Using analytical multidimensional isocratic HPLC methods of separation to isolate active...

Ori

gin

alP

aper

J. Sep. Sci. 2004, 27, 47–52 www.jss-journal.de i 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Victor WongAlan P. Sweeney1)

R. Andrew Shalliker

School of Science, Food andHorticulture, University ofWestern Sydney, HawkesburyCampus, Locked Bag 1797,Penrith South, NSW 1797,Australia

Using analytical multidimensional isocratic HPLCmethods of separation to isolate active constituentsin natural products

Multidimensional reversed-phase HPLC was employed to isolate the active constit-uents from a crude extract of Clerodendrum floribundum R. Br. These active constit-uents were known to have bioactivity against the enzyme xanthine oxidase andpotentially could be employed in the treatment of gout. Using a multidimensional sep-aration approach, rapid isolation of the active constituents was achieved from a com-plex sample matrix. As each separation dimension was isocratic, no equilibration timewas required and consequently the technique shows promise in the scale up to pre-parative levels where high throughput is important.

Key Words: Multidimensional HPLC; Clerodendrum floribundum;

Received: April 7, 2003; revised: October 10, 2003; accepted: October 10, 2003

DOI 10.1002/jssc.200301589

1 Introduction

Clerodendrum floribundum R. Br. (Verbenaceae) is a five-metre tall Australian native shrub found throughout thenorth of the continent, along coastal regions and someinland areas. The Aboriginals have a high regard for themedicinal qualities of C. floribundum, especially for thetreatment of inflammation, infection, coughs, colds, head-ache, fever, stomach or general body pains. In addition,C. floribundum is also used for numerous other illnessesincluding diarrhoea, minor skin complaints, and bronchialcongestion [1–6].

Sweeney et al. [7, 8] obtained an ethanol (EtOH)/water(H2O)/ethyl acetate (EtOAc) extract of C. floribundum andfound that this possessed significant biological activitytowards the enzyme xanthine oxidase (XO). XO is respon-sible for the formation of uric acid from hypoxanthine andxanthine within the body. Accumulation of uric acid withinjoints leads to the medical condition known as gout. Thus,inhibition of this enzyme shows potential in the treatmentof such a condition.

Purification of the extract media using liquid chromatogra-phy and solvent extraction procedures led Sweeney andcoworkers [7, 8] to isolate two significant regions of bioac-tive importance; their identities are yet to be elucidated. Inthe present study we aim to expedite the isolation process

of these two active constituents using an instrumentalmultidimensional separation approach. Our ultimate aimis to extend the protocol to preparatory scale fractionationof the bioactives.

Multidimensional chromatography (MD-HPLC), alsoknown as coupled column chromatography, is a powerfulmodern instrumental technique where fractions from oneseparation system are selectively transferred to one ormore secondary separation systems, commonly perform-ed through column switching valves while utilizing two ormore columns [9]. The increased separation space orpeak capacity of a multidimensional system affords betterresolution of components than a single dimensional sep-aration. Furthermore, a multidimensional approach to theseparation problem allows the chromatographer to focusa specific isolation towards target compounds and tunethis separation so that they may be rapidly isolated from acomplex mixture.

A number of workers have employed multidimensionalseparations for the isolation of specific components fromcomplex samples. For example, Posluszny and Weinber-ger [10] developed a MD-HPLC system for the detectionof drug substances in blood serum or plasma. The firstdimension consisted of a micellar mobile phase usingSDS as a modifier. This dimension functioned essentiallyas sample extraction and preconcentration. The seconddimension utilized a coupled online reversed phase med-ium with an appropriate modifier for analytical separation.Two solvent pumps and one six-port switching valve wereincorporated in their system. The drugs of interest werecarbamazepine, chloramphenicol, procainamide, furose-mide, quinidine, and propranolol. By coupling a restrictedaccess medium (RAM) bovine serum albumin (BSA) C18

Correspondence: R. Andrew Shalliker, School of Science, Foodand Horticulture, University of Western Sydney, HawkesburyCampus, Locked Bag 1797, Penrith South, NSW 1797, Australia.Fax: +61 2 4570 1621. E-mail: [email protected]) Present address: Novartis Animal Health Australasia Pty. Ltd.,

Yarrandoo R & D Centre, Department of Natural Products, 245Western Road, Kemps Creek, NSW 2171 Australia.

