h t h o s e o b s e r v e d f o r HPLC u s i n g bonded o c t a d e c y l o r η - p r o p y l amino columns, and a r e n o t y e t adequate f o r d e a l i n g w i t h complex c a r o t e n o i d m i x t u r e s i s o l a t e d from n a t u r a l s o u r c e s . A l s o , SFC-FID d e t e c t i o n l i m i t s w i l l need t o be improved by a f a c t o r o f 5-10 i n o r d e r t o match t h e low nanogram l e v e l c u r r e n t l y p o s s i b l e w i t h HPLC. T h u s , from a c h r o m a t o g r a p h i c p e r s p e c t i v e , HPLC w i l l r e m a i n t h e method o f c h o i c e u n t i l improvements i n SFC columns, as w e l l as i n SFC i n j e c t i o n methods and d e t e c t o r d e s i g n a r e a c h i e v e d . Present l i m i t a t i o n s o n t h e use o f p o l a r f l u i d s and thus t h e p o l a r i t y l i m i t s amenable t o SFC-MS, imposed by t h e c u r r e n t l y a v a i l a b l e s e l e c t i o n and s t a b i l i t y o f p o l a r s t a t i o n a r y p h a s e s , w i l l undoubtedly be eased by f u r t h e r r e s e a r c h i n s t a t e - o f - t h e - a r t column t e c h n o l o g y . The



224


100

η

|413

CH -CI 4

50 Η

SCANS 50-58

641

581 489

659

I

400 100413

5 6 7

I

6

599

A * ^ , , ^ M f

500

5

?

i M»

600

I 700

m/z

700

m/z

CH -CI SCANS 65-70 4

565 581

473

50-

400

623 y 6

™

500

600

F i g u r e 8· T o t a l i o n chromatogram, mass chroma t o g rams and s e l e c t e d scans o b t a i n e d f o r in-beam d e s o r p t i o n CH4-CI o f fucoxanthin*


FREW ET AL.

SFC-MS

of Carotenoid

Pigments

100 CH4-CI

581

641

ISOFUCOXANTHIN YL


50 i

800

F i g u r e 9· Methane c h e m i c a l i o n i z a t i o n mass s p e c t r a o f t h e fucopigments i s o f u c o x a n t h i n and f u c o x a n t h i n - 3 - a c e t a t e .



226

SUPERCRITICAL FLUID EXTRACTION AND CHROMATOGRAPHY

most p r o m i s i n g s t a t i o n a r y phases f o r c a p i l l a r y SFC o f complex n a t u r a l m i x t u r e s o f c a r o t e n o i d s appear t o be c y a n o p r o p y l p o l y 8 i l o x a n e and p o l y e t h y l e n e g l y c o l phases* A major advantage o f SFC a n a l y s i s o f c a r o t e n o i d s i s t h e comp a t i b i l i t y o f SFC w i t h mass s p e c t r o m e t r y . We have shown h e r e t h a t combined SFC-MS i s a u s e f u l t e c h n i q u e f o r t h e i d e n t i f i c a t i o n o f t h e s e compounds i n m i x t u r e s . I n comparison w i t h in-beam desorpt i o n c h e m i c a l i o n i z a t i o n t e c h n i q u e s , t h e SFC-MS method p r o d u c e s superior quality spectra at s i m i l a r s e n s i t i v i t y l e v e l s , p a r t i c u l a r l y f o r v e r y l a b i l e pigments s u c h as f u c o x a n t h i n and i t s d e r i v a tives. The lower t e m p e r a t u r e s p r e v a l e n t i n t h e i o n s o u r c e due t o t h e c o o l i n g e f f e c t o f CO2 e x p a n s i o n from t h e r e s t r i c t o r appear to r e s u l t i n extremely m i l d i o n i z a t i o n c o n d i t i o n s . With the exception of the fucopigments, CH4-CI fragmentation of the other c a r o t e n o i d s s t u d i e d i s m i n i m a l . The s i m p l i c i t y o f t h e s e s p e c t r a may be advantageous i n d e t e r m i n i n g low l e v e l d i s t r i b u t i o n s o f t h e s e pigments u s i n g t h e i r m o l e c u l a r i o n a b u n d a n c e s . Further structural c h a r a c t e r i z a t i o n would r e q u i r e more e n e r g e t i c i o n i z a t i o n c o n d i t i o n s o r tandem mass s p e c t r o m e t r y i n c o m b i n a t i o n w i t h c o l l i s i o n - i n d u c e d dissociation. F i n a l l y , direct s u p e r c r i t i c a l f l u i d injection-mass s p e c t r o m e t r y ( 4 0 , 4 1 ) , i n v o l v i n g no c h r o m a t o g r a p h i c s e p a r a t i o n , and t h e r e f o r e , s h o r t a n a l y s i s t i m e s , o f f e r s a v e r y a t t r a c t i v e a l t e r n a t i v e f o r t h e a n a l y s i s o f pure c a r o t e n o i d f r a c t i o n s and simple carotenoid m i x t u r e s . Acknowledgments We thank D r e . D . H . Répéta and J . E r t e l f o r p r o v i d i n g a u t h e n t i c c a r o t e n o i d s t a n d a r d s and f o r h e l p f u l d i s c u s s i o n s d u r i n g t h e c o u r s e o f t h i s work. T h i s r e s e a r c h has been s u p p o r t e d by t h e O c e a n i c C h e m i s t r y Program, O f f i c e o f N a v a l R e s e a r c h , under c o n t r a c t N00014-85-C-001. Woods H o l e Océanographie I n s t i t u t i o n C o n t r i b u t i o n N o . 6510. Literature 1. 2. 3. 4. 5. 6. 7. 8.

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