Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair HSCCC...
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Transcript of Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair HSCCC...
Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-
pair HSCCC
Sławomir Wybranieca,∗, Paweł Stalica a, Gerold Jerz b, Bettina Kloseb, Nadine Gebersb,Peter Winterhalterb, Aneta Spornaa, Maciej Szaleniecc, Yosef Mizrahi
a Faculty of Analytical Chemistry, Institute C-1, Department of Chemical Engineering and Technology, Cracow University of Technology, ul. Warszawska 24, 31-155 Cracow, Poland
Journal of Chromatography A Accepted 13 August 2009
Presenter : MinSeok Kang ( 2010 1 30 )
Contents
Ion-pair method Introduction
Experiments/Result
Further study
Ion-pair method on Chromatography
(Focus on RP - HPLC)
Ion-pair method on Chromatography(Focus on HPLC system)
Acid wih pKa of 8.0
Base wih pKa of 8.0
pKa ± 1.5
HA
A-
B
BH+
HA
B + H+
H+ + A-
BH+
Hydrophobic(more retained in RPC)
Hydrophilic(less retained in RPC)
Ion-pair method on Chromatography(Focus on HPLC system)
You can separate some ionic-samples by adjusting pH value
Ion-pair method on Chromatography(Focus on HPLC system)
RP – HPLC is somewhat restricted
Stable pH range of Silica-bonded column
2< pH < 8 Unstable Packing could be dissolved
What if pKa of your target compound is over this range?
Retention time was turned in the same pH value
Example
Packin
g M
ate
rial-COO-
-COO-
-COO-
-COO-
-COO-
Ion-pair method on Chromatography(Focus on HPLC system)
Ion-pair reagent
Cationic ion-pair reagent
Anionic ion-pair reagent
Tetraethylammonium
Tetrahexylammonium
Tetrabutylammonium
Hexadecyltrimethylammonium
Sodium decyl sulfate
Sodium 1-tetradecanesulfonate
Sodium 1-hexanesulfonate
Perfluorocarboxylic acid (TFA , HFBA…)
SDS
THA-Br
So, What happens if IPC-reagent was introduced in HSCCC?
Hylocereus polyrhizus (Cactaceae-Cacoideae) , 火龍果
Pitaya (spanish)Dragon fruit (English)
Activity : anti-oxidant
Hylocereus undatus Hylocereus megalanthus(Yellow pitaya)
Compound Structure
High Polarity & Ionization
Stability is extremely limited depending on pH values
Betalains
Betacyanin(Red , violet)
Betaxanthin (yellow,orange)
red and yellow indole-derived pigments
Low chemical stability (decomposition occurred)
Preparation
400g freezed-dried fruit In 1000mL 80% MeOH (3 times)
Filtered (filter cloth)
Through a layer of 0.040mm sillica gel& 0.2uM pore size filter
Lyophilized
Dissolved in DW (0.7% TFA)
Using prep-SPE (DW->
A CN : DW(0.7% TFA) = 4:96 elution)
Lyophilized
HSCCC Solvent system
Previous Method to separate some betacyanins from Phytolacca americana.(But, It didn’t work on this experiment !! )
Upper(Stationary
phase)
Lower(Mobile phase)
Shifting Kd value
Betalains + Ion-pair agents = Increased hydrophobicity
More lipophilic , KD increased
Low Viscosity , Sf factor increased
Rt, Sf were Enlarged
HSCCC Solvent system
But KD is insufficient for 0.5
Increasing concentration of acid above 1.0% cause degradation of pigments.
HSCCC procedurePharma-Tech
Research model CCC 1000
Total volume : 850mL(20mL sample loop)
Head to tail mode
Pump Upper organic phase(Stationary) into Coil
850-900mg sample was dissolved in 10mL each of phase
Injection before EqulibriumMobile phase : 3.0 mL
After eluants had decreased, EECCC started
(flow 5.0mL , 800rpm)
Rotation speed at 1000rpm
Effluent stream was monitored by UV-absorbance at 540nm
Upper(Stationary
phase)
Lower(Mobile phase)
Result
System 3 System 5
System 4
System 5
BetaxanthinBetacyanin
BetaxanthinBetacyanin
Result
Stationary phase retention determined resolution
Elution order : Betanin > Hylocerenin > Phyllocactin (HSCCC)Betenin > Phyllocactin > Hylocerenin (RP- HPLC)
Conclusion
Upper(Stationary
phase)
Lower(Mobile phase)
Shifting Kd value
Betalains + Ion-pair agents = Increased hydrophobicity
1. In case of highly water soluble compound , patition will occurred increasing hydrophobicity by using ion-pair reagent
2. Stationary phase retention is important part of resolution
3. EECCC approach is able to recover all pigment compound of H.polyrhizus ( all polarity scale )
Further study
Operating method
Important considerations for Ionic compound
Additive (pH)
Solvent Viscosity
Sample property
Compound structure
Solubility
Successful isolation
Thank you for your attention.