166
`
`trends in analytical chemistry, vol. 21, no. 3, 2002
`
`Capillary monoliths for the analysis of nucleic
`acids by high-performance liquid
`chromatography–electrospray ionization mass
`spectrometry
`Herbert Oberacher
`Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innrain 52a,
`A-6020 Innsbruck, Austria
`
`Christian G. Huber*
`Instrumental Analysis and Bioanalysis, University of Saarland, Im Stadtwald, D-66041,
`Saarbru¨cken, Germany
`
`Monolithic capillary columns prepared by copoly-
`merization of styrene and divinylbenzene inside a
`200 m i.d. fused silica capillary allow the rapid and
`highly efficient separation of single- and double-
`stranded DNA by ion-pair reversed-phase high-per-
`formance liquid chromatography. Hyphenation to
`electrospray ionization–mass spectrometry can be
`readily achieved. # 2002 Published by Elsevier
`Science B.V. All rights reserved.
`
`Keywords: Nucleic acids; Oligonucleotides; Ion-pair reversed-
`
`phase liquid chromatography; Monolithic capillary columns;
`
`Electrospray ionization–mass spectrometry (ESI–MS)
`
`1. Introduction
`
`The importance of nucleic acids in all areas of
`biosciences necessitates the development of
`effective techniques for their isolation, separation,
`quantitation, and structural analysis. Six major
`modes are utilized for separation of nucleic acid
`
`*Corresponding author. Tel.: +49 (0) 681 302 3433;
`Fax: +49 (0) 681 302 2963. E-mail: christian.huber@uibk.ac.at
`
`Abbreviations: ESI-MS, electrospray ionization-mass spectro-
`metry; HPLC, high-performance liquid chromatography;
`i.d.,
`inner diameter; IP-RP-HPLC,
`ion-pair reversed-phase HPLC;
`TEAA, triethylammonium acetate; TEAB, triethylammonium
`bicarbonate.
`
`0165-9936/02/$ - see front matter
`P I I : S 0 1 6 5 - 9 9 3 6 ( 0 2 ) 0 0 3 0 4 - 7
`
`mixtures by high-performance liquid chromato-
`graphy (HPLC)
`[1],
`including size-exclusion
`chromatography
`[2],
`anion-exchange HPLC
`[3,4], mixed-mode HPLC [5], reversed-phase
`HPLC, ion-pair reversed-phase HPLC (IP-RP-
`HPLC) [6], and affinity chromatography [7]. Of
`these,
`anion-exchange HPLC and IP-RP-
`HPLC represent the most commonly applied
`modes, mainly because of their high resolution
`capability and their flexibility to separate both
`single- and double-stranded nucleic acids rang-
`ing in size from a few nucleotides to several
`thousand base pairs [8].
`During the past decade, electrospray ioni-
`zation–mass spectrometry (ESI–MS) has become
`an important tool
`in the structural analysis of
`nucleic acids [9], and its on-line hyphenation to
`HPLC has been shown to greatly enhance the
`productivity and information content of both
`analytical methods [10,11]. Whereas the salt
`gradients of increasing ionic strength commonly
`applied in anion-exchange HPLC are rather
`incompatible with the ESI process,
`IP-RP-
`HPLC, employing non-polar stationary phases
`and volatile mobile phases of low ionic strength,
`is the mode of choice for on-line combination
`with ESI–MS [12].
`The availability of stationary phases with
`favorable mass transfer properties is a pre-
`requisite for successful HPLC separation of
`
`# 2002 Published by Elsevier Science B.V. All rights reserved.
`
`1
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`MTX1018
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`trends in analytical chemistry, vol. 21, no. 3, 2002
`
`167
`
`such as nucleic
`biological macromolecules,
`acids. Stationary phases based on microparticles
`have been successfully utilized as separation
`media for HPLC since its introduction in the
`mid-60s [13]. Nevertheless, the relatively large
`void volume between the packed particles and
`the slow diffusional mass transfer of solutes into
`and out of the stagnant mobile phase present in
`the pores of porous stationary phases are the
`major factors limiting the separation efficiency
`of conventional porous packing materials, espe-
`cially for biopolymers [14].
