Multichannel Image Detectors Volume 2 - American Chemical Society

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Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 28, 2018 | https://pubs.acs.org Publication Date: November 16, 1983 | doi: 10.1021/bk-1983-0236.ch007

Luminescence Measurements with an Intensified Diode Array J A M E S D. I N G L E , JR. and M A R Y A N D R I E U RYAN1 Department of Chemistry, Oregon State University, Corvallis, O R 97331

In the past, molecular luminescence spectrometry was always conducted with single channel systems involving a photomultiplier tube (PMT) as the detector. The a v a i l a b i l i t y of multichannel detectors with internal gain has provided a new powerful tool for luminescence measurements, and several types of applications have been reported (1-15). This paper i s concerned with the application of an intensified diode array (IDA) for dynamic molecular fluorescence and chemiluminescence measurements. In this paper the types of measurements and analytical systems for which multichannel detectors are used i n our laboratory are introduced. Next the specific IDA system used i s presented along with important hardware and software considerations. Third, the characteristics of the IDA detector are reviewed to give some perspective about i t s influence on the quality of measurements. Finally, some typical applications to chemical systems are presented to i l l u s t r a t e the advantages of multichannel detection.

1Current address: Tektronix, Inc., Beaverton, OR 97977.

0097-6156/83/0236-0155$06.00/0 © 1983 American Chemical Society

Talmi; Multichannel Image Detectors Volume 2 ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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Types of Chemical Systems Studied In fluorescence k i n e t i c s - b a s e d (FL KB) measurements, the analyte i s reacted with a s e l e c t i v e reagent to form a f l u o r e s c e n t product. Under s u i t a b l e c o n d i t i o n s the i n i t i a l r a t e , measured as a change i n the FL s i g n a l over a s e l e c t e d time p e r i o d , i s p r o p o r t i o n a l to the analyte c o n c e n t r a t i o n . Low d e t e c t i o n l i m i t s (ng/mL and sub ng/mL) are provided by the technique f o r inorganic and organic a n a l y t e s . E x c e l l e n t s e l e c t i v i t y a r i s e s due to the use of fluorescence monitoring (a small f r a c t i o n of molecules f l u o r e s c e , e x c i t a t i o n and emission wavelengths can be optimized), the use o f a s p e c i f i c optimized chemical r e a c t i o n s , and f i n a l l y the use of a k i n e t i c measurement ( d i s c r i m i n a t i o n against steady s t a t e background fluorescence and s c a t t e r i n g s i g n a l s and species which r e a c t at s i g n i f i c a n t l y different rates). Instrumentally, KB FL measurements are u s u a l l y made with a scanning emission monochromator which i s set to monitor a s p e c i f i c wavelength band during the course of a reaction. In chemiluminescence (CL) measurements the analyte i s mixed with s u i t a b l e reagents to cause a r e a c t i o n to occur i n which an intermediate or product i n an e x c i t e d e l e c t r o n i c s t a t e i s formed. The f l a s h of l i g h t produced i s q u a n t i t a t e d as a peak height or area. Metal analytes are u s u a l l y determined by t h e i r enhancement of a blank CL r e a c t i o n . In some cases the analyte can be the CL p r e c u r s o r . The technique i s s u i t a b l e f o r t r a c e determination of a wide v a r i e t y of species with o f t e n good s e l e c tivity. T y p i c a l l y no wavelength s e l e c t i o n device i s employed and r a d i a t i o n at a l l wavelengths i s d i r e c t e d to a PMT s i n c e a l l the CL a r i s e s from the r e a c t i o n of i n t e r e s t . For both types of measurements described above, the luminescence s i g n a l i s dynamic or changes with time. Because the r e a c t i o n s studied f o r KB FL measurements have h a l f - l i v e s o f 2-30 min, s i g n i f i c a n t i n t e n s i t y changes can occur over even a few seconds. The f l a s h of l i g h t observed i n many CL r e a c t i o n s t y p i c a l l y has a d u r a t i o n of s e v e r a l seconds or l e s s . C l e a r l y i n both s i t u a t i o n s an u n d i s t o r t e d spectrum cannot be acquired w i t h a conventional scanning monochromator. Instrumentation A m u l t i p l e wavelength spectrometer i s constructed by p l a c i n g a multichannel d e t e c t o r i n the f o c a l plane of a spectrograph so that each d e t e c t o r element i n t e r c e p t s a d i f f e r e n t small wavelength range. The multichannel detector used f o r t h i s work i s a Tracor Northern TN-1710-21 IDA. I t c o n s i s t s of a Reticon 512 element diode array (DA) with 0.45 mm high diodes separated by 50 ]M spacings. The DA i s cooled with a P e l t i e r thermoelect r i c c o o l e r . The DA i s mounted at the output of a microchannel p l a t e i n t e n s i f i e r (MCP) which has a photocathode with an extend-



