Versatility of an Optical Multichannel Analyzer as an HPLC Detector

5 Versatility of an Optical Multichannel Analyzer as an

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H P L C Detector J. RICHARD JADAMEC, WILLIAM A. SANER, and RICHARD W. SAGER U. S. Coast Guard Research and Development Center, Groton, CT 06340

High pressure liquid chromatography (HPLC) has been used by many investigators to concentrate and separate complex mixtures of polyaromatic hydrocarbons (PAH's) from environmental samples (1-6). The specific identification of the separated eluates (i.e, the separated HPLC fractions), until recently, has been achieved by using one or a combination of the following approaches: a) fraction collection and subsequent analysis of the collected fractions by various spectroscopic techniques; b) stop flow methods where the column flow is stopped and the UV absorption, fluorescence, or other spectroscopic data are obtained; c) relative chromatographic retention times; d) relative chromatographic retention times and absorbance ratios at two or more fixed wavelengths; e) spiking the environmental sample with known compounds of interest; f) selective fluorescence excitation and emission wavelengths. If a known PAH is being monitored, or the number of components present in the sample is relatively small, then any of the above approaches can be effectively used to identify or tentatively identify an HPLC fraction. However, as the number of PAH's increase within a given environmental sample, then each of these above approaches becomes ineffective and/or impractical. HPLC column technology has produced highly effective and efficient analytical columns and, as has been previously stated (7), this development has led to a demand for more sensitive and versatile detectors for HPLC systems. HPLC detector development within the past several years has been aimed at increasing sensitivity, as evidenced by the development of fluorescence detectors capable of quantitating subnanogram levels of PAH's. Similarly, UV/VIS detectors have been developed which can detect nanogram levels of PAH's. Recently, several investigators have demonstrated the utility of optoelectronic image devices as detectors for HPLC systems, allowing the analyst to obtain spectral information of the separated HPLC eluate on-the-fly" (8-13). This capability n

This chapter not subject to US copyright. Published 1979 American Chemical Society Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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i n t h e p a s t h a s b e e n l a r g e l y c o n f i n e d t o g a s c h r o m a t o g r a p h i c (GC) s y s t e m s , where t h e mass s p e c t r a o f GC f r a c t i o n s have b e e n r e c o r d e d o n - t h e - f l y " and s t o r e d f o r subsequent r e t r i e v a l and i d e n t i f i c a tion. O p t o e l e c t r o n i c image d e v i c e s a r e c u r r e n t l y a t t h e s t a g e o f d e v e l o p m e n t where t h e y c a n be u s e d t o c o n v e r t s t a n d a r d s p e c t r o m e t e r s i n t o p o l y c h r o m a t o r s f o r u s e a s d e t e c t o r s t o r e c o r d t h e UV/ V I S a b s o r p t i o n o r f l u o r e s c e n c e s p e c t r a o f HPLC e l u a t e s . n

T h i s r e p o r t w i l l d i s c u s s the r e s u l t s of a study i n which an o p t i c a l m u l t i c h a n n e l a n a l y z e r (OMA) was c o u p l e d t o s t a n d a r d s p e c t r o m e t e r s t o r e c o r d b o t h t h e UV/VIS a b s o r p t i o n and f l u o r e s cence e m i s s i o n s p e c t r a o f complex m i x t u r e s o f PAH's s e p a r a t e d by HPLC t e c h n i q u e s " o n - t h e - f l y " ( i . e . , one s e c o n d s p e c t r a l s c a n s o f t h e HPLC e f f l u e n t s t r e a m ) and s t o r e d o n a f l o p p y d i s c f o r subsequent r e t r i e v a l and d a t a a n a l y s i s . The s y s t e m d e s c r i b e d h a s t h e c a p a b i l i t y o f s t o r i n g 250 (500 p o i n t ) s p e c t r a and c a n r e a d i l y be u s e d t o i n c r e a s e t h e e f f e c t i v e n e s s o f HPLC a n a l y s i s by a l l o w i n g b o t h q u a n t i t a t i v e a n d q u a l i t a t i v e d a t a t o be o b t a i n e d . Experimental L i q u i d C h r o m a t o g r a p h . T h e l i q u i d c h r o m a t o g r a p h was c o m p r i s e d o f a W a t e r s 660 S o l v e n t P r o g r a m m e r , two W a t e r s 6000A pumps, a W a t e r s U 6 - K I n j e c t o r a n d a W a t e r s 440 a b s o r b a n c e d e t e c t o r (254 nm). Whatman m i c r o - c a p i l l a r y t u b i n g ( 0 . 0 0 7 ID) was u s e d t o t r a n s f e r t h e HPLC c o l u m n e f f l u e n t f r o m t h e 254 nm a b s o r p t i o n d e t e c t o r to the fluorescence d e t e c t o r . 11

