Starch glucans: Molecular and Supermolecular Characteristics in Aqueous Systems. Werner Praznik, Renate Löppert & Anton Huber

Transcription

1 Department of Chemistry Division of rganic Chemistry University of atural Resourses and Applied Life Science Vienna / Austria Starch glucans: Molecular and Supermolecular Characteristics in Aqueous Systems Werner Praznik, Renate Löppert & Anton uber IfC - Inst. f. Chemie PSI - PolySaccharide Initiative KFU - Karl-Franzens-Univ. Graz / Austria partners of European Polysaccharide etwork of Excellence

2 Content starch: characterization with SEC - concept potato starch part. hydr. waxy maize starch no branched glucan: synthetic amylose

3 Starch polysaccharide Geometry / Symmetry helical conformations 4)-αD-Glcp-(1 4)-αD-Glcp-(1 non-branched starch component 'amylose' hydrophilic / hydrophobic domains low intra-molecular stabilizing; retrogradation, gel formation supermolecular structures irregular / globular conf. lcb / scb starch glucans: (long-chain-branched / short-chain-branched) αd(1 4)-glucans + α(1 6) Glc x branches 'amylopectin' o pronounced hydrophilic / hydrophobic domains intra-molecular stabilizing: comparably high packing density + increased solubility

4 Polysaccharide Performance structuring (aqueous) phase space by means of molecular + supermolecular structures

5 Polysaccharide Performance V e = ip. md mc structuring (aqueous) phase space by means of molecular + supermolecular structures

6 Polysaccharide Performance V e = ip. md mc excluded (sphere volume equivalent) hydrodynamic radius R coherence length l coh inter- / intra- molecular stability structuring (aqueous) phase space by means of molecular + supermolecular structures

7 Polysaccharide Performance V e = ip. md mc excluded (sphere equivalent) volume molecular dimensions hydrodynamic radius R coherence length l coh inter- / intra- molecular stability MWD / dpd [η] D T visco-elasticity structuring (aqueous) phase space by means of molecular + supermolecular structures

8 Polysaccharide Performance V e excluded (sphere equivalent) volume hydrodynamic radius R coherence length l coh inter- / intra- molecular stability = ip. md molecular dimensions MWD / dpd [η] D T visco-elasticity mc molecular conformation branching pattern water content status of oxidation substitution pattern packing density hydrophobic / -philic domains structuring (aqueous) phase space by means of molecular + supermolecular structures

9 Polysaccharide Performance V e excluded (sphere equivalent) volume hydrodynamic radius R coherence length l coh inter- / intra- molecular stability = ip. interactive properties intra: Glc x / Glc x inter: Glc x / Glc y Glc / 2 Glc / any material md molecular dimensions MWD / dpd [η] D T visco-elasticity mc molecular conformation branching pattern water content status of oxidation substitution pattern packing density hydrophobic / -philic domains structuring (aqueous) phase space by means of molecular + supermolecular structures

10 SEC-Concept for Starch characteristics Separation criterion in SEC is excluded volume (V glucans Therefore, key characteristics which need to be determined are: branching characteristics; molecular weight distribution; supermolecular dimensions, coherent segment dimensions; e : excluded volume). Molecular weight are determined in several ways: calibrated applying reference glucan-materials (e.g. dextrans) via peak position calibration or broad standard calibration; absolute - light scattering (LS) combined with universal mass detection (SECmass/LS),viscosity combined with universal mass detection (SEC-mass/visc). Both absolute approaches are extraordinary sensitive towards high molecular components, in particular towards glucan-aggregates. absolute molare detection with endgroup labeling of glucan e.g UV/VIS or fluorescence of a unique chromophor in each molecule universal mass detection (SEC-mass/molar). and

11 Analytical SEC + multiple inline detection V_ret [ml]

12 Analytical SEC + multiple inline detection V_ret [ml] excluded volume fractionations of heterogeneous materials Ve(i) from SEC

13 Analytical SEC + multiple inline detection V_ret [ml] excluded volume fractionations of heterogeneous materials Ve(i) from SEC details on excluded volume fractions Ve (i) from triple-detection of mass, scattering, viscosity mass by refractive index DRI ( ) scattering intensity as Rayleigh Θ 0 ( o ), specific viscosity from differential pressure (dp) / inlet pressure (ip) ratio ( ).

