Quantitative Analysis of Inorganic Phosphates Using 31P

Chapter 3 Quantitative Analysis of Inorganic Phosphates Using P NMR Spectroscopy 31

1

Janice K Gard, David R. Gard, and Clayton F. Callis

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Phosphorus-31 nuclear magnetic resonance ( P NMR) is optimized with respect to accuracy, precision, and analysis time for the characterization of inorganic phosphates. Species determinations in commercial sodium tripolyphosphate are routinely achieved with an accuracy and precision (0.1-0.5%) comparable to that obtained by chromatographic methods as determined in interlaboratory analyses. The method has been completely automated using a robotic sample changer and an algorithm for data analysis. In a demonstration of the precision attainable, the hydrolysis kinetics of tripolyphosphate is obtained with a corre lation coefficient of 0.998. Extension of P NMR to the analysis of higher oligophosphate mixtures (i.e. sodium phosphate glass) has recently been examined using homonuclear 2DJ -resolved spectroscopy to separate the coupling constant and chemical shift information. Semi -quantitative analyses are achieved using curve deconvolution. 31

31

The ubiquitous nature and broad importance of phosphates demands exacting analytical methods for their charac terization. Phosphorus-31 nuclear magnetic resonance ( P NMR) has been used as a method for the quantitative analysis of small inorganic phosphates (1-4). Several potential advantages are offered by P NMR including observation of only the phosphorus-containing species, structural information which may complement or aid Retired 3l

31

1

0097H5156/92/0486-0041$06.00/0 © 1992 American Chemical Society In Phosphorus Chemistry; Walsh, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

PHOSPHORUS CHEMISTRY

42

q u a n t i t a t i o n , and q u a n t i t a t i o n w i t h an e l e m e n t a l a s o p p o s e d t o a m o l e c u l a r s t a n d a r d . L i m i t a t i o n s o f p h o s p h o r u s - 3 1 NMR include sensitivity, complexity of spectra for oligophosphates higher than tripolyphosphate, and, o c c a s i o n a l l y , slow r e l a x a t i o n times. L i t t l e appears i n the l i t e r a t u r e , however, c o n c e r n i n g t h e p r e c i s i o n o r a c c u r a c y of p h o s p h a t e a n a l y s i s by P NMR. Recent advances i n instrumentation have revolutionized NMR spectroscopy, p a r t i c u l a r l y w i t h r e s p e c t t o s e n s i t i v i t y and r e s o l u t i o n , and h a s p r o m p t e d t h i s r e e x a m i n a t i o n o f t h e q u a n t i t a t i v e c a p a b i l i t i e s of P NMR. 31


31

F u r t h e r e n h a n c e m e n t s o f t h e NMR method h a v e b e e n carried out in our laboratory by optimization of experimental parameters with respect to accuracy, precision, and analysis time u s i n g commercial sodium tripolyphosphate (STP). The NMR technique was then directly compared with chromatographic methods in a c o n t r o l l e d i n t e r l a b o r a t o r y study w i t h t h e use o f L o r e n t z i a n l i n e s h a p e a n a l y s i s t o improve p r e c i s i o n y e t f u r t h e r . The a c c u r a c y and p r e c i s i o n o f t h e NMR method i s i l l u s t r a t e d by the high value of the correlation coefficients in monitoring the kinetics of hydrolysis of sodium tripolyphosphate. With the i n s t a l l a t i o n of a r o b o t i c sample changer and customized robotic software the efficiency of NMR quantitation i s greatly enhanced. Demonstration of the complete automation of NMR quantitative analysis i s herein described, including f u l l d a t a r e d u c t i o n and g e n e r a t i o n o f t h e a n a l y t i c a l r e p o r t . E f f o r t s a r e c u r r e n t l y underway t o e x p a n d t h e u t i l i t y o f P NMR t o q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s i s o f much more c o m p l e x o l i g o p h o s p h a t e m i x t u r e s . A n o v e l a p p l i c a t i o n of homonuclear two-dimensional J-resolved (2DJ) s p e c t r o s c o p y o f sodium polyphosphate g l a s s i s shown t o effectively yield p- p decoupled spectra. Used in c o n j u n c t i o n w i t h L o r e n t z i a n l i n e s h a p e a n a l y s i s and c u r v e deconvolution, semiquantitative analyses of these mixtures has been a c h i e v e d . 3l

31

3l

A n a l y t i c a l Methods One-Diaensional Q u a n t i t a t i v e "P NMR Spectroscopy. S p e c t r a were c o l l e c t e d o n a u t o m a t e d V a r i a n XL-200 o r VXR300S Fourier transform NMR spectrometers, operating at p h o s p h o r u s f r e q u e n c i e s o f 80.98 and 121.42 MHz. The p h o s p h a t e s were t y p i c a l l y p r e p a r e d a s 2-5 w e i g h t p e r c e n t i n D 0 w i t h t h e pH m a i n t a i n e d n e a r 9 i n o r d e r t o o p t i m i z e t h e s i g n a l s e p a r a t i o n and t h e l o n g i t u d i n a l r e l a x a t i o n t i m e s (5,6). T v a l u e s were d e t e r m i n e d u s i n g t h e F a s t I n v e r s i o n Recovery Fourier Transform (FIRFT) method (7) and a c q u i s i t i o n p a r a m e t e r s were o p t i m i z e d t o m a x i m i z e the observable magnetization with r e s p e c t t o a n a l y s i s time ( 8 ) . S p e c t r a were a c c u m u l a t e d u s i n g a 20-25 d e g r e e p u l s e w i d t h , 0.50 sec a c q u i s i t i o n time, a 3 t o 5 second repetition 2

1

In Phosphorus Chemistry; Walsh, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

3.