`The different routes to enhance the mass
`transfer of biopolymers in stationary phases
`include: (1) increasing the pore diameter and
`decreasing the particle diameter [15]; (2) elim-
`inating the support pores by using non-porous
`stationary phases [16]; (3) decreasing the pore
`depth by using superficially porous stationary
`phases [17]; (4) introducing flow-through pores
`transversing the stationary phase particles [18];
`and, (5) using porous monoliths as the chro-
`matographic bed [19,20].
`In such monolithic columns, the separation
`medium is a single piece of a rigid, porous
`polymer
`that does not have any interstitial
`volume but has internal porosity comprising
`micro-, meso-, and macro-pores [20–23]. Because
`of the absence of intra-particular volume, all of
`the mobile phase is forced to flow through the
`pores of the separation medium [24]. According
`to theory, mass transport is enhanced by such
`convection [25,26] and has a positive effect on
`chromatographic efficiency.
`Monolithic columns can be synthesized using
`two principal approaches. First, columns packed
`with granular stationary phases are converted to
`monolithic columns by sintering or cross-linking
`of the microparticulate packing material [27].
`Second, monolithic chromatographic beds are
`prepared by polymerization or polycondensation
`of suitable monomers and porogens in a stain-
`less steel or fused silica tube, which acts as a mold
`[19,20,23]. In the preparation of organic mono-
`liths, a porous structure is achieved as a result
`of the phase separation, which occurs during
`the polymerization of a monomer or monomer
`mixture containing appropriate amounts of both
`
`a cross-linking monomer and a porogenic sol-
`vent or a mixture of porogenic solvents [28].
`The concept of monolithic stationary phases
`is especially suitable for the fabrication of capil-
`lary columns, because the immobilization of the
`monolith at
`the capillary wall eliminates the
`necessity to prepare a tiny retaining frit, which is
`one of the steps more tedious and difficult to
`control during the manufacture of packed-bed
`capillary columns [29]. Miniaturized chromato-
`graphic separation systems applying capillary
`columns of 10–500 mm i.d. are frequently the
`method of choice for the separation and charac-
`terization of biopolymer mixtures, when the
`amount of available sample is limited. Conse-
`quently, monolithic capillary columns have
`proved to be efficient for the separation of bio-
`polymers, such as peptides [30,31] and proteins
`[19,20,32–34]. Moreover, monolithic separation
`media with anion-exchange functional groups
`have been successfully applied to the separation
`of mononucleotides
`[35],
`oligonucleotides
`[36,37], and plasmid DNA [38].
`Recently, the concept of monolithic columns
`has been transferred to the separation of nucleic
`acids by IP-RP-HPLC [11]. In this paper, highly
`efficient separations of single-stranded oligo-
`nucleotides and double-stranded DNA fragments
`over a range of five nucleotides to 622 base pairs
`in monolithic poly(styrene/divinylbenzene)-based
`capillary columns are considered. The miniatur-
`ized separation system is on-line hyphenated to
`ESI–MS, and this allows nucleic acids to be
`identified and their masses to be accurately
`characterized.
`
`2. Structure and performance of
`micropellicular, monolithic separation
`media
`
`The role and importance of mass transfer in
`chromatographic processes is well understood
`[39]. Appropriate choice of stationary phase
`configurations that provide favorable mass-
`transfer properties is particularly important for
`the rapid and highly efficient separation of large
`molecules having low diffusivities. One approach
`
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`168
`
`trends in analytical chemistry, vol. 21, no. 3, 2002
`
`to alleviate the mass-transport problem is based
`on the application of stationary phases of the
`micropellicular configuration, which is char-
`acterized by a core of fluid-impervious support
`material covered by a thin, retentive layer of
`stationary phase [16,40]. Another approach to
`the problem of stagnant mobile phase mass
`transfer is the use of porous stationary phases
`that possess a bimodal pore size distribution,
`where macro-pores provide the channels for
`convective flow through the support material
`while meso- and micro-pores provide the surface
`for chromatographic interaction. This concept
`has been successfully realized in perfusion particles
`[18] and monolithic stationary phases [19,32].
