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Ground State acid dissociation constant determination of Organic Imidazolium cations and Ruthenium (II) complexes By Kane Logue 1

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Ground State acid dissociation constantdetermination of Organic Imidazolium cations and Ruthenium (II) complexesBy Kane Logue

Table of ContentsI. Abstractpg 3II. Introductionpg 3a. Compounds analyzed pg4b. Acid Base Theorypg 5c. Factors that affect pKapg 7d. UV-Vispg 8e. * transitionpg 11f. biological importance of acid dissociationIII. Methodpg 14IV. Materialspg 14

V. Results and Discussionpg 14VI. Conclusionpg 22VII. AppendixVIII. Sourcespg 23

I. AbstractSeveral compounds that fall under of the categories of imidazolium cations and Ru(II) complexes, are analyzed to determine the acid dissociation constant, pKa. The pKa determination is important because itaffects pH, absorbance of light, and the metabolism of the compound in the body1. In pharmaceutic aspects, pKaaffects solubility, lipophilicity, permeability, and protein binding inside the body. Ultraviolet-visible, (UV-Vis) spectrophotometry currently serves as one of the more widely used techniques in pharmaceutical analysis for determination of dissociation constant. Utilizing the UV-Vis, absorbance of different pH solution would be scanned and plotted. Thus an isosbestic point was determined where all the absorbance values at different absorbance values crossed at a single wavelength. The wavelength of the greatest change was then selected. From a plot of absorbance vs. pH the curve generated was fitted to a Boltzmann Sigmoidial non-linear fit, using GraphPad Prism software to determine the pKaII. Introduction:The value of the acid dissociation constant (pKa) is an important parameter that indicates the degree of ionization of molecules in solution at different pH values. The smaller the pKa the dissociated the the acid becomes. The pKa is a property of a compound that tells us how acidic it is. The lower the pKa means the stronger the acid. Many chemical, physical and biological properties of natural and synthetic compounds are influenced by acidic and basic groups. pKa controls many aspects of metabolism and even transport across membranes; therefore, its study is of significant interest in biology, pharmaceutics, medicine, and numerous other scientific fields. Several methods exist for determining the pKa of a compound include: potentiometric titration, conductometry, voltammetry, calorimetric, Nuclear Magnetic Resonance, solubility, fluorometry, polarimetry, kinetic methods, computational chemistry, and spectrophotometric titration. Out of these the most popular involves conducting a spectrophotometric titration via UV-Vis. The main advantage of for spectrophotometric titration is the ability to obtain a titration curve, which allows for estimation of pKa at any point. Potentiometric titration requires knowledge of the equilibrium concentrations of the reagents, which are not necessary in spectrophotometric titration because the ratio of the concentrations chromophore of the [A-]/[HA] at various pH values is obtained from the results of absorbance measurements. In order to determine pKavalues by UV-Vis, compounds must contain a to the ionization centers. The chromophore absorbs light from the UV-Vis which allows the absorbance to be calculated. Also, the compounds absorbance must change as a function of the degree of ionization.The pKa can be determined from the spectrophotometric data using nonlinear least squares regression software.A. Compounds analyzed:

NNRu(bpy)2NNCO2HbpyB2RuCacid2+2PF6-Three compounds named CAM, QM, and B2RuCacid2+ were analyzed by UV-Vis spectrometry. The structures are as follows:

Sites of activity were hypothesized, to infer what kind of pKa to expect.

