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A New Monolithic-Type HPLC Column For Fast SeparationsKarin Cabrera*, Dieter Lubda

Merck KGaA, R & D Biochemistry and Separation, Frankfurter Strasse 250, 64293 Darmstadt, Germany

Hans-Michael Eggenweiler

Merck KGaA, Medicinal Chemistry, Research Laboratories, Frankfurter Strasse 250, 64293 Darmstadt, Germany

H. Minakuchi, K. Nakanishi

Division of Material Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan

Ms received: September 18, 1999; accepted: December 1, 1999

Key Words:HPLC; monolithic column; silica rod column; fast separations

SummaryThe application of a new silica-based, monolithic-type HPLC-columnfor fast separations is presented. The column is prepared accordingto a new sol-gel process, which is based on the hydrolysis and poly-condensation of alkoxysilanes in the presence of water soluble poly-mers. The method leads to “rods” made of a single piece of poroussilica with a defined pore structure, i. e. macro- and mesopores. Themain feature of silica rod columns is a higher total porosity, about15% higher than of conventional particulate HPLC columns. Theresulting column pressure drop is therefore much lower, allowingoperation at higher flow rates including flow gradients. Conse-quently, HPLC analysis can be performed much faster, as it isdemonstrated by various applications.

1 Introduction

High performance liquid chromatography (HPLC) hasbecome one of the most widely used methods for the analysisof different compound mixtures. Especially in industry it isroutinely used,e.g.for the quality control of products, for themonitoring of analytes in biological matrices, or morerecently for the analysis of compound libraries synthesizedby combinatorial chemistry methods. Nowadays, the numberof samples to be analyzed is constantly increasing. Therefore,methods and tools are needed to drastically reduce analysistime in order to obtain a higher throughput.

One approach to the development of such methods has beento use monolithic HPLC columns. The motivation for thisapproach comes from the realization that monolithic materi-als with large throughpores or channels should lead to higherpermeabilities. A hydrodynamic flow could then be used tospeed up separations. On the other hand, the surface arearequired for the chromatographic adsorption- and desorptionprocess can be obtained by creating “mesopores” on the sur-face of the monolithic skeleton.

Monolithic materials for use in HPLC have recently beenintroduced by different groups [1–17, 29]. Hjerte´n et al. [1–2, 5, 10] prepared materials within a chromatographic tubebased on polyacrylamides. The columns were successfullyapplied for fast bioseparations. Frechetet al. [3–4, 6, 11–12,16–17] described the preparation of either polyacrylates orpoly(styrene-co-divinylbenzene) in the presence of porogensleading to monolithic materials with a permanent macropor-

ous structure. Their chromatographic suitability was demon-strated with the separation of proteins. Monolithic polymershave also been successfully prepared within capillaries andapplied for HPLC and electrochromatography [5, 16–17].

However, the use of polymeric materials in HPLC is accom-panied by several disadvantages. Most polymers are knownto swell in organic solvents. This frequently leads to a lack ofmechanical stability. Furthermore, the structure of polymersusually leads to micropores, which negatively effect the effi-ciency and peak symmetry of columns. Taking into accountall these aspects, monolithic porous inorganic materialsshould be the materials of choice for conventional HPLCapplications with low molecular weight compounds.

Recently, Nakanishiet al. [18–20] developed a new sol gelprocess for the preparation of monolithic silica rod columnswith a bimodal pore structure,i.e. with throughpores andmesopores. The method is based on the hydrolysis and poly-condensation of alkoxysilanes in the presence of water solublepolymers. Tanakaet al. [7–9, 13–14] demonstrated that thismethod allows the preparation of chromatographic columnswith high efficiencies and low column backpressures. This is aresult of the independent control of the sizes of the silica skele-ton and throughpores. Moreover, it was demonstrated that thenew developed monolithic-type HPLC column could be oper-ated at high flow rates maintaining a high efficiency.

Tanakaet al. [21–22] and others [23–25] have also reportedon the preparation of monolithic materials within the confinesof fused silica capillaries. They can be applied for LC andCEC separations. This approach has the substantial advan-tage, that no cladding procedure is needed for their practicaluse in liquid chromatography. Beside this, monolithic capil-laries show even higher permeabilities as compared to con-ventional silica rod columns. However, the general perfor-mance as well as the long term stability have to be improvedin the future.

Here, we report our studies on the suitability of conventionalsilica rod columns for fast separations. Variations of the flowrate, the use of flow gradients as well as its combination withsolvent gradients were used for the optimization of separa-tions with respect to time. Furthermore, silica rod columns

J. High Resol. Chromatogr.2000, 23, (1) 93–99 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 2000 0935-6304/2000/0101–0093$17.50+.50/0 93

Cabrera,Lubda,Eggenweiler, Minakuchi,Nakanishi

94 VOL. 23,JANUARY 2000 J.High Resol.Chromatogr.

wereusedfor theLC/MS analysisof compoundsobtainedbycombinatorial chemistry methods.