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ed red response to provide s e n s i t i v i t y i n the red part o f the spectrum. The MCP i s an e l e c t r o s t a t i c a l l y - f o c u s e d image i n v e r t i n g i n t e n s i f i e r with a nominal luminous g a i n o f 35,000. A s c i n t i l l a t o r i n f r o n t o f the photocathode determines the response i n the UV. The commercial IDA i s provided w i t h p r e a m p l i f i e r and timing e l e c t r o n i c s . We chose to b u i l d our own data a c q u i s i t i o n and c o n t r o l system, although such a system can a l s o be purchased from Tracor Northern. The use o f the IDA f o r luminescence s p e c t r a l measurements i s i l l u s t r a t e d i n F i g u r e 1. Fluorescence or chemiluminescence from a sample c e l l i s focused on the s l i t o f an f/3 spectrograph. The spectrograph disperses the r a d i a t i o n i n i t s f o c a l plane where the spectrum i s i n c i d e n t on the photocathode o f the IDA. Some o f the photons impingent on the photocathode cause e j e c t i o n o f photoelectrons from the photocathode. Each p h o t o e l e c t r o n passes i n t o a channel o f the MCP and emerges at the other end as a charge packet o f 10^ - 10^ e l e c t r o n s . The charge packet s t r i k e s the phosphor to produce a photon packet f o r every i n i t i a l p h o t o e l e c t r o n . The photon packet i s impingent on a p a r t i c u l a r diode element and causes many e l e c t r o n - h o l e p a i r s t o be c r e a t e d . A f t e r a s u i t a b l e viewing o r i n t e g r a t i o n time, the s i g n a l s from the i n d i v i d u a l diodes are read out i n a s e q u e n t i a l manner. The diode s i g n a l s are r e l a t e d to the amount o f charge required to destroy the e l e c t r o n - h o l e p a i r s created by impingent l i g h t o r thermal e f f e c t s . The charge pulse i s converted to a p r o p o r t i o n a l voltage pulse o f about 2 ps d u r a t i o n and a maximum peak height o f 10 V. The maximum value i s determined by the s a t u r a t i o n l e v e l o f the diodes (which i s l i m i t e d by the maximum number o f e l e c t r o n - h o l e p a i r s that can be generated). F i b e r o p t i c f a c e p l a t e s between the photocathode and input o f MCP and between the phosphor and the DA ensure that good s p a t i a l i n t e g r i t y i s obtained. Thus a photon o f a p a r t i c u l a r wavelength which s t r i k e s a p a r t i c u l a r part o f the photocathode w i l l e v e n t u a l l y r e s u l t i n a s i g n a l at a p a r t i c u l a r diode. The r e c i p r o c a l l i n e a r d i s p e r s i o n o f the spectrograph used i s about 25 nm/mm y i e l d i n g 1.3 nm/diode over the wavelength range 200 - 840 nm. Hardware. To employ the IDA, the user must supply a c l o c k s i g n a l , a begin scan s i g n a l , and data a c q u i s i t i o n hardware. The hardware to accomplish t h i s has been based on a PDP 11/20 m i n i computer o r a KIM microcomputer plus a d d i t i o n a l e x t e r n a l h a r d ware (13-15). The c l o c k s i g n a l determines the r a t e a t which the diodes are i n t e r r o g a t e d during readout and was 33 kHz with the minicomputer and 125 kHz with the microcomputer. This means the time to read out a l l 512 diodes and hence the minimum i n t e g r a t i o n time v a r i e s from 15 ms to 3.7 ms, r e s p e c t i v e l y . F o r the microcomputer, d i r e c t memory access (DMA) c i r c u i t r y was used t o increase the data a c q u i s i t i o n r a t e .



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sample cell

|

| < * > * A / V e x c i t a t i o n beam —

entrance

slit

hi/

to data ->

acquisition system

photocathode

concave holographic grating

photodiode array phosphor

microchannel plate, electron multiplier

Figure 1. Instrumental

configuration for fluorescence diode array.

measurements with an intensified



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The begin scan s i g n a l i s used to s t a r t the DA diode i n t e r r o g a t i o n and to c o n t r o l the i n t e g r a t i o n time o f a spectrum. The array i s c l e a r e d with one begin scan s i g n a l , the photon s i g n a l i s i n t e g r a t e d , and then another begin scan s i g n a l i s used f o r s i g n a l readout. The time between begin scan s i g n a l s determines the i n t e g r a t i o n time. The diode a r r a y s i g n a l s are output on a video l i n e . T h i s was connected to a f a s t sample-and-hoId and then a f a s t (2 ps) 12 b i t ADC. T h i s allows the peak s i g n a l v o l t a g e from each diode to be sampled and converted to a 12 b i t d i g i t a l r e p r e s e n t a t i o n to be stored i n computer memory. S e q u e n t i a l memory l o c a t i o n s are used f o r the s i g n a l from each diode with the s a t u r a t i o n s i g n a l corresponding to 1 0 * o r 4096. 2

