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Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France) Guadalajara, Mexico, 2-4 May 2017
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Page 1: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Introduction to polyelectrolytes and polysaccharide characterization

M.Rinaudo Biomaterials Applications

Grenoble (France)

Guadalajara, Mexico, 2-4 May 2017

Page 2: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

1.- Introduction to natural polymers and biopolymers 2.- Main characteristics of biopolymers 3.- Solubility and purification. Method of purification. Role of H-bond network & aggregation 4.- Water soluble polysaccharides and polyelectrolyte properties. 5.- Chemical structure of polysaccharides (chemical analysis, NMR…). Introduction to semi-rigid polymers and persistence length. Determination of MW and Lp from SEC and LS.

Program

Page 3: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

How to study a polysaccharide? Which are the main difficulties? Which are their main characteristics?

Page 4: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Natural polysaccharides (biopolymers) are often stereoregular and have original properties (looking other biopolymers as DNA or proteins). I focus on water soluble polysaccharides.

-They are often able to adopt helical conformation (usually extended helix) in given thermodynamic conditions (pH, temperature, salt concentration…)

-They are often semi-rigid polymers (liquid cristalline phase) good rheological performances in aqueous solution applications as thickeners or stabilisers or film forming

4

Page 5: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Biopolymer characteristics

-Water soluble H-bond and hydrophobic interactions (aggregates) play an important role -Ionic groups along the chains electrostatic interactions (polyelectrolyte) impose conformation, chain extension, counterion interaction -Stereoregular polymers conformational transition (helix-coil) -Transition from sol to gel states chain aggregation-cooperative interaction But in the solid state, often semi-cristalline polymers

5

Page 6: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

- Isolation & Fractionnation & Purification

- Solubility and preparation of a dilute solution

- Characterization:

-NMR analysis/partial or complete hydrolysis, gas chromatography, HPLC etc..

-SEC, MW distribution & dynamic LS

-Intrinsic viscosity (influence of Mw and C on the viscosity Stiffness, interacting system)

How to study a polysaccharide from natural resource

6

Page 7: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Main biopolymers covered: -Natural or pseudo-natural polysaccharides -Polysaccharides examined: alginate,gellan, hyaluronan, chitosan, galactomannan, - Polysaccharide derivatives:methylcellulose, alkylchitosan… -Same type of behaviour occurs, in aqueous solution, with deoxyribonucleic acids (-), proteins (+/-) or polypeptides (+ or -)(PLGA -) (e.s. interactions, conformational change….) 7

Page 8: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

First part

-Source, extraction, fractionation -Purification -Solubility

8

Page 9: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

General method proposed for a water-soluble polysaccharide purification.

The anionic polysaccharide is isolated under sodium salt form for better solubility (specific conditions for alginates and pectins).

For chitosan, precipitation at neutral pH from the acidic conditions( -NH3

+) 9

Page 10: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Purification

For ionic polysaccharides:

-avoid multivalent counterions which may cause some crosslinking.

-dry in ambiant air (no total dehydration; avoid freeze-drying or high temperature drying)

For water soluble neutral polysaccharides:

-filtration, precipitation with ethanol, isopropanol or acetone, drying in ambiant conditions (avoid complete drying)

-better solvent for characterization is often DMSO

Fractionation on purified samples: Precipitation may be selective and/or calibrated porous membrane filtration and/or followed by column chromatography

10

Page 11: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Example: solid state structure of Crustaceous Chitin.

Chitin + CaCO2+ proteins

[NaOH], [HCl] T°

Chitosan (DA,MW) 11

Page 12: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Example of chitin in the solid state.

*Solid state configuration controls chitin reactivity (solubility or chemical reaction) in relation with the H-bonds network

*Semi-cristalline morphology controls the chemical structure of derivatives from which chitosan (blockwise distribution of the –N-acetyl groups)

12

Page 13: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

-chitin: (a) ac projection; (b)

bc projection (lobster, crab &

shrimp shells)

-chitin: (a) ac projection; (b)

bc projection (rare; squid

pens)

-chitin is more reactive

Chitin with two main isomorphs (, )

13

Page 14: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Isolated Chitin is insoluble in majority of solvents (only DMAC/LiCl in precise conditions) Very difficult to characterize in MW or conformation

Good mechanical performance for fibers and films (H bonds)

Deacetylated chitin Chitosan soluble in acidic conditions

due to –NH2 protonation but properties depend on purity

(residual proteins & CaCl2) , on the average DA but also on

the distribution of –NH2 groups along the chains (blocks).

