sistema de separacion de betalainas.pdf

8/10/2019 Sistema de separacion de betalainas.pdf

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Journal of Chromatography B, 941 (2013) 5461

Contents lists available at ScienceDirect

Journal of Chromatography B

j o u rn a l h om epag e : www.e l sev i e r. com/ loca t e / ch romb

Versatile solvent systems for the separation of betalains fromprocessed Beta vulgaris L. juice using counter-current chromatography

Aneta Sprna-Kucab a , , Svetlana Ignatova b , Ian Garrard b , Sawomir Wybraniec aa Department of Analytical Chemistry, Institute C-1, Faculty of Chemical Engineering and Technology, CracowUniversity of Technology, ul. Warszawska 24,Cracow 31-155, Polandb Brunel Institute for Bioengineering, Brunel University, Uxbridge, Middlesex, United Kingdom

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Article history:Received 13 May 2013Received in revised form27 September 2013Accepted 1 October 2013Available online 10 October 2013

Keywords:BetaninBetalainsBetacyaninsCounter-current chromatographyBeta vulgaris L

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Two mixtures of decarboxylated and dehydrogenated betacyanins from processed red beet roots (Betavulgaris L.) juice were fractionated by high performance counter-current chromatography (HPCCC) pro-ducing a range of isolated components. Mixture 1 contained mainly betacyanins, 14,15-dehydro-betanin(neobetanin) and their decarboxylated derivatives while mixture 2 consisted of decarboxy- and dehydro-betacyanins. The products of mixture 1 arose during thermal degradation of betanin/isobetanin in mildconditions while the dehydro-betacyanins of mixture 2 appeared after longer heating of the juice from B.vulgaris L. Two solvent systems were found to be effective for the HPCCC. A highly polar, high salt concen-tration system of 1-PrOHACN(NH 4 )2 SO4 (satd. soln)water (v/v/v/v, 1:0.5:1.2:1) (tail-to-head mode)enabled the purication of 2-decarboxy-betanin/-isobetanin, 2,17-bidecarboxy-betanin/-isobetanin andneobetanin (all from mixture 1) plus 17-decarboxy-neobetanin, 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2-decarboxy-neobetanin and 2,15,17-tridecarboxy-neobetanin (from mixture 2). The othersolvent system included heptauorobutyric acid (HFBA) as ion-pair reagent and consisted of tert -butylmethyl ether (TBME)1-BuOHACNwater (acidied with 0.7% HFBA) (2:2:1:5, v/v/v/v) (head-to-tail mode). This system enabled the HPCCC purication of 2,17-bidecarboxy-betanin/-isobetanin andneobetanin (from mixture 1) plus 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2,17-bidecarboxy-2,3-

dehydro-neobetanin

and

2,15,17-tridecarboxy-neobetanin

(mixture

2). The

results

of

this

research

arecrucial in nding effective isolation methods of betacyanins and their derivatives which are meaningfulcompounds due their colorant properties and potential health benets regarding antioxidant and cancerprevention. The pigments were detected by LC-DAD and LCMS/MS techniques.

2013 Elsevier B.V. All rights reserved.

1. Introduction

Counter-current chromatography (CCC) is a liquidliquid chro-matography technique which was invented in the early 1960s[1,2] . In high-speed counter-current chromatography (HSCCC),high speed coil rotation around its own axis and a central axis(planetary motion) generates a centrifugal eld to retain the liquidstationary phase in the coil. The mobile phase is pushed through

Abbreviations: ACN, acetonitrile; BuOH, butanol; CCC, counter-currentchromatography; CID, collision induced dissociation; EtOH, ethanol; HFBA, hep-tauorobutyric acid; HPCCC, high-performance counter-current chromatography;HSCCC, high-speed counter-current chromatography; K D, partition coefcient;MeOH, methanol; PFCA, peruorocarboxylic acid; RP-HPLC, reversed phase high-performance liquid chromatography; PrOH, propanol; TBME, tert -butyl methylether; TFA, triuoroacetic acid.

Corresponding author: Tel.: +48 12 628 30 74.E-mail addresses: [email protected] , [email protected]

(A. Sprna-Kucab).

with a pump. The g-level produced is an effect from the coil rota-tion and for a typical HSCCC machine, it is between 55 and 80g-level [13] . High-performance counter-current chromatography(HPCCC) is the name given to a high g-level machine (240g) andwas introduced by the Brunel Institute for Bioengineering [4] .

The application of CCC to the fractionation and purication of natural plant pigments has been shown in numerous publications[511] .

