miniaturization of analytical systems || miniaturized systems for analytical separations i: systems...

4

Miniaturized Systems for AnalyticalSeparations I: Systems Based on a

Hydrodynamic Flow

4.1 Introduction

Analytical separation techniques play a prominent role in analytical science.Without the participation of these techniques, very few reliable analyses could becarried out. Separation techniques are involved in both the treatment of samples (useof the so-called nonchromatographic separation techniques, meaning techniqueswithout instrumental detection) and the detection of analytes after chromatographicor electrophoretic separation. Aspects related to the automation andminiaturizationof separation techniques used for sample treatment havebeen reviewed inChapter 3.Chapters 4 and 5 deal with miniaturized systems for analyte separation, using eithera hydrodynamic flow (Chapter 4) or an electroosmotic flow (Chapter 5). Therefore,these two chapters cover the miniaturization tendencies of both chromatographic(liquid chromatography (LC), basically) and electrophoretic separation techniques;they also connect with Chapter 8, which is devoted to the miniaturization of theentire analytical process through the mTAS approach.The miniaturization of analytical separation techniques can be reviewed in

parallel with the evolution of these techniques. Figure 4.1 summarizes this evolu-tion, from the classic modes to their current alternatives. LC and electrophoresisbasically involve the movement of fluids and/or analytes for the performance ofseparation. Planar and column chromatography, performed at atmospheric pressure(open- and macrocolumns), have given way to more efficient approaches such as

Miniaturization of Analytical Systems: Principles, Designs and Applications Angel Rios, Alberto Escarpa and Bartolome Simonet © 2009 John Wiley & Sons, Ltd. ISBN: 978-0-470-06110-7

high-performance think-layer chromatography (HPTLC) in the planar modeand high-performance liquid chromatography (HPLC) in the column mode. Theimprovements were based on the stationary phases, the detection system and the useof closed columns (reduced size with respect to classical LC) working at highpressure. Microcolumns were also needed in ulterior developments, and particu-larly for the development of new alternatives addressing the use of capillarycolumns. The replacement of microcolumns by capillaries has led to the develop-ment of very interesting modern alternatives in analytical separation science. Thus,capillary liquid chromatography (CLC), through so-called micro- and nano-HPLC,and capillary electrophoresis (CE), have introduced a true revolution in instrumen-tal separation techniques. Capillaries constitute a significant size reduction, butmore importantly have allowed the merging of chromatographic and electropho-retic techniques in interesting analytical modes. Modes such as micellar electroki-netic chromatography (MEKC) and capillary electrokinetic chromatography (CEC)are hybrid methodologies with electrophoretic and chromatographic foundations,providing a wide analytical potential. Thus, it is possible to cover everything frompure CLC to pure electrophoresis using the capillary zone electrophoresis (CZE)mode – a wide variety of separation alternatives with an impressive scope ofapplication. Despite the clear improvements and possibilities these modes repre-sent, miniaturization of the equipment as a whole is not truly evident. Miniaturiza-tion affects many of the components and devices integrated in the commercializedequipment, but the equipment itself is not considered miniaturized at all. The truepass to miniaturization is given when chromatographic or electrophoretic separa-tions are carried out in chips. In these cases, all the principles of micro- and

oflu

idic

fluid

ic

Mic

rN

ano

ANALYTICAL SEPARATION TECHNIQUES‘miniaturization’

Classic modes Contemporary modes Present modes / tendencies

G CG C

Planar & ColumnChromatography-Paper-Think layer HPTLC-(Macro)column

ConventionalElectrophoresis

Column ChromatographyHPLC

COLUMNHigh pressureFlow rate: ml/minSample volume: µl CAPILLARY

Capillary LiquidChromatography

Flow rate: µl/min nl/minSample volume: µl nl

µHPLC nano-HPLC

CapillaryElectrophoresis

CEHigh potential

Sample volume: nl to fl

CZE

MEKC

CEC

CLC

Micro-GC

CE-ChipCE chip

HPLC chip

Figure 4.1 A general view of analytical separation techniques and the tendency to mini-aturization

140 Miniaturization of Analytical Systems

nanofluidics described in Chapter 2 are involved. Thus, today HPLC chips and CEchips are truly miniaturized systems connecting to the lab-on-a-chip approach.Based on the evolution shown in Figure 4.1 toward the miniaturized hydrodynamicseparation techniques, in this chapter liquid chromatographic tendencies focussedon capillary HPLC and HPLC chip are reported. The common characteristicsassociated with LC techniques are summarized in Table 4.1.

4.2 The Earliest Example ofMiniaturization of aGas Chromatographand Some Other Developments

Because of its foundation and equipment requirements, gas chromatography (GC) isan exceptional case, aswas noted inChapter 1.Although the objective of this and thefollowing chapter is to describe the miniaturization of hydrodynamic analyticalsystems, in order to give a complete view of miniaturized chromatographicseparations, gas phase separations must first be briefly commented upon. Thereis not much in the literature about gas phase separation on chips, despite the fact thefirst working microchip-based chromatographic system was a miniaturized gaschromatograph in 1979 [1]. As J.P. Kutter said [2], this development was hardlypursued afterwards, probably because the analytical community was not yet readyto embrace this new technology. Unsatisfactory results, due to difficulties inproducing homogeneous stationary phases, may have contributed to this feeling.However, miniaturized gas chromatographs are of great interest in several applica-tion fields. Thesemicrosystems could potentially be used for breath analysis, indoorair-qualitymonitoring andwarfare agent detection, allowing the onsite and portablemonitoring of gases in a fast and reliable way.Miniaturization of a GC system requires the miniaturization of the gas transpor-

tation system that is necessary for the efficient extraction and manipulation of theanalytes in the gaseous samples. Due to the ineffective transportation produced bygas micropumps, micro-GC (mGC) systems have had to rely on off-chip, high-volume and high-powergas transportation systems, such as syringe pumps and largemechanical pumps. Such dependence on off-chip systems has introduced difficul-ties for the creation of a truly portable miniaturized GC analyser. H. Kim et al. have

