Eric Dannhäuser. Studienarbeit. Lehrstuhl für Umweltmeßtechnik am Engler-Bunte-Institut der Universität Karlsruhe (TH), Germany

Transcription

1 Characterisation of Hydrophobically Modified Copolymers of N-Isopropylacrylamide and Acrylic Acid and Study of their Solution Properties by Microcalorimetry and Fluorescence Spectroscopy Eric Dannhäuser Studienarbeit Lehrstuhl für Umweltmeßtechnik am Engler-Bunte-Institut der Universität Karlsruhe (TH), Germany Carried out at the Pharmacy Department of the Université de Montréal in Montréal, Québec, Canada Montréal, 04.October 2000

2 Abstracts The present work is based on copolymers of N-isopropylacrylamide, and acrylic acid, carrying hydrophobic perfluorinated C8-sidechains and pyrene labels. These copolymers have been characterised by means of IR and UV spectroscopy. The pyrene content was determined by UV absorption measurements. Isothermal titration calorimetry (ITC) was used to evaluate the ratio between NIPAM and PAA. The polymers behaviour in aqueous solution has been studied by differential scanning calorimetry (DSC) at different ph focussing on the changes of the lower critical solution temperature. The LCST was found to increase with increasing ph and this ph dependence grows stronger with increasing amount of PAA in the copolymer. However, the hydrophobic side chains do not seem to have any effect on the LCST. Fluorescence spectroscopy has been performed in order to determine whether hydrophobic microdomains or polymeric micelles occur in solutions of the polymer. The measurements showed that the pyrene does not form aggregations.

3 Contents 1 Introduction Principles DSC ITC Experimental Polymers and Chemicals Polymers Solvents Chemicals Instruments Spectroscopic measurements Calorimetric measurements Other Polymer stock solutions Buffers Measurements IR-spectra UV-spectra Isothermal titration calorimetry (ITC) Differential scanning calorimetry (DSC) Fluorescence spectroscopy Results and Discussion IR-Spectroscopy UV-Spectroscopy Calculation of pyrene content Peak-Valley-Ratio Isothermal titration calorimetry (ITC) Differential scanning calorimetry, DSC Discussion of the phase transition Dependence of LCST upon ph Double transition peak at high ph Dependence of phase transition enthalpy upon ph Fluorescence Spectroscopy Emission spectra I1/I3-ratio Excitation spectra and peak/valley-ratio Conclusions Acknowledgements References Appendix... 39

4 1 Introduction Water soluble hydrophobically modified polymers have been of great interest for medical and pharmaceutical research in the past years. Since they are capable of forming hydrophobic microdomains or polymeric micelles, they can be used as drug delivery agents in medical fields. Depending on the structure of the polymer, they can be sensitive to changes in their environment such as temperature, ph, concentration of salt etc. Therefore, it is possible to design hydrophobically modified copolymers in order to make them release the drugs under specific conditions. However, designing polymers towards specific properties requires intense research on their behavior upon all kinds of changes in the environment. For this purpose, various instrumental methods are commonly used such as spectroscopy, calorimetry, chromatography etc. The purpose of this work was to determine the solution properties of copolymers of N-isopropylacrylamide, NIPAM and acrylic acid carrying hydrophobic alkyl chains and pyrene labels. At the beginning of the following report, a brief description of the principles of the two calorimetric methods is given (chapter 2). The experimental section in chapter 3 describes the procedures and methods used for analysis. Chapter 4 (results and discussion) can be divided into two parts: First, the characterization of the polymers by means of UV and IR spectroscopy and isothermal titration calorimetry is described. The second part presents the results of differential scanning calorimetry (DSC) and fluorescence spectroscopy. Microcalorimetric experiments such as DSC provide a lot of information about the thermodynamics of the polymers phase transition. In particular the influence of the polymers structure and ph of the solutions was studied by means of this method. Fluorescence spectroscopy was used to investigate the formation of micelles or hydrophobic microdomains. The appendix provides detailed descriptions of some calculations as well as additional spectra and DSC-scans of the studied polymers.

5 2 Principles 2.1 DSC Differential scanning calorimetry (DSC) is a powerful tool for thermodynamic investigation on temperature related transitions of all kinds. The DSC-calorimeter consists of two identical cells which can be heated and cooled electrically by a Peltier-element. One cell contains the sample solution and the other one is used for the reference solution e.g. the solvent. During a scan both cells are heated separately and temperature is measured in each cell. By means of changing the heating power, the temperature in both cells is kept at the same level and the difference in power between the sample and the reference cell is plotted against temperature. Consequently, changings in the heat capacity of the sample, for example due to phase transitions or changes in the enthalpy, lead to a transition peak in the plot of differential power (DP) versus temperature. 2.2 ITC Isothermal titration calorimetry (ITC) can be used to study the thermodynamic behavior of various systems when adding another component to a sample. Like the DSC, the ITC-calorimeter consists of two cells for the sample and reference but in addition, it has an automatic pipette which is filled with the titrand. Before starting the experiment, the pipette is inserted into the cell compartment in order to deliver an exact amount of titrand into the sample cell at each injection (see Figure 2.1). Furthermore, the pipette closes the sample cell forming a kind of valve in order to keep the cell volume constant even though liquid is injected. In contrast to the DSC, the cells in the ITC apparatus are kept at constant temperature by heating or cooling the cells independently, which requires different electric power for each cell. In the raw data plot this differential power is plotted against time resulting in a peak for each injection. Exothermic reactions between the content of the cell and the titrand lead to a downward peak and endothermic reactions give rise to an upward peak. The ITC software automatically performs integration of all peaks over time which gives the change of enthalpy at each injection. In the final plot, enthalpy is plotted against the injected amount of titrand (molar ratio Rm.).

