Health Concerns of Bisphenols and the Determination of
their Acid-Dissociation Constants by Capillary
Electrophoresis

UREAP Report
Summer 2020

Name: Zahkary Barone
Supervisor: Dr. Kingsley Donkor
Date of Project: May-September 2020
Date of Submission: October , 2020

In a study by Lehmler H et al (2018) analyzed urinary bisphenols levels in 1808
adults and 868 children participating in the national health and nutrition examination
survey in 2013-2014 in order to investigate demographic and lifestyle factors associated
with urinary levels of bisphenols. Specifically, Bisphenol F (BPF), Bisphenol S (BPS)
and Bisphenol A (BPA) were studied as BPF and BPS are replacing BPA in the
manufacturing of products containing polycarbonates and epoxy resins.
BPA is a high-volume industrial chemical with a global consumption of about 7.7
million metric tons in 2015. It us used for the manufacturing of poly carbonates and
epoxy resins. Due to growing human health concerns the use of BPA based plastics in
food and beverage applications is a concern. This comes from its potential toxicity in
humans, causing for manufacturers to attempt to replace use of BPA with structurally
similar chemicals such as BPS and BPF.
This experiment used ANOVA to compare results from the survey. Differences in
urinary concentrations of BPA, BPF, and BPS (log transformed) were compared among
various categorical variables. Linear regression analyses were done by including all
variables simultaneously to mimic the independent effects of each variable. Also,
urinary creatinine levels were adjusted in the model to account for urine dilution.
From the survey, BPA, BPS and BPF were detected in 95.7%, 89.4% and 66.5%
of randomly selected samples from the survey respectively. Furthermore, medial levels
of BPA in the U.S for adults were measured at 1.24 ug/L, while BPF and BPS levels
were only 0.35 and 0.36 ug/L respectively. In children, median BPA levels were
measured at 1.25 ug/L with BPF and BPS levels being recorded at 0.32 and 0.29 ug/L
respectively. The limits of detection for BPA, BPF, and BPS were 0.2, 0.2 and 0.1 ug/L
respectively. Urinary levels showed correlations with gender, race, family income,
physical activity, smoking and alcohol intake that depended on the specific bisphenol.
These results indicate that exposure of the general U.S. population to BPA substitutes
is almost ubiquitous.
In a study conducted by Kinch C et al (2015), zebrafish were used to link
Bisphenol A (BPA) mechanistically to disease etiology. BPA is a ubiquitous endocrine
disruptor that is present in many household items. Studies have shown it has roles in
causing obesity as well as cancer, and more relevant to the current experiment,
childhood neurological disorders like anxiety and hyperactivity.
Many other effects of BPA exposure have been noted, in humans and rodent
models, BPA has been associated with an increased risk of developing social,
psychiatric, and behavioral challenges later in life. BPA is a compound used in the
production of diverse consumer products which makes it particularly dangerous to
adults as everyday exposure is common. Furthermore, the fetal brain is especially

vulnerable due to its immature xenobiotic metabolizing system and the blood-brain
barrier
Results showed that by treatment of embryonic zebrafish with very low doses of
BPA and Bisphenol S (BPS) a common analogue, resulted in 180% and 240%
increases in neuronal birth (neurogenesis) within the hypothalamus. Also, restricted
BPA/BPS exposure specifically during the neurogenic window caused hyperactive
behaviors in zebrafish larvae. While human results are still scarce, high maternal urinary
BPA during gestation has been linked to hyperactivity and other behavioral disturbances
in the child.
The findings of this experiment provided mechanistic support that the neurogenic
period may be a window of vulnerability and opens up other areas of how endocrine
disruptors might prevent early brain development. Furthermore, BPA may not be the
only problem and the results suggest the removal of all bisphenols from consumer
merchandise.
In another experiment, Ullah A et al (2018) evaluated the toxicity of Bisphenol A
(BPA) and its analogs, BPB, BPF and BPS. The effects of chronic exposure to low
bisphenol doses in male rats would have an effect on spermatogenesis with outcomes
on oxidative stress and the reproductive system. This is in questions because BPA is an
estrogen-mimic endocrine disrupting chemical, commonly used in the making of
polycarbonate plastics and epoxy resins with toxic effects for male reproductivity.
Because of this, manufacturers have begun to move towards its analogs as substitutes
in products, although now, analogs such as BPB, BPF and BPS are proving to be toxic
as well.
In this study, male rats (22 days old) were exposed to water containing 0.1%
ethanol as a control group and different concentrations of BPA or its analogs in doses of
5, 25 and 50 ug/L in the drinking water for 48 weeks. This formed 13 groups, each
group with its own bisphenol (BPA, BPB, BPF or BPS) at the for mentioned
concentrations plus the control group. Bisphenol solutions in the water bottles were
replaced daily with fresh solutions. After the experimental period, animals were
weighed, euthanized and had blood collected from the heart. Reproductive organs were
also dissected and weighed and used for testing.
Results of this study showed a large change in the gonadosomatic index (GSI)
and relative reproductive organ weights. Oxidative stress in the testis was significantly
elevated while sperm motility, Daily sperm production (DSP) and the number of sperm
in epididymis were reduced. Plasma testosterone, LG and FSH concentrations were
reduced and estradiol levels were high in the group receiving 50ug/L doses. These
results suggest that exposure to BPA and its analogues for chronic duration can induce
structural changes in testicular tissue and endocrine alterations in the male reproductive

