Electrophoresis 1986, 7, 187-190 Ionophoretic technique in the study of mixed ligand complexes 187

15-45 nL in one day. The analysis can therefore be carried out efficiently if such sensitivity is required. The gels are par- ticularly appropriate for the analysis of LMW proteins that are trapped in the dense end of the gel. The application de- scribed here is the analysis of proteins in kidney tubule fluid. LM W protein bands have been detected in normal renal tubule fluid and that obtained from rats with glomerulonephritis. An additional advantage is that proteins of higher molecular weights than albumin can enter the gel and be detected when their filtration is increased in glomerulonephritis. Therefore it is possible to analyze both tubule fluid and urine by the method of gradient gel electrophoresis and thereby facilitate com- parison of their respective protein compositions.

This work was supported by Veterans Administration re- search funds.

Received January 16, 1985; in revised form December 9, 1985

5 References

Omstein, L., Ann. N.Y. Acad. Sci. 1964,121,321-349. Davis, B. F. Ann. N.Y. Acad. Sci. 1964,121,404-427. Neuhoff, V. Arzneim. Forsch. 1968,18,35-39. Neuhoff, V., in: Neuhoff, V. (Ed.) Micormethods in Molecular Biology, Springer 1973, pp. 1-83. Oken, D. E. Microchem. J. 1970,15,557-563.

Satyendra Singh Arun K. Bajpai Sita R. Tripathi

161 Oken, D. E. and Flamenbaum, W., J. Clin. Invest. 1971, 50,

[71 Eisenbach, G. M., Van Liew, J. B. and Boylan, J. W., Kidney Int.

[Sl Baldamus, C. A., Galaske, R. G., Eisenbach G. M., Krause, H. P. and

[91 Galaske, R. G., Baldamus, C. A. and Stolte, M., Pfliigers Arch. 1978,

1101 Ruchel, R., Mesecke, S., Wolfrum, D. and Neuhoff, V., Hoppe- Seyler’s 2. Physiol. Chem. 1973,354, 1351-1368.

[ 1 1 1 Galaske, R. G., Van Liew, J. B. and Feld, L. G.,KidneyZnt. 1979,16,

[ 121 Ruchel, R., in: Allen, R. C. and Maurer, M. R. (Eds.), Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel, De Gruyter, Berlin

1131 Allen, R. C., Moore, D.J. and Dilworth, D.J., J. Histochem. and Cytochem. 1969,17,189-190.

[I41 Dames, W. and Maurer, H. R., in: Allen, R. C. and Maurer, H. R. (Eds.), Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel, de Gruyter, Berlin 1974, pp. 221-231.

1151 Cho, J., Galaske, R. G., Arbesman, H. and Van Liew, J. B., Renal Physiol. 1985,8, 8-18.

I161 Feld, L. G.,Van Liew, J. B., Galaske,R. G. and Boylan, J. W., Kidney Int. 1911,12,332-343.

[ 171 Hubbard, R. S. and Garbott, H. R., Am. J. Clin. Pathol. 1935, 5, 433-442.

I181 Lane, S. E. and Neuhaus, 0. W., Biochim. Biophys. Acta 1972,257, 461-470.

[ 191 Mendrick,D. L.,Noble,B.,Brentjens, J. R. and Andres,G. A.,Kidney Int. 1980, I S , 328-343.

I201 von Baeyer, H. B., Van Liew, J. B. and Boylan, J. W., Kidney Int.

1498- 1505.

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1976,10,425-436.

Ionophoretic technique in the study of mixed ligand complexes (M-nitrilotriacetate-threoninate systems)

Kanhaiya L. Yadava

Department Of Chemistry9 University of Allahabad

-

A novel ionophoretic technique for the study of complexes especially mixed ones has been described in the communications from this laboratory. Here with the help ofthis technique the stability constants of the complexes M-nitrilotriacetate-threoninates have been found to be 5.24, 5.01, 3.54 and 3.21, respectively.

