A Dual-Beam Photodiode Array Spectrometer System for Liquid

Jul 23, 2009 - Also, to facilitate the use of this detector an extensive computer software system has been written which allows the user almost unlimi...
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6 A Dual-Beam Photodiode Array Spectrometer System for L i q u i d Chromatographic Separation Methods Development

Downloaded by FUDAN UNIV on December 20, 2016 | http://pubs.acs.org Publication Date: June 21, 1979 | doi: 10.1021/bk-1979-0102.ch006

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W. G. NUNN and R. E. DESSY Virginia Polytechnic Institute & State University, Chemistry Department, Blacksburg, VA 24061 W. R. REYNOLDS National Center for Toxicological Research, Jefferson, AR 72074 A multiwavelength, dual beam optical system with 256 detectors operating in a free spectral range of 200 to 800 nm has been designed and implemented for use with a liquid chromatograph. Also, to facilitate the use of this detector an extensive computer software system has been written which allows the user almost unlimited freedom in the way that the data taken over the bandwidth of the detector may be plotted or displayed for visual examination. The linear photodiode arrays used as detectors have been shown to have a significant response in the ultraviolet region of the spectrum. Automation and computerization of laboratory equipment have had a profound effect on the manner in which three important areas of chemistry have been developed and used: 1. The addition of a computer to multiple gas-chromatograph installations permitted rapid data analyses and report generation, 2. The application of Fourier transform methods to nuclear magnetic resonance experiments improved the signal-to-noise ratio and permitted extraction of data heretofore inaccessible. 3. The use of a computer with high-speed high-resolution mass spectrometers allowed vast amounts of data to be collected rapidly and manipulated easily. In all of these cases, the instrument was designed long before the computer was attached. Increasingly, instruments will be designed from the beginning with automation and computerization in mind. The form of the instrument will be determined by the needs of the chemist for proper data, correlated to the needs of the computer. The purpose of this article is to describe an automated unit for liquid chromatography (LC) which utilizes state-of-the-art digital electronics in the instrument and in the associated computer equipment. It will describe the development of the system 'Current Address: Union Carbide Chemicals and Plastics, South Charleston, W. VA. 25303 'Request for reprints. 0-8412-0504-3/79/47-102-135$08.25/0 © 1979 American Chemical Society Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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L i q u i d chromatography has been h i n d e r e d by the l a c k o f a u n i v e r s a l d e t e c t o r - a n d a s y e t no e q u i v a l e n t f o r t h e GC/MS s y s t e m has been d e s c r i b e d . For s e v e r a l y e a r s i t has been e v i d e n t t h a t

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UV/VIS s p e c t r o s c o p y c o u l d p r o v i d e a v i a b l e d e t e c t o r l e n g t h d e t e c t i o n p r i n c i p l e s were e m p l o y e d .

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Many v a r i e t i e s o f r a p i d s c a n n i n g s p e c t r o m e t e r s y s t e m s are available. The f i r s t g e n e r a t i o n u n i t s r e l i e d on moving mechani c a l p a r t s to scan the spectrum. These are t y p i f i e d by the r o t a t i n g m i r r o r or v i b r a t i n g galvanometer types commercially available. The second g e n e r a t i o n i n s t r u m e n t u t i l i z e d V i d i c o n t u b e s o f some s o r t . These e l i m i n a t e d t h e c o n s t r u c t i o n and maintenance problems a s s o c i a t e d w i t h mechanical spectrometers, but t h e h i g h c o s t o f V i d i c o n s y s t e m s , a n d t h e i r t e n d e n c y t o show memory a n d b l o o m i n g makes them i n t e r i m s o l u t i o n s o n l y . The stage i s o b v i o u s l y set f o r a t h i r d generation instrument u s i n g l i n e a r p h o t o s e n s i t i v e a r r a y s as the d e t e c t o r f o r a r a p i d scanning/multiwavelength spectrometer for l i q u i d chromatographic use. An e x c e l l e n t r e v i e w o f m u l t i e l e m e n t p u b l i s h e d by T a l m i . 1 by

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s o l i d s t a t e l i n e a r a r r a y d e t e c t o r s b a s e d on c h a r g e c o u p l e d d e v i c e s , o r c h a r g e . i n j e c t i o n d e v i c e s may a l s o be o f interest. T y p i c a l of the f a m i l i e s of d e t e c t o r s , the R e t i c o n d e t e c t o r s are b u i l t i n a number o f e l e m e n t s / a r r a y sizes. Commercially a v a i l a b l e u n i t s h a v e a n y w h e r e f r o m 128 t o 1024 e l e m e n t s / a r r a y . Each i n d i v i d u a l e l e m e n t i n t h e a r r a y i s 1 x 1 m i l t o 1 x 100 m i l i n a r e a , and a r e s p a c e d on a p p r o x i m a t e l y 1 m i l c e n t e r s . The t r o m e t e r s y s t e m d i s c u s s e d i n t h i s a r t i c l e was b u i l t u s i n g Reticon-type The are

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3. O v e r t h e i n t e g r a t i o n t i m e ( t h e t i m e l i g h t s t r i k e s the a r r a y ) t h e c h a r g e o n t h e c a p a c i t o r d r o p s a n amount e q u i v a l e n t t o t h e amount o f i n t e g r a t e d l i g h t f a l l i n g o n t h e d e t e c t o r . 4 . When t h e e l e m e n t i s r e a d o u t a s o l i d - s t a t e d e v i c e c a l l e d a m u l t i p l e x e r a t t a c h e s the d e t e c t o r to a v o l t a g e source which c h a r g e s t h e c a p a c i t o r b a c k up t o a s t a n d a r d p o t e n t i a l . 5. A s i g n a l , c o r r e s p o n d i n g to the c u r r e n t - f l o w n e c e s s a r y r e c h a r g e t h e v o l t a g e s o u r c e , i s s e n t down a w i r e c a l l e d t h e

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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line. 6 . The m u l t i p l e x e r t h e n c a u s e s t h e v o l t a g e s o u r c e t o b e c o n n e c t e d to t h e n e x t element i n t h e d e t e c t o r , and the p r o c e s s i s repeated. The s i g n a l f r o m t h e r e c h a r g i n g o f t h e c a p a c i t o r s i s s e n t down t h e v i d e o l i n e a s a s e r i e s o f p e a k s where e a c h p e a k c o r r e sponds to a c a p a c i t o r , p h o t o d i o d e p a i r . The s i g n a l s e e n on t h e v i d e o l i n e i s i l l u s t r a t e d i n u p p e r t r a c e f i g u r e 2, where f i g u r e 3 i s the s i g n a l s t r i k i n g the a r r a y .

