Coupling of Open Tubular Liquid Chromatography to Electrospray

has the potential of becoming a powerful and generic tool for separation and quantification of various complex mixtures. We can demonstrate here how e...
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Anal. Chem. 1999, 71, 2915-2921

Coupling of Open Tubular Liquid Chromatography to Electrospray Mass Spectrometry with a Nanospray Interface Gustaf Hulthe,* Maria A. Petersson, and Elisabet Fogelqvist

Department of Chemistry, Analytical and Marine Chemistry, Go¨teborg University, SE-412 96 Go¨teborg, Sweden

The concept of interfacing open tubular liquid chromatography (OTLC) to electrospray ionization mass spectroscopy (ESI-MS) is here introduced. This combination has the potential of becoming a powerful and generic tool for separation and quantification of various complex mixtures. We can demonstrate here how easy and straightforward it is to connect an open capillary LC column to nano-ESI, simply by drawing the column through a stainless steel tube onto which the high voltage (HV) is supplied. This method is compared with applying the HV to a gold-coated column tip and the standard dynamic nanospray interface supplied by the ESI-MS manufacturer. Reliability and stability are documented. OTLC-ESIMS is demonstrated by separation of fatty acids in the negative mode. Over the past 5-10 years a few powerful separation concepts have been developed,1 which have proven to work excellently together with atmospheric pressure ionization mass spectrometry (API-MS). These include techniques such as capillary electrophoresis (CE)2, and capillary electrochromatography (CEC)3 as well as LC techniques giving extremely fast separations with short packed columns. Lately, turbulent-flow LC4 and nonporous silica columns5,6 at high back pressures have also been utilized, but the simple and straightforward concept of open tubular liquid chromatography (OTLC), already discussed in 1970,7 has virtually been forgotten. OTLC offers a considerable gain in column efficiency compared with packed columns in terms of time and global separation, which has been demonstrated theoretically and empirically.8 The Golay equation9 indicates that the column efficiency is enhanced quadratically by reducing the column inner diameter (i.d.). Column performance increases upon miniaturization if band broadening (1) Dorsey, J. G.; Cooper, W. T.; Siles, B. A.; Foley, J. P.; Barth, H. G. Anal. Chem. 1998, 70, 591R-644R. (2) Kriger, M. S.; Cook, K. D. Anal. Chem. 1995, 67, 385-389. (3) Jakubetz, H.; Czesla, H.; Schurig, V. J. Microcolumn Sep. 1997, 9, 421431. (4) Ayrton, J.; Dear, G. J.; Leavens, W. J.; Mallett, D. N.; Plumb, R. S. Rapid Commun. Mass. Spectrom. 1997, 11(18), 1953-1958. (5) Huber, C. G.; Oefner, P. J.; Bonn, G. K. Anal. Chem. 1995, 67(3), 578585. (6) MacNair, J. E.; Patel, K. D.; Jorgensen, J. W. Anal. Chem. 1999, 71(3), 700-708. (7) Nota, G.; Marino, G.; Buonocore, V.; Ballio, A. J. Chromatogr. 1970, 46, 103-106. (8) Go ¨hlin, K.; Buskhe, A.; Larsson, M. Chromatographia 1994, 39, 729-73. (9) Golay, M. J. E. In Gas Chromatography; Desty, D. H.; Ed.; Amsterdam Symposium 1958; Butterworths: London, 1958; pp 36-53, 53-55. 10.1021/ac981352f CCC: $18.00 Published on Web 06/11/1999