6 Lead Screw Sensor Plunger Rotation Outer Shield Injector Syringe Inner Shield Reference Cell Sample Cell Figure 2.1: Principle of syringe and cell system for ITC

7 3 Experimental 3.1 Polymers and Chemicals Polymers The polymers that have been analyzed in this work are copolymers of N-isopropylacrylamide, NIPAM and acrylic acid. Perfluorinated C8F17-side-chains have been attached 1 to the carboxylic group of the poly(acrylic acid). The polymers have also been marked with pyrene dyes for fluorescence measurements. Figures 1a to 1d show the structure of the polymers and their composition and properties are presented in Tables 3.1 and 3.2. The polymers were synthesized 2 in the laboratory of Dr. S. Bossmann at the Institute of Environmental Analysis Technology at the Engler- Bunte-Institute of the University of Karlsruhe, Germany. CH 2 CH CH 2 CH HN CH C O HO C PAA O l H 3 C CH 3 PNIPAM Figure 3.1 a: Structure of PNIPAM-PAA k

8 CH 2 CH CH 2 CH CH 2 CH HN CH C O HO C PAA O l O C CF 2 O H 3 C CH 3 PNIPAM k F 2 C CF 2 F 2 C CF 2 F 2 C CF 2 F 3 C m PAA-C 8 F 17 Figure 3.1 b: Structure of PNIPAM-PAA-C8F17 CH 2 CH CH 2 CH CH 2 CH HN CH C O HO C PAA O l HN H 2 C C O H 3 C CH 3 PNIPAM k Figure 3.1 c: Structure of PNIPAM-PAA-PY PAA-PY m

9 CH 2 CH CH 2 CH CH 2 CH CH 2 CH HN C O HO C O l O C O HN C O CH PAA CF 2 H 2 C H 3 C CH 3 PNIPAM k F 2 C CF 2 F 2 C CF 2 PAA-PY n F 2 C CF 2 F 3 C m Figure 3.1 d: Structure of PNIPAM-PAA-C8F17-PY PAA-C 8 F 17 used polymer abbreviation PNIPAM- PAA-90 PNIPAM- PAA-95 PNIPAM-PAA- PY-90 PNIPAM-PAA- PY-95 monomer content (k/l) [mol-%] 90.8/9.2 b 90.8/9.2 c 95.3/4.7 b 95.6/4.4 c 90.6/9.4 b 95.4/4.6 b M n [g/mol] M w [g/mol] Nr. of PY per monomer d (= 0,165 mol-%) 460 d (= 0,217 mol-%) determined by: a GPC, b elemental analysis, c titration of COOH, d UV-vis-spectroscopy Table 3.1: Composition and properties of non-fluorinated polymers

10 used polymer abbreviation PNIPAM-PAA- C8F17-90 PNIPAM-PAA- C8F17-95 PNIPAM-PAA- C8F17-PY-90 PNIPAM-PAA- C8F17-PY-95 monomer content (k/l/m) [mol-%] 90.8/4.6/4.6 a 90.8/4.2/5.0 b 95.3/2.4/2.3 a 95.3/2.2/2.5 b 90.8/4.6/4.6 a 90.8/4.2/5.0 b 95.4/2.3/2.3 a 95.4/2.2/2.5 b M n [g/mol] M w [g/mol] N of PY per monomer d (= 0,253 mol-%) 655 d (= 0,153 mol-%) determined by: a GPC, b elemental analysis, c titration of -COOH, d UV-vis-spectroscopy Table 3.2: Composition and properties of fluorinated polymers Solvents Unless otherwise stated, all substances were dissolved in deionized water prepared with a Millipore water purification system. The water had a specific resistance of 18 M cm (at 25 o C). For spectroscopic measurements spectrograde methanol and chloroform were used, both provided by American Chemicals ltd Chemicals 0.1 N standard solutions of hydrochloric acid and sodium hydroxide as well as sodium azide were purchased from Aldrich Chemical Co. Inc., sodium chloride and solid sodium hydroxide were obtained from J.T. Baker and citric acid was purchased from Sigma Chemicals. 3.2 Instruments Spectroscopic measurements Infrared spectra were measured with solid samples on a Bomem Hartmann & Braun FTIRspectrometer using Bomem Grams/32 software (version 4.04) for data processing. UV spectra were recorded on a Hewlett-Packard 8452A diode array spectrophotometer with HP8452Win system software based on Windows 3.1.

11 Fluorescence spectroscopy was carried out on a Aminco Bowman Series 2 Luminescence Spectrometer with AB2 Luminescence Spectrometer software version 4.2 based on OS/ Calorimetric measurements Differential scanning calorimetry (DSC) was performed on a MicroCal VP-DSC Micro Calorimeter using MicroCal Origin 5.0 with VPViewer 2000 DSC-plugin for data processing. Isothermal titration calorimetry (ITC) was carried out on a MicroCal VP-ITC Micro Calorimeter using the same software as for DSC but with the VPViewer 2000 ITC-plugin for controlling the measurements. All samples for DSC and ITC were degassed with a MicroCal ThermoVac degassing device before use Other The ph of the buffers was measured with a Corning ph-meter 430 equipped with an combined Glas-Ag/AgCl-electrode. 3.3 Solution preparation Polymer stock solutions The polymer stock solutions for fluorescence, UV spectroscopy and DSC (1 g/l for aqueous solutions and 0.5 g/l for solutions in methanol) were prepared by dissolving a weighed amount of polymer in water or methanol using a graduated conical flask. The solutions were kept in the refrigerator overnight in order to ensure complete dissolution of the polymer. The stock solutions were diluted to the final concentration using a digital pipette. The stock solutions for DSC were diluted to the final concentration of 0.1 g/l with citric buffer solution of which the ph had been checked and adjusted right before dilution. All solutions were kept at room temperature for at least 3 hours before use. For the ITC-experiment, the stock solution was prepared by dissolving mg PNIPAM-PAA-95 in 50 ml 0.1 M NaCl solution. To prepare the sample, 150 µl of

