1. Phase Separation of Water/Glycerol Binary Mixtures
Next to Lipid Monolayers:
An X-ray and Neutron Reflectivity Study
Luka Pocivavsek
James Franck Institute and Department of Chemistry,
The University of Chicago, Chicago IL, USA

2. Lungs present a unique and interesting mechanical problem
Surface tension of clean
air/water interface  72 mJ/m2
Surface tension at healthy alveolar surface  0-20mJ/m2
Schurch et al., BBA, (1998) 1408, 180.

3. Interfacial Elastic Stability Key to Surfactant Function
Surfactant monolayer ≈ 2 nm
Pocivavsek L. et al., Science 320, 912 (2008)
Alveolar lining fluid
≈ m
Role of SUBPHASE?
Schurch et al., BBA, 1408, 180 (1998) .

4. Simple Model Subphase for Alveolar Lining Fluid
Hyaluronan
disaccharide monomer
Glycerol molecule
Why glycerol over
HA to begin with?
1. simplicity -
simpler structure
makes for simpler
perturbation
2. cost - conduct
initial experiments
with less expensive
additive
3. purity - ideally
would like system
with little surface
active contaminants
glucuronic
acid
N-acetylglucosamine
glucose isomers
HA and glycerol both strongly hydrogen bonding molecules highly soluble in water

5. Lung surfactant models
Lung surfactant monolayer is primarily composed of lipids with small amounts of protein. A simple lipidic model for the system is a combination of two lipids on a deep water subphase.
DPPC
POPG
Langmuir trough with fluorescence microscopy (FM) “artificial lungs” captures the essential mechanical motions seen during breathing.
Subphase is water or glycerol

6. Non-intuitive response with small changes in viscosity
system: DPPC:POPG 7:3 monolayer at 25oC compressed at 0.1mm/sec
0% glycerol (1 mPa•sec)
40% glycerol (3 mPa•sec)
small amplitude folds
large amplitude folds
Large folds seen for >10% glycerol.
Non-intuitive at first why a small perturbation would give rise to an order of magnitude change in fold size.

7. Liquid Surface Reflectometry Probes Interface Structure
Principles behind a reflectivity (scattering) experiment:
air
liquid subphase  
Reflected radiation
collected at detector
Reflected radiation must be to first order a function of incident radiation wavelength, layer composition, and layer thickness : z y x
coherent nuclear scattering length

8. X-ray reflectivity performed at ChemMatCARS (sector 15, APS, ANL)
DPPC:POPG 7:3 at 25oC compressed to 30mN/m spread on water, 20%, 40%, and 64% wt/wt glycerol.
water
20%
head
40%
subphase
64%
tail
X-ray reflectivity shows that on glycerol the interface is thicker and a new layer appears between the headgroups and bulk subphase - what is the composition of this layer? Need better contrast between water and glycerol.

9. Neutron Reflectivity - SPEAR (Lujan Center, LANSCE, LANL)
Neutrons scatter from nuclei and the strength of interaction between a neutron and a particular nucleus is quantified in the atoms coherent scattering length.
neutrons
x-rays
coherent nuclear scattering length
Atomic number for our systems almost constant. Main contrast is between hydrogen (1) and all the rest C (6), N (7), O (8), P (15)
Sensitive to type of isotope, very useful in soft matter is the difference between hydrogen (-3.7 fm) and deuterium (6.6 fm).
Possibility to contrast molecules by isotope enrichment or depletion.

10. Neutron Reflectivity - SPEAR (Lujan Center, LANSCE, LANL)
DPPC-d75 at 25oC compressed to 30mN/m spread on water, 20%, and 64% wt/wt glycerol.
tail = 7.210-6 Å-2
headgroup = 6.510-6 Å-2 0%
20%
64%
Same trend is seen with DPPC-d75 as with DPPC-d64. A third layer ~10Å in length enriched in glycerol appears between the headgroups and the bulk subphase as glycerol is added to the bulk solution.
20% D2O/glyc: 20/80 = 5.3010-6 Å-2
D2O = 6.3510-6 Å-2
glycerol = 0.6010-6 Å-2
64% D2O/glyc: 64/36 = 2.9710-6 Å-2

11. Conclusions from X-ray and Neutron Reflectivity
Reflectivity spectra show clearly that when glycerol present in the subphase the solution de-mixes, creating a ~10Å layer enriched in glycerol adjacent to the headgroups. GID shows increased monolayer stability.
1. What are the possible consequence of this de-mixing and enrichment on the mechanical response of the monolayer?
2. What can the wrinkle-to-fold model tell us given this new structural insight about the monolayer covered interface?
glycerol
H2O/D2O

12. Connection between interface structure and folding?
Recall different folding behavior of water and glycerol solutions:
0% glycerol
40% glycerol
lipid
glycerol rich boundary layer
bulk fluid
Structural/Mechanical Sugar Hypothesis:
Increased thickness gives rise to larger folds
Need an understanding of relaxation times and viscosities of bound glycerol layer
XR, neutron reflectivity, and GID show glycerol enriched layer that stabilizes lipids  cross-linked hydrogen bond network induced by glycerol

13. Acknowledgments Ka Yee Lee, Enrique Cerda, Binhua Lin, Jarek Majewski
University of Chicago
USACH/UdeC
SPEAR, Lujan, LANL
Tom Witten
Kseniya Gavrilov
Eva Chi
Kathleen Cao
Shelli Frey
Dongxu Li
Brian Leahy
Sebastian Johnson
Nico Rivas
Jarek Majewski
Eric Watkins
ChemMatCARS, ANL/UChicago
Binhua Lin
Mati Meron
Tim Graber
Jim Viccaro