Starch Analysis
• Morphology
• Chemical compositions
• Physicochemical properties
• Molecular structure
Amylograph
Information on
pasting/gelatinizing behaviors
Instruments;
• Brabender
•
Rapid Visco Analyzer (RVA)
the worldwide standard for measuring the viscosity
of starch and starch containing products as a
function of temperature and time.
The principle
The sample is heated up within a rotating bowl and cooled down again, both
under controlled conditions. Pins in the bowl provide for good mixing and
prevent sedimentation. Use a simple heating - holding - cooling process, or
create your own complex temperature programs for specific needs.
A measuring sensor reaching into the sample is deflected according to the
viscosity of the sample in the bowl. This deflection is measured as torque mechanically against a spring in the Viscograph Pt 100, or electronically
with the Viscograph E.
Standard Procedure
• a water suspension of the tested starch is
heated from 25 C up to 95 C at the uniform rate
of temperature increase of 1.5 C/min and under
constant stirring (75 rpm)
• on attaining 95 C, the sample is maintained at
this temperature for 30 min (first holding
period) while being continuously stirred.
• the paste is then cooled down to 50 C at the
specfied rate and held at this temperature for
another 30 min (second holding period).
Effect of concentration
Effect of pH
THAI-PURPLE
Peak viscosity (RVU)
180
160
140
120
100
80
TP
60
TP-Ac 1.5%
40
TP-Ac 2.0%
20
TP-Ac 2.5%
0
2
4
6
pH
Effect of shear
THAI-PURPLE
Peak viscosity (RVU)
200
180
160
140
120
TP
100
TP-Ac 1.5%
80
TP-Ac 2.0%
60
TP-Ac 2.5%
40
0
160
320
Speed (rpm)
480
640
• Rapid
(high heating/cooling rate
• Small sample (25 ml)
หลักการ
Microprocessor จะควบคุมการจ่ายกระแสไฟฟ้ า
เพื่อขับเคลื่อนให้ใบพัดหมุนผ่านสารละลายแป้ ง
กระแสไฟฟ้ าที่ใช้จะถูกแปรเป็ นหน่วยความหนืด
Introducing the
PYRIS Diamond DSC
The only DSC that gives you the whole story about your
sample
What does a DSC measure?
 DSC measures the amount of energy (heat) absorbed
or released by a sample as it is heated, cooled or
held at constant temperature.
 A DSC precisely measures temperature.
 DSC is used to analyze

Melting

Purity

Crystallization

Specific Heat

Glass Transition

Kinetic Studies

O.I.T. (Oxidative Induction Time)

