Introduction to polyelectrolytes and polysaccharide characterization
M.Rinaudo Biomaterials Applications
Grenoble (France)
Guadalajara, Mexico, 2-4 May 2017
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
How to study a polysaccharide? Which are the main difficulties? Which are their main characteristics?
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
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
- 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
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
First part
-Source, extraction, fractionation -Purification -Solubility
8
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
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
Example: solid state structure of Crustaceous Chitin.
Chitin + CaCO2+ proteins
[NaOH], [HCl] T°
Chitosan (DA,MW) 11
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
-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
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
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
Schematic representation of polysaccharides in solution: interaction may be H-bonds , hydrophobic groups or divalent crosslinking
Phenomenon of aggregation
16
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
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
Second part
Single chain characteristics in the case of a charged polysaccharide
-Introduction to polyelectrolytes
19
* 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
-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
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
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
e.s.Potential determination
24
25
Titration of a polyacid and extrapolation to zero net charge to determine the pK0
26
Example of determination of the pK0 for
CMC (=1.38) and hyaluronan HA in water (=0.7)
pK ()
27 CMC at different concentrations.
Exemple d’un polypeptide (PGA)- helix-coil transition
28
29
30
31
32
Results obtained on CMC with different charge densities.
Example for Polyacrylate and carboxymethylcelluloses
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
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
Reduced viscosity of HA in water or in 10-4 M NaCl.
-peak location depends on the ionic concentration (Cp+Cs) 35
Reduced viscosity of HA in 10-4 M NaCl
-location of the peak is independent on the molar mass (chain length)
36
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
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
39 N.Nierlich et al, Journal de Physique 49, 701 (1979)
40
I.Morfin et al. J. Phys.II, 4, 1001 (1994)
Influence of external salt on diffusion
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
42
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
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
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
Third part
Single chain characteristics
-Chemical structure (NMR)
-Conformation (helix-coil)
-Persistence length
(Lp & conformational analysis, AFM)
-MW & Lp (SEC)
46
Exocellular bacterial polysaccharide, Succinoglycan (Rheozan)
-Stereoregular polysaccharide based on a 8 sugars repeat unit
47
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
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
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
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
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)
Xanthan
Succinoglycan
Stereoregular bacterial polyelectrolytes
53
NMR signals as a
funtion of the
temperature.
Role of temperature and ionic concentration on the xanthan conformation
helix
coil
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
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
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
13C NMR on the 4 samples (solid phase)
58
15N NMR
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
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
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
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
SEC experiments with 3 detectors on line.
Waters Alliance GPCV 2000 Wyatt MALLS
63
64
-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
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
-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
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
68
Importance of the persistence length
Influence of stiffness and the salt concentration
on the intrinsic viscosity.
69
Xanthan
Lp450Å
Slope 1
Lp10Å
Slope 0.5
Comparison between xanthan and PSS-Na
*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.
70
71
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.
72
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)
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.
73
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)
74
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
75
Good physical properties in solution (higher viscosity than synthetic polymer) and gel but also in solid state.