system. Furthermore, exposure for extended periods of time to BPA and its analogues
are capable of suppressing gonadotropins secretions from pituitary, exhibiting
estrogenic and antiandrogenic effects in mammals.
In this study, Moreman J et al (2017) analyzed the toxicity and teratogenic effects
of the bisphenols BPA, BPS, BPF and BPAF in zebrafish embryo-larvae and an
assessment on their estrogenic mechanism in an estrogen responsive transgenic fish.
This experiment targeted the heart, liver, somite muscles, fins, and corpuscles of the
Stannius. The effects of development and toxicity were studied in the wild type strain
while estrogenic potency ad tissue targets for estrogenic responses were analyzed in
the transgenic zebrafish.
BPA is a chemical incorporated into plastics and resins which contains
estrogenic activity and is associated with adverse health effects in humans and wildlife.
Because of this, similarly structured BPA analogues are widely used although their
potential toxicity and estrogenic activity in vivo so less known. They are also endocrine
disrupting chemicals (EDC’s) which possess structural similarities with endogenous
hormones that can alter hormone biosynthesis, biodegrading or excretions and
consistent exposure can alter biological homeostatic. This experiment shows that there
are differences in the effects and potencies between the different bisphenol analogues.
Furthermore, this study shows that BPA alternative induce similar toxic and estrogenic
effects as BPA, and that BPAF is actually more potent then BPA.
The results found that the toxicity was most prominent in BPAF, then BPA,
followed by BPF and lastly BPS. At concentrations between 1.0 and 200 mg/L (higher
than environmental levels) developmental deformities for larval exposures such as
cardiac edema, spinal malformation, and craniofacial deformities were observed. The
rank order for estrogen city was BPAF, followed by BPA and BPF, with BPS showing
the lowest levels. The bioconcentration factors were 4.5, 17.8, 5.3 and 0.067 for
exposure concentrations of 1.0, 1.0, 0.1, and 50 mg/L for BPA, BPF, BPAF and BPS
respectively. Final results show that the bisphenols tested are toxic to fish although
concentrations in the environment are lower than what was used in the lab.
Bisphenol A (BPA) is a well-known endocrine disrupting chemical with estrogenic
activity was studied by Ullah et at (2019) in this experiment. Widespread exposure of
individuals to BPA is suspected to affect a variety of physiological functions including
reproduction, development and metabolism. It is also used in many plastic consumer
products such as food container, water pipes, paper products, electronics, toys and
medical equipment. Humans can be exposed to it via dietary or non-dietary pathways.
While BPB, BPF, and BPS have been used as an alternative to BPA, data from many
agencies that monitor the environment have shown that these chemicals are going to
become a serious threat to both human and animal life.

This study observed rat models and the mechanism in which Bisphenol A (BPA)
and three of its analogues Bisphenol B (BPB), Bisphenol F (BPF) and Bisphenol S
(BPS) cause generation of reactive oxygen species (ROS), sperm DNA damage and
oxidative stress. Furthermore, the selective bisphenols will be compared on the toxic
effects they present to antioxidant enzymes of sperm.
In the in vitro section of this experiment, sperm were incubated with different
concentrations (1, 10, and 100 ug/L) of BPA and its analogues BPB, BPF, and BPS for
2 hours. Then, the species were observed for increases in DNA fragmentation,
formation of ROS, and affected levels of superoxide dismutase at higher concentration
groups. In the in vivo part of this experiment, the rats were exposed to different
concentrations 95, 25, and 50 mg/kg/day) of BPA, BPB, BPF, and BPS for 28 days.
It was observed that in the higher dose (50 mg/kg/day) groups treated with BPA,
BPB, BPF, and BPS, there was DNA damage although the motility of the sperm was not
affected. Results of the in vivo and in vitro studies showed that the selected bisphenols
exhibited altered DSP, affected sperm quality and DNA damage. Furthermore, evidence
of genotoxic potential and oxidative stress-inducing ability was present, which may be
due to the generation of ROS and LPO in sperm.
Bisphenol A (BPA) was studied by Zhang Y et al (2018) and is known to have
disrupting activity on the thyroid hormone signaling. Due to concerns regarding the
potential toxicities of alternatives, this study aims to investigate whether Bisphenol S
(BPS) and Bisphenol F (BPF), two leading analogues to BPA also present interference
with the thyroid hormone signaling pathway.
BPA is one of the highest volume chemicals used in the production of diverse
consumer products across the world. And its use has resulted in ubiquitous existence in
the environment and organisms. Studies have measured BPA concentrations in
picomolar and nanomolar levels in the water, and levels have even reached micromolar
levels in human blood and urine samples. Given the widespread human exposure and
possible adverse effects, BPA has been banned or is planning to be banned in many
products creating the need of a new substance to help create these products.
In this experiment, a series of in vitro and in vivo assays were conducted. The
results from the fluorescence competitive binding assay showed that BPS and BPF, like
BPA, bound to thyroid hormone receptors (TRa and TRb), although with less binding
potencies that BPA. Furthermore, the molecular docking data also showed their binding
potencies to the thyroid hormone receptors. Also, in the coactivator recruitment assay,
BPS and BPF recruited coactivator to TRB although not with TRa, also with weaker
potencies than BPA.