GARD ET AL

Quantitative Analysis of Inorganic Phosphates

43

d e l a y , g a t e d W a l t z d e c o u p l i n g , c o l l e c t i o n o f 16K data p o i n t s p r i o r t o z e r o - f i l l i n g t o 32K p o i n t s , and a p p l i c a t i o n of a 1.0 Hz exponential line broadening. In the i n t e r l a b o r a t o r y a n a l y s e s 1024 t r a n s i e n t s were g e n e r a l l y c o l l e c t e d r e q u i r i n g about 1 h r . of t o t a l scan time. A spectrum o f commercial sodium t r i p o l y p h o s p h a t e a c q u i r e d u n d e r t h e s e c o n d i t i o n s a t 80.98 MHz i s shown i n F i g u r e 1. N o n - l i n e a r l e a s t s q u a r e s L o r e n z i a n l i n e s h a p e a n a l y s i s was a l s o e x a m i n e d f o r c u r v e f i t t i n g and i n t e g r a t i o n s i n t h e interlaboratory analysis (9).


3l

R o b o t i c P NMR Assays. S p e c t r a were c o l l e c t e d on a V a r i a n VXR300S s p e c t r o m e t e r equipped with a Varian automatic s a m p l e management s y s t e m ( r o b o t ) w i t h a 50 s a m p l e t r a y u s i n g t h e parameters d e s c r i b e d above. Customized s o f t w a r e was d e v e l o p e d f o r r o b o t c o n t r o l p r o v i d i n g f u l l a u t o m a t e d work-up o f data including the calculation of weight p e r c e n t s and g e n e r a t i o n o f a n a l y t i c a l r e p o r t s . 31

Two-Dimensional S e m i q u a n t i t a t i v e P NMR. S p e c t r a were c o l l e c t e d on a V a r i a n U n i t y 400 F o u r i e r t r a n s f o r m NMR s p e c t r o m e t e r a t 161.90 MHz. The p h o s p h a t e s were p r e p a r e d a t 3-5 w e i g h t p e r c e n t i n w a t e r and a D 0 i n s e r t u s e d f o r l o c k i n g purposes. H o m o n u c l e a r 2 D J - r e s o l v e d s p e c t r a were a c c u m u l a t e d u s i n g an 8K Χ 0.2K d a t a s e t w i t h an a c q u i s i t i o n t i m e i n t h e F d i m e n s i o n o f 0.946 s e c , f o u r s t e a d y s t a t e pulses, 128 transients, and 200 increments i n the F domain. S p e c t r a were a n a l y z e d w i t h z e r o - f i l l i n g t o 16K X 0.5K and a p p l i c a t i o n o f a s i n e b e l l o r s h i f t e d s i n e b e l l w e i g h t i n g f u n c t i o n on a Sun M i c r o s y s t e m s S p a r c 1+ c o m p u t e r . 2

2

A

Chromatography. Separations by ion exchange column c h r o m a t o g r a p h y were p e r f o r m e d a c c o r d i n g t o ASTM methods (10,11). The T e c h n i c o n A u t o A n a l y z e r ( B r a n and L u e b b e ) (12,13) was e m p l o y e d f o r a n a l y s i s o f t h e e l u e n t f r o m ASTM method D 2761 ( 1 1 - 1 3 ) . ASTM method D 501 was f o l l o w e d a s w r i t t e n , u s i n g t h e ammonium m o l y b d a t e c o l o r i m e t r i c a n a l y s i s of the h y d r o l y z e d f r a c t i o n s .

Interlaboratory Tripolyphosphate

Analyses

of

Commercial

Sodium

An interlaboratory s t u d y was conducted to assess the capabilities of standard ion-exchange chromatographic methods and NMR a t 80.9 MHz f o r t h e a n a l y s i s o f sodium t r i p o l y p h o s p h a t e . Three well-mixed, but s e p a r a t e , samples o f c o m m e r c i a l s o d i u m t r i p o l y p h o s p h a t e were s u b m i t t e d t o each l a b o r a t o r y i n t r i p l i c a t e , r e p r e s e n t i n g t h r e e d i f f e r e n t levels of tripolyphosphate. P r o t o c o l was maintained c o n s i s t e n t among m e t h o d s / s i t e s . The r e s u l t s c o m p a r i n g t h e methods a r e p r e s e n t e d i n T a b l e I ( G a r d , D.R.; B u r q u i n , J . C . ; G a r d , J.K.; submitted for publication.). The A u t o A n a l y z e r and t h e NMR analyses were e a c h p e r f o r m e d by two d i f f e r e n t a n a l y s t s ; r e s u l t s by



32

7

p o -

L

Ρ,Ο '3»9

3-

4

2

0

-4

-6

-8

-10

-12

-14

-16

F i g u r e 1. A sample phosphorus-31 NMR spectrum ( a t 80.98 Hz) of a phosphate mixture c o n t a i n i n g p r i m a r i l y sodium t r i p o l y p h o s p h a t e .