`In an attempt
`to take advantage of
`the
`enhanced mass-transfer properties in stationary
`phases by combining both the micropellicular
`and the monolithic configuration, we used
`porogen mixtures that favor the formation of
`macro-pores and suppress the formation of
`micro-pores. 1-Decanol and tetrahydrofuran
`were utilized as macro- and meso-porogen,
`respectively, for the preparation of poly(styrene/
`divinylbenzene)-based monoliths [11], resulting
`in a stationary phase configuration that can
`
`be adequately described as a micropellicular
`monolith.
`Fig. 1 illustrates the structural differences
`between a column packed with micropellicular
`particles (Fig. 1a) and one packed with a
`micropellicular monolith (Fig. 1b). In the con-
`ventional column,
`the chromatographic bed
`consists of tightly packed, micropellicular parti-
`cles
`(Fig. 1a).
`Intra-particular mass-transfer
`resistances are confined to the very thin outer
`shell of the particles and, therefore, drastically
`reduced. The micropellicular, monolithic col-
`umn bed is made of one single piece of a por-
`ous solid with relatively large channels for
`convective flow (Fig. 1b). Mathematical model-
`ing has shown that increased pore connectivity,
`which represents the number of pores con-
`nected at a node of the porous network, plays a
`key role in improving the transport of
`the
`adsorbate molecules in the monolithic stationary
`phase configuration [41]. Moreover, mass transfer
`in a micropellicular monolith is further enhanced
`due to the absence of micro-pores. Comparing
`the electron micrographs in Fig. 1a and b, one
`can see that the number of channels penetrating
`the chromatographic bed is much higher in the
`
`Fig. 1. Structural characteristics of (a) packed, and (b) monolithic chromatographic beds.
`
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`

`trends in analytical chemistry, vol. 21, no. 3, 2002
`
`169
`
`monolith than in the column packed with
`micropellicular particles. In addition, the diam-
`eter of the channels is reduced in the monolithic
`structure, allowing a considerable decrease in
`the path-lengths required for mass transfer
`between the mobile and stationary phase.
`Fig. 2 compares the chromatographic perfor-
`mance obtained with a conventional capillary
`column packed with micropellicular particles
`and a monolithic capillary column, both having
`an i.d. of 200 mm, under gradient elution con-
`ditions. The proper choice of the detection
`volume has been shown to be critical to be able
`to detect the very sharp chromatographic peaks
`without band-broadening caused by extra-col-
`umn dead volumes [29]. Therefore, a capillary
`detection cell with 3 nl
`internal volume was
`used for this separation. It can be seen that the
`peak width at half height for a 12-mer oligo-
`deoxythymidylic acid, p(dT)12, decreased by
`almost a factor of 2 from 3.9 s with the con-
`ventional column (Fig. 2a) to 2.0 s with the
`monolithic column (Fig. 2b). Although the
`elution window between the smallest and the
`
`largest oligomer decreased slightly, the resolu-
`tion of all peaks was significantly better on the
`monolithic column (average resolution value of
`3.1 on the packed versus 5.1 on the monolithic
`column).
`
`3. Separation and analysis of single-
`stranded oligonucleotides
`
`IP-RP-HPLC is a well-established chromato-
`graphic technique for separating oligonucleo-
`tides on non-polar
`stationary phases
`[42].
`Retention of nucleic acids is effected upon
`addition of amphiphilic ions, such as triethyl-
`ammonium ions, to the hydroorganic mobile
`phase, usually water-acetonitrile. Adsorption of
`the amphiphiles onto the non-polar surface
`of the stationary phase results in the formation of
`a positive
`surface potential, which retains
`the negatively charged analytes. Subsequently, the
`nucleic acids are eluted by a gradient of
`increasing acetonitrile concentration [43].
