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Micellar catalyzed reactions for flow injection analysis

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Title:
Micellar catalyzed reactions for flow injection analysis
Creator:
Hernández Torres, María A., 1958-
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[s.n.]
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Language:
English
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x, 77 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Catalysis ( jstor )
Cyanides ( jstor )
Flow velocity ( jstor )
Fluorescence ( jstor )
Micelles ( jstor )
Reaction kinetics ( jstor )
Reagents ( jstor )
Signals ( jstor )
Standard deviation ( jstor )
Surfactants ( jstor )
Chemistry thesis Ph. D
Dissertations, Academic -- Chemistry -- UF
Instrumental analysis ( lcsh )
Micelles ( lcsh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1986.
Bibliography:
Bibliography: leaves 73-76.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by María A. Hernández Torres.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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17689643 ( OCLC )
AEW6261 ( NOTIS )
AA00004861_00001 ( sobekcm )

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MICELLAR CATALYZED REACTIONS
FOR FLOW INJECTION ANALYSIS






BY






MARfA A. HERNANDEZ TORRES


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1986




MICELLAR CATALYZED REACTIONS
FOR FLOW INJECTION ANALYSIS
BY
MARA A. HERNNDEZ TORRES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986


To my parents,
Saul and Carmen,
with all my love.


ACKNOWLEDGMENTS
I would like to express my gratitude and appreciation to my
research director, Dr. John G. Dorsey, for his invaluable assistance,
guidance and encouragement throughout the years. I was very lucky to
have him as a teacher. His special qualities helped me to develop as
a professional, and at the same time he allowed me to grow as an
individual. His view of science and life created a good and healthy
environment for learning. His lessons will always be remembered.
Special thanks are given to Dr. Morteza G. Khaledi, for his
helpful suggestions and advice at the beginning of this project.
I would like to express my gratitude to my colleagues and friends
in Dr. Dorsey's research group, for their friendship and daily
encouragement. I treasure very much the time spent at the lab with
all of them even when life was tough. I will miss them all!
I would like to thank the faculty members at the University of
Florida with whom I was in contact during my graduate years. Special
thanks are due to the members of my committee, for their contributions
to my learning experience.
I would like to mention Cindy Zimmerman, for her promptness and
accuracy in typing the final draft of this thesis.
There are no words to express how thankful I am to my family and
friends. Their love, support, encouragement, patience, and faith in
me made possible this new achievement in my career. I want to thank
i ii


my grandparents, Avelina and Jose Antonio, for their care throughout
the years.
iv


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
CHAPTERS
I INTRODUCTION 1
Principles of Flow Injection Analysis 1
Properties of Surfactants and Micelles in Aqueous
Solution 10
II THEORY AND BACKGROUND 17
Chemical Kinetics in a Flow Injection Analysis System....17
General Features of Micellar Catalysis 19
III EXPERIMENTAL: FIA SYSTEM FOR PYRIDOXAL DETERMINATION....23
Appara tus 23
Reagents 25
Procedure 25
IV MICELLAR CATALYSIS IN THE DETERMINATION OF PYRIDOXAL
BY FLOW INJECTION ANALYSIS 26
Results and Discussion 26
Measurements of Dispersion 55
V CONCLUSIONS AND FUTURE WORK 70
REFERENCES 73
BIOGRAPHICAL SKETCH 77
v


LIST OF TABLES
Table Page
IFigures of merit for pyridoxal determination 40
IIVariable parameters for the Modified Simplex Optimization
program 42
IIIOptimized and fixed variables for the determination of
pyridoxal 45
IVFigures of merit for pyridoxal determination with
conditions obtained by Modified Simplex program 47
VVariance and standard deviation values for pyridoxal
in aqueous media at 10% peak height 59
VIVariance and standard deviation values for pyridoxal
in aqueous media at 30% peak height 60
VIIVariance and standard deviation values for pyridoxal
in aqueous media at 50% peak height 61
VIIIVariance and standard deviation values for pyridoxal
in 0.05 M CTAB micellar media at 10% peak height 62
IXVariance and standard deviation values for pyridoxal
in 0.05 M CTAB micellar media at 30% peak height 63
XVariance and standard deviation values for pyridoxal
in 0.05 M CTAB micellar media at 50% peak height 64
XIAverage values for variance and standard deviation
for pyridoxal in 0.05 M CTAB micellar and aqueous media
at 10, 30, and 50% peak height for aqueous and
0.05 M CTAB systems 65
XII Dispersion values for aqueous and micellar system 68
XIII Measurement of dispersion versus CTAB concentration 69
VI


LIST OF FIGURES
Figure Page
1 Diagram of an FIA system (a) and typical recording (b) 3
2 Dispersion in an FIA system 6
3 Velocity profiles and shapes of injected sample bolus 9
4 Dill-Flory's representation of a normal micelle 12
5 A two dimensional schematic representation of the
regions of a spherical ionic micelle 14
6 FIA manifold for the determination of pyridoxal 24
7 Reaction and surfactant media used for the analysis
of pyridoxal 27
8 Absorbance versus wavelength (nm) in aqueous system
for the determination of pyridoxal 29
9 Absorbance versus wavelength (nm) in 0.05 M CTA8
micellar system for the determination of pyridoxal 30
10 Change in maximum absorbance (355 nm) versus time
(minutes) in aqueous media for the determination
of pyridoxal 31
11 Change in maximum absorbance (355 nm) versus time
(minutes) in 0.05 M CTAB micellar media for the
determination of pyridoxal 32
12 Log versus time (minutes) for pyridoxal
in aqueous system 34
13 Log (A^-AJ versus time (minutes) for pyridoxal
in 0.05 M CTAB system 35
14 Absorbance calibration plots for pyridoxal in aqueous
( ) and 0.05 M CTAB ( ) systems 36


15 Fluorescence calibration plots for pyridoxal
determination in aqueous ( ) and 0.05 M CTAB ( )
systems 38
16 Absorbance calibration plots for pyridoxal in aqueous
( ) and 0.09 M CTAB micellar ( ) media 46
17 Absorbance recordings of a series of pyridoxal
standards in aqueous media 48
18 Absorbance recordings of a series of pyridoxal
standards in 0.05 M CTAB micellar media 49
19 Absorbance recordings of a series of pyridoxal
standards in 0.09 M CTAB micellar media 51
20 Fluorescence recordings of a series of pyridoxal
standards in aqueous media 52
21 Fluorescence recordings of a series of pyridoxal
standards in 0.05 M CTAB micellar media 54
22 Measurement of peak width, W, and asyrmietry factor,
B/A, at 10, 30 and 50% peak height in an FIA peak 57
v i i i


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MICELLAR CATALYZED REACTIONS
FOR FLOW INJECTION ANALYSIS
3Y
MARIA A. HERNANDEZ TORRES
August, 1986
Chairman: Dr. John G. Dorsey
Major Department: Chemistry
In this study, the applicability of micellar carrier streams for
the catalysis of reactions in FIA was investigated. Flow Injection
Analysis (FIA) is an automated kinetic method of analysis. An FIA
system with low dispersion and fast reaction kinetics will provide low
limits of detection and high sampling rates.
Micelles exhibit the ability to solubilize hydrophobic compounds
on or within their structures. The rate of many reactions in micellar
media is altered due to the proximity of reagents and analyte, changes
in the microenvironment and orientation of solutes.
The advantages of combining the solubilization property and
micellar catalysis for a given reaction that is taking place in an FIA
system are demonstrated. The reaction of pyridoxal (a Bg vitamer)
with cyanide was investigated in aqueous and micellar media. The
cationic surfactant, hexadecyltrimethyl ammonium bromide (CTAB), was
chosen for the micellar carrier solution. The oxidation product of
ix


this reaction, 4-pyridoxolactone, was detected either fluorimetrically
or by ultraviolet absorbance. The reaction rates in the two media
were determined and compared. Calibration plots for pyridoxal were
made and the analytical figures of merit were compared for aqueous and
0.05 M CTAB micellar media.
A Simplex Optimization was carried out for the determination of
pyridoxal in micellar media. A new set of conditions for micellar
media was obtained from the Simplex program. With this new set of
conditions, a calibration plot for the micellar media was prepared and
subsequently compared to the calibration plot for the aqueous
system. The analytical figures of merit for the two carrier solutions
were calculated and compared.
The peak shape obtained in the FIA system was investigated. The
standard deviation and variance at 10, 30, and 50% peak height were
calculated by empirical equations. The agreement of these values
allows the peaks to be classified as exponentially modified gaussians.
Measurement of dispersion in both aqueous and micellar systems
was investigated. Dispersion values for both aqueous and micellar
systems were examined by two methods: by using the definition of
dispersion in FIA and by measuring the variance (second moment) at 10%
peak height. In both methods, higher values for dispersion were
obtained for micellar media.
x


CHAPTER I
INTRODUCTION
Principles of Flow Injection Analysis
Flow injection analysis (FIA) is now established as a fast,
precise, accurate, efficient and extremely versatile analytical
tool. The FIA technique is used by many analytical chemists working
in a variety of different industries and institutions (1-9).
A historical review of the development of FIA reveals that since
its conception in the early 1970s, many of the concepts of flow
injection analysis have been adopted from other fields and many
workers have contributed to its development (10). However, the
technique of FIA became known by the simultaneous appearance of the
work of Stewart, Beecher and Hare (11) in the United States and
Ruzicka and Hansen (12) in Denmark in 1975. The Danish group
developed the method using primarily instrumentation normally
associated with segmented flow analyzers. In contrast, the American
group based their initial work on high performance liquid
chromatography components. For this reason, FIA is considered a
hybrid of the two techniques.
In the past, it was generally assumed based on Skegg's concept
that air segmentation and attainment of a steady state signal were
essential for performing continuous flow analysis (6). The presence
of air bubbles in the analytical stream was thought to be necessary
1


2
to limit sample dispersion, to promote mixing of the sample with
reagents (by generating turbulent flow) and to scrub the walls of the
analytical conduits to prevent carryover of samples. However, it was
proven that analysis without air segmentation is not only possible but
also advantageous. In FIA, there is no air segmentation, the sample
is introduced as a plug via a valve or syringe, mixing is mainly by
diffusion-controlled processes, and the response curves do not reach
the steady-state plateau, but have the form of sharp peaks. The
absence of air segmentation leads to a higher sample throughput. The
presence of a sample carrier interface over which concentration
gradients develop during the course of analysis has opened new
analytical possibilities for continuous flow analysis. The
reproducibility is good, and there is no sample carryover. There is
no need to introduce and remove air bubbles, and an expensive high
quality pump is not necessary.
Flow injection analysis is based on the injection of a liquid
sample into a moving, nonsegmented reagent carrier stream. The
injected sample forms a zone that disperses and reacts with the
carrier on its way to a detector (5). The simplest FIA analyzer
(Figure la) consists of a pump (P) that propels the carrier stream
(R), an injector port (S), by which a well defined sample volume is
injected into a carrier stream, and a coil in which the sample zone
disperses and reacts with the components of the carrier stream,
forming a species to be sensed by a flow-through detector (D). A
typical recording has the form of a sharp peak (Figure lb) the height
of which is related to the concentration of analyte, and the time from


(a)
mL/min
2-30 s
Figure 1. Diagram of an FIA system (a) and typical recording (b)
(from 1).


injection to peak maxima is termed the residence time. Residence
times are typically from 3 to 30 seconds.
4
Between the points of injection and detection the sample plug
will have been physically dispersed to some degree and, in addition,
some chemical reactions will have taken place. The peak will reflect
both processes.
The injection of a sample lias to be done in such a way that by
inserting a discrete slug of sample into a continuously moving stream
the movement of the stream is not disturbed. The residence time of
the sample in the analytical manifold should ideally be identical for
each sample, and the conditions to which the sample is exposed during
processing should also be the same. The reason for this is that not
only the physical dispersion but the chemistry involved requires a
reproducible travel pattern of the sample from the point of injection
to detection. In an FIA system, neither the mixing nor the chemical
reaction is complete; an equilibrium for either process is not
attained (1). By monitoring the reaction at a fixed precise time, the
concept of the steady state can be abandoned. This measurement at a
fixed time is just as analytically significant as the steady state
signal. Any fluctuations in residence time of the sample, i.e.,
variation in flow rate, will result in imprecise measurement of the
signal.
When a sample is first injected, it forms a well-defined sample
plug in the stream. As the sample is swept downstream through the
analytical conduits of narrow bore tubing, the plug disperses into
and, thus, mixes with the carrier stream under laminar flow conditions


5
to form a gradient. The magnitude of this dispersion is dependent on
the operating parameters applied to the system, including sample
volume, tubing bore size, tubing length, flow rate, sample volume and
coil diameter (1,6). Varying the values of these parameters confers a
significant degree of control over the dispersion characteristics and
facilitates optimization of a flow injection system for many diverse
applications, so that the optimum response is obtained at minimum time
and reagent expense.
The response curve has the shape of a peak reflecting a continuum
of concentrations. In contrast to all previous methods of automated
assay, there is no single element of fluid that has the same
concentration as the neighboring one (7) (Figure 2).
The dispersion coefficient, D, is the ratio of the concentration
of sample solution before (C) and after (C) the dispersion process
has taken place (Figure 2). The dispersion coefficient can be defined
at any point of the curve but, for convenience in the majority of FIA
methods, the dispersion coefficient is defined at maximum peak height.
C
In cases where a reaction is developed as a result of mixing
effected by dispersion, the peak height will increase as the reaction
proceeds toward completion. The maximum response will then be
attained when an optimum balance is reached between dispersion and
reaction time.


CONCENTRATION
t or scan
O')
Figure 2. Dispersion in an FIA system (from 7).
RESPONSE


7
Flow injection analysis operates only in the laminar flow region
(3,13-16). In this region, FIA systems generate dispersion through
both diffusion and convection. Under such conditions of laminar flow,
the layer of liquid in contact with the tube surface is practically
stationary and the velocity of centrally placed molecules is twice the
mean velocity of the liquid. This gives rise to a parabolic velocity
profile (Figure 3a). In the absence of molecular diffusion, a sample
placed into a moving stream would have an infinitely long tail by the
time it reached the detector (Figure 3b). This results in
unacceptable carryover between samples. Diffusion of molecules
between the carrier and sample bolus serves to limit this convective
dispersion and effectively mixes sample and reagent. In coiled tubes,
it is the radial rather than the axial dispersion that contributes
most significantly to sample dispersion in FIA systems. This type of
dispersion, also called secondary flow, operates to move the fluid
both toward and away from the tubing wall and thus serves as an
efficient scrubbing mechanism (Figure 3d). A molecule located at the
center will tend to diffuse into a region of lower sample
concentration and by doing so it will move into a layer of liquid
moving at a slower longitudinal velocity. On the other hand, a
molecule located near the wall will diffuse toward the center of the
carrier solution and it will encounter a layer of faster moving
liquid, which will carry it away from the wall and toward the center
of the sample zone. This radial diffusion perpendicular to the
direction of the flow modifies the shape of the bolus head and the
result is low carryover and cross contamination. High sample


Figure 3. Velocity profiles and shape of an injected sample bolus:
(a) Laminar flow, parabolic velocity profile; (b) Sample
dispersion caused by laminar flow without diffusion; (c)
Sample shape resulting from laminar flow with molecular
diffusion; (d) Secondary flow pattern in the cross-section
of a tightly coiled tube (from 3).


9
tube wall


10
throughput is obtained because of the limited band spreading. When
samples under these conditions reach the detector they have a shape as
in Figure 3c.
The versatility and simplicity of FIA systems permits this
technique to be widely used for performing chemical assays. The
recent increase in publications dealing with new FIA methods as well
as in separate symposia on this topic indicate the popularity of this
relatively new technique (2). On the other hand, more has to be done
on the theoretical side, where the dispersion of the sample zone must
be investigated in much greater detail. The theory of dispersion,
although very useful, is far from being exact and complete (17). Only
deeper theoretical studies will lead to design of even more advanced
continuous flow techniques that will allow chemical analyses to be
performed in new ways.
Properties of Surfactants and Micelles in Aqueous Solution
Scientists from around the world have shown special interest in
surfactants because of their unique characteristics (18-24).
Surfactants, or surface active agents, are amphiphilic molecules
having both pronounced hydrophobic and hydrophilic properties.
Surfactants are classified as cationic, anionic, nonionic or
zwitterionic according to the hydrophilic part (polar head group).
The hydrophobic tail consists of a hydrocarbon chain usually from 8 to
20 carbon atoms in length. Furthermore, this hydrophobic tail can
contain unsaturated portions or aromatic moieties, can be partly or


11
completely halgena ted, and can be branched or consist of two or more
chains.
At low concentration, the surfactant is dispersed predominantly
as monomers, although dimers, trimers, etc. can exist. Over a narrow
concentration range, termed the critical micelle concentration (CMC),
surfactants have the important property of forming molecular
aggregates, called micelles. Above the CMC, there exists a dynamic
equilibrium between monomers and micelles. The amount of free monomer
remains approximately constant and equal to the CMC.
In aqueous solutions, surfactant molecules (typically from 60 to
100) associate to form a roughly spherical cluster (25) (Figure 4).
This micelle structure is such that the hydrophilic head groups are
directed toward and in contact with the aqueous solution, forming a
polar surface, while the hydrophobic tails are directed away from the
water forming a liquid-like hydrocarbon core. The microviscosity of
this core is considerably higher than in hydrocarbons. The micellar
surface is not uniform: some of the hydrocarbon chains are turned
towards the solvent or at least come into contact with it periodically
(26). On the whole, it may be supposed that the micellar surface is a
polar environment differing in properties from water itself.
Changes in temperature, concentration of surfactant, additives in
the liquid phase, and structural groups in the surfactant may cause
changes in the size, shape, and aggregation number of the micelle
(26-28).