© 1999 American Chemical Society

remains negligible and with the use of a concentration-sensitive detector. A problem with OTLC columns has been their small capacity factors, k′. A handy solution is to apply a thicker film of stationary phase, for which methods involving polyacrylate phases have recently been published.10,11 Swelling of the stationary phase was earlier shown to be an easy and convenient way of increasing capacity and affinity without a severe loss of efficiency due to a more liquidlike stationary phase.12,13 The preparation technique for open tubular columns with small inner diameters for LC use has been known for many years,7 but there is still a lack of commercially available OTLC columns. Despite the popular shift to open tubular GC columns during the last 10-20 years, a similar development within the LC field has been slow, largely due to the lack of good concentration-sensitive detectors. Ideal flow rates for OTLC columns are in the order of 1-20 nL min-1 (i.d. 1-5 µm).14-16 Such low flow rates entail compromising between efficiency and sensitivity17 when microdetectors based on laser10 and electrochemical techniques are used.18 These detectors often demand derivatization to add the fluorophores, chromophores, or electrochemically active groups necessary for sensitive detection. During the 1980s, thermospray19 and particle beam chemical ionization/electron impact mass spectrometry20-23 were introduced as techniques for interfacing capillary LC with MS. However, thermospray is not compatible with the low flow rates of an optimal OTLC separation, besides having other drawbacks. (10) Swart, R.; Elgersma, J. W.; Kraak, J. C.; Poppe, H. J. Microcolumn Sep. 1997, 9(8), 591-600. (11) Eguchi, S.; Kloosterboer, J. G.; Zeges, C. P. G.; Choemakers, P. J.; Tock, P. P. H.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1990, 516, 301-312. (12) Kirkland, J. J. J. Chromatogr. Sci. 1971, 9, 206-214. (13) Swarts, R.; Brouwner, S.; Kraak, J. C.; Poppe, H. J. Chromatogr., A 1996, 732, 201-207. (14) Knox, J. H.; Gilbert, M. T. J. Chromatogr. 1979, 186, 405-418. (15) Knox, J. H.; Saleem, M. J. Chromatogr. Sci. 1969, 7, 614-622. (16) Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 56, 483-486. (17) Tock, P. P. H.; Duijsters, P. P. E.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1990, 506, 185-200. (18) Tu ¨ do¨s, A. J.; Van Dyck, M. M. C.; Poppe, H., Kok, W. Th. Chromatographia 1993, 37, 79-85. (19) Blakeley, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750-754. (20) de Wit, J. S. M.; Parker C. E.; Tomer, K. B.; Jorgenson, J. W. Anal. Chem. 1987, 59, 2400-2404. (21) de Wit, J. S. M.; Tomer, K. B.; Jorgenson, J. W. J. Chromatogr. 1989, 462, 365-375. (22) de Wit, J. S. M.; Parker, C. E.; Tomer, K. B.; Jorgenson, J. W. Anal. Chem. 1987, 59, 2400-2402. (23) Escoffier, B. H.; Parker, C. E.; Mester, T. C.; de Wit, J. S. M.; Corbin, F. T.; Jorgensen, J. W., Tomer, K. B. J. Chromatogr. 1989, 474, 301-316.

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Introduction of the nanospray24 brought new life to the interfacing of miniaturized low-flow separation techniques with MS, although OTLC has not previously been interfaced with nanoESI-MS. Three different interface principles have so far been applied to other microseparation techniques such as small packed column chromatography, CE, and CEC. The first principle consists of applying high voltage to a gold-coated tapered spraying tip attached to the column. One practical drawback of this approach is the deterioration of the gold layer and thus the limited lifetime of the spraying tip. Although efforts have been made to circumvent this problem, the lifetime has not been prolonged by more than a few days.2 In the second approach, the HV is supplied via a sheath flow,25 which enables control of the chemical composition and stability of the spray. The sample is, however, consistently diluted. The third principle entails a stainless steel junction, which is in contact with the liquid, supplying the HV (e.g., the standard Micromass dynamic nanoflow). Other similar approaches have recently been demonstrated.26-28 For example, Emmet and Caprioli29 prepared a packed and tapered microcolumn, placed directly after the liquid junction, which served as the spray tip. Whether column efficiencies as high as those that OTLC columns offer are still needed, when detection is made by tandem mass spectrometry (MS-MS) with multiple reaction monitoring (MRM), depends on the nature of the application. High efficiencies are needed when complex mixtures are to be separated and coeluting compounds cause ion suppression and consequently low precision. Furthermore, isomers such as enantiomers and diastereoisomers interfere severely when detected by MRM and demand a chromatographic separation prior to the MS detection. If the objective is to achieve rapid chromatographic separations, for example, separation of the analytes from the matrix, OTLC may be the fastest chromatographic technique to use. The aim of this work was to utilize the advantages offered by OTLC and to overcome its detection problems by using ESI-MS as a concentration-sensitive detector. Avoiding losses in column efficiency and sensitivity, as well as seeking an interface robust enough to work unattended for long periods, were also important objectives here. EXPERIMENTAL METHODS Instrumentation. A Quattro-LC Micromass (Manchester, U.K.) equipped with a Z-spray ion source was used as the detector. The ionization block was kept at 80 °C. The only gas flow in the ESI interface was a gentle flow of cone gas (nitrogen,100 L h-1). The chromatographic system consisted of an isocratic Jasco PU980 HPLC pump (Great Dunmow, U.K.), samples being injected with a Gilson 234 autoinjector (Lewis Center, OH) with 20- and 4-µL loops, after which splitting was conducted. Column inlet and waste capillary were coaxially arranged between the injection valve and the splitting T-junction to minimize band broadening. Sharp(24) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167180. (25) Vahl, J. H.; Goodlett, D. R.; Udseth, H. R.; Smith, R. D. Anal. Chem. 1992, 64, 3194-3196. (26) Geremanos, S.; Philip, J.; Freckleton, G.; Tempst, P. Rapid Commun. Mass Spectrom. 1998, 12, 551-556. (27) Wang, H.; Hackett, M. Anal. Chem. 1998, 70, 205-212. (28) Vanhoutte, K.; Van Dongen, W.; Esmans, E. L. Rapid Commun. Mass Spectrom. 1998, 12, 15-25. (29) Emmet, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605613.

