LEE KONG CHIAN FACULTY

OF ENGINEERING AND
SCIENCE

Name: Choi Yu Hui

ID: 19UEB06715
Supervisor: Dr. Lee Sze Ying
Co-supervisor: Dr. Mah Shee Keat
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 INTRODUCTION
Spirulina Platensis
 Blue-green microalgae
 Easily reproduction and high
productivity
 Excellent source of
phycocyanin

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Figure 1: S. platensis derived products Figure 2: Biochemical composition of S. platensis
C-PHYCOCYANIN (CPC)
• Light –harvesting and pigment-binding protein

• Water soluble and insoluble in alcohol and

Jelly Milk product
esters Bubble gum Candy
Cake Fluorescent reagent
Fluorescent probe
Fluorescent tracer
CPC

Nail product
Lipstick
Eye shadow
Eye liner
Figure 3: Crystal structure and schematic of CPC
Anti-aging
Anti- Alzheimer
Anti- Parkinson
Anti-oxidant
Anti-inflammatory
Anti-tumor

Pharmaceutical
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Figure 4: Chemical Structure of CPC Figure 5: Application of CPC
 Extraction Methods Purification Methods
• Freezing and thawing method • Ammonium sulfate precipitation
• Ultrasonication • Ultrafiltration
• Lysozyme method • Ion exchange chromatography
• Water extraction
• Gel filtration chromatography
• Mechanical method

Table 1: Advantages and Disadvantages of Purification Methods

Purification Methods
Advantages Disadvantages
Ammonium sulfate • High yield • Long processing time
precipitation • Denaturation of CPC

Chromatography method • Effective • High packing material cost

(Ion exchange/ gel filtration)
Aqueous two-phase system
Conventional ATPS: polymer-
polymer and polymer-salt
• Long processing time
• No denaturation of biomolecules • Difficulty in separation of
• Easier to scale up CPC
• Flexibility in selection of phase former 4
• Ability to fractionate more than one compounds
PROBLEM STATEMENT
Not economically Scalability Low selectivity
competitive Not every cell Polymer-based ATPS 
Tedious downstream disruption/extraction or limited polarity range
process purification methods are between two phases
Energy intensive scalable Difficult in separation of
CPC from PEG
Time consuming
One-compounds
extraction

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OBJECTIVES
To study the feasibility of IL/ ATPS in extraction and fractionation
of CPC and other compounds

To identify the optimum salts and their concentration for the

fractionation of CPC

To evaluate the effect of IL alkyl chain length and IL anion on CPC

fractionation

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LITERATURE
REVIEW
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IONIC LIQUID-BASED AQUEOUS TWO
PHASE (IL-ATPS)
• Tunable physiochemical properties  hydrophobic and hydrophilic nature of IL

• Cation: Imidazolium, pyridinium, pyrrolidinium, phosphonium, ammonium and sulfonium

• Anion: halide ions, carboxylic acid, alkyl ester, amides and fluorinated anion
• Superior solvation behavior for both polar and non-polar compounds from microalgae
• Properties : Good electrochemical stability, negligible vapor pressure, good thermal stability

• Driving force for fractionation of biomolecules (Pei et al., 2009; Chang et al, 2018) :
- Imidazolium – aromatic 𝜋 system – stronger interaction with protein molecules
- Electrostatic interaction– above isoelectric point of molecules– charge state of

protein become negative – interact electrostatically with positive charge imidazolium

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 APPLICATION OF IL-ATPS
Kosmotropic Salts:
• Ionic hydration
• Stronger interaction with water molecules
• Salting out effect
• Phosphate salts: higher ability to form two phase

Table 2: Fractionation of Biomolecules from Microalgae using IL-ATPS

Microalgae Sources Targeted Compounds Phase-former components Purity/Extraction References
Efficiency (%)
Neochloris Iolilyte 221PG/ C6H5O7K3 82% (Suarez Garcia et al.,
oleoabundans Protein/ Carbohydrate 2018)

Isochrysis galbana Protein / Polysaccharides C8minCl/ K3PO4 100% /71.21% (Santos et al., 2018)

S.platensis CPC C8minBr / K2HPO4 99% (Chang et al., 2018)

S.platensis CPC BminCl / K2HPO4 93% (Zhang et al., 2015)
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METHODOLOGY
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 Overall Research Methodology Flow Diagram

Fractionation of
Crude Extract Construction of Phase compounds from
Preparation Diagram crude extract by IL-
ATPS

Quantification of compounds
Parameter studies
-CPC by UV-vis spectrophotometer
- Effect of inorganic salts and HPLC
- Effect of salt concentration -Total protein content by Bradford
- Effect of IL alkyl chain length Assay and BCA
- Effect of IL anion -Total Carbohydrate by phenol-
sulfuric acid method

Figure 5: Flow Diagram of Methodology 11

Crude Extract Preparation

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 Construction of Phase Diagram (Cloud Point Method)
Weight of :
 Initial IL solution (5g)
 Amount of salt solution added
 Amount of distilled water added

Addition of salt solution into IL dropwise until the Addition of distilled water until formation of
solution turn cloudy (formation of two phase) monophasic regime 13
Fractionation of Compounds from Crude Extract using IL-ATPS

5 g system

* Measure the volume of each

phase after separation

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 Effect Parameters on Extraction Efficiency
Effect of type of Salt Effect of IL Alkyl Chain Length