12 N HCl (standard solution) were added to 10 ml of this stock solution to drop the ph to The titrand for ITC-titration was prepared by diluting 15 ml of N NaOH (standard solution) to 50 ml to obtain a concentration of NaOH of M Buffers Buffers were prepared with 0.1 N aqueous solutions of citric acid and sodium hydroxide adding 0.06 % of solid sodium azide to keep solutions free from bacteria. Immediately before using the buffer, ph was checked and occasionally adjusted by adding citric acid or sodium hydroxide. 3.4 Measurements IR-spectra Samples were prepared by mixing the pulverised polymer with spectrograde potassium bromide in a ratio of approximately 1:5 and pressing a tablet using a hydraulic press at approximately 200 bar. Polymers that could not be pulverized because of their plastic behavior were dissolved in spectrograde chloroform. The saturated solution was applied on a KBr-tablet and the solvent was evaporated leaving a polymer film on the tablet. This procedure was repeated up to 20 times until the polymer film was thick enough to provide a good spectrum. IR spectra were recorded through a wavenumber range from 3600 to 500 cm -1. Reference spectra were taken without putting a tablet in the light path UV-spectra UV spectra were carried out with solutions of the polymers in water using concentrations of 1 g/l or in methanol at 0.5 g/l. Spectra were measured using 1.0 cm quartz cuvettes at room temperature without any special temperature control. The wavelength range was chosen from 300 to 360 nm according to the absorption range of pyrene. Pure solvent was used to record the respective reference spectrum which was subtracted from the sample spectrum. The absorbance values at the maximum absorption wavelengths were calculated with the UV data analyzing software.

13 3.4.3 Isothermal titration calorimetry (ITC) Prior to ITC measurements, the sample, reference solution and titrand were degassed for 7 minutes at 25 o C. The cells were rinsed with the reference solution before filling in the sample and reference. The syringe was purged with titrand (NaOH) three times. The volume of each injection was set to 2 µl with each injection lasting 10 s and with 400 s of spacing between two injections. The reference power was set to 25 µcal/sec ( µw) and the cells were thermostated at 25 o C. ITC-titration was carried out overnight performing 144 injections. The reference experiment was carried out under the same conditions injecting the titrand in NaCl solution without HCl and polymer Differential scanning calorimetry (DSC) Polymer solutions in buffers for DSC were prepared by diluting the stock solutions with the buffer after having verified its ph. The sample and reference solutions were degassed by putting them under vacuum for approximately 7 minutes and cooling to 15 o C. The calorimeter cells, which had a volume of ml each, were rinsed with buffer solution before filling in the sample. Measurements were carried out at a pressure of approximately 1.8 bar using upscanning mode at 1.5 K/min. The starting and final temperature of each scan were chosen to be at least 15 K below/above the expected transition temperature and the calorimeter was thermostated at starting temperature for 15 minutes before each scan. Reference scans were recorded using buffer solution in both cells. At least three scans were recorded of each reference and sample and the results of the first scan were discarded because of their incoherence to the following. Between two experiments with different buffers the cells were rinsed with dilute soap solution, water and methanol and dried with air Fluorescence spectroscopy Fluorescence spectra were recorded using dilute aqueous or methanol solutions at room temperature. The solutions were not degassed. Excitation wavelengths were chosen at 342 nm for methanol solutions and 344 nm for solutions in water according to the maximum UV absorption peak of pyrene. The high voltage for the photomultiplier tube was set to 850 V for aqueous solutions and 670 V for solutions in methanol. The

14 bandwidth of the excitation and emission monochromators were set at 2 nm. Emission spectra were recorded in a wavelength range between 345 and 600 nm scanning 2 nm/s. For excitation measurements, the emission monochromator was set to 396 nm according to the second large emission peak. The wavelength range for excitation spectra was 290 to 380 nm.

15 Transmittance (normalized for values corresponding to cm -1 ) Results and Discussion 4.1 IR-Spectroscopy Infrared spectra of the fluorinated and non-fluorinated polymers (the samples without pyrene) were recorded in order to determine the differences in the spectra due to the hydrophobic C8F17 side chains. The spectra were normalized in order to adjust the intensities and the spectrum of the fluorinated polymer was superimposed to the non-fluorinated one. Figures 4.1 and 4.2 show the superimposed spectra of the respective polymers in comparison. Apart from little differences in the intensity, the spectra are very similar. However, the fluorinated polymer has some absorption peaks that do not appear in the spectrum of the corresponding non-fluorinated polymer. 1,3 [µm] 2, ,2 1,1 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 PNIPAM-PAA-90 PNIPAM-PAA-C 8 F Wavenumber [cm -1 ] Figure 4.1: IR-spectrum of PNIMAM-PAA-90 and PNIPAM-PAA-C8F17-90

16 3287 Transmittance (normalized for values corresponding to 2160,1 cm -1 ) [µm] 2, ,1 1,0 0,9 0,8 PNIPAM-PAA-95 PNIPAM-PAA-C 8 F ,7 0,6 0, Wavenumber [cm -1 ] Figure 4.2: IR-spectrum of PNIMAM-PAA-95 and PNIPAM-PAA-C8F17-95 For both, the polymer with 90 % and the one with 95 % poly(acrylic acid), the fluorinated polymer spectrum shows a weak peak at 664 cm -1 and a strong one around 752 cm -1. They belong to the CF3 group of the perfluorinated chains. 3 The bands of CF2 should appear 3 between 1250 cm 1 and 1050 cm -1 but may be shifted to lower wavenumbers due to the C=O group of the ester. This would be a possible reason for the two little peaks appearing at 1031 cm -1 and 1017 cm -1 in the spectrum of PNIPAM-PAA-C8F17-90 (marked with a circle in Figure 4.1). However, due to lower intensity, these peaks are almost not visible in the spectrum of PNIPAM-PAA-C8F Three bands of the carboxylic acid appear between 3000 cm -1 and 2750 cm -1 for both, the fluorinated and non-fluorinated polymer. 3 The band around 2350 cm -1 belongs to atmospheric CO2 and depending on the CO2 signal of the blank compared to the sample, these peaks may go upwards or downwards. The broad peak at 3300 cm -1 in the spectra of the fluorinated polymers most certainly belongs to the secondary amide of NIPAM. In the same spectral region ( cm -1 ) a very broad band can be seen in the spectra of the non-fluorinated polymer which overlaps the NIPAM band. It is due to residual water in the polymer without fluorine. In fact, the fluorinated polymer obviously contains less water because of its hydrophobic perfluorinated alkyl chains. This is the reason for which the NIPAM s amide peak can only be seen in the fluorinated polymer.

