The Structure and Dynamics of Diacetylene-Lipid Langmuir Monolayers NICHOLAS JOSEPH CASTORANO. Submitted in partial fulfillment of the requirements

Transcription

1 The Structure and Dynamics of Diacetylene-Lipid Langmuir Monolayers by NICHOLAS JOSEPH CASTORANO Submitted in partial fulfillment of the requirements For the degree of Master of Science Thesis Adviser: Dr. J. Adin Mann Jr. Department of Chemical Engineering CASE WESTERN RESERVE UNIVERSITY August, 2010 i

2 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of candidate for the degree *. (signed) (chair of the committee) (date) *We also certify that written approval has been obtained for any proprietary material contained therein.

3 I would like to dedicate this thesis to my parents and brothers who have always been a source of support and inspiration to me. ii

4 Table of Contents Table of Contents... i List of Figures...ii List of Tables... v Acknowledgements...vi Abstract Introduction Background Overview of Langmuir Monolayers Lipid Molecule of Interest Data Analysis Theory Experimental Preparation of Sample KSV Trough System at Kent State University Brewster Angle Microscopy System at Kent State University KSV Trough System at Case Western Reserve University Surface Light Scattering Spectroscopy Results and Discussion Isotherms Measured at Kent State University Using Barrier System BAM Images Captured Simultaneously With Isotherm Data Collection Isotherms Measured at CWRU Using Addition Method APL Analysis of SLSS Data Conclusions and Further Work Conclusions Further Work Bibliography i

5 List of Figures Figure 1. Probable structure of the film when subjected to certain stresses, forming a multilayer film Ω is the domain, Ω can be defined as the periphera or frontier of the domain, Ω c is the compliment of the domain with respect to the entire surface, and ň is the normal to the periphera in the plane... 6 Figure 2. Diacetylene lipid structural drawing [23:2 Diyne PE 1,2-di-(10Z,12Ztricosadiynoyl)-sn-glycero-3-phosphoethanolamine]... 7 Figure 3. Sample BAM image of nucleation growth of the spiral structure witnessed by E. Mann s group at Kent State University... 8 Figure 4. Plot of critical surface pressures for several temperatures accompanied by BAM images showing structural formations... 9 Figure 5. Example of KSV Minitrough System Figure 6. Surface pressure-mean molecular area isotherms, where π represents the surface pressure and σ represents the mean molecular area, at multiple temperatures measured by Mann s group using their protocol Figure 7. Brewster Angle Microscopy (BAM) system Figure 8. Sample BAM Image of a monomolecular Langmuir film showing a dislocation pair in two-dimensions Figure 9. Zero reflectivity with the proper Brewster angle and the effect of adding a thin film on top of the substrate Figure 10. General schematic for Surface Light Scattering Instruments where u denotes the optical field function of the laser beam at each point: u(g) is the field ii

6 right after the grating, u(f) is the field at the Fourier Plane, u(-) is the field just above the surface, u(+) is the optical field for the incident beam as modulated by the grating and the surface amplitude fluctuation, see Mann, et al. [11] Figure 11. SLSS Diagram used in this research emphasizing the different beam orders. The system works on the principle outlined in Figure 10 except that the detected beam is that which is reflected back from the surface as shown here. The transmitted beam is not shown. Note that the value of the capillary ripple wavenumber, q ~ 500 cm-1, is related to the location of the first-order beam. The wavelength of the laser is nm (From Mann, private communication) Figure 12. Example of SLSS data correlation function. This function is approximated well by a Lorentzian function, equation (2.2), when the viscosity of the liquid phase is small, see Mann, et al. [11]. The quantity <E*(0)E(τ)> is the correlation function of the electric field as determined from the photocurrent of the detector for the system shown in Figure Figure 13. Example of power spectrum function that is a result of the Fourier transform of the correlation function outputted from the SLSS data. Only the ω>0 branch is observed. G L (ω) is the Fourier transform of equation (2.2); the spectrum function of an ideal liquid surface is Lorentzian, see Mann, et al. [11]. 33 Figure 14. Surface pressure-mean molecular area isotherms at multiple temperatures measured at Kent State University using new protocol. The KSV trough was used with barriers to control the area iii

7 Figure 15. BAM image taken during the acquisition of the surface pressure-mean molecular area isotherms using the alternative protocol. Image is from a 25 C isotherm at a surface pressure of approximately 0, after decompression Figure 16. Surface pressure-mean molecular area isotherms at multiple temperatures collected at CWRU using new protocol. The spreading pressure, π, was determined with the KSV Wilhelmy plate system Figure 17. Center frequency-mean molecular area isotherms calculated through analysis of SLSS data using APL analysis program. The center frequency was determined through fitting the correlation function or the spectrum with the Lorentzian Fourier transform pair, see equation (2.2) Figure 18. Damping factor-mean molecular area isotherms calculated through analysis of SLSS data using APL analysis program Figure 19. Standard deviation of center frequency-mean molecular area calculated through analysis of SLSS data using APL analysis program Figure 20. Standard deviation of damping factor-mean molecular area calculated through analysis of SLSS data using APL analysis program Figure 21. Signal to noise ratio-mean molecular area calculated through analysis of SLSS data using APL analysis program Figure 22. Surface pressure-mean molecular area isotherms calculated through analysis of SLSS data using APL analysis program iv

8 List of Tables Table 4.1 Outlines the effect of temperature on density and surface tension of pure water and the resulting ratio relevant to equation (4.1) Table 4.2 Details the effect of temperature on density and viscosity of pure water and the resulting ratio relevant to equation (4.2) v

