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J. Sep. Sci. 2004, 27, 843–852 www.jss-journal.de i 2004WILEY-VCH Verlag GmbH&Co. KGaA,Weinheim

Karin Cabrera

Merck KGaA, Frankfurter Str.250, D-64293 Darmstadt,Germany

Applications of silica-basedmonolithicHPLC columns

The recent invention and successive commercial introduction of monolithic silicacolumns has motivated many scientists from both academia and industry to studytheir use in HPLC. The first paper on monolithic silica columns appeared in 1996. Cur-rently about 200 papers have been published relating to applications and character-ization of monolithic silica columns, including monolithic capillaries. This reviewattempts to give an overview covering various aspects of this new column type in thefield of high throughput analysis of drugs and metabolites, chiral separations, analysisof pollutants and food-relevant compounds, as well as in bioanalytical separationssuch as in proteomics. Some of the applications are described in greater detail. Thenumerous publications dealing with the physicochemical and chromatographic char-acterization of monolithic silica columns are briefly summarized.

KeyWords:Monolithic column; Silica; Chromolith; Characterization; Applications; Review;

Received: April 19, 2004; revised: April 30, 2004; accepted: May 4, 2004

DOI 10.1002/jssc.200401827

1 Introduction

In the last 30 years high performance liquid chromatogra-phy (HPLC) has become one of the most frequently usedmethods for the analysis of mixtures of compounds in avariety of fields, including quality control of drugs, pharma-cokinetic studies, and determination of pollutants or foodadditives. The heart of each HPLC method is the column,which enables the resolution of compounds based uponselectivity and column performance. Generally, HPLC col-umns consist of a tube packed with 3–5 lm porous silicamicroparticles. The quality of such a column, i.e. its sep-aration performance, is mainly determined by the particlesize and its distribution and the quality of the packing ofthe particles within the column. In contrast to such HPLCcolumns packed with particulate materials, monolithic col-umns are made of a single piece of porous silica, which isalso called a “silica rod”. These rods are prepared by apolymerization process either in situ in a column tubesuch as in glass tubes or fused silica capillaries, or in a col-umn mould in which the monolith can later be replaced. Insitu preparation of monolithic columns has the advantagethat no further encapsulation of the porous monolith in asolvent and pressure resistant tube is needed. However,this approach is not compatible with monolithic silica col-umns having larger diameters such as 4.6 mm, due toshrinkage, which occurs during the sol-gel preparationprocess. Therefore in the second step, monolithic silica

rods with a conventional diameter have to be coated orclad with a suitable material such as poly(ether etherketone) (PEEK) to which the column end fittings can beattached for use in HPLC systems (Figure 1).

Monolithic HPLC columns are prepared from eitherorganic polymers, such as polymethacrylates, polysty-renes or from inorganic polymers, such as silica. Hjert�net al. [1, 2] were the first in 1989 to report a continuous-bed column made of compressed polyacrylamide. Sincethen, numerous groups have developed organic polymermonoliths using radical polymerization techniques withdifferent monomer compositions [2–10]. Their work hasbeen reviewed in various papers [11–13, 76] and will notbe discussed here.

The preparation of silica based monoliths started as longago as the early 1990s with the invention of silica rod col-umns [14–20]. A literature survey (1989–2003) revealedthat the number of published papers concerning silicabased monoliths is clearly increasing and this field hasbeen reviewed several times [74–75]. Figure 2 showsthe number of published papers on silica-based monolithsover the last years in comparison to the total amount ofpapers dedicated to all kinds of monoliths for HPLC. Theimpressive increase in the number of papers on silicabased monoliths was stimulated by the introduction of thefirst commercially available product, the ChromolithTM col-umns, on the market in 2000 [21–24]. This can also beinterpreted as a sign of the wide acceptance of this newtype of HPLC-columns. This review will focus only onthese silica basedmonolithic HPLC columns.

Correspondence: K. Cabrera, Merck KgaA, Head of R & D Ana-lytical Chromatography, Frankfurter Str. 250, D-64293 Darm-stadt, Germany. Fax: +49 6151 72917730.E-mail: [email protected].