17 Although some of the reported differences in the spectra can be related with certainty to the presence of perfluorinated alkyl chains, it is important to note that this is not an evidence for the fluorinated chains being attached to the copolymer backbone! However, during sample preparation, differences in the polymers consistency have been noticed which could indicate that the perfluorinated chains are, at least partially, attached to the polymer: in contrast to the non-fluorinated polymers, the ones with fluorine had a much more plastic consistency. Upon application of pressure and shear stress (e.g. in a mortar) the particles of the fluorinated polymer only flattened and could not be pulverized or broken into smaller pieces. This changed consistency might be interpreted as due to a covalent attachment of the fluorinated chains to the polymer backbone.

18 Absorption UV-Spectroscopy Ultraviolet absorption spectra of the polymers with attached 1-aminomethyl-pyrene were recorded for two purposes. First, the pyrene content per mole of polymer was determined by measuring the absorption of a polymer solution with known concentration. Second, the ratio between absorption peaks and valleys in the spectra can indicate pyrene association. A typical UV spectrum of PNIPAM-PAA-PY (0.652 g/l) in methanol is shown in Figure 4.3. The three characteristic absorption bands of aminomethyl-pyrene attached to the polymer are located at 312, 326 and 342 nm. 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0, [nm] Figure 4.3: UV absorption spectrum of PNIPAM-PAA-PY-90 in methanol Calculation of pyrene content The absorption value for the 3 rd peak at abs = 342 nm was measured for methanol solutions of all four pyrene-labeled polymers and the molar concentration of pyrene cpy was calculated using Lambert-Beer s law: ( ) c d A = (1). abs py

19 with an absorption coefficient = L (mol cm) -1. The pyrene content rpoly [mol-%] was calculated by equation (2): c py r poly = 100% (2) c poly where cpoly is the molar concentration of the studied polymer. It can be calculated from the mass concentration x [g/l] of the polymer using its molar weight Mpoly which, on the other hand, depends on rpy. The detailed calculation procedure for rpy is reported in Appendix 8.1. Table 4.1 shows the results of the calculation with 1/rpy being the statistical number of polymer molecules for one molecule of pyrene. Results of present work Results of S. Bossmann, Karlsruhe, Germany PNIPAM- PAA-PY-90 PNIPAM-PAA- C 8F 17-PY-90 PNIPAM- PAA-PY-95 PNIPAM-PAA- C 8F 17-PY-95 x poly [g/l] c py [mol/l] r py [mol-%] /r py r py [mol-%] /r py Table 4.1: Results of calculation of the pyrene content in the polymers As it can be seen from Table 4.1, the results for rpy obtained in this work are of the same order of magnitude as those obtained by Bossmann and co-workers in Karlsruhe, Germany. However, the differences could be explained by the fact that a different was used by Bossmann and co-workers for the calculations of the pyrene content Peak-Valley-Ratio The shape of the highest absorption peak of dilute polymer solutions can indicate to what extent interaction or association between pyrene molecules take place. Such associations of pyrene molecules may be a hint towards existence of hydrophobic microdomains or micelles within the polymer solution.

20 UV-Absorption Upon these associations of pyrene molecules, their absorption peaks become broader and lower. A convenient relative measure of this loss in resolution can be extracted from the ratio PA of the absorption intensity of the most intense band to that of the adjacent minimum at shorter wavelength. 5 Figure 4.4 illustrates this ratio PA and its values for the polymers in aqueous and methanol solutions are shown in Table ,6 0,5 A max 3 0,4 0,3 P A 0,2 A min 2 0,1 0, [nm] Figure 4.4: The way of measuring the peak/valley ratio PA in the UV-absorption spectrum PNIPAM- PAA-90 PNIPAM-PAA- C8F17-90 PNIPAM- PAA-95 PNIPAM-PAA- C8F17-95 PA in methanol PA in water Table 4.2: Peak/valley ratio PA of the UV absorbance spectrum in different solvents Without association, the value of PA is usually around 3 or higher and decreases with increasing association of the pyrene molecules. Consequently, the measured values for PA in Table 4.2 indicate that, over all, there are very little interactions between the pyrenes. However, the aqueous solutions show a smaller peak-to-valley-ratio compared to the methanol solutions. Since the polymers and pyrenes are more soluble in methanol, there might be fewer interaction among the pyrenes whereas, when dissolved in water, they might associate to some extend resulting in a lower PA. In addition to that, the PA value for the polymers containing perfluorinated chains are lower than these of the non-fluorinated polymers. This could be a hint towards a contribution of the hydrophobic chains to any association or hydrophobic interactions in water.

