MIAMI UNIVERSITY The Graduate School. Certificate for Approving the Dissertation

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1 MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the dissertation of Michelle Marie Wandstrat Candidate for the Degree: Doctor of Philosophy Prof. James A. Cox, Director Prof. Gilbert E. Pacey, Reader Prof. Neil D. Danielson, Reader Prof. Richard T. Taylor, Reader Prof. Jan M. Yarrison-Rice, Graduate School Representative

2 ABSTRACT MATERIALS AND MODIFICATION OF ELECTRODES FOR THE DETECTION OF BIOLOGICAL MOLECULES by Michelle Marie Wandstrat Sol-gels have been utilized in many applications due to many desirable properties. A limitation of sol-gels is the gradual collapse of their structure with time. To address this problem, sol-gels were formed from tetramethoxysilane in the presence and absence of generation-zero poly(amidoamine) dendrimers (G0-PAMAM). To test the hypothesis that G0-PAMAM, which serves as a crosslinking agent, will stabilize the structure, the pore volume was monitored over a 52-week period. The premise was that the crosslinking will result in rapid densification. Nitrogen adsorption data stabilized after 25 weeks when G0- PAMAM was present. In its absence, the total pore volume continued to decrease over the entire period. Phospholipids (PLs) determination is important in the food and pharmaceutical industries. Methods were developed for the detection of a PL, phosphatidylcholine (PC). Electrochemical oxidation of PC, was accomplished at a 4-aminothiophenol (ATP)- modified gold electrode coated with a layer-by-layer assembly of electrochemical catalyst (dirhodium phosphomolybdic acid), a trapping agent for PC (a cyclophane, CP, derivative, 1,4-xylylene-1,4-phenylene-diacetate), and a spacer (generation-4 polyamidoamine dendrimer, PAMAM). The hypothesized process was verified by quartz crystal microbalance measurements which showed that Au ATP CP PAMAM CP trapped (1.5 ± 0.4) 10-9 mol cm -2 of PC. The oxidation of PC at 0.16 V vs. Ag AgCl yielded a current that varied linearly with concentration over the range 1-50 µm.

3 Finally, an electrochemically assisted method for deposition of nm-scale films of porous silica was developed. In aqueous, un-buffered solution at a potential where H + is generated in a tetraethylorthosilicate sol, nm-scale, insulating deposits of silica on electrodes are formed. Inclusion of β-cyclodextrin in the sol provides conductivity, presumably by producing channels in the film. With the addition of a mediator, Rh II, the electrochemical oxidation of PC is demonstrated; the process occurs without the passivation that otherwise precludes the voltammetric determination of PLs. Voltammetric current proportional to PC concentration in the range µm is achieved with this electrode.

4 MATERIALS AND MODIFICATION OF ELECTRODES FOR THE DETECTION OF BIOLOGICAL MOLECULES A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry by Michelle Marie Wandstrat Miami University Oxford, Ohio 2006 Dissertation Director: Dr. James A. Cox

5 Table of Contents Table of Contents List of Tables List of Figures Dedication Acknowledgements Page ii vii viii xii xiii 1 Chapter 1 1 Sol-gel Chemistry 1.1 Introduction Sol-gel processing Hydrolysis and condensation Gelation Aging Drying Xerogels Ambigels Aerogels Stabilization Factors affecting sol-gel structure Basic sol-gel components: precursor, water, alcohol Catalyst type and concentration Temperature Additives Surfactants 16 ii

6 1.4 Characterization of sol-gel materials: surface area and pore size 18 distribution Adsorption and isotherms Surface area and porosity basics Surface area Pore size distribution 23 2 Chapter 2 26 Acceleration of the Densification of a Silica Sol-gel by Inclusion of Generation-zero Poly(amidoamine) Dendrimer 2.1 Introduction Dendrimers Densification Experimental Reagents Apparatus Sol-gel synthesis Results and discussion Moisture Isotherms Surface area Total pore volume Pore size distribution Conclusions 47 3 Chapter 3 48 Imprinting of Biological Molecules 3.1 Introduction Voltammetry Templated sol-gels Ambigels 53 iii

7 Ormosils Dopamine Experimental Reagents Apparatus Ambigel synthesis Ormosil synthesis and the templating process Results and discussion Ambigels Cyclic voltammetry of templated ormosils Square wave voltammetry of templated ormosils Aging of templated ormosils Conclusions 64 4 Chapter 4 67 Phospholipids 4.1 Introduction Types Fatty acids Separation and determination Thin-layer chromatography Gas chromatography Capillary electrophoresis High performance liquid chromatography Detectors Refractive index Ultraviolet Evaporation light scattering detection Mass spectrometry Electrochemical detection of phospholipids after chemical treatment Preliminary study of phospholipid detection by electrochemistry 82 iv

8 5 Chapter 5 84 Preconcentration and determination of a phospholipid at a surface modified by layer-by-layer assembly 5.1 Introduction Layer-by-layer Electrostatic layer-by-layer assembly Quartz crystal microbalance Cyclophane Polyoxometalates Electrochemical oxidation of phospholipids Experimental Reagents Apparatus Procedures Cyclophane synthesis Synthesis of dirhodium molybdophosphate acid Layer-by-layer assembly Electrochemical measurements Results and discussion Initial quartz crystal microbalance measurements Timed quartz crystal microbalance measurements Quartz crystal microbalance measurements of acetylcholine trap Quartz crystal microbalance measurements of phosphatidylcholine trap Electrochemical determination of phosphatidylcholine Conclusions Chapter Oxidation of a Phospholipid at an Electrode Modified with an Electrochemically Formed Sol-Gel Film Doped with Cyclodextrin 6.1 Introduction 110 v

9 6.1.1 Cyclodextrin Mass transport Electrodeposition Sol-gel modified electrodes Experimental Reagents Apparatus Procedures Synthesis of dirhodium molybdophosphate acid Electroplating sol-gel films Results and discussion β-cyclodextrin-templated films Mass transport Inclusion of Rh II in silica film Dependence of cyclodextrin concentration on film properties Oxidation of phosphatidylcholine Conclusion Chapter 7 Conclusions 143 vi

10 List of Tables Tables 1 Comparison of the surface coverage of the outer layers, CP and CP PAMAM, with different immersion times. 2 Comparison of the amounts of ACH trapped by the listed CP-containing LBL assemblies in a 30-min immersion in a 10-µM solution. 3 Comparison of the amounts of PC trapped by the listed CP-containing LBL assemblies in a 30-min immersion in a 10-µM solution Page vii

11 List of Figures Figure Page 1 Polycondensation of tetramethylorthosilicate 4 2 Different drying conditions 7 3 Phase diagram for tetraethylorthosilicate 11 4 Hydrolysis mechanism 12 5 Condensation mechanism 14 6 Templating sol-gels 17 7 Classification of isotherms 20 8 Structures of poly(amidoamine) dendrimers 28 9 Thermogravimetric study of GO-PAMAM doped sol-gel Thermogravimetric determination of solvent content of undoped TMOSderived 34 silica and a composite of G0-PAMAM 11 Isotherm of G0-PAMAM doped silica Isotherm of undoped silica BET plot of G0-PAMAM doped silica Influence of aging time on the surface area for undoped TMOS-derived 39 silica and a composite of G0-PAMAM with silica 15 Influence of aging time on the total pore volume for undoped TMOSderived 41 silica and a composite of G0-PAMAM with silica 16 Pore size distribution of composites of G0-PAMAM and TMOS-derived 43 silica 17 Pore size distribution of TMOS-derived silica Pore size distribution under 6 nm (3.2 to 6 nm) of G0-PAMAM doped 45 silica 19 Pore size distribution under 6 nm (3.2 to 6 nm) of TMOS-derived silica Waveform for square wave voltammetry Cyclic potential sweep 51 viii

12 22 Structure of dopamine Cyclic voltammogram of 0.1 mm dopamine at ITO electrode Verification of the deposition of a passivating film of silica Cyclic voltammograms at dopamine-templated TMOS-PTMOS-MTMOS 62 ITO electrode 26 Square wave voltammogram at TMOS-PTMOS-MTMOS ITO electrode Cyclic voltammograms at dopamine-templated TMOS-PTMOS-MTMOS 65 ITO electrode after 2 weeks of aging 28 Cyclic voltammograms of dopamine-templated TMOS-PTMOS-MTMOS 66 ITO electrode, with the addition of G0 PAMAM in the sol, after 2 weeks of aging 29 Types of phospholipids Saturated fatty acids Unsaturated fatty acids Deacylation of phosphatidylinositol Structures of rhodamine and ninhydrin Structure of 10-N-nonyl-3,6-bis(dimethylamino)acridine Cyclic voltammetry at dirhodium phosphomolybdic acid doped sol-gel 83 electrode 36 Structures of molecules used for the modification of layer-by-layer 86 substrates 37 Electrostatic layer-by-layer formation Diagram of quartz crystal microbalance Structures of acetylcholine and phosphatidylcholine Model of Wells-Dawson [P 2 Mo 18 O 62 ] 6- polyoxometalate Schematic of LBL assembly of PAMAM and CP on Au modified with a 98 self-assembled monolayer of MBA 42 Schematic of LBL assembly of CP and PAMAM on Au modified with a 99 self-assembled monolayer of ATP 43 Cyclic voltammogram of 5.0 mm Rh 2 PMA at an ITO electrode 107 ix

13 44 Cyclic voltammetric determination of PC at the assembly, Au ATP CP 108 PAMAM CP c PC Rh 2 PMA 45 Structure of the different types of cyclodextrin molecules Cavity of the different types of cyclodextrin molecules Mass transport Theoretically peak shapes of cyclic voltammograms undergoing diffusion Sol-gel modified ITO electrode with cyclodextin Electrochemical system for electroplating thin films of sol-gel Electroplating thin sol-gel films on an ITO electrode Cyclic voltammogram of 1 mm ferrocene at a silica-modified electrode 125 prepared from TEOS film without and with CD in electroplating solution. 53 Cyclic voltammogram of 1 mm ferrocene at ITO coated with CDtemplated 126 TEOS film 54 Influence of scan rate on the cyclic voltammetry of ferrocene at CD doped 127 TEOS electrode 55 Cyclic voltammogram of 1 mm ferrocene at ITO coated with CDtemplated 128 TMOS film 56 Influence of scan rate on the cyclic voltammetry of ferrocene at CD doped 129 TMOS electrode 57 Cyclic voltammogram of 1 mm K 4 Fe(CN) 6 at ITO coated with CDtemplated 131 silica 58 Influence of scan rate on the cyclic voltammetry of hexacyanoferrate Cyclic voltammograms of 0.1 M Bu 4 NPF 6 at silica-coated ITO plated 134 from CD-containing TEOS in the presence and absence of 10 mm Rh 2 Ac 60 Cyclic voltammogram at Rh II, CD-templated TEOS ITO electrode Influence of scan rate on the cyclic voltammetry of Rh II Cyclic voltammetry of 0.1 M Bu 4 NPF 6 at silica-coated Au plated from CD-containing TEOS in the presence of 10 mm Rh 2 Ac and 5 mm CD or 10 mm CD 138 x

14 63 Cyclic voltammograms at a gold electrode modified with a film of CDtemplated silica in the sequence: first, 0.1 M Bu 4 NPF 6, 100 µm PC, and 139 final, in 0.1 M Bu 4 NPF 6 64 Cyclic voltammograms at a gold electrode modified with a film of CDtemplated 140 silica in PC 65 Cyclic voltammograms at silica-coated Au plated from CD-containing 142 TEOS in the presence of 5 mm CD in PC 66 Separation and preconcentration using a nano-assembly 144 xi

15 Dedicated to Theresa, Matt, and Jaylin and in loving memory of my mother, Janet xii

16 Acknowledgements I would like to thank my advisor, Prof. James A. Cox, for his guidance and support. You helped me become a better scientist. There are several people I like to thank for helping me with my research: Dr. Anna Kijak for helping me get started with the phospholipid project, Dr. Tom Scott for helping me with the cyclophane synthesis, and Dr. Wolf Spendel for helping me with the cyclodextrin sol-gel project. I also like to thank Diep, Dorota, and Laisheng for all their help and friendship. I like to thank all my family including: Margie, for printing my dissertation for my committee; Jaylin, Makenzee, and Trinity for the little joys in life; and Grandma Wandstrat and my father for their support. I would especially like to thank my brother, Matt, for all his support and comic relief. Most of all, I like to thank my sister and best friend, Theresa, for all her support and especially for typing much of this dissertation. xiii

17 Chapter 1 Sol-gel Chemistry 1.1 Introduction Sol-gels are used today in many fields including sensors, coatings, and separations due to their numerous advantages over other materials. Sol-gels can be formed into films, fibers, powders, and monoliths. Also, the processing conditions of solgels can be manipulated. For instance, changing the type and concentration of sol components allows for sol-gels to be used for different detection methods based on size, charge, shape, and chemical affinity. Three basic states are represented in a typical solgel process: the sol or suspension of colloid particles, a viscous gel, and solid porous material. Although some form of sol-gels has been known since the 1600 s, sol-gels were not widely used until much later. Historically, colloids have been used for thousands of years, appearing in cave paintings dating back 17,000 years [1]. The formal beginning of sol-gel science originated with the discovery of water glass. Upon the addition of acid to a solution containing dissolved silicate materials in alkali, von Helmont [2], in 1644, discovered that the weight of the precipitate of silica equaled the weight of the original silicate material. In 1846, Ebelmen formed the first reported silicon alkoxide sol-gel. He found that when SiCl 4 and ethanol were exposed to the atmosphere, the solution slowly formed a gel at room temperature. This product underwent repeated condensation to form polysilanes. He hoped to utilize the sol-gel material in optical instruments [3]. However, the long drying time, approximately one year, resulted in little interest in this area until the early 1900 s. At this time, Patrick developed sol-gels as a means of constructing catalytic materials [4, 5]. Later, Geffcken and Berger [6, 7] of Schott Glass Company were searching for a glass covering with improved insulation characteristics. The glass was dip-coated in alkoxides, forming an antireflection coating. It was not until the 1930 s that scientists agreed on a model explaining sol-gel structure. In 1864, Graham [8] showed evidence that supported the model that sol-gels 1