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2 History

The first paper describing the application of porous silicarods for HPLC was published by Tanaka et al. in1996 [14]. They were prepared using a new sol-gel pro-cess developed by Nakanishi et al. [25–27], based on thehydrolysis and polycondensation of tetramethoxysilane(TMOS) in the presence of polyethylene oxide (PEO)used as a template. The phase separation during the poly-condensation step leads to monolithic materials with co-continuous silica structures having defined macro- andmesopores. The first porous silica rods had a columndiameter of about 7 mm and a length of about 83 mm.They were encased in heat shrinking PTFE tubes andtested in a module used for radial compression of col-umns [14]. With these first monolithic silica columns,Tanaka et al. [14] demonstrated their unique chromato-graphic properties, namely the simultaneous high perme-ability and high performance.

Another approach to silica monoliths was described byFields et al. [28] who prepared a silica xerogel from a

potassium silicate solution. In order to avoid the laboriousencasing of silica rods, he simply filled 320 lm ID fusedsilica capillaries with the xerogel and derivatized their sur-face in a subsequent step using dimethyloctadecylchloro-silane. The column possessed very low efficiency. How-ever, the separation of a mixture of polycyclic aromatichydrocarbons could be achieved.

In1998,Horvathetal. [29]publisheda thirdapproach,com-prisingpackingof fusedsilicacapillarieswithoctadecylated(ODS) 6 lm particles followed by a thermal treatment toform monolithic columns. After sintering, the monolithicpacking was reoctadecylated in situ with dimethyloctade-cylchlorosilane. These columnswere used for l-HPLCandcapillaryelectrochromatography (CEC)andexhibitedgoodefficiencies characterized by a theoretical plate height ofHmin = 16 lm for l-HPLC and 8 lm for CEC. A similarapproach to the preparation of monolithic silica capillariesbysinteringparticleswasusedbyAdamet al. [87].

Finally, Zare et al. [30] prepared monolithic silica capil-laries by incorporation of 3–5 lm ODS-particles into a


a) b)

Figure 1. a) Monolithic silica rods; b) Monolithic silica columns ChromolithTM clad with poly(ether ether ketone) and equipped withcolumn end fittings.

Figure 2. Total number of paperspublished on monolithic columnsfor HPLC during the time period1989–2003 and the share of pub-lications concerning silica-basedmonoliths.

Applications of silica-basedmonolithic HPLC columns 845

sol-gel solution, which was filled into a fused silica capil-lary. This approach led to columns that were used in CEC.They were tested with a mixture of aromatic compoundsand exhibited efficiencies of up to 80,000 plates/m. Sev-eral other authors [88–97] have also prepared monolithicfused silica capillaries by sol-gel bonding of particles in aslightly different way. Fused silica capillaries were firstpacked with silica particles, e.g. small-pore [90–92] andlarge-pore ODS [91, 93, 94], mixed-mode ODS/SCX [91,95], and C30-silica particles [96, 97]. In a second step, asol-solution was introduced into the particle packed capil-lary, thus forming a monolith by chemical bonding of theparticles. A column efficiency of 410,000 plates/m in CECwas obtained using a sol-gel-bonded 3 lm 1500 A ODScolumn.

In 1998 Tanaka et al. [31] reported successful in situ pre-paration of monolithic silica in 100 lm ID capillaries byadapting the original sol-gel method developed by Naka-nishi et al. [25–27] to that column format. A furtherimprovement of this technique used hybrid silanes suchas TMOS/MTMSmixtures, which allow the in situ prepara-tion of capillary monoliths with a diameter of up to 500 lm.

Currently, numerous ongoing activities are traceable, aim-ing at further development and improvement of monolithicsilica columns. All these activities focus on the preparationof monolithic silica capillaries in order to avoid the difficultencasing of the monolith. However, capillary columns canonly be used with special instrumentation, such as LC/MStechniques. Conventional HPLC systems, being widelydistributed all over the world, can usually be operated onlywith HPLC size columns with diameters in a range of 2–4.6 mm. Monolithic silica columns of this size cannot beprepared in situ. As a result, the Merck KgaA companydeveloped a proprietary technology for the cladding of4.6 mm ID silica rod with a resistant PEEK polymer, lead-ing to monolithic columns, which are encased in a solvent-and pressure resistant material. Monolithic silica capil-laries, which are presently also commercially availableunder the trade name ChromolithTM CapROD(100 lm6150 mm), are discussed elsewhere in thisissue.