Software. The software was t a i l o r e d to f i t our p a r t i c u l a r a p p l i c a t i o n . Some o f the important options are d e s c r i b e d below: 1. Successive 512 point s p e c t r a are stored i n s u c c e s s i v e blocks of memory where the i n t e g r a t i o n time ( t ) , time between s p e c t r a , and a number o f spectra are user chosen. 2. Under user c o n t r o l , s number o f s u c c e s s i v e scans can be summed i n the same memory l o c a t i o n s f o r s i g n a l averaging. 3. To save memory space, the data from the f i r s t η diodes can be ignored (not s t o r e d ) . 4. To save memory space and time, the l a s t m diodes can be clocked at a f a s t e r c l o c k rate and the data ignored. 5. A f t e r s p e c t r a are a c q u i r e d , they can be d i s p l a y e d on a graphics t e r m i n a l . Overlapping o f d i f f e r e n t s p e c t r a and s c a l e expansion allow easy comparison o f s p e c t r a . 6. The s i g n a l from one diode or the sum o f s i g n a l s from η diodes can be d i s p l a y e d . 7. The sum o r d i f f e r e n c e o f any two s p e c t r a o r o f s i g n a l s from one o r more diodes can be c a l c u l a t e d o r d i s p l a y e d . The d i f f e r e n c e o p t i o n can be used to c a l c u l a t e a r a t e o r subtract a blank. 8. The mean and standard d e v i a t i o n o f the s i g n a l s from any group o f diodes f o r a s e r i e s o f e q u i v a l e n t runs can be c a l c u l a t e d and d i s p l a y e d . 9. A wavelength c a l i b r a t i o n program allows readout o f s i g n a l information i n terms o f nanometers r a t h e r than diode numbers. Performance

Characteristics

Dark Signal C h a r a c t e r i s t i c s . The dark s i g n a l c h a r a c t e r i s t i c s are measured with no l i g h t impingent the IDA and are important because the dark s i g n a l l i m i t s the maximum i n t e g r a t i o n time and can l i m i t the S/N at low l i g h t l e v e l s . Without c o o l i n g , the DA reaches s a t u r a t i o n i n about 10 s, while i t takes about 50 s with c o o l i n g to about -10°C. Normally the heat from the high tempera ture side o f the P e l t i e r c o o l e r i s d i s s i p a t e d by ambient a i r movement. Considerable dark s i g n a l d r i f t i s observed throughout



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a day as the a i r temperature changes. We found i t e s s e n t i a l to use a c o o l i n g c o l l a r based on c i r c u l a t i o n o f tap water to d i s s i pate the heat. This lowered the dark s i g n a l by over a f a c t o r o f two, but more s i g n i f i c a n t l y i t lowered the d r i f t i n the dark s i g n a l . B e t t e r thermostating c o n t r o l of the P e l t i e r c o o l e r would reduce the d r i f t problem a l s o . It was determined that a f t e r a l a r g e dark s i g n a l had accumul a t e d , s e v e r a l scans were r e q u i r e d to reach a constant dark s i g n a l l e v e l f o r successive scans. This problem was a l l e v i a t e d by c o n t i n u a l l y scanning ( p r o v i d i n g begin scan p u l s e s ) every 20 ms between spectra to keep the dark s i g n a l accumulation at a small v a l u e . I n t e n s i f i e r C h a r a c t e r i s t i c s . The i n t e n s i f i e r gain can be v a r i e d between about 200 and 10^. Intensification i s essential for luminescence measurements to produce photon s i g n a l s above the dark and readout n o i s e o f the DA. The S/N under s i g n a l c a r r i e d noise s i t u a t i o n s ( s i g n a l shot or f l i c k e r n o i s e ) i s independent of the i n t e n s i f i e r g a i n . The i n t e n s i f i e r degrades the r e s o l u t i o n of the DA by about a f a c t o r o f 2 to 3.5 diode elements. At the lowest i n t e n s i f i e r g a i n s e t t i n g the dark s i g n a l c h a r a c t e r i s t i c s are e q u i v a l e n t to those obtained with the i n t e n s i f i e r b i a s voltage o f f . At f u l l i n t e n s i f i e r gain, the dark s i g n a l i s increased about 10% and the dark s i g n a l n o i s e i s doubled. T h i s i s suspected to be due to thermal e l e c t r o n s generated at the photocathode that r e c e i v e the same i n t e n s i f i e r gain as photoelectrons. When l i g h t i s removed from the photocathode o f the i n t e n s i f i e r , a decaying l i g h t s i g n a l i s observed due to r e s i d u a l phosphorescence from the phosphor used i n the i n t e n s i f i e r . I t appears to be composed o f two components with h a l f - l i f e s o f tens of m i l l i s e c o n d s and a few seconds. This behavior d i d not s i g n i f i c a n t l y a f f e c t our luminescence measurements. Because i n t e g r a t i o n times were t y p i c a l l y 1 s or g r e a t e r , the s h o r t e r l i v e d component response time i s i n s i g n i f i c a n t . In other experiments (not i n v o l v i n g luminescence) w i t h pulsed l i g h t sources, the phosphor l i f e t i m e d i d d i s t o r t s i g n a l s modulated a t frequencies greater than 10 Hz. The l o n g e r - l i v e d decay component e f f e c t was i n s i g n i f i c a n t i n our time sequence experiments because the r e l a t i v e change i n s i g n a l between s e q u e n t i a l spectra was a f r a c t i o n o f f u l l s c a l e . S i g n a l and S/N R e l a t i o n s h i p s . As a f i r s t approximation the f o l l o w i n g c h a r a c t e r i s t i c s are expected to apply: n,