18 were solid materials containing networks of pores. He replaced water with organic solvent in silica gel. However, at this time there was a competing postulate that the gel structure consisted of a coagulated sol with particles surrounded by a layer of bound water. It was not until the 1930 s, primarily due to Hurd s work [9] that Graham s model was widely accepted. He showed that the structure of silica gels consisted of a polymeric skeleton of silicia acid surrounded in a liquid phase. Also in the 1930 s, Kistler[10] developed a new type of gel called aerogels while studying the gel structure. Beginning in the late 1930 s, scientists were developing sol-gel methods to prepare homogeneous powders [11]. Roy [12, 13] synthesized ceramic powders including Al, Si, Ti, Mg, Ca, and Ba oxides for phase equilibrium studies. Independently, Levene and Thomas [14] and Dislich [15] developed multicomponent ceramic materials by controlling hydrolysis and condensation of alkoxides. In 1968, Stober et al. [16] determined that the final size and morphology of silica based powder could be controlled by manipulating the amount and type of components in the sol. The ability to produce monoliths by controlling the drying conditions of the gels was developed by Yoldas [17, 18] and Yamane et al. [19] in the 1970 s. Since then, interest in sol-gel technology has gained increasing interest. Sol-gels are widely used due to the numerous advantages over other materials. For example, sol-gels are usually synthesized at mild reaction conditions, e.g. low temperatures and relatively mild phs. Many thermal labile biological molecules have been entrapped within sol-gels and, most importantly, their functions were retained. Solgels are also highly porous, permitting the entrapment and controlled release of molecules [20-22]. In addition, most precursors are volatile and therefore high-purity starting materials can be used, leading to high-purity products. Also, chemically modifying sol-gels can be accomplished with many functional groups. In addition, numerous characteristics such as shape, average pore size, pore size distribution, surface area, refractive index, and polarity can be controlled by manipulations in sol-gel processing. Sol-gels have been utilized as catalysts, chemical sensors, membranes, optical components, coatings, and electrochemical matrices. 2

19 Although these numerous advantages have led to many applications, there are three main problems often encountered with sol-gels: shrinking, stress cracking, and time-consuming processing. Ways to address these problems will be discussed. 1.2 Sol-gel processing As stated previously, three unique states are involved in common sol-gel processing. First, a sol or colloidal suspension is formed. Colloids are 1 µm to 1 nm particles suspended in Brownian motion. The precursor undergoes a series of hydrolysis and polymerization reactions, forming a rigid wet gel in the shape of the vessel containing it. After drying and further reactions, the gel shrinks and converts into a solid. Several steps take place in between these states: hydrolysis, condensation, gelation, aging, drying, and stabilization. Most of the studies discussed in later chapters involve sol-gels made with silicates. Therefore, this chapter will focus mainly on silica sol-gels Hydrolysis and condensation Generally, the reactions during sol-gel processing are catalyzed by an acid or base and therefore undergo different pathways. Several other variables control the kinetics of these reactions including the nature and concentration of solvent and precursor. In this section, the general reaction equations will be given. The mechanisms will be discussed in section 3 of this chapter. During hydrolysis, the alkyl groups of the precursor react with water: Si(OR) 4 + nh 2 O Si(OR) 4-n (OH) n + nroh (1) where R is an alkyl group. Usually hydrolysis and condensation occur simultaneously. The hydrated silica tetrahedral molecules undergo condensation, forming a Si-O-Si bond and either alcohol or water. Si-OR + HO-Si Si-O-Si + ROH (2) Si-OH + HO-Si Si-O-Si + H 2 O (3) These molecules undergo further condensation or polycondensation eventually resulting in SiO 2 networks. An example of polycondensation is shown in Fig 1. of the precursor tetramethylorthosilicate (TMOS). Although water and alcohol are usually added to the 3

20 Fig. 1 Polycondensation of tetramethylorthosilicate. 4

21 sol, both are products of these reactions. In fact, gels can be prepared without alcohol by applying ultrasound [27]. The ratio of the hydrolysis to the condensation rate influences the structure of the gel. Linear polymers will form if the hydrolysis rate is faster than the condensation rate. If the opposite occurs (hydrolysis is slower compared to condensation), then highly crosslinked polymers will form [1, 23] Gelation The structure of a gel is established at the time of gelation. After numerous condensation reactions, large clusters form. These clusters expand and take the shape of the mold, in the case of a monolith. The viscosity rapidly increases so that the sol will not pour from the vessel containing it. However, there are many sol particles entrapped in the spanning clusters. Initially the gel has a high viscosity but a low elasticity. The elasticity of the gel continues to increase over time. Although it is difficult to measure the exact gel time, scientists study several properties of the developing sol-gel to estimate the time. Some methods are based on certain values of viscosity, others are based on the elasticity of the gel, and others, on a combination of both. Perhaps the most widely used method was developed by Sacks and Sheu [24, 25], where the gelation time is measured by the viscoelastic response of the gel as a function of shear rate Aging If a gel is allowed to set, it will undergo the aging process. In fact, the reactions previous described continue even after gelation. During the overall aging process, the gel shrinks to form a transparent solid monolith immersed in liquid. Four processes may be seen as a gel ages: polymerization, syneresis, coarsening, and phase transformation. Polymerization or further condensation takes place, increasing the number of linkages. This occurs due to the large concentration of labile hydroxyl groups. Spontaneous shrinkage of the gel and subsequent expulsion of liquid from pores, also known as syneresis, causes the gel to shrink. Syneresis is affected by the condensation reactions and the tendency to reduce the large solid/liquid interfacial area of the gel. As the gel begins to stiffen, the shrinkage rate decreases because it becomes harder to 5

22 squeeze liquid out of the pores. The rate of syneresis increases with the concentration of the precursor and temperature. Due to solubility difference, some of the small particles that previously dissolved in the gel will reprecipitate into larger particles. This is known as coarsening or ripening. Coarsening is affected by temperature, ph, concentrations and type of sol components. Finally, phase transformations may be noticed. The solid phase can separate from the liquid or the liquid can separate into two or more phases. An opaque gel can form if the refractive indexes of the two phases are sufficiently different. Generally, during the aging process, the pore size increases, while the surface area decreases [1, 23] Drying Drying involves further gel shrinkage and the loss of water and/or alcohol. The pore size and the rate of evaporation of the pore liquid can cause drying stress within the gel. Therefore, precise control of the sol-gel environment during the drying process is needed to prevent cracking. There are three different methods of drying, resulting in three unique types of sol-gels: aerogel, ambigel, and xerogel, as shown in Fig. 2 [26]. Aerogels are supercritically dried. Ambigels are dried after the pore liquid is exchanged for a low surface tension liquid. Xerogel are commonly dried at high temperatures. The following explanation of the drying process focuses on xerogels Xerogels The drying process of xerogels can often take months due to the reaction conditions. Xerogels dry at low temperatures. With this type of sol-gel, capillary pressure causes the majority of the shrinkage of the gel network, reducing the volume of the gel by a factor of 5 to 10. During the first stage, known as the constant rate period, the volume of the gel decrease equals the volume of liquid lost by evaporation. Due to the relatively rigid network of the sol-gel, transport of liquid through the pores is difficult. Many interparticle bonds in the sol-gel must be broken and reformed in order to expel the pore liquid. During this stage, the pores can collapse. The next stage begins at the critical point. At this point, shrinkage stops and cracking is most likely to occur due to the large 6

23 Fig. 2 Different drying conditions. Structures of a) xerogel, b) ambigel, c) aerogels. 7

24 pressure generated within the gel. The pressure increase is due to the increase of surface tension as the liquid recedes into the small pores as the gel shrinks. Some liquid continues to be transported through the pores to the surface; however, the process is less efficient than the previous stage. Hence, this stage is often called the first falling-rate period. In the final stage, the second falling-rate period, the rate of weight loss decreases further because liquid can no longer be transported to the surface. The liquid that remains within the pores, eventually evaporates and the vapors diffuse through the gel to the surface [1, 20, 27] Ambigels Solvents are exchanged during the drying process of ambigels. The pore liquid of the gel, e.g. water and alcohol, is replaced with a low surface tension liquid [28, 29] like an alkane. As discussed previously, surface tension during the drying process may cause the gel to crack. The capillary pressure is related to surface tension by the following equation: P C γ = R t (4) where P C is the capillary presure, γ is the surface tension, R is the pore radius, and t is the thickness of the surface adsorbed layer [23]. Therefore, the large capillary forces at liquid-gas interface that typically cause pores to collapse are minimized. In general, the pores and surface area of ambigels are larger than those of a xerogel, but smaller than those with of aerogels [26] Aerogels Aerogels are made by supercritical drying techniques, eliminating the capillary pressure. In the 1930 s, Kistler [10] was interested in demonstrating that a wet gel s structure consisted of a continuous solid network. If a gel is allowed to dry on its own, the gel will shrink and become a fraction of its original size, e.g. a xerogel. Kistler hypothesized that by removing the liquid within the pores of the gel and replacing it with gas molecules, he could study the structure without causing damage or shrinkage. A gel containing sodium silicate and ethanol was held at a pressure greater than the vapor 8

25 pressure, causing the temperature to increase. At the critical temperature, the pore liquid was replaced with air and the liquid-gas interface never formed. Teichner and Nicolaon [30] improved Kistler s method by eliminated the time-consuming salt removal step by using tetramethylorthosilicate (TMOS). Although aerogels are brittle, these gels have unique properties that are ideal for many applications. Aerogels have low density; air comprises up to 95% of the volume. Large pores and high surface areas are also characteristics of aerogels. As a result, aerogels exhibit low thermal conductivity and often are used for insulating applications. Aerogels are also used in other applications such as optics, sensors, and catalysts [31] Stabilization Stabilization includes structural relaxation and sintering. Sintering is the final step in the sol-gel process. Driven by surface energy, sintering is the process of densification or elimination of pores within the gel. Usually, densification is not halted by processing conditions. Some properties of the sol-gel continue to change for indefinite period. This is an important consideration for applications where changes in the surface area or pore volume of the sol-gels may affect results. As discussed in the next chapter, additives are used in the present study to increase the rate of densification. 1.3 Factors affecting sol-gel structure Usually, there are four basic components in a sol: precursor, water, alcohol, and catalyst. The ratios and types of these components can greatly affect the ultimate structure and properties of the sol-gel such as porosity, surface area, and surface functionality. Also, physical reaction conditions like pressure and temperature during solgel processing may cause modifications in the properties of the sol-gel Basic sol-gel components: precursor, water, and alcohol Precursors for sol-gel syntheses can be either inorganic salts or organic compounds. However, precursors are commonly metal alkoxides, predominantly silicates. For example, TMOS, a common precursor used today, was first used in 1884 by Grimaux [32, 33]. Also, metal alkoxides of Ti, V, Cr, Mo, and W have been synthesized. 9

26 Precursors with larger alkoxy groups cause slower reactions because of increased steric hindrances and overcrowding of the transition state. For instance, the hydrolysis rate of TMOS is faster than that of tetraethylorthosilicate (TEOS) [1]. Also, the bulkier the alkoxy groups, the larger the average pore diameter in the final sol-gel product [1] and slower rate constants are observed during processing [34]. The nature of the precursor, whether hydrophobic or hydrophilic, determines if a co-solvent is needed to achieve miscibility in water. For instance, TEOS and water are immiscible, so a co-solvent like alcohol is needed to facilitate hydrolysis as shown in Fig. 3 [1, 23]. The choice of a co-solvent can affect the reaction rates of hydrolysis, k H. Artaki et al. [35] showed that k H varies in the following solvents: acetonitrile > methanol > dimethylformamide > dioxane > formamide, with k H of acetonitrile being about 20 times larger than that in formamide. Alcohols are perhaps the most common choice for a cosolvent. However, it does not necessarily need to be included if the gel is sonicated during processing. The water:precursor molar ratio, r, has a significant affect on the product structure. The stoichiometric value of r for complete hydrolysis is 4. Theoretically, an r value greater than 2 is sufficient to complete the reaction because water is a product of the condensation reaction. If too little water is used, the hydrolysis rate slows down due to reduced reactant concentration, and gel time increases. Other reactants will be diluted if too much water is added, slowing the gel time Catalyst type and concentration The type of catalyst and its concentration can influence the structure and properties of the gel. Gels are usually catalyzed by an acid or base. Both acid and base catalyzed gels undergo a bimolecular nucleophilic substitution, S N 2, reaction, as shown in Fig. 4 [23]. The first step in an acid catalyzed hydrolysis reaction is the rapid protonated of an alkoxide group, causing electron density withdrawal from silicon. As a result, it is more electrophilic and is attacked by water, forming a pentacoordinate intermediate. The attack reduces the positive charge on the protonated alkoxide, making alcohol a good leaving group. The transition state decays in the final step involving the removal of 10

27 Fig. 3 Phase diagram for tetraethylorthosilicate in water and alcohol (based on ref. 1 and 23). 11

28 Acid catalysed H H RO H + RO OR H O + Si OR δ + O Si O RO H RO OR R δ + H HO OR OR Si + ROH H + OR Base catalysed HO - RO Si RO RO OR OR RO OR OR HO δ Si OR δ HO Si + OR - OR OR Fig. 4 Hydrolysis mechanism (based on ref. 23) 12