9 Acknowledgements I would like to thank my principal investigator: Dr. J. A. Mann Jr. The support and guidance he provided me throughout my graduate career has been remarkably beneficial to my development not only as a researcher but as a person as well. His knowledge and command over the subject area is something that cannot be matched elsewhere. Simultaneously, his willingness to teach the subject from several perspectives is what makes him an excellent mentor and teacher. I would also like to thank Dr. Donald Feke and Dr. Heidi Martin for serving as members of my defense committee. Their feedback and review of my thesis is much appreciated. I would also like to thank the research group at Kent State University that collaborated on this project. In particular, Dr. Elizabeth Mann for her continual feedback of new ideas and thoughts on the project. I would also like to thank Prem Basnet, Pritam Mandal, Fanindra Bhatta, and Dominic Malcolm for their help in the lab running the KSV/BAM system. I am also grateful for the undergraduate students who helped with much of the lab work throughout the research project. In particular, Andrew Swisher and Dan Wolak for their help with the Surface Light Scattering Spectroscopy (SLSS) system at CWRU as well as experimental work down at Kent State University. Grace Chen s assistance with the creation of many of the solutions used in this research is also much appreciated. vi

10 I would also like to give a special thanks to Craig Virnelson for his help with troubleshooting any lab equipment throughout the extent of my research. Another thanks to the group in the machine shop for their help in construction of custom designed equipment. Special thanks to the National Science Foundation who funded this research as a joint project between J. Mann, E. Mann, J. Alexander, and A. Bernoff. Collaborative Research: Dynamics of Interfacial Domains NSF: CBET vii

11 The Structure and Dynamics of Diacetylene-Lipid Langmuir Monolayers Abstract By NICHOLAS JOSEPH CASTORANO Investigation into the structure of nanoscale monolayer films at the air/water interface requires the integration of multiple research techniques. Langmuir films, which consist of organic molecules that are insoluble in water, such as a diacetylene-lipid, were used throughout this research. The research methods that were implemented in this research include the use of equipment from KSV Instruments Ltd., which will be referred to as the KSV trough system, Brewster Angle Microscopy (BAM), and Surface Light Scattering Spectroscopy (SLSS). The SLSS data obtained at several temperatures has provided an accurate and precise database that can be used in further research to determine characteristic material coefficients of the diacetylene lipid. Another product of this research was the design and implementation of an effective temperature control system for the replacement trough. The effect of varying protocols was also proven to be vital when researching this particular diacetylene lipid monolayer system. 1

12 1. Introduction The main objective of this research is to gain a better understanding of the structure and dynamics of Langmuir monolayer systems [1], in particular diacetylene lipid monolayers [2]. In order to achieve this greater understanding, it was determined that a multiple research method approach would best provide the understanding that is desired. The techniques that were implemented and that will be discussed further are Surface Light Scattering Spectroscopy (SLSS), Brewster Angle Microscopy (BAM), and the KSV Trough system (KSV Instruments Ltd.) for measuring the isotherm properties of Langmuir films, such as surface pressure as a function of mean molecular area. This research analyzes the diacetylene lipid system using all of these techniques individually, but not all simultaneously. In order to understand the structure and dynamics of these monolayer systems it would be beneficial to quantify or characterize them through parameters such as the interfacial tension, the surface elastic modulus, the surface viscosity coefficient, and the bending coefficient [3]. Another central aim of this research was to generate a database of precise and accurate SLSS data across several temperatures that can then be used to calculate the characteristic coefficients of the diacetylene lipid monolayer studied. The investigation into these types of systems proves useful for several applications. One of these applications is in the field of Alzheimer s disease research. The mechanism of Alzheimer s disease has been studied through the analysis of the aggregation of certain peptides. The Langmuir Monolayer approach is one of the excellent methods used to investigate the mechanism and 2

13 origin of Alzheimer s disease. Surface pressure-area isotherms provide information regarding the nature of short-range and long-range interactions between the molecules especially the lipids and peptides [4]. Another area of research that has a particular desire and use for understanding monolayers and their structure is that of biosensor development. Monolayer films, in particular self-assembled monolayer (SAM) films, have been proven to improve biosensors by providing a simple way to functionalize the surface of electrodes. Because there is such a vast amount of materials in which to choose what the monolayer will be comprised of, the applications can be spread across several categories. It is demonstrated with suitable examples that monolayer design plays a key role in controlling the performance of these SAM based biosensors, irrespective of the immobilization strategy and sensing mechanism [5]. The last major application of this research that will be discussed is the field of oil recovery. Although there has been a major push in recent years for the development of alternative energy methods, society is still heavily dependent upon oil, therefore placing a high importance on oil recovery technology. Most crude oils contain organic acids, so it is of great significance to study the effects of fatty acids on the interfacial tension of surfactant systems. Producing ultra-low interfacial tensions is one of the most important mechanisms relating to surfactant flooding for enhanced oil recovery [6]. This study of the structure and dynamics of diacetylene-lipid monolayer systems will be presented in the following form: Chapter 2 will present 3

14 background information on Langmuir Films and the diacetylene lipid molecules of interest. Chapter 3 will discuss the experimental methods, including the preparation of the sample as well as characterization techniques. Chapter 4 will present and discuss the results of the analysis. Chapter 5 will include conclusions and recommendations for further experiments that would be useful to carry out. 4

15 2. Background This chapter discusses background information that is significant to the research as a whole. In particular it will focus on the subject of Langmuir monolayers as well as the lipid molecules that are of interest within this project. It will also provide an overview of some of the mathematical relationships that will help quantify these monolayer systems into measured parameters. 2.1 Overview of Langmuir Monolayers The structure of monolayer films at the air/water interface is of particular importance when analyzing the effect these films have on certain characteristics of the system, such as surface tension. It is known that the surface tension at the air/water interface is altered when the water phase contains surfactants. These surfactants can exist in the form of various different materials. This particular research will examine the effect of diacetylene-lipid molecules. The diacetylene-lipid molecules create what is referred to as a Langmuir film. In general, these films exhibit a thickness of only a few nanometers and are comprised of organic molecules that exhibit an amphiphilic nature; that is, they consist of a hydrophilic head and a hydrophobic tail. This hydrophobic tail must be long enough that the molecules do not dissolve easily in water, meaning that all the molecules can be thought of as surfactants. At the same time, the long hydrocarbon chain must not be long enough for the molecule to crystallize. Many times there is confusion in the terminology in the difference between a Langmuir film and a Langmuir-Blodgett film. When several monolayers are built up into an 5