(1) (2)


Luminescence

7. INGLE AND RYAN n.

n. α L

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(3)

c, t , s

c

1/2

, t

161

1/2

, s

1/2

(4)

where o f f s e t s i g n a l ( f i x e d p a t t e r n "noise")


dark s i g n a l

CT

R

% σ

τ c

light

signal

total

signal

= readout n o i s e dark c u r r e n t n o i s e

= l i g h t signal noise t o t a l noise number o f channels added together

t

= i n t e g r a t i o n time

s

= number o f scans added together

Equations 1 and 2 j u s t i n d i c a t e that the t o t a l s i g n a l o r n o i s e i n counts i s due to the sum o f three independent e f f e c t s : the o f f s e t or readout process, the dark s i g n a l , and the l i g h t s i g n a l . R e l a t i o n s h i p 3 i n d i c a t e s that i f we add the s i g n a l s from two channels together, i n t e g r a t e twice as long, or sum the s i g n a l s from two scans, the dark and l i g h t s i g n a l s should double. For r e l a t i o n s h i p 4, i t i s assumed that s i g n a l l e v e l s are low enough that fundamental shot n o i s e i s l i m i t i n g , which i s p r o p o r t i o n a l to the square root o f the s i g n a l and hence the square root o f f a c t o r s d i r e c t l y p r o p o r t i o n a l to the s i g n a l . The above r e l a t i o n s h i p s were tested by v a r y i n g c, t , and s over a l a r g e range (14). F i r s t dark s i g n a l measurements were made and then measurements were made with the IDA photocathode i l l u m i n a t e d with uniform white l i g h t . The c o n t r i b u t i o n s o f dark and readout s i g n a l and n o i s e were subtracted from the t o t a l s i g n a l to o b t a i n the c o n t r i b u t i o n from only the l i g h t s i g n a l . Most of the above r e l a t i o n s h i p s are shown to be true and S/N s o f 500 were obtained i n some cases. The dependence o f s i g n a l s and noise on the number of channels added together i s approximate due to diode to diode v a r i a t i o n s i n dark s i g n a l and diode r e s p o n s i v i t y . The dark s i g n a l was not p r o p o r t i o n a l to t above about 25% o f DA s a t u r a t i o n ; a negative d e v i a t i o n was observed. The same a f f e c t was observed f o r the l i g h t s i g n a l i f the dark s i g n a l component reaches 25% s a t u r a t i o n i n the i n t e g r a t i o n time. Hence i t i s c r i t i c a l that the dark s i g n a l subtracted from the t o t a l s i g n a l to o b t a i n the net l i g h t i s measured with 1


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the same i n t e g r a t i o n time. In t h i s case, the net l i g h t s i g n a l i s p r o p o r t i o n a l to the i n c i d e n t l i g h t i n t e n s i t y . The l i g h t s i g n a l noise (στ,) was found not to be p r o p o r t i o n a l to the square root o f the number of channels unless the diodes chosen were separated by 9 diodes. The noise στ, increased f a s t e r than f a l l separations because the s i g n a l s from adjacent and near diodes are not independent. This i s b e l i e v e d to be due to the r e s o l u t i o n degradation caused by the i n t e n s i f i e r . Photons s t r i k i n g a 50 μη spot on the i n t e n s i f i e r photocathode cause an eventual burst of photons over s e v e r a l hundred micrometers. Thus the s i g n a l s from s e v e r a l diodes can t r a c k together the s i g n a l caused by photons a r r i v i n g at one spot on the photocathode.


o

r

s m

D e t e c t i o n L i m i t C o n s i d e r a t i o n s . For low l i g h t l e v e l measure ments, the S/N i s l i m i t e d by readout or dark n o i s e . The readout n o i s e (o%) i s about 3.5 counts. Above 1 s i n t e g r a t i o n times and with f u l l i n t e n s i f i e r g a i n , the dark s i g n a l noise becomes l i m i t i n g . The noise generated by the data a c q u i s i t i o n c i r c u i t r y i s i n s i g n i f i c a n t compared to the readout noise on the video l i n e generated by the IDA and i t s e l e c t r o n i c s . Since i n our system, 1 readout count i s e q u i v a l e n t to about 5000 e's or about 1-2 photoelectrons at f u l l i n t e n s i f i e r gain (^ 6000), the readout n o i s e i s about 1.7 χ 10^ e l e c t r o n s . Thus our system can detect the presence o f 4-8 photoelectrons i n one diode channel, and hence i s near the photon counting l i m i t . A p p l i c a t i o n s o f the

