Talonto Vol. 28, pp. 137 to 143

0 Pergamon Press Ltd 1981. Printed in Great Britain

0039-9140/81/030137-07102.00/0

POTENTIOMETRIC STUDIES ON THE COMPLEX FORMATION BETWEEN METHYLMERCURY(I1) AND

SOME KETO- AND AMINO-CARBOXYLIC ACIDS

MOHAMMAD JAWAID* and FOLKE INGMAN

Department of Analytical Chemistry, The Royal Institute of Technology, 100 44 Stockholm 70, Sweden

(Received 2 October 1980. Accepted 9 October 1980)

Summary-The protonation of pyruvic and laevulinic acid, L-serine, L-cysteine, L-methionine and L-asparagine, and their complex formation with methylmercury(I1) ions, have been studied by a poten- tiometric method, in l.OM sodium nitrate medium at 25”. The protonation and equilibrium constants were evaluated by using the computer program LETAGROP-ETITR. The limitations of the experimen- tal method are explained. The participation of different functional groups of the ligands in binding methylmercury(I1) and the biological significance of the complex formation are discussed.

The deleterious effects of organomercurials on man and the environment are now well documented1-5

and the use of these compounds as fungicides and in seed-dressing, etc. has been banned in many countries. Several studie&lo have shown that both inorganic and metallic mercury can be methylated in the pres- ence of certain bacteria found in sea sediments. More- over, it has also been shown that it is methylmercury species which are easily absorbed and accumulated by living organisms and thus pose potential danger to the health of man and animals.

lamine and some other sulphur-containing ligands by infrared spectrometry, PMR and potentiometry. X-Ray, infrared and Raman spectroscopy20-22 have also been used by some workers to investigate the structure of organomercurial complexes with some sulphur-containing ligands.

A study of the solution chemistry of organomer- curials, their complex formation with the ligands present in natural and biological systems, and the stability and specificity of the complexes so formed, can provide useful information about the nature and magnitude of binding between the organomercurials and such ligands, which, in turn, can be used to evalu- ate the methods of removing these undesirable sub- stances from biological systems employing chelation therapy. l1

Various techniques have been used for studying the solution chemistry of organomercurials. Simpson” employed solvent extraction and polarography to investigate interaction between methylmercury(I1) and organic ligands such as cysteine, glutathione and his- tidine. Solvent extraction was also used by Niitsu et a1.13 to study the distribution of R-Hg(I1) (R = methyl, ethyl or propyl) in the two-phase system l.OM (H,Na) (Cl)/benzene in presence of cysteine. Raben- stein and co-workers”” made use of NMR to study the hydrolysis of methylmercury(I1) and its complex formation with some selected amines, aminocarbox- ylic acids, glutathione, methionine, cysteine and DL- penicillamine. Hojo et ~1.” studied the complexes of methylmercury and phenylmercury with DL-penicil-

The work described here is a part of our systematic studies on methylmercury(I1) and its complex forma- tion with ligands that are present in natural and bio- logical systems. In previous publications we have reported the results on the hydrolysis of methylmer- cury(II),23*24 its complexes with the ligands found in natural waters23s25*26 and some simple carboxylic and aminocarboxylic acids24 and with EDTA.27 The present paper describes the results on the cgmplex formation between methylmercury(I1) and some simple keto-acids, namely pyruvic and laevulinic acid, and the representative aminocarboxylic acids L-serine, L-cysteine, L-methionine and L-asparagine. These acids have been chosen because they contain some model functional groups such as carboxylate, amino, thio-ether and sulph-hydryl, which can provide poten- tial co-ordination sites for binding metals to proteins, peptides, etc.

The method employed is based on potentiometric titrations covering a wide concentration range of the components, and the computer analysis of the col- lected data by the program LETAGROP-ETITR.2* The protolytic equilibria of these acids were also stud- ied under similar conditions (ionic medium, tempera- ture, etc.) and the protonation constants thus evalu- ated were used in the subsequent calculations.