21 Absorption Furthermore, for aqueous solutions of the fluorinated polymers, a red shift by 2 nm occurs in the absorption maxima (Figure 4.5) whereas this shift cannot be seen in methanol solution or for the non-fluorinated polymers. These small red shifts of the maxima positions often accompany 5 the above-mentioned broadening of the absorption peaks and the related decrease of PA. 1,0 0,9 0,8 0,7 0,6 0,5 0,4 PNIPAM-PAA-PY-90 PNIPAM-PAA-C 8 F 17 -PY-90 PNIPAM-PAA-PY-95 PNIPAM-PAA-C 8 F 17 -PY-95 0,3 0,2 0,1 0, [nm] Figure 4.5: Red shift of the UV-absorption maxima of the fluorinated polymers

22 kcal/mole of injectant kcal/mole of injectant µcal/sec µcal/sec 4.3 Isothermal titration calorimetry (ITC) ITC was used as an alternative titration method to investigate the monomer ratio of PNIPAM and PAA in the polymer. The ITC-titration was carried out on PNIPAM-PAA- 95, the polymer that was supposed to contain 5 mol-% of PAA. The polymer was dissolved in NaCl solution in order to maintain a constant ionic strength during titration. An excess of HCl was then added to protonate all carboxylic groups of the PAA. The remaining free HCl was then titrated back with NaOH as a titrand. For details on solution preparation and instruments settings, see and Like for a normal potentiometric titration, the results of an acid/base-titration by ITC lead to a S-shaped plot of enthalpy versus the injected amount of titrand, the latter being represented as a molar ratio Rm. 0-5 Time (min) Time (min) Molar Ratio R m Molar Ratio R m Figure 4.6: ITC-titration curve Figure 4.7: Reference titration

23 kcal/mole of injectant The result of titrating the solution of PNIPAM-PAA-95 can be seen in Figure 4.6 which shows a double-s shaped curve. In fact, the first part of the plot (from Rm=0 to the plateau marked with an arrow) corresponds to the real titration curve. The first injections result in a great release of enthalpy due to neutralization of HCl with NaOH. Around the neutral point the released enthalpy decreases sharply as it is common for neutralization of strong acids with a strong base. The plateau around Rm = corresponds to the end of the titration. As there is no free acid left but all previously injected NaOH has been used for neutralization and its concentration at this point is therefore equal to zero, further injections result in a dilution of the injected NaOH. Consequently, the second part of the plot is due to dilution enthalpy which decreases with increasing concentration of NaOH in the cell. In order to obtain the pure, single-s-shaped neutralization plot without the dilution process being overlaid, a reference experiment was performed injecting NaOH of the same concentration into the solvent (NaCl solution) without the polymer and without HCl. The obtained plot of the pure dilution process (see Figure 4.7) was then supposed to be graphically subtracted from the initial plot. However, as the plateau in plot 4.6 is due to an overlay of two processes (the end of titration and beginning of dilution), subtraction of the pure dilution curve did not yield satisfying results. In order to obtain the value of the molar ratio at the neutral point, the dilution part of Figure 4.6 was left apart (except 6 data points) and a sigmodial curve was fit to the remaining data leading to a molar ratio of Rm= R m neutr. = Figure 4.8: Sigmodial fit of titration curve Molar Ratio R m

24 Based on this fitting result (see Figure 4.8) and using the known concentrations for the polymer, acid and base, the amount of PAA in the polymer [mol-%] was calculated and found to be approximately 17.2 mol-%. The detailed calculation procedure is reported in Appendix 8.2. Bossmann and co-workers reported a content of PAA of 4.7 and 4.4 mol-% determined by elemental analysis and ordinary acid-base titration (see Table 3.1). The value obtained during this work turns out to be considerably higher than these values. However, the curve fitting of the raw data using different methods always gives similar results. In order to verify the result obtained by ITC, additional experiments would have been necessary. However, due to a lack of time and the fact that there was a great demand for the use of the instrument, not more than one ITC titration could be performed.

25 Cp (kcal/mole/k) 4.4 Differential scanning calorimetry, DSC It is a well-known fact that PNIPAM, among other water-soluble polymers, undergoes a phase transition when its aqueous solution is heated above the so called lower critical solution temperature (LCST). If ph sensitive groups are added to the polymer chain forming copolymers with NIPAM, the LCST will depend upon ph 4. During this work, differential scanning calorimetry was used in order to determine the LCST of the PNIPAM-PAA copolymers with and without C8F17-chains and their phase transition enthalpies at different values of ph. Each of the four polymer solutions was scanned at six different ph (2.5, 3.5, 4.0, 4.5, 5.0 and 5.5). Therefore, the polymer stock solutions were diluted with the buffer after having checked and adjusted its ph. After dilution, the ph of the solution was checked again and no significant change in the ph was noticed Discussion of the phase transition Figure 4.9 presents calorimetry scans for solutions of PNIPAM-PAA-95 at six different ph. Since the scans are quite similar for all polymers that have been studied, the one of PNIPAM-95 will be analysed here as an example whereas the diagrams of the remaining polymers as well as a table showing all transition temperatures and enthalpies can be found in Appendix ,8 PNIPAM-PAA-95 1,6 1,4 1,2 1,0 0,8 ph T m [ o C] H[cal/mole] ph ph ph ph ph ph ,6 0,4 0,2 0, Temperature ( o C) Figure 4.9: Phase transition plot for PNIPAM-PAA-95 at different ph

26 Figure 4.9 clearly shows that the position of the endothermic phase transition peaks depends on the ph of the solution. As the ph is increased and the PAA groups are progressively deprotonated, the phase transition temperature Tm increases. Furthermore, the transition peaks become broader and lower. On the molecular level this could be explained by a less co-operative phase transition among the polymer chains: at low ph, carboxylic groups can form hydrogen bonds with the amide of NIPAM and therefore establish connections between different polymer chains or within a chain. As a consequence, the polymer chains collapse more or less simultaneously. As ph increases, the carboxylic groups are progressively deprotonated and lose their ability to form hydrogen bonds and, consequently, the connections between or within the chains get lost. Therefore, independent polymer chains may collapse at slightly different temperatures leading to a broader transition on a macroscopic level. The broadening of the phase transition at high ph has also been described by F. M. Winnik and co-workers by means of turbidimetry for similar polymers: the decrease in transmittance takes place over an increasingly broader temperature range, until a ph is reached for which the solution retains its clarity over the entire temperature range scanned. 4 At this ph the thermodynamic transition peak measured by DSC is supposed to disappear completely meaning that all acid groups are deprotonated and ionised causing the polymer to be watersoluble at all temperatures because of its increased polarity Dependence of LCST upon ph The ph dependence of the phase transition temperature for the polymers studied during this work can be compared through Figure 4.10.

