Organic Phosphorus in the Hydrosphere - Advances in Chemistry

6 Organic Phosphorus in the Hydrosphere Characterization via P Fourier Transform Nuclear Magnetic Resonance Spectroscopy 31

Downloaded by PURDUE UNIV on March 12, 2016 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0237.ch006

M a r k A . Nanny and Roger A . M i n e a r Institute for Environmental Studies, University of Illinois at U r b a n a Champaign, Urbana, I L 61801

Phosphorus-31 Fourier transform nuclear magnetic resonance ( P FT-NMR) spectra of dissolved organic phosphorus (DOP) species, collected from the epilimnion of a small lake from September 1990 to May 1991, were used to identify and characterize soluble Ρ com pounds in lake water. Ultrafiltration and reverse osmosis concentra tion techniques were used to achieve a 2000-fold DOP concentration factor. The sensitivity of the NMR was further enhanced by the use of the spin-lattice relaxation agent iron ethylenediaminetetraacetate (FeEDTA). These techniques are briefly discussed, in addition to the effects of pH, ionic strength, concentrated humic matrix, and Fe EDTA on the Ρ FT-NMR spectra. Individual DOP species in lake water have not been conclusively identified with P FT-NMR spec troscopy. The 31P FT-NMR spectra indicate the presence of mono -and diester phosphates, and the presence of DNA is strongly sug gested. P FT-NMR spectra show seasonal changes that correlate to seasonal changes in the lake. Varying the sample pH and collecting the subsequent P FT-NMR spectra illustrates that not all of the DOP species's signal positions are pH-dependent. This independence in dicates possible DOP aggregate or micelle formation. 31

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D I S S O L V E D P H O S P H O R U S F R A C T I O N is t h e m o s t i m p o r t a n t a q u a t i c

phosphorus c o m p a r t m e n t i n terms o f biological g r o w t h i n a n aquatic system because it p r o v i d e s t h e major source o f available phosphorus to p h y t o p l a n k t o n . T o b e biologically useful t h e dissolved phosphorus c o m p o u n d s must 0065-2393/94/0237-0161$08.75/0 © 1994 American Chemical Society

Baker; Environmental Chemistry of Lakes and Reservoirs Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

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first be c o n v e r t e d into orthophosphate. T h e r e f o r e , to f u l l y u n d e r s t a n d the aquatic p h o s p h o r u s cycle, the i d e n t i t y a n d the c h e m i c a l a n d p h y s i c a l b e havior of the d i s s o l v e d organic phosphorus ( D O P ) m u s t b e k n o w n . T h e r e m o v a l rates a n d b e h a v i o r of D O P i n the p r e s e n c e o f o t h e r d i s s o l v e d a n d c o l l o i d a l material such as h u m i c a n d f u l v i c acids; clay c o l l o i d a l m a t e r i a l ; a n d various ions s u c h as C a , M g , a n d F e or F e m u s t also b e addressed.


2 +

2 +

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F o r these reasons, n u m e r o u s attempts have b e e n m a d e to i d e n t i f y a n d characterize D O P , b u t w i t h little success because it is u s u a l l y present i n v e r y l o w concentrations. T y p i c a l values i n lake waters range f r o m 5 to 100 μg o f P / L i n o l i g o t r o p h i c to e u t r o p h i c systems. C o l o r i m e t r i c m e t h o d s have b e e n u s e d extensively to detect a n d differentiate b e t w e e n soluble reactive p h o s p h o r u s (SRP) a n d soluble unreactive p h o s p h o r u s ( S U P ) at concentrations as l o w as 10 μ g of P / L (I). S R P is generally c o n s i d e r e d to consist o f o n l y orthophosphate c o m p o u n d s , whereas S U P is c o m p o s e d of a l l other phos p h o r u s species, p r i m a r i l y organic p h o s p h o r u s c o m p o u n d s . T h e s u m of S R P a n d S U P is e q u a l to the total soluble p h o s p h o r u s (TSP). T h e s e m e t h o d s w e r e u s e d to study the d y n a m i c s of b u l k p h o s p h o r u s fractionation b e t w e e n the sediments, s u s p e n d e d particulate matter, the biota, a n d the d i s s o l v e d frac t i o n (2). D e s p i t e these studies, v e r y little is k n o w n r e g a r d i n g the i d e n t i t y a n d characteristics of the D O P i n the h y d r o s p h e r e . A t t e m p t s to i d e n t i f y a n d characterize the D O P fraction have r e l i e d o n gel c h r o m a t o g r a p h y (Sephadex) (3-8), P a n d ^ P tracer studies (3-11), bioassays (9-12), e n z y m e bioassays (13, 14), a n d h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y ( H P L C ) w i t h a p o s t - c o l u m n reactor (15, 16). A consistent feature i n the gel c h r o m a t o g r a p h y studies is the appearance of a h i g h - m o l e c u l a r - w e i g h t ( H M W ) fraction ( > 3 0 , 0 0 0 - 5 0 0 0 daltons, d e p e n d i n g o n the exclusion l i m i t o f the gel e m p l o y e d ) , always at the u p p e r l i m i t of the sizeexclusion gel u s e d , a n d a l o w - m o l e c u l a r - w e i g h t fraction that coelutes w i t h orthophosphate. B e t w e e n the h i g h - a n d l o w - m o l e c u l a r - w e i g h t fraction peaks, w h i c h are distinct a n d p r o m i n e n t , a c o n t i n u u m of an i n t e r m e d i a t e m o l e c u l a r - w e i g h t fraction is often present i n l o w concentrations. S o m e t i m e s this fraction also is r e p r e s e n t e d b y a s h o u l d e r i n the l o w - m o l e c u l a r - w e i g h t e l u t i o n r e g i o n . T h e s e studies also indicate that S R P is sometimes present i n the H M W fraction. 3 2

I n c u b a t i o n of lake water w i t h P or P as tracers a n d subsequent g e l c h r o m a t o g r a p h y reveals that a major pathway exists b e t w e e n d i s s o l v e d or thophosphate a n d the particulate phase (3, 5 - 7 ) . L o w - m o l e c u l a r - w e i g h t p h o s p h o r u s forms i n the presence of bacteria a n d algae. S U P is present i n the l o w - m o l e c u l a r - w e i g h t fraction a n d is classified as i n d i v i d u a l D O P c o m p o u n d s unassociated w i t h particulate or c o l l o i d a l m a t e r i a l . T h e H M W frac t i o n f o u n d i n gel chromatography studies is c h a r a c t e r i z e d as a c o l l o i d that contains p h o s p h o r u s c o m p o u n d s or incorporates orthophosphate. T h e c o l loidal material t h e n releases orthophosphate, r e p l e n i s h i n g the d i s s o l v e d p h o s p h o r u s cycle. I n some e u t r o p h i c lakes the H M W S R P fraction can m a k e 3 2

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u p to 8 0 % of the total soluble p h o s p h o r u s (6). Studies b y K o e n i n g s a n d H o o p e r (17) a n d b y F r a n k o a n d H e a t h (18) d e m o n s t r a t e d the b i n d i n g of orthophosphate b y h u m i c m a t e r i a l i n the p r e s e n c e of ferric ions. T h i s h u m i e P 0 ~ - F e ( I I I ) c o m p l e x shows u p i n the H M W fraction w h e n a n a l y z e d w i t h gel chromatography. U p o n i r r a d i a t i o n w i t h u l t r a v i o l e t light, the phosphate is released a n d the ferric ions are r e d u c e d to ferrous ions. 4