A l l 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 s were made u s i n g a n ES I n d u s t r i e s 0 . 4 6 x 15 cm r e v e r s e - p h a s e c o l u m n p a c k e d w i t h E . M e r c k 5 ym L i c h r o s o r b R P - 1 8 . A Whatman p r e - c o l u m n ( 0 . 2 1 x 7 cm) p a c k e d w i t h C O : P E L L ODS ( R e e v e - A n g e l ) was u s e d t o p r o t e c t t h e a n a l y t i c a l column from b o t h p a r t i c u l a t e m a t t e r and i r r e v e r s i b l e b o n d i n g o f i n j e c t e d components o n t o t h e s t a t i o n a r y p h a s e . The p r e - c o l u m n i s a l s o u s e d t o dampen m o b i l e p h a s e p u l s a t i o n s f r o m t h e c h r o m a t o g r a p h i c pumps. A b s o r p t i o n and F l u o r e s c e n c e I n s t r u m e n t a t i o n . Absorption s p e c t r a were o b t a i n e d u s i n g a P r i n c e t o n A p p l i e d R e s e a r c h C o r p . (PARC) M o d e l 1208 p o l y c h r o m a t o r , a P e r k i n - E l m e r 8 y L a b s o r p t i o n f l o w - c e l l and a 50 w a t t d e u t e r i u m l i g h t s o u r c e . Fluorescence s p e c t r a were o b t a i n e d u s i n g a F a r r a n d Mark 1 S p e c t r o f l u o r o m e t e r ( p r e v i o u s l y d e s c r i b e d ( 1 3 ) ) a n d e i t h e r a 10 y L F a r r a n d m i c r o f l o w - c e l l , o r a P r e c i s i o n C e l l s , I n c . (Model N o . 8830) 20 y L flow-cell. A PARC M o d e l 1254 S I T d e t e c t o r , h a v i n g a UV s c i n t i l l a t o r , was mounted o n b o t h t h e a b s o r p t i o n p o l y c h r o m a t o r a n d fluorescence spectrofluorometer. S p e c t r a l coverage i n the a b s o r p t i o n a n d f l u o r e s c e n c e modes was 60 a n d 115 nm, r e s p e c tively. A l l a b s o r p t i o n a n d f l u o r e s c e n c e s p e c t r a were o b t a i n e d i n one s e c o n d , i . e . , 32 s c a n s o f t h e S I T t a r g e t .

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O p t i c a l M u l t i c h a n n e l A n a l y z e r S y s t e m (OMA 2 ) . The OMA 2 s y s t e m c o n s i s t e d o f a PARC M o d e l 1215 c o n s o l e , two PARC M o d e l 1254 S I T d e t e c t o r s , two PARC M o d e l 1216 d e t e c t o r c o n t r o l l e r s , a n d a PARC M o d e l 1217 f l e x i b l e d i s c d r i v e . The SIT d e t e c t o r i s c o n t r o l l e d b y t h e 1216 d e t e c t o r c o n t r o l l e r , w h i c h p r o v i d e s b o t h power and s c a n n i n g v o l t a g e s and p r o c e s s e s t h e s i g n a l f o r t r a n s m i s s i o n t o the OMA 2 c o n s o l e . T h e OMA 2 c o n s o l e p e r f o r m s a l l n e c e s s a r y c o n t r o l f u n c t i o n s , d a t a a c q u i s i t i o n s , d a t a p r o c e s s i n g and s t o r a g e o f spectra. T h e s y s t e m c a n s t o r e 250 (500 p o i n t s ) s p e c t r a l c u r v e s when e q u i p p e d w i t h t h e M o d e l 1217 f l e x i b l e d i s c d r i v e . Reagents. M i l l i p o r e Q system water and s p e c t r o q u a l i t y m e t h a n o l (MCBMX 475) were u s e d a s t h e m o b i l e p h a s e i n t h e l i q u i d chromatograph. F l u o r o p o r e f i l t e r s (FGLP 04700) h a v i n g a 0 . 2 2 ym p o r e d i a m e t e r were u s e d t o f i l t e r t h e m e t h a n o l p r i o r t o u s e . A l l a r o m a t i c s t a n d a r d s , e x c e p t b e n z e n e (MCBBX 2 1 5 ) , were o b t a i n e d f r o m Duke S t a n d a r d s C o . , P a l o A l t o , C a l i f o r n i a . Solutions of t h e s t a n d a r d s were p r e p a r e d i n s p e c t r o q u a l i t y m e t h a n o l . Glacial a c e t i c a c i d ( r e a g e n t g r a d e ) was u s e d t o a c i d i f y t h e s p e c t r o q u a l i t y m e t h a n o l t o p r e p a r e t h e o i l e x t r a c t i n g s o l u t i o n (0.4% a c e t i c a c i d i n methanol). Sample P r e p a r a t i o n . P e t r o l e u m o i l s were e x t r a c t e d w i t h a c i d i f i e d methanol, according to a procedure p r e v i o u s l y described (14). Samples o f s e a w a t e r c o n t a i n i n g s o l u b l e p e t r o l e u m o i l f r a c t i o n s were p r e p a r e d by g e n t l y s p r e a d i n g a n o i l l a y e r ( 1 - 2 mm t h i c k ) on f r e s h sea water contained i n a 4 l i t e r P y r e x ^ beaker. A g l a s s tube e x t e n d i n g from t h e bottom o f t h e b e a k e r t o above t h e w a t e r s u r f a c e , was u s e d t o c o n t a i n a T e f l o n * s i p h o n from w h i c h o i l f r e e w a t e r s a m p l e s were w i t h d r a w n f r o m t h e b o t t o m a t i n t e r v a l s o v e r a two week p e r i o d . The b e a k e r c o n t a i n i n g t h e o i l and w a t e r was n o t d i s t u r b e d n o r a g i t a t e d d u r i n g t h i s two week p e r i o d to preclude mixing or e m u l s i f i c a t i o n of the o i l / w a t e r layers. V a r i o u s s a m p l e v o l u m e s (50 o r 250 m l ) o f s e a w a t e r drawn f r o m t h e b o t t o m o f t h e b e a k e r were pumped t h r o u g h Whatman g u a r d c o l u m n s p a c k e d w i t h 33-44 ym C O : P E L L ODS a t a 10 m l / m i n . f l o w rate. T h e Whatman g u a r d c o l u m n s were t h e n c o n n e c t e d a h e a d o f t h e a n a l y t i c a l column f o r chromatographic a n a l y s i s . Separation Procedure. M e t h a n o l i c o i l e x t r a c t s were c h r o m a t o g r a p h i c a l l y s e p a r a t e d u s i n g a l i n e a r g r a d i e n t f r o m 50/50 m e t h a n o l i n w a t e r t o 100% m e t h a n o l i n 50 m i n u t e s a t a f l o w r a t e of 1 ml/min. (^ 1200 p s i ) . The a n a l y t i c a l c o l u m n was p r e c o n d i t i o n e d f o r 23 m i n u t e s w i t h 50% ( v / v ) m e t h a n o l i n w a t e r a t a f l o w r a t e of 1 ml/min. p r i o r to b e g i n n i n g the g r a d i e n t . T h e l o a d e d p r e - c o l u m n s were c o n n e c t e d b e t w e e n t h e i n j e c t o r and t h e a n a l y t i c a l c o l u m n . L i n e a r two segment g r a d i e n t s were u s e d ; 0% t o 50% ( v / v ) m e t h a n o l / w a t e r i n 20 m i n u t e s , f o l l o w e d b y 50% t o 100% ( v / v ) m e t h a n o l / w a t e r i n 50 m i n u t e s .


