Determination of Metal Binding Constants by Potentiometric Titrations Presentation by: Destinee K....
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Transcript of Determination of Metal Binding Constants by Potentiometric Titrations Presentation by: Destinee K....
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?