29 alcohol and the formation of the silica tetrahedron. In the first step of a base catalyzed reaction, water dissociates to produce nucleophilic hydroxide anions. A pentacoordinated intermediate state forms. The hydroxyl anion attacks the silicon atom, displacing the alkoxide group with the inversion of the silicon tetrahedron [20, 34]. Like hydrolysis, condensation reactions can be catalyzed by acid or by base. The condensation mechanisms are shown in Fig. 5 [23]. Both reactions proceed via the rapid formation of a charged intermediate involving a proton or hydroxide ion. The following step is slower, where the intermediate is attacked by another neutral silicon species [23, 36]. The type of polymers formed depends on the type of catalyst. With an acid catalyst, more linear polymers will form. The initial step of hydrolysis is fast; however, the second is slower. Alkoxy groups are more electron-donating than hydroxyl groups, so the transition state becomes less stabile as the alkoxy groups are replaced and the reaction rate decreases. Therefore, the condensation reaction between Si-OH and protonated Si-OR groups of non-hydrolyzed or partially hydrolyzed monomers will play a significant role in the reaction mixture. Chain elongation will occur because the Si-OR groups are more reactive than the Si-OH. As a result, the gel will have micropores. Unlike acid-catalyzed gels, fully hydrolyzed species undergo the fastest condensation reactions with base catalyzed gels because the transition state becomes more stable as more alkoxy groups are replaced with hydroxy groups. As a result, highly branched polymers form, and the product is mesoporeous. There are three ph domains: less than ph 2, ph 2-7, and greater than ph 7. The isoelectric point (IEP) and the point of zero charge (PZC) of silica both occur around ph 2, so this ph is used as a boundary. The gel time is the highest at this ph. At the PZC, the equilibrium species has zero net charge, and at the IEP, the electrical mobility of silica particles equals zero. Below ph 2, the solubility of silica is low. Particle growth is more likely to form due to aggregation than to ripening. Ripening contributes little to the growth of particles larger than 2 nm in diameter. In contrast, above ph 7 the solubility of silica is enhanced, depending strongly on particle size. Silica particles are ionized, so particles growth occurs without aggregation via Ostwald ripening [20, 34]. 13

30 Acid catalysed H + HO HO H OH Fast R + Si OH Si O+ + HO Si HO HO H R R OH Slow HO Si HO R O + Si OH R OH H 3 O + Base catalysed HO Si HO R Fast HO OH Si HO + OH - R + O - + HO Si HO R OH Slow HO Si HO R O + Si OH OH R H 2 O OH - Fig. 5 Condensation mechanism (based on ref. 23) 14

31 1.3.3 Temperature Normally sol-gels can be processed at relatively mild temperatures; however, changes in temperature can greatly influence the properties of base catalyzed gels. At basic phs, the main method of particle growth is by ripening, which depends on the high solubility of silica. At higher temperatures, the solubility of silica increases. Therefore, larger particles are generally formed at higher processing temperatures. Under basic conditions, Brinker and Scherer [23] estimated that increasing the temperature from 200 C to 295 C increased the estimated diameter of silica particles from 12 nm to 36 nm. Also, increasing the temperature, decreases gel time, t gel, according to Arrhenius equation: E In( t gel ) = A + (5) R T g where A is constant, R g is the ideal gas constant, T is the temperature, and E is the activation energy. As the gelation temperature increases, the porosity increases while the pore size distribution broadens Additives Additives such as surfactants, dendrimers (discussed in chapter 2), and biological molecules can be included in the sol to control sol-gel processes and affect one or more properties of the sol-gel. For example, drying control chemical additives (DCCA) are sometimes added to control the drying process. These additives can control the rate of hydrolysis and condensation. Templating sol-gels with different molecules are also used. Normally, the templating process involves two main steps (Fig. 6): first, forming the template within the gel and second, removing the templating agent. After removal, a cavity is created with the morphology and/or stereochemistry similar to the templating agent. However, the template can remain within the solid after the gel forms. In 1940, the first application of molecular templating to control the size and morphology of the sol-gel pores was reported by Dickey [37]. He used the dye, methyl orange, and its analogs e.g. ethyl, n-propyl, and n-butyl orange as templating agents. During gelation, the silica molecules organized tight networks around the dye molecules 15

32 due to non-covalent interactions such as van der Waals forces, hydrogen bonding, and interionic attractions. After the gels formed, methanol was used to remove the dye. The resulting gel showed a selective adsorption for each individual dye the gel was templated with over the other similar dyes with different alkyl groups. For instance, the relative adsorption for butyl orange was two times greater with gels prepared with this dye than with gels prepared with propyl orange. Other applications of additives have been reported since this time. For example, surfactants are often used to control pore size and shape and to reduce cracking by reducing the amount of shrinkage at the critical point. They are described in detail in the next section. Another example, organically modified silicate sol-gels (ormosils), are discussed in chapter Surfactants Surfactants reduce interfacial energy and decrease capillary stress. Surfactants are bifunctional with hydrophobic and hydrophilic portions. The amphiphilic nature of surfactants allows these molecules to form supramolecular arrays and spherical micelles e.g. from high concentrations at the interface between the surfactant solution and another phase. For example, cetyltrimethylammonium bromide, CH 3 (CH 2 ) 15 N(CH 3 ) + 3 Br -, will self-assemble in water to form spherical micelles, containing approximately 90 molecules, in which the hydrophilic ends point towards the outside and the hydrophobic ends point towards the center. The concentration of the surfactant molecules has the most influence on the extent of micellization, the shape of the micelles, and the aggregration of the micelles into liquid crystals. When the concentration of surfactant is very low, these molecules are distributed individually within the solution. At the critical micelle concentration (CMC1) surfactant molecules begin to aggregate, forming small spherical micelles. These micelles can coalesce to form elongated cylindrical micelles at higher concentration (CMC2). At even greater concentrations, a liquid-crystalline phase will form. Generally, surfactants are divided into four categories: 16

33 Fig. 6 Templating sol-gels a) the precursor(s) and templating molecules are mixed together, b) the templating molecules are removed, resulting in a cavity with the morphology and/or stereochemistry similar to the templating molecules. 17

34 1. Anionics-the hydrophilic portion of the molecule carries a negative charge. Examples of these surfactants include sulfates (C n H 2n+1 OSO 3, n = 12, 14, 16, 18), sulfonates (C 16 H 33 SO 3 H and C 12 H 25 C 6 H 4 SO 3 -Na), phosphates (C 12 H 25 OPO 3 H 2, C 14 H 29 OPO 3 K), and carboxylic acids (C 17 H 35 COOH and C 14 H 29 COOH). 2. Cationics-the hydrophilic portion of the molecule carries a positive charge. For example, alkylammonium salts, (C n H 2n+1 (CH 3 ) 3 NX where n > 5, X = OH, Cl, Br, HSO 4 ), cetylethylpiperidinium salts (C 16 H 33 N(C 2 H 5 )(C 5 H 10 ) + ), and dialkyldimethylammonium salts are all cationic surfactants. 3. Nonionics-the hydrophilic group is not charged. Examples of these surfactants include primary amines (C n H 2n+1 NH 2 ), octaethylene glycol monodecyl ether (C 12 EO 8 ), and octaethylene glycol monohexadecyl ether (C 16 EO 8 ) 4. Zwitterionic-a compound with both acidic and basic groups. In the early 1990 s, researchers at the Mobil Corporation made a breakthrough in material science by preparing (via surfactant templates) the first broad family of mesoporous molecular sieves, M41-S [38]. These molecules were used as a template in order to synthesis porous material. High surface areas, greater than 1000 m 2 /g, and tunable and uniform pores of 2-10 nm were achieved after removal of the template via calcination. Due to these desirable properties, surfactants have been increasing used in sol-gel. 1.4 Characterization of sol-gel materials: surface area and pore size distribution Surface area and porosity are perhaps the most frequently measured properties of porous material. These properties may be characterized with several different methods including gas adsorption, mercury porosimetry, transmission electron microscope (TEM), scanning electron microscope (SEM), and small angle x-ray scattering. Both TEM and SEM are rarely used for pore analysis. TEM gives information about pore connectivity. However, pore size can only be measure if the pores are ordered. Small angle x-ray scattering can be used for any pore size. However, this method is expensive. Because surface area and porosity often have complementary roles in any application of porous materials, it is convenient to measure these properties together. Gas adsorption/desorption offers a way to measure both properties. In fact, this method is the 18

35 most widely used. Based on gas adsorption and desorption, the surface area of a sample can be determined with the BET method, and the pore size distribution, with the BJH method. Before discussing these methods in detail, common concepts and calculations will be given Adsorption and isotherms When a gas molecule adheres to the surface of a solid, it is called adsorption. Desorption is the reverse process. There are two types of adsorptions: chemisorption and physisorption. Chemisorption is the process of chemically bonding the adsorbate and substrate. This process is often irreversible and may have activation barriers. On the other hand, physisorption is the process of the absorbent physically adsorbing to the surface by weak van der Waals attraction. Physisorption is a fast process where multilayer formation is possible. There are no activation barriers and the process is reversible. Isotherms represent the value of gas adsorbed as a function of gas pressure at a constant temperature. There are six classes [39], as shown in Fig. 7: Type I isotherms are concave to P/P 0. Microporous solids of less than 2 nm show this characteristic. Type II isotherms are exhibited by nonporous and macroporous (greater than 50 nm) solids. This type of isotherm shows unrestricted monolayer-multilayer adsorption. At the point where the amount adsorbed does not increase or increases very little versus P/P 0 signifies the conversion from monolayer coverage to multilayer adsorption. Type III solids are commonly nonporous. This type of isotherm is not common and is characterized by a convex curve to the P/P 0 axis. The adsorbate-adsorbate interaction plays an important role with Type III materials. Type IV isotherm also characterize mesoporous solids. However, Types IV and V represent isotherms with hysteresis loops, where the desorption curve derivates from the adsorption curve. 19

36 Fig. 7 Classification of isotherms (based on ref 39). 20

37 With Type IV isotherms, the amount adsorbed at some relative pressure is greater for the desorption process than the adsorption process. This is attributed to the capillary condensation taking place in the mesopores [40]. Sol-gels often are placed in this category. This type of isotherm is initially similar to Type II. However, at a certain point, the curve deviates upwards and then, at a certain relative pressure, the amount adsorbed starts to stabilize. Type V solids are not common. The adsorbate-adsorbate interactions are weak. Nonporous solids with almost completely uniform surfaces have Type VI isotherms Surface area and porosity basics Nitrogen is the most common gas used for gas adsorption analysis. However, other gases, such as argon, can be used. Other gases may be able to penetrate somewhat smaller pores than nitrogen and are used for microporous samples. Once the choice of gas is made, the sample must be degassed. The temperature and time of degassing depends on the sample. After degassing, the sample is ready for analysis. As gas is dosed into a sample chamber, some gas molecules adsorb to the sample, forming a monolayer (or more). However, other gas molecules do not adsorbed but form a residual pressure in the sample chamber. The relative pressure can be found by dividing the residual pressure by the saturated vapor pressure. The saturated vapor pressure is the boiling pressure of the liquid gas. It varies with temperature. Liquid nitrogen must be used to get accurate results because the saturation vapor pressure of the adsorbate gas must be known during the analysis. During an analysis, liquid nitrogen condenses, and the saturation vapor pressure changes. Therefore, the saturation vapor pressure must constantly be monitored to obtain accurate results. Helium is used in freespace calculation. It is calculated based on the amount of helium dosed according to the following equation: ( P P ) V Vd (6) M 1 M 2 M n = + Vd n 1 TM 760 where Vd n is the volume dosed, P M1 is the initial manifold pressure, P M2 is the final manifold pressure, V M is the manifold volume, T M is the manifold temperature, Vd n-1 is the volume dosed from previous data point, and / 760 is the standard temperature 21

38 and pressure conversion. This is the volume in the sample tube, which varies with the volume occupied by the sample. With the SA 3100 (Beckman Coulter Inc, Miami, FL), three helium data points are measured at incremental pressure to determine the freespace. The volume dosed is plotted versus the sample pressure to determine the freespace. The entire analysis depends on this number because the volume of gas absorbed by the sample is calculated by subtracting the volume of gas present in the freespace from the volume of gas pumped into the system according to the following equation: Vads = Vd ( P m b) (7) n n sn + where Vads n is the volume adsorbed, Vd n is the volume dosed, P sn is the sample pressure, m is the freespace measurement slope, and b is the freespace measurement intercept [41] Surface area Langmuir and others were trying to address the problem of how to determine surface areas of solids with complicated shapes, like powders. He used the basic principle behind the gas law, PV=nRT, to develop his method. Langmuir [42] developed the following equation to calculate surface area: P V s s = 1 + (8) A bv M P V M where V M is the volume of the monolayer, P S is the sample pressure, b is the Langmuir constant, and V A is the volume adsorbed. After calculating V M, the surface area may be calculated from: S N A V A M M L = (9) M V where S L is the Langmuir surface area, M V is the gram molecular volume, N A is Avogadro s number, and A m is the cross sectional area occupied by each adsorbate molecule. This equation was limited to homogeneous surface where adsorption sites were energetically identical and only applies to monolayer adsorption. Langmuir assumed that adsorbed gas molecules do not mutually interact. Brunauer, Emmett, and Teller [43] developed the BET method in 1938 by extending the Langmuir method to include multilayer adsorption. A BET instrument records changes in pressure as a gas, usually nitrogen, is pumped into a system. The 22

39 measurements are done at constant temperature (isotherm) in order to make the calculations easier. At constant temperature, the pressure in a closed system will proportionally increase as the number of gas molecules increases. However, it is not that simple because of adsorption. Brunauer et al. [43] made the assumption that further physisorption is possible after the initial first layer, i.e. multilayer adsorption. However, they assumed that there were no interactions between each adsorption layer. Like Langmuir, they assumed homogeneous adsorption sites. The BET method is useful for Types II and IV isotherms. The BET equation is often represented as: V P 1 ( C 1)( Ps / Po = + P P ) V C V C A ( 0 s ) s m m where P s is the sample pressure, V A is the volume adsorbed, P 0 is the saturation pressure, P s is the sample pressure, V m is the volume of monolayer, and C is the constant related to the enthalpy of adsorption. A line is made by plotting 1 Ps / Po V 1 Ps / Po (10) versus the relative pressure, P S /P 0. From this plot the surface area is calculated using the following equation: S V N M M A M = (11) V A where S is the surface area, measured in m 2 /g, N A is Avogadro s number, A m is the cross sectional area occupied by each adsorbate molecule (0.162 nm 2 for nitrogen), and M v is the gram molecular volume. One of the disadvantages of the BET method is that only surface area can be determined. The BET method does not give information about pore size. For this reason, the BET method is often coupled with the BJH method, described below Pore size distribution The characterization of Type IV isotherms has played an essential role in the development of adsorption theory. In 1911, Zsigmondy [44] used the principle of Thomson s work [45] on thermodynamics to develop his capillary condensation theory. He based his theory on the idea that at the same temperature the vapor pressure, P S, over 23