12
Figure 4. Dill-Flory's representation of a normal micelle. The ionic
head groups are indicated by the circles, and the
hydrocarbon chains are pointing toward the center of the
micelle (from 25).


13
In micelles of ionic surfactants, the charged head groups and the
counterions are located in a compact region, known as the Stern layer,
which extends from the core to within a few angstroms of the shear
surface of the micelle. Beyond the Stern layer, the remainder of the
counterions are located in the Gouy-Chapman electrical double layer
where they are completely dissociated from the charged aggregate and
are able to exchange with ions in the bulk of the solution (26)
(Figure 5).
The driving force for micelle formation in aqueous media is due
primarily to the hydrophobic effect, and the electrostatic repulsion
between the ionic head group limits the size that a micelle can
attain, thereby keeping the micelle size small.
One of the most important properties of micellar systems is their
ability to solubilize a variety of species (18-24,26-28).
Solubilization may be defined as the spontaneous dissolving of
substance (solid, liquid or gas) by reversible interaction with the
micelles of a surfactant in a solvent to form a thermodynamically
stable isotropic solution with reduced thermodynamic activity of the
solubilized material (24). The importance of the phenomenon of
solubilization from the practical point of view is that it makes
possible the dissolving of substances in solvents in which they are
normally insoluble or slightly soluble. Solubilization is a dynamic
equilibrium process and depends on temperature, nature of solute,
surfactant concentration and type of micellar system employed (26).
There are several possible solubilization sites and orientations
available in a micellar system and the site occupied by a solubilizate


14
Figure 5. A two dimensional schematic representation of the regions
of a spherical ionic micelle (from 24).


15
depends upon the nature of both the solute and the micelle. There is
a rapid equilibrium between various possible sites and also between
the solubilized state and the free state in the aqueous medium. The
site of solubilization is a topic of current debate. Some contend, if
the solute is nonpolar, it may pass completely into the hydrophobic
core or penetrate a particular depth into the surface layer. Others
contend it may be adsorbed at a hydrophobic region on the surface
(13).
Micellar systems are very useful in the field of analytical
chemistry due to the unique properties of these organized
assemblies. Micelles can lead to the modification and improvement of
existing procedures and to the development of new methods of chemical
analysis. Micellar structures offer means to overcome solubility
problems, to speed reaction rates of analytical reactions and to
reduce side reactions, to shift acid base or redox equilibria, to
change spectral distribution or intensities, and to improve
selectivity and efficiency in extraction and chromatographic
methods. Several reviews on the use of surfactants in analytical
chemistry have appeared (29-31).
Our studies are directed mainly to observing the behavior of
combining micellar media with the technique of FIA. This represents
the first attempt to demonstrate the advantages of combining the
solubilization property and micellar catalysis for a given reaction
that is taking place in an FIA system. Higher sensitivity and/or
lower limits of detection are expected for a given FIA system when the
reaction is carried out with the appropriate micellar media. Kinetic


16
studies and measurement of the analytical figures of merit will be
compared for reactions taking place in aqueous and micellar media.
v


CHAPTER II
THEORY AND BACKGROUND
Chemical Kinetics in a Flow Injection Analysis System
It is well recognized that flow injection analysis yields a
response curve which is the result of two processes, both kinetic in
nature: the physical process of dispersion and the chemical process
of formation of reaction products (1-6,32).
The kinetics of physical dispersion, or incomplete mixing, has
been described in a number of papers (14-16,33-35) while attempts to
describe the effect of chemical kinetics has been practically
ignored. The shape of the transient peak profiles of FIA
determinations have been described only in terms of the dispersion
process. However, there has been an attempt recently to explore in
more detail the complexity of the overall kinetic process taking place
inside the sample plug and its boundaries in FIA systems and the
contribution of this kinetic chemistry to the peak profiles. Ruzicka
and Hansen recognized that "it is obvious that the comprehensive
theory of the flow method will eventually combine the theory of mixing
of liquids in continuous moving streams with the theory of chemical
kinetics" (36). From now on, more attention will be paid to the
chemistry process that is taking place (37-42).
In developing a flow injection method, one of the primary goals
is to maximize response together with sample throughput. These
17


13
parameters are intimately related to the sensitivity of the method and
the number of injections possible per hour.
Since there exists an interdependence between the reaction rate
and the rate of dispersion, both factors have to be weighed when
designing a new FIA system. The longer a sample stays within the
system, the greater will be the signal due to the chemistry that is
taking place. Longer residence times mean more time is allowed for
the reaction to take place, therefore more product is formed. This
increase is balanced against the point where dispersion will overcome
the formation of the product and cause the signal to decrease. The
longer the sample stays between the points of injection and detection,
the higher will be the dispersion and the signal measured (usually
peak height) will decrease. Another factor to be considered is time
needed for each determination. Longer residence times allow maximum
sensitivity of measurement but at the expense of decreasing sample
throughput. Therefore, short residence times are often preferred.
Fast chemical reactions are required for performing a simple FIA
analysis at a continuous carrier pumping rate with practical residence
times of about 30 seconds. Slow reactions must presently be performed
by the stopped flow mode or in a packed reactor (17). In stopped flow
mode, an electronic timer is used to cease the movement of the carrier
stream containing the sample zone in order to allow enough time to
produce an adequate amount of detectable product, and then the sample
is pumped through the flow cell while the peak is recorded in the
usual manner. Reactors packed with inert materials, such as glass,


19
enhance micromixing of sample and reagents in the carrier stream
without loss of peak height or loss of sample frequency.
General Features of Micellar Catalysis
It has been reported that with the proper choice of surfactant
the rate of a chemical reaction is substantially enhanced in a
micellar solution relative to that in the corresponding aqueous system
(18,24,26,27,31,43-45).
Research on the effect of surfactants on the kinetics of organic
reactions has demonstrated that it is the micelle structure, not the
individual molecules, that is responsible for the catalysis or
inhibition of these reactions. In accordance with this fact, the term
micellar catalysis has been applied to this phenomenon. Rate
acceleration or inhibition of organic reactions in micellar solution
arises from different rates of reaction of the substrate in the
micellar phase and in the bulk solution and the distribution of the
substrate between these two phases (26,32). Then, a prerequisite to
understanding reaction kinetics in micellar systems is to understand
the structure and solubilization properties of the micelles themselves
(vide supra).
The kinetics of organic reactions occurring in micellar systems
are dominated by electrostatic and hydrophobic interactions between
the micelle structures, reactants, transition states and products.
The two physicochemical factors responsible for the efficiency of
micellar catalysis are


20
(1) the change in the reactivity of reagents on transfer from
water to the micellar phase and
(2) the concentration of reactants into the micellar phase.
The first factor, the differences in reactivity, can be explained by
the difference in the distribution of a substrate between these two
phases and by the difference in the degree and nature of substrate-
micelle binding. When a solute is solubilized in a micellar system,
the microenvironment about it is very different compared to that in
the bulk solvent. Micellar systems have the ability to change the
effective microenvironment and the microscopic properties of
solubilized solutes to that of aqueous media favoring the acceleration
of some organic and inorganic reactions.
For catalysis to occur, it is necessary that the substrate be
solubilized by the micelle and the site of solubilization be such that
the reactive site of the substrate is accessible to the attacking
reagent. It is here that hydrophobic interactions become important,
because they determine the extent and the site of solubilization in
the micelle. In general micellar effects on reactions follow several
rules, although there are exceptions. A hydrophobic reactant is
attached to a micelle by hydrophobic interactions, independently of
the charge on the micelle. If the second reactant is oppositely
charged to the micelle, it will be bound to the micelle and the
reaction is usually accelerated. When micelles and reactant ions bear
like charges, the reaction is inhibited due to the repulsion forces
between the ions and the micelle's surface. Nonionic or zwitterionic
micelles, generally, have no significant influence on the rates of


21
these reactions. The rate of certain organic reactions is unaffected
when one of the reactants is incorporated into the micellar phase and
the other is excluded from it. Exceptions to these rules can be
explained by the fact that sometimes hydrophobic effects overcome the
electrostatic repulsions and even when the micelle's surface charge
does not favor the reaction, catalysis does occur.
The orientation of the reagents in micellar media is different
from the bulk aqueous phase due to the different microenvironment that
the reagents experience on or within the micelles. If this micro
environment is more attractive to reagents, the reagents are going to
spend more time on this phase; therefore, they will be concentrated in
this region. Also, the micelle structure provides a very specific and
reduced region where the reagents are being solubilized. If the
reagents are localized within this small region, they are being
concentrated and are closer to each other inducing the reaction to go
faster. These are called proximity and concentration effects.
Quite generally, increasing the hydrophobic character of the
surfactant, having longer alkyl chains, increases its efficiency as a
catalyst. At equal concentration of two surfactants, the more
hydrophobic may appear to be the better catalyst (or inhibitor) simply
because it has greater affinity for the substrate. Variation in
substrate structure has a profound influence, in many cases, on the
magnitude of micellar catalysis. The general rule seems to be that
the more hydrophobic the substrate, the more pronounced the micellar
catalysis.


22
The multiphase profile of surfactant concentration on the
reaction rate is as follows: below the CMC, the rate constants are
independent of surfactant concentration; above the CMC, the rate
constants rise rapidly with increasing surfactant concentration, level
off, and finally decrease with increasing concentration of
surfactant. This profile can be rationalized by the fact that the
rate constant increases as the concentration of micellar bound
reactants increases, but eventually an increase in surfactant
concentration dilutes the reactants in the micellar pseudophase, with
a decrease in the rate constant (18).
The influence of electrolytes on micellar catalysis is less
predictable. For most reactions micellar catalysis is inhibited by
counterions and the larger the ion, the greater the effect. This
behavior has been rationalized by assuming a competition between the
reactant and the electrolyte for a binding site on or in the
micelle. This salt inhibition may be explained principally by the
displacement of one reactant from the micellar surface by the
electrolyte. Enhancement of the micellar catalyzed reaction rate by
counterions has been suggested to be due to changes in micellar
structure by the salts and this new configuration of the surfactants
promotes the reactions.
Another advantage of using micellar media is that of favorable
substrate partitioning and binding in specific orientations and
configurations on the micelle structure. This makes the reaction very
selective to that substrate by decreasing the probability of other
interfering species competing for the place of the substrate.


CHAPTER III
EXPERIMENTAL: FIA SYSTEMS FOR
PYRIDOXAL DETERMINATION
Apparatus
The flow injection manifold used is shown in Figure 6. The
reagent streams were pumped by an Isco (Lincoln, NE) Tris model
peristaltic pump. Samples were introduced with a Rheodyne (Cotati,
CA) model 7125 sample injection valve with a 10 ul loop. All tubing
was Teflon from the Anspec Company, Inc. (Ann Arbor, MI) with 0.5 mm
internal diameter. The reaction coil, 200 cm long, was tnermostated
by immersing it in a water bath with a Techne TE-7 circulator
(Cambridge, England). For fluorescence measurement, a Varian (Palo
Alto, CA) model Fluorichrom detector with a 25 ul total volume flow
cell was used. A combination of Varian filters was selected for
355 nm excitation wavelength and 435 nm emission wavelength. A Kratos
(Ramsey, NJ) model Spectroflow 757 absorbance detector with a 12 ul
flow cell was set at a wavelength of 355 nm to measure the absorbance
of the product. The output signals were recorded on a strip chart
OmniScribe recorder, Houston Instrument (Austin, TX). The pH of the
reagent solutions were measured with a Corning (Medfield, MA) model pH
meter 130.
For kinetic studies, the reaction was followed spectrophoto-
metrically by measuring the rate of change in the absorbance of
23


PYRIDOXAL
10 \l\
WASTE
Figure 6. FIA manifold for the determination of pyridoxal.


25
4-pyridoxolactone, at 355 nm using a Hewlett Packard (San Diego, CA)
model 3450A Diode Array spectrophotometer connected to a Hewlett
Packard model 7470A plotter.
Reagents
All reagents were used as received and prepared either in
deionized water or in surfactant solutions. The cationic surfactant
was hexadecyltrimethylammonium bromide (CTAB, purum grade) from Fluka
Chemical (Hauppauge, NY). Standard solutions of pyridoxal, Sigma
grade (Sigma Chemical Company, St. Louis, MO) and solutions of
potassium cyanide, certified ACS, from Fisher Scientific Company
(Fair Lawn, NJ) were used for this study. Phosphate buffer solutions
(0.6 M) certified ACS from Fisher Scientific Copmany were prepared and
the pH was adjusted with concentrated hydrochloric acid, ACS
certified, from Mallinckrodt (Paris, KY).
Procedure
The appropriate weight of surfactant was dissolved in distilled
water and the solution then filtered through a 0.45 urn nylon-66
membrane filter (Rainin Instruments, Woburn, MA). Appropriate amounts
of pyridoxal and cyanide were dissolved either in distilled H20 or
micellar solution. All reported values are averages of at least four
determinations.


CHAPTER IV
MICELLAR CATALYSIS IN THE DETERMINATION OF
PYRIDOXAL BY FLOW INJECTION ANALYSIS
Results and Discussion
Pyridoxal is one of the three substances designated as vitamin
Bg. Determination of pyridoxal and its derivatives is of great
interest, especially in clinical chemistry. A fundamental role for
pyridoxal has been postulated in the mechanism for active transport of
amino acids and metals ions across the cell membrane (46).
The analysis of pyridoxal in biological material has proven to be
difficult and unsatisfactory. Pyridoxal has been analyzed via high
pressure liquid chromatography (HPLC) with amperometric, enzymatic-
fluorometric or photometric detectors and via radiochemical means.
The most common detection procedure for pyridoxal is fluorimetry, in
volving the use of Zn-glycine or formation of hydrazone derivatives
(47).
P. Linares and coworkers (47) reported a fluorimetric method for
determination of pyridoxal by flow injection analysis. This method
was based on the oxidation of pyridoxal in the presence of cyanide to
yield 4-pyridoxolactone (see Figure 7a).
There is no spectral evidence of reaction between cyanide and the
other vitamin Bg derivatives. These results indicate that the
carbonyl is the only group in the pyridoxal molecule capable of
reacting with cyanide (48-50).
26


27
HOCH
CHO
ch3
OH
pyridoxal cyanide
4-pyridoxolactone
(a)
CTAB:
CH3( CH2)|5 N+(CH3 >3 Br
(b)
Figure 7. Reaction and surfactant media used for the analysis of
pyridoxal. Reaction for pyridoxal with cyanide (a) and
CTAB's molecular formula (b).


23
Preliminary studies on the behavior of this reaction are shown in
Figures 8 and 9. At a wavelength interval of 280 to 400 nm, the
change in absorbance versus time was recorded for aqueous and micellar
media. The cationic surfactant, CTAB, was chosen to attempt to
promote the rate of this reaction (see Figure 7b). A CTAB
concentration of 0.05 M was used which is safely above the CMC of
0.0013 M at 25C (30). Maximum absorbance was found to be at 355 nm
for the pyridoxal-cyanide reaction product for both aqueous and
micellar media. In agreement with the expected results, the cationic
surfactant, CTAB, promotes the rate of the reaction. Greater changes
in absorbance were absorbed in micellar media during the same amount
of time. This increase in the rate of the reaction can be explained
by electrostatic attraction forces between the positive charge at the
micelle surface and the negative charge of the cyanide. Also, the
hydrophobic forces between the pyridoxal molecule and the nonpolar
portion of the micelle caused the reaction to proceed at faster
rate. Not only the solubilization and proximity effects contribute
(24,43) to micellar catalysis, but probably the stabilization of some
intermediate species with partial negative charge at the positive
micelle surface favored the formation of 4-pyridoxolactone.
Kinetics studies were performed to measure the rate constants for
this oxidation reaction in aqueous and 0.05 M CTAB media. Figures 10
and 11 show the curves of the change in absorbance versus time at a
maximum absorbance wavelength of 355 nm. For micellar media, the
reaction reached the plateau of the curve after 15 minutes, whereas in
aqueous media the plateau was reached after 30 minutes.


WAVELENGTH (nm)
Figure 8. Absorbance versus wavelength (nm) in aqueous system for the determination of pyridoxal.
Phosphate buffer (0.6 M), pH 7.3, 0.61 ppm pyridoxal, KCN 7.5X103 M, 1 cm cell, at room
temperature. Spectra were run at 1 minute intervals.


ABSORBANCE
WAVELENGTH (nm)
Figure 9. Absorbance versus wavelength (nm) in 0.05 M CTAB micellar system for the determination of
pyridoxal. Phosphate buffer (0.6 M), pH 7.3, 0.61 ppm pyridoxal, KCN 7.5X10^ M, 1 cm cell,
at room temperature. Spectra were run at 1 minute intervals.


ABSORBANCE
TIME (minutes)
Figure 10. Change in maximum absorbance (355 nm) versus time (minutes) in aqueous media for the
determination of pyridoxal. Pyridoxal 0.93 ppm, phosphate buffer 0.6 M, pH 7.3, KCN
7.5X10 M, 1 cm cell, at room temperature.