47

48 Wong, Sweeney, Shalliker

column to a conventional C18 column, Cass and cowor-kers [11] employed MD-HPLC in the analysis of amoxycil-lin (an antibiotic substance) in human plasma. The firstdimension RAM column served as an analyte preconcen-trator while separation occurred in the second dimensionanalytical C18 column. A unique feature of such a systemis that undesired macromolecules from the sample matrixare excluded while the hydrophobic micromolecules areselectively retained. As with Posluszny and Weinber-ger [10], two solvent pumps and one six-port switchingvalve were incorporated into their system design. The pre-ceding system designs demonstrated the high selectivityachieved in the first dimension (the preconcentrationstep). However, such a system is incapable of heart-cut-ting a target section of a complex chromatogram for elu-tion into the next dimension, at least in a time efficient pro-cess. To achieve this, two or more solvent pumps andswitching valves must be incorporated into the systemdesign.

Stopher and Gage [12] employed a system that utilizedtwo switching valves, three solvent pumps and three col-umns in the determination of voriconazole (a broad spec-trum antifungal substance) in human plasma. Sampleswere first injected into a size exclusion column to separatethe plasma from the target analyte and an internal stan-dard, after which the eluate was transferred into a precon-centrating column. The trapped and concentrated ana-lytes were subsequently loaded into an analytical C18 col-umn for further separation. Apart from the occasionalblockages reported by the authors, the system has provento be very reliable. Sweeney et al. [13] demonstrated theuse of a MD-HPLC approach in the separation of lowmolecular weight polystyrene oligomers and stero-isomers. The system employed a heart-cutting techniquewhere the first separation process incorporated a C18 col-umn and a methanol mobile phase. Target oligomerswere heart-cut into the second separation process incor-porating a carbon-clad zirconia column and an acetonitrilemobile phase. Two solvent pumps and switching valveswere incorporated into the system design. The automatedtwo dimensional separation was found to be comparableto manual sample manipulation in each individual separa-tion step. The group also highlighted the high reproducibil-ity achieved for routine continuous operation over aneight-hour period.

In the present study we employ a MD-HPLC system com-prising two solvent delivery systems and two switchingvalves for the isolation of bioactives from an Australiannative plant. In order to achieve the goal of a rapid separa-tion process, isocratic conditions were employed to avoidre-equilibration time following gradient elution. The sep-aration of complex naturally occurring samples, such asthose derived from plant species, usually leads to a multi-tude of components, with large variations in retention fac-

tors. That was the case in the present work and hencecareful consideration of stationary phase surfaces for themultidimensional system was required prior to the suc-cessful application of the separation protocol. This wasdiscussed in previous studies by Sweeney et al. [8] andforms the starting point for the current study.

2 Experimental

2.1 Chemicals and chromatography columns

HPLC grade acetonitrile (ACN) was obtained from Mal-linckrodt, Australia. Milli-Q water was obtained in-houseand filtered through a 0.2 lm filter. A Luna CN (nitrile) col-umn (15064.6 mm, 5 lm Pd) (Phenomenex, PennantHills, NSW 1715, Australia) was used in the first dimen-sion and a C18 Nucleosil column (15064.6 mm, 3 lm Pd)(Alltech Associates, Baulkham Hills, NSW 2153, Austra-lia), prepared in-house using the downward slurry tech-nique [14], was employed in the second dimension.

2.2 Plant material

Plant material was collected from Mount Annan BotanicGarden, Mt Annan, New South Wales, Sydney, Australia.Plant material prior to solvent extraction was air-dried atroom temperature and crushed using a laboratory cuttingmill (Fritsch GmbH, Idar-Oberstein, Germany). Plant sam-ples were solvent extracted at room temperature in etha-nol (EtOH)/water (H2O)(80%/20%) for 3–5 days. Theextract was filtered and the filtrate concentrated to dry-ness in vacuo using a rotary evaporator.

2.3 Equipment

Chromatographic experiments were performed on aWaters LC system incorporating two 600 controllers, a717plus autosampler, two 2487 dual 9 UV detectors andMillennium32 version 4.00 software running on a CompaqEVO D500 Pentium 4 1.6 GHz PC with 256 Mb RAM. Theoperating system (OS) was Windows 2000 Professional(Service Pack 2). Column switching was achieved usingtwo 6-port, 2-position switching valves fitted with micro-electric two position valve actuators (Valco InstrumentsCo. Inc., Houston, TX, USA). Valve switching was con-trolled via the onboard Millennium32 software. The dia-grams in Figure 1 illustrate the design of the system.

