Time-Resolved Fluorescence Measurements on Dissolved Marine

Chapter 10

Time-Resolved Fluorescence Measurements on Dissolved Marine Organic Matter P. J. Milne, D. S. Odum, and Rod G. Zika

Downloaded by PENNSYLVANIA STATE UNIV on March 8, 2013 | http://pubs.acs.org Publication Date: December 8, 1987 | doi: 10.1021/bk-1987-0327.ch010

Rosenstiel School of Marine and Atmospheric Sciences, Division of Marine and Atmospheric Chemistry, University of Miami, Miami, FL 33149

The fluorescence decay rates of samples of dissolved organic matter of marine and terrestrial origin have been measured. The lifetimes were all closely similar (2-2.4 ns) despite their widely different origins and histories. Implications of this similarity to other physical and chemical properties of humic/fulvic substances is discussed as is the interpretation of the deactivation pathways of the excited states of marine chromophores. Q u a n t i t a t i v e measurement o f t h e p h o t o p h y s i c s o f t h e d i s s o l v e d o r g a n i c chromophores found i n n a t u r a l water i s needed t o b e t t e r u n d e r s t a n d the r a t e s o f energy t r a n s f e r from t h e i r e x c i t e d s t a t e s and t o p r o v i d e i n f o r m a t i o n as t o t h e k i n e t i c s o f e x c i t e d s t a t e r e a c t i o n s they may undergo. S p e c i f i c areas o f i n t e r e s t i n n a t u r a l w a t e r c h e m i s t r y a r e t h e k i n e t i c s o f quenching i n t e r a c t i o n s and energy t r a n s f e r p r o c e s s e s t o o t h e r c h e m i c a l s p e c i e s ; t h e e f f e c t o f such i n t e r a c t i o n s on t h e i n i t i a t i o n o f secondary photochemical r e a c t i o n s i s p a r t i c u l a r l y important ( I ) . These measurements may a l s o a i d i n t h e c h a r a c t e r i z a t i o n o f t h e p h y s i c a l and c h e m i c a l p r o p e r t i e s o f t h e humic and f u l v i c a c i d f r a c t i o n s o f d i s s o l v e d o r g a n i c m a t t e r . The p r e s e n t study d e s c r i b e s an e x p e r i m e n t a l setup f o r d e t e r m i n a t i o n o f luminescence l i f e t i m e s and p r e s e n t s v a l u e s o b t a i n e d f o r some n a t u r a l o r g a n i c m a t e r i a l s . F l u o r e s c e n c e Decay Rates Time r e s o l v e d f l u o r e s c e n c e measurements o f s i m p l e o r g a n i c m o l e c u l e s a r e r e g u l a r l y used t o a n a l y z e a wide range o f c h e m i c a l and b i o l o g i c a l processes (_2). T h i s i s p o s s i b l e by v i r t u e o f the ways i n w h i c h a number o f phenomena c a n i n f l u e n c e t h e r a t e a t w h i c h a m o l e c u l e f l u o r e s c e s , t h a t i s undergoes an e l e c t r i c d i p o l e t r a n s i t i o n f r o m an e x c i t e d e l e c t r o n i c s t a t e t o a l o w e r , u s u a l l y t h e g r o u n d , s t a t e of t h e same m u l t i p l i c i t y . The k i n e t i c parameter t h a t i s measured r e f l e c t s t h e sum o f the r a t e s o f p r o c e s s e s d e p o p u l a t i n g

0097-6156/87/0327-0132$06.00/0 © 1987 American Chemical Society

In Photochemistry of Environmental Aquatic Systems; Zika, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


10.

Time-Resolved Fluorescence Measurements

MILNE ET AL.

