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

By Kane Logue

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Table of Contents

I. Abstract pg 3

II. Introduction pg 3

a. Compounds analyzed pg4

b. Acid Base Theory pg 5

c. Factors that affect pKa pg 7

d. UV-Vis pg 8

e. π π* transition pg 11

f. biological importance of acid dissociation

III. Method pg 14

IV. Materials pg 14

V. Results and Discussion pg 14

VI. Conclusion pg 22

VII. Appendix

VIII. Sources pg 23

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I. Abstract

Several 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 it affects pH, absorbance of light, and the metabolism of the compound in

the body1. In pharmaceutic aspects, pKa affects 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 pKa 

II. 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,

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N

NRu(bpy)2

N N

CO2H

bpy

B2RuCacid2+

2PF6-

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 pKa values

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:

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.

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N

N

N

N

N

CH3

H3C

QM

PF6-

NN

NCH3

H3C

H2N

PF6-

CAM

NN

NCH3

H3C

H2N

PF6-

CAM+

NN

NCH3

H3C

H3N

PF6-

CAM+2

H+

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 π to π bonds. Uses of these flourophores could include use as a dye for staining of

certain structures, such as a substrate of enzymes, 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:1

HA + H 2O A−¿¿ + H 3 O+ ¿¿ (1)

The HA transfers a proton to water, and it becomes the anion ( A−¿¿. This anion tends to

retrieve the proton and behave as a base; therefore A−¿¿ is referred to as the “conjugate base” of

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N

NRu (bpy)2

CO2H

B2RuCacid 2+

N

NRu (bpy)2

CO2-

B2RuCacid +

OH-

QM2+ H+QM3+ Ka2

QM+ H+QM2+ Ka1

acid HA, and HA and A−¿¿ 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 H 3 O+¿¿ 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:1

Ka = ¿¿ (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 CT becomes very acidic, the entire indicator exists as HA.

The absorbance of the solution at a given wavelength λ is given by the following equation:1

AHA= ε HA * b *CT (3)

ε HA is the molar absorptivity of HA at wavelength (λ) 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

AA−¿ ¿= ε A−¿ ¿ * b *CT (4)

ε A−¿ ¿ is the molar absorptivity of A−¿¿. At an intermediate pH, the absorbance is given by:

AT= ε HA∗b∗¿ CHA + ε A−¿∗b∗¿¿ C A−¿ ¿ (5)

Where the total concentration can be defined in any condition as:1

CT=C HA+CA−¿ ¿ (6)

For a given CT, Equations (3)-(6) can be combined to obtain the following:1

¿¿ = CA−¿

CHA=

A−A HA

¿¿ ¿ (7)

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This relationship must be evaluated at multiple wavelengths, including one where HA

absorbs substantially but A−¿¿ does not, one where A−¿¿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 A−¿¿are present in

appreciable concentrations. Ka can be evaluated graphically by converting Equation (2) to

logarithmic form:1

−log Ka=−pH−log ¿¿-Log(Ka)= -log¿ + -log¿¿ (8)

This can be re written after algebraic manipulations into the following equation from the classic

Henderson Hasselbach equation:1

pKa= -log¿ (9)

In addition, the combination of Equation (7) and the definition equation of pKa=−log[Ka]

results in the following equation:1

log ¿ (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 ε HA = ε A−¿ ¿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

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absorbance=Bottom+(Top−Bottom )

1+e( p ka− pH

slope ) (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

λ = hcv =( En−E0 ) (14)

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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

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The wavelength (λ) 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

λ = cv=2πc

ω (15)

where c is the speed of light. The angular frequency ω = 2πv (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. π π* transition

For molecules that possess π bonds like alkenes, alkynes, aromatics, acryl compounds or

nitriles, light can promote electrons from a π bonding molecular orbital to a π anti-bonding

molecular orbital. This is called a π π* transition and is usually strong (high extinction

coeffcient ε). Groups of atoms involved in π bonding are thus often called chromophores. The

transition energy (or absorption wavelength) can be an indication for different types of π bonds

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(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

μ0 n=e ∫ ψ0 ( r⃑ ) rψ 0m ( r⃑ ) (16)

Where ψn is the wavefunction of the electronic state, n and ψ0 is the ground state

wavefunction. Equation 16 is as a measure for the overlap between orbital’s 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: 3

|μ0 n|2=k∫ ε ( λ )

λdλ=kε( λmax ) Δλ

λmax (17)

Whereλmax=centralwavelengt h, Δλ=full widt hat half maximum, and ε

( λmax ) extinction coeffcient at λmax

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.

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Figure 3

Figure 4

C. biological importance of acid dissociation

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

visualized.

In this compound analyzed, carbon to carbon π bonds can be seen in

the aromatic rings, and the carbon to nitrogen π bond

During 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

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

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 H 2 SO4 (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,

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The diagram shows how the UV Vis works and

(courtesy Google images)

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 H 2 S O4∧NaOH ,stir bar, and stir

plate.

V. Results & Discussion:

Compound Cam PF6- QM B2RuCacid2+

pKa 8.19 6.00 4.13

Ka 1.25E-9 1.00E-6 7.41E-5

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Figure 7

Figure 8

VI. Conclusion:The application of spectrophotometric titration allowed the acid dissociation constant of the three compounds; CAM, QM and B2RuCacid2+.to 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

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compounds with carboxylic acids tend to fall in the 4-5 range for pKa. The wavelength which CAM pKa was found at 338, QM’s pKa was indicated at the 285 wavelength and B2RuCacid2+was at 340 wave length.

IV Appendix:

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215 265 315 365 4150

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Absorbance

303

254

200 300 400 500 600 700 8000

0.5

1

1.5

2

2.5

Wavelength (nm)

Abso

rban

ce

18

200 250 300 350 400 450 500 550 6000

0.5

1

1.5

2

2.5

3

3.5

Wavelength (nm)

Abso

rban

ce

VII. Resources

1. Salgado, L.E.V. and Vargas-Hernández, 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.

<http://www.graphpad.com/guides/prism/5/user-guide/prism5help.html?

reg_classic_boltzmann.htm>.

3. Reijenga, Jetse, Arno Van Hoof, Antonie Van Loon, and Bram Teunissen.

"Development of Methods for the Determination of PKa Values." Anal Chem

19

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.

<http://www.chemagine.co.uk/resources/pka.htm>.

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.

<http://ishigirl.tripod.com/pchem/pka_sample.pdf>.

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

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CONSTANT."International Journal of Pharmacy 4.1 (2014): 278-

85. Pharmascholars. Web. 23 Mar. 2015.

10. Babic, Sandra, Alka J.M. Horvat, Dragana Mutavdzˇic´ Pavlovic, and Marija

Kasˇtelan-Macan. "Determination of pKa Values of Active Pharmaceutical

Ingredients." Trends in Analytical Chemistry 26.11 (2007): 1043-061.

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