Photodiode array detection was obtained using a Shi-madzu LC system (Shimadzu Scientific Instruments,Rydalmere, NSW, Australia) incorporating a LC-10Aipumping system, SPD-M10Avp diode array detector,SCL-10Avp system controller and Shimadzu Class-VPversion 5.03 software onboard a Pentium III 500 MHz PCsystem with 64 Mb RAM running Windows 98 Second Edi-tion.


Using analytical multidimensional isocratic HPLC methods 49

2.4 Chromatographic separations

Plant extracts were dissolved in methanol and injecteddirectly into the LC system without any form of filtration.Injection volumes were typically 10 lL. All flow rates were1.0 mL/min. Exact operation of the multidimensional sep-arations is described in the appropriate section of the text.

3 Results and discussionA crude extract from the plant species Clerodendrum flor-ibundum R. Br., was obtained by extracting the dried plantmaterial in an 80%/20% EtOH/H2O solvent. Subsequentanalysis of the crude extract using one-dimensional gradi-ent elution HPLC employing a C18 column and MeOH/H2O mobile phase yielded the chromatogram shown inFigure 2. Detection was recorded using a photodiodearray detector in order to gain a greater understanding ofthe sample complexity. The peaks labeled as CF1 andCF2 have previously been identified as the bioactive spe-cies of interest [8], and they are the target components ofthis work. Although CF1 and CF2 are major componentsof this plant species the lengthy run time and complexityof the sample in the region of interest would make suc-cessful purification time consuming and difficult, espe-cially considering that an additional 10 minutes of separa-tion time following completion of the gradient is required toelute the late eluting species. This could perhaps bedecreased by employing a ternary solvent system, but at

the expense of simplicity. The wide range in retention fac-tors in this sample would prohibit isocratic separationsemploying a single separation dimension.


Figure 1. Schematic diagram of two-dimensional HPLC column switching system. (P1-P2): Low pressure qua-ternary solvent delivery systems; (V1-V2): 6-port 2-position switching valves; (C1): Column in first separationdimension; (C2): Column in second separation dimension. (a) System configuration for elution on C1 and C2(back-flush of sample loop). (b) System configuration for the elution of a band from C1 onto sample loop.

Figure 2. Chromatogram of a crude extract (80%EtOH/20%H2O) from C. floribundum obtained using gradient elu-tion chromatography on a C18 column. The active constitu-ents are indicated as CF1 and CF2. Mobile phase (bi) of 95%H2O/5% MeOH running to a final composition (bf) of 100%MeOH over 19 minutes. Flow rate was 1.0 mL/min, with aninjection volume of 20 lL. Detection at 270 nm. Column:ValuePak C18 5 lm (25064.6 mm).


3.1 Instrumentation

The multidimensional HPLC system that was used in thisstudy is illustrated in Figure 1. A variety of different sys-tems could be configured with different numbers of valvesetc., however, when doing so due consideration should begiven to the ultimate aim of scaling the separation processto preparative levels. In that regard, systems that minimisethe cycle time will be the better performing systems.These systems require each dimension be operated inde-pendently. Hence two solvent delivery systems are prefer-able. An acetonitrile (ACN)/H2O mobile phase was used inthe first dimension (Nitrile column) in order to separate thecomponents of the crude C. floribundium sample into sev-eral regions (Figure 1.a). Isolation of the components CF1and CF2 into pure bands using an isocratic system thatemployed a nitrile column was not possible. The samplecomponents to be further fractionated following elutionfrom C1 were transported to a sample loop (Figure 1.b)situated between C1 and C2. The system was thenreverted to the position shown in Figure 1.a where furtherseparation of the heart cut section in the second dimen-sion occurred. Separations in both dimensions occurredconcurrently, speeding the separation process comparedto gradient separations in a single dimension, or even mul-tidimensional systems, which have only a single solventdelivery system [8].

3.2 Separation of Clerodendrum floribundiumcrude extract

Figure 3 illustrates the isocratic chromatographic separa-tion of a crude sample of C. floribundium on a nitrile col-umn, using a mobile phase composition (bi) of 40% H2O/60% ACN. Phosphoric acid was added to the mobilephase to eliminate irreversible adsorption and to improvepeak shape on the medium polar stationary phase. Poorseparation was observed for almost all components in thesample and co-elution of CF1 and CF2 (along with numer-

ous components) was observed in this separation. Thetotal run time was less than eight minutes. No separationwith suitable resolution of the target components wasachieved under isocratic or gradient conditions whenemploying the nitrile column. However, the separation ofCF1 and CF2 could be improved through the applicationof gradient elution using a C18 column (Figure 2).Although the separation shown exceeds thirty minutes(not including re-equilibration time) this could be reducedby applying step gradients (20%/min) [15], but even then,the late eluting components that required removal in100% acetonitrile resulted in cycle times around 25 min-utes.