Although all three of these compounds fall under the categories of weak acids, an acid dissociation constant is needed to characterize these organic compounds, since they are intended to become fluorophores, largely due to the amount of aromatic rings and other forms of conjugated to bonds. Uses of these flourophores could include use as a dyeforstainingof certain structures, such as a substrate ofenzymes, or as a probe or indicator when its fluorescence is affected by environment such as polarity and ions. The acid dissociation constant would tell how a compound will be ionized in the body.B. Acid Base Theory:When an acid (HA) is dissolved in water, equilibrium becomes established by the following equation:1HA + + (1)The HA transfers a proton to water, and it becomes the anion (. This anion tends to retrieve the proton and behave as a base; therefore is referred to as the conjugate base of acid HA, and HA and are referred to as conjugate pairs. A shift of the equilibrium in Equation (1) to the right or left depends on the strengths of HA and acids. How strong an acid is, refers its tendency to transfer protons, and one method of standardizing its force is to compare the protonation state when it interacts and dissolves in water. The result of this comparison is expressed as the acid dissociation constant, Ka, as follows:1Ka = (2)Equation (2) implies that Ka is a constant of the stoichiometric equilibrium defined in terms of the concentration ratio [A]/[HA],which can be determined spectrophotometrically. If a solution with a total concentration of indicator becomes very acidic, the entire indicator exists as HA. The absorbance of the solution at a given wavelength is given by the following equation:1= * b * (3) is the molar absorptivity of HA at wavelength () and b is the width of the cell containing the solution. If the solution is too basic, the same concentration is converted entirely into A and the absorbance at the same wavelength is given by the following equation:1= * b *(4) is the molar absorptivity of . At an intermediate pH, the absorbance is given by:= + (5)Where the total concentration can be defined in any condition as:1(6)For a given , Equations (3)-(6) can be combined to obtain the following:1 = (7)This relationship must be evaluated at multiple wavelengths, including one where HA absorbs substantially but does not, one where is much more absorbent than HA, and another where the absorbance of the two species is approximately the same.1 The pH of the solutions must be in the transition range of the indicator so that both HA and are present in appreciable concentrations. Ka can be evaluated graphically by converting Equation (2) to logarithmic form:1-Log(Ka)= -log + -log (8)This can be re written after algebraic manipulations into the following equation from the classic Henderson Hasselbach equation:1pKa= -log (9)In addition, the combination of Equation (7) and the definition equation of pKa=log[Ka] results in the following equation:1 (10). When HA is a strong acid, a value for Ka in aqueous solutions cannot be defined, because HA molecules cannot be detected; the value of Ka is therefore very high or infinite1. However, a very low value indicates that the dissociation Ka involves a very small fraction of the total acid present. The isosbestic point is where the wavelength at which = thus a constant appears and eliminates the isosbestic point for pKa .The appearance of an isosbestic point is evidence that only two species are involved (the conjugate pairs).From a titration curve, pKa can be found to be half the equivalence point. Graph pad prism utilizes the Boltzman sigmoidal equation:2 (11)The top and bottom indicates the limits of the sigmoidal plot. The slope is calculated from the curve, and the pKa is the halfway point the slope.

Figure 1: Shows and example of an isosbestic point where the absorbance values all meet a particular wavelength at different pH.A. UV-Vis Electronic orbitals of atoms and molecules have characteristic energies, giving rise to a set of discrete energy levels. An electron is able to change from an occupied orbital to another orbital, gaining or losing energy only in amounts exactly corresponding to the difference between two levels: The transition from the ground state at energy E0 to a higher level at energy En is possible if the molecule absorbs electromagnetic radiation of the corresponding wavelength.4 The equation is as follows:4 = = (14)Excited states will exist only for a very short period of time since the higher energy state is for more unstable. As a result the extra energy is lost through relaxation processes such as an emission of light.4 Generally, the energy difference between the ground and the first excited levels of many molecules corresponds to electromagnetic waves of the ultra-violet (UV) and visible regions of the electromagnetic spectrum.The UV-visible range is only a small part of the total electromagnetic spectrum, and is generally defined from wavelengths of 190 nm at the high energy UV end to approximately 750 nm. At the low energy which is commonly referred to as the red end of the spectrum. Light in other regions of the spectrum gives rise to different types of transitions and is the subject of different types of spectroscopy. For example, IR radiation is usually not energetic enough to cause electronic transitions but can excite vibrations of molecules. Figure 2: shows where the visible spectrum lies,Image courtesy of Google images