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Discussion F l u o r e s c e n c e Measurements. A p r e v i o u s s t u d y (13) described t h e u s e o f a n OMA s y s t e m t o r e c o r d t h e f l u o r e s c e n c e e m i s s i o n s p e c t r a o f p e t r o l e u m o i l a r o m a t i c h y d r o c a r b o n s s e p a r a t e d by HPLC. I n t h i s e a r l i e r s t u d y , t h e f l u o r e s c e n c e s p e c t r a were r e c o r d e d b y e i t h e r p h o t o g r a p h i n g a monitor scope or p l o t t i n g the s p e c t r a on a c o n t i n u o u s l y moving s t r i p c h a r t r e c o r d e r at a p l o t r a t e of 16.4 s per spectrum. Both of these approaches are however, i m p r a c t i c a l , s i n c e t h e y a l l o w e d o n l y enough time to r e c o r d c h r o m a t o g r a p h i c p e a k maxima a s a r e s u l t o f t h e c o m p a r a t i v e l y l o n g r e c o r d i n g t i m e r e l a t i v e t o t h e s h o r t o b s e r v a t i o n t i m e (1 s ) . The OMA 2 s y s t e m u s e d i n t h e p r e s e n t s t u d y i s c a p a b l e o f s t o r i n g a s p e c t r u m w i t h i n 10 s e c o n d s , a l l o w i n g f o r a more e f f e c t u a l m o n i t o r i n g o f t h e HPLC e f f l u e n t s t r e a m . T h i s 10 s e c o n d i n t e r v a l c a n be r e d u c e d t o l e s s t h a n 3 s e c o n d s b y m o d i f y i n g t h e s o f t w a r e ( w r i t t e n i n FORTH) u s e d t o c o n t r o l t h e OMA 2 s y s t e m . A s e c o n d p r o b l e m e n c o u n t e r e d i n t h e e a r l i e r s t u d y was a l o s s i n s e n s i t i v i t y and a n i n c r e a s e i n s c a t t e r a s s o c i a t e d w i t h t h e u s e o f a 10 y L c y l i n d r i c a l f l o w - c e l l . F i g u r e 1 compares s p e c t r a o b t a i n e d f o r a 2 ppm n a p h t h a l e n e s o l u t i o n i n t h e 10 y L f l o w c e l l and i n a s t a n d a r d 10 x 10 mm c u v e t t e ( 3 . 7 m l ) . Figure 2 c o m p a r e s t h e f l u o r e s c e n c e r e s p o n s e o f a 1 ppm s o l u t i o n o f c h r y s e n e o b t a i n e d u s i n g a 1 x 1 x 21 mm r e c t a n g u l a r f l o w - c e l l and a s t a n d a r d 10 x 10 mm c u v e t t e . T h e s e s p e c t r a were o b t a i n e d u s i n g a F a r r a n d Mark I S p e c t r o f l u o r o m e t e r . The f l u o r e s c e n c e i n t e n s i t i e s i n m i c r o a m p e r e s a t s e v e r a l p o i n t s o n t h e two s p e c t r a i n d i c a t e t h a t a s u b s t a n t i a l i n c r e a s e i n s i g n a l i s o b t a i n e d w i t h the 1 x 1 x 21 mm f l o w - c e l l . The s o l v e n t s i g n a l b l a n k was 0 . 0 0 1 microamperes f o r t h e c u v e t t e and 0 . 0 0 3 m i c r o a m p e r e s f o r t h e f l o w - c e l l . The peak to peak v a r i a t i o n i n the p h o t o m u l t i p l i e r d e t e c t o r r e s p o n s e a t a f l o w r a t e o f 1 m l / m i n u t e f o r b o t h t h e f l o w - c e l l and s t a n d a r d c u v e t t e s p e c t r a were t h e same, i n d i c a t i n g t h a t a f l o w i n g ( n o n s t a t i c ) c o n d i t i o n does not i n d u c e n o i s e . S i m i l a r f l o w - c e l l and c u v e t t e c o m p a r i s o n s were made u s i n g 1 ppm s o l u t i o n s o f p y r e n e , 2 - m e t h y l n a p h t h a l e n e , and f l u o r e n e . In each case the f l u o r e s c e n c e s i g n a l o b t a i n e d f r o m t h e f l o w - c e l l was g r e a t e r t h a n t h a t f r o m the standard c u v e t t e . S p e c t r a l r e s p o n s e d i f f e r e n c e s were f o u n d to v a r y w i t h the a r o m a t i c s t a n d a r d used and a r e p r o b a b l y a t t r i b u t a b l e t o i n n e r f i l t e r e f f e c t s and q u e n c h i n g . The 1 x 1 x 21 mm f l o w - c e l l i s shown i n F i g u r e 3 . A lens a s s e m b l y r e f o c u s e s t h e e x i t s l i t image o f t h e e x c i t a t i o n monochromator onto the f l o w - c e l l . This r e f o c u s i n g e s s e n t i a l l y reduces t h e o p t i c a l window o f t h e HPLC e f f l u e n t s t r e a m b e i n g m o n i t o r e d by 2/3 ( f r o m 20 mm t o a p p r o x i m a t e l y 6 mm, v e r t i c a l h e i g h t ) . This r e g i o n i s shown a s t h e d a s h e d a r e a i n F i g u r e 3 . The HPLC e f f l u e n t f l o w t h r o u g h t h i s c e l l i s f r o m b o t t o m t o t o p , and a t a f l o w r a t e o f 1 m l / m i n , t h e n 16.7 yL o f the e f f l u e n t stream has p a s s e d t h r o u g h t h i s r e g i o n i n one s e c o n d . Assuming an e l u t i o n volume o f 500 y L ( w h i c h a t a 1 m l / m i n f l o w r a t e r e q u i r e s 30 s ) t h e n