27 T m PNIPAM-PAA-90 PNIPAM-PAA-C 8 F PNIPAM-PAA-95 PNIPAM-PAA-C 8 F ,5 3,0 3,5 4,0 4,5 5,0 5,5 Figure 4.10: ph dependence of the phase transition temperature Tm for the four polymers ph Obviously, the ratio between PNIPAM and PAA seems to have an influence on the slope of the plot since the latter is considerably steeper at high ph for the polymer with 10 mol-% of PAA than for the one containing only 5 mol-%. This observation can easily be explained by the fact that PAA is the ph sensitive part of the copolymer and its content in the polymer chain must have an effect on the degree of ph dependence. At high ph the polarity of the polymer and its solubility in water are increased yielding in a higher value of the phase transition temperature. On the other hand, in comparison to their non-fluorinated counterparts, the fluorinated polymers are supposed to contain only half the amount of free acid groups, the other half being blocked by the C8F17 chain which is attached by an ester to the PAA (see Figure 3.1 b). Consequently, it should be possible to notice a difference in the ph dependence of Tm between corresponding polymers with and without fluorine. However, Figure 4.10 shows almost no such difference. Apart from this discord, it is interesting to note that, for the lowest ph, the values for Tm are not only very similar for all four polymers but also appear to be close to the transition temperature of pure NIPAM 4 which is about 31 C and independent of ph.

28 Cp (kcal/mole/k) Double transition peak at high ph One more interesting fact can be noted regarding the transition peaks at ph 5.0 and 5.5: these peaks show a kind of shoulder towards higher temperatures. This asymmetry of the peaks could be found as a reproducible result for any of the studied polymers. It may indicate a kind of double transition with two superimposed Gauss-shaped peaks yielding to an asymmetric transition. 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0, Temperature ( o C) Figure 4.11: 2-peak fitting into transition plot for PNIPAM-PAA-C8F17-95 at ph 5.0 In order to confirm this theory, a multiple-peak fit (Figure 4.11) has been attempted on the transition peaks at ph 5.5 yielding remarkable results regarding the fitting precision. In Figure 4.11, the dashed line is the fitted curve representing the sum of the two single transitions (dotted lines). However, a suitable interpretation of this double-transition effect could not yet be found Dependence of phase transition enthalpy upon ph The phase transition enthalpy H was calculated in calories per mol of NIPAM-monomer. It is found to be approximately constant over the studied range of ph (Figure 4.12).

29 H [cal/(mol of NIPAM)] PNIPAM-PAA-90 PNIPAM-PAA-C 8 F PNIPAM-PAA-95 PNIPAM-PAA-C 8 F ,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 ph Figure 4.12: ph dependence of the phase transition enthalpy for the four polymers However, towards higher ph (5.0 and 5.5) a decrease in the enthalpy can be noticed. It is important to note that this decrease is most certainly an artefact of the determination of the baselines used for peak integration: Since the baselines had to be adjusted manually during data processing, increasingly broader peaks yield to increased uncertainty during baseline construction. Therefore, especially the values of H at ph 5.5 are supposed to be imprecise. On the molecular level, the NIPAM part of the copolymer is responsible for the phase transition. For NIPAM, no changes occur from the chemical point of view and hence the transition enthalpy should remain constant through variation of ph.

30 4.5 Fluorescence Spectroscopy Under certain conditions, hydrophobically modified polymers form microdomains isolating their hydrophobic chains against the polar environment. Fluorescence is a common method to investigate whether such kinds of microdomains are present in a polymer solution. In such experiments, a fluorescent dye is added either by covalent binding to the polymer or by simply dissolving the polymer in a solution containing the dye. In this work, pyrene was used as a dye and it was attached to the polymers (see Figure 3.1 c and 3.1.d). The presence of hydrophobic microdomains with a high local concentration of pyrene can be detected by the occurrence of a broad excimer emission peak in the fluorescence spectrum (maximum intensity around 480 nm) in addition to the pyrene monomer emission. Excimers are excited state complexes formed by association of one pyrene molecule in excited state and one in its ground state. 5 Excimer formation therefore requires a relatively high local concentration of pyrene as it is found when pyrene molecules aggregate within hydrophobic microdomains Emission spectra Fluorescence spectra were measured of the pyrene labelled polymers in aqueous and methanol solutions. The order of magnitude for the polymer concentrations was 0.05 g/l for methanol solutions and 0.1 g/l for aqueous solutions except for PNIPAM-PAA-C8F17- PY-90 which was diluted to 0.05 g/l because of its exceptionally high pyrene content (In order to avoid saturation of the PMT, it has been found preferable to use a lower concentration instead of decreasing the high voltage of the PMT). Figures 4.13 and 4.14 show the fluorescence emission spectrum of polymer solutions in water (excitation wavelength ex = 344 nm) and in methanol ( ex = 342) nm. It should be mentioned that the absolute intensity of the spectra has no significance since it depends on the concentration of pyrene which is different for each polymer.

