EDTA Titrations Introduction 1.) Metal Chelate Complexes Any reagent which reacts with an analyte in a known ratio and with a large equilibrium constant can potentially be used in a titration. Complexation Titrations are based on the reaction of a metal ion with a chemical agent to form a metal-ligand complex. Metal Ligand Metal-Ligand Complex Note: multiple atoms from EDTA are binding Mn2+ Metal – Lewis Acid or Electron-pair acceptor Ligand – Lewis Base or Electron-pair donor

EDTA Titrations Introduction 1.) Metal Chelate Complexes Complexation Titrations are essentially a Lewis acid-base reaction, in which an electron pair is donated from one chemical to another The ligands used in complexometric titrations are also known as chelating agents. Ligand that attaches to a metal ion through more than one ligand atom Most chelating agents contain N or O Elements that contain free electron pairs that may be donated to a metal Fe-DTPA Complex

EDTA Titrations Metal Chelation in Nature 1.) Potassium Ion Channels in Cell Membranes Electrical signals are essential for life Electrical signals are highly controlled by the selective passage of ions across cellular membranes Ion channels control this function Potassium ion channels are the largest and most diverse group Used in brain, heart and nervous system K+ is chelated by O in channel channel contains pore that only allows K+ to pass K+ channel spans membrane Opening of potassium channel allows K+ to exit cell and change the electrical potential across membrane Current Opinion in Structural Biology 2001, 11:408–414 http://www.bimcore.emory.edu/home/molmod/Wthiel/Kchannel.html

EDTA Titrations Metal –Chelate Complexes 1.) Formation Constant (Kf) The equilibrium constant for the reaction between a metal ion (M+n) and a chelating agent (L-P) is known as a formation constant or stability constant. Applying different and specific names to the general equilibrium constant is a common occurrence Solubility (Ksp), acid-base (Ka, Kb), water dissociation (Kw), etc Chelate effect: ability of multidentate ligands to form stronger metal complexes compared to monodentate ligands. Kf = 8x109 Kf = 4x109 2 ethylenediamine molecules binds tighter than 4 methylamine molecules

EDTA Titrations Metal –Chelate Complexes 2.) Chelate Effect Usually chelating agents with more than one electron pair to donate will form stronger complexes with metal ions than chelating agents with only one electron pair. Typically more than one O or N Larger Kf values Multidentate ligand: a chelating agent with more than one free electron pair Stoichiometry is 1:1 regardless of the ion charge Monodentate ligand: a chelating agent with only one pair of free electrons Multidentate ligand that binds radioactive metal attached to monoclonal antibody (mAb). mAb is a protein that binds to a specific feature on a tumor cell delivering toxic dose of radiation.

EDTA Titrations EDTA 1.) EDTA (Ethylenediaminetetraacetic acid) One of the most common chelating agents used for complexometric titrations in analytical chemistry. EDTA has 6 nitrogens & oxygens in its structure giving it 6 free electron pairs that it can donate to metal ions. High Kf values 6 acid-base sites in its structure

EDTA Titrations EDTA 2.) Acid-Base Forms EDTA exists in up to 7 different acid-base forms depending on the solution pH. The most basic form (Y4-) is the one which primarily reacts with metal ions. EDTA-Mn Complex

EDTA Titrations EDTA 2.) Acid-Base Forms Fraction (a) of the most basic form of EDTA (Y4-) is defined by the H+ concentration and acid-base equilibrium constants Fraction (a) of EDTA in the form Y4-: where [EDTA] is the total concentration of all free EDTA species in solution aY4- is depended on the pH of the solution

EDTA Titrations EDTA 3.) EDTA Complexes The basic form of EDTA (Y4-) reacts with most metal ions to form a 1:1 complex. Other forms of EDTA will also chelate metal ions Recall: the concentration of Y4- and the total concentration of EDTA is solution [EDTA] are related as follows: Note: This reaction only involves Y4-, but not the other forms of EDTA where aY4-is dependent on pH

EDTA Titrations EDTA 3.) EDTA Complexes The basic form of EDTA (Y4-) reacts with most metal ions to form a 1:1 complex.

EDTA Titrations EDTA 3.) EDTA Complexes Substitute [Y4-] into Kf equation If pH is fixed by a buffer, then aY4- is a constant that can be combined with Kf where [EDTA] is the total concentration of EDTA added to the solution not bound to metal ions Conditional or effective formation constant: (at a given pH)

EDTA Titrations EDTA 3.) EDTA Complexes Assumes the uncomplexed EDTA were all in one form at any pH, we can find aY4- and evaluate Kf’

EDTA Titrations EDTA 4.) Example: What is the concentration of free Fe3+ in a solution of 0.10 M Fe(EDTA)- at pH 8.00?

