Luminescence Applications - American Chemical Society

Chapter 8 Peroxyoxalate

Chemiluminescence Reaction

Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: December 30, 1989 | doi: 10.1021/bk-1989-0383.ch008

A Versatile Method for Activation of Fluorophore Luminescence Richard S. Givens, Richard L. Schowen, John Stobaugh, Theodore Kuwana, Francisco Alvarez, Nikhil Parekh, Bogdan Matuszewski, Takao Kawasaki, Osborne Wong, Mirko Orlović, Hitesh Chokshi, and Kenichiro Nakashima Center for Bioanalytical Research and Department of Chemistry, University of Kansas, Lawrence, KS 66045 Investigations have shown that the peroxyoxalate chemiluminescence reaction can be effectively used for a postcolumn detector in high-performance liquid chromatography (HPLC). The reaction generates the fluorescent state of certain suitable acceptors, such as 2-cyanobenz[f]isoindole (CBI) derivatives of amino acids, peptides, amines, and related primary amines. The derivatization method makes use of the reaction of 2,3-naphthalenedicarboxaldehyde and cya nide (NDA/CN) as a new fluorogenic and chemilumigenic reagent for analysis of primary amine func tions. While the method has proven to be superior to standard fluorescence assays for low-level de tection of amino acids and peptides, optimizing the method's sensitivity remains the current goal of this research. For this reason, the contributing parameters for efficient generation of chemilumi nescence for the assay of CBI derivatives based on oxalate-hydrogen peroxide activation have been iden tified and investigated. Among the important factors are: (i) the nature of the leaving group on the oxa late ester, (ii) the fluorescence properties of the NDA/CN analyte, (iii) the fluorescence-quenching capa bilities of the oxalate ester and its reaction pro ducts, (iv) solvent effects, and (v) the effects of added catalysts. In order to optimize the chemiluminescence re sponse, we have investigated the mechanism of the com plex reactions leading to chemical generation of chemiluminescence. A new peroxyoxalate-hydrogen per oxide reaction mechanism has emerged from our prelim inary studies on the five contributing factors listed above. Two kinetic models are discussed, one for the NOTE: Dedicated to the Memory of Professor Takeru Higuchi (1916-1987) β

0097-6156/89A)383-0127$08.00/0 1989 American Chemical Society

Goldberg; Luminescence Applications ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