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The r e s p o n s e o f a n a r r a y t o l i g h t i s w a v e l e n g t h d e p e n d e n t . A s c a n be s e e n i n f i g u r e 4 t h e a r r a y h a s t h e g r e a t e s t r e s p o n s e to n e a r IR and the s m a l l e s t t o r a d i a t i o n i n t h e u l t r a v i o l e t . A t r u e d o u b l e beam s p e c t r o m e t e r r e q u i r e s s e p a r a t e o p t i c a l p a t h s f o r s a m p l e and r e f e r e n c e , and i n v o l v e s s i m u l t a n e o u s c o m p a r i s o n o f the two. I t i s p o s s i b l e t o c o n s t r u c t a p s e u d o - d o u b l e beam s p e c t r o m e t e r , e m p l o y i n g o n l y one d e t e c t o r and o p t i c a l p a t h , t a k i n g advantage o f the c a p a b i l i t i e s o f a computer system attached to the d e t e c t o r . A l l t h a t i s n e e d e d t o make s u c h a d e t e c t o r i n t o a M U L T I WAVELENGTH /RAP ID SCANNING s p e c t r o m e t e r f o r l i q u i d c h r o m a t o g r a p h i c purposes i s to d i s p e r s e the l i g h t e x i t i n g from an LC c e l l o n t o t h e s u r f a c e o f t h e p h o t o d i o d e a r r a y b y means o f a monochromater. In order to p r o p e r l y c o n d i t i o n the s e r i a l p u l s e t r a i n e x i t i n g f r o m t h e common v i d e o l i n e o f t h e d e t e c t o r f o r i n p u t t o a computer s y s t e m , i t i s normal to r u n i t t h r o u g h an i n t e g r a t e , sample/hold a m p l i f i e r system. Each p u l s e i s i n t e g r a t e d over a major f r a c t i o n o f i t s w i d t h , l e a d i n g to a r e d u c t i o n i n n o i s e by the a n a l o g i n t e g r a t i n g o p e r a t i o n . This i n t e g r a l i s "sampled" and t h e p o t e n t i a l " h e l d " a t t h e end o f t h e i n t e g r a t i o n p e r i o d , so t h a t the o u t p u t o f the system r e a l l y l o o k s l i k e F i g u r e 2 bottom. T h i s c a n be compared t o the o u t p u t t h a t m i g h t be e x p e c t ed f r o m a s t a n d a r d p h o t o m e t e r u n i t i n a r e g u l a r s p e c t r o m e t e r ( F i g u r e 3). U n d e r n o r m a l c o n d i t i o n s t h i s c o u l d be d i s p l a y e d d i r e c t l y o n t o a n o s c i l l o s c o p e , and a r a p i d s c a n n i n g s p e c t r o m e t e r has been c o n s t r u c t e d . I t i s o b v i o u s t h a t w i t h the l a r g e d a t a throughput a computer w i l l be n e c e s s a r y t o t a k e advantage o f a l l t h e d a t a a v a i l a b l e . T h e r e f o r e t h e o u t p u t shown i n F i g u r e 2 i s i n p u t t o a n a n a l o g / d i g i t a l c o n v e r t e r , and d i g i t i z e d ; i . e . , c o n v e r t e d f r o m a n a l o g domain t o d i g i t a l domain. T h i s p r o v i d e s a d i g i t a l number t h a t c a n be a c c e p t e d b y a c o m p u t e r and s t o r e d . A s h u t t e r a n d p u r e s o l v e n t i n t h e s a m p l e compartment o f t h i s p s e u d o - d o u b l e beam s p e c t r o m e t e r p e r m i t s a c q u i s i t i o n o f b o t h a d a r k c u r r e n t (0% T ) and a n e m m i s i v i t y / r e s p o n s i v i t y (100% T) d a t a array. These c o n s i s t o f d i g i t i z e d responses from each of the p i x e l s i n t h e a r r a y u n d e r c o n d i t i o n s where t h e i n t e g r a t i o n t i m e and t h e s p e e d o f r e a d - o u t i s i d e n t i c a l t o t h a t p l a n n e d f o r t h e measurement o f t h e s p e c t r u m o f a s a m p l e . The d a r k - c u r r e n t d a t a w i l l p r o v i d e i n f o r m a t i o n as to the s h o t - n o i s e and o t h e r i n h e r e n t

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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A spectrum falling on a 10 element section of a Reticon linear photodiode array (4)

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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noise i n a n o n - i l l u m i n a t e d spectrum. T h e 100% T d a t a w i l l c h a r a c t e r i z e the e m m i s i v i t y o f the l i g h t s o u r c e and the r e s p o n s i v i t y of the d e t e c t o r . T h i s w i l l permit compensation f o r the v a r y i n g r e s p o n s i v i t i e s o f the p i x e l s as w i l l be d i s c u s s e d . It i s p o s s i b l e t o c h o o s e a l i g h t s o u r c e whose e m i s s i o n c o m p l e m e n t s t h e d e t e c t o r r e s p o n s e , s o t h a t a r e l a t i v e l y f l a t 100% T c u r v e is obtained. T h e s e d a t a a r r a y s a r e s t o r e d i n c o m p u t e r memory, and a s a m p l e s u b j e c t e d t o m e a s u r e m e n t . With the three arrays i n memory i t i s a s i m p l e m a t t e r t o c a l c u l a t e a b s o r b a n c e v a l u e s f o r the m a t e r i a l under e x a m i n a t i o n . T h i s p s e u d o - d o u b l e beam s p e c t r o m e t e r r e q u i r e s t h a t t h e s o u r c e be s t a b l e o v e r t h e measurement o f t h e t h r e e r e q u i r e d arrays. I t a l s o consumes a l a r g e amount o f c o m p u t a t i o n a l t i m e f o r b i d d i n g the v e r y r a p i d a c q u i s i t i o n of spectra.4l A l t h o u g h s u c h s p e c t r o m e t e r s h a v e b e e n b u i l t and u s e d i n o u r l a b o r a t o r y , the i n t e n t of t h i s a r t i c l e i s to d e s c r i b e the t r u e d o u b l e beam v e r s i o n we h a v e c o n s t r u c t e d f o r t h e N a t i o n a l C e n t e r for T o x i c o l o g i c a l Research. The s a m p l e and r e f e r e n c e a r r a y s w o r k i n i d e n t i c a l f a s h i o n and a r e d r i v e n b y a common c l o c k ; t h u s when t h e s a m p l e a r r a y i s o u t p u t t i n g a s i g n a l c o r r e s p o n d i n g to a c e r t a i n element, the reference array i s also outputting i t s corresponding array element. The c l o c k t h a t i s u s e d to d r i v e the a r r a y s i s a twop h a s e c r y s t a l c l o c k o p e r a t i n g a t t w e n t y k H z . T h e two p h a s e s a r e n i n e t y degrees out o f phase w i t h the f i r s t phase b e i n g used to d r i v e the a r r a y s . The s e c o n d p h a s e i s d e l a y e d b y 25 m i c r o s e c o n d s and i s u s e d a s a n e x t e r n a l s t a r t s i g n a l f o r t h e c o m p u t e r ' s analog-to-digital converter. In the r a p i d scanning environment of l i q u i d chromatography c o n t i n u o u s o p e r a t i o n of the d e v i c e i s u n n e c e s s a r y . One t h o u s a n d s p e c t r a / s e c o n d p r o v i d e s more d a t a t h a n c a n e i t h e r be u s e d o r h a n d l e d by a computer. In p r a c t i c e t h e r e f o r e , i t i s u s u a l to i n i t i a t e d a t a c o l l e c t i o n o n l y p e r i o d i c a l l y . The c o n c e p t s i n v o l v ed a r e a s f o l l o w s . D u r i n g a d e a d p e r i o d , when t h e d e t e c t o r i s not b e i n g u s e d , the l i g h t s t r i k i n g the d e t e c t o r ' s s u r f a c e s l o w l y l e a d s to s a t u r a t i o n of a l l the elements. In o r d e r to prepare f o r t h e g a t h e r i n g o f s p e c t r a l i n f o r m a t i o n , t h e a r r a y must be "dummy read". T h i s means a r e a d p u l s e i s a p p l i e d w h i c h s t a r t s t h e d r i v e e l e c t r o n i c s , and a l l e l e m e n t s i n t h e a r r a y a r e r e c h a r g e d b a c k t o t h e i r s t a r t i n g charge s t a t e . The l i g h t t h a t s t r i k e s e a c h e l e m e n t of the a r r a y d u r i n g the time p e r i o d b e f o r e the next s t a r t p u l s e i s i n t e g r a t e d as d e s c r i b e d p r e v i o u s l y . The i n t e g r a t i o n o r exposure time of the a r r a y s i s c o n t r o l l e d b y t h e c l o c k and a s e r i e s o f c o u n t e r s . The minimum i n t e g r a t i o n t i m e a t room t e m p e r a t u r e o f a n a r r a y r u n n i n g w i t h a 20 kHz c l o c k i s 15 m i l l i s e c o n d s ; t h e maximum i n t e g r a t i o n t i m e i s 205 m i l l i seconds. The minimum t i m e i s l i m i t e d b y t h e s c a n t i m e o f t h e a r r a y ; t h e maximum t i m e b y t h e r m a l c h a r g e . S i g n a l s p r o v i d e d by the a r r a y s a r e an e n d - o f - s c a n p u l s e and a b e g i n n i n g - o f - s c a n p u l s e . The e n d - o f - s c a n (EOS) p u l s e

