Determination of Metal Binding Constants by

Potentiometric Titrations

Presentation by: Destinee K. Johnson

Research Mentor: Dr. Grossoehme

EDTA

EDTA-Zn Complex

ITC Data

Background of Zinc titrated into EDTA in HEPES

buffer

Goal of Project

To use potentiometric titrations to extract the metal binding constants of Zn for various buffers

Buffer: HEPES

HEPES HEPES-Zn Complex

Rorabacher, D.B. Journal of Inorganic Biochemistry Vol. 99 Issue 8, August 2005, Pages 1653-1660

Buffer: Pipes

Pipes Pipes-Zn Complex

Calculation of Binding Constants

Method: Potentiometry

Why? -Zinc is a spectroscopically silent metal

-The progress of the metal-ligand complex formation can be monitored by pH measurements

Hypothesis

It will be more difficult for protons to bind to the ligand (HEPES/PIPES buffer) in the presence of Zn. This competition will influence the pH.

B + H+ BH+

+

Zn2+

B-Zn2+

Procedure

Appropriate volumes of ligand solution, metal-ion solution, ionic salt, and metal free water are transferred into a beaker.

The solution is stirred magnetically.

Increments of titrant are added and the pH is recorded using a pH meter after the addition of each increment.

HEPES Experiments

HEPES in HNO3

0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12

HEPES Concentration

pH

…with Zn present

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10

HEPES Concentration

pH

Titration of 50 mL of 5 mM HNO3 , 100 mM KNO3 with 39.899 mM HEPES, 100 mM KNO3

Titration of 50 mL of 5 mM Zn, 5 mM HNO3 , and 100 mM KNO3 with 39.899 mM HEPES, 100 mM KNO3

HEPES ExperimentsHEPES Concentration

pH

HEPES Experiments

HNO3 in HEPES

0

1

2

3

4

5

6

7

8

9

10

0 2 4 6 8 10 12 14

Concentration of HNO3

pH

…with Zn present

Titration of 50 mL of 5 mM HEPES and 100 mM KNO3 with 5 mM HNO3

Titration of 50 mL of 5 mM Zn, 5 mM HEPES, 100 mM KNO3 with 5 mM HNO3

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12 14

Concentration of HNO3

pH

Pipes Experiments

Pipes in HNO3

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12 14 16

PIPES Concentration

pH

…with Zn present

Titration of 50 mL of 5 mM HNO3 and 100 mM KNO3 with 50 mM Pipes and 100 mM KNO3

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10 12

PIPES Concentration

pHTitration of 50 mL of 5 mM Zn, 5 mM HNO3 and 100 mM KNO3 with 50 mM Pipes and 100 mM KNO3

Pipes ExperimentsPipes Concentration

pH

Pipes Experiments

HNO3 in Pipes

0

1

2

3

4

5

6

7

0 2 4 6 8 10

HNO3 Concentration

pH

…with Zn present

Titration of 50 mL of 5 mM Pipes and 100 mM KNO3 with 50 mM HNO3 and 100 mM KNO3

0

1

2

3

4

5

6

7

8

0 2 4 6 8 10

HNO3 Concentration

pH

Titration of 50 mL of 5 mM Zn, 5 mM Pipes and 100 mM KNO3 with 50 mM HNO3 and 100 mM KNO3

Conclusion

A metal-ligand complex did not form between Zn and Pipes or HEPES at the working concentrations

Zinc becomes diluted

Other Experiments

Determination of formation constants in the Cu2+ -BCS system

Determination of formation constants in the Cu+-BCS system with Cu+ stabilized in acetonitrile

BCS Experiments

HNO3 in BCS

0

1

2

3

4

5

6

7

8

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

HNO3 Concentration

pH

…with Cu2+present

Titration of 25 mL of .6255 mM BCS with 6.1328 mM HNO3

Titration of 25 mL of .31 mM Cu(II) and .6255 mM BCS with 6.1328 mM HNO3

0

1

2

3

4

5

6

7

0 0.5 1 1.5 2 2.5

HNO3 Concentration

pH

BCS Experiments

Stability Constants

-The constant K1 is called a stability constant

]][[

][21

LM

MLK M2+ + L- ML+

Metal Complex Formation

M2+ + L- ML+

ML+ + L- ML2

]][[

][21

LM

MLK

]][[

][ 22

LML

MLK

β1 = K1

β2 = K1K2

Bjerrum Method

From pH measurements and knowledge of quantities originally added it is possible to calculate the stability constants

Θ, the average number of ligand molecules bound per metal ion

Cu + 2BCS Cu(BCS)2

Cu + BCS Cu(BCS)

22

2 ]][[

])([

BCSCu

BCSCu

]][[

)]([1 BCSCu

BCSCuK

TotalCu

BCSCuBCSCu

][

])([2)]([ 2

Fit Data

Fit parameters:

Y-int 2.07 ± 0.06Slope 0.17 ± 0.07

K1 = 2.07 ± 0.06 x 106

β2 = 0.17 ± 0.07 x 1011

Credits: Dr. Nicholas Grossoehme

Actual

Expected

BCS-Cu+ Experiments

Titration of 25 mL of .4 mM Cu(I), 50 mM MeCN, and .66 mM BCS with 5 mM HNO3

0 0.2 0.4 0.6 0.8 1 1.2 1.40

1

2

3

4

5

6

7

8

9

10

HNO3 Concentration

pH

Prospects for the Future

Develop a Cu+ stabilizing system to investigate the thermodynamic properties that control Cu+ binding and selectivity.

References

The Journal of Biological Chemistry Vol.286, NO.13, pp. 11047-11055, April 1, 2011

Synthesis and Technique in Inorganic Chemistry Third Edition, pp. 219-231

Quinn, Colette.“Analyzing ITC Data for the Enthalpy of Binding Metal Ions to Ligands.”

“Calculation of Binding Constants.” Excel for Chemists: A Comprehensive Guide. E. Joseph Billo pp.349-351

Journal of Inorganic Biochemistry Vol. 99 Issue 8, August 2005, Pages 1653-1660

Acknowledgements

The Grossoehme Lab

Winthrop University Chemistry Department

Winthrop University Summer Undergraduate Research Experience

Idea Network for Biomedical Research Excellence

Questions?