1. PittCon 2001: Paper #1338 Mark G. Hartell and W. Charles Neely
STUDY OF THE CHEMICAL, PHYSICAL AND OPTICAL PROPERTIES OF A LIPOPHILIC HYDROXYCOUMARIN ANALOG IN MIXED MONOLAYERS
Mark G. Hartell and W. Charles Neely
Department of Chemistry
Vitaly Vodyanoy
Department of Anatomy, Physiology and Pharmacology
Auburn University
Dr. S. Pathirana Prof. S.A. de Silva
FAA * USDA * NSF * DARPA * U.S. Army

2. I. Monolayer Studies Objectives
Two different Fluorophores, A & B
Both are indicators of pH
Different molecular structure
Different mechanism of fluorescence
Surface Properties of Mixed Monolayers with Ca-Arachidate
Compare and contrast properties of the two molecules
Introduce a third molecule (C) which differs only in positioning of fatty alkyl chain
Goals:
Determine qualitative set of characteristics which differentiate each molecule
Determine some quantitative criteria from which a more stable film may be constructed
R = fatty alkyl chain
Determine parameters to make a better sensor

3. The Langmuir-Blodgett Technique
Molecule of interest is spread into a monolayer film
Area dimensions are controlled by a movable barrier
Molecular orientation well controlled in 2-dimensions
Can be used to engineer highly ordered, molecularly thin layers

4. Langmuir-Blodgett System
SYSTEM SUMMARY
Self-contained system
Trough is milled from solid Teflon block
Computer controlled
Allows for the simultaneous data collection of surface’s area, pressure and potential
KSV 3000 LB System
Isotherm Parameters
3-4 trials at 20  0.1 C
25 mm/min compression
Subphase pH ~ 10.04
(0.3 mM CaCl2 & 0.4 mM NaHCO3)
Record  and V

5. Surface Pressure Measurements
Define net force exerted on plate downward:
F = p g l w t + 2 ( t+w)cos2 - Dl g l w d
2 º 0 when plate is completely wet:
 = - = -[ l g t w h/2(t + w)] h
  -½ l g t w h  h when t « w

6. Surface Potential Determinations Yield a Measure of Dipole Moment
SPECIFICATIONS
Built in-house for LB system with ionizing electrode
(210Po, ½ = 140 d)
Simultaneous data collection inputted into native KSV software
Source may be exchanged with fresh 210Po
Define surface dipole moment in units of mD:
 = (1/4)  (V/n)
Define A as film concentration in 2/molecule:
 = AV/12
210Po
Ag/AgCl

7. Thermodynamic Definitions
Isotherm Parameters 
Free Energy of Compression (pure monolayer):
Free Energy of Compression (mixed monolayer): A a

8. Arachidic Acid Baseline Data
Comparison of Dipole Moment with -A Isotherm
Change in dipole moment indicates rearrangement at 40 Å2/molecule before any notable rise in surface pressure
Molecular orientation appears to have been assumed long before L1 and L2 state LS L2 L1 G
CS state yields reduced area of approximately 20 Å2/molecule

9. pH Sensor Molecule: 10-C18AE
Diethanolamine moiety is proton receptor
Fluorescence activated by PET-based mechanism
Synthesized as part of a novel class of fluorescent pH indicators: proton turns fluorescence on
LUMO e-
LUMO e-
HOMO
HOMO
HOMO
HOMO
Symmetric Fluoroionophore
Excited FL
Free amine
Excited FL
H+-amine

10. Mixed Isotherms of 10-C18AE at 20oC
Pure 10-C18AE yields CS state not stable at high .
max = mN/m
Lower mol% mixtures indicate a phase separation in condensed states
10-C18AE may be slipping out of the plane of the film
Higher surface pressure phase resembles reduced areas of pure arachidic acid
Mixtures Spread in Hexane
Shift towards
pure arachidic acid
Fluorophore only stable in mixtures when compressed below 30 mN/m

11. Isotherms of 10-C18AE versus C18AE
Symmetrically placed alkyl chain
Asymmetrically placed alkyl chain
Similar two-phase isotherm yielding stable mixture below 30 mN/m