I Introduction

A number of technique viz. spectrophotometry, polaro- graphy, electrometry, etc., are generally used in studies of complexation processes. Many of these are specific and cum- bersome. In recent years the ionophoretic technique has been applied to study metal complexes in solution, and attempts have been made to determine the stability constant ofthe com- plex species I l-21. The usual procedure is to study the mobil- ity of a metal cation spot on a paper strip soaked with back-

Correspondence: Professor Kanhaiya L. Y adava, Electrochemical Labo- ratorics, Department of Chemistry, University of Allahabad, Allahabad - 21 1002. India

Abbreviations: NTA, nitrilotriacetic acid

ground electrolyte buffered as a fixed pH containing a pro- gressively increasing concentration of the ligand. In our labo- ratory the procedure has been drastically modified [3-61 by keeping the concentration of liganding sample constant and progressively decreasing the hydrogen ion concentration of the background electrolyte by addition of an alkali solution. Thus the previous technique failed to elucidate the effect ofthe change of relative concentration of the different ionic species of a ligand. The use ofthe ionophoretic technique with a single ligand seems to be well established, but there is no systemat- ic study of the formation of mixed ligand complexes using this technique. Publications [7-91 from our laboratory deal with a new method for the study of mixed complexes. The ion- ophoretic technique usually suffers from a number of defects viz. temperature during electrophoresis, capillary flow on paper strip, electroosmosis, adsorption, molecular sieving,

0 173-0835/86/0404-0 187 %02.50/0 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1986

188 S. Singh et al. Electrophoresis 1986, 7, 187-190

etc., which affect the mobility of charged moieties. The tech- nique described here is almost free from these vitiating fac- tors. The technique is simple and gives results in good agree- ment with literature values. In the present paper the novel ionophoretic technique has been employed to determine the nature and stability constants of some ternary complexes viz. Cu(II)/UO,(II)/Co(II)/Zn(II)-nitrilotriacetate-threoninate.

2 Materials and methods

2.1 Instruments

The electrophoresis equipment (Systronics model 604 India) consisted of a built-in power supply (AC-DC) directly con- nected to a paper electrophoresis tank. In most electropho- resis instruments no attention is paid to the control oftemper- ature. The electric current generates heat which causes eva- poration of background electrolyte from the paper strips, resulting in serious errors. In order to eliminate this drawback the paper strips were placed between two hollow metallic plates, coated with thin plastic, and thermostated with circu- lating water (35 "C).

2.2 Chemicals

Metal perchlorates were prepared by precipitation of the metal carbonate from their metal nitrate with sodium bicarbonate, which were thoroughly washed with boiling water and treated with calculated amounts of 1 % perchloric acid, followed by heating and filtration. The metal contents of the filtrates were determined and the final concentrations were kept at 0.005 M. 1-(2-Pyridylaz0)-2-naphthol (PAN), 0.1 % w/v in ethanol was used for detecting the metal ions. A saturated solution of silver nitrate in acetone was sprayed on the paper and subse- quently fumed with ammonia to detect glucose in the spot.

2.3 Background electrolyte

Stock solution of 2.0 M perchloric acid, 2.0 M sodium hy- droxide and 0.1 M threonine were prepared from BDH An- alar reagents. A 0.01 Mnitrilotriacetic acid (NTA) solution (E. Merck, Darmstadt, FRG) was prepared. The background e- lectrolyte in the study of binary complexes consists of 0. l M perchloric acid and 0.0 1 M threonine/0.00 1 M NTA, while in the study ofternary complexes,itconsistsofO. 1 M sodiumper- chlorate, 0.00 1 M NTA and varying amount of 0.0 1 M thre- onine, maintained at pH 8.5 by addition of sodium hydroxide.

2.4 Procedure

2.4.1 Binary complexes

After leveling the base plate with spirit level, 150 mL of back- ground electrolyte were placed into each tank of the electro- phoretic apparatus. The levels of the solution in the two tanks were made equal by siphoning to avoid any gravitational and hydrodynamic flow. Paper strips (Whatman No. 1) of 30 x 1 cmz size were soaked in the background electrolyte and blot-

On duplicate strips, metal ions and glucose solution (0.01 M) were applied in the center with a micropipette. In order to minimize capillary action, the strips were placed between the thermostated metalic plates for 30 min, with the ends dipping into the reservoir solutions. Electrophoresis was carried out for 60 min, followed by drying the strips and horizontal plate and spot detection. Electrophoresis was repeated at different pH's of the background electrolyte, adjusted by addition of a solution of sodium hydroxide. The distance recorded in duplicated differed within 5 %. Duplicates were noted for calculations. The distances migrating towards the anode were assumed to be negative and those towards the cathode to be positive. The average actual distance of sample spot takes into account the migration of glucose, employed as reference substance. The potentialgradient throughthe strips was found to be 7.57 V/cm. Dividing the migration distance by thepoten- tial gradient yields the mobilities plotted in Figs. 1 and 2.