133

the e x c i t e d s t a t e of the fluorophore b e i n g m o n i t o r e d . On a m o l e c u l a r l e v e l , the i n t r i n s i c r a t e of d e - e x c i t a t i o n of a g i v e n f l u o r o p h o r e i s s u b j e c t to a number of f a c t o r s i n c l u d i n g symmetry r e q u i r e m e n t s , the f r e q u e n c y of the f l u o r e s c e n t r a d i a t i o n , the i n t e g r a t e d a r e a under the a b s o r p t i o n curve as w e l l as on s p a t i a l r e s t r i c t i o n s imposed by the n e c e s s i t y of m o l e c u l a r o r b i t a l o v e r l a p of the e x c i t e d and ground s t a t e s . T y p i c a l l y , y^alues ^ f J^he r a t e c o n s t a n t f o r f l u o r e s c e n c e are i n the range of 1 0 - 1 0 s (3)· The i n t e n s i t y of f l u o r e s c e n c e from an e x c i t e d m o l e c u l e w i l l be dependent on the f l u o r e s c e n t decay r a t e and o t h e r n o n - r a d i a t i v e p r o c e s s e s s u c h as i n t e r s y s t e m c r o s s i n g t o t h e t r i p l e t s t a t e , i n t e r n a l conversion to the ground s t a t e , and any p h o t o c h e m i c a l r e a c t i o n s . In condensed media, v i b r a t i o n a l r e l a x a t i o n ^from^upper v i b r a t i o n a l l e v e l s of e x c i t e d s t a t e s i s u l t r a f a s t ( >10 s ) so t h a t v i b r o n i c r e l a x a t i o n i s complete b e f o r e e l e c t r o n i c r e l a x a t i o n can t a k e p l a c e . I n t e r n a l c o n v e r s i o n i s a p r o c e s s t h a t i s a l s o v e r y f a s t ( p i c o s e c o n d s ) at h i g h excess e n e r g i e s , but l e s s i m p o r t a n t f o r lower l y i n g l e v e l s of 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 . In t h i s way, the main competing p r o c e s s to f l u o r e s c e n c e i s o f t e n intersystem c r o s s i n g to the t r i p l e t m a n i f o l d of energy l e v e l s . W r i t i n g e q u a t i o n s f o r these p r o c e s s e s a l l o w s d e f i n i t i o n of the f l u o r e s c e n c e quantum y i e l d , 0, and the f l u o r e s c e n c e decay t i m e , t , i n terms of sums of the v a r i o u s f i r s t o r d e r r a t e c o n s t a n t s (nb. o t h e r p o s s i b l e d e a c t i v a t i o n pathways a l s o e x i s t ) . f

Pathway

Rate Constant

M + hv — > *M*

—>

1 * M —> l

M*

J

X

M*

I

M + hv

k

3 * M

k isc

f

—>

Μ + Δ

k

—>

(products)

k^

ic

1 * M

U s i n g the steady s t a t e a p p r o x i m a t i o n l e a d s 0_ = k. / ( k . + k, + k. + k ) f f f isc ic ρ

to:

and t. f

- ( k- + k, + k, + k f isc ic ρ

F r o m t h i s f r a m e w o r k i t ^ can be s e e n t h a t t h e fluorescence i n t e n s i t y o f a m o l e c u l e i s d e p e n d e n t upon t h e m a g n i t u d e o f k^ r e l a t i v e to the sum of a l l o t h e r d e a c t i v a t i o n path r a t e s . I t i s a l s o c l e a r t h a t l i f e t i m e measurements themselves do not g i v e the i n d i v i d u a l r a t e c o n s t a n t s f o r the p r o c e s s e s of i n t e r e s t , however i n c o n j u n c t i o n w i t h the e m i s s i o n and product quantum y i e l d s , they do p r o v i d e a way i n which t h i s can be c a l c u l a t e d .