In the in vivo assay, the three bisphenols like T3 induced thyroid hormone
response gene transcription in tadpole, although the presence of T3 altered T3 induced
gene transcription in a biphasic concentration response manner. These results
demonstrated that BPS and BPF, have the potential to interfere with the thyroid
hormone signaling pathway like BPA. In conclusion, they found that generally,
bisphenols could act as thyroid hormone signaling agonists in the absence of T3;
however, in the presence of T3, they would agonize or antagonize thyroid hormone
actions or display biphasic concertation-response under certain conditions, highlighting
the potential risks of using BPS and BPF as alternatives to BPA.
The experiment by P.M. Nowak et al (2018) used capillary electrophoresis to
determine the acid-base dissociation/deprotonation constant (pKa) of several
cathinone’s (2-methylmethcathinone, 3-methylmethcathinone, 4-methylmethcathinone,
a-pyrrolidinovalerophenone, methylenedioxypyrovalerone, and ephedrine as well as 1phenylethylamine. This study shows an effective method for enhancing the reliability of
parameters and estimating the magnitude of errors that result from insufficient
dissipation of Joule heating. Lack of sufficient temperature control is a source of error in
the determination of pKa values. This method relies on the relationship between
electrophoretic mobility and actual temperature, and performing pKa determination with
corrected mobility values.
The method proposed for minimizing the impact of Joule heating on the accuracy
of pKa values determined is based on the estimation of actual temperature reached at
various temperatures of coolant, using the current values and then, plotting the obtained
electrophoretic mobilities against these values. Then, after function fitting the corrected
electrophoretic mobility values corresponding to the temperature of interest can be
observed by interpolation. Comparing the data obtained from trials without any
correction to ramping and Joule heating effects with data with correction methods, the
magnitude of the systematic errors attributed to both effects can be estimated. The
ramping effect can be measured by comparing the non-corrected and rampingcorrected trials while comparing the ramping-corrected and fully-corrected trials shows
the temperature rise effect.
Results showed that the average pKa error caused by Joule heating noted at
25°C was relatively small, only 0.04-0.05 pH units. Also, it was demonstrated that the
thermal dependencies of electrophoretic mobility, modelled during the correction
procedure may be useful to determine the optimal temperature for providing maximum
separation efficiency.
The study conducted by Foulon C et al (2007) showed the use of capillary
electrophoresis to determine the acidity constants of benzoxa-, benzothia-, and
benzoselena-zolinone derivatives. This study also compared the results acquired to the

results of determining acidity constants by potentiometry as well as spectrophotometry
experiments. This experiment utilized heteroatom or 6-benzoyl substituted derivatives to
induce changes in acidity constants to determine pKa values. Acidity constants were
determined simultaneously for two compounds characterized by different electrophoretic
mobility and pKa in the presence of a neutral marker.
The knowledge of a substance’s acidity constant is a key parameter for
understanding its chemical interactions between the compound of interest and its
pharmacological target. Relationships between the acidity constant and structure can
prove useful in drug design and explaining the biopharmaceutical properties of
substances like the ones used in this experiment. Many biologically active molecules
are fully or partially ionized at physiological pH and it has often been shown that the
presence of ionizable groups is necessary for biological activity and solubility. This is
the importance of accurate determination of pKa values for the discovery of new
molecules.
The results of this study showed that the acid-base dissociation constants of ten
weak acids containing benzoxa-, benzothia-, and benzoselena-zolinone rings were
determined. The use of capillary electrophoresis allowed for convenient, quick
determination of the pKa values of these compounds. The results obtained were in good
agreement with values obtained using bot the potentiometric and spectrometric
methods.
Beach D et al (2018) used a CE-MS/MS technique that is well suited for
analyzing polar marine toxins and offers very high-resolution separation. This method
was developed with a custom-built interface for sensitive multiclass analysis of paralytic
shellfish toxins, tetrodotoxins, and domoic acid in seafood. A highly acidic background
electrolyte (BGE) (5 M formic acid) was developed to maximize protonation of analytes
and to allow a high degree of sample stacking to improve the limits of detection in this
experiment. The experiment used very low pH BGE where the EOF of the CE was
almost entirely suppressed.
Recently, the CE-MS/MS method for the analysis of neurotoxin B-Nmethylamino-L-alanine (BMMA) was successful at separating the analyte from
potentially interfering matrix components including several isomers by the same group,
which helped create the method for this experiment. This includes the utility of CEMS/MS as a multiclass analytical method by using a highly acidic BGE.
This method was applied to a wide range of regulated toxin analogues which
exhibited a high degree of selectivity between toxin isomers and matrix interference.
And minimal ionization suppression was observed when the response from neat and
mussel-matrix-matched standards was corrected with multiple internal standards. The
method was then transferred to a commercial CE-MS/MS system to demonstrate its