128

LUMINESCENCE APPLICATIONS

mechanism of the reaction in organic solvents and the second for mixed aqueous-organic solvents. The latter model is particularly adaptable to quantitative rate studies and has become the focus of efforts to develop the peroxyoxalate chemiluminescence reaction as a postcolumn detector for HPLC. Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counter parts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. The potential for improved chemiluminescent de tection is large, since the efficiency for activation of the acceptor is less than 0.01%. A thousand-fold increase in signal could be anticipated from this reaction. The C e n t e r f o r B i o a n a l y t i c a l R e s e a r c h was e s t a b l i s h e d i n 1983 t o f u r t h e r the a l r e a d y e x i s t i n g s t u d i e s i n the areas o f b i o a n a l y t i c a l and p h a r m a c e u t i c a l c h e m i s t r y a t the U n i v e r s i t y o f K a n s a s , w h i c h were d i r e c t e d toward the d i s c o v e r y and development o f e x q u i s i t e l y s e n s i t i v e methods f o r b i o a n a l y s i s . Among the areas o f i n t e r e s t were f l u o r o g e n i c methods f o r the assay o f p r i m a r y amines, amino a c i d s , o l i g o p e p t i d e s , and p r o t e i n s . Methods f o r p r i m a r y amines and amino a c i d s have been e x t e n s i v e l y s t u d i e d , f o r example, and a number o f e x c e l l e n t s e n s i t i v e f l u o r o g e n i c reagents have emerged. One o f the most p o p u l a r i s the c o m b i n a t i o n o f o r t h o - p h t h a l a l d e h y d e (ΟΡΑ) and 2 - m e r c a p t o e t h a n o l ( 2 - M E ) , d i s c o v e r e d by R o t h ( I ) , examined by Simons and Johnson ( 2 , 3 ) , and developed f o r precolumn o r p o s t c o l u m n d e r i v a t i z a t i o n o f amino a c i d s i n h i g h - p e r f o r m a n c e l i q u i d chromotography (HPLC) a n a l y s e s by a number o f w o r k e r s , i n c l u d i n g Bensen and Hare (4). A number o f drawbacks i n the a p p l i c a t i o n o f the 0PA/2-ME r e a g e n t system i n c l u d e the i n s t a b i l i t y o f the f l u o r e s c e n t i s o i n d o l e d e r i v a t i v e ( 5 - 7 ) ; the use o f the noisome reagent 2 - m e r c a p t o e t h a n o l ; the low and s o l v e n t - d e p e n d e n t f l u o r e s c e n c e e f f i c i e n c i e s ( 8 , 9 ) o f the i s o i n d o l e ; and—perhaps the most l i m i t i n g — t h e e f f e c t i v e r e s t r i c t i o n o f the ΟΡΑ assay t o p r i m a r y a l i p h a t i c amines and t o amino a c i d s . Most o l i g o p e p t i d e s and p o l y p e p t i d e s do not g i v e f l u o r e s c e n t a d d u c t s . (However, see R e f . 1 0 . ) The i n i t i a l d i s c o v e r i e s o f the e x t e n s i o n o f the a r o m a t i c r i n g o f the o r t h o - p h t h a l a l d e h y d e (ΟΡΑ) t o a n a p h t h a l e n e - 2 , 3 - d i c a r b o x a l d e h y d e (NDA) and the s u b s t i t u t i o n o f c y a n i d e (CN~) f o r 2-ME as the n u c l e o p h i l e have p r o v i d e d the C e n t e r w i t h a much more v e r s a t i l e reagent system ( 5 , 1 1 ) , w h i c h m a i n t a i n s the s e n s i t i v i t y f o r p r i m a r y a l i p h a t i c amines and amino a c i d s , and now i s known t o form f l u o r e s c e n t p r o d u c t s w i t h o l i g o p e p t i d e s , p r o t e i n s , and o t h e r r e l a t e d a n a l y t e s t h a t p o s s e s s a p r i m a r y amine f u n c t i o n ( E q u a t i o n 1 ) .


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129

Activation of Fluorophore Luminescence

CN +


Naphthalene-2,3d i c a r b o x a l d e h y d e (NDA)

H 0 2

(1)

2-Cyano-l-substitutedb e n z [ f ] i s o i n d o l e (CBI)

The d e r i v a t i z a t i o n p r o c e s s (5) i s a c c o m p l i s h e d i n aqueous media at b a s i c pH (pH 7-10) i n a m a t t e r o f a p p r o x i m a t e l y 15 min to y i e l d a 2 - c y a n o b e n z [ f ] i s o i n d o l e ( C B I ) , which i s s t a b l e f o r 10 t o 12 h r i n s o l u t i o n . As shown i n F i g u r e 1, the a b s o r p t i o n c h a r a c t e r i s t i c s o f the CBI adducts are a l s o r e a d i l y a c c e s s i b l e f o r assay by s t a n d a r d f l u o r e s c e n c e or u l t r a v i o l e t d e t e c t i o n . I n a d d i t i o n t o the a b s o r p t i o n between 200 and 300 nm, t h e r e are two maxima i n the v i s i b l e spectrum at a p p r o x i m a t e l y 420 and 440 nm a c c e s s i b l e f o r f l u o r e s c e n c e o r ultraviolet detection. A p r o b a b l e mechanism (5,11) f o r the CBI f o r m a t i o n i s i l l u s t r a t e d i n Scheme 1. The s t a b i l i t y o f the CBI d e r i v a t i v e i s s u f f i c i e n t f o r i t s i s o l a t i o n and complete c h a r a c t e r i z a t i o n ( I I ) , an accomplishment t h a t i s not r e a l i z e d w i t h most ΟΡΑ a d d u c t s . Thus, the CBI d e r i v a t i v e s o f a number o f r e p r e s e n t a t i v e amino a c i d s and amines have been i s o l a t e d and t h e i r f l u o r e s c e n t p r o p e r t i e s determined as a f u n c t i o n o f the media and o t h e r r e l e v a n t parameters encountered i n r e v e r s e - p h a s e HPLC (RP-HPLC). Perhaps most e n c o u r a g i n g i n these d i s c o v e r i e s was the o b s e r v a t i o n t h a t NDA/CN" worked e q u a l l y w e l l f o r d e r i v a t i z a t i o n o f d i p e p t i d e s and h i g h e r homologues o f the p r i m a r y amino a c i d s e r i e s . Again, a s t a b l e , f l u o r e s c e n t , i s o l a t a b l e d e r i v a t i v e was o b t a i n e d . One o f the most i m p o r t a n t i n i t i a l f i n d i n g s was the h i g h f l u o r e s c e n c e e f f i c i e n c y o f the CBI adduct ( 1 2 ) . T a b l e s 1 and 2 l i s t the e f f i c i e n c i e s f o r a r e p r e s e n t a t i v e group o f mono-, d i - , and t r i p e p t i d e s and a l i m i t e d comparison o f the CBI e f f i c i e n c i e s w i t h the more t r a d i t i o n a l ΟΡΑ (8) and d a n s y l (9) d e r i v a t i v e s , r e s p e c t i v e l y . As n o t e d , the f l u o r e s c e n c e e f f i c i e n c i e s o f the CBI adducts are i n v a r i a b l y g r e a t e r than those o f ΟΡΑ o r d a n s y l a n a l o g s , g e n e r a l l y r a n g i n g from 0 . 4 t o 0 . 8 , w i t h the e x c e p t i o n o f the b i s - C B I adduct o f

