i

A STUDY OF ORGANOGELS AND THEIR SOLUTE INTERACTIONS

BY

MELISSA ANN STOUFFER

A Thesis Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS & SCIENCES

In Partial Fulfillment of the Requirements

For the Degree of

MASTER OF SCIENCE

In the Department of Chemistry

May 2009

Winston-Salem, North Carolina Approved By: Willie Hinze, Ph.D., Advisor ____________________________ Examining Committee: Brad Jones, Ph.D., Chair ____________________________ Christa Colyer, Ph.D. ____________________________

ii

ACKNOWLEDGMENTS

First of all, I would like to acknowledge Dr. Willie Hinze for his time and dedication

to this project. I would like to thank him for his persistence and patience throughout these

years. Without his tenacity I would have not been able to finish this thesis. I would also like

to thank Dr. Brad Jones and Dr. Christa Colyer for serving on my committee and being ready

for the examination on such short notice. Furthermore, I would like to thank the Graduate

Office for ensuring my continued enrollment when I was unable to enroll in person and Dean

Moore for granting my extension. I would also like to thank fellow students, Jen Rust and

Amanda Davis for being good friends during my time at Wake Forest University. I enjoyed

their company during my tenure in Salem Hall. I would also like to thank the TAA program

for supporting me financially during the time I attended school. Finally, I would like to thank

Performance Fibers, my current employer for allowing me to work on my thesis and to take

time off work for my defense.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS ……………………………………………………………….. ii TABLE OF CONTENTS ………………………………………………………………. iii LIST OF TABLES ……………………………………………………………...… v LIST OF FIGURES …………………………………………………………….… vi ABSTRACT …………………………………………………………….... vii Chapter 1 INTRODUCTION TO REVERSE MICELLES AND ORGANOGELS ........ 1

1.1 Surfactants ………………………………………………………………... 1

1.2 Normal Micelles ……………………………………………………………...… 1

1.3 Reverse Micelles ……………………………………………………………...… 3

1.4 Organogels ……………………………………………………………..... 13

1.5 Statement of Thesis Research Objectives ……………...……………………..... 21

Chapter 2 EXPERIMENTAL …………...………………………………………….…. 23

2.1 Materials ………………………………………………………………………. 23

2.2 Equipment & Instrumentation …………...…………………………………...… 24

2.3 Organogel Preparation ………………………...……………………………….. 24

2.4 Organogel Stability in Different Solvent Systems …………...………………… 26

2.5 Density Determination ………………………..………………...…………...… 27

2.6 Analyte Solution Preparation …………………………...……………………… 27 2.7 Determination of Solute – Organogel Partition Coefficients and Organogel Extraction Efficiency …………...……………………………………… 27 2.8 Determination of the Time Course for the Absorption Process …..……………. 29 2.9 Data Analysis ………………………………………………………….…… 30

iv

Chapter 3 RESULTS AND DISCUSSION……………………………………………. 31

3.1 Brief Characterization Studies ………………..……………………………...… 31

3.2 Interaction of Organic Solutes with AOT Organogels ……………………..….. 34

3.3 Extraction of Polar Analytes from Nonpolar Solvents using AOT Organogels ………………………………………………………………. 41 3.4 Conclusions ………………………………………………………………. 49 Chapter 4 FUTURE STUDIES …………………...……………………………………..…. 51 4.1 Recommendations ……...………………………………………………………. 51 REFERENCES ………………………………………………………………………. 53 VITA ………………………………………………………………………. 58

v

LIST OF TABLES

1 CMC and Aggregation Numbers of AOT in Various Solvents……………………… 5 2 Solubilities of some Compounds in Bulk Solvent and Reverse Micellar Systems…... 6

3 Results of Some Enantioselective Enzymatic Reactions in Organogels.................... 19 4 Solute in Solution Spectral Parameters…………………………………………...… 28

5 Appearance of Organogels after Exposure to Different Solvent Systems……...…… 33 6 Swelling of Organogels when in Contact with Different Media……………………. 34

7 Partition Coefficients of Solutes in AOT Reverse Micelles and AOT Organogels…. 36 8 Summary of Solutes, their AOT Organogel-Hexane Partition Coefficients and their

Abraham Solvation Descriptor Values…………………………………………….... 38

9 Interaction Coefficients for Solute Partitioning into AOT Organogel and AOT Reversed Micellar Solutions……………………………………................................ 40

10 Pseudo-Second Order Parameters for the Sorption of Solutes on AOT Organogels .. 45

11 Extraction Efficiency and Concentration Factors Achieved for the Extraction of

Analytes from Hexane with the AOT Organogel Materials….................................... 47

vi

LIST OF FIGURES

1 General Diagram of a Surfactant Molecule………………………………………….. 1

2 Simple Representation of an Aqueous Normal Micelle……………………...……… 2

3 Schematic Representation of a Reverse Micelle……………………………………... 4

4 Structure of Bis(ethylhexyl) Sodium Sulfosuccinate (AOT)…………………...……. 5

5 Schematic Representation of the Production of Long Chain Lactones from HHA Catalyzed by the Enzyme Lipase, E, in a Reverse Micellar System……………….... 8

6 The Reaction of of 1-Fluoro-2, 4-Dinitrobenzene with an Amine to Form the

Corresponding N-(2,4-Dinitrophenyl)-Piperidine……………………………........… 9

7 Schematic of Experimental DNA Amplification Procedure……………………...… 11 8 Proposed Structure of Organogel where 1 represents the W/O

Microemulsion-Type Droplets and 2 the Gelatin/Water Channels………………..... 14

9 SEM and TEM Micrographs of Organogel……………………………………….... 15 10 Photo of an AOT Organogel that had been in Contact with a 0.50 M NaCl Solution

for 7 days ………………………………………………………………………….... 32

11 Plot of the Solute – AOT Reverse Micelle Binding Constant, Kb (M-1) versus the Solute – AOT Organogel Partition Coefficient …………......…………………...… 37

12 Comparison of the Predicted and Experimentally Observed Values of the

Partition Coefficients for Solute Incorporation into AOT Organogels…………...… 41

13 Plot of the Absorbance of 2-Nitroaniline at 376 nm in Hexane versus Time (minutes) after exposure to 3.945 grams of AOT Organogel at 25.0o C………….… 42

14 Plot of the Amount of 2-NA (mmol/gram gel) Sorbed onto the AOT

Organogel as a Function of Time (minutes)…………………………………...…… 43

15 Plot of the Second-Order Sorption Model Equation (Eq.7), t/Qt as a function of Time for the Solute 2-NA………………………………………….....… 44

16 Plot of Absorbance (at 318 nm) of 4-Nitroaniline that Sorbed onto the

Organogel versus Time at 25.0o C……………………………………………….…. 46

vii

ABSTACT

A brief background on bis(2-ethylhexyl) sodium sulfosuccinate (AOT) reversed

micelles and organogels and survey of their general properties and some possible

applications is presented. The AOT organogels are characterized in terms of their relative

stability when contacted with different solvent systems (water, salt water, alcohols, alkanes).

The gels were found to retain their structure in salt (NaCl) water and nonpolar solvents

(hexane, heptanes). The density of the AOT organogel materials was found to be 1.05 g/ml.

The partition coefficients for the interaction of solute molecules (substituted phenols,

anilines, naphthols) with the organogels were found to be in the range of 1.3 to 500. These

partition coefficients are roughly 40 times less relative to the partitioning of the same solutes

to AOT reverse micelles in solution. The AOT organogels were found to function as a

sorbent for the extraction of nitroanilines, phenols, and 2-naphthol from a bulk hexane

solution. The extraction efficiencies ranged from 72 – 97% with enrichment factors from 3 –

13 depending upon the amount of AOT organogel employed. A pseudo-second order kinetic

model was found to best fit the sorption the data for the solutes examined (2- and 4-

nitroaniline, 2,4-dinitroaniline, and 2-naphthol). Finally, the Abraham solvation model was

applied and binding to the AOT organogels was found to be dominated by hydrogen bond

basicity and acidity with a secondary contribution from solute polarizability.

1

Chapter 1

INTRODUCTION

Reverse Micelles and Organogels: Brief Background Description and Applications 1.1 Surfactants

Surfactants, or surface-active agents, are molecules that have distinct polar and

nonpolar regions (Figure 1). The apolar tail of the surfactant molecule is typically a

hydrocarbon chain consisting of eight or more carbon atoms. The surfactant charge-type,

i.e., anionic, cationic, zwitterionic and nonionic, is dictated by the nature of the polar head

group.1 Surfactants are considered amphipathic molecules because of their dual nature of

polarity. Soaps and detergents are examples of surfactants. Surfactant molecules have been

employed in a variety of analytical chemistry and separation science applications.2

Figure 1: General Diagram of a Surfactant Molecule

1.2 Normal Micelles

In an aqueous environment, surfactants can form aggregates. In the interior of these

aggregates the nonpolar regions are associated together where they are shielded from the

aqueous solvent and the polar regions are oriented into the solvent.1 These aggregates,

shown in Figure 2, are termed aqueous or normal micelles.

Polar head regionNonpolar, typicallyhydrocarbon tail

2

Figure 2: Simple Representation of an Aqueous Normal Micelle

Such micellar systems are characterized by two parameters, aggregation number (N)

and critical micelle concentration (CMC). N is defined as the average number of surfactant

molecules that comprise the aggregate. This number generally ranges from 40 to 140

surfactant molecules.1 The aggregation number depends upon both the nature of the micelle

forming surfactant (hydrocarbon chain length, head group, counter ion for ionic surfactants)

and the experimental conditions (ionic strength, temperature, presence and concentration of

other additives, etc.).2

The CMC is defined as the minimum concentration of surfactant needed for

micellization. The CMC for most surfactants in pure water are in the range of 0.01 – 10 mM

depending upon the charge type and nature of the specific surfactant molecule involved. For

instance, the CMC values for nonionic surfactants are lower relative to charged surfactants

and as the alkyl chain length of the surfactant molecule increases, its CMC value decreases.1

The presence of additives in water can lead to an increase or decrease in a surfactant’s CMC

H H

O

H H

O

H H

O

H H

O

H H

O

3

value relative to that observed in pure water; i.e., the CMC value of a surfactant typically

decreases with added salt while it increases in the presence of added organic solvents (such

as an alcohol).1,2

Therefore, a surfactant’s N and CMC values are dependent upon both nature and

charge type of the surfactant and the experimental conditions. CMC values are typically

experimentally determined using surface tension, conductivity, or various spectroscopic

methods.1

1.3 Reverse Micelles

1.3.1 Description

In an organic nonpolar solvent, surfactants can also form aggregates in which the

nonpolar tail region of the surfactants are oriented toward, and in contact with bulk nonpolar

solvent while the polar moieties are located in the interior of the aggregate where they are

shielded from the bulk nonpolar solvent. These aggregates are termed reverse micelles or

inverted micelles (Figure 3) since they are inverted (or reversed) compared to the normal

aqueous micelle.3 If prepared in the presence of trace amounts of water, the reverse micelles

can contain a water pool in the interior of the micelle. These inner water molecules are

shielded from the bulk organic solvent. Depending on the amount of water present, such

systems are also referred to as water-in-oil microemulsions (w/o μE). When the system

contains just enough or less water to hydrate the hydrophilic groups of the surfactant, it is

considered a true reverse micelle.3 W/O μE’s contain more water than necessary for

surfactant head group hydration and have an increased micelle diameter and larger

aggregation numbers.3 This distinction between reverse micelles and w/o μE’s is

characterized by the water to surfactant ratio, which is typically designated as wo or Ro and is

4

equal to the concentration of water present divided by the concentration of the reverse

micelle forming surfactant . For convenience, the term reverse micelle will be typically used

although the system may actually be considered a w/o μE.

Figure 3: Schematic Representation of a Reverse Micelle

Compared to normal micelles the aggregation number and CMC are more difficult to

determine for surfactants in nonpolar solvents.1 The problem with determining the

aggregation number arises from the small number of surfactant molecules that form such

reverse micelle aggregates, about 4-20.1 Thus, aggregation numbers are typically smaller for

reverse micelles than normal micelles. The determination of CMC using surface tension is

not feasible in an organic solvent due to the small surface activity of these solutions.1 The

CMC value for a commonly used surfactant., bis(2-ethlyhexyl) sodium sulfosuccinate

(Aerosol OT or AOT), shown in Figure 4, has been determined using a positron annihilation

technique.4 The CMC value was found to be between 2.0 and 2.2 mM in benzene. CMC

H H

O

H H

O

H H

O

H H

O

5

values for AOT previously reported in the literature range from 0.4 to 2.8 mM (Table 1)

depending upon the specific solution conditions (bulk nonpolar solvent employed) and the

detection technique.3, 4 On the other hand, some have even argued against the existence of a

CMC for surfactants in non-aqueous solvents.1 Although this is an ongoing debate, newer

methods have been used to study reverse micellar formation. These include fluorescence,

dynamic light scattering, and NMR spectroscopy, among others.3

Figure 4: Structure of Bis(ethylhexyl) Sodium Sulfosuccinate (AOT)

Table 1 CMC and Aggregation Numbers of AOT in Various Solvents

Solvent CMC (mM)a Na Heptane None detected 13-16 Hexane None detected 15 Benzene 0.4 – 2 11-24

Cyclohexane 0.5 17 a Values taken from the literature1.

Reverse micelles were thought to be spherically shaped but some studies have shown

that they may actually be discs or elongated discs.3 Using dynamic light scattering

OO

S

O

OO

Na

O

O

6

measurements, it was found that the shape of AOT aggregates are dependent upon the

amount of water present, i.e., the wo value.5 When the wo value was greater than 20, the

aggregate resembles deformed discs, whereas at wo values less than 20, they are spherical in

shape.5 Thus, the amount of water present dictates the shape (and size) of reverse micelles.5

1.3.2 Selected Applications

1.3.2.1 Solubilization

One of the most basic and important applications of reverse micelles is their ability to

increase the solubility of compounds that would otherwise be insoluble or sparingly soluble

in a nonpolar solvent. Reverse micelles can solubilize alcohols, amines, alkynes and aqueous

solutions of acids, bases, and buffers.3 Table 2 compares the solubilities of some substances

in organic solvents to those in reverse micelle systems. From the data in Table 2, it can be

seen that the solubilities of these compounds are greatly improved in the reverse micellar

system. AOT is one of the most commonly employed reversed micelle forming surfactants

because it can solubilize a large amount of water relative to other surfactants.

Table 2

Solubilities of Some Compounds in Bulk Solvent and Reverse Micellar Systems

Substrate Solubility in Bulk Solvent Solubility in Reverse Micelle

Pyridine-2-azo-p-dimethylaniline

1.5 X 10-4mol/L (in H2O) 1.9 X 10-3mol/L (in heptane) 3.3 X 10-3 mol/L a

1-(Pyridyl-2-azo)-2-naphthol (PAN) 3.3 X 10-3mol/L (in heptane) 7.0 X 10-3 mol/L a

Lysozyme < 5.0 X 10-6 mol/Lb 2.2 X 10-4 mol/La a In AOT/heptane/H2O reverse micelle; molar ratio of water to surfactant concentration is equal to ten.3 bEstimated from the literature.6

7

Reverse micelles can also solubilize, with retention of activity, a number of

hydrophilic biomolecules, which is of particular importance in biochemistry, biotechnology

and medicine.5,7 Proteins and enzymes can be selectively solubilized in reverse micelles for

extractive separation purposes with subsequent recovery achieved by various techniques.8,9

In most cases, binding is thought to occur due to electrostatic interactions between the

charged surface of the biomolecule and the reverse micelle surfactant charged head group.10

Therefore, a particular protein or enzyme can be selectively extracted from an aqueous

solution mixture to the water pool of the reverse micelle if the biomolecules have different

isoelectric points.3 For example, Vinogradov et al. have selectively extracted two different

enzymes from inclusion bodies grown in E.coli and shown that they both refolded to their

native active conformations in a reverse micelle system.11 These inclusion bodies contain

mainly misfolded and inactive products that are otherwise difficult to extract and activate by

conventional techniques.11 Therefore the use of reverse micelles is advantageous in this

regard. The primary disadvantage of this technique is in the recovery process where it is

often difficult to recover pure protein void of any water or surfactant. However, a new

recovery technique has been investigated using pressurized CO2 with increased purity of

precipitated protein.10 Numerous other examples of the utilization of reverse micelles for

solubilizaton and extractive separations have been reported.9, 10, 11

1.3.2.2 Synthesis

In addition to solubilization, reverse micelles can be used as novel reaction media. In

many situations, the utilization of reverse micelles for biosynthesis is more desirable than the

use of aqueous solutions because biomolecules often exhibit enhanced activity in their

8

presence and many chemical reagents have poor solubility or are insoluble in water.7 In

reverse micelles the size of the “nanoreactor” water pool can be controlled, and once the

reactants and reagents are contained reaction conditions can be manipulated for the desired

products. An example of a reaction that is more desirable in reverse micelles than in an

aqueous solution is the production of long-chain lactones that are used as perfume

ingredients.12, 13 Figure 5 shows a representation of the lactonization of 16-

hydroxyhexadecanoic acid (HHA) catalyzed by the enzyme lipase in a reverse micellar

system.13

Figure 5: Schematic Representation of the Production of Long Chain Lactones from HHA Catalyzed by the Enzyme Lipase, E, in a Reverse Micellar System. Reproduced with permission from article by Rees, Robinson and Stephenson13

In aqueous solutions intermolecular esterification between long-chain hydroxy acid

molecules is favored over the desired intramolecular reaction. In reverse micelles these

lactones have been successfully synthesized without this problem.12

9

Another example is the reaction of 1-fluoro-2, 4-dinitrobenzene with n-butylamine or

piperidine to form n-butyl-2, 4-dinitroaniline or n-(2, 4-dinitrophenyl) piperidine,

respectively (Figure 6). The kinetics of the reaction were examined in reverse micelles and

compared to the reaction in bulk hexane. The reaction rates for this reaction were increased

by at least 2 orders of magnitude in the reversed micelle medium relative to that in bulk

hexane.14

Figure 6: The Reaction of of 1-Fluoro-2, 4-Dinitrobenzene with an Amine to Form the Corresponding N-(2,4-Dinitrophenyl)-Piperidine. Reprinted with permission from an article by Correa, Durantini and Silber14

Reverse micelles have also been utilized in the amplification of DNA. PCR is an

important technique used for amplifying DNA from a small amount of initial template DNA.

Traditional PCR theoretically can occur with only a single template molecule. However, this

can be complicated because reactions have to take place with single or extremely low

concentration of reactants and can produce undesired products.15

Many PCR modifications have been explored to overcome these complications.

These modifications include such techniques as Nested PCR, Booster PCR, and Homo-

primer PCR.15 All three techniques have drawbacks because they include the use of carefully

designed primers. In the case of Nested PCR and Booster PCR there is also a chance of

10

contamination because they require a two step processes where the reaction vessel must be

opened during amplification.15

Micro-devices have been utilized for single molecule PCR. This technique requires

special equipment, which will increase the cost of amplification. Another limitation of using

micro-devices is the possibility of the template DNA being absorbed by the reaction vessel

wall. Along this same line of thought, reverse micelles have been explored as the micro-

device for single molecule PCR where their water pools are used as a micro-reactor without

the added cost of specialized equipment.15

In a study by Nakano et al., single molecule PCR was explored using reverse

micelles.15 The two step method proposed is simple compared to other single molecule PCR

methods. First, the template DNA is emulsified into the bulk oil phase and amplified in the

water droplets after the addition of PCR substrates. After amplification the mixture is

centrifuged so the water pools are merged and the unused PCR substrates can be employed

for further amplification.15 A schematic representation of this procedure is shown in Figure

7. This technique allows the use of only one DNA template and amplification only occurs in

a single water pool so that excess PCR substrates cannot form any undesired products.

Another advantage of this technique is that opening of the reaction vessel is not necessary.

Therefore, the possibility of contamination is eliminated.15

11

Figure 7: Schematic of Experimental DNA Amplification Procedure.

Reproduced with permission from an article by Nakano, et. al.15

Reverse micelles have also been examined as a medium for enantioselective

synthesis. Chiral products have been produced using reverse micelles containing chiral

surfactants. Wu and Zhang reported a synthetic approach for preparation of optically active

amino acids in which an enantiomeric excess of 59.5% was attained.16 In another example

of a stereospecific conversion, Zhang and Sun reported on the reduction of prochiral ketones

to form optically active alcohols (enantiomeric excesses in the range of 3.8 – 26.6% were

achieved depending upon the exact reaction conditions).17

Enzyme mediated enantioselective synthesis in the presence of reverse micelles can

also produce chiral molecules.7,18 Optically active epoxides are used in the production of β-

adrenergic receptor blocking agents, or β-blockers.18 Large scale production of these types

of epoxides is difficult because many substrates have limited solubility, can further

metabolize into undesired products, and the biocatalysts can be sensitive to the reaction

environment.18 However the whole cell mycobacterium sp. strain M156 exhibited enhanced

catalytic activity when incorporated in a reversed micellar water pool resulting in chiral

12

epoxide production (enantiomeric excess of 86%). However, there are limitations to using

reverse micelles as reaction media for epoxide production including the difficulty of

recovering the desired pure chiral product, ability to recycle the enzyme (or biomaterial), and

the inactivation of the enzyme after about 150 minutes.18

1.3.2.3 Size Tailoring / Nanoparticle Synthesis

Compared to their regular or micro-sized counterparts, nanoparticles often exhibit

unique magnetic, optical and mechanical properties. Nanoparticles are employed for a

variety of uses, including ceramics, UV stabilizers, biomedical applications, and in the

manufacturing of electronics, among others.19 Due to the ideal properties of nanoparticles

much attention has recently been given to their synthesis. The hydrophilic microcore (water

pool) of reverse micelles can be controlled which in turn facilitates the production of

nanoparticles which are more homogeneous in size relative to their production in a bulk

solvent medium. The reversed micellar approach has advantages over the traditional

methods of nanoparticle production in terms of size control, size distribution and purity.19

Two representative examples of the many different nanoparticles that have been

successfully synthesized in reverse micelles are production of ZnS particles by Zhang et al.20

and AgI particles by Tamura et al.21 ZnS nanoparticles have superior properties compared

to that of the bulk zinc sulfide. Zhang et al. showed that the reverse micelle water pool size

can be varied (increased or decreased) by merely varying the wo ratio (larger ratio results in

larger water pool and vice versa).20 Thus, the water pool size can be tailored to dictate the

size of a desired particular nanoparticle. This was demonstrated for the synthesis and size

control of ZnS nanoparticles in reverse micelles.20 Likewise, silver iodide nanoparticles

13

with properties closer to that of high conductivity α-AgI (which are characterized by a phase

transition at 147 0C from the β-AgI of the bulk material) were successfully synthesized in a

reverse micelle medium.21

1.4 Organogels

1.4.1 Description

Organogels are formed by the addition of gelatin at an elevated temperature to a

reverse micellar system that contains the surfactant AOT. Upon addition of gelatin the entire

system forms a stable organogel. This phenomenon was a surprising result discovered by

Luisi and Haering.22 They were expecting the gelatin that was present in the reverse micelle

to form a gel within the water pool but instead gelation of the entire AOT/isooctane/water

reverse micelle system was observed. Since this initial discovery, AOT organogels have

been the subject of many physicochemical and application studies.21-28

Organogels have a number of advantageous characteristics and properties. For

instance, they can exist intact for several months if stored in a closed container without the

loss of physical properties and they exhibit high electrical conductivity and viscosity.22 They

are optically transparent, which facilitates their utilization in spectroscopic applications.2

Organogels can also be prepared from the zwitterionic surfactant lecithin without the use of

gelatin.22 Lecithin organogels exhibit similar properties to AOT - gelatin based gels.25

The formation of organogels depends on the wo, the nature of the surfactant, and the

amount of gelatin present. It was determined by Luisi and Haering that the minimum wo

ratio required was 20 using AOT, independent of the amount of surfactant present.22 This is

probably the consequence of insufficient water to dissolve or hydrate the gelatin.22 Other

14

surfactants (cationic CTAB, CTAC) have been investigated for formation of gelatin

organogels but have been unsuccessful in the absence of AOT or other cosurfactant.25

The exact structure of organogels is still an open question although several structures

have been proposed. The basic structure is a network of tubules, as shown in Figure 8.

These tubules consist of a network of gelatin and water coated with the surfactant. The

tubules are surrounded by the solvent and microemulsion water droplets.22 Scanning

electron microscopy (SEM) and transmission electron microscopy (TEM) micrographs of

AOT organogels are shown in Figure 9.22 These micrographs clearly indicate a network

system in which the major components of the organogel have surprisingly relatively high

mobilities given the viscosity of the gel.22

Figure 8: Proposed Structure of Organogel where 1 represents the W/O Microemulsion-Type Droplets and 2 the Gelatin/Water Channels. Reproduced with permission from an article by Kantaria, Rees and Lawrence and Luisi26

15

Figure 9: SEM and TEM Micrographs of Organogel [TEM: A & B; SEM: C & D; panels A & C are of gels that contained 9 g gelatin per 100 mL and panels B&D are of gels that contained 4.5 g gelatin per 100 mL]. Reproduced with permission from an article by Haering and Luisi22

1.4.2 General Properties and Applications

Although based on limited data, it appears that organogels exhibit many of the same

general basic properties as reverse micelle solutions and thus can be utilized for similar

purposes and applications. The difference between organogels and reverse micelles is that

organogels are in a “solid” form because of gelation and can be used in situations that would

otherwise be difficult with a liquid, i.e. the reversed micelle solution. This “solid” form

allows for easier handling and reuse. Also, they are stable in many different types of solvents

which is useful in analyte (or product) recovery schemes where a product can be extracted

16

without the loss of physical properties or formation of emulsion systems that results when

reverse micelles in solution are utilized. These features have led to their application in

transdermal drug delivery, as fluorescent sensor platforms and as a catalytic medium for

synthetic reactions, particularly for enzyme mediated reactions and production of pure chiral

molecules.26-28

1.4.2.1 Transdermal Drug Delivery

In order to employ organogels for the purpose of transdermal drug delivery

pharmaceutically acceptable alternatives must replace the common use of AOT surfactant

and hydrocarbon solvents. This has been achieved using a variety of formulations including

the use of Tween 21, 81, and 85 surfactants with isopropyl myristate (IPM) as the solvent.26

In this example the use of AOT as a co-surfactant was greatly reduced but not eliminated.26

Such organogels can solubilize pharmaceuticals and have been successfully used in pig skin

models and the different factors impacting their drug delivery ability have been

investigated.26. The advantages of using organogels over other methods for transdermal drug

delivery include simple preparation and handling, resistance to microbial contamination, and

ease of use.

1.4.2.2 Fluorescent Sensors Fluorescent indicators incorporated into organogels have been successfully used as

humidity and oxygen sensors. 27,28 By adding the fluorescent dye sulforhodamin 101 (S101)

into a reverse micelle solution and then preparing an organogel, an organogel film humidity

sensor or optode membrane can be fabricated.27 S101 fluoresces at 608 and 588 nm in

17

solution and the luminescent intensity decreases with an increase in the water vapor

concentration. This same behavior was demonstrated when this dye molecule was

incorporated into an AOT organogel film optode.27 This AOT sensor system was

successfully employed for the determination of the relative humidity and the method was

validated by comparison of the results to that obtained using a hygrometer. 27

Another fluorescent indicator, tris-(2,2’-bipyridyl)ruthenium(II) complex

[Ru(bpy)3]2+, is highly fluorescent at 580 nm and its fluorescence intensity decreases in the

presence of increasing oxygen concentration in organic solvents.28 This indicator was also

incorporated into an AOT organogel membrane and evaluated for the quantification of

dissolved oxygen in hexane, toluene, and isooctane. 28

In both cases the organogel indicators performed as desired and the results were

comparable to those obtained with traditional methods. Also, the membranes were

completely reversible and reproducible allowing them to be reused repeatedly without the

loss of sensitivity.27, 28

1.4.2.3 Synthesis

The most frequent application of surfactant based organogels is as a medium for the

immobilization of enzymes for use in enzyme catalyzed reactions, especially in

enantioselective reactions for the production of optically pure chiral molecules. Enzymes

can be easily incorporated, i.e. solubilized, into the organogel system by either being

introduced in the solvent/surfactant or water/gelatin component before they are mixed

together.25 Once the enzyme is incorporated into the organogels, enzyme catalyzed reactions

can be performed. In the literature, most of the enzymatic reactions reported involve

18

hydrolytic or esterification reactions. Although a variety of different enzymes have been

utilized for an array of substrates, lipase seems to be the most frequently employed enzyme.

In addition, enzyme catalyzed reactions in AOT organogel systems have also been

employed to synthesize desired stereospecific products. Table 3 lists some examples of these

syntheses and the percent enantiomeric excess (%EE) found. As can be observed, very high

enantiomeric excesses have been reported.

Zhou et al. examined the lipase-catalyzed esterification of octanoic acid with 1-

octanol, which formed octyl octoate in an AOT/isooctane organogel system.29 The organogel

immobilized lipase was effective with recycling (after 10 rounds of esterification, only an

8.6% reduction in the percent conversion was observed) and stable upon storage (after 10

months of storage, only a 1.7% decrease in conversion was noted).29 This indicates that the

enzyme is contained in the stable organogel microenvironment with little loss of activity and

can be reused without modification. Similar results were also reported by Soni and

Madamwar who investigated the lipase-catalyzed esterification of n-caprylic acid with

ethanol to form ethylcaprylate in AOT organogels.30

19

Table 3

Results of Some Enantioselective Enzymatic Reactions in Organogels

Substrate Reactant Reaction Product Enzyme Reverse Micellar

System %EE Ref

Hexanol Hexanoic acid/ Dodecanoic acid (-)2-Hexanol

Chromobacterium viscosum (C.V.)

Lipase AOT/Hexane 96 31

Octanol Hexanoic acid / Dodecanoic acid (-)2-Octanol C.V Lipase AOT/Hexane 99 31

Phenylethanol Hexanoic acid/ Dodecanoic acid

(+)1-Phenyl-1-ethanol C.V. Lipase AOT/Hexane 98 31

Decanoic acid (-) 2-Octanol (-)1-Methyl-heptyl decanoate C.V. Lipase AOT/Heptane 92 32

(RS)-cyano(3-phenoxyphenyl)methyl butyrate

1-butanol (S)2-hydroxy-(3-

phenoxy) phenylacetonitrile

Lipase AOT/Isooctane/ Glutaraldehyde >99 33

20

Synthesis using enzymes immobilized in organogels offers advantages over the same

reaction in bulk organic solvent or aqueous solutions.23 For instance, enzymes can suffer a

loss of activity or may not be soluble in organic solvents. However, when solubilized in

reverse micelles or immobilized in organogels, they retain their native activity or even

exhibit enhanced activity relative to that seen in bulk solvent systems. Enzymes

encapsulated in organogels can be easily recycled and reused without a significant loss of

activity, which is not possible with reversed micelle solutions. Also, product recovery is

much easier when using enzymes immobilized in organogels since it is a “solid” entity and

thus the product can be extracted from the bulk external organic solvent. Attempted

extraction of a desired product(s) from a similar reverse micelle solution results in

troublesome microemulsion formation. 34

1.4.3 Advantages and Limitations of Organogels Relative to Reverse Micelle

Solutions

The unique properties of reverse micelles and organogels allow for a variety of uses.

These systems have advantages over classical methods of separation and synthesis. The

reaction conditions can be easily manipulated for size tailoring and nanoscale synthesis.

Also, in these systems, enzymes and proteins are easily solubilized along with other

compounds that may not be soluble in one particular bulk solvent or mixture of solvents.

In the case of reverse micelles, there is a major disadvantage compared to the

traditional methods of synthesis or separation. Product recovery is more difficult with

reverse micelles. First, there is a problem with the purity of the recovered sample. It is often

difficult to recover pure sample without surfactant contamination. The further purification of

21

the sample may be more time consuming or costly than the conventional methods. Second,

the destruction of the system may be necessary to recover a sample and in the case of enzyme

catalyzed synthesis, the enzyme may be deactivated. This leads to the use of more starting

materials, which also may be more costly. Finally, conventional methods may have a greater

percent yield of recovered product. Despite this disadvantage reverse micellar systems are

being studied extensively and product recovery may improve in the near future.

On the other hand, the use of organogels may solve many of these problems

encountered with reverse micelles. Organogels are in a “solid” form and are easier to

manipulate/handle but still retain the ability to incorporate enzymes/reactants. Enzymes

retain their activity or even show enhanced activity when immobilized in organogels. Since

the organogel is a “solid” gel, it can be extracted using bulk nonpolar solvents without the

formation of microemulsions as in the case of reverse micelle solutions. This facilitates

product recovery and the ability to recycle and reuse the organogel containing enzyme, etc..

1.5 Statement of Thesis Research Objectives

This research was undertaken with the aim to: (1) briefly characterize AOT

organogels with respect to their stability in aqueous solution (literature reports indicate that

they swell and leak their components when in contact with water); (2) determine their

density; (3) determine the partition coefficients for the interaction of a number of model

solute molecules with the AOT organogel; (4) determine whether or not any correlations

exist between the partition coefficient of the same solute for the AOT organogel relative to

its reported partition coefficient to an AOT reversed micelle solution; (5) evaluate the

feasibility of using AOT organogels to serve as an “extractant” medium for the extraction of

22

some environmentally relevant polar organic molecules from a bulk nonpolar solvent; and (6)

determination of the pertinent extraction parameters.

23

Chapter 2

EXPERIMENTAL 2.1 Materials

Sodium bis(2-ethylhexyl) sulfosuccinate (AOT) (f.w. 444.56 g/mol) and

gelatin (type A, 300 Bloom) were obtained from Sigma-Aldrich (St. Louis, MO) and used as

supplied. Heptane (99% laboratory grade) was also obtained from Sigma-Aldrich.

Isooctane and n-hexane (>98% purity) were obtained from Fisher Scientific (Raleigh, NC)

and used as received. Distilled, deionized water was obtained from a Milli-Q System

(Millipore Corp., Billerica, MA.). Solvents used for analyte preparation and organogel

stability studies included n-propanol, heptane, heptanol, carbon tetrachloride, and

chloroform; all reagent grade materials obtained from Fisher Scientific or Sigma-Aldrich.

Sodium chloride (analytical grade) was also obtained from Fisher Scientific Company. A

potassium phosphate / sodium hydroxide pH 6.0 buffer solution was prepared using

potassium phosphate monobasic from Sigma-Aldrich and sodium hydroxide from Fisher

Scientific. Both were used as supplied.

Model analytes employed in the partitioning and extraction studies included para-

nitrophenol (4-NP; f.w. 139.1 g/mol), para-bromoaniline (4-BrA; f.w. 172.0), ortho-

nitroaniline (2-NA; f.w. 138.1 g/mol), 2,4 dinitroaniline (2, 4-dNA; f.w. 183.1), phenol (f.w.

94.1), meta-nitroaniline (3-NA; f.w. 138.1) which were received from Fisher Scientific; para-

nitroaniline (4-NA; f.w. 138.1), tryptophan butyl ester (f.w. 296.8), tryptophan ethyl ester

(f.w. 268.7), and Nile Red (NR; f.w. 318.38) obtained from Sigma-Aldrich; 1-napthol and 2-

24

napthol (1-Nap or 2-Nap; f.w. 144.17), 2,4-dichlorophenol (f.w. 163.0), 2,4,5-trichlorophenol

(f.w. 197.45), 3-nitroaniline (m-NA; f.w. 138.1), phenol blue (f.w. 226.28) obtained from

Aldrich (Milwaukee, WI), and para-iodoaniline (f.w. 219.0) received from Eastman

Chemical Corp. (Kingsport, TN). All of these analytes were used as supplied from the

manufacturer. For the enzyme experiments, laccase, derived from Agaricus bisporus was

obtained from Sigma-Aldrich and used as supplied.

2.2 Equipment & Instrumentation

Standard laboratory equipment and glassware were used for all procedures.

Organogels were sectioned and cut on a glass plate or weighing paper with a single-edged

razor blade. A Mettler balance was employed for mass measurements.

Absorbance measurements were made using a Hewlett-Packard UV/Vis model 8452

diode array spectrophotometer. Standard one-centimeter Fisher quartz cuvettes were used for

all absorbance measurements.

2.3 Organogel Preparation

2.3.1 Standard Organogel Preparation Organogels were prepared by adding 2.4 ml water to 1.41 g gelatin type A in a 30 ml

beaker. In a separate 30 ml beaker, 6.0 ml of heptane were added to 0.89 g AOT. Both

beakers were heated in a water bath to 550C and allowed to remain at that temperature for a

few minutes until the gelatin was completely dissolved. The solutions were then removed

from the water bath and the AOT solution was poured into the beaker containing the gelatin

solution. The mixture was stirred vigorously until a homogeneous solution formed. The

solution was then allowed to cool to room temperature. After cooling, the gel was covered

25

with SealView laboratory film and placed in a refrigerator (4 ± 20C). All gels were allowed

to age for at least 3 days to allow for complete gelation prior to their utilization.

2.3.2 Alternative Organogel Preparation Organogels employed for some of the analyte absorption studies were prepared

according to the standard method except the amounts of the components were scaled up (i.e.,

3.75 ml water, 2.29 g gelatin (type A), 9.3 ml of heptane and 1.39 g AOT were utilized). In

addition, 50 ml sized beakers, instead of 30 ml, were employed. This organogel preparation

was altered from the standard method due to the need for more organogel materials for some

of the analyte absorption studies. This alteration allowed the gel thickness to remain

consistent with the standard method.

2.3.3 Organogels with Enzyme Preparation

Organogels were prepared in which the enzyme laccase was incorporated into the gel.

A solution of 3.75 ml 5.0 X 10-2 M potassium phosphate monobasic- sodium phosphate, pH

6.0, buffer solution, containing 1.9 X 10-2 g laccase was substituted for the water in the

alternative organogel preparation method. Alternatively, organogels with laccase were

formed by adding 1.50 ml of the buffer solution to 1.4 g of the gelatin in a 50 ml beaker. In a

separate 50 ml beaker 5.3 ml of isooctane were added to 0.89 g AOT. Both beakers were

heated to 550C simultaneously and allowed to remain heated until the gelatin dissolved. The

solutions were removed from the water baths and the AOT solution was poured into the

gelatin solution. Immediately afterwards, a solution of 0.90 mL of the buffer and 2.9 X 10-3 g

laccase was poured into the hot solution. The mixture was stirred vigorously until a

homogeneous solution formed. The solution was then allowed to cool to room temperature.

26

After cooling the laccase containing gel was covered with Seal View laboratory film and

placed in a refrigerator (4± 20C). The laccase gel was stored for a minimum of 3 days in

order to ensure complete gel formation before use.

2.4 Organogel Stability in Different Solvent Systems

The prepared organogels were sectioned to approximately one-centimeter cubes using

a razor blade. A cube was then placed in a flask containing the test solvent and then

stoppered. The gels were visually inspected as to their size, shape, color, etc. in order to

ascertain their stability in the test solvent or solution. The solvent systems examined

included n-propanol, heptane, aqueous 0.10 M NaCl, aqueous 0.25 M NaCl, aqueous 0.5 M

NaCl and bulk water. The organogel cubes were initially visually observed and inspected

and again inspected after three days of storage in the different solvent systems. The

procedure was repeated with an additional set of organogel cubes prepared in the same

manner.

A third set of gels was also thus inspected for stability but their uptake of solvent

was also quantified. This was performed by recording the initial mass of each organogel

cube prior to its immersion in the solvent system under study. Each organogel cube was then

submerged in a flask containing 10 ml of the particular solvent under study. The flasks were

then stoppered. Changes in size and appearance were visually noted over a 2 week period.

After the 2 weeks, the organogels were removed from the test solutions and their masses

again determined.

27

2.5 Density Determination

Organogel density measurements were made using approximately one centimeter

cubes of the organogel. The mass of the organogel cube was determined and then the gel

was carefully dropped into a 10.00 ml volumetric flask containing heptane. The displaced

solvent was removed from the flask and the volume of the displaced solvent was measured in

a graduated cylinder. Density measurements were repeated for a second set of gels that were

stirred for approximately 18 hours in 100 ml hexane. The density of the gel was also

measured after absorption of 2, 4-dinitroaniline in hexane.

2.6 Analyte Solution Preparation Solute solutions were prepared by accurately massing out the specific analyte,

transferring to a volumetric flask, and diluting to final volume using the bulk solvent.

Typically, the concentration of the stock solutions was on the order of 10-5 to 10-4 M. In the

case of sparingly soluble solutes, starting concentrations were between 3.3 x 10-7 and 2.6 x

10-5 M. In these latter cases, a saturated solution was prepared, left overnight, filtered and the

supernatant was used for analysis. Appropriate dilutions were made to obtain solutions at a

desired analyte concentration. The flasks containing these solutions were wrapped in Al foil

and stored in the dark when not in use.

2.7 Determination of Solute – Organogel Partition Coefficients and Organogel Extraction

Efficiency

In the majority of experiments performed, the organogels were cut into 8 to 12

sections of appropriate size for each analysis, massed on an analytical balance and placed

into a 25 or 50 ml beaker. The total gel mass ranged from ca. 1 to 4 grams. Appropriate

28

blanks were also prepared; i.e., a solution containing a section of organogel without analyte,

and another flask containing the analyte but without an added organogel, i.e. a control.

Typically, 10 to 50 ml of the analyte containing solution was then added to the gel containing

beakers and aliquots periodically taken, analyzed spectroscopically and immediately returned

to the beaker. The solutions were monitored until a constant absorbance was achieved

which indicated equilibration of the system. Table 4 summarizes the spectral data

(wavelength maximum and molar absorptivity values) for the solutes in bulk solvent and

reverse micellar systems.

Table 4

Solute in Solution Spectral Parameters

Solute Solubility, M

Reverse Micelle Bulk Organic Solvent λ,

nmε, M-

1cm-1 System λ, nm

ε, M-

1cm-1 Solvent

2-Nitroaniline 3.2 X 10-3 Hexanea 398 5500 AOT/Hexanea 376

3725370 5250

Hexanea

Isooctaneb 3-Nitroaniline ------- --- --- ---- 340 1300 Isooctaneb

4-Nitroaniline 2.3 X 10-4 Hexanea 358 18200 AOT/Hexanea

320318316

15500 10500 14100

Heptane Hexanea

Isooctaneb 2,4-

Dinitroaniline 3.1 X 10-5

Hexanea 334 17000 AOT/Hexanea 310 15500 Hexanea

Phenol ----- -- ---- ------ 269271

1410 2150

Isooctaneb Hexane/CCl4

d

4-Nitrophenol 4.6 X 10-4 Isooctanec 307 AOT/Isooctanec 288

27810500 10000

Heptane Isooctaneb

2,4-Dichlorophenol ---- ---- ---- ------ 285 2050 Carbon

tetrachloride 1-Naphthol ---- ---- ---- ------ 230 4890 Heptane 2-Naphthol ---- ---- ---- ------ 330 2210 Heptane

aData taken from literature.35 bData taken from literature.36

cValues taken from literature.37

dValues taken from literature.38

29

The partition coefficient (P) was defined as the solute concentration in the organogel

divided by the solute concentration in the bulk external solvent.32 To simplify the

calculations, Equation 1 was used for determination of the partition coefficient:

( )VV

AAAP

g

s

f

fi ×−

= (1)

where Ai is absorbance of the initial analyte containing solution prior to the addition of the

organogel, Af is the corrected absorbance of analyte solution with gel at equilibrium, Vs is

volume of analyte solution used, Vg is volume of the gel. All experiments were conducted at

room temperature (25 ± 2oC).

The extraction efficiency (percent extraction) was determined using an identical

experimental procedure as detailed for the solute partition coefficient determinations.

The extraction efficiency (%E) was calculated using Equation 2:

VVPPE

og/100(%)+

= (2)

where P is the partition coefficient from Equation 1, V is the volume of the analyte solution

used, and Vog is the volume of the organogel used.

2.8 Determination of the Time Course for the Absorption Process

The experimental protocol for determination of the time course for solute sorption to the

organogel was the same as noted for the partition coefficient determination except that

absorbance measurements of the solute containing solutions in contact with the gel were

taken at regular time intervals. From the data, the absorbance versus time profile could be

plotted.

30

In a few instances, absorption was monitored by measuring the solute absorbance

within the organogel. This was done by first preparing the organogel using the alternative

gel preparation method. After the gel formed it was reheated and poured into a quartz

cuvette allowing a thin layer, about 0.5 mm thick, to form on the inside face of the cuvette.

The solute containing solution was then contacted with the gel in the cuvette for a specified

time period, then the solute solution was removed and the absorbance due to the solute that

had absorbed into the gel was measured. This process was repeated at regular time intervals.

The blank measurement was made employing a similar gel lined cuvette that had not been

contacted with the solute containing solution. The data allowed for the determination of the

solute absorbance in the gel versus time profile.

2.9 Data Analysis

Required linear regression, multiple linear regression analyses, and statistical

calculations were performed using Excel (Microsoft, USA).

31

Chapter 3

RESULTS AND DISCUSSION

3.1 Brief Characterization Studies 3.1.1 Organogel Stability Applications involving the utilization of AOT organogels depend in part on their

robustness and ability to maintain their integrity under a variety of conditions. Previously,

AOT organogels have been shown to be quite stable when stored in closed containers and

when contacted with nonpolar organic solvents (hydrocarbons such as isooctane,

cyclohexane, n-heptane, etc.)22,32,39 However, the literature is sparse in regards to organogel

stability in polar organic solvents although it has been noted that organogels may be less

stable in more polar media, particularly in bulk water. 22,39

In this work, sections of organogels were contacted with selected solvents or aqueous

solutions and monitored over a one week time period with changes in size, appearance and

optical clarity noted. The swelling ratio was also determined. The swelling ratio, Esw, is

defined as Esw = [(We – Wo) / Wo], where We is the mass of the organogel after equilibration

in the external solvent system and Wo is the initial mass of the organogel before immersion.

The results obtained are summarized in Tables 5 and 6.

As can be observed from the Tables, in the presence of distilled water, the gel

significantly swelled (swelling ratio ca. 22, Table 6), became opaque and lost its integrity.

Such behavior has been previously noted in the literature.25 However, in the presence of salt

(NaCl), the gels appeared to be much more stable and did not significantly swell (swelling

ratio, 1.2 to 1.7 depending upon the salt concentration). It appears that AOT organogels

32

when contacted with aqueous salt solutions are stable and thus may also be utilized in such

aqueous solutions in addition to nonpolar organic solvents. This should serve to extend the

scope of applications possible with such materials. Figure 10 shows a photograph of the

appearance of a 1cm square cube AOT organogel after it had been immersed in a 0.50M salt

water (NaCl) solution for one week.

Figure 10: Photo of an AOT Organogel that had been in Contact with a 0.50 M NaCl Solution for 7 days

In the presence of propanol, the AOT organogel appeared unchanged after 1 day and

did not swell. However, after one week, it appeared to have shrunk in size and it became

more opaque. Likewise after one week’s exposure to isopropanol, the AOT organogel

shrank and became slightly opaque. These findings are in agreement with reports that AOT

organogels are not stable in polar organic solvents.39,25

When in contact with the nonpolar solvents cyclohexane and heptane, the AOT

organogels remained intact and maintained their integrity. Although there appeared to be

some swelling after 24 hours, the AOT swelling ratio was only 0.09 when in the presence of

33

cyclohexane and actually slightly negative if in bulk heptane. These observations are in

agreement with prior literature reports. 22,32,39

Table 5

Appearance of Organogels after Exposure to Different Solvent Systems

Solvent Observations

After 24 hours After one week

Propanol No observable change Slight whitening of gel

Isopropanol Slight whitening Shrinking of gel, slight whitening

Heptane Slight swelling Gel and solution cloudy / more swelling

Cyclohexane Some swelling / very clear almost transparent Some swelling, clearing

Water Whitening, swelling Much more swelling,

almost translucent, lost shape

Water (+ 0.50 M NaCl) Some gel swelling Greater swelling

34

Table 6

Swelling of Organogels When in Contact with Different Media

Solvent Initial Massa (Wo)

End Mass (We)

Swelling Ratio (Esw)

Water 0.0942 2.1435 21.75

Isopropanol 0.5082 0.4096 -0.19

NaCl 0.1M 0.0978 0.2255 1.31

NaCl 0.25M 0.1021 0.2646 1.59

NaCl 0.5M 0.0864 0.2307 1.67 NaCl (Saturated, ca.

5.3 M) 0.0941 0.2078 1.21

Cyclohexane 0.9591 1.0481 0.09

Heptane 0.9548 0.7800 -0.18 aInitial mass of gel prior to contacting it with the indicated solvent system. 3.1.2 AOT Organogel Density

Density represents another important property of organogel materials. The density of

AOT organogels prepared with hexane as the nonpolar solvent was found to be 1.05 g/mL (n

= 7; standard deviation 0.08 g/mL). To the best of my knowledge, there has been no prior

density value for AOT organogels reported in the literature.

3.2 Interaction of Organic Solutes with AOT Organogels

3.2.1 Determination of Partition Coefficients

Similar to AOT reverse micelles in solution, AOT organogels possess both ionic and polar

binding sites. Although a vast amount of literature is available regarding the binding or

partitioning of inorganic, biochemical and organic solutes to AOT reverse micelles in

solution40, there has been only one prior report concerning the binding of solutes to AOT

35

organogels.32 Consequently, in this work, the partition coefficients for the interaction of

twelve solutes with AOT organogels were determined. The results are summarized in Table

VII. Also included for comparison purposes are available literature values for binding of the

same solutes to AOT reverse micelles in solution. As can be observed, the values for the

partitioning of all solutes to the AOT reverse micelles in solution are greater (by factors of

roughly about 6 to 40) relative to that observed for their partitioning to the AOT organogels.

In addition, for the series of isomeric nitroanilines, the trends are the same; i.e., the 2-

nitroaniline exhibits the smallest partition coefficient while the 4-nitroaniline exhibits the

greatest (see Table 7) with respect to their interaction with AOT reverse micelles or AOT

organogels. For the nitroaniline derivatives, the order of their solubility in the nonpolar

solvent hexane follows the trend: 2,4-dinitroaniline (3.1 x 10-5 M) < 4-nitroaniline (2.3 x 10-

4 M) < 2-nitroaniline (3.2 x 10-3 M).6 Their binding/partitioning to either AOT reverse

micelles in solution or the AOT organogels appears to be inversely related to their solubility

in a nonpolar organic solvent like hexane; i.e., the greater the solubility in hexane, the

smaller is its binding or partitioning to the AOT reverse micelle/organogel. Thus, the more

nonpolar the solute, the lesser is its partitioning interaction with the AOT organogel. In

agreement with this hypothesis, two other nonpolar solutes, p-xylene and nitrobenzene, did

not exhibit any apparent partitioning to the AOT organogels (Table 7).

36

Table 7

Partition Coefficients of Solutes in AOT Reverse Micelles and AOT Organogels

Solute Kb M-1 (AOT RM) P (AOT RM)a P(AOT gel) Ref

2-Nitroaniline 45-70 35 119 – 185 17.9 ± 7 3-Nitroaniline ------ -- ------- 32.9 4-Nitroaniline 350-400 35 922 – 1054 122.4 ± 57

2,4-Dinitroaniline 3100-3350 35 8159 – 8817 276.5 ± 224 Phenolc 327-350 37 862 - 922 24.9

4-Nitrophenolb 3670-7510 37 9659 – 19737 512.3 ± 347 2,4-dichlorophenolc ------- -- --------- 1.31 ± 0.6

2,4,5-trichlorophenolc ------- -- -------- 3.9 ± 1.7 1-Naphthol ------- -- -------- 17.7 ± 0.9 2-Naphtholb 140-335 44 369 - 883 25.4 ± 5.3 Nitrobenzene -------- -- -------- 0

p-Xylene -------- -- ------- 0 aP represents the partition coefficient for solute distribution between AOT organogel and bulk solvent phase (hexane). P was calculated from Kb using the Berezin equation7; i.e., Kb = [P-1] v, where v is the molar volume of the AOT surfactant [v was taken to be 0.380 M-1 which is the average of literature values].42,43 bBulk solvent was heptane. cBulk solvent was carbon tetrachloride.

Figure 11 shows the plot of the solute – AOT reverse micelle binding constants, Kb

(median of reported values), versus their partition coefficient, P for interaction with the AOT

organogels obtained in this work. As can be seen, a good correlation (n = 6; R2 = 0.97) was

observed. Thus, if the solute – AOT reverse micelle binding constant is known, one can get a

rough estimate of that solute’s anticipated partition coefficient with AOT organogels using

such a plot.

37

0

1000

2000

3000

4000

5000

6000

0 100 200 300 400 500 600P value (AOT organogel)

K b (M

-1) (

AO

T R

ever

se M

icel

les)

Figure 11: Plot of the Solute – AOT Reverse Micelle Binding Constant, Kb (M-1) versus the Solute – AOT Organogel Partition Coefficient

3.2.2. Characterization of AOT Organogel Phase

In order to gain greater insight into the interactions that are involved in solute

partitioning to the organogel, the Abraham solvation parameter model was employed to

characterize the AOT organogel material.45-49 The Abraham model utilizes a linear free

energy equation that relates a dependent variable to a series of interaction parameters of the

solute molecule (general equation (3)):

log SE = c + r R2 + s π2H + a α2

H + b β2H + v V (3)

The dependent variable (SE) may be a chromatographic retention factor (log k’), partition

coefficient (log P), binding constant (log Kb), or Ostwald solubility coefficient (log L),

etc.45,46 Each solute molecule possesses a unique set of descriptors that are dependent upon

38

its specific structural features, which in turn governs the types of solute – host organogel

interactions that it may undergo. These descriptors include the solute’s excess molar

refraction (R2), polarizability (or dipolarity) (π2H), hydrogen bond acidity (α2

H), hydrogen

bond basicity (β2H ), and McGowan molar volume (V).45-47 These descriptors for all probe

molecules employed in this study are summarized in Table 8. It should be noted that in

addition to the eleven solute partition coefficients determined in this study, literature values

are included for octanol and decanoic acid32 in order to expand the data set.

Table 8

Summary of Solutes, their AOT Organogel-Hexane Partition Coefficients and their Abraham Solvation Descriptor Values

Solute Pa log Pa R2 π2

H α2H β2

H V Phenolb 24.90 1.40 0.81 0.89 0.60 0.30 0.775

4-nitrophenolb 512.32 2.71 1.07 1.72 0.82 0.26 0.949

2,4-dichlorophenolb 1.31 0.12 0.96 0.84 0.53 0.19 1.02

2,4,5-trichlorophenolb 3.87 0.59 1.07 0.92 0.73 0.10 1.14

2-nitroanilineb 17.93 1.25 1.18 1.37 0.30 0.36 0.99

3-nitroanilineb 32.90 1.52 1.20 1.71 0.40 0.35 0.99

4-nitroanilinec 128.87 2.11 1.22 1.93 0.46 0.35 0.99

1-naphtholc 17.74 1.25 1.52 1.05 0.60 0.37 1.144

2-naphtholc 25.45 1.41 1.52 1.08 0.61 0.40 1.144

Octanolc 0.94e -.03 0.20 0.42 0.37 0.48 1.30

Decanoic Acidc 1.02e .01 0.12 0.64 0.62 0.45 1.59

4-Xylenec,d 0 --- 0.61 0.52 0 0.16 1.00

Nitrobenzeneb,d 0 --- 0.87 1.11 0 0.28 0.89

aValues calculated from experimental data from Table 7. bSolvation parameters taken from the literature.47 cSolvation parameters taken from the litetarure.48

dValue was not used in the regression analysis ePartition coefficient taken from the literature.32

39

In this approach, the AOT organogel phase is characterized by independently

determining, from spectroscopic measurements, the partition coefficients for the interaction

of a number of solute molecules with the organogel. Using the measured partition

coefficients and multiple linear regression analysis (Eq. 3), the interaction parameters of the

AOT organogel (r, s, a, b v) can be determined. The coefficient r represents the ability of

the AOT organogel phase to interact with pi- and nonbonding electrons of the solute, s

indicates the polarizability/dipolarity of the organogel phase, a and b represent the AOT

organogel phase hydrogen bond acidity and basicity, respectively, and v represents its

hydrophobicity.

The calculated interaction parameters of the AOT organogel phase are summarized in

Table 8. In addition, the interaction parameters for AOT reverse micelles in solution as

reported by Zingaretti et al.49 are included for comparison purposes. In the case of AOT

reverse micelles in solution, solute incorporation is strongly favored by increasing solute

hydrogen bond acidity and slightly disfavored by increasing solute molar

volume/hydrophobicity (see Table 9). In contrast, for the AOT organogels, solute

incorporation is dominated by hydrogen bond basicity and acidity with a secondary

contribution from solute polarizability. It is also negatively impacted by increasing the solute

molar volume. For the reverse micelles in solution, there is no contribution to partitioning

due to solute hydrogen bond basicity whereas that is the dominant term in AOT organogels.

An explanation for this behavior might lie with the gelatin that is present in the organogel

system (and not solution reverse micelle). Each gelatin present contains a number secondary

amine and carbonyl oxygen moieties that contain lone pairs of electrons that are capable of

accepting a hydrogen bond, thus enhancing hydrogen bond basicity.

40

It should be cautioned that the interaction coefficients determined for both the reverse

micelles in solution and organogel systems were based on rather limited solute data sets (nine

amines and nine alcohols for the AOT reverse micelle systems and eleven solutes for the

AOT organogels) and thus probably reflect only crude approximations of the true interaction

coefficients.

Table 9

Interaction Coefficients for Solute Partitioning into AOT Organogel and AOT Reversed Micellar Solutions

aAOT Organogel values as determined in this work. bAOT Reverse Micelle values based on amine solutes reported in the literature.49

cAOT Reverse Micelle values based on alcohols as reported in the literature.49

A plot of the predicted partition coefficients (as calculated using the AOT organogel

interaction coefficients and equation 3) versus the experimentally observed partition

coefficients for solute incorporation into the AOT organogels is shown in Figure 12. The R2

value for this plot is 0.97, indicating a fair correlation. Thus, the partition coefficient for a

desired solute could be predicted with some confidence using equation 3 and the determined

AOT organogel interaction parameters provided that the solute’s descriptors are known. As

an example, the predicted partition coefficients for the solutes p-xylene and nitrobenzene are

calculated to be 0.025 and 0.60, respectively. The experimentally determined values were

zero within experimental error.

AOT System c r s a b v R2

Organogela -0.94 0.10 1.20 2.75 3.63 -1.93 0.975

Reverse Micelleb -1.4 0 0 7.0 0 -1.9 0.985

Reverse Micellec -2.8 0 0 12.7 0 -0.76 0.98

41

-0.30

0.30.60.91.21.51.82.12.42.7

-0.3 0.1 0.5 0.9 1.3 1.7 2.1 2.5log P observed

log

P pr

edic

ted

Figure 12: Comparison of the Predicted and Experimentally Observed Values of the Partition Coefficients for Solute Incorporation into AOT Organogels.

3.3 Extraction of Polar Analytes from Nonpolar Solvents using AOT Organogels

3.3.1 Extraction Time Profiles and Sorption Kinetics

Figure 13 shows the time course profile for the removal of the polar aromatic

solute 2-nitroaniline from hexane due to the presence of an AOT organogel extractant phase.

As can be observed, equilibrium is achieved fairly rapidly; i.e., within three hours and the

AOT organogel served to extract a significant amount of the 2-NA from the hexane solution.

42

0

0.2

0.4

0.6

0.8

1

1.2

0 25 50 75 100 125 150 175Time (min)

Abs

orba

nce

Figure 13: Plot of the Absorbance of 2-Nitroaniline at 376 nm in Hexane versus Time (minutes) after exposure to 3.945 grams of AOT Organogel at 25.0o C.

The amount of any solute absorbed by the organogel at any time, t, Qt (mmol

solute/gram organogel), was determined via use of equation (4):17

Qt = (Co – Ct) V / W (4)

where Co and Ct are the organic liquid phase solute concentrations (molar or millimolar) at

time equals zero (prior to addition of the gel to the hexane solution) and time t, respectively,

V is the volume (in mL) of the hexane solution, and W is the mass (in grams) of the AOT

organogel. Knowledge of the solute molar absorptivity in the organic solvent allows for

conversion from the absorbance reading to Qt value at each measurement time, t. Figure 14

shows the corresponding plot of the amount of 2-NA sorbed onto the AOT organogel as a

function of time.

43

0.0E+00

1.0E-04

2.0E-04

3.0E-04

4.0E-04

5.0E-04

6.0E-04

7.0E-04

8.0E-04

0 25 50 75 100 125 150 175Time (min)

Qt

Figure 14: Plot of the Amount of 2-NA (mmol/gram gel) Sorbed onto the AOT Organogel as a Function of Time (minutes) [other conditions are as noted in Figure 13 legend].

In order to identify the rate-controlling step for sorption of solutes onto the organogel,

three potential rate-limiting steps are typically considered:51

(i) Mass transfer of the polar solute from the bulk organic solution to the AOT

organogel surface

(ii) Sorption of the organic solute onto organogel sites, and

(iii) Internal diffusion of the organic solutes in the organogel

If the rate limiting step is due to solute mass transfer (i), then the first order equation first

proposed by Lagergren, equation (5) should best fit the data (from Fig. 14): 51,52

log (Qe – Qt) = log Qe - k1 (t) / 2.303 (5)

where k1 is the first-order sorption rate constant and Qe is the equilibrium sorption capacity

which is calculated from equation 6 as:

Qe = (Co – Ce) V / W (6)

where Ce is the liquid phase solute concentration (millimolar or molar) at equilibrium and the

other terms as defined in Equation 4.

44

0.0E+00

5.0E+04

1.0E+05

1.5E+05

2.0E+05

2.5E+05

0 50 100 150 200Time (min)

t/Qt

If the rate determining step is due to solute adsorption onto organogel sites (ii), then the

sorption data should best fit the following pseudo-second-order sorption model equation:51

t / Qt = 1 / (k2 Qe2) + t / Qe (7)

where k2 is the pseudo-second-order sorption rate constant.

Alternatively, if internal intraparticle diffusion (iii) is the rate limiting step, the

experimental data should best fit the Weber-Morris equation:

Qt = kp t0.5 (8)

where kp is the pore diffusion rate constant.20 Treatment of the 2-NA data shown in Figures

13 and 14 utilizing these three treatments revealed that 2-NA sorption onto the AOT

organogel sites (Eq. 7) best fit the data (see Fig. 15, correlation coefficient 0.9980).

Figure 15: Plot of the Second-Order Sorption Model Equation (Eq.7), t/Qt as a function of Time for the Solute 2-NA

45

The pseudo-second order sorption rate constant for 2-NA was found to be 139.5 g/mmol/min.

The correlation coefficients for treatment of the same data by the first order (Eq. 5) and

particle diffusion (eq. 8) models were 0.8663 and 0.8811, respectively.

The same treatment was applied to several other solutes (4-nitrophenol, 2-naphthol,

and 2,4-dinitroaniline) and in each instance, the pseudo-second order model (Eq. 7) was

found to best fit (gave the greater R2 value for) the experimental data. Table 10 summarizes

these data (i.e., the best fit model, rate constants, equilibrium sorption capacity, and the

correlation coefficient for each solute). There has not been any prior report in the literature

concerning an analysis of the sorption kinetics for solute partitioning to AOT organogels.