16 ensemble, it is generally referred to as a Langmuir-Blodgett film. Consequently, a floating monolayer itself is generally referred to as a Langmuir film. Below is a general schematic for the structure of such a Langmuir film. It is shown that the hydrophilic head of the molecule is submerged in the water while the hydrocarbon tail protrudes in the positive k direction, towards the air. When the film is compressed, it must structurally react in a way to conserve its insoluble nature resulting in the multi-layer domain seen below. The domain is formed in a head-to-head and tail-to-tail nature. In this research the focus is put on the system as it acts as a monolayer. Figure 1. Probable structure of the film when subjected to certain stresses, forming a multilayer film Ω is the domain, Ω can be defined as the periphera or frontier of the domain, Ω c is the compliment of the domain with respect to the entire surface, and ň is the normal to the periphera in the plane [7]. 6

17 2.2 Lipid Molecule of Interest The motivation to use the surfactant that has been chosen for this research lies in several different justifications. The particular lipid molecule that was used in this research was [23:2 Diyne PE 1,2-di-(10Z,12Z-tricosadiynoyl)-snglycero-3-phosphoethanolamine]. The melting temperature for this material is known to be 38 ± 0.5 C. Figure 2 below shows the structural drawing of the diacetylene lipid of interest. Figure 2. Diacetylene lipid structural drawing [23:2 Diyne PE 1,2-di- (10Z,12Z-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine] [8]. One of the several reasons for the choice of this lipid is because of previous work that had been conducted by E. Mann and her group at Kent State University with this material. Their previous work provided interesting results and insight into the 7

18 structure of this monolayer system when subjected to different stresses at various temperatures. Figure 3 below shows Brewster Angle Microscopy (BAM) images that provide a more detailed account of the nucleation growth of the spiral structure exhibited by the monolayer. A further discussion of BAM is in section 3.3. As shown below the spiral pattern grows radially from the nucleation center. The rate at which the structure grows is approximately constant, but increases with an increase in temperature. When the system is decompressed, the spiral patterns disappear [9]. It should also be noted that the spiral domains grow outwardly until they touch another growth. For a more detailed discussion of this structural formation, see Basnet et al [9]. Figure 3. Sample BAM image of nucleation growth of the spiral structure witnessed by E. Mann s group at Kent State University [From E. Mann]. Figure 4 below from E. Mann s group shows the BAM images of these unique structural formations at different temperatures and corresponding surface pressures. It is evident that the unique spiral structure does not form until the temperature is increased above 30 C. When the temperature is between 30 C and the melting temperature of 38 C, the monolayer exhibits the spiral structure 8

19 at the corresponding surface pressures. As the temperature is increased past the melting temperature, the monolayer no longer exhibits a spiral structure, but instead more of a branched pattern. Figure 4. Plot of critical surface pressures for several temperatures accompanied by BAM images showing structural formations [From E. Mann] Another reason for the choice of this lipid as the molecule of interest is due to previous work by Lando and Sudiwala [10]. They investigated the structure of a similar molecule [23:2 Diyne PC 1,2-di-(10Z,12Z-tricosadiynoyl)-snglycero-3-phosphocholine], that differs only in the end group located at the head of the molecule. In their research they studied the crystal structure and 9

20 mechanism for tubule formation. They were able to determine the structure of the film as it formed multiple layers when subjected to certain conditions or at certain surface pressures [10]. The work of Lando and Day also provided some motivation as to the use of this particular molecule as the surfactant [2]. Their research provided insight into the ability of diacetylene compounds to polymerize in the monolayer setting, resulting in a highly ordered structure. They took this work even further by showing that two monolayers sandwiched together retain their individual crystalline morphologies and act to reinforce one another, resulting in an extremely strong bilayer membrane [2]. Another more general reason for the choice of this lipid for investigation is for biological reasons. It is widely known that membranes are typically made up of lipid molecules. Therefore, understanding the structures that these monolayers conform to, and the way in which they conform to them, will help provide insight into the methods by which these membranes operate. Proper investigation may also lead to the understanding of why these membranes degrade over time when subjected to certain stresses. 2.3 Data Analysis Theory Both the surface light scattering spectroscopy data and surface pressure isotherm data that was collected as part of this research can be used to investigate the structure and dynamics of the diacetylene lipid monolayer system. For example, the Gibbs elasticity, K g, can be determined directly from the monolayer isotherm data using the relationship shown below [11]. K g = A π A (2.1) 10

21 where π is the surface pressure, and A is the area. The calculation of the Gibbs elasticity was not one of the goals of this research and it was not performed, but the data generated in this research would allow for it. The analysis program, which will be referred to as the APL analysis, was written in the high-level programming language APL (named after the book A Programming Language by K. Iverson). This APL analysis program determines the center frequency and damping factor through a fitting algorithm. This theory behind the program and implementation itself were the result of previous work done by J. Mann. The equation that governs the fitting of the correlation function is shown below. The center frequency is represented by ω c, and the damping factor, Γ c. R L (Γ c, ω c ; t) = Ae Γc t cos(ω c t) + B (2.2) For more details on the mathematical theory behind the use of the SLSS data in the calculation of material coefficients, refer to Mann et al [11]. Free energy functionals formulated by Mann also provide relationships between the data obtained in this research and the characteristic material coefficients [12]. For example, the values obtained from the APL analysis fitting can be used in the following equations in order to calculate the bending coefficient, B e. Y 1 = ρω c 2 γ q 2 q (2.3) γ = γ + gδρ q 2 + B eq 2 (2.4) where γ is the thermodynamic surface tension (which can be measured from the KSV Wilhelmy plate), g is the gravity term, Δρ is the difference in density between the upper and lower phase, and q is the wavenumber. Y 1 can be taken 11

22 as essentially constant with respect to γ *, meaning that the renormalized surface tension scales with the square of the center frequency. Equation (2.4) defines the renormalized surface tension in terms of the thermodynamic surface tension, γ, the gravity term, which includes the difference in densities of the upper and lower phase, and also the wavenumber, q. The last term in equation (2.4) is what allows us to estimate the bending coefficient, B e, from the data. 12