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Figure 1. Comparison of fluorescence spectra from a standard cuvette and a 10 fiL flow-cell of 2 ppm naphthalene in methanol: 100 (A-B) OMA accumulations, Em bandwidth 0.5 nm.

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an " o n - t h e - f l y " o b s e r v a t i o n t i m e o f 1 s i s t h e e q u i v a l e n t o f 1/30 t h o f t h e e l u t i o n v o l u m e . The a v e r a g e c o n c e n t r a t i o n o f t h e s o l u t i o n w i t h i n t h e m o b i l e p h a s e p a s s i n g t h r o u g h t h e f l o w - c e l l a t p e a k maximum i s a p p r o x i m a t e l y t w i c e t h e i n j e c t e d amount o f m a t e r i a l d i v i d e d b y t h e e l u t i o n volume (15). T h e r e f o r e , i f 1 n a n o g r a m o f s o l u t e were i n j e c t e d a n d o n - c o l u m n d i l u t i o n o f 500 y L o c c u r r e d , t h e n t h e c o n c e n t r a t i o n o f the s o l u t e p a s s i n g through the f l o w - c e l l at peak maximum w o u l d be 0 . 0 0 4 n g p e r m i c r o l i t e r , o r 4 p p b , a c o n c e n t r a t i o n e a s i l y d e t e c t e d by f l u o r e s c e n c e s p e c t r o s c o p i c t e c h n i q u e s . It i s o b v i o u s t h a t as the e l u t i o n volume d e c r e a s e s (chromatographic e f f i c i e n c y i n c r e a s e s ) the c o n c e n t r a t i o n o f the s o l u t e at peak maximum w i t h i n t h e f l o w c e l l w i l l i n c r e a s e . F i g u r e 4 ( A - B ) shows the f l u o r e s c e n c e spectrum of chrysene obtained i n 1 second (at t h e p e a k maximum) when 4 ng was i n j e c t e d a t a f l o w r a t e o f 1 m l / min. T h i s s p e c t r u m was o b t a i n e d u s i n g t h e 10 y L f l o w - c e l l a n d OMA s y s t e m p r e v i o u s l y d e s c r i b e d ( 1 3 ) . A p p a r e n t l y , improved d e t e c t a b i l i t y ( i . e . , i d e n t i f i a b l e spectrum) i s a t t a i n a b l e , even when u s i n g a s m a l l v o l u m e f l o w - c e l l , a s e v i d e n c e d b y t h e s p e c t r a shown i n F i g u r e s 1 and 2 . The o p t i c a l l e n s a s s e m b l y , i n a d d i t i o n t o r e d u c i n g t h e o p t i c a l window o n t h e f l o w - c e l l , a l s o s e r v e s a s a mask t o l i m i t t h e s l i t image h e i g h t a t t h e e x i t p l a n e o f t h e e m i s s i o n m o n o c h r o m a t o r t o a p p r o x i m a t e l y 6 mm. T h i s masking e f f e c t p r e c l u d e s the need t o use the f u l l d i o d e a r r a y o f the SIT d e t e c t o r and prevents v e r t i c a l d i s t o r t i o n s (pincushion e f f e c t s which are p r e sent i n the SIT d e t e c t o r ) , from a d v e r s e l y a f f e c t i n g the s p e c t r a l data. UV A b s o r p t i o n M e a s u r e m e n t s . w a v e l e n g t h , u s i n g a n OMA s y s t e m , a

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where Io i s t h e i n c i d e n t r a d i a n t e n e r g y , I t h e r a d i a n t e n e r g y t r a n s m i t t e d b y t h e s a m p l e , and x t h e d a r k c u r r e n t o f t h e o p t o e l e c t r o n i c image d e v i c e . A p r e v i o u s l y d e s c r i b e d OMA s y s t e m (13) a u t o m a t i c a l l y s u b t r a c t s d a r k c u r r e n t i n t h e r e c o r d i n g o f I o and I v a l u e s . T h e OMA 2 s y s t e m a l s o h a s a d a r k c u r r e n t w h i c h becomes c o n s t a n t a f t e r t h e S I T d e t e c t o r h a s r e a c h e d e q u i l i b r i u m w i t h the ambient temperature. However, t h i s d a r k c u r r e n t i s not a u t o m a t i c a l l y removed. I t must be r e c o r d e d and s u b t r a c t e d f r o m Io and I f o r c a l c u l a t i n g t r u e a b s o r b a n c e s . The OMA 2, h o w e v e r , c a n be programmed t o r e c o r d d a r k c u r r e n t r e a d i n g s t h r o u g h o u t a chromatographic a n a l y s i s . An o p t o e l e c t r o n i c image d e v i c e h a s b e e n u s e d a s a n HPLC d e t e c t o r t o o b t a i n UV a b s o r p t i o n s p e c t r a o f s h a l e o i l a r o m a t i c hydrocarbons separated i s o c r a t i c a l l y (10). Since i s o c r a t i c s e p a r a t i o n s m a i n t a i n a c o n s t a n t m o b i l e phase c o m p o s i t i o n , the