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o c c u r s immediately a f t e r t h e l a s t element o f t h e a r r a y has been switched onto the v i d e o l i n e . I t i s used by the f r o n t - e n d p r o c e s s o r w h i c h w i l l be d i s c u s s e d . T h e b e g i n n i n g - o f - s c a n (BOS) s i g n a l i s used to s t a r t t h e p r o c e s s by which each element i s r e a d and p u t onto t h e v i d e o l i n e . T h i s s i g n a l i s a l s o used i n the f r o n t - e n d processor. Analog Front-End Processor. The sample and r e f e r e n c e v i d e o s i g n a l s a s w e l l a s t h e BOS a n d EOS p u l s e a r e t r a n s m i t t e d v i a c o a x i a l cables to a front-end analog processor ( F i g . 5). The f i r s t s t a g e o f t h e f r o n t - e n d p r o c e s s o r i s a 10 kHz l o w p a s s f i l t e r t o remove n o i s e t h a t i s t h e r e s u l t o f F E T s w i t c h r i n g i n g a s i l l u s t r a t e d i n f i g u r e 6. A f t e r t h e sample and r e f e r e n c e s i g n a l s have been f i l t e r e d the s i g n a l s a r e p l a c e d i n t o v o l t a g e a m p l i f i e r s . The a m p l i f i e r u s e d w i t h t h e r e f e r e n c e s i g n a l may b e u s e d i n e i t h e r a u n i t y or v a r i a b l e g a i n c o n f i g u r a t i o n , c o n t r o l l e d by a two p o s i t i o n s w i t c h t o t h e f r o n t p a n e l . The a m p l i f i c a t i o n i s v a r i a b l e from a g a i n o f 0.5 t o a p p r o x i m a t e l y 2 . 0 . The v a r i a b l e a m p l i f i c a t i o n i s used t o b a l a n c e t h e sample and r e f e r e n c e signals. The o u t p u t f r o m t h e u n i t y g a i n f o l l o w e r s i s i n p u t t o two additional amplifiers. The f i r s t i s a d i f f e r e n t i a l a m p l i f i e r which s u b t r a c t s t h e r e f e r e n c e s i g n a l from t h e sample s i g n a l . The output o f an a r r a y i s a t r a n s m i t t a n c e s i g n a l and i f t h e r e f e r e n c e i s t a k e n t o i n d i c a t e one hundred p e r c e n t t r a n s m i t t a n c e t h e n t h e o u t p u t o f t h e d i f f e r e n t i a l a m p l i f i e r w i l l have a d i r e c t c o r r e spondence w i t h t r a n s m i t t a n c e . The second a m p l i f i e r i n t o w h i c h t h e s a m p l e and r e f e r e n c e s i g n a l s a r e i n p u t i s a l o g - r a t i o amplifier. The o u t p u t o f t h e a m p l i f i e r c a n be c o n s i d e r e d a s absorbance. W i t h i n the f r o n t - e n d processor i s an i n t e g r a t o r that w i l l i n t e g r a t e the output of the l o g - r a t i o - a m p l i f i e r over the p e r i o d between t h e b e g i n n i n g and e n d - o f - s c a n p u l s e s . This i s the e q u i v a l e n t o f t o t a l i o n c u r r e n t i n GC/MS; t h e v a l u e i n L C / U V , V I S i s termed a t o t a l a b s o r p t i o n chromatogram o r T A C . The f r o n t - e n d p r o c e s s o r a l s o p r o v i d e s a v i s u a l warning f l a g when t h e r e f e r e n c e a r r a y s a t u r a t e s . T h i s i s done by c o m p a r i n g the s i g n a l l e v e l r e c e i v e d from the r e f e r e n c e a r r a y w i t h a manually set upper l i m i t . Saturation of the reference array i s n o t a p r o b l e m when t h e d e t e c t o r s y s t e m i s r u n w i t h a r e v e r s e phase o r any s i n g l e s o l v e n t system. T h e p r o b l e m becomes v e r y r e a l when g r a d i e n t e l u t i o n s y s t e m s a r e e m p l o y e d . Optical Design. T h e most common d e s i g n f o r a n u l t r a - v i o l e t s p e c t r o m e t e r i s t h e C z e r n y - T u r n e r m o d i f i c a t i o n o f t h e E b e r t mount for plane grating. T h i s t y p e o f mount was d i s c o u n t e d b e c a u s e t h e d e t e c t o r s b e i n g used r e q u i r e the f a s t e s t o p t i c a l system p o s s i b l e ( f / 3 . 0 o r f a s t e r ) . T h e e a s i e s t way t o s i m p l i f y t h e o p t i c a l s y s t e m i s by r e p l a c i n g the plane g r a t i n g w i t h a concave one. A concave g r a t i n g w i t h a f o c a l l e n g t h o f 200 mm a n d a n a p e r a t u r e o f f / 3 . 0

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

REF

hv

SAMPLE CELL

hv

•2

RETICON ARRAY

RETICON ARRAY

Figure 5.

Journal of Chromatographic Science

INTEGRATOR

RESET

INTEGRATOR

TRANSMITTANCE

•ABSORBANCE

The general structure of the analog front-end processor (4)

LO PASS FILTER

LORASS FILTER

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6.

