Report
On-Line Coupling of Capillary Separation Techniques with 1 H N M R evelopments in separation methods in the last decade have focused primarily on miniaturization. Capillary electrophoresis (CE) and capillary HPLC (CHPLC) represent milestones in this respect; the recently introduced capillary electrochromatography (CEC)—a hybrid of CE and CHPLC—complements these fascinating developments. (Glossary on p 320 A.) Miniaturization of separation methods offers distinct advantages over conventional techniques: less sample is required, less eluent is consumed, and the separation time in CE and CEC is shorter. These factors are of particular importance in natural compound chemistry, pharmacokinetic studies of drug metabolism, and combinatorial chemistry. In these areas, capillary separation methods have found wide acceptance. Although UV-vis, fluorescence, and electrochemical detectors provide sensitive and simple detection with these techniques the information is generally not sufficient for unequivocal characterization or structural elucidation of unknown compounds especially in complex multicomponent mixtures Electrosprav MS (ESIMS) in which the analyte is Drotonated or deprotonated has proven hiehly successful for the analysis of peptides proteins and nucleotides (1) The on line forma '
tary techninue for ionizing nonpolar compounds (2). Nonetheless, the information provided by MS and tandem MS is not
Petra Gfrörer Jens Schewitz Klaus Pusecker Ernst Bayer Universitat Tubingen (Germany)
always sufficient for determining the unknown compound structure because it does not reveal stereochemistry. The most frequently used method for determining the chemical, dynamic, and spatial structural properties of organic compounds is NMR spectroscopy (3). Because NMR spectra are already measured in solution, no phase transfer is necessary when coupling with LC techniques. Additionally, NMR is a nondestructive detection technique, allowing the analyte to be submitted to other methods of characterization. On-line coupling of HPLC and NMR spectroscopy has been investigated for 20 years. The first studies were publlshed by Watanabe et al. (4) and Bayer et al. (5). For many years, however, the method languished, primarily because of the low sensitivity oo oMR spectrometers. The introduction of magnets with higher field strength, smaller-diameter receiver coils with greater dynamic range, suitable flow cells, and versatile solvent suppression techniques led to enormous gains in sensitivity. HPLC/NMR has now reached a stage where instruments are commercially available and are being successfully used for investigating' drug metabolites and natural products {6-8)
Improved sensitivity, deuterated solvents, and less sample make this approach feasible for many applications.
The major limitations of conventional HPLC/NMR coupling remain the relatively large amount of sample required and the high rate of solvent consumption. The latter restricts the use of deuterated solvents for economic reasons, thus necessitating the application of solvent suppression techniques. Solvent suppression, however, leads to the masking of part of the NMR spectrum. In addition, cross signals in twodimensional (2D) experiments (discussed later) will not appear if they couple to a signal in the suppressed area. Miniaturization of both the separation
Analytical Chemistry News & Features, May 1, 1999 3 1 5 A
Report and the NMR components is an elegant way to overcome the problems associated with solvent signals. The low rate of solvent consumption in capillary methods makes using fully deuterated solvents economically feasible and this, in turn, allows the entire range of the XH NMR scale to be used. In addition, there is no longer a loss of information in either one-dimensional and 2D experiments. The supplementary saving of material is particularly relevant for valuable samples. Finally miniaturization of NMR detection is essential for coupling to CE or CEC. In 1994, Wu et al. (9,10) described the first system in which NMR was coupled directly to a CE system using fused-silica capillaries of 75- to 530-um i.d. High sensitivities were reported, but the system was not suitable for routine operation. Another approach, based on the experience gained from coupling conventional HPLC to NMR, was reported in 1996 by Behnke et al. (11) and used for various applications (12,13). This led to the development of a modified configuration (14) that is equally suitable for CHPLC CE and CEC. Several reported applications have demonstrated the practicality of this system for the analysis of natural compounds (14 15) drug's (16) and their metabolites (17 18) In this Report. we summarize the work in this new
field and consider the prospects for further developments and applications. Miniaturized separation techniques Miniaturized HPLC has been an area of growing interest in recent years. The low sample consumption is particularly attractive for combinatorial chemistry and pharmaceutical applications. In many fields, the coupling of CHPLC with MS has displaced conventional HPLC, and systems are commercially available. CE (19) has found wide application in the separation of charged compounds, which are difficult to separate by hydrophobic reversed-phase HPLC, such as nucleic acids by capillary gel electrophoresis (20). The on-line coupling of CE to ESI-MS is routine nowadays (21) even though coupling is more difficult than with miniaturized HPLC ESI-MS. Examples of coupling CE with lH NMR have been published (9 10,14-18). CEC is a developing separation method, combining the high speed and efficiency of CE with the selectivity of HPLC (22-24). In CEC, the mobile phase is driven by the electroosmotic flow (EOF) generated by an applied voltage or by a combination of EOF and pressure. If reversed-phase materials