31 Fluorescence Fluorescence 120 I I 3 PNIPAM-PAA-PY-90 PNIPAM-PAA-C 8 F 17 -PY-90 PNIPAM-PAA-PY-95 PNIPAM-PAA-C 8 F 17 -PY-95 solvent: methanol ex = 342 nm [nm] Figure 4.13: Fluorescence emission spectrum for the polymers in methanol solution I 3 I 1 PNIPAM-PAA-PY-90 PNIPAM-PAA-C 8 F 17 -PY-90 PNIPAM-PAA-PY-95 PNIPAM-PAA-C 8 F 17 -PY-95 solvent: water ex = 344 nm [nm] Figure 4.14: Fluorescence emission spectrum for the polymers in aqueous solution The spectra shown in Figures 4.13 and 4.14 present the characteristic monomer emission of pyrene, which consists of two sharp peaks around 375 and 395 nm and two more or less

32 resolved small peaks in-between. However, no excimer emission was detected in any spectrum. In addition to the measurements of the polymers in aqueous solutions, emission spectra were recorded of PNIPAM-PAA-PY-95 and the corresponding fluorinated polymer dissolved in buffer solutions at ph 2.5 and 5.5. The spectra (see Appendix 8.4) were identical for solutions of both values of ph and no excimer emission could be found. Consequently, the pyrene molecules in any of the tested solutions are far apart from each other and cannot form excimers. This indicates that no pyrene aggregates are present in the polymer solutions. However, this result does not mean that the polymer solutions do not form any hydrophobic microdomains or micelles. Such assemblies could be formed by the fluorinated side chains of the polymer without incorporation of pyrene molecules. Several reasons for this can be found: first, the inter-molecular forces between aromatic pyrene and aliphatic perfluoro-alkyl chains are considerably different and therefore, pyrene molecules do not stay in close proximity to the C8F17-chains although both are hydrophobic. Second, given the fact that the pyrene labels are attached to the polymer backbone and are therefore relatively far apart from the fluorinated side chains, aggregation of the latter is possible without participation of the pyrene. Evidently, hydrophobic microdomains formed only by the perfluoro-alkyl chains can not be detected by fluorescence spectroscopy I1/I3-ratio One additional piece of information can be extracted from the fluorescence spectrum of pyrene: the ratio of intensity of the first and third vibronic peak (see arrows in Figures 4.13 and 4.14), also called I1/I3, gives a hint on the polarity of the pyrenes environment. 6 The ratio decreases with decreasing polarity. Table 4.3 shows the value of I1/I3 obtained from the spectra in Figures 4.13 and PNIPAM- PAA-PY-90 PNIPAM-PAA- C8F17-PY-90 PNIPAM- PAA-PY-95 PNIPAM-PAA- C8F17-PY-95 I1/I3 in methanol I1/I3 in water Table 4.3: Fluorescence intensity ratio I1/I3 of the polymers in water and methanol

33 Fluorescence The values in Table 4.3 indicate a very polar environment of the pyrene molecules. Hence, it is unlikely that hydrophobic microdomains with pyrene molecules inside are present in the polymer solution. Evidently, the values of I1/I3 for the methanol solutions are slightly smaller since methanol is less polar than water Excitation spectra and peak/valley-ratio Fluorescence excitation spectra were recorded, the wavelength range being limited to the region between 300 and 360 nm. The excitation spectra were very similar to the UV absorption spectrum as it can be seen in Figures 4.15 and PNIPAM-PAA-PY-90 PNIPAM-PAA-C 8 F 17 -PY-90 PNIPAM-PAA-PY-95 PNIPAM-PAA-C 8 F 17 -PY-95 solvent: water em = 396 nm [nm] Figure 4.15: Fluorescence excitation spectrum for aqueous polymer solutions

34 Fluorescence PNIPAM-PAA-PY-90 PNIPAM-PAA-C 8 F 17 -PY-90 PNIPAM-PAA-PY-95 PNIPAM-PAA-C 8 F 17 -PY-95 solvent: methanol em = 396 nm [nm] Figure 4.16: Fluorescence excitation spectrum for polymer solutions in methanol It can be seen that excitation spectra of PNIPAM-PAA-C8F17-PY-90 also show a slight red shift as it was previously noticed in the absorption spectra. However, the shifts in absorption are much stronger and were noticed for both fluorinated polymers, unlike the ones of fluorescence excitation. The peak-to-valley ratio PEx of the excitation spectra are listed in Table 4.4. Fluorescence excitation UV absorption PEx in water PEx in methanol PA in water PA in methanol PNIPAM-PAA-PY PNIPAM-PAA-C 8F 17-PY PNIPAM-PAA-PY PNIPAM-PAA-C 8F 17-PY Table 4.4: Peak/valley ratios PEx and PA for excitation and absorption spectra

35 The values for PEx are very similar to the corresponding values for the absorption spectra PA. However, in contrast to PA, there is no significant difference in PEx between the fluorinated and the non-fluorinated polymers.

36 5 Conclusions The experiments carried out during this work revealed interesting facts about the structure of the given polymers and the properties and behavior of their aqueous and methanol solutions under various conditions: Infrared spectroscopy was used to confirm the presence of fluorinated alkylic chains in the polymer but, however, could not give evidence whether these are covalently attached to the polymer. The pyrene content of the labeled polymer samples was determined by UV absorbance spectroscopy yielding values between 0.20 and 0.47 mol-% of pyrene. The peak-tovalley ratio calculated from the absorption spectra did not give any clear hint towards aggregation of pyrene molecules. ITC was used to determine the content of PAA of one polymer yielding a value of 17.2 mol-% of PAA. Aqueous solutions of the polymers underwent a phase transition upon heating. DSC measurements revealed an increase of the phase transition temperature with increasing ph until above approximately ph 6, where the polymers remain soluble over the entire temperature range. Along with this increase of LCST goes a more and more sluggish phase transition possibly due to a loss of intermolecular association. The ph dependence of the LCST was found to be more pronounced for solutions of polymers with higher percentage of PAA. However, the fluorinated side chains did not seem to have any effect on the LCST nor on its dependence upon ph. The phase transition enthalpy was constant over the tested ph range. Fluorescence spectra indicated that pyrene labels reside in a polar environment and do not aggregate. However, aggregation of the fluorinated alkyl chains without participation of pyrene could be possible.