ABSORBANCE
TIME (minutes)
Figure 11. Change in maximum absorbance (355 nm) versus time (minutes) in 0.05 M CTAB micellar media
for the determination of pyridoxal. Pyridoxal 0.93 ppm. phosphate buffer 0.6 M, pH 7.3,
KCN 7.5X10"'-5 M, 1 cm cell, at room temperature.


33
Assuming the reaction is pseudo first order, the rate constant
can be calculated by using the following equation:
-kt
log (Aco-At) = 2<303 + log (Aoo-A()) (eq. 4.1)
where Am, AQ, At are the absorbance at infinite, initial and time t,
respectively; t is time in minutes; and k is the rate constant in
minutes-1 (51). From the slope of the curve (taking the negative and
multiplied by 2.303) the rate constants for the reaction of pyridoxal
taking place in water and 0.05 M CTAB media were 0.0490 and 0.0971
minutes-1 (see Figures 12 and 13). The ratio of these rate constants,
k0.05M CTAB^H^ 1S eclual 1*98. This value means that the
reaction is taking place at double the velocity in micellar CTA3 than
in H20. Micellar catalysis does occur for this particular reaction.
To demonstrate the advantages of combining the technique of FIA
with micellar catalysis, measurements of the pyridoxal-cyanide
reaction product using absorbance and fluorescence detection were
carried out. Figure 14 shows the calibration plot for the pyridoxal
determination in aqueous and 0.05 M CTAB media. Comparing the slope
of both curves, 2.54X10-'1 and 3.26X10-^ absorbance units/ppm of
pyridoxal, one can observe that by using a CTAB micellar media the
sensitivity of the system is increased 1.3 times. The differences
between the ratio of rate constants found for water and 0.05 M CTAB,
1.98, and the ratio of sensitivities of these two systems by FIA, 1.3,
may be explained by the fact that the kinetics throughout the entire
sample plug is not constant. The work of Paiton and Mottola (37)


Log (.3756-At)
Figure 12. Log (A^-Aj.) versus time (minutes) for pyridoxal in aqueous system. Absorbance measured at
355 nnC pyridoxal 0.93 ppm, in 1 cm cell at room temperature, phosphate buffer (0.6 M), pH
7.3, KCN 7.5X10"3 M.
OJ


-.60
.65
-.70
-.75
oo -.80
oo
CM
ro
O -.85 -
CP
o
_l
-.90 -
-.95
-1.00 -
-1.05
0
8
10
TIME (minutes)
Figure 13. Log (A^-A^.) versus time (minutes) for pyridoxal in 0.05 M CTAB system. Absorbance measured
at 355nm, pyridoxal 0.93 ppm, in 1 cm cell at room temperature, phosphate buffer (0.6 M),
pH 7.3, KCN 7.5X10"3 M.
C71


Absorbance Units (water)
pyridoxal (ppm)
Figure 14. Absorbance calibration plots for pyridoxal in aqueous ( ) and 0.05 iA CTAB ( )
systems. Absorbance measured at 355 mn, flow rate 1.4 ml/min, temperature 45C, pH 7.3.
phosphate buffer (0.6 M), KCN 1.5X10- M.
co
cr>
Absorbance Units (CTAB)


37
demonstrated that the assumption of having a constant rate coefficient
throughout the entire body of the sample plug is invalid. The
kinetics involved within the sample plug are more complex. Paiton and
Mottola suggested that the rate coefficient changes with time. This
has been rationalized by assuming that each fluctuation in rate
coefficient with time corresponds with one of three regions within a
sample plug, namely, the leading region, the central region, and the
trailing region. In both the leading and trailing regions, the
carrier/sample interfaces induce molecular diffusion, while the
velocity profile induces convection. In the central region, where no
sample/carrier boundary exists, convection becomes the primary
dispersion force. Because the physical dispersion in these three
regions of the sample plug differ from one another, the rate
coefficients along the length of the plug are expected to vary with a
wave pattern. The fact that the reaction rate varies throughout the
sample plug may mean also that the kinetic order is not constant
within the sample plug.
Figure 15 shows the calibration plot with a fluorescence detector
for aqueous and micellar systems. From the slope of the curves, the
calculated sensitivity of the pyridoxal system is three times greater
when using CTAB micellar media compared with the same system in
aqueous media. The sensitivities for micellar and aqueous solutions
were 0.294 and 0.102 cm (peak height) per ppm of pyridoxal,
respectively. A greater change in the ratio of sensitivities was
observed for fluorescence determination due to the fact that not only
micellar catalysis was taking place but fluorescence enhancement was


FLUORESCENCE (WATER)
(peak height, cm)
38
CD
<
O.
~ E
L o
O
Z-C
UJ 2*
O a>
CO-C
L
oS
u.
Pyridoxal (ppm)
Figure 15. Fluorescence calibration plots for pyridoxal determination
in aqueous ( ) and 0.05 M CTAB ( ) systems. Flow rate
1.4 ml/min; temperature 45C; phosphate buffer (0.6 M); pH
7.3; KCN 1.5X10_ M; 355 and 435 nm excitation and
emission wavelengths, respectively.


39
also observed. In mi cellar-enhanced fluorescence, the emission
intensity of the analyte is usually many times greater than in the
corresponding homogeneous media (52,53). This increase in sensitivity
of solutes in solutions containing micellar aggregates has been
explained by the diminution of deactivation processes for the excited
states. These phenomena occur due to a decrease in polarity, and an
increase in viscosity and shielding against quenching in micellar
media (29).
Table I summarizes the analytical figures of merit for both
aqueous and micellar systems. Very good coefficients of correlation
were obtained for the four curves, all being 0.999. These calibration
curves recorded under working conditions are linear over a wide range
of concentrations. The linear range for the aqueous system with a
fluorescence detector was found to be from 0.42 ng to 2.0X103 ng and
from 94 ng to 2.0X10J ng of pyridoxal for absorbance measurement. For
0.05 M CTAB media the linear dynamic range was from 0.17 ng to 1.1X103
ng of pyridoxal for fluorescence and from 77 ng to 2.0X10 ng of
pyridoxal for absorbance. Due to the large concentration range for
the recorded curves, it was necessary to work at several values of the
instrument sensitivity. At higher concentrations of pyridoxal, the
fluorescence intensity is beyond the spectrofluorimeter range. The
reproducibility of the system was measured by manual injection of 11
replicates of pyridoxal solution at 25.53 ppm. Relative standard
deviation of peak height in percent was calculated and was found to
vary between 0.97 and 3.25 for the studied systems. Limits of
detection were found to be lower for the reaction taking place in


40
Table I. Figures of merit for pyridoxal determination. Fluorescence
detector: excitation 355 nm, emission 435 nm, range 500, or
variable UV-visible absorbance detector at 355 nin, range .1,
1.5X10"2 M cyanide, pH 7.3, phosphate buffer 0.6 M, flow
rate 1.4 ml/min, temperature 30C, chart speed 1 cm/min,
10 ul sample loop, pyridoxal in deionized water, tube length
200 cm, i.d. 0.5 mm.
Fluorescence
Absorbance
Aqueous
0.05 M CTAB
Aqueous
0.05 M CTAB
Sensitivity3
0.102
0.294
2.54X10"3
3.26X10'3
Coefficient of
correlation
0.9996
0.9992
0.9999
0.9999
Limits of
detection (ng)
0.42
0.17
94
77
Relative standard
deviation U)b
2.06
3.25
0.97
1.76
a Slope of the calibration plot for fluorescence, cm/ppm of pyridoxal
and absorbance units/ppm of pyridoxal for absorbance measurements.
b Eleven determinations at 25.53 ppm of pyridoxal.


41
micellar solutions. A more significant change in limits of detection
was not obtained due to an increase in the background signal for
micellar solutions. This needs further study in the future the
examination of other systems in micellar media by FIA, since a
significant change in limits of detection usually accompanies micellar
catalysis (52).
Optimum conditions may be different for aqueous and micellar
systems. The reason for this difference is that micelles can change
the microenvironment of the solubilized molecules (22). Then, an
optimization of conditions for FIA system is required.
The output signal, in this case peak height, is influenced by the
dispersion of the sample in the reagent stream and the degree of
completeness of the reaction taking place. These two are affected by
experimental parameters such as flow rate, reagent concentrations,
length of the reaction coil, etc. Since these experimental parameters
interact with each other, optimization of FIA methods using univariate
design (optimization of every parameter by separate studies) is time
consuming and may be inadequate to determine the best set of experi
mental conditions (54). Sequential simplex optimization procedures
have been found to be valuable in development of new FIA methods
(54-56).
A modified variable size simplex method (57,58) was used for
optimization of pyridoxal determination. The optimization of this
system was performed by changing four variables: pH, temperature,
flow rate and surfactant concentration. Table II shows the initial,
final and increment values for each changing parameter. The reason to


42
Table II. Variable parameters for the Modified Simplex Optimization
program.
Parameter
Initial
Value
Range
Value
Increment
Va 1 ue
1.
pH
7.3
6.5-3.0
0.5
2.
Flow rate (ml/min)
1.4
1.0-1.7
0.1
3.
Temperature (C)
45
30-50
5
4.
CTAB concentration (M)
0.05
0.05-0.15
0.05


43
include pH as one of the parameters to be optimized is that the
reaction of pyridoxal with cyanide is pH dependent. It is reported in
the literature that micelles affect the pH of solutions, by changing
acid dissociation constants (59). The range of pH from 6.5 to 8.0 was
chosen because previous studies showed this to be the optimal pH range
for the oxidation reaction. An increase in temperature increased the
reaction rate in such a manner that higher values for the response
function resulted, up to a certain point where the signal started to
decrease as the temperature increased due to deactivation of
fluorescence. At temperatures below 30C, the response of the system
(peak height) is greatly diminished; at temperatures above 50C,
bubble formation in the FIA system can prove detrimental to
reproducibility (47). Flow rate is closely related with the output
signal (vide supra). Faster flow rates will usually lead to a
decrease in signal because less time is available for the reaction to
take place and vice versa. The surfactant concentration was included
as a variable since increasing the number of micellar structures
increases the number of sites available for solute solubilization and,
therefore, promotes the formation of 4-pyridoxolactone. If the
surfactant concentration is increased too much, the reaction will
occur at a lower rate due to the dilution factor. In other words, as
the number of micelle structures increases, solute molecules will be
solubilized on different micelle structures apart from each other.
There will be a physical impediment for a pyridoxal molecule to
encounter a cyanide ion preventing the oxidation reaction to
proceed. The cyanide concentration and the length of reaction coil


44
were kept constant. At a cyanide concentration of fivefold the
pyridoxal concentration, the intensity of the output signal is not
influenced by a change in the cyanide concentration (47). The simplex
was finished after 13 experiments (one reflection, two expansions and
four contractions). Optimum values found, together with those
variables kept constant throughout the development of the simplex, are
summarized in Table III.
With these new optimum values for absorbance measurement in CTA6
media, a set of standard pyridoxal solutions were run and a
calibration plot was recorded and compared with the calibration plot
for the water system run with previous conditions (see Figure 16). A
ratio of 1.8 was found, by comparing the slopes of the two curves, for
water and CTAB, 2.09X10"^ and 3.84X10^ absorbance units per parts per
million of pyridoxal. As was expected, a higher sensitivity was
observed. This can be explained by the fact that now the conditions
are more favorable for the reaction to take place. Table IV shows the
analytical figures of merit for the reaction in optimized CTAB
conditions and previous conditions for aqueous system. Relative
standard deviation, measured by 11 determinations of 24.14 ppm of
pyridoxal, were 1.06 and 2.12% for CTAB and water systems. Very good
coefficients of correlation were observed for both curves, 0.999,
assuring the linearity of the curve. Lower limits of detection were
obtained by using 0.09 M CTAB and 49C, 64 ng, compared with the
aqueous system, 86 ng.
Figures 17 to 21 show a set of pyridoxal standard solutions run
by FIA under different studied conditions.


45
Table III. Optimized and fixed variables for the determination of
pyridoxal.
Variables Fixed
in the Optimization
Optimized Variables
1. 10 ul sample: Pyridoxal:
1. pH: 6.74
9.18X10"4 M in 0.05 M CTAB
2. Cyanide concentration:
2. Flow rate: 1.3 ml/min
1.5X10"2 M
3. Phosphate buffer solution: 0.6 M
3. Temperature: 49C
4. Length of reaction coil: 200 cm
4. CTA3 concentration: 0.09 M


ABSORBANCE UNITS (CTB)
45
PYRIDOXAL (ppm)
Figure 16. Absorbance calibration plots for pyridoxal in aqueous
( ) and 0.09 M CTAB micellar ( ) media. Absorbance
measured at 355 nm, flow rate 1.3 ml /min, temperature
49C, pH 6.74, phosphate buffer (0.6 M), KCN 1.5X10' M.
ABSORBANCE UNITS (water)


47
Table IV. Figures of merit for pyridoxal determination with
conditions obtained by Modified Simplex program. Variable
UV-visible absorbance detector at 355 nm, range .1, 0.09 M
CTA8, 1.5X10-2 M cyanide, pH 6.74, 0.6 M phosphate buffer,
flow rate 1.3 ml/min, temperature 49C, chart speed
1 cm/min, 10 ul sample loop, pyridoxal in deionized water,
tube length 200 cm, i.d. 0.5 mm.
Aqueous
CTAB
Sensitivity
,absorbance units*
'ppm of pyridoxal
2.09X10"3
3.84X10"3
Coefficient of correlation
0.9994
0.9998
Limits of detection (ng)
86
64
Relative standard deviation (?)a
2.12
1.06
a Eleven determinations at 24.14 ppm of pyridoxal.


48
Figure 17. Absorbance recordings of a series of pyridoxal standards
in aqueous media. Pyridoxal: (a) 8.10 ppm, (b) 24.31
ppm, (c) 40.52 ppm, (d) 81.04 ppm, (e) 105.4 ppm.
Absorbance measured at 355 nm, range 0.05, sample volume
10 ui, all tubes 0.5 mm I.D., flow rate 1.4 ml/min,
temperature 45C, phosphate buffer 0.6 M, pH 7.3, KCN
1.5X10"2 M, chart speed 0.25 cm/min.


Figure 19. Absorbance recordings of a series of pyridoxal standards
in 0.09 M CTAB micellar media. Pyridoxal: (a) 8.10 ppm,
(b) 24.31 ppm, (c) 40.52 ppm, (d) 81.04 ppm, (e) 105.4
ppm. Absorbance measured at 355 nm, range 0.05, sample
volume 10 ul, all tubes 0.5 mm I.O., flow rate 1.3 ml/min,
temperature 49C, phosphate buffer 0.6 M, pH 6.73, KCN
1.5X10"2 M, chart speed 0.25 cm/min.


TIME (min)
ABSORBANCE


52
TIME (min)
Figure 20. Fluorescence recordings of a series of pyridoxal standards
in aqueous media. Pyridoxal: (a) 8.10 ppm, (b) 24.31
ppm, (c) 40.52 ppm, (d) 81.04 ppm, (e) 105.4 ppm. Sample
volume 10 yl; all tubes 0.5 mm I.O.; flow rate 1.4 ml/min;
temperature 45C; phosphate buffer 0.6 M; pH 7.4; KCN
1.5X102 M; range 1000; chart speed 0.25 cm/inin; 355 nm
and 435 nm excitation and emission wavelengths,
respectively.


Figure 21. Fluorescence recordings of a series of pyridoxal standards
in 0.05 M CTAB micellar media. Pyridoxal: (a) 8.10 ppm,
(b) 24.31 ppm, (c) 40.52 ppm, (d) 81.04 ppm. Sample
volume 10 yl; all tubes 0.5 mm I.O.; flow rate 1.4 ml/min;
temperature 45C; phosphate buffer 0.6 M; pH 7.4; KCN
1.5X10^ M; 355 nm and 435 nm excitation and emission
wavelengths, respectively; range 1000; chart speed 0.25
cm/min.