IDA

Several chemical systems were studied with the s p e c t r o f l u o rometer with IDA d e t e c t i o n to l e a r n about the p o t e n t i a l advantages provided by multichannel d e t e c t i o n . A d e t e c t i o n l i m i t o f 0.2 ng/mL was obtained f o r measurements o f the steady s t a t e fluorescence o f quinine s u l f a t e (12). Here an 8 s i n t e g r a t i o n time was employed and the s i g n a l s from the 6 diodes (7.5 nm range) centered at the wavelength o f peak emission were averaged. The low d e t e c t i o n obtained i n d i c a t e s that the IDA i s a powerful q u a n t i t a t i v e t o o l f o r t r a c e fluorescence measure ments. The d e t e c t i o n with the IDA i s only about ten times higher than obtained with the same spectrofluorometer with monochromator wavelength s e l e c t i o n and PMT d e t e c t i o n (the s l i t width was about 4 l a r g e r than used with the IDA) · Although only information from 6 o f the 512 diodes was used f o r q u a n t i t a t i v e information i n t h i s case, the complete emission spectrum was obtained i n a matter o f seconds. T h i s i s about two orders o f magnitude l e s s than the time necessary to scan a spectrum with a conventional spectrofluorometer. The a b i l i t y to acquire s p e c t r a r a p i d l y provides a c o n s i d e r a b l e time savings, p a r t i c u l a r l y i n s i t u a t i o n s where i t i s necessary to survey the fluorescence c h a r a c t e r i s t i c s o f a number o f compounds. The



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wavelengths of emission maxima and r e l a t i v e fluorescence i n t e n s i t i e s at a number of d i f f e r e n t e x c i t a t i o n wavelengths can be e s t a b l i s h e d i n a r e l a t i v e l y short time. For measurement of steady-state f l u o r e s c e n c e , the r a p i d a c q u i s i t i o n of s p e c t r a i s a convenience but not e s s e n t i a l . However, the r a p i d a c q u i s i t i o n of s p e c t r a l information i s e s s e n t i a l f o r measurement of t r a n s i e n t species or where the luminescence s i g n a l c o n t i n u a l l y changes with time. This w i l l be demonstrated f i r s t f o r k i n e t i c - b a s e d luminescence measurements and then chemiluminescence measurements. Fluorescence Kinetic-Based Measurements. Our s t u d i e s of the r e a c t i o n r a t e determination of thiamine (vitamin B l ) w i l l be used to demonstrate the unique c a p a b i l i t i e s of r a p i d a c q u i s i t i o n of s p e c t r a i n k i n e t i c measurements. The k i n e t i c method i s based on the o x i d a t i o n of thiamine by H g i n b a s i c s o l u t i o n s to h i g h l y f l u o r e s c e n t thiochrome (16). The i n i t i a l r a t e , taken as the change i n fluorescence s i g n a l at 444 nm that occurs i n a f i x e d time a f t e r mixing the sample and reagents, i s d i r e c t l y p r o p o r t i o n a l to the thiamine c o n c e n t r a t i o n . Figure 2 shows emission s p e c t r a o f a s o l u t i o n c o n t a i n i n g thiamine and r i b o f l a v i n (vitamin B2) taken with the IDA at v a r i o u s times. Curve A i s the spectrum of the o r i g i n a l v i t a m i n sample s o l u t i o n a c i d i f i e d to pH 2 with H g added. Under computer c o n t r o l t h i s i n i t i a l spectrum i s acquired i n 2 s and then pH 12.2 b u f f e r i s i n j e c t e d i n t o the sample c e l l to r a i s e the r e a c t i o n mixture pH to i n i t i a t e the thiochrome r e a c t i o n . Curves B, C, and D are the emission s p e c t r a obtained at 16, 40, and 60 s a f t e r i n i t i a t i o n of the r e a c t i o n with a 2 s i n t e g r a t i o n time. These s p e c t r a are now used to i l l u s t r a t e a number o f important p o i n t s . 1. The growth of the thiochrome peak centered at 444 nm i s obvious· 2. The d i f f e r e n c e i n the sum o f the s i g n a l s from the 10 diodes (533-545 nm) centered at the wavelength of peak emission o f thiochrome i s a u t o m a t i c a l l y c a l c u l a t e d from the stored spectra to give a number p r o p o r t i o n a l to the r a t e . The d e t e c t i o n l i m i t f o r thiamine i s 6 χ 10"^ M and i l l u s t r a t e s the d e t e c t i v i t y of the IDA. This i s only a f a c t o r of 2 greater than the d e t e c t i o n l i m i t obtained with an e q u i v a l e n t system based on PMT d e t e c t i o n . 3. The f i r s t order and second order (not shown) R a y l e i g h s c a t t e r i n g peaks of the 365 nm e x c i t a t i o n l i g h t are obvious. The d e t e c t i v i t y of the IDA i s a l s o i l l u s t r a t e d by the d i s t i n c t water Raman band at 417 nm. 4. The i n i t i a l fluorescence peak centered at 550 nm i s due to the i n t r i n s i c fluorescence o f r i b o f l a v i n . Its intensity i s decreased when the pH i s increased. This i l l u s t r a t e s the use o f the IDA as a d i a g n o s t i c t o o l s i n c e p o t e n t i a l s p e c t r a l i n t e r f e r e n c e s i n a r e a l sample can be r e a d i l y i d e n t i f i e d 2 +

