Characterization of Starch Polysaccharides in Aqueous Systems: de facto molar masses vs supermolecular structures

Transcription

1 Characterization of Starch Polysaccharides in Aqueous Systems: de facto molar masses vs supermolecular structures Werner Praznik and Anton uber Department of Chemistry BKU - University of Natural Resourses and Applied Life Science Vienna / Austria Institute of Chemistry KF-University Graz / Austria

2 Content Introduction Analysis of molecular dimension by SEC-analysis by means of multiple detection systems Synthetic amylose ncb glucan Partially hydrolized starch scb glucan ( waxy maize, ) Potato starch glucans long-chain branched (lcb) - glucan / amylose short-chain branched (scb) - glucan / amylopectin

3 Kinds of starch glucans: non-branched (nb) / long-chain branched (lcb) glucans / amylose (α(1 4)-D-Glc) 30 dp 30 symmetry / order: conformation of α -elices hydrophilic / hydrophobic domains intra-molecular stabilizing: low pronounced tendency for retrogradation, gel formation (supramolecular structures) instance / model : synthetic amylose short-chain branched (scb) glucans / amylopectin (α(1 4)-D-Glc) with α(1 6)linkages dp 43 α(1 4) + α(1 6) linked branches α(1 6) positions are symmetry breaker forming irregular structures - more or less compact coils no hydrophilic /-phobic domains intra-molecular stabilizing: high comparably increased packing density + solubility instance / model: partially hydrolized glucan from waxy maize

4 Molecular dimension by size exclusion chromatography (SEC) Distribution of glucan excluded volume (V e = ip. md mc ) by means of size exclusion chromatography (SEC) - an entropy controlled separation on gel columns systems SEC combined with specific molar detection (e.g Fluorescence of an unique chromophor in a molecule) and universal mass detection (SEC-mass/molar). Information about molar mass of constituting glucans SEC - combined with light scattering (LS) and universal mass detection (SEC-mass/LS) Sensitive towards high molecular components, in particular towards glucan-aggregates Information about supramolecular structure

5 Absolute Molar Mass Distribution Quantitative labeling of terminal hemiacetals + SEC - mass / molar detection: DRI / fluorescence λ V_ret [ml]

6 Absolute Molar Mass Distribution SEC mass / scattering intensity / excluded volume - detection SEC - DRI / LS / viscosity

7 Synthetic amylose ncb glucan

8 Synthesis of 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,... procedure: 1 g glucose-1-p and 4.5 mg maltoheptaose in citrat puffer p 6.1 (50 ml) % NaN 3 were incubated at 40 for 30 min. Addition of 3 ml of chromatographically purificated potato phosphorylase and incubation at 40 C. For analysis equivalents were taken at increasing incubation times

9 [mv] Synthesis of amylose by catalytic action of potato phosphorylase molar mass [Dalton] D = M [ g / mol] = m[ g / L] n[ mol / L] 70 starter of synthesis: maltoheptaose 0,077 μmol/ml % turnover time h 1 h synthesis: 36% turnover μg amylose / ml M = 3450 μg /0,077μmol = [g/mol] determination of free phosphat: calculation of turnover of glucose 1 P calculation of mass of amylose μg / ml 3 h synthesis: 59% turnover μg amylose / ml M = 5590 μg /0,077μmol = [g/mol] DRI Glu-1-P 120 synthetic amylose citrate puffer V_ret [ml]

10 [mv] Synthesis of amylose by catalytic action of potato phosphorylase SEC analysis of synthetic amyloses: DRI synthetic amyloses Glu-1-P citrate puffer mass mass/molar fraction fraction V_ret [ml] M [g/m] period of synthesis SEC-results turnover calculated results h % turnover M w M n M w / M M n n

11 Molecular dimension of synthetic amyloses Quantitative pyridylamination of terminal hemi acetal groups of amylose = AP-amyloses SEC-analysis of AP-amylose by means of mass (RI) and molar (flourescence) detection: dissolved in DMS/water. SEC- eluent : 0.05 M NaCl SEC-profil analysis of AP-amyloses by mass (RI), scattering intensity (LS) and viscosity detection: dissolved in DMS/water/ M Na 2 C 3. SEC-eluent: M Na 2 C 3

12 Quantitative pyridylamination of terminal hemi-acetal group of amylose amylose n C N N aminopyridine - 2 amylose-imid n C 2 C N N + 2 Na-cyanoborhydride AP-amylose n C 2 2 C N N Procedure: Part of fresh centrifugated amyloses (precipitate from solution of synthesis + 10% of n-bu) + 1 ml 2 + 1g aminopyridine + 0.7mL conc. Cl (p=7) stirred at 62 C for 24 h. Then 0.2g NaB 3 CN was added and further stirred at 62 C for 24 h. Part of the solution added to Me and precipitated AP-glucan were purified with Me and acetone. Precipitates were redissolved in 2 ml DMS ml 2 for SEC-analysis.