Advantage of chitin among cellulose: specific C-2 position

allowing specific modifications.

14

Page 15: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Solubility and single chain behaviour

-Preparation of a dilute solution (C<C*) to be able to

get MW and chain characteristics in relation with

-the semi rigid character depending on the conformation and determination of the persistence length Lp (conformational analysis & experiments)

- the chemical structure or distribution of substituents for polysaccharide derivatives

Problems in relation with interchain interactions 15

Page 16: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Schematic representation of polysaccharides in solution: interaction may be H-bonds , hydrophobic groups or divalent crosslinking

Phenomenon of aggregation

16

Page 17: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

1 10 100 10000,0

0,2

0,4

0,6

0,8

1,0

am

plit

ude, a.u

.

hydrodynamic radius, nm

1.2x10-3 monomol/L solution of chitosan (DA= 0.12) in 0.3 М CH3COOH in the presence of 0.05 M CH3COONa.

DLS on chitosan solution to test the « quality » of solution

unimers

Aggregates

How much is engaged?

(fraction less than 0.1)

LS in the bulk is very difficult to analyze safely.

17

Page 18: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Influence of the chemical structure of the membranes

on solution properties:

Dynamic LS shows elimination of aggregates on 0.1 m membrane

made of nitrocellulose

18

Page 19: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Second part

Single chain characteristics in the case of a charged polysaccharide

-Introduction to polyelectrolytes

19

Page 20: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

* Definition and charge parameter ()

* Activity coefficient of counterions (mono and divalent) osmotic pressure and effective charge density

* pK and conformation on single chain

* Viscosity and radiation scattering at low ionic concentration (isoionic dilution)

•Worm-like chain and local stiffness (persistence length)

Specific problems implied:

20

Page 21: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

-Attractive interactions: polyion- counterions distribution ionic selectivity -Repulsive interactions:* short range -conformation(extension) - pKa, pK0 *long range (Debye length -1) -peak in viscosity and in radiation scattering -exclusion from gel in SEC Monovalent electrolyte -1 ~ Cs -1/2; 10-4M~ 30nm; 10-2M ~3nm

Main electrostatic interactions

21

Page 22: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Characterization of a polyelectrolyte

-when DP>15, properties are controlled by b, the distance between two charged groups.

-influence of the charge distribution along the backbone (block?)

is the charge parameter;

it imposes the activity of

counterions and

electrostatic interactions.

depends on the

stereoregular

conformation.

b

coil helix

Helix-coil transition is induced usually by temperature or external salt

b

b

22

Page 23: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Polyelectrolyte model proposed by A.Katchalsky (dilute solution).

Electrostatic potential around the polyelectrolyte imposes the counterions (and coions) distribution and ion pairs formation (ionic selectivity); described by PB equation.

(r) and (a)

23

Page 24: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

e.s.Potential determination

24

Page 25: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

25

Page 26: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Titration of a polyacid and extrapolation to zero net charge to determine the pK0

26

Page 27: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Example of determination of the pK0 for

CMC (=1.38) and hyaluronan HA in water (=0.7)

pK ()

27 CMC at different concentrations.

Page 28: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Exemple d’un polypeptide (PGA)- helix-coil transition

28

Page 29: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

29

Page 30: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

30

Page 31: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

31

Page 32: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

32

Results obtained on CMC with different charge densities.

Example for Polyacrylate and carboxymethylcelluloses

Page 33: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Chain extension due to nearby ionic sites but also interchain interactions Viscosity and scattering radiation anomalies

33

Water dilution

Role of excess of external salt or dilution in water on the chain extension

Page 34: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

electrostatic interchain interactions(e.s network?)

Isolated chains

+Expansion of the chain due to

electrostatic repulsions between two

ionic sites on the same chain is not

the most important

10-5 M

10-4 M

155mL/g 34

Viscosity of dilute solution

Page 35: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Reduced viscosity of HA in water or in 10-4 M NaCl.

-peak location depends on the ionic concentration (Cp+Cs) 35

Page 36: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Reduced viscosity of HA in 10-4 M NaCl

-location of the peak is independent on the molar mass (chain length)

36

Page 37: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Isoionic dilution for HA in NaCl for different total ionic concentrations is the only way to determine the intrinsic viscosity of polyelectrolyte at low ionic concentration.