Beta vulgaris L. is increasingly utilized as a source of naturalfood dyes due to a growing interest of consumers in its potentialhealth benets (antioxidant, anticarcinogenic) and the non-toxicfeatures of betalains.Since somesynthetic pigmentsare consideredas toxic and harmful [12] there is a demand for natural equiva-lents. Choosing a suitable solvent system for betalains puricationis challenging due to their low stability in some physicochemicalconditions [1214] .

A few pathways of betalain degradation and transformation areknown, such as decarboxylation, dehydrogenation, hydrolysis anddeglycosylation. Decarboxylation of betalains can occur at either

1570-0232/$ seefrontmatter 2013 Elsevier B.V. All rights reserved.

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A. Sprna-Kucab et al. / J. Chromatogr. B 941 (2013) 5461 55

Fig. 1. Steps of betanin and itsdiastereomerisobetanin thermal degradation path-ways.

C-2, C-15 or C-17 carbon positions, however, usually occurs at C-2and C-17. The dehydrogenation is observed at C-2,3 and C-14,15.The productsof betanin degradation are usually morestable, whichmakes theminterestingmaterial for further application in thephar-maceutical and food industries ( Fig. 1) [12,1517] .

Preparative isolationof unstablebetalains byHPLCis oftenprob-lematic due to the catalytic action of the solid stationary phasecausing pigment degradation, therefore, new separation methodssuch as counter-current chromatography create an important pos-

sibility of obtainingpure pigments.CCC enables the use of differentstationary phases through the application of different solvent sys-tems without the need to buy a new column. In addition, modernCCC technology is as easy as HPLC to scale up to preparative andpilot levels.

Hitherto, the rst successful isolation and purication of morehydrophobic betalains by HSCCC was carried out in a solvent sys-temconsisting of TBMEBuOHACNwater (acidied withion-pairreagents TFA or HFBA) [69] . The addition of ion-pair reagentsresults in a different chromatographic behavior of betalains e.g.longer retention time of betalains in RP-HPLC [6,18] . The additionof ion-pair additives to the CCC solvent systems changes the parti-tion coefcient ( K D) of betalains and efciently shifts them to theorganic phase, creating a new possibility for separation of these

highly polar plant pigments [6,8,18] .

The novelty of this contribution is a fractionation and isolationof decarboxylatedand dehydrogenated derivatives of betanin fromprocessed B. vulgaris L. juice using HPCCC. These mixtures of beta-lains have never been separated by CCC, which would be a usefultechnique for the separation as its liquid stationary phase does notcatalyze degradation or cause irreversible adsorption and loss of the components, in the way solid stationary phases may do. Thedifferences in elution proles traced in theHPCCCand HPLCsepara-tions were of special interest andwere indicatedby recentbetalainseparations [6,8,18] . The HPCCC process was accomplished usingtwo different types of solvent systems: an ion-pair system and ahigh salt concentration system. The high salt solvent systems wereused for the rst time in order to separate betalains. Whilst ion-pair solvent systems have been reported before for the separationof non-decarboxylatedandnon-dehydrogenatedbetalains,nothingis known about their efciency in the separation of decarboxy-lated and dehydrogenated betacyanins. Furthermore, the presenceof toxic ion-pair agents makes these systems less attractive for usein the food industry [69] .

2. Experimental

2.1. Reagents

HPLC-grade acetonitrile (ACN), 1-propanol (1-PrOH), ethanol(EtOH), 1-butanol (1-BuOH), ammonium sulphate, tert -butylmethyl ether (TBME), TFA and HFBA were obtained from FisherChemicals (Loughborough, UK). Water was deionized (Purite,Thames, Oxon, UK). HPLC-grade formic acid, methanol (MeOH)were obtained from POCH (Gliwice, Poland).

2.2. The preparation of the crude pigment extracts

Two groups of betacyanins with different decarboxylation anddehydrogenation levels were obtained by thermal treatment of B.vulgaris L. juice and then analyzed by LC-DAD and LCMS/MS. The juice was obtained from red beet roots (purchased as whole beetroots from the local market, Krakw, Poland) which were washed,hand-peeled,cut into small piecesand squeezed in a juice extractor(Zelmer, Rzeszw, Poland) ( Table 1 ).

The heating of betalain mixtures in the juice was performedat 85 C for 30 min (mixture 1) and 60min (mixture 2), bothacidied with 0.2% (v/v) formic acid according to a previous proce-dure [12] . The mixtures were separately puried on a preparativesolid-phase extraction (SPE) column packed with C-18 reversedphase material (Merck, Darmstadt, Germany) [12] . The eluates inaqueous-acetonitrile solution were then concentrated by rotaryevaporator and then freeze-dried for the HPLC analysis and theHPCCC experiments.