Table 4.1 Common characteristics associated with LC techniques

Name of technique Column i.d. Flow rate Injection volume level

Conventional LC 3–6 mm 0.5–2.0 ml/min mlMicrobore LC 1–3 mm 100–500 ml/min mlMicro-LC 0.2–1.5 mm 10–100 ml/min mlCapillary-LC (micro) 150–500 mm 1–10 ml/min mlNano-LC 10–150 mm 10–1000 nl/min Nl

Miniaturized Systems for Analytical Separations I 141

developed a micropump-driven high-speed MEMS GC system for volatile organiccompound (VOC) separation and detection [3]. Micropump operation is based onthe use of a four-stage micropump with integrated microvalves, working as aperistaltic micropump. Commonly, micromachined GC channels are coupled withdevices for preconcentration, injection and/or detection of gas phase analytes [4–6].But, obviously, microcolumns are the heart of the GC technique, and will be formGC systems too. The first works were concentrated on columns arranged with aspiral geometry, which produce lower dispersion than serpentines in CE micro-chips [7,8]. More recently, in several published articles, MEMS have beenconsidered as columns for mGC [9–12]. R.I. Masel et al. have compared micro-columns fabricated with different geometries and have demonstrated that, for mGC,serpentine columns give the lowest dispersion, since the small spiral columnsgenerate on overall higherDeanvorticewhen integrated over the entire column [13].On the other hand, an efficient stationary phase contained within a microfabricatedchannel is crucial to the development of a complete mGC. The deposition of thestationary phase into the microchannel can be a complicated task. The mostcommon types of deposition reported for open-tubular microfabricated columnsuse traditional static and dynamic coatings [14,15] and vapour deposition of apolysiloxane phase [16]. Fullerenes and carbon nanotubes have been demonstratedto have very interesting properties as stationary phases in GC [17]. In particular,multiwall carbon nanotubes have been shown to perform well as a GC stationaryphase, in both packed and open-tubular approaches. In fact, compared to traditionalstationary phases, they have the higher surface-to-volume ratio characteristic ofnanostructurated materials, as well as better thermal and mechanical stability[18,19]. Because of the high thermal stability, this material is ideal for temperature-programmed separation. In addition, carbon nanotube stationary phases can bedeposited easily by lithography. In this way, O. Bakajin and coworkers havedeveloped an ultrafast GC methodology based on the use of single-wall carbonnanotube stationary phases in microfabricated channels [20]. They used anintegrated heater for temperature programming and a synchronized dual-valvefor rapid injections. The authors see this development as a field-test sensor micro-system that can be used to continuously monitor the gas composition of atmos-pheric environments.The integration of all these elements allows the fabrication ofmGCmicrosystems.

Researchers from the Engineering Research Center for Wireless Integrated Micro-Systems (WIMS ERC) have developed an integrated microanalytical system forcomplex vapour mixtures, which is a good example of a microfabricated gaschromatograph [21]. This particular mGC is illustrated in Figure 4.2. These authorsdeveloped a WIMS mGC prototype, which includes the following parts:

(i) An inlet particle filter, consisting of a macroporous silicon membrane withtortuous pores, which exhibits a high filtering efficiency.


(ii) A calibration vapour source, using a dual-layer structure whose basecontains a reservoir for retaining the volatile-liquid calibrant. This micro-device produces a constant vapour generation for several days.

(iii) Smart latching valves, manipulating transportation into the system.(iv) Dual-separation microcolumns, based on a convolved square–spiral geom-

etry (3.0 m length each).Wafer-level low-pressure anodic bonding is used toseal the channel with a Pyrex cover plate, and fused-silica capillaries areepoxied into recessed side ports for fluidic connections. The compounds ofthe sample are separated according to their reversible partition equilibriumsbetween the mobile phases and the thin polymeric stationary phases liningthe walls of the columns.

(v) An integrated chemiresistor sensor array coated with thin films of differentgold-thiolate monolayer-protected nanoparticles (MPNs), whose responsesvary with the nature of the analyte vapour.

(vi) Commercial microspectrometers, which can be interfaced to the micro-column.

(vii) A distributed vacuum pump for micropumping.(viii) A preconcentrator.

Figure 4.2 Scheme and specific images of the analytical components of the WIMS mGCprototype. (From [21] with permission of IEEE, Copyright 2007)


Figure 4.3 shows a view of theWIMS mGC prototype, as well as an example of achromatogram in which 19 air contaminants were separated in 4 minutes.Microfabricated GC has the potential to achieve superior performance to

traditional GC. Other advantages lie in its capability to perform parallel analysiswith low cost, low power consumption, small thermal mass (which allows fasttemperature programming rates), portability and short analysis time.

4.3 Capillary Liquid Chromatography (CLC)

‘Capillary liquid chromatography’ (CLC) is a general term that includes, in manycases, mLC and nano-LC. This definition is accepted in this chapter, because theseparation is carried out into capillaries in both cases. Table 4.1 reports the featuresof CLC modes. The high resolution intended in CLC with the downsizing of theseparation element containing the stationary phase (which follows the sequenceshown in Figure 4.4) has the limitation of the pressure drop across the initial packedcolumns. Although open-tubular capillary columns and packed microcapillarycolumns present a higher permeability, and have allowed the development of basicCLC separations with a higher resolution, the problem of the pressure still remains.

