Analysis of molecular
structure of starch
Physicochemical properties/
Chemical composition
Molecular
structure
Genes
Biosynthesis
(enzymes)
Molecular Structure of Amylose
Property
Thai green
canna
Thai purple
canna
Mung
bean
1650
1590
2200
1550
1440
2110
Average chain length, CL
471
420
350
Average number of chain,NC
3.5
3.8
6.3
Linear amylose (mole %)
84
84
84
Branched amylose (mole% )
16
16
16
NC of branched amylose
17
17
34
Number average DP, DPn
Colorimetric method
Labeling method
Molecular Structure of Amylopectin
Property
Thai green
canna
Thai purple
canna
Mung
bean
Isoamylolysis
26
28
24
Labeling method
25
27
23
Average chain length, CL
Average chain-length and amount (mole %)
of the fractions of amylopectin unit chain
Source
Average chain - length
A
B1
B2+B3
Unit chain (mole %)
A
B1
B2+B3
Thai green canna
14
52
84.3
15.7
Thai purple canna
15
53
84.0
16.0
10 21
48
59.1 31.4
9.5
Mung bean
Molecular characterization
Number average degree of
polymerization (molecular size, DP)
Average chain length (CL)
Amylose
Average number of chain (NC)
Linear amylose fraction (mole%)
Branched amylose fraction (mole%)
Molecular characterization
Average (branch) chain length, overall
Average (branch) chain length, of A-chain
Amylopectin
Average (branch) chain length, of B1-chain
Average (branch) chain length, of B2-chain
Unit chain (A, B1, B2, B3,..) fraction (mole%)
Methods of Analysis
 Colorimetric methods
 chemical reaction
 chemical reaction + enzyme reaction
 Chromatographic Techniques
 without enzyme reaction
 with enzyme reaction
Low-angle laser-light-scattering photometer
Detector
Refractive index detector
Pulsed amperometric detector
Fluorescence detector
Colorimetric methods (chemical reaction)
Determine: Total sugar/ Reducing end/Non-reducing end
Average degree of polymerization =
total sugar
(molecular size, DP)
reducing end sugar
Average chain length (CL)
=
total sugar
non-reducing end sugar
Average number of chain (NC)
=
DP/CL
Total sugar:
• Anthrone-H2SO4
• Phenol-H2SO4
Reducing end sugar
Modified Park-Johnson’s method
Ref; 1. J. Park and M.J. Johnson, J. Biol. Chem., 181 (1949), 149-151.
2. S. Hizukuri, Y.Takeda, M. Yasuda, Carbohydrate Research, 94 (1981), 205-213.
Non-reducing end sugar
Rapid Smith Degradation method
Ref; 1. J.K. Hamilton and F. Smith, J. Am. Chem. Soc., 78 (1956), 5907-5909.
2. S. Hizukuri and S. Osaki, Carbohydrate Research, 63 (1978), 261-264.
Colorimetric methods
(chemical reaction + enzyme reaction)
Isoamylase/pullulanase
Branch chain length (CL)
=
total sugar
non-reducing end sugar
Hydrolyze -1,6 by isoamylase/pullulanase
Determine reducing end sugar by Modified Park & Johnson’s method
Experimental Procedure
Chromatographic Techniques with Enzyme Reaction
Amylopectin structure studied by HPSEC
Starch
Fractionation
(selective precipitation)
Amylose
Molecular analyses
(HPSEC)
Amylopectin
Debranched
Chromatographic Techniques with Enzyme Reaction
A
B
B
A
C
A
B
A
Isoamylase or pullulanase.
Chromatographic Techniques with Enzyme Reaction
Column
|S| |M| |L|
Mobile
Phase
Detectors
Solvent
Delivery
System
Injector
Injection of
debranched
amylopectin
Response
Recorder
L
M
S
Retention time
Chart record
Figure 6 Block diagram showing the component of an HPSEC
instrument.
Experimental Procedure
Amylopectin structure studied by HPSEC









Column: Zorbax PSM 60S ( 2)
MW range: 5  102 – 104
Column dimension: 6.2 mm ID  250 mm
Loading size: 40 μl
Eluent: 90% DMSO
Flow rate: 0.5 ml/min
Pressure: <3,000 psi
Column temperature: 50oC
Standard: maltoheptaose, pullulan6000 and
pullulan12000 (MW 1,170, 5,900,
11,800, respectively)
Results & Discussion
Refractive index respond
MW 1,170,
17.845 min
Pullulan12000
Pullulan6000
Maltoheptaose
MW 11,800,
12.639 min
MW 5,900,
13.727 min
6
8
10
12
14
16
18
20
22
24
Retention time (min)
Figure 7 High-performance size exclusion chromatography of
maltoheptaose, pullulan6000 and pullulan12000.
Results & Discussion
5.0
4.5
Pullulan12000
Log MW
4.0
Pullulan6000
3.5
Maltoheptaose
3.0
Log MW = -0.1867(Retention time; min) + 6.3882
R2 = 0.9906
2.5
2.0
8
10
12
14
16
18
Time (min)
Figure 8 Standard curve for Zorbax PSM60S ( 2).