37

Page 38: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

38

Model of Odijk for polyelectrolyte in dependence of concentration Long range e.s. interactions and supramolecular organisation

d<L

d and q* are calculated from polymer concentration Role of Lp, the persistence length

Page 39: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

39 N.Nierlich et al, Journal de Physique 49, 701 (1979)

Page 40: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

40

I.Morfin et al. J. Phys.II, 4, 1001 (1994)

Influence of external salt on diffusion

Page 41: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Characterization of polyelectrolytes must be performed in the presence of salt excess (0.1M NaCl) to avoid « anormal scattering »

(a) f (polymer concentration) (b)~slope ½ in agreement with a hexagonal packing.

Light and neutron scattering in salt free solution (q is the scattering wave vector; q max is related to the distance between two interacting chains – see Katchalsky model)

Increasing polymer concentration

41

Page 42: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

42

Page 43: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

How to study polyelectrolyte specific properties? *dilute solution *absence of external salt (or controlled low 1-1 salt content) *homoionic polymer (monovalent counterions) *control of the degree of neutralisation for weak polybase or polyacid (no buffer)

43

Page 44: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Conclusions -Electrostatic interactions play a large role on solution properties of polyelectrolyte -Debye length is important and its estimation must include external salt +polyelectrolyte contribution -Donnan equilibrium controls the osmotic pressure -Abnormal behaviour in viscosity and radiation scattering is mainly due to long range electrostatic interactions (suppressed for Cp/Cs ~2or 2).

44

Page 45: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Conditions needed to characterize a polyelectrolyte in absence of long range e.s.interactions:

-solvent : 0.1M electrolyte 1-1 to screen long range electrostatic repulsions (-1~1nm) ( minimum Cp/Cs~2 or 2)

-the reduced viscosity f(C) is linear in dilute solution and allows to extrapolate for intrinsic viscosity with Huggins constant k’~0.4 but [] value depends on the salt concentration (as well as the constants of Mark-Houwink []=KMa due to intrachain electrostatic interactions)

-SEC analysis is valid -pKa is closed to pK0

45

Page 46: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Third part

Single chain characteristics

-Chemical structure (NMR)

-Conformation (helix-coil)

-Persistence length

(Lp & conformational analysis, AFM)

-MW & Lp (SEC)

46

Page 47: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Exocellular bacterial polysaccharide, Succinoglycan (Rheozan)

-Stereoregular polysaccharide based on a 8 sugars repeat unit

47

Page 48: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Succinoglycan (Rheozan) showing a very cooperative transition (1) in water (D2O)(2) in 0.1M NaCl

1H NMR spectrum to control the structure (quantitative amount of substituents) T~85°C/D2O

Influence of temperature NMR Signal

48

Helix Coil

Page 49: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Activity coefficient of counterions ~1/2 for monovalent

~1/4 for divalent

combined with molar mass determination

Allows to conclude on the nature of the conformational transition of stereoregular polysaccharide in solution:

-Is it a single or a double helix ??

-Is it a double helical structure formed by a single chain or two chains?

ordered conformation (helical) or the helix-coil transition are shown by DSC, optical rotation, circular dichroïsm …potentiometry… This behaviour is frequent with bacterial polysaccharides which are stereoregular and rich in H-bonds (just as with DNA or proteins)

49

Page 50: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Specific optical rotation as a function

of temperature.C=1 g/L in water (1)

and 0.1M NaCl (2)

Relative viscosity as a function opf

temperature. Heating-cooling cycle on

succinoglycan in 0.1M NaCl

(1) Mw=3. 106 ; []= 7600 mL/g; Lp= 35 nm (helical conformation)

(2) Mw=3. 106 ; []= 2130 mL/g ; Lp= 5 nm (coiled conformation)

1

2

Two conformational states:

50

Page 51: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Experimental thermodynamic characteristics on a single chain polysaccharide

Succinoglycan on both sides of the conformational transition (Tm)

Succinoglycan

calc. (Na+)

calc

(Ca+2)

exp.

(Na+)

exp. (Ca+2)

c = 0.68

0.933

---

0.97

----

h = 0.74

0.92

0.62

0.93

0.6 ¤

¤ helical conformation from optical rotation experiment. Mc ~Mh Succinoglycan is a helical single chain

2 Mc = Mh Gellan is a helical dimer

51

Page 52: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

2 Mc = Mh Gellan is a helical dimer

52

Conclusion on succinoglycan characterisation Using different techniques (light scattering, viscosimetry, NMR, optical rotation…), it comes: - At low temperature: it is a semi-rigid chain with a helical conformation

- Helical conformation is stabilized in presence of

external salt (screening of electrostatic repulsions)

- Single chain helix –coil transition is induced by temperature increase (MW is the same for the two conformations i.e. coil and helix)

Page 53: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Xanthan

Succinoglycan

Stereoregular bacterial polyelectrolytes

53

Page 54: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

NMR signals as a

funtion of the

temperature.

