Supercritical Fluid Chromatography with Fourier Transform Infrared

Chapter 13 Supercritical Fluid Chromatography with Fourier Transform Infrared Detection Richard C. Wieboldt and James A. Smith 1

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Spectroscopy Research Center, Nicolet Instrument Corporation, Madison, WI 53711 Winton Hill Technical Center, The Procter & Gamble Company, Cincinnati, OH 45224

Downloaded by NATL TAIWAN UNIV on July 1, 2015 | http://pubs.acs.org Publication Date: March 17, 1988 | doi: 10.1021/bk-1988-0366.ch013

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This paper describes the design of a packed column and a capillary column SFC/FT-IR system using a flow-through FT-IR analysis cell and their application to citrus oil and pyrethrin analyses. FT-IR detection with density programmed SFC separations is enhanced using a modified Gram-Schmidt algorithm. Infrared absorption bands due to the density changes in the supercritical carbon dioxide mobile phase are removed by spectral subtraction. FT-IR spectra collected across an unresolved chromatographic peak are used to resolve and quantitate the components of a peak in the citrus oil extract. Pyrethrin II is separated from a pyrethrin extract without thermal degradation using mild SFC conditions. FT-IR spectra have sufficient signal-to-noise ratio to clearly distinguish the cinerin and pyrethrin components. Infrared spectroscopy i s probably the most widespread a n a l y t i c a l spectroscopic technique for i d e n t i f i c a t i o n and characterization of organic compounds. Because of this i d e n t i f i c a t i o n capability/ infrared spectroscopy i s desirable as a detection technique for chromatographic separations. With the advent of Fourier transform infrared spectroscopy, the speed and s e n s i t i v i t y of infrared detection i s greatly enhanced making such applications feasible. FT-IR detection has been widely accepted as a detector for gas chromatography (GC/FT-IR) ( 1 ) and has been applied with l i m i t e d success to l i q u i d chromatography (LC/FT-IR) ( 2 ) , and more recently to s u p e r c r i t i c a l f l u i d chromatography (SFC/FT-IR) ( 3 ) . The recent review a r t i c l e s c i t e d here provide excellent introduction and references to current state-of-the-art i n these areas. In addition to i d e n t i f i c a t i o n , FT-IR i s equally useful i n i t s a b i l i t y to function as a chemically s p e c i f i c detector for chromatographic effluents. The presence of absorption bands i n c e r t a i n regions of the infrared spectrum are c h a r a c t e r i s t i c of 0097-6156/88/0366-0229506.00/0 ©

1988

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

In Supercritical Fluid Extraction and Chromatography; Charpentier, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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p a r t i c u l a r f u n c t i o n a l groups. For example, the C=0 bond s t r e t c h ( c a r b o n y l f u n c t i o n a l group) causes an a b s o r p t i o n band between 1825-1645 c m i n the m i d - i n f r a r e d spectrum. The e x a c t p o s i t i o n , say 1760 c m v e r s u s 1720 c m , serves to f u r t h e r d i s t i n g u i s h between a c a r b o x y l i c a c i d and a k e t o n e . There are many such c h a r a c t e r i s t i c r e g i o n s i n i n f r a r e d s p e c t r a , and i t i s the p r e s e n c e or absence o f a b s o r p t i o n bands i n t h e s e r e g i o n s which makes i n f r a r e d so u s e f u l f o r s t r u c t u r a l e l u c i d a t i o n . The FT-IR s p e c t r o m e t e r can be used t o m o n i t o r any o r s e v e r a l of these c h a r a c t e r i s t i c regions i n r e a l time. I n t h i s mode the FT-IR i s not o n l y c o l l e c t i n g f u l l i n f r a r e d s p e c t r a f o r l a t e r i d e n t i f i c a t i o n , b u t i t i s a l s o a c t i n g as a f u n c t i o n a l group s p e c i f i c detector. T h i s i s somewhat analogous t o s i n g l e i o n m o n i t o r i n g i n mass s p e c t r o m e t r y w i t h the advantage t h a t an i n f r a r e d a b s o r p t i o n band i s o f t e n more c h a r a c t e r i s t i c o f a c l a s s o f compounds t h a n i s a s i n g l e mass i o n . FT-IR i s o f t e n compared w i t h mass s p e c t r o s c o p y as a chromatographic d e t e c t o r . Each t e c h n i q u e p r o v i d e s u n i q u e i n f o r m a t i o n which i s o f t e n complementary. IR p r o v i d e s c l e a r c h e m i c a l i n f o r m a t i o n w h i l e MS p r o v i d e s c l e a r m o l e c u l a r w e i g h t and s t r u c t u r a l information. Taken t o g e t h e r , the i n f o r m a t i o n g r e a t l y s i m p l i f i e s the d e t e r m i n a t i o n o f unknowns and almost always provides s u f f i c i e n t information for p o s i t i v e i d e n t i f i c a t i o n . -1


