Introduction to polyelectrolytes and polysaccharide characterization

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

2 Program 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.

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

4 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

5 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 aggregationcooperative interaction But in the solid state, often semi-cristalline polymers 5

6 How to study a polysaccharide from natural resource - 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) 6

7 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

8 First part -Source, extraction, fractionation -Purification -Solubility 8

9 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( -NH 3+ ) 9

10 For ionic polysaccharides: Purification -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

11 Example: solid state structure of Crustaceous Chitin. Chitin + CaCO 2 + proteins [NaOH], [HCl] T Chitosan (DA,MW) 11

12 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

13 Chitin with two main isomorphs (, ) -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 13

14 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 NH 2 protonation but properties depend on purity (residual proteins & CaCl 2 ), on the average DA but also on the distribution of NH 2 groups along the chains (blocks). Advantage of chitin among cellulose: specific C-2 position allowing specific modifications. 14

15 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

16 Phenomenon of aggregation Schematic representation of polysaccharides in solution: interaction may be H-bonds, hydrophobic groups or divalent crosslinking 16

17 amplitude, a.u. DLS on chitosan solution to test the «quality» of solution 1,0 0,8 0,6 0,4 Aggregates How much is engaged? (fraction less than 0.1) 0,2 unimers 0, hydrodynamic radius, nm LS in the bulk is very difficult to analyze safely. 1.2x10-3 monomol/l solution of chitosan (DA= 0.12) in 0.3 М CH 3 COOH in the presence of 0.05 M CH 3 COONa. 17

18 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

19 Second part Single chain characteristics in the case of a charged polysaccharide -Introduction to polyelectrolytes 19

20 Specific problems implied: * 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) 20

21 Main electrostatic interactions -Attractive interactions: polyion- counterions distribution ionic selectivity -Repulsive interactions:* short range -conformation(extension) - pka, pk 0 *long range (Debye length -1 ) -peak in viscosity and in radiation scattering -exclusion from gel in SEC Monovalent electrolyte -1 ~ Cs -1/2 ; 10-4 M~ 30nm; 10-2 M ~3nm 21

22 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

23 Polyelectrolyte model proposed by A.Katchalsky (dilute solution). (r) and (a) Electrostatic potential around the polyelectrolyte imposes the counterions (and coions) distribution and ion pairs formation (ionic selectivity); described by PB equation. 23

24 e.s.potential determination 24

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26 Titration of a polyacid and extrapolation to zero net charge to determine the pk 0 26

27 Example of determination of the pk 0 for CMC ( =1.38) and hyaluronan HA in water ( =0.7) pk ( ) CMC at different concentrations. 27

28 Exemple d un polypeptide (PGA)- helix-coil transition 28

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32 Example for Polyacrylate and carboxymethylcelluloses Results obtained on CMC with different charge densities. 32

33 Water dilution Role of excess of external salt or dilution in water on the chain extension Chain extension due to nearby ionic sites but also interchain interactions Viscosity and scattering radiation anomalies 33

34 10-5 M Viscosity of dilute solution 10-4 M Isolated chains electrostatic interchain interactions(e.s network?) +Expansion of the chain due to electrostatic repulsions between two ionic sites on the same chain is not the most important 155mL/g 34

35 Reduced viscosity of HA in water or in 10-4 M NaCl. -peak location depends on the ionic concentration (Cp+Cs) 35

36 Reduced viscosity of HA in 10-4 M NaCl -location of the peak is independent on the molar mass (chain length) 36

37 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 d<l Long range e.s. interactions and supramolecular organisation d and q* are calculated from polymer concentration 38 Role of Lp, the persistence length

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

40 Influence of external salt on diffusion I.Morfin et al. J. Phys.II, 4, 1001 (1994) 40

41 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) (a) f (polymer concentration) Increasing polymer concentration (b)~slope ½ in agreement with a hexagonal packing. Characterization of polyelectrolytes must be performed in the presence of salt excess (0.1M NaCl) to avoid «anormal scattering» 41

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43 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

44 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

45 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 [ ]=KM a due to intrachain electrostatic interactions) -SEC analysis is valid -pka is closed to pk 0 45

46 Third part Single chain characteristics -Chemical structure (NMR) -Conformation (helix-coil) -Persistence length (Lp & conformational analysis, AFM) -MW & Lp (SEC) 46

47 Exocellular bacterial polysaccharide, Succinoglycan (Rheozan) -Stereoregular polysaccharide based on a 8 sugars repeat unit 47

48 1 H NMR spectrum to control the structure (quantitative amount of substituents) T~85 C/D 2 O NMR Signal Influence of temperature Helix Coil Succinoglycan (Rheozan) showing a very cooperative transition (1) in water (D 2 O)(2) in 0.1M NaCl 48