widespread applicability for use in both R and D and routine regulatory settings.
Accuracy of this method was evaluated through quantitative analysis of mussel matrix
(certified) reference materials and spiked samples.
The analysis of shellfish matrix reference materials and spiked samples
demonstrated good accuracy and precision. The limits of detection in mussel tissue
were 0.0052 mg/kg for tetrodotoxins, 0.169 mg/kg for domoic acid and between 0.0018
and 0.120 mg/kg for paralytic shellfish toxins in which all showed good linearity.
In a study by Zrnčić M et al (2015), the determination of the thermodynamic acid
dissociation constant (pKa) of 22 common pharmaceuticals by capillary electrophoresis
in aqueous media was done. The species of interest belong to different pharmacological
groups such as macrolides, fluoroquinolones, sulfonamides, B-lactams, tetracyclines,
and other miscellaneous pharmaceuticals. The electrophoretic mobilities of the
investigated analytes were monitored in a pH range of 2.00 to 10.82. Obtained data
were plotted on effective mobility vs pH graphs. And mobility measurements were done
in triplicate.
The acid dissociation constant is an important factor in pharmaceuticals as it can
allow for better prediction of there fate and behavior in an environment. Furthermore,
the acid-base property of a pharmaceutical substance is one of several crucial
properties that can be used to estimate the adsorption, distribution, metabolism and
excretion of compounds in biological systems. And, the retention and electrophoretic
behavior of ionizable compounds strongly depends on the pKa of the compound and
thee pH of the mobile phase. Also, the knowledge of an analytes pKa is useful in the
optimization of machines like capillary electrophoresis for the separation of compounds.
This study utilized sodium dihydrogen phosphate and disodium hydrogen
phosphate the determination of the thermodynamic pKa values was done by
extrapolations to zero ionic strength. And experimentally obtained acid dissociation
constants were interpreted using structural formulae of investigated analytes and the
moieties corresponding to specific pKa were identified.
While the pKa values for many of the pharmaceuticals are not well known, the
results of this experiment were in good agreement with the analytes that have been
determined and published. They were also compared to predicted values using
ChemSpider and/or ACD Labs computational programs. Most r2 values obtained were
higher than 0.99. Also, the %RSD values for the effective mobilities calculated were less
than 1.5% for all compounds.
This experiment done by MORI M et al (2001) focused on the application of
capillary electrophoresis (CE) using sulfated B-cyclodextrin (SCD) for the separation of
various sizes of alkylphenols as well as bisphenol A and S. In this study the application

of CE using SCD was examined to separate a mixture of BPA, BPS and six
alkylphenols with varied chain lengths from ethyl to heptyl phenol. This system was also
applied to the separation of 4-octylphenol (OP) and 4-nonylphenol (NP) isomers
The addition of sulfated B-cyclodextrin was crucial to the study as in trials without
SCD, all the phenols migrated with the same velocity as the electroosmotic flow. This is
because most of the compounds used are electrically neutral, making them difficult to
separate by only running a buffer. Upon addition of SCD, separation of alkylphenols
was achieved based on the capabilities of each analyte to bind to do hydrophobic cavity
of B-cyclodextrin. This method is known as micellar electro kinetic capillary
electrophoresis (MEKC) using sodium dodecyl sulfate (SDS) which separates neutral
compounds on the bases of their distribution into the micelles. Furthermore, CD is used
as an additive with SDS to improve separating in MEKC.
Binding constants between analyte phenols and SCD were determined from
Benesi-Hildebrand plots obtained by CE and UV-visible measurements and showed that
the greater the hydrophobicity of phenols the largest the binding constants. This system
of using SCD was also effective for the separation of 4-octylphenol and 4-nonyl phenol
isomers having longer alkyl chains. The binding constants between SCD and analyte
phenols were also estimated and the effect of organic solvents on the separation of
alkylphenols was examined.
Results showed that using CE and SCD together would prove to be a good
method for the separation of alkylphenols and bisphenols, specifically highly
hydrophobic alkylphenols. Also, the migration order in the running buffer was correlated
with the length of the alkyl chain in the phenols. Addition of an organic solvent cause a
decrease in resolution between alkylphenols but improved separation between BPA and
BPS. This also provided complete separation of OP and NP’s.
In a study by Konášová R et al (2015), the acid dissociation constants (pKa) of
six widely used triazole fungicides (cyproconazole, epoxiconazole, flusilazole,
tebuconazole, penconazole, and propiconazole) were determined by pressure assisted
capillary electrophoresis (pCE). The pKa values were determined from the dependence
of effective electrophoretic mobility of the triazole fungicides on p[H+] of the background
electrolyte (BGE) by non-linear regression analysis. Due to BGE’s being strongly acidic,
and there for having an increased ionic strength, p[H+] was chosen over pH for this
experiment. Before pKa calculations were done, the measured effective electrophoretic
mobilities were corrected to a reference temperature of 25°C and constant ionic
strength of 25 mM.
The acid dissociation constant of a weak base is important to determine as it
provides information about a form of the compound (neutral or protonated) in a solution.
The protonation of a weak base can increase its solubility in water and may change its