-2

-18

-20

ppm

• • • • ( ( • • • l > i > ( | i i i i l i i i i | i i i i | i i i i | i i i i | i i i > | i i i i | i i i i | i i i i | i i i i | i i i i | i i i T | i r T i | i i i i | i i i i ( i i i i | i r r i | r T T i j T i i i | i r T i | i i i i | T i i i | i i T i f r i i r j i i i i ] i

ΡΟ4

Réf.

Ρ, ο 10


d O S w

O

W

o

3

3. GARD ET A L

Quantitative Analysis ofInorganic Phosphates

each a n a l y s t a r e l i s t e d s e p a r a t e l y i n Table s h a p e a n a l y s i s a n d c u r v e f i t t i n g r o u t i n e was one s e t o f NMR d a t a (row 4 o f T a b l e I ) ; t h e s e p r e s e n t e d i n row 6 o f T a b l e I . IECC d e s i g n a t e s D 501.

45

I . The l i n e employed on results are ASTM method

Table I I n t e r l a b o r a t o r y Assay


% Total

o f Sodium

Tripolyphosphate

P A as-

M e a n / S t a n d a r d D e v i a t i o n o f 9 Samples TripolyPyroOrthophosphate phosphate phosphate

Method AutoAn. AutoAn. IECC P NMR* P NMR* P NMR** 31

31

X

0.31/0.09 0.25/0.11 0.55/0.16 0.35/0.11 0.36/0.15 0.51/0.28

7.36/0.73 6.60/0.38 7.66/0.17 7.35/0.48 7.20/0.56 6.68/0.05

92.19/0.78 92.84/0.34 91.03/0.23 91.86/0.51 92.09/0.61 92.33/0.35

Trimetaphosphate 0.14/0.12 0.30/0.03 0.73/0.10 0.44/0.14 0.37/0.18 0.51/0.17

* Separate i n t e g r a t i o n o f t h e t r i p o l y p h o s p h a t e d o u b l e t and the pyrophosphate s i n g l e t ** U s i n g

line

shape a n a l y s i s and c u r v e

fitting

T w o - t a i l e d t e s t s o f t h e n u l l hypothesis a r e used t o i d e n t i f y s i g n i f i c a n t d i f f e r e n c e s among t h e methods ( 1 4 ) . The I E C d e t e r m i n a t i o n s a r e c o n s i s t e n t l y t o o n e e n d o f t h e range o f r e s u l t s . The d e t e r m i n a t i o n o f t r i p o l y p h o s p h a t e by I E C i s l o w e r t h a n f o r t h e o t h e r methods w h i l e t h a t f o r the other species i s higher. H y d r o l y t i c degradation during the IEC a n a l y s i s would account f o r t h i s d i f f e r e n c e s i n c e tripolyphosphate i s t h e s p e c i e s most susceptible t o h y d r o l y s i s . T h i s hypothesis i s independently supported by t h e f a c t t h a t l i t t l e o r no orthophosphate (ahydrolysis p r o d u c t ) c o u l d b e d e t e c t e d b y NMR i n s e p a r a t e a n a l y s e s o f t e t r a s o d i u m pyrophosphate (£0.1%, d e t e c t i o n l i m i t -0.01%), w h i l e s i g n i f i c a n t c o n c e n t r a t i o n s (0.5%) were o b s e r v e d b y I E C . D i f f e r e n c e s i n t h e a c c u r a c y between d i r e c t a b s o r b a n c e measurements f o r ASTM D 501 v s p e a k i n t e g r a t i o n f o r t h e A u t o A n a l y z e r may a l s o b e i m p o r t a n t . S i g n i f i c a n t d i f f e r e n c e s i n the AutoAnalyzer results are -1% d i f f e r e n c e i n t h e d e t e r m i n a t i o n o f t h e major s p e c i e s between t h e two s e t s o f d a t a . This i s mainly t h o u g h t t o r e f l e c t e r r o r i n peak i n t e g r a t i o n ; a l t h o u g h t h e means o f t h e A u t o A n a l y z e r d é t e r m i n a t i o n s f o r p y r o - a n d tripolyphosphate are closer i nthe f i r s t interlaboratory analysis, the standard d e v i a t i o n i s larger. On t h e o t h e r



46 31

h a n d , t h e two s e t s o f r e s u l t s f r o m P NMR using simple e l e c t r o n i c i n t e g r a t i o n a r e i n c l o s e agreement w i t h each other, e s p e c i a l l y c o n s i d e r i n g d i f f e r e n t a n a l y s t s performed t h e a n a l y s e s . R e s u l t s b y P NMR a g r e e w e l l w i t h one s e t o f the AutoAnalyzer r e s u l t s , the only s i g n i f i c a n t d i f f e r e n c e being i n the determination of trimetaphosphate. 31