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FT-IR D e t e c t i o n Methods. T h e r e are two methods f o r m o n i t o r i n g the i n f r a r e d a b s o r p t i o n of a chromatographic e f f l u e n t : flow-through c e l l (4,5) and e f f l u e n t d e p o s i t i o n ( 6 ) . F l o w - t h r o u g h c e l l d e t e c t i o n r e q u i r e s t h a t the background remain c o n s t a n t t h r o u g h o u t the e x p e r i m e n t . W i t h GC/FT-IR t h i s i s not a problem because the c a r r i e r gas stream i s an i n e r t gas h a v i n g no i n f r a r e d absorbance i n the mid-IR. T h i s r e q u i r e m e n t i s a s e v e r e r e s t r i c t i o n f o r l i q u i d chromatography where the m o b i l e phase s o l v e n t t y p i c a l l y has s i g n i f i c a n t i n f r a r e d a b s o r p t i o n o f i t s own. As a r e s u l t , the u s e f u l n e s s o f f l o w - c e l l LC/FT-IR i s v e r y l i m i t e d . The o t h e r method f o r o b t a i n i n g IR s p e c t r a o f c h r o m a t o g r a p h i c e f f l u e n t s i s t o d e p o s i t the e l u t e d m a t e r i a l on a s u r f a c e , d r i v e o f f the m o b i l e phase, and examine the r e s i d u a l sample by FT-IR. T h i s a p p r o a c h c i r c u m v e n t s the m o b i l e phase a b s o r p t i o n problem b u t i s cumbersome t o c a r r y out i n p r a c t i c e . T e c h n i q u e s such as t h e s e have been employed f o r LC/FT-IR a n a l y s i s (7,8,9) and d e m o n s t r a t e d w i t h SFC/FT-IR ( 1 0 ) . One o f the o b j e c t i v e s o f t h i s s t u d y i s t o demonstrate the f e a s i b i l i t y o f u s i n g f l o w - t h r o u g h c e l l d e t e c t i o n w i t h SFC/FT-IR. The s u c c e s s or f a i l u r e o f f l o w c e l l d e t e c t i o n depends on the a b i l i t y t o cope w i t h the a b s o r p t i o n due t o the s u p e r c r i t i c a l f l u i d m o b i l e phase. Because c a r b o n d i o x i d e i s used as the s u p e r c r i t i c a l f l u i d i n the m a j o r i t y o f a p p l i c a t i o n s , i t i s i m p o r t a n t t o demonstrate i t s v i a b i l i t y w i t h FT-IR d e t e c t i o n . The a b s o r p t i o n spectrum o f s u p e r c r i t i c a l c a r b o n d i o x i d e i n the m i d - i n f r a r e d i s c o m p a t i b l e w i t h the d e t e c t i o n o f most c h e m i c a l species. F i g u r e 1 shows the a b s o r p t i o n bands f o r s u p e r c r i t i c a l c a r b o n d i o x i d e i n the mid-IR. The r e g i o n s from 3800-3500 cm and 2500-2200 cm" are t o t a l l y b l o c k e d by the c a r b o n d i o x i d e and no u s e f u l i n f o r m a t i o n about sample a b s o r p t i o n i s a v a i l a b l e . A -1

