Starch AnalysisStarch 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 sedi

- mentation. Use a simple heating holding- cooling process, or create your own com

plex temperature programs for specific n eeds.

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 mecha

nically against a spring in the Viscograph Pt 100, or electronically with the Viscogr

aph 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

0

20

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180

2 4 6pH

Pea

k vi

scos

ity (R

VU

)

TP

TP-Ac 1.5%

TP-Ac 2.0%

TP-Ac 2.5%

THAI-PURPLE

40

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0 160 320 480 640

Speed (rpm)

Peak

vis

cosi

ty (R

VU)

TP

TP-Ac 1.5%

TP-Ac 2.0%

TP-Ac 2.5%

Effect of shear

• Rapid (high heating/cooling rate

• Small sample (25 ml)

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Introducing the Introducing the PYRIS Diamond DSCPYRIS Diamond DSC The only DSC that gives you the whole story about your The only DSC that gives you the whole story about your samplesample

Introducing the Introducing the PYRIS Diamond DSCPYRIS Diamond DSC The only DSC that gives you the whole story about your The only DSC that gives you the whole story about your samplesample

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

What does a DSC measure?

Melting

Crystallization

Glass Transition

O.I.T. (Oxidative Induction Time)

Polymorphism

Purity

Specific Heat

Kinetic Studies

Curing Reactions

Denaturation

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

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.

Power- Compensation Principle

Sample ReferencePlatinum Alloy

PRT Sensor

Platinum

Resistance Heater

Heat Sink

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

Power - Compensation DSC

Heat flux furnace

Power compensation furnace

DSC 204 Phoenix® -180 … 700°C

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

DSC204-e/02.01

DSC 204 Phoenix® -180 … 700°C

Technical Specificationof the DSC 204 Phoenix

gas outlet

air cooling

protective gasreferencesampleheat-flux sensorfurnace block (gold-plated)heating elementpurge gasLN2/GN2 coolingcirculating cooling

insulation

DSC204-e/02.01

DSC 204 Phoenix® -180 … 700°C

Technical Specification

Standard crucibles

Al (-180 ... 600°C)Pt (for the entire temperature

range)

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 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.

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.

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.

The carbohydrate binding site in Concanavalin A is highlighted in green. Note how it is formed from surface loop structures

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.

SpectrophotometryStarch-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.

This method is rapid but it does not consistently correlate with more accurate measures of milled rice amylose content.

  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.

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.

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 ProcedureAmylose content determination by

SEC

Nongranular starch

Starch

Size Exclusion Chromatography

Total carbohydrate (Phenol-H2SO4, Dubois et al., 1956)

& blue value (I2 binding)

Experimental ProcedureAmylose 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

Figure 4 Sepharose CL-2B Chromatograms of ICI maize starches developed at different temperature (Lu et al., 1996).

Base line

Results & Discussion

Figure 3 Size exclusion chromatography of nongranular normal rice starch.

0

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Fraction

Glu

cose

concentratio

n (m g

/ml)

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1.0

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Rela

tive b

lue v

alu

e

Glucose concentrationRelative blue value

Amylopectin

Amylose

31.49%

100area Totalamylose of Area

(%) Amylose