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ening of fused-silica and stainless steel capillaries was performed on a Struers rotating wet sanding machine (Rodove, Denmark) equipped with a home-built second axis capillary holder rotating on its axis. Gold sputtering was performed on a modified Speedivac coating unit, Edwards 12EA/604 (Crawley, West Sussex, U.K.). The original Micromass dynamic nanospray probe was modified so that the xyz board was left to hold the capillary column, supporting high voltages, and allowing capillary adjustment. Since commercial OTLC columns are not available, those used here were made in-house. Materials and Reagents. For column preparation, fused silica was purchased from Polymicro Technologies (Phoenix, AZ) (5 and 20 µm i.d., 150 µm o.d). Columns were statically coated according to Farbrot et al.30 Stationary phases PS 255 (copolymer of dimethylsiloxane with 1-3% methylvinylsiloxane) and PS 264 (copolymer of 92-96% polydimethylsiloxane, 3-7% diphenylsiloxane, and 0.5-1% methylvinylsiloxane) and dicumyl peroxide were purchased from Sigma (St. Louis, MO). Results shown are from two typical columns. The 20 µm × 2.25 m column was coated with 0.75 µm of nonswollen PS264 stationary phase, and the 5 µm × 0.8 m column was coated with 0.012 µm of PS255 stationary phase swollen with n-heptane. As test substances, we chose fatty acids (also from Sigma) as they require derivatization to be detected with conventional OTLC detectors or analyzed by GC. The negative mode for fatty acids in ES is very suitable, and this mode also allows detection of unwanted glow discharge. Standard mixtures (1 mg/mL of each of the four fatty acidsslauric (C12:0), myristic (C14:0), palmitic (C16:0), and stearic (C18:0) acids) were prepared in tetrahydrofuran (THF), from which the test solutions were prepared by dilution with the mobile phase. Chromatography. The elution was isocratic with 75% acetonitrile and 0.1% acetic acid (v/v) in water. When chromatography was performed on the 5 µm column, the mobile phase was saturated with n-heptane for swelling of the stationary phase. As the chromatography was run isocratically, injection times had to be kept short, i.e., 0.05-1 s. MS Interfacing. Three different nanospray interfaces were compared (Figure 1). (1) Standard Dynamic Nanospray. This option (Figure 1A) consists of a 0.25-mm-bore Valco stainless steel 1/16-in. junction, to which the column and the fused-silica spray needle are connected. The fused-silica capillaries are attached by use of pieces of polyetheretherketone (PEEK) or poly(tetrafluoroethylene) (PTFE) tubings. HV is supplied to the liquid via this junction. (2) Gold-Coated Column End. The end of the column is sharpened symmetrically like a pencil or angled like a pen on a rotating sanding plate (see above) after which it is covered with a layer of gold, about 0.5 µm thick, by sputtering. The gold-coated part of the column is drawn through a conductive elastomer (supplied by Micromass) to hold the column and supply HV so that a spray can be established (Figure 1B). Stainless Steel Liner. The column is drawn through a piece of stainless steel tubing, to which the HV is directly applied (Figure 1C). The column end is untreated, neither tapered nor sharpened; i.e., the polyimide coating is still present. A Taylor cone is built (30) Farbrot, A.; Folestad, S.; Larsson, M. J. High Resolut. Chromatogr. Chromatogr. Commun. 1986, 9, 117-119.