3 Physical and chromatographiccharacterization

3.1 Permeability

The above-described sol-gel process [25–27] typicallyinvolves the phase separation due to the polymer tem-plate and sol-gel transition due to polycondensation of thesilica precursor, which leads to a co-continuous silica net-work with a defined pore structure. Further thermal treat-ment results in monolithic silica columns with a bimodalpore structure consisting of macro- or through-pores andmesopores in the silica skeleton. A typical SEM picture of


a)

b)

c)

Figure 3. SEM-picture of the typical porous structure ofmonolithic silica columns (a), the mesoporous structure ofthe silica skeleton (b), and the macropores or throughpor-es (c).

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the porousmonolithic silica structure is shown in Figure 3.The sol-gel process developed by Nakanishi et al. [25–27] allows independent control of the pore size. In otherwords, it is possible to prepare monolithic silica columnswith smaller or larger macropores in the range of 1–8 lmwhile keeping the size of mesopores constant at 13 nmand vice versa. This makes the monolithic silica columnsunique compared to their packed counterparts.

Comparison of monolithic and packed columns revealsthat the macropores can be seen as the equivalent of theinterparticle void volume of a particle packed column.Consequently, the permeability or column back pressureis determined by the macropores on the one hand and theinterparticle voids on the other hand. In the case of theparticle packed columns the interparticle volume dependsstrongly on the particle diameter (dp), i.e. columns withsmall particles (e.g. a 3 lm) show small permeabilitieswhereas columns with big particles (e.g. >11 lm) showbig permeabilities. On the other hand the particle sizedetermines the performance of a column, i.e. the platenumber (N) is inversely proportional to the particle diam-eter. Therefore, performance and permeability cannot becontrolled independently with particle packed columns.With monolithic columns it is possible to control indepen-dently both permeability and performance. This can bedone by control over the macro- and mesopore size. Thisunique possibility with monolithic columns has led Guio-chon [32] to make the following statement “The recentinvention and development of monolithic columns is amajor technological change in column technology, indeedthe first original breakthrough to have occurred in this areasince Tswett invented chromatography, a century ago.”

The most widely used monolithic silica columns includemacropores with a size of around 2 lm and mesopores ofabout 13 nm in size. The total porosity of such a column isabout 85% which is 15–20% higher compared to a typicalcolumn packed with 5 lm particles. The total column por-osity et is the sum of the internal porosity ei due to themesoporous structure and the external porosity ee arisingfrom the throughpores:

et = ei + ee (1)

The internal and external porosity can be simply deter-mined by inverse size-exclusion chromatography (ISEC).Guiochon et al. [32] confirmed the bimodal pore structurethrough their experiments. Furthermore, they showed that80% of the total porosity can be ascribed to the macro-pores and 15% to the mesopores. Therefore, the externalporosity of monoliths is nearly twice that found in conven-tional particle packed columns, whereas the internal por-osity is slightly higher in particulate silica columns. In atheoretical approach with equivalent particle dimensions,Tallarek et al. [33] have found that monolithic silica col-umns exhibit a permeability equivalent to a column packed

with 11 lmbeads. This is in accordance with experimentalstudies where columns having different particle sizeswere used [34].

A direct result of the high external porosity typical of mono-lithic silica columns is their extremely low column backpressure. Figure 4 shows the column back pressure atdifferent flow rates for a monolithic silica column and com-pares it to conventional columns packed with 3.5 and5 lm particles. Clearly, the back pressure is lowest for themonolithic columns and they can therefore be operated athigh flow rates leading to fast separations. Figure 5shows the separation of five drugs at different flow ratesfrom 1 to 9 mL/min. It is possible to separate the five com-pounds within oneminute at the highest flow rate.

3.2 Separation efficiency

Another unique feature of monoliths is the high columnefficiency, even at high linear flow velocities. Figure 6shows the van Deemter curve for a monolithic silica col-umn in comparison to conventional ones. The columnpacked with 3.5 lm particles cannot be operated at highervelocities because of the high flow resistance leading tohigh column back pressure. The minimum of the vanDeemter plot of the monolithic column represents a theo-retical plate height H of 8 lm, which corresponds to125,000 plates/m. This efficiency equals that of good col-umns packed with 3.5 lm particles. Tallarek et al. [33]used their theoretical approach including equivalent parti-cle dimensions and calculated that the performance ofmonolithic columns is equivalent to that of columnspacked with 1 lm particles. Indeed, the author of thispaper believes that many improvements are possible inthe field of monolithic columns with respect to column effi-ciency. It seems realistic to expect that monolithic col-umns with 200,000 plates/m will be available in the near


Figure 4. Comparison of back pressure in the monolithic andconventional column packed with silica particles at differentflow rates.


future. One prerequisite for this is an optimal radial homo-geneity of the silica rods, which is probably not achievedat the moment.