EDTA Titrations EDTA 5.) pH Limitation Note that the metal –EDTA complex becomes less stable as pH decreases Kf decreases [Fe3+] = 5.4x10-7 at pH 2.0 -> [Fe3+] = 1.4x10-12 at pH 8.0 In order to get a “complete” titration (Kf ≥106), EDTA requires a certain minimum pH for the titration of each metal ion End Point becomes less distinct as pH is lowered, limiting the utility of EDTA as a titrant

EDTA Titrations EDTA 5.) pH Limitation Minimum pH for Effective Titration of Metal Ions EDTA 5.) pH Limitation By adjusting the pH of an EDTA titration: one type of metal ion (e.g. Fe3+) can be titrated without interference from others (e.g. Ca2+)

EDTA Titrations EDTA Titration Curves 1.) Titration Curve The titration of a metal ion with EDTA is similar to the titration of a strong acid (M+) with a weak base (EDTA) The Titration Curve has three distinct regions: Before the equivalence point (excess Mn+) At the equivalence point ([EDTA]=[Mn+] After the equivalence point (excess EDTA)

EDTA Titrations EDTA Titration Curves 2.) Example mmol of Mg2+ What is the value of [Mn+] and pM for 50.0 ml of a 0.0500 M Mg2+ solution buffered at pH 10.00 and titrated with 0.0500 m EDTA when (a) 5.0 mL, (b) 50.0 mL and (c) 51.0 mL EDTA is added? Kf = 108.79 = 6.2x108 aY4- at pH 10.0 = 0.30 mL EDTA at equivalence point: mmol of EDTA mmol of Mg2+

EDTA Titrations EDTA Titration Curves 2.) Example moles of Mg2+ (a) Before Equivalence Point ( 5.0 mL of EDTA) Before the equivalence point, the [Mn+] is equal to the concentration of excess unreacted Mn+. Dissociation of MYn-4 is negligible. moles of Mg2+ originally present moles of EDTA added Original volume solution Volume titrant added Dilution effect

EDTA Titrations EDTA Titration Curves 2.) Example Original volume of (b) At Equivalence Point ( 50.0 mL of EDTA) Virtually all of the metal ion is now in the form MgY2- Original volume of Mn+ solution Moles Mg+ ≡ moles MgY2- Original [Mn+] Original volume solution Volume titrant added Dilution effect

EDTA Titrations EDTA Titration Curves 2.) Example (b) At Equivalence Point ( 50.0 mL of EDTA) The concentration of free Mg2+ is then calculated as follows: Initial Concentration (M) 0.0250 Final Concentration (M) x 0.0250 - x Solve for x using the quadratic equation:

EDTA Titrations EDTA Titration Curves 2.) Example Volume excess (c) After the Equivalence Point ( 51.0 mL of EDTA) Virtually all of the metal ion is now in the form MgY2- and there is excess, unreacted EDTA. A small amount of free Mn+ exists in equilibrium with MgY4- and EDTA. Calculate excess [EDTA]: Volume excess titrant Original [EDTA] Excess moles EDTA Original volume solution Volume titrant added Dilution effect

EDTA Titrations EDTA Titration Curves 2.) Example Original volume of (c) After the Equivalence Point ( 51.0 mL of EDTA) Calculate [MgY2-]: Original volume of Mn+ solution Moles Mg+ ≡ moles MgY2- Original [Mn+] Only Difference Original volume solution Volume titrant added Dilution effect

EDTA Titrations EDTA Titration Curves 2.) Example (c) After the Equivalence Point ( 51.0 mL of EDTA) [Mg2+-] is given by the equilibrium expression using [EDTA] and [MgY2-]:

EDTA Titrations EDTA Titration Curves 2.) Example Final titration curve for 50.0 ml of 0.0500 M Mg2+ with 0.0500 m EDTA at pH 10.00. Also shown is the titration of 50.0 mL of 0.0500 M Zn2+ Note: the equivalence point is sharper for Zn2+ vs. Mg2+. This is due to Zn2+ having a larger formation constant. The completeness of these reactions is dependent on aY4- and correspondingly pH. pH is an important factor in setting the completeness and selectivity of an EDTA titration