2.3. Apparatus

A semi-preparative Spectrum HPCCC J-type modern hydro-dynamic CCC instrument was used (Dynamic Extractions, Slough,UK) for the separation of betanin/isobetanin and their decarboxy-and dehydro-derivatives (mixtures 1 and 2).

The Spectrum HPCCC had a maximum rotation speed of ca.1600rpm ( R=75mm, 240 g eld). The instrument was equippedwith two columns of 143.5ml total capacity, 71m longand 1.6 mmi.d. The mobile phase was pumped in the tail-to-head direction(system AI ) and head-to-tail direction for system BIV (Table 2 ).

The initial scouting runs were performed on the analytical sizeMini HPCCC instrument ( systems AI AIII, and BIBIV ) supplied byDynamic Extractions (Slough, UK). The Mini HPCCC was equippedwith a single 7cm diameter column made with 0.8 mm i.d. poly-

tetrauorethylene (PTFE) tubing: 18.2ml

capacity, column length


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56 A. Sprna-Kucab et al./ J. Chromatogr. B 941 (2013) 5461

Table 1Chromatographic, spectrophotometric and mass spectrometric data of the pigments identied in the crude mixtures submitted for the HPCCC separations.

Peak no. Compound Symbol Rt [min] max [nm] m/ z [M+H] + m/ z from MS/MS of [M+H] +

1 Betanin a Bt 14.3 538 551 3892 17-Decarboxy-betanin a 17-dBt 15.1 505 507 3451 Isobetanin a IBt 15.6 538 551 3892 17-Decarboxy-isobetanin a 17-dIBt 16.5 505 507 3453 2-Decarboxy-isobetanin a 2-dIBt 18.7 533 507 3453 2-Decarboxy-betanin a 2-dBt 18.7 533 507 345

4

2,17-Bidecarboxy-betanina

2,17-dBt 20.2 507 463 3014 2,17-Bidecarboxy-isobetanin a 2,17-dIBt 20.2 507 463 3015 17-Decarboxy-neobetanin b 17-dNBt 20.5 446 505 343; 299; 2556 2,15,17-Tridecarboxy-2,3-dehydro-neobetanin b 2,15,17-dec-2,3-dHNBt 21.3 394 415 2537 14,15-Dehydro-betanin (neobetanin) a NBt 22.0 468 549 387; 3438 2,17-bidecarboxy-2,3-dehydro-neobetanin b 2,17-dec-2,3-dHNBt 22.8 409 459 2979 2,15,17-Tridecarboxy-neobetanin b 2,15,17-dNBt 23.5 451 417 255

10 2-Decarboxy-neobetanin b 2-dNBt 26.0 483 505 343; 299; 255

a Pigments from themixture 1.b Pigments from the mixture 2 (tentatively identied).

36m. The column was mounted in a cantilever rotor containinga counterweight for balance when rotating. The distance betweenthe holder axis of the coil and the central axis of the instrumentwas 50mm (revolution radius R). The maximum rotation speed

was 2049rpm (240g eld). The CCC machines were connected toa thermostat, which enabled maintaining a constant temperatureduring the separation process (20 C). During all CCC runs a K-501Knauer (Berlin, Germany) pump, UV-ViS detector Shimadzu (Lyon,France)and fractioncollectorFoxyJr. fromKnauercompany(Berlin,Germany) were used.

The positive ion electrospray mass spectra were recorded on aThermoFinnigan LCQ Advantage (electrospray voltage 4.5kV; cap-illary 250 C; sheath gas: N 2 ) coupled to a ThermoFinnigan LCSurveyor pump utilizing the HPLC systems. The MS was controlledandtotal ionchromatograms andmass spectra wererecordedusingThermoFinnigan Xcalibursoftware (San Jose,CA, USA). Heliumwasused to improve trapping efciency and as the collision gas forCID experiments. The relative collision energies for MS/MS anal-yses were set at 30% (according to a relative energy scale). For theLCMS/MS analyses, a 25cm 3.0mm, 5 m Luna C18 (2) Pheno-menex chromatographic column was used.

HPLC analyses were carried out using a Gynkotek HPLC systemwith UVD340U Gynkotek HPLC Pump Series LPG-3400A and ther-mostat (Gynkotek Separations, H. I. Ambacht, The Netherlands).The software package Chromeleon 6.0 (Gynkotek Separations) wasapplied for the data acquisition. For the CCC fraction analysis byHPLC, a 10cm 2.1mm, 2.7 m Supelco (C-18) column was used.