adsorption in the soil. Furthermore, the ionized form has different abilities to enter into
organisms as compared to the neutral form. The knowledge of pKa of weak electrolytes
is important in analytical chemistry as it provides information on how to optimize
experimental conditions for separation and analysis. Specifically, the motivation to
determine the pKa of triazole fungicides were to better estimate potential risk to the
environment by these compounds, and to fill in missing or ambiguous values in
literature to be used in future experiments.
CE was found to reduce the time on the CE analysis of the strongly acidic BGE’s
and increased the throughput of pKa determination by CE. Also, attachment of the
substituents with different structures in the position B to the triazole ring resulted in the
decreased electron density of the ring and decreased basicity of the above triazole
fungicides as compared to the unbound molecule.
Results showed that the pKa values were found to be in the range of 1.05 – 1.97
which are confirmed by values calculated by SPARC online pKa calculator. It was also
determined that the analyzed triazole fungicides are very weak bases that remain in the
non-protonated form in the soil at natural environmental pH.
In 2018, Qian C et al studied the effects of adding external pressure to analytes
in the process of capillary electrophoresis (CE) which usually results in an increased
band broadening, especially if the pressure induced flow is significant. This negatively
effects the resolution in pressure assisted capillary electrophoresis (PACE). Although,
frontal analysis (FA) can potentially benefit from using an external pressure while
avoiding the negative effects in other modes of CE.
In CE, charged molecules with different electrophoretic mobility are separated in
the presence of an electric field and species are driven by an electroosmotic flow (EOF).
This produces a flat shaped flow profile of the EOF driven flow. On the other hand,
pressure driven laminar flow in an open tube has a parabolic flow profile. This parabolic
profile has a negative effect on the separation efficiency making it uncommon that a
significant large pressure is applied during a CE process. The addition of pressure in
the capillary can have many effects. In PACE the pressure is applied on the same
direction with the analyte migration. This can be used for accelerating the migration of
low-mobility molecules.
Frontal analysis, when combined with CE, requires a much larger amount of
sample than traditional CE methods. In CE-FA, only the front of the plateaus, instead of
the peaks, are measured in the electropherograms, and the concentrations of ligands
are determined with a pre-made calibration curve. As a result, CE-FA is not dependent
on baseline resolution of the analytes, thus more tolerant to band broadening caused by
the application of an external hydrodynamic pressure.

In this experiment, the possible impact from the external pressure was simulated
by COMSOL Multiphysics. Results showed that under a typical CE-FE set up, the
detected concentrations of analyte will not be significantly affected by and external
pressure lower than 5 psi. Furthermore, based on experimental results, it was
concluded that PACE-FA can reduce the time of binding analysis while maintaining
accuracy of the measurements.
Qian C et al studied a popular method for determining binding affinities of
molecular interactions in 2017 known as capillary electrophoresis frontal analysis (CEFA). Consequently, the current data processing method requires specific mobilities of
the binding pair in order to obtain accurate binding constants.
CE based methods can be roughly categorized into two groups, there is affinity
CE and vacancy affinity CE. In these methods the binding constant is deterred by the
shift in mobility of ligand molecules while in the Hummel-Dreyer method, vacancy peak
method and CE frontal analysis, the binding constant is determined by concentration
change of related species due to the binding interaction. Although it is not common yet,
the method of CE with frontal analysis can be a better choice for high-throughput
determination of binding constants because of the simplicity in procedure and its ability
to determine binding stoichiometries.
The purpose of this study is to show that significant errors can result when the
mobilities of interacting pieces do not meet the requirements. This lowers the
applicability of CE-FA in many real-world applications. In this experiment, an
electrophoretic mobility-based correction method is developed based on the flux of each
species. The equations and effectiveness of this method were evaluated via a
simulation program and a pair of model compounds.
In this experiment, ibuprofen and hydroxypropyl-B-cyclodextrin are used to
demonstrate the differences in the obtained binding constant by CE-FA when different
calculations methods are used. The results are compared with those obtained by affinity
capillary electrophoresis (ACE). The results suggest that CE-FA, with the mobility-based
correction method can be generally applicable. Specifically, for the binding pairs that did
not fulfill the prerequisites of the current CE-FE data processing method; the new
equation derived in this work has proved effective in correcting the systematic error
caused by mobility difference of various species. This mobility-based correction method
has also shown no restrictions to the type of molecules involved in the interaction.
In this experiment by TAKAYANAGI T et al (2016), the acid-base equilibrium of
hexamethylenetetramine (hexamine) was analyzed with its effective electrophoretic
mobility by capillary zone electrophoresis (CZE). Hexamine, is degradable in weak
acidic aqueous solutions and produces ammonia and formaldehyde. CZE is an
alternative method for the analysis of acid-base equilibrium in homogenous solutions

