Analysis of molecular

structure of starch

Physicochemical properties/
Chemical composition

Molecular
structure

Genes

Biosynthesis
(enzymes)

Molecular Structure of Amylose

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

B1

B2+B3

Unit chain (mole %)

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-Johnsons 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 & Johnsons 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|

Mobile
Phase

|M|

|L|

Solvent
Delivery
System

Injector
Injection of
debranche
d
amylopecti
n

Response

Recorder

Detectors

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 l
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

MW 1,170,
17.845 min
MW 11,800,
12.639 min

MW 5,900,
13.727 min

Figure 7
High-performance size exclusion
chromatography of maltoheptaose, pullulan6000

Results & Discussion

Pullulan12000
Pullulan6000
Maltoheptaose

Log MW = -0.1867(Retention time; min) + 6.3882

R2 = 0.9906

Figure 8

Standard curve for Zorbax PSM60S ( 2).

Results & Discussion

100000

100000

R e fra c tiv e n d x re s p o n d R e fra c tiv e n d x re s p o n d R e fra c tiv e n d x re s p o n d

Normal rice

10

10

10

12

12

12

14

14

16

16

Retention time (min)

16

10000

1000

1000

100

100

10

10

1
100000
208

18

Normal potato

14

10000

Normal corn
18

18

Waxy rice

10

12

14

16

Waxy corn
18

20

1
100000

10000

10000

1000

1000

100

100

10

10

1
100000
208

10

12

14

16

18

Waxy potato

20

1
100000

10000

10000

1000

1000

100

100

10

10

1
208

1
10

12

14

16

Retention time (min)

Figure 9
High-performance size exclusion
chromatography of isoamylolyzate of

18

20

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

mass(RIresponse)
response) byRelative
the corresponding
Relative
mole
Correspond
ingmolecularweight
molecular weight.

Results & Discussion

Degree of polymerization
150 100 80 60

40 30

20

Degree of polymerization
5

10

150 100 80 60

Normal rice

20

10

Waxy rice

RI detector response
Mole

R e fra c tiv e n d e x r s p o n d R e fra c tiv e n d e x r s p o n d R e fra c tiv e n d e x r s p o n d

M o le ( R I re s p o n s e / M W ) M o le ( R I re s p o n s e / M W )

RI detector response
Mole

40 30

150 100 80 60

10
12
RI detector
response
Mole

150 100 80 60

10
12
RI detector
response
Mole

10

12

40 30

20

14

40 30

10

16

20

150 100 80 60

18
20
8
Normal
corn

10

150 100 80 60

8
18 potato
20
Normal

14

16

14

16

Retention time (min)

10
12
RI detector
response
Mole

18

20
8

10
12
RI detector
response
Mole

10

12

40 30

20

14

40 30

10

16

20

18 corn
20
Waxy

10

14

16

14

16

18 potato
20
Waxy

18

Retention time (min)

Figure 10 High-performance size exclusion

chromatography of isoamylolyzate of

20

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 m) 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 (7.575 mm)
(Tosoh Co., Tokyo, Japan), connected in series
TSK guard column PWH (7.575 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.

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 (8300 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

4
3

Property

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