3

O t h e r methods u s e d to characterize a n d i d e n t i f y D O P i n v o l v e bioassays w i t h Chlorella to study the b i o l o g i c a l availability a n d b i o u p t a k e of the H M W S R P fraction (4, 6). T h e s e bioassays indicate that the algal g r o w t h responds s i m i l a r l y to H M W S R P a n d to P O ^ " . A preference for P 0 ~ was d e t e c t e d , a n d not a l l of the reactive H M W fraction was u s e d . E n z y m a t i c assays u s e d b y H e r b e s et a l . (13) tentatively i d e n t i f i e d i n o s i t o l hexaphosphate as part of the D O P U s i n g an anion-exchange H P L C system w i t h a p h o s p h o r u s - s p e c i f i c p o s t - c o l u m n reactor, M i n e a r a n d co-workers (15,16) p o s s i b l y have d e t e c t e d inositol hexaphosphate, D N A , a n d n u c l e o t i d e fragments i n lake waters. O n l y a few D O P species have b e e n c o n c l u s i v e l y i d e n t i f i e d i n natural fresh waters. T h e s e species are D N A b y M i n e a r (19) a n d D e F l a u n et a l . (20), R N A a n d D N A b y K a r l a n d B a i l i f f (21), a n d 3 ' , 5 ' - c y c l i c adenosine monophosphate b y F r a n k o a n d W e t z e l (22). Jefferey (23) d e t e c t e d the pres ence of p h o s p h o l i p i d s i n sea water. O t h e r s have p r o v i d e d c i r c u m s t a n t i a l e v i d e n c e for i n o s i t o l hexaphosphate i n lake water (24-27) a n d i n aquatic sediments (13).


3

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D e s p i t e the variety of analytical methods u s e d a n d the a m o u n t of effort e m p l o y e d , the p a u c i t y of i n f o r m a t i o n r e g a r d i n g D O P a n d its i m p o r t a n c e i n aquatic ecosystems indicates a n e e d for n e w tools i f the k n o w l e d g e c o n c e r n i n g D O P is to b e e x p a n d e d . F o u r i e r transform n u c l e a r magnetic reso nance ( F T - N M R ) spectroscopy holds p r o m i s e , for it is already b e c o m i n g a h i g h l y beneficial tool i n the area of e n v i r o n m e n t a l analysis. C F T - N M R spectroscopy has b e e n u s e d since 1976 (28) to examine h u m i c a n d f u l v i c acids. S i F T - N M R spectroscopy was r e c e n t l y a p p l i e d i n the d e t e c t i o n of polyorganosiloxanes i n the e n v i r o n m e n t (29). Ρ F T - N M R spectroscopy was r e c e n t l y a p p l i e d to the characterization of organic p h o s p h o r u s present i n the e n v i r o n m e n t b y e x a m i n i n g organic p h o s p h o r u s i n soils (30-34) a n d h u m i c m a t e r i a l f r o m soils (35, 36), m a r i n e sediments (37), a n d wastewater-treatm e n t - p l a n t activated sludge (38-41). M o n o - a n d diester a n d i n o r g a n i c p o l y phosphates a n d occasionally phosphonates w e r e detected i n these samples. 1 3

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Because the a p p l i c a t i o n of N M R spectroscopy to e n v i r o n m e n t a l samples is r e l a t i v e l y n e w , w e focused o u r studies o n the i d e n t i f i c a t i o n a n d charac terization of D O P b y P F T - N M R spectroscopy. U l t r a f i l t r a t i o n a n d reverse osmosis concentration techniques w e r e e m p l o y e d to increase the d i s s o l v e d organic p h o s p h o r u s concentrations to the d e t e c t i o n l e v e l of Ρ F T - N M R techniques (approximately 1 0 - 2 0 m g of P / L ) . W i t h these c o n c e n t r a t i o n methods a D O P concentration factor of u p to 2000 is obtainable. T h i s chapter reports the use of Ρ F T - N M R spectroscopy i n the analysis o f D O P . I n 3 1

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contrast to soil a n d s e d i m e n t P F T - N M R studies, w e a v o i d e d t h e use o f strong base extraction techniques to m a i n t a i n D O P i n t e g r i t y . I n d i v i d u a l D O P c o m p o u n d s have not y e t b e e n c o n c l u s i v e l y i d e n t i f i e d , b u t w e have d e t e c t e d D O P as m o n o - a n d diester phosphates i n a n e n g i n e e r e d lake i n C h a m p a i g n C o u n t y , Illinois. W e also o b s e r v e d t e m p o r a l changes i n t h e Ρ N M R spectra o v e r t h e p e r i o d o f S e p t e m b e r t h r o u g h M a y . F r o m these r e sults, w e c o n c l u d e d that P F T - N M R spectroscopy is a v i a b l e t e c h n i q u e w i t h p o t e n t i a l to i d e n t i f y a n d characterize t h e D O P i n v o l v e d i n t h e aquatic phosphorus cycle. 3 1

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Background NMR Theory T h e basic p r i n c i p l e o f N M R spectroscopy is that a signal arises from t h e magnetic d i p o l e o f n u c l e i u n d e r g o i n g a transition b e t w e e n e n e r g y l e v e l s . A m a g n e t i c d i p o l e is created b y t h e i n t r i n s i c s p i n n i n g m o t i o n o f t h e n u c l e u s a n d t h e electrical charges o f its protons (42). T h e n u c l e a r s p i n c a n b e d e s c r i b e d b y t h e s p i n q u a n t u m n u m b e r , I, w h i c h m u s t b e e q u a l to o r greater than ¥2 f o r a magnetic d i p o l e to b e present. I n a d d i t i o n , t h e n u c l e u s m u s t have a n u n p a i r e d s p i n a r i s i n g from e i t h e r a n o d d n u m b e r o f protons o r a n o d d n u m b e r o f neutrons w i t h a n e v e n n u m b e r o f protons present. M a n y o f the most c o m m o n n u c l e i e x a m i n e d (such as * H , C , a n d P ) have s p i n q u a n t u m n u m b e r s e q u a l to ¥2. I n t h e presence o f an external magnetic field, H , t h e magnetic d i p o l e of the n u c l e u s orients itself i n discrete positions relative to H , c o r r e s p o n d i n g to specific e n e r g y levels. T h e e n e r g y difference b e t w e e n these levels is g i v e n by e q 1 1 3

3 1

0

()

AE =

(1)

w h e r e Δ Ε is t h e e n e r g y difference b e t w e e n each p a i r o f levels, μ is t h e m a g n e t i c m o m e n t o f t h e n u c l e u s expressed as n u c l e a r magnetons, Β is t h e n u c l e a r m a g n e t o n constant (5.049 Χ 1 0 " erg/G), a n d H is t h e a p p l i e d external field. A s c a n b e seen f r o m e q 1, t h e a p p l i e d external m a g n e t i c field a n d t h e e n e r g y difference b e t w e e n t h e p o s i t i o n a l levels are d i r e c t l y p r o p o r t i o n a l . I n t h e absence o f H , t h e r e is n o difference b e t w e e n t h e e n e r g y l e v e l s . F o r a nucleus w i t h a s p i n q u a n t u m n u m b e r o f I = ¥2 i n a n a p p l i e d external magnetic field, t h e magnetic d i p o l e has o n l y t w o discrete positions: it c a n b e a l i g n e d w i t h o r against H . 24