Figure 4.3 Photography of the WIMS mGC prototype (top) and separation of a set of commonair contaminants (FID detection) (bottom). (From [21] with permission of IEEE, Copyright 2007)


As T. Takeuchi recognized [22], one possible reason for the limited development ofCLC (which began with Ishii’s group, Japan, in 1974 [23]) lies in the use ofelectrically-driven pumping (Figure 4.4) for separation methods such as CZE andthe other electrochromatographic modes (described in Chapter 5). In fact, theseelectrochromatographic techniques have the potential to produce much highertheoretical plates than pressure-driven separation methods.The features of CLC are attributed to the use of smaller-diameter columns and

lower eluent flow rate. The latter results in the saving of solvents, reagents andpacking materials, in comparison with conventional LC. Mass sensitivity isgenerally improved due to the small volume detection in the capillary, which isof particular interest when only very low sample size is available (biologicalsamples, for instance). The low flow rate in CLC is another advantage for the widermanipulation of the mobile phases, as well as for connection to detectors requiringlow flow of sample. This is the case with CLC–MS coupling, presenting a highercompatibility than conventional HPLC–MS systems. Finally, the low heat capacityof the capillary columns facilitates the control of column temperature and hencemore effective and easier temperature programming. All these features andadvantages are summarized in Figure 4.5, adapted from reference [24].As stated before, bioanalytical applications are one of the biggest potential uses

of CLC, particularly when it is coupled to a mass spectrometry (MS) detector.Recently, J.M. Saz and M.L. Marina have reviewed the application of CLC to thedetermination and characterization of bioactive and biomarker peptides [25].The combination of CLC with MS is very selective and sensitive, enabling theanalysis of new (even not-yet-discovered) peptides and permitting the simulta-neous analysis of a great number of peptides. Both CLC and MS are frequentlyinterfaced with micro- or nano-electrospray ionization (ESI). Miniaturization ofthe ESI interface improves sample ionization efficiency in the MS system.Additionally, miniaturized ESI devices work with low flow rates of the sameorder of magnitude as those provided by the CLC system. Here, the use of capillarycolumns improves separation efficiency and, hence, increases the overall selectivity

Conventional LC - - - CLC (micro- and nano-) - - - -Electrochromatography modes -- - - -Conventional CE (CZE) - - - -

COLUMN CAPILLARY CHIP(microchannels)

High-pressure pumps and micropumpsElectrically-driven pumping and electro-osmotic flow

LC chip

CE chip

Figure 4.4 Key factors for downsizing LC systems and main analytical techniques


of the CLC–ESI–MS arrangement. In this review [25], an interesting example wasselected to show the potential of CLC–ESI–MS for the analysis of succinylated ortrimethylammonium (TMAB)-labelled endogenous peptides extracted from thepituitaries of mice [26]. To detect and quantitate the labelled peptides, a micro-HPLC (300mm inner diameter (ID), C18 3mm particles)–ESI–MS system was used.To identify the peptides, a trapping column (5mm· 300mm ID, C18 5mm, 100A

�) to

preconcentrate and desalt the sample and a nano-HPLC (150mm· 75mm ID, C18

5mm, 300A�)–ESI–MS system were used. As expected, due to the larger mass

difference between the heavy and light forms, peptides labelled with the TMABreagent generally showed much better resolution in the MS than the succinylatedpeptides, as Figure 4.6 illustrates. In each of these CLC–ESI–MS systems it is verycommon to use online preconcentration steps following the scheme shown inFigure 4.7, as already described by A.J. Oosterkamp et al. [27]. Particularly atnano-level LC, the use of precolumns is highly recommended (in addition to thecleanup and preconcentration objectives), since capillaries can easily be blocked atthe inlet when real samples have to be analysed.The special compatibility of CLC with particular detectors has been studied by

P. Chaminade and coworkers [28]. They compare three commercial universaldetectors that allow a direct detection of lipids. These detectors are: the chargedaerosol detector (CAD), the evaporative light-scattering detector (ELSD) and theion trap (IT) mass spectrometer with atmospheric pressure chemical ionization(APCI) and ESI (see above) sources. The detectors are compared in terms ofresponse intensity, linearity and limit of detection, while working at high tempera-tures. CLC offers interesting possibilities for the determination of lipids, as thesecompounds are not very sensitive in other instrumental separation techniques.

SMALLER COLUMN DIAMETER(CAPILLARY)

Improved mass sensitivity

LOW FLOW RATE

LOW HEAT CAPACITY Temperature programming

Low consumption of mobile phase

Easier manipulation of mobile phase

Better compatibility with MS detectors

SMALLER PARTICLE SIZE(stationary phase)

Lower stationary phase, solvent and sample volume consumption

Figure 4.5 The main effects, and the corresponding advantages, produced by the reduction ofthe column (capillary) diameter


Figure 4.6 Mass spectra of labelled peptides obtained from CLC–ESI–MS analysis ofsuccinylated or TMAB-labelled endogenous peptides extracted from the pituitaries of mice.Left-column spectra correspond to a phosphorylated fragment of chromagranin B. Middle-column spectra correspond to joining peptide-Gly-Lys-Arg. Right-column spectra correspond toVS-Gly-Lys-Arg. The upper panels correspond to succinylated peptides and the lower panels toTMAB-labelled peptides. From [26], reproduced with permission of John Wiley & Sons, Ltd

Figure 4.7 Typical setup for online SPE preconcentration CLC–ESI–MS (adapted from [27])


