Electrochemical Studies of Biological Systems

tion as function of total NTA concentration. Cad mium concentration = 2.67 X .... Credit. This research was supported by the Oceanography. Section, Na...
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12 Differential Pulse Polarographic Analysis for Ethylenediaminetetraacetate ( E D T A ) and Nitrilotriacetate ( N T A ) i n Phytoplankton Media RICHARD J. STOLZBERG

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Harold Edgerton Research Laboratory of the New England Aquarium, Central Wharf, Boston, Mass. 02110 In t h e c o u r s e o f investigating t h e effects o f t r a c e m e t a l s p e c i a t i o n on p h y t o p l a n k t o n p r o d u c t i v i t y , we have found it desirable t o measure t h e concen­ tration of small q u a n t i t i e s o f strong organic l i g a n d s . These i n c l u d e artificial l i g a n d s such as e t h y l e n e ­ d i a m i n e t e t r a a c e t a t e (EDTA), nitrilotriacetate (NTA), and t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e ( t r i s ) added by the e x p e r i m e n t e r and some l e s s w e l l - d e f i n e d e x t r a ­ cellular m e t a l b i n d i n g o r g a n i c s (EMBO) added by t h e phytoplankton. Artificial l i g a n d s have traditionally been added by algal p h y s i o l o g i s t s because a wide variety o f a l g a e c a n be grown in media c o n t a i n i n g complexed t r a c e m e t a l s . In a d d i t i o n , precipitation of t h e medium is reduced, e n a b l i n g t h e e x p e r i m e n t e r t o p r e p a r e a more r e p r o d u c i b l e medium ( 1 ) . The h y p o t h e s i s has been made t h a t p l a n k t o n might a c t i v e l y produce EMBO f o r much t h e same r e a s o n - t o improve the medium f o r growth e i t h e r by d e t o x i f y i n g potentially t o x i c m e t a l s such as copper (2) o r by making i r o n a v a i l a b l e as a s o l u b l e c h e l a t e d s p e c i e s (3,4). Our work c u r r e n t l y i n v o l v e s correlating t h e c o n c e n t r a t i o n o f complexed and uncomplexed s p e c i e s o f copper w i t h p h y t o p l a n k t o n p r o d u c t i v i t y and w i t h t h e p r o d u c t i o n o f EMBO. W e l l d e f i n e d artificial media t h a t l e n d themselves t o c o n v e n i e n t c h e m i c a l manipu­ lation and which s u p p o r t a good growth o f a l g a e a r e used. Table I g i v e s the composition o f both the s y n t h e t i c seawater (SSW) used in t h e analytical development work and t h e artificial medium d e s i g ­ nated Cu-IV. In media c o n t a i n i n g l e s s than 5 χ 10 M EDTA o r NTA, a c c u r a t e measurement o f specific l i g a n d concen­ t r a t i o n i s q u i t e important. Small v a r i a t i o n s i n l i g a n d c o n c e n t r a t i o n may i n c e r t a i n c a s e s produce -6

194

Sawyer; Electrochemical Studies of Biological Systems ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

12.

EDTA

STOLZBERG

and NTA

in Phytoplankton

TABLE Composition

of

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I

Synthetic

Component

195

Media

Seawater

and

Cu-IV

SSW

Cu-IV

NaCl

4.3

χ

ΙΟ"

1

M

4.3

χ

ΙΟ"

1

M

KC1

9.4

χ

1(Γ

3

M

9.4

χ

ΙΟ"

3

Μ

Μ

3.7

χ

10~

Μ

7.5

χ

ΙΟ"

1.2

χ

10~

3

Μ

5

Μ

MgS0

4

2.7

χ

10~

CaCl

2

9.5

χ

ΙΟ"

NaN0

2

3

3

2

Μ Μ

3

NaH P0

4

4.8

χ

10~

Na Si0

3

2.6

χ

ΙΟ"