The p r e c i s i o n f o r the determination of the major p h o s p h a t e s p e c i e s was s i g n i f i c a n t l y increased with the a p p l i c a t i o n o f l i n e s h a p e a n a l y s i s . The t r i p o l y p h o s p h a t e c o n t r i b u t i o n i s , however, w e i g h t e d more a t t h e e x p e n s e o f p y r o p h o s p h a t e u s i n g l i n e s h a p e a n a l y s i s and c u r v e f i t t i n g f o r t h e P NMR, c o m p a r e d t o manual i n t e g r a t i o n s . More accurate determinations are achieved s i n c e curve fitting b e t t e r a c c o u n t s f o r s i g n a l o v e r l a p , b a s e l i n e n o i s e , and contribution of the wings of the Lorentzian peaks, e s p e c i a l l y f o r the lower c o n c e n t r a t i o n s p e c i e s (15,16).


31

A p p l i c a t i o n o f "P

NMR

to Kinetics 31

The h i g h p r e c i s i o n a n d a c c u r a c y o f P NMR f o r t h e a n a l y s i s of sodium t r i p o l y p h o s p h a t e i n d i c a t e d t h e t e c h n i q u e might a l s o be s u i t a b l e f o r a p p l i c a t i o n t o p r e c i s e k i n e t i c s t u d i e s involving oligophosphates. The h y d r o l y t i c d e g r a d a t i o n o f 0.035 M sodium t r i p o l y p h o s p h a t e was examined in the p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f Me NCl a t 60.3°C. The h y d r o l y s i s was f o l l o w e d o v e r a p p r o x i m a t e l y two h a l f - l i v e s . Even w i t h l a r g e changes i n t h e r e l a t i v e c o n c e n t r a t i o n s o f phosphate s p e c i e s over time, e x c e l l e n t c o r r e l a t i o n f o r p s e u d o - f i r s t order k i n e t i c p l o t s are obtained allowing d i s t i n c t i o n s t o be made among t h e r a t e s u n d e r d i f f e r e n t Me NCl c o n c e n t r a t i o n s ( F i g u r e 2 ) . The h i g h c o r r e l a t i o n f o r t h e s e p l o t s i l l u s t r a t e s t h e combined h i g h a c c u r a c y and p r e c i s i o n o f t h e NMR method ( T a b l e I I ) . Values f o r the r a t e c o n s t a n t s a r e c o n s i s t e n t w i t h t h o s e o f Watanabe e t a l (17) and C r o w t h e r a n d Westman ( 1 8 ) . 4

4

Table II H y d r o l y s i s o f Sodium T r i p o l y p h o s p h a t e w i t h T e t r a m e t h y l a m m o n i u m C h l o r i d e a s F o l l o w e d by (35mM STP,

Me.NCI 0 0.43 1.38

(M}

10

3

k

pH

9.0,

fhr-M

7.56 7.29 6.83

Added Ρ NMR

60.3°C) Correlation coeff. (R) 0.998 0.998 0.998




3. GARD ETAL

47

In ( [STP]o / [STP]t ) 1.6 3

4

1.2

+ χ

1

M MeNCI k(10 )hr 7.56 0.00 0.43 7.29 1.38 6.83

R 0.998 0.998 0.998

^

^ ^ ^ ^ ^ ^ ^ ^

0.8

0.4

I

ι

600

1000 Hours

l—

1500

2000

F i g u r e 2. P s e u d o - f i r s t order k i n e t i c s p l o t o f t h e h y d r o l y s i s o f 35 mM sodium t r i p o l y p h o s p h a t e (STP) as followed by P NMR (pH 9.0, 60.3°C) i n the presence o f v a r i o u s concentrations o f Me NCl. Me NCl concen t r a t i o n s , observed r a t e constants, and c o r r e l a t i o n c o e f f i c i e n t s (R) are given i n the i n s e t . 31

4

4

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

In Phosphorus Chemistry; Walsh, E., et al.; Washington, D.C. ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


