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

The end

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