Q

0

Q

W h e n energy e q u i v a l e n t to t h e difference b e t w e e n t h e e n e r g y levels is a p p l i e d to t h e system, a transition f r o m t h e l o w e r to t h e h i g h e r e n e r g y l e v e l occurs. I n N M R spectroscopy, t h e a p p l i e d e n e r g y that allows this n u c l e a r magnetic d i p o l e transition to o c c u r is a r a d i o - f r e q u e n c y magnetic field, H w h i c h is a p p l i e d p e r p e n d i c u l a r l y to H . u

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T h e intensity o f the signal p r o d u c e d b y this transition is p r o p o r t i o n a l to the n u m b e r o f n u c l e i (n) that change f r o m the l o w e r to t h e h i g h e r e n e r g y state. A t e q u i l i b r i u m , the p o p u l a t i o n difference b e t w e e n these energy states can b e d e s c r i b e d b y the B o l t z m a n n e q u a t i o n

(2) w h e r e k is the B o l t z m a n n constant a n d Τ is the absolute t e m p e r a t u r e . A t r o o m t e m p e r a t u r e i n an H t y p i c a l o f m o d e r n N M R spectrometers, this p o p u l a t i o n difference is v e r y s m a l l . Because the p o p u l a t i o n difference varies w i t h the strength o f H , larger a n d larger magnets are b e i n g d e v e l o p e d . H o w e v e r , this d e v e l o p m e n t is v e r y difficult a n d expensive. T h u s N M R spec troscopy is l i m i t e d to concentrated samples, o r it r e q u i r e s n u m e r o u s r e p e a t e d acquisitions to p r o v i d e a viable s p e c t r u m . 0


0

A t a specified H , the f r e q u e n c y o f the H that i n d u c e s transitions is an i n t r i n s i c p r o p e r t y o f the n u c l e u s . T h e H magnetic f i e l d c a n b e a l t e r e d b y the e l e c t r o n d e n s i t y s u r r o u n d i n g the n u c l e u s . T h u s , e v e n t h o u g h H m a y be at t h e p r o p e r f r e q u e n c y to i n d u c e n u c l e a r magnetic d i p o l e transitions, the nucleus m a y b e r e c e i v i n g a magnetic field that is slightly greater o r smaller than H as a result of the e l e c t r o n s h i e l d i n g . Because of this s h i e l d i n g , each nucleus o f a g i v e n e l e m e n t w i l l b e excited at a s i m i l a r b u t discrete f r e q u e n c y . T h u s n u m e r o u s signals w i l l b e present i n t h e s p e c t r u m , w i t h positional differences d e p e n d e n t u p o n t h e e l e c t r o n d e n s i t y s u r r o u n d i n g the nucleus b e i n g e x a m i n e d . T h e signal p o s i t i o n , r e p o r t e d as t h e c h e m i c a l shift, is g i v e n i n parts p e r m i l l i o n (ppm) a n d d e f i n e d as 0

Y

x

x

x

c h e m i c a l shift =

o b s e r v e d shift (Hz) Χ 1 0

6

spectrometer f r e q u e n c y ( H z )

(3)

T h e o b s e r v e d shift is t h e difference b e t w e e n the o b s e r v e d signal a n d a reference signal. P N M R spectroscopy often uses the signal p r o d u c e d b y 8 5 % H P 0 as the reference signal a n d assigns i t the c h e m i c a l shift o f 0.00 ppm. C o u p l i n g is a p h e n o m e n o n that affects t h e signal p a t t e r n (singlet, d o u b let, t r i p l e t , etc.). T h i s c o u p l i n g is caused b y the i n t e r a c t i o n b e t w e e n the magnetic fields f r o m the e x a m i n e d nucleus a n d electrons s u r r o u n d i n g o t h e r nearby n u c l e i . F o r example, the p h o s p h o r u s n u c l e i f r o m m o n o - a n d diester phosphates can c o u p l e w i t h the protons present i n t h e phosphate g r o u p , g i v i n g rise to c o m p l e x signal patterns. T o s i m p l i f y the P F T - N M R s p e c t r u m , N M R e x p e r i m e n t s are r u n d e c o u p l e d (i.e., the protons are subjected to a separate radio-frequency f i e l d that matches the f r e q u e n c y o f the proton's resonance). T h i s treatment eliminates the proton's c o n t r i b u t i o n to c o u p l i n g so that the p h o s p h o r u s N M R signals are not split, b u t appear as a single peak. 3 1

3

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In a c o n t i n u o u s - w a v e N M R ( C W N M R ) e x p e r i m e n t the Η γ f r e q u e n c y is s l o w l y scanned a n d each nucleus resonates at its specific f r e q u e n c y , w h i c h is a f u n c t i o n o f the e x a m i n e d nucleus a n d the e l e c t r o n d e n s i t y that s u r r o u n d s it. T h i s e x p e r i m e n t can be l o n g a n d tedious because i f h i g h r e s o l u t i o n is d e s i r e d , the frequencies must be swept v e r y slowly. If the c o n c e n t r a t i o n o f the e x a m i n e d nucleus is l o w , a large n u m b e r of r e p e a t e d sweeps m a y b e n e e d e d to obtain a detectable signal. F o u r i e r transform N M R spectroscopy, o n the other h a n d , p e r m i t s r a p i d scanning o f the sample so that the N M R s p e c t r u m can b e o b t a i n e d w i t h i n a f e w seconds. F T - N M R e x p e r i m e n t s are p e r f o r m e d b y subjecting the sam ple to a v e r y intense, b r o a d - b a n d , H p u l s e that causes a l l of the e x a m i n e d n u c l e i to u n d e r g o transitions. A s the excited n u c l e i relax to t h e i r e q u i l i b r i u m state, t h e i r relaxation-decay pattern is r e c o r d e d . A F o u r i e r transform is p e r f o r m e d u p o n this relaxation-decay pattern to p r o v i d e the N M R spectra. T h e relaxation-decay pattern, w h i c h is i n the t i m e d o m a i n , is t r a n s f o r m e d into the t y p i c a l N M R s p e c t r u m , the f r e q u e n c y d o m a i n . T h e t i m e r e q u i r e d to a p p l y the H p u l s e , a l l o w the n u c l e i to r e t u r n to e q u i l i b r i u m , a n d have the c o m p u t e r p e r f o r m the F o u r i e r transforms o n the relaxation-decay p a t t e r n often is o n l y a few seconds. T h u s , c o m p a r e d to a C W N M R e x p e r i m e n t , the t i m e can be r e d u c e d b y a factor of 1000-fold or m o r e b y u s i n g the F T N M R technique.