40 a concave meniscus, is less than the saturation vapor pressure, P 0. In other words, liquid condenses in the pores of a solid, even when the relative pressure is less than unity. Zsigmondy [44] used the isotherm of Type IV solids to explain his theory. Initially, adsorption is restricted to a thin layer on the walls. At the point where the hysteresis loop begins, the finest pores are filled with liquid by the process of capillary condensation. The pores are continuously filled until P S /P 0 reaches near unity. At this point, all mesopores and macropores are filled with liquid. Barrett, Joyner, and Halenda (BJH) [46] developed a method to determine pore size distribution based on the Kelvin equation, which relates the core radius of the pores filled by capillary condensation. Some assumption are made with this method including that the shape of the pore is cylindrical, the pores are open-ended, and no pore networks are present. The Kelvin equation relates the relative pressure in equilibrium with a porous solid to the size and shape of the pores as follows: P RT ln P V R S M = 2γ (12) 0 K where R is the gas constant, T is the boiling point of nitrogen, γ is the adsorbate surface tension at T, V M is the molar volume of nitrogen, and R K is the Kelvin radius. Adsorption or desorption isotherms can be used. Prior to condensation, some adsorption has taken place on the walls of the pore. The thickness of the adsorbant layer, t, is calculated by the modified Halsey equation: 1/3 5 t = 3.54 (13) P log P s After determining the thickness of the film, the actual radius of the pore, R P, can be calculated by adding the Kelvin radius, R K to t. 24

41 Pore size is characterized into three categories according to the International Union of Pure and Applied Chemistry (IUPAC) [39]: 1) micropores are smaller than 2 nm in diameter 2) mesopores are between 2 to 50 nm in diameter 3) macropores are larger than 50 nm in diameter. 25

42 Chapter 2 Acceleration of the Densification of a Silica Sol-gel by Inclusion of Generation-zero Poly(amidoamine) Dendrimer 2.1 Introduction The preparation of sol-gels by processing under ambient conditions is important to applications that require porosity or that involve thermally labile dopants. The disadvantage of this approach relative to sintering is that the aging process that is accompanied by densification of the solid is not halted by the processing condition. Instead, a change in the surface area and the pore structure continues for an indefinite period. For applications that are sensitive to parameters such as pore diameter, this gradual change in internal structure is problematic. Zink and co-workers [47] used the blue shift of a fluorescence probe, ReCl(CO) 3 bpy, with rigidity of the matrix to investigate the densification of sol-gels during the gelation, aging, and drying periods. The study not only established the utility of this probe molecule for such measurements but also demonstrated a difference between the evolving internal environments of silicon and mixed aluminum-silicon alkoxides. They demonstrated that densification of the latter continued over a period of at least 10 3 h. In a study aimed at development of an optical sensor for dioxygen based on fluorescence of tris(4,7-diphenyl-1,10-phenthroline)ru II, Ru(dpp) 3, Bright and coworkers [48] observed that sol-gel platforms derived from pure tetraethoxysilane (TEOS) exhibited a decrease in sensitivity with age for at least 11 months, which was attributed to shrinkage of the solid and collapse of the pore structure with time. Significant fracturing of the sol-gel film was also observed. That influence of age of a sol-gel thin film on sensor performance was consistent with a previous report that employed both TEOS and tetramethoxysilane in conjunction with a pyrene probe [49]. The influence of age on the formation of organically modified silica, specifically organosilsequioxane materials, by sol-gel processing was studied by Cerveau et al. [50]. 26

43 When tetrahydrofuran was the solvent, the nature of the catalyst had a marked influence on the physical structure of the products. An interesting observation was that increasing the aging time favored the formation of a mesoporous solid under the conditions they employed, which is in contrast to the collapse of pore structure with time that is typical of sol-gel processing with inorganic precursors and with water-alcohol mixtures as the solvent [23]. The study [50] was limited to 30 days in most cases. In an extension of this work, Cerveau et al. [51] showed that the temperature of the processing and the nature of the leaving group (methoxy or ethoxy) had a profound influence on the structure of the product Dendrimers Dendrimers have highly branched structures with numerous multifunctional end groups. The term dendrimer was derived from the Greek words, dendron (tree) and meros (part). This is a relatively new field, becoming popular in the 1990 s. One of the earliest examples was the synthesis performed in the 1980 s by Tomalia [52]. He developed a series of polyamidoamine (PAMAM) compounds known as starburst dendrimers (Fig. 8). PAMAM dendrimers usually have an ammonia or pentaerythritol core. Amide groups, carboxyl groups or other functional groups are attached to the terminal nitrogen atoms. Each progressive branch represents one generation. Generations 0 to 4 are normally used in sol-gel applications. Around the same time Newkome et al. [53] developed compounds that had a different constitution for each layer or generation. These were called arborol systems. Three structural components comprise these molecules: a core, interior branch cells, and terminal branch cells. Some compounds follow the divergent approach and grow outwards from the core. Both the PAMAM dendrimers and the arborol systems are constructed by the divergent approach, while others begin at what will ultimately become the surface of the dendrimer, working inwards by gradually linking surface units together with more monomers. This is the convergent approach. The limit of a dendrimer to increase exponentially as a function of generation is referred to as starburst limit. At some point, the increase in size is so great that the dendrimer can no longer grow due to lack of space. Newkome et al. [53] and Tomalia 27

44 Fig. 8 Structures of poly(amidoamine) dendrimers: a) Generation-0, b) Generation-4. 28

45 and coworkers [54] pointed out that the steric crowding of higher generation dendrimers causes them to adopt a globular conformation. Factors such as the type of solvent and the dendrimer s constitution determine whether the branch ends on the dendrimers lie throughout the entire structure or on the surface of the molecule. Cavities are created in the latter. The internal surface area of the higher generation, e.g. > 2, dendrimers have greater internal surface area compared with the external surface area and therefore are idea for applications requiring high surface areas. The use of dendrimers in sol-gels synthesis began to appear in literature in the late 1990 s. Boury et al. [55] prepared hybrid xerogel from dendrimers with methoxysilane groups and arborols with octadecyl and phenyl groups. They found that gels formation did not occur with the first generation dendrimers. Porous silica gels were obtained with generation 2 dendrimers and arborols. Kriesel and Tilley [56, 57] also prepared dendrimer-based xerogels with higher generational dendrimers than Bourys et al. [55]. Second and third generation alkoxysilylterminated carbosilane dendrimers were used as building blocks for the synthesis of solgels. They found that the total surface area and pore volume of the xerogel observed was greater with the xerogel produced with the higher generation dendrimers than with lower generations. With generation 2, the surface area was measured as 600 m 2 g -1, whereas the surface area of the generation 3 sol-gel was 800 m 2 g -1. Dendrimers have been use for many applications, including as a host for dyes and other molecules, molecular recognition in layer-by-layer assemblies, catalysts, and as templates. This chapter will focus on the use of dendrimers as cross-linking agents in solgels Densification Treatment with high temperature, for example up to 1200 o C, is the typical method of densification [58]; however, methods other than conventional heating have been used. Jiwei et al. [59] used laser irradiation to densify selected regions of silicatitania composite sol-gel films. By micro-raman spectroscopy and other methods, they verified that the densified zone corresponded to the laser beam dimension. Imai et al. [60] studied the densification of sol-gel thin films by ultraviolet irradiation. With silica sol- 29

46 gels, energies higher than 9 ev produced significant changes in film thickness even though the increase in the temperature of the substrate did not exceed 40 o C. Densification also has been achieved by crosslinking sol-gels with dendrimers. Gong et al. [61] synthesized a composite from a TEOS precursor and Generation 3 poly(amidoamine), G3-PAMAM, using 3-glycidoxypropyltrimethoxysilane as a coupling agent. The study was focused on evidence of covalent crosslinking and the optical properties of the composite rather than the pore structure and the influence of age on the properties. An earlier report on a dispersion of PAMAM in a TEOS-derived sol-gel demonstrated high porosity but did not consider the influence of age systematically [62]. The present study is an extension of our report that inclusion of G0-PAMAM in a TMOS-derived sol-gel yielded a microporous product with enhanced mechanical strength and resistance to fracturing upon immersion of monolithic products in water [63]. Our hypothesis is that the G0-PAMAM crosslinks the strands of silica, thereby yielding a mechanically stable material. The crosslinking is expected because the G0-PAMAM has multiple positive sites, namely the protonated amines, and the silica carries a partial negative charge. Consistent with that model is the prediction that the crosslinking will increase the rate of densification of the silica, a conjecture that is tested by the experiments described herein. The influence of age on the total pore volume, pore size, surface area, and residual solvent is investigated over a 52-week period during which the humidity was controlled. 2.2 Experimental Reagents The 10% (wt.) solution of Generation-0 poly(amidoamine) dendrimer, G0- PAMAM (molecular mass, 517 Da; 4 surface amines), in methanol was purchased from Aldrich Chemical Co. (Milwaukee, WI). The tetramethoxysilane, TMOS, was obtained from Aldrich at 99% purity. All other chemicals were Reagent Grade. Water used in this study was house-distilled that was further purified with a Barnstead NANOpure II system. 30

47 2.2.2 Apparatus Surface Area, total pore volume, and pore size were measured using an SA 3100 Surface Area Analyzer (Beckman Coulter Inc, Miami, FL). The surface area was determined by the Brunauer, Emmett, and Teller (BET) method, whereas the pore structure was elucidated by the Barrett, Joyner, and Halenda (BJH) approach. Nitrogen adsorption and desorption were evaluated and sample pressure / saturation pressure (P S / P 0 ) was set at Hydrogen was used to determine the free space as explained in Chapter 1. Monoliths were crushed and degassed for 90 min, at 40 C, prior to analysis. A sample size of approximately 0.2 g was used. After degassing, the sample tube was placed in liquid nitrogen for analysis. First, the free space in the sample tube was calculated. Then, adsorption was measured to determine the surface area as outlined in chapter 1. Pore size was determine based on desorption data. Thermal gravimetric analysis (TGA) experiments were performed on a Perkin Elmer TGA 7 system (Boston, MA). The same monolith crushed for surface analyses were used in the TGA experiments. The sol-gel monoliths were crushed prior to analysis. The sample size was approximately 20 mg. The samples were held at 25 C for 1 min, after which the temperature was increased to 120 C at a rate of 20 C min -1. Samples were held at 120 C for 5 min. All TGA analyses were carried out under nitrogen Sol-gel synthesis The undoped silica sol-gels were prepared from a mixture that contained equal volumes of TMOS, methanol, 0.1 M HCl, and 0.2 M KCl. After magnetically stirring for approximately 30 min, 2-mL aliquots were transferred to 38 / 25 mm hexagonal polystyrene weigh dishes from Fisher Scientific (Pittsburgh, PA). The containers were covered with Parafilm, in which a 2-4 mm hole was made in the center, to control the rate of drying. The sol-gels were aged at ambient temperature in a humidistat, which comprised a Fisher brand Unit Desiccator Cabinet (Fisher Scientific), controlled at 9% humidity with a saturated solution of KOH. The preparation of silica with G0-PAMAM as the dopant was by the same except that the methanol contained 10-mM dendrimer. 31

48 2.3 Results and discussion The experimental strategy was to prepare large sets of monoliths that were equally divided between sol-gels derived from TMOS alone and those with G0-PAMAM as a dopant. At eight periods (2, 4, 8, 17, 25, 35, 45, 52 weeks), six samples of each type were removed from the humidistat and analyzed by nitrogen adsorption and TGA methods. Due to the large quantity of gels needed and limited space, the overall period of study was 90 weeks Moisture The data set was analyzed first in terms of the residual solvent in the sol-gels as determined by TGA. An example of a TGA study of GO-PAMAM doped sol-gel at 2 weeks is showed in Fig. 9. The black line shows the decrease in mass versus time, while the gray line shows the temperature profile of the study. The solvent in the pore of the sol-gel slowly evaporates as the temperature increases. The maximum temperature chosen needed to be greater than the boiling temperature of the pore liquids but not so great as to degrade the gel. The pore liquids in these gels are water and methanol. Therefore, a maximum temperature of 120 C was chosen. The percent mass loss was calculated by first determining the difference in masses before and after the temperature program and then dividing by the initial mass. A summary of the results are shown in Fig. 10. As expected, the solvent decreased from ca. 15% to 7% over a 52-week period for both the silica alone and a composite of silica with the G0-PAMAM. As the gel ages, the pore liquid, e.g., water and methanol is expelled or evaporates. Statistical t-tests were carried out at 95% confidence level on the data in Fig. 10. The percent mass loss for the composite and the undoped silica are statistically the same value at each time period except at 45 weeks. A typical result was that after 17 weeks the percents of the mass lost from the composite and the undoped silica corresponded to 11 ± 2 (6 trials) and 12 ± 2 (6 trials), respectively, yielding a pooled standard deviation of 2.0. Although each subset showed a lower mean solvent content for the composite, the results for each measurement period were identical at the 95% confidence level when compared by the t-test except between 2 and 4 weeks. A statistical difference in moisture content is seen between 45 and 52 weeks for the undoped silica. The data show that the undoped silica continues to 32

49 Mass, mg Temperature, C Time, min Fig. 9 Thermogravimetric study of GO-PAMAM doped sol-gel showing mass versus time (black) and temperature profile line (gray). 33

50 Mass Loss, Percent Composite Undoped Silica Aging Time, Weeks Fig. 10 Thermogravimetric determination of solvent content of undoped TMOS-derived silica (gray squares) and a composite of G0-PAMAM with silica (black squares) as a function of aging time. Each bar for the composite and undoped silica represents the mean of 6 trials. 34