2 Experimental

Silica rod columns of the dimension 10064.6mm and5064.6mm will besooncommercially availableproductsofMerck KGaA (Germany). They are preparedaccording to aprocedure published elsewhere[7, 13]. TheparticulateHPLCcolumnsusedin this studyarecommercially available fromMerck KGaA (Germany), namely LiChroCART PurospherRP-18e, 5 lm, 12564 mm and LiChroCART PurospherSTAR RP-18e,3 lm, 5564 mm.

Chromatographywascarriedout using a conventional HPLCsystemfrom Merck/Hitachi (Germany, Japan)consisting of aLaChromL 7100pump,a LaChromautosamplerL 7200, anda LaChromDAD L 7450detector. The systemwasoperatedwith a dataprocessorworking with a LaChrom interface D7000andtheLaChromD 7000HSM software.

LC/MS measurements were carriedout with a systemfromHewlett Packard (USA), HP 1100 SeriesLC/MSD with thefollowing features: Ion-source: electrospray, positive mode;scan:100–1000m/z; fragmentor voltage:60 V; gastempera-ture: 3008C, DAD: 220nm. A splitter was used after theDAD in orderto reducethe flow rateto 0.75mL/min beforeentranceinto theMS.

All solventsusedfor the preparation of the mobile phaseswereof LiChrosolv gradefrom Merck KGaA (Germany).

3 Resultsand Discussion

3.1 Description andChromatographicCharacterization ofSilica RodColumns

Silica rod columns (Figure 1) are monolithic silicas with abimodalporestructure,i. e. with macro-andmesopores[15].

SEM images(Figure 2) of a silica rod-crosssection reveals,that the macroporescanbedescribedasthroughpores,whichpreferentially determine the permeability propertiesof thecolumn. Typically, thesizeof themacroporesis around2 lm.Themesoporeswith a sizeof about13 nm arelocatedon thesilica skeletonand are the oneswhich provide the surfaceareaneeded(ca 300m2/g) for a sufficient chromatographicseparation process.Further featuresof silica rod columnsarelistedin Table 1.

It hasbeenpointedout earlier that monolithic silica rod col-umns possessa much higher total porosity than particulatecolumns [8]. In fact, about15% higher porosity is obtaineddueto the presenceof large throughpores.As a result,silicarod columns possessa much lower column backpressure.Figure 3 showsthe column backpressure at different flowrates.Evenwith a flow rateof 10.42mm/s(corresponding to9 mL/min) a column backpressure of only 154bar isobserved. Consequently, silica rod columns can be operatedat higherflow rates.

Figure 1. Monolithic silica rodcolumnsversussilica particles.

Figure 2. SEMimagesof a crosssectionof silica rodcolumnsshowingtheporousstructurewith throughporesandsilica skeletons.

A NewMonolithic-TypeHPLCColumnForFastSeparations

J.High Resol.Chromatogr. VOL. 23,JANUARY 2000 95

Figure3 showsthe H/u curve of a typical silica rod column,which wasmodified by derivatizationwith C-18 moietiesandendcapping (RP-18e). The column was operated underreversed phase mode. Plate heights of 8.4–9.5lm areobservedat a speedof 0.87–2.92mm/s (corresponding to0.8–2.5 mL/min). Even at a flow rate of 5 mL/min a plateheight of 14.4lm is obtainedwith a corresponding columnback pressureof 87bar. As being pointed out also by otherauthors[7–8, 13], thesedataconfirm that monolithic silicarodcolumnspossessa very high performance.

Theselectivityof silica rod columns (RR-18e) wascomparedto that of a conventional particulate5 lm column (LiChro-CART PurospherRP-18e,12564 mm). Figure 4 shows theseparationof four nonpolar compounds:butyl- andamylben-zeneto determinetheCH2-selectivity ando-terphenyl andtri-phenylene for the steric selectivity. Interestingly, the elutionorderaswell asthea values(aamylbenzene/butylbenzene= 1.54for thesilica rod column and 1.55 for the particulate column) arevery similar on bothcolumns.

3.2 Applicationof Silica RodColumnsfor Fast Separations

3.2.1 HigherFlow Rates

A uniquefeature of silica rod columns is that they providesimultaneouslyhigh separation efficienciesand low columnbackpressures(high permeabilities). This is intrinsically notpossible with particulate columns becausean increaseof

separation efficiency obtainedby reduction of the particlesize resultsnecessarily in an increaseof column back pres-sure.Moreover, as is shown in Figure3, silica rod columnskeeptheir high performanceevenat higherflow rates.There-fore, this type of monolithic column is ideally suited foroperationat higherflow rates. Figure 5 shows theseparationof five b-blocking drugs at different flow rates. As can beseen,at a flow rateof 9 mL/min it is possible to separatethefive componentsin lessthan1 min.