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p a i r o f weak a b s o r p t i o n bands a t 1381 and 1277 cm"* a r e a l s o present i n the spectrum. These a r e c a u s e d b y a F e r m i r e s o n a n c e i n t e r a c t i o n between t h e O - C - 0 symmetric s t r e t c h , v ^ , a t 1330 cm" . The fundamental i s n o t IR a c t i v e w h i c h i s why t h e r e i s n o t a s t r o n g a b s o r p t i o n band i n t h i s r e g i o n . The Fermi resonance a b s o r p t i o n b a n d s , w h i c h o b v i o u s l y a r e IR a c t i v e , increase i n i n t e n s i t y with d e n s i t y of the s u p e r c r i t i c a l CO2 f l u i d ( 1 1 ) . T h i s a b s o r p t i o n becomes t h e l i m i t i n g f a c t o r i n s e l e c t i n g the p a t h l e n g t h f o r the flow-through a n a l y s i s c e l l . In o r d e r t o use t h i s r e g i o n o f t h e IR s p e c t r u m , t h e r e must be enough a n a l y t i c a l r a d i a t i o n remaining a f t e r the s u p e r c r i t i c a l f l u i d absorbance t o o b t a i n adequate s e n s i t i v i t y f o r a p a r t i c u l a r application. The s u p e r c r i t i c a l CO2 a b s o r p t i o n bands change i n i n t e n s i t y as a f u n c t i o n o f d e n s i t y b u t t h e band shape does n o t change - a t l e a s t n o t a t t h e 8 c m " s p e c t r a l r e s o l u t i o n t y p i c a l l y used f o r this application. As a r e s u l t , i t i s a s i m p l e m a t t e r t o s u b t r a c t the s u p e r c r i t i c a l c a r b o n d i o x i d e a b s o r p t i o n spectrum from an F T - I R d a t a f i l e c o l l e c t e d d u r i n g an S F C / F T - I R e x p e r i m e n t . The s u b t r a c t i o n f a c t o r i s a d j u s t e d t o e x a c t l y compensate f o r t h e Fermi resonance a b s o r p t i o n . The r e s u l t i n g spectrum w i l l t h e n c o n t a i n o n l y a b s o r p t i o n bands due t o o t h e r components, i f a n y , e n t r a i n e d i n the s u p e r c r i t i c a l f l u i d . The r e g i o n s from 3800-3500 c m " and from 2500-2200 c m appear as gaps i n t h e spectrum because t h e s u p e r c r i t i c a l c a r b o n d i o x i d e absorbs a l l t h e a v a i l a b l e i n f r a r e d r a d i a t i o n i n these r e g i o n s . 1


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Experimental Two d i f f e r e n t S F C / F T - I R systems were used f o r t h i s work. System 1 was used f o r t h e packed column a n a l y s i s o f v o l a t i l e c i t r u s o i l components. System 2 was used f o r t h e p e s t i c i d e and p y r e t h r i n extract analyses. System 1. T h i s system c o n s i s t s o f an HP1082B l i q u i d chromatograph ( H e w l e t t - P a c k a r d , P a l o A l t o , CA) m o d i f i e d f o r s u p e r c r i t i c a l C 0 o p e r a t i o n , and a 60SX F T - I R s p e c t r o m e t e r ( N i c o l e t I n s t r u m e n t C o r p . , M a d i s o n , WI). The HP1082B system uses a manual b a c k p r e s s u r e r e g u l a t o r t o m a i n t a i n p r e s s u r e t h r o u g h o u t t h e SFC system. As a r e s u l t , i t i s l i m i t e d t o a p p l i c a t i o n s w h i c h do n o t r e q u i r e d e n s i t y o r p r e s s u r e programming. The h i g h p r e s s u r e UV f l o w - t h r o u g h c e l l s u p p l i e d w i t h t h e chromatograph was used f o r F T - I R d e t e c t i o n a f t e r r e p l a c i n g t h e s t a n d a r d q u a r t z windows w i t h i n f r a r e d t r a n s p a r e n t z i n c s e l e n i d e windows. T h i s c e l l was p l a c e d i n t h e 60SX microbeam sample compartment w h i c h uses 6x beam c o n d e n s i n g o p t i c s and a narrow band MCT detector. The system a l l o w e d one i n f r a r e d spectrum t o be c o l l e c t e d p e r second w i t h 8 i n t e r f e r o g r a m s a v e r a g e d p e r s p e c t r u m . The i n j e c t o r (Rheodyne #7520, 0.5 u l sample l o o p ) and column oven were mounted next t o t h e F T - I R s p e c t r o m e t e r t o reduce p o s t - c o l u m n dead volume. 2