Figure 6: Effect of Inorganic Salts Figure 7: Effect of IL Alkyl Chain Length

Effect of IL Anion

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Figure 8: Effect of IL Anion
 CPC Quantification

UV-vis Spectrophotometer Reversed-phase High performance

Liquid Chromatography (RP-HPLC)
nm (Suarez Ruiz et al., 2018a).
Measuring the absorbance at 620nm and
652nm with accordance with the equation
 To Identify the CPC in the top and bottom
of Bennett and Bogorad (1973):
phase.
Concentration:  Condition:
Flow rate :1ml/min
Sample volume: 20 ul
mg A620 − 0.474 ∙ A652
CCPC = Pre-equilibrated with 20 % (v/v) ACN solution
mL 5.34 Eq (1) containing 0.1 % (v/v) TFA.
Linear gradient: 20 to 100 % (v/v) aqueous ACN
Purity: (containing 0.1 % TFA)
Retention time: 45 min.
mg A620 Emission wavelength: 580nm and 640nm
Purity = Eq (2)
g A280
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 Protein Quantification
Bradford Assay
Plotting of calibration curve
- protein bovine serum albumin (BSA)
Preparation of Bradford Reagent (BR)
- 0.1g of Brilliant Blue G-250 + 50ml ethanol +
100ml H3PO4 + 850ml water
Preparation of Samples
100 μL top/bottom phase + 5ml BR (stirring)
Measurement of Protein Content
- UV-vis spectrophotometer at 595nm
Figure 9: Bradford Assay Procedures
Bicinchoninic Acid Assay (BCA)
Plotting of calibration curve
- protein bovine serum albumin (BSA)
Preparation of Samples
- 25 μL top/bottom phase + 200μL BCA
reagent in 96-well-microplate
Measurement of Protein Content
- Incubated for 30min for 37°C in microplate
reader
- Measure absorbance at 562nm using UV-vis
spectrophotometer
Figure 10: BCA Procedures 17
Carbohydrate Quantification–Phenol Sulfuric Acid Method
Plotting of calibration curve
-Using Glucose Standard

Preparation of Sample
- 80 μL of top /bottom phase + 150 μL phenol solution (5% (w/v))+ 1 mL of
concentrated H2SO4
- Placed in Water bath for 10min at at 100 °C

Measurement of Carbohydrate Content

- Measure the absorbance at 490nm using UV-vis spectrophotometer.

Figure 11: Phenol Sulfuric Acid Procedures 18

 Extraction Analysis
Partition coefficient (K 𝑖 ): Selectivity (S):

K 𝐶𝑃𝐶 Eq (5)
Ct,i S=
K𝑖 = Ki
Cb,i Eq (3)

Extraction Efficiency (E): Volume Ratio (V):

Eq (4) Eq (6)
Ct,i Vt,i Vt,i Ei
Ei (%) = Vr = =
C𝐶𝐸,𝑖 V𝐶𝐸,𝑖 Vb,i K i (1 − Ei )

Ct,i = Concentration of compounds at top phase

Cb,i = Concentration of compounds at bottom phase
C𝐶𝐸,𝑖 = Concentration of compounds in the crude extract
Vt,i = Volume of Compound at top phase
Vb,i = Volume of Compound at bottom phase 19
REFERENCES
CHANG,Y. K., SHOW, P. L., LAN, J. C., TSAI, J. C. & HUANG, C. R. 2018. Isolation of C-phycocyanin from Spirulina
platensis microalga using Ionic liquid based aqueous two-phase system. Bioresour Technol, 270, 320-327.

SANTOS, J. O. H., TRIGO, J. O. P., MARICATO, E. L., NUNES, C. U., COIMBRA, M. A. & VENTURA, S. N. P. 2018.
Fractionation of Isochrysis galbana Proteins, Arabinans, and Glucans Using Ionic-Liquid-Based Aqueous Biphasic
Systems. ACS Sustainable Chemistry & Engineering, 6, 14042-14053.

UAREZ GARCIA, E., SUAREZ RUIZ, C. A., TILAYE, T., EPPINK, M. H. M., WIJFFELS, R. H. & VAN DEN BERG, C. 2018.
Fractionation of proteins and carbohydrates from crude microalgae extracts using an ionic liquid based-aqueous
two phase system. Separation and Purification Technology, 204, 56-65.

SUAREZ RUIZ, C. A., EMMERY, D. P., WIJFFELS, R. H., EPPINK, M. H. & VAN DEN BERG, C. 2018a. Selective and mild
fractionation of microalgal proteins and pigments using aqueous two-phase systems. J Chem Technol Biotechnol, 93,
2774-2783.
SUAREZ RUIZ, C. A., VAN DEN BERG, C., WIJFFELS, R. H. & EPPINK, M. H. M. 2018b. Rubisco separation using
biocompatible aqueous two-phase systems. Separation and Purification Technology, 196, 254-261.

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 Amin C2minC C4minCl C4minBF C2minOA C2minN(CN C4minB C6minB C8minB C2min C2minN
Cl l 4 c )2 r r r oAc (CN)2

C6H5O7K3 √ √ √ √

K3PO4 √ √ √ √ √ √ √ √ √ √
K2HPO4 √ √ √ × √ √ √ √ √ √
KH2PO4 √ ×
K2CO3 √ √ √ √ √
Phosphate √ √ √ √ √

buffer
NaOH √
Na2HPO4 √

KH2PO4, ×
K2SO4, ×
(NH4)2SO ×
4
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