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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
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