Crystallization Bioseparation Engineering. Crystallization crystal formation 1. Supersaturation...
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Transcript of Crystallization Bioseparation Engineering. Crystallization crystal formation 1. Supersaturation...
Crystallization
Bioseparation Engineering
Crystallizationcrystal formation
1. Supersaturation
Figure 1. Regions of supersaturation.
Supersaturated solution: thermodynamically unstable
Metastable region:solute will deposit on existing crystals but no new crystal nuclei formed
Intermediate zone:both growth of the existing crystals and the formation of new nuclei occur simultaneously
Labile zone:nuclei are formed
2. Purityis important
3. Nucleationcrystal growth; genesis of new crystal
Homogeneous nucleation: result of supersaturation
Heterogeneous nucleation: insoluble material initiates crystal growth
Secondary nucleation: induced by the contact between different crystals
Nucleation Rate
B
cckdt
dN in
* where: c – concentration in solutionc* – concentration at saturationkn and i – empirical parameters
crystal formation equation
cluster embryo nucleus crystal
[1]
4. Single Crystal Growthin nonagitated system, diffusion limited
*cckAdt
dM [2]
where: A – crystal areak – mass transfer coefficient
Agitation increase the relative velocity betweenthe solution and the crystal
*11
cc
k
A
dt
dM
κ – surface reaction rate (affected by cooling)k – mass transfer coefficient (affected by viscosity and agitation)A – crystal areaM – crystal mass
where:
[3]
Characteristic crystal length
(assume cubic crystal)A
Ml
6
For cubic crystal of side, s
3sM 26sA sl
For spherical particle, l is the sphere diameter
Define:3lM v
26 lA A
φi - geometric factors characteristic of the crystal shape
Gdt
dl
cckcc
kdt
dl
cc
k
ll
dt
d
gv
A
Av
**11
2
*11
6 23
crystal growth equation
Batch Crystallization
Figure 2. Stirred tank batch crystallizers.
• solution is cooled to produce supersaturation• seeding crystal can be added• cooling rate can be controlled
(cooling curve) – key idea
How to obtain the cooling curve?Assume: a single crystal size
change insupersaturation
change fromaltered temperature
change fromcrystal growth
change fromnucleation= + +
Batch crystallization happens at metastable zone,thus – change in supersaturation is small
– nucleation occurs by seeding
*11
*0 cc
k
A
dt
dcV
A – total crystal areac* – saturation concentration = f(temp.)
where:
dt
dT
dT
dc
dt
dc **
2
36 Gtl
l
M
crystal
areacrystalsofnumberA
sAsv
S
MS – total mass of the seed crystallS – initial size of the seed crystal
where:
2
3
3*
Gtll
G
dTdc
VM
dt
dTs
s
S
Integrating
2
0 3
11
3*
sss
S
l
Gt
l
Gt
l
Gt
dTdc
VM
TT
or in the form of
TP – final temperatureT0 – temperature at which crystal begins to formMP = [(T0 – TP)V dc*/dt]
Maximum mass of crystalline product minus that in the seedη = (lp – lS) /lS
Fractional increase in product size per seed sizeτ – actual time divided by the total time
where:
2
0
0
3
113
p
S
P M
M
TT
TT
Batch Scale-up
Important factor in large scale batch type,secondary nucleation
Scaling up needs experiences and secondary nucleation should be considered
Example 3.12
Briefly analyze the large-scale purification and crystallization of lipase from Geotricbum candidum.
2011.11.14 심세나
Introduction
Lipase from Geotrichum candidum
Two step isolation
Isoelectric focusing
Specificity for oleic acid
Crystallization
X- ray
Method: Purification of lipase
• Chromatographic two-step purification
1) Q-Sepharose FF column (4 X 30 cm) - Anion exchange chromatography
2) Phenyl-Sepharose CL-4B column (4 X 30 cm) -Hydrophobic interaction chromatography
Results: Lab-scale isolation
Raw enzyme 12 g - Q-Sepharose FF column (4 X 30 cm) - Phenyl-Sepharose CL-4B column (4 X 30 cm)
Results: Lab-scale isolation
StepTotal
protein(mg)
Total ac-tivity
Specific activ-ity
(U/ mg pro-tein)
Yield (%) Purification factor
Raw enzyme 1180 94700 80 100 1.0
Q-Sepharose 183 93000 508 98 6.4
Phenyl-Sepharose 38 39500 1052 42 13.2
Results: Pilot-scale isolation
Raw enzyme 300 g
- DEAE-Sepharose FF column (19 X 25.2 cm) - Phenyl-Sepharose CL-4B column (11.3 X 17 cm)
Results: Isoelectric focusing
Specificity for oleic acid
At mole conversion 27
Specificity E is maximum= prefer methyl oleate
Crystallization of GC-4 lipase
• GC-4 lipase - crystallized in the presence of polyethylene glycol. - size of crystals is dependent on the molecular weight of agent.
11 % PEG4000
at pH 4~5.55 % PEG20000 at pH 4~5.5
11 % PEG20000 at pH 4~5.5
X-ray
a = 53.1 Åb = 83.5 Åc = 57.8 Å
ß = 100°
∴ space-filling coefficient = 2.3 Å3 per dalton (based on 61600 MW)
Bioseparation Engineering
: presentation
2011-2 Prof. Young Je Yoo
Chang Hyeon Song
2011-21042
서울대학교화학생물공학부
School of Chemical and Biological Engineering
Example 10.1-1 , 10.1-2
Example 10.1-1 Crystallization of adipic acid
Question ) Determine the weight of crystals recovered in this operation
90℃ 35℃
Adipic acid 10kg
Water 13.1 kg
10% water evaporated
0.05 kg adipic acid/kg water
filteredcrystallized
solubilized
Question ) Determine the weight of crystals recovered in this operation
Example 10.1-1 Crystallization of adipic acid
1. Set up two mass balance equation
water : water in = water in liquor + water evaporated
adipic acid : crystals in = crystals formed + remaining in mother liquor
2. Calculation of water in liquor
water in = water in liquor + water evaporated
13.1 kg = water in liquor + 13.1 kg×0.1
∴ water in liquor = 11.79 kg
3. Calculation of crystals formed
crystals in = crystals formed + remaining in mother liquor
10 kg = crystals formed + 0.05×11.79 kg
crystals formed = 9.41 kg
Example 10.1-2 Separation of soy sterols
Question ) Determine the β value for this separation
2040 kg of Stigmasterol & sitosterol
crystallization
Stigmasterol86.5 %
sitosterol13.5 %
Stigmasterol96.6 %
sitosterol3.4 %
Stigmasterol74.6%
sitosterol25.4 %
Question ) Determine the β value for this separation
Example 10.1-2 Separation of soy sterols
1. Calculation of E value for stigmasterol and sitosterol
2. Calculation of βvalue for this separation (purification)
According to the equation,
β value for this separation is 9.6 and is quite large so separation is effective
2011 Bioseparation engineering
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