With the currently available monolithic columns is possibleto obtain high efficiencies by serial coupling of severalunits. Thus fourteen ChromolithTM Performance RP-18ecolumns representing a length of 1.4 m have been con-nected in series for the separation of six alkylben-zenes [86]. The measured plate number of this long col-umnwas 108,000 with a corresponding column back pres-sure of only 11.7 MPa at a flow rate of 1 mL/min. The cou-pling of several monolithic columns leading to high columnefficiency permits the separation of complex analyte mix-turesandsimilar compoundssuchas isomers.Someappli-cationsof this kindarealreadyavailable [35–37, 72, 77].

Clearly, the monolithic silica columns possess a muchhigher permeability than packed columns with a compar-able efficiency, which makes them more valuable. Thisfact can be confirmed using separation impedance Edefined in Eq. (2), which was originally derived by Knoxet al. [38]:

E = Dp6 to/N 2 6 g (2)

where Dp is the pressure drop, to is the time, N is the platenumber, and g is the viscosity of the mobile phase. Fig-ure 7 shows the plots of E at different linear flow velocitiesfor monolithic columns and, for comparison, for columnspacked with 3.5 and 5 lm particles. The lowest values ofE are obtained for the monolithic column. A value like thiscannot be reached with particulate columns since theirinterparticular volume cannot be controlled independently


Figure 5. Separation of five b-blocking drugs using a monolithic silica column Chromolith Performance RP-18e, 10064.6 mm atdifferent flow rates leading to a separation time of 1 min.

Figure 6. Comparison of the van Deemtercurves for the monolithic and conventional col-umn packed with silica particles of differentdiameters.

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of the particle size, which in turn determines the columnefficiency.

3.3 Adsorption capacity

Comparative studies of the adsorption and saturationcapacity of monolithic and packed silica columns usingfrontal analysis for four different compounds reveal a 30–40% higher capacity of monolithic columns while the bind-ing constants are nearly the same for both types [39]. Thissuggests that the effective or accessible surface area ofthe monolithic column is larger than that of the packed col-umn while the chemistry of both surfaces are very similar,leading to the same elution order for many analytes. How-ever, due to the fact that the density of monolithic columnsis much lower, the loadability of a conventional column ofthe same size is much higher.

4 Applications of monolithic silicacolumns

Numerous papers [33, 40–50] have compared packedand monolithic silica columns in different applications.Most of the authors arrived at the conclusion that the col-umns are comparable with respect to their performanceand selectivity. However, monolithic columns clearly pos-sess an advantage in their faster operation leading toshorter analysis times. This has been proven in manyapplications from different fields, including analysis ofdrugs and metabolites, analysis of environmentally rele-vant substances, food additives, chiral separations, aswell as bioanalytical separations. Generally, separationsusing monolithic silica columns can be achieved 5 to 10times faster than with packed columns. Despite the factthat each monolithic column is independently produced asa single unit, the manufacturing process is reproducible,which makes these columns a real alternative to conven-tional ones. In the following, some application fields will bedescribed in more detail.

4.1 High throughput analysis of drugs andmetabolites

As a result of their high permeability monolithic silica col-umns are especially suitable for high throughput analysis.This is typically needed in pharmacokinetic studies wherethe decreasing concentration of a given drug is monitoredand correlated with the increasing formation of metabo-lites. Usually these applications are carried out usingmass spectrometry as the detection method, which allowsmore information to be obtained about the chemical struc-ture. A typical example of this application was describedby Barbarin et al. [51]. The authors developed a LC/MSmethod for the determination of methylphenidate, a cen-tral nervous stimulant and its de-esterified metabolite, rita-linic acid, in rat plasma. A separation of these two com-pounds was achieved in 15 s at a flow rate of 3.5 mL/minusing a ChromolithTM Flash RP-18e column. Overall, 768protein-precipitated samples were analysed within 3 hand 45 min. Hsieh et al. [52] described a similar methodfor the determination of a drug discovery compound andits metabolite in blood plasma using the same monolithiccolumn and LC/MS/MS. Baseline separation of the twocompounds was achieved with run times of 24 or 30 sunder isocratic or gradient conditions. The method wasused for a quantitative study and compared to a methodbased on a packed column. The results obtained on themonolithic column and conventional packed column werein good agreement, within 10% error, which demonstratesthe robustness of the monolithic columns for daily routineanalysis.