23 3. Experimental This chapter provides details on the experimental techniques used to investigate the structure and dynamics of the diacetylene lipid monolayer systems. In particular, it discusses the preparation of the diacetylene lipid sample, the technique of Surface Light Scattering Spectroscopy (SLSS), the use of Brewster Angle Microscopy (BAM), and the KSV Trough system used for measuring the isotherm properties of the monolayer films. 3.1 Preparation of Sample When analyzing such a sensitive system as the monolayer film described above, several precautionary steps must be taken. In order to maintain the integrity of the measurements, a clean substrate is imperative, whether it is water or some mix of water and an organic. In addition, any instruments that will be in contact with the monolayer system must be properly cleaned in order to deny contaminants from entering the system, as it is known that contaminants can affect system characteristics greatly. In addition to the precautions that must be taken when preparing the substrate that will be used, the cleanliness of the lipid sample itself is also highly important. As stated before the lipid that was used in this research was [23:2 Diyne PE 1,2-di-(10Z,12Z-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine]. The material was purchased from Avanti Polar Lipids Inc. As purchased from Avanti, the material exhibits a purity of 99%, and there was no further purification performed before the sample was used. Cholesterol and other impurities may change domain shapes through line activity, see Figure 1, and may contribute to 13

24 some spiral shaped domains. Cholesterol is less likely to be a contaminant in this synthetic lipid, and indeed, thin layer chromatography on these lipids demonstrated that they contained less than 0.1% of any such contaminants [9]. During the preparation of the solution, the dry material was held under argon, and the dry material was also stored at a temperature of -17 C. Sigma Aldrich HPLC grade chloroform was used as the solvent to produce the diacetylene lipid solution that would be deposited. The solution concentration used in this research was mg/ml, but was not of particular importance in this research. The mean molecular area was of a much greater importance. Henceforth, the volume of the diacetylene lipid deposited was adjusted based off of the concentration to obtain a certain mean molecular area. The diacetylene lipid of interest in this research is known to photopolymerize when subjected to UV light; therefore extra caution was taken when preparing the samples by using a yellow light. The substrate used for the Langmuir films was a pure water substrate. Often substrates may consist of water and an organic, but in this research only de-ionized ultra filtered pure water was used. In the laboratory at Kent St. University this was obtained from an in-house unit (PurelabPlus/UV system, >18.2MΩ-cm) [9]. In the laboratory work done at Case Western Reserve University the water was purchased from Fischer Scientific. In both settings the cleanliness of the substrate was tested via a shake test, in which all bubbles must break at the surface immediately after shaking to prove that there is no contamination of the substrate. 14

25 3.2 KSV Trough System at Kent State University The KSV system that is used for the measuring the isotherm properties of the diacetylene lipid monolayer must be discussed in two different sections. The reason for this being that the KSV Trough system was implemented in two different ways. The method in which it was used in the laboratory at Kent State University will be discussed in this section in conjunction with the Brewster Angle Microscopy, while the method in which it was used in the laboratory at Case Western Reserve University will be discussed later in conjunction with the Surface Light Scattering Spectroscopy. In regards to the KSV Trough system in the laboratory at Kent State University, the system resembles that of the Minitrough system shown in Figure 5. The dimensions of the trough measure 364 x 175 mm. There is a dipping well in the center of the trough that measures 70 mm deep. The well size is 37 x 37 mm. The trough is constructed out of solid PTFE (Teflon) with a bottom thickness of 1.5 mm. It is mounted on an aluminum plate that is used for temperature control. One of the advantages of the system is the two symmetrically movable barriers that are automatically controlled by the user through the software. This allows for a uniform compression and expansion of the monolayers at a rate that is chosen by the user. The barriers are made of Delrin, a hydrophilic material, in order to decrease leakage during compression. In order to maintain a high order of cleanliness in the system, the barriers are removable which allows for complete cleaning before every trial. Another feature of the KSV trough system is the surface balance that uses a Wilhelmy plate to 15

26 measure the surface pressure. The Wilhelmy plate is constructed of sandblasted aluminum. Typical protocol calls for the plate to be partially submerged in the solution in order to obtain accurate readings. The surface pressure is then calculated by an electrobalance based on the force with which the plate pulls downward. In addition, the Wilhelmy Plate is removable, allowing for optimal cleaning. The force measured by the Wilhelmy Plate is directly related to the bare surface tension, γ, independent of the bending coefficient. As stated before, the trough is mounted on an aluminum plate that has channels built into it, allowing for the heating or cooling of the system. An automated temperature controller using a water bath was used in this particular research to heat and cool the system. In addition to these features a suction system was implemented in order to remove the fluid from the trough or to skim the top off of the substrate. The tips used on the suction apparatus are dispensable ensuring a clean tip is used during each experimental run. When combining all of these features it provides the ability to obtain surface pressure measurements while the monolayer is being compressed. Coupling these measurements with the knowledge of area of the monolayer allows for the construction of surface pressure-mean molecular are isotherms. Again, the temperature control feature allows the user to obtain these isotherms at any desired temperature, although the trough itself should not be operated at a temperature higher than 60 C. Overall, the KSV Trough system allows for control of several system parameters that can help to maximize the reproducibility of resulting measurements. 16

27 Figure 5. Example of KSV Minitrough System [13]. The first step in operating the KSV Trough system is to ensure that all the components are properly cleaned in order to maintain the integrity of the resulting measurements. When dealing with monolayer systems of this sort, any small amount of contamination can ruin an experimental run instantly. Therefore, the first step in the process is to clean the trough itself. Since it is removable from the apparatus, it was easily be cleaned without disrupting any of the instrumentation connected to the apparatus. As stated before the barriers are also removable, which is the next component to be cleaned in the process. As each component is cleaned, it is inserted back into the apparatus in order to reduce the risk of contamination. The Wilhelmy plate is the last component to be cleaned and inserted back into the system. After all components are back in place, the water lines are attached to the bottom of the trough for which the 17