ELSD is the primary detector for the analysis of lipids because of its compatibilitywith a large range of solvents and elution gradients. ELSD can detect all solutes thatare less volatile than the mobile phase. Recently, CAD has been developed as a newdetector. It is a universal hybrid detector useful for HPLC applications, initiallypresented by Dixon and Peterson in 2002 [29]. CAD works in two steps: the firstinvolves nebulizing the LC column effluent and evaporating the solvents (similarlyto ELSD); the second is the ionization of the aerosol particles by impact with thepositively charged nitrogen cation obtained by corona discharge. The amount of ioncharged is afterwards detected by an electrometer (similar to the principle of APCIin MS). It is interesting to compare the sensitivity (through the limits of detection,LOD, estimated on the basis of three times the signal-to-noise ratio; S/N¼ 3) forrepresentative lipid compounds using the different detectors [28]. Table 4.2 re-produces these results.Without any doubt, these less popular detectors such as CADand ELSD openvery interesting possibilities when used for CLC separations. Thus,ELSD has been coupled to CLC since 1999 [30], is already commercialized and,particularly for lipids, presents analytical advantages with respect MS detection (asTable 4.2 demonstrates). Again, biological samples not available in large amountscan be analysed by CLC–ELSD. An interesting application has been developed byL. Quinton et al. [31]. These authors propose a microanalytical system, based onCLC–ELSD, for the separation of stratum corneum ceramides. Stratum corneumlipids were obtained from the volar side of forearm skins of healthy subjects. Asample injection volume, as low as 1 ml, was used for the chromatographic analysis.Figure 4.8 shows the separation of the different lipid classes of a stratum corneumsample. The main groups of lipids and the chromatographic region for the analysisof ceramide classes can be seen.

Table 4.2 LOD a for three representative lipids (in ng) obtained with different detectorscoupled to CLC. (Reprinted from [28] with permission from Elsevier)

Detector Temperature (�C) Cholesterol Ceramide III B Squalene

ELSD 100 16.00� 1.10 30.00� 2.12 1.20� 0.08125 10.00� 1.00 20.00� 0.87 1.00� 0.01150 5.00� 0.20 10.00� 1.00 0.60� 0.02

CAD 100 15.00� 1.20 40.00� 1.90 1.00� 0.10125 7.50� 0.80 36.00� 0.30 0.70� 0.02150 2.50� 0.10 30.00� 1.69 0.18� 0.01

ESI-MS (full scan) 100 N/Aa 4.00� 0.12 N/Aa

125 N/Aa 1.50� 0.06 N/Aa

150 N/Aa 1.00� 0.01 N/Aa

API-MS (full scan) 100 N/Aa 0.03� 0.01 0.50� 0.10125 N/Aa 0.04� 0.02 0.80� 0.40150 N/Aa 0.05� 0.01 0.84� 0.30

aNot detected with this detector.


Separation capabilities are not only associated with the reduction size of thechromatographic column. The manipulation of the stationary phase can signifi-cantly improve the selectivity of many applications. Basically, three main disposi-tions of stationary phase in chromatographic columns can be identified for CLC:open-tubular capillary columns [32], packed microcapillary columns [33] andmonolithic silica capillary columns [34]. Additionally, fused silica capillaries [35]can be used for electrochromatographic methods. Among these, columns of packedparticles are still the most popular for HPLC because of their great utility, excellentperformance and wide variety. Recently, Q.-S. Qu et al. have proposed the use ofgold microspheres (AuMSs) modified with octadecanethiol for CLC [36]. Goldnanoparticles (AuNPs) are becoming increasingly attractive in many scientificfields because of their long-term stability, high surface area-to-volume ratio andease of chemical modification. With this background, AuMSs have great potentialto become substitutes for silica-basedmaterials as stationary phase for CLC. This isdue to their stability at high pH, reasonable rigidity and ease of manipulation from aphysicochemical point of view. This pure AuMS-based C18 stationary phase maybe a promising alternative to conventional C18 material for CLC.F. Fanali and coworkers have reviewed recent applications of CLC (nano-LC for

these authors; they refer to the use of capillaries of 10–100 mm ID) [37]. As theyreport, the main areas of application of CLC are proteomic, pharmaceutical andenvironmental. One of the most exciting applications of CLC is in proteomics,especially in peptide mapping, protein sequencing and the study of proteins.Nanoscale proteomics studies commonly require CLC–MS technology, employingthe interfaces reported above (ESI or APDC) as well as CLC–tandem massspectrometry (CLC–MS/MS) [38]. Even the use of nano-HPLC–ICPMS for thequantification of sulfur-containing peptides has been described [39]. Figure 4.9shows the high capacity for separating 24 different peptides using a combination ofnano-HPLC–ICP IDMS with nano-HPLC–ESI MS/MS.

Free fatty acids

Ceramides classes

min

ChoTriglycerides

SqmV375

300

225

150

75

–150.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

Figure 4.8 Separation of lipid classes of a stratum corneum sample. Sq, squalene; Cho,cholesterol. (Reprinted from [31] with permission from Wiley VCH)


Figure 4.9 (a) Sulfur mass chromatogram obtained from human serum albumin (HAS) trypticdigest by precolumn isotope dilution analysis with nano-HPLC–ICP–MS. (b) Table with theassignments of the peaks. (From [39] with permission, Copyright ACS)

4.4 Liquid Chromatography on Microchips

The miniaturization of the column inner diameter (Table 4.1 and Figure 4.5) andvolumetric flow rates in LC (HPLC) is an ongoing trend that is mainly driven by theneed to handle small volumes of complex sample, particularly in the context of high-throughput screening technologies. Recent progress toward HPLC in lab-on-a-chipincludes the integration of individual operations (basically, reaction,preconcentration, separation and the corresponding detection) into mass-producedand low-cost device development. This recent progress toward separations inmicrochip HPLC has the potential to become a more powerful tool than nano-LC for the analysis of complex samples [40]. This is of particular interest forLC–MS or LC–tandem mass spectrometry (LC–MS/MS) analyses, because of itscompatibility with the flow-rate requirements of a nano-electrospray interface forthe online coupling of the microchip HPLC to MS. This coupling facility is oneimportant advantage over microchip CE, which has clear difficulties with interfac-ing to MS. Agilent Technologies now markets a microfluidic chip that integrates atrapping column, separation column and electrospray sourcewithin a single device.