3.2.2 Flow Gradients

Flow gradients have not had any practical relevance withconventional particulate HPLC columnsbecauseof the lim-

Table1. Generalfeaturesof silica rodcolumns.

Dimension: 10064.6mm; 5064.6mmSilica type: highpurity (total metalcontenta 10ppm)Macroporesize: 2 lmMesoporesize: 13nmPorevolume: mL/gSurfacearea: 300m2/gSurfacemodification C-18,endcappedSurfacecoverage 17%C

Figure 3. Plotsof columnbackpressureandplateheightagainstlinearvelocity of mobilephase.Col-umn:silica rod column(RP18e),10064.6mm; mobilephase:acetonitrile/water(60/40;v/v); sample:anthracene.

Figure 4. Chromatographyof non polar compoundson particulatesilica column and silica rod column. Columns: a) PurospherRP18e,5 lm, 12564 mm, b) silica rod column RP 18e,10064.6mm; mobilephase:methanol/water(80/20; v/v); detection:UV 254nm; samples:1)butylbenzene,2) o-terphenyl,3) amylbenzene,4) triphenylene;the earlyelutingpeaksareimpurities.



itations imposedby the column back pressure:higher backpressurewith smaller particles. In contrast to monolithicsilica rod columns, the separation efficiency of particulatesilica columns decreasesdramatically at higher flow rates.This makesthemunsuitable for flow gradients. As silica rodcolumnsshow high performanceeven at higher flow rates,they are ideally suited for the operation of flow gradientsinsteadof solvent gradients.

With solventgradients theelution powerof themobile phaseis graduallyincreased by increasing thecontentof theorganicmodifier. Thusa mixture of compoundswith extremely dif-ferentadsorption properties towardsthe stationary phasecanbe separatedwithin a reasonabletime. However, in order tostarta secondchromatographicrun of thesametype,thesys-tem has to be re-equilibratedto the original state,which istime consuming.

On silica rod columnsflow gradientscan be appliedfor theelution of stronger retardedcompounds.The pump can besimply programmedin sucha way, that chromatographyisfirst performed with “slow” flow ratesfor the separationofcompoundswith low adsorption properties.Af terwards“fast”flow ratescan be usedfor the elution of compoundswithstrongeradsorption properties.A typical example is shownin

Figure 6. Nine b-blocking drugs are baseline-separatedwithin 6 min starting with a flow rate of 2 mL/min. After3 min the flow rate is changedto 5 mL/min and kept foranother3 min. Af terwards,thepumpis programmedto start-ing conditions for a secondrun. No time for re-equilibrationbetweentwo chromatographic runsis needed.

Figure 5. Chromatographicseparationof five b-blocking drugson a silica rod columnat different flow rates:Column:silica rodcolumn RP 18e, 5064.6mm; mobile phase:acetonitrile/0.1%TFA in water (20/80; v/v); flow rate: 1–9 mL/min, detection:UV254nm,samples:1. atenolol,2. pindolol,3. metoprolol,4. celiprolol, 5. bisoprolol.

Figure 6. Chromatographicseparationof b-blocking drugson a silicarod column with a flow gradient.Column: silica rod column RP 18e,10064.6mm, mobile phase:methanol/0.02m phosphatebuffer pH 3(30/70; v/v), flow gradient:0–2 min/2 mL/min, 2–3 min/2–5 mL/min,3–6 min/5 mL/min, detection:UV 254nm;samples:1. bisoprolol-hemi-furate,2.pindolol,3. nadolol,4. pafenolol,5. metoprolol,6. celiprolol, 7.carazolol,8. bisoprolol,9. alprenolol,10.propranolol.



3.2.3 CombinedFlow andSolvent Gradients

Despitethe considerableadvantages of flow gradientsoversolvent gradients, it is sometimes necessary to work withcombined flow and solvent gradientsin order to obtain therequiredselectivitywithin shorttime periods.Figure7 showsthe base-lineseparationof a mixture of phenol derivativeswithin 5 min. The starting flow rate was 3 mL/min; after2.5min it was changed to 5 mL/min. The starting mobilephasecompositionwas65% of a phosphate buffer and35%acetonitrile. Af ter 1.8min it waschangedto 46%acetonitrileandfinally after2.5min to 80%acetonitrile. Re-equilibrationwasthenperformedat thehigh flow ratefor 1 min.

3.2.4 LC/MS with Short Silica RodColumns

The introduction of combinatorial chemistry [26] has pro-vided the medicinal chemist with new tools to accelerate thesynthesis of pharmaceutical compounds,most notably tosynthesize large“lib raries”of compoundsthrough automatedsolid- andsolution-phaseparallel synthesis.