28 heating/cooling water will run through. The trough was then filled with the deionized ultra filtered water substrate. In order to ensure a clean surface to deposit the diacetylene lipid onto, the surface of the substrate was skimmed off the top multiple times using the clean suction mechanism. With a clean substrate and apparatus, deposition is the next step. A note must be made that the protocol in which the deposition and compression of the material was performed differed in comparison to what E. Mann s group had used previously. This difference in protocol is because of a desire to have matching protocol with the system in place at Case Western Reserve University. The major difference was that E. Mann s group raised the temperature of the substrate to 50 C, deposited the diacetylene lipid, cooled to the temperature of interest, and then started the compression of the monolayer. Figure 6 below shows E. Mann s group s surface pressure (π) mean molecular area (σ) isotherms collected using their protocol. 18

29 Figure 6. Surface pressure-mean molecular area isotherms, where π represents the surface pressure and σ represents the mean molecular area, at multiple temperatures measured by Mann s group using their protocol [From E. Mann] The protocol that was used in this research was to raise the temperature to the desired temperature, deposit the diacetylene lipid, and start compression after a time of waiting for the monolayer to properly equilibrate. The reason for this difference in protocol is that the temperature control in the laboratory at CWRU could not achieve a substrate temperature of 50 C consistently. Therefore, the next step in the process is raising the temperature to the desired value. In this research the following temperatures were of interest: 20 C, 25 C, 28 C, 30 C, 32 C, 34 C, and 37 C. These temperatures were chosen in order to mimic the work done previously by E. Mann s group that obtained some 19

30 interesting results with regards to the structure of the monolayer during compression. Once the temperature of interest was achieved, the electrobalance was zeroed out in order to obtain the correct reading of surface pressure. The diacetylene lipid was then deposited via a micro-syringe. A moment of time was then given for the monolayer to properly spread and reach a state of equilibrium. When the system was determined to be stable, that is by verifying the surface pressure was no longer changing, compression was commenced. The rate of compression used in this research was 10 mm/min. This rate was slow enough to give the monolayer time to respond to the compression, but still fast enough to allow for experimental runs to be performed within a reasonable amount of time. When the compression of the film reached its maximum point, the system was set to wait 10 seconds before decompression of the monolayer film commenced. Decompression was performed at the same rate as the compression, 10 mm/min. During the process, the temperature was monitored and controlled via the water bath system. If any adjustments were needed to maintain the desired temperature they were done so through the software controller. After decompression is complete, the sample was then removed from the trough via the suction apparatus. The temperature control system was shut down, and the hoses disconnected from the bottom of the trough. The trough, barriers, and Wilhelmy plate are then all cleaned stored properly. The surface pressure-area data that is generated via the Wilhelmy plate and barrier system can be monitored in real-time during compression and decompression. The software system collects and plots the data on a set of axes that was defined by the user. 20

31 3.3 Brewster Angle Microscopy System at Kent State University In the laboratory at Kent State University, Brewster Angle Microscopy (BAM) is another measurement technique is used simultaneously with the KSV trough system. The BAM system is used in conjunction with the KSV trough in order to allow a direct correlation between the surface pressure-area isotherms and the images generated from the BAM. The BAM provides a direct way of examining the film during real-time compression and relaxation of the film. In other words, the BAM technique allows for the study of monolayer films in situ. Figure 7 below is a schematic that shows the design of the BAM instrumentation for a system in which water is the substrate. It also shows how the system is integrated so that the BAM can run simultaneously with the KSV Trough measurement system. The Wilhelmy plate is positioned in a way that does not obstruct the BAM laser. The BAM unit that was used for this research was a home-made unit constructed in the laboratory at Kent State University. The BAM uses a red semiconductor (668 nm) Argon-ion laser to illuminate the surface. A CCD camera (Panasonic GP-MF 602, 480 x 709 pixels) working at 30 fps, with a field-of-view of 12 mm x 10 mm was used to capture the images. The resolution is approximately the pixel size, 20 µm [9]. It should be noted that the absorption coefficient at 668 nm is small enough that there is no localized heating of the monolayer at the location of the laser beam. Also, the wavelength of the beam is not in the absorption band of the diacetylene that will cause polymerization of the diacetylene lipid molecules. 21

32 Figure 7. Brewster Angle Microscopy (BAM) system (From E. Mann) The BAM can provide insight and visualization into the different structures that the film will conform to and the patterns that will result from these different structural forms. The optical properties of the monolayer can be analyzed using BAM as well [14]. It has been shown that shearing an initially isotropic film by a rotating disk can lead to an optical anisotropy at the interface [14].The BAM is exquisitely sensitive to variations in layer thickness, density, roughness, and optical anisotropy [9]. This method does not, however, distinguish easily between most of these different film parameters. Only in-plane optical anisotropy can be tested separately, by passing the reflected light through an analyzer to test for polarization changes [9]. Below is an example of such an image. It is noted that the scale of the image is on the order of µm. The patterns that are 22

33 created are evident and show that the film is undergoing a stress caused by the compression of the film. A full continuum of these images can be seen throughout the process of compression and relaxation in real-time. This can be related to the surface pressure data being taken simultaneously. Together these two measurement techniques can provide some very interesting images correlated with experimental data. Figure 8. Sample BAM Image of a monomolecular Langmuir film showing a dislocation pair in twodimensions [15] The following section will note the protocol in which the BAM was used in this research. It is important to note that the BAM/KSV Trough setup includes the use of an anti-vibration table due to the importance of not disturbing the film 23

34 during operation. Therefore, the first step in the process was to turn on the gas to the table. The next action was turning on the water for the laser. The water must be run for a few minutes before the laser can be used in order to ensure proper cooling. The power to the laser was then turned on and the shutter opened. The camera that was used to capture the images was controlled by a computer and turned on at this time in the process. It is important to note that the lights in the room are turned off during operation in order to not cause any damage to the camera. The lights were off during the entire process due to the photosensitivity of the diacetylene lipid as well. In order to obtain a high-quality image, there were many parameters that need to be adjusted, the first being the angle of the apparatus. Different substrates require different critical Brewster angle settings. The Brewster angle is dependent upon the refractive indexes of the two phases. For example, an air/glass interface requires an angle of 57, and an air/diamond interface requires an angle of In this research the interface was one between air and water, therefore the critical Brewster angle needed to be set to 53. After adjusting the angle properly, the polarizer must then be adjusted to ensure that the beam consists of only p-polarized light. When the correct angle is coupled with a beam consisting of only p-polarized light, no reflection should occur off the surface. Figure 9 below shows an effective Brewster angle alignment and the effect of adding a thin film on top of the substrate. Another parameter that affects the quality of the BAM images is the height or substrate fill level. Therefore, when adjusting for the best quality images the water substrate was either removed via the suction apparatus or 24