Role of temperature and ionic concentration on the xanthan conformation

helix

coil

Page 55: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Experimental thermodynamic characteristics

Xanthan at 25°C.

Xanthan

calc.

(Na+)

calc

(Ca+2)

exp.

(Na+)

exp. (Ca+2)

c

=1.03

0.587

0.293

0.650

-----

h =

1.13

0.535

0.267

----

0.295 ¤

2h

=2.26

0.267

0.134

¤ helical conformation from optical rotation experiment. Mc~Mh and activity coefficient Native xanthan is a helical single chain

2 Mc = Mh Gellan is a helical dimer

determination by conductivity and potentiometry. 55

Page 56: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Xanthan: 5/1 helix viewed (a) perpendicular to and (b) down the helix axis (X-ray diffraction and computer building modeling)

R.Moorhouse, M.D.Walkinshaw, S.Arnott (1977)

Possible H-bonds that may stabilize the molecule.

56

Page 57: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

57

Example of chitin and chitosan: NMR characterization

1H NMR only on perfectly soluble sample (chitosan DA< 0.5 in acidic medium HCl)

Determination of DA from O (sample D) to 1(sample A, chitin) on 4 different samples using different NMR

Page 58: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

13C NMR on the 4 samples (solid phase)

58

15N NMR

Page 59: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

C1

O5

C5

O

O4

C4

NH2

HO

OH

OH

NHHO

O

O

F

Y

Conformational analysis of chitin and chitosan

*Dimensions of the chain

*Intrinsic viscosity

*Persistence length Lp

K.Mazeau,S.Pérez, M.Rinaudo J.Carbohydr. Chem 19, 2000, 1269-1284

Prediction

Specific viscosity

59

Page 60: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Chitin

Chitosan

Chitin and chitosan conformations are stabilized by a H bond network (sensitive to

temperature); the polymer behaves as a semi-rigid polysaccharide in solution.

Chitosan is soluble in aqueous system when pH<6.5; it is the only positively

charged biopolymer. 60

Page 61: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Determination of the persistence length of pure chitin (N-acetylated form) and pure chitosan ( D- glucosamine repeat unit)

Conclusion: there is not a dramatic change in the chain stiffness during deacetylation but difficult to get experimentally!

61

Page 62: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

MW & Molar Mass distribution

Using SEC with three detectors on line:

-viscometer (Waters)

-differential refractometer (Waters)

-multiangle laser light scattering detector (Wyatt)

Porous columns used depend on the range of MW studied; for succinoglycan or HA , two columns in series are used :Shodex OH-pack 805 and 806

Eluent is 0.1M NaNO3 (added of 0.2g/L Na-azide for antibacterial effect)

Temperature is 30°C; flow rate is 0.5mL/mn

Samples are filtrated on 0.2m porous membranes before to be injected.

(these conditions are not valid for chitosan)

62

Page 63: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

SEC experiments with 3 detectors on line.

Waters Alliance GPCV 2000 Wyatt MALLS

63

Page 64: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

64

Page 65: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

-0.02

0.00

0.02

0.04

0.06

0.08

0 5 10 15 20 25

LS

#1

1,

AU

X1

, A

UX

2

Volume (mL)

11

Peak ID - HA Bioniche

LS #11

AUX1

AUX2

0.0

0.4

0.8

1.2

1.6

1.0x104 1.0x105 1.0x106 1.0x107

Dif

fere

ntia

l W

eig

ht

Fra

cti

on

Molar Mass (g/mol)

Dif f erential Molar Mass HA BionicheNorm = Log1st order

0.0

0.2

0.4

0.6

0.8

1.0

1.0x104 1.0x105 1.0x106 1.0x107

Cu

mu

lati

ve

We

igh

t F

racti

on W

Molar Mass (g/mol)

Cumulativ e Molar Mass HA Bioniche

SEC analysis:

3 traces analyzed as a function of the elution volume.