2 +






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simultaneously as the primary a n a l y t i c a l information i s obtained. In t h i s case there i s a small s p e c t r a l overlap o f the t a i l o f the r i b o f l a v i n peak with the maximum of thiochrome f l u o r e s c e n c e . 5. The computer was programmed to not only c a l c u l a t e the r a t e o f the thiochrome r e a c t i o n but to subtract a p r e v i o u s l y stored blank spectrum from spectrum A to o b t a i n a s i g n a l p r o p o r t i o n a l to the r i b o f l a v i n c o n c e n t r a t i o n . A detection l i m i t of 7 χ Ι Ο " ^ M was obtained f o r r i b o f l a v i n . Thus the m u l t i p l e wavelength c a p a b i l i t y o f the IDA was used f o r simultaneous determination of vitamins i n one vitamin p i l l sample to provide a s i g n i f i c a n t time savings over using two a n a l y t i c a l procedures on two separate samples. The d i a g n o s t i c c a p a b i l i t i e s of IDA d e t e c t i o n were f u r t h e r revealed when applying the thiochrome r a t e method to other matrices such as urine and c e r e a l (13). Fluorescence spectra o f samples can be r a p i d l y obtained at d i f f e r e n t pH's or e x c i t a t i o n wavelengths. S h i f t s i n the maxima of emission spectra under these d i f f e r e n t c o n d i t i o n s r e v e a l the presence o f a d d i t i o n a l f l u o r e s c e n t s p e c i e s . Spectra of p o t e n t i a l i n t e r f e r e n t s can be compared to the spectrum o f the sample to confirm the presence of c e r t a i n s p e c i e s . In the case o f u r i n e samples, a f l u o r e s c e n t compound was formed at a r a t e comparable to the thiochrome r e a c t i o n when the pH was increased. The appearance o f t h i s fluorescence peak was r e a d i l y observed when the d i f f e r e n c e i n spectra taken at d i f f e r e n t times was c a l c u l a t e d and d i s p l a y e d . This information could not be obtained with a conventional scanning spectrofluorometer. C l e a r l y by o b t a i n i n g the complete fluorescence spectrum of sample, unique or unexpected i n t e r f e r e n c e s or problems can be i d e n t i f i e d which might a f f e c t the q u a l i t y o f the measurement. D i f f e r e n c e s i n the magnitude of the Rayleigh s c a t t e r peak or the general background b a s e l i n e are o f t e n i n d i c a t i v e o f s c a t t e r i n g components i n the sample. This can e x p l a i n a decrease i n measurement p r e c i s i o n due to an increase i n background n o i s e . Chemiluminescence Measurements. For chemiluminescence measurements, the IDA i s used as a fundamental and d i a g n o s t i c t o o l rather than a q u a n t i t a t i v e a n a l y s i s t o o l . This i s because the l i g h t l e v e l s i n CL are very low, and normally no wavelength s e l e c t i o n i s employed. Even with a monochromator and a PMT d e t e c t o r , d e t e c t i o n l i m i t s are s e r i o u s l y degraded. For most chemiluminescence r e a c t i o n s , a multichannel spectrometer i s e s s e n t i a l to o b t a i n s p e c t r a information because with d i s c r e t e sampling ( i n j e c t i o n of the f i n a l reagent to i n i t i a t e the r e a c t i o n ) , the CL r e a c t i o n l a s t s only at most a few seconds. Some chemiluminescence r e a c t i o n s proceed f o r minutes or even hours, but s t i l l there are u s u a l l y s i g n i f i c a n t i n t e n s i t y changes over a minute such that a d i s t o r t e d spectrum would be



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obtained with a scanning monochromator. We have used the IDA to o b t a i n s p e c t r a during s i x d i f f e r e n t CL r e a c t i o n s (12,15). Knowledge o f the CL spectrum, and hence wavelength o f peak emission provides the f o l l o w i n g advantages. 1. F o r l a t e r q u a n t i t a t i v e a n a l y s i s with a PMT, the PMT photocathode response can be chosen f o r maximum detectability. 2. The CL spectrum can be compared to the absorption s p e c t r a o f r e a c t a n t s , products, and suspected i n t e r f e r i n g species to determine i f s p e c t r a l absorption i n t e r f e r e n c e s o r i f self-absorption i s significant. 3. The CL spectrum can be compared to fluorescence spectra o f intermediates o r products to i d e n t i f y the e x c i t e d species r e s p o n s i b l e f o r CL, and hence provide mechanistic information. The spectrum shown i n Figure 3 i s f o r the r e a c t i o n o f 0C1~ with H2O2. The two peaks a t 636 and 703 nm c l e a r l y i n d i c a t e that s i n g l e t O2 i s the species r e s p o n s i b l e f o r CL. 4. The CL spectrum o f the blank r e a c t i o n can be compared to the CL spectrum obtained with the analyte to e s t a b l i s h i f the analyte i s an a c t i v a t o r that increases the r a t e o f the blank CL r e a c t i o n o r induces a new CL r e a c t i o n t o occur. For CL s p e c t r a we found i t c r i t i c a l to o b t a i n a "dark" spectrum immediately before automated i n i t i a t i o n o f the r e a c t i o n by i n j e c t i o n o f the f i n a l reagent and a c q u i s i t i o n o f the CL spectrum. S u b t r a c t i o n o f the dark spectrum from the CL spectrum compensates f o r dark s i g n a l d r i f t . A l s o , s i g n a l averaging o f many s p e c t r a (one from each r e a c t i o n run) i s o f t e n e s s e n t i a l to achieve a reasonable S/N. Conclusions Our s t u d i e s have demonstrated that a molecular luminescence spectrometer with IDA multichannel d e t e c t o r i s a powerful q u a n t i t a t i v e , fundamental, and d i a g n o s t i c t o o l . F o r molecular fluorescence i t allows survey spectra to be obtained q u i c k l y . The simultaneous wavelength coverage i s e s s e n t i a l f o r o b t a i n i n g fluorescence s p e c t r a during k i n e t i c r e a c t i o n s . F o r steady s t a t e o r k i n e t i c measurements, complete fluorescence s p e c t r a l information can be c r i t i c a l i n i d e n t i f y i n g s p e c t r a l o v e r l a p problems i n r e a l samples which can go unnoticed i n conventional measurements where only one narrow wavelength range i s monitored. F o r CL measurements, the s p e c t r a l information i s u s e f u l f o r diagnosis o f s p e c t r a l absorption problems, as w e l l as f o r p r o v i d i n g mechanistic information. A molecular absorption spectrophotometer based on diode a r r a y d e t e c t i o n has been a v a i l a b l e f o r many years. Although some commercial molecular fluorescence spectrometers have p r o v i s i o n f o r a t t a c h i n g a multichannel d e t e c t o r , i t i s