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

143

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was s p e c i f i c a l l y d e s i g n e d b y J - Y O p t i c a l ( I n s t r u m e n t s S . A . ) f o r use w i t h l i n e a r photodiode a r r a y s . The f i n a l o p t i c a l p a t h u s e d i s shown i n F i g u r e 7 . The l i g h t s o u r c e i s a d e u t e r i u m lamp w i t h a power o u t p u t r a n g e o f 30 t o 60 w a t t s . As p r o v i d e d by O r i e l i t i n c l u d e s a 35 m i l l i m e t e r S u p e r s i l q u a r t z c o n d e n s e r s e t u s e d a s a c o n d e n s e r . A beam s p l i t t e r i s p l a c e d i n t h e l i g h t beam s u c h t h a t i t i s b e t w e e n t h e f o c a l p o i n t o f t h e c o n d e n s e r and t h e c o n d e n s e r . The l i g h t beam i s d i v i d e d i n t o two beams w h i c h w i l l b e u s e d a s a s a m p l e and r e f e r e n c e beam. A f t e r t h e s a m p l e and r e f e r e n c e cells, q u a r t z l e n s e s a r e used to f o c u s on entrance s l i t s . The s p e c t r o g r a p h s a r e composed o f a c a s t a l u m i n u m h o u s i n g w h i c h c o n t a i n s an e n t r a n c e s l i t , a f r o n t s u r f a c e f o l d i n g m i r r o r , and a c o n c a v e g r a t i n g . The g r a t i n g was d e s i g n e d t o h a v e a f o c a l l e n g t h o f 200 mm and a d i a m e t e r o f 70 mm. T h i s w i l l y i e l d an f-number o f a p p r o x i m a t e l y 3. T h e l i n e s p a c i n g i s 75 l i n e s / m i l l i m e t e r r e s u l t i n g i n a l i n e a r d i s p e r s i o n of 1.2 nanometer p e r one t h o u s a n d t h o f a n i n c h . Computer H a r d w a r e . The u s e o f a c o m p u t e r f o r d a t a a c q u i s i t i o n and c o n t r o l i s mandatory b e c a u s e o f the c l o c k f r e q u e n c y u s e d b y t h e R e t i c o n a r r a y (20 k H z ) and t h e v o l u m e o f d a t a t h a t i t i s capable of o u t p u t t i n g . A P D P - 8 e c o m p u t e r made b y D i g i t a l E q u i p m e n t C o r p o r a t i o n , w i t h 12 K w o r d s o f memory was e m p l o y e d . An E x t e n d e d A r i t h m e t i c ISlement (EAE) i n s t r u c t i o n s e t was installed. Two RK05 m o v i n g h e a d c a r t r i d g e d i s k d r i v e s p r o v i d e mass s t o r a g e c a p a b i l i t i e s of 3.2 m i l l i o n words. The most e c o n o m i c s o l u t i o n to long term storage i s magnetic tape. A 9 track, 800 b i t s p e r i n c h , 45 i n c h e s p e r s e c o n d t a p e d r i v e was u s e d . The ADC c o n v e r t e r h a d a n a n a l o g i n p u t r a n g e o f 0 t o +5 v o l t s a n d a d i g i t a l r a n g e o f 0 t o 1024 (10 b i t s ) . The D / A c o n v e r t e r s u s e d f o r s c o p e d i s p l a y were 10 b i t b i p o l a r c o n v e r t e r s . Two a d d i t i o n a l p i e c e s o f hardware were c o m m e r c i a l l y u n a v a i l a b l e . The f i r s t i t e m was a n e x t e n d e d r a n g e f i x e d p e r i o d c l o c k w i t h a r e s o l u t i o n o f 0.01 seconds and a r a n g e o f 1 6 7 , 7 7 2 . 1 6 s e c o n d s . In u s e , t h i s c l o c k i s s t a r t e d at the b e g i n n i n g o f a chromatographic r u n ; then as each spectrum i s t a k e n f o r s t o r a g e , the time a t w h i c h i t was t a k e n i s r e a d f r o m t h e d a y c l o c k . The s e c o n d h a r d w a r e u n i t was a n i n t e r r u p t , s k i p - c h e c k c o n t r o l u n i t f o r the R e t i c o n a r r a y s . T h i s u n i t a l l o w s the computer t o see the e n d - o f - s c a n and b e g i n n i n g - o f - s c a n p u l s e s g e n e r a t e d by the a r r a y s and a l l o w s the computer to have an o n / o f f c o n t r o l o f the e x t e r n a l c l o c k which i s used to s t a r t the a n a l o g to d i g i t a l converter. Applications

array

Software

S e v e r a l c o m m e r c i a l s i n g l e beam v e r s i o n s o f t h i s t y p e o f detector are a v a i l a b l e . They a r e o r i e n t e d toward the

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Figure 7.

Ray diagram for the optical bench (4)

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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MULTICHANNEL IMAGE DETECTORS

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s i n g l e e v e n t - r a p i d e v e n t e x p e r i m e n t , and h a v e v e r y l i m i t e d moving s t o r a g e c a p a b i l i t y . E s s e n t i a l t o any w i d e s p r e a d a p p l i c a t i o n o f t h e s e s p e c t r o m e t e r s i s an i n t e g r a t e d s e t o f a p p l i c a t i o n s software. W i t h o u t t h i s k e y i t e m p r o v i d e d and s u p p o r t e d b y a commercial s o u r c e , the a r e a w i l l not reach i t s p r o p e r development and a p p l i c a t i o n . The s p e c i f i c t a r g e t i n s t a l l a t i o n f o r t h e d o u b l e beam spectrometer d e s c r i b e d i s the r e s e a r c h l a b o r a t o r y of a l a r g e g o v e r n m e n t l a b o r a t o r y e n g a g e d i n t h e d e t e c t i o n and i d e n t i f i c a t i o n o f m e t a b o l i t e s i n the u r i n e o f mice b e i n g f e d p o t e n t i a l c a r c i n o gens. O v e r 400 m e t a b o l i t e s c a n be s e p a r a t e d i n s u c h u r i n e samples. The p r o j e c t i n v o l v e d c h a r a c t e r i z a t i o n o f e a c h o f t h e s e by U V / V I S s p e c t r o s c o p y . The u l t i m a t e g o a l was t o a t t e m p t t o c o r r e l a t e o n c o l o g i c a l events with c e r t a i n " d e s c r i p t o r s " obtained by o b s e r v a t i o n o f t h e U V / V I S r e c o r d . A t t h e o u t s e t , i t was i m p o s s i b l e to d e f i n e the exact r e g i o n s of the s p e c t r a t h a t would be i m p o r t a n t o r how t h e y s h o u l d be d i s p l a y e d b e s t f o r t h e o p e r a tor. I t was e s s e n t i a l t h a t a l l raw d a t a be s t o r e d f i r s t o n d i s k , and s e c o n d on b a c k - u p m a g n e t i c t a p e s o t h a t t h e u s e r c o u l d r e p l a y any g i v e n e x p e r i m e n t i n a v a r i e t y o f i n t e r a c t i v e modes. Theref o r e , s o f t w a r e was d e v e l o p e d w h i c h w o u l d p e r m i t t h e s p e c t r o m e t e r t o be s e t up b y p e r s o n n e l r e l a t i v e l y u n s k i l l e d i n c o m p u t e r o p e r a tion. A f t e r t h e d a t a was c o l l e c t e d i t c o u l d be s c a n n e d b y " c i n e m a t o g r a p h i c " l i k e t e c h n i q u e s , v i e w i n g each successive s p e c t r a i n a f o r w a r d r a p i d s c a n n i n g mode; s t o p p i n g d i s p l a y a t a f r a m e o f i n t e r e s t ; and r e v e r s i n g t h e d i s p l a y . Certain sections o f t h e s p e c t r a c o u l d be i n t e g r a t e d t o p r o v i d e t h e e q u i v a l e n t o f r u n n i n g the experiment w i t h a narrow band p a s s f i l t e r , a l l o w i n g c o m p a r i s o n o f an A b s o r b t i o n Band C h r o m a t o g r a m (ABC) i n c o n t r a s t t o t h e T A C , w h i c h i s an i n t e g r a l o f t h e T o t a l A b s o r b t i o n Chromatogram. T h e ABC c o u l d be c o n s t r u c t e d f r o m a s i n g l e p i x e l , o r t h e e n s e m b l e o f a l l p i x e l s , a t w h i c h p o i n t t h e ABC i s e q u i v a l e n t to the T A C . Any o f t h e s e d i s p l a y s c o u l d be p r e s e n t e d o n a CRT w i t h i n t e r a c t i v e c u r s o r s f o r d e t e r m i n a t i o n o f w a v e l e n g t h o r maxima, and a n y d i s p l a y c o u l d be d i r e c t e d t o a h a r d c o p y d i g i t a l p l o t t e r . T h i s c o n s t i t u t e s a minimum s e t o f s o f t w a r e p r o g r a m s t h a t s h o u l d be p r o v i d e d by any v e n d o r . T y p i c a l c o m p u t e r / u s e r d i a l o g u e s a r e shown i n f i g u r e s 8 and 9. S e v e r a l i m p o r t a n t e n t r i e s w i l l be d i s c u s s e d . A r e q u e s t f o r "SPECTRAL R A T E : " i s m a d e . * When a n o n - a v e r a g i n g method i s u s e d * a n y number may be g i v e n a s i n p u t t o t h e request for s p e c t r a l r a t e . T h e command d e c o d e r w i l l t h e n r e q u e s t "ENDING T I M E : " . The r e s p o n s e t o t h i s w i l l d e t e r m i n e t h e t i m e a t w h i c h d a t a a c q u i s i t i o n w i l l be a u t o m a t i c a l l y t e r m i n a t e d . Data *The p r o p e r r e s p o n s e i s dependent upon the d a t a a c q u i s i t i o n method u s e d . T h e means t o s e l e c t a s p e c i f i c method w i l l be discussed. I f a n e n s e m b l e a v e r a g i n g method i s u s e d , t h e n t h e s p e c t r a l r a t e i s t h e number o f s p e c t r a t o b e a v e r a g e d .