EXPERIMENTAL

Reagents

* Present address: National Institute of Environmental Sodium pyruvate and laevulinic acid (analytical grade) Medicine, 104 01 Stockholm, Sweden. and L-serine, L-cysteine, L-methionine and L-asparagine

TN,. 28/3-A 137

138 MOHAMMAD JAWAID and FOLKE INGMAN

(Merck biopure grade) were assayed by potentiometric titrations.” Carbon dioxide-free sodium hydroxidea was

c& = the molar concentration of sodium hydroxide in the titrant

standardized against hydrochloric acid by the Gran method. Methylmercury hydroxide (Ventron Alfa, Den- Vo = the initial volume of the solution in the titra-

mark) was assayed as described previously.z4 Sodium tion vessel nitrate (p.a., Merck) was recrystallized once before use. All v = the voldme of titrant added the solutions were prepared in freshly boiled doubly dis- tilled water. To illustrate the computer calculations, we shall here-

Potentiometric titrations after use CHcalr: and dHexp for CH in equations (3) and

The titrations were performed in a 200-ml (Metrohm EA (6), respectively.

880) glass vessel with a lid having five inlets for burette, The program LETAGROP-ETITR, when supplied . . . electrodes, degassing tubes, etc. The cell used can be with an mltlal estimate of &,,,,?. and the known quanti- written as -1

Reference I

u ml of l.OM Na(OH,NOJ Glass half-cell V0 ml of l.OM (H,MeHg,Na) (L,NO,) electrode

where v is the volume of the titrant added and V, the initial volume of the solution in the titration vessel. MeHg and L represent methylmercury and fully deprotonated ligand, respectively.

The electrodes used were combined glass and silver/ silver chloride electrodes (Ingold, micro-design). The emf was read to 0.1 V with a digital voltmeter. Thetemperature was kept at 25 k 0.05” with an oil-bath.

[H’] was calculated from the equation

E = Eb + 59.156 log[H+] + j[H’] (1)

The practical details and meaning of the symbols have already been given.“’

CALCULATION METHODS

We assume that the components H, MeHg and L react to form a complex of composition (H),(MeHg),,,(L),. The equilibrium constant, Blmn of the reaction is given by the expression

Blmn = C(H), (MeW&)~ICW -‘CMeHgl -‘“CL1 -” (2)

The charges are omitted for convenience. The mass-balance equations are

CH = CHI + ~~~~,,CHl’CMeHgl”CLl” = CHCalc (3)

C MeHg = [MeHgl + Cn&,,,CH1’CMeHdmCL1 (4)

G. = P-1 + ~nB~m.CH1lCMeHglmCLl” (5)

where C stands for the total analytical concentration

and the square brackets for the equilibrium concen- tration of the components/complexes. If there is more than one ligand in the solution, the model can be extended accordingly, by inclusion of further mass- balance equations.

For potentiometric titrations, the following re-

lationship is valid for the proton excess.

C” = (C:, v, - c&, . v)/( v, + 0)

= Gexp (6) where

Cfi = the initial concentration of the ionized and potentially ionizable hydrogen

ties CMeHg, CL [equations (4) and (5), respectively] and

[H] (hereafter called h) from equation (l), can calcu-

late, for each experimental point, the values of CHEalc from equation (3) and minimize the error-square sum

U = &&,,,, - CH,~p)2 (7) 1

where Np is the number of experimental points avail-

able and CHexp is calculated from equation (6) by the procedure BDTV in the program.

The program is used to test a series of probable chemical models describing various complexes that

may be formed in a solution of known composition. The chemical model which gives the minimum error- square sum, explains the data satisfactorily within the limits of experimental error, and is most plausible from co-ordination chemistry considerations, is accepted as the “best” model.