l

l

T h e major advantage of this d r a m a t i c t i m e r e d u c t i o n lies i n the a b i l i t y to scan the sample repeatedly, c o m b i n e the relaxation-decay patterns c o l l e c t e d f r o m each scan, a n d t h e n p e r f o r m a F o u r i e r transform u p o n the final c o m p o s i t e relaxation-decay p a t t e r n . T h i s t e c h n i q u e , i n essence, increases the spectral sensitivity b y a l l o w i n g the N M R signals a c q u i r e d f r o m each scan to be c o n s t r u c t i v e l y a d d e d to each other w h i l e the noise cancels itself d e c o n s t r u e d v e l y . T h i s approach greatly increases the sensitivity of the i n s t r u m e n t a n d allows N M R e x p e r i m e n t s to b e p e r f o r m e d o n samples that have l o w concentrations of the d e s i r e d nucleus (i.e., for P , 20 m g of P / L is a feasible concentration w i t h i n s t r u m e n t t i m e o f hours to a f e w days). It is v e r y i m p o r t a n t that there be sufficient t i m e b e t w e e n pulses i n F T N M R e x p e r i m e n t s so that the n u c l e i can r e t u r n to the o r i g i n a l e q u i l i b r i u m state. If the e q u i l i b r i u m state has not b e e n r e a c h e d before the next H p u l s e , the still-excited n u c l e i w i l l not participate i n the transition a n d thus w i l l p r o d u c e a decreased signal intensity relative to the p r e v i o u s signal. A s the e x p e r i m e n t proceeds a n d m o r e pulses are a p p l i e d , m o r e n u c l e i w i l l r e m a i n i n the exited state u n t i l e v e n t u a l l y none of the n u c l e i w i l l b e i n the l o w e r e n e r g y state w h e n p u l s e d . A t this p o i n t the sample is saturated a n d w i l l not p r o d u c e a signal. T h e l e n g t h of t i m e r e q u i r e d for the n u c l e i to relax is c a l l e d the spin-lattice or T relaxation t i m e . T o reduce the nuclei's Τ relaxation t i m e , relaxation agents are u s e d . Relaxation agents are usually transition m e t a l complexes, p r i m a r i l y f e r r i c ethylenediaminetetraacetate or c h r o m i u m acetylacetonate. Transfer of e n 3 1

t

l

λ


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ergy f r o m the excited nucleus to the m e t a l i o n s lone p a i r of electrons p r o vides a relaxation pathway a n d allows the e x a m i n e d n u c l e u s to relax at a m u c h greater rate, thus p e r m i t t i n g m o r e scans p e r u n i t t i m e . T h e relaxation agent must be selected carefully. If the relaxation rate is increased too m u c h , signal l i n e b r o a d e n i n g w i l l occur, e v e n to the extent that the signal b e c o m e s undetectable, because the signal w i d t h is i n v e r s e l y p r o p o r t i o n a l to the re laxation t i m e . T h e c h e m i c a l shift associated w i t h * H n u c l e i indicates the type of func tional group c o n t a i n i n g the p r o t o n (e.g., alkane, alkene, carboxylic a c i d , p h e n o l i c , a n d alcohol). E v i d e n c e of c o u p l i n g w i t h n e a r b y protons also p r o vides i n f o r m a t i o n about the n u m b e r of adjacent protons a n d t h e i r i d e n t i t y . C N M R spectroscopy p r o v i d e s i n f o r m a t i o n r e g a r d i n g the t y p e of c a r b o n atoms present (e.g., aliphatic, aromatic, o r carboxylic), as s h o w n b y the c h e m i c a l shift. P N M R spectroscopy p r o v i d e s the same i n f o r m a t i o n as C N M R spectroscopy. H o w e v e r , most of the p h o s p h o r u s i n e n v i r o n m e n t a l samples is l i k e l y to be i n the forms of orthophosphate or its m o n o - a n d diesters. T h e r e f o r e , i n the P N M R e x p e r i m e n t , the effect of the n u m b e r and t y p e of R groups present i n 0 - P O ( O R ) _ o n the p h o s p h o r u s n u c l e u s w i l l d e t e r m i n e the signal's p o s i t i o n . O r t h o p h o s p h a t e a n d m o n o e s t e r phos phate P N M R signals appear i n the 0 - 1 0 - p p m r e g i o n , d e p e n d i n g u p o n the sample p H . D i e s t e r phosphate signals appear i n the 0- to - 5 - p p m r e g i o n , whereas triester phosphates are not l i k e l y to be d e t e c t e d i n the e n v i r o n m e n t because of concentration limitations related to c o m p o u n d s o l u b i l i t y . P o l y phosphates appear i n the r e g i o n of - 5 to - 1 0 p p m ; phosphonates, w h i c h are characterized b y the C - P b o n d b e t w e e n the phosphate p h o s p h o r u s atom and an R g r o u p , have signals a p p e a r i n g at 2 0 - 2 5 p p m . A l l of these c h e m i c a l shifts are relative to 8 5 % p h o s p h o r i c a c i d .


1 3

3 1

1 3

3 1

; 3

x

3 1

Methodology T o achieve Ρ N M R spectra of D O P , ultrafiltration ( U F ) a n d reverse osmosis (RO) m e m b r a n e concentration techniques a n d T relaxation agents w e r e e m p l o y e d . C o n c e n t r a t i o n factors of 2000 or m o r e are n e e d e d to reach the N M R detection t h r e s h o l d for p h o s p h o r u s , a n d use of a relaxation agent p e r m i t s m o r e scans p e r u n i t t i m e . P F T - N M R studies of soil a n d s e d i m e n t do not r e q u i r e such h i g h concentration factors because soil a n d s e d i m e n t phosphorus concentrations are m u c h h i g h e r a n d extraction t e c h n i q u e s are easily e m p l o y e d . A caveat w i t h soil a n d s e d i m e n t extraction m e t h o d s is that a strong base is r e q u i r e d ; thus o n l y base-soluble organic p h o s p h o r u s is iso lated. P o t e n t i a l h y d r o l y s i s o f the organic p h o s p h o r u s b y the strong base is an e v e n greater p r o b l e m w i t h these extraction m e t h o d s . 3 1

x

3 1

T h e use of U F a n d R O m e m b r a n e s as a c o n c e n t r a t i o n p r o c e d u r e (com p a r e d to other concentration techniques for D O P s u c h as freeze-concentrat i o n , f r e e z e - d r y i n g - r e c o n s t i t u t i o n , a n i o n - e x c h a n g e - s m a l l - v o l u m e elutions,


168


and lanthanide p r e c i p i t a t i o n - s e q u e n t i a l dissolution) is p r e f e r r e d . It sepa rates D O P a c c o r d i n g to m o l e c u l a r size, d e p e n d i n g o n t h e n o m i n a l m o l e c u l a r w e i g h t cutoff (i.e., pore size) o f the m e m b r a n e u s e d . O u r samples are fractionated i n t o a H M W fraction (30,000 daltons), an i n t e r m e d i a t e - to l o w m o l e c u l a r - w e i g h t fraction (1000 daltons), a n d a l o w - m o l e c u l a r - w e i g h t frac tion (approximately 300 daltons) ( F i g u r e 1). T h e y are p u r p o s e f u l l y fraction ated so as to isolate a n d concentrate the various D O P fractions o b s e r v e d i n


Water Sample 250 - 500 L

0.2 um tangential flow membrane

Cation exchange Sodium form

30 Κ Dalton tangential flow membrane 30 Κ retentate Final vol. 1 L 1 Κ Dalton tangential flow membrane Ι Κ retentate Final vol. 1 L Reverse Osmosis Bench top unit R,0, retentate Final vol. 1.8 L

To Batch Pressure Ultrafiltration units Figure 1. Ultrafiltration

and reverse osmosis concentration

system.


