The physical characterisation of polysaccharides in solution. Stephen Harding University of Nottingham

Transcription

1 The physical characterisation of polysaccharides in solution Stephen Harding University of Nottingham

2 The physical characterisation of polysaccharides in solution Viscometry SEC-MALLs Analytical Ultracentrifugation Stephen Harding University of Nottingham Imaging

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6 Physical characterisation 1. Viscosity, stability 2. Heterogeneity, Molecular weight & distribution, stability 3. Conformation in solution 4. Interactions

7 Physical characterisation 1. Viscosity, stability 2. Heterogeneity, Molecular weight & distribution 3. Conformation in solution 4. Interactions 1: Viscometry. 2: SEC-MALLs & analytical ultracentrifugation. 3: Viscometry, SEC-MALLs & analytical ultracentrifugation. 4. Analytical ulttracentrifugation & atomic force microscopy

8 1. Viscosity from precision viscometry

9 1. Viscosity by Precision viscometry [η] Intrinsic viscosity, ml/g

10 Types of Viscometer: Ostwald Viscometer

11 Types of Viscometer: 1. U-tube (Ostwald or Ubbelohde) Ostwald Viscometer

12 Types of Viscometer: 1. U-tube (Ostwald or Ubbelohde) Extended Ostwald Viscometer

13 Types of Viscometer: 1. U-tube (Ostwald or Ubbelohde) 2. Cone & Plate (Couette) Couette-type Viscometer

14 Types of Viscometer: 1. U-tube (Ostwald or Ubbelohde) 2. Cone & Plate (Couette) 3. Pressure imbalance on-line viscometer

15 Auto-timer Coolant system Density meter Solution Water bath o C

16 Definition of viscosity: For normal (Newtonian) flow behaviour: viscosity τ = (F/A) = η. (dv/dy) shear rate shear stress η = τ/(dv/dy) units: (dyn/cm 2 )/sec -1 At 20.0 o C, η(water) ~ 0.01P = dyn.sec.cm -2.. = POISE (P)

17 Viscosity of biomolecular solutions: A dissolved macromolecule will INCREASE the viscosity of a solution because it disrupts the streamlines of the flow:

18 We define the relative viscosity η r as the ratio of the viscosity of the solution containing the macromolecule, η, to that of the pure solvent in the absence of macromolecule, η o : η r = η/η o no units For a U-tube viscometer, η r = (t/t o ). (ρ/ρ o )

19 Reduced viscosity The relative viscosity depends (at a given temp.) on the concentration of macromolecule, the shape of the macromolecule & the volume it occupies. If we are going to use viscosity to infer on the shape and volume of the macromolecule we need to eliminate the concentration contribution. The first step is to define the reduced viscosity η red = (η r 1)/c If c is in g/ml, units of η red are ml/g

20 The Intrinsic Viscosity [η] The next step is to eliminate non-ideality effects deriving from exclusion volume, backflow and charge effects. We measure η red at a series of concentrations and extrapolate to zero concentration: [η] = Lim c 0 (η red) units [η] = ml/g sometimes researchers use dl/g so 200 ml/g = 2 dl/g

21 irradiated (10kGy) guar Huggins η red or η inh (ml/g) [η] Kraemer c (mg/ml)

22 Viscosity probe: chitosan stability Fee et al, 2006

23 2. Heterogeneity and molecular weight: SEC-MALLs

24 2. Heterogeneity and Molecular Weight: SEC-MALLs

25 Molecular Weight: Light scattering photodiode detectors incident beam transmitted beam

26 Molecular Weight: Zimm plot 10 6.(Kc/R θ ) 1/M w R g from slope sin 2 (θ/2) + kc (k=100)

27 Molecular Weight: SEC MALLS

28 Molecular Weight: SEC MALLS Colonic mucin

29 Can also couple a pressure imbalance type of viscometer on-line

30 irradiated (10kGy) guar Huggins η red or η inh (ml/g) [η] Kraemer c (mg/ml)

31 irradiated (10kGy) guar Huggins Solomon-Ciuta η red or η inh (ml/g) Kraemer c (mg/ml)

32 Analytical Ultracentrifugation

33 Optima XLA/ XLI

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37 Sedimentation Velocity Centrifugal force Top view, sector of centrifuge cell Air Solvent Solution conc, c Rate of movement of boundary sed. coeff distance, r s o 20,w Sedimentation coefficient, S

38 Chitosan G213, 0.5 mg/ml meniscus Cell base

39 Chitosan G213, 0.5 mg/ml meniscus Cell base

40 Chitosan G213, 0.5 mg/ml Sedimentation g(s*) plot : from analysis of the change with time of the whole concentration profile

41 Guar, 0.75 mg/ml

42 Wheat starch, 8 mg/ml

43 stability studies SAN02 Freeze-thaw (1.16mg/mL) g*(s) Control 10cycles 20cycles 25cycles s(s)