including conventional potentiometric and spectrophotometric titrations. In CZE, the
equilibrium reactions are analyzed in an aqueous solution without any organic cosolvents and without any side reaction, such as a partition to the stationary phase.
In CZE, the acid-dissociation constants (pka) values are determined though
measurements of the effective electrophoretic mobility of the equilibrium species of
interest. Because this is still a separation technique, other substances and impurities
are allowed to coexist and are resolved.
To determine the pKa value, N-ethyl quinolinium ion was used as an internal
standard of the electrophoretic mobility. The CZE signal of hexamine became closer to
the EOF with increasing pH of the separation buffer suggesting hexamine came to be
neutral form acidic to neutral pH conditions. Changes in the effective electrophoretic
mobility of hexamine were plotted against the pH of the separation buffer where the
effective electrophoretic mobility of hexamine was standardized with that of the N-ethyl
quinolinium ion.
In this study, the effective electrophoretic mobility of hexamine was measured in
the range of pH 2.8 to pH 6.9. The acid-base dissociation equilibrium of the protonated
hexamine was analyzed based on mobility change. The monoprotic acid-base
equilibrium of hexamine was confirmed through comparisons of its electrophoretic
mobility with the N-ethyl quinolinium ion and with the monocationic N-ethyl derivative of
hexamine. This was also compared to the slope analysis of the dissociation equilibrium.
The results of this experiment determined the acid dissociation constant to be pKa =
4.93 +- 0.01 at an ionic strength of 0.020 mol dm-3-.
In this study by Takatangi T et al (2015), the acid dissociation constant of
pravastatin was determined under degraded conditions. It was determined with residual
pravastatin through its effective electrophoretic mobility in order to study the ability of
CZE analysis. Pravastatin was degraded in an acidic solution of pH = 2.0 for 5 hours.
The degraded solution was subjected to the measurement of the effective
electrophoretic mobility by capillary zone electrophoresis. The results of this experiment
agree with some reported values.
Pravastatin, is a cholesterol-lowering compound that completely inhibits the
microsomal enzyme 3-hydroxy-3-methylglutaryl-co enzyme A (HMG-CoA) reductase.
Pravastatin is degradable under acidic pH conditions, and its lactonized and/or C-6epimerized forms are supposed to be generated. The acid dissociation constant (Ka)
has been determined by capillary zone electrophoresis (CZE) in pressure-assisted
mode using the effective electrophoretic mobility with changing pH of the separation
buffer.

CZE analysis is convenient using the effective electrophoretic mobility of an
analyte with changing pH of the separation buffer. CZE includes the electrophoretic
separation, therefore like normal capillary electrophoresis, is a separation method that
will resolve impurities or degraded species from the analytes of interest. In this
experiment, the degraded species are neutral and therefor migrated at the EOF time.
CZE separation was utilized to determine the pKa by electrophoretic ally separating the
degraded species.
The results of this experiment found that the Ka value was successfully
determined by the CZE analysis or residual pravastatin. The pKa value determined was
4.46 +/- 0.03 (I = 0.01 – 0.02; error: standard error) which agreed with one obtained
freshly prepared solution and reported values. The signal height of the
electropherogram of pravastatin was decreased by the degradation and the degraded
species were detected by and MEKC format.
The ionization constant (pKa) and limiting ionic mobility of 3-chlorotyrosine (CT)
and 3-nitroyrosine (NT) were determine by capillary one electrophoresis (CZE) by Ren
H et al (2013). Furthermore, the diameter of the hydrated ion was calculated as well. CT
and NT are oxidation products of reactive oxidative species (ROS) and other radicals
under inflammatory conditions. Chlorination of tyrosine decreases the ionization
constant of the phenolic group which results in an altered protein conformation and
function similar to the way that nitration of tyrosine can alter enzymatic activity or protein
function.
The determination of the ionization constant is important as pKa plays an
important role in biochemical activity and electrophoretic behavior along with other
physiochemical parameters. And CZE is a useful tool for the determination of these
values. In this study, it is assumed that a cationic capillary, with revered and detectable
EOF, should be more suitable for cations at low pH due to electrostatic repulsion; this
was based on the conclusion that electrostatic interactions can bring variations in pKa
values.
When attempting to determine the limiting ionic mobilities and pKa values by
CZE, the team conducting this experiment found that great caution should be taken,
especially when monitoring experimental conditions or determining the parameters used
in the calculations. They also suggested that the charge of both the analyte and the
capillary inner surface should be taken into account when calculating pKa and ionic
mobility, and that electrostatic interaction should be avoided. Furthermore, some
formulas in this paper are helpful to use if the diameter of a compound is unknown.
This experiment was done over a wide pH range, measurements were carried
out in a polyionic liquid (PIL) modified capillary at a low pH (1.80-4.00) and a bare fused
capillary at an upper pH of 3.00-11.00. The electrostatic interaction between analytes