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studies u s i n g g e l c h r o m a t o g r a p h y . A t present, o n l y the retentate f r o m the 1000-dalton, tangential-flow U F m e m b r a n e has a h i g h e n o u g h D O P c o n centration to facilitate P F T - N M R spectroscopy. A n a d d i t i o n a l b e n e f i t of ultrafiltration a n d reverse osmosis concentration m e t h o d s is that the sample does not u n d e r g o drastic p h y s i c a l or c h e m i c a l transformations a n d thus m a i n tains its i n t e g r i t y .


3 1

T h e ΤΊ relaxation agent increased the sensitivity o f the N M R i n s t r u m e n t b y decreasing several of the m o n o - a n d diester phosphate relaxation t i m e s b y factors of 2 - 5 . I n this w a y the delay t i m e b e t w e e n scans was decreased (43). T h i s change p e r m i t s an increase i n the n u m b e r o f scans o b s e r v e d p e r u n i t t i m e . A l t h o u g h the p r e s e n c e of f e r r i c ions creates the p o t e n t i a l for p r e c i p i t a t i o n of p h o s p h o r u s - i r o n complexes, the a d d i t i o n of a large m o l a r excess of ethylenediaminetetraacetate ( E D T A ) relative to f e r r i c ions p r e v e n t e d p r e c i p i t a t i o n , e v e n o v e r a large p H r e g i o n (44).

Experimental Section Sample C o l l e c t i o n . Pelagic lake-water samples were collected at Crystal Lake, an engineered mesotrophic lake i n C h a m p a i g n C o u n t y , Illinois. It has an average depth of 10 feet w i t h a maximum depth of 13 feet and is fed by ground water from a 200-foot-deep w e l l . F o r each sample 2 5 0 - 5 0 0 L of water was filtered w i t h a plankton net and stored i n 5 5 - L polyethylene (Nalgene) containers u n t i l processed i n the laboratory approximately 30 m i n later. Concentration. Samples were concentrated by using ultrafiltration and reverse osmosis membranes (Figures 1 and 2). T h e water was first filtered w i t h a 0.2-μιη tangential flow filter to remove algal cells, bacteria, and colloidal and suspended solids. T h e water then passed through a N a cation-exchange c o l u m n containing 6.2 d m of 50-100-mesh resin (Dowex 50 X 8) i n the sodium form to remove C a and M g and then onto a second tangential-flow filtration unit consisting of either a 30,000-dalton polysulfone or a 1000-dalton cellulose acetate membrane. The retentate was continuously recycled to the second tangentialflow filter while the filtrate passed to a reverse osmosis bench-top spiral-wound membrane unit (Millipore), i n which various membranes were installed. T h e membranes used were 50% and 80% N a C l rejection reverse osmosis cellulose acetate membranes, a 99% N a C l rejection reverse osmosis polyamide membrane, and a 1000-dalton cellulose acetate ultrafiltration membrane. T h e retentate from this filtration was recycled back to the spiral-wound membrane. T h e final v o l u m e of the second tangential-flow unit retentate was usually 0.8 L , and for the spiralw o u n d reverse osmosis unit the final volume was usually 1.5 L . T h e spiral-wound reverse osmosis retentate was passed through a second smaller cation-exchange column containing the same resin type as the first cation-exchange unit to remove any remaining C a and M g . Samples were further concentrated to a final volume of 8 m L w i t h a sequence of decreasing-volume batch pressure ultrafil tration units (Amicon) containing cellulose acetate filters. Effective pore sizes of 500 or 1000 daltons were used for the low- and intermediate-molecular-weight fractions; 5000- and 10,000-dalton membranes were e m p l o y e d w i t h the H M W fraction. +

3

2 +

2 +

2 +

2 +


ENVIRONMENTAL CHEMISTRY O F L A K E S A N D RESERVOIRS


170

25 mm dla.

C

Concentrated Sample

Figure 2. Pressure filtration

batch concentration system.

Sample P r e p a r a t i o n . Samples for P F T - N M R analysis were prepared b y adding sodium azide to form a 0 . 1 % solution and to prevent microbial growth, and an appropriate amount of ferric E D T A to provide a phosphorus : iron m o lecular ratio of 1 . 5 : 2 . 0 . C o n t r o l studies have shown that the addition of sodium azide has no effect upon the P F T - N M R spectra. T h e 0 . 0 1 0 3 M ferric E D T A relaxation agent solution had an E D T A : i r o n ratio of approximately 3 0 a n d was made from ferric nitrate (Fischer) and tetrasodium ethylenediaminetetraacetate ( E D T A , Sigma) dissolved i n deionized water. Total soluble phosphorus and soluble reactive phosphorus concentration measurements were done by the phosphate molybdate test with the oxidation step achieved via potassium persulfate oxidation (J). Samples for p H and ionic strength studies were prepared b y making solutions of 1 5 0 m g of P / L of each model compound. T h e model compounds chosen were mi/o-inositol hexaphosphate (IHP, Sigma), inositol monophosphate (IMP, Sigma), D N A from degraded herring sperm (Sigma), choline phosphate (Sigma), serine phosphate (Sigma), and potassium dihydrogen orthophosphate (J. T. Baker). F o r the p H studies, the sample p H was modified w i t h either dilute H C 1 or N a O H . T h e ionic strength was modified b y the addition of N a C l . I n the ionic strength studies, the p H was h e l d fairly constant by the use of either an acetic a c i d - s o d i u m acetate buffer or a sodium carbonate-sodium bicarbonate buffer. T h e contribution of the buffers to the ionic strength was taken into consideration. 3 1

3 1


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Hydrosphere

Concentrated natural humic matrix was obtained from o l d , concentrated natural water samples i n w h i c h the dissolved phosphorus concentration was either too low to be measured or i n which it had all hydrolyzed to orthophosphate. N M R Spectroscopy. A l l P F T - N M R spectra were collected on one of two instruments ( G N 300 narrow bore or G N 300 wide bore) at the University of Illinois, School of C h e m i c a l Sciences Molecular Spectroscopy Laboratory. A l l p H , ionic strength, concentrated humic matrix, and relaxation agent spectra were obtained w i t h the G N 300 narrow-bore spectrometer. C r y s t a l L a k e samples were scanned for 2 4 - 4 8 h w i t h the G N 300 wide-bore instrument or over several 12-h periods with the G N 300 narrow-bore N M R , c o m p i l i n g the individual free induction decay patterns ( F I D ) into a single F I D . A F o u r i e r transformation was then performed w i t h the composite F I D to obtain the final spectrum. T h e P F T - N M R spectra, obtained at 121.648 M H z , were generated b y a pulse w i d t h of 2 0 - 2 4 μς with a pulse delay of 3 - 6 s, depending upon the sample. A l l spectra were proton decoupled, and a spectral w i d t h of 10,000 H z was used. Magnetic shimming and signal phasing were done by computer, and all chemical-shift measurements were measured relative to 8 5 % H P 0 . Samples were placed i n 10-mm glass N M R tubes ( W i l m a d Corporation) w i t h inserts containing deute rium oxide (Sigma). Longitudinal (T ) relaxation measurements were obtained by using the inversion recovery method (i.e., a 1 8 0 ° - f - 9 0 ° pulse sequence. 3 1