SEC also gives Rg(M) and

[](M) relationships but valid only on perfect solution

65

Page 66: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

COLLECTION INFORMATION Example on xanthan SEC

Instrument type : DAWN DSP-F

Cell type : K5

Laser wavelength : 632.8 nm

Solvent name : water

Solvent RI : 1.332

Calibration constants

DAWN : 7.6000e-06

» AUX1 : 4.7200e-05

Flow rate : 0.500 mL/min

Results Volume (mL) : 10.667 - 16.400

Slices : 345

A2 (mol mL/g²) : 0.000e+00

Fit degree : 1

Injected Mass (g) : 3.5900e-05

Calc. Mass (g) : 5.3146e-06 14.8% soluble

dn/dc (mL/g) : 0.157

Polydispersity(Mw/Mn) : 1.003±0.029 (2.9%)

Polydispersity(Mz/Mn) : 1.006±0.050 (5%)

Molar Mass Moments (g/mol)

Mn : 1.403e+06 (2.1%)

Mw : 1.408e+06 (2.0%)

Mz : 1.412e+06 (4%)

R.M.S. Radius Moments (nm)

Rn : 84.8 (0.8%)

Rw : 84.7 (0.8%)

Rz : 84.7 (0.7%) 66

Page 67: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

-0.4

0.0

0.4

0.8

1.2

0 10 20 30 40

LS

#11,

AU

X1,

AU

X2

Volume (mL)

1122

Peak ID - 10mr09-04_01

LS #11

AUX1

AUX2

HA

Proteins

SEC chromatography of a synovial fluid (LS-7M, stade III) allowing the determination of soluble protein and HA molecular weights and concentrations.T=30°C, eluent 0.1M NaNO3. LS is the light scattering signal; refractive index signal gives concentration.

__ LS

__ Conc.

___ Viscosity

67

Influence of the hydrodynamic volume for separation in Ve, high molar mass gives high LS signal

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Schematic representation of salt effect on polyelectrolyte chains with different stiffness (Lp)

Advantage of stereoregular

polysaccharides:

-Higher viscosity in solution at a

given Mw

-Low salt sensitivity

-Possible liquid crystalline structure

at moderate concentration

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Importance of the persistence length

Page 69: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

Influence of stiffness and the salt concentration

on the intrinsic viscosity.

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Xanthan

Lp450Å

Slope 1

Lp10Å

Slope 0.5

Comparison between xanthan and PSS-Na

Page 70: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

*Wayne Reed ** K.Haxaire,M.Rinaudo et al.Glycobiology,10,587 (2000)

Application of the worm like chain model on HA. Influence of salt concentration and molar mass on the radius of giration.

-Lp=80 Å in agreement with molecular modeling**

-Le~ Cs-1 (T.Odijk)

SEC experiment* allows the determination of Rg (M) and Lp.

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Page 71: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

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Persistence length from Neutron scattering on CMC.

I~ q-2 for q<q*; I~q-1 at q>q* Validation of the isoionic dilution Q* =Constant at Ct=3x10-3 M/L LT= constant

Moan &Wolff, Polymer,16,776 (1975)

Other determinations of the persistence length.

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Influence of the degree of neutralisation on CMC (-COO-): Q* decreases when increases Persistence length (here b) increases up to a limit (condensation or effective charge density- charge buffer effect)

Moan &Wolff, Polymer,16,776 (1975)

Page 73: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

AFM experiment on succinoglycan which confirms

Lp determination from Rg

E. Balnois, S. Stoll, K.J. Wilkinson, J. Buffle, M. Rinaudo & M. Milas "Conformations of

succinoglycan as observed by atomic force microscopy", Macromolecules, 33, 2000, 7440-7447.

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Conclusions.

*The characterization of few polysaccharides (natural biopolymers) is

described using general techniques which are able to be used on many

different systems (especially other biopolymers).

*We focused on the chemical control by NMR, on the difficult problem of

solubility which is essential for valuable polymer characterization

*The semi-cristalline organization in the solid state is related to the

stereoregularity of the polysaccharides favouring chain interaction

*Stereoregularity allows the stabilisation of stiffer helical conformation in

given thermodynamic conditions (single or double helix)

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Page 75: Introduction to polyelectrolytes and polysaccharide ... · Introduction to polyelectrolytes and polysaccharide characterization M.Rinaudo Biomaterials Applications Grenoble (France)

cooperative interactions (aggregation and gel formation stabilized

by junction zones rigid gels discussed later)

-natural (but not the chemically modified ) polysaccharides are usually

semi-rigid polymers having a relatively high persistence length in the

helical conformation

high viscosity even in dilute regime and large stability of this

viscosity in salt excess

favouring interchain interactions

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Good physical properties in solution (higher viscosity than synthetic polymer) and gel but also in solid state.


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