Some authors have transferred existing HPLC methodsfor profiling of drug impurities to monolithic silica columns.The major advantage is time saving. Van Nederkassel etal. [53] transferred threemethods to reduce analysis timesfrom 15–30 min on conventional packed columns to 48 s,1.8 min, and 3 min using ChromolithTM columns with flowrates up to 9 mL/min. The robustness and repeatability of


Figure 7. Effect of flow rate on the sep-aration impedance for the monolithic andconventional column packed with silicaparticles of different diameters.


these separations was found acceptable. Despite the useof high flow rates and frequent mobile phase changes withpH values varying from 3.5 to 7, the column performancewas found to be rather constant and the column agingminimal. Dear et al. [54] also transferred a LC/MS/MS-method for the separation of six metabolites from a con-ventional packed silica column to a monolith. They couldseparate the six isomers in 1 min. The resolution andselectivity achieved with the short monolithic silica columnof 5 cm length were comparable to those obtained in con-ventional analytical chromatography while the analysistime per sample was reduced from 30 to 5 min. Recently,Gerber et al. [73] have demonstrated practical aspects offast reversed-phase HPLC using columns packed with3 lm particle and monolithic columns in development andproduction of pharmaceuticals working under currentgood manufacturing practice (GMP) mode. The authorsconcluded that monolithic columns enable dramaticreduction in the analysis time by a factor of up to 6.Furthermore, they claimed that monolithic columns notonly have repeatability and reproducibility comparable topacked columns but are also easy to handle on conven-tional HPLC systems and have a very good stability.

Monolithic silica columns have also been shown to besuperior in the analysis of drugs of abuse. Pihlainen etal. [55] described the development of a LC/MS/MS-method for the identification of 14 forensically interestingcompounds. All these compounds were eluted within2.5 min and the total analysis time was 5 min including thetime for column re-equilibration. Additionally, a libraryincluding MS-MS spectra and retention times was com-piled for 476 standard samples. Then, 50 authentic sam-ples could be unambiguously identified using the library. Aquantitative method was also developed for the samplesusing external calibration. The evaluation process show-ed good linearity of the method and reasonable repeat-ability. Limits of detection ranged from 10 to 50 ng/mL.

4.2 Separation of environmentally relevantsubstances and food additives

Monolithic silica columns have also been applied in thearea of environmental and food analysis. Someauthors [56–63] have developed methods for the fastseparation of pollutants or ions in rain water. For example,Koal et al. [57] presented a fast method for the analysis ofpesticides in water samples using online SPE/LC/MS/MS.Separation of 30 pesticides in trace levels was obtainedon a 5064.6 mmChromolithTM SpeedROD column in lessthan 14 min. The method had a good reproducibility andextremely low detection limits in a range of 0.1–1 ng/L.Hatsis et al. [63] used a monolithic silica column for theultrafast separation of common inorganic anions in indus-trial water requiring only 15 s. Separations were perform-ed using ion-interaction chromatography with tetrabutyl-

ammonium phthalate as the ion reagent and either directconductivity or indirect absorbance detection. The repro-ducibility of the validated method was 0.4 and 2%RSD forretention time and peak area, respectively, at a 1 min sep-aration time and 2.8 and 3–15% for a separation time of15 s. Xu et al. [58] described the use of a monolithic ODScolumn for the ion chromatography of mono- and divalentcations in rain water in a different way. The authors coatedthe column with lithium dodecylsulfate and separated H+

from Na+, NH4+, K+, Mg2+, and Ca2+ within 1 min at a flowrate of 1.5 mL/min using conductimetric detection.