2.4. Solvent systems

The solvent systems initially investigated for the HPCCC sepa-ration are listed in Table 2 and were divided into two groups: A

highly polar solvent systems containing ammonium sulphate salt( AI AIII) and B ion-pair solvent systems containing an ion-pair

agent ( BIBIV ). The ion-pair solvent systems were prepared in aseparator funnel by mixing appropriate solvents then, after equil-ibration, the phases were separated and sonicated before HPCCCseparations.

The biphasic highly polar solvent systems, containing saturatedammoniumsulphate solution,were preparedas describedby Faheyet al. [19] . Saturated ammonium sulphate was made by dissolvingthesalt in boiling water, letting this cool down to 78 C anddecant-ing the supernatant. The saturated ammonium sulphate was thenmixed with theremaining solvents in theratiodescribed in Table 2 .The solvent systems were equilibratedat 20 C and thenthe phaseswere separated and sonicated in order to remove dissolved gases.

2.5. Separation of betalains by HPCCC

Determination of stationary phase retention for each solventsystem and preliminary separation studies were performed on theanalytical Mini HPCCC instrument. The solvent systems were pre-

paredaccordingto Section 2.4 . TheMiniHPCCCinstrumentwas runataowrateof0.25ml/mininbothnormalphase( systems AI AIII)and reversed phase ( systems BI BIV ) modes. The sample (15 mg)was dissolved in 1.5 ml of stationary phase ( systems AI AIII) ormobile phase ( systems BI BIV ). The choice of the injection solventwas primarily a result of betalain solubility. The chromatographiccolumn was rst entirely lled with the stationary phase and themobile phase was pumped while the coil was rotating at 2049 rpmat constant temperature of 20 C. The retention of the stationaryphase measured for each solvent systems was as follows: 64.3%(system AI ), 42.3%( system AII )and50.0%( systemAIII ),52.6%(sys-tem BI ), 49.0% (systemBII ),69.8%( systemBIII ),and60.5%( systemBIV ).

The semi-preparative separation of the betalain mixtures was

performed on the Spectrum HPCCC. Using either solvent system AI or BIV , the centrifuge was run at a ow rate of 1.0ml/min.

Table 2Composition of thesolvent systems tested forbetalainsseparation by HPCCC.

System no. Composition

A Highly polar solvent systems with saltI 1-PrOHACNsaturated (NH 4 )2 SO4 H 2 O (v/v/v/v, 1:0.5:1.2:1II EtOHACN-1PrOHsaturated (NH 4 )2 SO4 H 2 O (v/v/v/v/v, 0.5:0.5:0.5:1.2:1)III EtOH-1BuOHACNsaturated (NH 4 )2 SO4 H2 O (v/v/v/v/v, 0.5:0.5:0.5:1.2:1)

B Ion-pair solvent systemsI TBME-1BuOHACNH 2 O (0.7% TFA) (v/v/v/v, 2:2:1:5)II TBME-1BuOHACNH 2 O (1.0% TFA) (v/v/v/v, 2:2:1:5)III TBME-1BuOHACNH 2 O (0.4% HFBA) (v/v/v/v, 2:2:1:5)IV TBME-1BuOHACNH 2 O (0.7% HFBA) (v/v/v/v, 2:2:1:5)


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The sample (15mg) was dissolved in 1.5 ml of stationary phase(system AI ) or mobile phase ( system BIV ). As with the MiniCCC runs, the chromatographic coil was rst entirely lled withthe stationary phase and the mobile phase was pumped whilethe coil was rotating at 1600rpm at constant temperature of 20 C. The retention of the stationary phase was measured onthe Spectrum instrument as follows: 80.5% ( system AI ) and81.2% (system BIV ). The system AI enabled purication of 2-decarboxy-betanin/-isobetanin (3.24mg), 2,17-bidecarboxy-betanin/-isobetanin (3.42 mg) and neobetanin (0.47mg) (mixture1) plus 17-decarboxy-neobetanin (1.65 mg), 2,15,17-tridecarboxy-2,3-dehydro-neobetanin (1.88 mg), 2-decarboxy-neobetanin(6.39 mg) and 2,15,17-tridecarboxy-neobetanin (0.87mg) (mixture2) and the system BIV was effective for 2,17-bidecarboxy-betanin/-isobetanin (3.74 mg) and neobetanin (0.44mg) (mixture1) plus 2,15,17-tridecarboxy-2,3-dehydro-neobetanin (2.4 mg),2,17-bidecarboxy-2,3-dehydro-neobetanin (1.5 mg) and 2,15,17-tridecarboxy-neobetanin (1.2 mg) (mixture 2).