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Io i n t h e a b o v e e q u a t i o n r e m a i n s c o n s t a n t . However, d u r i n g a g r a d i e n t e l u t i o n , Io i s c o n t i n u o u s l y c h a n g i n g s i n c e t h e c o m p o s i t i o n o f the m o b i l e phase v a r i e s d u r i n g the chromatographic analysis. T h i s becomes a c r i t i c a l c o n s i d e r a t i o n when u s i n g t h e OMA s y s t e m a s a d e t e c t o r t o a n a l y z e l o w c o n c e n t r a t i o n s o f a r o m a t i c hydrocarbons s e p a r a t e d by g r a d i e n t e l u t i o n t e c h n i q u e s . In t h i s s t u d y , ( I o - x) v a l u e s f o r e v e r y 5% change i n t h e m e t h a n o l / w a t e r g r a d i e n t ( f r o m 50 t o 100% m e t h a n o l ) were s t o r e d i n memory a n d u s e d t o d e t e r m i n e t h e a b s o r p t i o n s p e c t r a o f t h e HPLC e l u a t e s a t t h e appropriate gradient composition. Another approach i s c u r r e n t l y u n d e r d e v e l o p m e n t (16) i n w h i c h two m i c r o f l o w - c e l l s w i l l b e i n t e r c h a n g e d i n t o t h e o p t i c a l p a t h , one c o n t a i n i n g t h e HPLC e f f l u e n t s t r e a m ( f r o m t h e o u t l e t o f t h e a n a l y t i c a l c o l u m n ) and t h e s e c o n d c o n t a i n i n g o n l y the changing m o b i l e phase c o m p o s i t i o n ( p r i o r to t h e i n j e c t o r and c o n s e q u e n t l y d e v o i d o f s a m p l e ) to a l l o w b o t h (Io - x) and I - x) v a l u e s t o be o b t a i n e d f o r n e a r l y i d e n t i c a l gradient compositions. (The l a g t i m e b e t w e e n s a m p l e m o b i l e p h a s e c o m p o s i t i o n and b l a n k m o b i l e p h a s e c o m p o s i t i o n w i l l b e m i n i m a l and s h o u l d n o t p r e s e n t a p r o b l e m u n l e s s v e r y f a s t g r a d i e n t r u n times over l a r g e and/or r a p i d m o b i l e phase c o m p o s i t i o n a l changes are employed.) Another f a c t o r which can cause s p e c t r a l d i s t o r t i o n s i s the f l u c t u a t i o n of the output energy of the r a d i a n t s o u r c e . The OMA 2 system i n c o r p o r a t e s a c a p a b i l i t y to c o r r e c t s p e c t r a l data f o r v a r i a t i o n s i n source output energy (17). The s o u r c e c o m p e n s a t i o n mode c o n t i n u o u s l y m o n i t o r s t h e o u t p u t e n e r g y o f t h e lamp a n d automatically c o r r e c t s incoming s p e c t r a l data f o r source i n t e n s i t y f l u c t u a t i o n s p r i o r to s t o r i n g t h a t d a t a . HPLC/OMA S y s t e m . I n t h i s s t u d y , t h e HPLC e f f l u e n t s t r e a m was p a s s e d t h r o u g h t h r e e f l o w - c e l l s c o n n e c t e d i n s e r i e s w i t h d i f f e r e n t l e n g t h s o f Whatman m i c r o - c a p i l l a r y ( 0 . 0 0 7 " I . D . ) tubing. T h e f i r s t f l o w - c e l l m o n i t o r e d t h e a b s o r p t i o n a t 254 nm and was u s e d t o k e y t h e r e c o r d i n g o f a b s o r p t i o n and f l u o r e s c e n c e s p e c t r a o f t h e e f f l u e n t s t r e a m p a s s i n g t h r o u g h t h e s e c o n d and t h i r d f l o w - c e l l s , r e s p e c t i v e l y , a t peak maxima. F i g u r e 5 shows a n HPLC a b s o r p t i o n c h r o m a t o g r a m o f what a p p e a r s t o be a two component m i x t u r e o f b e n z e n e a n d e t h y l benzene separated i s o c r a t i c a l l y . F i g u r e s 6 and 7 a r e t h e a b s o r p t i o n and f l u o r e s c e n c e s p e c t r a o f t h e f i r s t e l u t i n g compound ( b e n z e n e ) o b t a i n e d as t h e HPLC e f f l u e n t s t r e a m p a s s e d t h r o u g h t h e s e c o n d and t h i r d f l o w - c e l l s , r e s p e c t i v e l y . Each spectrum was o b t a i n e d i n one s e c o n d , ( i . e . , 32 s c a n s o f t h e S I T d e t e c t o r ) . I t c a n be s e e n i n F i g u r e s 6 and 7 t h a t t h e a b s o r p t i o n and f l u o r e s c e n c e s p e c t r a o b t a i n e d u s i n g t h e OMA s y s t e m a g r e e s w i t h p u b l i s h e d benzene s p e c t r a . F i g u r e s 8 and 9 a r e t h e OMA a b s o r p t i o n and f l u o r e s c e n c e s p e c t r a o b t a i n e d a t t h e p e a k maxima o f t h e s e c o n d f r a c t i o n ( e t h y l b e n z e n e ) a s t h e HPLC e f f l u e n t s t r e a m p a s s e d t h r o u g h t h e


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Figure 5. HPLC absorption chromatogram (254 nm) of a contaminated two component mixture separated isocratically


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Figure 6. Comparison of benzene absorption spectrum in methanol (32 OMA accumulations) with published absorption spectrum of benzene in water

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300 WATER 275

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Figure 7. Comparison of fluorescence spectrum of benzene in methanol (32 OMA accumulations) with published fluorescence spectrum of benzene in water


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233

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Figure 8. Comparison of ethylbenzene absorption spectrum in methanol (32 OMA accumulations) with published fluorescence spectrum in cyclohexane. Arrows indicate spectral region of difference.