44 Multi-Gaussian fit estimates proportions of each species too:

45 2 types of AUC Experiment: Sedimentation Velocity Centrifugal force Top view, sector of centrifuge cell Air Solvent Sedimentation Equilibrium Centrifugal force Diffusion Solution conc, c Rate of movement of boundary sed. coeff distance, r s o 20,w 1S=10-13 sec conc, c distance, r STEADY STATE PATTERN FUNCTION ONLY OF MOL. WEIGHT PARAMETERS

46 Extraction of M w,app from sedimentation equilibrium and MSTAR analysis Chitosan G213 M w,app cell bottom

47 Extraction of M w,app from sedimentation equilibrium and MSTAR analysis xanthan M w = ( )x10 6 g/mol

48 3. Conformation in solution

49 Conformation in solution 1. Experimental data required Molecular weight [η], s, R g 2. Modelling strategies General conformation type (rod, coil or sphere etc.) Measure of flexibility the persistence length, L p, If ~ rigid then aspect ratio.

50 Conformation in solution 1. Experimental data required Molecular weight: SEC-MALLs reinforced by sed. equilibrium [η], s, R g 2. Modelling strategies General conformation type (rod, coil or sphere etc.) Measure of flexibility the persistence length, L p, If ~ rigid then aspect ratio.

51 Sedimentation Velocity Centrifugal force Top view, sector of centrifuge cell Air Solvent Solution conc, c Rate of movement of boundary sed. coeff distance, r s o 20,w Sedimentation coefficient, S

52 s o 20,w extraction κ-carrageenan 1/s 20,w (S -1 ) slope=k s /s o 20,w 1/s o 20,w c (mg/ml)

53 Molecular Weight: Zimm plot 10 6.(Kc/R θ ) 1/M w R g from slope sin 2 (θ/2) + kc (k=100)

54 irradiated (10kGy) guar Huggins η red or η inh (ml/g) [η] Kraemer c (mg/ml)

55 Haug Triangle

56 Sphere [η] ~ M 0 Rod [η] ~ M 1.8 Coil [η] ~ M s o 20,w ~ M0.67 R g ~ M 0.33 s o 20,w ~ M0.15 R g ~ M 1.0 s o 20,w ~ M R g ~ M

57 Mark-Houwink-Kuhn-Sakurada Power law plot Galactomannans a=

58 Change in Conformation Rollings, 1992

59 Sphere [η] ~ M 0 Rod [η] ~ M 1.8 Coil [η] ~ M s o 20,w ~ M0.67 R g ~ M 0.33 s o 20,w ~ M0.15 R g ~ M 1.0 s o 20,w ~ M R g ~ M k s /[η] ~1.6 k s /[η] <1 k s /[η] ~1.6

60 Conformation Zoning: Pavlov, Harding & Rowe., 1997

61 Conformation Zoning: Extra-rigid Rod: Schizophyllan Rigid-rod: DNA Semi-flexible coil: cellulose derivatives, chitosans Random coil: Dextran, glycoproteins from mucus Globular/Branched: Proteins/glycogen

62 Pectins: Morris et al., 2007

63 Worm-like Chain Flexibility parameter: Persistence length L p Contour Length Kuhn-statistical length λ -1 = 2L p

64 Worm-like Chain Flexibility parameter: Persistence length L p Theoretical limits: Random coil L p = 0 Rigid rod L p = infinity Practical limits: Random coil L p ~ 1-2nm Rigid rod L p ~ 200nm

65 [ ] 2 1/ 2 1/ 3 1/ 0 3 1/ 0 3 1/ 2 2 w L p L w M M L B M A M Φ + Φ = η ( ) = / / p L w p L w A L L M M A A L M M N v M s πη ρ Bohdanecky relation Yamakawa-Fujii relation

66 800 Global plot: xyloglucan 700 M L (g. mol -1. nm -1 ) L p (nm) Patel et al, 2007

67 .or if you know the mass per unit length Target function, Δ L p (nm) Patel et al, 2007

68 Flexibilities of carbohydrate polymers Carbohydrate Polymer Pullulan Xyloglucan L p (nm) Pectins DNA Schizophyllan Scleroglucan Xanthan

69 4. Interactions

70 Atomic Force Microscopy: chitosan Sedimentation coefficient s o 20,w ~ 1S Deacon et al, 2000

71 chitosan-mucin complex Sedimentation coefficient s o 20,w ~ 2000S

72 chitosan-mucin complex Sedimentation coefficient s o 20,w ~ 2000S very strong, irreversible interaction

73 bioactive arabinoxylan very weak, reversible interaction Patel et al, 2007

74 bioactive arabinoxylan very weak, reversible interaction

75 Physical characterisation 1. Viscosity, stability 2. Heterogeneity, Molecular weight & distribution 3. Conformation in solution 4. Interactions

76 Physical characterisation 1. Viscosity, stability 2. Heterogeneity, Molecular weight & distribution 3. Conformation in solution 4. Interactions Thanks to: Prof. Arthur Rowe, Drs. Gordon Morris, Yanling Lu, Trushar Patel (NCMH) & Professor Jose Garcia de la Torre (Murcia)

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