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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. R ZZOO MODE ?0 V P I & SU MULTIPLE WAVELENGTH DETECTION SYSTEM

THIS SYSTEM I S COMPOSED OF XXX SUBPROGRAMS WHICH ACQUIRE DATA FROM A DUAL BEAM MULTIPLE WAVELENGTH DETECTOR IN A CONFIGURATION FOR USE WITH A HIGH PRESSURE LIQUID CHROMATOGRAPH.

THE MODE OF OPERATION I S SELECTED BY ENTERING THE NUMBER CORRESPONDING TO THE OPERATION DESIRED IN THE FOLLOWING L I S T IN RESPONSE TO ' M O D E ? 1

THE 0. 1. 2. 3.

MODES OF OPERATION A R E : DESCRIPTION OF SYSTEM. DATA A C Q U I S I T I O N . EXAMINATION AND PLOTTING OF INDIVIDUAL SPECTRA. VIEW FROM TIME A TO TIME B (WITH INTEGRATION)

4. 5. 6. 7. 8.

TRANSFER DATA FROM MAGTAPE TO DISK TRANSFER OF DATA FROM DISK TO MAGTAPE FOR STORAGE. DETAILED INSTRUCTIONS FOR SYSTEM OPERATION. GENERATION OF PATTERN RECOGNITION DESCRIPTORS. SYSTEM ALIGNMENT AND ADJUSTMENT.

*******************^

* A L L INPUT FROM THE KEYBOARD I S ENDED BY HITTING * THE RETURN K E Y . OTHER ACTIONS CAUSE ERRORS *************^ MODE

* *

?

Figure 8.

Overall system description given by the primary monitor

American Chemical Society Library 1155 16th St. N. W.

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979. Washington, D. C. 20036

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MULTICHANNEL IMAGE DETECTORS

. R ZZOO MODE ?1

DATA ACQUISITION MODE DESCRIPTION

OF COMMANDS, Y OR N :

?Y

COMMAND SET

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FILENAME:-

(MUST B E 6 CHARACTERS.)

COMMENTS: (MAY BE 3 L I N E S OF 72 CHARACTERS E A C H . THEY MAY BE TERMINATED A T ANY POINT BY TYPING AN - @ - . ) SPECTRAL R A T E : SPECTRA/MINUTE = 1 / ( S P E C T R A L RATE * 50 M I L L I S E C . ) ENDING T I M E : (TIME AT WHICH RUN I S TO B E ENDED, GIVEN IN MULTIPLES OF A THOUSAND SECONDS, I . E . 1=1000 SECONDS 1 5 = 1 5 , 0 0 0 SECONDS. A RESPONSE OF ZERO - 0 - WILL BE TAKEN AS ENDING TIME = I N F I N I T Y . ) METHOD: A. B.

SINGLE SPECTRA ENSEMBLE AVERAGE WITH HARDWARE BACKGROUND SUBTRACTION

C.

ENSEMBLE AVERAGE WITH HARDWARE AND SOFTWARE BACKGROUND SUBTRACTION (IN METHODS B AND C THE VALUE OF SPECTRA/MINUTE IS USED AS THE NUMBER OF SPECTRA TO BE AVERAGED.)

EXECUTE,

Y OR N : (Y WILL START DATA ACQUISITION N WILL CAUSE A RETURN TO SYSTEM MONITOR.)

DATA ACQUISITION MAY BE STOPPED A T ANY TIME BY SETTING B I T 11 EQUAL TO ONE

Figure 9.

Description of input to data acquisition command decoder

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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a c q u i s i t i o n may a l s o b e t e r m i n a t e d f r o m t h e t e r m i n a l . The command d e c o d e r w i l l t h e n r e q u e s t " T H R E S H O L D : . The t h r e s h o l d i s used by the d a t a a c q u i s i t i o n programs to determine w h e t h e r o r n o t d a t a i s t o be s a v e d . I f the t h r e s h o l d i s set to 1 5 , t h e d a t a a c q u i s i t i o n p r o g r a m w i l l compare 15 w i t h t h e v a l u e o f the s p e c t r a l i n t e g r a t o r (TAC). I f the s p e c t r a l i n t e g r a t o r is l e s s t h a n 15 t h e n t h a t s p e c t r u m w i l l be r e j e c t e d a s c o n t a i n i n g baseline data. Thus s t o r a g e o f z e r o i n f o r m a t i o n i s a v o i d e d . The f i n a l p i e c e o f i n f o r m a t i o n r e q u i r e d b y t h e command d e c o d e r w i l l be p r o m p t e d b y t h e r e q u e s t " M E T H O D : " . The r e s p o n s e t o t h i s q u e r y w i l l d e t e r m i n e what method o f d a t a a c q u i s i t i o n w i l l be u s e d . The a l l o w a b l e r e s p o n s e s are: A - s i n g l e spectrum a c q u i s i t i o n B - ensemble average C - ensemble a v e r a g e w i t h the background s u b t r a c t i o n . The f i n a l o b v i o u s a c t i o n w i l l be t o p r i n t " E X E C U T E — Y o r N:". I f a n " N " i s e n t e r e d t h e command d e c o d e r w i l l terminate i t s e l f and t h e p r i m a r y m o n i t o r w i l l be s t a r t e d . A response of " Y " w i l l cause the a c q u i s i t i o n of s p e c t r a l data to b e g i n .

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11

Methods o f Data A c q u i s i t i o n . Any o f t h r e e m e t h o d s o f d a t a a c q u i s i t i o n may be s e l e c t e d . T h e f i r s t method i s t h e s i n g l e spectrum a c q u i s i t i o n . T h e mass s t o r a g e d e v i c e s w i l l be z e r o e d and a h a r d w a r e s t o p w a t c h i s started. When i t i s t i m e t o a c q u i r e a s p e c t r u m t h e c u r r e n t e l a p s e d time i s r e c o r d e d , the spectrum i s s t o r e d i n a temporary b u f f e r , and t h e v a l u e o f t h e s p e c t r a l i n t e g r a t o r i s c o m p a r e d t o t h e manually set t h r e s h o l d . I f the v a l u e i s l e s s than the t h r e s h o l d then t h i s spectrum i s i g n o r e d . At t h i s p o i n t the program b e g i n s t o c o u n t a n d s k i p s p e c t r a a c c o r d i n g t o t h e number g i v e n t o t h e q u e r y , "SPECTRAL R A T E " . When t h e a p p r o p r i a t e number o f s p e c t r a have been s k i p p e d the p r o c e s s w i l l r e p e a t i t s e l f . I f a t some p o i n t i n t h i s l o o p the v a l u e o f the s p e c t r a l i n t e g r a l i s g r e a t e r t h a n t h e t h r e s h o l d t h e s p e c t r a w i l l be s t o r e d on one o f t h e mass storage devices. S p e c t r a w i l l c o n t i n u e t o be s a v e d u n t i l t h e v a l u e of the i n t e g r a t i o n f a l l s below t h a t of the t h r e s h o l d . I n t h e f o l l o w i n g d i s c u s s i o n o f d a t a a c q u i s i t i o n b y methods B a n d C t h e l o g i c o r d e r o f e v e n t s i s t h e same a s u s e d i n method A. M e t h o d B i s a n e n s e m b l e a v e r a g e r t h a t u s e s t h e number e n t e r e d f o r s p e c t r a l r a t e a s t h e number o f s p e c t r a t o be a v e r a g e d . The s u m m a t i o n o f s p e c t r a i s d i v i d e d b y t h a t number t o g i v e a n average. S i g n a l t o n o i s e r a t i o improvement e q u a l t o the s q u a r e r o o t o f t h e number a v e r a g e d i s a c h i e v e d . Method C o f the d a t a a c q u i s i t i o n r o u t i n e s i s a l s o an ensemble a v e r a g e r ; however, a s o f t w a r e r o u t i n e has been added to do b a c k g r o u n d s u b t r a c t i o n . At the b e g i n n i n g of the r u n the s a m p l e and r e f e r e n c e c e l l must c o n t a i n p u r e s o l v e n t o n l y . The b a c k g r o u n d s u b t r a c t i o n i n method C w o r k s b y t a k i n g t h e f i r s t ensemble and s u b t r a c t i n g i t f r o m each o f the f o l l o w i n g ensembles b e f o r e t h e y a r e s t o r e d on a mass s t o r a g e d e v i c e . T h i s compen-