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PHOTOCHEMISTRY OF ENVIRONMENTAL AQUATIC SYSTEMS


E x p e r i m e n t a l Methods The e x p e r i m e n t a l setup employed i n t h i s work used c o n v e n t i o n a l r i g h t angled v i e w i n g o f the time course o f the f l u o r e s c e n c e i n t e n s i t y of a sample c o n t a i n e d i n a s m a l l volume (0.2 ml) f l o w cell. The f l u o r e s c e n c e f l o w c e l l was f i l l e d f r o m a s e p a r a t e r e s e r v o i r v i a a p e r i s t a l t i c pump. S o l u t i o n parameters such as pH c o u l d be monitored d i r e c t l y from t h i s l a r g e r r e s e r v o i r , as c o u l d t h e oxygen p a r t i a l p r e s s u r e by b l a n k e t i n g o r b u b b l i n g t h e c e l l c o n t e n t s w i t h n i t r o g e n o r oxygen. The e x c i t a t i o n s o u r c e was a l a s e r (PAR 2100 D y e s c a n , m o d i f i e d t o a l l o w use of the primary l i n e ) a t a w a v e l e n g t h o f 337.lnm. The f l u o r e s c e n t e m i s s i o n was d e t e c t e d by a m o d i f i e d side-on p h o t o m u l t i p l i e r (PMT Hammamatsu R928), a f t e r passage through a monochromator (ISA HR 320). P a r t i c u l a r a t t e n t i o n was p a i d t o t h e w i r i n g o f t h e v o l t a g e d i v i d e r network o f the anodes o f t h e PMT t o ensure f a s t e s t p o s s i b l e response and l e a s t d i s t o r t i o n o f the f l u o r e s c e n t photon f l u x . The s i g n a l from the PMT was a m p l i f i e d t h r o u g h a h i g h - s p e e d , wide-band ( t o 0.5GHz) s i n g l e t r a c e a m p l i f i e r ( T e k t r o n i x 7A29) and monitored on a t r a n s i e n t waveform d i g i t i z e r ( T e k t r o n i x 7912AD). The d i g i t i z e r was i n t e r f a c e d t o a l a b o r a t o r y computer ( T e k t r o n i x 4502) f o r d a t a a c q u s i t i o n , c o n t r o l and p r o c e s s i n g of the e x p e r i m e n t a l r e s u l t s . A f a s t photodiode (EG&G E l e c t r o - o p t i c s FND-100Q) monitored the f i r i n g o f the l a s e r p u l s e ( u s u a l l y a t a r e p e t i t i o n r a t e of 2Hz) and p r o v i d e d an e x t e r n a l t r i g g e r s i g n a l f o r the d i g i t i z e r . Deconvolution o f the observed decay p u l s e was n e c e s s a r y because o f the f i n i t e w i d t h (approx. 1.9ns FWHM) o f the e x c i t a t i o n p u l s e . T h i s was c a r r i e d out u s i n g a F o u r i e r t r a n s f o r m method ( 4 ) . D e s p i t e the n o n - t r i v i a l c o m p u t a t i o n a l requirements and t h e need t o f i l t e r out the h i g h frequency components of the F o u r i e r d i v i s i o n b e f o r e t a k i n g t h e i n v e r s e t r a n s f o r m ( 5 ) i t was f e l t t h a t t h i s method was t h e most d i r e c t way o f g e n e r a t i n g t h e t r u e i m p u l s e r e s p o n s e f u n c t i o n o f t h e system. The r e s u l t i n g f l u o r e s c e n c e decay curves were f i t t o a s i n g l e ( o r double) e x p o n e n t i a l . The phase p l a n e p l o t method o f Demas ( 6 ) and o t h e r s ( 2 ) , was a l s o used f o r d e c o n v o l u t i o n but w i t h l e s s s u c c e s s . Two t e s t s of the r e l i a b i l t y o f the d e c o n v o l u t i o n procedure u s e d i n t h i s s t u d y were c a r r i e d o u t . I n t h e f i r s t o f t h e s e , a n a l y t i c f u n c t i o n s o f the form * (-b*t) y=a*exp were generated and c o n v o l v e d , by complex m u l t i p l i c a t i o n of the two F o u r i e r t r a n s f o r m s i n the frequency domain, w i t h e x p e r i m e n t a l l y determined i n s t r u m e n t response c u r v e s . The i n s t r u m e n t response c u r v e s were approximated as the time course o f l a s e r p u l s e f l a s h e s r e c o r d e d from the same c e l l and c e l l geometry, a t an i n t e n s e Raman s c a t t e r i n g frequency of the s o l v e n t , which f o r water a t our e x c i t a t i o n frequency was 380.6 nm. T h i s a p p r o x i m a t i o n was based on c o n s i d e r a t i o n o f t h e time course o f t h e o f f - r e s o n a n c e Raman s c a t t e r as b e i n g t h e same as t h e e x c i t i n g l i g h t p u l s e on a t i m e s c a l e s i g n i f i c a n t l y s h o r t e r than the f l u o r e s c e n c e time s c a l e under investigation (7)· The resulting synthetic decay curves,



10.

