Phase Separation of Aqueous Biopolymer Mixtures Yapeng Fang and Liangbin Li

My current work Phase separation Associative/segregative phase separations of gelatin/  - carrageenan Drying and rehydration project Use phase separation know-how to design rehydratable structure

Part I. The phase separation of gelatin/  -carrageenan Materials used Gelatin: Type B bovine gelatin, Mw = 120 kDa (LS), pI = 4.9 (Nanosizer).  -carrageenan:  -fraction = 0.93, Mw = 550 kDa (LS), Na = 0.540%, K = 0.180%, Ca = 0.010%, Mg = 0.010%.

Objectives To investigate the associative phase separation behaviors of gelatin/  -carrageenan system To explore if a second segregative phase separation could coexist; if so, how they complete in terms of experimental conditions To create microstructures by the phase separations fish gelatin/ k-carrageenan 60 o C20 o C

Attempts to identify phase boundaries Turbidity titration method failed: no inflexion points Centrifuge and composition analysis failed: no bulk phase separation even at 6*10 4 G force at 45 degree Necessities of using confocal Raman spectroscopy Carr. gelatin Experimentally inaccessible for preparation Fix at 5% gelatin

Mapping out the state diagram NaCl titration of turbidity: pH=7; Cgel=Ccarr=0.75% Derived state diagram of gelatin/ k-carrageenan C associative  compatible C compatible  segregative More qualitative than quantitative!

Temperature scan of turbidity: pH=7; Cgel=Ccarr=0.75% Samples that have no pre-phase- separations (associative) Samples that have pre-phase- separations (associative)

DSC of mixture: pH=7; Cgel=Ccarr=0.75% Modified state diagram of gelatin/ k-carrageenan compatible associative segregative coexisting carr. ordering induced segregative PS gelatin ordering induced segregative PS

State diagrams at different pHs pH increase

Microstructures created by different phase separations: pH=7; Cgel=Ccarr=0.75% ABCD 262*262 µm

Microstructure images: pH=5; Cgel=Ccarr=0.75% 262*262 µm66*66 µm segregative PS segregative-co-associative PS

pH titration of turbidity and charge density: Cgel=Ccarr=0.75%

pH induced structure transitions A B C 262*262 µm

Stoichiometric interaction between gelatin/k-carrageenan: Cgel+Ccarr=0.75%, I=30mM pH 6pH 5 r max =0.65

Stoichiometric interaction gelatincarr. individual gelatin saturated with carr. r<<r max Intermediate cluster r=r max individual carr. saturated with gelatin r>>r max Huge network clusters r<r max

Understanding rehydration based on Multi-length scale structure Yapeng, Chiharu, Liangbin, Rob, Ingrid Eduardo (Delft), Dmytro (AMOLF)

Biopolymer Structures in Multi-length scales Molecular scale(WAXS)Meso scale (SAXS) Micro-scale (CLSM) Macro-scale

Molecular scale (WAXS) Meso scale (SAXS) Molecular and mesoscopic structure Unlike synthetic polymer gels with permanent chemical crosslink, biopolymer gels are generally built on reversible physical crosslink. The size and number of the junction zones changes during dehydration and rehydration, which is one of the most important factors controlling the rehydratability.

Combination of SAXS and WAXS SAXS WAXS Spacing along molecular chain Spacing between molecular chain

Molecular and mesoscopic structure dehydration rehydration

The crosslinks dissolving (or melting) follows a homogeneous nucleation process!

Food systems are generally built by multi components, which may not be miscible with each other in certain PH, ionic strength, temperature and concentrations. Phase separations lead to different phases in a length scale from nanometer to millimeter. Different morphologies is expected to have different dehydration and rehydration properties. (CLSM) Micro-scale

Alginate (2.0%) Gelatin (1.0%) pH 10.5 pH 7.0pH 4.0pH 3.5 Increasing extent of phase separation Mixing at 50 o C Adjusting pH Dropping into 1.0% CaCl 2 solution Air dryingRehydration

Rehydrations of beads prepared at different pHs pH 10.5 compatible; preventing aggr. pH 3.5 Local overconcentration of alginate pH 10.5 pH 7.0pH 4.0pH 3.5 Increasing extent of phase separation

Why do the mixture beads rehydrate faster at the beginning, but more slowly at the late stage compared with the control beads?

Control Mixture The presence of gelatin does not influence the crosslink domain of alginate regardless of different extents of phase separation. SAXS of fresh gel beads:

WAXD of dried gel beads: Control Mixture Gelatin could form another network in addition to alginate network

Spacing along molecular chain Spacing between molecular chain

What is the next for: Alginate and gelatin system 1)Alginate and gelatin are in sol states (without network) 2)Alginate in gel state, Gelatin in sol state (fish gelatin, CaCl 2, single network) 3)Gelatin in gel state, Alginate in sol state (bovin gelatin, single network) 4)Gelatin and alginate both in gel states (bovin gelatin, CaCl 2, double networks)

Molecular interactions & Topological constraint on network building Effect of G/M ratio 0%CaCl 2 Effect of CaCl 2 concentration

Combinatorial effects or specific molecular interactions Flory-Huggins

Planned experiments 1)DSC measurements (systematical mapping, micro- calorimetric, Colworth) 2)Rheological and DMA characterization (sol-gel transition, synergistic interactions in both sol (molecular interactions) and gel) 3)X-scattering on samples with high concentrations 4)NMR or IR on molecular interactions

Couplings & Competitions Gelation, Phase Separation & Evaporation What happen during dehydration of biopolymers? Three phase transitions may occur simultaneously! S1S1 P1P1 P2P2 associative type PS segregative type PS S1S1 P1P1 P2P2 S1S1 P1P1 P2P2 Temperature Ionic strength/PH Sol-gel

Phase separation (spinodal decomposition) Remaining water Gelation

A simple setup for controlled drying: Air cylinder Flow meter Valve 1 Valve 2 Humidity meter Drying container Sample Water Completely dried air High moisture air

9/14/12/1 1/1 1/2 1/4 1/9 Gelatin/maltodextrin total 4%, Dry 5% humidity Flow rate 5 L/h

Gelatin/Maltodextrin total 4%, Dry 5% humidity Flow rate 5 L/h 9/1 4/1 2/1 1/1 1/2 1/41/9

Macroscopic scale Lump formation during rehydration Hydrated layer with high viscoelasticity. Feature: high concentration gradient concentration varies from 100% (core) to 0% (interface) in 1 mm scale. Can we control this? Dry core

Molecular scale (WAXS) Meso scale (SAXS) Micro-scale (CLSM) Macro-scale Control rehydration through Multi-length scale structural design Experimental conditions PH, ionic strength, temperature