The efuent from the outlet of the HPCCC was monitored usinga UV-ViS detector (Gilson, Middleton, WI, USA) and collected intotest tubes with a fraction collector at 6min intervals (ow rate0.25ml/min and 1 ml/min). The elution -mode was stopped whenall the pigments had been eluted as shown by the UV-ViS detector.

Where necessary, this wasfollowedby the extrusion -mode withthe pumping of the stationary phase at a ow rate 0.5ml/min(analytical scale) and 2.0ml/min (semi-prep scale) during the coilrotation.

2.6. HPLC analysis (LC-DADESIMS/MS)

To prevent dissolved salt in the fractions from adversely affect-ing the HPLC analysis, a precipitation of the salt bulk from thesamples was accomplished with methanol. LC-DAD analyses of mixtures 1 and 2 and HPCCC fractions were carried out using agradient elution mode at 40 C with methanol (A) and 2% aqueousformicacid (B) system: 5%A inB at0 min,a gradientto 7%A inB at2min and 20% A in B at 8min then 40% A in B at 10min and 80%Ain B at 12min, returning to the start conditions in 0.6 min. For theLCMS/MS analyses, a solvent system: 7%A in B at 0 min a gradientto 30% A in B at 35 min (A, methanol; B, 2% formic acid in water)was used. The injection volume was 70 l and the ow rate was0.5ml/min (LC-DAD and LCMS/MS systems).

2.7. Freeze drying

The HPCCCfractions were diluted withdeionisedwater becausethey contained large amounts of solvents. The diluted fractionswere then frozen and lyophilized. The fractions containing higheramounts of solvents were partially evaporated by speed vacuumcentrifuge at room temperature to minimize compound degrada-tion and then freeze dried.

3. Results and discussion

3.1. Analysis of decarboxylated and dehydrogenated derivativesof betanin/isobetanin

For the experiments, two different mixtures of betanin andits derivatives, differing in decarboxylation and dehydrogenationproducts, were obtained as a result of the different heating timesof the acidied betanin extract ( Table 1 ). Mixture 1 containedmainly betanin, isobetanin, neobetanin and decarboxy-betaninswhile mixture 2 consisted of decarboxy- and dehydro-betanins.The mixtures differed in the pigment polarities and physico-

chemical properties determining their chromatographic behavior.

For example, the compounds in mixture 1 were more unsta-ble than in mixture 2 and the HPLC retention times of thedecarboxylated and dehydrogenated derivatives were longer incomparison to their corresponding betacyanins, due to theirlower polarity. Betanin, as well as 2-, 17-, and 2,17-bidecarboxy-betanins detected by HPLC and LCMS/MS were identiedaccording to the standards isolated in previous studies [20] ,and were monitored according to their retention times, and ViSabsorption maxima

max (538, 533, 505, 507nm for betanin,

2-monodecarboxy-, 17-monodecarboxy- and 2,17-bidecarboxy-betanins, respectively). The other compounds were mostlytentatively identied based on their max (446, 394, 468, 409, 451,483nm for 17-decarboxy-neobetanin, 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, neobetanin, 2,17-bidecarboxy-2,3-dehydro-neobetanin, 2,15,17-tridecarboxy-neobetanin and 2-decarboxy-neobetanin, respectively)as wellas their protonated molecularandfragmentation ions ( Table 1 ) according to a previous discussion[21,22] .

3.2. HPCCC separations of betacyanins and their derivatives

This study is a rst attempt of a complete HPCCC separationof betacyanins and their decarboxylated/dehydrogenated deriva-tives obtained during thermal treatment of red beet juice. Findingan appropriate phase system for the successful CCC separation of polar betacyanins is problematic [69] . However, studies on beta-lains from Phytolacca americana [6] and Bougainvillea glabra [9]suggested that an effective separation of the less polar compounds(e.g. acylated-betacyanins) could be achieved in solvent systemswith ion-pair reagents.

The use of hydrophilic solvent systems containing ammoniumsulphate for the purication of anionic glucosinolates from crudeplant homogenates [19] suggested that they were appropriate sys-tems fora purication of polar compounds, however a similar polarsystem consisting of EtOHACN(NH 4 )2 SO4 (satd. soln)water(1:0.5:1.2:1, v/v/v/v) was unsuccessfully used for separation of betanin and isobetanin [11] as these compounds were co-eluted.

In order to investigate the separation of new betacyanin groups(the decarboxylated and dehydrogenated derivatives) by HPCCC,both types of solvent systems were tested, i.e.:

(A) Highly polar solvent systems containing a high concentrationof ammonium sulphate to enable the formation of two phasesin solvent systems containing water in both phases.