20
22
Results & Discussion
12
14
16
Retention time (min)
1100000
18
Normal
corn 208
Refractive index respond
Refractive index respond
10000
8
10
12
14
Retention time (min)
Normal potato
16
18
1000
100
10
Refractive index respond
Refractive index respond
10
12
14
Retention time (min)
16
18
10
12
14
16
Retention time (min)
1
100000
208
1000
100
10
1
208
18
Waxy
corn 20
1
100000
10000
1000
100
10
10
12
14
16
Retention time (min)
10000
8
10
Waxy potato
18
20
Molecular weight (dalton)
10
100
Molecular weight (dalton)
10
100
1000
1
100000
10000
Molecular weight (dalton)
8
1000
10000
Molecular weight (dalton)
Refractive index respond
Refractive index respond
10000
100000
Waxy rice
1000
100
10
Molecular weight (dalton)
100000
Molecular weight (dalton)
Normal rice
1
10
12
14
16
18
20
Retention time (min)
Figure 9 High-performance size exclusion chromatography of
isoamylolyzate of amylopectin from starches.
Results & Discussion
Yuan et al. (1993)
 Refractive index response is proportional to
the mass of the eluted material.
 The relative mole was derived by dividing
the relative mass (RI response) by the
corresponding molecular weight.
Relative mass (RI response)
Relative mole 
Correspond ing molecular weight
Results & Discussion
Degree of polymerization
Degree of polymerization
40 30
20
5
10
10
12
14
16 Normal
18
RI detector
response
Mole
Retention time (min)
corn208
Degree of polymerization
150
8
8
100 80 60
40 30
20
5
10
Mole (RI response/MW)
8
100 80 60
Mole (RI response/MW)
Normal rice
RI detector response
Mole
150
150
10
12
14
16
Normal18potato208
RI detector
response
Mole
Retention time (min)
10
12
14
16
Retention time (min)
18
20
8
100 80 60
40 30
20
5
10
Waxy rice
RI detector response
Mole
Mole (RI response/MW)
5
10
Degree of polymerization
150
100 80 60
40 30
20
5
10
10
12
14
16
RI detector
response
Mole
Retention time (min)
18 corn 20
Waxy
Degree of polymerization
150
100 80 60
40 30
20
5
10
Mole (RI response/MW)
20
Mole (RI response/MW)
40 30
Refractive index respond Refractive index respond Refractive index respond
Refractive index respond Refractive index respond Refractive index respond
100 80 60
10
12
14
16 Waxy18potato20
RI detector
response
Mole
Retention time (min)
10
12
14
16
18
Mole (RI response/MW)
Degree of polymerization
150
20
Retention time (min)
Figure 10 High-performance size exclusion chromatography of
isoamylolyzate of amylopectin from starches.
High performance anion-exchange chromatography
with pulsed amperometric detection (HPAEC-PAD)
System: Model 4000i Dionex BioLC system
Column: Dionex HPIC-AS6 (now called CarboPac PA-1) 250  4 mm
(10 µm) with AG6 guard column (50  4 mm)
Detector: Model 2 PAD system
Individual members of the
components can be obtained
Ref: 1. Koizumi K. and Fukuda M., Estimation of the distributions of chain length of
amylopectins by HPAEC-PAD, J. of Chromatography, 585 (1991), 233-238.
2. Hanashiro, I., Abe, J., & Hizukuri, S. (1996). A periodic distribution of the
chain length of amylopectin as revealed by high-performance anion-exchange
chromatography. Carbohydrate Research, 283, 151-159.
Cannot determine the individual glucans directly by use of their
peak areas in the chromatogram, as the responses of a pulsed
amperometric detector to glucans having different DPs
were different.
High performance size-exclusion chromatography
(HPSEC) with fluorescence detector
System: HPLC
Column: For amylose
TSK gel G6000PW, G4000PW and G3000PW (7.575 mm)
(Tosoh Co., Tokyo, Japan), connected in series
TSK guard column PWH (7.575 mm)
Temp. 37 C, Eluent: 0.1 M phosphate buffer (pH 6.1)
containing 0.02% sodium azide
Detector: Fluorescence Detector
Refractive index detector
Fluorescent reagent: 2-aminopyridine (aromatic primary amine)
Std. amylose: AS-110 (DP 521), AS-320 (2320), AS-1000 (4400)
Ref: Hanashiro, I., & Takeda, Y. (1998). Examination of number-average degree
of polymerization and molar-based distribution of amylose by fluorescent
labeling with 2-aminopyridine. Carbohydrate Research, 306, 421-426.
Fluorescent labeling of amylose with 2aminopyridine
DPn
=
RI response (RI)
fluorescence response (F)
Chromatograms of Fluorescence-labeled Amyloses
DP
Fluorescence
RI
DPsample = (RI/F)sample
(RI/F)std.
x DPstd.
Column for amylopectin (unit chain)
Sample: Isoamylolyzate
Column: Shodex OHpak SB-803HQ and
SB-802.5HQ x 2 (8300 mm)
Eluent: Aq. Me2SO (50%) containing 50 mM NaCl
Column Temperature: 50 C
Std. amylose: G6, AS-10 (52), AS-30 (141), AS-70 (440)
Beta-Amylolysis of Amylose Molecule
Branch molecule
Linear molecule
Reducing end
Glucose
alpha-1,4
alpha-1,6
-amylolysis
Swelling of starch granule
Increase viscosity of starch paste
Action of amylase on starch
6
5
Phosphorus
1
4
3
Property
2
Thai green
canna
Thai purple
canna
Mung
bean
Total organic P (ppm)
397
420
100
Link to C-6 (ppm)
275
299
15
Phosphorus at C-6 (%)
69
71
15
Phosphorus (P)
Sugar Phosphate