51 change after 52 weeks, while the composite gel seems to remain relatively stable after 4 weeks Isotherms The adsorption and desorption isotherms for G0-PAMAM modified silica and unmodified silica are shown in Figs. 11 and 12. These isotherms were measured after 2 weeks of aging. The pore volume adsorbed was measured versus the relative pressure, P S / P 0, between 0 and The desorption of the pore volume was measured from P S / P 0 = 0.98 to 0.4. The isotherms obtained for both these materials represent Type I features, which are characteristic of microporous solids (pore radii of less than 2 nm). The volume adsorbed at the lowest relative pressure represents about ~50% of the total pore volume for the G0-PAMAM doped silica (Fig. 11), which indicates a large volume of extremely small pores. This value is greater for the undoped silica, ~70% (Fig. 12), showing that the material contains less micropores than the doped silica. A hysteresis loop is not observed in the desorption branch for either silica materials, suggesting smooth and cylindrical pores Surface area The surface areas of the sol-gels were also examined. An example of the BET plot is shown in Fig. 13, where the BET function or 1 Ps / Po V 1 P / P s o is plotted versus the relative pressure, P S /P 0. The equation of the line is y = 0.01 x , with a R 2 value of From this plot, the surface area can be calculated using Equation 11. The monolayer volume was determined to be 100 cc / g, and the surface area was calculated to be 440 m 2 / g. Fig. 14 showed that the surface area of the GO-PAMAM doped silica rapidly decreased from 110 m 2 / g at 17 weeks to 1 m 2 / g after 25 weeks. The data in Fig. 14 were analyzed statistically by the t-test at the 95% confidence level. No statistical difference is seen between weeks 25 and 35 for the doped silica. The surface area of the undoped sol-gel gradually decreases over 45 weeks, going from 140 m 2 / g after 25 weeks to 20 m 2 / g after 35 weeks, eventually decreasing to 1 m 2 / g after 45 weeks. Although it may appear that there is a difference between weeks 25 and 35 for the undoped silica, 35

52 150 Pore Volume, cc / g Relative Pressure, P S / P 0 Fig. 11 Isotherm of G0-PAMAM doped silica showing both adsorption (P S / P 0 = 0 to 0.98) and desorption (P S / P 0 = 0.98 to 0.4). 36

53 160 Pore Volume, cc / g Relative Pressure, P S / P 0 Fig. 12 Isotherm of undoped silica showing both adsorption (P S / P 0 = 0 to 1) and desorption (P S / P 0 = 1 to 0.4). 37

54 BET Function Relative Pressure, P S / P 0 Fig. 13 BET plot of G0-PAMAM doped silica at 2 weeks, n = 1. The equation of the line is y = 0.01 x , with a R 2 value of

55 600 Composite Undoped Silica Surface Area, sq. m/g Aging Time, Weeks Fig. 14 Influence of aging time on the surface area for undoped TMOS-derived silica (gray squares) and a composite of G0-PAMAM with silica (black squares). The data were obtained by the BET method. 39

56 statistically these values are the same. The large standard derivation at 25 weeks of 140 m 2 / g prevented a statistical difference from being observed. At 25 weeks, some of the samples had much larger surface areas than the average. The pores in these materials did not yet collapse. However, it appears that the pores collapsed very soon after this time since at 35 weeks all the surface areas of the sol-gels decreased to around 20 m 2 / g. A difference in the surface areas of the doped and undoped silica from weeks 2 to 25 is observed. During this time, the surface areas of the undoped silica are greater than the doped silica. After 35 weeks, the surface areas of these materials are statistically the same. Hence, the surface area studies showed no densification enhancement resulting from the inclusion of GO-PAMAM in the sol Total pore volume A more direct approach to study of densification is to measure the total pore volume as a function of aging time. A graphic comparison of this behavior for TMOSderived silica and a composite of that substance a G0-PAMAM is shown in Fig. 15. Because the time intervals between the entries were not constant, a precise determination of when the most rapid change in the pore structure occurred is not possible; however, the general trend in the data demonstrates that the total pore volume undergoes a more rapid decrease with the composite that with the undoped sol-gel. Specifically, with the composite the densification is completed at some point between weeks 17 and 25. With undoped TMOS-derived, the limiting value is approached between weeks 35 and 45, but a decrease in total pore volume continues for the entire 52-week period. The data in Fig. 15 were analyzed statistically by the t-test at the 95% confidence level. Two points are apparent regarding the pore volume of the densified material. With the composite, the value is constant over the range weeks, and the densified composite and undoped silica have the same limiting value for the total pore volume. The molecular size of G0-PAMAM does not determine the pore structure; instead, the general characteristics of the TMOS-derived sol-gel that is made by acid-catalyzed processing determines the structure. This characteristic is different from the influence of crosslinking of organic polymers such as polystyrene crosslinked with divinylbenzene (DVB) where 40

57 0.26 Total Pore Volume, ml/g 0.13 Composite Undoped Silica Aging Time, Weeks Fig. 15 Influence of aging time on the total pore volume for undoped TMOS-derived silica (gray squares) and a composite of G0-PAMAM with silica (black squares). The data were obtained by the BJH method. 41

58 the presence and concentration of DVB profoundly impacts the density of the resulting polymer Pore size distribution Further evidence that the G0-PAMAM did not influence the internal structure was obtained from pore size distribution data, which are summarized in histograms in Figs. 16 and 17. The general patterns for the composite (Fig. 16) and the undoped silica (Fig. 17) were similar, including the influence of aging time on the percent of pore diameters in any given range. For example, after 2 weeks, with the composite the percent of the pore diameters below 6 nm was 50 ± 5; with undoped silica, the same result (50 ± 5) was observed. Each set had 6 samples. After 52 weeks, the percents in this range, below 6 nm, for the composite and the undoped silica were 29 ± 3 and 30 ± 2, respectively. Figs. 18 and 19 show a closer look at the pore distribution of the smallest range of pores (3.2 to 6 nm). In this range both the composite and the undoped silica showed similar distributions throughout the study. For instance, after 2 weeks, with the composite the percent of the pore diameters between 3.19 and 3.48 nm was 16 ± 2, and with undoped silica, the percent was 9 ± 2. After 52 weeks, the percents in this range, between 3.19 and 3.48 nm, for the composite and the undoped silica were 8.7 ± 0.7 and 2.7 ± 0.4, respectively. The decrease of the population of pores in the smallest diameter range with aging time in Figs. 16 and 17 is inconsistent with the expectation that densification leads to materials with smaller pores. The data also may seem surprising in that sol-gel processing of silica under ambient conditions at ca. ph 2 is known to yield microporous solids from simple inorganic precursors such as TMOS [23]. However, when it is considered that the resolving power of the instrument used in this study limits the identification of pores to those with diameters above 3.2 nm, these results are reasonable. That is, the data domains that can be identified do not include the boundary between mesoporous and microporous sol-gels. Because the goal of this investigation was to determine whether the presence of G0-PAMAM influenced primary structure of the sol-gel as a function of time (the point at which it collapsed into a stable form on the bulk level), determination of the distribution of pore sizes in the final structure was not important. Indeed, from other studies, this information is known; the addition of G0-PAMAM leads to formation of 42

59 60.00 Percent Week 2 Week 4 Week 8 Week 17 Week 25 Week 35 Week 45 Week Over 80 Pore Size, nm Fig. 16 Pore size distribution of composites of G0-PAMAM and TMOS-derived silica as a function of aging time. Each histogram represents the mean of the pore size determined by the BJH analysis of nitrogen adsorption data with 4 6 individual samples (typical number, 6). 43

60 60.00 Percent Week 2 Week 4 Week 8 Week 17 Week 25 Week 35 Week 45 Week Over 80 Pore Size, nm Fig. 17 Pore size distribution of TMOS-derived silica as a function of aging time. The conditions are those in Fig

61 9.00 Percentage 6.00 Week 2 Week 4 Week 8 Week 17 Week 25 Week 35 Week 45 Week Pore Size, nm Fig.18 Pore size distribution under 6 nm (3.2 to 6 nm) of G0-PAMAM doped silica as a function of aging time. The conditions are those in Fig

62 Percentage Week 2 Week 4 Week 8 Week 17 Week 25 Week 35 Week 45 Week Pore Size, nm Fig. 19 Pore size distribution under 6 nm (3.2 to 6 nm) of TMOS-derived silica as a function of aging time. The conditions are those in Fig

63 microporous silica under processing at ambient temperature [63-65]. Also, the isotherms previously described support the view that the doped silica is microporous. 2.4 Conclusions Processing silica sol-gel derived from tetramethylorthosilicate at ambient temperature leads to a gradual collapse of the structure that makes its surface area and total pore volume a function of time for at least 52 weeks. By including generation-zero poly(amidoamine) dendrimer in the sol, this process is more rapid; after about 25 weeks it stabilizes. The influence of this dendrimer is attributed to crosslinking of the structure. The amine sites are positive whereas the silica backbone has a partial negative charge on the oxygen sites. Although the pore size distribution in the range below 3.2 nm cannot be elucidated by the nitrogen adsorption instrumentation available in our laboratory, the isotherm of the silica observed and previously reported atomic force microscopic [63] and electrochemical data [64] suggest that with this dendrimer present, the overall structure is microporous. Portions of the text and figures originally appeared in the Journal of Non- Crystalline Solids, 351, M.M. Wandstrat, J.A. Cox, Acceleration of the densification of a silica sol gel by inclusion of generation-zero poly(amidoamine) dendrimer,

64 Chapter 3 Imprinting of Biological Molecules 3.1 Introduction The ability to make materials with tailor-made pore sizes and shapes is important for many applications [66]. Molecular imprinting or templating is one way to achieve this [67, 68]. During imprinting, polymerizable monomers are arranged around a template. The interaction between the template and the monomers can either be noncovalent such as hydrogen bonding, ion pair, or van der Waals forces or covalent interaction. After forming the imprint around the templating agent, this agent can be removed, leaving behind microcavities with specific pore sizes, shapes and/or chemical functional groups comparable to the original templating agent in the host. In general, covalent bonding between the monomers and templating agent results in homogeneous binding sites. Noncovalent interactions result in a more flexible range of functionalities, which can be targeted [68] and are commonly used for biological applications [69]. Perhaps the first example of molecular templating to control pore size and shape was reported by Dickey [37] in 1949, with the dyes methyl orange and its homologues. He found that during gelation, the monomers (silicate species) organized around the dye molecules due to noncovalent interactions. After the templating agent was removed with methanol, the gel imprinted with methyl orange showed a higher adsorption for methyl orange dye compared to other analytes such as ethyl, n-propyl, or n-butyl orange. Since then, many other molecules have been templated including antibody-like molecules [69], active ingredients in pharmaceuticals [70], polymers [71] and enantiomers for chiral separation [72] Voltammetry In this and subsequent chapters, electrochemistry is used. Therefore, a brief introduction is given. Electrochemistry is based on the oxidation or reduction of a species. The half-cell potential, E, is represented in the Nernst equation below: 48

65 E = E o RT nf A ln A b B a A where E is the standard reduction potential, R is the gas constant, T is the temperature, n is Faraday constant, and A is the activity of species. All electrochemical processes occur at the boundary of the electrode surface. When E is applied, the argln rearranges, yielding current, which is measured versus E. Commonly, this current is measured with a three electrode system. The working electrode is where the analytical reaction occurs. The reference electrode is used to determine the potential at the working electrode because the potential is applied between two electrodes. The auxiliary or counter electrode is used to provide a current path across the solution, which does not require current to flow through the reference electrode. (14) Voltammetry is a set of common electrochemical techniques where the relation of current and voltage is observed. During square wave voltammetry, a linear scan of E is superimposed on a staircase potential, as shown in Fig. 20. The sensitivity is enhanced because the oxidation and reduction of the same analyte species is repeated. In cyclic voltammetry, a triangular waveform is applied as shown in Fig. 21, and current versus applied potential is plotted. The anodic current increases as a positive potential relative to that of the standard potential is applied, reaching a peak. The current then begins to decrease as the diffusional distance is increased. Normally, this decrease in current is slow (decays with t -1/2, where t is the time past the peak potential) because the surface concentration of the analyte at the electrode is replenished by diffusion of the analyte from the bulk solution. The potential scan direction is then switched and an increase in current (cathodic), i pc, is observed as the applied negative potential approaches the standard potential. In a reversible process, the cathodic and anodic peak currents are the same: i i pa pc = 1 where i pa is the anodic peak current and i pc is the cathodic peak current. (15) 49

66 Fig. 20 Waveform for square wave voltammetry. 50

67 Fig. 21 Cyclic potential sweep. Point a is the switching time, where the applied potential changes directions. 51

68 Current depends on the concentration of the analyte, the number of electrons, the area of the electrode, and the mass transport coefficient. Mass transport is the flux of analyte from the bulk solution to the electrode surface. This process will be discussed in more detail in chapter 7. Theoretically, the peak current is proportional to v 1/2, according to the Randles-Sevcik equation: i p (2.95*10 5 ) n 3 AD 1 v 1 C a, bulk = (16) where i p is the peak current, n is number of electrodes, A is the area of the electrode, D is the diffusion coefficient, v is the scan rate, and C Abulk is the concentration of A in the bulk solution [73] Templated sol-gels The concept of molecular imprinting in sol-gels is experiencing increased interest for several reasons. Sol-gels can be made highly porous, and other properties of sol-gels can be manipulated [23]. Also, the mild reaction conditions allow for many molecules to be templated without thermal or chemical decomposition, which could have an effect on the structure of the imprint [74]. Diaz-Garcia and Laino [75] recently wrote a review on applications of molecularly imprinted sol-gel materials. Applications discussed included imprinted sol-gel sorbents for metal ions, molecular recognition, catalysis, and chemical sensors. Monolithic sol-gels have been used in chromatography. Monoliths are generally prepared by pouring the sol into a container, allowing it to gel in the shape of the container, aging, and drying. Wei et al. [71] used inorganic molecularly imprinted polymers in monolithic sol-gels, in conjunction with HPLC analysis, to determine caffeine and its structural analogs. The selectivity for lisinopril dehydrate, a pharmaceutically active ingredient, with solid phase extraction, was increased using finely ground templated sol-gel particles over that of other non-imprinted materials [70]. Although monoliths are useful for separation, thin films would be more useful for electrochemical applications because of the decrease in path length and, therefore the resistance. However, the disadvantage of using thin films is that they usually are less porous than monoliths. One way to make more porous films is by the process of templating. Fireman-Shoresh et al. [72] spin coated thin films containing templating 52