48 Robotic

31

P NMR

A s s a y s o f Sodium T r i p o l y p h o s p h a t e


31

A l t h o u g h P NMR y i e l d s r a p i d , r e p r o d u c i b l e q u a n t i f i c a t i o n of o l i g o p h o s p h a t e s , q u a n t i t a t i v e s p e c t r a r e q u i r e l o n g e r accumulations than simple survey spectra. Manual d a t a r e d u c t i o n o f t h e s p e c t r a i s a s u b j e c t i v e p r o c e s s , and c a n be t i m e - c o n s u m i n g , especially when l a r g e numbers o f samples a r e i n v o l v e d . In o r d e r t o improve spectrometer e f f i c i e n c y , t h e p h o s p h a t e a s s a y s a r e now b e i n g p e r f o r m e d on a VXR300S e q u i p p e d w i t h a r o b o t i c s a m p l e changer. Utilizing easy-to-use menu-driven software, laboratory p e r s o n n e l d e s i g n a t e t h e p r e f e r r e d s o l v e n t , d u r a t i o n and t y p e o f shimming ( m a g n e t i c f i e l d o p t i m i z a t i o n ) , a u t o m a t i o n o p e r a t i n g p a r a m e t e r s e t ( e n v i r o n m e n t ) , and p l o t t e r c h o i c e . Initiation of the automation r u n i s s i m p l y begun by c l i c k i n g a s i n g l e menu b u t t o n . The c u s t o m i z e d s o f t w a r e then a u t o m a t i c a l l y accumulates the data, s t o r e s i t i n a common a r e a f o r r o b o t i c r u n s , a n d p e r f o r m s t h e F o u r i e r t r a n s f o r m a n d i n t e g r a t i o n , and c a l c u l a t e s t h e r e l a t i v e d i s t r i b u t i o n o f phosphate s p e c i e s . S p e c t r a comparable t o that i n Figure 1 are generated without f u r t h e r operator i n t e r v e n t i o n . Improvements i n t h e s e n s i t i v i t y a r e o b s e r v e d a t t h e h i g h e r f i e l d (121.4 MHz) o f t h e VXR300S, r e s u l t i n g i n shorter o v e r a l l accumulations. The d a t a r e d u c t i o n s o f t w a r e F o u r i e r t r a n s f o r m s and phases t h e d a t a a f t e r z e r o - f i l l i n g and a p p l i c a t i o n o f t h e d e s i g n a t e d e x p o n e n t i a l f i l t e r f u n c t i o n . The u s u a l c h e m i c a l s h i f t range o f t h e peaks i s noted, as t h e system s e a r c h e s for the primary phosphorus-containing species. A signalt o - n o i s e check i s p e r f o r m e d o v e r t h a t r e g i o n , and peaks g e n e r a t i n g a v a l u e l e s s than t h r e e a r e d e s i g n a t e d as not b e i n g p r e s e n t ( o r a p p r o x i m a t e l y 0.2 w e i g h t p e r c e n t , t h e l o w e r c o n c e n t r a t i o n l i m i t f o r t h e r o u t i n e P a s s a y ) . Once the peaks are found, the spectrum i s automatically integrated. The i n t e g r a l v a l u e s a n d m o l e c u l a r w e i g h t o f each of the s p e c i e s are then used i n the computation of the r e l a t i v e weight percent d i s t r i b u t i o n of the phosphate species. The c o m p u t e r - d e r i v e d d a t a r e d u c t i o n c o m p a r e s q u i t e f a v o r a b l y t o t h a t p e r f o r m e d by human a n a l y s t s a s s e e n in Table I I I . The two methods commonly d i f f e r b y a r e l a t i v e e r r o r o f l e s s t h a n 0.2 w e i g h t p e r c e n t f o r t h e major s p e c i e s . The mean f o r e a c h s p e c i e s i s shown t o i l l u s t r a t e t h e l a c k o f b i a s b e t w e e n t h e two t e c h n i q u e s . The m a i n a d v a n t a g e s o f t h e c o m p u t e r method a r e i t s s p e e d and lack of subjectivity. The automated quantitative analyses are normally performed a t n i g h t , a l l o w i n g access t o t h e s p e c t r o m e t e r d u r i n g t h e d a y , when demand f o r t h e instrument i s high. 31


3. GARD ET A L

49


Table I I I M a n u a l v s . R o b o t i c (ASM) D a t a R e d u c t i o n C o m p a r i s o n o f STP A s s a y s R e l a t i v e Wt % a s Sodium