35 added. It should also be noted that when performing experiments at higher temperatures, the evaporation of the substrate during the experiment will require that more substrate be added. It is important that this is done in a manner that does not disturb the film. Therefore, it is added outside the barriers. The imaging system is controlled by the computer software and is saved in order to correlate with the KSV Trough isotherm data that is taken simultaneously. Figure 9. Zero reflectivity with the proper Brewster angle and the effect of adding a thin film on top of the substrate 3.4 KSV Trough System at Case Western Reserve University The KSV trough system used to measure the isotherm properties of the monolayer films at Case Western Reserve University differs slightly from the setup at Kent State University. Because the KSV mini trough was inoperable during the time of this research, a replacement trough had to be used. The replacement trough, made of a single block of Teflon, has a rectangular shape that measures approximately 100 x 75 mm. The trough has a depth of 10 mm with a glass window located in the center. The trough is constructed out of the 25

36 same material as the trough used in the setup at Kent State University, solid PTFE (Teflon). One main feature that is missing from the replacement trough system is the ability to change the area of the monolayer through movement of the barrier system. In this case the area of the trough is fixed and the achievement of different surface densities is done through a volume addition method. This method will be discussed later in this section. The system still incorporates the Wilhelmy plate and electrobalance to measure the static surface pressure through the software on the computer. Both the replacement trough and the Wilhelmy plate are easily removed for cleaning purposes just as done at Kent State University. Due to the fact that a replacement trough was used, originally there was no way to control the temperature of the substrate. In order to combat this problem, a system was designed in which the trough would be clamped down to a copper plate with channels cut out of it for heating or cooling water to run through. The water bath system was then adjusted by the user in order to achieve the proper temperature of the water. Copper was chosen as the construction material for this heating/cooling plate due to its high degree of thermal conductivity and availability. A problem that arose immediately during the efficiency testing of the heating/cooling system was that the bottom thickness of the Teflon trough was limiting the ability to heat the substrate. The system was able to achieve temperatures on the lower end of the desire spectrum, such as 28 C and possibly 30 C, but not effective enough to reach the higher temperatures that were needed. In order to remedy this problem the trough was preheated in an oven before the substrate was added in order to raise the 26

37 temperature of the substrate to the desired value. The duty of the copper plate heating mechanism then became to maintain the temperature of the trough as well as it could. This combined temperature control mechanism allowed for the system to reach all the desirable temperatures, and maintain the system at that temperature, with variation of ± 0.5 ºC, for a period long enough to perform experimental runs. As discussed previously, cleanliness is an extremely important factor when dealing with any sort of surface chemistry research. Therefore, the first step in to protocol for running the KSV Trough system at CWRU is to properly clean the Teflon trough and Wilhlemy plate. When trying to operate at higher temperatures, the trough was then placed in the oven for typically an hour to an hour and a half, at a temperature between 70 C and 85 C, depending on the desired temperature of the substrate. It was later discovered that even with the preheating of the trough and the circulation of the heating water, the highest desired temperature was not consistently achieved. Therefore, when trying to achieve the maximum desired temperature, the water substrate was also preheated in the oven for approximately 15 minutes at the same temperature. The water bath system is also turned on prior to addition of the substrate to the trough in order to achieve proper water heating temperature in the copper plate. The clean trough was then placed into the apparatus and secured tightly to the copper plate. The de-ionized ultra filtered water substrate was then added to the trough, and given time to equilibrate with regards to the temperature. 27

38 When the system is equilibrated and the surface pressure readings from the Wilhelmy plate become stable, deposition of the diacetylene lipid was performed. As noted before, the barrier system that controls the area of the Langmuir film is not operational due to the use of the replacement trough. Therefore, the protocol for deposition of the lipid is different than that used in the laboratory at Kent State University. The method that was used in this research will be referred to as the addition method. The basic idea behind this method is that a typical KSV isotherm measurement generates a surface pressure-surface density plot. Without the ability to change the area that the monolayer film covers, the surface density can then only be changed by the amount of material spread over that constant area. Consequently, instead of decreasing the area of the monolayer to increase the surface density, addition of more diacetylene lipid molecules to the system will provide the same effect. If planned correctly, this addition technique can generate an isotherm similar to that generated from the technique used with KSV trough system in the laboratory at Kent State University. Before any addition of the diacetylene lipid was made to the system, the electrobalance was zeroed out in order to have pure water be the reference state that the surface pressure was calculated from. The concentration of the diacetylene lipid solution was mg/ml The first volume of lipid added to the system, 10 µl, was calculated based off of the surface density desired for the first point on the isotherm. After each stage of deposition, the system takes a short amount of time to equilibrate as the monolayer spreads to cover the entire 28

39 surface. As the system achieves stability, the next volume of lipid was added. The following amounts of lipid were added in succession: 5 µl, 2.5 µl, 2.5 µl, 1 µl. These volumes were chosen in order to move up the isotherm in a timely manner, since it was known that there was minor surface pressure changes at low surface densities. The addition of 1 µl of the lipid was repeated until the system showed a state of saturation. Saturation of the system can be seen visually when depositing the material onto the surface, as well as numerically when monitoring the surface pressure. After completion of the isotherm data collection, the temperature control system was shut down. The trough and the Wilhelmy plate were removed from the apparatus and cleaned properly. The data collected and plotted by the software is simply surface pressure over time. The surface density at each successive addition of lipid was calculated by hand in order to create a complete surface pressure-surface density isotherm. 3.5 Surface Light Scattering Spectroscopy The fluctuations that are evident at the air/water interface can be related to material coefficients of that which is involved at the interface [11]. Through surface light scattering spectroscopy (SLSS), it is possible to relate the changes in elevation on the surface to scattered light [3]. For a more detailed discussion of the measurement of surface fluctuations through surface light scattering spectroscopy, see Mann, et al. [11]. The ability to measure this scattered will allow for the determination of the material coefficients that are related to such fluctuations. Figure 10 below shows the general schematic of how the SLSS system operates. 29