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135


c o r r e s p o n d i n g to a c o n v o l u t i o n of the i n s t r u m e n t response f u n c t i o n and exact f l u o r e s c e n t decays of v a r i o u s known l i f e t i m e s , were then d e c o n v o l v e d . For known l i f e t i m e s down t o 500ps, the c a l c u l a t e d v a l u e s were w i t h i n 5%, the e r r o r growing to some 30% f o r known l i f e t i m e s of lOOps. These e s t i m a t e s then, are the lower l i m i t s of measurement a t t a i n a b l e w i t h our o b s e r v a t i o n system. The main l i m i t a t i o n of the system i s seen to be the p u l s e w i d t h of the l a s e r flash. I n t h e s e c o n d t e s t , a number o f f l u o r e s c e n t compounds o f r e l a t i v e l y w e l l known l i f e t i m e s i n the nanosecond time range (8,9) were used as standards, allowing evaluation of both the i n s t r u m e n t a l and c o m p u t a t i o n a l a s p e c t s of the measurement. Table I shows the v a l u e s o b t a i n e d f o r 2 , 5 - d i p h e n y l o x a z o l e (PPO), anthracene and q u i n i n e b i s u l p h a t e . A l l chemicals were a n a l y t i c a l grade and not f u r t h e r p u r i f i e d b e f o r e use. Anthracene and PPO were d i s s o l v e d i n c y c l o h e x a n e , q u i n i n e i n 0.1N H^SO^; s o l v e n t s were not degassed. The c a s e of q u i n i n e i s of i n t e r e s t b e c a u s e o f i t s common use as a s t a n d a r d f o r f l u o r e s c e n c e measurements, d e s p i t e i t s complex decay k i n e t i c s ( 10) . I n a g r e e m e n t w i t h p r e v i o u s w o r k ( 1 0 ) we f o u n d s a t i s f a c t o r y f i t s of our deconvolved d a t a to a b i e x p o n e n t i a l r a t h e r than a s i n g l e e x p o n e n t i a l model. TABLE I .

Fluorescence

decays of s t a n d a r d

substances

Compound

l(nm)

t(ns)

solvent

reference

PPO

440 440 400

1.2 1.3 1.4

cyclohex. cyclohex. ethanol

( t h i s work) (Lampert et a l , 8 ) (Zuker et a l , 9 )

Anthracene

420 410 415 380

4.1 4.1 4.0 5.3

cyclohex. cyclohex. cyclohex. ethanol

( t h i s work) (Lampert et a l , 8 ) (Rayner et a l , 1 1 ) (Zuker e t al,9)

Quinine*

l(nm)

t (ns)

t (ns)

reference

400 450 500 550

5.0 5.0 5.2 5.4

17.2 19.7 21.2 21.9

( t h i s work)

400 450 500 550

2.6 3.6 10.3

18.2 19.1 19.7

(O'Connor et

* s o l v e n t used was

L

0.1N

2

al,10)

-

H„S0, 2 4

Sample P r e p a r a t i o n A number of samples of d i s s o l v e d o r g a n i c m a t e r i a l were used i n t h i s i n v e s t i g a t i o n . The f i r s t of these was a commercial p r e p a r a t i o n of "humic a c i d " ( A l d r i c h ) which was d i s s o l v e d (lmg/lOOml) i n d i l u t e