Sample No,

Orthophosphate manual ASM

A Β C D Ε F Mean

Pyrophosphate manual ASM

Salts

TripolyptlQSPhate manual ASM

TrimetaphQSPhate manual ASM

0.35 0.17 0.24 0.11 0.14 0.23

0.39 0.15 0.21 0.08 0.12 0.18

8.63 6.62 4.29 6.82 6.31 5.41

8.62 6.61 4.35 6.72 6.16 5.36

89.39 90.99 93.56 91.05 91.60 92.76

89.48 91.18 93.68 91.31 91.84 93.01

1.63 2.22 1.91 2.03 1.95 1.60

1.51 2.05 1.76 1.90 1.87 1.45

0.21

0.19

6.35

6.30

91.56

91.75

1.89

1.76

Hcmonuclear Two-Dimensional

J-Resolved Spectroscopy

Due t o t h e c o m p l e x i t y o f t h e o n e - d i m e n s i o n a l s p e c t r a o f o l i g o p h o s p h a t e g l a s s e s , s p e c i e s d e t e r m i n a t i o n by c u r v e f i t t i n g has been s u c c e s s f u l l y a p p l i e d o n l y t o r e l a t i v e l y pure samples o f i n d i v i d u a l o l i g o p h o s p h a t e s p e c i e s ( 1 9 ) . In an e f f o r t t o r e s o l v e t h e o v e r l a p p i n g m u l t i p l e t s i n c o m p l e x s p e c t r a , t w o - d i m e n s i o n a l NMR i s b e i n g e x a m i n e d . A o n e - d i m e n s i o n a l P spectrum o f a sodium phosphate g l a s s w i t h a n a v e r a g e c h a i n l e n g t h _ o f 4.11 i s shown i n F i g u r e 3. (The a v e r a g e c h a i n l e n g t h , n, i s d e t e r m i n e d b y e l e m e n t a l analysis.) A s c a n be o b s e r v e d , a l a r g e number o f c l o s e l y s p a c e d r e s o n a n c e s a r e o b t a i n e d . An e x p a n s i o n o f t h e " e n d s " r e g i o n i s a l s o s e e n i n t h e i n s e t o f F i g u r e 3. Although some o f t h e p e a k s a r e s e p a r a t e d a t h i g h f i e l d , a s s i g n m e n t s , and t h e r e f o r e q u a n t i t a t i o n , o f e a c h o f t h e v a r y i n g c h a i n l e n g t h r e s o n a n c e s , i s c o m p l i c a t e d by t h e p r e s e n c e o f PP c o u p l i n g . Removing t h i s c o u p l i n g w o u l d g r e a t l y s i m p l i f y the spectrum. S i m p l e h o m o n u c l e a r d e c o u p l i n g w o u l d be i n s u f f i c i e n t , as t h e a b i l i t y t o simultaneously observe a l l p h o s p h o r u s - c o n t a i n i n g s p e c i e s would t h e n be l o s t . 31

31

3l

31

P h o m o n u c l e a r 2DJ e x p e r i m e n t s were p e r f o r m e d o n t h e g l a s s t o e f f e c t i v e l y d e c o u p l e t h e s p e c t r u m . A 2DJ s p e c t r u m o f t h e p h o s p h a t e e n d s r e g i o n i s shown i n F i g u r e 4. The e n d s r e g i o n was u s e d f o r t h e p h o s p h a t e s p e c i e s d e t e r m i n a t i o n because o f i t s r e l a t i v e s i m p l i c i t y and f i r s t o r d e r nature i n comparison w i t h t h e middles r e g i o n . The p h o s p h o r u s c h e m i c a l s h i f t s a r e s e e n on one a x i s , a n d t h e P- P c o u p l i n g c o n s t a n t s on t h e o t h e r . P a i r s ( o r more) o f c o n t o u r s a t one c h e m i c a l s h i f t a r e t h e p e a k s f o r one ( c h a i n l e n g t h ) s p e c i e s , s e p a r a t e d by t h e c o u p l i n g c o n s t a n t . A v e r t i c a l p r o j e c t i o n o f t h e spectrum onto t h e chemical s h i f t a x i s c l e a r l y d e l i n e a t e s the newly-separated resonances, 31

31



"ends"

ppm

"middles"

-6.5

4

2

0

31

-E -4 -6 -8 -10 -12 -14 -16 -18 F i g u r e 3. B a s e s p e c t r u m - 161.9 MHz P NMR s p e c t r u m o f s o d i u m p h o s p h a t e g l a s s w i t h n= 4.1. Inset- Expansion o f t h e "ends" r e g i o n . Each s p e c i e s i s d e s i g n a t e d by t h e number o f p h o s p h o r u s atoms i n t h e m o l e c u l e .

-88

I I 1 1 I 1 I I I I I I I 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Γ I I I I I I I I I I 1 I I I I' I I I

-22

ppm

111 111 111 1111 111 111 111 1111 11 II 11 II 11 111 111 1111 111 111 111 1111 111 111 1111 111 111 111 II 11 111 111 1111 μ ι 111 1111 111 111 1111 M I l'| ι 111 11 M 111 111 1111 111 111 11

ortho


1

S

Ο

S

ο

3. GARD ET A L


51


r e s u l t i n g i n a phosphorus-phosphorus decoupled spectrum as shown a t t h e t o p o f F i g u r e 4. A monotonie i n c r e a s e i n chemical s h i f t i s seen with i n c r e a s i n g phosphorus c h a i n length species(20). I n an e f f o r t t o q u a n t i f y t h e amounts o f t h e v a r i o u s c h a i n l e n g t h s , t h i s p r o j e c t i o n was s u b j e c t e d t o s p e c t r a l curve-fitting. I n F i g u r e 5 i s seen t h e r e s u l t s o f t h i s a n a l y s i s , w i t h t h e e x p e r i m e n t a l spectrum a t t h e bottom, and t h e c o m p u t e r - g e n e r a t e d f i t above i t . The d e t e r m i n a t i o n o f t h e phosphate s p e c i e s by i n t e g r a t i o n o f t h e s i g n a l a r e a s o f t h e f i t t e d spectrum i s l i s t e d i n T a b l e I V . The average c h a i n l e n g t h d e t e r m i n e d b y 2DJ NMR i s 4.13, w e l l w i t h i n experimental error o f t h e 4.11 v a l u e determined by elemental a n a l y s i s . A c o m p a r i s o n i s a l s o made i n T a b l e I V o f t h e 2DJ r e s u l t s w i t h t h a t p r e v i o u s l y f o u n d f o r a s o d i u m p h o s p h a t e g l a s s d e t e r m i n e d b y p a p e r c h r o m a t o g r a p h y t o be n= 4.0 ( 2 1 ) . T h e r e s u l t s a r e c u r r e n t l y c o n s i d e r e d t o be s e m i - q u a n t i t a t i v e , a s t h e 2DJ e x p e r i m e n t c a n g i v e r i s e t o a r t i f a c t s ; t r u e i n t e n s i t i e s d o n o t a l w a y s r e s u l t when p r o j e c t i o n s o n t o t h e c h e m i c a l s h i f t a x i s a r e made b e c a u s e of poor l i n e s h a p e s . More a c c u r a t e q u a n t i f i c a t i o n c o u l d p o s s i b l y be a c h i e v e d by u s i n g volume i n t e g r a l s o r by u t i l i z i n g t h e methods o f Xu e t a l ( 2 2 ) t o h e l p e l i m i n a t e these shortcomings.