37 6 Acknowledgements I would like to thank my parents for supporting my stay in Canada since this work would not have been possible without their aid. My supervisor, Prof. Françoise M. Winnik deserves great thanks for having generously accepted me to join her group at the University of Montréal and for the fruitful discussions during my stay. Great acknowledgements are also made to Dr. S. H. Bossmann from the Department of Environmental Analysis at the University of Karlsruhe, Germany who kindly established the contact to Canada and supplied the polymers. I especially owe great thanks to all members of the research group at the Faculty of Pharmacy, namely the post-docs Céline, Raju and Sébastian for the numerous and fruitful discussions and for always being available in case I needed help. Thanks also to the rest of the team, Victoria, Denis, Piotr, Kazu and Roger for the great time during my stay.

38 7 References 1 Pokhrel, M. R.; Bossmann, S. H.: J. Phys. Chem. 2000, 104, Winnik, F. M.: Polymer, 1990, 31, Dean, J. A.: Lange s Handbook of Chemistry, 14 th ed., p. 766, table Principi, T.; Goh, E.; Liu, R. C. W.; Winnik, F. M.: Macromolecules 2000, 33, Winnik, F. M.: Chem. Rev. 1993, 93, Kalyanasundaram, K.; Thomas, J. K.: J. Am. Chem. Soc. 1977, 99, 2039

39 8 Appendix 8.1 Calculation of pyrene content of the polymers from UV absorption Lambert-Beer-Law: A ( ) c d = (1) c py c py pyrene content: r py = 100% = M poly 100% (2) c x poly abs py poly Molar weight of polymer: M poly = k M NIPAM + l M PAA + mm PAA C F + n M 8 17 PAA PY (3) k, l, m reflect the percentage of NIPAM, PAA and PAA-C8F17 in the polymer and are given in Tables 3.1 and 3.2. n is equal to rpoly. It is now necessary to eliminate k, l and m which can be done by considering the fact that k + l + m + n =1 (4) Now, for the four different polymers, k, l, and m can be written as shown in the following table. (The values written in italic correspond to the GPC-Result from Karlsruhe (see Table 3.1) which yields l = m for the fluorinated polymers.) PNIPAM- PAA-PY-90 PNIPAM-PAA- C8F17-PY-90 PNIPAM- PAA-PY-95 PNIPAM-PAA- C8F17-PY-95 k l n ½ n n ½ n m 0 (no fluorine) ½ n 0 (no fluorine) ½ n n n = rpy n = rpy n = rpy n = rpy Table 8.1: Values for k, l and m for all four polymers Replacing k, l, and m in equation (3) with the values of Table 8.1 and inserting the result in equation (1) leads to an equation for rpy with cpy and xpoly as parameters. Thus, for every polymer there is one equation:

40 PNIPAM-PAA-PY-90: r py = x c poly py ,24 PNIPAM-PAA-C8F17-PY-90: r py = x c poly py PNIPAM-PAA-PY-95: r py = x c poly py PNIPAM-PAA-C8F17-PY-90: r py = x c poly py

41 8.2 Calculation of the content of PAA from ITC result The molar ratio obtained from ITC titration is Rm = Rm is the molar ratio of the remaining free HCl in the cell to the amount of added HCl: R m N HCl, free = and therefore N HCl, added N HCl, prot 1 Rm =. N HCl, added NHCl, prot is the amount of HCl that has been used to protonate the carboxylate groups. The content of PAA that is to be calculated is Evidently, k N PAA = with N N Poly N NIPAM + N PAA Poly cell N PAA N HCl, prot = N COOH = ( 1 Rm ) N HCl, added = (1) = (2) 6 where N HCl, added = chcl, cell Vcell = mol as it can be found from the values given in section Npoly, cell, the number of moles of polymer in the cell, can be calculated, but one must consider the fact that the average molar weight of the monomer depends on k and therefore has to be left variable: A N Poly, cell = (3) with A = g summarising all occurring constant values M Poly like the mass of the polymer and the volumes of solution occurring during dilution process (see section for details). Finally, M = ( 1 k) M + k M (4) Poly NIPAM with MNIPAM and MAA being the molar weights of NIPAM and acrylic acid monomers respectively. Inserting equations (2) and (3) with (4) into equation (1) yields (1 R ) m k = (1 k) M NIPAM g mol AA k N AA. (5) Simplifying equation (5) and inserting the molar weights MNIPAM and MAA one obtains a simple equation for k: k = mol% (1 R m ) With the result from ITC titration Rm = one obtains a content of PAA of k 17.2 mol-%..

42 Cp (kcal/mole/k) Cp (kcal/mole/k) 8.3 DSC scans at different ph 1,8 PNIPAM-PAA-90 1,6 1,4 1,2 1,0 0,8 ph T m [ o C] H[cal/mole] ph ph ph ph ph ph ,6 0,4 0,2 0, Temperature ( o C) 1,6 PNIPAM-PAA-C 8 F ,4 1,2 1,0 0,8 ph T m [ o C] H[cal/mole] ph ph ph ph ph ph ,6 0,4 0,2 0, Temperature ( o C)

43 Cp (kcal/mole/k) Cp (kcal/mole/k) 1,8 PNIPAM-PAA-95 1,6 1,4 1,2 1,0 0,8 ph T m [ o C] H[cal/mole] ph ph ph ph ph ph ,6 0,4 0,2 0, Temperature ( o C) 1,6 PNIPAM-PAA-C 8 F ,4 1,2 1,0 0,8 ph T m [ o C] H[cal/mole] ph ph ph ph ph ph ,6 0,4 0,2 0, Temperature ( o C)