RESULTS AND DISCUSSION

Some experimental data for the titration of L-serine,

r_-cysteine, L-methionine and L-asparagine, in presence of the same amount of methylmercury(I1) are plotted in,Fig. 1, as Z = (H - Il)/Ch(eHg as a function of pH. For almost similar solution composition, the Z-curves of, the biprotic acids L-serine and L-methionine are nearly superimposable over the whole pH range,

whqeas that for L-asparagine deviates slightly from the 4her two at higher pH. This probably indicates

that the methylmercury(I1) complex of L-asparagine is less stdble than those of the other two amino-acids. For L-cysteine the formation curve is similar in shape but lies well above the other three, because L-cysteine was considered a triprotic acid for calculating the analytical proton excess.

The results of the computer calculations are sum- marized in Table 1, which includes the equilibrium constants for the formation of various complexes of methylmercury(I1) with these ligands as well as the

protonation constants of the acids in the same ionic medium. The chemical models given in the table were accepted as the “best” in each case for the present

Methylmercury complexes 139

Fig. 1. Titration of 2.50 x lo-‘M solution of (a) L-serine, (b) L-cysteine, (c) L-methionine and (d) L-asparagine, in presence of 3.32 x lo-‘M methylmercury solution, shown as Z = (H - h)/Chc,Hp as a function of pH. Note that L-cysteine is taken as a triprotic acid for calculating

the analytical proton excess. H = CHerp

experimental conditions. All the other models tried gave a rather high error-square sum and systematic deviations. In the calculations, the formation of hy- drolysed methylmercury species was taken into account, by use of the equilibrium constants reported earlier.24

The potentiometric titration method used in this work implies the titration of a weak acid with a strong base. If metal ions that form complexes with the ligand are present in the solution the ligand will appear to be a conditionally stronger acid than if it were present alone. This strengthening effect is used to evaluate the complexation between the metal ions and the ligand. However, when the complexation is very strong, as in the case of cysteine, the acid will appear conditionally strong, that is, completely dis- sociated, and still stronger complexation will not affect the titration curve further. Thus, there is a limit to the strength of complexes that can be evaluated by this method.

For the MeHg(II)-cysteine system, the limiting value for log fioll is 9-10. The analysis of the experi- mental data obtained by titrating a solution of methylmercury(I1) in presence of cysteine alone will therefore always yield this limiting value, and although the fit to the model will be very good the results so obtained will be entirely erroneous.

One way to increase the range is to introduce a second ligand into the system, that can compete for the metal ions. We have used iodide as an appropriate auxiliary complexing agent because it is an aprotic ligand that forms a fairly strong 1: 1 complex with methylmercury(II).3’ The results obtained by analys- ing the experimental data from titration of a solution of methylmercury(I1) in presence of cysteine and an excess of iodide are also given in Table 1.

The situation can be clarified by using the concept of conditional constants and side-reaction coeffi- cients.32 Let the main reaction characterizing the titration of a weak acid HA with strong base (omit- ting charges) be

HL+H+L(+OH+L+H,O); K==w

If the metal ion M is now introduced, forming complexes with L, the following side-reaction will occur :

W-1 L+M-+ML KML= ___ CM1 CL1

The side-reaction coefficient is given by

CL’1 CL1 + CMLI Ei = aL(M’ = CL1

= 1 + [M]KhlL 2 1

The conditional constant for the main reaction is

K, = CHIP’1 CHlCLIaL(M) PC 8 Ml WI

= KaaL(M)

Since KL ; K,, the main reaction will proceed further to the right upon the introduction of M, and the acid will consequently be conditionally stronger. As the value of aLcMj is increased, the titration curve approaches that for the titration of a strong acid and a further increase of aLcMj will have no effect on the titration curve. In other words, the value of KL for HL can never be greater than CHL, the total concentration of HL.

Example. Titration of a O.OlM solution of acid HL having pK, = 8. The pH at the half-neutralization point will be -8. For a titration of a O.OlM solution of a strong acid (e.g., pK, = -5) the corresponding pH is -2.3. Since K: can never become greater than C the limiting value of aLcMj is in this case C::,K. = 10-2/10-* = 106, corresponding to K ML - 10g, assuming [M] - 10m3M. Further in- crease of aLcM) will have no more influence on the titration curve.