40 Figure 10. General schematic for Surface Light Scattering Instruments where u denotes the optical field function of the laser beam at each point: u(g) is the field right after the grating, u(f) is the field at the Fourier Plane, u(-) is the field just above the surface, u(+) is the optical field for the incident beam as modulated by the grating and the surface amplitude fluctuation, see Mann, et al. [11] [3] The lens L1 forms a Fourier transform of the grating at the Fourier plane. Lens L2 Fourier transforms onto the surface where an image of the grating is formed. The field u(+) includes a superposition of the field on the surface modulated by the surface fluctuations. The first order diffraction spot is then positioned to be aligned optimally with the detector, D. The optical system acts as a band pass filter in wave number space. In its entirety, the system amounts to a homodyne 30

41 optical system for resolving the surface fluctuations with a particular wave number, q. For a more detailed discussion see Mann, et al. [11]. Figure 11. SLSS Diagram used in this research emphasizing the different beam orders. The system works on the principle outlined in Figure 10 except that the detected beam is that which is reflected back from the surface as shown here. The transmitted beam is not shown. Note that the value of the capillary ripple wavenumber, q ~ 500 cm-1, is related to the location of the first-order beam. The wavelength of the laser is nm (From Mann, private communication) Figure 11 above is a general schematic of the experimental setup used in this research. The system consists of several lenses as shown, and the distance between the second lens and the surface of interest is enough to allow for manipulation of the solution. The reflected light is then passed through another lens as it finally reaches the detector. It is important to note that the first order 31

42 diffraction beam is what needs to be focused onto the detector. This is one of the difficulties present when acquiring data. The ability to focus the first order spot correctly is crucial in minimizing the amount of noise within the signal. Figure 12. Example of SLSS data correlation function. This function is approximated well by a Lorentzian function, equation (2.2), when the viscosity of the liquid phase is small, see Mann, et al. [11]. The quantity <E*(0)E(τ)> is the correlation function of the electric field as determined from the photocurrent of the detector for the system shown in Figure 11. The data that is generated by such a SLSS system is in the form of a correlation function. This function is defined in terms of time elapsed space, sometimes called lap times. In order to adequately analyze the data, it must first be put through a Fourier transform in order to achieve the function in frequency 32

43 space. Figure 12 above shows an example of what an ideal correlation function would looks like. It is important to note the symmetry of the curve and its uniformity. Figure 13. Example of power spectrum function that is a result of the Fourier transform of the correlation function outputted from the SLSS data. Only the ω>0 branch is observed. G L (ω) is the Fourier transform of equation (2.2); the spectrum function of an ideal liquid surface is Lorentzian, see Mann, et al. [11]. Because judging the quality of the data from the correlation function may be difficult, it is also expressed in terms of the power spectrum function which is derived from the correlation function by the use of a Fourier transformation. When presented in the form of a power function, it is easier to perform preliminary analysis by first glance before running the data through the APL 33

44 analysis program discussed in background section 2.3. This first glance analysis involves looking at the symmetry of the function around the center frequency/maximum peak, as well as the definition of the peak. If these are not ideal, a decision can be made whether to take another set of measurements. A sample of the power function discussed is shown above in Figure 13. Once this has been accomplished, the function may then be processed through a non-linear least squares fitting algorithm. After performing these manipulations, it is then possible to determine the material coefficients that were said to be related to the surface fluctuations. These coefficients include the interfacial tension, the surface elastic modulus, and the surface viscosity coefficient. These all apply to an isotropic surface, which is assumed upon the system [3]. When describing the protocol used in this research it is important to note that the SLSS measurements were taken simultaneously with the KSV trough isotherm data. One reason this becomes so important is the timing of measurement readings and addition of more lipid to the system. The first step in the process is to turn on the power to the laser itself. The batteries that power the detector must also be connected, as well as both preamplifiers turned on. In this particular research both preamplifiers were not set to the same settings. The filter on both 5113 EG&G preamplifiers was set to 3 khz 100 khz. The course gain on first preamplifier set to 20, and the second was set to 10. The system grating was 500 cm -1 with a magnification of 27 out of 50. The settings of the preamplifiers were empirically found to yield a high quality signal. After the 34

45 preamplifiers have been powered on and adjusted properly, a measurement was done to ensure that the fluid was 120 mm from the face of the final lens. At this time the computer software that controls the recording of the signal is turned on. The last step in the acquisition of the SLSS data, and the most difficult as noted previously, is aligning the first order diffraction beam with the diode photodetector. This is a very delicate process that can take some time to achieve, and certainly takes several hours of practice to master. SLSS data of pure water was acquired at the beginning of each experimental run. The collection of pure water is important during the analysis procedure to refine the parameters in the instrument function and the value of the wave number, q, at the surface [3]. SLSS readings were taken after time was given for equilibration of the monolayer after each incremental volume deposition. After all the SLSS readings were completed the computer software was shut down along with the power to the laser, preamplifiers, and the batteries powering the detector. The software records the data in a file format that is consistent with the analysis program that is used in this research. 35

