HYPHENATED TECHNIQUES 498 CHIMIA 1999, 53, No. 10

Chimia 53 (1999) 498-500 © Neue Schweizerische Chemische Gesellschaft ISSN 0009-4293

Capillary - Challenges and Opportunities for Coupling with Mass Spectrometry

Silke Luedtke* and Klaus K. Unger

Abstract. Capillary electrochromatography (CEC) has attracted considerable interest within recent years because of its potential to generate very high efficiencies within relatively short analysis time. Since CEC combines the attributes of both capillary electrophoresis (CE) and high-performance liquid (HPLC). neutral as well as charged analytes can be separated. Usually. CEC is performed with UV detection, but mass spectrometry (MS) is becoming a more common detection method because additional information about the molecular weight and the structure of an analyte can be obtained. Due to the low flow rates in the packed capillary and the small sample amounts that are required. CEC is ideally suited for the implementation into miniaturized systems and for coupling with MS. While numerous advantages have been made in CEC/MS. the coupling technique is still in a development and growth stage. So far, the development of the technique seems to be limited by the lack of robust and automated specially designed CEC instruments and CEC interfaces. As soon as these practical constraints have been solved, CEC/MS will be a powerful separation/ detection technique with unrivaled sensitivity and specifity. This article aims at highlighting the potential of CEC as coupling technique with mass spectrometry.

Introduction the EOF. The EOF has its origin in the Advantages of CEC vs. HPLC electrical double layers that exist at the Capillary electrochromatography (CEC) solid-liquid interfaces of the fused-silica A characteristic that distinguishes elec- is a merger of capillary electrophoresis capillary and the mobile phase, and, addi- troosmotic flow from pressure-driven flow (CE) and high-performance liquid chro- tionally, of the packing material and the is the difference in the flow profile. Be- matography (HPLC) and combines ad- mobile phase. Studies on the influence of cause EOF is generated by the electrical vantageous features of both techniques, the packing material on the EOF showed field action on mobile-phase counterions namely the high efficiency of CE and the that the contribution of the capillary wall at the solid-liquid interface, the flow ve- outstanding selectivity of HPLC [1]. It has to the EOF is negligible and that the flow locity is independent of the diameter ofthe been invented in the early seventies by is almost exclusively generated by the flow channels between adjacent station- Pretorius et al. [2] but has not attracted silica beads [3]. The magnitude of the ary-phase particles, and between particles considerable interest until the last few EOF depends on the surface charges of the and the capillary wall. This holds as long years. Usually, CEC is performed on a particles as well as on the properties of the as the channels are large enough to prevent modified CE equipment with fused-silica eluent, i.e., the dielectric constant and overlap of the oppositely charged ions of capillaries packed with 3 J..Ul1 reversed- viscosity of the mobile phase, the temper- the electrical double layer [4]. The near- phase material or mixed-mode phases. ature, and the concentration of the electro- constant flow velocity across the capi Ilary Eluents comprise aqueous buffers mixed lyte. The ideal electroosmotic-flow pro- diameter produces a flat flow profile. In with organic modifiers such as acetonitrile file is plug-like so that the flow velocity is contrast, the flow profile in HPLC is par- and methanol and should have an ade- the same across the inner diameter of the abolic reflecting the flow inequalities quate dielectrical constant to stabilize the capillary. In CEC, separation can be among flow channels of different diame- electroosmotic flow (EOF). The mobile achieved by adsorptive solute-surface in- ter. The differences in flow increase the phase is driven through the capillary by teractions of the analyte with the mobile degree of solute dispersion, i.e., the eddy and the stationary phase, as well as by diffusion, resulting in significantly great- electromigration. Depending on their er band broadening compared to CEC. As charge and their size, ionized compounds no pressure limitation exists in CEC, po- can elute ahead or after the EOF marker. rous and nonporous micron and submi- 'Correspondence: Dr. S. Luedtke Department of Due to the small dimensions of the capil- cron silica beads with an average particle University of Mainz lary and the buffer/sample vials, CEC is diameterdp < 3 J..Ul1 have been investigated Duesbergweg 10-14 advantageous where only small sample as stationary phases [5]. It has been found D-55099 Mainz Tel.: +496131 394679. Fax: +495131392710 volumes are available, i.e., biological and that the EOF is independent of the particle E-Mail: [email protected] combinatorial library samples. diameter dp in the range between 0.2 and HYPHENATED TECHNIQUES 499 CHIMIA 1999.53. No. 10

