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 oC 20 oC

Attempts to identify phase boundaries

Turbidity titration method failed: no inflexion points

Centrifuge and composition analysis failed: no bulk phase separation even at 6*104 G force at 45 degree

Necessities of using confocal Raman spectroscopy

Carr.

gelatin

Experimentally inaccessible for

preparation

70

75

80

85

90

95

100

105

110

0 0.02 0.04 0.06 0.08 0.1 0.12

K-carrageena concentration (%)

Tran

smit

tan

ce (

%)

T490

T800

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

Cassociativecompatible Ccompatiblesegregative

0

50

100

150

200

250

300

350

400

450

500

550

600

650

0 5 10 15 20 25 30 35 40 45 50 55 60

Temperature (oC)

Na

cl C

on

ce

ntr

ati

on

(m

M)

compatible region

segregative phase separation

associative phase separation

associative-co-segregative phase separation

0

0.05

0.1

0.15

0.2

0.25

0.3

0 100 200 300 400 500 600 700

NaCl Concentration (mM)

A50

0

50 oC40 oC30 oC20 oC

More qualitative than quantitative!

Temperature scan of turbidity: pH=7; Cgel=Ccarr=0.75%

0

0.2

0.4

0.6

0.8

1

1.2

5 10 15 20 25 30 35 40 45 50

Temperature (oC)

A5

00

500mM

450mM

400mM

350mM

300mM

250mM

200mM

0

0.2

0.4

0.6

0.8

1

1.2

5 10 15 20 25 30 35 40 45 50

Temperature (oC)A

50

0

200mM

150mM

100mM

50mM

0mM

Samples that have no pre-phase-separations (associative)

Samples that have pre-phase-separations (associative)

DSC of mixture: pH=7; Cgel=Ccarr=0.75%

0

100

200

300

400

500

600

0 10 20 30 40 50 60

Temperature (oC)N

aCl c

on

c. (

mM

)

Onset of turbidity

Carr. Ordering temp.

gelatin ordering temp.

-2

-1.5

-1

-0.5

0

0.5

5 10 15 20 25 30 35 40

Temperature (oC)

Exot

herm

ic H

eat F

low

-0.1

-0.05

0

0.05

0.1

0.15

400mM

300mM

200mM

100mM

0mM

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

0

100

200

300

400

500

600

700

10 20 30 40 50 60

Temperature (oC)

Na

Cl C

on

ce

ntr

ati

on

(m

M)

pH 7

pH 6

pH 5

pH 7

pH 6pH 5

pH 4

pH 4

pH

increa

se

pH increase

Microstructures created by different phase separations: pH=7; Cgel=Ccarr=0.75%

0

50

100

150

200

250

300

350

400

450

500

550

600

650

0 5 10 15 20 25 30 35 40 45 50 55 60

Temperature (oC)

Nac

l Co

nce

ntr

atio

n (m

M)

AB

CD

segregative phase separation

associative phase separation

compatible region

associative-co-segregative phase separation

A B

C D

262*262 µm

Microstructure images: pH=5; Cgel=Ccarr=0.75%

262*262 µm 66*66 µm66*66 µm

segregative PSsegregative-co-associative PS

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

0

0.5

1

1.5

2

2.5

3

2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5

pH

A50

0

I=25 mM

I=50mM

I=75mM

I=100mM

I=150mM

I=200mM

0

0.04

0.08

0.12

0.16

0.2

0.24

6 7 8 9 10

pHA

500

I=25 mM

I=50mM

I=75mM

I=100mM

I=150mM

I=200mM

3

4

5

6

7

8

9

10

0 50 100 150 200 250

NaCl Concentration (mM)

pH liquid coacervate

solid coacervate

soluble complex

pH induced structure transitions

A B

C

262*262 µm-80

-60

-40

-20

0

20

40

2 3 4 5 6 7 8 9 10 11

pH

Zet

a P

ote

nti

al (

mV

)

gelatin

k-carrageenan

Ano cationic group (pH>pK a)

BOverall negative

COverall positive

0

0.5

1

1.5

2

2.5

3

2 3 4 5 6 7 8 9 10 11

pH

A50

0

Asoluble complex

Bliquid coacervate C

solid coacervate

Stoichiometric interaction between gelatin/k-carrageenan: Cgel+Ccarr=0.75%, I=30mM

0

0.02

0.04

0.06

0.08

0.1

0.01 0.1 1 10 100 1000

r (Gelatin/K-carrageenan)

A5

00

0

0.05

0.1

0.15

0.2

0.25

0.01 0.1 1 10 100 1000

r (Gelatin/K-carrageenan)

A5

00

pH 6 pH 5

-80

-60

-40

-20

0

20

40

2 3 4 5 6 7 8 9 10 11

pH

Zet

a P

ote

nti

al (

mV

)

gelatin

k-carrageenan

Ano cationic group (pH>pK a)

BOverall negative

COverall positive

rmax=0.65

Stoichiometric interaction

Size Distribution by Volume at 50 oC

0

5

10

15

20

25

30

1 10 100 1000 10000

Size (nm)