55
Measurements of Dispersion
In flow injection analysis, both sample throughput and sample
dilution are directly related to dispersion. The dispersion process
which takes place during the transport of the sample from the
injection device toward the detector is one of the less understood
aspects of FIA (vide supra). In analogy with chromatographic systems,
Poppe (60) observed that the total peak broadening in FIA is the sum
of the contributions from the injection process, the flow through
reactors and connectors, the holdup volume of the flow through
detector, and the time constants of associated electronics. These
processes can be described by the individual peak variances:
2 2 2 2
o = a. .. + a.. + a, .
overall injection flow detector
Provided that the detector and electronics are well designed, the
variance of detection may be neglected (if it is at least five times
smaller than the standard deviations due to injection and
transport). One of the models frequently used to describe the
dispersion process is the tank in series model. According to this
model, the flow reactor can be considered as a series of M ideal
mixers. If the number of mixing stages, N, is sufficiently high, the
resulting curve has a Gaussian shape. However, in FIA, peaks mostly
show a tailing character (1,42). In 1981, Reijn and coworkers (33)
described the distribution curve of an FIA to be a modified Gaussian
function. Mote that only the physical aspects of dispersion are taken
into consideration. It was assumed that there is no contribution to


56
dispersion due to chemical reaction between the sample and reagent
stream. This assumption, however, is known to be invalid (38,42).
For our system, the FIA peaks were examined for their resemblance
to Gaussian, exponentially modified Gaussian and other peak shapes by
measurement of the variance, and the standard deviation of the
parent Gaussian function, ctq. Foley and Dorsey (61) demonstrated that
the type of peak shape can be assigned by the agreement of the values
D
M2 and determined from the asymmetry factor, /A, and peak width,
W, at 10, 30, and 50% peak height (see Figure 22). Equations 4.2 to
4.7 were used to calculate ¡2 and at different peak heights.
__ V 1
= I (eq. 4.2)
1.764(8/A)g l 11.15(B/A)0 ]_ + 28
W
2
0.3
-3.85(B/A)q 3 + 23(B/A)g 3 47.9(3/A)Q 3 + 38.7
(eq. 4.3)
W
0.5
M =
8.23(3/A)g 5 + 41.8(B/A)q g 72.3(B/A)Q g + 44.5
(eq. 4.4)
aG = 3.27(B/A)q 1 + 1.2
(eq. 4.5)
^0.3
G = 2.8(B/A)0 3 + 0.48
(eq. 4.6)


ABSORBANCE UNITS
57
Q.
JO
X
o
L
X
*
<
L
£L
TIME
Figure 22. Measurement of peak width, W, and asymmetry factor, B/A,
at 10, 30 and 50$ peak height in an FIA peak.


and
(eq. 4.7)
aG 2.5(8/A)0 5
Tables V to X show the calculated values for the variance and
standard deviation for pyridoxal in aqueous and micellar media at
different peak heights. For this study, the variable wavelength
absorbance detector was set at 292.6 nm, the maximum absorbance
wavelength for pyridoxal. Triplicate injections of 10 ul of
pyridoxal, 2.81X10-4 M in aqueous solution, were made into the aqueous
stream and peaks were recorded. Manual measurement of peak height, h,
peak width and asymmetry factor at 10, 30 and 50% peak height were
done for each peak and substitution of these values into equations 4.2
to 4.7 gave M^ and 0q. Averages of three values were calculated
for M2 and ctq at each peak height. The same procedure was followed
for 2.83X10-4 M pyridoxal in 0.05 M CTAB injected into 0.05 M CTA8
stream. Table XI shows the average values for M2 and Oq at 10, 30 and
50% peak height. The relative standard deviation of the values from
the three peak heights was calculated for pyridoxal in the aqueous
system to be 6.06% for M2 and 4.88% for relative standard deviation was 5.68% for M2 and 5.20% for ctq.
According to the agreement of the values calculated at the three
heights, these peaks can be classified as exponentially modified
Gaussians. Foley and Dorsey (61) reported a relative standard
deviation of 7.7% for M2 or *3.2% for values calculated at the
three heights was necessary to ensure the validity of the


59
Table V. Variance and standard deviation values for pyridoxal in
aqueous media at 10? peak height. Variable UV-visible
absorbance detector at 292.6 nm, range .1, temperature 45C,
flow rate 1.4 ml/min, tube length 200 cm, i.d. 0.5 mm, 10 pi
sample loop, pyridoxal 2.81X10-4 M, chart speed 10 cm/min.
h (Peak height in cm)
14.15
14.35
14.15
Wq.i (Width at 10% peak height in cm)
2.70
2.70
2.70
A
1.09
1.10
1.08
B
1.61
1.60
1.62
B/A (Asymmetry ratio)
1.48
1.45
1.50
Mo (Variance in cm2)a
0.47
0.47
0.48
X = 0.473 cm2
RSDb = 1.48%
J = 0.448 cm2
RSD = 1.35%
a 1 cm2 = 36 s2.
b Relative standard deviation.


50
Table VI. Variance and standard deviation values for pyridoxal in
aqueous media at 302 peak height. Variable UV-visible
absorbance detector at 292.6 nm, range .1, temperature
45C, flow rate 1.4 ml/min, tube length 200 cm, i.d.
0.5 mm, 10 Ml sample loop, pyridoxal 2.81X10-4 M, chart
speed 10 cm/min.
h (Peak height in cm)
14.15
14.35
14.15
Wq.3 (Width at 30% peak height in cm)
1.90
2.00
1.90
A
0.80
0.85
0.80
B
1.10
1.15
1.10
B/A (Asymmetry ratio)
1.38
1.35
1.38
Mo (Variance in cm2)a
0.57
0.55
0.57
X = 0.563 cm2
RSDb = 2.052
aG (Standard deviation in cm^) 0.437 0.469 0.437
I = 0.448 cm2
RSD = 4.12%
a 1 cm2 = 36 s2.
b
Relative standard deviation.


51
Table VII. Variance and standard deviation values for pyridoxal in
aqueous media at 50% peak height. Variable UV-visible
absorbance detector at 292.6 nm, range .1, temperature
45C, flow rate 1.4 ml/min, tube length 200 cm, i.d.
0.5 mm, 10 ul sample loop, pyridoxal 2.81X10-4 M, chart
speed 10 cm/min.
h (Peak height in cm)
14.15
14.35
14.15
Wq.s (Width at 50% peak height in cm)
1.40
1.40
1.40
A
0.65
0.65
0.65
B
0.75
0.75
0.75
3/A (Asymmetry ratio)
1.15
1.15
1.15
Mo (Variance in cm2)a
0.47
0.47
0.47
X = 0.47 cm2
RSDb = 0%
(Standard deviation in cm^) 0.487 0.487 0.487
T = 0.487 cm2
RSD = 0%
a 1 cm2 = 36 s2.
b
Relative standard deviation.


52
Table VIII. Variance and standard deviation values for pyridoxal in
0.05 M CTA8 micellar media at 10% peak height. Variable
UV-visible absorbance detector at 292.6 nm, range .1,
temperature 45 C, flow rate 1.4 ml/min, tube length
200 cm, i.d. 0.5 mm, 10 yl sample loop, pyridoxal
2.33X10"4 M in 0.05 M CTAB, chart speed 10 cm/min.
h (Peak height in cm)
13.60
13.50
13.70
Wq.i (Width at 10% peak height in cm)
2.85
2.90
2.90
A
1.10
1.15
1.15
B
1.75
1.75
1.75
B/A (Asymmetry ratio)
1.59
1.59
1.59
Mp (Variance in cm2)a
0.55
0.55
0.55
X = 0.55 cm2
RSDb = 0%
(Standard deviation in cm2) 0.445 0.470 0.470
I = 0.462 cm2
RSD = 3.12%
a 1 cm2 = 36 s2.
b
Relative standard deviation.


53
Table IX. Variance and standard deviation values for pyridoxal in
0.05 M CTA8 micellar media at 30% peak height. Variable
UV-visible absorbance detector at 292.6 nm, range .1,
temperature 45C, flow rate 1.4 ml/min, tube length 200 cm,
i.d. 0.5 mm, 10 ul sample loop, pyridoxal 2.83X10-4 M in
0.05 M CTAB, chart speed 10 cm/min.
h (Peak height in cm)
13.60
13.50
13.70
Wq.3 (Width at 30% peak height in cm)
2.10
2.10
2.10
A
0.95
0.95
0.95
B
1.15
1.15
1.15
8/A (Asymmetry ratio)
1.21
1.21
1.21
M2 (Variance in cm2)a
0.58
0.58
0.58
X = 0.58 cm2
RSDb = 0%
aG (Standard deviation in cm2) 0.543 0.543 0.543
Y = 0.543 cm2
RSD = 0%
a 1 cm2 = 36 s2.
b Relative standard deviation.


64
Table X. Variance and standard deviation values for pyridoxal in
0.05 M CTAB micellar media at 50% peak height. Variable In
visible absorbance detector at 292.6 nm, range .1,
temperature 45C, flow rate 1.4 ml/min, tube length 200 cm,
i.d. 0.5 mm, 10 ul sample loop, pyridoxal 2.83X10"4 M in
0.05 M CTAB, chart speed 10 cm/min.
h (Peak height in cm)
13.60
13.50
13.70
Wq 5 (Width at 50% peak height in cm)
1.60
1.70
1.60
A
0.75
0.80
0.75
B
0.80
0.90
0.85
8/A (Asymmetry ratio)
1.13
1.125
1.13
Mo (Variance in cm2)a
0.59
0.667
0.59
X = 0.616 cm2
RSDb = 7.2%
(Standard deviation in cm2) 0.566 0.544 0.566
J = 0.559 cm2
RSD = 2.36%
a 1 cm2 = 36 s2.
b Relative standard deviation.


55
Table XI. Average values for variance and standard deviation for
pyridoxal in 0.05 M CTAB micellar and aqueous media at 10,
30, and 502 peak height for aqueous and 0.05 M CTAB
systems.
102
302
502
X
(cm2)
RSD
(2)
0.05 M
CTAB
m2
(cm^)
0.55
0.58
0.616
0.582
5.68
ctG
(cm2)
0.462
0.559
0.543
0.521
5.20
h2o
m2
(cm2)
0.473
0.559
0.473
0.503
6.06
aG
(cm2)
0.448
0.448
0.487
0.461
4.88


66
exponentially modified Gaussian peak shape. Proven that the FIA peaks
in our system were exponentially modified Gaussian, studies of the
dispersion in aqueous and micellar system were performed by using
equation 4.2. Variance measurements at 10% peak height are more
precise than those calculated at 30% and 50% peak height (61).
Given the conditions in Tables V and VIII, the dispersion between
aqueous and micellar media was compared in terms of the variance,
i^* The values for L were found to be 0.473 and 0.55 cm^ for
aqueous and 0.05 M CTAB media, respectively.
The dispersion of both aqueous and micellar systems was
calculated also by using equation 1.1, which is the definition of FIA
dispersion. The concentration of pyridoxal before the dispersion
process was measured by passing through the detector a solution of
pyridoxal in water (2.81X10-4 M) or in 0.05 M CTAB (2.83X10-4 M) and
measuring the response of the detector. The response of the detector
was obtained by the average of six measurements. The concentration of
pyridoxal after the dispersion process has taken place was obtained by
measuring the maximum peak height of 11 injections of 10 U1 of
pyridoxal solutions (above) into pure aqueous or 0.05 M CTAB. Note
again that dispersion occurring under this circumstance is only due to
physical contributions; no chemical reaction is taking place. The
ratio of the detector response before and after the dispersion process
occurred was 17.62 for the aqueous system and 19.14 for the micellar
system. Contrary to what was expected higher dispersion was found for
micellar media. Due to the higher viscosity of 0.05 M CTAB solution
compared with water, the dispersion was expected to be less. To prove


67
the validity of these results, the same type of experiment was
performed on two different days and the same results were obtained
(see Table XII).
To investigate the effect of surfactant concentration on the
dispersion process, a set of dispersion measurements (M?) versus CTAB
concentration were performed. The concentration of CTAB was varied
from 5X10 ,1 to 5X10 M. This range of concentration includes
solutions above and below the critical micelle concentration. Ten
microliters of pyridoxal (2.81X10-^ M) aqueous solution was injected
into the stream and from the recorded peaks the variance at 10% peak
height was calculated. From Table XIII, one can say that for a
solution of 5X10^ M the dispersion obtained is very similar to that
calculated for water. Above this concentration an increase in
surfactant concentration had no effect on the dispersion up to a
concentration of 5X10^ m CTAB, at which point an increase in
dispersion was observed. Micellar concentration changes the
dispersion of the FIA system. This may be explained by the fact that
the pyridoxal molecules are localized on or within the micelle
structure. Micellar media is more viscous than aqueous media (43);
therefore, the mass transfer in a radial direction decreases. An
increase in peak dispersion results because a decrease in mixing
across the stream tends to increase dilution of the solute by
longitudinal dispersion (42). The increase of dispersion with
micellar media needs further study.


68
Table
XII. Dispersion
values
for aqueous and micellar
systems.
0.05 M CTAB
Aqueous
m2
m2
C/Cmax
(cm2)
c/cmax
(cm2)
1.
19.54
0.55
17.62
0.47
2.
19.14
0.56
17.05
0.45
3.
19.26
0.54
17.25
0.45
C/Cmax = ratio of concentration of pyridoxal before and after the
dispersion process has taken place.
O
M2 = variance or second moment in cm .


69
Table XIII. Measurement of dispersion versus CTAB concentration.
Variable UV-visible absorbance detector at 292.6 nm,
range .1, temperature 45C, flow rate 1.4 ml/min, tube
length 200 cm, i.d. 0.5 mm, 10 ul sample loop, pyridoxal
2.81X10-4 M in deionized water, chart speed 10 cm/min.
k2
(cm2)
m2
(cm2)
m2
(cm2)
M2 Average
(cm2)
CTAB (M)
5.0X10"6
0.44
0.43
0.46
0.44
2.5X10"5
0.52
0.46
0.52
0.50
5.0X10'5
0.47
0.52
0.49
0.49
2.5X10'4
0.48
0.50
0.46
0.48
5.0X10"4
0.49
0.46
0.52
0.49
2.5X10-3
0.52
0.50
0.49
0.50
5.0X10-3
0.50
0.52
0.52
0.51
2.5X10-2
0.47
0.52
0.49
0.49
5.0X10'2
0.52
0.57
0.53
0.54
h2o
0.43
0.44
0.48
0.45
= variance or second moment in cm
2


CHAPTER V
CONCLUSIONS AND FUTURE WORK
The applicability of combining the technique of Flow Injection
Analysis with micellar catalysis has been shown. The results obtained
from the determination of pyridoxal in micellar media compared to
those in aqueous media are promising. Higher response, as measured by
peak height, was recorded at all times for micellar carrier
solutions. Higher sensitivities and lower limits of detection were
obtained for the micellar system when the oxidation product of
pyridoxal and cyanide was detected either fluorimetrically or by UY
absorbance. Higher sensitivity ratios, for micellar to aqueous
systems, were obtained when using fluorescence detection. In this
case, not only is the reaction taking place at a faster rate but the
solubilization of reagents within the micelle structure contributes to
increase the signal due to the shielding effect.
With this example, it has been proven that due to the kinetic
nature of the FIA technique the use of micellar media can be very
advantageous. To support this investigation, the utility of using
micellar carrier solution in an FIA system, it will be necessary to
perform similar experiments by running different reactions taking
place in aqueous and micellar media. Keeping in mind the kind of
surfactant that will catalyze the specific reaction (anionic,
70


71
cationic, nonionic or zwitterionic), the rate of the reaction should
be measured and if it is favorable applied to FIA.
One of the reactions that will be very interesting to look at is
the determination of metals by their complexation with dyes in the
presence of micelle media (62-68). Some characteristics observed for
these metal-dye complexes in micellar media are an increase in molar
absorptivity and red shifts in the wavelength of maximum absorbance
(53). These substantial changes in the UV visible spectrometry of
these complexes together with the technique of FIA can be developed as
a new spectrophotometrie method for determining micro amounts of metal
ions. The resulting method should be fast, easy and inexpensive.
Lower background signals were found for the aqueous systems
compared to CTAB micellar systems. Therefore, the limits of detection
for micellar media were not as low as expected. This increase in
background noise for micellar media needs further study to elucidate
if the increase in noise is due to the presence of micelles or if it
is just observed with this specific reaction.
Due to the specific interactions between micelles and solutes the
selectivity of a particular reaction can be increased, reducing the
amount and kind of interferences. An investigation on this topic will
be very valuable especially for the determination of very small
amounts of analytes.
It has been mentioned that catalysis of organic and inorganic
reactions also occur in apolar media in the presence of reversed
micelles (19,69). Reversed micelles offer similar and at the same
time different characteristics from normal micelles. Future research


72
on the application of reversed micelles for reactions occurring in
apolar media in combination with the technique of FIA will be very
interesting.
According to the agreement of the values for the second moment
and the standard deviation measured at 10, 30 and 50% peak height, the
peaks recorded from an FIA system can be designated to be
exponentially modified gaussian.
Higher values for dispersion were found for micellar media.
These results were not as expected and need further study to
understand better the reason for this increase in dispersion. More
detailed experiments should be performed under different conditions,
e.g., varying the type and concentration of surfactant, temperature,
etc.
The contribution of a chemical reaction to the dispersion of a
peak in FIA is not well understood. 3y comparing the dispersion of
different reactions taking place in different types of surfactants to
the dispersion obtained in aqueous media could help to explain the
contribution of the chemistry to the total dispersion process. The
differences in rate of reaction between different micellar and aqueous
systems can help to explain this phenomena. By taking a reaction
whose rate of reaction varies in the presence of anionic, cationic,
nonionic and aqueous media, the contribution of chemical kinetics may
be clarified.


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BIOGRAPHICAL SKETCH
Mara A. Hernandez Torres was born in Mayagez, Puerto Rico, on
May 29, 1958. She received her elementary and high school education
at the Colegio de La Milagrosa, Mayagez, Puerto Rico. In 1980, she
completed her Bachelor of Science degree in chemistry at the
University of Puerto Rico Mayagez Campus (magna cum laude). In 1983,
she pursued a Master of Science degree in analytical chemistry at the
University of Florida. She is now receiving a Doctor of Philosophy
degree in analytical chemistry at the University of Florida.
77


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
G
orsey, Chairman
Professor of Chemistr
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
S?. kJ
imes D. Winefordnei
Graduate Research l/rofessor of Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
4
jterf-To-
Anna F. Brajterf-Toth
Assistant Professor of Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Martin Vala
Professor of Chemistry


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
This dissertation was submitted to the Graduate Faculty of the
Department of Chemistry in the College of Liberal Arts and Sciences
and to the Graduate School and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.
August, 1986
Dean, Graduate School


Full Text
UNIVERSITY
OF FLORIDA


MICELLAR CATALYZED REACTIONS
FOR FLOW INJECTION ANALYSIS
BY
MARÍA A. HERNÁNDEZ TORRES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986

To my parents,
Saul and Carmen,
with all my love.