3.0 /lffi using the same n-alkyl-bonded and form a pseudo-stationary phase with- coupling of pseudo-capillary electrochro- phase and identical experimental condi- in the capillary.'Unfortunately, micelles matography, a combination of both pres- tions. This result encourages the applica- lack selectivity, cause high background, sure-driven and electroosmotically driven tion of submicron silica beads since small particularly with laser-induced fluores- chromatography, with mass spectrome- particles enable high separation efficien- cence, as well as high currents at high try. In 1994 and 1995, Gordon et al. re- cies and short analysis times [6]. Accord- voltages and cannot be used in a mass ported on CEC combined with coupling to ing to the modified van Deemter equation, spectrometric system as they would clog both continuous-flow fast atom bombar- the influence of the A- and the C-term will up the ion source. As there is no stationary dement (CF-FAB) [9] and ESI mass spec- diminish when submicron silica beads are phase in CE, the selectivity of a separation trometry [10]. All work published since applied leaving the B-term (axial diffu- is entirely controlled and adjusted by the has dealt with CEC interfaced to ESI-MS. sion) as the only factor contributing to mobile-phase composition. Several classes of substances have been band spreading. This will result in ex- investigated, e.g., peptides [11], DNA- tremely sharp peaks of the order of almost adduct mixtures [12], corticosteroids [13], 1 million plates per meter. Advantageous Features of CEC and surrogate tags [14]. Wu et al. [15] with Respect to Coupling with MS analyzed protein-digest samples by ion- Equation. Modified van Deemter equation trap storage/reflection time-of-f1ight mass Mass spectrometry has become estab- spectrometry (IT/reTOF). So far, CEC has BDm Cdp2 H = A dpul/3 +---+ ---u lished as an extremely powerful adjunct not been coupled with atmospheric-pres- u Dm to chromatography techniques. The sensi- sure chemical ionization (APCl) which tivity that mass spectrometry brings to has already been successfully interfaced separation techniques is well established to capillary electrophoresis [16]. CEC has A is the eddy-diffusion term, B is the when combined with gas chromatogra- attracted reasonable interest within recent axial-diffusion term, C is the mass-trans- phy, high-performance liquid chromatog- years because it is ideally suited for imple- fer term, Dm the diffusion coefficient of raphy, supercritical fluid chromatography, mentation into miniaturized systems. In the analyte in the mobile phase, and dp the and capillary electrophoresis. Numerous CEC, the EOF moves through the packed particle diameter. Calculations of theoret- applications of HPLC/MS as well as GC/ capillary at a flow rate of about several ical H vs. u curves based on the modified MS and CE/MS have been described. De- hundred nl/min depending on the type of van Deemter equation for 1.0 /lffi and 0.5 spite the progresses that have been made packing material, the composition of the /lffi particles clearly indicate that at mod- within the last years, it is sometimes still mobile phase and the electrical field erate EOF velocities (-3 mmls) the plate necessary to split typical