The introduction of an aprotic ligand such as iodide will lower the free concentration of M with a consequent reduction of aLcMj as a result:

M+I+MI; WI -- KM’ - [M][I]

%(I) = ‘CM’1 = cM3 + CM11 = I + [I]K cM1 CM1

MI 2 I

aLtM) = 1 + [M]Kd, = 1 + CM’1 K - ML h(I)

1 4 0 M O H A M M A D J A W A I D a n d F O L K E I N G M A N

g

O"

o

H

+1

- i

. o

O z n~

o

E

O

. o

! ~0

o

8

ff

~D

. o

"O

+1 O

8

d +1

T

e~

I

il

+1

~.( o

g

O

"O

x~

+1+1+1+1+1+1+1+1+1+1+1+1+1+1+1+1+1+1+1+1

~ & & 6 ~ 6 & & g & & 6 &

." - : . . . . ¢~ o ~ o ~ .

~ , e-i .--: e,-~ e,g ,-,-.i e,4 t--i m eq e-- i ~ e - ~

b o b

b b ~ 0 0 Z ~ ~ ~- z

, ~ o ~ E ~ ~"

O

"8

H

O

.B

O

c~

. o

oo rl

O

',-.

8 +

I---,

8 8

. ~ . -

. . ~ o o

-~N o N ~ - ~ - N

Methylmercu~(I1) complexes 141

The same total concentration of M will now p’roduce a lower value of aLoe.

Table 2 summarizes the results for the complex for- mation between methylmercury(I1) and some car- boxylic and aminocarboxylic acids reported by various workers. Most of the results, obtained by dif- ferent methods, are comparable; the small differences in some cases can be attributed to the different ionic media and the temperatures. However, in the case of L-cysteine the differences are more significant. One reason is that different experimental methods were used and some workers were not aware of the limita- tions of their chosen method. The greatest differences can be seen between some results obtained by poten- tiometric titration using the glass electrode,‘9 and results obtained by liquid/liquid distribution experi- ments.“J3 The difference is about 8 log~ithmic units. This agrees fairly well with what can be pre- dicted from the discussion above on potentiometric titration of cysteine. This is evident from Table 1, where the values found by the present authors, both with and without iodide as an auxiliary complexing agent, are listed.

Attention also needs to be paid to the significance of the acid dissociation constants of cysteine. If no complexation occurs, the sulph-hydryl proton will dissociate slightly before the proton on the amino- group. Once the sulph-hydryl proton has dissociated, the amino-group proton will be more strongly retained, giving rise to the macroscopic protonation constants listed in Table 1. Since, however, the bond between sulphur and mercury is particularly strong, ~mplexation of cysteine with methylmercu~(I1) will block the sulph-hydryl group and evaluation of an acid-base titration of the complex will yield a value for the microscopic protonation constant of the amino-group. This protonation has been studied by Rabenstein and by Hojo and co-workers, by NMR methods.

It is known that an approximate relationship exists between the first dissociation constants of a series of acids and the stability constants of their 1 :l com- plexes with a given metal. Such a relationship can be used to estimate the stability constants of the metal complexes of closely related substances if their pK, values and one KML value are known. Figure 2 dem- onstrates such a relationship for the methylmer- cury(I1) complexes of some simple carboxylic, keto- and amino-carboxylic acids, with a correlation coeffi- cient of 0.99 if L-cysteine and hydroxide are excluded. The greater deviation in the case of these last two can be attributed to the fact that they are not closely related to the others and the complexation takes place in a different manner; in the case of cysteine it is the deprotonated sulph-hydryl group which binds meth- ylmercury most strongly.