136


b a s e (0.01M NaOH). T h i s s u b s t a n c e i s d e r i v e d f r o m a p e a t l i k e m a t e r i a l . These s o l u t i o n s were u l t r a s o n i c a t e d (30sec) t o speed d i s s o l u t i o n and f i l t e r e d through 0.2um p o l y c a r b o n a t e (Nuclepore) membrane f i l t e r s t o ensure homogeneity o f the s o l u t i o n . Two other samples were marine material isolated by u l t r a f i l t r a t i o n o f n a t u r a l seawater samples through a Nuclepore 500 MWCO membrane, which has a nominal r e t e n t i o n o f a l l compounds o f m o l e c u l a r weight g r e a t e r than 500 D a l t o n s (see however Staub e t a l , 12). U l t r a f i l t r a t i o n was c a r r i e d out i n an T e f l o n coated u l t r a f i l t r a t i o n c e l l (Amicon 2000B) under a n i t r o g e n p r e s s u r e o f 5 0 p s i . The two samples were chosen t o r e p r e s e n t two q u i t e d i f f e r e n t l i g h t regimes o f t h e ocean. One sample was taken o f f s h o r e from Whitewater Bay ( 1 3 ) and r e p r e s e n t s a near s h o r e , terrestrially i n f l u e n c e d s u r f a c e water. T h i s sample was c o n c e n t r a t e d from an i n i t i a l volume o f a p p r o x i m a t e l y 17 l i t e r s t o a f i n a l volume o f 1000ml ( x l 7 ) . The second sample was taken from deep (1500m) w a t e r , w e l l below the p h o t i c zone, a t a s i t e i n the Tongue o f t h e Ocean, Bahamas and r e p r e s e n t s a sample o f marine o r g a n i c m a t t e r t h a t has n o t been exposed t o s u n l i g h t f o r some t i m e . T h i s sample, which was kept i n t h e dark throughout sampling and p r e p a r a t i o n , was c o n c e n t r a t e d from a p p r o x i m a t e l y 17 l i t e r s t o 900ml ( x l 9 ) . F u r t h e r c o n c e n t r a t i o n o f these samples was d e l i b e r a t e l y avoided t o guard a g a i n s t any a g g r e g a t i o n o f the o r g a n i c m a t t e r . The f i n a l two s a m p l e s i n v e s t i g a t e d c o n s i s t e d o f a m a r i n e f u l v i c a c i d i s o l a t e d by XAD-2 r e s i n a b s o r p t i o n ( 1 4 ) o f a b u l k s a m p l e o f near s u r f a c e seawater c o l l e c t e d from a s i t e i n the G u l f o f M e x i c o ( 1 5 ) and a l s o a s o i l f u l v i c a c i d e x t r a c t e d f r o m an h o r i z o n Β podsol ( 1 6 ) . These samples were d i s s o l v e d i n f i l t e r e d G u l f Stream seawater and d e i o n i z e d ( M i l l i Q ) water r e s p e c t i v e l y , w i t h the a d d i t i o n o f s u f f i c i e n t 0.1M NaOH t o e f f e c t d i s s o l u t i o n o f the s o l i d s o i l f u l v i c a c i d . Results S i n g l e e x p o n e n t i a l f l u o r e s c e n c e decay times f o r t h e d i f f e r e n t d i s s o l v e d o r g a n i c chromophores are t a b u l a t e d i n Table I I . R e p l i c a t e d e t e r m i n a t i o n s o f t h e l i f e t i m e s o f these samples was g e n e r a l l y b e t t e r than 5%. D e s p i t e t h e d i v e r s i t y o f t h e sources o f these m a t e r i a l s , t h e i r f l u o r e s c e n t l i f e t i m e s are s i m i l a r . I t was not found necessary t o model any o f the decay f u n c t i o n s w i t h more t h a n a s i n g l e e x p o n e n t i a l , p e r h a p s i n d i c a t i n g some u n d e r l y i n g s i m i l a r i t y i n the nature o f the f l u o r o p h o r e s t o be found i n t h e s e m o l e c u l e s . T h i s i s i n disagreement w i t h the work o f Lapen and S e i t z (17) who suggested t h a t t h e l i f e t i m e o f a s o i l f u l v i c a c i d they i n v e s t i g a t e d was best f i t by a double e x p o n e n t i a l , ( w i t h components l i f e t i m e s o f 1.0 and 6.0ns) r e f l e c t i n g the h e t e r o g e n e i t y of a complex m i x t u r e . Recent work ( L a n g f o r d p e r s o n a l communication) on t h e f l u o r e s c e n c e l i f e t i m e s o f a w e l l c h a r a c t e r i z e d Armedale h o r i z o n Β s o i l f u l v i c a c i d m a t e r i a l has suggested t h a t m a t e r i a l t o have t h r e e components c o n t r i b u t i n g t o t h e o v e r a l l f l u o r e s c e n c e o f the sample. While i t i s u n c e r t a i n , w i t h the time r e s o l u t i o n a t t a i n a b l e w i t h o u r measurement system, t h a t a component o f t h e o r d e r o f lOOps c o u l d be adequately time r e s o l v e d , t h e non-


10.