14 mass fraction SEC - mass / LS / visc SEC: excluded volume calibration mass light scattering viscosity V_ret [ml] Re [nm] potato starch glucans mix of lcb+scbmolecules lcb: helical conformation scb: irregular coil

15 SEC + multiple detection mass, light scattering, viscosity

16 SEC + multiple detection mass, light scattering, viscosity lg ( M [g/mol] ) V_ret [ml] mass(i) + laser light scattering(i) potato starch glucan absolute molar mass(i) M (app) approx g/mol

17 SEC + multiple detection mass, light scattering, viscosity lg ( M [g/mol] ) eta_intrinsic [ml/g] V_ret [ml] V_ret [ml] mass(i) + laser light scattering(i) potato starch glucan absolute molar mass(i) M (app) approx g/mol mass(i) + viscosity(i) potato starch glucan intrinsic viscosity(i) [eta ] ml/g

18 Chemistry + SEC-dual-detection

19 Chemistry + SEC-dual-detection absolute and de facto molar masses by combining SEC elution profiles of mass fractions [g/l] + molar fractions [mol/l]

20 Chemistry + SEC-dual-detection absolute and de facto molar masses by combining SEC elution profiles of mass fractions [g/l] + molar fractions [mol/l]

21 Chemistry + SEC-dual-detection absolute and de facto molar masses by combining SEC elution mass fractions [g/l] + molar fractions [mol/l] profiles of

22 Chemistry + SEC-dual-detection absolute and de facto molar masses by combining SEC elution profiles of mass fractions [g/l] + molar fractions [mol/l]

23 100 µm 100 µm Starch Glucans reductive pyridyl-amination (PA) of starch free terminal hemiacetals: AP-glucans Praznik W., uber A., Journal Chromatography B, 824, , 2005

24 Starch Glucans 100 µm 100 µm reductive pyridyl-amination (PA) of starch free terminal hemiacetals: AP-glucans Praznik W., uber A., Journal Chromatography B, 824, , 2005 R 2 C R C R

25 Starch Glucans 100 µm 100 µm reductive pyridyl-amination (PA) of starch free terminal hemiacetals: AP-glucans Praznik W., uber A., Journal Chromatography B, 824, , 2005 R 2 C R C R idyl-amination (PA) of starch free terminal hemiacetals

26 Starch Glucans 100 µm 100 µm reductive pyridyl-amination (PA) of starch free terminal hemiacetals: AP-glucans Praznik W., uber A., Journal Chromatography B, 824, , 2005 R 2 C R C R idyl-amination (PA) of starch free terminal hemiacetals diss. of g glucan + 500mg 2-aminopyridine + 1mL 2 for 3 60 C

27 100 µm 100 µm Starch Glucans reductive pyridyl-amination (PA) of starch free terminal hemiacetals: AP-glucans R R diss. of g glucan + 500mg 2-aminopyridine + 1mL 2 for 3 60 C C + 2 R Praznik W., uber A., Journal Chromatography B, 824, , 2005 neutralization to p 6-7 with 4 M Cl for 1 60 C C

28 100 µm 100 µm Starch Glucans reductive pyridyl-amination (PA) of starch free terminal hemiacetals: AP-glucans R R diss. of g glucan + 500mg 2-aminopyridine + 1mL 2 for 3 60 C C + 2 R Praznik W., uber A., Journal Chromatography B, 824, , 2005 neutralization to p 6-7 with 4 M Cl for 1 60 C C reduction to stabilized secondary amine with cyanoboro hydride for 2 60 C

29 100 µm 100 µm Starch Glucans reductive pyridyl-amination (PA) of starch free terminal hemiacetals: AP-glucans R R diss. of g glucan + 500mg 2-aminopyridine + 1mL 2 for 3 60 C C + 2 R Praznik W., uber A., Journal Chromatography B, 824, , 2005 neutralization to p 6-7 with 4 M Cl for 1 60 C C reduction to stabilized secondary amine with cyanoboro hydride for 2 60 C precipitation of AP-Glc n in Me excess acetone washing 2 -steam drying

30 potato starch part. hydr. waxy maize starch

31 Disintegration of Supermolecular structures Endgroup-labelled Potato Starch SEC-DRI/FL: mass fractions [mv] DRI V_ret [ml] AP potato starch dissolved in 0.05M acl (eluent) 10mg/mL 65 C; centrifuged; 0.3 ml injected volume at SECsystem (Superose 6 / Fractogel W 40 (Toyoperl); eluent: 0.05M acl; DRI detection; redissolved in eluent n-bu precipitate lcb (amylose type starch glucans) supernatant scb (amylopectin type starch glucans)

32 Iodine staining of potato starch fractions AP- potato starch supernatant scb (amylopectin type starch glucans) n-bu precipitate lcb (amylose type starch glucans)

33 Starch AP-glucans: de facto molar mass partially hydrolized scb (waxy maize starch); potato starch: scb-fraction, lcb-fraction lg ( M [g/mol] ) V_ret [ml] mass fraction part. hydr. scb Mw = g/mol scb-fraction Mw = g/mol lcb-fraction Mw = g/mol V_ret [ml]

34 starch AP-glucans: de facto molar mass distribution 1.2 Mn Mw Mn Mw 1.2 mass fraction molar fraction mass fraction molar fraction lg(m) [g/mol] part. hydr. scb starch (waxy maize) Mw g/mol Mn g/mol Mw / Mn lg(m) [g/mol] lcb fraction (potato starch amylose) Mw g/mol Mn g/mol Mw / Mn 1.8