(B) Ion-pair, aqueous-organic solvent systems including ion-pairreagents (TFA, HFBA).

3.2.1. Highly polar solvent systems containing ammoniumsulphate 3.2.1.1. Analytical scale separation of decarboxylated and dehydrogenated betanins. The initial experiments were carried out in an

analytical machine with highly polar solvent systems containingammonium sulphate ( Table 2 , systems AI AIII). The pH of thesesolvent systems is ca. 5.5 and at this pH betalains are more sta-ble. The results of the separation of decarboxylated ( Fig. 2) anddehydrogenated ( Fig. 3) betanins in the three solvent systems arecompared. In this mode of separation (tail-to-head), the mobilephase is the upper phase (organic phase) and the stationary phaseis the lower phase (aqueous phase), therefore, the more hydropho-biccompounds are eluted rst as expected. The high concentrationof ammonium sulphate in the aqueous phase enhances the reten-tion of the stationary phase in CCC by increasing the difference indensity between the two phases. The stationary phase retention inCCC inuences peak resolution; the larger amount of the station-ary phase in the coil the higher resolution. In system AI , the highly

polar Bt/IBt ( 1/1 ) are retained longer in the HPCCC coil and are


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Fig. 2. Reconstructed HPLC chromatograms of betalains (mixture 1) in three highsalt-solvent systems separated by analytical HPCCC (composition of the solventsystems, see Table 2 ).

eluted with 17-dBt/-dIBt ( 2/2 ) during elution extrusion process(Fig. 2). The separation of NBt ( 7) is very successful in the appliedconditions. 2,17-dBt/-dIBt ( 4/4 ) are eluted as the rst decarboxy-betacyanins, partially resolved from 2-dBt/-dIBt ( 3/3 ).In the groupof decarboxylated betacyanins (mixture 1) separated in solventsystem AII , 2,17-dBt/-dIBt ( 4/4 ) are eluted as rst, virtually coin-cident with 2-dBt/-dIBt ( 3/3 ). Neobetanin ( 7 ) is eluted next andis relatively pure. Yet the nal four compounds 17-dBt/-dIBt ( 2/2 )and Bt/IBt( 1/1 ) show a considerablepeak overlap,withnone beingpure. System AIII gives good results for 2,17-dBt/-dIBt ( 4/4 ) and 2-

dBt/-dIBt ( 3/3 ) separation, however, 17-dBt/-dIBt ( 2/2 ) and Bt/IBt(1/1 ) are eluted during elution extrusion process less separatedthan in system AI (Fig.2 ). The elution order of betalainsis the samefor all solvent systems and mainly depends on their polarity. Themore hydrophobic compounds are eluted rst, followed by morepolar pigments. The applied solvent systems have different polar-ity, the most polar being system AII , then system AI and system AIII. In system AII , betalains are eluted too fast and therefore withpoor resolution. Neobetanin ( 7 ) is eluted signicantly earlier in themost polar system AII than in the remainingsystems.In the case of 2,17-dBt/-dIBt ( 4/4 ) and 2-dBt/-dIBt ( 3/3 ), the situation is similar.Theseparation of these pigments clearly depends on thepolarityof the solvent systems and is more effective in less polar solvent sys-tems AI and AIII. Theefciency of separation is also associated with

retention of the stationary phase, which is the highest in systems

Fig. 3. Reconstructed HPLC chromatograms of betalains (mixture 2) in three highsalt-solvent systems separated by analytical HPCCC (composition of the solventsystems, see Table 2 ).

AI and AIII. The retention of the stationary phase and polarity of the solvents presumably inuence separationof 17-dBt/-dIBt ( 2/2 )from Bt/IBt ( 1/1 ) which is the most effective in solvent system AI .In this case, the retention of the stationary phase is the highest(64.3%).

The separation of dehydrogenated betacyanins (mixture 2) byanalytical HPCCCis presented in Fig.3 . The best results areobtainedfor systems AI and AIII where the separation of the majority of dehydro-derivatives is quite effective, with a very good separationof 17-dNBt ( 5 ) from the rest of the compounds, as a result of thehighest stationary phase retentionin these solventsystems anddue

to lower polarity of 5 . In solvent system AI , only a partial overlapin the groupof 2,15,17-dec-2,3-dHNBt( 6), 2,17-dec-2,3-dHNBt ( 8),2,15,17-dNBt ( 9), and 2-dNBt ( 10 ) is observed. Solvent system AII ,except for 17-dNBt ( 5), gives no pure fractions, with the other fourpeaks substantially overlapped. In this group of compounds testedin system AIII , only a relatively good separation of 2,15,17-dec-2,3-dHNBt ( 6 ) and 2,15,17-dNBt ( 9) is observed. In system AII , thepigments are mostly co-eluted except of 17-dNBt (5) in spite of its early elution with the other compounds. The best results areobtained for system I where the retention of the stationary is thehighest ( Fig. 3).