C i t r u s O i l Sample. A s y n t h e t i c t e s t m i x t u r e o f 10 v o l a t i l e o i l components was p r e p a r e d . The h i g h e s t m o l e c u l a r w e i g h t


citrus

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compound was d - l i m o n e n e (136.24 g / m o l e ) and t h e l o w e s t m o l e c u l a r w e i g h t compound p r e s e n t was w a t e r . Limonene was 85% o f t h e sample w e i g h t and t h e 0 . 5 u l sample was i n j e c t e d n e a t onto a 5 m i c r o n p a r t i c l e s i z e PRP-1 column ( H a m i l t o n , 4 mm i . d . χ 15 cm l e n g t h ) . S u p e r c r i t i c a l c a r b o n d i o x i d e was pumped a t a r a t e o f 1 m l / m i n a t a c o n s t a n t system t e m p e r a t u r e o f 5 0 ° C . The column head p r e s s u r e was 1750 p s i w h i l e t h e system b a c k p r e s s u r e was m a i n t a i n e d a t 1400 psi. System 2. T h i s system c o n s i s t s o f a Model 501 s u p e r c r i t i c a l f l u i d chromatograph (Lee S c i e n t i f i c , S a l t Lake C i t y , UT) and a N i c o l e t 20 SXC F T - I R s p e c t r o m e t e r . The S F C / F T - I R i n t e r f a c e i s a p r o t o t y p e d e s i g n c o n t a i n i n g a 600 urn I . D . b y 5mm p a t h l e n g t h S F C / F T - I R f l o w - t h r o u g h c e l l , narrow band MCT d e t e c t o r , and o p t i c s d e s i g n e d t o match t h e c o l l i m a t e d beam from t h e main bench t o t h e f l o w c e l l and d e t e c t o r . The S F C / F T - I R f l o w c e l l i s c o n n e c t e d i n - l i n e a t t h e end o f t h e c a p i l l a r y SFC s e p a r a t i o n column and b e f o r e t h e e n d - o f - c o l u m n restrictor. The column and r e s t r i c t o r a r e l o c a t e d i n t h e chromatograph oven compartment. The f l o w c e l l i s housed i n an i n t e r f a c e module between t h e s p e c t r o m e t e r and c h r o m a t o g r a p h . It i s c o n n e c t e d by two l e n g t h s o f 100 urn I . D . d e a c t i v a t e d f u s e d s i l i c a t u b i n g u s i n g a s e t o f z e r o dead volume f i t t i n g s ( S c i e n t i f i c G l a s s E n g i n e e r i n g , A u s t i n , T X ) . The t r a n s f e r l i n e s and c e l l body were n o t h e a t e d f o r t h e s e e x p e r i m e n t s . The 20SX F T - I R s p e c t r o m e t e r was s e t up t o c o l l e c t 8 c m " r e s o l u t i o n s p e c t r a w i t h 8 scans coadded p e r f i l e . D a t a f i l e s were s t o r e d on magnetic d i s k w i t h a time r e s o l u t i o n between f i l e s o f 2.27 s e c o n d s . 1

P e s t i c i d e Sample. The p e s t i c i d e m i x t u r e c o n s i s t s o f a l d i c a r b ( 5 . 6 m g / m l ) , methomyl ( 5 . 3 m g / m l ) , c a p t a n ( 5 . 0 m g / m l ) , and phenmedipham ( 5 . 1 mg/ml) p r e p a r e d i n d i c h l o r o m e t h a n e . Samples were p r o v i d e d c o u r t e s y o f Lee S c i e n t i f i c . T h i s sample was s e p a r a t e d u s i n g an S B - M e t h y l - 1 0 0 10 meter 100 m i c r o n I . D . column w i t h a 0 . 5 m i c r o n f i l m (Lee S c i e n t i f i c ) . The chromatograph was programmed from an i n i t i a l d e n s i t y o f 0.180 g / m l t o 0.360 g / m l a t 0.010 g / m l / m i n a f t e r a 6.0 minute i n i t i a l h o l d . The program r a t e was t h e n i m m e d i a t e l y i n c r e a s e d t o 0.040 g / m l / m i n t o a f i n a l d e n s i t y o f 0.600 g / m l and h e l d f o r 10 m i n u t e s . The oven t e m p e r a t u r e was m a i n t a i n e d a t 100°C t h r o u g h o u t t h e e x p e r i m e n t . The sample was d e l i v e r e d t o t h e column u s i n g a 200 nL i n j e c t i o n l o o p and a 2 2 : 1 split ratio. P y r e t h r i n sample. The p y r e t h r i n sample c o n s i s t s o f 54 mg o f a c o m m e r c i a l l y a v a i l a b l e 20% p y r e t h r i n e x t r a c t , p r o v i d e d c o u r t e s y o f Adams V e t e r i n a r y L a b o r a t o r y , p r e p a r e d i n 1.0 ml m e t h a n o l . This sample was s e p a r a t e d u s i n g t h e same column d e s c r i b e d a b o v e . The chromatograph was programmed from an i n i t i a l d e n s i t y o f 0.180 g / m l t o 0.700 g / m l a t 0.020 g / m l / m i n a f t e r a 6.0 minute i n i t i a l h o l d . The f i n a l d e n s i t y was h e l d f o r 5.0 m i n u t e s . The oven t e m p e r a t u r e was m a i n t a i n e d a t 100°C t h r o u g h o u t t h e e x p e r i m e n t . The sample was d e l i v e r e d t o t h e column u s i n g a 200 nL i n j e c t i o n l o o p and a 25:1 s p l i t r a t i o .