`Fig. 3 demonstrates the high resolving power
`of micropellicular, monolithic capillary columns
`for
`the separation of single-stranded oligo-
`nucleotides by IP-RP-HPLC. The sample was
`
`Fig. 2. Comparison of the chromatographic performance
`of (a) a column packed with micropellicular particles and
`(b) a micropellicular monolithic column for the separation
`of oligonucleotides. Column, (a) 60  0.2 mm i.d., packed
`with 2.3 mm PS/DVB-C18 particles, (b) 60  0.2 mm, PS/
`DVB monolith; mobile phase, (A) 100 mM TEAA, pH 7.00,
`(B) 100 mM TEAA, pH 7.00, 20% acetonitrile; linear gra-
`dient, 25–60% B in 10 min; flow-rate, (a) 2.7 ml/min, (b) 3.1
`ml/min; temperature, 50 C; sample, 2.5 ng (dT)12–(dT)18.
`
`Fig. 3. High-resolution capillary IP-RP-HPLC separation of
`a mixture of 120 phosphorylated and dephosphorylated
`deoxyadenylic acids. Column, PS-DVB monolith, 60  0.20
`mm i.d.; mobile phase, (A) 100 mM TEAA, pH 7.00, (B)
`100 mM TEAA, pH 7.00, 20% acetonitrile; linear gradient,
`5–35% B in 5.0 min, 35–40% B in 5.0 min, 40–45% B in 6.0
`min, 45–52% B in 14.0 min; flow-rate, 2.1 ml/min; tem-
`perature, 50 C; detection, UV, 254 nm; sample, hydrolyzed
`p(dA)40–p(dA)60 spiked with 2.5 ng p(dA)12–p(dA)18.
`
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`

`170
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`trends in analytical chemistry, vol. 21, no. 3, 2002
`
`generated by partial hydrolysis of a commer-
`cially available oligonucleotide ladder of phos-
`phorylated oligodeoxyadenylic acids
`(40–60-
`mers, p(dA)40-p(dA)60), which gave two series
`of either phosphorylated or dephosphorylated
`oligonucleotides that may range in size from 1
`to 60 nucleotide units. Separation was accom-
`plished by applying a gradient of 1.0–7.0% ace-
`tonitrile in 5.0 min, 7.0–8.0% acetonitrile in 5.0
`min, 8.0–9.0% acetonitrile in 6.0 min, and 9.0–
`10.4% acetonitrile in 14.0 min in 100 mM
`triethylammonium acetate at a flow rate of
`2.1 ml/min. For correct peak assignment, the
`hydrolysate was spiked with p(dA)12–p(dA)18.
`The analytes were separated into 98 peaks
`within 28 min, which meant elution of one peak
`every 17 s. The peak widths at half height
`ranged from 1.5–3.2 s for the oligomers up to
`the 18-mer and did not exceed 11.2 s for oligo-
`mers up to the 60-mer. The increase in peak
`width with increasing size of the oligomers is a
`consequence of the decreasing gradient steep-
`ness that was necessary to ensure adequate
`resolution of the longer oligonucleotides. From
`Fig. 3, it can be deduced that the whole series
`both of phosphorylated and dephosphorylated
`oligodeoxyadenylic acids could be resolved to
`baseline with
`single-nucleotide
`resolution.
`However, occasional overlapping of the signals
`of both series resulted in only partial resolution
`or coelution of some of the oligonucleotides,
`which explains why only 98 peaks instead of the
`maximum of 120 could be observed in the
`chromatogram. As already found previously, the
`dephosphorylated oligomers eluted later than
`the phosphorylated analogs of the same length
`[42]. The separation efficiency observed for
`monoliths clearly surpasses that obtainable on
`columns packed with micropellicular PS/DVB-
`C18 particles, where baseline resolution of
`dephosphorylated oligodeoxyadenylic acids was
`possible up to about the 50-mers (see Fig. 7 in
`[42]).