are used, the retention of uncharged analytes is governed solely by their hydrophobic interaction with the stationary phase, whereas the retention of charged analytes is additionally influenced by their electrophoretic mobility in the mobile phase. Because the flow rates in pressurized CEC are compatible with the requirements of ESIMS, coupling of these methods is easy and does not require a special interface (25-27). The coupling of CEC with NMR is also possible (14 16-18). Microcoils Coupling these separation techniques with NMR required the simultaneous development of new capillary NMR probes and detection cells. To date, two different microcoil NMR configurations have been used for on-line coupling of capillary separation techniques with XH NMR One approach is based on a solenoid rf coil wound directly on the capillary (Figure la) (9,10,28,29). A new coil has to be prepared for every separation capillary, which makes changing the capillary cumbersome. Each sample is externally loaded, after which the probe is inserted and shimmed. In NMR sensitivity is related to the perfect filling factor—the ratio of the detection volume (V) to the volume of the NMR coil
Figure 1 . Different microcoils for coupling capillary separation techniques with NMR. (a) Solenoidal rf coil, (b) saddle-type rf coil, and (c) influence of the electric current-induced magnetic field around the capillary on the main magnetic field. 316 A
Analytical Chemistry News & Features, May 1, 1999
(Vc). With this coil configuration, it is possible to detect samples down to 160 pmoll The other configuration is based on a saddle-type rf coil (Figure lb). The detection unit consists of a coil affixed directly to a glass tube, into which the detection capillary is inserted. This setup has a considerable advantage in that the coil is not permanently attached to the separation capillary, thus allowing the capillary to be easily exchanged without the risk of damaging the rf coil. In addition, the probe does not require removal before measurement. On the other hand, the internal diameter of the coil is fixed and the filling factor is lower than with the solenoid setup. Moreover the close proximity of the metal rf coil and the sample could lead to susceptibility gradients within the sample which result in increased linewidths and the loss of resolution This problem is avoided by increasing the distance between tlip sample and the coil These two coil forms also diifer in another important aspect: The axis of the detectton capillary is parallel to the permanent magnetic field (B0) with the saddle-type coil, ana it is at right angles to B0 for the solenoid coiil The induced magnetic field (Bj) generated by the current in the capillary affects the B0fieldof the NMR in different ways, as shown in Figure lc. The magnetic field B{ of the solenoid coil system must be added to B0, and this affects the *H NMR signals. .n the saddletype arrangement, B{ has no component in the B0 direction and thus has no effect. When coupling electrophoretic techniques line broadening resulting from the interactions of the main magnetic field with the magnetic field induced by the electric current in the capillary does not occur Interfacing NMR to capillary separations
Signal-to-noise ratio. The limiting factor in coupling capillary separation techniques with NMR is usually the detection limit of the system. The parameters that affect S/N, and, therefore, the detection limit of an NMR measurement, are
S/N = yN 1(1+/)
(BQ\2
—
W
/
/QVS\\
(1)
\ b Jn-l
In the above equation, y represents the mag-
Figure 2. Coupling of capillary separation techniques with NMR.
netogyric ratio; N is the number of nuclei in the detection volume; lis the spin quantum number; T is the temperature;/is the filling factor; Q is the quality factor of the resonating circuit; and b is the receiver band wiith (6). With a given NMR instrument operattng under similar conditions for the acquisition of the NMR data, most of the variables are, in fact, constants. The filling factor (/= VJV) and V sre thus the erucial parameters that can be influenced. The value of/ for a solenoid arrangement is very favorable; detection volumes between 5 and 200 nL can be reached because the coil fits directly onto the detection capillary
poor because the diameter of the fused-silica capillary is limited by the chromatographic and electrophoretic requirements. For example, CE separations require the use of capillaries with an internal diameter of less than 100 um. In the case of CEC and CHPLC separations, the ratio of the internal diameter of the separation column (100- to 300-um i.d.) to the internal diameter of the transfer/detection capillary should be greater than four. To improve/ and thus the sensitivity of the system, the detection volume of the standard fusedsilica capillaries (50- to 100-um i.d. and 360-um o.d.) has to be increased.