12. Reduced Molecular Areas of 10-C18AE
Higher surface pressure states notable only in more dilute mixtures - reduced areas are identical to pure arachidic acid
Lower mol% mixtures appear to be comparable to ideal mixture calculations
>70 mol% indicates threat of fluorophore aggregation during analysis
Average standard deviation of the mean ~ 0.3 2
Reduced Molecular Areas of Stable Lower Surface Pressure Component
Reduced molecular areas indicates stability at more dilute mixtures

13. Reduced Molecular Areas of 10-C18AE vs. C18AE
Symmetrically placed alkyl chain
Asymmetrically placed alkyl chain
Symmetrically placed alkyl chain yields reduced areas slightly closer to ideality

14. Dipole Moment Determinations at 20oC
10-C18AE has higher dipole moment than simple fatty acid
Higher mol% yields larger contribution to net dipole moment of the film
Concentration function is not simply linear - suggests at least one minima at 70 to 80 mol%
Large data spread may be due to small variation in film due to minute impurities - molecule custom synthesized
Higher concentrations resemble
pure sensor molecule
Lower concentrations resemble
pure arachidic acid

15. Thermodynamic Measurements: G of Mixing for 10-C18AE
Close to ideal behavior observed for mixtures less than 70 mol%
Higher concentrations indicate that condensed packing assumes lower energy
Concentrations relevant to sensor construction (low mol%) exhibit ideal behavior if deposited with a constant  <30 mN/m
G of mixing confirms ideal behavior observed at lower mol% mixtures

16. G of Mixing for 10-C18AE vs. C18AE
Symmetrically placed alkyl chain
Asymmetrically placed alkyl chain
Symmetrically placed alkyl chain yields energy curves closer to ideal mixture at concentrations <70 mol%

17. pH Sensor Molecule #2: a Cellular Probe
Hydroxycoumarin derivative: “HC”
Novel fluorescent probe: fluorescence changes as function of pH
Used as cellular pH probe
Used as measure of local surface potentials by measuring shift in pKa
May be functional in sensor system to detect a binding event

18. HC-Arachidic Acid Mixed Isotherms at 20oC
Pure fluorophore stable at high surface pressures
max = mN/m
Can resolve condensed liquid state in higher mol%
Do not see two-phase separation into pure arachidic acid at lower mol%
Isotherms imply a more stable mixed monolayer versus 10-C18AE
Mixtures Spread in Chloroform
Shift towards
pure arachidic acid
Mixtures of HC appear to yield more stable isotherms at higher surface pressures compared to 10-C18AE

19. -A Isotherms of HC Mixtures are Reversible
Two Different Reversibility Studies
(1) Compression-Expansion:
Isotherms are consistently reversible
(2) Compression-expansion-compression-expansion:
Isotherms are continuously reversible when compressed multiple times
A,B,C are Study (1) and B,C are Study (2)

20. Determine Loss of Fluorophore During Isotherm Experiments
 vs. A  A vs. t
Measure loss of A at constant 
Calculate loss of A vs. time

21. Examples of Raw Kinetic Data
25 mN/m
30 mN/m
Two Studies Performed
(1) 15, 20, 25 mN/m
(2) 25, 30, 35 mN/m
35 mN/m
Worst-Case Determination
Calculate %-Loss Assuming 12 min. at Compressed State
Determine Decay Function
Fit 30 min. Decay to 1st Order Kinetics

22. Summary of Two Separate Decay Experiments
Negligible Loss of Material During Experiment - Molecular Areas Not Altered
Summary of Two Separate Decay Experiments
const t=12 min.
15 mN/m %
20 mN/m 0.69%
25 mN/m 0.74%
25 mN/m 1.5%
30 mN/m 0.38%
35 mN/m 0.38%
Data assumes unrealistic worst-case scenario: 12 min. under compressed conditions
Reality: Compressed state maintained for only last few minutes of experiment
Conclusion: Loss is negligible for a realistic 12 min. isotherm experiment

23. Reduced Molecular Areas of HC Probe
Avg. Standard Deviation of the mean from 3 to 4 trials is ~ 0.3 2
Molecular areas of fluorophore and arachidic acid differ by only 3 Å2/molecule
Pure HC Area confirmed by literature data - rationalized by molecular modeling
Lower mol% mixtures appear to be comparable to ideal mixture calculations
Observed expanding effect may be artificially emphasized due to small axis scaling
Reduced molecular areas indicate stability at more dilute mixtures