69 molecules on indium tin oxide (ITO) electrodes. Two enantiomer pairs were used: D-3,4- dihydroxyphenylalanine or (L)-3,4-dihydroxyphenylalanine and (R)-N,N - dimethylferrocenylethylamine or (S)-N,N -dimethylferrocenylethylamine. Cyclic voltammetry and square wave voltammetry showed that very good chiral recognition was observed with the templated films for the appropriate analyte Ambigels Two other ways to increase the pore size observed in common sol-gels (xerogels) is by manipulating the drying conditions such as with ambigels or by including additives such as with ormosils. As discussed in chapter 1, in theory ambigels would be more suited for templating than xerogels. Ambigels are prepared by replacing the typical pore fluid (water and methanol) with a low surface tension liquid [28, 29]. The liquid is then allowed to evaporate at ambient or reduced pressure. Most of the initial porosity and high surface area of the wet gel are retained during this process, unlike xerogels [76] Ormosils Organically modified silicate sol-gels (ormosils) may have advantages over conventional sol-gels in creating a template. The addition of an organic site can improve the chemical specificity by increasing the porosity, changing the polarity, and/or the surface functionality [77, 78]. For example, the R-groups of the precursors can be changed to increase the affinity of the template to the monomers. Makote and Collinson [79, 80] added phenyltrimethoxysilane (PTMOS) to increase the hydrophobic of the gel and the affinity for an aromatic template (dopamine) Dopamine Dopamine (Fig. 22) belongs to the catecholamine family. This molecule is produced in the human body; it functions as both a neurotransmitter and a neurohormone. Dopamine can also be used as a medication, such as by increasing heart rate and blood pressure. 53

70 Fig. 22 Structure of dopamine. 54

71 Dopamine is often associated with the reward center of the brain, providing feelings of joy. But dopamine influences the way the brain controls movement. A person with a shortage of dopamine may lose the ability to control movements such as in Parkinson s disease. In fact, L-dihydroxyphenylalamine (L-DOPA), a precursor to dopamine is prescribed to Parkinson s patients. L-DOPA is given in place of dopamine because dopamine cannot pass the blood-brain barrier but L-DOPA can. On the other hand, an increase in dopamine, up to 10 times the normal amount, is seen in people who are addicted to cocaine, amphetamines, heroin, alcohol, and nicotine [81]. It is of importance to be able to distinguish between the individual neurotransmitter species in the presence of interferents including ascorbic acid [82]. One way to do this is by electrochemistry. Wightman is one of the leaders in the area of electrochemical detection of dopamine [82-86]. He pioneered the use of microelectrodes for the detection of catecholamine neurotransmitters (dihydroxyphenylacetic acid (DOPAC), L-DOPA, dopamine) and differentiated dopamine in the present of numerous interferents, including ascorbic acid. In our study, two types of sol-gels were evaluated as platforms for dopamine as a templating agent: ambigels and ormosils. Ambigels were synthesized; however, due to the brittle nature of these gels, an alternative type of gel (ormosil) also was tested. Templated films were studied to determine their usefulness in detecting dopamine. 3.2 Experimental Reagents Tetraethylorthosilicate (TEOS) at 99+% purity, tetramethylorthosilicate (TMOS) at 99+% purity, phenyltrimethoxysilane (PTMOS) at 97% purity, methyltrimethoxysilane (MTMOS) at 99% purity, 3-hydroxytyramine hydrochloride (dopamine) at 98% purity, sodium permanganate (97%), fumaric acid (99+%), ethoxyethanol (99%), cyclohexane (99+%) were obtained from Aldrich (Milwaukee, WI). The 10% (wt.) solution of Generation-0 poly(amidoamine) dendrimer, G0-PAMAM (molecular mass, 517 Da; 4 surface amines), in methanol was purchased from Aldrich Chemical Co. (Milwaukee, 55

72 WI). Other chemicals were Reagent Grade. Water used in this study was house-distilled that was further purified with a Barnstead NANOpure II system Apparatus Beckman Coulter Inc. (Miami, FL) SA 3100 Surface Area Analyzer was used to measure surface area, total pore volume, and pore size by nitrogen adsorption and desorption. The surface area was determined by the Brunauer, Emmett, and Teller (BET) method. The Barrett, Joyner, and Halenda (BJH) approach was used to evaluate the pore structure. P S / P 0 was set at and hydrogen was used to determine the free space in the sample tube. Monoliths were crushed and approximately 0.2 g of sample was degassed for 90 min, at 40 C, prior to analysis. The sample tube was placed in liquid nitrogen during analysis. Adsorption was measured to determine the surface area while pore size was determine based on desorption data. Electrochemical experiments were carried out with the CH Instruments (Austin, TX) Models 400, 660B or 800 electrochemical workstations. The working electrode (slide) is indium tin oxide (ITO). All potentials were measured versus an Ag AgCl, 3 M KCl reference electrode from Bioanalytical Systems (West Lafayette, IN). Platinum gauze was the counter electrode. ITO slides were purchase from Delta Technologies, Limited (Stillwater, MN). The ITO slides were cut into 2.5 cm by 2.5 cm sections, rinsed with ethanol, and dried under nitrogen prior to use as electrode platforms Ambigel synthesis Gels were prepared based on previous work [76]. Briefly, fumaric acid was added to a 0.2 M solution of sodium permanganate in a 1:3 molar ratio. After vigorously stirring, the solution was degassed under vacuum for approximately 10 min to remove CO 2. Following degassing, 2-mL aliquots were transferred to 38 / 25 mm hexagonal polystyrene weigh dishes from Fisher Scientific (Pittsburgh, PA) and allowed to age for 24 h. Byproducts were removed by rinsing the gels with water. Next, the gels were soaked in 1 M H 2 SO 4 for 24 hr. H 2 SO 4 and Mn (II) salts were removed by rinsing the gels with water. The gels were then rinsed with acetone, followed by cyclohexane to replace 56

73 the high surface tension pore liquid. For 24 hr, the gels were vacuum dried at 60 C. Gels were vacuum dried with Isotemp Vacuum Oven Model 281 (Fisher Scientific, Pittsburgh, PA) Ormosil synthesis and the templating process Film synthesis was based on previous papers [79, 80] with some modifications. Most films were prepared from a sol containing 3.0 ml of TMOS, 0.37 ml of PTMOS, 0.30 of MTMOS, 3 ml of ethoxyethanol, 0.7 ml of water, and 1.0 ml of 0.1 M hydrochloric acid. The preparation of silica with G0-PAMAM as the dopant was the same except that 3 ml of 10 mm dendrimer PAMAM in ethoxyethanol prepared from the stock solution in methanol was included in the sol, in place of 3 ml of ethoxyethanol. Dopamine-templated films were formed from 0.1 mm dopamine in the sol. After stirring 1-2 h, 100 µl of the sol was spin-coated on ITO slides. A piece of tape was placed down the slide, prior to spin coating, covering approximately 0.5 cm to prevent coating on the ITO where this electrode would be attached to the electrochemical station. A Headway Research, Inc. (Garland, Texas) spin-coater was used. Parameters for spin-coating were set at 2000 rpm for 120 s. Films were allow to dried at ambient conditions for 24 h. Films were placed in 0.1 M phosphate buffer, ph 7 to remove dopamine. 3.3 Results and discussion Ambigels The primary purpose of the present study was to design a stable template through sol-gel processing, remove the templating agent, and then detect the analyte. The analyte would be the same biological molecule as the templating agent. Because ambigels are generally more porous than xerogels, we hypothesized that ambigels would serve as a better templating material than xerogels. Initially, ambigels were synthesized as stated previously by Long et al. [76]. After the sol was allowed to dry, a brown gel formed. During the period the gels soaked in 1 M H 2 SO 4, some of the gel began to break apart. By the time the gels were rinsed with cyclohexane to replace the pore liquid, most of the gels 57

74 were in pieces. Following drying for 24 h at 60 C, very fragile brown monoliths were formed. For several reasons, ambigels were not used in further studies. For one reason, the monoliths were very fragile. Many were in too many pieces to be useful. In fact, the gel was so fragile that many of the pores collapsed. A surface area of 130 m 2 g -1 was measured for one gel. The surface areas of several gels were less than 10 m 2 g -1. Long et al. [76] reported a surface area of 210 m 2 g -1, pore volume of 1.6 m1 g -1, and average pore diameter of 29 nm. In the gel synthesized, the pore size was 0.7 ml g -1, and the average diameter was approximately 19 nm. These values are much smaller than expected, which supports the conjecture that the pores were collapsing. Second, the required solvent exchange with acetone or cyclohexane may remove the templating molecule during the formation of the ambigel, resulting in fewer imprints. These solvent may also degrade biological templating molecules, creating distorted imprints. Finally, the present gels were nontransparent so UV-VIS methods could not be used. Hence, this approach was abandoned Cyclic voltammetry of templated ormosils For the above reasons, ormosils were used in the remaining studies. To determine the oxidation potential of dopamine at an ITO electrode, the cyclic voltammogram of 0.1 mm dopamine in 0.1 M phosphate buffer, ph 7, was measured (Fig. 23). As expected, the oxidation of dopamine at the bare electrode was seen at 0.25 V vs. Ag AgCl, and the electrochemical process was reversible. Initially electrochemical experiments were performed with ormosils that were prepared as described, in detail, in the Experimental section. The components in the sol were included for reasons described by Makote and Collinson [79, 80]. For instance, due to the hydrophobicity of PTMOS and its likely affinity for the aromatic functionality on the template, it was utilized as the functionalized monomer; whereas ethoxyethanol was included due to its both polar and nonpolar solvating properties. MTMOS was also included due to its hydrophobicity and to add stability to the gel. 58

75 3 Current, µa Potential, V Fig. 23 Cyclic voltammogram of 0.1 mm dopamine in 0.1 M phosphate buffer, ph 7 at ITO electrode. Scan rate 100 mv / s. 59

76 Films were made by spin-coating 100 µl of the sol at 2000 rpm for 120 s. As described in the Experimental section the dopamine was leached out in phosphate buffer for 24 h. During removal of the template, part of the film came off in the solution. Also, some cracking in the film was seen. As a result, the peak currents were inconsistent for different films. However, the peak current was always higher for the templated films than with the untemplated films. Cyclic voltammograms of these electrodes were studied in Fig. 24 after aging for 24 h. Scan a is of 0.1 mm dopamine in 0.1 M phosphate buffer, ph 7, at an untemplated silica electrode. A passivating film was observed, as demonstrated by the blocking of the voltammetry of dopamine. Repeating the above experiment but with dopamine templated in the silica resulted in a film that did not block the voltammetry of the analyte, dopamine (Fig. 24, scan b). To determine if the templating agent, dopamine, was removed from the sol-gel before analysis, the templated electrode was scanned (Fig. 25, scan b) in only the phosphate buffer, ph 7. An oxidation peak current for dopamine is not observed. In fact, the increase in current (less than a 10% increase) was very small compared to the blank (Fig. 24, scan a). When this electrode was placed in 0.1 mm dopamine, the oxidation of dopamine can be seen (Fig. 25, scan b). These scans proved cavities were formed in the silica resulting in an increased ability to detect the analyte, dopamine. Another observation is that the peak current shifted to a more positive potential from 0.25 V (Figs. 24 and 25) to 0.5 V at the templated electrode compared to the bare ITO electrode (Fig. 23). This may have been caused by the resistance in the sol-gel Square wave voltammetry of templated ormosils Fig. 26 shows the square wave voltammogram of templated and untemplated silica. Scan a shows the current at an untemplated silica film in 0.1 mm dopamine in phosphate buffer, ph 7. A current peak is not observed. The baseline increases slightly in scan b at a dopamine-templated silica-coated electrode in phosphate buffer. This shows that more than 80% of the template was removed. A peak is seen at 0.15 V in scan c at templated silica in 0.1 mm dopamine in phosphate buffer. These scans show that with the 60

77 80 b Current, µa 40 0 a Potential, V Fig. 24 Verification of the deposition of a passivating film of silica. Cyclic voltammograms of 0.1 mm dopamine at ITO electrodes a) without dopamine imprint, b) with dopamine imprint. Scan rate 100 mv/s, initial potential -0.2 V in 0.1 M phosphate buffer, ph 7. 61

78 80 b Current, µa 40 0 a Potential, V Fig. 25 Cyclic voltammograms at a dopamine-templated TMOS-PTMOS-MTMOS ITO electrode of a) 0.1 M phosphate buffer, ph 7, b) 0.1 mm dopamine in 0.1 M phosphate buffer, ph 7. Scan rate 100 mv/s, initial potential -0.2 V. Dopamine removed by soaking in 0.1 M phosphate buffer for 24 h. 62

79 75 Current, µa c b a Potential, V Fig. 26 Square wave voltammogram of a) 0.1 mm dopamine in 0.1 M phosphate buffer at untemplated electrode, b) 0.1 M phosphate buffer at dopamine-templated electrode, c) 0.1 mm dopamine in 0.1 M phosphate buffer at dopamine-templated electrode. 63

80 templated silica, more dopamine was detected than without a template, consistent with the previously described cyclic voltammetry data Aging of templated ormosils Films were aged for 2 weeks to determine their stability over time. Fig. 27 showed the cyclic voltammograms at these electrodes. A defined peak for the oxidation of dopamine is not seen. However, the anodic current is 4-times greater for the templated film than the untemplated film in 0.1 mm dopamine. At 0.5 V, there is a 4-times decrease in current after aging 2 weeks at the templated film. In an effort to determine if adding PAMAM would strengthen and stabilize the films, as previously reported [63, 87], films were made as previously described; however, the sol also contained 3 ml of 10 mm PAMAM in ethoxyethanol prepared from the stock solution in methanol. Fig. 28 shows the cyclic voltammograms at these electrodes. At 0.5 V the current is over 2 times greater than without the PAMAM. These data show that the pores of the films were more stable when PAMAM was included in the sol than without. 3.4 Conclusions Ambigels were not useful for the present study. Dopamine-templated ormosil films on an ITO electrode results in a conductive coating rather than the insulating film. The template provides cavities for the electrochemical detection of the analyte, dopamine. During removal of the template, part of the film came off in the solution. This led to inconsistent peak currents to be observed. However, the peak current was always higher for the templated films than with the untemplated films. By including generationzero poly(amidoamine) dendrimer in the sol, a more stable film was formed. The current obtained with the films with this dendrimer was greater than that of the films without the dendrimer after aging for 2 weeks. 64