ACKNOWLEDGMENTS
I would like to express my gratitude and appreciation to my
research director, Dr. John G. Dorsey, for his invaluable assistance,
guidance and encouragement throughout the years. I was very lucky to
have him as a teacher. His special qualities helped me to develop as
a professional, and at the same time he allowed me to grow as an
individual. His view of science and life created a good and healthy
environment for learning. His lessons will always be remembered.
Special thanks are given to Dr. Morteza G. Khaledi, for his
helpful suggestions and advice at the beginning of this project.
I would like to express my gratitude to my colleagues and friends
in Dr. Dorsey's research group, for their friendship and daily
encouragement. I treasure very much the time spent at the lab with
all of them even when life was tough. I will miss them all!
I would like to thank the faculty members at the University of
Florida with whom I was in contact during my graduate years. Special
thanks are due to the members of my committee, for their contributions
to my learning experience.
I would like to mention Cindy Zimmerman, for her promptness and
accuracy in typing the final draft of this thesis.
There are no words to express how thankful I am to my family and
friends. Their love, support, encouragement, patience, and faith in
me made possible this new achievement in my career. I want to thank
i ii

A
my grandparents, Avelina and Jose Antonio, for their care throughout
the years.
iv

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT ix
CHAPTERS
I INTRODUCTION 1
Principles of Flow Injection Analysis 1
Properties of Surfactants and Micelles in Aqueous
Solution 10
II THEORY AND BACKGROUND 17
Chemical Kinetics in a Flow Injection Analysis System....17
General Features of Micellar Catalysis 19
III EXPERIMENTAL: FIA SYSTEM FOR PYRIDOXAL DETERMINATION....23
Appara tus 23
Reagents 25
Procedure 25
IV MICELLAR CATALYSIS IN THE DETERMINATION OF PYRIDOXAL
BY FLOW INJECTION ANALYSIS 26
Results and Discussion 26
Measurements of Dispersion 55
Y CONCLUSIONS AND FUTURE WORK 70
REFERENCES 73
BIOGRAPHICAL SKETCH 77
v

LIST OF TABLES
Table Page
IFigures of merit for pyridoxal determination 40
IIVariable parameters for the Modified Simplex Optimization
program 42
IIIOptimized and fixed variables for the determination of
pyridoxal 45
IVFigures of merit for pyridoxal determination with
conditions obtained by Modified Simplex program 47
VVariance and standard deviation values for pyridoxal
in aqueous media at 10% peak height 59
VIVariance and standard deviation values for pyridoxal
in aqueous media at 30% peak height 60
VIIVariance and standard deviation values for pyridoxal
in aqueous media at 50% peak height 61
VIIIVariance and standard deviation values for pyridoxal
in 0.05 M CTAB micellar media at 10% peak height 62
IXVariance and standard deviation values for pyridoxal
in 0.05 M CTAB micellar media at 30% peak height 63
XVariance and standard deviation values for pyridoxal
in 0.05 M CTAB micellar media at 50% peak height 64
XIAverage values for variance and standard deviation
for pyridoxal in 0.05 M CTAB micellar and aqueous media
at 10, 30, and 50% peak height for aqueous and
0.05 M CTAB systems 65
XII Dispersion values for aqueous and micellar system 68
XIII Measurement of dispersion versus CTAB concentration 69
VI

LIST OF FIGURES
Figure Page
1 Diagram of an FIA system (a) and typical recording (b) 3
2 Dispersion in an FIA system 6
3 Velocity profiles and shapes of injected sample bolus 9
4 Dill-Flory's representation of a normal micelle 12
5 A two dimensional schematic representation of the
regions of a spherical ionic micelle 14
6 FIA manifold for the determination of pyridoxal 24
7 Reaction and surfactant media used for the analysis
of pyridoxal 27
8 Absorbance versus wavelength (nm) in aqueous system
for the determination of pyridoxal 29
9 Absorbance versus wavelength (nm) in 0.05 M CTA8
micellar system for the determination of pyridoxal 30
10 Change in maximum absorbance (355 nm) versus time
(minutes) in aqueous media for the determination
of pyridoxal 31
11 Change in maximum absorbance (355 nm) versus time
(minutes) in 0.05 M CTAB micellar media for the
determination of pyridoxal 32
12 Log versus time (minutes) for pyridoxal
in aqueous system 34
13 Log (A^-AJ versus time (minutes) for pyridoxal
in 0.05 M CTAB system 35
14 Absorbance calibration plots for pyridoxal in aqueous
( â–¡ ) and 0.05 M CTAB ( â–  ) systems 36

15 Fluorescence calibration plots for pyridoxal
determination in aqueous ( â–¡ ) and 0.05 M CTAB ( â–  )
systems 38
16 Absorbance calibration plots for pyridoxal in aqueous
( □ ) and 0.09 M CTAB micellar ( • ) media 46
17 Absorbance recordings of a series of pyridoxal
standards in aqueous media 48
18 Absorbance recordings of a series of pyridoxal
standards in 0.05 M CTAB micellar media 49
19 Absorbance recordings of a series of pyridoxal
standards in 0.09 M CTAB micellar media 51
20 Fluorescence recordings of a series of pyridoxal
standards in aqueous media 52
21 Fluorescence recordings of a series of pyridoxal
standards in 0.05 M CTAB micellar media 54
22 Measurement of peak width, W, and asyrmietry factor,
B/A, at 10, 30 and 50% peak height in an FIA peak 57
v i i i

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MICELLAR CATALYZED REACTIONS
FOR FLOW INJECTION ANALYSIS
3Y
MARIA A. HERNANDEZ TORRES
August, 1986
Chairman: Dr. John G. Dorsey
Major Department: Chemistry
In this study, the applicability of micellar carrier streams for
the catalysis of reactions in FIA was investigated. Flow Injection
Analysis (FIA) is an automated kinetic method of analysis. An FIA
system with low dispersion and fast reaction kinetics will provide low
limits of detection and high sampling rates.
Micelles exhibit the ability to solubilize hydrophobic compounds
on or within their structures. The rate of many reactions in micellar
media is altered due to the proximity of reagents and analyte, changes
in the microenvironment and orientation of solutes.
The advantages of combining the solubilization property and
micellar catalysis for a given reaction that is taking place in an FIA
system are demonstrated. The reaction of pyridoxal (a Bg vitamer)
with cyanide was investigated in aqueous and micellar media. The
cationic surfactant, hexadecyltrimethyl ammonium bromide (CTAB), was
chosen for the micellar carrier solution. The oxidation product of
ix

this reaction, 4-pyridoxolactone, was detected either fluorimetrically
or by ultraviolet absorbance. The reaction rates in the two media
were determined and compared. Calibration plots for pyridoxal were
made and the analytical figures of merit were compared for aqueous and
0.05 M CTAB micellar media.
A Simplex Optimization was carried out for the determination of
pyridoxal in micellar media. A new set of conditions for micellar
media was obtained from the Simplex program. With this new set of
conditions, a calibration plot for the micellar media was prepared and
subsequently compared to the calibration plot for the aqueous
system. The analytical figures of merit for the two carrier solutions
were calculated and compared.
The peak shape obtained in the FIA system was investigated. The
standard deviation and variance at 10, 30, and 50% peak height were
calculated by empirical equations. The agreement of these values
allows the peaks to be classified as exponentially modified gaussians.
Measurement of dispersion in both aqueous and micellar systems
was investigated. Dispersion values for both aqueous and micellar
systems were examined by two methods: by using the definition of
dispersion in FIA and by measuring the variance (second moment) at 10%
peak height. In both methods, higher values for dispersion were
obtained for micellar media.
x

CHAPTER I
INTRODUCTION
Principles of Flow Injection Analysis
Flow injection analysis (FIA) is now established as a fast,
precise, accurate, efficient and extremely versatile analytical
tool. The FIA technique is used by many analytical chemists working
in a variety of different industries and institutions (1-9).
A historical review of the development of FIA reveals that since
its conception in the early 1970s, many of the concepts of flow
injection analysis have been adopted from other fields and many
workers have contributed to its development (10). However, the
technique of FIA became known by the simultaneous appearance of the
work of Stewart, Beecher and Hare (11) in the United States and
Ruzicka and Hansen (12) in Denmark in 1975. The Danish group
developed the method using primarily instrumentation normally
associated with segmented flow analyzers. In contrast, the American
group based their initial work on high performance liquid
chromatography components. For this reason, FIA is considered a
hybrid of the two techniques.
In the past, it was generally assumed based on Skegg's concept
that air segmentation and attainment of a steady state signal were
essential for performing continuous flow analysis (6). The presence
of air bubbles in the analytical stream was thought to be necessary
1

2
to limit sample dispersion, to promote mixing of the sample with
reagents (by generating turbulent flow) and to scrub the walls of the
analytical conduits to prevent carryover of samples. However, it was
proven that analysis without air segmentation is not only possible but
also advantageous. In FIA, there is no air segmentation, the sample
is introduced as a plug via a valve or syringe, mixing is mainly by
diffusion-controlled processes, and the response curves do not reach
the steady-state plateau, but have the form of sharp peaks. The
absence of air segmentation leads to a higher sample throughput. The
presence of a sample carrier interface over which concentration
gradients develop during the course of analysis has opened new
analytical possibilities for continuous flow analysis. The
reproducibility is good, and there is no sample carryover. There is
no need to introduce and remove air bubbles, and an expensive high
quality pump is not necessary.
Flow injection analysis is based on the injection of a liquid
sample into a moving, nonsegmented reagent carrier stream. The
injected sample forms a zone that disperses and reacts with the
carrier on its way to a detector (5). The simplest FIA analyzer
(Figure la) consists of a pump (P) that propels the carrier stream
(R), an injector port (S), by which a well defined sample volume is
injected into a carrier stream, and a coil in which the sample zone
disperses and reacts with the components of the carrier stream,
forming a species to be sensed by a flow-through detector (D). A
typical recording has the form of a sharp peak (Figure lb) the height
of which is related to the concentration of analyte, and the time from

(a)
mL/min
2-30 s
Figure 1. Diagram of an FIA system (a) and typical recording (b)
(from 1).

injection to peak maxima is termed the residence time. Residence
times are typically from 3 to 30 seconds.
4
Between the points of injection and detection the sample plug
will have been physically dispersed to some degree and, in addition,
some chemical reactions will have taken place. The peak will reflect
both processes.
The injection of a sample lias to be done in such a way that by
inserting a discrete slug of sample into a continuously moving stream
the movement of the stream is not disturbed. The residence time of
the sample in the analytical manifold should ideally be identical for
each sample, and the conditions to which the sample is exposed during
processing should also be the same. The reason for this is that not
only the physical dispersion but the chemistry involved requires a
reproducible travel pattern of the sample from the point of injection
to detection. In an FIA system, neither the mixing nor the chemical
reaction is complete; an equilibrium for either process is not
attained (1). By monitoring the reaction at a fixed precise time, the
concept of the steady state can be abandoned. This measurement at a
fixed time is just as analytically significant as the steady state
signal. Any fluctuations in residence time of the sample, i.e.,
variation in flow rate, will result in imprecise measurement of the
signal.
When a sample is first injected, it forms a well-defined sample
plug in the stream. As the sample is swept downstream through the
analytical conduits of narrow bore tubing, the plug disperses into
and, thus, mixes with the carrier stream under laminar flow conditions

5
to form a gradient. The magnitude of this dispersion is dependent on
the operating parameters applied to the system, including sample
volume, tubing bore size, tubing length, flow rate, sample volume and
coil diameter (1,6). Varying the values of these parameters confers a
significant degree of control over the dispersion characteristics and
facilitates optimization of a flow injection system for many diverse
applications, so that the optimum response is obtained at minimum time
and reagent expense.
The response curve has the shape of a peak reflecting a continuum
of concentrations. In contrast to all previous methods of automated
assay, there is no single element of fluid that has the same
concentration as the neighboring one (7) (Figure 2).
The dispersion coefficient, D, is the ratio of the concentration
of sample solution before (C°) and after (C) the dispersion process
has taken place (Figure 2). The dispersion coefficient can be defined
at any point of the curve but, for convenience in the majority of FIA
methods, the dispersion coefficient is defined at maximum peak height.
C°
In cases where a reaction is developed as a result of mixing
effected by dispersion, the peak height will increase as the reaction
proceeds toward completion. The maximum response will then be
attained when an optimum balance is reached between dispersion and
reaction time.

CONCENTRATION
t or scan
O')
Figure 2. Dispersion in an FIA system (from 7).
RESPONSE

7
Flow injection analysis operates only in the laminar flow region
(3,13-16). In this region, FIA systems generate dispersion through
both diffusion and convection. Under such conditions of laminar flow,
the layer of liquid in contact with the tube surface is practically
stationary and the velocity of centrally placed molecules is twice the
mean velocity of the liquid. This gives rise to a parabolic velocity
profile (Figure 3a). In the absence of molecular diffusion, a sample
placed into a moving stream would have an infinitely long tail by the
time it reached the detector (Figure 3b). This results in
unacceptable carryover between samples. Diffusion of molecules
between the carrier and sample bolus serves to limit this convective
dispersion and effectively mixes sample and reagent. In coiled tubes,
it is the radial rather than the axial dispersion that contributes
most significantly to sample dispersion in FIA systems. This type of
dispersion, also called secondary flow, operates to move the fluid
both toward and away from the tubing wall and thus serves as an
efficient scrubbing mechanism (Figure 3d). A molecule located at the
center will tend to diffuse into a region of lower sample
concentration and by doing so it will move into a layer of liquid
moving at a slower longitudinal velocity. On the other hand, a
molecule located near the wall will diffuse toward the center of the
carrier solution and it will encounter a layer of faster moving
liquid, which will carry it away from the wall and toward the center
of the sample zone. This radial diffusion perpendicular to the
direction of the flow modifies the shape of the bolus head and the
result is low carryover and cross contamination. High sample

Figure 3. Velocity profiles and shape of an injected sample bolus:
(a) Laminar flow, parabolic velocity profile; (b) Sample
dispersion caused by laminar flow without diffusion; (c)
Sample shape resulting from laminar flow with molecular
diffusion; (d) Secondary flow pattern in the cross-section
of a tightly coiled tube (from 3).

9
tube wall

10
throughput is obtained because of the limited band spreading. When
samples under these conditions reach the detector they have a shape as
in Figure 3c.
The versatility and simplicity of FIA systems permits this
technique to be widely used for performing chemical assays. The
recent increase in publications dealing with new FIA methods as well
as in separate symposia on this topic indicate the popularity of this
relatively new technique (2). On the other hand, more has to be done
on the theoretical side, where the dispersion of the sample zone must
be investigated in much greater detail. The theory of dispersion,
although very useful, is far from being exact and complete (17). Only
deeper theoretical studies will lead to design of even more advanced
continuous flow techniques that will allow chemical analyses to be
performed in new ways.
Properties of Surfactants and Micelles in Aqueous Solution
Scientists from around the world have shown special interest in
surfactants because of their unique characteristics (18-24).
Surfactants, or surface active agents, are amphiphilic molecules
having both pronounced hydrophobic and hydrophilic properties.
Surfactants are classified as cationic, anionic, nonionic or
zwitterionic according to the hydrophilic part (polar head group).
The hydrophobic tail consists of a hydrocarbon chain usually from 8 to
20 carbon atoms in length. Furthermore, this hydrophobic tail can
contain unsaturated portions or aromatic moieties, can be partly or

11
completely halógena ted, and can be branched or consist of two or more
chains.
At low concentration, the surfactant is dispersed predominantly
as monomers, although dimers, trimers, etc. can exist. Over a narrow
concentration range, termed the critical micelle concentration (CMC),
surfactants have the important property of forming molecular
aggregates, called micelles. Above the CMC, there exists a dynamic
equilibrium between monomers and micelles. The amount of free monomer
remains approximately constant and equal to the CMC.
In aqueous solutions, surfactant molecules (typically from 60 to
100) associate to form a roughly spherical cluster (25) (Figure 4).
This micelle structure is such that the hydrophilic head groups are
directed toward and in contact with the aqueous solution, forming a
polar surface, while the hydrophobic tails are directed away from the
water forming a liquid-like hydrocarbon core. The microviscosity of
this core is considerably higher than in hydrocarbons. The micellar
surface is not uniform: some of the hydrocarbon chains are turned
towards the solvent or at least come into contact with it periodically
(26). On the whole, it may be supposed that the micellar surface is a
polar environment differing in properties from water itself.
Changes in temperature, concentration of surfactant, additives in
the liquid phase, and structural groups in the surfactant may cause
changes in the size, shape, and aggregation number of the micelle
(26-28).

12
Figure 4. Dill-FIory's representation of a normal micelle. The ionic
head groups are indicated by the circles, and the
hydrocarbon chains are pointing toward the center of the
micelle (from 25).