EDTA Titrations Auxiliary Complexing Agents 1.) Metal Hydroxide In general, as pH increases a titration of a metal ion with EDTA will have a higher Kf. Larger change at the equivalence point. Exception: If Mn+ reacts with OH- to form an insoluble metal hydroxide Auxiliary Complexing Agents: a ligand can be added that complexes with Mn+ strong enough to prevent hydroxide formation. Ammonia, tartrate, citrate or triethanolamine Binds metal weaker than EDTA Fraction of free metal ion (aM) depends on the equilibrium constants (b) or cumulative formation constants: Use a new conditional formation constant that incorporates the fraction of free metal:

EDTA Titrations Auxiliary Complexing Agents 2.) Illustration: CuSO4 Titration of Cu+2 (CuSO4) with EDTA Addition of Ammonia Buffer results in a dark blue solution Cu(II)-ammonia complex is formed Addition of EDTA displaces ammonia with corresponding color change CuSO4 Cu-ammonia Cu-EDTA

EDTA Titrations Metal Ion Indicators 1.) Determination of EDTA Titration End Point Four Methods: Metal ion indicator Mercury electrode pH electrode Ion-selective electrode Metal Ion Indicator: a compound that changes color when it binds to a metal ion Similar to pH indicator, which changes color with pH or as the compound binds H+ For an EDTA titration, the indicator must bind the metal ion less strongly than EDTA Similar in concept to Auxiliary Complexing Agents Needs to release metal ion to EDTA Potential Measurements End Point indicated by a color change from red to blue (red) (colorless) (colorless) (blue)

EDTA Titrations Metal Ion Indicators 2.) Illustration Addition of EDTA Titration of Mg2+ by EDTA Eriochrome Black T Indicator Addition of EDTA Before Near After Equivalence point

EDTA Titrations Metal Ion Indicators 3.) Common Metal Ion Indicators Most are pH indicators and can only be used over a given pH range

EDTA Titrations Metal Ion Indicators 3.) Common Metal Ion Indicators Useful pH ranges

EDTA Titrations EDTA Titration Techniques 1.) Almost all elements can be determined by EDTA titration Needs to be present at sufficient concentrations Extensive Literature where techniques are listed in: G. Schwarzenbach and H. Flaschka, “Complexometric Titrations”, Methuen:London, 1969. H.A. Flaschka, “EDTA Titrations”, Pergamon Press:New York, 1959 C.N. Reilley, A.J. Bernard, Jr., and R. Puschel, In: L. Meites (ed.) “Handbook of Analytical Chemistry”, McGraw-Hill:New York, 1963; pp. 3-76 to 3-234. Some Common Techniques used in these titrations include: Direct Titrations Back Titrations Displacement Titrations Indirect Titrations Masking Agents

EDTA Titrations EDTA Titration Techniques 2.) Direct Titrations Analyte is buffered to appropriate pH and is titrated directly with EDTA An auxiliary complexing agent may be required to prevent precipitation of metal hydroxide. 3.) Back Titrations A known excess of EDTA is added to analyte Free EDTA left over after all metal ion is bound with EDTA The remaining excess of EDTA is then titrated with a standard solution of a second metal ion Approach necessary if analyte: precipitates in the presence of EDTA Reacts slowly with EDTA Blocks the indicator Second metal ion must not displace analyte from EDTA

EDTA Titrations EDTA Titration Techniques 4.) Displacement Titration Used for some analytes that don’t have satisfactory metal ion indicators Analyte (Mn+) is treated with excess Mg(EDTA)2-, causes release of Mg2+. Amount of Mg2+ released is then determined by titration with a standard EDTA solution Concentration of released Mg2+ equals [Mn+] Requires:

EDTA Titrations [Total EDTA] = [MYn-4] + [Y4-] EDTA Titration Techniques 5.) Indirect Titration Used to determine anions that precipitate with metal ions Anion is precipitated from solution by addition of excess metal ion ex. SO42- + excess Ba2+ Precipitate is filtered & washed Precipitate is then reacted with excess EDTA to bring the metal ion back into solution The excess EDTA is titrated with Mg2+ solution [Total EDTA] = [MYn-4] + [Y4-] complex free Known determine Titrate

EDTA Titrations EDTA Titration Techniques 6.) Masking Agents A reagent added to prevent reaction of some metal ion with EDTA Demasking: refers to the release of a metal ion from a masking agent Al3+ is not available to bind EDTA because of the complex with F- Requires: