micelle enhanced analytical chemistry by lin zhang …€¦ · cmc critical micelle concentration n...
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MICELLE ENHANCED ANALYTICAL CHEMISTRY
BY
LIN ZHANG
A Thesis Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES
in Partial Fulfillment of Requirements
for the Degree of
MASTER OF SCIENCE
Chemistry
May 2014
Winston-Salem, North Carolina
Approved By:
Willie L. Hinze, Ph.D., Advisor
Christa L. Colyer, Ph.D., Chair
Rebecca Alexander, Ph.D.
ii
ACKNOWLEDGEMENT
I wish to express my heartfelt thanks and appreciation to Dr. Willie Hinze for his patience,
continuous and prompt encouragement and helpful criticism during my study, research work and
thesis. I am sure that the completion of this work could not have been accomplished without his
support and that of other people. Also, I wish to thank Dr. Christa Colyer for exposing me to new
things, being willing to hear out my ideas, and offering useful critique during my work in her lab
and my stay here.
In addition, I would like to thank my committee member Dr. Rebecca Alexandra and all of
the other faculty members for their teaching, advising, and motivating me during my years at Wake
Forest. I would also like to thank the staff and my fellow graduate students in the Chemistry
Department for their friendship and encouragement during my stay.
I would like to acknowledge the support from the graduate school of Wake Forest through
a teaching assistantship.
Finally, I would give my special thanks to my husband, Yuyang and my parents for their
love, understanding, and support.
iii
TABLE OF CONTENTS
PAGE
TABLE OF CONTENTS ........................................................................................................... iii
LIST OF TABLES AND FIGURES ............................................................................................ v
ABBREVIATIONS .................................................................................................................. ix
ABSTRACT ............................................................................................................................... x
CHAPTER 1 Brief Introduction to Micelle Systems, Their Properties and Utilization in Chemical
Analysis and Separation Science Applications ........................................................ 1
1.1 Surfactant and Micelle Systems ......................................................................... 1
1.2 Properties of Aqueous (Normal) Micelle Systems.............................................. 5
1.3 Vesicles ......................................................................................................... 10
1.4 Applications in Chemical Analysis and Separation Science Systems ............... 11
1.5 References ....................................................................................................... 14
CHAPTER 2 Examination of the Factors that Influence Fluorescence Quenching for Luminescent
Probes by Copper (II) ion in the Presence of Surfactant Micelles .......................... 18
2.1 Introduction .................................................................................................. 18
2.2 Experimental ................................................................................................. 20
2.3 Results and Discussion .................................................................................. 24
2.4 Conclusions .................................................................................................. 38
2.5 References .................................................................................................... 40
CHAPTER 3 Determination of Some Pertinent Physical Parameters for Triton X-114 Micellar
Solutions and Their separated Systems ................................................................. 44
3.1 Introduction .................................................................................................. 44
iv
3.2 Experimental ................................................................................................. 46
3.3 Results and Discussion .................................................................................. 48
3.4 Conclusions .................................................................................................. 58
3.6 References .................................................................................................... 59
CHAPTER 4 Conclusions and Future Work .............................................................................. 62
SCHOLASTIC VITA .............................................................................................................. 64
v
LIST OF TABLES AND FIGURES
TABLES
CHAPTER 1 PAGE
1. Micelle parameters for representative surfactant micelle systems………………………..3
2. Variation of SDS micelle parameters (CMC, N) with temperature………………………4
3. Cloud point temperature of nonionic triton surfactants under different conditions………5
4. Solubility of aromatic solutes in water and different aqueous micellar media…………...6
5. Binding constant for the interaction of a few aromatic compounds and cations with
different charge-type micelles…………………………………………………………….8
CHAPTER 2
1. The fluorescence wavelengths and some other properties of the fluorescent probe
molecules………………………………………………………………………………...23
2. Summary of the measured Stern-Volmer constants for the quenching of two aromatic
fluorophores by copper (II) in different anionic surfactant media………………………27
3. Effect of NaTDS surfactant concentration on the quenching of NMA fluorescence by
copper (II) ions…………………………………………………………………………..29
4. Summary of observations in attempted determination of the Stern-Volmer Quenching
Constants for the PSF – Copper (II) – NaTDS System………………………………….30
5. Effect of temperature on the quenching of PSF fluorescence by copper (II) ions in SDS
micelles…………………………………………………………………………………..31
6. Effect of NMA probe concentration on the quenching efficiency in SDS micelles……..34
7. Effect of DHP vesicle concentration on the quenching of NMA fluorescence by copper (II)
ion………………………………………………………………………………………. .35
8. Influence of NMA fluorescent probe concentration upon standard deviation of blank signal
and limit of detection for determination of copper (II) ions in SDS micelles……………..36
vi
9. Summary of recovery data for determination of copper (II) ions based upon their
fluorescence quenching of NMA or Naphthalene in SDS micelles……………………….38
CHAPTER 3
1. Summary of the effect of incubation time and temperature upon the volume of the
surfactant-rich phase of TX-114 following phase separation……………………………51
2. Summary of the actual temperature observed following incubation of the aqueous
surfactant TX-114 solutions at the specified temperature in a constant temperature water
bath for the indicated incubation time immediately following the 2.00 minute
centrifugation step………………………………………………………………………. 53
3. The cloud point temperature of the Triton X-114 solutions at the four indicated TX-114
concentrations (in percent mass)…………………………………………………………55
4. Summary of the volume ratio obtained for aqueous solutions of TX-114 upon surfactant
concentration and centrifugation time The CPT as function of surfactant concentration
TX-114…………………………………………………………………………………...57
vii
FIGURES
CHAPTER 1
1. Schematic cartoon illustration of micelle formation……………………………………...2
2. Artistic representation of an idealized cross section of an aqueous normal micelle……....7
3. Artistic representation of a dynamic equilibrium process of solutes association (binding) to
the micellar aggregate……………………………………………………………………..8
4. Effect of sodium dodecyl sulfate on the equilibrium constant of formation of
benzylideneaniline………………………………………………………………………...9
5. The Schematic cartoon illustration of DHP vesicle………………………………………11
6. Fluorescence of pyrene as a function of its concentration in different homogeneous solvent
and micellar systems……………………………………………………………………..12
CHAPTER 2
1. Stern-Volmer plot of (𝐼0
𝐼− 1) vs. Cu2+ concentration in the prescence of 0.010 M SDS
micelle and in bulk water alone…………………………………………………………..25
2. The dependence of the KSV due to quenching of PSF fluorescence by copper (II) ion upon
the [NaTDS] at 27oC……………………………………………………………………..28
3. Fluorescence emission spectra of 1.00 10-6 M solutions of NMA in the presecnce of 0.010
M SDS at the following copper (II) ion concentrations: 0.00 M (blue line), 0.099 mM (red
line), 0.198 mM (grey line) and 0.495 mM (yellow line)………………………………..32
4. Temperature dependence of Critical Micelle Concentration for the Surfactant SDS…….33
CHAPTER 3
1. Schematic representation of the phase separation behavior exhibited by an aqueous solution
of a nonionic surfactant micellar system upon temperature alteration……………………45
2. Experimentally determined phase diagram for aqueous solutions of Triton X-114………49
viii
3. Plot of surfactant-rich phase volume as a function of the total TX-114 surfactant
concentration……………………………………………………………………………..50
4. Dependence of the volume ratio in the phase separated solutions of the surfactant TX-114
present at the indicated concentrations as a function of the incubation temperature……..55
5. Effect of ionic strength at three temperatures at a fixed TX-114 concentration of 7.20 wt. %
upon the volume ratio following incubation for 18 hours………………………………..56
ix
ABBREVIATIONS
SDS Sodium Dodecylsulfate
NaDS Sodium Decylsulfate
NaTDS Sodium Tetradecylsulfate
CTAB Cetyltrimethylammonium bromide
CTAC Cetyltrimethylammonium Chloride
TX-114
TX-100
Triton X-114
Triton X-100
CMC Critical Micelle Concentration
N Aggregation Number
CPT Cloud Point Temperature
NMA N-methylacridone
PSF Phenosafranine
Ksv Stern-Volmer Constant
LOD Detection Limit
DHP
NaC
SB-12
Brij-35
MLC
HPLC
CE
Dihexydecylphosphate
Sodium Cholate
Sulfobetaine 12
Polyethylene (23) glycol octadecyl ether
Micelle Liquid Chromatography
High Performance Liquid Chromatography
Capillary Electrophoresis
x
ABSTRACT
The ability of surfactant micellar aggregates to enhance collisional quenching between a
model cation analyte, copper (II) ion, and two fluorescent probes, phenosafranin and N-
methylacridone (NMA), was studied. The intensity of the fluorescence emission observed from
these two probe molecules was monitored as a function of added copper (II) ion quencher and the
data evaluated in terms of the Stern-Volmer equation in three anionic surfactant micellar systems
(sodium decylsulfate (NaDS), sodium dodecylsulfate (SDS) and sodium tetradecylsulfate
(NaTDS)). The presence of these anionic micellar systems enhanced the Stern-Volmer quenching
constant, Ksv, relative to that observed in water alone (by a factor of ca. 80 for SDS as the micelle
and NMA as fluorescent probe). The SDS micelle system appeared to be best in terms of the
enhancement factor observed and the reproducibility of the results relative to NaDS or NaTDS.
Temperature was found to have only a moderate effect on the KSV values. While the effect of the
luminescent probe concentration upon the KSV value was negligible, lower signal/noise levels and
hence lower limits of detection were attained as the concentration of the probe molecule was
increased. A detection limit of 9.3 10-6 M Cu2+ was obtained for the system in which NMA was
the probe molecule in the presence of 10.0 mM SDS surfactant molecules. A major limitation of
this approach is that many other cations will interfere since they also quench these probe molecules.
For real world samples, the use of micelle enhanced fluorescence quenching as a method for
detection of cations will be limited to that for indirect detection following HPLC or CE separation
of those analytes.
Additionally, the phase separation properties of the nonionic surfactant Triton X-114 were
examined. This surfactant has been extensively employed in surfactant mediated extractive (cloud
point extraction, CPE) schemes for a variety of analytes. Upon temperature alteration, solutions of
nonionic surfactants like Triton X-114 exhibit phase separation behavior giving rise to a small
volume surfactant-rich (coaceravate) phase and a larger volume surfactant-lean phase. This forms
xi
the basis for the extraction of hydrophobic analytes that bind TX-114 as they become concentrated
in the surfactant-rich phase. However, larger biomolecules are excluded from this small volume
surfactant-rich phase and can be extracted into the surfactant-learn phase. This work determine the
conditions (surfactant concentration, temperature, equilibration time, gravity versus centrifugation
for phase separation) necessary to obtain a phase separated system such that the surfactant-lean
phase occupies a smaller volume relative to the surfactant-rich phase so that such large
biomolecules can be concentrated in that phase. Conditions were identified (lower temperature and
greater surfactant concentrations) under which a concentration (enrichment) factor of 10 could be
achieved for such CPE based upon the excluded volume effect.
1
Chapter 1- Brief Introduction to Micelle Systems, Their Properties and Utilization in
Chemical Analysis and Separation Science Applications
1.1 Surfactant and Micellar Systems
Micelles are self-assembling aggregates composed of surfactant molecules. Typically, a
surfactant molecule consists of two distinct chemical moieties: the hydrophilic head group, which
is a water-loving moiety (such as a sulfate, quaternary ammonium or hydroxyl group) and a
hydrophobic water-fearing moiety (typically C-8 to C-18 alkyl groups). Surfactant and micellar
systems can be classified as either ionic (i.e. anionic or cationic) or neutral (zwitterionic or nonionic)
depending on the nature of the hydrophilic moiety that is bound to the hydrophobic backbone.
Anionic surfactants contain a negatively charged moiety as their polar head group. Some typical
types include sodium alkylsulfates (e.g., sodium dodecyl sulfate (SDS), sodium decyl sulfate
(NaDS) and sodium tetradecyl sulfate (NaTDS)) or sodium alkylcarboxylates. Cationic surfactants
have a positively charged head group with common types including alkyl pyridinium halides and
alkyl ammonium halides, such as hexadecyl (or cetyl) trimethylammonium bromide (CTAB) or
cetyltrimethylammnoium chloride (CTAC). Non-ionic surfactants have a polar, but uncharged,
head group and include such surfactants as polyoxyethylene glycol octylphenol ethers or
polyoxyethylene glycol tert-octylphenol ethers, which differ in the number of their ethylene oxide
repeat units. Popular examples are the Triton X series of surfactants such as Triton X-100
(polyoxyethylene (9.5) octyl phenyl ether) and Triton X-114 (polyethylene (7.5) tert-octylphenyl
ether). Zwitterionic (amphoteric) surfactants contain both a cationic and an anionic polar
headgroup. Some typical zwitterionic surfactants include alkyl ammonium ethyl sulfates,
phosphobetaines, lecithins or sulfobetaines (such as N-dodecyl-N, N-dimethyll-3-ammonio-1-
propanesulfonate, SB-12), etc.
When surfactant molecules are dissolved in water, they are initially in monomeric form
(individual surfactant molecules in the aqueous solution). However, when the added surfactant
2
concentration achieves a critical value (termed the critical micelle concentration, CMC), they
spontaneously form aggregated colloidal assemblies termed micelles (aqueous or normal micelles,
Figure 1). In the micelle, the surfactant hydrophilic “head” group is on the exterior surface thus
maximizing contact with the surrounding water molecules while the hydrophobic alkyl tails all
flock together in the interior region to minimize the unfavorable contact with water and maximize
hydrophobic interaction between their alkyl tails.
Figure 1. Schematic cartoon illustration of micelle formation.1 The blue circles represent the
surfactant heads (hydrophilic moieties) and the curved lines represent the surfactant tails
(hydrophobic moieties).
As shown in Figure 1, the micelle aggregate is a dynamic entity. That is, there is an
equilibrium situation with surfactant monomers exchanging with surfactant molecules in the
micelle aggregate. Unlike polymers which are formed by covalent bonding of individual
monomeric repeat units, micelles are labile entities formed by noncovalent interactions between
the individual surfactant molecules. Two key micellar parameters are the CMC and the aggregation
number (N), which is the number of surfactant molecules that comprise the micelle aggregate (in
the micelle cartoon shown in Figure 1, the aggregation number is 9). The Krafft temperature (also
known as Krafft point) is the minimum temperature at which surfactants form micelles. Below the
3
Krafft temperature, there is no value for the critical micelle concentration (CMC), i.e., micelles
cannot form.2 The CMC, N and Krafft point values for some common surfactant systems in pure
water as solvent at 25oC are summarized in Table 1. The specific numeral values of the CMC and
N are surfactant dependent as well as dependent upon the
Table 1. Micelle Parameters for Representative Surfactant Systems
Charge-Type Name
CMC, mM
(25oC) N
Krafft Temperature
(oC)
Reference
Nonionic Triton X-100 0.3 0.6a
43-66, 100-140 64.3 3,5,6,8
Nonionic Triton X-114 0.35 -------- 28 3,5,6
Cationic CTAB
0.92
0.31 b 61 25 7-9
Cationic CTAC 1.3
0.28c
78 23-25 7,8,10
Anionic NaDS 30-32 38-64 8 4-6
Anionic SDS 8.0-8.4 1.1, 15 d
62-92 10-15 4-6,8
Anionic NaTDS 1.8-2.0 120-138 21-30 4-6
Zwitterionic Sulfobetaine
SB-12 3.3
69-71 < 0 5,6
Zwitterionic
Sulfobetaine
SB-14 0.3
83 --------- 5-7
a. The CMC of Triton X-100 changed from 0.3 mM to 0.6 mM in the presence of 3.0 M urea. b. The CMC of CTAB decreased from 0.92 mM in water to 0.31 mM in the presence of 1.0
M sulfuric acid. c. The CMC of CTAC decreased from 1.3 mM in water to 0.28 mM in the presence of 0.010
M NaOH. d. The CMC of SDS decreased from 8.1 mM in water to 1.1 mM in the presentence of 0.125
M NaCl whereas that increased to 15 mM in the presence of 6.0 M urea.
4
solution conditions (pH, ionic strength, presence of other molecules/species) and temperature (see
Table 2).
Table 2. Variation of SDS Micelle Parameters (CMC, N) with Temperature.a
Temperature (oC) CMC (mM) N
20 7.94 0.2 69
25 8.05 0.2 64
30 8.50 0.2 54
35 8.97 0.2 47
40 9.57 0.2 40
a. Data taken from Reference 10.
Unlike ionic surfactant solutions, nonionic and zwitterionic surfactant micelle solutions
exhibit phase separation behavior upon temperature alteration. For example, upon heating, aqueous
solutions of many nonionic surfactants become turbid at a temperature known as the cloud point
temperature (CPT), above which there is a separation of the solution into two phases. One phase
is referred to as the “bulk aqueous phase” (really aqueous surfactant-poor phase) while the other is
the small volume surfactant-rich phase. This physical separation of the phases is due to differences
in their density. Such clouding phenomenon has been exploited in separation science for the
development of extraction, purification and preconcentration schemes for a variety of analytes.5
The phase separation process is reversible and, upon cooling to a temperature below the cloud
point, the two phases merge to form an isotropic, homogeneous solution.11-13 The cloud point
temperatures of several nonionic surfactants in the absence and presence of additives are
summarized in Table 3.
5
Table 3. Cloud Point Temperature of Nonionic Triton Surfactants under Different Conditions
Surfactant
Additive CPT (oC) Ref.
Triton X-100
None 63.7
5, 6
0.30 M Urea 65.7
0.50 M Urea 68.1
0.30 M 1,1-Diethylurea 78.4
0.50 M 1,1-Diethylurea 87.2
2% Sodium Azide (pH 7) 61
0.5 M Sodium Chloride (pH 7) 56
1.0 M Sodium Chloride (pH 7) 47
0.29 mM NPE8 + 48 mM KCl 40
Triton X-114
None 28 3
1 M NaCl 14
1 M NaI 41
1.2 Properties of Aqueous (Normal) Micelle Systems
The unique properties of micelle systems have been exploited in a number of chemical
analysis applications.15-17 A key factor in all successful applications of micelles lies in the ability
of micelles to solubilize and bind various solutes. Once a micelle aggregate is formed in water, it
can serve to enhance the solubility of sparingly water soluble organic solutes. Some representative
solubility data for several aromatic hydrocarbons is summarized in Table 4 along with their
solubility in water alone. The presence of micelle aggregates in water can enhance the aqueous
solubility of some solutes by several orders of magnitude depending upon the specific surfactant
6
employed and its concentration. Typically, the increase in solute solubility (at surfactant
concentrations above the CMC) is directly proportional to the total surfactant concentration.18-20
Table 4. Solubility of Aromatic Solutes in Water and Different Aqueous Micellar Media.
Analyte molecule Solubility in indicated medium Reference
N-methylacridone
3.45 10-5 M (water)
19
3.2 10-4 M (0.01 M CTAC micelles)
2.4 10-3 M (0.10 M CTAC micelles)
1.0 10-3 M (0.40 M CTAC micelles)
1.1 10-4 M (0.01 M SB-12 micelles)
7.8 10-4 M (0.10 M SB-12 micelles)
1.7 10-3 M (0.20 M SB-12 micelles)
Naphthalene
(2.2-2.6) x 10-4 M (water)
19, 22 1.3 x 10-2 M (0.20 M NaC, pH 8-9)
0.38 M (0.04 M SDS)
1.11 M (0.02 M CTAB)
Pyrene
4.0-8.0 10-7 M (water)
18, 19, 23
7.1 10-2 M (hexane)
7.0 10-2 M (0.06 M SDS)
4.1 10-1 M (0.04 M CTAB)
9.8 10-5 M (3 mM CPC)a
2.8 10-4 M (10 mM CPC)a
2.2 10-3 M (0.5 M potassium dodecanoate) a. CPC = cetylpyridinium chloride
Depending upon the nature of the solute (ionic, polar, hydrophobic), the micelle aggregate
offers several potential binding sites as illustrated in Figure 2. An ionic species could be
electrostatically repelled from the micelle surface if the surfactant is of the same charge as that of
the ion (A) or attracted if of the opposite charge (B in the cartoon). Nonpolar solutes (C) can
interact with the alkyl tails of the particular surfactant of the micelle in the core region. A polar
molecule (D) could be located so that its nonpolar moiety is aligned between the surfactant
molecule’s alkyl tails while the solute’s polar moiety is oriented towards the micelle interface
and/or in contact with water.7
7
Figure 2. Artistic representation of an idealized cross section of an aqueous normal micelle. Note:
The polar headgroups and hydrophobic tails are schematically indicated to show only their relative
locations (the counterions, which would be associated with an ionic micelle, are not shown). The
dotted line encompasses most the hydrophobic core region of the micelle. (Adapted with
permission from Elsevier Publisher).8
Solute association (binding) to the micellar aggregate is a dynamic equilibrium process with
the solute molecule exchanging between the micelle and the bulk aqueous phase (see Figure 3).
The strength of solute binding (typically expressed as the solute-micelle binding constant, Kb)
depends upon the nature (charge type and hydrophobicity of the surfactant comprising the micelle
aggregate) in relation to that of the solute since these factors determine the net hydrophobic and/or
electrostatic interactions possible for a particular solute - micelle combination.21 Binding
constants for the interaction of a few aromatic compounds and inorganic cations with different
types of micelles are given in Table 5. The difference in Kb values for the same solute micelle
system (for instance, Kb ranging 350 to 2,500 M-1 for interaction of naphthalene with SDS micelles)
has been attributed to the different analytical methods employed for measurement of the binding
8
constant.
Figure 3. Artistic representation of the dynamic equilibrium process of solute association (binding)
to the micellar aggregate.
Table 5. Binding Constant for the Interaction of Aromatic Compounds and Cations with Different
Charge-Type Micelles
Analyte Micelle System Binding Constant, Kb Reference
Naphthalene
SDSa 350-400; 2500 M-1
19, 24 CTABb 1500-9100 M-1
NaCc 2000-4800 M-1
Pyrene
CTAB 1.02 107 M-1
19, 21,
23-26
SDS 1.7 105-2 106 M-1
n-beta-Octyl glucoside 1.5 104 M-1
NaC (1.5-4.7) 105 M-1
N-Methylacridone
CTACd 3.3 104 M-1
19 SB-12e 1.6 104 M-1
Brij-35f 1.0 104 M-1
Copper (II) ion SDS 1400 – 10,000 M-1 27, 28
Terbium (III) ion SDS 500 M-1 29 a. Sodium dodecyl sulfate b. Hexadecyl trimethylammonium bromide c. Sodium Cholate d. Cetyltrimethylammnoium chloride e. N-Dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate or Sulfobetaine 12 f. Polyethylene (23) glycol octadecyl ether
Binding to micelles alters the effective microenvironment (microscopic polarity,
fluidity/viscosity) that the solute experiences. For example, solutes C and D (Figure 2) would
experience a less polar, more hydrophobic microenvironment when bound to a micellar aggregate
9
relative to what they experience when dissolved in bulk water alone. This in turn can influence
that solute’s spectral properties (such as wavelength of maximum absorption, luminescence
emission wavelength, luminescence quantum yield, etc.).7, 8, 18 Bound solutes are also less mobile
(less fluid) when bound to the micellar entity and experience a more viscous local microscopic
environment, which can also impact that solute’s spectral properties, among of others.
Additionally, micelles can serve as a reaction medium in which the rate of chemical
reactions can be altered relative those that observed in bulk water alone.30 That is, the apparent rate
of reaction can be catalyzed or inhibited. For instance, in the equilibrium reaction for the
preparation of benzylideneaniline (equation 1),
the presence of SDS micelles shift the reaction more towards the product side (Figure 4).
Figure 4. Effect of sodium dodecyl sulfate on the equilibrium constant of formation of
benzylideneaniline. Experimental conditions: 20oC, 5 vol% of methanol, 0.04 M borate buffer;
concentrations of reagents: aniline from 2.0 10-3 to 1.6 10-2 M, benzaldehyde from 1.0 10-3 to
5.4 10-3 M, benzylideneaniline 1 10 -4 to 2 10-4 M. (Adapted with permission from Elsevier
Publisher).30
The apparent equilibrium constant Kapp was increased from 6 M-1 (without detergent) to around 60
M-1 in the presence of SDS micelles.30
10
In addition to these useful properties, aqueous micellar solutions are generally considered
to be environmentally safe. The surfactants employed are generally nontoxic and their solutions
are nonvolatile and nonflammable relative to organic solvents. Since only small amounts of
surfactant are required for form micelles, the micellar solutions are predominantly aqueous and the
cost per volume solution is low.
1.3 Vesicles
In addition to formation of micelles, some synthetic surfactants can also from vesicles.
Namely, aqueous solutions of surfactant molecules that contain two long alkyl tails, when sonicated
above their phase transition temperature, can form bilayer type structures termed vesicles.31, 32 The
vesicle structure33 (represented by the cartoon structure, Fig.5) consists of a bilayer, whose outer
and inner surfaces are hydrophilic while the central bilayer itself is hydrophobic. The vesicle
aggregate also contains an inner water core region while its outer surface polar headgroups are in
contact with the bulk solvent water molecules. Compared to micelle aggregates, surfactant vesicle
aggregates are less fluid, more static entities whose individual surfactant molecules do not
exchange with surfactant monomers in the bulk water. The vesicle aggregates can have the same
four charge-types as described for surfactant micelles. Anionic surfactant vesicles, such as those
formed from dihexyldecylphosphate (DHP), have the same general properties as just outlined for
surfactant micelles. Solutions of surfactant vesicles take more time to prepare compared to micelles
since one must sonicate their solutions for a certain period of time above their phase transition
temperature. Thus, their use is somewhat less convenient compared to that of micelles.
11
Figure 5. The schematic cartoon illustration of DHP vesicle33
1.4 Applications in Chemical Analysis and Separation Science Systems
As a consequence of the general properties of aqueous based surfactant micelle and vesicle
systems just summarized, they have found widespread utilization in chemical analysis and
separation science applications. These include just their use to enhance the aqueous solubility of
target analytes or required analytical reagents in water (solubility enhancement agents) thereby
eliminating the need for organic co-solvents for solubility purposes. In addition, the presence of
micelles can aid sample preservation. For instance, aromatic compounds such as pyrene can adsorb
to the surfaces of its container thus decreasing its solution concentration. The addition of surfactant
micelles in the solution can greatly minimize this problem.8
As noted above, micellar bound solutes experience an altered microenvironment. This in turn
can impact their spectral parameters. For instance, water typically diminishes the fluorescence of
aromatic molecules. However, when bound to micelles, the solute’s exposure to water is
diminished, which can result in increased fluorescence emission.34 In addition, when bound, the
solute will be in a “more rigid” less fluid microenvironment, which can also improve fluorescence
efficiency. Figure 6 illustrates the use of different micelles to enhance the fluorimetric
determination of pyrene.34
12
Figure 6. Fluorescence of pyrene as a function of its concentration in different homogeneous
solvent and micellar systems [λex = 241 nm, λem = 390 nm. a: water; b: ethanol; c: SDS 3.5 10-2
M; d: HTAC 6.25 10-3 M; e: Triton X100 4 10-3 M]. (Adapted with permission from Taylor &
Francis Online.)34
Depending upon the specific micelle employed; the analytical sensitivity was improved by factors
of 3 to 16. Numerous other examples of such use of micelles as luminescence enhancement agents
are reported in the analytical literature.8, 34
Different solutes bind to a particular micelle to different degrees depending upon their
polarity (Table 5). Such differential binding of solutes to micelles provided the basis for
development of the technique of micelle liquid chromatography (MLC) in which the traditional
reversed phase mobile phases (methanol-water or acetonitrile-water) were simply replaced by
aqueous micelle solutions.35, 36 Solute retention can be manipulated by merely adjusting the
surfactant concentration: increasing the surfactant concentration leads to formation of more
micelles in solution, which is akin to increasing the methanol or acetonitrile concentration in
conventional reversed phase HPLC. The use of micellar mobile phases in HPLC and TLC is now
widely employed and considered routine. The differential binding of solutes to micelles (or
13
vesicles) also led to development of the technique known as electrokinetic capillary
chromatography or micelle (or vesicle) enhanced capillary chromatography,37-39 which allowed for
the CE separation of neutral analytes.
In addition to the application of micelle solutions in spectroscopic and chromatographic
methods, detailed investigations on the electrochemical processes occurring in the presence of
surfactant micelles as well as on their application in electroanalytical chemistry have been reported
in the last thirty years as well.18, 40-42 Important advantages are related to the use of surfactant
aggregates, which include the stabilization of unstable chemical species (e.g. ion-radicals)
produced at electrodes by coulombic and hydrophobic interactions with micelles, the alteration of
the product distribution arising from electrodes processes due to partitioning of electro active
reagents and/or products and favorable catalytic effects due to the increase in the electron transfer
rate (usually resulting in a sensitivity increase).41
In my thesis work, the general properties of micelles were exploited for analytical purposes.
Namely, in Chapter 2, the use of micelles to enhance the detection limits possible for the
determination of copper (II) ions via fluorescence quenching was evaluated and the different
experimental conditions that influence quenching examined. In Chapter 3, some basic properties
of the phase behavior of Triton X-114 micelle solutions were determined with the aim of providing
data that would be useful for its use in surfactant mediated (also termed cloud point) extraction,
techniques that are based upon the excluded volume effect.
14
CHAPTER 1 REFERENCES
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Chapman and Hall Publishers: New York, 1983.
2. JR., Robert. Manual of Symbols and Terminology for Physicochemical Quantities and
Units, Appendix II: Definitions, Terminology and Symbols in Colloid and Surface
Chemistry. Pure & Appl. Chem. 1972, 31, 577-613.
3. Koshy, L.; Saiyad, A. H.; Rakshit, A. K. The effects of various foreign substances on the
cloud point of Triton X 100 and Triton X-114. Colloid Polym. Sci., 1996, 274, 582-587.
4. Balouch, A.; Zhang, L.; Bhanger, M.I.; Thimmaiah, K.N.; Hinze, W. L. Factors
Affecting the Use of Organized Surfactant Assemblies for Amplification of Fluorescence
Quenching with Application to the Quenchiofluorimetric Determination of Copper (II)
Ion. This manuscript will be shortly submitted for publication in Talanta.
5. Hinze, W. L.; Pramauro, E. A critical review of surfactant-mediated phase separations
(cloud-point extractions): theory and applications. Critical Reviews in Analytical
Chemistry, 1993, 24, 133-177.
6. Ahuja, E. S.; Preston, B. P.; Foley, J. P. Anionic-zwitterionic mixed micelles in micellar
electrokinetic chromatography: sodium dodecyl sulfate-N-dodecyl-N,N-
dimethylammonium-3-propane-l-sulfonic acid. Journal of Chromatography B, 1994,
657, 271-284.
7. Fluorescence in Organized Assemblies: Electronic Absorption and Luminescence.
Encyclopedia of Analytical Chemistry, ed.; John Wiley & Sons, Ltd.: 2008, 10364-10447
8. Hinze, W.L; Singh, H. N.; Baba, Y.; Harvey, N. G. Micellar enhanced analytical
fluorimetry. Trends in analytical chemistry, 1984, 3, 193-199.
9. Vautier-Giongo, G.; Bales, B. L. Estimate of the Ionization Degree of Ionic Micelles
Based on Krafft Temperature Measurements. J. Phys. Chem. B, 2003, 107, 5398-5403.
15
10. Chen, Z; Greaves, T. L.; Fong, C.; Caruso, R. A. Lyotropic liquid crystalline phase
behavior in amphiphile–protic ionic liquid systems. Phys. Chem. Chem. Phys., 2012, 14,
3825–3836.
11. Shah, S. S.; Jamroz, N. U. Colloids and surfaces: Micellization parameters and
electrostatic interactions in micellar solution of sodium dodecyl sulfate at different
temperature. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2001,
178, 199-206.
12. Myers, D. Surfaces, Interfaces, and Colloids: Principles and Applications; VCH: New
York, 1991.
13. Attwood, D. Surfactant System: Their Chemistry, Pharmacy and Biology; Chapman and
Hall: New York, 1983.
14. Rosen, M. J. Surfactants and Interfacial Phenomena; Wiley-Interscience: New York,
1978.
15. Hinze, W. L. Bile Acid/Salt Surfactant Systems; JAI Press: Stamford, CT, 2002.
16. Hinze, W. L.; Srinivasan, N., Smith, T. K.; Igarashi, S. Organized Assemblies in
Analytical Chemiluminescence Spectroscopy: An Overview, In Advances in
Multidimensional Luminescence; McGown, L. B., Ed.; JAI Press: Greenwich, CT, 1990.
17. Pramauro, E.; Pelizzetti, E. Analytical Applications of Organized Molecular Assemblies,
Anal. Chim. Acta, 1985, 169, 1-29.
18. Attwood, D.; Florence, A. T. Surfactant Systems: Their Chemistry, Pharmacy and
Biology; Chapman & Hall: New York, 1983.
19. Hinze, W. L.; Reihl, T. E.; Singh, H. N. Micelle-enhanced chemiluminescence and
application to the determination of biological reductants using lucigenin. Analytical
Chemistry, 1984, 56, 2180-2191.
20. Almgren, M.; Grieser, F.; Tomas, J. K. Dynamic and static aspects of solubilization of
neural arenes in ionic micellar solutions. J. Am. Chem. Soc., 1979, 101, 279-291.
16
21. Fendler, J. H. Membrane Mimetic Chemistry; Wiley: New York, 1982.
22. Akimova, E. I.; Snegov, M. I. Photoxidation of Aminocoumarines in Aqueous Micellar
Solutions. Opt. Spectros. (USSR), 1987, 63, 41-42.
23. Mukerjee, P.; Cardinal, J. R. Solubilization as a Method for Studying Self-Association
Solubility of Naphthalene in Bile-Salt Sodium Cholate and Complex Pattern of its
Aggregation. J. Pharm. Sci., 1976, 65, 882-885.
24. Nelson, G.; Patonay, G.; Warner, I. M. Effects of Selected Alcohols on Cyclodextrin
Inclusion Complexes of Pyrene Using Fluorescence Lifetime Measurements. Anal.
Chem., 1988, 60, 274-279.
25. Quina, F. H.; Alonso, E. O.; Farah, J. P. S. Incorporation of Nonionic Solutes into
Aqueous Micelles: A Linear Solvation Free Energy Relationship Analysis. J. Phys.
Chem., 1995, 99, 11708-111714.
26. Sugioka, H.; Moroi, Y. Micelle Formation of Sodium Cholate and Solubilization into
Micelle. Biochim. Biophys. Acta, 1998, 1394, 9-110.
27. Almgren, M. Migration and Partitioning of Pyrene and Perylene between Lipid Vesicles
in Aqueous Solution Studied with a Fluorescence Stopped-Flow Technique. J. Am.
Chem. Soc., 1980, 102, 7882-7887.
28. Itoh, H.; Ishido, S. Nomura, M.; Hayakawa, T.; Mitaku, H. S.: Estimation of the
Hydrophobicity in Microenvironments by Pyrene Fluorescence Measurements: N--
Octyl-glucoside Micelles. J. Phys. Chem., 1996, 100, 9047-9053.
29. Ziemiecki, H.; Cherry, W. R. Association constants and reaction dynamics of metal ions
bound to anionic micelles. J. Am. Chem. Soc., 1981, 103, 4479-4483.
30. Martinek, K., Yatsimirski, A. K., Osipov, A. Micellar Effects on Kinetics and
Equilibrium of Synthesis and Hydrolysis of Benzylideneaniline. Tetrahedron, 1973, 29,
963-969.
17
31. Eendler, J. H. Membrane Mimetic Chemistry; John Wiley: NY, 1982, Chapter 6, pp. 158-
181.
32. Camona-Ribeiro, AM; Hix, S. pH Effects on properties of dihexyldecyl Phosphate
Vesicles, J. Phys. Chem., 1991, 95, 1812-1817.
33. http://www.chemguide.co.uk/CIE/section113/learningb.html
34. Singh, H. N.; Hinze, W. L. Micellar Enhanced Spectrofluorimetric Methods: Application
to the Determination of Pyrene. Anal. Lett., 1982, 15, 221-243.
35. Hinze, W. L.; Armstrong, D. Ordered Media in Chemical Separations, 342th ACS
Symposium; American Chemical Society: Washington, DC, 1987, Chapter 1, 3-6.
36. Borgerding, M.; Williams, R.; Hinze, W. L.; Quina, F. New Perspective in Micellar
Liquid Chromatography. J. Liq. Chromatogr., 1989, 12, 1367-1406.
37. Terabe, S.; Otsuka, K.; Tsuchiya, A.; Ando, T. Electrodeposition of actinides in a mixed
oxalate-chloride electrolyte Anal. Chem., 1984, 56, 113-116.
38. Terabe, S.; Otsuka, K.; Ando, T. Electrokinetic Separations with Micellar Solutions and
Open-tubular Capillaries. Anal. Chem., 1985, 57, 834-841.
39. Bui, HH; Khaledi, MG. Determination of Vesicle-Water Partition Coefficients by
Electrokinetic Chromatography. J. Colloid Interface Sci., 2002, 397-401
40. Hinze, W. L. Solution Chemistry of Surfactant; Plenum Press: New York, 1979.
41. Mcintire, G. L. Micelles in analytical chemistry. Crit. Rev. Anal. Chem., 1990, 21, 257-
278.
42. Rusling, J. F. Electroanalytical Chemistry; Dekker: New York, 1994.
18
Chapter 2- Examination of Some Factors that Influence Fluorescence Quenching of
Luminescent Probes by Copper (II) ion in the Presence of Surfactant Micelles
2.1 Introduction
Fluorescence methods are very popular owing to their inherent sensitivity relative to
ultraviolet – visible absorption methods.1, 2, 3 Fluorimetric methods are, however, restricted to
molecules that contain either an aromatic moiety or an extensive conjugated pi electron system.
Nonfluoresent analytes can be chemically converted to a fluorescent species via appropriate
chemical derivatization.4-7 Alternatively, some non-fluorescent analytes can be indirectly
determined by measuring their ability to quench the fluorescence of a suitable fluorescent molecule.
Such an approach is referred to as a fluorescence quenching method.8 Quenching methods are
rapid, simple and convenient due to the fact that no chemical derivatization (or complexation)
reactions are necessary.
The basis of such dynamic quenching method arises from the interaction of a fluorescent
probe molecule in its excited state (FP*) with the quencher analyte species with resultant
deactivation of the probe molecule’s excited state without the emission of a photon as shown in
equation 19:
FP* + A → A + FP (eq. 1)
The quenching process is typically represented by the Stern-Volmer equation8:
[Io / I] – 1 = kq τo [A] (eq. 2)
where Io represents the fluorescence intensity of the fluorescent probe in the absence of any
quencher, I represents its fluorescence intensity at a particular concentration of quencher species
([A]), kq represents the bimolecular quenching rate constant and τo represents the singlet excited
state lifetime of the fluorescent species in the absence of quencher. This equation is often rewritten
as:
19
[Io / I] – 1 = Ksv [A] or [Io / I] = Ksv [A] + 1 (eq. 3)
where Ksv is the Stern-Volmer quenching constant and equals to the product of (kq τo). A plot
of the left-hand term of eq. 3 vs. quencher concentration [A] serves as the calibration plot for such
fluorescence quenching methods.
Fluorescence quenching methods were first reported in the 1960’s and this approach has
found application for the analysis of inorganic, organic and biochemical species,10-14 and as a means
of detection following capillary electrophoretic or chromatographic separations.15 However,
fluorescence quenching methods based on collisional (dynamic) quenching are limited due to their
lack of sensitivity (detection limit in the mM range) and selectivity.16 Additionally, many of the
potentially useful aromatic probe molecules (fluorophores) exhibit limited water solubility, which
means that mixed organic-aqueous solvent systems must be employed.
Fluorescence quenching of micelle-solubilized fluorescent probes by a variety of
quenchers has been reported in the micelle literature for the determination of dynamic behavior and
characteristic parameters of aqueous micelles as well as of other surfactant aggregates.17, 18 Steady-
state and time-resolved fluorescence quenching studies have been employed in order to provide
information on such micelle properties as the mean aggregation number (N), critical micelle
concentration (CMC) and fluidity as well as the location and partition coefficients of fluorophores
and quenchers bound to the surfactant micelle aggregate.19-21 The quenching of various aromatic
fluorophores by copper (II) ions in aqueous solutions both in the absence and presence of organized
surfactant assemblies has been the subject of numerous reports in the surfactant and micelle
literature.17, 22 The results of such studies clearly indicate that the quenching of fluorescent probe
molecules by quencher species is often much greater in surfactant micelles relative to that observed
in bulk solution.
20
More recently, a number of publications have concerned micelle amplified fluorescence
quenching “sensors” for the determination of a number of analyte species, including copper (II).17,
23-25 Almost all of this published work focused only on determination of the magnitude of the
fluorescence quenching enhancement possible in sodium dodecylsulfate micelles (relative to that
in water alone) for a host of different fluorescent probe molecules. However, the effect of different
experimental parameters (such as surfactant hydrophobicity, surfactant concentration, temperature)
and the analytical parameters (such as limit of detection and recovery data) were not reported. The
determination of a number of transition metal cations by fluorescence quenching of naphthalene in
an anionic micelle medium has been recently reported.22
In this chapter, some of the different factors (surfactant hydrophobicity and concentration,
temperature) that can influence the degree of quenching observed from several fluorescent probe
molecules due to cupric ion in the presence of appropriate types of surfactant micelle are assessed.
In addition, some analytical characteristics (detection limit and recovery data) for the
quenchofluorimetric method for determination of copper (II) ion are reported. Lastly, the use of
micelle media will be demonstrated to modestly address the previously noted general limitations
(sensitivity, solubility of probe molecule) of fluorescence quenching methods.
2.2 Experimental
2.2.1 Instrumentation
Fluorescence spectra and intensity measurements were made using an Aminco-Bowman model J4-
8960 Spectrophotoflurometer (American Instrument Co., Silver Spring, MD). In addition, a Perkin-
Elmer model LS-55 or LS-50B Luminescence Spectrometer (Perkin-Elmer Corporation, Waltham,
MA) was employed for some measurements. Quartz cuvettes (pathlength 1.00 cm) were used for
all fluorescence measurements. Unless otherwise noted, the excitation, emission and detector slit
widths were set at 1.0 mm. Appropriate blank solutions (which contained all components except
21
for the copper (II) quencher) were employed in order to correct for any fluorescence background.
A Fisher Model 300 sonicator (probe tip type) (Fisher Scientific Co., Raleigh, NC) was used to
sonicate appropriate surfactant solutions in the preparation of synthetic vesicles.
2.2.2 Materials
Phenosafranin, N-methylacridone, tetradecylsulfate sodium salt (95%), decyl sodium
sulfate and dihexydecylphosphate (DHP) were obtained from Sigma Aldrich Chemical Co.
(Milwaukee, WI); copper (II) chloride or nitrate from Fischer Scientific Co. (Raleigh, NC); sodium
dodecylsulfate was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN); and
absolute ethanol (AAPER Alcohol & Chemical Company) was utilized as received without any
further purification. All glassware (volumetric flasks, pipettes, etc.) employed in the preparation of
solutions was initially cleaned by soaking in an acidified (6.0 M nitric acid) aqueous solution
followed by thorough rinsing with deionized, distilled water and then dried for at least one hour in
an oven at 120oC prior to use.
2.2.3 Preparation of Stock Solutions
Stock solutions of the fluorescent probes (N-methylacridone, phenosafranin, naphthalene)
were prepared by weighing out the solid material followed by dissolution in absolute ethanol so
that their final concentrations were in the range of 0.4 – 0.6 mM. Working solutions were prepared
by appropriate serial dilution of these concentrated stock solutions with deionized, distilled water,
ethanol or appropriate aqueous surfactant stock solutions. The final percentage of ethanol in the
probe surfactant micelle solutions used in the quenching experiments was usually less than 0.5%.
These fluorescent probe stock and working solutions were stored in the dark when not in use. Stock
solutions of the analyte copper (II) ion (typically in the 0.0501.00 M concentration range) were
prepared by weighting out the appropriate amounts of either copper (II) nitrate (or chloride)
followed by dissolution with deionized, distilled water. Working solutions were prepared by serial
22
dilution of the stock solution. Concentrated surfactant micelle stock solutions were prepared by
dissolution of the appropriate amount of the solid anionic surfactants in distilled water. In some
instances, the stock solutions had to be slightly heated in order to facilitate dissolution.
Dihexyldecylphosphate (DHP) surfactant vesicles were prepared using modified
literature26 products. First, 20 mg of DHP were dissolved in 10.00 mL of an aqueous solution at pH
7.5 and then this heterogeneous solution was heated to ca. 64oC in a constant temperature bath and
sonicated with a probe tip sonicator for two, 5.0 minute periods at 40 watts power, with an
intervening 5.0 minute cooling period. This heating – cooling cycle was repeated until a constant
absorbance reading at 540 nm was obtained for the translucent solution. This stock solution was
then stored in the dark at room temperature and used within one week of preparation. Desired lower
concentrations were prepared by appropriate dilution of the stock solution using an aqueous pH 7.5
buffer solution.
2.2.4 Procedures
2.2.4.1 Quenching Studies. Fluorescence quenching studies were conducted in the following
manner. Typically, 10.00 mL of the surfactant (SDS, NaDS, NaTDS or DHP vesicle) solution
was added to a 10.00 mL volumetric flask followed by addition of a small fixed aliquot (typically
20 - 100 µL) of the fluorescent probe molecule (N-methylacridone, phenosafranin or naphthalene)
under study. Next appropriate volumes (0 – 100 µL) of a copper (II) solution (final concentration
typically ranged from 0.000 – 0.0009 M) were added along with sufficient distilled water so that
each flask had the same final volume (usually 10.09 to 10.20 mL). After gently shaking to mix,
the solutions were kept in a constant temperature water bath (temperature ranging from room
temperature to 45oC) until any bubble (foam) formation had subsided before the fluorescence
intensity of the solutions was measured at the excitation and emission wavelengths for the probe
being employed (Table 1 gives these wavelength values). The fluorescence intensity of each
solution was measured as a function of the [Cu2+].
23
Prior to these measurements, the instrument was set to “zero” fluorescence intensity via
use of a blank solution that contained the surfactant being employed along with sufficient ethanol
so that the final percentage of ethanol was the same as it was in the probe containing solutions.
Such blank corrects for any background fluorescence due to impurities that may be present in any
of the reagents and solutions. The measurements were typically done in triplicate. The Stern-
Volmer constants were determined from a plot of the data according to Eq. 3. The procedure for
the determination of the KSV values in water (or water-ethanol) was the same as that outlined above
except that no surfactant was added and the copper (II) concentrations employed were greater.
The above protocol was employed in order to determine the effect of variation of (i) the
surfactant hydrophobicity (achieved by varying the alkyl chain length from C-10 (NaDS) to C-12
(SDS) to C-14 (NaTDS); (ii) probe molecule (N-methylacridone, phenosafranin, naphthalene) ; (iii)
temperature (ranged from 25oC to 45oC); and (iv) probe concentration ([NMA] ranged from 0.5
10-6 to 5.0 10-6 M) upon the Stern-Volmer quenching constant and (v) DHP vesicle concentration
(ranged from 0.46 10-4 M to 0.92 10-4 M) upon the Stern-Volmer quenching constant.
Table 1. The flourescence wavelengths and some other properties of the fluorescent probe
molecules.17, 27
Probe Molecule λex, nm λem, nm t, nseca Kmwb, M-1 Solubilityc, µM
N-Methylacridone 384 422 13-16 13000 + 1500 34.5
Phenosafranin 520 584 1-2.4 6.45 105 0.26 a. Single excited-state lifetimes in 1:1 (v/v) ethanol-water as solvent b. Association (binding) constant for interaction of the indicated probe molecule with sodium
dodecylsulfate (SDS) micelles c. Solubility of indicated fluorescent probe molecule in water
24
2.2.4.2 Determination of detection limit and recovery data. The detection limit was calculated as
“3σ/m”: three times the standard deviation of the background (“blank”) signal divided by the slope
of the analytical calibration curve (KSV).15 In fluorescence quenching, the “blank” refers to the
fluctuations in the fluorescence signal of the probe molecule in the absence of quencher. This was
determined by preparing replicates (n = 10 typically) of the probe molecule in the micelle (or water)
solvent system and measuring the fluorescence intensity. The mean fluorescence intensity was
then calculated and the standard deviation of the signal (Iaverage / Iindividual reading) determined.
Recovery data were determined using the same general protocol previously outlined. Copper
(II) solutions of known concentrations were prepared and their fluorescence intensity measured.
Their concentrations were then determined via use of the Stern-Volmer calibration curve prepared
for the same system (fluorescence probe – surfactant micelle combination). The % error or %
recovery was then calculated by use of Eq. 4 or Eq. 5:
Error % = [𝐶𝑢2+]𝑘𝑛𝑜𝑤𝑛 − [𝐶𝑢2+]𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑
[𝐶𝑢2+]𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑 × 100% (eq. 4)
Recovery % = [𝐶𝑢2+]𝑐𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑒𝑑
[𝐶𝑢2+]𝑘𝑛𝑜𝑤𝑛 × 100% (eq. 5)
2.3 Results and Discussion
2.3.1 Influence of surfactant charge-type, hydrophobicity and concentration upon quenching ability.
The ability of copper (II) ions to quench the fluorescence of the probe molecule, N-methylacridone,
NMA (structure I), was first examined since previous work in our group had indicated that NMA
was a good probe.
Structure I: N-methylacridone
25
As shown in Fig. 1, linear plots of the Stern-Volmer equation (Eq. 3) were observed in the presence
of 0.010 M SDS micelles as well as in bulk water alone. The KSV value in the presence of SDS
micelles is ca. 80 times greater compared to that in water.
Figure 1. Stern-Volmer plot of (𝐼0
𝐼− 1) vs. Cu2+ concentration in the prescence of 0.010 M SDS
micelle and in bulk water alone. Conditions: [NMA] = 1.00 10-6 M; 25oC
This reflects the micellar catalysis of the collisional quenching process. In the micellar literature,
there are reports of micellar catalysis of great number of reactions, including that of bimolecular
chemical and photochemical reactions.28 Such apparent catalysis is the result of the micellar
aggregate’s ability to “bring together” and concentrate the reactants (so as to provide a greater local
concentration of reactant species relative to that in water). As shown in Fig. 2 (Chapter 1), an
anionic surfactant micelle (SDS) attracts cations, such as Cu2+ ions, and solubilizes and binds
neutral fluorophores, such as NMA, providing for greater concentrations of these species and an
apparent enhancement in the bimolecular collisional rate constant (kq) and greater value for Ksv.
Regarding the specific mechanism of dynamic quenching of a fluoreophore’s (like NMA’s)
singlet excited state by a metal ion (such as Cu2+), the literature provides two routes29: (i)
paramagnetic heavy metal ions can enhance spin-orbit coupling which results in increased in
intersystem crossing from the excited singlet state to the excited triplet state or (ii) complete (or
26
partial) change transfer between the fluorophore (donor) and metal ion acceptor.29 In both instances,
the number of fluorophore molecules in their excited singlet state is diminished.
Reports in the micellar literature had also reported that the magnitude of micellar catalysis
in some reactions is more pronounced for surfactants that are more hydrophobic.30 It was thought
that the greater rate constants in micelles formed from surfactants with longer alkyl moieties
stemmed from their increased charge density at the micelle surface. In addition, one report noted
that same trend in Ksv values for the quenching of the probe phenosafrarin with copper (II) ions.25
Thus, the Stern-Volmer constants for the quenching of the probe NMA due to copper (II) ions was
also determined in micelles formed from the surfactants sodium dodecylsulfate and sodium
tetradecylsulfate. The results (Table 2) show that there was an approximately 3-fold increase in the
Ksv value for the C-12 SDS surfactant compared to that observed for the C-10 NaDS surfactant,
which is in agreement with prior micellar literature.
Difficulties were encountered with the experiments with the more hydrophobic C-14 NaTDS.
While the quenching of phenosafranin by copper (II) in the presence of NaTDS at room temperature
(25oC) had been previously reported,32 it was not possible to prepare a concentrated stock solution
of NaTDS at that temperature. The Krafft temperature of NaTDS has been reported to be in the
range of 21 – 48oC 31-34 This wide range could be due to differences in the purity of the surfactant
preparation. A temperature of 30oC was required in this work to prepare the NaTDS solutions.
At this temperature, a decrease in Ksv due to Cu2+ quenching of NMA fluorescence was observed
(Table 2) relative to that in SDS even though the NaTDS is the more hydrophobic surfactant.
Since a different temperature was employed, this complicates direct comparisons and it is not
possible to say whether or not the trend of increasing Ksv with surfactant hydrophobicity holds for
copper (II) quenching of NMA.
27
Table 2. Summary of the measured Stern-Volmer constants for the quenching of two aromatic
fluorophores by copper (II) in different anionic surfactant media
a. Probe concentrations: [NMA] 1 10-6 M; [PSF] 4 10-6 M. b. 25oC, [NaDS] = 0.035 M c. 25oC, [SDS] = 0.010 M d. 30oC, [NaTDS] = 0.002 M e. 25oC, [SDS] = 0.010 M
The reason for this is that at 30oC, the micellar parameters (CMC and N) for NaTDS are different
than they are at 25oC. This in turn means that the NaTDS concentration required for optimal
quenching is different than that at room temperature. For most fluorophore – quencher – surfactant
micelle systems, the optimal quenching efficiency is observed at surfactant concentrations that are
just above their aqueous CMC values (under those experimental conditions).17, 25, 32 A typical KSV
vs. [surfactant] profile is shown in Fig. 2. From reported profiles like that in Fig. 2, the optimal
surfactant concentrations (at 25oC) for sodium decylsulfate (NaDS), sodium dodecylsulfate (SDS)
and sodium tetradecylsulfate (NaTDS) are approximately 35.0 mM, 10.0 mM and 2.00 mM,
respectively.17, 32
Fluorescent Probea Anionic surfactant Media KSV KSV in water
NMA NaDSb. 1050
38 NMA SDSc. 3270
NMA NaTDSd. 2020
PSF SDSe 2450 1.5
28
Figure 2. The dependence of the KSV due to quenching of PSF fluorescence by copper (II) ion as a
function of [NaTDS] at 27oC (Adapted and taken with permission from Elsevier publisher).32
Based on such considerations, additional experiments were conducted at 30 oC to determine
the KSV values for the quenching of NMA with Cu2+ in NaTDS micelles formed using different
fixed NaTDS concentrations. The results are summarized in Table 3. Reproducible data (linear
plots of the Stern-Volmer equation with R2 = 0.9980 or better) were obtained at three different
NaTDS concentration levels. However, for reason(s) still unknown, non-reproducible results were
observed at two other NaTDS concentrations examined. The results from the other three
concentrations of NaTDS seem to indicate that the concentration of NaTDS over this concentration
range does not appreciably alter the KSV values which suggests that the elevated temperature is not
the reason why KSV for NaTDS was not greater than that for SDS (Table 2) even though the former
is the more hydrophobic surfactant.
29
Table 3. Effect of NaTDS surfactant concentration on the quenching of NMA fluorescence by
copper (II) ionsa
NaTDS Micelle System
Surfactant Concentration (mM) Ksv (M-1)
1.80 NR
1.90 2020
1.93 1910
2.33 NR
2.67 2260
a. The concentration of the fluorescent probe, N-methylacridone, was kept constant (1.00
10-6 M) in all of the experiments; all fluorescence measurements were made at a temperature of 30oC.
b. NR means that the results were not reproducible.
In view of these results, attempts were made to reproduce the published work32 concerning the
quenching of phenosafranin (PSF, Structure II) by copper (II) in NaTDS.
Structure II: Phenosafranin
In the reported reference,32 all experiments were conducted at room temperature (around 27oC).
But based on my results (Table 4), this experiment could not be reproduced. The formation of some
small needle-like crystals was observed in the surfactant solutions upon addition of copper (II) ions
(at the higher levels of added copper (II) in the determination of the Stern Volmer data) at which
30
point the data points failed to adhere to the plot of data according to Eq. 3. The same general
experiment was then repeated at higher temperature, with the same result as well as those solutions
turned cloudy in appearance. It is unclear why the published literature results could not be
reproduced.
Next, another attempt was made to reproduce the literature report,32 but this time for the
quenching of phenosafranin by copper (II) in the presence of SDS surfactant micelles.
Table 4. Summary of observations in attempted determination of the Stern-Volmer quenching
constants for the PSF – Copper (II) – NaTDS Systema.
a. The phenosafranin concentration was fixed at 4.00 10-6 M in all experiments. b. ND refers to not determined due to nonlinear plots of the data according to Eq. 3.
This proved successful and the result (Table 2) indicated that the KSV value for phenosafranin as
the probe molecule (2450 M-1) is less than that if N-methylacridone is the probe (K = 3270 M-1).
Based upon these results, it appears that SDS should be considered the surfactant of choice for
micelle amplified fluorescence quenching methods since it provided consistant, reproducible
results (KSV values) with different fluorescence probe molecules. In addition, it is much less
expensive than NaTDS and has a Krafft point that is well below room temperature.
NaTDS Micelle System
Temperature (oC ) Ksv (M-1) Observations
27 NDb Crystal Formed
30 ND Cloudy and Crystal
35 ND Cloudy
31
2.3.2 Influence of temperature upon fluorescence quenching in SDS micelles. In order to examine
the influence of temperature, the quenching of the fluorescence emission of micelle-solubilized
phenosafranin (PSF) by copper (II) ions at a fixed concentration of sodium dodecyl sulfate over the
temperature range from 25.5 to 45oC was evaluated. The dependence of the Ksv values upon
solution temperature are tabulated in Table 5. The Stern-Volmer values were essentially constant
over the range from 25.5 to 35.0oC and then increased slightly as the temperature was increased
from 35 to 45oC. At first, these results were a bit surprising since it is known that Ksv values
increase with an increase in temperature if the quenching process is due to dynamic (collisional)
quenching (with the opposite trend observed if the quenching is due to static (ground state complex
formation)).1 Typically, for dynamic quenching in water (or other solvent system), the Stern-
Volmer constant roughly doubles for each 10oC increase in temperature.1
Table 5. Effect of temperature on the quenching of PSF fluorescence by copper (II) ions in SDS
micelles.a
a. Conditions: [PSF] = 4.00 10-6 M and [SDS] = 10.0 mM.
It should be noted that examination of the visible absorption and fluorescence emission spectra
of the SDS solubilized fluorescent probe, NMA, indicated these wavelength maxima did not shift
as a function of added copper (II) ion quencher, which is indicative of a dynamic or collisional
quenching process. A similar observation has been reported for the probe PSF in SDS micelles.25
Dynamic quenching affects only the excited states of the fluorescent probe molecule without
formation of any ground state complex as is the case in static quenching and as a result, no changes
Temperature (oC ) Ksv (M-1)
25.5 2450
35.0 2420
40.0 2690
45.0 2870
32
in the appearance of the probe absorption or emission spectra profiles are expected1, 25 nor were
observed (Figure 3).
Figure 3. Fluorescence emission spectra of 1.00 10-6 M solutions of NMA in the presecnce of
0.010 M SDS at the following copper (II) ion concentrations: 0.00 M (blue line), 0.099 mM (red
line), 0.198 mM (grey line) and 0.495 mM (yellow line). Conditions: Measurements made at 25.0oC
with λex =422 nm.
The most likely explanation for the atypical dependence of KSV with temperature for the
copper (II) quenching of PSF (Table 5) is that increasing temperature causes alterations in the
micellar parameters (CMC and N), which in turn could alter the magnitude of the probe binding
constant so that the surfactant concentration required for optimal quenching is changed at the
elevated temperature relative to that at 25.5oC. As shown in Fig. 3, the CMC value for SDS
increased from 7.94 mM at 20oC to 9.57 mM at 40oC35 while its aggregation number (N) decreased
from 69 to 40 over this temperature range (Table 2, Chapter 1).35 The concentration of micelles can
be calculated from eq. 6:
[Micelle] = ([surfactant] – CMC) / N (eq. 6)
where [surfactant] represents the total surfactant concentration. Thus, as one goes from 25.5oC to
40.0oC, the concentration of SDS micelles (under the conditions of the experiment summarized in
Table 5) decreased from ca. 3.1 10-5 M to 1.07 10-5 M. For the results in Table 5, the increased
0
20
40
60
80
100
120
410 415 420 425 430 435 440
Flu
ore
scen
ce In
ten
sity
Wavelength (nm)
33
quenching at the elevated temperature was offset by the fact that the micelle concentration was
lowered (below optimum) so that the net effect observed was only a modest increase in KSV over
the indicated temperature range.
Figure 4. Temperature dependence of Critical Micelle Concentration for the surfactant SDS
(Adapted with permission from Elsevier publisher)35
2.3.3 Effect of variation of the fluorescence probe concentration upon quenching in SDS micelles.
A brief study was undertaken to explore if the concentration of the fluorescent probe molecule
affected the KSV values. Namely, the quenching of the fluorescence emission of NMA (present at
different fixed concentrations) by copper (II) ions in the presence of SDS micelles was determined.
As is evident from the data (Table 6), the Stern-Volmer quenching constant was essentially the
same (3230 M-1) over this concentration range of NMA. However, as will be detailed in the next
section, the NMA fluorescence probe concentration did significantly influence the detection limit
for the quenchofluorimetric method for determination of copper (II) ions in the SDS micellar
medium.
34
Table 6. Effect of NMA probe concentration on the quenching efficiency in SDS micelles.a
NMA Concentration (M) Ksv (M-1)
5.00 10-7 3240
1.00 10-6 3260
2.50 10-6 3200
a. Conditions: [SDS] = 10.0 mM; all the experiments were conducted at room
temperature (around 25-27oC).
2.3.4 Effect of DHP concentration on the quenching of NMA fluorescence by copper (II) ion. A
brief study was undertaken to explore if the presence of DHP vesicles affected the KSV values. The
quenching of the fluorescence emission of NMA by copper (II) ions in the presence of DHP vesicles
was determined. As is seen from the data (Table 7), the Stern-Volmer quenching constants (26.2
and 17.8 M-1) were low in the presence of DHP vesicles, even lower than the KSV values in water
(38 M-1 from Table 2). It is unclear why the values are lower in the DHP vesicles. Potential
speculative reasons could include pH and temperature effects which influence the vesicle structure
and properties (such as NMA and copper (II) ion binding)36 all of which might serve to lower the
Ksv value for copper (II) quenching. In addition, some reports indicate that DHP vesicles are subject
to cation permeability37 (i.e. ion goes from outer surface (refer to Figure 5 in Chapter 1) to the inner
water pool surface). If this occurs in our system, then such copper (II) ions would not be available
to quench the NMA.
35
Table 7. Effect of DHP concentration on the quenching of NMA fluorescence by copper (II) iona
DHP Concentration, (M) Ksv, (M-1)
0.92 10-4 26.2
0.46 10-4 17.8
a. Conditions: all the experiments were conducted at room temperature (around 25-27oC);
[NMA] = 1.00 10-6 M.
2.3.5 Analytical performance and selectivity. The limit of detection for the determination of
copper (II) ions based upon their quenching of the fluorescence of NMA in SDS micelles was
determined at different probe concentrations. The results are summarized in Table 8. The detection
limit is observed to decrease with an increase in the NMA fluorescent probe concentration over the
range of 1.00 10-6 to 5.00 10-6 M (Table 8). An approximate 5-fold increase in sensitivity was
obtained by just altering the NMA probe concentration. A similar result had previously been
reported in the literature by Goodpaster and McGuffin8 for the fluorescent quenching detection of
nitrated explosives in HPLC using different polycyclic aromatic hydrocarbons as the fluorescence
probe molecule (which was present in the mobile phase). By comparison, the detection limit of
copper (II) using NMA as the fluorescence probe in water (with 1% added ethanol) is 5.5 10-3
M.40 The use of anionic SDS as the analytical reaction medium serves to increase the sensitivity of
the NMA quenching method for determination of copper (II) ion by over two orders of magnitude.
It is unclear why the standard deviation for the solution containing the highest NMA
concentration (1.00 10-5 M) exhibited the greatest standard deviation for its blank signal. One
reason could be that the slit widths employed for this highest NMA concentration had to be
decreased in order to keep the fluorescence intensity signal on scale. Another possible reason is
36
that there were insufficient SDS micelles present to solubilize all of the NMA at that concentration
level and that any microcrystalline NMA present would lead to increased light scattering and
fluctuations in the background fluorescent signal. To the naked eye, the solutions appeared to be
clear and homogeneous.
Table 8. Influence of NMA fluorescent probe concentration upon standard deviation of blank
signal and limit of detection for determination of copper (II) ions in SDS micelles.a
NMA Concentration, (M) Standard Deviationb Detection Limit,c(M)
1.00 10-6 0.05 4.6 10-5
2.00 10-6 0.04 3.7 10-5
5.00 10-6 0.01 9.3 10-6
1.00 10-5 0.06 5.5 10-5, d
a. Conditions: [SDS] = 10.0 mM; measurements made a room temperature. b. Standard deviation based upon ten replicates at the indicated NMA concentration. c. Limit of detection calculated 3σb / KSV. d. The detector slit width was set at 0.5 mm for this run while in all other experiments, it
was set at 1.0 mm.
One issue that none of the previous publications in the literature on micelle amplified
quenching has addressed is that of selectivity. Just as copper (II) ions quench fluorescent probes
like NMA or PSF, many other cations (cobalt, lead, iron, etc.) will also quench the fluorescence of
these probes. Consequently, the micelle enhanced fluorescence quenching methods can only be
used if the samples are very simple (contain only the target cation, such as Cu2+) or they may be
employed as a means of detection in chromatographic (or electrophoretic) methods. The use of
aqueous SDS solutions as the mobile phase in reversed phase liquid chromatography or in the run
buffer for capillary electrophoresis is well documented.34 To employ fluorescence quenching as an
37
indirect detection method in HPLC (or CE) would just require addition of the fluorescent probe
molecule to the mobile phase (or run buffer).
While interference from other cations would pose problems for the determination of copper
(II) ion based upon its quenching of NMA in SDS micelles, the magnitude of interferences due to
anions would be expected to be diminished. Anions, such as iodide or bromide ions, would be
electrostatically repelled from the SDS micelle surface (and the bound NMA probe), so their Stern-
Volmer constants would be less in the presence of SDS relative to that in water with the net result
that they would have to be present at much greater concentrations to cause an interference. In this
sense, the use of SDS micelles offers some modest improvements with respect to potential
interference from anionic quenchers.
Lastly, a brief recovery study was performed. The Stern-Volmer calibration plot (eq. 3) of
{[Io / I] – 1} vs. [Cu2+] was prepared in the usual fashion and was then employed to experimentally
determine the concentration of aqueous solutions that had been spiked with known amounts of Cu2+.
The amount of copper (II) as experimentally determined from the calibration curve was then
compared to the actual amount that had been added. The results are summarized in Table 9. With
one notable exception, the recovery were fair. The one very high recovery value is likely due to
some experimental error and that data should be repeated.
38
Table 9. Summary of recovery data for determination of copper (II) ions based upon their
fluorescence quenching of NMA or Naphthalene in SDS micelles
Probe Micelle Solution [Cu2+]original [Cu2+]measured % Error % Recovery
NMAa SDSa
0.000075 0.000069 8.0% 92.0%
0.00012 0.00021 -71.5% 171.5%
0.00035 0.00038 -9.0% 109.0%
NMAc SDSd 0.000075 0.000088 -18.7% 118.7%
0.00012 0.00015 -22.1% 122.1%
Naphthalenec SDSc 0.00012 0.00011 7.6% 92.4%
0.00028 0.00028 -1.7% 101.7%
a. Conditions: [NMA] = 5.00 x 10-7 M; [SDS] = 10.0 mM.
b. Conditions: [NMA] = 2.50 x 10-6 M; [SDS] = 10.0 mM. c. Conditions: [Naphthalene] = 1.01 x 10-5 M; [SDS] = 10 mM; [HNO3] = 15.8 mM.20
2.4 Conclusions.
The use of an anionic SDS micellar medium for the quenchfluorimetric determination of
copper (II) ion based upon its ability to quench the fluorescence of the luminescent probe, NMA,
was found to lead to an increase of the KSV value by a factor of ca. 80 relative to that observed in
water alone as solvent under the same conditions. The concentration of the fluorescent probe,
NMA, has a fairly significant effect on the limit of detection as greater probe concentrations yielded
smaller fluctuations in the background fluorescence signal (smaller standard deviations) with
resultant improved detection limits. Under optimal conditions, the limit of detection for copper
(II) in SDS micelles is over two orders of magnitude better relative to that in water as solvent. In
the SDS micellar medium (at fixed SDS concentrations), temperature only slightly influences the
KSV value over the temperature range from 25 – 35oC. Although increased KSV values had been
reported if SDS was replaced by the more hydrophobic surfactant, NaTDS, those results could not
be reproduced in this study. Due to the greater Krafft temperature of NaTDS (relative to SDS),
the experimental is more involved as a temperature bath is required and results were not
reproducible. In addition, the cost of NaTDS is greater than that for SDS. Based on our work, SDS
39
appears to be the anionic micelle forming surfactant of choice for determination of copper (II)
cation. Many other cations would be expected to also quench NMA and thus interfere with the
method for Cu2+ ion although interference from anions would be expected to be less severe relative
to water as solvent for its determination.
40
CHAPTER 2 REFERENCES
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principles and practice; Pergamon Press, Eds.; Academic: New York, 1977, pp 100-105.
2. Krul, I. S.; Deyl, Z.; Lingeman, H. General strategies and selection of derivatization
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3. Fukushima, T.; Usui, N.; Santa, T.; Imai, K. Recent progress in derivatization methods
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5. Sawicki, E.; Stanley, T. W.; Elbert, W. C. Quenchofluorometric analysis for
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6. Sawicki, E.; Johnson, H.; Kosinski, K.Chromatographic separation and spectral analysis
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7. Ho, C. N.; Patonay, G.; Warner, I. M. Bioanalytical applications of fluorescence. Trends
Anal. Chem., 1986, 5, 37-43.
8. Goodpaster, J. V.; McGuffin, V. L., Fluorescence quenching as an indirect detection
method for nitrated explosives. Anal Chem 2001, 73, 2004-2011.
9. Seitz, W. R. Treatise on Analytical Chemistry; Elving, P. J., Ed.; John Wiley & Sons,
Inc.: New York, 1981; Vol.7, pp 200 – 206.
10. Guilbault, G. G. Practical Fluorescence; Marcel Dekker, Inc. Eds.; Academic: New
York, 1973.
11. Rakicioglu, Y.; Young, M. M.; Schulman, S. G. Limitations of quenching as a method of
fluorometric analysis of non-fluorescent analytes. Anal Chim Acta, 1998, 359, 269-273.
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12. Mallick, A.; Mandal, M. C.; Haldar, B.; Chakrabarty, A.; Das, P.; Chattopadhyay, N.
Surfactant-induced modulation of fluorosensor activity: a simple way to maximize the
sensor efficiency. J Am Chem Soc, 2006, 128, 3126-3127.
13. Bandyopadhyay, P.; Saha, K. Surfactant-induced fluorescent sensor activity enhancement
of tryptophan at various pH. Chem Phys Lett, 2008, 457, 227-231.
14. Das, P.; Mallick, A.; Sarkar, D. Application of anionic micelle for dramatic enhancement
in the quenching-based metal ion fluorosensing. J Colloid Interface Sci, 2008, 320, 9-14.
15. Malliaris, A.; Lang, J.; Zana, R. Dynamics of micellar solutions of ionic surfactant by
fluorescence probing. J Phys Chem, 1986, 90, 655-660.
16. Quina, F. H.; Lissi, E. A. Photoprocesses in microaggregates. Acc Chem Res, 2004, 37,
703-710.
17. Abuin, E.; Lissi, E. A. Fluorescence quenching in the study of micellar and vesicular
system. Bol Coc Chil Quim, 1997, 41, 113-114.
18. Lunardi, C. N.; Bonilha, J. B. S.; Tedesco, A. C. Stern-Volmer quenching and binding
constants of 10-alkyl-9(10H)-acridone probes in SDS and BSA. J Luminescence, 2002,
99, 61-71.
19. Armstrong, D. W.; Spino, L. A. Micelle-Medicated Resonance Raman Spectroscopy: A
new approach for characterizing low levels of luminescent compounds. J Am Chem Soc,
1986, 108, 5646-5647.
20. Spino, L. A.; Armstrong, D. W.; Alak, A. A.; Vo-Dinh, T. Resonance Raman analysis of
fluorescent compounds using micellar solutions and ultraviolet laser excitation. Appl
Spectro, 1987, 41, 771-773.
21. Tarek, M.; Zaki, M.; Esmail, L. F. M.; El-Sayey, A. Y. Fluorimetric determination of iron
(III) by quenching the luminescence of the zinc-morin-Triton X-100 System. Fresenius Z
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22. Bandyopadhyay, P.; Ghosh, A. K. Recent developments in micelle induced fluorescent
sensors. Sensor Lett, 2011, 9, 1249-1264.
23. Hinze, W., Riehl, TE., Singh, HN, Baba, Y. Micelle-enhanced chemiluminescence and
application to the dertermination of biological reductants using lucigenin. Anal Chem,
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24. Memon, N.; Baouch, A.; Hinze, W. Fluorescence in organized assemblies. In
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25. Paramita, D.; Arabinda, M.; Deboleena, S.; Nitin, C. Application of anionic micelle for
dramatic enhancedment in the quenching-based metal ion fluorosensing. Journal of
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26. Eendler, J. H. Membrane Mimetic Chemistry; John Wiley: NY, 1982, Chapter 6, pp. 158-
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27. McNaught, A. D., Wilkinson, A.: IUPAC. Compendium of Chemical Terminology, 2nd
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30. Dunlap RB, Cordes EH (1968) Secondary valence force catalysis. IV. Catalysis of
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31. Shinoda, K.; Hiruta, S.; Amaya, K. The heat of solution and of wetting of ionic
surfactants close to the Krafft Points. J. Colloid Interface Sci., 1966, 21, 102-106.
32. Shinoda, K.; Soda, T. Partial Molal Volumes of surface active agents in micellar, single
dispersed, and hydrated solid states. J. Phys. Chem.,1963, 67, 2072.
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33. Koshy, L.; Saiyad, A. H.; Rakshit, A. K.: The effects of various foreign substances on the
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34. Wu, J.; Lee, C.; Harwell, J. H.; O’Rear, E. A. Adsorbed Surfactant Bilayers as two-
dimensional solvents, in Surfactant-Based Separation Processes; Scamehorn, J. F., Harwell,
J. H., Eds.; Surfactant Science Series: New York, Vol. 33, 1989, pp 184.
35. Shah, S., Jamroz, N., Sharif, Q. Micellization parameters and electrostatic interactions in
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Surfaces A: Physicochemical and Engineering Aspects, 2001, 178, 199-206.
36. Camona-Ribeiro, AM; Hix, S. pH Effects on properties of dihexyldecyl Phosphate Vesicles,
J. Phys. Chem., 1991, 95, 1812-1817.
37. Tricot, Y. M.; Furlong, D. N.; Sasse, W. H. F. Dihexadecyl phosphate vesicles:
Permeability to cationic components in solar photolysis systems. Aust, J. Chem., 1984, 37,
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38. Berthod,A.; Garcia C. A. Micellar Liquid Chromatography; Marcel Dekker, publisher;
Chromatographic Science Series: New York, 2000; Vol. 83.
39. Vargas, L.; Brandao, T. A. S.; Fiedler, H. D.; Quina, F. H.; Nome, F. Determination of
environmentally important metal ions by fluorescence quenching in anionic micellar
solution. Analyst, 2008, 130, 241-246.
40. . Balouch, A.; Zhang, L.; Muhammad, I.; Thimmaiah, K. N.; Hinze, W. L. (2014) Factors
affecting the use of organized surfactant assemblies for amplification of fluorescence
quenching with application to the quenchiofluorimetric determination of copper (II).
Manuscript In Preparation.
44
Chapter 3- Determination of some Phase Separation Properties of Aqueous Solutions of the
Nonionic Surfactant Triton X-114
3.1 Introduction
Compared to ionic surfactant micelles, aqueous solutions of nonionic surfactants can exhibit
unique phase behavior. At temperatures below their cloud point (defined as the temperature at
which the surfactant micelle solution turns turbid), aqueous micelle solutions consist of a
homogeneous, isotropic phase. If the temperature is increased and exceeds the cloud point, the
solution will undergo a macroscopic phase separation to yield (after equilibration) two phases, a
surfactant-lean (also called micelle-poor or aqueous) phase and a surfactant-rich (micelle-rich or
coacervate) phase.1-3 The surfactant concentration in the surfactant-lean phase is typically around
the CMC value. Typically the density differences are such that the surfactant-lean phase is the top
layer and the surfactant-rich phase is the bottom, although there are exceptions.1 The schematic
cartoon (Fig. 1) gives a pictorial representation of this phase separation behavior exhibited by
nonionic surfactants. Any species that binds to the nonionic micelle in solution would, after phase
separation, be concentrated in the micelle-rich phase.
Such nonionic surfactant phase phenomenon was first employed by Watanabe2 to extract
metal cations from water. After addition of a metal chelating agent, a nonionic surfactant was
added so that micelles of that surfactant were present in solution. If the metal complex binds to the
nonionic micelle aggregate and the temperature is increased above the cloud point, the metal chelate
will be extracted and present in the small volume of the surfactant-rich phase. Later, Bodier3
employed such an approach for the extractive separation of hydrophobic and hydrophilic proteins.
The hydrophobic proteins would be present in the surfactant-rich phase with the hydrophilic
proteins in the surfactant-lean phase. Such extractions, termed as surfactant mediated or cloud
point extractions (CPE), have become very popular with thousands of publications in the literature.
45
Figure 1. Schematic representation of the phase separation behavior exhibited by an aqueous
solution of a nonionic surfactant micellar system upon temperature alteration (Adapted with
permission from Wiley Online Library Publisher).4
Most recently, a variation of the mentioned cloud point extraction has been employed for
the enrichment of large biomolecules.4, 5 Blankschtein and coworkers have shown that large
biomolecules can be concentrated in the surfactant-lean phase because partitioning is primarily
dictated by repulsive steric excluded volume interactions between the large biomolecule and the
nonionic surfactant micelle.4-13 The theory for this excluded volume CPE has been worked out and
the enrichment (preconcentration) factor shown to be given by Eq. 1:
Enrichment factor ≈ 1 + 1 / (Vt / Vb) (Eq. 1)
where Vt and Vb represent the volumes of the top and bottom phases, respectively.6, 7, 8 The use of
nonionic surfactants, such as Triton X-114, for such excluded volume extractions has been reported
46
for proteins, viruses and bacteria.9, 10, 11 The use of aqueous nonionic surfactants offers a
convenient and mild extraction medium as it typically does not denature the biological species.
In the traditional CPE, the ideal conditions are those that result in a very small volume
element for the surfactant-rich phase since that will result in the greatest enrichment factor.
However, in the excluded volume CPE, the opposite is true as the greatest enrichment factor is
possible under conditions that give rise to the smallest volume for the surfactant-lean phase. There
is a much greater volume of literature available on conditions necessary to achieve the smallest
volume for the surfactant-rich phase in comparison to that on attainment of the smallest volume for
the surfactant-lean phase. So the primary objective of this project was to determine conditions
necessary to obtain small volume surfactant-lean phases which would be useful to those who wish
to utilize the excluded volume CPE in their work.
3.2 Experimental
3.2.1 Equipment and Materials:
A Model 6-K Precision Micro Centricone Centrifuge (Precision Scientific Co., Chicago, IL)
was employed for centrifugation. An Isotemp Incubator (Fisher Scientific Co., Raleigh, NC) was
used for incubating the centrifuge at 37.0oC. The centrifugation rate is ca. 1500 rpm. Typically,
capped, graduated 5.00 mL glass centrifuge tubes were employed in the phase separation
experiments. A model B14 Isotemp 2150 water bath (Fisher Scientific Co.) was employed for
temperature control.
Triton X 114 (TX-114) was obtained from Sigma Aldrich. A 105.6 g/L stock solution of
Triton X-114 was prepared by weighting out the appropriate amount and diluting it to the final
volume with distilled water.
Standard buffer solutions, pH 4.63 acetate buffer (50 mM acetic acid and 50 mM sodium
acetate trihydrate), pH 9.00 borate buffer (100 mM boric acid and 50 mM potassium hydroxide)
47
and Dulbecco’s PBS buffer (137 mM sodium chloride, 2.7 mM potassium chloride, 8.1 mM
disodium hydrogen phosphate, 0.9 mM calcium chloride and 0.49 mM magnesium chloride) were
all obtained from Fisher Scientific Company. Sodium chloride (>99%) was obtained from Sigma
Aldrich Co. Distilled water was employed in the preparation of all required solutions. All other
solvents and reagents employed were of the best commercial grade available.
3.2.2 Procedures:
3.2.2.1 Density determination. The density of Triton X-114 was determined by accurately
massing the amount present in a 5.00 mL volumetric flask. The density was taken as the mean
value of 3 determinations.
3.2.2.2 Determination of the Cloud Point Temperature. The cloud point of the different aqueous
solutions of Triton X-114 over the concentration range from 0.5 – 10% (v/v) was determined by
visually observing the temperature required for the solution to turn cloudy or turbid upon heating
and also by noting the temperature at which the turbid solution turned clear again upon cooling.
The average of these two temperatures was taken as the cloud point of that specific TX-114 solution.
The rate of the temperature change for these experiments was 0.5oC around the cloud point. The
phase diagram was obtained by measuring the cloud point temperature as a function of the TX-114
concentration.
3.2.2.3 Determination of the surfactant-rich and surfactant-lean phase volumes. Aqueous solutions
of TX-114 at different specified concentrations were prepared and added to 5.00 mL graduated
glass centrifuge tubes (preparation occurring at a temperature below the cloud point for that
solution). They were then incubated for a specific amount of time at a fixed temperature in a
constant temperature water bath. The solutions were then either (i) centrifuged for a specified
period of time or (ii) kept in the constant temperature water bath for 18 or 24 hours in order to
affect phase separation. Following this phase separation step, the centrifuge tube was taken and
48
the volumes of the top surfactant-lean phase and the bottom surfactant-rich phase determined from
the marking on the graduated centrifuge tube. The same procedure was employed for the
determination of the volumes of the respective phases for solutions of TX-114 that contained
different buffers or salt in order to determine the influence of ionic strength on the phase ratio. The
reported results are the average of at least 3 determinations.