MILNE ET AL.


TABLE I I .

137

Time-Resolved Fluorescence Measurements F l u o r e s c e n c e l i f e t i m e s of d i s s o l v e d o r g a n i c m a t t e r (seawater samples) and humic a c i d ( A l d r i c h ) measured under d i f f e r e n t s o l u t i o n c o n d i t i o n s

Sample

l(nm)

Whitewater Bay

460

2.4

pH 8.0 S= 33.62

Tongue of the Ocean

460

2.3

pH 8.2 S= 34.65

Fulvic

460

2.4

alk.

Yucatan (XAD-2)

460

2.1

alk. S=35

Humic a c i d

460

2.0

pH

8

Humic a c i d

460

2.3

pH

11

Humic a c i d

460

1.8

pH

1

Humic a c i d

460

2.0

pH 8 latm

acid

Humic a c i d

460

lifetime(ns)

s o i n . cond.

0

2

pH 8 0.1M KI

1.9

o b s e r v a t i o n of a l o n g e r (6-7 ns) decay i n the samples measured i n t h i s work i s of i n t e r e s t . Work to f u r t h e r e l u c i d a t e the number of i d e n t i f i a b l e components i n the same samples i s c l e a r l y i n d i c a t e d . I n a n o t h e r r e p o r t of the measurement of l i f e t i m e s of humic m a t e r i a l s (18) a s i m i l a r constancy of t f o r samples of apparently w i d e l y d i f f e r e n t s o u r c e s was a l s o n o t e d . The effects of some important solution variables on f l u o r e s c e n c e l i f e t i m e s of ( A l d r i c h ) humic a c i d were a l s o b r i e f l y i n v e s t i g a t e d . V a l u e s o b t a i n e d as a f u n c t i o n of pH i n d i c a t e d t h a t the l i f e t i m e was somewhat l o n g e r at pH 11 than at e i t h e r pH 8 or pH 1. I t i s p e r h a p s i n t e r e s t i n g to s p e c u l a t e on the t h e possible i n v o l v e m e n t of i o n i z a b l e f u n c t i o n a l g r o u p s , which i o n i z e at h i g h pH, b e i n g p a r t of the chromophore. Phenols f o r i n s t a n c e , i o n i z e at h i g h e r pH v a l u e s and a r e known t o be p r e s e n t as p a r t o f t h e complex s t r u c t u r e of these m o l e c u l e s . Temperature v a r i a t i o n s may have an e f f e c t on the l i f e t i m e s and yields of luminescence p r o c e s s e s . Measurements made here were c a r r i e d out at room temperature (22+ 2C) f o r the most p a r t . Some l i m i t e d temperature runs over the range of 5-60 C, gave l i n e a r Arrhenius plots of relative fluorescence intensity versus temperature. f


