Peter Winterhalterb, Aneta Spornaa, Maciej Szaleniecc, Yosef Mizrahi 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 pKa ± 1.5 A- Acid wih pKa of 8.0 HA Base wih pKa of 8.0 B HA H+ + A- BH+ B + H+ BH+ Hydrophobic (more retained in RPC) Hydrophilic (less retained in RPC) Ion-pair method on Chromatography (Focus on HPLC system)

Ion-pair method on Chromatography 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 Unstable Packing could be dissolved 2< pH < 8 What if pKa of your target compound is over this range? Ion-pair method on Chromatography (Focus on HPLC system)

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

Ion-pair method on Chromatography Ion-pair reagent Anionic ion-pair reagent Sodium decyl sulfate Sodium 1-tetradecanesulfonate SDS Sodium 1-hexanesulfonate Perfluorocarboxylic acid (TFA , HFBA…) Cationic ion-pair reagent Tetraethylammonium Tetrahexylammonium Tetrabutylammonium THA-Br Hexadecyltrimethylammonium Ion-pair method on Chromatography (Focus on HPLC system)

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

Hylocereus megalanthus Hylocereus polyrhizus (Cactaceae-Cacoideae) , 火龍果 Pitaya (spanish) Dragon fruit (English) Activity : anti-oxidant 부아나가의 학명은 Hylocereus Undatus입니다. 식물학적인 계층을 살펴보면 속씨식물문(Magnoliophyta), 쌍떡잎식물강(Magnoliopsida), 석죽목(Caryophyllales), 선인장과(Cactaceae)에 속해 있습니다. 약재로 쓰이는 대표적인 선인장과의 식물들로는 불면, 히스테리, 정신질환을 치료하기 위한 페요테가 있으며, 이뇨약이나 이질약에 쓰이는 선인장이 있습니다. 이 밖에도 선인장과의 식물들은 볼거리를 치료하기 위한 약재로 쓰기도 하며, 과자나 치약에도 쓰이고 있습니다. 부아나가는 멕시코, 중앙아메리카, 남아메리카를 그 원산지로 하고 있습니다. 이후 열대 지방으로 확산되어 베트남, 필리핀, 말레이시아등과 같은 동남아 지역과 대만, 오끼나와, 이스라엘, 중국 남부 등지에서도 만날 수 있는 과일입니다. 부아나가는 보통 pitaya라 불리며, strawberry pear, thanh long, dragon fruit, huo long guo(火龍果)라고도 불립니다. 밤에만 꽃을 피운다고 하여 night blooming cereus, moonflower, Queen of the night라는 별명도 가지고 있습니다. huo long guo(火龍果)라는 이름은 부아나가의 열매가 달린 모양이 용이 여의주를 물고 있는 모양이라고 하여 붙여졌다고 합니다. 또한 열매가 인체에 매우 유익하면서 귀한 것이라 하여 동양에서 상서로운 동물로 알려진 용에 비할만한 가치가 있다 하여 붙여진 이름이라고도 합니다. 부아나가는 등반성인 삼각선인장의 일종으로 나무나 암반을 타고 올라가는 덩굴성 식물입니다. 밤에 향기가 강한 하얀 꽃이 활짝 피고 나서 열리는 열매는 갑옷처럼 단단한 껍질을 가지고 있습니다. 부아나가는 붉은색의 껍질에 초록색의 잎을 많이 가지고 있습니다. 육질은 하얀색인데 군데군데 검은 깨 같은 씨를 가지고 있습니다. 그 맛은 정말 키위와 배를 섞어 놓은 듯 달고 약간 시며, 신선한 맛을 지니고 있습니다. 인도네시아의 부아나가는 하얀 육질의 빨간 껍질을 가지고 있는 Hylocereus Undatus가 대부분입니다. 이외에도 빨간 육질에 빨간 껍질을 가진 Hylocereus polyrhizus와 하얀육질의 노란껍질을 가진 Selenicereus Megalanthus도 알려져 있습니다. [출처] ● 인도웹 ● - http://www.indoweb.org/love/bbs/board.php?bo_table=health_info&wr_id=21 Hylocereus undatus Hylocereus megalanthus (Yellow pitaya)

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

HSCCC Solvent system Rt, Sf were Enlarged More lipophilic , KD increased Betacyanins는 water soluble하다.!! 고정상의 양은 peak resolution에 영향 Low Viscosity , Sf factor increased But KD is insufficient for 0.5 Increasing concentration of acid above 1.0% cause degradation of pigments.

HSCCC procedure Pharma-Tech Research model CCC 1000 Pump Upper organic phase(Stationary) into Coil Rotation speed at 1000rpm 850-900mg sample was dissolved in 10mL each of phase Injection before Equlibrium Mobile phase : 3.0 mL Total volume : 850mL (20mL sample loop) Upper (Stationary phase) Lower (Mobile phase) Head to tail mode After eluants had decreased, EECCC started (flow 5.0mL , 800rpm) Effluent stream was monitored by UV-absorbance at 540nm

Result System 4 System 5 System 3 System 5 Betaxanthin Betacyanin

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

2. Stationary phase retention is important part of resolution 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 )

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

Thank you for your attention.