In the solid phasesynthesis approach, the target compoundsare synthesized on crosslinked polymers (i. e. beads)[19].After completed synthesis the products arecleavedfrom theresin, e.g. by treatmentwith trifl uoroacetic acid (TFA) indichloromethane (DCM). Sinceno purification of intermedi-atesin a multistep reaction sequence is possible on the solidphase,oneof the fundamental requirementsof this techniqueis that each reactionstep has to go to completion, ideallywithout the formation of unwanted side products. However,not all synthetic stepsproceedequally efficiently and theyields and purities can vary dramatically. Consequently, theneedhasbeencreated for developingexquisitely rapidmeth-odsfor thecharacterizationof combinatoriallibraries[28].

Figure 7. Chromatographicseparationof phenolson a silica rod col-umn with a flow- and solventgradient.Column: silica rod column RP18e,10064.6mm, mobile phase:acetonitrile(A)/phosphatebuffer (B)at 0 min 65% A/35% B, at 1.8min 54% A/46% B, at 2.5min 20% A/80% B; flow rate: 3 mL/min (0–2.5min), after 2.5min of chromato-graphicrun5 mL/min; detection:UV 254nm;samples:1) 2,4,6-trichloro-phenol,2) pcp, 3) 2,4-dinitrophenol,4) 4-chlorophenol,5) 2-chlorophe-nol, 6) 2-methyl-4.6-dinitrophenol.

Figure 8. LC/MS on silica rod columnswith samplesof combinatorialchemistry. Column: silica rod column RP 18e, 5064.6mm, detection:eitherUV at 220nm (upperchromatogramm)or MS documentedasTIC(total ion current)andsinglemassspectraof theelutedcompounds;sam-ples:1.4min UV (respective1.6min TIC): startingmaterial(molecularweight 268), 2.2min UV (2.3min TIC): side product (MW 401.9),2.9min UV (3.0min TIC) product(MW 450).



LC-MS providesthemedicinal chemistwith automatedtoolsfor the fast qualitative characterization of pharmaceuticalcompounds. Automated, semi-quantitative assessment ofcombinatorial libraries is most readily accomplishedby cou-pling HPLC with UV detectionand ESI-MS. Usually, thechromatography is carried out using a fast gradient contain-ing waterandacetonitrile asthe organic modifier, often withthe addition of small amounts of TFA. The peaks in theobtainedchromatogramscan represent the desiredproduct,remaining starting material or side products respectively.With the additional information provided by ESI-MS, it isnow easyto assign the target compound to a peakand esti-mateits relativepurity. Traditional HPLC methodshowever,do not provide the throughput necessary to handlethe sizeandcomplexity of combinatoriallibraries.

This problem is addressed by the silica rod columns. Theyallow for a substantial increaseof sample throughput withoutcompromising the quality of the separationscompared to“slow” gradientson particulatecolumns.Figure 8 shows theanalysisof a typical sample obtainedby solid phasesynthesisusing short silica rod columns (5064.6mm) for LC/MS.Three peakswere observed representingthe product, sideproduct,andstartingmaterial. They aremonitored by a UVdetectorandsimultaneouslywith the total ion current (TIC)

comingfrom theMS detector. Thecorrespondingmassspec-tra are listed below. Each chromatographic run takes ca3.5min with an injection-to-injection cycle of ca 5 minincluding re-equilibration, reporting, etc. The sameanalysiswasperformed with a small particulatesilica column(Puro-spherSTAR, 3 lm, 5564 mm) using the same instrumenta-tion. 10 min wereneededfor oneanalysis indicatingthat thenumberof samplesto beanalyzedon silica rod columnsis bya factor of 2 higher (Figure 9). The reason is that the silicarod column couldbeoperatedwith a flow rateof 2.2mL/minwith a corresponding column backpressureof pmax = 85 barascompared to the particulate columnwith 0.75mL/min andacorresponding pmax = 215bar.

4 Conclusions

Monolithic silica rod columns canbepreparedwith indepen-dentcontrol of the sizeof silica skeletonsandthroughpores.As a resultchromatographiccolumns with highertotal poros-ity as comparedto particulate ones are obtained. The silicarod columns discussedhere possessseparation efficienciescomparable to 4 lm particulatesilica columns.Furthermore,theymaintain thehigh performanceevenat higherflow rates.Therefore,silica rod columnsareideally suited for fast chro-

Figure 9. Comparisonof a particulatesilica columnanda silica rod column for usein LC/MS with samplesof combinatorialchemistry. Column:PurospherSTAR RP18e,3 lm (upperchromatogram),5564 mm andsilica rodcolumnRP18e,506 4 mm.



matographic separationsusing the flow rate of the mobilephase as one parameter for optimizing separations withrespectto time.

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