138



Oxygen M o l e c u l a r oxygen i s an e f f i c i e n t quencher of the f l u o r e s c e n c e of a r o m a t i c hydrocarbons (19) e s p e c i a l l y i n non-aqueous s o l v e n t s where the solubility of oxygen and i t s diffusion coefficient are p r o p o r t i o n a t e l y h i g h e r than i n aqueous s o l u t i o n . In these systems quenching by oxygen approaches a d i f f u s i o n c o n t r o l l e d p r o c e s s , w h e r e i n e a c h e n c o u n t e r o f an o x y g e n m o l e c u l e w i t h an e x c i t e d f l u o r o p h o r e r e s u l t s i n quenching. I t i s known t o quench b o t h the s i n g l e t and t r i p l e t e x c i t e d s t a t e s , and q u e n c h i n g c a n be b o t h c h e m i c a l or p h y s i c a l i n n a t u r e (3)· The p h o t o c h e m i c a l p r o d u c t i o n of s i n g l e t oxygen s p e c i e s from t e r r e s t r i a l humic m a t e r i a l s has been noted (20,2J.,22). Measurement of the decay times f o r s o l u t i o n s of humic a c i d t h a t were s a t u r a t e d ( l a t m ) w i t h oxygen d i d not show any r e d u c t i o n o v e r those at e q u i l i b r i u m w i t h a i r o r s a t u r a t e d w i t h n i t r o g e n . G i v e n t h a t the maximum oxygen c o n c e n t r a t i o n i n s o l u t i o n o b t a i n a b l e u n d e r 760mm 0^ i s of the o r d e r of 10 M, and assuming a 10Çg e f f i c i e n c y of quenching at a d i f f u s i o n c o n t r o l l e d l i m i t of 10 s , then the product of the d i f f u s i o n c o e f f i c h e n t ^ a n d the quencher c o n c e n t r a t i o n w i l l be l e s s than or e q u a l t o 10 s , so t h a t e i t h e r s i n g l e t or t r i p l e t s t a t e s having lifetimes of the o r d e r of nanoseconds w i l l at b e s t o n l y be s l i g h t l y quenched i n even oxygen saturated s o l u t i o n s . I t appears then that there is little i n t e r a c t i o n , e i t h e r p h y s i c a l or c h e m i c a l , of oxygen w i t h the e x c i t e d s i n g l e t s t a t e s produced by i r r a d i a t i o n of o r g a n i c m a t t e r , at l e a s t a t t h i s e x c i t a t i o n wavelength. Work on oxygen quenching i n p r o t e i n systems (23) has shown t h a t f l u o r o p h o r e s a t t a c h e d to p r o t e i n m o l e c u l e s may be i n a c c e s s i b l e to t h e d i f f u s i o n o f a s m a l l , u n c h a r g e d and n o t p a r t i c u l a r l y h y d r o p h i l i c m o l e c u l e such as oxygen. These workers used e l e v a t e d pressures (up to lOOatm) of oxygen to achieve significant q u e n c h i n g , and p o i n t e d out t h a t s i n c e oxygen does not form any complexes w i t h the m o l e c u l e s they s t u d i e d , quenching would r e q u i r e a c t u a l short-range i n t e r a c t i o n w i t h the f l u o r o p h o r e . Quenchers which do form complexes w i t h the f l u o r o p h o r e , or o t h e r p a r t s of the m o l e c u l e , s h o u l d be expected to have a w i d e r r a d i u s of a c t i o n f o r energy t r a n s f e r . The o n l y o t h e r p o t e n t i a l quencher b e s i d e s oxygen measured i n t h i s study was i o d i d e i o n , which a t a s o l u t i o n c o n c e n t r a t i o n of 0.1M d i d show a s m a l l r e d u c t i o n i n fluorescent l i f e t i m e . Further work on the s t a t i c and dynamic quenching of these m o l e c u l e s i s under i n v e s t i g a t i o n . Conclusion I n i t i a l measurement of the f l u o r e s c e n c e l i f e t i m e s of samples of m a r i n e o r g a n i c m a t t e r and a t e r r e s t r i a l humic a c i d have suggested an u n d e r l y i n g s i m i l a r i t y i n t h i s p h y s i c a l p r o p e r t y of a l l of these m o l e c u l e s . T h i s i s i n accord w i t h the apparent homogeneity of certain other of the spectral properties (fluorescence and a b s o r p t i o n s p e c t r a , p h o t o s e n s i t i z i n g c h a r a c t e r i s t i c s e t c ) observed f o r a number of n a t u r a l water chromophores. At the same time t h i s i s at v a r i a n c e w i t h o b s e r v a t i o n s of o t h e r p r o p e r t i e s , such as m e t a l


10.

MILNE ET AL.