Volmer et al. [37] published an interesting paper showingthe use of monolithic silica columns for the fast LC/MSanalysis of several azaspiracid biotoxins. The columnswere operated with flow rates of 1–8 mL/min, thus reduc-ing the chromatographic run times to 30 s. Additionally, acomplex biotoxin mixture was separated on a 40 cm longcolumn obtained by connection of four standard 10 cmChromolithTM Performance RP-18e. This shows again thehigh resolution power of monolithic silica columns coupledin series to obtain high plate numbers.

In the field of food analysis, the mycotoxin ochratoxin Awas analysed in different wines using LC/MS/MS and ashort monolithic silica column (5064.6 mm). With the useof MS detection in multiple reaction monitoring (MRM)mode, Zollner et al. [64] achieved a detection limit of0.5 ppb. At higher flow rates the authors were able toreduce the analysis time by a factor of three. Castellari etal. [65] described the determination of phenolic com-pounds in wines. Separation was carried out on a mono-lithic silica column using a binary gradient elution anddiode array detection. They separated and quantified 17monomeric phenolic compounds such as hydroxybenzoicacids, hydroxycinnamic acids, hydroxycinnamyltartaricacids, flavanol, flavonol, and stilbenes in a single run andin a very short time. Jakab et al. [66] used a short mono-lithic column for the identification of oils in peanuts, pump-kin and sesame seeds, soybean, and wheat germs. Plantoil triacylglycerols were identified with LC/MS using atmo-spheric pressure chemical ionization. Principal compo-nent analysis indicated that the different plant oils couldbe clearly differentiated according with their triacylglycerolcomposition.

4.3 Separation of enantiomers

Monolithic silica columns have also been introduced in thefield of chiral separations. Three different approacheswere successful so far. The first one involves the additionof chiral selectors to the mobile phase, which are dynami-cally coated via hydrophobic interactions on the ODSmodified monolithic silica column, thus creating a chiralsurface. The second approach uses chiral selectorschemically bonded by an in-situ derivatization of mono-lithic silica columns with chiral silanes. Finally, the third


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approach consists in activation of pore surface in mono-lithic silica columns with amino-, thiol-, or epoxide func-tionalities onto which the chiral selector is absorbed orchemically bonded. Since the permeability of chiral mono-lithic columns is similar to that of the ODS types, fast sep-arations of enantiomers can be obtained.

Chankvetadze et al. [67] described the baseline enantio-separation of 2,2,2-trifluoro-1-(9-anthryl)ethanol on amonolithic silica column coated with cellulose tris(3,5-dimethylphenylcarbamate). The first enantiomer waseluted in 7.2 s, the second in 18.5 s. The complete separa-tion was accomplished in 30 s, which may be the shortesttime in which baseline HPLC enantioseparations havebeen reported. Lubda et al. [68] reported the enantiose-paration of drugs on amonolithic silica column with chemi-cally bonded b-cyclodextrin. They transferred existingmethods validated on packed columns with chemicallybonded b-cyclodextrin and achieved a tenfold reduction inanalysis time. Similar results were obtained usingdynamic coating of b-cyclodextrin on a monolithic col-umn [69].

Massolini et al. [70, 71] described the preparation of achromatographic enzyme reactor by immobilizing penicil-lin G acylase (PGA) on aminopropyl monolithic silica col-umn. The column was used to determine enantioselectiv-ity of the PGA catalyzed hydrolysis of some 2-aryloxyalka-noic acid methyl esters and isosteric analogs. Studies withthe racemic esters were also carried out to unambiguouslyclarify the substrate specificity and the enantioselectivityof PGA.

4.4 Separation of complex biological samples

The analysis of complex biological samples such as trypticdigests is a real challenge in HPLC. Usually hundreds oreven thousands of components have to be separated andidentified. This requires HPLC columns with a high resolu-tion power on the one hand and sophisticated detectionsystems typically mass spectrometry on the other. Cur-rently, monolithic silica capillaries with a length of up to1 m seem to be the most promising devices in thisfield [78]. Plate numbers of more than 100,000 for a singlecolumn provide a high resolution power. However, someauthors also used conventional monolithic silica columnsfor the separation of tryptic digests.