Curing Reactions

Polymorphism

Denaturation
Types of DSC instruments
 Heat flux DSC:
Measures temperature differential between sample side
and reference side using single, large mass furnace.
Needs mathematical equations to get the heat flow.
 Power compensation DSC:
Directly measures heat flow between sample side and
reference side using two separate, low mass furnaces
Power- Compensation Principle
 An exothermic or endothermic change occurs in the sample
 Power (energy) is applied or removed from the furnace to compensate
for the energy change occurring in the sample.
 The system is maintained in “Thermal Null” state all the times.
 The amount of power required to maintain the system in equilibrium
is directly proportional to the energy changes.
Platinum Alloy
Sample
Reference
PRT Sensor
Platinum
Resistance
Heater
Heat Sink
Power - Compensation DSC
 The power - compensation DSC uses ultra low mass
furnaces (< 1g) which provide the fastest controlled
heating and cooling rates up to 500 C/min
 A heat flux furnace is 30 to 200 times larger and
therefore reacts more slowly to temperature changes
Heat flux furnace
Power compensation furnace
Technical Specification
Wide temperature range
–180 ... 700°C
great variety of applications
Fast linear heating
and cooling rates
high sample throughput
fast response time of the
measuring signal
High reproducibility /
accuracy
stable baselines over the entire
temperature range
precise temperature
precise enthalpy
DSC 204 Phoenix®
-180 … 700°C
DSC204-e/02.01
Technical Specification
of the DSC 204 Phoenix
gas outlet
air cooling
protective gas
reference
sample
heat-flux sensor
furnace block (gold-plated)
heating element
purge gas
LN2/GN2 cooling
circulating cooling
insulation
DSC 204 Phoenix®
-180 … 700°C
DSC204-e/02.01
Technical Specification
Standard crucibles
Al
Pt
DSC 204 Phoenix®
(-180 ... 600°C)
(for the entire temperature
range)
-180 … 700°C
DSC204-e/02.01
Determination of Amylose content in starch
Measurement’s Techniques
Spectrophotometry
Potentiometric/Amperometric Titration
Chromatographic Technique
Chemical complexation
(amylopectin precipitation)
DSC
Lectin concanavalin A interacts with nonreducing terminal -D-glucosyl groups.
Reaction with amylopectin, is not as strong
as with glycogen, and amylose produces no
turbidity, since the single (or few) nonreducing end group per molecule does not
allow multivalent association.
Protein or glycoprotein substances, usually of plant origin, that bind to
sugar moieties in cell walls or membranes and thereby change the
physiology of the membrane to cause agglutination, mitosis, or other
biochemical changes in the cell.
Ref: “Estimation and fractionation of the essentially unbranched (amylose) and branched
(amylopectin) components of starches with Concanavalin A”, Norman K. Matheson and
Lynsey A. Weish., 1987.
“Estimation of amylose content of starches after precipitation of amylopectin by
Concanavalin A”, Yun S. and Norman K. Matheson, Starch/Starke, 1990.
The carbohydrate binding site in Concanavalin A is highlighted in
green. Note how it is formed from surface loop structures
Spectrophotometry
Starch-Iodine-Blue Value Analysis (late 1950's)
Halick, J.V. and Keneaster, K.K. 1956. The use of a starch-iodine-blue
test as a quality indicator of white milled rice. Cereal Chem 33:315-319.
Milled rice is ground into a flour, water is added and the solution is
heated. The solution is then filtered and iodine and hydrochloric
acid solutions are added to the filtrate. A complex then forms
between the iodine and the amylose. The intensity of the resulting
blue color is measured in a spectrophotometer as the iodine-blue
value.
This method is rapid but it does not consistently correlate with more
accurate measures of milled rice amylose content.
Apparent Amylose Content Determination (early 1970's)
Juliano, B.O. 1971. A simplified assay for milled-rice amylose.
Cereal Sci Today 16:334-336, 338, 360.
Milled rice is ground into a flour and then dispersed in water by first treating it with
ethanol and sodium hydroxide. The solution is heated for an hour or allowed to set at
room temperature overnight. The pH is then adjusted using acetic acid and a
solution of iodine is added. The amylose present in the rice forms a complex with the
iodine. The color change (measured using a spectrophotometer) in the solution is
correlated to the amount of the iodine-amylose complex that is formed. Samples
(standards) with known amounts of amylose are also run at the same time. Results
are calculated by comparing the sample's color change to that of the standards.
This method is relatively rapid because protein and lipids do not need to
be removed from the rice prior to using this method. Also, a very small
quantity of sample is required.
 the colored amylose-iodine complex was sensitive to changes of pH
in the alkaline/neutral region.
 Fatty acids derived from fat during starch dispersion reduce the
starch-iodine blue color by competing with iodine in complexing with
amylose.
 The blue color is unstable at higher pH but a greenish blue color is
obtained at low pH.
 Acetic acid has the advantage of buffering action and lower
variation than hydrochloric acid.
 the blue amylose-iodine complex was stable in acidic medium,
however, hydrochloric, sulfuric, nitric acids could not be used,
because they precipitated the amylose-iodine complex.
 Using dilute trichloroacetic acid, no precipitation of the colored
complex occurred, even after long standing at RT. The color was more
stable, and less sensitive to experimental conditions, than that
developed in neutral or alkaline medium.
Chromatographic Technique
Ref: Effect of amylose molecular size and amylopectin branch chain
length on paste properties of starch, Jay-Lin Jane and Jen-Fang Chen,
Cereal Chem., 1992.
Gel preparation:
Soak the gel (Sephacryl S-400 HR/S-500 HR,
Sepharose CL-2B) with water overnight
Decant the water
Wash the gel with DW (2 times)
Size Exclusion Chromatography
Figure 2 Illustrative description of separation of size exclusion
chromatography (SEC).
Experimental Procedure
Amylose content determination by SEC
Starch
Nongranular starch
Size Exclusion Chromatography
Total carbohydrate (Phenol-H2SO4, Dubois et al., 1956)
& blue value (I2 binding)
Experimental Procedure
Amylose content determination by SEC
 Packing bed: Sepharose CL-2B
 MW range: 105 – 2  107 (dextrans)
 Column dimension: 2 cm ID  90 cm
 Loading size: 2 ml (contained starch 15 mg)
 Eluent: 10 mM NaOH + 50 mM NaCl + 0.02%
NaN3
 Flow rate: 30-40 ml/hr
 Flow direction: descending mode
 Volume/fraction: 2.25 ml
Results & Discussion
Base line
Figure 4 Sepharose CL-2B Chromatograms of ICI maize starches
developed at different temperature (Lu et al., 1996).
Results & Discussion
Amylopectin
Glucose concentration
Relative blue value
350
300
Area of amylose
 100
Total area
 31.49%
Amylose (%) 
250
1.2
1.0
0.8
0.6
200
150
0.4
100
Amylose
50
Relative blue value
Glucose concentration ( mg/ml)
400
0.2
0
0.0
0
10
20
30
40
50
60
70
80
90
100
Fraction
Figure 3 Size exclusion chromatography of nongranular normal
rice starch.