and the inner wall were suppressed. The parameters were calculated form a simple
theoretical model. Results were compared and agreed with predictions from the
database of Peakmaster software.
Nowak P et al (2016) conducted an experiment in which the acid dissociation
constant (pKa) of 20 structurally diverse coumarin derivatives were determined by
capillary electrophoresis; for many of these compounds the pKa values were determined
for the first time. Coumarins acidity strongly depends on chemical structure making
them very diverse and a good modal for theoretical investigation of structure0acidity
relationships.
Notably, in this experimental an amine permanently coated capillary was used
instead of the bare silica or other coated capillaries for the determination of pKa as it
ensured good precision and shorter migration times. Also, the traditional methodology
relying on measurements in a broad pH range and fitting of a sigmoidal function was
compared to an alternative simplified method that requires less measurements. This
reported for the first time where only two electrophoretic mobility values suffice for pKa
estimation. In this method, the first value corresponds to the partially ionized form and
its measured experimentally while the second one corresponds to the totally ionized
form and is either measured experimentally by the two-valve method, or directly from
the molecular mass (one-valve method). The reliability of this method has been
confirmed by the analytical predictions, comprising resolutions, migration times and
order, and peaks overlapping.
For the determination of pKa in this study, the traditional method utilized the
relations between the effective electrophoretic mobility and pH. In the case of the new
approach, the mobility values related to partial ionization and total ionization were used
while experimentally measuring electrophoretic mobility.
The values obtained in this experiment varied between 4.16 – 9.10 pH units. This
suggests some interesting structure-acidity relationships. Furthermore, it was shown,
despite a limited measurements number, that the alternative approach may be
consistent with traditional methodology, yielding the relatively low pKa deviation. In
conclusion, the use of an amine capillary companied with the simplified calculation
method is a fast and reliable method for pKa determination.
In this experiment, Tůmová T et al (2016) determined the thermodynamic acidity
constants (pKa), and actual ionic mobilities of polycationic antimicrobial peptides (AMPs)
by capillary electrophoresis. Bacterial resistance to conventional antibiotics is a major
reason for intensive research into new therapeutics. And the determination of
physiochemical properties of potent drug candidates is important for physical modeling
of pharmacokinetics and can reduce time taken in the early stages of drug development

especially when solubility, permeability, acid-base properties, lipophilicity and protein
binding data is already known.
In this study, the effective electrophoretic mobilities of AMPs were measured with
a series of background electrolytes within a pH range of 2.00-12.25 at a constant ionic
strength of 25 mM at ambient temperature. Furthermore, a polybrene coated fused
silica capillary was used in order to suppress sorption of cationic AMPs to the capillary
wall. The Haarhoff-Van der Linde peak fitting function was used to calculate correct
migration times of some AMPs peaks that had been altered by electromigration
dispersion. Furthermore, the measured effective mobilities were corrected to 25°C.
Also, mixed acidity constants and ionic mobilities of the AMPs were determined by the
nonlinear regression analysis of pH dependence of their effective mobilities, and the
mixed acidity constants were recalculated to thermodynamic pKa using the DebyeHuckel theory.
The results of this experiment found that the thermodynamic pKa of imidazolium
group of histidine residues was in the range of 3.72 – 4.98. And the pKa of the a-NH3+
group was in the range of 6.14 – 6.93. And finally, that the pKa of the ε-NH3+ group of
lysine was in the range of 7.26 – 9.84 and varied depending on the particular amino
acid sequence of the AMPs.
The actual ionic mobilities of AMPs with positive charges from one to six
elementary units achieved values of 9.8 – 36.5x10-9 m2V-1s-1; also, the actual ionic
mobilities of peptides from the first to the third protonation degree are growing
proportionally to the increasing effective charge. This is untrue for tetra to hexavalent
AMPs though as the growth of their effective and ionic mobilities is much lower, which
maybe be due to the condensation of counter ions.
Matyjaszczyk K et al (2017) determined the thermodynamic acid dissociation
constants (pKa) of 16 anthracycline antibiotics by the analysis of the dependence
between measured electrophoretic mobility and the pH of the buffer using the capillary
zone electrophoresis (CZE) method. pKa determination in anthracyclines is important as
it can lead to optimization of their efficacy/toxicity ratio. Furthermore, it can also
influence drugs uptake into tumor cells, as well as their penetration into the
cardiomyocyte and vascular endothelium.
CZE is a well-known approach for pKa determination. Often, it is done by
measuring the differences in the effective electrophoretic mobility of an ionizable
compound in a series of buffers with different pH and constant ionic strength. The
analytes will show maximum electrophoretic mobility while fully ionized and lack mobility
when not ionized allowing for the separation of species.