3.3 Heart-cutting column-switching studies

Using a 40% H2O/60% ACN solvent composition in thefirst dimension (nitrile column) and 55% H2O/45% ACN inthe second dimension (Nucleosil C18 column), a series ofmoving-average 200 lL heart-cut fractions from the firstdimension to the second were conducted. These fractionsspanned the region between 1.80 min and 2.40 min asshown in Figure 3.

The series of chromatograms depicted in Figure 4 illus-trate the change in relative component composition offractions CF1 and CF2 following separation on the seconddimension. Note in the chromatograms illustrated in Fig-ure 4 there is a progressive increase in the retention timesof all components due to the subsequent delay in heartcutting each of the sections from the first dimension. Forvisual clarity we have not corrected for this time, butemphasise that the change in retention time is a systemartifact generated by the change in the heart-cut time. The


Figure 3. Chromatogram of a crude extract (80%EtOH/20%H2O) from C. floribundum obtained using isocratic elu-tion chromatography on a nitrile column. Mobile phase 40%H2O/60% ACN. Flow rate was 1.0 mL/min, injection volume10 lL and detection at 370 nm. Column: Luna CN column(15064.6 mm, 5 lm Pd).

Figure 4. Chromatograms in the second dimension illustrat-ing the relative change in the concentrations of CF1 and CF2following heart cutting from the first dimension. Mobilephase: first dimension 40% H2O/60% ACN, second dimen-sion 55% H2O/45% ACN. Columns: first dimension Luna CNcolumn (15064.6 mm, 5 lm Pd), second dimension Value-Pak C18 5 lm (25064.6 mm). Injection volume 10 lL. Heartcut volume 200 lL. Other conditions as per Figure 3. Chro-matograms a, b, c, d, and e show heart-cut time intervals of1.80–2.00 min, 1.90–2.10 min, 2.00–2.20 min, 2.10–2.30 min, and 2.20–2.40 min respectively.

Using analytical multidimensional isocratic HPLC methods 51

maximum response for CF1 was observed to occur for afraction cut at 2.10 min to 2.30 min (Figure 4, Curve d),while that for CF2 was in the region cut at 2.20 min to2.40 min (Figure 4, Curve e). This result shows the het-erogeneity of CF1 and CF2 in the first dimension, andcould ultimately have ramifications in a scale up to pre-parative conditions if the aim is to isolate both componentsin a single separation process and maximise samplerecovery. The total run time for this analysis was in theorder of eight minutes although recovery of the fractionsof interest from the injected sample was at best 41% forCF1 and 58% for CF2, based on the total amount of CF1and CF2 in the injection plug.

In general, experiments involving 200 lL cuts into the sec-ond dimension yielded good separation of our target com-ponents within the time-frame of less than eight minutesfor both separation dimensions. However, in the majorityof instances, band widths were still broad, possibly as aconsequence of solvent effects or an overload in theapparent injection volume. As mentioned before, this wasdeemed acceptable given our ultimate scale-up objective.In any case, a series of heart-cutting exercises were con-ducted on an analytical scale by eluting only 100 lL loads,between 2.00 min and 2.50 min, from the first dimensioninto the second dimension. Solvent composition in the firstdimension was maintained at 40% H2O/60% ACN, while60% H2O/40% ACN was employed in the second dimen-sion. Figure 5 shows the resulting heart cut separationsfor the components CF1 and CF2. Peak shape wasimproved. The maximum responses for CF1 and CF2were observed to occur for a fraction cut at 2.20 min to2.30 min (Figure 5, Curve c). By decreasing the volume ofthe cut fraction to 100 lL recoveries decreased to 38%

and 46% for CF1 and CF2 respectively. Purity determina-tion for CF1 and CF2 was performed between the wave-lengths of 200 nm and 800 nm. The purity of both fractionswas in the order of 95% [15].

While we have illustrated the successful heart-cuttingmultidimensional separation of active constituents fromthis plant material on an analytical scale, the preparativecollection of the same active constituents using this tech-nique is currently being investigated. The importance ofthe current method, however, lies in its potential ability todetermine the appropriate separation conditions for largerscale separations. Such separations may be performedwith minimal sample preparation and consequentlyreduce the possibility of sample lose or degradation duringextraction processes. At any rate, an important issue thatneeds to be addressed in the scale up process is improv-ing the recovery of sample. This may limit the separationprocess to the isolation of one active only per separation.In that regard, the medical significance of the recoveredconstituent would undoubtedly drive the experimental out-come.

