Vibrational spectroscopy study of starch and cell wall polysaccharide structures N Wellner Institute of Food Research Norwich, UK
Outline IR spectroscopy, normal modes and molecular structure Composition of plant tissue Functional imaging of film fracture Investigation of starch granules
IR spectra of polysaccharides Wheat starch Microcrystalline cellulose
IR spectra of polysaccharides
IR/Raman State of molecules described by Schrödinger Equation HY = EY Born-Oppenheimer approximation separates terms for electrons and nuclei, and also allows separation of the wave function into distinct components Oscillator potential (symmetric/asymmetric) and energy levels Interaction with light <y * m y > FTIR Absorption depends on dipole moment change Raman scattering depends on polarisability tensor (Source: Wikipedia)
IR spectra of polysaccharides Wheat starch Microcrystalline cellulose
Absorbance Interpreting the spectra OH stretch normal mode Structure modelled from Popov et al. Macromolecules 2009, 42, 1167-1174. 0.18 0.13 0.08 0.03-0.02 3800 3400 3000 2600 2200 1800 Wavenumber (cm -1 ) 1400 1000 600
Absorbance Interpreting the spectra 940 cm -1 band Structure modelled from Popov et al. Macromolecules 2009, 42, 1167-1174. 0.18 0.13 0.08 0.03-0.02 3800 3400 3000 2600 2200 1800 Wavenumber (cm -1 ) 1400 1000 600
Absorbance Structure and spectrum Methyl-a-Dglucopyranoside CH 2 OH O OH OH OCH 3 OH 0.06 0.05 0.04 0.03 Methyl-a-Dmannopyranoside 0.02 0.01 CH 2 OH O OH OH OH OCH 3 0.00 1,800 1,600 1,400 1,200 Wavenumber (cm -1 ) 1,000 800
Absorbance Mixtures spectra of plant materials 0.60 cellulose + hemicellulose 0.50 0.40 lignin straw 0.30 protein starch maize kernel 0.20 0.10 0.00 water CWM + starch carrot 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 Wavenumber (cm -1 )
Carbohydrate metabolism in arabidopsis sucrose invertase glucose + fructose sucrose synthase UDPglucose + fructose cellulose synthase cellulose
Wavenumber ( cm -1 ) FT-IR imaging: spatially resolved chemical composition FTIR microscope Focal plane array detector with 128 x 128 elements Each 5 x 5 mm 2 element records a spectrum Spectra from regions or Images obtained by colouring pixels according to spectra information Y X
Changes in cell wall metabolism - sucrose synthase mutants Barratt et al., PNAS 2009, 106/31, 13124 13129
FTIR/Raman State of molecules described by Schrödinger Equation HY = EY Born-Oppenheimer approximation separates terms for electrons and nuclei, and also allows separation of the wave function into distinct components Oscillator potential (symmetric/asymmetric) and energy levels Interaction with light <y * m y > FTIR Absorption depends on dipole moment change Raman scattering depends on polarisability tensor Directional: strongest when parallel to electric vector of the incident light (Source: Wikipedia)
Delta Absorbance Molecular orientation in HPC films 0.070 0.060 0.050 0.040 0.030 1040 63 %rh 45 %rh 36 %rh Hydroxypropyl cellulose 0.020 0.010 0.000 0% 10% 20% 30% 40% 50% 60% Strain Parallel/ perpendicular in stretched film
Fracture in HPC film > 0.35 0.30-0.35 0.25-0.30 0.20-0.25 0.15-0.20 0.10-0.15 0.05-0.10 < 0.05 0.1 mm
Fracture in HPC film > 0.35 0.30-0.35 0.25-0.30 0.20-0.25 0.15-0.20 0.10-0.15 0.05-0.10 < 0.05 0.1 mm
Fracture in HPC film > 0.35 0.30-0.35 0.25-0.30 0.20-0.25 0.15-0.20 0.10-0.15 0.05-0.10 < 0.05 0.1 mm
Fracture in HPC film > 0.35 0.30-0.35 0.25-0.30 0.20-0.25 0.15-0.20 0.10-0.15 0.05-0.10 < 0.05 0.1 mm
Fracture in HPC film > 0.35 0.30-0.35 0.25-0.30 0.20-0.25 0.15-0.20 0.10-0.15 0.05-0.10 < 0.05 0.1 mm
Fracture in HPC film > 0.35 0.30-0.35 0.25-0.30 0.20-0.25 0.15-0.20 0.10-0.15 0.05-0.10 < 0.05 0.1 mm
Fracture in HPC film > 0.35 0.30-0.35 0.25-0.30 0.20-0.25 0.15-0.20 0.10-0.15 0.05-0.10 < 0.05 0.1 mm
- 970-933 - 924 Absorbance - 1126-1105 - 1153-1178 - 1050-1044 - 1002-1023 FTIR microscopy of maize starch Maize WT and ae mutant 0.9 0.8 wt 0.7 ae 0.6 0.5 0.4 0.3 0.2 0.1 0-0.1 1200 1150 1100 1050 1000 950 900 Wavenumbers (cm -1 ) Iodine-stained micrograph and Cross-polarised micrograph FTIR map of starch structure in maize sections monitored by the height of the 1002 cm -1 shoulder relative to the 1022 cm -1 peak maximum. Top: Wild type sections, Bottom: ae mutant maize 0.1 mm
Maize starch granules: Raman microscopy image - crystallinity wt ae 10 mm 10 mm
Maize starch granules: Raman microscopy image - branching wt ae 10 mm 10 mm
Maize starch granules: Raman microscopy image 860 cm -1 wt ae 10 mm 10 mm
Raman microscopy of WT maize starch granules protein map 153 carotene 0 20 40 60 860 cm-1 combined 0 20 40 60 0 20 40 60 50 100 150 200 250 300 350 400 450 500 map 153 starch ratio 50 100 150 200 250 300 350 400 450 500 map 153 combined 50 100 150 200 250 300 350 400 450 500 800 600 400 200 1800 1600 1400 1200 1000 800 600 400 200
Orientation in ae maize starch granules 20 40 60 80 100 120 20 40 60 80 100 120 10 mm
Spectroscopy of polysaccharides Composition Orientation Interactions Location Composition of mutant Arabidopsis tissue Functional imaging of HPC film fracture IR and Raman investigation of starch granules in maize kernels
Thanks to IFR Mary Parker Yuri Alexeev Vic Morris Reg Wilson JIC Paul Derbyshire Alison Smith And you!