`The chromatographic separation system was
`readily on-line hyphenated to ESI–MS, as
`demonstrated in Fig. 4 by the separation of the
`p(dA)4060 ladder with ESI–MS detection. In
`order to increase detection sensitivity, aceto-
`
`nitrile was added post-column as sheath liquid
`through the triaxial electrospray ion source [44].
`Moreover, the concentration of ion-pair reagent
`was reduced from 100 to 25 mM, which resul-
`ted in an additional
`improvement of analyte
`detectability at the cost of only a slight decrease
`in chromatographic
`resolving power
`[12].
`Therefore, the total ion chromatogram showed
`an envelope of
`incompletely resolved peaks
`(Fig. 4a). Because of the low abundances of the
`long-chain oligomers in the sample, eluting
`analytes were difficult to distinguish from the
`noise. Nevertheless, upon extraction of charac-
`teristic ion traces at the selected m/z values
`
`Fig. 4. IP-RP-HPLC-ESI–MS analysis of single-stranded oli-
`gonucleotides: (a) reconstructed total ion chromatogram;
`(b) selected ion chromatograms of p(dA)40–p(dA)60; and,
`(c) extracted mass spectrum of p(dA)60. Column, PS-DVB
`monolith, 60  0.20 mm i.d.; mobile phase, (A) 25 mM
`TEAB, pH 8.4, (B) 25 mM TEAB, pH 8.4, 20% acetonitrile;
`linear gradient, 15–20% B in 10 min; flow-rate, 3.0 ml/min;
`temperature, 50 C; scan, 2000–4000 amu; electrospray
`voltage, 3.4 kV; sheath gas, 40 units; sheath liquid, aceto-
`nitrile; flow rate, 3.0 ml/min; sample, 500 ng p(dA)40–
`p(dA)60.
`
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`

`trends in analytical chemistry, vol. 21, no. 3, 2002
`
`171
`
`Table 1
`Mass-to-charge ratios (m/z) chosen for the selected ion
`traces in Fig. 4 and molecular masses of phosphorylated
`oligodeoxyadenylic acids p(dA)n
`
`n
`
`m/z
`
`Measured
`mass
`
`n
`
`m/z
`
`Measured
`mass
`
`40
`41
`42
`43
`44
`45
`46
`47
`48
`49
`50
`
`2508.1
`2570.8
`2633.5
`2696.1
`2758.6
`2821.3
`2884.1
`2946.6
`3009.4
`3072.1
`3134.8
`
`12,546
`12,859
`13,173
`13,486
`13,798
`14,112
`14,426
`14,738
`15,052
`15,366
`15,679
`
`51
`52
`53
`54
`55
`56
`57
`58
`59
`60
`
`3197.5
`3259.9
`3322.6
`3385.5
`3447.8
`3510.5
`3573.1
`3635.8
`3698.8
`3134.4
`
`15,993
`16,305
`16,618
`16,933
`17,244
`17,558
`17,871
`18,184
`18,499
`18,812
`
`given in Table 1, clear chromatographic profiles
`were obtained for all individual oligonucleotides
`(Fig. 4b). The eluting species were unequivocally
`identified on the basis of their molecular mas-
`ses, which were calculated from the extracted
`mass spectra (Table 1). Fig. 4c illustrates, as an
`example,
`the mass spectrum of the 60-mer,
`which was the least abundant analyte in the
`sample. Because of efficient charge state reduc-
`tion in only slightly basic eluents [12,45], only
`one or two signals of multiply-charged species
`were observed in the mass spectra. The charge
`state of the multiply-charged species could be
`calculated from the m/z shift of mono-potas-
`sium adducts, and this enabled the calculation
`of the molecular masses with an accuracy of
`0.001–0.01%.
`
`4. Separation and analysis of
`double-stranded DNA fragments
`
`Rapid developments in DNA technology,
`such as DNA-cloning
`techniques, DNA-
`restriction analysis, DNA sequencing,
`in-situ
`hybridization, polymerase chain reaction (PCR),
`and mutation-screening methods, have created
`the need for fast analytical methods to charac-
`terize minute
`amounts of double-stranded
`DNA molecules.