4 ConclusionMultidimensional reverse-phase HPLC incorporating acolumn-switching technique was employed in the analyti-cal fractionation of two known biologically active compo-nents (CF1 and CF2) found in the Australian native plant,Clerodendrum floribundum R. Br. In general, the tech-nique shows a high degree of promise, although improve-ments in the recovery of the sample need to be addressedif the preparative scale is to be realised.

Acknowledgements

Two of the authors (VW and APS) wish to acknowledgethe receipt of an Australian Postgraduate Award (APA).All authors also wish to acknowledge the assistance ren-dered by Ms. K. Mayfield for proofreading this manuscript.In addition, the authors would like to extend their gratitudeto Mr. Richard Johnstone of Mount Annan Botanical Gar-dens, Sydney, Australia, for supplying the necessary plantmaterials.

References[1] Aboriginal Communities of the Northern Territory of Austra-

lia, Traditional Aboriginal Medicines in the Northern Territoryof Australia, Conservation Commission of the Northern Ter-ritory, Darwin, Northern Territory, Australia, 1993.

[2] E.V. Lassak, T. McCarthy, Australian Medicinal Plants,Reed Books Australia, Kew, Victoria, Australia, 1997.

[3] J. Brock, Native Plants of Northern Australia, Read Austra-lia, Chatswood, New South Wales, Australia, 1993.

[4] A.B. Cribb, J.W. Cribb, Useful Wild Plants in Australia, Wil-liam Collins Pty. Ltd., Sydney, New South Wales, Australia,1982.


Figure 5. Chromatograms in the second dimension illustrat-ing the relative change in the concentrations of CF1 and CF2following heart cutting from the first dimension. Mobilephase: first dimension 40% H2O/60% ACN, second dimen-sion 60% H2O/40% ACN. Heart cut volume 100 lL. Otherconditions as per Figure 4. Chromatograms a, b, c, d, and eshow heart-cut time intervals of 2.00–2.10 min, 2.10–2.20 min, 2.20–2.30 min, 2.30–2.40 min, and 2.40–2.50 min, respectively.


[5] G. Wightman, D. Jackson, L. Williams, Alawa EthnobotanyAboriginal Plant Use from Minyerri Northern Australia, Con-servation Commission of the Northern Territory, Darwin,Northern Territory, Australia, 1991.

[6] G. Wightman, J.G. Roberts, L. Williams, Mangarrayi Ethno-botany Aboriginal Plant Use from the Elsey Area NorthernAustralia, Conservation Commission of the Northern Terri-tory, Darwin, Northern Territory, Australia, 1992.

[7] A.P. Sweeney, S.G. Wyllie, R.A. Shalliker, J.L. Markham, J.Ethnopharmacology 2001, 75, 273–277.

[8] A.P. Sweeney, ,Studies Towards the Development of TwoDimensional High Performance Liquid ChromatographicSystems for the Separation of Complex Samples’, Ph.D. the-sis, University of Western Sydney, Australia, 2003.

[9] K. Robards, P.R. Haddad, P.E. Jackson, Principles andPractice of Modern Chromatographic Methods, AcademicPress, 1994.

[10] V.J. Posluszny, R. Weinberger, Anal. Chem. 1988, 60,1953–1958.

[11] Q.B. Cass, R.F. Gomes, S.A. Calafatti, J. Pedrazolli, J.Chromatogr. A 2003, 987, 235–241.

[12] D.A. Stopher, R. Gage, J. Chromatogr. B 1997, 691, 441–448.

[13] A.P. Sweeney, S.G. Wyllie, R.A. Shalliker, J. Liq. Chroma-togr. Rel. Technol. 2001, 24, 2559–2581.

[14] A.P. Sweeney, V. Wong, R.A. Shalliker, Chromatographia2001, 54, 24–30.

[15] V. Wong, R.A. Shalliker, ,The Isolation of the Active Consti-tuents in Natural Materials Using ,Heart-cutting’ IsocraticReversed Phase-Reverse Phase Two-Dimensional LC ’,Presented at Prep2003, San Francisco June 28–July 2,2003.


Using analytical multidimensional isocratic HPLC methods of separation to isolate active...

Documents

Transcript of Using analytical multidimensional isocratic HPLC methods of separation to isolate active...