3 1

3

4

{

Results and Discussion M a t r i x Effects. pH. N u m e r o u s factors such as sample p H , i o n i c strength, h u m i c substances, a n d relaxation agents can m o d i f y t h e N M R s p e c t r u m . F o r example, monoester phosphate c h e m i c a l shifts are ρ H - d e p e n d e n t (44-46), a n d w e s h o w e d (44) for several monoester phosphates that as the sample p H is increased, t h e i r c h e m i c a l shifts also increase ( F i g u r e 3). T h i s b e h a v i o r is caused b y the ability of monoester phosphates to u n d e r g o p r o t o n a t i o n - d e p r o t o n a t i o n . Because the monoester phosphate c h e m i c a l shift is p H - d e p e n d e n t , the curves r e s u l t i n g f r o m p l o t t i n g p H versus c h e m i c a l shift are analogous to a titration c u r v e . T h u s , monoester phosphate p K values can b e m e a s u r e d f r o m these p H - c h e m i c a l shift curves (44-46). a

A c c o r d i n g to the theory of L e t c h e r a n d V a n W a z e r (47), changes i n t h e chemical-shift p o s i t i o n d e p e n d o n three factors: the difference i n t h e elec tronegativity o f the P - X b o n d , the change i n t h e e l e c t r o n o r b i t a l o v e r l a p , a n d t h e change i n b o n d angles b e t w e e n t h e atoms attached to p h o s p h o r u s . U p o n deprotonation of a monoester phosphate m o l e c u l e , t h e e l e c t r o n d e n s i t y on the deprotonated phosphate oxygen atom changes. A f t e r d e p r o t o n a t i o n , the oxygen atom contains a negative charge, increasing t h e e l e c t r o n density s u r r o u n d i n g the oxygen nucleus. T h i s increase i n t u r n decreases t h e e l e c t r o n density o f the phosphorus nucleus. T h e p h o s p h o r u s n u c l e u s , w i t h less elec tron s h i e l d i n g f r o m the external magnetic f i e l d H than it p r e v i o u s l y h a d w h i l e the oxygen was protonated, resonates at a greater f r e q u e n c y , a n d its signal appears at a h i g h e r chemical-shift value. T h e other t w o factors affecting the chemical-shift position are c o n s i d e r e d to b e m i n o r relative to that caused 0


172

β Pu

£


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34-

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0 Ο

ο-ο

Δ-Δ

1+

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Serine phosphate

Ο-ο Inositol monophosphate • - • Adenosine monophosphate

0 0

1

Η

2

h

3

4

5

6

+ 7•

8

9

10 11 12 13

14

PH Figure 3. Effect of pH on the chemical shift of several monoester phosphate compounds. Serine phosphate and inositol monophosphate are in a pure water matrix, and adenosine monophosphate is in a concentrated humic-FeEDTA matrix. b y t h e changes i n t h e P - X bond's electronegativity. T h e d e p r o t o n a t i o n p r o t o n a t i o n o f a monoester phosphate p r o b a b l y does not greatly i n f l u e n c e the TT-electron o r b i t a l d e n s i t y o f t h e P = 0 b o n d , a n d changes i n t h e b o n d angles are b e l i e v e d to have o n l y v e r y small effects u p o n t h e signal p o s i t i o n . A n o t h e r p H - i n d u c e d effect is change i n t h e signal p a t t e r n o f c o m p o u n d s c o n t a i n i n g m u l t i p l e monoester phosphate groups. A n e x a m p l e o f this is inositol hexaphosphate ( I H P ) . A t l o w p H values ( 2 - 5 ) the P N M R s p e c t r u m of I H P exhibits four i n d i v i d u a l singlets. T h e intensity o f the t w o i n n e r peaks is t w i c e t h e intensity o f each o f the t w o o u t e r peaks ( F i g u r e 4). Increasing the p H causes this signal p a t t e r n to u n d e r g o dramatic changes u n t i l t h e o r i g i n a l four-peak pattern returns w h e n p H 12 is r e a c h e d . I n t h e p H 2 - 5 r e g i o n the I H P m o l e c u l e maintains t h e chair configuration i n w h i c h t h e phosphate groups 2 , 6 a n d 3 , 5 ( F i g u r e 5), a l l i n t h e axial p o s i t i o n , are i n v o l v e d i n h y d r o g e n b o n d i n g . T h e s e t w o sets give rise to t h e t w o large c e n t r a l peaks; phosphate groups 1 a n d 4 give rise to t h e t w o smaller outer signals ( F i g u r e 4). D e p r o t o n a t i o n o f I H P disrupts t h e c h a i r configuration because o f steric r e p u l s i o n b e t w e e n t h e lone e l e c t r o n pairs f r o m the n e w l y d e p r o t o n a t e d phosphate groups a n d the p r o t o n a t e d phosphate groups. C h a n g e s i n t h e m o l e c u l a r configuration a n d t h e c h e m i c a l shift w i l l c o n t i n u e u n t i l a l l o f t h e p h o s p h o r u s groups have lost a n e q u i v a l e n t n u m b e r o f protons so t h e m o l ecule c a n r e t u r n to t h e chair configuration. 3 I



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I—ι ι ι I ι ι ι—ι—ι—ι—ι—|—ι—ι—ι—ι—r—i—r— 8 6 4 £ 0 -2 PPM Figure 4. Effect of pH on the Ρ FT-NMR spectrum of inositol hexaphosphate in a concentrated humic-FeEDTA matrix. 31

Ionic Strength. T h e sample's i o n i c strength also influences the c h e m i c a l shift a n d signal pattern o f t h e N M R spectra. Increasing t h e N a C l c o n centration o f the solution causes t h e N M R signals to shift slightly d o w n f i e l d ( F i g u r e 6). D o w n f i e l d shifts of the signal are caused b y a decrease i n e l e c t r o n charge o f the phosphorus nucleus. I n t u r n , t h e decrease is p r o b a b l y caused b y t h e large n u m b e r o f positive cations c l u s t e r i n g a r o u n d t h e oxygen a t o m , a l l o w i n g it to contain a greater electron d e n s i t y . Increasing t h e N a C l c o n centration also affects the signal pattern o f c o m p o u n d s c o n t a i n i n g m u l t i p l e monoester phosphate groups. A t p H 9.8, I H P displays a P N M R s p e c t r u m 3 1


174



PO(OH)

2

Figure 5. Chair configuration of myo-inositol hexaphosphate, illustrating hydrogen bonding between phosphate groups 2 and 6. Hydrogen bonding between groups 3 and 5 is not shown for clarity. consisting of an intense singlet at a p p r o x i m a t e l y 4.1 p p m w i t h a large b r o a d s h o u l d e r u p f i e l d f r o m it ( F i g u r e 7). A s the N a C l concentration is increased, this b r o a d s h o u l d e r narrows, increasing i n intensity, u n t i l it b e c o m e s a peak. A t 1.00 M N a C l the s p e c t r u m displays three separate peaks w i t h an e m e r g i n g f o u r t h peak. T h e s e signal pattern changes result f r o m the c l u s t e r i n g of cations a r o u n d the oxygen atoms, protonated a n d d e p r o t o n a t e d . T h e electrostatic radius is decreased such that steric h i n d r a n c e b e t w e e n the phosphate groups is r e d u c e d a n d the I H P m o l e c u l e can b e g i n to r e t u r n to the chair c o n f i g u ration. Humic Matrix and T Relaxation Agent. T h e effects of the concentrated h u m i c matrix a n d the ferric relaxation agent o n the P N M R s p e c t r u m are s i m i l a r to that o f increasing i o n i c strength. W h e n orthophosphate is d i s s o l v e d i n a concentrated h u m i c matrix, its signal p o s i t i o n is shifted slightly d o w n f i e l d relative to a p u r e water matrix at an e q u i v a l e n t p H ( F i g u r e 8). T h e a d d i t i o n of F e E D T A to the concentrated h u m i c matrix causes this d o w n f i e l d shift to b e e v e n m o r e p r o n o u n c e d . T h e increase i n the d o w n f i e l d shift o f signals 2

3 1



6.