3.2.1.2. Semi-preparative scale separation of decarboxylated anddehydrogenated betanins. The system AI was used to separate mix-

tures1 and2 usinga semi-preparative machine.The applied solvent


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Fig.4. HPCCCchromatogram ofbetalains(mixture 1) aftertheseparation in thehighsalt-solvent system by semi-prep HPCCC ( system AI , see Table 2 ). Peak numbersrefer to compoundsshownin Table1 .

system enables a signicantly better separation of betalains dueto a higher retention of the stationary phase (80.5%). The HPCCCinstrument used has almost twice the coil length of the analyti-cal instrument, plus a wide bore (1.6mm) to reduce any plug oweffects.TheHPCCCchromatogram monitoredat 500nm (systemAI )for mixture 1 is shown in Fig. 4 . The rst two peaks (2,17-dBt/-dIBt(4/4 ) and 2-dBt/-dIBt ( 3/3 )) are partially resolved with a resolu-tion of 0.67 and the third compound (NBt ( 7)) is eluted as a singlepeak.The mostpolarcompoundpairs (17-dBt/-dIBt( 2/2 )andBt/IBt(1/1 )) appear to co-elute in fractions 4 and 5 (elution extrusionmode). However, the reconstructed HPCCCchromatogram ( Fig.5 A)of mixture 1 shows a tendency of a separation of the pairs 1/1 and2/2 . The extrusionof thecolumn content results in fractions highlyrich in either Bt/IBt ( 1/1 ) or 17-dBt/-dIBt ( 2/2 ) (Fig. 5A). Compar-ison of Fig. 2 and Fig. 5A and B demonstrates that polarity of thesolvent systems is the main factor determining the resolution of the compounds. The separation of mixture 1 is not much effectivedespite considerably higher retention of the stationary phase onSpectrum CCC. TheHPLC chromatogramsof the crude mixtures andthe puried fractions are depicted in Fig. 6.

Fig. 7 demonstrates the HPCCC chromatogram of mixture 2(monitored at 500nm ) separated in system AI with only partial

Fig. 6. HPLC chromatograms of betalains (mixture 1) before separation by semi-prep HPCCC (a) and selected fractions after the separation in the high salt-solventsystem( AI, see Table 2 ) (bf).

Fig.7. HPCCCchromatogramof betalains(mixture2) aftertheseparation inthe highsalt-solvent system by semi-prep HPCCC ( system AI , see Table 2 ). Peak numbersrefer to compounds shown in Table 1 .

Fig. 5. Reconstructed HPLC chromatograms of betalainsafterthe separation in thehigh salt-solventsystem (a mixture 1, solvent system AI , b mixture 2, solvent system

AI)

and ion-pair solvent system with 0.7% HFBA (c mixture 1, solvent system BIV , d mixture 2, solvent system BIV , see Table 2 ) by semi-prep HPCCC.


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Fig.8. HPLCchromatogramof betalains(mixture2) before separationby semi-prepHPCCC(a) and selectedfractionsafter theseparation in the highsalt-solventsystem( AI, see Table 2 ) (bf).

overlap of the rst four components and a complete separa-tion of 17-dNBt ( 5) eluted as a single irregular broad peak. Twopartially resolved dehydrogenated betacyanins (2-dNBt ( 10 ) and2,17-dec-2,3-dHNBt ( 8 )) are eluted in the rst and second peak,respectively, and are well separated from 2,15,17-dec-2,3-dHNBt(6 ) and 2,15,17-dNBt ( 9). Fig. 8 shows the HPLC chromatogram of the crude mixture 2 plus selected fractions from the HPCCC puri-cation run.

3.2.2. HPCCC solvent systems containing ion-pair reagents (TFA,HFBA)

In this study, four solvent systems ( Table 2 , systems BI BIV )containing TFA or HFBA were compared. The presence of TFA orHFBA in the solvent systems at different concentrations inuencesthe stationary phase retention (which is higher at a lower concen-tration of the acids).