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

14 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: =0.005 Na 2 C 3 /0.05M NaCl; 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

15 Partially hydrolized starch scb glucan ( waxy maize, )

16 mass fraction lg(m) Partially hydrolized starch glucan ( waxy maize, ) short chain branched glucan (scb glucan) 50 mg scb glucan were solved in 5 ml 0.05M NaCl ml n-butanol 65 C, 40h. Then + 0.2mL n-butanol were added, room temperature, 5 h and cool 5-6 C, 5h. Not any precipitation scb-glucan was precepitated with Me:Aceton (1:1), washed with Me/Aceton and derivatisied with aminopyridine. AP-amylopectin sample for SEC was solved in 1mL DMS ml water Column system: Superose 12 (10x300mm) + Superose 6 (10x300mm) + Fractogel W40 (10x300mm); eluent: 0.05M NaCl; DRI- and Fl (315nm/380nm)-detection V_ret [ml] Mw= Mn = Mw/Mn = 2.4 AP scb glucan solved in 1 ml DMS +0.5mL 65 C 8h. Profil of normalized mass fractions, area = 1.0; calibration with absolut mass/molar detection

17 Partially hydrolized starch glucan ( waxy maize, ) short chain branched glucan (scb glucan) mass (DRI) and LALLS detection: solved in DMS/water/ M Na 2 C 3 mass fraction lg(m) [g/mol] SEC-mass/molar detection Mw= g/mol Mn = g/mol Mw/Mn = 1.9 SEC-mass/LALLS detection Mw= g/mol Mn=53700 g/mol Mw/Mn=2.8 Mean Intrinsic Viscosity [η] = 30 ml/g corresponds roughly to constituting molecules Mw similar for both approaches minor supramolecular structures

18 Potato starch glucans lcb glucan (amylose) scb glucan (amylopectin)

19 mass fraction lg(m) SEC-analysis of potato starch glucans: lcb glucan (amylose) + scb glucan (amylopectin) precipitate with Me 100 µm Mw = Mn = Mw/Mn = Potato starch disssolved in DMS/eluent/n- Butanol, precipitated with Me and derivatizied with aminopyridine V_ret [ml] AP- glucans dissolved in 0.3 ml DMS 65 C 20h. Profil of normalized mass fractions, area = 1.0; calibration with absolut mass/molar detection Column system: Superose 12 (10x300mm) + Superose 6 (10x300mm) + Fractogel W40 (10x300mm); eluent: 0.05M NaCl

20 Iodine staining of potato starch fractions native potato starch

21 Preparation of potato starch glucans after aqueous dissolving Potato starch 100 µm 50 mg potato starch were dissolved in 5 ml 0.05M NaCl ml n-butanol 65 C, 40h. Then + 0.2mL n-butanol were added, room temperature, 5 h and cool 5-6 C, 5h. Precipitate of lcb glucan (amylose) rpm, 10 min. Precipitate washed three times with n-bu/water and derivatizied with aminopyridine. AP-amylose sample for SEC was solved in 1mL DMS ml water. Amylopectin in supernatant precepitated with Me Precipitate washed three times with Me and derivatizied with aminopyridine. AP-amylopectin sample for SEC was dissolved in 1mL DMS ml water.

22 mass fraction lg(m) SEC-analysis of potato starch glucans: precipitate with n-butanol lcb glucan (amylose) 0.24 Mw = Mn = Mw/Mn = V_ret [ml] AP- lcb glucan (amylose) solved in 1 ml DMS +0.5mL 65 C 8h. Profil of normalized mass fractions, area=1.0; calibration with absolut mass/molar detection; Column system: Superose 12 (10x300mm) + Superose 6 (10x300mm) + Fractogel W40 (10x300mm);

23 Iodine staining of potato starch fractions lcb glucan (amylose) precipitate with n-butanol

24 SEC-profil analysis of AP- lcb glucan (amylose) with mass (DRI) and LALLS detection: solved in DMS/water/ M Na 2 C 3 bulk analysis of potato AP-amylose with low angle scattering information about glucan-aggregates building process - supramolecular structure apparent Mw = g/mol 30 times the constituting molecules supramolecular structures in the applied aqueous system Column system: Superdex 75 (10x300mm); eluent: =0.005 Na 2 C 3 /0.05M NaCl; Mean Intrinsic viscosity [η] = 80 ml/g roughly corresponds with constituting molecules

25 lg(m) SEC-analysis of potato starch glucans: precipitate with Me scb glucan (amylopectin) mass fraction V_ret Mw = Mn = Mw/Mn = % Mw = Mn = Mw/Mn = % [ml] AP- scb glucan (amylopectin) solved in 1 ml DMS +0.5mL 65 C 8h. Profil of normalized mass fractions, area=1.0; calibration with absolut mass/molar detection; Column system: Superose 12 (10x300mm) + Superose 6 (10x300mm) + Fractogel W40 (10x300mm);

26 Iodine staining of potato starch fractions scb glucan (amylopectin) precipitate with Me

27 SEC-profil analysis of AP- scb glucan (amylopectin) with mass (DRI) and LALLS detection: solved in DMS/water/ M Na 2 C 3 bulk analysis of potato AP-amylopectin with low angle scattering information about glucan-aggregates building process - supramolecular structure apparent Mw = g/mol 300 times the constituting molecules supramolecular structures in the applied aqueous system Column system: Superdex 75 (10x300mm); eluent: =0.005 Na 2 C 3 /0.05M NaCl; Mean Intrinsic viscosity [η] = 70 ml/g Roughly corresponds to constituting molecules

28 Conclusion: Instance / Model for ncb-glucan (synthetic amylose) forms supramolecular structures (aggregates) in aqueous systems more or less immediately high tendency for inter-molecular interaction. Instance / Model for low molar mass scb-glucan (partially hydrolized waxy maize starch) forms minor supramolecular structures in aqueous systems preferred intra-molecular interaction and intra-molecular stabilization. de facto molar mass of potato lcb-glucan (native amylose) from molar mass distribution: Mw = , Mn = , Mw/Mn = 2.2; tendency to form supramolecular structures is very high; apparent Mw from light scattering g/mol. Potato scb-glucan (native amylopectin) is partially dissolved in applied solvent system only; molar mass in the range g/mol; a significant fraction (approx. 70%) of molecules gets not dissolved truely in the aqueous system and remain / form supermolecular structures: apparent Mw LS g/mol.