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Figure 3. Chemiluminescence spectrum of H 0 - OCT singlet oxygen reaction. duced from Ref. 15. Copyright 1981, American Chemical Society.) 2

2


(Repro-


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s u r p r i s i n g that none have been designed t o t a l l y around an i n t e n s i f i e d diode a r r a y . T h i s should occur i n the f u t u r e . I t i s n a t u r a l to compare the performance o f an optimized spectrofluorometer with PMT d e t e c t i o n and photon counting s i g n a l processing to a spectrofluorometer with IDA d e t e c t i o n f o r r o u t i n e q u a n t i t a t i v e measurements. Here the sum o f the s i g n a l s from a few diodes d e f i n e s a s p e c t r a l bandpass equivalent to that defined by a monochromator with PMT d e t e c t i o n . With our system, some l o s s o f d e t e c t a b i l i t y occurs i n c e r t a i n s i t u a t i o n s . D e t e c t a b i l i t y i s sometimes l i m i t e d by the readout n o i s e , the dark s i g n a l n o i s e , o r the s i z e o f the i n d i v i d u a l diode a r r a y d e t e c t o r elements. For short i n t e g r a t i o n times (< 1 s ) , read out noise can be l i m i t i n g f o r very weak s i g n a l s . Others (17) have demonstrated that with proper design o f the e l e c t r i c a l r e a d out c i r c u i t r y , the readout noise can be reduced a f a c t o r o f 10 compared to our system, and s i n g l e photoelectron d e t e c t i o n can be achieved. This would be p a r t i c u l a r l y c r i t i c a l f o r CL measure ments. Often f o r FL measurements a short i n t e g r a t i o n time i s used because diode s a t u r a t i o n would occur with longer i n t e g r a t i o n times. Thus the present magnitude o f the readout noise i s o f t e n not l i m i t i n g because photon c a r r i e d noise i s dominant. For longer i n t e g r a t i o n times used with weaker s i g n a l s ( o r i f the readout noise was reduced), dark s i g n a l noise can be limiting. In our system, the dark s i g n a l corresponds to about 80 photoelectrons/s o r about a f a c t o r o f 10 g r e a t e r than achieved with a good cooled PMT system. T h i s means the r e l a t i v e dark s i g n a l noise i s greater with the IDA than a PMT. Reduction of the dark s i g n a l r a t e , e s p e c i a l l y the component from the i n t e n s i f i e r photocathode, would h e l p . Cooling the i n t e n s i f i e r photocathode i s p o s s i b l e but would be inconvenient. T h i s again would be most c r i t i c a l f o r CL measurements. We have observed many s i t u a t i o n s where a CL s i g n a l was e a s i l y observed with a PMT but was not d e t e c t a b l e with the IDA. F o r many s i t u a t i o n s with fluorescence measurements, the background fluorescence or s c a t t e r i n g s i g n a l count r a t e i s greater than the dark s i g n a l count r a t e such that dark s i g n a l noise i s not l i m i t i n g . The readout and dark s i g n a l noise problems are aggravated by the small s i z e o f the diode d e t e c t o r element compared to a PMT photocathode. In some instances the PMT photocathode views about 100 times more photons/s than a diode d e t e c t o r element ( f a c t o r i n g out the i n t e n s i f i e r g a i n ) . T h i s i s the p r i c e paid for m u l t i p l e wavelength coverage. This disadvantage can, o f course, be reduced by u s i n g a l a r g e r spectrograph entrance s l i t width and summing the s i g n a l s from η diodes. In the l a t e r case, the readout o r dark s i g n a l noise increases by νίΓ. As p r e v i o u s l y mentioned, the S/N near the d e t e c t i o n l i m i t i s o f t e n l i m i t e d f o r complex r e a l samples by background s i g n a l n o i s e , and above the d e t e c t i o n l i m i t , where the analyte f l u o r e s cence s i g n a l i s g r e a t e r than the background s i g n a l , by a n a l y t i c a l s i g n a l n o i s e . I f shot n o i s e i s l i m i t i n g , the smaller detec-