Composition

T a b l e IV o f Sodium P h o s p h a t e

% Total

Length,

* Ref.

5

Ρ NMR

1.2 9.4 13.8 24.9 17.5 19.7 7.8 2.8 2.9

Glass = 4.0*

H>

0 6.6 28.2 27.4 16.9 9.4 5.7 2.7 1.8 1.2

η =

Elemental A n a l y s i s 2DJ P NMR 31

2

31·

Ortho Pyro Tripoly Tetrapoly Pentapoly Hexapoly Heptapoly Octapoly Nonapoly Higher Average Chain

P 0 as2DJ

Phosphate Species

Glass

4.11 4.13

21



52


9 765

Fl CHz)-

4

3

2

*

-15-J -10

J

-5"

0

•

T

0

A 9

5^

ο

15-

20" TlllllII II II 11 II 111111IIH11 ITlf II 1II1111 II 1IIIΜ Γ

1

-4.S

-4.6

-5.β F2

31

-5.4

-5.8

-6.8

(ppm)

31

F i g u r e 4. p- p homonuclear 2DJ-resolved NMR spectrum of the same g l a s s whose one-dimensional spectrum i s shown i n F i g u r e 3. The c h a r a c t e r i s t i c J c o u p l i n g p a t t e r n i s observed f o r each s p e c i e s . A projection onto the chemical s h i f t a x i s i s shown, above. The p r o j e c t i o n f o r each s p e c i e s i s designated by the number of phosphorus atoms i n the molecule. The l i n e s h a p e of the octaphosphate was not d i s t i n c t enough t o allow f o r independent f i t t i n g .


GARD ET AL

53


id φ

Η Η ο ο ο ο ο ο ο ο ο ο ο ο ι+++ + + Η Η U Η Η Η ιο ο η η η νο 00 as Η οο η ιη «Η νο σ* νο r* Φ I Λ O 00 CM CM ^ m ΓΟ •M u

Si


······ "ί Ν

^

χ:

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Ο

Η

Η

VO Η

^

ιη

ιη

CM * Μ

•H (U w e Ί! ta Φ c α «χ U

Φ 4J Γ-

xi *

ο Φ in in CM ο ο as σ\ as CM CM η η νο ιη

· φ Ο ο ο ο ο ο ο

Φ

αι te β J

•Η

cS »ο φ

Φ

χ

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Τ-ϊ

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r>

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0 σ\ r-t ·

Οι Ο ·Η

ιη

^

ΓΜ .

«Μ

ο

se CM Û4 ·

(Ν rH u I

Ο -Ρ

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WW

(0 •Ρ (0

C CM

1

Ο

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γΗ

Ο

2

VO

C I

o ^ o v o n o miovoeo^n

I I I I I I

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ο •Η

α>


ιη

ο • Η ··»