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s a t e s f o r beam s p l i t t e r n o n - e q u a l i t i e s the p h y s i c a l arrangement.

and o t h e r

aberrations

in

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Examination of Spectral Data. C o n t r o l of the system w i l l r e t u r n t o t h e p r i m a r y m o n i t o r when d a t a a c q u i s i t i o n h a s b e e n concluded. At t h i s p o i n t i f a 2 i s g i v e n i n response to the q u e r y "MODE?", t h e n the s e c o n d a r y m o n i t o r w i l l be s t a r t e d so t h a t i t can o v e r s e e the u s e o f the x - y p o i n t - p l o t - d i s p l a y and a Calcomp p l o t t e r . The f i r s t a c t i o n o f t h e s e c o n d a r y m o n i t o r w i l l b e t o p r i n t t h e que " S C O P E , PLOTTER MONITOR, DESCRIPTION AND INSTRUCTION S E T ? — Y E S OR N O ? " , a s shown i n f i g u r e 1 0 . When t h e r e s p o n s e i s " N O " i t w i l l go d i r e c t l y t o t h e n e x t q u e r y " S C O P E , PLOTTER, OR E X I T — S , P , OR E ? " Upon a r e s p o n s e o f " S " , i t i s assumed t h a t t h e s p e c t r a l d a t a i s t o be examined. An a b b r e v i a t e d l i s t of the scope commands i s p r i n t e d and t h e d i s p l a y o f t h e s p e c t r a d a t a w i l l begin. F i g u r e 11 c o n t i n u e s t h e s c o p e commands t h a t a r e p r i n t e d . The r a n g e o f t h e s p e c t r a l d a t a d i s p l a y e d on t h e scope i s 230 n a n o m e t e r s a t t h e l e f t edge o f t h e s c r e e n t o 537 nanometers at the r i g h t edge. The number i n t h e u p p e r most l e f t h a n d c o r n e r i s t h e t i m e a t w h i c h t h e s p e c t r a was t a k e n . D i r e c t l y below t h i s number i s a s e c o n d number w h i c h r e p r e s e n t s t h e w a v e l e n g t h . This w a v e l e n g t h i s t i e d t o a m o v a b l e c u r s o r w h i c h may b e p o s i t i o n e d w i t h t h e l i n e f e e d and b a c k s p a c e k e y s a t any p o i n t i n t h e s p e c trum. I f the d i s p l a y o f s p e c t r a l d a t a i s moving e i t h e r forward o r backward i n time the d i s p l a y o f the wavelength w i l l be t u r n e d off. O n l y when t h e s c o p e i s d i s p l a y i n g a s i n g l e s p e c t r u m c o n t i n u o u s l y w i l l t h e w a v e l e n g t h d i s p l a y be a c t i v a t e d . I n t h e command l i s t f o r t h e s p e c t r u m d i s p l a y p r o g r a m i s t h e " P " command. T h i s command i s u s e d t o t a g t h e s p e c t r u m w h i c h i s c u r r e n t l y b e i n g d i s p l a y e d f o r p l o t t i n g on the Calcomp p l o t t e r . In the n o r m a l sequence o f e v e n t s , c o n t r o l would be p a s s e d to the p l o t t i n g programs. These programs w i l l be d i s c u s s e d later. I f a " P " i s t y p e d i n r e s p o n s e t o t h e query "SCOPE, PLOTTER, OR E X I T " t h e n c o n t r o l w i l l b e g i v e n t o a n a s s e m b l y l a n g u a g e program t h a t i s d e s i g n e d t o format the s p e c t r a l d a t a so c h r o m a t o g r a p h s may b e p l o t t e d . The f i r s t a c t i o n o f t h e program w i l l be t o a s k " S I N G L E , INTEGRAL, OR E X I T — S , I , OR C T R L / D ? " . When a n " S " i s t y p e d t h e p r o g r a m w i l l go i n t o t h e s i n g l e w a v e l e n g t h mode and a c h r o m a t o g r a p h o f s i g n a l a m p l i t u d e v e r s u s t i m e may be g e n e r a t e d . The p r o gram w i l l g i v e q u e r i e s f o r w a v e l e n g t h , s t a r t i n g t i m e , and e n d i n g time. If an " I " i s e n t e r e d , an i n t e g r a l w i l l be p e r f o r m e d . The p r o g r a m w i l l a s k f o r a s t a r t i n g and e n d i n g w a v e l e n g t h a s w e l l a s t h e s t a r t i n g and t h e e n d i n g t i m e . A f t e r the chromatograms have been f o r m e d , whether by t h e s i n g l e o r i n t e g r a l w a v e l e n g t h method, a r e q u e s t w i l l be made a s t o t h e t y p e o f f i l t e r i n g t h a t i s t o

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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. R ZZOO MODE ?2 S C O P E , PLOTTER MONITOR DESCRIPTION AND INSTRUCTION S E T ? — Y E S OR NO?YES THIS PROGRAM SERVES AS A COORDINATOR OF THE USE OF THE OSCILLOSCOPE AND CALCOMP P L O T T E R . WILL RUN IN TWO MODES, SCOPE AND PLOTTER

SCOPE MODE ? C P E

IT

COMMAND SET CINE FORWARD IN DATA SET HOLD CURRENTLY DISPLAYED SPECTRA CINE BACKWARDS IN DATA SET RETURN TO S C O P E , PLOTTER MONITOR TAG CURRENT SPECTRA FOR PLOTTING E X I T , PLOT ANY TAGGED SPECTRA AND RETURN TO S Y S . MON.

************^ * T H E ABOVE COMMANDS DO NOT ECHO TO THE PRINTER* ************^^ THE NUMBER IN THE UPPER L E F T OF THE SCREEN IS THE TIME AT WHICH THE SPECTRA WAS TAKEN TO THE NEAREST ONE-HUNDREDTH OF A SECOND.

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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PLOTTER MODE THE PLOTTER FUNCTIONS

BY ASKING A SERIES OF

QUESTIONS

COMMAND SET SINGLE WAVELENGTH OR AN INTEGRAL OR WAVELENGTHS?—YES

OR NO

SINGLE MODE WAVELENGTHS? STARTING TIME? ENDING TIME? E X E C U T E — Y E S OR NO INTEGRAL MODE STARTING WAVELENGTH? ENDING WAVELENGTH? STARTING TIME? ENDING TIME? E X E C U T E — Y E S OR NO AN ANSWER OF ' 0 ' TO THE 'ENDING T I M E ' QUESTION WILL SET THE ENDING TIME TO THAT OF THE LAST SPECTRA

* U S E C TO INTERRUPT

TAKEN.

