N Wellner

Institute of Food Research

Norwich, UK

Vibrational spectroscopy study of starch

and cell wall polysaccharide structures

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

Interpreting the spectra

OH stretch normal mode

Structure modelled from Popov et al. Macromolecules 2009, 42, 1167-1174.

-0.02

0.03

0.08

0.13

0.18

60010001400180022002600300034003800

Ab

so

rba

nc

e

Wavenumber (cm-1)

Interpreting the spectra 940 cm-1 band

Structure modelled from Popov et al. Macromolecules 2009, 42, 1167-1174.

-0.02

0.03

0.08

0.13

0.18

60010001400180022002600300034003800

Ab

so

rba

nc

e

Wavenumber (cm-1)

Structure and spectrum Methyl-a-D-

glucopyranoside

Methyl-a-D-

mannopyranoside

0.00

0.01

0.02

0.03

0.04

0.05

0.06

8001,0001,2001,4001,6001,800

Ab

sorb

ance

Wavenumber (cm-1)

CH2OH

OOH OH

OH OCH3

CH2OH

OOH

OHOH OCH3

Mixtures – spectra of plant materials

0.00

0.10

0.20

0.30

0.40

0.50

0.60

800900100011001200130014001500160017001800

Ab

sorb

anc

e

Wavenumber (cm-1)

straw

maize kernel

carrot

lignin

cellulose + hemicellulose

protein

starch

water CWM + starch

Carbohydrate metabolism in

arabidopsis

sucrose

invertase sucrose synthase

glucose + fructose UDPglucose + fructose

cellulose

cellulose synthase

FT-IR imaging: spatially resolved

chemical composition

FTIR microscope

Focal plane array detector

with 128 x 128 elements

Each 5 x 5 mm2 element

records a spectrum

Spectra from regions or

Images obtained by

colouring pixels according

to spectra information X

Y

Waven

um

ber

(cm

- 1)

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 polarisabilitytensor

Directional: strongest when parallel to electric vector of the incident light

(Source: Wikipedia)

Molecular orientation in HPC films

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0% 10% 20% 30% 40% 50% 60%

Strain

De

lta

Ab

so

rba

nc

e63 %rh

45 %rh

36 %rh

Hydroxypropyl

cellulose

Parallel/ perpendicular

in stretched film

1040

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

FTIR microscopy of maize starch

Maize WT and ae mutant

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

90095010001050110011501200

Wavenumbers (cm-1

)

Ab

so

rb

an

ce

wt

ae

- 1

15

3

- 1

12

6

- 1

10

5

- 1

17

8

- 1

05

0-

10

44

- 1

02

3

- 1

00

2

- 9

33

- 9

24

- 9

70

Iodine-stained micrograph and Cross-polarised micrographFTIR 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

10 mm10 mm

wt ae

Maize starch granules: Raman

microscopy image - branching

10 mm10 mm

wt ae

Maize starch granules: Raman

microscopy image 860 cm-1

10 mm10 mm

wt ae

map 153 carotene

50 100 150 200 250 300 350 400 450 500

0

20

40

60

map 153 starch ratio

50 100 150 200 250 300 350 400 450 500

0

20

40

60

map 153 combined

50 100 150 200 250 300 350 400 450 500

0

20

40

60

20040060080010001200140016001800200

400

600

800

Raman microscopy of WT maize starch

granules

protein

860 cm-1

combined

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 JIC

Mary Parker

Yuri Alexeev

Vic Morris

Reg Wilson

Paul Derbyshire

Alison Smith

And you!