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t o r element s i z e can s t i l l cause a S/N disadvantage since the S/N i s p r o p o r t i o n a l to the number o f photons viewed per u n i t time. The S - s e r i e s Reticon DA has diodes about 5 times t a l l e r (2.5 mm) than the DA we used, and thus should help t h i s problem. Looking t o the f u t u r e , i t would be u s e f u l to have diode arrays with d e t e c t o r elements 5-10 mm high and with only 100 diodes per inch (250 pm s p a c i n g ) . T h i s would i n c r e a s e the number o f pho tons impingent on a diode a r r a y element t o be more n e a r l y comparable to that impingent on a PMT. The l a r g e r diode width would reduce r e s o l u t i o n f o r the same wavelength coverage but i n molecular luminescence high r e s o l u t i o n i s not o f t e n r e q u i r e d and t y p i c a l l y we sum the s i g n a l s from 5-10 diodes f o r q u a n t i t a t i v e measurements. H o p e f u l l y t h i s new diode array could be c o n s t r u c t ed without a s i g n i f i c a n t i n c r e a s e i n the dark s i g n a l accumula t i o n r a t e and with a l a r g e r s a t u r a t i o n l e v e l . F i n a l l y , i t should be noted that with our present system and c e r t a i n samples, background o r analyte photon s i g n a l fluxes a r e l a r g e enough that measurements are f l i c k e r noise l i m i t e d (noise p r o p o r t i o n a l to the s i g n a l ) . In t h i s case, the IDA already provides d e t e c t i o n l i m i t s e q u i v a l e n t t o that obtained with PMT detection. Acknowledgment s Acknowledgment i s made t o the NSF (Grant No. CHE-76-16711 and CHE-79-21293) f o r p a r t i a l support o f t h i s r e s e a r c h , and one o f us (M. A. R.) g r a t e f u l l y acknowledges an NSF graduate f e l l o w s h i p and an American Chemical S o c i e t y , D i v i s i o n o f A n a l y t i c a l Chemistry Summer F e l l o w s h i p sponsored by General Motors Research Laboratory. Literature

Cited

1. Johnson, D. W.; C a l l i s , J . B.; Christian, G. D. Anal. Chem. 1977, 49, 747A. 2. Kohen, E.; Kohen, C.; Salmon, J. M. Mickrochim. Acta. 1976, II, 195. 3. F i l l a r d , J . P.; deMurcia, M.; Gasiot, J . ; Chor, S. J . Phys. E. 1975, 8, 993. 4. Jadamec, J . R.; Saner, W. Α.; Talmi, Y. Anal. Chem. 1977, 49, 1316. 5. Conney, R. P.; Vo-Dinh, T.; Winefordner, J . D. Anal. Chim. Acta. 1977, 89, 94. 6. Conney, R. P.; Vo-Dinh, T.; Walden, G.; Winefordner, J . D. Anal. Chem. 1977, 49, 939. 7. Steinhart, H.; Sandmann, J . Anal. Chem. 1977, 49 950. 8. Johnson, D. W.; C a l l i s , J . B.; Christian, G. D., "Higher Order Strategies for Fluorescence Analysis Using an Image Detector", Talmi, Y. Ed.; ACS Symposium Series No. 102, ACS: Washington, DC, 1979; p. 97.



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9. Jadamec, J . R.; Sanner, W. Α.; Sager, R. W., "Versatility of an Optical Multichannel Analyzer as an HPLC Detector", Talmi, Y. Ed.; ACS Symposium Series No. 102, ACS: Washington, DC, 1979; p. 115. 10. Hirschberg, J. G.; Wouters, A. W.; Kohen, E.; Kohen, C; Thorell, B.; Eisenberg, B.; Salmon, J. M.; Ploem, J . S., "A High Resolution Grating Microspectrofluorometer with Topographic Option for Studies i n Living Cells", Talmi, Y. Ed.; ACS Symposium Series No. 102, ACS: Washington, DC, 1979; p. 263. 11. Talmi, Y.; Baker, D. C.; Jadamec, J. R.; Saner, W. A. Anal. Chem. 1978, 50, 936A. 12. Ryan, Μ. Α.; Miller, R. J.; Ingle, J. D., J r . Anal. Chem. 1978, 50, 1772. 13. Ryan, Μ. Α.; Ingle, J . D., J r . Talanta 1981, 28, 225. 14. Ryan, Μ. Α.; Ingle, J. D., Jr. Anal. Chem., submitted. 15. Marino, D. F.; Ingle, J . D., J r . Anal. Chem. 1981, 53, 645. 16. Ryan, Μ. Α.; Ingle, J . D., Jr. Anal. Chem. 1980, 52, 2177. 17. Simpson, R. W. Rev. S c i . Instrum. 1979, 50, 730. R E C E I V E D May 23, 1983


Multichannel Image Detectors Volume 2 - American Chemical Society

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