13
In micelles of ionic surfactants, the charged head groups and the
counterions are located in a compact region, known as the Stern layer,
which extends from the core to within a few angstroms of the shear
surface of the micelle. Beyond the Stern layer, the remainder of the
counterions are located in the Gouy-Chapman electrical double layer
where they are completely dissociated from the charged aggregate and
are able to exchange with ions in the bulk of the solution (26)
(Figure 5).
The driving force for micelle formation in aqueous media is due
primarily to the hydrophobic effect, and the electrostatic repulsion
between the ionic head group limits the size that a micelle can
attain, thereby keeping the micelle size small.
One of the most important properties of micellar systems is their
ability to solubilize a variety of species (18-24,26-28).
Solubilization may be defined as the spontaneous dissolving of
substance (solid, liquid or gas) by reversible interaction with the
micelles of a surfactant in a solvent to form a thermodynamically
stable isotropic solution with reduced thermodynamic activity of the
solubilized material (24). The importance of the phenomenon of
solubilization from the practical point of view is that it makes
possible the dissolving of substances in solvents in which they are
normally insoluble or slightly soluble. Solubilization is a dynamic
equilibrium process and depends on temperature, nature of solute,
surfactant concentration and type of micellar system employed (26).
There are several possible solubilization sites and orientations
available in a micellar system and the site occupied by a solubilizate

14
Figure 5. A two dimensional schematic representation of the regions
of a spherical ionic micelle (from 24).

15
depends upon the nature of both the solute and the micelle. There is
a rapid equilibrium between various possible sites and also between
the solubilized state and the free state in the aqueous medium. The
site of solubilization is a topic of current debate. Some contend, if
the solute is nonpolar, it may pass completely into the hydrophobic
core or penetrate a particular depth into the surface layer. Others
contend it may be adsorbed at a hydrophobic region on the surface
(13).
Micellar systems are very useful in the field of analytical
chemistry due to the unique properties of these organized
assemblies. Micelles can lead to the modification and improvement of
existing procedures and to the development of new methods of chemical
analysis. Micellar structures offer means to overcome solubility
problems, to speed reaction rates of analytical reactions and to
reduce side reactions, to shift acid base or redox equilibria, to
change spectral distribution or intensities, and to improve
selectivity and efficiency in extraction and chromatographic
methods. Several reviews on the use of surfactants in analytical
chemistry have appeared (29-31).
Our studies are directed mainly to observing the behavior of
combining micellar media with the technique of FIA. This represents
the first attempt to demonstrate the advantages of combining the
solubilization property and micellar catalysis for a given reaction
that is taking place in an FIA system. Higher sensitivity and/or
lower limits of detection are expected for a given FIA system when the
reaction is carried out with the appropriate micellar media. Kinetic

16
studies and measurement of the analytical figures of merit will be
compared for reactions taking place in aqueous and micellar media.
v

CHAPTER II
THEORY AND BACKGROUND
Chemical Kinetics in a Flow Injection Analysis System
It is well recognized that flow injection analysis yields a
response curve which is the result of two processes, both kinetic in
nature: the physical process of dispersion and the chemical process
of formation of reaction products (1-6,32).
The kinetics of physical dispersion, or incomplete mixing, has
been described in a number of papers (14-16,33-35) while attempts to
describe the effect of chemical kinetics has been practically
ignored. The shape of the transient peak profiles of FIA
determinations have been described only in terms of the dispersion
process. However, there has been an attempt recently to explore in
more detail the complexity of the overall kinetic process taking place
inside the sample plug and its boundaries in FIA systems and the
contribution of this kinetic chemistry to the peak profiles. Ruzicka
and Hansen recognized that "it is obvious that the comprehensive
theory of the flow method will eventually combine the theory of mixing
of liquids in continuous moving streams with the theory of chemical
kinetics" (36). From now on, more attention will be paid to the
chemistry process that is taking place (37-42).
In developing a flow injection method, one of the primary goals
is to maximize response together with sample throughput. These
17

13
parameters are intimately related to the sensitivity of the method and
the number of injections possible per hour.
Since there exists an interdependence between the reaction rate
and the rate of dispersion, both factors have to be weighed when
designing a new FIA system. The longer a sample stays within the
system, the greater will be the signal due to the chemistry that is
taking place. Longer residence times mean more time is allowed for
the reaction to take place, therefore more product is formed. This
increase is balanced against the point where dispersion will overcome
the formation of the product and cause the signal to decrease. The
longer the sample stays between the points of injection and detection,
the higher will be the dispersion and the signal measured (usually
peak height) will decrease. Another factor to be considered is time
needed for each determination. Longer residence times allow maximum
sensitivity of measurement but at the expense of decreasing sample
throughput. Therefore, short residence times are often preferred.
Fast chemical reactions are required for performing a simple FIA
analysis at a continuous carrier pumping rate with practical residence
times of about 30 seconds. Slow reactions must presently be performed
by the stopped flow mode or in a packed reactor (17). In stopped flow
mode, an electronic timer is used to cease the movement of the carrier
stream containing the sample zone in order to allow enough time to
produce an adequate amount of detectable product, and then the sample
is pumped through the flow cell while the peak is recorded in the
usual manner. Reactors packed with inert materials, such as glass,

19
enhance micromixing of sample and reagents in the carrier stream
without loss of peak height or loss of sample frequency.
General Features of Micellar Catalysis
It has been reported that with the proper choice of surfactant
the rate of a chemical reaction is substantially enhanced in a
micellar solution relative to that in the corresponding aqueous system
(18,24,26,27,31,43-45).
Research on the effect of surfactants on the kinetics of organic
reactions has demonstrated that it is the micelle structure, not the
individual molecules, that is responsible for the catalysis or
inhibition of these reactions. In accordance with this fact, the term
micellar catalysis has been applied to this phenomenon. Rate
acceleration or inhibition of organic reactions in micellar solution
arises from different rates of reaction of the substrate in the
micellar phase and in the bulk solution and the distribution of the
substrate between these two phases (26,32). Then, a prerequisite to
understanding reaction kinetics in micellar systems is to understand
the structure and solubilization properties of the micelles themselves
(vide supra).
The kinetics of organic reactions occurring in micellar systems
are dominated by electrostatic and hydrophobic interactions between
the micelle structures, reactants, transition states and products.
The two physicochemical factors responsible for the efficiency of
micellar catalysis are

20
(1) the change in the reactivity of reagents on transfer from
water to the micellar phase and
(2) the concentration of reactants into the micellar phase.
The first factor, the differences in reactivity, can be explained by
the difference in the distribution of a substrate between these two
phases and by the difference in the degree and nature of substrate-
micelle binding. When a solute is solubilized in a micellar system,
the microenvironment about it is very different compared to that in
the bulk solvent. Micellar systems have the ability to change the
effective microenvironment and the microscopic properties of
solubilized solutes to that of aqueous media favoring the acceleration
of some organic and inorganic reactions.
For catalysis to occur, it is necessary that the substrate be
solubilized by the micelle and the site of solubilization be such that
the reactive site of the substrate is accessible to the attacking
reagent. It is here that hydrophobic interactions become important,
because they determine the extent and the site of solubilization in
the micelle. In general micellar effects on reactions follow several
rules, although there are exceptions. A hydrophobic reactant is
attached to a micelle by hydrophobic interactions, independently of
the charge on the micelle. If the second reactant is oppositely
charged to the micelle, it will be bound to the micelle and the
reaction is usually accelerated. When micelles and reactant ions bear
like charges, the reaction is inhibited due to the repulsion forces
between the ions and the micelle's surface. Nonionic or zwitterionic
micelles, generally, have no significant influence on the rates of

21
these reactions. The rate of certain organic reactions is unaffected
when one of the reactants is incorporated into the micellar phase and
the other is excluded from it. Exceptions to these rules can be
explained by the fact that sometimes hydrophobic effects overcome the
electrostatic repulsions and even when the micelle's surface charge
does not favor the reaction, catalysis does occur.
The orientation of the reagents in micellar media is different
from the bulk aqueous phase due to the different microenvironment that
the reagents experience on or within the micelles. If this micro¬
environment is more attractive to reagents, the reagents are going to
spend more time on this phase; therefore, they will be concentrated in
this region. Also, the micelle structure provides a very specific and
reduced region where the reagents are being solubilized. If the
reagents are localized within this small region, they are being
concentrated and are closer to each other inducing the reaction to go
faster. These are called proximity and concentration effects.
Quite generally, increasing the hydrophobic character of the
surfactant, having longer alkyl chains, increases its efficiency as a
catalyst. At equal concentration of two surfactants, the more
hydrophobic may appear to be the better catalyst (or inhibitor) simply
because it has greater affinity for the substrate. Variation in
substrate structure has a profound influence, in many cases, on the
magnitude of micellar catalysis. The general rule seems to be that
the more hydrophobic the substrate, the more pronounced the micellar
catalysis.

22
The multiphase profile of surfactant concentration on the
reaction rate is as follows: below the CMC, the rate constants are
independent of surfactant concentration; above the CMC, the rate
constants rise rapidly with increasing surfactant concentration, level
off, and finally decrease with increasing concentration of
surfactant. This profile can be rationalized by the fact that the
rate constant increases as the concentration of micellar bound
reactants increases, but eventually an increase in surfactant
concentration dilutes the reactants in the micellar pseudophase, with
a decrease in the rate constant (18).
The influence of electrolytes on micellar catalysis is less
predictable. For most reactions micellar catalysis is inhibited by
counterions and the larger the ion, the greater the effect. This
behavior has been rationalized by assuming a competition between the
reactant and the electrolyte for a binding site on or in the
micelle. This salt inhibition may be explained principally by the
displacement of one reactant from the micellar surface by the
electrolyte. Enhancement of the micellar catalyzed reaction rate by
counterions has been suggested to be due to changes in micellar
structure by the salts and this new configuration of the surfactants
promotes the reactions.
Another advantage of using micellar media is that of favorable
substrate partitioning and binding in specific orientations and
configurations on the micelle structure. This makes the reaction very
selective to that substrate by decreasing the probability of other
interfering species competing for the place of the substrate.

CHAPTER III
EXPERIMENTAL: FIA SYSTEMS FOR
PYRIDOXAL DETERMINATION
Apparatus
The flow injection manifold used is shown in Figure 6. The
reagent streams were pumped by an Isco (Lincoln, NE) Tris model
peristaltic pump. Samples were introduced with a Rheodyne (Cotati,
CA) model 7125 sample injection valve with a 10 ul loop. All tubing
was Teflon from the Anspec Company, Inc. (Ann Arbor, MI) with 0.5 mm
internal diameter. The reaction coil, 200 cm long, was tnermostated
by immersing it in a water bath with a Techne TE-7 circulator
(Cambridge, England). For fluorescence measurement, a Varian (Palo
Alto, CA) model Fluorichrom detector with a 25 ul total volume flow
cell was used. A combination of Varian filters was selected for
355 nm excitation wavelength and 435 nm emission wavelength. A Kratos
(Ramsey, NJ) model Spectroflow 757 absorbance detector with a 12 ul
flow cell was set at a wavelength of 355 nm to measure the absorbance
of the product. The output signals were recorded on a strip chart
OmniScribe recorder, Houston Instrument (Austin, TX). The pH of the
reagent solutions were measured with a Corning (Medfield, MA) model pH
meter 130.
For kinetic studies, the reaction was followed spectrophoto-
metrically by measuring the rate of change in the absorbance of
23

PYRIDOXAL
10 \l\
WASTE
Figure 6. FIA manifold for the determination of pyridoxal.

25
4-pyridoxolactone, at 355 nm using a Hewlett Packard (San Diego, CA)
model 3450A Diode Array spectrophotometer connected to a Hewlett
Packard model 7470A plotter.
Reagents
All reagents were used as received and prepared either in
deionized water or in surfactant solutions. The cationic surfactant
was hexadecyltrimethylammonium bromide (CTAB, purum grade) from Fluka
Chemical (Hauppauge, NY). Standard solutions of pyridoxal, Sigma
grade (Sigma Chemical Company, St. Louis, MO) and solutions of
potassium cyanide, certified ACS, from Fisher Scientific Company
(Fair Lawn, NJ) were used for this study. Phosphate buffer solutions
(0.6 M) certified ACS from Fisher Scientific Copmany were prepared and
the pH was adjusted with concentrated hydrochloric acid, ACS
certified, from Mallinckrodt (Paris, KY).
Procedure
The appropriate weight of surfactant was dissolved in distilled
water and the solution then filtered through a 0.45 urn nylon-66
membrane filter (Rainin Instruments, Woburn, MA). Appropriate amounts
of pyridoxal and cyanide were dissolved either in distilled H20 or
micellar solution. All reported values are averages of at least four
determinations.

CHAPTER IV
MICELLAR CATALYSIS IN THE DETERMINATION OF
PYRIDOXAL BY FLOW INJECTION ANALYSIS
Results and Discussion
Pyridoxal is one of the three substances designated as vitamin
Bg. Determination of pyridoxal and its derivatives is of great
interest, especially in clinical chemistry. A fundamental role for
pyridoxal has been postulated in the mechanism for active transport of
amino acids and metals ions across the cell membrane (46).
The analysis of pyridoxal in biological material has proven to be
difficult and unsatisfactory. Pyridoxal has been analyzed via high
pressure liquid chromatography (HPLC) with amperometric, enzymatic-
fluorometric or photometric detectors and via radiochemical means.
The most common detection procedure for pyridoxal is fluorimetry, in¬
volving the use of Zn-glycine or formation of hydrazone derivatives
(47).
P. Linares and coworkers (47) reported a fluorimetric method for
determination of pyridoxal by flow injection analysis. This method
was based on the oxidation of pyridoxal in the presence of cyanide to
yield 4-pyridoxolactone (see Figure 7a).
There is no spectral evidence of reaction between cyanide and the
other vitamin Bg derivatives. These results indicate that the
carbonyl is the only group in the pyridoxal molecule capable of
reacting with cyanide (48-50).
26

27
HOCH
CHO
ch3
OH
pyridoxal cyanide
4-pyridoxolactone
(a)
CTAB:
CH3( CH2)|5 N+(CH3 >3 Br”
(b)
Figure 7. Reaction and surfactant media used for the analysis of
pyridoxal. Reaction for pyridoxal with cyanide (a) and
CTAB's molecular formula (b).

23
Preliminary studies on the behavior of this reaction are shown in
Figures 8 and 9. At a wavelength interval of 280 to 400 nm, the
change in absorbance versus time was recorded for aqueous and micellar
media. The cationic surfactant, CTAB, was chosen to attempt to
promote the rate of this reaction (see Figure 7b). A CTA3
concentration of 0.05 M was used which is safely above the CMC of
0.0013 M at 25°C (30). Maximum absorbance was found to be at 355 nm
for the pyridoxal-cyanide reaction product for both aqueous and
micellar media. In agreement with the expected results, the cationic
surfactant, CTAB, promotes the rate of the reaction. Greater changes
in absorbance were absorbed in micellar media during the same amount
of time. This increase in the rate of the reaction can be explained
by electrostatic attraction forces between the positive charge at the
micelle surface and the negative charge of the cyanide. Also, the
hydrophobic forces between the pyridoxal molecule and the nonpolar
portion of the micelle caused the reaction to proceed at faster
rate. Not only the solubilization and proximity effects contribute
(24,43) to micellar catalysis, but probably the stabilization of some
intermediate species with partial negative charge at the positive
micelle surface favored the formation of 4-pyridoxolactone.
Kinetics studies were performed to measure the rate constants for
this oxidation reaction in aqueous and 0.05 M CTAB media. Figures 10
and 11 show the curves of the change in absorbance versus time at a
maximum absorbance wavelength of 355 nm. For micellar media, the
reaction reached the plateau of the curve after 15 minutes, whereas in
aqueous media the plateau was reached after 30 minutes.

WAVELENGTH (nm)
Figure 8. Absorbance versus wavelength (nm) in aqueous system for the determination of pyridoxal.
Phosphate buffer (0.6 M), pH 7.3, 0.61 ppm pyridoxal, KCN 7.5X10”3 M, 1 cm cell, at room
temperature. Spectra were run at 1 minute intervals.

ABSORBANCE
WAVELENGTH (nm)
Figure 9. Absorbance versus wavelength (nm) in 0.05 M CTAB micellar system for the determination of
pyridoxal. Phosphate buffer (0.6 M), pH 7.3, 0.61 ppm pyridoxal, KCN 7.5X10-5 M, 1 cm cell,
at room temperature. Spectra were run at 1 minute intervals.

ABSORBANCE
TIME (minutes)
Figure 10. Change in maximum absorbance (355 nm) versus time (minutes) in aqueous media for the
determination of pyridoxal. Pyridoxal 0.93 ppm, phosphate buffer 0.6 M, pH 7.3, KCN
7.5X10 M, 1 cm cell, at room temperature.

ABSORBANCE
TIME (minutes)
Figure 11. Change in maximum absorbance (355 nm) versus time (minutes) in 0.05 M CTAB micellar media
for the determination of pyridoxal. Pyridoxal 0.93 ppm, phosphate buffer 0.6 M, pH 7.3,
KCN 7.5X10"'-5 M, 1 cm cell, at room temperature.