4.4.1 The Agilent HPLC Chip

H. Yin and K. Killeen, from Agilent Technologies, have recently reviewedthe fundamental aspects and applications of the Agilent HPLC Chip [41]. Thisis a polyimide device which is simultaneously a nano-electrospray interface to amass spectrometer, an analytical chromatographic column of appropriate size tothe nano-electrospray flow rate, and an enrichment column for online sampleconcentration in advance of the analytical column. In this device, there are no


Figure 4.10 View of the commercial Agilent HPLC Chip (top right) and traditional nano-LCcomponents (around the imageof the chip). Equivalent locations are indicatedbyarrows.Detailsof the electrospray tip are shown at the top of the figure as the HPLC Chip–MS interface. Thephotography of the Agilent nano-CLC–MS equipment shows the Agilent HPLCChipCubewheremicrochip is loaded. (Reproduced by permission of Agilent Technologies)

fittings, adapters, connectors or any other dispersive flow elements negativelyaffecting the performance in nano-LC. The commercial Agilent HPLC Chip isshown in Figure 4.10; traditional nano-LC components are displayed around thisimage, indicating their location in the chip. The figure is completed with the overallAgilent equipment in which the chip must be assembled, and details of theelectrospray tip. Polyamide was chosen as the chip substrate because it is anextremely heat- and cold-stable material and has good dimensional stability(coefficient of thermal expansion of 20 ppm/�C). It is quite insoluble in mostorganic media and is not appreciably basic or acidic, which is a fundamentalrequirement for reversed phase LC. In contrast to glass or fused silica, channels canbe formed by a laser ablation process, as this material absorbs the light. Thus,according to the information given by these authors [41], the microfabricationprocess consists of laser ablation of the channels, holes, chambers and columns. Thesample-enrichment column and the analytical column are slurry packed with avariety of chromatography media. The basic HPLC Chip has an LC channel of50mm (d)· 75mm (w) · 50mm (l), with a 40 nl enrichment channel. Particlesconforming the stationary phase are retained in the column space by the ‘keystoneeffect’, avoiding the need for frits, under operating pressure of over 120 bar [42].The chip-packing process is adapted and modified from methods originallydeveloped for the packing of fused silica capillaries.Both the enrichment and the chromatographic channels are packed using an input

capillary that is connected to the chip by a chip-valve interface [43]. Transfer


volume between the enrichment column and the analytical column (or other on-chipfunctions) is minimized by installing the HPLC Chip within a two-position rotaryvalve. Thus, the chip is sandwiched between the stator and the rotor of the valve,establishing a micro-to-macro interface. The valve assembly, the support plate andthe attached valve are mounted on a two-axis stage via a rotating bracket that allowsthe chip to be loaded into the valve assembly (Figure 4.11a and Figure 4.11b), andthen to be rotated into place such that the chip extends into a spray chambermountedon the mass spectrometer (Figure 4.11c). The two-axis stage allows vertical andaxial adjustment of the chip spray tip with respect to the cones on the massspectrometer inlet (Figure 4.11d).The chip is interposed between the rotor and the stator with enough precision so

that three basic running steps can be performed. In the initial position, mobile phaseis pumped to the chip, passing through the enrichment unit and separation column

Figure 4.11 (a) Schemeof the chipwith clamp inopenposition, including the stator, clampandvalve assembly. (b) Photography showing the chip in the clamped position ready for rotation intothe spray chamber. (c) Details of the chip and valve assembly in operational position on the MSinterface. (d) Photographyof the spray formed in the nano-LC–MS interface after the flowexits bythe electrospray tip. (Reproduced from [43], Copyright 2007 American Chemical Society, andby permission of Agilent Technologies)


and then reaching the spray (base-line signal); whereas sample flow is driven towaste (Figure 4.12a). In the enrichment position of the valve, sample is introducedto the chip flow from the autosampler, driven through the enrichment column, andthen to waste (Figure 4.12b). When the rotor is again switched 60� (injection of thepreconcentrated analytes), the flow from the nanopump enters the enrichmentcolumn and sweeps the analytes into the analytical column (Figure 4.12c). At theend of this column the flow passes electrical contacts which allow the biasing of theeffluent for electrospray.Agilent has developed the HPLC ChipCube to optimize the performance of the

entire system. The valve is designed to form a face seal with the HPLC Chip withminimum mechanical wear on the valve rotor. Figure 4.13 shows details of theentrance of the chip into the cube through the sandwiched position between thestator and the rotor of the valve.The chromatographic performance of the analytical column on an HPLC Chip

was studied by G. Rozing et al. [44]. The results obtained for a group of peptidesshowed that chromatographic peaks resulting from HPLC Chip were narrower andmore symmetrical than those obtained from a packed fused-silica capillary column.This fact is in agreement with the recordings shown in the article published by S.Ehlert and U. Tallarek [40], reproduced from a presentation by Vollmer andMiller [45]. These are given in Figure 4.14. As can be seen, the HPLC Chip offersincreased resolution and superior peak shape compared to conventional nano-LC

Figure 4.12 Scheme of the chip–rotor interface in the LC run mode (a) and in the loadingprocess: the sample-loading configuration (b) and the LC-running configuration (c) of the rotorchannels. The upper-right part of the figure shows the scheme of the LC run mode with the six-way positions of the rotary valve. (Reproduced from [43], Copyright 2007 American ChemicalSociety, and by permission of Agilent Technologies)


Figure 4.13 Introduction and sandwiched position of the chip in the stator–rotor unit.(Reproduced by permission of Agilent Technologies)

Figure 4.14 Comparison of microchip LC–MS with nano-LC–MS by analysis of a yeast gelband. (Reproduced from [40] with permission from Springer)


using the same adsorbent particles and similar packed-bed dimensions. The worstresults were obtained using a longer nano-LC column packed with smaller adsor-bent particles.