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233

and D i s c u s s i o n

Comparison o f FID and F T - I R D e t e c t i o n . F i g u r e 2a shows t h e flame i o n i z a t i o n d e t e c t o r (FID) chromatogram from t h e c a p i l l a r y SFC s e p a r a t i o n of the p e s t i c i d e mixture. T h i s t r a c e was o b t a i n e d w i t h o u t t h e S F C / F T - I R f l o w c e l l by c o n n e c t i n g t h e c a p i l l a r y s e p a r a t i o n column d i r e c t l y t o t h e e n d - o f - c o l u m n r e s t r i c t o r mounted i n t h e F I D . T h i s s e r v e s as a r e f e r e n c e t o show t h e chromatographic s e p a r a t i o n obtained before connection to the SFC/FT-IR i n t e r f a c e . F i g u r e 2b i s t h e Gram-Schmidt r e c o n s t r u c t e d chromatogram (GSR) g e n e r a t e d from t h e s t o r e d F T - I R d a t a c o l l e c t e d d u r i n g t h e separation. Gram-Schmidt r e c o n s t r u c t i o n i s a t e c h n i q u e used i n G C / F T - I R a p p l i c a t i o n s t o produce a chromatogram by comparing i n f r a r e d d a t a c o l l e c t e d d u r i n g a G C / F T - I R r u n w i t h background d a t a c o l l e c t e d at the beginning of the run (12). The Gram-Schmidt response i s completely n o n - s p e c i f i c ; t h a t i s , i t produces a r e s p o n s e whenever t h e r e i s any change i n t h e i n f r a r e d s i g n a l which i s d i f f e r e n t from t h e background s c a n s . I n t h i s c a s e , t h e GSR responds p r i m a r i l y t o t h e c h a n g i n g d e n s i t y o f t h e s u p e r c r i t i c a l carbon d i o x i d e . The two programmed d e n s i t y ramps and t h e i n i t i a l and f i n a l h o l d p e r i o d s a r e c l e a r l y d i s p l a y e d . I t i s easy t o d e t e c t t h e s o l v e n t peak a t 13.23 minutes b u t d i f f i c u l t t o d e t e c t the p r e s e n c e o f t h e f o u r p e s t i c i d e p e a k s . C l e a r l y some o t h e r a p p r o a c h i s r e q u i r e d t o enhance d e t e c t i o n . F i g u r e 3 shows t h e GSR (a) w i t h a m o d i f i e d Gram-Schmidt r e c o n s t r u c t e d chromatogram (b) a f t e r compensating f o r t h e c h a n g i n g CO2 d e n s i t y . The m o d i f i c a t i o n c o n s i s t s o f i n c l u d i n g i n f r a r e d d a t a c o l l e c t e d a t t h e h i g h e r d e n s i t y i n t h e s e t o f background c o n d i t i o n s u s e d f o r t h e Gram-Schmidt c a l c u l a t i o n ( 1 3 ) . Because h i g h d e n s i t y s u p e r c r i t i c a l CO2 i s now d e f i n e d as p a r t o f t h e background c o n d i t i o n s , t h e Gram-Schmidt a l g o r i t h m i g n o r e s t h e changing s u p e r c r i t i c a l f l u i d d e n s i t y . The r e s u l t i s a chromatogram w h i c h enhances t h e o t h e r c h r o m a t o g r a p h i c peaks and c l e a r l y r e v e a l s the e l u t i o n of the four p e s t i c i d e s . For lack of any b e t t e r t e r m , t h i s chromatogram i s r e f e r r e d t o as t h e Gram-Schmidt P l u s r e c o n s t r u c t i o n (GSP). The p r i m a r y f u n c t i o n o f t h e GSP chromatogram i s t o d e t e r m i n e w h i c h o f t h e hundreds o f S F C / F T - I R d a t a f i l e s c o n t a i n F T - I R s p e c t r a c o l l e c t e d d u r i n g e l u t i o n of chromatographic peaks. The r e c o n s t r u c t i o n i n F i g u r e 3 b , f o r example, i n d i c a t e s t h a t F T - I R s p e c t r a f o r t h e peak a t 24.09 minutes a r e c o n t a i n e d i n f i l e s 653-665. W i t h t h i s i n f o r m a t i o n one c a n q u i c k l y r e t r i e v e t h e F T - I R spectrum o f t h i s component. F i g u r e 4b shows t h e %T IR spectrum f o r t h e peak a t 24.09 minutes. Because o f t h e d e n s i t y programming, t h i s spectrum was c o l l e c t e d a t a h i g h e r CO2 d e n s i t y t h a n t h a t o f t h e background scans o b t a i n e d a t t h e b e g i n n i n g o f t h e r u n . As a r e s u l t , t h e dominant s p e c t r a l f e a t u r e s a r e t h o s e o f t h e c a r b o n d i o x i d e . F i g u r e 4a i s t h e spectrum o f s u p e r c r i t i c a l CO2 (same as F i g u r e 1) p l o t t e d t o v i s u a l l y compare t h e two s p e c t r a . I t i s important to r e a l i z e t h a t t h e Gram-Schmidt P l u s a l g o r i t h m removes t h e CO2 i n t e r f e r e n c e from t h e r e c o n s t r u c t e d chromatograms o n l y . I t does n o t a f f e c t t h e a c t u a l F T - I R s p e c t r a i n any way.