Table 10

Pseudo-Second Order Parameters for the Sorption of Solutes on AOT Organogels

Solute Best Fit Model

k2 (g/mmol/min) R2 Qe

a

Observed Qe

a

Calculatedb

2-Nitroaniline 2nd order (Eq.3.5) 139.5 0.998 7.96E-04 7.17E-04

4-Nitroaniline 2nd order (Eq.3.5) 3709.9 0.983 5.54E-04 5.27E-04

2,4-Dinitroaniline 2nd order (Eq.3.5) 306.1 0.999 2.68E-04 3.06E-04

2-Naphthol 2nd order (Eq.3.5) 92.2 0.999 2.19E-03 2.27E-03

aUnits for Qe are mmol*mL/g bValues calculated from the slope of the best fit model, equation 7. It should be noted that AOT organogels are optically transparent and thus in addition

monitoring the solute absorbance decrease in the bulk external organic solvent (as shown in

Figure 13), one can also monitor the absorbance due to solute incorporation into the

organogel directly. Figure 16 illustrates the time profile for the incorporation of the solute

46

0

0.2

0.4

0.6

0.8

1

1.2

0 50 100 150 200Time (min)

Abs

orba

nce

4-nitroaniline into an AOT organogel that was obtained by measuring the 4-NA absorbance

in the gel directly.

Figure 16: Plot of Absorbance (at 318 nm) of 4-Nitroaniline that Sorbed onto the Organogel versus Time at 25.0o C. 3.3.2 Extraction Parameters and Considerations Although a wide variety of sorbent materials have been employed for extraction of

organic solutes from aqueous solutions, a more limited number have been reported for

extraction of polar organic solutes from nonpolar organic solvents. Thus, the utilization of

AOT organogels for the extraction of polar aromatic solutes from nonpolar solvents was

evaluated. The extraction efficiencies achieved for different substituted anilines and phenols

are summarized in Table 11. As can be observed, the extraction efficiencies ranged from 72

to 98 % for the analytes examined for a single step extraction. The percent extraction

achieved roughly parallels the magnitude of the respective partition coefficients for the

47

analytes examined. Table 11 also gives the concentration factor achieved by the extraction,

which ranged from ca. 3 to 13 depending upon the amount of extractant AOT organogel that

was employed. A greater concentration factor can be achieved if one uses a smaller amount

of the gel (in this work, ca. 1.0 gram AOT organogel was the smallest amount of gel that was

employed for an extraction).

Table 11

Extraction Efficiency and Concentration Factors Achieved for the Extraction of Analytes from Hexane with the AOT Organogel Materials

Solute %Ea Concentration Factorb

2-Nitroanaline 71.7 5.3 3-Nitroanaline 75.8 --- 4-Nitroanaline 94.6 2.9 – 10c

2, 4 - Dinitroanaline 96.8 12.9 4-Nitrophenol 77.8 ---

2-Naphthol 74.8 8.3 Phenol 82.6 7.3

aCalculated using equation 2. bConcentration (or enrichment) factor was calculated as the ratio of the solute concentration in the AOT organogel phase to the original solute concentration in the bulk organic phase. cDepends upon the mass of gel (2.9 if 10 grams and 10.0 if 1 gram organogel employed).

These preliminary results indicate that the AOT organogel can serve for the extractive

preconcentration of polar organic analytes initially present in nonpolar organic solvent.

Previously, such polar analytes as anilines, nitroanilines, and phenols had been extracted

from nonpolar solvents using salt water as the extractant phase or different ion exchange

resins.54-56 None of these literature reports indicated the extraction efficiencies or enrichment

factors that were attained using these approaches.

48

It is anticipated that such AOT organogels will most likely be employed for the

extractive purification of nonpolar organic solvents rather than for analyte preconcentration

prior to its quantification. Previously, one report noted the utilization of AOT organogel

films for the extractive removal of wastewater pollutants and their detoxification.57 The

organogel films contained appropriate entrapped (immobilized) enzymes that allowed for the

in situ conversion of the original toxic pollutant material into a less toxic (or nontoxic)

product(s) within the film. A problem with this approach is that with extended use, the

organogel materials swelled and lost their integrity. This problem might be overcome if the

ionic strength of the contaminated water were increased by addition of salt prior to addition

of the enzyme-containing organogel material (refer to section 3.1.1, p. 31).

In this work, the incorporation of the enzyme laccase into the organogel with a

potassium phosphate buffer solution was performed. The uptake of some phenols into the

enzyme containing gel was measured with the intent of the enzyme detoxifying the phenols.

Little or no absorbance into the organogel was observed.

One consideration in the utilization of AOT organogels for extractions and/or

purifications is that of the subsequent desorption and recovery of the target analyte or

contaminant if required. In this context, the ability to use the organogels in salt water might

prove very beneficial. Some preliminary experiments revealed that when an AOT organogel

was contacted with an aqueous solution (containing NaCl) of 4-nitroaniline, the gel only

bound a small amount of the 4-nitroaniline. In other words, the partition coefficient for the

sorption of 4-NA from salt water as external solvent was only ca. 2.2. This means that if an

AOT organogel that had been previously employed for the removal of this analyte from a

nonpolar organic solvent were then subsequently immersed in a salt water solution, most of

49

the 4-NA should then be desorbed. Of course, experiments to conclusively demonstrate this

need to be conducted but this preliminary work bodes well for the likely success of this

desorption approach.

3.4 Conclusions For organogels to be a viable medium for solute extraction they must be stable and

maintain their integrity in the relevant solvent. In this work it was found that organogels are

stable in a variety of nonpolar solvents and varying strengths of aqueous sodium chloride

solutions. The stability in salt solutions is significant because this allows the possibility of

developing novel uses for organogels in such solutions. Conversely, the organogels were not

stable in water alone as was demonstrated by the large swelling ratio. This indicates that the

organogels absorb water and may be useful in extracting water from organic solvents.

The determined density of the AOT organogels is important in that it allows for the

interconversion from mass of the gel used to the corresponding volume. The volume is

required for some calculations that might involve such gels, as in the determination of the

sorption kinetics and/or calculation of extraction parameters, such as the enrichment factor.

The determined partition coefficient for the interaction of a number of solutes to AOT

organogels extends the data pool available in the literature. More important, the relative

binding/partitioning trends observed for the binding of solutes to AOT reverse micelles is

mirrored for the AOT organogels. This allows for rough estimates to be made if

corresponding reverse micelle binding data is available in the literature. Whereas, the more

nonpolar the solute the less it may be absorbed by the organogel.

Building upon the partition coefficient, further insight into the characterization of

organogel interactions was gained. The Abraham solvation model was employed to

50

determine the interaction parameters for the AOT organogel. The dominant terms for

organogels were determined and compared to those of AOT reverse micelles. It was also

noted that these interactions for both AOT containing systems were based on limited data and

represent only an estimate of the true systems.

Furthermore, this work determined the rate constants of uptake into the organogels

for a few solutes. The pseudo-second order sorption model was found to best fit the solute

sorption data for the four solutes examined in this study. Lastly, some extraction

parameters were determined for the extraction of polar aromatic analytes from hexane (or

heptane) as the nonpolar solvent. The extraction efficiencies were in the range of >70% with

enrichment factors of ca. 10 achieved via use of 1.0 gram of the AOT organogel as the

extractant phase.

51

Chapter 4

FUTURE STUDIES

4.1 Recommendations Given the findings in this work, there are a few recommendations for future studies

involving organogels. These recommendations may promote potential new applications and

further extend the characterization of organogels.

Although it was demonstrated that the organogels were unstable in water it would be

interesting to investigate the possibility of employing organogels for extraction of water from

organic or other non aqueous solutions (serving as a dehydration agent). Along with this,

one may want to quantify the maximum amount of water that can be extracted prior to the

organogel losing its integrity, i.e. the organogel cannot be removed from the solvent.

Furthermore, if the organogels are stable in salt water (such as NaCl), the minimum

salt concentration required to maintain organogel stability should be determined. Once this is

established, an investigation as to whether or not organogels can extract a target solute

initially present in an aqueous salt solution should be undertaken. In addition, what are the

factors (salt concentration, solute polarity) that impact such extraction? It might be expected

that relative to water as the initial phase, the AOT organogels might prove useful for the

extraction of “more” nonpolar analytes. Even if organogels prove to be ineffective for the

removal of analytes initially present in water, such data would be useful in the context of

using salt water for the desorption of analytes that had been removed from a nonpolar

52

organic solvent (as detailed in this work). It would be beneficial to determine the extent of

desorption possible and effectiveness of the subsequent reuse (recycling) of the spent

organogel after contact with an appropriate aqueous salt solution.

Due to the limited number of solutes examined in the Abraham treatment, the

determination of partitioning data for a wider range of solutes (particularly various

nonaromatic organic molecules such as aliphatic aldehydes, ketones, amines, alcohols,

carboxylic acids, etc.) would allow for further refinement of the AOT organogel interaction

parameters. Lastly, some extractive purification applications should be attempted. For

instance, ethers are subject to slow build up of peroxides that may become explosive. The

removal and detoxification of such peroxides initially present in bulk solvents have been

reported.58,59 Organogels have been shown to be stable in ethers,60 and the possibility of

organogels absorbing and detoxifying organic peroxides should be studied.

53

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58

VITA

Melissa Ann Stouffer was born on April 24th 1972 in Hagerstown, Maryland. She

completed her undergraduate work at Lenoir-Rhyne College in Hickory, North Carolina, and

received a Bachelor of Science in Chemistry in 2000 while working at Alcatel N.A. as an

associate chemist. Melissa entered Wake Forest University in 2001 as a part time student.

After a reduction in workforce at Alcatel she was able to attend full time thanks to the

support of the TAA program. She is now employed with Performance Fibers in New Hill,

North Carolina as a quality control chemist.