NANNY & MINEAR

l.OOOE-4


0.001

0.010

Hydrosphere

0.100

1.000

175

10.000

NaCl Molarity Figure 6. Effect of ionic strength on the chemical shift of inositol monophosphate (IMP), inositol hexaphosphate (IHP), choline phosphate, and DNA at various pH values. Key: 1, IMP, pH 9.9; 2, choline phosphate, pH 10.8; 3, IMP, pH 4.7; 4, IHP, pH 5.0; 5, DNA, pH 9.8; and 6, DNA, pH 4.4.

d u e to t h e presence o f concentrated h u m i c matrix o r t h e relaxation agent occurs w i t h monoester phosphates s u c h as c h o l i n e phosphate ( F i g u r e 9). T h e reason for this b e h a v i o r lies i n the i o n i c - s t r e n g t h - i n d u c e d d o w n f i e l d shifts. I n a concentrated h u m i c matrix, possible i n t r a m o l e c u l a r h y d r o g e n b o n d i n g occurs b e t w e e n h y d r o x y l a n d carboxylic groups a n d t h e phosphates. T h e ferric relaxation agent, F e E D T A , has b e e n p r o p o s e d b y E l g a v i s h a n d G r a n o t (48) to f o r m a p h o s p h a t e - F e - E D T A c o m p l e x . I f this is the case, there is little d o u b t that the phosphate oxygen atoms are i n v o l v e d i n c o m p l e x i n g w i t h F e E D T A , shifting electron d e n s i t y f r o m t h e p h o s p h o r u s n u cleus. Lake-Water Samples. T e n lake-water samples w e r e c o l l e c t e d f r o m S e p t e m b e r 1990 to M a y 1991. T h e total soluble p h o s p h o r u s c o n c e n t r a t i o n for the concentrated samples r a n g e d f r o m 23.8 to 60.8 m g o f P / L , a n d t h e soluble reactive phosphorus concentrations r a n g e d f r o m 1.0 to 18.1 m g of P / L (Table I). D i s s o l v e d organic carbon concentration values for t h e c o n centrated samples ranged f r o m 5000 to 20,000 m g o f C / L . T h e signal-tonoise ratios from 1 2 - 1 4 - h runs a c h i e v e d for t h e N M R spectra range f r o m 3.0 to 7.0. T h e p H of the concentrated samples after t h e a d d i t i o n o f F e E D T A fell b e t w e e n the values of 7.00 a n d 8.00. A d d i t i o n of the F e E D T A i n c r e a s e d the p H b y o n l y a f e w tenths o f a p H u n i t .



176


η—ι—ι—j—j—j—ι—ι—j—ι—ι—ι—j—r~T—ι—j—Î—

8

6

4

2

Figure 7. Effect of ionic strength on the Ρ FT-NMR hexaphosphate at pH 9.8. 31

0 ppm spectrum of inositol

C o m p a r i s o n o f the P N M R spectra reveals several similarities ( F i g u r e 10). A l l spectra contain p r o m i n e n t signals i n t h e monoester r e g i o n ( 0 . 0 0 5.00 p p m ) a n d less distinct signals b e t w e e n 0.00 a n d - 2 . 0 0 p p m , t h e diester phosphate r e g i o n . Phosphonates a n d polyphosphates w e r e not d e t e c t e d i n any samples. 3 1

A t y p i c a l spectral pattern o f D O P usually consists o f four basic c o m ponents: • a small broad envelope spanning from approximately 4 . 0 0 - 5 . 0 0 ppm. • an intense single peak i n t h e r e g i o n f r o m 2.50 to 4 . 0 0 p p m . • a large, intense, b r o a d e n v e l o p e s p a n n i n g 0 . 0 0 - 2 . 0 0 p p m ; often a p p e a r i n g to consist o f t w o o r m o r e o v e r l a p p i n g peaks. • several smaller, sometimes o v e r l a p p i n g , signals i n t h e r e g i o n f r o m - 2 . 0 0 to 0.00 p p m .


6.

NANNY & M I N E A R

·-·

177


Pure water

O-o FeEDTA/Conc. Humics


o-o Cone. Humics

10 11 12 13 14

Figure 8. Effect of pH on orthophosj>hate's chemical shift in various matrices. The sample concentration was 150 mg of P IL. Pure water data are taken from reference 46.

5

0-1 0

1 1

h —I 2 3 5

1 1 1 1 1 1 4 5 6 7 8 9

1 10

1 1 11 12

pH Figure 9. Effect of pH on choline phosphate's chemical shift in various matrices. The sample concentration was 150 mg of P/L.


178


Table I. Summary of Crystal Lake

3 1

P F T - N M R Spectra SRP

Sample A B C b

b d

D

R F

E

d

G

d

H Ρ

C


r

Date 9/5/90 9/12/90 9/26/90 10/24/90 11/9/90 1/16/91 2/11/91 3/11/91 4/3/91 5/2/91

S:N

a

3.2 3.0 3.8 2.4 2.6 5.6 4.6 4.0 3.7 4.9

pH c c c c

7.90 7.92 7.28 7.53 7.72 7.68

(mgP/L) 1.0 2.4 3.0 5.2 6.0 18.1 3.4 2.8 2.5 1.8

TSP

(mgP/L) 23.8 25.6 46.0 48.6 25.0 60.8 50.0 23.5 29.3 36.7

"Signal-to-noise ratio. 'The membrane used was 80% NaCl rejection reverse osmosis cel lulose acetate. No data were collected. The membrane used was 99% NaCl rejection reverse osmosis polyamide. T h e membrane used was 1000-dalton, tangential-flow polysulfone. c

rf

Studies o f several m o d e l monoester phosphate c o m p o u n d s (44), each o f w h i c h are p o t e n t i a l l y present i n lake water, w e r e p e r f o r m e d b y e x a m i n i n g signal p o s i t i o n , a n d signal pattern i f appropriate, o v e r a p H range to o b t a i n an i d e a o f w h i c h c o m p o u n d s c o u l d b e present i n the sample. A l l m o d e l c o m p o u n d s w e r e d i s s o l v e d i n a concentrated h u m i c matrix w i t h F e E D T A present. T h e s e studies indicate that the signal e n v e l o p e f r o m 4 . 0 0 to 5.00 p p m c o u l d result f r o m 3'-adenosine m o n o p h o s p h a t e , i n o s i t o l m o n o p h o s phate, serine phosphate, o r c h o l i n e phosphate (Figures 3 a n d 9) because P N M R signals for a l l o f these c o m p o u n d s appear i n this r e g i o n at p H 7. 3 1

T h e intense singlet that appears phosphate. C o r r e l a t i o n o f the sample p H - d e p e n d e n t chemical-shift c u r v e h u m i c matrix w i t h F e E D T A ( F i g u r e

b e t w e e n 2.50 a n d 4 . 0 0 p p m is o r t h o p H a n d signal p o s i t i o n w i t h that o f t h e o f orthophosphate i n a concentrated 8) confirms this peak's i d e n t i t y .