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i o n c o m p l e x a t i o n , s t r u c t u r a l d e t e r m i n a t i o n s based on d e g r a d a t i v e s t u d i e s e t c which seem t o i m p l y much g r e a t e r h e t e r o g e n e i t y a t a m o l e c u l a r l e v e l (24)· The q u e s t i o n o f how s e n s i t i v e a parameter the time r e s o l v e d f l u o r e s c e n c e p r o p e r t i e s o f t h e s e c h r o m o p h o r e s c a n be t o b o t h s t r u c t u r a l d i f f e r e n c e s and photochemical r e a c t i v i t y o f these m o l e c u l a r assemblages remains u n c l e a r . The o b s e r v a t i o n t h a t oxygen, at n a t u r a l l e v e l s , i s not l i k e l y t o be a s i g n i f i c a n t quenching agent f o r t h e e x c i t e d s i n g l e t s t a t e s o f these chromophores i s o f i n t e r e s t to the i n t e r p r e t a t i o n o f the p h o t o s e n s i t i z i n g a b i l i t y and d i r e c t p h o t o r e a c t i v i t y of t h i s important c l a s s of m a t e r i a l s . Ac knowledgmen t s T h i s work was c a r r i e d out w i t h t h e support o f the O f f i c e o f N a v a l R e s e a r c h u n d e r Navy G r a n t N00014-85C-0020. We t h a n k D r . G.R.Harvey f o r k i n d l y s u p p l y i n g some o f h i s humic e x t r a c t s . We a l s o thank Dr. C.H.Langford f o r making t h e r e s u l t s o f h i s s t u d i e s a v a i l a b l e t o us and f o r h i s h e l p f u l comments on t h e m a n u s c r i p t .

Literature Cited

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Zika, R.G. In "Marine Organic Chemsitry"; Duursma, E.K. and Dawson, R., Eds.; Elsevier: Amsterdam, 1981. Demas, J.N. "Excited state lifetime measurements"; Academic Press: New York, 1983. Turro, N.J. "Modern Molecular Photochemistry"; Benjamin Cummings: Menlo Park, CA., 1978. Andre, J.C.; Vincent, L.M.; O'Connor, D.; Ware, W.R. J. Phys. Chem. 1979, 83, 2285. Wild, U.; Holzworth, Α.; Good, H.P. Rev. Sci. Instrum. 1977, 48, 1621. Demas, J.N.; Adamson, A.W. J. Phys. Chem. 1971, 75, 2463. Kinoshita, S.; Kushida, T. Rev. Sci. Instrum. 1982, 52, 469. Lampert, R.A.; Chewter, L.A.; Phillips, D.; 0Connor, D.; Roberts, A.J.; Meech, S.R. Anal. Chem. 1983, 55, 68. Zuker, M.; Szabo, A.G.; Bramall, L.; Krajcarski, D.T.; Selinger, B.K. Rev. Sci. Instrum. 1985, 56, 14. O'Connor, D.V.; Meech, S.R.; Phillips' D. Chem. Phys. Lett. 1982, 88, 22. Rayner, D.M.; McKinnon, A.E.; Szabo, A.G.; Hackett, P.A. Can. J. Chem. 1976, 54, 3246. Staub, C; Buffle, J.; Haerdi, W. Anal. Chem. 1984, 56, 2843. Moffett, J.W.; Zika, R.G. 1986, (this volume). Stuermer, D.H.; Harvey, G.R. Deep Sea Res. 1977, 24, 303. Harvey, G.R.; Boran, D.A.; Chesal, L.A.; Tokar, J.M. Marine Chemistry. 1983, 12, 119. Gamble, D.S.; Schnitzer, M. In "Trace Metal and Metal Organic Interactions in Natural Water"; Singer, P.S., Ed.; Ann Arbor Science: Ann Arbor, 1973. 17. Lappen, A.J.; Seitz, W.R. Anal. Chim. Acta. 1982, 134, 31. 18. Fischer, A.M. Ph.D. Thesis, University of California, Santa Cruz, 1985. T


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Berlman, I.B. "Handbook of Fluorescence Spectra of Aromatic Molecules"; Academic Press: New York, 1965, p. 35. Zepp, R.G.; Wolfe, N.L.; Baughman, G.L.; Hollis, R.C. Nature 1977, 267, 421. Woolf, C.J.M.; Halmans, M.T.H.; van der Hiejde, H.B. Chemosphere 1981, 10, 59. Haag, W.R.; Hoigne, J.; Gassman, E.; Braun, A. Chemosphere 1984, 13, 641. Lakowicz, J.R.; Weber, G. Biochemistry 1973, 12, 4161. Aiken, G.R.; McKnight, D.M.; MacCarthy, P. "Humic Substances in Soil, Sediment, and Water"; Wiley-Interscience: New York, 1985.

RECEIVED June 24, 1986


Time-Resolved Fluorescence Measurements on Dissolved Marine

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