HPLC flow rates strength. This low flow rate is directly height H is indeed controlled by the axial of about I ml/min to those compatible compatible with nanospray emitters with- diffusion whereas the intluence of the A- with electrospray mass spectrometry (ESI/ out the need for either splitting the flow or and the C-term can be neglected [7]. Since MS) devices. The advent of capillary sep- supplementing it with a sheath liquid. the H vs. u curve is still descending at these aration techniques, such as capillary elec- Therefore, no dilution of the sample oc- velocities, a further decrease in analysis trophoresis and capillary electrochroma- curs which is beneficial as the mass spec- time can be attained by increasing the tlow tography, offer more compatible flow rates, trometer is a concentration-sensitive de- velocity, i.e., increasing the electrical field though still not being ideal. Conversely, tector. A significant increase of sensitivity strength, without compromising the effi- the flow rates are too low for the existing of several orders in magnitude can be ciency. Unfortunately, this cannot be real- MS devices and require a supplementary attained compared to LC/MS and regular ized using current commercial instruments sheath liquid. The invention of microelec- CEC/electrospray mass spectrometry. because they only generate field strengths trospray (IlESI/MS) and nanoelectrospray Due to the reduced band-spreading ef- up to 1000 V/cm. Therefore, the potential (nESI/MS) mass spectrometry, with flow fects and the high efficiencies that can be of submicron silica beads cannot yet be rates of of 20-100 nJ/min for nanospray attained in CEC, narrow peaks are ob- fully exploited with modified CE instru- and several hundred nanolitres per minute tained. They are quite beneficial because ments. for microspray, facilitates the coupling narrower chromatographic peaks produc- with CEC. CEC/MS has successfully been es higher mass flux leading to an increased applied by a number of groups using var- sensitivity of the MS. The full electro- Advantages of CEC vs. CE ious types of ionization techniques. In phoretic integrity of the separation is main- 1991, Verheij et al. [8] reported on the tained in the mass spectrometer. In CEC as Since the EOF is more or less exclu- sively generated by the packing material, a more reproducable flow is established Table. Characteristic Data of Packed CEC Capillaries across the entire diameter of the capillary. dp(j.tml Llmm] i.d. [mm] Vmllil] EOF [mm/s] Fe (j.tVmln] V [nl] This allows the use of much wider capil- s laries leading to greater sensitivity due to 3 100-295 0.05-0.1 0.16-1.86 1.60 0.6 0.5-3.3 increased sample loading and, concerning optical detection, increased path length. 100-295 0.05-0.1 0.16-1.86 1.60 0.15 0.3-2.0 Unlike CE, CEC enables the separation of neutral and charged compounds due to the dp particle size L Lenght hydrophobic stationary phase in the packed i.d. internal diameter capi IIary. In CE, the separation of neutral Vm volume mobile phase analytes can only be achieved when mi- EOF electroosmotic flow celles are used (MECC). The micelles Fe flow mobile phase consist of anionic or cationic surfactants Vs volume stationary phase HYPHENATED TECHNIQUES 500