46 4. Results and Discussion The following chapter will present the data that was obtained using the experimental techniques discussed in the previous chapter. It will also provide a discussion and analysis of the data. As mentioned before surface chemistry research puts a great emphasis on cleanliness, therefore much importance was given to making sure the data was accurate of the monolayer system. The experiments that generated the data in this section were repeated when possible in order to ensure reproducibility. Also, to ensure accuracy of the measurements, pure water surface tension measurements were taken and verified to be accurate. 4.1 Isotherms Measured at Kent State University Using Barrier System The surface pressure-mean molecular area isotherms that were measured at Kent State University were measured for multiple reasons. The first reason being an attempt to discover a difference in the isotherms measured through E. Mann s group s protocol and that which was used in this research. The protocol used by E. Mann s group may ensure that the diacetylene lipid is completely isotropic as a two-dimensional liquid due to the fact that they raise the temperature above the three-dimensional, crystal melting temperature of the material for deposition. The other reason being to provide a baseline set of data that would be used to help design the set of experiments that would be run at CWRU. The surface pressure-mean molecular area isotherm data would also be compared to that calculated through the analysis of the SLSS data. Figure 6 presented previously shows the surface pressure-mean molecular area 36

47 isotherms that were measured by E. Mann s group using their protocol. Their protocol differs from that which was used in this research in the fact that the substrate is heated to a temperature of 50 C, the diacetylene lipid was deposited, the system was cooled to the desired temperature, and then compression was started. In this research, the substrate was immediately heated to the temperature of interest, the diacetylene lipid deposited, and then compression was started. When analyzing the isotherms that were measured by E. Mann s group, refer to Figure 6, it is evident that not until the temperature is raised above 30 C is there any plateau formation in the isotherm curve. Structurally, this plateau in the isotherm corresponds to a phase transition that the monolayer is undergoing. At mean molecular areas greater than the plateau, the monolayer is in a gaseous state. That is the monolayer has a density that is characteristic of a gaseous system. As the monolayer system reaches the state represented by the plateau, the monolayer starts its transition and takes the form of a gas-liquid state. As the isotherm begins to increase after the plateau, the system has achieved a liquid state. It should be noted that the 34 C isotherm exhibits more of a diagonal shaped plateau than a completely flat/horizontal shape like the other isotherms. This is abnormal and should be studied further. Regardless of the temperature, all of the isotherms seem to start this transition around the same mean molecular area, 0.6 nm 2 /molecule. The difference between the different temperatures is the surface pressure value at which the monolayer begins this transition. It is apparent that as the temperature is increased, the surface pressure at which the 37

48 transition begins increases as well. In other words, the surface pressure increases more quickly when subjected to higher temperatures. The surface pressure-mean molecular area isotherms measured using the alternative protocol at CWRU exhibited slightly different curves in comparison to those calculated by E. Mann s group using the older protocol. Figure 14 below shows the surface pressure-mean molecular area isotherms calculated at CWRU using the alternative protocol. When comparing to the isotherms shown in Figure 6, there are definitely differences but there are also some similarities. In particular, the isotherms measured below 30 C exhibit the same shape with no plateau similar to those calculated by E. Mann s group. The major difference between the two sets of isotherms is that the new protocol only exhibited the characteristic phase transition plateau at one temperature, 34 C. A similarity that can be extracted, is where the phase transition begins in regards to mean molecular area. The 34 C isotherm calculated using the new protocol also exhibits the beginning of the phase transition at roughly 0.6 nm 2 /molecule. It is apparent that after the 30 C temperature was eclipsed the monolayer began to react differently. When focusing on the 32 C isotherm, it is evident that the surface pressure began rising more quickly than the lower temperature isotherms, although it did not exhibit the phase transition plateau. As stated before, the 34 C isotherm included the phase transition plateau that is characteristic of the isotherm E. Mann s group collected at this temperature. When analyzing the 37 C isotherm, the surface pressure increases linearly as 38

49 the mean molecular area is decreased. This is unlike any of the other isotherms taken at other temperatures Degrees 25 Degrees 28 Degrees π (mn/m) Degrees 32 Degrees 34 Degrees 37 Degrees σ (nm^2/molecule) Figure 14. Surface pressure-mean molecular area isotherms at multiple temperatures measured at Kent State University using new protocol. The KSV trough was used with barriers to control the area. Overall the resulting set of surface pressure-mean molecular area isotherms measured using the new protocol do show significant differences from that which was measured by E. Mann s group using the original protocol. Some similarities can be extracted from the two, such as the mean molecular area in which the phase transition begins, as well as the behavior of the monolayer when the temperature is less than 30 C. The isotherms measured using the new protocol also helped to establish a baseline set of data that was used to design 39

50 the experiments that were run at CWRU. In particular, they provided insight into the mean molecular area values that are of interest, as well as providing some isotherms to compare to the SLSS isotherm data. 4.2 BAM Images Captured Simultaneously With Isotherm Data Collection As noted in the experimental section, one advantage of the setup at Kent State University is the ability to obtain surface pressure-mean molecular area isotherms using the KSV trough system while imaging the monolayer film using the BAM simultaneously. It was also stated before that E. Mann s group at Kent previously had witnessed some interesting structural formations while compressing the diacetylene lipid monolayer system. Figures 3 and 4 documented these structural formations previously. As discussed before, the spiral structures begin to form when the surface pressure-mean molecular area isotherms reach the phase transition plateau. Since the isotherms that were collected using the new protocol did not exhibit the phase transition plateau, except for the 34 C, it is expected that the system would not show the same spiral structural formation as E. Mann s group witnessed. Figure 15 below supports this expectation that the system at no point formed the spiral structure as shown in Figure 3. In fact the monolayer tends to show a solid state immediately after deposition. This is much different than what E. Mann s group witnessed using their original protocol. 40

51 Figure 15. BAM image taken during the acquisition of the surface pressuremean molecular area isotherms using the alternative protocol. Image is from a 25 C isotherm at a surface pressure of approximately 0, after decompression. 4.3 Isotherms Measured at CWRU Using Addition Method As stated before, the surface pressure-mean molecular area isotherms measured at Kent State University using the KSV trough system were of interest partly in helping to design the experiments that would be run in the laboratory at CWRU. The results of these isotherm measurements define what mean molecular area value should be the starting point of the isotherm. Since the addition method was used in this part of the research, the mean molecular area was calculated from the trough area, the concentration of the diacetylene lipid solution, and the volume of solution delivered to the water surface. Also, because it was witnessed that the surface pressure does not change greatly 41