Starch Analysis

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Starch Analysis

• Morphology
• Chemical compositions
• Physicochemical properties
• Molecular structure

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Amylograph
Information on pasting/gelatinizing behaviors

• Instruments
• Brabender
• Rapid Visco Analyzer (RVA)

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the worldwide standard for measuring the
viscosity of starch and starch containing
products as a function of temperature and time.
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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.
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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.
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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).

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Effect of concentration
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Effect of pH
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Effect of shear
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• Rapid (high heating/cooling rate
• Small sample (25 ml)

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Introducing the PYRIS Diamond DSC The only DSC
that gives you the whole story about your sample
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What does a DSC measure?

• DSC measures the amount of energy (heat)
  absorbedor released by a sample as it is heated,
  cooled orheld at constant temperature.
• A DSC precisely measures temperature.
• DSC is used to analyze

• Melting
• Crystallization
• Glass Transition
• O.I.T. (Oxidative Induction Time)
• Polymorphism

• Purity
• Specific Heat
• Kinetic Studies
• Curing Reactions
• Denaturation

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Types of DSC instruments

• Heat flux DSC Measures temperature differential
  between sample sideand 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 andreference side using
  two separate, low mass furnaces

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Power- Compensation Principle

• An exothermic or endothermic change occurs in the
  sample
• Power (energy) is applied or removed from the
  furnace to compensatefor 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 equilibriumis directly proportional to
  the energy changes.

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Power - Compensation DSC

• The power - compensation DSC uses ultra low mass
  furnaces (lt 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
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Technical Specification
Wide temperature range 180 ... 700C 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
DSC204-e/02.01
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Technical Specificationof 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
DSC204-e/02.01
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Technical Specification
Standard crucibles Al (-180 ... 600C) Pt (for
the entire temperature range)
DSC204-e/02.01
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Determination of Amylose content in starch
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Measurements Techniques

• Spectrophotometry
• Potentiometric/Amperometric Titration
• Chromatographic Technique
• Chemical complexation
• (amylopectin precipitation)
• DSC

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Lectin concanavalin A interacts with non-reducing
terminal ?-D-glucosyl groups. Reaction with
amylopectin, is not as strong as with glycogen,
and amylose produces no turbidity, since the
single (or few) non-reducing 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.
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The carbohydrate binding site in Concanavalin A
is highlighted in green. Note how it is formed
from surface loop structures
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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
33315-319.
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.
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Apparent Amylose Content Determination (early
1970's) Juliano, B.O. 1971. A simplified assay
for milled-rice amylose. Cereal Sci Today
16334-336, 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.
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• 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.

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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)
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Size Exclusion Chromatography
Figure 2 Illustrative description of separation
of size exclusion chromatography (SEC).
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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)
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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

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Results Discussion
Figure 4 Sepharose CL-2B Chromatograms of ICI
maize starches developed at different temperature
(Lu et al., 1996).
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Results Discussion
Amylopectin
Amylose
Figure 3 Size exclusion chromatography of
nongranular normal rice starch.