33
Assuming the reaction is pseudo first order, the rate constant
can be calculated by using the following equation:
-kt
log (Aco-At) = 2<303 + log (Aoo-AQ) (eq. 4.1)
where Am, AQ, At are the absorbance at infinite, initial and time t,
respectively; t is time in minutes; and k is the rate constant in
minutes-1 (51). From the slope of the curve (taking the negative and
multiplied by 2.303) the rate constants for the reaction of pyridoxal
taking place in water and 0.05 M CTAB media were 0.0490 and 0.0971
minutes-1 (see Figures 12 and 13). The ratio of these rate constants,
k0.05M dAB^^O’ 1S eclual 1*98. This value means that the
reaction is taking place at double the velocity in micellar CTA3 than
in H20. Micellar catalysis does occur for this particular reaction.
To demonstrate the advantages of combining the technique of FIA
with micellar catalysis, measurements of the pyridoxal-cyanide
reaction product using absorbance and fluorescence detection were
carried out. Figure 14 shows the calibration plot for the pyridoxal
determination in aqueous and 0.05 M CTAB media. Comparing the slope
of both curves, 2.54X10-'1 and 3.26X10-^ absorbance units/ppm of
pyridoxal, one can observe that by using a CTAB micellar media the
sensitivity of the system is increased 1.3 times. The differences
between the ratio of rate constants found for water and 0.05 M CTAB,
1.98, and the ratio of sensitivities of these two systems by FIA, 1.3,
may be explained by the fact that the kinetics throughout the entire
sample plug is not constant. The work of Paiton and Mottola (37)

Log (.3756-At)
Figure 12. Log (A^-Aj.) versus time (minutes) for pyridoxal in aqueous system. Absorbance measured at
355 nnC pyridoxal 0.93 ppm, in 1 cm cell at room temperature, phosphate buffer (0.6 M), pH
7.3, KCN 7.5X10"3 M.
CO
-P.

-.60
.65
-.70
-.75
oo -.80
oo
CM
ro
O -.85 -
CP
o
_l
-.90
-.95 “
-1.00 -
-1.05
0
8
10
TIME (minutes)
Figure 13. Log (A^-A^) versus time (minutes) for pyridoxal in 0.05 M CTAB system. Absorbance measured
at 355°nm, pyridoxal 0.93 ppm, in 1 cm cell at room temperature, phosphate buffer (0.6 M),
pH 7.3, KCN 7.5X10"3 M.
OJ
C71

Absorbance Units (water)
pyridoxal (ppm)
Figure 14. Absorbance calibration plots for pyridoxal in aqueous ( â–¡) and 0.05 iA CTAB ( â– )
systems. Absorbance measured at 355 mn, flow rate 1.4 ml/min, temperature 45°C, pH 7.3.
phosphate buffer (0.6 M), KCN 1.5X10-¿ M.
co
cr>
Absorbance Units (CTAB)

37
demonstrated that the assumption of having a constant rate coefficient
throughout the entire body of the sample plug is invalid. The
kinetics involved within the sample plug are more complex. Paiton and
Mottola suggested that the rate coefficient changes with time. This
has been rationalized by assuming that each fluctuation in rate
coefficient with time corresponds with one of three regions within a
sample plug, namely, the leading region, the central region, and the
trailing region. In both the leading and trailing regions, the
carrier/sample interfaces induce molecular diffusion, while the
velocity profile induces convection. In the central region, where no
sample/carrier boundary exists, convection becomes the primary
dispersion force. Because the physical dispersion in these three
regions of the sample plug differ from one another, the rate
coefficients along the length of the plug are expected to vary with a
wave pattern. The fact that the reaction rate varies throughout the
sample plug may mean also that the kinetic order is not constant
within the sample plug.
Figure 15 shows the calibration plot with a fluorescence detector
for aqueous and micellar systems. From the slope of the curves, the
calculated sensitivity of the pyridoxal system is three times greater
when using CTAB micellar media compared with the same system in
aqueous media. The sensitivities for micellar and aqueous solutions
were 0.294 and 0.102 cm (peak height) per ppm of pyridoxal,
respectively. A greater change in the ratio of sensitivities was
observed for fluorescence determination due to the fact that not only
micellar catalysis was taking place but fluorescence enhancement was

FLUORESCENCE (WATER)
(peak height, cm)
38
CD
<
O—.
~ E
Lü o
O
Z-C
UJ 2*
O a>
CO-C
LÜ
oS
u.
Pyridoxal (ppm)
Figure 15. Fluorescence calibration plots for pyridoxal determination
in aqueous ( □ ) and 0.05 M CTAB ( • ) systems. Flow rate
1.4 ml/min; temperature 45°C; phosphate buffer (0.6 M); pH
7.3; KCN 1.5X10_¿ M; 355 and 435 nm excitation and
emission wavelengths, respectively.

39
also observed. In mi cellar-enhanced fluorescence, the emission
intensity of the analyte is usually many times greater than in the
corresponding homogeneous media (52,53). This increase in sensitivity
of solutes in solutions containing micellar aggregates has been
explained by the diminution of deactivation processes for the excited
states. These phenomena occur due to a decrease in polarity, and an
increase in viscosity and shielding against quenching in micellar
media (29).
Table I summarizes the analytical figures of merit for both
aqueous and micellar systems. Very good coefficients of correlation
were obtained for the four curves, all being 0.999. These calibration
curves recorded under working conditions are linear over a wide range
of concentrations. The linear range for the aqueous system with a
fluorescence detector was found to be from 0.42 ng to 2.0X103 ng and
from 94 ng to 2.0X10J ng of pyridoxal for absorbance measurement. For
0.05 M CTAB media the linear dynamic range was from 0.17 ng to 1.1X103
ng of pyridoxal for fluorescence and from 77 ng to 2.0X10 ng of
pyridoxal for absorbance. Due to the large concentration range for
the recorded curves, it was necessary to work at several values of the
instrument sensitivity. At higher concentrations of pyridoxal, the
fluorescence intensity is beyond the spectrofluorimeter range. The
reproducibility of the system was measured by manual injection of 11
replicates of pyridoxal solution at 25.53 ppm. Relative standard
deviation of peak height in percent was calculated and was found to
vary between 0.97 and 3.25 for the studied systems. Limits of
detection were found to be lower for the reaction taking place in

40
Table I. Figures of merit for pyridoxal determination. Fluorescence
detector: excitation 355 nm, emission 435 nm, range 500, or
variable UV-visible absorbance detector at 355 nin, range .1,
1.5X10"2 M cyanide, pH 7.3, phosphate buffer 0.6 M, flow
rate 1.4 ml/min, temperature 30°C, chart speed 1 cm/min,
10 ul sample loop, pyridoxal in deionized water, tube length
200 cm, i.d. 0.5 mm.
Fluorescence
Absorbance
Aqueous
0.05 M CTAB
Aqueous
0.05 M CTAB
Sensitivity3
0.102
0.294
2.54X10"3
3.26X10'3
Coefficient of
correlation
0.9996
0.9992
0.9999
0.9999
Limits of
detection (ng)
0.42
0.17
94
77
Relative standard
deviation U)b
2.06
3.25
0.97
1.76
a Slope of the calibration plot for fluorescence, cm/ppm of pyridoxal
and absorbance units/ppm of pyridoxal for absorbance measurements.
b Eleven determinations at 25.53 ppm of pyridoxal.

41
micellar solutions. A more significant change in limits of detection
was not obtained due to an increase in the background signal for
micellar solutions. This needs further study in the future the
examination of other systems in micellar media by FIA, since a
significant change in limits of detection usually accompanies micellar
catalysis (52).
Optimum conditions may be different for aqueous and micellar
systems. The reason for this difference is that micelles can change
the microenvironment of the solubilized molecules (22). Then, an
optimization of conditions for FIA system is required.
The output signal, in this case peak height, is influenced by the
dispersion of the sample in the reagent stream and the degree of
completeness of the reaction taking place. These two are affected by
experimental parameters such as flow rate, reagent concentrations,
length of the reaction coil, etc. Since these experimental parameters
interact with each other, optimization of FIA methods using univariate
design (optimization of every parameter by separate studies) is time
consuming and may be inadequate to determine the best set of experi¬
mental conditions (54). Sequential simplex optimization procedures
have been found to be valuable in development of new FIA methods
(54-56).
A modified variable size simplex method (57,58) was used for
optimization of pyridoxal determination. The optimization of this
system was performed by changing four variables: pH, temperature,
flow rate and surfactant concentration. Table II shows the initial,
final and increment values for each changing parameter. The reason to

42
Table II. Variable parameters
program.
for the
Modified Simplex Optimization
Initial
Range
Increment
Parameter
Value
Value
Va 1 ue
1.
pH
7.3
6.5-3.0
0.5
2.
Flow rate (ml/min)
1.4
1.0-1.7
0.1
3.
Temperature (°C)
45
30-50
5
4.
CTAB concentration (M)
0.05
0.05-0.15
0.05

43
include pH as one of the parameters to be optimized is that the
reaction of pyridoxal with cyanide is pH dependent. It is reported in
the literature that micelles affect the pH of solutions, by changing
acid dissociation constants (59). The range of pH from 6.5 to 8.0 was
chosen because previous studies showed this to be the optimal pH range
for the oxidation reaction. An increase in temperature increased the
reaction rate in such a manner that higher values for the response
function resulted, up to a certain point where the signal started to
decrease as the temperature increased due to deactivation of
fluorescence. At temperatures below 30°C, the response of the system
(peak height) is greatly diminished; at temperatures above 50°C,
bubble formation in the FIA system can prove detrimental to
reproducibility (47). Flow rate is closely related with the output
signal (vide supra). Faster flow rates will usually lead to a
decrease in signal because less time is available for the reaction to
take place and vice versa. The surfactant concentration was included
as a variable since increasing the number of micellar structures
increases the number of sites available for solute solubilization and,
therefore, promotes the formation of 4-pyridoxolactone. If the
surfactant concentration is increased too much, the reaction will
occur at a lower rate due to the dilution factor. In other words, as
the number of micelle structures increases, solute molecules will be
solubilized on different micelle structures apart from each other.
There will be a physical impediment for a pyridoxal molecule to
encounter a cyanide ion preventing the oxidation reaction to
proceed. The cyanide concentration and the length of reaction coil

44
were kept constant. At a cyanide concentration of fivefold the
pyridoxal concentration, the intensity of the output signal is not
influenced by a change in the cyanide concentration (47). The simplex
was finished after 13 experiments (one reflection, two expansions and
four contractions). Optimum values found, together with those
variables kept constant throughout the development of the simplex, are
summarized in Table III.
With these new optimum values for absorbance measurement in CTA6
media, a set of standard pyridoxal solutions were run and a
calibration plot was recorded and compared with the calibration plot
for the water system run with previous conditions (see Figure 16). A
ratio of 1.8 was found, by comparing the slopes of the two curves, for
water and CTAB, 2.09X10"^ and 3.84X10“^ absorbance units per parts per
million of pyridoxal. As was expected, a higher sensitivity was
observed. This can be explained by the fact that now the conditions
are more favorable for the reaction to take place. Table IV shows the
analytical figures of merit for the reaction in optimized CTAB
conditions and previous conditions for aqueous system. Relative
standard deviation, measured by 11 determinations of 24.14 ppm of
pyridoxal, were 1.06 and 2.12% for CTAB and water systems. Very good
coefficients of correlation were observed for both curves, 0.999,
assuring the linearity of the curve. Lower limits of detection were
obtained by using 0.09 M CTAB and 49°C, 64 ng, compared with the
aqueous system, 86 ng.
Figures 17 to 21 show a set of pyridoxal standard solutions run
by FIA under different studied conditions.

45
Table III. Optimized and fixed variables for the determination of
pyridoxal.
Variables Fixed
in the Optimization
Optimized Variables
1. 10 ul sample: Pyridoxal:
1. pH: 6.74
9.18X10-4 M in 0.05 M CTAB
2. Cyanide concentration:
2. Flow rate: 1.3 ml/min
1.5X10"2 M
3. Phosphate buffer solution: 0.6 M
3. Temperature: 49°C
4. Length of reaction coil: 200 cm
4. CTA3 concentration: 0.09 M

ABSORBANCE UNITS (CTÁB)
45
PYRIDOXAL (ppm)
Figure 16. Absorbance calibration plots for pyridoxal in aqueous
( â–¡ ) and 0.09 M CTAB micellar ( â–  ) media. Absorbance
measured at 355 nm, flow rate 1.3 ml /min, temperature
49°C, pH 6.74, phosphate buffer (0.6 M), KCN 1.5X10'¿ M.
ABSORBANCE UNITS (water)

47
Table IV. Figures of merit for pyridoxal determination with
conditions obtained by Modified Simplex program. Variable
UV-visible absorbance detector at 355 nm, range .1, 0.09 M
CTA8, 1.5X10-2 M cyanide, pH 6.74, 0.6 M phosphate buffer,
flow rate 1.3 ml/min, temperature 49°C, chart speed
1 cm/min, 10 ul sample loop, pyridoxal in deionized water,
tube length 200 cm, i.d. 0.5 mm.
Aqueous
CTAB
Sensitivity
,absorbance units*
'ppm of pyridoxal
2.09X10"3
3.84X10"3
Coefficient of correlation
0.9994
0.9998
Limits of detection (ng)
86
64
Relative standard deviation (%)a
2.12
1.06
a Eleven determinations at 24.14 ppm of pyridoxal.

48
Figure 17. Absorbance recordings of a series of pyridoxal standards
in aqueous media. Pyridoxal: (a) 8.10 ppm, (b) 24.31
ppm, (c) 40.52 ppm, (d) 81.04 ppm, (e) 105.4 ppm.
Absorbance measured at 355 nm, range 0.05, sample volume
10 vi, all tubes 0.5 mm I.D., flow rate 1.4 ml/min,
temperature 45°C, phosphate buffer 0.6 M, pH 7.3, KCN
1.5X10"2 M, chart speed 0.25 cm/min.

49
TIME (min)
Figure 18. Absorbance recordings of a series of pyridoxal standards
in 0.05 M CTAB micellar media. Pyridoxal: (a) 8.10 ppm,
(b) 24.31 ppm, (c) 40.52 ppm, (d) 31.04 ppm, (e) 105.4
ppm. Absorbance measured at 355 nm, range 0.05, sample
volume 10 ul, all tubes 0.5 mm I.D., flow rate 1.4 ml/min,
temperature 45°C, phosphate buffer 0.6 M, pH 7.3, KCN
1.5X10'¿ M, chart speed 0.25 cm/min.

Figure 19. Absorbance recordings of a series of pyridoxal standards
in 0.09 M CTAB micellar media. Pyridoxal: (a) 8.10 ppm,
(b) 24.31 ppm, (c) 40.52 ppm, (d) 81.04 ppm, (e) 105.4
ppm. Absorbance measured at 355 nm, range 0.05, sample
volume 10 ul, all tubes 0.5 mm I.O., flow rate 1.3 ml/min,
temperature 49°C, phosphate buffer 0.6 M, pH 6.73, KCN
1.5X10-2 M, chart speed 0.25 cm/min.

TIME (min)
ABSORBANCE

52
TIME (min)
Figure 20. Fluorescence recordings of a series of pyridoxal standards
in aqueous media. Pyridoxal: (a) 8.10 ppm, (b) 24.31
ppm, (c) 40.52 ppm, (d) 81.04 ppm, (e) 105.4 ppm. Sample
volume 10 yl; all tubes 0.5 mm I.D.; flow rate 1.4 ml/min;
temperature 45°C; phosphate buffer 0.6 M; pH 7.4; KCN
1.5X10“2 M; range 1000; chart speed 0.25 cm/inin; 355 nm
and 435 nm excitation and emission wavelengths,
respectively.

Figure 21. Fluorescence recordings of a series of pyridoxal standards
in 0.05 M CTAB micellar media. Pyridoxal: (a) 8.10 ppm,
(b) 24.31 ppm, (c) 40.52 ppm, (d) 81.04 ppm. Sample
volume 10 yl; all tubes 0.5 mm I.O.; flow rate 1.4 ml/min;
temperature 45°C; phosphate buffer 0.6 M; pH 7.4; KCN
1.5X10“^ M; 355 nm and 435 nm excitation and emission
wavelengths, respectively; range 1000; chart speed 0.25
cm/min.