49 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

50 1 2 Specific optical rotation as a function of temperature.c=1 g/l in water (1) and 0.1M NaCl (2) Two conformational states: Relative viscosity as a function opf temperature. Heating-cooling cycle on succinoglycan in 0.1M NaCl (1) Mw= ; [ ]= 7600 ml/g; Lp= 35 nm (helical conformation) (2) Mw= ; [ ]= 2130 ml/g ; Lp= 5 nm (coiled conformation) 50

51 Experimental thermodynamic characteristics on a single chain polysaccharide Succinoglycan on both sides of the conformational transition (Tm) Succino glycan calc. (Na + ) calc (Ca +2 ) exp. (Na + ) exp. (Ca +2 ) c = 0.68 h = helical conformation from optical rotation experiment. Mc ~Mh Succinoglycan is a helical single chain 51

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) 52

53 Xanthan Succinoglycan Stereoregular bacterial polyelectrolytes 53

54 NMR signals as a funtion of the temperature. coil Role of temperature and ionic concentration on the xanthan conformation helix

55 Experimental thermodynamic characteristics Xanthan at 25 C. Xanthan calc. (Na + ) calc (Ca +2 ) exp. (Na + ) exp. (Ca +2 ) c =1.03 h = h = helical conformation from optical rotation experiment. Mc~Mh and activity coefficient Native xanthan is a helical single chain determination by conductivity and potentiometry. 55

56 Possible H-bonds that may stabilize the molecule. 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) 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 57

58 13 C NMR on the 4 samples (solid phase) 15 N NMR 58

59 Conformational analysis of chitin and chitosan OH O HO O 5 F C 1 NH 2 HO O 4 C 4 Y C 5 O NH O Prediction OH *Dimensions of the chain *Intrinsic viscosity Specific viscosity *Persistence length Lp K.Mazeau,S.Pérez, M.Rinaudo J.Carbohydr. Chem 19, 2000,

60 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

61 Determination of the persistence length of pure chitin (Nacetylated 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

62 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 0.2 m porous membranes before to be injected. (these conditions are not valid for chitosan) 62

63 SEC experiments with 3 detectors on line. Waters Alliance GPCV 2000 Wyatt MALLS 63

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Cumulativ e Weight Fraction W LS #11, AUX1, AUX2 Dif f erential Weight Fraction SEC analysis: 3 traces analyzed as a function of the elution volume. 0.08 0.06 Peak ID - HA Bioniche LS #11 AUX1 AUX2 1.

65 Cumulativ e Weight Fraction W LS #11, AUX1, AUX2 Dif f erential Weight Fraction SEC analysis: 3 traces analyzed as a function of the elution volume Peak ID - HA Bioniche LS #11 AUX1 AUX Dif f erential Molar Mass HA Bioniche Norm = Log 1st order Volume (ml) x x x x10 7 Molar Mass (g/mol) Cumulativ e Molar Mass HA Bioniche SEC also gives Rg(M) and [ ](M) relationships but valid only on perfect solution x x x x10 7 Molar Mass (g/mol) 65

66 COLLECTION INFORMATION Example on xanthan SEC Instrument type : DAWN DSP-F Cell type : K5 Laser wavelength : nm Solvent name : water Solvent RI : Calibration constants DAWN : e-06» AUX1 : e-05 Flow rate : ml/min Results Volume (ml) : Slices : 345 A2 (mol ml/g²) : 0.000e+00 Fit degree : 1 Injected Mass (g) : e-05 Calc. Mass (g) : e % soluble dn/dc (ml/g) : 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

67 LS #11, AUX1, AUX2 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 NaNO 3. LS is the light scattering signal; refractive index signal gives concentration HA Peak ID - 10mr09-04_01 Proteins LS #11 AU X1 AU X2 LS Conc. Viscosity Volume (ml) Influence of the hydrodynamic volume for separation in Ve, high molar mass gives high LS signal 67

68 Importance of the persistence length 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

69 Influence of stiffness and the salt concentration on the intrinsic viscosity. Xanthan Lp 450Å Slope 1 Comparison between xanthan and PSS-Na Lp 10Å Slope

70 Application of the worm like chain model on HA. Influence of salt concentration and molar mass on the radius of giration. SEC experiment* allows the determination of Rg (M) and Lp. -Lp=80 Å in agreement with molecular modeling** -Le~ Cs -1 (T.Odijk) *Wayne Reed ** K.Haxaire,M.Rinaudo et al.glycobiology,10,587 (2000) 70

71 Other determinations of the persistence length. 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 L T = constant Moan &Wolff, Polymer,16,776 (1975) 71

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) 72

73 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,

74 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

75 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 Good physical properties in solution (higher viscosity than synthetic polymer) and gel but also in solid state. 75

Question 1. Identify the sugars below by filling in the table below (except shaded areas). Use the page or a separate sheet

Question 1. Identify the sugars below by filling in the table below (except shaded areas). Use the page or a separate sheet Sugar name aworth projection(s) of the corresponding pyranose form (Six membered