In this study, doxorubicin (DOX) and daunorubicin (DAU), their epimers,
epidoxorubicin (EDOX) and epidaunorubicin (EDAU), as well as novel anthracycline
derivatives containing piperidine (FPIP), morpholine (FMOR) and hexamethylenoimine
(FHEX) rings in the formamidine group of the daunosamine moiety were the species of
interest. Modifications in the daunosamine structure of anthracycline caused a
significant change in the pKa determined in this experiment. Two pKa values were
determined for each molecule according to the assumption of two ionized states of each
analyte. The greatest differences between the set of pKa values obtained was observed
for EDOX-FPIP and DAU-FPIP while the parent drugs showed the greatest similarities.
The results from this experiment confirmed the ampholytic character of
anthracyclines with at least two ionization states. The pKa values were determined to be
in the range of 8.36 – 9.28 and 9.38 – 11.48 for pKa1 and pKa2 respectively. These
values come from the ionization of amino (pKa1) and phenolic groups (pKa2). Structural
modifications in the daunosamine moiety of the studied anthracyclines affected their
pharmacological properties, such as antiproliferative activity.
In this study a Malý M et al (2020) created new method for determining the data
of a weak electrolyte by nonlinear regression of effective electrophoretic mobility versus
buffer composition dependence when measured in a set of background electrolytes
(BGEs) with various pH. This method differs from traditional approaches as the
nonlinear regression is performed on limiting mobility data calculated by Peak Master’s
correction engine, instead of the raw experimental mobility data. This removes the
requirement to perform all measurements at a constant ionic strength. Corrections for
experimental data for zero ionic strength were done via the extended Debye Huckel
model and Onsager-Fuoss law.
This study was done to devise the computer program AnglerFish that performs
the necessary calculations in a user-friendly fashion. This includes calculating all
thermodynamic pKa values and limiting electrophoretic mobilities for arbitrarily charged
substances with any number of ionic forms in just one run. It is also equipped with the
buffer composition of the set of BGEs and experimentally measured effective mobilities
of the inspected weak electrolyte.
Thermodynamic acidity constants (also known as acid or acid-base dissociation
constants or ionization constants) and limiting ionic mobilities are fundamental
physiochemical characteristics of a weak electrolyte, that is, a weak acid or weak base
or ampholyte. And acidity dissociation constant and electrophoretic mobilities are
fundamental physiochemical constants that characterize and ionic constituent. Both
constants determine the effective mobility of the analytes in the electromigration
movement in capillary electrophoresis and therefore have a direct impact on the quality

of separation in these methods. And the ability to accurately quantify these constants is
essential to multiple fields in chemistry.
Finally, in an experiment conducted by Tanikami Y et al in 2020, the acid
dissociation constants (pKa) of nine kinds of flavin (FL) analogues as molecular catalyst
candidates were determined by capillary zone electrophoresis (CZE). The effective
electrophoretic mobility of the flavin analogue of interest has been measured with the
residual substance. The pKa values of the flavin analogues were analyzed through the
changes in effective electrophoretic mobility with varying pH of the separation buffer. In
some cases, specifically analogues possessing carboxylic acid moiety, a pressureassisted CZE was utilized for the estimation of pKa values; this method also aided
determinations of values at weakly acidic pH conditions where the EOF is slow.
Acid dissociation constants have traditionally been determined by potentiometric
and spectrophotometric titrations. Although, the homogeneous titrations are not
applicable to such substances as these are not pure or degradable. The spectrum shift
is indispensable for the pKa determination by the spectrophotometric titration. The
advantage of CZE for determining the pKa of the selected analytes is that its method is
based on the change in the effective electrophoretic mobility with
protonation/deprotonation reactions under varying pH (pH 7 in this study). Furthermore,
CZE analysis is applicable to substances that contain impurities or are degradable.
As a result of this study, the acid dissociation constants were determined by CZE
for a series of molecular catalysts candidates of FL analogues. Even though some of
the FL analogues were degradable under alkaline conditions and/or light exposure, the
pKa values were successfully determined. This was done by analyzing changes in
electrophoretic mobility by the protonation or deprotonation in CZE. CZE was also
useful for the separation of the target analyte from the surrounding matrix.

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