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Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair HSCCC Sławomir Wybranieca, ∗, Paweł Stalica a, Gerold Jerz.

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Presentation on theme: "Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair HSCCC Sławomir Wybranieca, ∗, Paweł Stalica a, Gerold Jerz."— Presentation transcript:

1 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, Cracow, Poland Journal of Chromatography A Accepted 13 August 2009 Presenter : MinSeok Kang ( )

2 Contents Ion-pair methodIntroduction Experiments /Result Further study

3 Ion-pair method on Chromatography (Focus on RP - HPLC)

4 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-A- B BH + HA B + H + H + + A - BH + Hydrophobic (more retained in RPC) Hydrophilic (less retained in RPC)

5 Ion-pair method on Chromatography (Focus on HPLC system) You can separate some ionic-samples by adjusting pH value

6 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?

7 Retention time was turned in the same pH value Example Packing Material -COO -

8 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

9 So, What happens if IPC-reagent was introduced in HSCCC?

10 Hylocereus polyrhizus (Cactaceae-Cacoideae), 火龍果 Pitaya (spanish) Dragon fruit (English) Pitaya (spanish) Dragon fruit (English) Activity : anti-oxidant Hylocereus undatus Hylocereus megalanthus (Yellow pitaya) Hylocereus megalanthus (Yellow pitaya)

11 Compound Structure High Polarity & Ionization Stability is extremely limited depending on pH values Betalains Betacyanin (Red, violet) Betacyanin (Red, violet) Betaxanthin (yellow,orange) Betaxanthin (yellow,orange) red and yellow indole-derived pigments Low chemical stability (decomposition occurred)

12 Preparation 400g freezed-dried fruit In 1000mL 80% MeOH (3 times) 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 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) Using prep-SPE (DW-> A CN : DW(0.7% TFA) = 4:96 elution) Lyophilized

13 HSCCC Solvent system Previous Method to separate some betacyanins from Phytolacca americana. (But, It didn’t work on this experiment !! ) Upper (Stationary phase) Upper (Stationary phase) Lower (Mobile phase) Shifting Kd value Betalains + Ion-pair agents = Increased hydrophobicity

14 More lipophilic, K D increased Low Viscosity, Sf factor increased Rt, Sf were Enlarged HSCCC Solvent system But K D is insufficient for 0.5 Increasing concentration of acid above 1.0% cause degradation of pigments.

15 HSCCC procedure Pharma-Tech Research model CCC 1000 Total volume : 850mL (20mL sample loop) Head to tail mode Pump Upper organic phase(Stationary) into Coil mg sample was dissolved in 10mL each of phase Injection before Equlibrium Mobile phase : 3.0 mL Injection before Equlibrium Mobile phase : 3.0 mL After eluants had decreased, EECCC started (flow 5.0mL, 800rpm) 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) Upper (Stationary phase) Lower (Mobile phase)

16 Result System 3 System 5 System 4 System 5 Betaxanthin Betacyanin Betaxanthin Betacyanin

17 Result Stationary phase retention determined resolution Elution order : Betanin > Hylocerenin > Phyllocactin (HSCCC) Betenin > Phyllocactin > Hylocerenin (RP- HPLC)

18 Conclusion Upper (Stationary phase) 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 )

19 Further study Operating method Important considerations for Ionic compound Additive (pH) Solvent Viscosity Sample property Compound structure Solubility Successful isolation

20 Thank you for your attention.


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