4.4.2 Other Approaches to Microchip HPLC

Other microchip LCs have been described in the literature. T.D. Lee and coworkersdeveloped amicrofluidic chip that integrates all the fluidic components of a gradientLC system [46], designed as a platform for an LC–MS/MSmicrosystem. This chipwas batch fabricated on a silicon wafer using photolithographic processes, withParylene as the main structural material. The chip includes three electrolysis-basedelectrochemical pumps, one for loading the sample and the other two for deliveringthe solvent gradient; platinum electrodes for delivering current to the pumps andestablishing the electrospray potential; a low-volume static mixer; a column packedwith silica-based reversed phase support; integrated frits for bead capture; and anelectrospray nozzle. Figure 4.15 shows a photograph of the chip and the corre-sponding schemes. As Figure 4.15b illustrates, the chip is basically conformed on a500 mmsiliconwafer layerwith various layers on top deposited for the fabrication ofthe different chip elements (50 mm thicknesses). In this top layer is packed thechromatographic column (RP column), the mixer, the exit for the electrospray andthe chamber for the pump or the sample. The chip is mounted into a polyetherimidejig that couples a port at the front of the column to Teflon tubing via a poly-(dimethylsiloxane) (PDMS) gasket. A slurry of the stationary phase material isforced into the column through an access port between it and the mixer until itsentire length is filled. Once packed, the chip is mounted in a different holder thatutilizes a 5 mm-thick polyetherimide cover. Chambers are machined into this coverpiece to form reservoirs for the sample and solvents. The PDMS gasket allows the

Figure 4.15 (a) Photograph of the LC chip. (b) Diagram showing the placement of the differentelements. (c) Chip-holder assembly for the inlet in an Agilent MSD ITMS. (Reproduced from[46] with permission of American Chemical Society, Copyright 2005)


sealing between the cover and the chip. For the analyses, the completed chipassembly is mounted on an Agilent electrospray ion source housing using amodified probe that incorporates a 3D positioner (Figure 4.15c).A. Ishida and coworkers have developed amicrochip for reversed phase LC using

porous monolithic silica [47]. The chip consists of a double T-shaped injector and a40 cm separation channel (in a serpentine configuration). Figure 4.16 shows aphotograph and details of this LC chip. This microchip is fabricated using glasswafer as the substrate and the cover plates. Standard photolithography,wet chemicaletching and bonding techniques are used in the different steps of the fabricationprocess. The layout and the dimensions of the different sections of the chip areshown in Figure 4.16b. Two photomasks are prepared for the fabrication. The firstdefines the double T-type injector and separation channel pattern, and the seconddefines the grooves which will serve as connectors. The overall dimensions of thischip are 35mm · 35mm. The typical channel width is about 400 mm, and thechannel depth is about 30mm. The connection grooves are etched to a depth of150mm. After rinsing with water, the etched glass chip is aligned to the cover plate

Figure 4.16 (a) Photograph of the LC chip. (b) Schematic layout of the chip with details of thechannels and inlet/outlet positions. (c) Overall configuration including the LC chip. S, sample;MP, mobile phase; ED, electrochemical detector; W1, mobile phase waste; W2, sample drain;V1, mobile phase inlet valve; V2, mobile phase outlet valve; V3, sample inlet valve; V4, sampleoutlet valve. (Reprinted from [47] with permission from Elsevier)


and then thermally bonded in a programmable oven. Then fused silica capillaries areinserted into the grooves.Monolithic stationary phase in themicrochip is created byintroducing reactant solutions by syringes. Figure 4.16c shows the schematicconfiguration of the chip LC system. The ends of the capillaries inserted in themicrochip are connected to two-way valves (V1,V3 andV4 in Figure 4.16c) via heat-shrinkable tubes. Valves V1 and V3 are connected to syringes allowing theintroduction of the mobile phase (V1) and the sample (V3), whereas valves V2

and V4 drive to waste. The end of the separation channel in the microchip isconnected to an electrochemical detector (ED) using conventional amperometricdetection. Different catechin compounds can be separated in this chip with goodresolution, although long analysis times are required.The sample-loading capacity of microchip LC systems can be enhanced by

introducing parallel multichannel flow pass, analogous to multicapillaries or fibre-packed capillaries, as T. Greibrokk and coworkers pointed out [48]. This possibilitywill increase the sample throughput and the speed of the analysis because of thebetter resolution of separation in microchips.