275

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Figure 9. Comparison of ethylbenzene fluorescence spectrum in methanol (32 OMA accumulations) with published fluorescence spectrum in cyclohexane. Arrows indicate spectral region of difference.


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s e c o n d and t h i r d f l o w - c e l l s , r e s p e c t i v e l y . H o w e v e r , i t c a n be s e e n i n F i g u r e s 8 and 9, t h a t t h e UV a b s o r p t i o n and f l u o r e s c e n c e s p e c t r a o b t a i n e d f o r e t h y l b e n z e n e d i d n o t compare e x a c t l y t o t h e p u b l i s h e d s p e c t r a o f e t h y l b e n z e n e ; the s p e c t r a l r e g i o n s where d i f f e r e n c e s o c c u r a r e i n d i c a t e d by a r r o w s . T h e s e d i f f e r e n c e s were c a u s e d by a t r a c e amount o f n a p h t h a lene contamination. The 5 y L s y r i n g e t h a t was u s e d t o i n j e c t t h e m i x t u r e (32 y g e a c h o f b e n z e n e and e t h y l b e n z e n e ) had p u r p o s e l y n o t been c l e a n e d p r i o r to the i n j e c t i o n of t h i s m i x t u r e . The c h r o m a t o g r a m i n F i g u r e 5 c l e a r l y i n d i c a t e s a two component m i x t u r e , w i t h no p e a k s h o u l d e r s and good p e a k s y m m e t r y . No i n d i c a t i o n o f t h e t r a c e contaminant i s present i n the second e l u t i n g peak. However, p e a k symmetry i s n o t a good i n d i c a t o r o f p e a k p u r i t y ( 1 8 ) . A l t h o u g h s i n g l e w a v e l e n g t h m o n i t o r i n g o f t h e HPLC s t r e a m by e i t h e r a b s o r p t i o n or f l u o r e s c e n c e d e t e c t o r s i s an e f f e c t i v e approach to q u a n t i t a t i n g known c o m p o n e n t s , i t c a n n o t s o l e l y be u s e d t o d e t e r m i n e p e a k p u r i t y r e l i a b l y , e s p e c i a l l y when a p o o r l y r e s o l v e d contaminant i s present at t r a c e l e v e l s . F i g u r e 10 i s a n e n l a r g e ment o f t h e OMA f l u o r e s c e n c e e m i s s i o n s p e c t r u m o f t h e e t h y l b e n z e n e p e a k c l e a r l y s h o w i n g t h e e x t r a n e o u s e m i s s i o n due t o t h e p r e s e n c e of the naphthalene contaminant. F i g u r e 11 shows t h e p u b l i s h e d UV a b s o r p t i o n s p e c t r a f o r b o t h e t h y l b e n z e n e and n a p h t h a l e n e . The s p e c t r a l b a n d w i d t h and l o c a t i o n o f t h e s t a n d a r d f i x e d w a v e l e n g t h UV a b s o r p t i o n HPLC d e t e c t o r (254 nm) a r e shown, i n a r e g i o n where b o t h compounds absorb. However, s i n c e the chromatographic r e t e n t i o n times o f t h e s e two components a r e n e a r l y i d e n t i c a l ( u n d e r t h e c h r o m a t o g r a p h i c c o n d i t i o n s employed), the standard s i n g l e wavelength a b s o r p t i o n i s a c t u a l l y t h e sum o f two components a b s o r b i n g i n t h i s r e g i o n , and m o n i t o r i n g a r e g i o n o f s p e c t r a l o v e r l a p f o r two p o o r l y r e s o l v e d compounds m i n i m i z e s t h e c h a n c e s o f d e t e c t i n g the minor component. The c o n t a m i n a n t p e a k c o u l d be d e t e c t e d using m u l t i p l e f i x e d absorption wavelengths, p a r t i c u l a r l y i n nono v e r l a p p i n g s p e c t r a l r e g i o n s , but t h i s r e q u i r e s m u l t i p l e d e t e c t o r s or repeated chromatographic i n j e c t i o n s . The OMA s y s t e m c a n be used to r e c o r d m u l t i p l e wavelength a b s o r p t i o n or f l u o r e s c e n c e chromatograms ( i n a d d i t i o n to the s p e c t r a of the e l u t i n g f r a c t i o n s ) s i n c e these r e p r e s e n t o n l y s l i c e s of the s p e c t r a l windows. S t o p - f l o w , o r f r a c t i o n c o l l e c t i o n , a l s o c o u l d be u s e d t o i d e n t i f y the presence of naphthalene i n the second e l u t i n g f r a c t i o n , but o n l y at a c o n s i d e r a b l e s a c r i f i c e i n time r e l a t i v e t o t h e r a p i d s c a n n i n g c a p a b i l i t y o f t h e OMA. H o w e v e r , a s t h e complexity of a chromatographic separation i n c r e a s e s , these a p p r o a c h e s become i m p r a c t i c a l i n t e r m s o f t h e t i m e r e q u i r e d t o s p e c t r a l l y d e t e r m i n e the peak p u r i t y of each f r a c t i o n . F i g u r e 12 shows t h e 254 nm a b s o r p t i o n c h r o m a t o g r a m o f a complex m i x u t r e of P A H s e x t r a c t e d from a marine d i e s e l f u e l and s e p a r a t e d on a r e v e r s e - p h a s e C-18 c o l u m n u s i n g a m e t h a n o l / water g r a d i e n t . F o r t h i s a n a l y s i s t h e OMA 2 s y s t e m was p r o g r a m f