The wavelength () is the distance between adjacent cent peaks (or troughs) in the time-frozen electromagnetic wave, and is measured in nanometers. Visible wavelengths cover a range from approximately 400 to 750 nm. The frequency (v) is the number of wave cycles that travel past a fixed point per unit of time, and is usually given in cycles per second, or Hertz (Hz).Frequency and wavelength are related via = (15)where c is the speed of light. The angular frequency = 2v (radians s-1) is often used instead of v. When polychromatic or 'white' light passes through or is reacted by a colored substance, a characteristic portion of the spectrum is absorbed. The remaining light will then exhibit the complementary color to the wavelengths absorbed. The absorption of blue light between 420-430 nm renders a substance yellow, and absorption of green, 500-520 nm light makes it red. Green, to which our eyes are most sensitive, is unique in that it can be created by absorption close to 400 nm as well as absorption near 800 nm. When the compound Cam was placed in solution phase, a green fluorescent color was noticed. As the pH increased the solution become brighter, and when the pH decreased the solution become almost slight yellow clear tint. B. * transitionFor molecules that possess bonds like alkenes, alkynes, aromatics, acryl compounds or nitriles, light can promote electrons from a bonding molecular orbital to a anti-bonding molecular orbital. This is called a * transition and is usually strong (high extinction coeffcient ). Groups of atoms involved in bonding are thus often called chromophores. The transition energy (or absorption wavelength) can be an indication for different types of bonds (carbon-carbon, carbon oxygen or carbon-nitrogen in a nitrile group). The probability of an electronic transition is proportional to the square of the electronic transition dipole moment, which is defined as: 3(16)Where is the wavefunction of the electronic state, n and is the ground state wavefunction. Equation 16 is as a measure for the overlap between orbitals in the ground state and in the excited state. In solution, interactions with the solvent can modify the energy gap of individual molecules leading to a distribution of transition energies. Vibrational excitations also contribute to the broadening of an electronic transition. The overall transition probability should be independent of these broadening effects, and is extracted by integrating over the absorption band. This integral provides is experimental measure for the transition dipole moment: 3d=k (17)Where, , and Charge transfer transitions: Much stronger absorption is found when complexing the metal ion with some suitable organic chelating agent to produce a charge-transfer complex. Electrons may be transferred from the metal to the ligand or vice versa.

Figure 3

In this compound analyzed, carbon to carbon bonds can be seen in the aromatic rings, and the carbon to nitrogen bond

Figure 4

In this compound the metal (Ru) attached to the ligand can be visualized.

C. biological importance of acid dissociationDuring dissociation, only the unionised form of a drug can partition across biological membranes due to hydrophobic lipids repelling ions off the membranes. While the ionised form tends to be more water soluble, and will become picked up by plasma. If the pH shifts the balance of dissociation towards the unionized form, the drug would be absorbed. If the pH shifts the balance of dissociation towards the ionized form, the drug would not be absorbed. Because most drugs are ionizable at different body pH ranges, the percent of ionization must be taken into consideration for when a drug is going to be synthesized. Using equation (9), the ratio of ionization can be calculated. From a calculated pKa value, the lipophilicity can be determined, from and then where the drug will be absorbed and what target tissue will reach.

Figure 5

Figure 5 shows drug as a weak acid, with a pKa of 4 inside the stomach.

The diagram shows how the UV Vis works and (courtesy Google images)Figure 6

III. Method:A 1.5 liter solution of the compound is placed in deionized water, placed on a stir plate and allowed to stir. In order to maintain a constant temperature the solution was kept far away from windows and vents. Once a pH was established, various concentration of (sulfuric acid) is added one drop at a time. The change in pH is not to exceed 0.1 units. A time period of at least 15 minutes is allotted to allow the pH to equilibrate in the solution. A scans are run after adding each drop in the UV-Vis spectrometer. Each of the following steps will repeated, and the pH was brought down to as low as the solution could go with until 18M sulfuric acid is used. Then NaOH (sodium hydroxide) will be added to bring the pH up to a basic pH range. Each individual graphs are plotted as absorbance versus pH, and where the absorbance values cross at a particular wavelength indicates the isosbestic point. After the isosbestic point is determined, the wavelength in the graph where the greatest change would be located, and a graph of the pH versus the absorbance at the specific wavelength would be analyzed in graphpad prism using a Boltzman sigmoidal fit. The function V50 would then indicate the pKa value as derived in the theory section. IV. Materials:Instrument: UV-visible spectrophotometer, three matched quartz cuvettes with 1 cm path length, Digital pH meter, deionized water, various concentrations of stir bar, and stir plate.V. Results & Discussion:CompoundCam PF6- QM B2RuCacid2+