T a b l e I . E f f e c t s o f P e p t i d e S u b s t i t u e n t s on F l u o r e s c e n c e Quantum E f f i c i e n c i e s (Φ^,) o f 1-Cyano-JV-substituted B e n z [ f ] i s o i n d o l e s (CBI) i n Aqueous A c e t o n i t r i l e CBI d e r i v a t i v e

CN Γ

ΊΓ

T^NCHRCOONa

R

-Gly -Gly-Gly -Gly-Gly-Gly -Ala -Ala-Ala -Lys -Lys-CBI

*F 0. 73 0.,48 0.,59 0.,75 0..54 0..55 0..03


130


Fluorescence E x c i t a t i on

Emi ss i on


CN

CC&-* 400

600

800

λ(ηχη)

200

400

800

600 λ (nm)

F i g u r e 1. A b s o r p t i o n and f l u o r e s c e n c e s p e c t r a o f t h e cyanobenz[ f ] i s o i n d o l e chromophore: C B I - A l a .

CN

o H *CN

a

r

=

j

II» CN

ÇN

CN

cet- = coi» — QXr OH

CBI

Scheme 1.

P r o b a b l e mechanism f o r CBI f o r m a t i o n .


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131

Table I I . A Comparison o f F l u o r e s c e n c e Quantum E f f i c i e n c i e s (Φ^,) o f ΟΡΑ, D a n y s l , and CBI D e r i v a t i v e s i n Aqueous-Organic Mixed S o l v e n t s L a b e l e d Amino A c i d


Ala Gly Tyr Val Lys GlyGly G l y G l y Gly

ΟΡΑ (β) 0..40 0..39 0,.33 0,.34 0..34 0,.14 0,.081

Danysl (9)

0,.065 0..068 0..091

CBI (12) 0..75 0..73

0..55 0..48 0..59

lysine. T h i s amino a c i d has two p r i m a r y amine f u n c t i o n s , b o t h o f which r e a c t w i t h NDA/CN". The weak f l u o r e s c e n c e o f the b i s - C B I adduct suggests t h a t an i n t r a m o l e c u l a r complex o f the two newly formed chromophores may r e s u l t i n a s i g n i f i c a n t i n c r e a s e i n the i n t e r n a l decay o r quenching o f the f i r s t e x c i t e d s i n g l e t s t a t e ( 1 3 ) . E f f o r t s t o c i r c u m v e n t the quenching e f f e c t o r t o improve on the e m i s s i o n e f f i c i e n c y o f the l y s i n e b i s - a d d u c t have l a r g e l y been i n e f f e c t i v e . I f r e a c t i o n time i s kept s h o r t , however, the d e r i v a t i z a t i o n p r o c e s s can be i n t e r c e p t e d a t the mono-adduct form, w h i c h i s s u f f i c i e n t l y f l u o r e s c e n t f o r assay p u r p o s e s . I t s h o u l d be noted t h a t the f l u o r e s c e n c e e f f i c i e n c i e s o f the CBI adducts are r e l a t i v e l y i n s e n s i t i v e t o the water c o n t e n t o f the s o l v e n t m i x t u r e ( 2 2 , 1 2 ) i n c o n t r a s t w i t h e a r l i e r r e p o r t s on the d a n s y l d e r i v a t i v e s , which l o s e an o r d e r o f magnitude o f e f f i c i e n c y i n aqueous-based s o l v e n t s y s t e m s ( 9 ) . F i g u r e 2 i s a chromatogram o f 18 p r i m a r y amino a c i d s t h a t were f i r s t c o n v e r t e d t o the CBI adducts w i t h NDA/CN" i n b o r a t e b u f f e r (pH 9 . 5 ) and then s e p a r a t e d by RP-HPLC ( 5 , 2 4 ) . The t r a c e r e p r e s e n t s 20 pmol o f each amino a c i d i n j e c t e d f o l l o w e d by e l u t i o n t h r o u g h an U l t r a s p h e r e 0DS ( 4 . 6 x 250 mm) column w i t h a g r a d i e n t t h a t c o n s i s t e d i n i t i a l l y o f 10% t e t r a h y d r o f u r a n / p h o s p h a t e b u f f e r (pH 6 . 8 ) ( s o l v e n t A ) , t o w h i c h was added a 55% a c e t o n i t r i l e / 1 0 % methanol/phosphate b u f f e r ( s o l v e n t Β ) , p r o d u c i n g a f i n a l m i x t u r e o f 40% s o l v e n t A and 60% s o l v e n t B . The l i m i t s o f d e t e c t i o n and the r e l a t i v e s e n s i t i v i t y o f the assay are c o n s i d e r a b l y enhanced w i t h l a s e r - i n d u c e d f l u o rescence ( L I F ) d e t e c t i o n , as r e p o r t e d e a r l i e r by Roach and Harmony (25). At a s i g n a l - t o - n o i s e r a t i o of 2, a conventional fluorescence d e t e c t o r ( K r a t o s FS-980) i s s e n s i t i v e t o l e v e l s as low as 10 t o 30 fmol f o r the amino a c i d s l i s t e d i n T a b l e I I I . However, w i t h L I F , the improvement i n d e t e c t i o n l i m i t s i s a p p r o x i m a t e l y two o r d e r s o f magni t u d e , i . e . , 0.1 t o 0.4 f m o l . A f u r t h e r comparison o f the l i m i t s f o r NDA/CN" and those f o r 0PA/2-ME u s i n g L I F i s g i v e n i n T a b l e I I I . In t h i s d i r e c t comparison o f the two t a g g i n g r e a g e n t s , the NDA/CN" method i s a p p r o x i m a t e l y t h i r t y f o l d b e t t e r . The v e r s a t i l i t y o f the reagent system i n the assay o f s m a l l p e p t i d e s i s n i c e l y i l l u s t r a t e d i n F i g u r e s 3 through 5 . Neurotensin, a p o l y p e p t i d e composed o f 13 amino a c i d s , and t h r e e fragments from p a r t i a l h y d r o l y s i s were d e r i v a t i z e d w i t h NDA/CN" and s e p a r a t e d by


132



have a l s o undertaken a s y s t e m a t i c i n v e s t i g a t i o n o f


2

w

e

the r e a c t i o n c o n d i t i o n v a r i a b l e s t h a t are encountered assays.

i n t y p i c a l HPLC

Φ = ώ · ώ ·ώ CL ™CHEM *EXCH VFL where

Φ__ CL

*CHEN ^EXCH

v

(2) j

i s the t o t a l chemiluminescence quantum e f f i c i e n c y , i s the e f f i c i e n c y o f the c h e m i c a l reaction, i s the e f f i c i e n c y o f the energy exchange from c h e m i c a l p o t e n t i a l t o e l e c t r o n i c e x c i t a t i o n e n e r g y , and i s the f l u o r e s c e n c e e f f i c i e n c y .

More r e c e n t work on the analogous d i o x e t a n e s (2 i n F i g u r e 7) and d i o x e t a n o n e s (3 i n F i g u r e 7) by T u r r o ( 2 0 ) , S c h u s t e r ( 2 2 , 2 2 ) , and Adam (23) has shown t h a t e x c i t a t i o n o f the a c c e p t o r can o c c u r by two d i f f e r e n t p r o c e s s e s : d i r e c t energy t r a n s f e r t o the a c c e p t o r from d i o x e t a n e , o r o x i d a t i o n o f the a c c e p t o r by d i o x e t a n o n e , f o l l o w e d by c h e m i c a l d e c o m p o s i t i o n o f the d i o x e t a n o n e and e l e c t r o n t r a n s f e r back t o the r a d i c a l c a t i o n o f the a c c e p t o r , y i e l d i n g i t s s i n g l e t e x c i t e d state. That n e i t h e r o f t h e s e two mechanisms a l o n e accounts f o r the p e r o x y o x a l a t e system was c l e a r l y i n d i c a t e d by the e a r l i e r work o f T u r r o ( 2 0 ) , who found no c o r r e l a t i o n o f chemiluminescence i n t e n s i t y t o e i t h e r the e x c i t a t i o n energy o r the o x i d a t i o n p o t e n t i a l o f the acceptor (Figure 7). Indeed, our own e x a m i n a t i o n (24) o f the time course o f the T C P 0 - H 0 - i n d u c e d chemiluminescence o f d i p h e n y l a n t h r a c e n e (DPA) i n e t h y l acetate with varying concentrations of triethylamine revealed a biphasic emission p r o f i l e , a r e s u l t that i s inconsistent with a single reactive (light-producing) intermediate i n t h i s reaction ( F i g u r e 8 ) . W i t h i n c r e a s i n g amine c o n c e n t r a t i o n (from curve a t o b t o c ) , the b i p h a s i c p r o f i l e i s s h i f t e d toward a s i n g l e maximum, but the a n a l y t i c a l form o f the l i g h t i n t e n s i t y decay i s b i e x p o n e n t i a l , i n d i c a t i n g t h a t more than one a c t i v e i n t e r m e d i a t e r e m a i n s . In f a c t , 2

2



GIVENS ET AL.


Oxidation Potential or Singlet Energy in volts or in kcal per mol

Ye

Intensity vs Oxidation Potential

ο—ο

3

t t ο—ο

2

^Intensity vs Singlet Energy

1 F i g u r e 7. Absence o f a l i n e a r c o r r e l a t i o n o f e i t h e r o x i d a t i o n p o t e n t i a l o r s i n g l e t energy t o chemiluminescence e f f i c i e n c y f o r the p e r o x y o x a l a t e r e a c t i o n .


142


π

Τ"

4.0

=l


TCPO 0.7 mM in Ethyl Acetate a: 0.05 mM TEA b: 0.10 mM TEA c: 0.20 mM TEA

3.0

2.0

1.0

0.0

200

400

600

Time, s F i g u r e 8. Observed decay c u r v e s f o r T C P 0 - H 0 - D P A c h e m i l u m i n e s cence. TEA, t r i e t h y l a m i n e ; a . u . , a r b i t r a r y u n i t s . (Reproduced from R e f . 2 4 . C o p y r i g h t 1986 American C h e m i c a l S o c i e t y . ) 2

2


8.

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143

Activation of Fhiorophore Luminescence

c a r e f u l s c r u t i n y o f the p u b l i s h e d t i m e - c u r v e s i n the s e m i n a l r e p o r t of Rauhut e t a l . (26) r e v e a l s p a r a l l e l b e h a v i o r f o r s i m i l a r chemil u m i n e s c e n t r e a c t i o n s t h a t t h e y examined. To c o n f i r m t h a t the e m i s s i o n under each o f the two maxima d e t e c t e d i n our s t u d y was i n f a c t t h a t o f DPA, the w a v e l e n g t h de pendence was m o n i t o r e d as a f u n c t i o n o f the i n t e n s i t y w i t h each one. As shown i n F i g u r e 9 , the e m i s s i o n s p e c t r a are those o f the DPA fluorescent excited state. From t h e s e r e s u l t s , we have c o n s t r u c t e d a m a t h e m a t i c a l model (24) f o r the time-dependent e m i s s i o n w h i c h i n c o r p o r a t e s three r e active intermediates:


I =

1

χ

= ι° ΐο

{(4=fc)

(

β

L i y V

' ^ -

(

β

"

V*x>

ν

)

+

(k -k ) y

T

(k -k )\ b

x

where [ 0 X ] i s the i n i t i a l o x a l a t e c o n c e n t r a t i o n , hv i s the photon c o u n t , f i s the f r a c t i o n o f X t h a t goes t o X ' , g i s the f r a c t i o n o f Y t h a t goes t o Y ' , k^, k^ k^> k^ and k^ are the r a t e c o n s t a n t s f o r 0

9

9

the r e a c t i o n s shown i n Scheme 3 , and k^ i s the sum o f k^ and k^. X , Χ ' , Υ, Y ' , and Ζ r e p r e s e n t the n e c e s s a r y p u t a t i v e c h e m i c a l i n t e r mediates shown i n the r e a c t i o n s scheme. Two o f t h e s e i n t e r m e d i a t e s (X and Y) l e a d t o the c h e m i l u m i n e s c e n t p r o c e s s , and a t h i r d (Z) s e r v e s as a b r i d g e o r "way s t a t i o n " between the two l i g h t - i n d u c i n g s p e c i e s . A s c h e m a t i c r e p r e s e n t a t i o n based on t h i s k i n e t i c model i s shown i n Scheme 3 . T h i s model c o r r e c t l y s i m u l a t e s the s e r i e s o f time-dependent i n t e n s i t y c u r v e s , as i s seen by comparing F i g u r e 10 t o F i g u r e 8. Thus, the c h e m i c a l gener a t i o n o f the " r e a c t i v e i n t e r m e d i a t e ( s ) " i s a much more complex p r o c e s s t h a n o r i g i n a l l y e n v i s i o n e d by Rauhut ( 2 6 ) . T h i s f i n d i n g i s i n agreement w i t h r e c e n t work by C u n d a l l ( 2 5 ) , b u t c o n t r a s t s w i t h e a r l i e r r e p o r t s , which r e p e a t e d l y proposed a s i n g l e i n t e r m e d i a t e , i.e., the d i o x e t a n e d i o n e mechanism ( 2 6 - 2 9 ) . F o r t u n a t e l y , as the r e a c t i o n i s t r a n s f e r r e d from a p u r e l y o r g a n i c s o l v e n t system t o mixed o r g a n i c - a q u e o u s m e d i a , w h i c h are employed i n most RP-HPLC s e p a r a t i o n s , the apparent m u l t i p l i c i t y o f maxima i n the time p r o f i l e o f the i n t e n s i t y dependence seems t o be suppressed o r t o c o l l a p s e t o a r e a s o n a b l y s i m p l e b i e x p o n e n t i a l - l i k e dependence. As shown i n F i g u r e 1 1 , s i m p l y changing the s o l v e n t from e t h y l a c e t a t e t o 95% aqueous a c e t o n i t r i l e and the c a t a l y s t from t r i e t h y l a m i n e t o i m i d a z o l e produces a s i n g l e maximum p r o f i l e , one t h a t i s more e a s i l y modeled m a t h e m a t i c a l l y , as d e f i n e d i n E q u a t i o n 4:


144


^million

ipbclro


ία-

400.

425.

450.

475.

500.

Wavelength , nm

F i g u r e 9. Chemiluminescence e m i s s i o n s p e c t r a from the two pulses w i t h DPA as the a c c e p t o r ; i n s e t i s DPA f l u o r e s c e n c e spectrum. (Reproduced from R e f . 24. C o p y r i g h t 1986 American C h e m i c a l S o c i e t y . )

0-gJkb

hν

hv

Scheme 3. G e n e r a l mechanism f o r the T C P 0 - H 0 - D P A c h e m i l u m i nescence. (Reproduced from R e f . 24. C o p y r i g h t 1986 American Chemical S o c i e t y . ) 2

2



8.

GIVENS ET A L .