Vo

lum

e (%

)

r=0.044

r=0.258

r=0.652

r=2.28

r=23.8

gelatincarr.

individual gelatin saturated with carr.

r<<rmax

Intermediate cluster

r=rmax

individual carr. saturated with gelatin

r>>rmax

Huge network clusters

r<rmax

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

0.3 0.6 0.9 1.2 1.5

Iq2 (

arb.

uni

ts)

q (nm-1)10 15 20 25

(002)

(110)

0.405nm0.81nm

(001)

I (ar

b. u

nits

)

2 (degree)

SAXSWAXS

Spacing along molecular chain

Spacing between molecular chain

Molecular and mesoscopic structure

0.3 0.6 0.9 1.2 1.5

Iq2 (

arb

. uni

ts)

q (nm-1)

0% 68% 90%

dehydration

rehydration

10 15 20 25

Rehy

Fresh

Dehy

110

002

I (a

rb. units

)

2(degree)

001

10 15 20 25900min

0min

I (a

rb.

un

it)

2 theta (degree)0 200 400 600 800

0.0

0.2

0.4

0.6

0.8

1.0

cro

sslin

ks

rehydration time (min)

experimental exponential fitting

)]239

(exp[067.1t

c !0n

consaTR )(

The crosslinks dissolving (or melting) follows a

homogeneous nucleation process!

])(exp[ 1 n

coc

t

)()()(

tTRdt

td nTaTR )(

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 7.0 pH 4.0 pH 3.5

Increasing extent of phase separation

Mixing at 50 oC

Adjusting pH

Dropping into 1.0% CaCl2 solution

Air drying Rehydration

Rehydrations of beads prepared at different pHs

0

5

10

15

20

25

0 100 200 300 400

Time (min)

(Wt-W

0)/W

0 pH 3.5

pH 4.0

pH 7.0

pH 10.5

pH 10.5compatible;

preventing aggr.

pH 3.5Local

overconcentration of alginate

pH 10.5 pH 7.0 pH 4.0 pH 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?

0

5

10

15

20

25

30

0 100 200 300 400 500

Time (min)

(Wt-W

0)/W

0

0.5% NaCL1.0% Alg

1.0% Alg/0.5% Gel

0.3 0.6 0.9 1.2 1.5

Alginate 1%

Iq2

(arb

. u

nits

)

q (nm-1)

PH 10.5

PH 7

PH 4

PH 3.5

PH 3

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Alginate 1%

Gelatine 0.5%

PH 10.5

PH 7

PH 4

PH 3.5

Iq2

(arb

. u

nits

)

q (nm-1)

PH 3

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:

5 10 15 20 25

PH10.5

PH4

I (ar

b. u

nits

)

2 (degree)

PH3

5 10 15 20 25

PH10.5

PH7

PH4

I (ar

b. u

nits

)2 (degree)

PH3

gelatin 1.1 nm

WAXD of dried gel beads:

ControlMixture

Gelatin could form another network in addition to alginate network

5 10 15 20 25

Al+Gel control

001 110

I (ar

b. u

nits

)

2 (degree)

002

PH=10.5

5 10 15 20 25

I (ar

b. u

nits

)

2(degree)

Al+Gel

control

001 110

002

PH=4

5 10 15 20 25

I (ar

b. u

nits

)

2(degree)

Al+Gel control

001

110

002

PH=3

Sp

acing

alon

g

mo

lecular ch

ain

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, CaCl2, 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, CaCl2, double networks)

Molecular interactions & Topological constraint on network building

10 20 30 40 50 60

5% gelatin+1%LG

5% gelatin+1%MG

5% gelatin+1%HG

5% gelatin

End

o>

temperature (oC)10 20 30 40 50 60

0% CaCl2

0.05% CaCl2

0.5% CaCl2

5% CaCl25% gelatin+HG

End

o>temperature (oC)

Effect of G/M ratio 0%CaCl2

Effect of CaCl2 concentration

0 20 40 60 80 100

0.0

0.1

0.2

0.3

gelatin

visc

osity

(P

a s)

volume (%)

HG MG LG

10% gelatin2% alginate

alginate

0 20 40 60 80 100

0.0

0.2

0.4

0.6

0.8

1.0

gelatin

visc

osity

(P

a s)

volume (%)

HG MG LG

10% gelatin2% alginate

alginate

Combinatorial effects or specific molecular interactions

02112222

21

11

1 /)(lnln VTVNVNTk

F

B

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!

S1

P1P2

associative

type PS

segregative

type PS

S1

P1P2

S1

P1P2

Temperature

Ionic strength/PH

Sol-gel

Sol-gel

0 200 400 600 800

0.0

0.2

0.4

0.6

0.8

1.0

time

rem

aini

ng w

ater

0

1x104

2x104

3x104

4x104

L (arb. units)

3

1

tL

Phase separation (spinodal decomposition)

)exp(

ntwater

Remaining water

Gelation

)exp(1 mc ktx

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

9/1 4/1 2/1 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

9/14/1 2/1

1/1

1/21/41/9

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