ο ΦΟ U Φ·Η IT Ο ϋ •Η ^ φ En α·Γϊ -Ρ


54 Conclusions


P h o s p h o r u s - 3 1 NMR p r o v i d e s an a c c u r a t e and p r e c i s e method f o r t h e a n a l y s i s o f s o l u b l e p h o s p h a t e s and t h e i r m i x t u r e s . The a c c u r a c y and p r e c i s i o n a r e c o m p a r a b l e t o accepted c h r o m a t o g r a p h i c m e t h o d s . The r e s u l t s s i g n i f y t h a t t h e NMR method might also be successfully applied to less s t r a i g h t f o r w a r d s y s t e m s w i t h h i g h a c c u r a c y and p r e c i s i o n and w i t h t h e a d d i t i o n a l a d v a n t a g e s c h a r a c t e r i s t i c o f NMR w h i c h were e n u m e r a t e d e a r l i e r i n t h e a r t i c l e . The most s i g n i f i c a n t s o u r c e o f e r r o r i n t h e NMR method i s i n i n t e g r a t i o n of the s i g n a l areas, e s p e c i a l l y f o r resonances w i t h c h e m i c a l s h i f t s l y i n g c l o s e t o one a n o t h e r . This s o u r c e o f e r r o r i s a l l e v i a t e d i n l a r g e measure by e m p l o y i n g c u r v e d e c o n v o l u t i o n and l i n e s h a p e a n a l y s i s . Application of quantitative robotic analysis g r e a t l y improves the e f f i c i e n c y , w h i l e r e l i a b l y p r o d u c i n g r e s u l t s e q u i v a l e n t t o t h o s e o b t a i n e d b y manual s e l e c t i o n o f t h e i n t e g r a l r e g i o n s . L i t t l e more t h a n c l i c k i n g a s i n g l e menu b u t t o n i s r e q u i r e d t o i n i t i a t e an a u t o m a t i o n run. By removing much of the analyst subjectivity, more r e p r o d u c i b l e and r e l i a b l e a s s a y s a r e o b t a i n e d . By e x t e n d i n g t h e a p p l i c a t i o n o f t h e h o m o n u c l e a r 2DJ experiment t o the realm of oligophosphate assays, twod i m e n s i o n a l NMR i s s e e n t o be a p r o m i s i n g e x t e n s i o n f o r unravelling complicated oligophosphate s p e c t r a and in quantitative analysis. Coupled w i t h the use o f l i n e s h a p e a n a l y s i s and c u r v e f i t t i n g , r e a s o n a b l e s e m i q u a n t i t a t i v e results are obtained that compare well with other established a n a l y t i c a l techniques. Acknowledgments NMR l i n e shape a n a l y s i s s o f t w a r e d e v e l o p e d f o r use at M o n s a n t o was k i n d l y s u p p l i e d by Mr. N.G. H o f f m a n , R e s e a r c h C o m p u t i n g C o n s o r t i u m , M o n s a n t o Co., and P r o f . A . J . Duben, Southeast M i s s o u r i State U n i v e r s i t y . The a u t h o r s w o u l d also like t o t h a n k Mr. Brad Herman o f t h e Research C o m p u t i n g C o n s o r t i u m , M o n s a n t o Co., f o r h i s d e v e l o p m e n t o f t h e a u t o m a t i c s a m p l e management s o f t w a r e , Dr. W i l l i a m W i s e o f t h e P h y s i c a l S c i e n c e s C e n t e r ( P S C ) , M o n s a n t o Co., f o r his many valuable discussions on homonuclear 2DJ s p e c t r o s c o p y , and Mr. J o h n B u r q u i n ( M o n s a n t o PSC) f o r h i s assistance i n the development o f the robotic assay. A p p r e c i a t i o n i s a l s o e x p r e s s e d t o t h e many a n a l y s t s who p a r t i c i p a t e d i n the study.



3. GARD ET AL

55 Quantitative Analysis of Inorganic Phosphates

Literature Cited 1. Colson, J.G.; Marr, D.H. Anal. Chem. 1973, 45, 370. 2. Gurley, T.W.; Ritchey, W.H. Anal. Chem., 1975, 47, 1444. 3. Sojka, S.A.; Wolfe, R.A. Anal. Chem., 1978, 50, 585. 4. Stanislawski, D.A.; Van Wazer, J.R. Anal. Chem., 1980, 52, 96. 5. Ruben, I.B. Anal. Lett., 1984, 17, 1259. 6. Glonek, T.; Wang, P.J.; Van Wazer, J.R. J . Amer. Chem. Soc., 1976, 98, 7968. 7. Hanssum, H. J . Magn. Reson., 1981, 45, 461. 8. Becker, E.D.; Ferretti, J.A.; Gambhir, P.N. Anal. Chem., 1979, 51, 1413. 9. Press, W.H.; Flannery, Β.Ρ.; Teukolski, S.A.; Vetterling, W.T. Numerical Recipes: The Art of Scientific Computing, Cambridge Univ. Press, Cambridge, MA, 1986; Chapter 14. 10. ASTM Method D 501, Standard Test Methods of Sampling and Chemical Analysis of Alkaline Detergents, Annual Book of ASTM Standards, American Society for Testing and Materials, Philadelphia, 1989. 11. ASTM Method D 2761, Standard Test Method for Analysis of Sodium Triphosphate by the Simplified Ion Exchange Method, Annual Book of ASTM Standards, American Society for Testing and Materials, Philadelphia, 1989. 12. Lundgren, D.P.; Loeb, N.P. Anal. Chem., 1961, 33, 366. 13. Condensed Phosphates by Column Separation, Industrial Method No.317-74I. Bran and Luebbe, Technicon Industrial Systems, Elmsford, NY. 14. Longley-Cook, L.H. Statistical Problems and How to Solve Them, Barnes & Noble, New York, N.Y., 1970; Chapter 14. 15. Sotak, C.H.; Dumoulin, C.L.; Levy, G.C. Anal. Chem., 1983, 55, 782. 16. Weiss, G.H.; Ferretti, J.A. J . Magn. Reson., 1983, 55, 397. 17. Watanabe, M.; Matsuura, M.; Yamada, T. Bull. Chem. Soc. Japan, 1981, 54, 738. 18. Crowther, J.P.; Westman, A.E.R. Can. J . Chem., 1954, 32, 42. 19. MacDonald, J.C.; Mazurek, M. J . Magn. Reson., 1987, 72, 48. 20. Glonek, T.; Costello, A.J.R.; Myers, T.C.; Van Wazer, J.R. J . Phys. Chem. 1975, 79, 1214. 21. Van Wazer, J.R. Phosphorus and Its Compounds, Interscience Publishers, New York, 1958, Vol. 1, Chap. 12. 22. Xu, P.; Wu, X-L.; Freeman, R. J . Magn. Reson., 1991, 95, 132. RECEIVED December 10, 1991


Quantitative Analysis of Inorganic Phosphates Using 31P

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