View
215
Download
1
Category
Preview:
Citation preview
Ground State acid dissociation constant determination of Organic Imidazolium cations and Ruthenium (II) complexes
By Kane Logue
1
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
2
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,
3
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.
4
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
5
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)
6
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
7
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)
8
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
9
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
10
(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.
11
Figure 3
Figure 4
C. biological importance of acid dissociation
12
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
13
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,
14
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
15
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
16
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:
17
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
20
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.
21
Recommended