In Fig. 3, the equilibrium distribution of various species in the methylmercury(II)-cysteine-iodide sys- tem are shown as functions of pH. The calculations, done with the computer program HALTAFALL,33

x

4

2 4 6 8 10 12 14

P&

Fig. 2. Relationship between the first dissociation constant of the acids and the stability constant of their 1: 1 com- plexes with methylmercury( Some data were taken from

ref. 24. pK, = 13.61 in l.OM NaNO, ionic medium.

Fig. 3. The equilibrium distribution of the species in the methylmercury(II~ysteine-iodide system as a function of pH. Curve 1 is for MeHgI, 2 for MeHgL- and 3 for MeHgLH. C,,,, = 1.00 x lO-+M, c, = 1.00 x 10-2&f,

c, = 0.W.

Tab

le 2

. The

equ

ilibr

ium

co

nsta

nts,

lo

g K

, fo

r th

e fo

rmat

ion

of m

ethy

lmer

cury

co

mpl

exes

w

ith s

ome

carb

oxyl

ic,

keto

- an

d am

ino-

carb

oxyl

ic

acid

s in

dif

fere

nt

syst

ems

Aci

d/lig

and

Equ

ilibr

ium

re

actio

ns

log

K(a

) Io

nic

med

ium

M

etho

d R

ef.

Form

ic

acid

Ace

tic a

cid

Pyru

vic

acid

L

aevu

linic

ac

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Gly

cine

aAla

nine

/I

-Ala

nine

dl

-Val

ine

L-S

erin

e L

-Cys

tein

e(bl

L-M

ethi

onin

e M

eHgC

l +

HL

H =

M

eHgL

H

+

Hf

+ C

l M

eHg’

+

L-

c M

eHgL

L-A

spar

agin

e M

eHg+

+

L-

e M

eHgL

MeH

g’

+ L

- Z

$ M

eHgL

MeH

g+

+ L

- Z

$ M

eHgL

MeH

g+

+ L

- z$

MeH

gL

MeH

g+

+ L

- e

MeH

gL

MeH

g’

+ L

- $

MeH

gL

MeH

g+

-t L

- +

M

eHgL

M

eHg+

+

L-

+

MeH

gL

MeH

g+

+ L

- +

M

eHgL

MeH

g+

+ L

- +

M

eHgL

M

eHg+

+

LH

- $

MeH

gLH

MeH

g+

+ H

LH

e

MeH

gLH

+

H

+

MeH

g+

+ L

2-

zz M

eHgL

- M

eHg*

+

H+

+ L

*-

+

MeH

gLH

M

eHgL

- +

HC

e

MeH

gLH

2.61

2.

68 )

0.

01

3.6

3.18

3.

20 f

0~

04

2.31

+ 0

.08

2.51

+ 0

.04

7.88

*

0.05

6.

07

1.52

+ 0

.05

1.52

f

0.05

7.

56 +

0.0

7 7.

41 f

0.

01

7.27

f

0.06

6.

93 _

t 0.0

6 15

.7

15.1

6”’

7.19

’d’ /

14

.81

& O

X#

,‘dr

r,

6:69

+ 0

.12

15.7

0 7

0.12

24

.96

+ 0

.08

9.05

8.

92

9.25

+ 0

.14

1.71

7.

40 +

0.0

1 7.

17 f

0.

16

6.32

f

0.06

0.4M

? l.O

M N

aNO

s un

defi

ned

0.4M

? L

OM

NaN

Oa

l.OM

NaN

O,

l.OM

NaN

O,

0.4M

? un

defi

ned

l.OM

NaN

O,

l.OM

NaN

Os

0,4M

? 0.

4M?

l.OM

NaN

O,

l.OM

NaN

O,

O.lM

? l.O

M (

Na,

H)C

l un

defi

ned

l.OM

NaN

Oa

O.iM

? l.O

M (

Na,

H)C

l l.O

M N

aNO

, l.O

M N

aN03

l.O

M N

aNO

, 0.

4M?

unde

fine

d l.O

M N

aNO

s l.O

M (

Na,

H)C

l 0.