3.3 Results and Discussion
3.3.1 Determination of the Density and Phase Diagram for TX-114
Since different surfactant preparations can sometimes give different results, the density and
phase diagram for the Sigma TX-114 preparation were determined and compared to available
literature values. The density for the as received TX-114 surfactant was determined to be 1.056
g/mL (at 25.5oC), which is in good agreement with a reported literature value of 1.058 g/mL (at
28oC).14
The cloud point as a function of the TX-114 concentration was determined. The resulting phase
diagram (coexistence curve) is shown in Fig. 2. This curve is in good agreement with such curves
published in the literature.1, 15 At any given concentration, the aqueous solution of TX-114 will
separate into two macroscopic phases if the temperature exceeds the cloud point temperature. The
minimum in such curve is referred to as the critical point and is characterized by a critical surfactant
concentration ([surfactant]c) and critical temperature (Tc).1 For the TX-114 used in this work, [TX-
114]c is ca. 1.05 % (w/w) and Tc is 27.8oC. These results are in good agreement with published
literature values1 such as Koshy who reported 1.00 % (w/w) and 28.0oC as the critical point.15
49
Figure 2. Experimentally determined phase diagram for aqueous solutions of Triton X-114. Over
the range of temperatures and TX-114 concentrations shown, the homogeneous one-phase region
occurs at conditions that are below the curve while the phase separated two-phase region occurs at
conditions that are above the curve.
3.2.2 Determination of the phase ratio behavior of aqueous solutions of Triton X-114 under
different experimental conditions.
3.2.2.1 Determination of surfactant-rich (micelle-rich) phase volume under conditions (relatively
high temperature with low surfactant concentrations) employed for the traditional cloud point
extraction. The volume of the surfactant-rich phase formed after 5.00 mL TX-114 solutions ( [TX-
114] in the range from 3.4 – 69.7 mM) were incubated in an 80oC constant temperature water bath
for 5.0 minutes and centrifuged for 2.0 minutes was determined. All solutions phase separated at
this temperature and the volume of the surfactant-rich phase increased with an increase in the
surfactant TX-114 concentration over the concentration range examined (Fig. 3) in agreement with
the literature.1 The greatest extraction enrichment factors are obtained by use of lower
concentrations of TX-114 in the CPE method. For example, at 20.0 mM TX-114, the volume of
the surfactant-rich phase is ca. 0.12 mL, which corresponds to a phase ratio (volume of the
25
26
27
28
29
30
31
32
33
0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00%
CP
T (o
C)
[TX-114] wt%
Two phases
One phase
50
surfactant-rich phase divided by the volume of the surfactant-lean phase = 0.12/4.88) of 0.025.
The maximum possible enrichment factor is just the reciprocal of this phase ratio (40 in this
example). By comparison, the maximum enrichment factor expected at 10.0 mM TX-114 would
be 99.
Figure 3. Plot of surfactant-rich phase volume as a function of the total TX-114 surfactant
concentration. Conditions: 5.00 mL samples in graduated centrifuge tubes were incubated for 5.0
minutes at 80oC and then taken out of the bath and immediately centrifuged for 2.0 minutes.
Next, the same general experiment was repeated using just two TX-114 concentrations
(34.9 and 69.7 mM) in order to determine the influence of the incubation time at different fixed
temperatures (each maintained in a constant temperature water bath) upon the volume of the
surfactant-rich phase. The solutions were all incubated for 5.0, 10.0 or 20.0 minutes in the water
bath and then the surfactant-rich phase volumes determined after centrifugation for 2.0 min. The
data are summarized in Table 1. At both concentration levels, there were only small changes
observed in the volume of the surfactant rich phase (ca. 0.012 – 0.106 mL) as the incubation time
was increased from 5 to 20 minutes at the different fixed incubation temperatures. In most reported
0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
0 20 40 60 80
Vo
lum
e o
f M
icel
le-R
ich
Ph
ase
(mL)
[TX-114] mM
51
CPE methods, the incubation time was evaluated in terms of the extraction efficiency obtained for
the target analyte species rather than in terms of the phase ratio (and impact on the magnitude of
the enrichment factor).1, 16 Over the temperature range examined, the slight changes in the volume
of the surfactant-rich phase as a function of incubation time would have only a minimal impact on
the maximum enrichment factor possible in CPE.
Table 1. Summary of the effect of incubation time and temperature upon the volume of the
surfactant-rich phase of TX-114 following phase separation.a
[TX-114]
(mM)
Temp (oC )
80 70 50 35
Time
(min) Volume of Micelle-Rich Phase (mL)
34.9
5 0.200 ± 0.000
0.218 ± 0.006
0.331 ± 0.013
0.912 ± 0.002
10 0.197 ± 0.005
0.211 ± 0.007
0.407 ± 0.009
20 0.212 ± 0.022
0.203 ± 0.003
0.421 ± 0.010
69.7
5 0.400 ± 0.000
0.487 ± 0.005
0.633 ± 0.012
10 0.310 ± 0.008
0.404 ± 0.006)
0.713 ± 0.013
20 0.318 ± 0.001
0.381 ± 0.007
0.721 ± 0.018
a. All solutions were incubated at the indicated temperature in constant temperature water bath
for the noted times after which there were removed and centrifuged for 2.0 min at 1550 rpm
and the volume of the surfactant-rich phase determined. Reported values are the average of
replicates (n= 3 or 4).
The other clear trend in Table 1 is that as the incubation temperature increases, the volume of the
surfactant-rich phase decreases regardless of the incubation time. This is the main reason why
temperatures well above the surfactant cloud pint temperature have been employed for the cloud
point extraction of inorganic and organic species since this leads to larger maximum enrichment
factors.1, 17 This decrease in the surfactant-rich phase volume with an increase in temperature has
52
been attributed to a decrease in the water content of the surfactant-rich phase due to dehydration of
the surfactant’s polyoxyethylene moieties (in the case of Triton X-114, Structure III, there are on
average 7.5 oxyethylene moieties).17
Structure III: Triton X-114 (n = 7.5)
In the traditional CPE, after incubation at elevated temperature, the solutions are typically
centrifuged for 2 – 10 minutes to facilitate separation of the two phases. The centrifugation step is
almost always conducted at room temperature (ca. 25oC), so the two separated phases are cooling
during the centrifugation step. There appears to have been no previous attempt made to determine
the magnitude of cooling that occurs during centrifugation. Thus, experiments were conducted as
described above except that instead of determining the volume of the surfactant-rich phase
following the centrifugation step (as in Table 1), the temperature of the phase separated “solution”
was directly measured. The results are summarized in Table 2. As can be seen, appreciable cooling
occurred during the 2.00 minute centrifugation step. For example, for solutions initially incubated
at 35oC, the temperature of the surfactant-rich phase following centrifugation was decreased by ca.
6.5oC and a ca. 36.3oC decrease was observed for solutions initially incubated at 80oC. Such cooling
during the centrifugation step could potentially lead to variations in the final volume of the
surfactant-rich phase and reduce precision of CPE, especially at the higher incubation temperatures.
3.2.2.2 Determination of conditions necessary for maximizing the volume of the surfactant-lean
phase as is required in CPE in the excluded volume format. In the reports of the CPE technique
based upon excluded volume effects, no centrifugation step was employed. Rather, the solutions
being extracted were kept in a constant temperature bath for 18 hours and allowed to phase separate
due to gravity.8, 18, 19 Relative to the conditions in traditional CPE, in excluded volume CPE
53
incubation temperatures much nearer to the cloud point temperature were employed as well as
much greater concentrations of the surfactant, TX-114 employed. Data such as that presented in
Fig. 3 and Table 1 point to those conditions since in the excluded volume CPE, a smaller surfactant-
lean phase (and larger surfactant-rich phase) is desired in order to improve the extraction
enrichment factors. Lower incubation temperatures (Table 1) and greater surfactant concentrations
(Fig. 1) result in greater surfactant-rich phase volumes (and smaller surfactant-lean phase volumes).
Only a small handful of data has been reported on the actual incubation temperature – TX-114
concentration combinations required to achieve volume ratios in the range of 0.10 – 1.1 for
excluded volume CPE.18, 19 One goal of this research was to experimentally compile a much larger
data set of such information for the separation science community.
Table 2. Summary of the actual temperature observed following incubation of the aqueous
surfactant TX-114 solutions at the specified temperature in a constant temperature water bath for
the indicated incubation time immediately following the 2.00 minute centrifugation step. a
[TX-114]
mM
Temp (oC )
80 70 50 35
Time (min)
Actual Temperature of Surfactant-Rich Phase Volume
34.9
5 43.10 ± 0.70
41.27 ± 0.50
30.28 ± 0.87
28.35 ± 0.45
10 42.56 ± 0.10
40.73 ± 0.66
28.50 ± 0.40
69.7
5 44.37 ± 0.23
40.18 ± 0.71
30.45 ± 0.00
28.73 ±
0.000
10 44.52 ± 0.16
40.62 ± 0.33
31.89 0.87
a. Values are the average of triplicate determinations.
54
The volume ratio (volume of the surfactant-lean or micelle-poor top phase divided by the
volume of the surfactant-rich bottom phase) was determined for aqueous solutions of Triton X-114
(present at four different TX-114 concentration levels: 5.30, 6.02, 7.20 and 9.50 % (w/w)) after
incubation for 18 hours at different temperatures (over the temperature range of ca. 27 to 40oC).
The lowest temperature at which volume ratio measurements could be made is dictated by the cloud
point temperatures at the four different TX-114 concentrations (Table 3 summarizes the cloud
points). The dependence of the volume ratio upon temperature is summarized in Fig. 4. The
general trends are as follows: (i) at a fixed incubation temperature, the volume ratio decreased with
an increase in total surfactant concentration and (ii) at fixed TX-114 concentration, the volume
ratio decreases decreased as the incubation temperature decreased. In both instances, the decrease
in volume ratio is a consequence of the fact that the volume of the surfactant-rich phase increases
while that of the surfactant-lean phase decreases. At 32oC and for the 9.90% (w/w) TX-114 solution,
the volume ratio was 0.07 (which means that the maximum enrichment factor would be ca. 14).
The data presented in Fig. 4 allows for quick estimation of the incubation temperature and
surfactant concentration required in order to achieve a desired volume ratio for cloud point
extraction of large biomolecules based on the excluded volume effect. When comparing of some
of our results (Fig. 4) with some of the few reported literature values, our experimentally
determined values were found to be lower. Then it was realized that our results were obtained in
aqueous solution with just the added TX-114 surfactant (in the absence of buffers for pH control
or salts for ionic strength control). In fact, literature reports indicate that inert salts are often added
prior to conducting CPE in order to facilitate the phase separation process.20-23 It should be noted
that addition of salts can also alter the cloud point temperature relative to that seen in its absence
24, 25 (refer to Table 3, chapter 1).
55
Figure 4. Dependence of the volume ratio in the phase separated solutions of the surfactant TX-
114 present at the indicated concentrations as a function of the incubation temperature. After
preparation, all solutions were initially cooled to 4oC (well below their cloud point temperature) in
order to have a homogeneous one phase solution present for mixing prior to being placed in a
constant temperature water bath for 18 hours. Immediately after removal from the water bath, the
volumes of the surfactant-rich and surfactant-lean phases were measured and the volume ratio
calculated (as the volume of the top surfactant-lean phase divided by the volume of the bottom
surfactant-rich phase).
Table 3: The cloud point temperature of the Triton X-114 solutions at the four indicated TX-114
concentrations (in percent mass).
A limited study was made to gauge the impact of buffer and salt additives upon the volume
ratio of aqueous solutions of 7.20 % (w/w) prepared in just distilled water (ionic strength, I = 0),
0
0.5
1
1.5
2
2.5
3
27 29 31 33 35 37 39
VO
LUM
E R
ATI
O,
INCUBATION TEMPERATURE, OC
5.30% w/w
6.02% w/w
7.20% w/w
9.90% w/w
[TX-114] .% (w/w) Cloud Point Temperature (oC)
5.3% 27.6
6.0% 28.2
7.2% 29.3
9.9% 30.0
56
pH 4.63 buffer (I = 0.10), pH 9.0 buffer (I = 0.15), Hyclone DPBS buffer (I = 0.17) and 0.25 M
NaCl (I = 0.25) after incubation for 18 hours in a constant temperature bath at fixed temperatures
of 32, 34 and 38oC. The results are summarized in Fig. 5. As can be observed, the greater the
ionic strength of the surfactant containing solution, the greater is the volume ratio at any fixed
incubation temperature. For instance, at an incubation temperature of 32oC, the volume ratio is
0.45 in aqueous solutions of 7.2% (w/w) TX-114 whereas in aqueous 0.25 M NaCl solutions at the
same concentration of TX-114, the ratio is just over 1.1.
Figure 5. Effect of ionic strength at three temperatures at a fixed TX-114 concentration of 7.20
wt. % upon the volume ratio following incubation for 18 hours. The ionic strengths were: water (I
= 0); pH 4.63 buffer (I=0.10); pH 9.00 (I = 0.15); Hyclone DPBS (I = 0.17); and 0.25 M NaCl (I =
0.25).
Lastly, a brief study was conducted to see if the 18 hour incubation step could be replaced
by centrifugation. Although not specifically stated in any of the excluded volume CPE reports,6, 8,
12, 13, 18, 19 the 18 hour incubation was most likely employed because the sample will cool during the
centrifugation step (as shown by our data in Table 2) and resultant drop in temperature would alter
the volume ratio (Fig. 4). Since the temperatures required for the minimum volume ratio are very
0.1
0.6
1.1
1.6
2.1
30 31 32 33 34 35 36 37 38 39 40
VO
LUM
E R
ATI
O
INCUBATION TEMPERATURE , OC
0.25 M NaCl
pH 4.63pH 9.00Hyclone
Water
57
close to the cloud point temperatures of the surfactant, such temperature drop during centrifugation
could be such that the solution ends up being below the CPT with the phases then beginning to
coalesce and becoming more homogeneous. Therefore, in this study, the prepared TX-114
solutions were placed in the centrifuge and then the entire centrifuge was placed in an Isotemp
Incubator so that both the solution incubation step and centrifugation step were conducted at the
same fixed temperature. The results are summarized in Table 4. The time of centrifugation (2.0 vs.