T h e i d e n t i t y o f t h e large b r o a d peak s p a n n i n g f r o m 0.00 to 2.00 p p m is an e n i g m a . P N M R spectra o b t a i n e d at p H 7 . 0 0 - 8 . 0 0 suggest that this e n v e l o p e c o u l d b e a t t r i b u t e d to i n o s i t o l hexaphosphate. H o w e v e r , spectra o b t a i n e d at o t h e r p H values ( w h i c h w i l l b e d e s c r i b e d i n d e t a i l later) show that this e n v e l o p e has drastically different p H b e h a v i o r t h a n i n o s i t o l hexa phosphate. G l y c e r o p h o s p h o r y l c h o l i n e a n d g l y c e r o p h o s p h o r y l ethanola m i n e , e v e n t h o u g h they are diester phosphates, have b e e n s h o w n b y o t h e r researchers (49) to p r o d u c e signals i n this r e g i o n at this p H . B o t h a r e d e g radation products o f structural components o f c e l l u l a r m e m b r a n e s (50) a n d thus w o u l d b e e x p e c t e d to b e fairly u b i q u i t o u s i n the aquatic e n v i r o n m e n t . B u t these species are susceptible to f u r t h e r h y d r o l y s i s a n d attack b y l i p o phosphatases, to y i e l d c h o l i n e phosphate, ethanolamine phosphate, a n d g l y c 3 1



6.

NANNY & MINEAR

179


I I ι ι » ; ι ι ι ι I ι ι ι ι I ι ι ι ι I ι ι ι ι I I 1 1 ι ; I I « » 15 tO S 0 - 5 -10 -IS -20

Ε

! I

n 15

11/9/90

10

ι S

0

-

5

-10

-15

F

-20

1/16/91

Figure 10. P FT-NMR spectra of Crystal Lake samples. Letters correspond to sample designations in Table I. (Continued on next page.) 31

e r o l phosphate. T h u s it w o u l d seem h i g h l y u n l i k e l y to detect large amounts of these g l y c e r o p h o s p h o r y l c o m p o u n d s . P F T - N M R spectra of soils a n d sediments (31-38), no signals are present b e t w e e n 1.50 a n d 0.00 p p m . T h e final r e g i o n f r o m - 2 . 0 0 to 0.00 p p m is a t t r i b u t e d to diester phos phates such as R N A , D N A , n u c l e o t i d e fragments, a n d p h o s p h a t i d y l c o m p o u n d s . Signals occur i n this r e g i o n for b o t h D N A a n d p h o s p h a t i d y l c h o l i n e i n a concentrated h u m i c matrix w i t h F e E D T A present ( F i g u r e 11). P F T N M R e v i d e n c e s u p p o r t i n g the presence of D N A is p r o v i d e d b y the s p e c t r u m of a sample that has b e e n o x i d i z e d b y alkaline b r o m i n a t i o n ( F i g u r e 12). 3 1

3 I



180

< ' 20

1

"


1 " 1S

• ' I " 10

"

5

I "

"

0

t '

1

» U ' ' " I ' " -3 -10

I

• I ' ' " -IS -20

< ' ' ' ' > ' ' ' ' I ' ' ' ' I ' ' ' * 1 ' ' ' ' I ' ' ' I ' ' ' > 20 IS 10 S O —S -10 -IS 1

4/3/91 Figure 10.

1

J

-20

5/2/91

Continued.

A l k a l i n e b r o m i n a t i o n oxidizes organic p h o s p h o r u s , except for I H P (51) a n d D N A (52), to orthophosphate. A f t e r alkaline b r o m i n a t i o n , t h r e e d i s t i n c t signals are present. T h e signal at 6.5 p p m is p r e s u m a b l y caused b y o r t h o phosphate, e v e n t h o u g h at the sample p H of 10.3 orthophosphate w o u l d b e expected to appear b e t w e e n 3.0 a n d 4.0 p p m . T h e s e c o n d signal at 1.5 p p m is p r o b a b l y r e m a i n i n g u n o x i d i z e d D O P that originally gave rise to the large signal e n v e l o p e . T h e t h i r d signal, i n the diester phosphate r e g i o n , is at - 1 . 5 p p m a n d is p r o b a b l y from D N A .

pH Studies. I n f u r t h e r attempts to characterize a n d b e t t e r d e f i n e the structure of the signal envelopes, the p H of the samples o b t a i n e d i n A p r i l a n d M a y was v a r i e d a n d P N M R spectra w e r e o b t a i n e d ( F i g u r e 13). T h e p H b e h a v i o r was s i m i l a r for b o t h samples. T h e 4 . 0 0 - 5 . 0 0 - p p m e n v e l o p e b e h a v e d as a t y p i c a l monoester phosphate; at p H > 7 it was present b e t w e e n 4.00 a n d 5.00 p p m , b u t at l o w e r p H values it m o v e d u p f i e l d a n d m e r g e d into the large 0 . 0 0 - 2 . 0 0 - p p m e n v e l o p e . T h e large singlet, w h i c h appears b e t w e e n 4.00 a n d 2.50 p p m a n d is a t t r i b u t e d to orthophosphate, was not d e t e c t e d i n these samples. 3 1


6.

NANNY & MINEAR


181

Hydrosphere

• - · DNA in pure water matrix • - A Phosphatidyl choline in cone, humic / FeEDTA


•A

6

7

8

— •

10

12

11

PH Figure 11. Effect of pH on the chemical-shift position of DNA and phosphatidyl choline. DNA is in a pure water matrix and phosphatidyl choline is in a concentrated humic-FeEDTA matrix. y

T h e p o s i t i o n of the large, b r o a d e n v e l o p e b e t w e e n 0.00 a n d 2.00 p p m is o n l y slightly affected b y p H changes. T h i s b e h a v i o r indicates that this signal e n v e l o p e is not caused b y I H P , for i f I H P w e r e present t w o large singlets w o u l d appear at 2.3 a n d 3.0 p p m at p H 9, a n d at p H 10 the t w o peaks w o u l d be shifted to 4.2 a n d 3.2 p p m ( F i g u r e 4). N o n e of these peaks appear w h e n the sample p H is increased; i n fact, no signals are v i s i b l e i n the r e g i o n b e t w e e n 2.00 a n d 4.00 p p m . W h e n the p H is decreased to 4, the t y p i c a l four-peak pattern of inositol hexaphosphate (rather t h a n the b r o a d envelope) w o u l d be expected f r o m the P F T - N M R spectra of I H P s p i k e d into a concentrated h u m i c matrix w i t h F e E D T A present. O n the basis of the sample's l o w - p H data ( p H

Organic Phosphorus in the Hydrosphere - Advances in Chemistry

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