US


Time (a.u.) F i g u r e 10. S i m u l a t e d decay curves from the k i n e t i c a n a l y s i s o f the T C P 0 - H 0 - D P A chemiluminescence. A r b i t r a r y u n i t s ( a . u . ) used on b o t h a x e s . (Reproduced from R e f . 24. C o p y r i g h t 1986 American Chemical S o c i e t y . ) 2

2

10.0

ι

•

•

•

1

•

• max

•

•

1

•

•

•

j

Time, s

F i g u r e 11. Time-dependent e m i s s i o n f o r aqueous a c e t o n i t r i l e chemiluminescence o f T C P 0 - H 0 - i m i d a z o l e system measured a t 430 nm. 2

2


146

LUMINESCENCE APPLICATIONS Kt)

=

(e"

r t

f t

- e" )

(4)

f-r where

I(t) M is r is f is

and

i s the e m i s s i o n i n t e n s i t y , the ( t h e o r e t i c a l ) i n i t i a l maximum i n t e n s i t y , the r i s e r a t e c o n s t a n t , the f a l l r a t e c o n s t a n t .

T h i s model p e r m i t s a d e t e r m i n a t i o n o f the r a t e c o n s t a n t s f o r the r i s e of the chemiluminescence i n t e n s i t y and i t s subsequent decay and, more i m p o r t a n t l y , a l l o w s a q u a n t i t a t i v e assessment o f the e f f e c t s o f r e a c t i o n c o n d i t i o n s , such as s o l v e n t v a r i a t i o n , t e m p e r a t u r e , o r a d d i t i v e s , on the r a t e s ( r and f ) , the time r e q u i r e d i^ Equation Downloaded by UNIV LAVAL on July 13, 2016 | http://pubs.acs.org Publication Date: December 30, 1989 | doi: 10.1021/bk-1989-0383.ch008

max

5 ) , the i n t e n s i t y ( J , E q u a t i o n 6 ) , and the e f f i c i e n c y ( Y , E q u a t i o n 7) of a p a r t i c u l a r chemiluminescent r e a c t i o n (see a l s o F i g u r e 1 2 ) . X

max

=

I

l n

f

r

f

r

( / )]/( " )

J = M(r/f)

(Time r e q u i r e d f o r the e m i s s i o n t o r e a c h maximum)

(5)

(Maximum i n t e n s i t y )

(6)

(Quantum e f f i c i e n c y )

(7)

00

Y = ÎI(t)dt = M/f 0

Such an a n a l y s i s has been conducted f o r the T C P 0 - H 0 - D P A r e a c t i o n by v a r y i n g the s o l v e n t , the t e m p e r a t u r e , and the concent r a t i o n s o f the r e a c t a n t s . Some o f the r e s u l t s from t h i s study are presented i n Table V I I . The r a t e c o n s t a n t s f o r p h e n o l r e l e a s e ( 2 , 4 , 6 - t r i c h l o r o p h e n o l ; TCP) were determined s p e c t r o p h o t o m e t r i c a l l y a t 298 nm i n b o t h the presence and the absence o f the f l u o r o p h o r e (DPA) and were shown t o be independent o f DPA a t the c o n c e n t r a t i o n s employed. The t i m e dependent r e l e a s e o f TCP m o n i t o r e d a t 298 nm, w h i c h i s shown i n 2

Table V I I .

E f f e c t of H 0 2

[H 0 ] (mM)

R i s e Rate Constant, r (xiO-s" )

1.25 2.50 5.0 10.0 30.0 120.0

3.0 3.2 3.4 3.7 11.0 22.5

2

2

1

2

C o n c e n t r a t i o n on the T C P 0 - H 0 - D P A Reaction 2

F a l l Rate Constant, f (X102S )

[TCPO] = 0 . 7 5 mM [DPA] = 0.075 mM [ I m i d a z o l e ] = 2.5 mM

2

- 1

3.6 3.5 3.2 2.6 2.2 3.0

2

Maximum Intensity, J (au)

Quantum Efficiency, γ (ψχιο )

1.7 1.8 2.5 3.5 4.9 5.9

2.9 3.2 4.5 6.7 10.0 8.4

4

25°C pH 7.0



8. GIVENS ET AL.


147

Mi(i-c-") f-r

Figure 1 2 . model.

Efficiency,

l i f e t i m e , and i n t e n s i t y o f single-maximum

American Chemical Society Library 1155Luminescence 16th St. H.w. Goldberg; Applications ACS Symposium Series; American Chemical Washington, O.C. Society: 20036 Washington, DC, 1989.


148


F i g u r e 13, i n d i c a t e s t h a t the f i r s t mole o f p h e n o l i s r e l e a s e d i n

Luminescence Applications - American Chemical Society

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