5.0 minutes) did not appreciably alter the volume ratios obtained, which is in agreement with prior
literature reported for regular CPE.26 More important is a comparison of these results obtained
after equilibration at 37.0oC in an incubator for 30.0 minutes followed by a 2.0 or 5.0 minute
centrifugation step with the results obtained after incubation at 37.0oC in a water bath for 18.0 hours
with gravity settling. At a TX-114 concentration of 5.3% (w/w), the volume ratio obtained were
2.00 (or 2.04) (Table 4) which compares to a volume ratio value of 2.17 obtained after 18 hours.
At a TX-114 concentration of 9.9 % (w/w), the ratios were 0.62 (or 0.63) for 30 minutes
equilibration followed by centrifugation, which compares well to the volume ratio of 0.60 obtained
after 18 hours.
It seems that the CPE based on excluded volume effects could be greatly speeded up if a
short equilibration time followed by a centrifugation step is employed compared to the 18 hours
incubation with phase separation achieved due to gravity. The main caution, however, is that the
equilibration, incubation, and centrifugation step must be conducted at the same constant
temperature in order to avoid cooling during the centrifugation step. Such an approach should
make excluded volume CPE a more viable approach for the extraction of large biomolecular
analytes.
58
Table 4. Summary of the volume ratio obtained for aqueous solutions of TX-114 upon surfactant
concentration and centrifugation time.a
Centrifuge Time (min)
2.00 5.00
[TX-114] wt.% Volume Ratio in the TX-114 System
5.3% 2.00 2.04
9.9% 0.62 0.63
a. Conditions: Solutions of TX-114 at the indicated two concentrations were equilibrated at
37.0oC in an incubator for 30.0 minutes after which they were centrifuged (centrifuge also in
incubator) for 2.00 or 5.00 minutes. The volumes of the respective phases were determined
immediately after the centrifugation step. The values represent the mean of 4 trials.
3.4 Conclusions
Conditions were identified under which the surfactant-lean (micelle-poor) phase in phase
separated solutions of TX-114 are at a minimum (thus resulting in maximum enrichment factors).
In addition, an alternative to the current use of an 18 hour incubation step was identified. Namely,
a shorter equilibration time in conjunction with a centrifugation step was found to give essentially
the same volume ratio as was observed after 18 hours incubation. These two main results should
prove valuable to separation scientists who might wish to employ the cloud point extraction based
upon excluded volume effects for the extraction and enrichment of large biomolecules.
59
CHAPTER 3 REFERENCES
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theory and applications. Crit. Rev. Anal. Chem., 1993, 24, 1733-177.
2. Watanabe, H.; Tanaka, H. A nonionic surfactant as a new solvent for liquid-liquid
extraction of zinc (II) with 1-(2-pyridylazo)-2-nahthol. Talanta, 1978, 25, 585-589.
3. Bordier, C. Phase separation of integral membrane proteins in Triton X-114 solutions. J.
Biol. Chem., 1981, 256, 1604-1607.
4. Lue, L.; Blankschtein, D. A Liquid-State Theory Approach to Modeling Solute
Partitioning in Phase-Separated Solutions. Ind. Eng. Chem. Res., 1996, 35 (9), 3032–
3043.
5. Nikas, Y. J.; Puvvada, S.; Blankschtein, D. Surface tensions of aqueous nonionic
surfactant mixture. Langmuir 1992, 8, 2680-2689.
6. Mashayekhi, F.; Meyer, AS.; Shiigi, S. A.; Nguyen, V.; Kamei, D.T. Concentration of
Mammalian Genomic DNA Using Two-Phase Aqueous Micellar Systems. Biotechnol.
Bioeng. 2009, 102, 1613-1623.
7. Rangel-Yagui, CO; Pessoa, A.; Blankschtein, D. Two-Phase Aqueous Micellar Systems
– An alternative Method for Protein Purification. Brazilian J. Chem. Engineering, 2004,
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8. Mashayekhi, F.; Chiu, RYT; Le, AL; Chao, FC; Wu, AM.; Kamei, D.T. Anal. Bioanal.
Chem., 2010, 398, 2955-2961.
9. Albertsson PA. Separation of Cell and Cell Organelles by Partition in Aqueous Polymer
Two-Phase systems. Methods Enzymol 1989, 171, 532-549.
10. Albertsson PA.; Cajarville A; Brooks, DE; Tjerneld F. Partition of Proteins in Aqueous
Polymer Two-Phase Systems and The Effect of Molecular Weight of The Polymer.
Biochim Biophys Acta 1987, 926, 87-93.
60
11. Capezio L; Romanini D; Pico GA; Nerli B.: Partition of Whey Milk Proteins in Aqueous
Two-Phase Systems of Polyethylene Glycol-Phosphate as A Starting Point to Isolate
Proteins Expressed in Transgenic Milk. J Chromatogr B Analyt Technol Biomed Life Sci.
2005, 819, 25-31.
12. Kamei, DT.; King, JA; Wang, DC.; Blankschtein, D. Understanding Viral Partitioning in
Two-Phase Aqueous Nonionic Micellar Systems: 2. Effect of Entrained Micelle-Poor
Domains Biotechnol. Bioeng. 2002, 78, 203-216.
13. Kamei, D. T.; King, J. A.; Wang, D. C.; Blankschtein, D. Separating Lysozyme from
Bacteriophage P222 in Two-Phase Aqueous Micellar Systems. Ind. Eng. Chem.
Res., 2002, 80, 233-236.
14. Helenius A.; Simons K. Solubilization of Membranes by Detergents. Biochim Biophys
Acta. 1975, 415, 29-79.
15. Koshy, L.; Saiyad, A.H.; Rakshit, A.K. The Effects of Various Foreign Substances on the
Cloud Point of Triton X 100 and Triton X 114. Colloid Polym Sci, 1996, 274,582-587.
16. Chen,J.; Teo,K. C. Determination of cobalt and nickel in water samples by flame atomic
absorption spectroscopy after cloud point extraction. Anal. Chim. Acta, 2001, 434, 326-
330.
17. Ferrera,Z. S.; Sanz,C.P.; Santana, C.M.; Rodriguez, JJS. Use of micellar systems in
extraction and preconcentration of organic pollutants in environmental samples. Trends
Anal. Chem., 2004, 22, 469-479.
18. Mashayekhi,F.; Meyer, A.S.; Shiigi, S. A.; Nguyen, V.; Kamei, D. T. Concentration of
Mammalian Genomic DNA using Two Phase Aqueous Micellar Systems. Biotechnol.
Bioeng. 2009, 102, 1613-1623.
19. Shiigi, S. A.; Meyer, A. S.; Kamei, D. T. Enhancing the detection of urinary tract infections
using two-phase aqueous micellar systems. The UCLA USJ, 2009, 22, 47-56.
61
20. Akita, S.; Takeuchi, H. Cloud-Point Extraction of Organic Compounds from Aqueous
Solutions with Nonionic Surfactant. Sep.Sci.Technol. 1995, 30, 833-846.
21. Stangl, G.; Niesner, R. Michellar Extraction-A New Step for Enrichment in the Analysis
of Napropamide. Int. J. Environ. Anal. Chem. 1995, 58, 15-22.
22. Fernandez, A. E.; Ferrera, Z. S.; Rodriguez, J. J. S. Determination of Polychlorinated
Biphenyls by Liquid Chromatography Following Cloud-Point Extraction. Anal. Chim. Acta
1998, 358, 145.
23. Fernandez, A. E.; Ferrera, Z. S.; Rodriguez, J. J. S. Application of Cloud Point
Methodology to the Determination of Polychlorinated Dibenzofurans in Sea Water by
High-Performance Liquid Chromatography. Analyst 1999, 124, 487-491.
24. Watanabe, H.; Tanaka, H. A non-ionic surfactant as a new solvent for liquid—liquid
extraction of zinc(II) with 1-(2-pyridylazo)-2-naphthol. Talanta, 1978, 25, 585-589.
25. Hoshino, H.; Saitoh, T.; Taketomi, H.; Yotsuyanagi, T.: Watanabe, H.; Tachikawa, K.
Weak binding of N-alkylpyridinium ions to nonionic surfactant micelles as studied by
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62
Chapter 4- Conclusions and Future Work
In Chapter 2, the use of an anionic SDS micellar medium for the quenchfluorimetric
determination of copper (II) ion based upon its ability to quench the fluorescence of the luminescent
probe, NMA, was found to lead to an increase of the KSV value by a factor of ca. 80 relative to that
observed in water alone as solvent under the same conditions. The concentration of the fluorescent
probe, NMA, has a fairly significant effect on the limit of detection as greater probe concentrations
yielded smaller fluctuations in the background fluorescence signal (smaller standard deviations)
with resultant improved detection limits. Under optimal conditions, the limit of detection for copper
(II) in SDS micelles is over two orders of magnitude better relative to that in water as solvent. In
the SDS micellar medium (at fixed SDS concentrations), temperature only slightly influences the
KSV value over the temperature range from 25 – 35oC. Although increased KSV values had been
reported if SDS was replaced by the more hydrophobic surfactant, NaTDS, those results could not
be reproduced in this study. Due to the greater Krafft temperature of NaTDS (relative to SDS),
the experimental is more involved as a temperature bath is required and results were not
reproducible. In addition, the cost of NaTDS is greater than that for SDS. Based on our work, SDS
appears to be the anionic micelle forming surfactant of choice for determination of copper (II)
cation. Many other cations would be expected to also quench NMA and thus interfere with the
method for Cu2+ ion although interference from anions would be expected to be less severe relative
to water as solvent for its determination.
In regards to future work in this area, additional experiments that could be performed include:
(i) repeating the Ksv vs. temperature studies at a constant SDS micelle concentration to see if the
KSV values would then increase with temperature as is observed in non-micellar homogeneous
solvent systems; (ii) conducting the quenching experiments with NaTDS at much greater
temperatures well above the highest reported Krafft point (> 48oC) to see if then the previously
reported results with PSF could be reproduced;25 (iii) conducting additional experiments in the
63
[NMA] concentration range from 5.0 10-6 M to 1.0 10-5 M to see if the trend of lower detection
limit with increased [NMA] is observed in that range; and (iv) obtaining more complete recovery
results; perhaps employing some known commercial copper (II) reference standard solutions.
In Chapter 3, conditions were identified under which the surfactant-lean (micelle-poor) phase
in phase separated solutions of TX-114 are at a minimum (thus resulting in maximum enrichment
factors). In addition, an alternative to the current use of an 18 hour incubation step was identified.
Namely, a shorter equilibration time in conjunction with a centrifugation step was found to give
essentially the same volume ratio as was observed after 18 hours incubation. These two main
results should prove valuable to separation scientists who might wish to employ the cloud point
extraction based upon excluded volume effects for the extraction and enrichment of large
biomolecules.
Regarding future research on this topic, the following experiments might prove interesting: (i)
determination of the volume ratio of a solution(s) containing a greater amount of TX-114 (12.0 %
(v/v)) to further expand the current data in Figure 4; (ii) conduct many more experiments to
determine the volume ratio of solutions of TX-114 using the equilibration incubation –
centrifugation step to expand the type of data reported in Table 4 (at more different TX-114
concentration levels, at many more temperatures, in both water and in buffer/salt, etc.); and (iii)
select a system and conduct a excluded volume cloud point extraction for a model biomolecule
using the equilibration incubation – centrifuge step and compare those extraction results to that
obtained using the previously published 18 hour incubation approach.
64
SCHOLASTIC VITA
LIN ZHANG
BORN: March 30, 1987
An’shan, China
UNDERGRADUATE STUDY: Shaanxi Normal University Xi’an, China
B.S., Chemistry, 2010
GRADUATE STUDY: Wake Forest University
Winston-Salem, North Carolina
Master, Analytical Chemistry, present
SCHOLASTIC AND PROFESSIONAL EXPERIENCE:
Compliance Regulatory Intern, Inmar, Winston-Salem, NC, 2013 – present
Scientific Associate, Novartis, Boston, MA, summer 2013
Teaching Assistant, Wake Forest University, Winston-Salem, NC, 2011-2013
PUBLICATIONS:
“A Unique Au-Ag-Au Triangular Motif in a Trimetallic Halonium Dication: Silver Incorporation in a Gold (I) Catalyst.” Yuyang Zhu, Cynthia Day, Lin Zhang, Katarina J.
Hauser, Amanda C. Jones*, A. C. Chem. Eur. J. 2013, 19, 1226412271.
“Factors Affecting the Use of Organized Surfactant Assemblies for Amplification of Fluorescence Quenching with Application to the Quenchiofluorimetric Determination of
Copper (II) Ion.” Aamna Balouch, Lin Zhang, Muhammad I., Bhanger, KN Thimmaiah
and Willie Hinze* Manuscript In Preparation
PRESENTATIONS:
“Use of Micelles to Enhance Fluorescence Quenching.” Lin Zhang and Willie Hinze, 14th
ACS Poster & Vendor Night, Greensboro, NC, 2014
“NMR Analysis to Study Intramolecular H-bonds in Small Molecules.” Lin Zhang, Global
Discovery Chemistry Department Meeting, Novartis, Boston, MA, 2013
65
“Ligand-Silver Interaction in a Homogeneous Gold (I) Catalyst.” Yuyang Zhu, Lin Zhang
and Amanda C. Jones, 245th ACS National Meeting, New Orleans, LA, 2013
“Ligand-Silver Interaction in a Homogeneous Gold(I) Catalyst.” Yuyang Zhu, Lin Zhang
and Amanda C. Jones, 13th Annual Graduate Student and Postdoctoral Research Day,
Wake Forest University, NC, 2013