Figure 1. (A) Standard Micromass dynamic nanospray interface. Schematic drawing of the Valco junction to which high voltage is supplied. The theoretical dead volume of this junction is 5 nL. (B) Gold-coated column interface. The OTLC fused-silica column is drawn through the conductive elastomer in one piece. Schematic picture showing the application of high voltage to the gold-sputtered column end. The column sticks out approximately 20 mm from the HV supply. (C) Stainless steel liner interface. Schematic picture of the liner interface in which high voltage is applied to the liquid through the liner and presumably a liquid film at the column surface.

up at the flat surface of the column tip,31 and a spray is formed (Figure 2). Two different stainless steel tubes have been used, sharpened and nonsharpened. The column moves freely in the liner. The position of the column end is easily adjusted by pulling it back and forth through the liner. MS Detection. Selected-ion recording (SIR) of the four strongest negative ions from the test substances, with 4-6 total scans/s, was performed. Safety Considerations. Apart from the handling of thin fusedsilica capillaries (especially when sharpened) and the caution required for work with high voltages, any person familiar with mass spectrometry should be able to repeat all the experiments described in this paper without any danger. Chemicals should be handled according to instructions from the suppliers. (31) Kebarle, P.; Ho, Y. In Electrospray Ionization Mass Spectrometry; Cole, R. B., Ed.; John Wiley & Sons: New York, 1997.

RESULTS AND DISCUSSION Coupling OTLC to ESI-MS. The standard Micromass interface theoretically has a dead volume in the liquid junction of 5 nL (Figure 1A), which can certainly be significantly larger due to imperfectly cut PEEK tubings. There is also a dead volume between the fused silica and the hole in the PEEK tubing. As expected, the standard interface turned out to be less successful for our application, mainly due to band broadening at flow rates that were a factor of 10 lower than the manufacturer’s recommendations. If the injected amount was increased by a factor of 10 compared to Figure 3A and C, it was possible to obtain a chromatogram from the 20-µm column whereas it was impossible to do so from the 5-µm column. Another problem with this interface was the instability of the spray performance at the low flow rates, at least in the negative mode, due to electrolytic Analytical Chemistry, Vol. 71, No. 14, July 15, 1999

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Figure 2. Photograph of a spraying column with i.d. of 5-µm, o.d. of 150 µm, drawn through a sharpened 40-mm-long stainless steel liner with the dimensions 170 µm i.d. and 1.6 mm o.d. The flow rate is 50 nL min-1, with an applied voltage of -3 kV. The distance between the column tip and the liner is 0.1 mm.

formation of hydrogen in the junction, leading to a pulsating spray (about every second, a flow of gas bubbles was observed). Gold-Coated Column End. To circumvent the problems of dead volumes in an interface in which the OTLC column end is connected to a separate spraying tip, the column tip was coated with a thin layer of gold sputtered onto the last 4 cm of the column (Figure 1B). The gold-coated column end provided a stable spray at flow rates down to 5 nL min-1. Our experiences are that a stable spray is achieved only when the column has in some way been sharpened, either symmetrically like a pencil or angled like a pen. This interface provided the high separation efficiency expected with no postcolumn band broadening, both for the 5- and 20-µm OTLC columns (Figure 3A and B). However, an important consideration and a significant drawback related to the gold-coated capillary interface is its short lifetime (a couple of days) due to mechanical weakness of the gold layer. This leads first to instability and then to malfunction, which has also been observed by others, and solutions to the problem have been proposed.2,32 Stainless Steel Liner. The poor long-time stability of the goldcoated column makes it unsuitable for routine analysis. This together with the time-consuming coating procedure, which frequently has to be repeated, rouse the idea of the stainless steel liner interface (Figure 1C). To investigate the performance of this interface it was compared with the interface that had worked best so far, the gold-coated column. Comparison showed that the stainless steel liner interface provided a spray equally stable to that generated by the gold-coated column. Chromatography under identical conditions with the same columns gave the same results (Figure 3) for the two interfaces. None of them seemed to affect the chromatography negatively by adding postcolumn band broadening. A sensitivity comparison between the two interfaces showed that there was no significant difference in signal intensities. The lower limit of flow rate for obtaining a stable spray was very similar for the two interfaces. The stainless steel liner interface did not (32) Barnidge, D.; Hjort, K.; Markides, K. Poster Presented at 15th Montreux Symposium on Liquid Chromatography/Mass Spectrometry, 11-13 November 1998.

