Fractionation of Starch

References:1. Chapter 14, “Commercial separation of amylose and amylopectin

from starch”, Starch Production Technology, edited by J.A. Radley.

2. Chapter 8, “Fractionation of starch”, Austin H. Young, Starch: Chemistry and Technology, edited by R.Y Whistler.

Amylose VS Amylopectin

• Molecular size

• Molecular arrangement

• Functional groups

Methods of Fractionation

Aqueous leaching of gelatinized granules

Dispersion of the granule and fractionation

with complexing agents (selective precipitation)

Fractionation by retrogradation

Fractional precipitation (salting out)

Raw Materials

• Cereal starches have a great disadvantage as a raw materials because they have a high content of fatty compounds.

• Potato starch is very suitable for fractionation process, for it contains no fatty substances. The phosphate groups, bound only to the amylopectin molecules, increase the difference between both starch components.

• Waxy starches contain practically no amylose, so that it is not necessary to fractionate them.

• Amylomaize can be subjected to fractionation.

Aqueous leaching of gelatinized granules

Leaching occurs from swelling starch granules in water at temperatures of 57 – 100 °C. Mobile amylose molecules diffuse out of the swallen granule while the granule is intact.

Most of the amylopectin remains H-bonded or crystallized in the granule residue.

25 g starch + 25 ml cold water

Add slowly with stirring 150 ml of water at 80 °C

Maintain at 70 °C for 5 mins

Pour into 1200 ml of water at 60 °C

Stir very slowly at this temperature for 4 h

(avoid rupture of the swollen granule)

Cool, centrifuge

Supernatant amylose

Gelatineous deposit amylopectin

Ref:

Supernatant Gelatineous deposit

Add methanol into supernatant

(to get 20% alcohol solution)

precipitate

centrifuge

Grind precipitates in mortar with 95% ethanol

Filter and dry in vacuum oven

Amylose

Grind in mortar with

95% ethanol

Filter and dry

in vacuum oven

Amylopectin

defatting starch geanules by heating in aqueous methanol enhances leaching of the amylose. (useful for starch which strongly resists swelling and gelatinization.

removal of oxygen from the water reduced degradation of starch.

low temperature pretreatment of granules induces further crystallization, resulting in higher yield of extraction without granule rupture.

(for example; froze at -78 °C for 1 min)

Dispersion of the granule and fractionationwith complexing agents (selective

precipitation)

• Defattation

• Dispersion

• Inclusion complex formation

• Separation of complexRef: Schoch T.J., 1941

Defatting procedureReflux the starches for several hours with 85% methanol. After five extractions the lipids are sufficiently removed.

Dispersion

Disperse 1-3% of starch in water at 105 – 109 °C for 2-3 h

Dispersion by chemicals

1 N NaOH/KOH at 25 °C

Dimethyl solfoxide at 25 °C

DMSO is a powerful H-bond acceptor, thereby breaking association H-bond in polysaccharide and in water.

CH3

Starch-OH ----- -O – S+

CH3

Inclusion complex formation

“precipitating the amylose as a complex with complexing agents (organic compounds)”

Starch + Complexing agent Complex

K = [complex]

[starch] [complexing agent]

K = complex formation constant

Kam >> Kamp

Aliphatic Alcohols; isopropyl, n-butyl, isoamyl, methyl

Lower aliphatic ketone

Lower aliphatic fatty acids

Benzenoid derivatives having aldehyde groups

Alkyl halides

Cyclic alcohols

Phenol

Esters

Complexing agents

FRACTIONATION OF AMYLOSE AND AMYLOPECTIN

STARCH+DMSO

BOILED, STIRRING ( UNDER N2)

MIXTURE STORED 00 C

CENTRIFUGATION

REDISSLOVED IN DMSO

PRECIPITATE DISPERSE IN WATER

REFLUX 3 hr.

CENTRIFUGATION

PRECIPITATE SUSPEND IN 10% 1-butanol

REFLUX 1h.

ADD ETOH

ADD 1-butanol3-methyl-1-butanol

water

Cool, keep overnight at RT.And 8 0C 24 hr.

Supernatant 1 ( AP)

Cool, keep overnight at RT.And 8 0C 24 hr.

CENTRIFUGE

PRECIPITATE DISPERSE IN WATER

ULTRACENTRIFUGATION

SUPERNATANT + WTER +1-butanol

Reflux 1h.

Cool, keep overnight at RT. and 8 0C 24 hr.

CENTRIFUGE

PRECIPITATE DISPERSE IN 10% 1-butanol 1 liter

REFLUX 10 min.

FILTERED THROUGH GLASS FILTER G-5

REFLUX 10 min.

Cool, keep overnight at RT. and 8 0C 24 hr.

*1RECRYTALLISATION*

*2 RECRYTALLISATION**3

REC

RYTA

LLIS

ATIO

*

CHECK PURITY OF AMYLOSEGPC TOYOPEARL HW-75 F1 PEAK

CENTRIFUGE

GROUND WITH ETOH

WASH WITH Diethyl Ether

DRY IN VACUUM AT RT. OVER CaCl2

AMYLOSE

SUPERNATANT 2 (AP )

Fractionation by retrogradation

A 7-9% slurry of potato starch is gelatinized at 85 °C and homogenized in a blender. Sufficient energy is given the system to cause starch dissolution.

The mixture is then centrifuged and cooled so that the amylose separates in the form of globules from a liquid containing the amylopectin.

Fractional precipitation (salting out)

“industrially acceptable method”A commercial process salted out the amylose from

potato starch with magnesium sulfate.

This method was based upon the fractional crystallization of amylose from a 10% by weight aqueous solution of potato starch in the presence of 10-13% magnesium sulfate.

In 1960, the Avebe Company in Holland produced 5.4 ton/day of amylose and 15.4 tons/day of amylopectin.

10% starch in 10-13% MgSO4 solution is solubilized

by heating to 160 °C

Cool down the solution (a phase separation

occurs )

The amylose separates in the form of small droplets and the amylopectin remains in the solution

By cooling down to lower temperatures the droplets of amylose solution retrograde rapidly, forming small particles of

amylose gel.

Amylose gel is separated by centrifugation at 1000 g for 5 min

Wash out the salt by cold water

AMYLOSE

Amylopectin solution

Treat with more MgSO4 to precipitate all starch

(including amylose remained)

Ripen for 8 h at RT, the flocculent precipitate become insoluble

Wash the precipitate with water

AMYLOPECTIN

Amylopectin fraction is less pure than the amylose fraction