234

SUPERCRITICAL FLUID EXTRACTION AND CHROMATOGRAPHY


LO

WRVENUMBER F i g u r e 1. I n f r a r e d spectrum o f s u p e r c r i t i c a l c a r b o n showing l o c a t i o n o f major a b s o r p t i o n b a n d s .

dioxide

F i g u r e 2. a) FID chromatogram from c a p i l l a r y SFC s e p a r a t i o n o f pesticide mixture, b) Gram-Schmidt r e c o n s t r u c t e d chromatogram o f t h e same s e p a r a t i o n .



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WIEBOLDT AND SMITH

(ΓΟΟ

SFC with FTIR

Detection

235

δ! 0 0 Ϊ Ο . ' ό ο ' " ' 'Ϊδ'.Όο' ' ' 20.00' "25. 00' RETENTION TIME (MIN) 134t

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700

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926

35.00 ÎO^O

F i g u r e 3. a) Gram-Schmidt r e c o n s t r u c t i o n from F i g u r e 3b. b) Gram-Schmidt P l u s r e c o n s t r u c t e d chromatogram o f t h e same separation.

in

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2000 2^00 2000 NAVENUMBER

1600

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F i g u r e 4. a) I n f r a r e d spectrum o f s u p e r c r i t i c a l c a r b o n dioxide, b) FT-IR spectrum from peak a t 24.09 minutes i n p e s t i c i d e SFC/FT-IR s e p a r a t i o n .


236


Because the s p e c t r a l f e a t u r e s o f s u p e r c r i t i c a l c a r b o n d i o x i d e a r e w e l l d e f i n e d , i t i s a s i m p l e m a t t e r t o remove them, e i t h e r m a n u a l l y o r a u t o m a t i c a l l y , by s p e c t r a l s u b t r a c t i o n . F i g u r e 5 i s t h e spectrum from F i g u r e 4b w i t h the CO2 absorbance removed. There i s c l e a r l y s u f f i c i e n t s p e c t r a l i n f o r m a t i o n t o r e v e a l the c h a r a c t e r i s t i c a b s o r p t i o n bands w h i c h i d e n t i f y t h i s component as aldicarb. I t i s i m p o r t a n t t o p o i n t out t h a t the upper b l a n k e d r e g i o n o f CO2 does n o t b l o c k d e t e c t i o n o f t h e N-H s t r e t c h absorbance.