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necessitate sharpening of the column tip. Several designs were tested, but no obvious improvements could be observed in comparison with the simplest and virtually untreated column end. Not even the polyimide layer, if left on the column end, deteriorated the performance (see above). We have also used it without problems for nanospray infusion applications, interchangeably within seconds to OTLC operation (unpublished work). When various columns are tested during development of analytical methods, e.g., new stationary phases, this simplifies the handling drastically. The liner interface performs well also at high flow rates (>700 nL min-1). From all these considerations, it was obvious that the liner interface was the superior choice when coupling low-flow OTLC with ESI-MS and why this interface was chosen for further studies. Long-Time Stability. A major question is whether OTLC-ES is a practically working technique, i.e., whether it is stable enough to handle thousands of samples even if unattended. To investigate this, a OTLC-ESI-MS setup with the stainless steel liner interface was left to work overnight with automatic injection of the same standard solution 420 times. The standard solution used in the experiment illustrated in Figure 3 was used also for this purpose, but a smaller amount was injected, namely, 2 fmol of each acid. The chromatographic setup was the same as in Figure 3D, except that we used a 4-µL injection loop and a flow rate of 25 nL min-1. Chromatograms throughout the test were similar, regarding k′, t0, and peak areas (Figure 4). Lauric and myristic acids were used for statistical evaluation. The area ratios of the two [M - H]- peaks of acids, as well as the absolute peak areas of the [M - H]- ions of lauric acid, were recorded in order to simulate fast separations with and without internal standards, respectively. The results are plotted in Figure 5A and B. The stability during the test period was (9% (relative standard deviation, RSD ) (s/xj) × 100%, where s is the standard deviation and jx is the mean) in absolute peak areas and (8% (RSD) in the ratios of two substances (internal standard mode). Part of the uncertainty is probably due to the fact that we recycled the mobile phase (split flow 1:218000) and thereby increased the background signal and decreased the S/N ratio slightly. Practical Considerations. A main advantage of miniaturized ES methods with flow rates below 50 nL min-1 is that cleaning of the sample cone has shown to be unnecessary over a time period of several months. Column clogging due to dirt or precipitation has not been experienced with the two direct on-column OTLC interfaces. An unattended spray of 20 nL min-1 from the liner interface continued to function for several days. This was observed by monitoring the acetate ion (m/z 59), whose signal remained constant within (10% (RSD). We have operated the OTLC separation at slightly higher linear flow rates than the optimum of the HETP curve indicates, and we avoided a nonaqueous mobile phase in order to stabilize the spray. This led to a slight decrease in separation efficiency and resolution. The Taylor cone is not so large that it covers the entire column end, but only the outlet hole, somewhat depending on the applied voltage, as observed by microscope inspection (see Figure 2).

Figure 3. Chromatographic comparison of the gold-coated and the stainless steel liner interfaces for the separation of four fatty acids at ambient temperature, detected by SIR of [M - H]- at 4.17 scans s-1; mobile phase 75% acetonitrile in water with 0.1% HAc. Injection made at t ) 0 min. (a) Gold-coated column interface. OTLC column 20 µm × 2.25 m, stationary phase, 0.74-µm-thick PS264; flow rate 170 nL min-1 (540 mm min-1); 7.6 pg of each analyte injected. (B) Gold-coated column interface. OTLC column 5 µm × 0.8 m, stationary phase 12-nm-thick PS255 swollen with n-heptane; flow rate 15 nL min-1 (760 mm min-1); 2.7 pg of each analyte injected. (C) Stainless steel liner interface. OTLC column 20 µm × 2.25 m, stationary phase 0.74-µm-thick PS264; flow rate 170 nL min-1 (540 mm min-1); 7.6 pg of each analyte injected. (D) Stainless steel liner interface. OTLC column 5 µm × 0.8 m, stationary phase 12-nm-thick PS255 swollen with n-heptane; flow rate 15 nL min-1 (760 mm min-1); 2.7 pg of each analyte injected.

A small Taylor cone is important in order to maintain minimal band broadening when column inner diameters are reduced. When low flow rates were used (