2.4.2 Ternary complexes

Metal were applied in duplicate on strips pretreated as de- scribed in Section 2.4.1. An additional strip was spotted with glucose. Electrophoresis was carried out for 60 min at the

13.2t

P H

Figure I . Mobility curves for the [M(II)-Threoninel System. Temperature 35 O C , ionic strength 0.1.

13.2 t

- 4 . 4 1

Figure2. Mobility curves for the I M(I1)-NTAI system. Temperature 35 'C, ted with dry filter paper sheets to revmove any exess solution. ionic strength 0.1.

Electrophoresis 1986, 7, 187-190 Ionophoretic technique in the study of mixed ligand complexes 189

iog(Threonlne1 - 0 - 8 - 7 - 6 - S -4 - 3 -2

- - 2 . 2 - -

Figure 3. Mobility curves for the [M-NTA-Threoninel system. Tem- perature 35 "C, ionic strength 0.1.

same potential gradient as in the case of binary complexes. A solution of threoninate, maintained at pH 8.5, was added in varying amounts and ionophoretic mobility was recorded. A plot of mobility against log [threoninel is shown in Fig. 3.

3 Results and discussion

3.1 Metal-threonine binary systems

The plot of overall electrophoretic mobility of a metal spot against pH gives a curve with a number of plateaus (Fig. 1). A plateau indicates a pH range with practically constant veloci- ty, where a particular complex is formed. Thus every plateau corresponds to the formation of certain complex species. The first plateau, at low pH, with amaximum of highly protonated, non-complexing species of threonine, corresponds to un- complexed metal ions. Beyond this range,the metal ions ex- hibit decreasing mobility, indicating complexion with other ionic species of threonine whose concentration increases with increasing pH. The second plateaus in each case, with positive

mobility, indicate the formation of 1 : 1 complexes of cationic nature. Further increases in pH give rise to the third plateau with zero mobility corresponding to electrically neutral 1 :2 metal complexes, formed by two anionic species of threonine with one bivalent metal ion. Literature prominent liganding property is assigned also to umprotonated anionic species of threonine acid, however ruling out such property for zwit- terions [10-121. Further increase of pH has no effect on mobility of metal ions. The complexation of metal(M) ion with threonine anion (L) may be represented as:

ML+ t L- ~ Kz ML2 The overall mobility is given by Eq. (1):

u= Z U n f n (1)

where u, and fn are the mobility and mole fraction of a par- ticular complex species. Eq. (1) is transformed into the follow- ing form on taking into consideration different equilibria:

uo t u 1 K, [L-] t u, K, K, [L-]' 1 + K , [L-] + K , K , [L-],

U =

where uo, u1 and uz are mobilities of uncomplexed metal ions, 1 : 1 metal complex and 1 :2 metal complex, respectively.

Eq. (2) has been used for calculating stability constants of the complex of metal ions with the threonine anion. For calcu- lating the first stability constant, k,, the region between the first and second plateau is pertinent. The overall mobility, U, will be equal to the arithmetic mean of the mobility of the un- complexed metal ion, uo, and that of the first complex uI at a pH wereK, =/[L-l. Withthehelpofthedissociationconstants of threonine ( K , = 102.'o; kz = 108.84, [ 131) the concentration of the threoninate anion (L-) can be determined for the pH from which k, can then be calculated. The stability constants kz of

Table 1. Stability constants of some binary and ternary complexes of Cu(II), UOz (II), Co(I1) and Zn(I1) (Temperature 35 OC; ionic strength O.l)*)

Calculated value of stability constants Literature values Metal

log KIM-NTA log K! -N TA M-NTA-L

ions M M M log KIM-NTA M log KIML log KZMLZ log K ~ - ~ ~ ~ M-NTA-L log KIML log KZMLZ