A n a l y s i s o f V o l a t i l e C i t r u s O i l Components F i g u r e 6 i s a Gram-Schmidt r e c o n s t r u c t e d chromatogram o f a c i t r u s o i l components t e s t m i x t u r e . The major peak i n t h e chromatogram i s due t o limonene w h i c h i s e s s e n t i a l l y a s o l v e n t . Note t h a t the a n a l y s i s time i s e x t r e m e l y s h o r t , l e s s t h a n 5 m i n u t e s / and i s a c c o m p l i s h e d under m i l d temperature c o n d i t i o n s , 50°C. The r e s o l u t i o n i s n o t superb on a l l the compounds p r e s e n t . However, i n f r a r e d d e t e c t i o n can be used t o r e s o l v e o v e r l a p p i n g c h r o m a t o g r a p h i c b a n d s . For example, t h e peak p r i o r t o limonene i n t h e chromatogram (denoted w i t h an *) appears t o be pure w i t h no s h o u l d e r s e v i d e n t . C l o s e e x a m i n a t i o n o f the i n f r a r e d s p e c t r a o f t h e peak shows t h a t t h e peak a c t u a l l y c o n s i s t s o f two compounds. An i n f r a r e d spectrum a b s t r a c t e d from the f r o n t s i d e o f t h e peak, F i l e #452, matches the r e f e r e n c e l i b r a r y spectrum o f myrcene as i s shown i n F i g u r e 7. W h i l e the i n f r a r e d spectrum o f F i l e #464 from t h e b a c k s i d e o f t h e peak, i s a v e r y good match w i t h * - p i n e n e (Figure 8). T h u s , a t l e a s t two compounds a r e p r e s e n t i n the peak. Pyrethrin Extract Analysis. P y r e t h r i n s a r e a group o f n a t u r a l l y o c c u r r i n g p e s t i c i d e s e x t r a c t e d from "Chrysanthemum c i n e r a n i a e f o l i u m . " As w i t h any p e s t i c i d e , r e g u l a t i o n s r e q u i r e q u a n t i t a t i v e measurements and p o s i t i v e i d e n t i f i c a t i o n o f the a c t i v e i n g r e d i e n t s . C u r r e n t l y , gas chromatography i s used f o r the a n a l y s i s . P y r e t h r i n I and I I must be s e p a r a t e d from t h e o t h e r components i n t h e e x t r a c t under m i l d temperature c o n d i t i o n s . P y r e t h r i n I I , w h i c h d i f f e r s by o n l y t h e p r e s e n c e o f a t e r m i n a l m e t h y l e s t e r g r o u p , r a p i d l y decomposes a t t e m p e r a t u r e s r e q u i r e d t o a f f e c t t h e s e p a r a t i o n . T h i s makes q u a n t i t a t i o n e x c e e d i n g l y difficult. SFC i s a l o g i c a l a l t e r n a t i v e f o r t h i s a p p l i c a t i o n because i t uses much m i l d e r temperature c o n d i t i o n s t h a n GC. T h i s e l i m i n a t e s the p o s s i b i l i t y o f t h e sample d e g r a d a t i o n caused by temperature. F i g u r e 9 i s the FID chromatogram o f a c a p i l l a r y SFC s e p a r a t i o n o f a c o m m e r c i a l l y a v a i l a b l e p y r e t h r i n e x t r a c t . The a c t i v e components a r e P y r e t h r i n I a t 22.54 min and P y r e t h r i n I I a t 23.91 m i n . B o t h show good peak shape w i t h no e v i d e n c e o f t h e r m a l decomposition. The group o f peaks e l u t i n g b e f o r e r e t e n t i o n time 19 m i n u t e s a r e from t h e p e t r o l e u m based e x t r a c t m a t r i x . The s o l v e n t peak a t 8.79 min i s m e t h a n o l . The s m a l l peaks a t 21.98 and 23.45 m i n u t e s a r e C i n e r i n I and I I r e s p e c t i v e l y . These a r e r e l a t e d components i n the e x t r a c t h a v i n g t h e same s t r u c t u r e as p y r e t h r i n e x c e p t f o r t h a t the t e r m i n a l methylene i n t h e p y r e t h r i n s i s r e p l a c e d by a m e t h y l g r o u p . F i g u r e 10 shows the S F C / F T - I R s p e c t r a f o r a) C i n e r i n I I and


13.

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F i g u r e 5. F T - I R spectrum from same peak as F i g u r e 4b w i t h s u p e r c r i t i c a l c a r b o n d i o x i d e absorbance removed.

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SFC Density Program


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WAVENUMBER F i g u r e 10. a) F T - I R spectrum f o r c i n e r i n I I peak a t 23.45 minutes i n p y r e t h r i n S F C / F T - I R s e p a r a t i o n , b) F T - I R spectrum f o r p y r e t h r i n I I peak a t 2 3 . 1 9 m i n u t e s .


13.