Thebest separation results formixture 1 ( Fig.5 C) were obtainedin the system with 0.7% HFBA ( system BIV ) (despite a smaller sta-tionary phase retentionthanin the systemwith0.4% HFBA ( systemBIII)). Comparison of systems BIII and BIV (data not shown) leadsto a conclusion that the amount of acid is a more signicant factordetermining the resolution of the compounds than the retentionof the stationary phase. Most of betalains from mixture 1 are notseparated with solvent systems BI, BII nor BIII at all (data notshown). TFA forms less lipophilic ion-pairs than HFBA with beta-lains which are eluted too early from the coil which makes theireffective separation impossible. For further experiments on semi-

preparative scale, only system BIV was taken. The separation of the dehydrogenated pigments (mixture 2) on analytical scale is notsuccessful in the solvent systems containing TFA ( systems BI-BII )and all compounds are co-eluted (data not shown). HFBA createsmore lipophilic ion-pairs than TFA, therefore, the presence of HFBAsignicantly shifts the analytes to the organic phase, improvingK D values. However, comparing the systems BIII and BIV revealsthat only system BIV could be useful for the dehydrogenated beta-cyanins separation (data not shown), considering that the amountof acid inuences betalains separation.

Based on the initial results obtained for the analytical systems,system BIV wasused ona semi-preparative scale forthe separationof the two groups of betacyanins (the retention of the stationaryphaseis 81.2% in the operated apparatus). Forthe mixture 1, almost

pure fractions are obtained for the less polar neobetanin ( 7) and for

Fig.9. HPLCchromatogramof betalains(mixture1) before separationby semi-prepHPCCC (a)and selected fractionsafterthe separation in theion-pairsolvent systemwith 0.7% HFBA ( BIV , see Table 2 ) (be).

the more polar 2,17-dBt/-dIBt ( 4/4 ) (Fig. 5C). It can be noticed thatneobetanin ( 7 ) is eluted much faster than betanin/isobetanin ( 1/1 )and decarboxy-betacyanins ( 2/2 , 3/3 , 4/4 ). The faster elution of neobetanin ( 7 ) results from theweaker formationof ion-pairs withthe anions due to a lower protonation of its structure.

Bt/IBt (1/1 ) is eluted with just a minor contamination from NBt(7), however, its tailing peak co-elutes with unresolved pairs of 17-dBt/-dIBt ( 2/2 ) and 2-dBt/-dIBt ( 3/3 ) (Fig. 5C). For the puricationof 17-dBt/-dIBt ( 2/2 ) and 2-dBt/-dIBt ( 3/3 ) the high salt system AI (Fig. 5A) is recommended instead. The HPLC chromatograms of the crude injection material and selected puried fractions frommixture 1 can be seen in Fig. 9.

Comparing Figs. 5C and 9 , the elution proles of betalainsobtained from the CCC with ion-pair solvent systems (reversedmode) are completely different from the proles observed in theHPLC system (working also in the reversed mode). For mixture 1,the following elution order in the HPLC system ( Fig. 9) is usuallyobserved: Bt ( 1), 17-dBt ( 2), IBt( 1 ), 17-dIBt ( 2 ), 2-dBt/-dIBt ( 3/3 ),2,17-dBt/-dIBt ( 4/4 ) and NBt ( 7), whereas in the CCC system it is:7 , 1/1 , 2/2 , 3/3 , and 4/4 (Fig. 5C), indicating that 1/1 and 2/2 areeluted as pairs in contrast to HPLC elution. The studied differencesresult from different effectiveness of the interactions betweenselected betalainsand the ion-pair reagents,which inuences theirseparation and elution order. In particular, the differences in ion-ization properties are observed between the decarboxylated anddehydrogenated betacyanins (e.g. very fast elution of NBt ( 7)). Elu-tion of betalains in HPLC is based on their polarity, the more polarpigments are eluted as rst. In CCC the situation is similar, morepolar betanins and decarboxy-betanins are eluted depending ontheir polarity except NBt ( 7). Neobetanin (less positively charged

pigment) ( 7 ) presumably does not create stabile ion-pairs withHFBA,therefore, its polarity is not signicantly changed during theCCC separation.

For mixture 2, a goodseparation of dehydrogenated betacyaninsis observed in system BIV (Fig. 5D) on the semi-preparative scale.Interestingly, the compounds are eluted in different order than inthe high salt system AI (Fig. 5B). The different elution order isobserved due to different separation modes (tail-to-head versushead-to-tail) but also because of a formation of ion-pairs withbetalains. Especially a completely different relative retention isobserved for 17-dNBt ( 5) and 2,15,17-dec-2,3-dHNBt ( 6) in boththe systems. Only a slightly higher overlap is observed for 17-dNBt(5) and 2-dNBt( 10 ) in systemBIV and a good separation of 2,15,17-dec-2,3-dHNBt ( 6 ), 2,17-dec-2,3-dHNBt ( 8 ) and 2,15,17-dNBt ( 9 ) is

accomplished.


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