TIME (min)
FLUORESCENCE INTENSITY (peak height,cm)

55
Measurements of Dispersion
In flow injection analysis, both sample throughput and sample
dilution are directly related to dispersion. The dispersion process
which takes place during the transport of the sample from the
injection device toward the detector is one of the less understood
aspects of FIA (vide supra). In analogy with chromatographic systems,
Poppe (60) observed that the total peak broadening in FIA is the sum
of the contributions from the injection process, the flow through
reactors and connectors, the holdup volume of the flow through
detector, and the time constants of associated electronics. These
processes can be described by the individual peak variances:
2 2 2 2
o = a. . . . + + a, J
overall injection flow detector
Provided that the detector and electronics are well designed, the
variance of detection may be neglected (if it is at least five times
smaller than the standard deviations due to injection and
transport). One of the models frequently used to describe the
dispersion process is the tank in series model. According to this
model, the flow reactor can be considered as a series of M ideal
mixers. If the number of mixing stages, N, is sufficiently high, the
resulting curve has a Gaussian shape. However, in FIA, peaks mostly
show a tailing character (1,42). In 1981, Reijn and coworkers (33)
described the distribution curve of an FIA to be a modified Gaussian
function. Mote that only the physical aspects of dispersion are taken
into consideration. It was assumed that there is no contribution to

56
dispersion due to chemical reaction between the sample and reagent
stream. This assumption, however, is known to be invalid (38,42).
For our system, the FIA peaks were examined for their resemblance
to Gaussian, exponentially modified Gaussian and other peak shapes by
measurement of the variance, » ancl the standard deviation of the
parent Gaussian function, the type of peak shape can be assigned by the agreement of the values
— O
M2 and determined from the asymmetry factor, /A, and peak width,
W, at 10, 30, and 50% peak height (see Figure 22). Equations 4.2 to
4.7 were used to calculate Ñ¡2 and crG at different peak heights.
V » 1
= I (eq. 4.2)
¿ 1.764(8/A)g l - 11.15(B/A)0 ]_ + 28
W
2
0.3
-3.85(B/A)q 3 + 23(B/A)g 3 - 47.9(3/A)Q 3 + 38.7
(eq. 4.3)
M
2
—8.28(3/A)q 5 + 41.8(B/A)g g
72.3(B/A)0 5 + 44.6
(eq. 4.4)
aG = 3.27(B/A)Q 1 + 1.2
(eq. 4.5)
^0.3
°G = 2.8(B/A)0 3 + 0.48
(eq. 4.6)

ABSORBANCE UNITS
57
X.
<
LÜ
CL
TIME
Figure 22. Measurement of peak width, W, and asymmetry factor, B/A,
at 10, 30 and 50$ peak height in an FIA peak.

and
(eq. 4.7)
aG 2.5(8/A)0 5
Tables V to X show the calculated values for the variance and
standard deviation for pyridoxal in aqueous and micellar media at
different peak heights. For this study, the variable wavelength
absorbance detector was set at 292.6 nm, the maximum absorbance
wavelength for pyridoxal. Triplicate injections of 10 ul of
pyridoxal, 2.81X10-4 M in aqueous solution, were made into the aqueous
stream and peaks were recorded. Manual measurement of peak height, h,
peak width and asymmetry factor at 10, 30 and 50% peak height were
done for each peak and substitution of these values into equations 4.2
to 4.7 gave M^ and 0q. Averages of three values were calculated
for M2 and aG at each peak height. The same procedure was followed
for 2.83X10-4 M pyridoxal in 0.05 M CTAB injected into 0.05 M CTA8
stream. Table XI shows the average values for M2 and Oq at 10, 30 and
50% peak height. The relative standard deviation of the values from
the three peak heights was calculated for pyridoxal in the aqueous
system to be ±6.06% for M2 and ±4.88% for relative standard deviation was ±5.68% for M2 and ±5.20% for ctq.
According to the agreement of the values calculated at the three
heights, these peaks can be classified as exponentially modified
Gaussians. Foley and Dorsey (61) reported a relative standard
deviation of ±7.7% for M2 or ±3.2% for values calculated at the
three heights was necessary to ensure the validity of the

59
Table V. Variance and standard deviation values for pyridoxal in
aqueous media at 10? peak height. Variable UV-visible
absorbance detector at 292.6 nm, range .1, temperature 45°C,
flow rate 1.4 ml/min, tube length 200 cm, i.d. 0.5 mm, 10 pi
sample loop, pyridoxal 2.81X10-4 M, chart speed 10 cm/min.
h (Peak height in cm)
14.15
14.35
14.15
Wq.i (Width at 10% peak height in cm)
2.70
2.70
2.70
A
1.09
1.10
1.08
B
1.61
1.60
1.62
B/A (Asymmetry ratio)
1.48
1.45
1.50
Mo (Variance in cm2)a
0.47
0.47
0.48
X = 0.473 cm2
RSDb = 1.48%
J = 0.448 cm2
RSD = 1.35%
a 1 cm2 = 36 s2.
b Relative standard deviation.

50
Table VI. Variance and standard deviation values for pyridoxal in
aqueous media at 302 peak height. Variable UV-visible
absorbance detector at 292.6 nm, range .1, temperature
45°C, flow rate 1.4 ml/min, tube length 200 cm, i.d.
0.5 mm, 10 Ml sample loop, pyridoxal 2.81X10-4 M, chart
speed 10 cm/min.
h (Peak height in cm)
14.15
14.35
14.15
Wq.3 (Width at 30% peak height in cm)
1.90
2.00
1.90
A
0.80
0.85
0.80
B
1.10
1.15
1.10
B/A (Asymmetry ratio)
1.38
1.35
1.38
Mo (Variance in cm2)a
0.57
0.55
0.57
X = 0.563 cm2
RSDb = 2.052
aG (Standard deviation in cm^) 0.437 0.469 0.437
I = 0.448 cm2
RSD = 4.12%
a 1 cm2 = 36 s2.
b
Relative standard deviation.

51
Table VII. Variance and standard deviation values for pyridoxal in
aqueous media at 50% peak height. Variable UV-visible
absorbance detector at 292.6 nm, range .1, temperature
45°C, flow rate 1.4 ml/min, tube length 200 cm, i.d.
0.5 mm, 10 ul sample loop, pyridoxal 2.81X10-4 M, chart
speed 10 cm/min.
h (Peak height in cm)
14.15
14.35
14.15
Wq.s (Width at 50% peak height in cm)
1.40
1.40
1.40
A
0.65
0.65
0.65
B
0.75
0.75
0.75
3/A (Asymmetry ratio)
1.15
1.15
1.15
Mo (Variance in cm2)a
0.47
0.47
0.47
X = 0.47 cm2
RSDb = 0%
(Standard deviation in cm^) 0.487 0.487 0.487
T = 0.487 cm2
RSD = 0%
a 1 cm2 = 36 s2.
b
Relative standard deviation.

52
Table VIII. Variance and standard deviation values for pyridoxal in
0.05 M CTA8 micellar media at 10% peak height. Variable
UV-visible absorbance detector at 292.6 nm, range .1,
temperature 45 °C, flow rate 1.4 ml/min, tube length
200 cm, i.d. 0.5 mm, 10 yl sample loop, pyridoxal
2.33X10"4 M in 0.05 M CTAB, chart speed 10 cm/min.
h (Peak height in cm)
13.60
13.50
13.70
Wq.i (Width at 10% peak height in cm)
2.85
2.90
2.90
A
1.10
1.15
1.15
B
1.75
1.75
1.75
B/A (Asymmetry ratio)
1.59
1.59
1.59
Mp (Variance in cm2)a
0.55
0.55
0.55
X = 0.55 cm2
RSDb = 0%
(Standard deviation in cm2) 0.445 0.470 0.470
I = 0.462 cm2
RSD = 3.12%
a 1 cm2 = 36 s2.
b
Relative standard deviation.

53
Table IX. Variance and standard deviation values for pyridoxal in
0.05 M CTA8 micellar media at 30% peak height. Variable
UV-visible absorbance detector at 292.6 nm, range .1,
temperature 45°C, flow rate 1.4 ml/min, tube length 200 cm,
i.d. 0.5 mm, 10 ul sample loop, pyridoxal 2.83X10-4 M in
0.05 M CTAB, chart speed 10 cm/min.
h (Peak height in cm)
13.60
13.50
13.70
Wq.3 (Width at 30% peak height in cm)
2.10
2.10
2.10
A
0.95
0.95
0.95
B
1.15
1.15
1.15
8/A (Asymmetry ratio)
1.21
1.21
1.21
M2 (Variance in cm2)a
0.58
0.58
0.58
X = 0.58 cm2
RSDb = 0%
aG (Standard deviation in cm2) 0.543 0.543 0.543
Y = 0.543 cm2
RSD = 0%
a 1 cm2 = 36 s2.
b Relative standard deviation.

64
Table X. Variance and standard deviation values for pyridoxal in
0.05 M CTAB micellar media at 50% peak height. Variable In¬
visible absorbance detector at 292.6 nm, range .1,
temperature 45°C, flow rate 1.4 ml/min, tube length 200 cm,
i.d. 0.5 mm, 10 ul sample loop, pyridoxal 2.83X10-4 M in
0.05 M CTAB, chart speed 10 cm/min.
h (Peak height in cm)
13.60
13.50
13.70
Wq 5 (Width at 50% peak height in cm)
1.60
1.70
1.60
A
0.75
0.80
0.75
B
0.80
0.90
0.85
B/A (Asymmetry ratio)
1.13
1.125
1.13
Mp (Variance in cm2)a
0.59
0.667
0.59
X = 0.616 cm2
RSDb = 7.2%
J = 0.559 cm2
RSD = 2.36%
a 1 cm2 = 36 s2.
b Relative standard deviation.

55
Table XI. Average values for variance and standard deviation for
pyridoxal in 0.05 M CTAB micellar and aqueous media at 10,
30, and 502 peak height for aqueous and 0.05 M CTAB
systems.
10%
30%
50%
X
(cm2)
RSD
(%)
0.05 M
CTAB
m2
(cnr)
0.55
0.58
0.616
0.582
5.68
ctG
(cm2)
0.462
0.559
0.543
0.521
5.20
h2o
m2
(cm2)
0.473
0.559
0.473
0.503
6.06
aG
(cm2)
0.448
0.448
0.487
0.461
4.88

66
exponentially modified Gaussian peak shape. Proven that the FIA peaks
in our system were exponentially modified Gaussian, studies of the
dispersion in aqueous and micellar system were performed by using
equation 4.2. Variance measurements at 10% peak height are more
precise than those calculated at 30% and 50% peak height (61).
Given the conditions in Tables V and VIII, the dispersion between
aqueous and micellar media was compared in terms of the variance,
The values for ^ were found t° be 0.473 and 0.55 cm^ for
aqueous and 0.05 M CTAB media, respectively.
The dispersion of both aqueous and micellar systems was
calculated also by using equation 1.1, which is the definition of FIA
dispersion. The concentration of pyridoxal before the dispersion
process was measured by passing through the detector a solution of
pyridoxal in water (2.81X10-4 M) or in 0.05 M CTAB (2.83X10-4 M) and
measuring the response of the detector. The response of the detector
was obtained by the average of six measurements. The concentration of
pyridoxal after the dispersion process has taken place was obtained by
measuring the maximum peak height of 11 injections of 10 U1 of
pyridoxal solutions (above) into pure aqueous or 0.05 M CTAB. Note
again that dispersion occurring under this circumstance is only due to
physical contributions; no chemical reaction is taking place. The
ratio of the detector response before and after the dispersion process
occurred was 17.62 for the aqueous system and 19.14 for the micellar
system. Contrary to what was expected higher dispersion was found for
micellar media. Due to the higher viscosity of 0.05 M CTAB solution
compared with water, the dispersion was expected to be less. To prove

67
the validity of these results, the same type of experiment was
performed on two different days and the same results were obtained
(see Table XII).
To investigate the effect of surfactant concentration on the
dispersion process, a set of dispersion measurements (M?) versus CTAB
concentration were performed. The concentration of CTAB was varied
from 5X10 ,1 to 5X10 M. This range of concentration includes
solutions above and below the critical micelle concentration. Ten
microliters of pyridoxal (2.81X10-^ M) aqueous solution was injected
into the stream and from the recorded peaks the variance at 10% peak
height was calculated. From Table XIII, one can say that for a
solution of 5X10“^ M the dispersion obtained is very similar to that
calculated for water. Above this concentration an increase in
surfactant concentration had no effect on the dispersion up to a
concentration of 5X10”^ m CTAB, at which point an increase in
dispersion was observed. Micellar concentration changes the
dispersion of the FIA system. This may be explained by the fact that
the pyridoxal molecules are localized on or within the micelle
structure. Micellar media is more viscous than aqueous media (43);
therefore, the mass transfer in a radial direction decreases. An
increase in peak dispersion results because a decrease in mixing
across the stream tends to increase dilution of the solute by
longitudinal dispersion (42). The increase of dispersion with
micellar media needs further study.

68
Table
XII. Dispersion
values
for aqueous and micellar
systems.
0.05 M CTAB
Aqueous
m2
m2
C7Cmax
(cm2)
C°/Cmax
(cm2)
1.
19.54
0.55
17.62
0.47
2.
19.14
0.56
17.05
0.45
3.
19.26
0.54
17.25
0.45
C°/Cmax = ratio of concentration of pyridoxal before and after the
dispersion process has taken place.
— O
¡^2 = variance or second moment in cm .

69
Table XIII. Measurement of dispersion versus CTAB concentration.
Variable UV-visible absorbance detector at 292.6 nm,
range .1, temperature 45°C, flow rate 1.4 ml/min, tube
length 200 cm, i.d. 0.5 mm, 10 ul sample loop, pyridoxal
2.81X10-4 M in deionized water, chart speed 10 cm/min.
k2
(cm2)
m2
(cm2)
m2
(cm2)
M2 Average
(cm2)
CTAB (M)
5.0X10"6
0.44
0.43
0.46
0.44
2.5X10"5
0.52
0.46
0.52
0.50
5.0X10'5
0.47
0.52
0.49
0.49
2.5X10'4
0.48
0.50
0.46
0.48
5.0X10"4
0.49
0.46
0.52
0.49
2.5X10"3
0.52
0.50
0.49
0.50
5.0X10-3
0.50
0.52
0.52
0.51
2.5X10-2
0.47
0.52
0.49
0.49
5.0X10'2
0.52
0.57
0.53
0.54
h2o
0.43
0.44
0.48
0.45
= variance or second moment in cm
2

CHAPTER V
CONCLUSIONS AND FUTURE WORK
The applicability of combining the technique of Flow Injection
Analysis with micellar catalysis has been shown. The results obtained
from the determination of pyridoxal in micellar media compared to
those in aqueous media are promising. Higher response, as measured by
peak height, was recorded at all times for micellar carrier
solutions. Higher sensitivities and lower limits of detection were
obtained for the micellar system when the oxidation product of
pyridoxal and cyanide was detected either fluorimetrically or by UV
absorbance. Higher sensitivity ratios, for micellar to aqueous
systems, were obtained when using fluorescence detection. In this
case, not only is the reaction taking place at a faster rate but the
solubilization of reagents within the micelle structure contributes to
increase the signal due to the shielding effect.
With this example, it has been proven that due to the kinetic
nature of the FIA technique the use of micellar media can be very
advantageous. To support this investigation, the utility of using
micellar carrier solution in an FIA system, it will be necessary to
perform similar experiments by running different reactions taking
place in aqueous and micellar media. Keeping in mind the kind of
surfactant that will catalyze the specific reaction (anionic,
70

71
cationic, nonionic or zwitterionic), the rate of the reaction should
be measured and if it is favorable applied to FIA.
One of the reactions that will be very interesting to look at is
the determination of metals by their complexation with dyes in the
presence of micelle media (62-68). Some characteristics observed for
these metal-dye complexes in micellar media are an increase in molar
absorptivity and red shifts in the wavelength of maximum absorbance
(53). These substantial changes in the UV visible spectrometry of
these complexes together with the technique of FIA can be developed as
a new spectrophotometrie method for determining micro amounts of metal
ions. The resulting method should be fast, easy and inexpensive.
Lower background signals were found for the aqueous systems
compared to CTAB micellar systems. Therefore, the limits of detection
for micellar media were not as low as expected. This increase in
background noise for micellar media needs further study to elucidate
if the increase in noise is due to the presence of micelles or if it
is just observed with this specific reaction.
Due to the specific interactions between micelles and solutes the
selectivity of a particular reaction can be increased, reducing the
amount and kind of interferences. An investigation on this topic will
be very valuable especially for the determination of very small
amounts of analytes.
It has been mentioned that catalysis of organic and inorganic
reactions also occur in apolar media in the presence of reversed
micelles (19,69). Reversed micelles offer similar and at the same
time different characteristics from normal micelles. Future research

72
on the application of reversed micelles for reactions occurring in
apolar media in combination with the technique of FIA will be very
interesting.
According to the agreement of the values for the second moment
and the standard deviation measured at 10, 30 and 50% peak height, the
peaks recorded from an FIA system can be designated to be
exponentially modified gaussian.
Higher values for dispersion were found for micellar media.
These results were not as expected and need further study to
understand better the reason for this increase in dispersion. More
detailed experiments should be performed under different conditions,
e.g., varying the type and concentration of surfactant, temperature,
etc.
The contribution of a chemical reaction to the dispersion of a
peak in FIA is not well understood. 3y comparing the dispersion of
different reactions taking place in different types of surfactants to
the dispersion obtained in aqueous media could help to explain the
contribution of the chemistry to the total dispersion process. The
differences in rate of reaction between different micellar and aqueous
systems can help to explain this phenomena. By taking a reaction
whose rate of reaction varies in the presence of anionic, cationic,
nonionic and aqueous media, the contribution of chemical kinetics may
be clarified.

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BIOGRAPHICAL SKETCH
Maria A. Hernandez Torres was born in Mayagüez, Puerto Rico, on
May 29, 1958. She received her elementary and high school education
at the Colegio de La Milagrosa, Mayagüez, Puerto Rico. In 1980, she
completed her Bachelor of Science degree in chemistry at the
University of Puerto Rico Mayagüez Campus (magna cum laude). In 1983,
she pursued a Master of Science degree in analytical chemistry at the
University of Florida. She is now receiving a Doctor of Philosophy
degree in analytical chemistry at the University of Florida.
77

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
G
orsey, Chairman
Professor of Chemistr
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
S?. kJ
imes D. Winefordnei
Graduate Research professor of Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
4
Kj
jterf-To-
Anna F. Brajterf-Toth
Assistant Professor of Chemistry
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
Martin Vala
Professor of Chemistry

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the degree
of Doctor of Philosophy.
This dissertation was submitted to the Graduate Faculty of the
Department of Chemistry in the College of Liberal Arts and Sciences
and to the Graduate School and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.
August, 1986
Dean, Graduate School

UNIVERSITY
OF FLORIDA



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