Cu(1I) 7.96 13.93 12.70 5.24 7.90 [13] 1458 [13] 13.60 [13] 7.85 [13] 14.71 [13] 13.30 [13] 7.90 [13] 14.58 [13] 12.75 [13] 7.88 [13] 14.46 [13] - 8.03 [13] 14.77 [13] - 7.86 13.85 [17] - 6.00 [13] - 7.88 [13] 7.32 [I31 14.20 [I31 9.50 [13] 4.38 [13] 9.01 [13] 10.38 [13] 4.71 [13] 8.73 [13] 10.45 [13] 3.40 [IS] 4.67 [13] 8.66 [13] 10.69 [13] 4.64 [13] 859 [13] - 4.60 [13] 8.52 [13] - 4.65 [17] 7.35 [17] -

OH NH2

uoz (11) 7.85 13.82 9.85

co (11) 4.40 7.91 10.43 Zn(I1) 4.70 7.21 10.59

5.01

3.54 3.24

I 1 a) NTA anion, N(CHZCOO);~; threonine anion (L), = CH3 - CH - CH - COO-

190 S. Singh et a/. Electrophoresis 1986, 7, 187-190

the second complex can be calculated by taking into con- sideration the region between the second and third plateau of the mobility curve (Table 1).

3.2 Metal nitrilotriacetate binary systems

For the overall mobility of metal spotsin the presence ofNTA, at different pH values, with all five metal ions two plateaus are obtained (Fig. 2). The mobility of the second plateau lies in the negative region showing the negative charge ofthe complexes. Only one NTA acid anion is assumed to combine with one bivalent metal ion to give a 1 : 1 M-NTA- complex which is in agreement with other findings 14-16]. The stability con- stants of the complex with NTA were calculated as described in Section 3.2 (Table 1).

3.3 Metal-nitrilotriacetate-threoninate ternary systems

The study ofthis system has purposely been done at pH 8.5 . It can be seen from the mobility curve of m(I1)-threonine and M(I1)-NTA binary systems that binary complexes are formed at a pH < 8.5. Therefore, the transformation of the M-NTA complex into the M-NTA-threoninate complex was studied at pH 8.5 in order to avoid any additional interaction. The plot of mobility against logarithm of concentration of added threo- nine gives a curve with two plateaus at both ends (Fig. 3). The mobility in the range ofthe first plateau corresponds to mobil- ities of 1 : 1 M-NTA complexes, in agreement with results in binary M-NTA systems. The mobility of the second plateau indicates the formation of a more negatively charged complex. Since the mobility does not correspond to the mobility of 1 : 1 and 1 :2 metal-threonine complexes ofour binary M-threonine system, the formation of 1 : 1 : 1 mixed complexes (M-NTA- threoninate) is inferred:

K' M-NTA- + L- . - M-NTA-L*-

'= ' 0 fM-NTA "1 fM-NTA-L

The overall mobility is given by:

(3 ) where UO, u1 and f.,.NTA and fM NTA.L are the mobilities and the mole fractions of M-NTA- and M-NTA-L- complexes, re- spectively. Substituting the values of mole fractions the overall mobility is given in Eq. (4).

uo t u1 K' [L-] 1 +K' [L-] U = (4)

where uo and u, are the mobilities in the regions of the two plateaus of the curve. From the figure,the concentration of threonine, for which the overall mobility is the mean of the mobilities of both plateaus, can be determined. The concentra- tion of anionic species ofthreonine at pH 8.5, for this threonine concentration, k'is calculated and is obviously equal to 1 :[L-l (Table 1). The calculated stability constants are in good agree- ment with those determined by others [ 13, 171, the variations being partly due to differences in temperature and ionic strength.

3.4 Precision

The precision of the method, roughly 5 %, is limited to that of paper electrophoresis. Obviously this method cannot replace more reliable methods but it provices a new technique worth developing and, with further refinements, may prove valuable.

4 Concluding remarks

The ionophoretic technique, as modified in our laboratory, is simple as compared to other techniques. It helps determine the composition of mixed complexes and its stability constants with a fair degree of accuracy. It has itslimitationsinnot being applicable in the cases of uncharged complex species.

Received August 7, 1985; in revised form December 12, 1985

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