CHIMIA 1999, 53, No. 10

length of ~25 em. Furthermore, most of the CEC interfaces are sub-optimal. They require even longer capillaries or addi- tional transfer capillaries. Transfer capil- 400 4 laries often cause postcolumn dispersion reducing the performance of the separa- tion capillary, whereas long separation capillaries cause longer analysis times 1 digitoxigenine due to the diluted field strength. The de- velopment of CEC/MS has been limitited 300 2 digitoxigenine-bis-digitoxoside 3 digitoxigenine-mono-digitoxoside by the lack of purpose-built automated CEC instruments, CEC/MS interfaces, and 5' 2 4 digitoxine « special CEC capillaries. In-house con- E- structed CEC systems often lack robust- O) ness and automation capabilities, although (,) 3 c their potentia] has clearly been demon- m 200- .0•... strated. The coupling of commercial CE 0 l/) instruments with mass spectrometry is not .0 m optimal due to the reasons outlined above. To implementCEC/MS as routine method in chemical and pharmaceutical industry,

100 these problems have to be overcome in future.

Received: June 26. 1999

o ~ [1] M. Dittmann, G. Razing, K. Wienand, F. Bek, LC GC maga::.ille 1995, 13, 800. o 2 4 [2] V. Pretorius, B.J. Hopkins, J.D. Shieke, J.Chromatogr. 1974, 99,23. time [min] [3] M. Dittmann, G. Rozing, J. Microcolulllll Separatioll 1997, 9, 399. Fig. Separation of four cardioactive substances. Capillary: 8.5 (38) cm x 100 IJm, packed with [4] c.L. Rice, R. Whitehead, J. Phys. Chelll. 1 IJm C8; Mobile phase: 60/40 ACNfrRIS-HCI 12 mM, pH 6, 30°, -790 Vlcm, detection: 1965,69,4017. 254 nm UV. [5] T. Adam, S. Luedtke, K.K. Unger, Cllro- matographia 1999,49,49. [6] J.H. Knox, Chrolllatographia 1988, 26, 329. well as in CEC/MS, short capillaries and beads of dp 111111.Most of the capillaries = [7] K.K. Unger, S. Luedtke, M. Gruen, LC GC high voltages are desirable for rapid sepa- that are commercially available are either magazille 1999, 12, 370. rations. Several authors have already per- packed with a 3-1lITI C18 material or a [8] F.R. Verheij, U.R. Tjaden, W.M.A. Nies- formed fast CEC separations using capil- mixed-mode phase. Mixed-mode phases sen, J. van del' Greef, J. Chromatogr. 1993, laries with a packed bed < I0 em at a field have both C18 alkyl chains and strong 647,375. strength of about 800 V/cm. The Figure cation-exchange groups attached to the 19] D.B. Gordon, G.A. Lord, D.S. Jones, Rapid displays the separation of four cardioac- surface of the silica. Since strong cation- Commull. Mass Spectrom. 1994,8,544. [10] D.B. Gordon, G.A. Lord, I.W. Tetler, C.M. tive substances. The separation has been exchange groups have a permanent nega- Carr,./. Chroma/agr. 1995, 700,27. achieved packing the outlet side of the tive charge even at low pH values, they [II] K. Schmeer, B. Behnke, E. Bayer, Allal. capillary up to the detection window (us- maintain a relatively stable EOF [18]. As Chem. 1995,67,3656. ing an Hp3DCE cartridge holder). The low-pH buffer systems are typically used [12] J. Ding, P. Vourous,Allal. Clte/1/.1997,69, analysis was carried out in less than two with mass spectromeric detection, the 379. minutes. mixed-mode phases are advantageous. The [13] R.N. Warriner, A.S. Craze, D.E. Games, To avoid deplenishment of the mobile application of small particles, short capil- S.J. Lane. Rapid Commutl. Mass Spectrom. 1998, 12, 1143. phase, the zwitterionic buffer TRTS (tris- laries, and high field strengths in CEC/MS [14] S.J. Lane, A. Pipe, Rapid COlllmllll. Mass (hydroxymethyI)aminomethane) has been will offer the potential for a rapid and Spectrom. 1998, 12,667. used as electrolyte. It is well established efficient separation/detection technique ap- [15J J.T. WU,P. Huang,M.X. Li,D.M. Lubman, that zwitterionic buffers tend to give a proaching the efficiency of GC/MS but Allal. Chem. 1997,69, 2908. high background signal which can reduce without its limitations regarding volatility [16] I.M. Johanson, R. Pavelka, J.D. Henion, mass spectrometric detection sensitivity. and thermal lability of the samples. J. Chrol11atogr. 1991, 559, 515, Some authors have already used involatile The column length is one ofthe major [17] S.J. Lane, R. Boughtflower, C. Paterson, T. buffer systems which have not been prob- restrictions in CECIMS, especially when Underwood, Rapid COI11I1WII.Mass Spec- trol11.1995, 9, 1283. lematic if operated at nanolitre flow rates commercial CE instruments are used. As [] 8J T. Adam, Ph.D.Thesis, University ofMainz, [ 171. demonstrated, there is no need for CEC 1998. To improve the efficiency of the sepa- capillaries with a packed bed longer than ration, the capillary was packed with po- 10 cm, but most of the instruments that are rous n-octyl-bonded submicron silica commercially available require a total