81 40 Current, µa 20 0 a b Potential, V Fig. 27 Cyclic voltammograms at a dopamine-templated TMOS-PTMOS-MTMOS ITO electrode after 2 weeks of aging of a) 0.1 mm dopamine in 0.1 M phosphate buffer, ph 7, b) 0.1 M phosphate buffer, ph 7. Scan rate 100 mv/s. 65

82 80 Current, µa 40 0 a b Potential, V Fig. 28 The conditions are the same as those in Fig. 27 but with the addition of G0 PAMAM in the sol a) 0.1 mm dopamine in 0.1 M phosphate buffer, ph 7, b) 0.1 M phosphate buffer, ph 7. 66

83 Chapter 4 Phospholipids 4.1 Introduction Phospholipids (PLs) are amphiphilic molecules that form into a bilayer to makeup biological membranes. There are several different types of PLs, and the amount of each varies in different animals and food products. Chromatography methods, mainly high performance liquid chromatography (HPLC), are used to determine PLs. PLs are essential for healthy cells because they form a barrier around the cell creating an environment within the cell for the proper function of proteins. Changes in PL content may affect cellular functions and viability. For example, the protein, kinase C, can only be activated in the presence of phosphatidylserine (PS) [88]. All living organisms and most natural fats contain PLs. In fact, PLs comprise approximately 60% of the lipid mass of a eukaryotic cell membrane [89]. PLs also serves as a source of energy as triglycerides. The determination of PLs is carried out in several fields including the food and pharmaceutical industries [90]. In the food industry, PLs contribute to the flavor of cereals legumes, and meats and they are used for emulsification [91, 92], antioxidation [93], and stabilization [94-96]. In the pharmaceutical industry, PLs are used to reduce blood cholesterol levels, as an antioxidant, as fat emulsifiers, and in drug delivery systems [97, 98]. Because PLs are rich in choline and fatty acids, they are also marketed as health food additives. 4.2 Types PLs can be divided into two main groups: glycerophospholipids and sphingophospholipids. Because the following chapters discuss the detection of glycerophospholipids, sphingophospholipids will only be briefly mentioned. In general, an amine group of sphingophospholipids links the fatty acid and phosphate group. 67

84 Sphingophospholipids play a role on cell differentiation and apoptosis. For example, they suppress certain tumors. Glycerophospholipids are usually made of three main components: glycerol, phosphate group at sn-3 position of the glycerol, and two fatty acids at the sn-1 or sn-2 positions. However, diphosphatidylglycerols (DPGs or cardiolipins) have two glycerols and four fatty acids in their structures. The arrangement generally results in a structure with a nonpolar tail part (fatty acids) and a polar head group (attached to the other end of the phosphate group). This unique structure is important because it allows PLs selfassemble into bilayers in water. PLs are broken into different classes based on the attachment to the phosphate group. Fig. 29 shows the structures of several PLs. One of the most abundant PL in animal is phosphatidylcholine (PC) [99]. Other PLs include phosphatidylethanolamine (PE), phosphatidylinositol (PI), DPG, PS and phosphatidylglycerol (PG). Some PL species can have an acyl group or an ether or vinyl ether linkage at the sn-1 position, for instance, alkenylacyl-glycerophospholipids. The outer part of the bilayer of a cellular membrane is mostly made-up of PC while the inner part is made up of PE and PS [100]. These classes are further broken into species based on the type of fatty acids attached [101]. Generally, more saturated fatty acid chains are attached on the sn-1 position compared to the sn-2 position. For example, in animal cells, palmitic or stearic acid is almost always attached at the sn-1 position of PC whereas oleic and linoleic acids are usually linked at the sn-2 position. 4.3 Fatty acids Fatty acids are made of carbon chains, commonly with the length of 12 to 24 carbons and with 0 to 6 double bonds. There are two main types of fatty acids. Saturated fatty acids contain no double bonds, while unsaturated fatty acids contain at least one double bond. Two examples of saturated fatty acids are palmitic and stearic acid (Fig. 30). A fatty acid is said to be monounsaturated if only one double bond is present and polyunsaturated fatty acids if more than one double bond is present. Fig. 31 shows two examples of unsaturated fatty acids: oleic (monounsaturated) and linoleic 68

85 Fig. 29 Types of phospholipids: phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, diphosphatidylglyerol, phosphatidylserine, and phosphatidylglycerol. 69

86 Fig. 30 Saturated fatty acids: palmitic and stearic. 70

87 Fig. 31 Unsaturated fatty acids: oleic (monounsaturated) and linoleic (polyunsaturated). 71

88 (polyunsaturated). Unsaturated fatty acids are very susceptible to oxidation. Oxygen attacks the double bonds of the fatty acids to form peroxide linkages [102]. 4.4 Separation and determination PLs are difficult to detect because they lack conjugated double bonds and have unreactive aliphatic functional groups in their structures. Many different methods have been studied for PL separation and determination including: thin-layer chromatography (TLC), mass spectrometry (MS), capillary electrophoresis (CE), nuclear magnetic resonance (NMR), gas chromatography (GC), and high performance liquid chromatography (HPLC). Although there are many known methods, they have drawbacks that need to be addressed Thin-layer chromatography TLC methods use a plate (thin piece of glass, metal, or plastic) coated with a thin layer of solid adsorbent (usually silica gel, alumina, or cellulose). A small amount of the sample is spotted near the bottom of the plate. The plate is then placed in a chamber where only the bottom edge of the plate is in a solvent reservoir. The solvent moves up the plate by capillary action. The different components in the sample move up the plate at different rates because of the differences in their partitioning behavior between the mobile liquid phase and the stationary phase. Detection usually is carried out visually or with ultraviolet (UV) or fluorescence spectroscopy. To describe the separation of different components, the retention factor (or R f values) are measured. d c R f = (17) d s where d c is the distance traveled by the compound and d s is distance traveled by the solvent. The types of solvent(s) and adsorbent(s), the thickness of adsorbent, the amount of material spotted, and the temperature can all affect the R f values. The first separation methods for PLs involved TLC. The most common stationary phase used for PL separation is silica gel on glass plates; however, aluminum is also used. Different modifications to the silica gel have been shown to improve separation of PLs. For instance, the separation of all the PL classes was improved by coating the plate 72

89 with magnesium silica or oxalate [103]. Kaulen [104] found that the addition of ammonium sulfate improved the resolution of PS and PL separation. In order to separate different PLs classes, it may be necessary to remove the fatty acids, because fatty acids may cause some interference [105]. Fig. 32 shows the hydrolyzed PL, phosphatidylinositol. Once the fatty acids are removed, separation based on the structural differences in the different head groups of the PLs is possible. One deacylation procedure involves using monomethylamine to hydrolyze the fatty acids [106]. Detection is commonly approached by determining the phosphorus content of the spots on a silicon plate [107]. In order to view the spots, a coloring agent such as 2,7- dichlorofluorescein can be added [107]. Also, phosphomolybdic acid [108] or naphthol sulphuric acid [107] is commonly added and then the sample is charred to develop the PLs spots on the TLC plate. A ninhydrin reagent can be used to show PLs with amino groups such as PE and PS [107]. Rhodamine 6G or primulin can also be added to visualize the spots for UV detection in a non-destructive determination. The structures of some of these dyes are shown in Fig. 33. Iodine vapor can also be used to detect PLs that do not contain double bonds [109]. However, none of these methods are suited to quantitative determination. There are several disadvantages to using TLC for PL separation and determination. For example, the structure of PL may be destroyed during the visualization step(s), preventing further study of the chemical details of the PLs [110]. The reproducibility of TLC separation methods is poor compared to HPLC and GC [111] because changes in temperature, humidity, and solvent equilibration greatly affect it. With HPLC methods, better resolution can be obtained than with TLC, as discussed later. Unlike GC and HPLC, TLC cannot be attached to an auto-sampler system, and detection is more difficult [90]. One alternative to classical TLC is the use of silica-coated quartz rods, called chromarods in conjunction with a flame ionization detector [112]. PLs quantified by this method were in the range of 0.1 to 20 µg with a relative standard deviation of 4.4 to 7.2 %. However, the response deviates from linearity, and the reproducibility is limited by rod-to-rod variability [90]. 73

90 Fig. 32 Deacylation of phosphatidylinositol. 74

91 a) b) Fig. 33 Structures of the dyes a) rhodamine 6G, b) ninhydrin. 75

92 4.4.2 Gas chromatography GC is another separation method that is used for volatile analytes. A carrier gas, normally helium, is used to transport the analyte through a column. Analytes are separated based on their partition between the gaseous mobile phase and the stationary phase (which may be a nonvolatile liquid bonded to the inside of a column or solid particles packed into a column). GC is normally used to separate only the individual fatty acids present in the PLs, which are derviatized. The different types of PLs are not usually separated with GC because they are non-volatile. Usually, phospholipase C is used to hydrolyze PLs to diacylglycerols, followed by methyl transesterification. The fatty acid methyl esters are then separated by GC [113] Capillary electrophoresis CE is a separation method based on the migration of ions in a buffer solution, down a capillary tube, under the influence of an applied dc electric field. The mobility depends on several factors including the field strength, temperature, and characteristic properties of the ion and electrolyte solution. The electrophoretic mobility µ ep is defined as: µ ep = ν ep E -1 (18) where ν ep is the electrophoretic velocity and E is the electric field strength. Based on different elechrophoretic mobility values, corresponding to the charge-to-size ratio or q r -1 s, ions can be separated. In conjunction with UV-VIS or fluorescent detection, CE has been used to study cardiolipin [114, 115]. Danielson and coworkers [115] isolated cardiolipin from different parts of the mitrochondrial membrane by CE. They used on-line determination of cardiolipin without prederivatization by including the dye, 10-N-nonyl-3,6- bis(dimethylamino)acridine (NAO), in the mobile phase. The structure of the dye, NAO, is shown in Fig. 34. UV-VIS detection at 497 nm was possible due to the increase absorbance of the cardiolipin-nao complex compared to NAO alone. A detection limit of 0.05 µm for cardiolipin was observed, and a linear calibration curve was found in the range of µm. 76

93 Fig. 34 Structure of 10-N-nonyl-3,6-bis(dimethylamino)acridine. 77

94 A less common method is micellar electrokinetic chromatography (MEKC). Here, the analytes are separated based on their distribution between a mobile and a pseudostationary phase. Szucs et al. [116] separated and detected PLs in lecithins. They compared two separation methods: MEKC and HPLC. They found that with MEKC, more PLs were able to be separated and detected with UV at 200 nm than with HPLC in conjunction with a UV detector at 206 nm due to the higher peak capacity of MEKC. In fact, individual species of PLs may be separated based on fatty acid chain differences. They also looked at the reproducibility of the two methods and found that the HPLC method was more reproducible than MEKC (relative standard derivation of 1.5 3% versus 2.5 5%). Because compounds move at different velocities, it is more complicated to compare peak areas in MEKC than with HPLC [90]. The integrated signal for an apparently faster-moving analyte will be lower than that of the slower-moving analyte, assuming equal concentrations and response factors High performance liquid chromatography There are several types of LC including normal phase, reversed phase, bonded phase, size exclusion, and ion chromatography. Both normal phase [101, 117] and reversed phase [101, 118, 119] LC can be used for PL analysis. Analytes are separated based on their partition between the mobile and the stationary phases. For example, PC was separated from other PLs in egg yolk using a silica column and different amounts of isopropanol, hexane, and methanol (such as 55:36:9 v/v/v) in the mobile phase [117]. With reverse phase, the mobile phase is a solvent(s) that is more polar than the stationary phase. The reverse is true for normal phase. Different PLs classes are normally separated using normal phase LC. With this method, separation is based on the different charges on the polar heads of the PL. Different molecular species within one PL class are usually detected with reversed phase LC. Here the species elute according to fatty acid composition [119]. The risk of autoxidation of PLs is reduced with HPLC compared to TLC because the exposure of the sample to atmospheric oxygen is decreased. One disadvantage of HPLC is that separation using reverse-phase chromatography of molecular species can be difficult due to the wide range of polarities. Hence, mobile phase gradients are often 78

95 needed to separate molecular species of PLs by HPLC [120, 121]. This often results in peak and baseline disturbances, making resolution of minor components difficult. 4.5 Detectors The detectors described below can all be used in conjunction with HPLC. Most of the methods described below are HPLC-based because the following chapters discuss preconcentration and detection methods that may be used with HPLC in the future. There are several detectors that can be used with HPLC, including refractive index (RI), UV, evaporative light scattering detection (ELSD), fluorescence, and mass spectrometry (MS) Refractive index There are many drawbacks to using RI detection. For instance, this detector is not very sensitive and fluctuations in temperature, pressure, flow rate, and solvents can cause variability in detection [121]. Also, only isocratic methods can be used with RI, limiting the ability to determine all of the PLs [109]. For all of these reasons, RI detection is not often used with HPLC when the goal is quantitative determination Ultraviolet The most common detectors for HPLC of PLs are ELSD [94, 122, 123] and UV [101, 124]. Neither detection method is selective. UV detection usually occurs at nm since phospholipids have no chromophoric groups [91]. This limits the type of solvent that can be used because most solvents have UV cutoffs that fall above 210 nm [125]. Therefore, chloroform, ethyl ether and acetone cannot be used. Acetonitrile, methanol, water, hexane, and isopropanol all can be used as the mobile phase [103]. Also, UV detection is affected by the number of double bonds in the PLs because carbonyl and the carbon-carbon double bonds in the fatty acids are the only UV absorbing functional groups [109]. PLs samples have been derivatized with benzoates, dinitrobenzoates, pentafluorobenzoates, and nicotinic acid to improve UV detection [126]. 79