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310 275

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390

Figure 10. Enlargement of spectral region where differences exist in the fluorescence spectrum of ethylbenzene (Figure 9) indicating the presence of naphthalene

Figure 11. Absorption spectra of ethylbenzene and naphthalene indicating region of overlap at 254 nm



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med t o t a k e f l u o r e s c e n c e s p e c t r a o f t h e e f f l u e n t s t r e a m e v e r y 10 seconds throughout the chromatographic r u n . F i g u r e 13 i s a n e n l a r g e m e n t o f t h e r e c t a n g u l a r p o r t i o n o f t h e c h r o m a t o g r a m shown i n F i g u r e 12. T h i s segment r e p r e s e n t s a s e v e n t y (70) second p o r t i o n of t h i s chromatographic a n a l y s i s . F i g u r e 14 shows t h e f l u o r e s c e n c e e m i s s i o n s p e c t r a t a k e n a t 10 s e c o n d i n t e r v a l s o v e r t h i s t i m e f r a m e ( e a c h s p e c t r u m r e p r e s e n t s a one s e c o n d s c a n o f the e f f l u e n t stream). The s p e c t r a l window c o v e r s 115 nm, f r o m 295 nm t o 410 nm. I d e n t i f i c a t i o n o f t h e s e components c o u l d be a c c o m p l i s h e d by c o m p u t e r i f l i b r a r i e s o f f l u o r e s c e n c e (and/or a b s o r p t i o n ) s p e c t r a were i n e x i s t e n c e (as i n t h e c a s e o f GC-MS and G C - F T I R s y s t e m s ) . T h i s a p p r o a c h has p r o v e n u s e f u l i n t h e a n a l y s i s of e n v i r o n m e n t a l samples f o r the p r e s e n c e of hazardous c o n t a m i n a n t s . Figure 15 shows t h r e e c h r o m a t o g r a m s ; t h e l o w e r c h r o m a t o g r a m i s a t r a c e e n r i c h m e n t o f uncontaminated sea w a t e r ; the m i d d l e chromatogram i s o f a methanol e x t r a c t o f a Bunker C f u e l o i l ; the upper chromatogram i s a t r a c e enrichment o f contaminated sea water a f t e r a one d a y o i l / w a t e r c o n t a c t t i m e . The t r a c e e n r i c h m e n t c h r o m a t o grams were o b t a i n e d by pumping t h e two s e a w a t e r s a m p l e s t h r o u g h Whatman g u a r d c o l u m n s ( 2 . 1 x 70 mm) p a c k e d w i t h C O : P E L L ODS and a n a l y z e d (as d e s c r i b e d u n d e r t h e e x p e r i m e n t a l s e c t i o n ) u s i n g a 2 segment w a t e r t o m e t h a n o l g r a d i e n t . The OMA s y s t e m was u s e d t o r e c o r d b o t h t h e UV a b s o r p t i o n and f l u o r e s c e n c e s p e c t r a o f t h e s e p a r a t e d e l u a t e s a t peak maxima's o n l y . A t o t a l o f 145 a b s o r p t i o n and f l u o r e s c e n c e s p e c t r a were o b t a i n e d d u r i n g t h e HPLC a n a l y s i s of the contaminated sea water sample. This represents w a t e r s o l u b l e f r a c t i o n s o f t h e o i l s i n c e no a g i t a t i o n o f t h e o i l / water mixture o c c u r r e d . T h i s c a p a b i l i t y a l l o w s f o r the r a p i d i d e n t i f i c a t i o n o f s o l u b l e P A H ' s by c o m p a r i s o n w i t h p u b l i s h e d spectral data. Conclusions O p t o e l e c t r o n i c image d e v i c e s c a n be u s e d t o i n c r e a s e t h e v e r s a t i l i t y o f s t a n d a r d HPLC a b s o r p t i o n and f l u o r e s c e n c e d e t e c t o r s by p r o v i d i n g s p e c t r a l i n f o r m a t i o n o f t h e HPLC e f f l u e n t s t r e a m on-the-fly during a chromatographic a n a l y s i s . In a d d i t i o n , conventional m u l t i p l e f i x e d wavelength a b s o r p t i o n or fluorescence c h r o m a t o g r a m s c a n a l s o be o b t a i n e d b y p l o t t i n g n a r r o w b a n d w i d t h s w i t h i n t h e m o n i t o r e d s p e c t r a l w i n d o w s . F u r t h e r , t h e OMA s y s t e m c a n e f f e c t i v e l y be u s e d t o a s c e r t a i n c h r o m a t o g r a p h i c p e a k p u r i t y d u r i n g the e l u t i o n o f a band by c o n t i n u o u s l y m o n i t o r i n g i t s s p e c t r u m f r o m l e a d i n g e d g e , t h r o u g h p e a k maximum, t o t a i l i n g e d g e . The c a p a b i l i t y o f t h e OMA s y s t e m t o m o n i t o r a w i d e s p e c t r a l w i n d o w , r a t h e r t h a n a n a r r o w b a n d , i n c r e a s e s n o t o n l y t h e amount of data generated, but a l s o the u t i l i t y of that d a t a i t s e l f . f l

t f

C u r r e n t methods o f i d e n t i f y i n g P A H ' s s e p a r a t e d by HPLC t e c h n i q u e s become i n c r e a s i n g l y i m p r a c t i c a l as t h e s a m p l e s become more c o m p l e x ; s t o p - f l o w t e c h n i q u e s a r e s u i t a b l e o n l y f o r


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-H

10

1

1

1

1

1—

20

30

40

50

60

Figure 12. HPLC absorption chromatogram of a marine diesel fuel oil

Figure 13. Enlargement of a seventy (70) second portion of the chromatogram shown in Figure 12


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1.0 AUF8

Figure 15. Three HPLC absorption chromatograms: lower is trace enrichment chromatogram of clean (uncontaminated) sea water; middle chromatogram is a methanol extract of a Bunker C oil; upper chromatogram is a trace enrichment chromatogram of sea water after 1 day contact with Bunker C oil.