Xiong et al. [79] has evaluated a ChromolithTM Perform-ance RP-18e column for the separation of tryptic digest ofbovine cytochrome c to determine the impact of separa-tion time on column efficiency, analyte recovery, and ana-lyte purity. Analyte purity was determined using matrix-assisted laser desorption ionisation mass spectrometry.The authors concluded that the monolithic silica column isof great value for the analysis of peptides. Hennessy etal. [80] also developed a procedure for the generation of

peptide mapping of various cytochrome c species usingmonolithic silica columns. They found that the use of silicamonoliths led to significantly reduced separation times.Furthermore, the existing methods could be easily trans-ferred to the monolith. The reproducibility of the chromato-graphic profiles enabled monitoring of sequence varia-tions in protein homologues whilst determination of theputative binding domains could be ascertained from therespective tryptic peptides obtained after digestion.

Shah et al. [81] has published an interesting paper evalu-ating separation methods for the determination of aminoacids, which serve as neurotransmitters in the central ner-vous system. The determination of amino acids in braintissue and extracellular fluid was used to develop effectivetreatment strategies for neuropsychiatric and neurode-generative diseases and for the diagnosis of such patholo-gies. The authors coupled a 5 and 10 cm long Chromo-lithTM column to achieve the separation of 17 amino acidswith post column derivatization and fluorescence detec-tion in less than 10 min.

Pham-Tuan et al. [82] have developed a HPLC methodfor high throughput profiling and metabonomic studies ofbiofluids. The motivation for this work was the desire tobetter understand the impact of food ingredients on con-sumer health. The authors created fingerprints of urinesamples using a short monolithic column in combinationwith mass spectrometry.

One of the most recent developments in HPLC are two-dimensional methods for the analysis of complex mix-tures [83–85]. Generally, two HPLC columns are involvedin such a system. For the separation in the first dimension,15 or 25 cm long columns with high resolution power andselectivity are needed. On the contrary, the seconddimension requires shorter columns with a high resolutionpower enabling fast separation of fractions obtained fromthe first column. Tanaka et al. [83] have demonstrated thisconcept. Each fraction from the first 15064.6 mm columnpacked with fluoroalkylsilyl bonded silica particles wassubjected to separation in the second dimension usingone or two ODS derivatized 3064.6 mm monolithic col-umns that were eluted at a flow rate of up to 10 mL/min.The separation time of 30 s met the requirement that afraction collected every 15–30 s from the first dimensionoperated at a flow rate of 0.4–0.8 mL/min is analysed inthe second dimension.

5 ConclusionsSince monolithic silica columns became commerciallyavailable, they are being used in many different fields.Monolithic silica columns exhibit a tailor-made bimodalpore structure with both macropores or through pores andmesopores. The most unique feature of these columns istheir high permeability, which is nearly twice as high as



that of packed columns. Therefore, monolithic silica col-umns can be operated at high flow rates of up to 10 mL/min, thus allowing fast separations of various mixtures.Many applications, originally developed using packed col-umns, can be performed with a monolith while reducingthe analysis time by a factor of 5–10. Monolithic silica col-umns are suitable for high throughput analysis as well asfor two-dimensional HPLC methods. Many authors havecompared monolithic silica columns and conventionalpacked silica columns with respect to their physical andchromatographic properties. All of them came to the con-clusion that both types of the silica-based columns arecomparable with respect to performance, selectivity, andreproducibility. Some authors even claim that monolithicsilica columns are more stable than packed ones due tothe rigid silica structure. Therefore, monolithic silica col-umns seem to display a great promise for the near futurebecause further improvements may lead to enhanced effi-ciency which is needed in the challenging field of highthroughput and bioanalytical analysis.

AcknowledgmentsThe author would like to thank the ChromolithTM R & D-and production team at Merck KgaA (K. Czerny, Dr. K.-H.Derwenskus, L. Friedrich, T. Gruner, G. Jung, P. Knoell,A. Kraus, K. Kreher, A. Leinert, D. Lubda, W. Neuroth, F.Paesler, Dr. G. Pieper, H.-D. Pohl, A. Portl, Dr. A. Rauh,Dr. T. Zwing) for their dedication. Furthermore, the authorwould like to express her gratitude to the Japanese coop-eration partners (Dr. N. Ishizuka, Dr. H. Minakuchi, Prof.Nakanishi, and Prof. Tanaka) for their permanent supportthroughout the development of ChromolithTM columns.The author would also like to thank Prof. F. Svec for read-ing the manuscript and making many valuable sugges-tions.

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