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.