PLOTTING AT YOUR OWN R I S K *

************^

Figure TO. Command set description given by secondary monitor

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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153

SCOPE MODE

MOVE FORWARD HOLD CURRENT SPECTRA MOVE BACKWARDS RETURN TO SCOPE, PLOTTER MONITOR TAG CURRENT SPECTRA FOR PLOTTING PLOT ANY TAGGED SPECTRA & RETURN TO S Y S . MON.

? CTRL/C P E

SCOPE, PLOTTER MONITOR DESCRIPTION AND INSTRUCTION S E T ? — Y E S OR NO? Figure 11.

Non-echoing commands for spectral display program

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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MULTICHANNEL IMAGE DETECTORS

b e u s e d i n r e m o v i n g any h i g h f r e q u e n c y o r p e r i o d i c n o i s e . The types of f i l t e r s are l i s t e d i n Table I w i t h the response that causes i t s a c t i v a t i o n . TABLE A L i s t of

the D i g i t a l

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Response

I

Filters Filter

0 1 2 3 4

W h i c h May Be U s e d type

Do n o t f i l t e r 7 point Savizky-Golay f i l t e r 13 p o i n t S a v i t z k y - G o l a y f i l t e r 21 p o i n t S a v i t z k y - G o l a y f i l t e r Tukey E x p l o r a t o r y F i l t e r ! 2

I t s h o u l d b e n o t e d t h a t t h e s e p r o g r a m s do n o t make u s e o f s t a n d a r d l o g i c i n t h e way t h a t t h e y h a n d l e t h e d a t a . The n o r m a l s e q u e n c e w o u l d b e t o b r i n g a s much d a t a i n t o memory a s p o s s i b l e and move t h e f i l t e r t h r o u g h t h e d a t a . However, because o f the s i z e of the data base t h i s i s not f e a s i b l e . It i s necessary to move t h e d a t a t h r o u g h t h e f i l t e r . A f t e r the chromatographic d a t a has been f i l t e r e d a query o f "PREVIEW, P L O T , OR E X I T — V , P , OR E ? " w i l l b e made. A response of " E " w i l l r e t u r n c o n t r o l to the secondary m o n i t o r , w h i l e t y p i n g a " V " w i l l cause the e x e c u t i o n of a program t h a t d i s p l a y s the chromatograph on the x - y p o i n t p l o t d i s p l a y . The b e h a v i o r o f t h e d i s p l a y may b e c o n t r o l l e d b y t h e commands l i s t e d i n f i g u r e 12. T h e o n l y p u r p o s e o f t h i s method i s t o a l l o w t h e p r e v i e w i n g o f the chromatograph b e f o r e i t i s p l o t t e d on t h e Calcomp P l o t t e r . T y p i n g e i t h e r t h e command " C T R L / P " o r t h e command " P " i n r e s p o n s e t o t h e q u e r y "PREVIEW, PLOT OR E X I T ? " w i l l c a u s e t h e e x e c u t i o n o f a peak p i c k e r . I t works on the b a s i s o f d e t e r m i n i n g t h e mean n o i s e l e v e l o f t h e d a t a a c c o r d i n g t o t h e e q u a t i o n X

=

N Z i=2

| P -P

| /N

A f t e r the n o i s e l e v e l has been c a l c u l a t e d then the d i f f e r e n c e b e t w e e n two a d j a c e n t d a t a p o i n t s w i l l b e compared t o t h e n o i s e level. I f the d i f f e r e n c e i s g r e a t e r than the n o i s e then the p r o gram w i l l assume t h a t a p e a k h a s b e g u n . A t t h i s p o i n t an i n t e g r a l i s begun t o determine the a r e a under the p e a k . The end p o i n t o f the peak i s determined i n the r e v e r s e manner. At the end o f e a c h p e a k t h e s t a r t i n g , c e n t e r , a n d e n d i n g t i m e s o f t h e peak a r e p r i n t e d as w e l l as the a r e a under the p e a k . Calcomp P l o t t e r , Use and C o n t r o l . As can be seen from the command l i s t f o r p l o t t i n g s p e c t r a l d a t a i n f i g u r e s 13 and 14 a n y o f s e v e r a l p r o g r a m s may b e c h o s e n t o g i v e t h e d e s i r e d p l o t . For example, i f i n p l o t t i n g s p e c t r a l d a t a the program "USPEC.LD" i s chosen then a l l o f the s p e c t r a p r e v i o u s l y tagged f o r p l o t t i n g

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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PLOTTER PREVIEW PROGRAM COMMAND SET (NON-ECHOING)
?

AC Ap AF AD AN

MOVE FORWARD IN TIME MOVE BACKWARD IN TIME HOLD CURRENT DISPLAY RETURN TO SCOPE,PLOTTER MONITOR TO PLOTTER ROUTINE ACTIVATE FILTER ROUTINE FOR ONE PASS TURN DISPLAY ON TURN DISPLAY OFF

SCOPE, PLOTTER MONITOR DESCRIPTION AND INSTRUCTION SET?—YES OR NO? Figure 12.

Non-echoing commands for plotter preview program

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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MULTICHANNEL IMAGE DETECTORS

SPECTRUM PLOTTER INSTRUCTIONS,

Y OR N

?Y

THERE ARE THREE PROGRAMS WHICH MAY BE USED TO PLOT A SPECTRUM. SPEC.LD SPECG.LD UNSPEC.LD

THEY A R E :

PLOTS A L L SPECTRA, NO GRID PLOTS A L L SPECTRA WITH A GRID PLOTS A L L SPECTRA UNSCALED

IN RESPONSE TO THE PERIOD TYPE THE FOLLOWING

INSTRUCTIONS

. R FRTS *XXXX.ZZ/G *BLOCKl.DA/l *PLOTZl.DA/2

WHERE X X X X . Z Z

Figure 13.

IS

THE SPECTRUM PLOTTER TO BE USED

Instruction for using spectrum plotter

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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6.

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CHROMATOGRAPH PLOTTER INSTRUCTIONS,

(Y OR N)

?Y

IN RESPONSE TO THE PERIOD, FOLLOWING INSTRUCTIONS

TYPE

IN

. R FRTS *CHRMG.LD/G *BLOCK.DA/l *OUTPUT/2 Figure 14.