4.4.3 Some Selected Applications

LC analysis using themicrochip approach is practically addressed to the analysis ofvery small sample sizes, in which appropriate resolutions and sensitivity must beachieved.As the rest of the auxiliary elements, including the detector, are commonlynot miniaturized, portability is not the objective of such analytical microsystems.Therefore, biological or related bioanalytical applications have received the mainattentionofmicrochipLC,mainlytheuseofMSdetectionforreliablescreeningofthenumeroustargetcompoundsinthesesamples.Inthissection,someselectedexamplesillustrate the practical potential of microchip LC systems.In the pharmaceutical field, for example drug metabolism and pharmacokinetic

studies, it is common to use small animals for testing prior to human assays. Theanimal size sets the limit on the blood volume that can be drawn per unit time, andserial bleeding of a few or even only one animal, which reduces the variability in thepharmacokinetic profile, results in even lower volumes per bleed. Consequently,studies with small animals set extreme demands on the analytical system withrespect to both the capacity to handle small sample volumes and the detectionsensitivity. For these studies, LC–MS is often used for the analysis of samples.Here, the HPLC chip/MS system fulfils these requirements. In this context, S.Buckenmaier et al. have used the HPLC Chip interfaced to a triple quadrupole(QQQ)mass spectrometer for the monitoring of atenolol, atropine, metroprolol andimipranine in blood plasma samples [49].M. Vollmer and S. Buckenmaier have coupled the HPLC Chip with a time-of-

flight (TOF) mass spectrometer detector for the determination of dextromethor-phan (DEM) and its metabolites in human plasma and urine samples [50]. The


metabolism of DEM has been extensively investigated and has been used asa model drug to distinguish between ‘poor’ and ‘efficient’ cytochrome P450D6metabolizers. During the metabolism (Figure 4.17a), DEM (m/z¼ 272) is eitherdemethylated to 3MM (m/z¼ 258) or converted to the isobar dextrorphan (DOR).The latter compound may be further metabolized to 3OM (m/z¼ 244) or directlyglucoronidated to dextrorphan glucoronide (DORGlu, m/z¼ 434). 3OM is furthermetabolized to 3-hydroxymorphinan glucuronide (3OMGlu, m/z¼ 420). Thesamples are analysed with two different gradients. Figure 4.17b shows the typicalelution profile for the fast gradient.Proteomics is a field in which the LC microchip is paid more attention. Initially,

proteomics had the objective of identifying the proteins of biological systems, butmore and more this discipline is moving to targeted strategies aiming at identifyingkey proteins (biomarkers, for instance) that can provide reliable diagnostic andprognostic indicators of disease progression or treatment effects. The problem isthat biological samples are complex mixtures of proteins and the detection ofbiomarkers (existing at very low abundance) in these mixtures needs sensitive androbust analytical methods. One of the conventional and most powerful proteomicsapproaches involves combining high-resolution separations with high-accuracyMS. Since LC is complementary to MS/MS with respect to the population ofpeptides thatmay be detected, an interesting approach is to combine different piecesof information obtained by these two techniques. Thus, for instance, the retentiontime (RT) obtained by CLC clearly defines a peptide, as does its mass; thecharacterization of a peptide by a couple of pieces of data (mass measurement

Figure 4.17 (a) Metabolism of DEM by citochrome P4502D6. (b) Analysis of DEMmetabolitesby HPLC Chip–TOFMS (100nM in vitro assay; concentration on column 100 fmol; gradientlength 2.5 minutes; total run time 9.5 minutes). (Reproduced from [50] with permission fromAgilent Technologies)


and the corresponding RT, noted as mass/retention time, MRT, for m/z-RT) is veryattractive. In this way, a protein will be characterized by different MRT values,which constitute a specific peptide map. Therefore, both the RTand the m/z valuesmust be as accurate and reproducible as possible. The replacement of the CLCequipment by a HPLC Chip system can be of great value in these cases, asmicrofluidics chromatographic separations provide high separation efficiency anddeliver precise and repeatable flow rates. Using these advantages of HPLCChip–MS systems, J. Hardouin et al. have investigated different biomarkerssignatures [51]. In this case, an ITMS was used. These authors carried out theidentification of human autoantigens after obtaining autoantigen mixtures byaffinity separation (in order to simplify the complexity of the sample) of Caco-2cell proteins on the immunoglobulins from a group of healthy volunteers. Thesemixtures were digested by trypsin, and just the tryptic digest-resulting solutionswere then analysed by HPLC Chip–MS coupled with an IT. In each sample, around20 proteins were identified using the software package MASCOT. The measure-ment of MRTs revealed the identity of a peptide in different situations, such as thelack of fragment information,measuredm/z above the authorizedmass tolerance, orthe presence of different peptides sequences with close m/z ratios.Anti-doping laboratories use a test to detect the misuse of recombinant erythro-

poietin (rhEPO) based on its different migration pattern on isoelectric focusing(IEF) gel compared to endogenous human erythropoietin (hEPO), which can beexplained by structural differences.While there is definitely a need to identify thosedifferences by LC–MS/MS, the extensive characterization that was achieved for therhEPO was never performed on hEPO because its standard is not available insufficient amount. Taking this problem into account, P.E. Groleau and coworkersdeveloped an analytical method to detect pmol amounts of N-linked and O-linkedglycopeptides of the recombinant hormone (used as a model), using a nanoflowHPLCChip–ESI–ITMS [52]. The diagnostic ion atm/z 366 of oligosaccharideswasmonitored in the product ion spectra to identify the four theoretical glycosylationsites, Asn24, Asn38, Asn83 and Ser126 on glycopeptides 22–37, 38–55, 73–96 and118–136, respectively. The method described by these authors provides a means todetect glycopeptides from commercially available pharmaceutical preparations ofrhEPOwith the sensitivity required to detect pmol amounts of hEPO, which allowsthe identification of structural differences between the recombinant and the humanforms of the hormone.Proteins in general do not act individually in biological processes. Most cellular

events are controlled by multiple proteins, involving – in many cases – non-covalent interactions between proteins and other molecules. Traditional methodsused to study noncovalent protein interactions include ultracentrifugation, calo-rimetry and various types of spectroscopy. Commonly, these methods require largeamounts of sample, are relatively slow and can be fairly nonselective. For thesereasons, ESI–MS has recently been used to study noncovalently-associated