pKa8.19 6.00 4.13

Ka1.25E-9 1.00E-6


Figure 7

Figure 8

VI. Conclusion:The application of spectrophotometric titration allowed the acid dissociation constant of the three compounds; CAM, QM and be found. The pKa of Cam was calculated to be 8.19, QM was 6.00 and B2RuCacid2+ 4.10. Notably QM only saw one pKa value. It hypothesized that the second pKa value is located at a low pH (less than zero) the pH probes would not be able to obtain proper pH readings. In the QM, the pKa being relatively close to pyridine (5.21) indicates the protanation site on the pyridine ring. Future trials will be done to validate these findings, to create an average and standard deviation, and obtain better accuracy and precision. B2RuCacid2+ pKa was comparative to that of acetic acid, (4.75), which seems reasonable as compounds with carboxylic acids tend to fall in the 4-5 range for pKa. The wavelength which CAM pKa was found at 338, QMs pKa was indicated at the 285 wavelength and B2RuCacid2+was at 340 wave length.

IV Appendix:

VII. Resources1. Salgado, L.E.V. and Vargas-Hernndez, C. (2014) Spectrophotometric Determination of the pKa,Isosbestic Point and Equation of Absorbance vs. pH for a Universal pH Indicator. American Journal of Analytical Chemistry, 5, 1290-1301.

2. "GraphPad Prism 5 Help." GraphPad Prism 5 Help. Graphpad Sofware, 1 Jan. 2007. Web. 27 Mar. 2015. .

3. Reijenga, Jetse, Arno Van Hoof, Antonie Van Loon, and Bram Teunissen. "Development of Methods for the Determination of PKa Values."Anal Chem Insights(2013): 53-71.US National Library of Medicine National Institutes of Health. Web. 23 Mar. 2015.4. Physikalisch, -. UV/VIS Spectroscopy. 07 Sept. 2014.

5. "How to Measure PKa by UV-vis Spectrophotometry." : A Chemagination Know-How Guide. Chemagination, 2009. Web. 23 Mar. 2015. .

6. Keiichiro Fuwa, B. L. Valle. The Physical Basis of Analytical Atomic Absorption Spectrometry. The Pertinence of the Beer-Lambert Law. Anal. Chem.; 1963; 35(8); 942- 946.

7. Mukerjee, Pasupati and Banerjee, Kalyan. A Study of the Surface pH of Micelles Using Solubilized Indicator Dyes J. Phys. Chem., 68, 12, 3567 - 3574, 1964

8. Wong, Flory, and Roxanne Cheung.CHEM 335: Physical Biochemistry Lab PKa of a Dye: UV-VIS Spectroscopy.Ishigirl.tripod. N.p., n.d. Web. 23 Mar. 2015. .

9. Pathare, Bebee, Vrushali Tambe, Shashikant Dhole, and Vandana Patil. "AN UPDATE ON VARIOUS ANALYTICAL TECHNIQUES BASED ON UV SPECTROSCOPY USED IN DETERMINATION OF DISSOCIATION CONSTANT."International Journal of Pharmacy4.1 (2014): 278-85.Pharmascholars. Web. 23 Mar. 2015.

10. Babic, Sandra, Alka J.M. Horvat, Dragana Mutavdzic Pavlovic, and Marija Kastelan-Macan. "Determination of pKa Values of Active Pharmaceutical Ingredients."Trends in Analytical Chemistry26.11 (2007): 1043-061.