4M?

l.OM

NaN

O,

l.OM

NaN

O,

NM

R

GE

PO

L

NM

R

GE

G

E

GE

N

MR

N

MR

/GE

G

E

GE

N

MR

N

MR

G

E

GE

D

IST

R

DIS

TR

PM

R/G

E

GE

D

IST

R

DIS

TR

G

E

GE

G

E

NM

R

PMR

/GE

G

E

DIS

TR

N

MR

G

E

GE

15

24

12

15

24

Thi

s w

ork

Thi

s w

ork

16

19

;: 16

16

24

Thi

s w

ork

12

13

19

Thi

s w

ork

l2

13

Thi

s w

ork

Thi

s w

ork

Thi

s w

ork

17

19

Thi

s w

ork

13

18

Thi

s w

ork

Thi

s w

ork

(a)

Tem

pera

ture

25

” if

not

oth

erw

ise

spec

ifie

d.

(b)

HL

H:

= H

SCH

,CH

(NH

:)C

OO

H;

HL

H

= H

SCH

,CH

(NH

:)C

OO

-;

HL

- =

HSC

H#Z

H(N

Ha)

CO

O-

LH

- =

-SC

H,C

H(N

H;)

CO

O-

and

L2-

=

-SC

H,C

H(N

H,)

CO

O-

(c)

Cal

cula

ted

valu

e as

sum

ing

(MeH

g+

+ C

l-

Z$

MeH

gCl)

, lo

g K

= 5

.33,

‘“6)

and

pK o

f th

e cy

stei

ne

thio

l gr

oup

(p&

n)

= 8

.12.

(d

) A

t 22

”.

(e)

Cal

cula

ted

valu

e.

(f)

Cal

cula

ted

with

the

equ

ilibr

ium

co

nsta

nts

give

n in

(c)

abo

ve.

Methylmercury complexes 143

are based on the equilibrium constants given in Table 1.

The results show that of the potential co-ordination sites available in amino-acids, the deprotonated sulph-hydryl group can bind methylmercury(I1) most strongly. The complexation, however, depends pre- dominantly on the pH of the solution, owing to protonation of the ligand and hydrolysis of methyl- mercury(I1). In neutral solution the following order of binding power is most likely to prevail:

sulph-hydryl > amine > hydroxide > carboxylate

Only the 1: 1 complexes are formed in presence of an excess of the ligand, because methylmercury(I1) has very little Lewis acidity after co-ordinating with a sulph-hydryl or amino-group. However, with an excess of methylmercury( L-cysteine may also form a 2:l complex, (log fi = 12.40),19 the second methyl- mercury ion being co-ordinated to the amino-group. The formation of such a species was considered to be very unlikely under the experimental conditions used in the present studies and was not of interest because in biological systems the ligand is always present in excess.

Since cysteine residues are the integral component of many sulphur-containing proteins, the present results support the conclusion that the sulph-hydryl group is the main target in metal poisoning, because after co-ordinating with methylmercury it can disrupt the enzyme structure and consequently impede its normal catalytic functions.

1.

2. 3. 4.

5. 6.

REFERENCES

T. Takeuchi, Minamata Disease. A Study of Toxic Sys- tems by Organic Mercury, Univ. of Kumamato, 1966. K. Irukavama Adu. Water Polk. Rex. 1967. 3. 153. FAO Fisheries Rept., No. 99, Rome, 1971. ’ K. Borg H. Wanntorp, K. Erne and E. Hanko, .J. Appl. Ecol., 1971, 3, 171. J. M. Wood, Adv. Environ. Sci. Technol., 1971, 2, 39. J. M. Wood, F. S. Kennedy and C. G. Rosen, Nature, 1968, 220, 173.

7. 8. 9.

10.

11.

12. 13.

14.

15. 16.

17.

18.

19.

20.

21.

22.

23.

24.

25. 26.

27. 28.

29.

30.

31.

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