`Fig. 5 depicts the IP-RP-HPLC separation of
`a mixture of DNA-restriction fragments coming
`
`Fig. 5. High-resolution capillary IP-RP-HPLC separation of
`a mixture of double-stranded DNA fragments in a mono-
`lithic capillary column. Column, PS-DVB monolith, 60 
`0.20 mm i.d.; mobile phase, (A) 100 mM TEAA, pH 7.00,
`(B) 100 mM TEAA, pH 7.00, 25% acetonitrile; linear gra-
`dient, 35–55% B in 6.0 min, 55–70% B in 9.0 min; flow-
`rate, 2.2 ml/min; temperature, 50 C; detection, UV, 254
`nm; sample, 4.9 ng pBR322 DNA-Hae III digest and 4.1 ng
`pBR322 DNA-Msp I digest.
`
`from a pBR322 DNA-Hae III and a pBR322
`DNA-Msp I digest. The fragments ranged in
`size from 51 to 622 bp and were separated
`by applying a gradient of 8.75–13.75% aceto-
`nitrile in 6.0 min, followed by 13.75–15.75%
`acetonitrile in 9.0 min in 100 mM triethy-
`lammonium acetate at a flow rate of 2.2 ml/min.
`The sample containing 37 fragments was frac-
`tionated at least partially into 33 peaks. The total
`amount of DNA analyzed in this run was only
`9.0 ng, corresponding to about 1.7 fmol for
`each DNA fragment. Compared to the 1.15 mg
`DNA that were analyzed on a conventional
`analytical column of 504.6 mm i.d. dimensions
`(see Fig. 7 in [46]), this represents about a 120-
`fold reduction of sample required for analysis in
`a 200 mm i.d. capillary column. The peak widths
`at half height were between 2.3 and 4.3 s for the
`fragments up to a length of 217 bp, and
`between 5.6 and 8.5 s for the longer fragments.
`Compared to the previously published sepa-
`rations of the same mixture in an analytical [46]
`or a capillary column [29] packed with PS/
`DVB-C18 particles, the separation efficiency is
`higher, because better, or at least equivalent,
`resolution was achieved in a shorter period
`of time. This result clearly demonstrates the
`
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`

`172
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`trends in analytical chemistry, vol. 21, no. 3, 2002
`
`higher resolving power of this new monolithic
`stationary phase compared to conventional,
`granular stationary phases.
`Retention data can yield valuable information
`about the size of DNA fragments [46]. How-
`ever, measurement of the molecular mass cer-
`tainly is a very powerful
`tool for confident
`identification and characterization of nucleic
`acids. The analysis of 12 fmol of restriction
`fragments from an HaeIII digest of the cloning
`vector pUC18 is illustrated in Fig. 6. Although
`resolution in the reconstructed total ion chro-
`matogram (Fig. 6a) was not perfect because of
`the low concentration of ion-pairing reagent in
`the eluent and overloading of the capillary col-
`umn [11], high-quality mass spectra could be
`extracted for characterization of the individual
`DNA fragments. For example,
`in the mass
`spectrum of the 102-mer (Fig. 6b), multiply-
`charged ions with charge states from 29- to 46-
`were observed deconvoluted into a molecular
`mass of 63,122.1, corresponding very well to
`the theoretical mass of 63,067.3. Moreover,
`confident mass data were obtained for DNA
`fragments up to the 434-mer and the relative
`deviations of
`the measured from the theo-
`retical molecular masses did not exceed 0.1%
`(Table 2).