complexes. Despite the advantages of technique for this purpose, care must betaken to ensure the protein complex does not denature in the solvents used for ESI.Moreover, other instrumental parameters (temperature, voltage and pressure) mustbe controlled to avoid fragmentation of the complex. For identification purposes,high-resolution, accurate-mass and high-mass scanning are critical features in theanalysis of macromolecular assemblies. TOFMS posseses all of these capabilities,and hence is a good choice for the study of noncovalent associations. In addition tothese advantages of TOF detectors, HPLC Chip provides an extremely stablenanospray and significantly improves upon the ease of use and reliability ofnanoflow ESI. G.W. Kilby demonstrated this suitability for the study of apomyo-globin and myoglobin noncovalently-associated complexes using an HPLCChip–ESI–TOFMS arrangement [53]. The analysis was carried out in less thanfive minutes.N. Tang and P. Goodley carried out the characterization of protein phosphoryla-

tion using HPLC Chip electron transfer dissociation (ETD) ITMS [54]. Proteinphosphorylation is a type of post-translational modification which plays animportant role in the regulation of many cellular functions. The precise determina-tion of phosphorylation sites within a protein is crucial to the understanding of cellregulationmechanisms. Collision-induced dissociation (CID) and ETD can be usedin the same LC–MS run to analyse samples for protein phosphorylation studies.Figure 4.18a depicts the scheme for the ETCprocess. TheETC reactant is generatedby a small negative chemical ionization (NCI) source that is mounted directly uponthe inlet section of the second octapole ion guide on the ITMS. The NCI source isfilled with fluoranthene, which is sublimed and combined with methane gas in thepresence of electrons emitted from a filament. The electrons are slowed by collisionwith the methane gas and captured by the fluoranthene molecules to makefluoranthene radical anions. From the ESI chamber, the peptide ions are generatedfrom the nanospray chip tip. All the positive ions are allowed to enter the MS ioninlet, through the ion optics and ultimately into the IT. The positive multiply-charged peptide ions are isolated and all other ions are ejected. During this process,the flow of the reactant radical anions is closed by a voltage-gating process.Alternatively, the NCI source can be gated to allow negative ions to enter the ITas a packet. The two packets of ions (negative and positive) exist in the ITat the samepoint in time. After a few milliseconds, the positive peptide ions and the negativeions become interactive and the electron from the fluoranthene radical anions istransferred to the positive peptide ion. The electron transfer is rapid and sufficientlyenergetic, yielding a series of positive amino acid residue fragments. The IT issubjected to the scan process in the same manner as CID and the positive ETDproduct ions are scannedout of the trap, yielding theETDMS/MSspectrum [54].Asan example of the results obtained by this methodology, Figure 4.18b illustrates thedifferences between CID and ETD fragmentation for the synthetic phosphopeptideTTHyGSLPQKusingHPLCChip nanospray infusion.As can be seen, CIDhas very


few fragment ions, while ETD produces nearly complete sequence coverage, withindication of the phosphotyrosine location.The majority of the protein drugs in existence are glycoproteins. It has been

demonstrated that the efficacy of a glycoprotein drug (commonly used to treat andprevent diseases) critically depends on the type and extent of glycosylation of theprotein. Therefore, it is of great interest to characterize glycoproteins as an essentialpart of drug quality control. P.D. Perkins has explored the ability of HPLC Chipcoupled to a trap XCT ultra-MS system to detect and identify oligosaccharides andN-linked glycans cleaved from two commercially available glycoproteins [55]. TheHPLC Chip used in this case contains a porous graphitized carbon column

Figure 4.18 (a) Diagram of the Agilent 6340 ITMS with ETD module. (b) Comparative spectraobtained with CID and ETD for the phosphopeptide TTHyGSLPQK (m/z¼ 404.7) using theHPLC Chip. (Reproduced from [54] with permission from Agilent Technologies)


Table 4.3 Relative percentage amounts of the IgG N-linked glycans identified by HPLCChip/Trap XCT Ultra MS system from Agilent Technologies. (Reproduced from [55]with permission from Agilent Technologies)

Glycan(including isoforms) Structure

m/z valueof alditol [Mþ2H]2þ

Peak height ofaveraged spectrum

Relativepercent

G0 733.4 46886 14.3

G1 814.4 103172 31.4

G0 þ bisecting GlcNAc 835.2 30969 9.4

G2 895.4 35483 10.8


G2 þ bisecting GlcNAc - fuc 924.0 7623 2.3

G1 þ NeuAc 959.9 16101 4.9


G2 þ NeuAc 1041.0 38886 11.8

10 12 14 16 20 22

A: Isoform of GoB: Isoforms of G1C: Isoform of G2

orBPG, 700-2200 m/zGlycans from 1 pmolstarting glycoprotein

G 1

G 2

G 0

Time (min)18

GalManGlcNAcNeuAcFuc

AB C

Figure 4.19 Base peak chromatogram of released reduced N-linked glycans from humanserum IgG. (Reproduced from [55] with permission from Agilent Technologies)


specifically designed for oligosaccharide separation applications. The combinationof the chip with the sensitivity and scan speed of the trap MS detector provide theappropriate platform to characterize N-linked glycans. After a reasonable reductionof the number of plausible candidate compounds, base peak chromatograms, suchas that shown in Figure 4.19, were obtained. As can be seen in this figure, the knownmajor glycans in IgG, designated by G0, G1 and G2, were found. The two linkageisoforms of G1 were separated, due to the high resolving power of the porousgraphitized carbon column for oligosaccharide separation. Some minor isoformswere detected whose MS/MS spectra also matched those for G0, G1 and G2.Additionally, the data produced by this systemenabled the calculation of the relativedistribution of glycans in the sample. Thus, Table 4.3 shows the identities andrelative percentage distributions of the IgG N-linked glycans.

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