WIEBOLDT AND SMITH

SFC

with FTIR

Detection

241

b) P y r e t h r i n I I and t h e i r s t r u c t u r e s . The I I s e r i e s a r e d i e s t e r s h a v i n g b o t h a t e r m i n a l m e t h y l e s t e r group and t h e e s t e r l i n k a g e between t h e two r i n g s . T h i s g i v e s r i s e t o t h e complex C - 0 s t r e t c h absorbance bands between 1300-1100 cm*" . The o n l y f e a t u r e w h i c h can be used t o d i s t i n g u i s h p y r e t h r i n from c i n e r i n i s t h e C - H out-of-plan b e n d i n g absorbance f o r t h e t e r m i n a l methylene group i n p y r e t h r i n w h i c h o c c u r s a t 912 c m " . The p r e s e n c e o f t h i s a b s o r p t i o n band i s c l e a r e v i d e n c e o f t h e t e r m i n a l =CH2 g r o u p . 1

1


Conclusions These r e s u l t s demonstrate t h e f e a s i b i l i t y o f u s i n g a f l o w - t h r o u g h c e l l f o r F T - I R d e t e c t i o n o f c a r b o n d i o x i d e SFC e f f l u e n t . The c e l l d e s i g n i s d i c t a t e d by t h e r e s t r i c t i o n s imposed b y t h e F e r m i resonance absorbance o f C O 2 , n o t t h e c h r o m a t o g r a p h i c separation. As a r e s u l t / t h e d e s i g n works e q u a l l y w e l l f o r e i t h e r p a c k e d - c o l u m n o r c a p i l l a r y SFC s e p a r a t i o n s . W i t h packed column SFC s e p a r a t i o n s , F T - I R d e t e c t i o n c a n be u s e f u l i n s p e c t r a l l y r e s o l v i n g components w h i c h may n o t be r e s o l v e d by t h e c h r o m a t o g r a p h i c column. F T - I R a l s o shows good s e n s i t i v i t y f o r the smaller q u a n t i t i e s of m a t e r i a l s encountered w i t h c a p i l l a r y SFC a p p l i c a t i o n s . Spectral quality i s sufficient f o r i d e n t i f i c a t i o n and f o r d i s t i n g u i s h i n g s u b t l e d i f f e r e n c e s between r e l a t e d compounds. Acknowledgments The a u t h o r s thank D . Wymer o f t h e P r o c t e r & Gamble Company f o r assistance with the c i t r u s o i l s SFC/FT-IR analyses, J . F r e a l of Adams V e t e r i n a r y R e s e a r c h L a b o r a t o r i e s f o r t h e p y r e t h r i n e x t r a c t , G . Adams and K . Kempfert o f N i c o l e t I n s t r u m e n t C o r p o r a t i o n f o r a s s i s t a n c e w i t h t h e S F C / F T - I R i n t e r f a c e and p y r e t h r i n e x t r a c t analysis respectively. Literature 1. 2. 3. 4. 5. 6. 7. 8. 9.

Cited

Herres, W. HRGC-FT-IR: Capillary Gas Chromatography Fourier Transform Infrared Spectroscopy; Huthig: Heidelberg, 1987. Hellgeth, J.W.; Taylor, L.T. J. Chromatogr. Sci. 1986, 24, 519-528. Jinno, K. Chromatographia 1987, 23, 55-62. Olesik, S.V.; French, S.B.; Novotny, M. Chromatographia 1984, 18, 489-495. Johnson, C.C.; Jordan, J.W.; Taylor, L.T.; Vidrine, D.W. Chromatographia 1985, 20, 717-723. Shafer, K.H.; Pentoney, S.L.; Griffiths, P.R. Anal. Chem. 1986, 58, 58-64. Kuehl, D.T.; Griffiths, P.R. Anal. Chem. 1980, 52, 1394-1399. Kalasinsky, K.S.; Smith, J.A.S.; Kalasinsky, V.F. Anal. Chem. 1985, 57, 1969-1974. Fujimoto, C.; Jinno, K.; Hirata, Y. J. Chromatogr. 1983, 258, 81.


242


10. Pentoney, S.L.: Shafer, K.H.; Griffiths, P.R. J. Chromatogr. Sci. 1986, 24, 230-235. 11. Morin, P.; Caude, M.; Richard, H.; Rosset, R. Chromatographia 1986, 21, 523-530. 12. de Haseth, J.A.; Isenhour, T.L. Anal. Chem. 1977, 49, 1977-1981. 13. Wieboldt, R.C.; Hanna, D.A. Anal. Chem. 1987, 59, 1255-1259. October 9, 1987


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Supercritical Fluid Chromatography with Fourier Transform Infrared

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