`
`Fig. 6. IP-RP-HPLC-ESI–MS analysis of double-stranded
`DNA fragments: (a) reconstructed total ion chromatogram;
`(b) extracted mass spectrum of the double-stranded 102-
`mer. Column, PS-DVB monolith, 60  0.20 mm i.d.;
`mobile phase, (A) 25 mM TEAB, pH 8.4, (B) 25 mM
`TEAB, pH 8.4, 20% acetonitrile; linear gradient, 5–16% B in
`3.0 min, 16–25% B in 12 min; flow-rate, 3.0 ml/min; tem-
`perature, 25 C; scan, 1000–3000 amu; electrospray vol-
`tage, 3.4 kV;
`sheath gas, 40 units;
`sheath liquid,
`acetonitrile; flow rate, 3.0 ml/min; sample, 20.4 ng pUC 18
`DNA Hae III digest.
`
`Table 2
`Molecular masses of double-stranded DNA fragments form the pUC18 DNA-Hae III digest
`
`Theoretical
`
`Relative
`deviation (%)
`
`Fragment
`length (bp)
`
`Molecular mass
`
`Measureda
`49,535 89 (7)
`80
`49,475.4
`0.1
`63,123110 (10)
`102
`63,067.3
`0.09
`107,640131 (10)
`174
`107,566.3
`0.07
`158,220254 (10)
`0.001
`256
`158,221.7
`165,221239 (13)
`267
`165,018.1
`0.1
`184,262242 (22)
`298
`184,198.4
`0.03
`268,341399 (24)
`434
`268,240.4
`0.04
`458
`n.d.
`283,002.8
`n.d.
`587
`n.d.
`362,706.1
`n.d.
`aMolecular mass given as average standard deviation of the molecular masses calculated from the signals of the individual
`charge states present in the ESI mass spectrum, the numbers in brackets indicate the number of charge states used to calculate
`the average molecular mass.
`n.d., not determined.
`
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`

`trends in analytical chemistry, vol. 21, no. 3, 2002
`
`173
`
`5. Conclusions
`
`separation media hold great
`Monolithic
`potential for the on-line hyphenation of capillary
`HPLC with ESI–MS for the characterization of
`nucleic acids. The major advantage of
`the
`monolithic stationary phases rests within their
`excellent chromatographic separation efficiency
`for biopolymers, which significantly exceeds the
`efficiency of
`the currently available granular
`stationary phases. Moreover, the lack of retaining
`frits and the mechanical and chemical stability
`of capillary columns packed with polymeric
`monoliths greatly contribute to the stability, rug-
`gedness and longevity of the hyphenated system.
`Through the use of miniaturized columns in
`the capillary format, very small amounts of bio-
`logical samples are amenable to analytical charac-
`terization. This is expected to make HPLC-
`ESI–MS applicable not only in specialized aca-
`demic laboratories, but also for routine analysis
`in industrial environments.
`The accurate masses obtained from HPLC-
`ESI–MS measurements yield important infor-
`mation about
`the identity and structure of
`nucleic acids and will help to characterize
`synthetic nucleic acids [45] as well as to detect
`sequence variations in genomic DNA [47,48].
`
`Acknowledgements
`
`This work was supported by a grant from the
`Austrian Science Fund (P14133-PHY).
`
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`
`Christian Huber studied chemistry at the University of Innsbruck
`and received his Ph.D. in 1994 for a work entitled ‘‘Development
`and application of chromatographic methods for the analysis of
`biopolymers’’. In 1996, he was involved in the development of
`
`capillary electrochromatographic instrumentation for gradient elu-
`tion in the laboratories of Professor Csaba Horva´th at
`the
`Department of Chemical Engineering, Yale University. Between
`1997 and 2002 he was an associate professor of analytical
`chemistry at the Institute of Analytical Chemistry and Radio-
`chemistry, Leopold-Franzens-University of Innsbruck. Since April
`2002, he has been professor of analytical chemistry at the Uni-
`versity of Saarland, Saarbru¨cken, Germany. His research interests
`include
`the development
`of monolithic
`separation media,
`hyphenation of capillary chromatography with mass spectrometry,
`and biological mass spectrometry. Herbert Oberacher studied
`chemistry at the University of Innsbruck and graduated in 1999.
`In 2002, he finished his Ph.D. thesis about the application of
`monolithic separation systems for LC–MS analysis of nucleic
`acids.
`
`9
`
`