96 Jungalwala et al. [127] were one of the first to use HPLC to separate PLs. They used a silica column (average particle diameter of 10 µm). The amino groups of the PLs were derivatized to biphenylcarbonyl so that detection of PLs with UV could be carried out at a longer wavelength (268 nm) than with un-derivatized PLs. At 268 nm there is less noise and a wider range of solvents can be used. The PLs, PE, PS, and lysophosphatidylethanolamine were then separated by HPLC. The limit of detection found with this method was pmol for the derivatized PLs. Kang and Row [120] separated PLs in soybeans by HPLC and detected PE, PI, and PC with UV at 208 nm and 210 nm without derivatization. A mobile phase gradient was used with Nova-Pak column (4 µm, Waters). Three solvents were used in the mobile phase: hexane, methanol, and isopropanol, with the mobile phase becoming more polar with time (5 % to 100 % v/v of methanol) Evaporation light scattering detection The three main steps involved with using ELSD are nebulization, evaporation, and detection. First, the eluent is transformed into a fine mist by the nebulizer. Next, the analyte and solvent molecules are separated using a heated evaporation tube. Finally, the detection is based on the light scattering by the solute particles remaining after evaporation of the mobile phase. One advantage of ELSD is that solvent gradients can be used without base line changes. For example, Stith et al. [121] used varying concentrations of chloroform, methanol, and ammonium hydroxide in the mobile phase (with increasing amounts of methanol over time) with a silica column to separated PG, PE, PI, PC, PS, and sphingomyelin. With this method, they were able to alter the elution times of the PLs so PI and PS peaks were separated from the PE and PC peaks. Usually, PE and PC peaks are so large that the smaller peaks representing PI and PS are obsured. On the other hand, ELSD is only compatible with volatile solvents and the response is rarely linear. For example, Coboni et al. looked at PLs from cooked beef with HPLC and ELSD. They found, at low concentrations ( µg), the calibration curve is exponential [128]; the response is usually sigmoidal in shape [129]. 80

97 4.5.4 Mass spectrometry Mass spectrometry (MS) is used to determine PLs based on their mass-to-charge ratio. Mass-to-charge ratio is obtained by dividing the molecular mass of an ion, m, by the number of charges, z, that the ion has. MS can be used alone or with HPLC or GC. Ionization can occur at either the phosphate group or on the head group of the PL. PLs can also be identified using both negative and positive ion modes. Usually, due to the nature of the PLs head groups, PC and PE are identified with the positive ion mode. HPLC separation may be necessary prior to MS detection because PC often overshadows the response of PE. PG, PS, and PI can be detected using the negative ion mode [130, 131]. Moe et al. [132] used negative electrospray ionization tandem quadrupole mass spectrometry to characterize the complete structure of intact PLs, and to determine the molecular mass of the fatty acid substituents. For example, PC form adducts with small anions, X -, e.g. Cl -, shown below: M + X - M-CH CH 3 X (19) where a methyl group is lost from the choline moiety of PC. PC is identified by the product ions at m/z 168 and 224, whereas m/z 140 and 196 designate the ethanolamine group of PE. In order to determine the position of the double bond (unsaturated fatty acid), derivation was carried out by converting the olefinic sites to their 1,2- dihydroxylated derivatives. This derivation provided information on the position of the hydroxyl groups; hence, the position of the double bond could be found Electrochemical detection of phospholipids after chemical treatment Klein et al. [133] looked at the electrochemical detection of free choline and choline metabolites, including acetylcholine and phosphocholine in rat brain and body fluids. First, hydrophilic and lipophilic choline containing compounds were separated, followed by the hydrolysis of the lipophilic species to free choline. Separation was carried out using HPLC. The amount of hydrogen peroxide formed from the reaction of choline with the choline oxidase and acetylcholine on a reactor column was electrochemically detected. The amount of choline contained in phospholipids was carried out by separating these phospholipids using thin layer chromatography. The free 81

98 choline was released after an acidic treatment. They found that the intracellular levels of glycerophosphocholine (1.15 mm) and phospholcholine (0.59 mm) in the brain were higher than their concentrations in the central nervous system (2.83 and 1.70 µm). Ikarashi et al. [134] determined the amount of bound choline that was hydrolyzed from phospholipids using the enzyme, phospholipase D, and compared this to the amount of choline free in rat plasma. The liberated choline was separated with liquid chromatography and detected electrochemically. They found that the amount of free choline (10 ± 2 nmol ml -1 ) was less than the amount of bound choline (1300 ± 100 nmol ml -1 ). 4.6 Preliminary study of phospholipid detection by electrochemistry Although PLs are known to be chemically oxidized [135], direct electrochemical detection methods cannot be carried out because of strong adsorption of the PL, which causes surface passivation. In initial studies in our group [136], sol-gel films with dirhodium phosphomolybdic acid (Rh 2 POM) were formed on an indium tin oxide (ITO) electrode. The presence of PC in solution was detected by an increase of the current at about 1 V vs. Ag / AgCl that corresponded to the oxidation of PC catalyzed by the dirhodium catalyst. Fig. 35 shows the oxidation of 10 µm PL at the silica modified ITO electrode. A calibration curve from µm of PC was obtained, with a R 2 of Although detailed experiments were not performed, the results suggested that PC does not strongly adsorb to the sol-gel and that Rh 2 POM can catalyze its oxidation. The chapters that follow (chapters 5 and 6) discuss extension of this study. Chapters 5 will discuss the preconcentration and electrochemical detection of a PL on a nanocomposite prepared by layer-by-layer (LBL) electrostatic assembly. The LBL contains an cyclophane derivative, 4-xylylene-1,4-phenylenediacetate, which is evaluated as a trapping agent for PC. Rh 2 POM is used as a catalyst after entrapment. A cyclodextrin templated sol-gel electrode will be described in Chapter 6 as an electrochemical detector for PLs. The catalyst, Rh 2 POM, or a rhodium(ii) acetate dimer (Rh 2 Ac) is contained within the sol-gel and used for the oxidation of PC. 82

99 blank 10µM PL 20 I / µa E / V Fig. 35 Cyclic voltammetry at dirhodium phosphomolybdic acid doped sol-gel electrode. 83

100 Chapter 5 Preconcentration and determination of a phospholipid at a surface modified by layer-by-layer assembly 5.1 Introduction Phospholipids (PLs) are building blocks of biologically membranes [99]. The determination of PLs is important not only in the biological field but also in the food and pharmaceutical industry [90]. In the food industry, PLs are used for emulsification [91,92], as anti-oxidants [93], and for general stabilization [94-96]. In the pharmaceutical industry they are additives with such roles as fat emulsification and drug delivery [97, 98]. Generally, PLs have two nonpolar fatty acid tails and a phosphate head group. An outcome of this structure is that PLs self-assemble into bilayers in water and at interphases. There are several classes of PL, depending on the attachment to the phosphate group [99], which are further separated into species based on the nature of the fatty acids [101]. The combination of CP and phosphatidycholine (PC) was selected for the present study to serve as surrogates for future work of on PLs such as cardiolipin for which the development of methods for trace-level determinations is needed Layer-by-layer Layer-by-layer (LBL) fabrication is a technique involving the sequential assembly (usually by ion exchange of macromolecules layers) on a solid substrate. LBL approaches are attractive for several reasons. For instance, highly tailored polymer thin films can be created. Also, LBL procedures are relatively simple to carry out and inexpensive to make. Many functional groups can be incorporated. Polymers, organic dyes, electrochemically active species, proteins, DNA, and other biological molecules have been incorporated into LBL assemblies [ ]. Applications of the LBL method include: nanobiorectors, drug delivery systems, electronic devices, artificial cells, and electrochromic thin films, as discussed in references 137 and

101 Electrostatic layer-by-layer assembly There are several LBL approaches: interlayer interaction (electrostatic), affinity, covalent, and hydrogen binding, although many LBL approaches may involve more than one type of interaction. Electrostatic approaches are by far the most popular. Affinity approaches are usually used in biological applications. Electrostatic LBL assembly was first developed in 1990 s by Decher and coworkers [ ]. In an electrostatic approach, layers formed on a charged substrate. The substrate may need to be modified in order to be charged. For instance, gold may be modified with a thiol containing an amine or hydroxyl group. In the procedure described in this chapter, aminothiophenol (ATP) or mercaptobenzoic acid (MBA) is used to modify the gold substrate with the required charge, whereas 3- aminopropyltriethoxysilane (APTES) is used to modify indium tin oxide (ITO). Fig. 36 shows the structures of these molecules. The charged substrate is immersed in a solution containing a polyelectrolyte of the opposite charge to that of the substrate. For instance, a layer of poly(styrene sulfonate) can form on a positively charged surface [143]. The substrate is then rinsed and dried. Next, the substrate is placed in another solution containing a polyelectrolyte of the opposite charge to that of the first polyelectrolyte solution. For example, poly(allylamine hydrochloride) can be used. The substrate is again rinsed and dried. This procedure can be repeated to form multi-layers, as shown in Fig Quartz crystal microbalance There are several techniques used to study LBL formation including atomic force microscopy, X-ray reflectometry, UV/VIS spectroscopy, cyclic voltammetry, infrared spectroscopy, and quartz crystal microbalance (QCM). QCM is used to monitor the LBL formation in this chapter. QCM is used to measure shifts in frequency caused by mass charges. In 1959, Sauerbrey [144] showed that the frequency shift of a quartz crystal resonator is directly proportional to mass changes. Fig. 38 shows a diagram of a QCM, based on a model 400 Electrochemical QCM (CHI 400), from CH Instruments (Austin, TX.) [145]. A thin gold 85

102 a) b) c) Fig. 36 Structures of molecules used for the modification of layer-by-layer substrates a) 4-aminothiophenol, b) 4-mercaptobenzoic acid, c) 3-aminopropyltriethoxysilane 86

103 Fig. 37 Electrostatic layer-by-layer formation a) the charged substrate (positive) is immersed in a solution containing a polyelectrolyte (negative), b) the substrate is placed in another solution containing a polyelectrolyte (positive), c) multi-layer formation of (a + b) n, where n, 3, is the number of times placed in the positive polyelectrolyte solution (a) followed by the negative polyelectrolyte solution (b). 87

104 Fig. 38 Diagram of quartz crystal microbalance (based on ref. 145). 88

105 disk is coated on both sides of the piezoelectric quartz crystal. A contact pin is use to connect the working electrode (only one side) to the oscillator box. The crystal oscillates at a certain frequency, which is monitored. An increase in mass will cause the frequency to decrease proportionally based on the Sauerbrey equation: f = 2 2 f 0 m A µρ (20) where f is the measured frequency shift, f 0 is the resonant frequency of the fundamental mode of the crystal, A is the piezo-electrically active area, µ is the shear modulus of quartz, 2.947x10 11 g cm -1 s -2, ρ is the density of quartz, g cm -3, and m is the mass change per unit area (g cm -2 ). The frequency difference between the working crystal and the reference crystal is measured with instruments that use the time-resolved mode. With the CHI 400 instrument, the reference crystal has an oscillation frequency of MHz; whereas the working crystal oscillation frequency should be MHz. Other values with this instrument are: ρ = g cm -3, µ = 2.947x10 11 g cm -1 s -2, and A = cm 2. Therefore, a mass change of 0.14 ng will result in a frequency charge of 0.1 Hz Cyclophane In 1951, Cram and Steinberg [146] reported the synthesis of a compound with two benzene rings that were held face-to-face by methylene bridges. They named the compound [2.2] paracyclophane, which led to the development of the cyclophane nomenclature. Cyclophanes were known before this time under other names, such as ansa (latin for handle) compounds, which characterized the bridges as handles on a ring system [147]; however, it was not until two decades later that molecules with at least one aromatic ring bridged by at least one aliphatic n-membered bridge were called cyclophanes [148]. A variety of molecules including cations (inorganic and organic), anions, and neutral molecules have been trapped with these molecules [149]. The investigation of the inclusion of quaternary ammonium cation has gained a considerable interest mainly because of its biological implications. One example is acetylcholine, a neurotransmitter (Fig. 39). Roelens and colleagues [150, 151] have shown that 89

106 a) b) Fig. 39 Structures of a) acetylcholine, b) phosphatidylcholine. Different fatty acids may be attached to the phosphate group of this phospholipid. 90

107 acetylcholine and other quaternary ammonium cations bind to cyclophane host, specifically1,4-xylylene-1,4-phenylenediacetate Polyoxometalates Polyoxometalates (POMs) are molecular clusters of metal oxides of nm-sized usually containing vanadium, molybdenum or tungsten; although niobium, tantalum, rhenium, and iodine have also been synthesized [152]. These molecules are important electrochemical catalysts, which undergo reversible, multi-electron transfer reactions. Interest in POMs also include applications such as corrosion resistant coatings, dyes, recording materials, protein precipitation agents, and staining agents. These application and others are discussed in a review article by Katsoulis [153]. There are two main types of POMs: isopolyanions [M m O y ] P- and heteropolyanions [X x M m O y ] q-, where x m [154]. Heteropolyanions can be further divided into Anderson [XM 6 O 24 ] q-, Keggin [XM 12 O 40 ] q-, and Dawson-Wells [X 2 M 18 O 62 ] q- ; although there are other types. In this experiment, indirect electochemical detection of the analyte is accomplished with a POM mediator. The bifunctional catalyst in this experiment consists of the dirodium substituted Dawson-Wells POM, [Rh 2 Mo 18 O 62 ] 6-, (Fig. 40) [155]. Cox and workers [156] were the first to synthesize this molecule, based on previous synthesis of bis(acetato)dirhodium-11-tungstophosphate, [(PO 4 )W 11 O 35 {Rh 2 (OAc) 2 }] 5- by Pope and coworkers [157] and Cox and coworkers [158], exchanging the transition metal from tungsten to molybdenum Electrochemical oxidation of phospholipids Phospholipids are readily oxidized chemically [135]; however, the development of methods based upon electrochemical oxidation has been slowed because the penchant for PLs to strongly adsorb to surfaces, which results in passivation of conventional electrodes. The strong adsorption was exploited in a tensammetric method for the determination of PC among other PLs at a mercury electrode; it was used in conjunction with both flow-injection methodology and HPLC [159, 160]. Adsorption also was employed in an indirect amperometric detection method, in which the signal was the 91

108 Fig. 40 Model of Wells-Dawson [P 2 Mo 18 O 62 ] 6- polyoxometalate (based on ref. 155). 92