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r e l a t i v e l y simple mixtures. P r e s e n t r e s e a r c h e f f o r t s are b e i n g d i r e c t e d toward d e v e l o p i n g a p o l y c h r o m a t o r s y s t e m u t i l i z i n g a n OMA d e t e c t o r t o r e c o r d b o t h a b s o r p t i o n a n d f l u o r e s c e n c e s p e c t r a o f a n HPLC e f f l u e n t f r o m a s i n g l e flow c e l l . T h i s w i l l a l l o w f o r the n e a r - s i m u l t a n e o u s r e c o r d i n g o f b o t h t h e a b s o r p t i o n and f l u o r e s c e n c e s p e c t r a o f a n eluate. S i n c e o n l y one f l o w - c e l l w i l l be u t i l i z e d , any d e g r a d a t i o n i n s e p a r a t i o n r e s u l t i n g from band b r o a d e n i n g ( a t t r i b u t a b l e t o t h e f l o w - c e l l ) w i l l be e l i m i n a t e d . T h i s i s n o t t h e c a s e when s e v e r a l f l o w - c e l l s a r e u s e d i n s e r i e s (19) . Studies currently i n p r o g r e s s i n d i c a t e t h a t the use of m u l t i p l e f l o w - c e l l s ( i n tandem) n o t o n l y c a u s e s a n i n c r e a s e i n band b r o a d e n i n g b u t a l s o i n c r e a s e s peak skewness. T h i s r e s u l t s not only i n a decrease i n d e t e c t a b i l i t y but a l s o a l o s s i n chromatographic r e s o l u t i o n . In the a n a l y s i s of environmental samples, o p t o e l e c t r o n i c image d e v i c e s a l l o w f o r r e a l t i m e s p e c t r a l a c q u i s i t i o n a n d r a p i d i d e n t i f i c a t i o n by c o m p a r i s o n t o p u b l i s h e d s p e c t r a . It i s not u n r e a l i s t i c t o p r o j e c t t h a t w i t h i n 5 t o 10 y e a r s s u i t a b l e s p e c t r a l l i b r a r i e s w i l l be a v a i l a b l e f o r i d e n t i f y i n g HPLC e l u a t e s by computer s e a r c h r o u t i n e s s i m i l a r to those p r e s e n t l y i n use w i t h MS and F T I R s y s t e m s .

The o p i n i o n s o r a s s e r t i o n s c o n t a i n e d h e r e i n a r e the p r i v a t e o n e s o f t h e w r i t e r s a n d a r e n o t t o b e c o n s t r u e d as o f f i c i a l o r r e f l e c t i n g t h e v i e w s o f t h e Commandant o r t h e C o a s t G u a r d a t large.


5. JADAMEC ET AL.


LITERATURE CITED 1. F. Eisenbeiss, H. Hein, R. Joester, and G. Naundorf, Chromatogr. Newsletter. 6, 8 (1978). 2. C. G. Creed, Research/Development, Sep. 40 (1976). 3. D. Kasiske, K. D. Klinkmuller, and M. Sonneborn, J. Chromatogr., 149, 703 (1978). 4. S. A. Wise, S. N. Chester, H. S. Hertz, L. R. Hilpert, and W. E. May, Anal. Chem., 49, 2306 (1977). 5. A. D. Thruston, J. Chromatogr. Sci. 16, 254, (1978). 6. W. E. May, and S. P. Wasik, Anal. Chem., 50, 997 (1978). 7. R. D. Conlon, Anal. Chem., 41, 107A, (1969). 8. A. E. McDowell, and H. Pardue, Anal. Chem., 48, 1815 (1976). 9. A. E. McDowell and H. Pardue, Anal. Chem., 49, 1171 (1977). 10. L. N. Klatt, Abstracts, 173rd ACS National Meeting, New Orleans, LA, March 1977. 11. R. E. Dessy, W. G. Nunn, C. A. Titus, and W. R. Reynolds, J . Chromatogr. Sci., 14, 195 (1976). 12. M. J. Milano, S. Lam, and E. Gruska, J. Chromatogr. 125, 315 (1976). 13. J . R. Jadamec, W. A. Saner; and Y. Talmi, Anal. Chem. 49, 1318, (1977). 14. W. A. Saner, G. E. Fitzgerald, and J . P. Welsh, Anal. Chem. 48, 1747, (1976). 15. J. J. Kirkland, "Modern Practice of Liquid Chromatography" Wiley-Interscience, New York, 1971, p. 101. 16. U. S. Governmental Contract DOT-CG-81-78-1963 awarded to Farrand Optical Co., Inc., Oct. (1978). 17. Y. Talmi, D. C. Baker, J. R. Jadamec, and W. A. Saner, Anal. Chem., 50, 936 A (1978). 18. E. D. Pellizzari, and C. M. Sparacino, Anal. Chem., 45, 378, (1973). 19. T. J. Porro, R. D. Conlon, J. L. Dicesare, Abstracts, 29th Pittsburgh Conference. Cleveland, Ohio, Feb. 1978. RECEIVED

February 5, 1979.


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Versatility of an Optical Multichannel Analyzer as an HPLC Detector

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