4.4

χ

10~

to

5 χ

ΙΟ"

6

Μ

to

5 χ

1θ"

β

Μ

to

8 χ

10~

2

2

Boron EDTA

5 χ

ΙΟ"

ΝΤΑ

5 χ

ΙΟ""

Tris

8 χ

ΙΟ"

7

7

5

Μ

4

5

3

Μ

Μ

Μη

2.2

χ 1θ"

Ζη

2.2

χ

1θ""

7

Μ

5.4

χ

10~

8

Μ

3.7

χ

10~

9

Μ

Co

(inorganic)

Vitamin

Β

1 2

Sawyer; Electrochemical Studies of Biological Systems ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

6

Μ

Downloaded by UNIV OF ARIZONA on April 26, 2017 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0038.ch012

196

ELECTROCHEMICAL

STUDIES O F B I O L O G I C A L

SYSTEMS

i m p o r t a n t changes i n copper s p e c i a t i o n . Loss of l i g a n d due t o p h o t o d e g r a d a t i o n i s a d i s t i n c t p o s s i b i l i t y when c u l t u r i n g a l g a e , p a r t i c u l a r l y i n l o n g term experiments under h i g h l i g h t i n t e n s i t y . The s e n s i t i v i t y of ferric-EDTA to photodegradation has b e e n known f o r o v e r two d e c a d e s (5) . The s u s c e p t i b i l i t y o f b o t h f e r r i c - and c u p r i c - N T A t o p h o t o d e g r a d a t i o n has been documented r e c e n t l y {6,]_,S). Sorption of ligands can a l s o present experimental d i f f i c u l t i e s i n dense a l g a l c u l t u r e s . Changes i n m e t a l s p e c i a t i o n c a n be e x p e c t e d f r o m any o f t h e s e mechanisms t h a t m i g h t r e d u c e l i g a n d o r m e t a l c o n c e n trations. F i n a l l y , t h e p r o d u c t i o n o f EMBO b y t h e c e l l s s h o u l d be t a k e n i n t o c o n s i d e r a t i o n when calculating speciation. T h e q u a n t i t y o f EMBO p r o d u c e d by t h e c e l l s c o u l d be d e t e r m i n e d by t h e d i f f e r e n c e in t o t a l c o m p l e x i n g c a p a c i t y (9^) a n d t h e q u a n t i t y o f EDTA o r NTA p r e s e n t . The a n a l y t i c a l methodology f o r s p e c i f i c and s e n s i t i v e d e t e r m i n a t i o n o f EDTA a n d NTA i n s a l i n e waters i s not w e l l developed i n s p i t e o f the wide r a n g e o f t e c h n i q u e s d e v e l o p e d f o r NTA i n f r e s h w a t e r and sewage s l u d g e (100 . The most w i d e l y used s p e c i f i c t e c h n i q u e s i n n o n - s a l i n e w a t e r a r e gas chromatography (11-13) and e l e c t r o c h e m i s t r y (14-18). The p r e s e n c e o f l a r g e q u a n t i t i e s o f d i s s o l v e d salts i n seawater c l e a r l y favors e l e c t r o c h e m i c a l techniques. E l e c t r o c h e m i c a l r e d u c t i o n o f t h e CdNTA c o m p l e x , f i r s t u s e d a n a l y t i c a l l y f o r t h e d e t e r m i n a t i o n o f NTA i n EDTA (14^) a n d t h e n l a t e r a d a p t e d f o r NTA d e t e r m i n a t i o n i n l a k e and r i v e r w a t e r s ( 1 6 , 1 7 ) , i s t h e system of c h o i c e . R e d u c t i o n o f NTA c o m p l e x e s o f l e a d , b i s m u t h , a n d i n d i u m (_15,1_8) h a s b e e n u s e d a n a l y t i c a l l y , b u t t h e CdNTA {19_,2C0 a n d C d E D T A ( 2 1 , ,22,23) e l e c t r o c h e m i s t r y h a s b e e n c h a r a c t e r i z e d i n d e t a i l and appears r e l a t i v e l y w e l l behaved. C l a s s i c a l DC p o l a r o g r a p h y h a s b e e n u s e d t o m e a s u r e 1 t o 10 ppm NTA i n l a k e w a t e r (16^) , b u t t h i s t e c h n i q u e c o u l d n o t be u s e d i n s e a w a t e r . The r e d u c t i o n c u r r e n t due t o t h e l a r g e q u a n t i t y o f cadmium a d d e d t o d i s p l a c e c a l c i u m f r o m t h e NTA w o u l d swamp t h e s m a l l c u r r e n t i n c r e m e n t s d u e t o CdNTA r e d u c t i o n . D i f f e r e n t i a l pulse polarography (DPP) i s more s e n s i t i v e t h a n DC p o l a r o g r a p h y , and i t c a n be u s e d t o measure s m a l l c u r r e n t s a t a p o t e n t i a l c a t h o d i c o f an e l e c t r o c h e m i c a l l y a c t i v e s p e c i e s p r e s e n t a t much g r e a t e r c o n c e n t r a t i o n .

Sawyer; Electrochemical Studies of Biological Systems ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

12.

EDTA

STOLZBERG

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Theoretical

and NTA

in Phytoplankton

197

Media

Considerations

T h e r e d u c t i o n o f t h e CdNTA c o m p l e x a t - 0 . 9 V (vs SCE) i s i r r e v e r s i b l e a n d d i f f u s i o n controlled (20). A t pH 8 t h e r e d u c t i o n o f t h e CdEDTA c o m p l e x a t - 1 . 2 t o - 1 . 3 V i s more i r r e v e r s i b l e t h a n t h a t for CdNTA. I t i s d i f f u s i o n c o n t r o l l e d o n l y when t h e c o n c e n t r a t i o n o f t h e s u p p o r t e l e c t r o l y t e i s h i g h (2_1) . S e p a r a t i o n o f t h e uncomplexed Cd (at - 0 . 6 V ) , the CdNTA, and t h e CdEDTA waves w i l l p r e s e n t no p r o b l e m s u s i n g DPP. I n s e a w a t e r , t h e CdEDTA c o m p l e x r e d u c t i o n c u r r e n t i s e x p e c t e d t o be d i f f u s i o n c o n t r o l l e d due to the presence of a high c o n c e n t r a t i o n of salts, and t h e s e n s i t i v i t y s h o u l d be s u f f i c i e n t f o r t h e determination of micromolar q u a n t i t i e s of ligand. The a d d i t i o n o f a l a r g e e x c e s s o f cadmium w i l l cause a l a r g e f r a c t i o n o f b o t h l i g a n d s t o be p r e s e n t i n t h e b u l k o f t h e s o l u t i o n as t h e cadmium complex (see below). E q u a t i o n 1 d e s c r i b e s the g e n e r a l i z e d competition r e a c t i o n b e t w e e n c a d m i u m a n d c o m p e t i n g m e t a l (M) for EDTA o r NTA ( L ) . Charges have been o m i t t e d f o r clarity. C o n c e n t r a t i o n s t a b i l i t y c o n s t a n t s and m o l a r c o n c e n t r a t i o n s are used throughout. CdL + Μ ^

Cd + ML,

K„ = C

w

h

a

n

e

d

r

=

e

.

TmTTTlT

=

CdL

[ [ C

C

d

L

( l a )

(lb)

]

d]

(D

CdL

[L]

Using Ringbom's concept o f c o n d i t i o n a l s t a b i l i t y c o n s t a n t s a n d h i s n o t a t i o n (24^) , t h e c o m p e t i t i o n s t a b i l i t y c o n s t a n t , K , c a n be r e w r i t t e n as a conditional competition constant c

K'

=

'ML K '

K

c

d

[Cd'] [M