24. Fluorophore Comparison of Reduced Molecular Areas
>70 mol% 10-C18AE presents unique packing arrangement yielding a more condensed area
When observed on similar scale HC also indicates strong adherence to ideal areas up to 80 mol%

25. Dipole Moment Determinations at 20oC
Higher concentrations resemble
pure sensor molecule
HC has higher dipole moment than simple fatty acid
Higher mol% yields larger contribution to net dipole moment of the film
Concentration function is quite linear
Smaller data spread may be due to higher purity of this commercially available molecule vs. 10-C18AE
Lower concentrations resemble
pure arachidic acid

26. Thermodynamic Measurements: G of Mixing for HC Probe
Close to ideal behavior for very dilute solutions
Packing arrangements of some concentrations yield higher than ideal energies
Data suggests compositions which are energetically more stable for sensor design:
0 to 20, 50 and 80 mol%
G of mixing indicates optimal compositions for sensor design

27. II. Fluorescence Studies Objectives
Two different Fluorophores, A & B
Both are indicators of pH
Different molecular structure
Different mechanism of fluorescence
Optical Properties of Solid State Films
Determine fluorescence response vs pH
Compare molecules in solid state with data from free solution
Goals:
Determine qualitative set of characteristics which differentiate each molecule and compare with solution data
Determine some quantitative parameters to incorporate fluorophore B into a viable solid-state sensor system
R = fatty alkyl chain
Determine parameters to test a solid-state sensor

28. LB Film Deposition System
SYSTEM SUMMARY
System contained in laminar flow hood
Trough is milled from solid Teflon block
Computer controlled
KSV 2200 LB System
Deposition Parameters
20  0.1 C set = 30 mN/m
25 mm/min compression to set
Subphase pH 7.41
55 mM KCl, 4 mM NaCl, 0.1 mM CaCl2, 1 mM MgCl2 & 2 mM MOPS

29. HC Probe Film Deposition
DEPOSITION SUMMARY
Quartz slides placed back-to-back: deposit material on one side only
Compress 20 mol% HC to a constant 30 mN/m at 20 C
~8 layers deposited
Transfer ratios indicate mixed X-Y type multi-layers
Position of quartz slides during deposition
2 sets of back-to-back slides
Thin layer of 20 mol% HC fluorophore in arachidic acid successfully deposited

30. Positioning of Sample for Fluorescence Measurements Important
Quartz slide positioned in sample chamber of spectrometer
Quartz slides custom made to fit diagonal of standard 1 cm cuvette
Spectra collected at 90o angle through backside of slide
Reflective scatter is directed away from detector
Source Light
Fluorophore
Signal
Reflective
Scatter

31. C18AE: Solution vs. Solid-State Fluorescence
Free Solution
10 mol% Solid-State Film

32. HC: Solution Fluorescence
UN-IONIZED FORM
ex = ~360 nm
IONIZED FORM
ex = ~385 nm
Solution pH titration data consistent with literature data

33. HC: Solid-State Fluorescence
UN-IONIZED FORM
ex = ~360 nm
IONIZED FORM
ex = ~400 nm
Shift in pKa consistent with literature data in micelles

34. I. Conclusions
Determination of surface properties provides unique understanding of molecular orientation and monolayer stability
Data provides insight towards understanding dependence of surface properties on molecular structure and packing arrangements
Data provides knowledge base to make more informed choices in the construction of thin film sensors
Data has been used successfully to yield optically viable sensors whose solid state optical properties mimic that of a free solution model
Hartell, M.G., Pathirana, S.T., de Silva, S.A., Neely, W.C., Vodyanoy, V. “Dependence of Surface Properties of Flourophores and Calcium Arachidate on Mixed Monolayer Composition.” 13th International Symposium on Surfactants in Solution. Seminar. University of Florida, Gainesville, FL, June 11-16, 2000.
Hartell, M.G., Neely, W.C., Vodyanoy, V. “Study of the Chemical, Physical and Optical Properties of a Lipophilic Hydroxycoumarin Analog in Mixed Monolayers.” Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. Abstract #1338. Seminar. New Orleans, LA, March 4-9, 2001.