Instruction for using chromatograph plotter

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w i l l be p l o t t e d i n a n u n s e a l e d f o r m a t . I f on the o t h e r hand, e i t h e r o f the programs " S P E C . L D " o r " S P E C G . L D " i s chosen the s p e c t r a l d a t a w i l l be i n d i v i d u a l l y s c a l e d . "SPECG.LD" w i l l o v e r l a y t h e p l o t w i t h a s e t o f g r i d l i n e s s p a c e d a t one i n c h i n t e r v a l s while "SPEC.LD" w i l l not. T h e two p r o g r a m s f o r p l o t t i n g c h r o m a t o g r a p h s , " C H R M . L D " a n d "CHRMG.LD" a r e i d e n t i c a l i n t h e i r methods o f t r e a t i n g t h e d a t a . Both programs a r e d e s i g n e d t o s c a l e the d a t a . Both programs w i l l g e n e r a t e p l o t s o f v a r y i n g l e n g t h up t o a maximum o f f o r t y i n c h e s . T r a n s f e r o f D a t a From D i s k t o M a g n e t i c T a p e . Transfer of the s p e c t r a l d a t a from d i s k to magnetic tape i s a c c o m p l i s h e d by r e s p o n d i n g t o t h e p r i m a r y m o n i t o r ' s que w i t h a 5 . Each time t h i s mode o f o p e r a t i o n i s e n t e r e d two f i l e s w i l l b e w r i t t e n t o magtape. T h e f i r s t f i l e w i l l c o n t a i n one r e c o r d o f f i v e h u n d r e d t w e l v e b y t e s , where a b y t e i s s i x b i t s . The i n f o r m a t i o n w r i t t e n w i l l b e t h e s a m p l e name and comments w h i c h w e r e g i v e n t o t h e d a t a a c q u i s i t i o n command d e c o d e r . The f i l e s a r e t e r m i n a t e d w i t h an end o f f i l e m a r k . C o n t r o l w i l l b e p a s s e d t o a new p r o g r a m whose f u n c t i o n i s to t r a n s f e r s p e c t r a l data from the d i s k to the magtape. One s p e c t r u m i s c o n t a i n e d i n e a c h r e c o r d o f 512 b y t e s . Results D e t e c t o r P e r f o r m a n c e and C o n c l u s i o n s . T h e p e r f o r m a n c e o f t h e d e t e c t o r was c h e c k e d b y c o n n e c t i n g i t t o a S p e c t r a - P h y s i c s M o d e l 3500 l i q u i d c h r o m a t o g r a p h . T h e L C i s s e t t o do b o n d e d p h a s e c h r o m a t o g r a p h y w i t h a s p h e r i s o r b ODS c o l u m n a n d a 3 : 1 m e t h a n o l , water s o l v e n t system. S i g n a l to Noise R a t i o . The n a t u r e o f the d a t a c o l l e c t e d and t h e d i f f e r e n t ways i n w h i c h i t may b e h a n d l e d y i e l d 3 t y p e s o f s i g n a l to n o i s e r a t i o s . They a r e the s i g n a l to n o i s e r a t i o f o r an i n d i v i d u a l s p e c t r u m ; the s i g n a l t o n o i s e r a t i o o f a c h r o m a t o gram a t a g i v e n w a v e l e n g t h , a n d t h e s i g n a l t o n o i s e r a t i o o f a chromatogram o v e r a s p e c t r a l b a n d w i d t h . The s i g n a l t o n o i s e r a t i o f o r a n i n d i v i d u a l s p e c t r u m i s given i n Table II. The methods o f a c q u i s i t i o n were e i t h e r A ( s i n g l e s p e c t r u m a c q u i s i t i o n ) o r B (ensemble averaged s p e c t r u m ) . TABLE Signal Method A B B B

to Noise Ratios SNR 174 206 253 271

II

f o r an I n d i v i d u a l

Spectrum

No. of Spectra Averaged 0 2 4 8

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159

The s i g n a l t o n o i s e f i g u r e s f o r e n s e m b l e a v e r a g i n g i n d i c a t e t h a t some n o i s e s o u r c e o t h e r t h a n t h e r m a l n o i s e i s a c t i v e . The s o u r c e o f t h i s n o i s e was u l t i m a t e l y f o u n d t o b e t h e c o m p u t e r b u s and t h e r e a d - o u t e l e c t r o n i c s . These s i g n a l to n o i s e r a t i o s can be i m p r o v e d g r e a t l y b y u s e o f t h e " S " S e r i e s a r r a y s w h i c h h a v e much l a r g e r a r e a s and d y n a m i c r a n g e s o f 9 0 , 0 0 0 / 1 , a s w e l l a s b y using read-out e l e c t r o n i c s which minimize switching n o i s e . Comm e r i c a l equipment i s a v a i l a b l e to p e r f o r m t h i s f u n c t i o n . The s i g n a l t o n o i s e r a t i o s f o r c h r o m a t o g r a m s a r e g i v e n i n Table III. I n t h i s t a b l e " M e t h o d " r e f e r s t o t h e means b y w h i c h t h e s p e c t r a l d a t a were t a k e n , " S N R " i s t h e s i g n a l t o n o i s e r a t i o o f a chromatogram g e n e r a t e d by s p e c i f y i n g a s i n g l e w a v e l e n g t h , and " S N R 2 o ^ 8 l n o i s e r a t i o o f a chromatogram g e n e r a t e d b y i n t e g r a t i n g 20 a r r a y e l e m e n t s o r 24 n a n o m e t e r s .

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TABLE Signal Method A B B B

SNR 348 438 576 869

to N o i s e R a t i o s C

III for

SNR 2Q C

906 1006 1217 1765

Chromatograms N

o

«

Averaged 0 2 4 8

E f f e c t s o f P o s t Run D i g i t a l F i l t e r i n g . I n an e f f o r t to i m p r o v e t h e s i g n a l t o n o i s e r a t i o a 21 p o i n t S a v i t z k y - G o l a y 2 . d i g i t a l f i l t e r was i n s t a l l e d i n t h e s o f t w a r e s y s t e m . However, t h i s f i l t e r p r o v e d t o h a v e a n a d v e r s e e f f e c t u p o n p e a k s p a c e and amplitudes. T h e e f f e c t i s i l l u s t r a t e d i n f i g u r e s 15 and 16 w h i c h a r e p l o t s o f the e f f e c t of the d i g i t a l f i l t e r w i d t h upon peak a r e a and p e a k h e i g h t r e s p e c t i v e l y . The f i l t e r has i t s g r e a t e s t e f f e c t upon peak a r e a . T h i s response of a s i g n a l to f i l t e r i n g i s t y p i c a l o f a l l m e t h o d s o f f i l t e r i n g when t h e number o f d a t a p o i n t s i n the f u l l w i d t h a t h a l f h e i g h t of the peak d i v i d e d by the f i l t e r w i d t h i s l e s s than 1 . 3 . The b e h a v i o r o f t h e T u k e y l e x p l o r a t o r y f i l t e r i s e x c e l l e n t i n t h a t the change i n s i g n a l to n o i s e r a t i o approximates t h a t o f a 9 p o i n t S a v i t z k y - G o l a y w i t h o u t the c o r r e s p o n d i n g e f f e c t s upon peak h e i g h t and a r e a . I n o p e r a t i o n , the Tukey f i l t e r i s a 3 p o i n t m e d i a n r e p l a c e m e n t s e r i e s i n w h i c h t h e f i l t e r window moves through the data r e p l a c i n g the c e n t e r p o i n t of the three w i t h the v a l u e w h i c h i s n u m e r i c a l l y l o c a t e d between the o t h e r two. The r e s u l t s o f s u c h a f i l t e r a r e shown i n f i g u r e s 1 7 , 1 8 , a n d 1 9 . F i g u r e 17 i s a s e t o f t e s t d a t a ; f i g u r e 18 i s t h e r e s u l t o f f i l t e r i n g t h e t e s t s e t w i t h a 21 p o i n t S a v i t z k y - G o l a y f i l t e r ; a n d f i g u r e 19 i s t h e same d a t a f i l t e r e d b y t h e T u k e y a l g o r i t h m . W h i l e the s i g n a l to n o i s e r a t i o of the S a v i t z k y - G o l a y f i l t e r i s b e t t e r t h a n t h a t o f T u k e y , i t h a s a l s o c a u s e d a 30 p e r c e n t d r o p i n t h e s i g n a l a m p l i t u d e w h i l e the Tukey f i l t e r g i v e s an 8 p e r c e n t

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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MULTICHANNEL IMAGE DETECTORS

Figure 15.

The effect of the Savitzky-Gohy filters upon peak areas

Talmi; Multichannel Image Detectors ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

NUNN AND DESSY

Liquid Chromatographic Separation

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16 -

Q.