35 mass fraction starch AP-glucans lg([eta]) [ml/g] Re [nm] lg(m) [g/mol] dimensions R e distributions from V e D part. hydr. scb starch lcb-fraction conformation (Staudinger / Mark / ouwink plot) a (part. hydr. scb starch) = 0.66 a (lcb) = 0.80

36 starch AP-glucans: apparent & de facto molar mass mass / light scattering - mass / fluorescence lg ( M [g/mol] ) lg ( M [g/mol] ) V_ret [ml] V_ret [ml] partially hydrolized AP-starch scb glucans / DMS absolute molar mass mass / light scattering g/mol mass / fluorescence g/mol AP-labelled scb-fraction of potato starch / DMS absolute molar mass mass / light scattering g/mol mass / fluorescence g/mol

37 Starch: nb-glucans Synthetic Amylose

38 Synthesis of nb-glucans (amylose) by catalytic action of potato phosphorylase C 2 maltoheptaose C 2 5 C n C 2 glu-1-p - P - potato phosphorylase citrate puffer p 6,1 C 2 amylose C 2 (5 + n) C n - - P - n = 1,2,3,...100,...

39 ev_mass mass fraction lg(m) SEC-analysis of AP-amylose by means of universal mass (DRI) and molar concentration 380 nm) detection DRI-detection FD nm mass/molar fraction V_ret [ml] M [g/m] mg of AP amylose solved in 2 ml DMS 24h at 62 C + 0.5mL eluent, 300µl Injection DRI 8x, FD: 380nm / att.10 column system: Superose12+Superose 6 + Fractogel W 40 eluent: 0.05 M acl V_ret [ml] Mw= g/mol Mn = g/mol Mw/Mn = 1.6

40 SEC-profil analysis of AP-amylose by means of mass (DRI), viscosity and scattering intensity (LALLS) detection mass fraction V_ret [ml] intrinsic viscosity [ml/g] V_ret [ml] scattering intensity 5 [g/ (ml mol )] V_ret [ml] Column system: Superdex 75 (10x300mm); eluent: a 2 C 3 / 0.05M acl; Mean Intrinsic Viscosity [η] = 70 ml/g corresponds roughly with constituting molecules bulk analysis of AP-amylose: scattering intensity at low angle of detection (5 ) sensitive information about glucan-aggregation / supramolecular structures apparent Mw = g/mol 10 times the constituting molecules supramolecular structures in the applied aqueous system

41 Conclusions: instances / models for nb-glucans (synthetic amylose) form supermolecular structures (aggregates) in aqueous systems more or less immediately high tendency for inter-molecular interaction. instances / models for low molar mass scb-glucans (partially hydrolized waxy maize starch) form minor supermolecular structures in aqueous systems prefered intra-molecular interaction and intra-molecular stabilization. de facto mean values of molar mass for potato lcb-glucans (native amylose): Mw = , Mn = , Mw/Mn = 1.8; high tendency to form supermolecular structures; apparent Mw from light scattering g/mol. Potato scb-glucan (native amylopectin) can get dissolved in applied solvent system incomplete only; apparent molar mass ranges g/mol; a significant fraction (approx. 70%) of molecules gets not dissolved truely in the aqueous system and form supermolecular structures: apparent Mw LS g/mol.

42 General remarks Polysaccharide Qualities: high performance 'advanced polymeric' materials

43 General remarks Polysaccharide Qualities: high performance 'advanced polymeric' materials with high variability on molecular level, and hence, manifold superímposed heterogeneities with efficient sensor quality for varying (aqueous) milieu conditions with pronounced response capability upon detected / applied stress assembled and transformed by molecular tools and molecular-level-library based command sequences (genetic code)

44 General remarks Polysaccharide Qualities: high performance 'advanced polymeric' materials with high variability on molecular level, and hence, manifold superímposed heterogeneities with efficient sensor quality for varying (aqueous) milieu conditions with pronounced response capability upon detected / applied stress assembled and transformed by molecular tools and molecular-level-library based command sequences (genetic code) Polysaccharides dynamically structurized aqueous phase space

45 General remarks Polysaccharide Qualities: high performance 'advanced polymeric' materials with high variability on molecular level, and hence, manifold superímposed heterogeneities with efficient sensor quality for varying (aqueous) milieu conditions with pronounced response capability upon detected / applied stress assembled and transformed by molecular tools and molecular-level-library based command sequences (genetic code) Polysaccharides dynamically structurized aqueous phase space by varying superimposed heterogeneities are minor affected by de facto molecular dimensions (e.g. degree of polymerization) however strongly dependent on conformational aspects (kind of glycosidic linkage, branching pattern, symmetries / domains, packing density,... ) and dominantly controlled by dynamic interactions supermolecular structures