Detection capillary. Whereas the
The use of extended light paths for UV detection is a common practice for both CE and CHPLC. Because the signal is sirecdy proportional to the length of the llght path, Z-cells, U-cells, or bubble-cells can be esedd This approach cannot be adopted for NMR detection, however, because the symmetry of the detection cell would not work for NMR Instead, we have developed a CE capil-
solenoid coil configuration uses a standard fused-silica capillary for detection, the saddle-type coil system requires a special detection capillary. The smallest saddle-type rf coil used at present has a diameter of 2 mm and a length of 9 mm. If this soil is used directly with the fused-silica capillaries common in CE instruments, the value of / i s
Analytical Chemistry News & Features, May 1, 1999 317 A
Report This treatment in creases the internal diameter of the unheatedpartofthe capillary by —5-10 um, and the heated part can be enlarged in a per fectly symmetrical manner UD to 250 um. Moreover the detection cellwall thickness is re duced which aids detection and the increased detection-
It is obvious that the solenoid arrangement has great potential for samples that are avail able in limited amounts. Data indicate that the mass sensitivity of the solenoid coil system is twice that of the saddle-type coil setup. On the other hand, the concentrations necessary for detection are typically 30-fold higher than in the case of the saddle-type coil. Therefore, the saddle-type system seems to be more appropriate for the coupling of capillary tech niques with NMR experiments especially in cases in which an average peak concentra tion in excess of 30 mM is difficult to achieve Further reducing the size of the coil relative to that of the capillary could improve /and hence increase sensitivity
cell volume mini mizes the line broad-
Saddle-type NMR setup
t i m t i o in f l i e r**i11
Additionally, the larger cell facilitates shimming—the opti mization of field ho mogeneity by manu ally adjusting the elec tric current in shim coils to obtain the best line shapes and the maximum signal intensities because the lock signal is more stable and the Figure 3. Static TOCSY spectrum of a 1 % solution monitored proton (10 nmol, 4.4 μg) of the protected dipeptide signal has a greater Fmoc-Val-Tmob-Gly-OH. intensity This results in better line shape and better S/N lary with an integrated, symmetrical detec tion cell that prevents accidental leakage of Detection limit. The detection limits liquids onto the NMR magnet This integrated of the two NMR coupling systems were com system is fabricated from a 2-m length of pared using arginine as sample. In 1995, 75-um i.d., 360-um o.d. fused-silica capillary, Sweedler et al. (29) reported a detection which is rinsed with 2 M HC1 followed by limit of 34.2 ng (arginine hydrochloride) and 2 M NaOH for 15 min each to clean the inter a corresponding concentration of 32.4 mM nal surface. The detection cell is prepared by for an arginine solution (acquisition time of heating a 15-mm length of the capillary to 1 min, detection volume of 5 nL, S/N = 3)) 90 ° C while drawing a 10% HF solution For these measurements, a 300-MHz spec through the capillary by means of an aspira trometer and a solenoid rf coil wound on a tor vacuum. Excess HF is flushed out with standard fused-silica capillary were used. distilled water In the detection area the Using a special capillary with saddle-type rf polyimide coating is burned off and the coil and a detection volume of —400 nL in exposed surfaced is cleaned with methanol conjunction with a acquisition 600-MHz spectrometer 70 ng of arginine (acquisition The internal diameter of the detection time 45 s) corresponding to a concentration cell is measured under a microscope by of 1 mM can be detected comparing it with an untreated capillary. 318 A
Analytical Chemistry News & Features, May 1, 1999
Figure 2 shows the configuration for the coupling of CHPLC, CE, and CEC with NMR (14). A key element is the one-piece detec tion capillary, which facilitates handling and allows the safe use of the high voltages typi cal of electrophoretic techniques. The CE and CEC equipment must be positioned ~3 m from the magnet. The inlet and outlet vial, constructed of poly (ether ether ketone) (PEEK), can be positioned directiy under the NMR magnet. For CE measurements both ends of the detection capillary are im mersed in buffer reservoirs Voltage was applied by a modular CE system When op erating in the CHPLC mode a packed col umn was connected to the detection capillary hv means of a Teflon tube outside the NMR A gradient HPT JP wstem was rnnnprtpH via a 250-um i d capillary to the injprtion device For CEC measurements CE and
were
Stopped-flow measurements. Mul tidimensional NMR spectra are essential for structural elucidation of compounds. These techniques normally require more time because an analyte solution resides in the detection cell under continuous moni toring conditions, and the flow is stopped when the target substances are in the de tection cell. After acquiring the spectra, continuous flow is resumed. Homonuclear correlation spectroscopy (COSY) can typically identify spin-coupled pairs of protons separated by as many as three bonds. In the contour plot in this 2D experiment, the normal spectrum (as pro jected on the axis) is represented as con-
tours along the diagonal of the plot with cross-peak contours appearing if spin coupling is present. From the connectivities indicated by the COSY spectrum, a chemical structure can often be derived. A variation of COSY, total correlation spectroscopy (TOCSY) is a long-range, shift-correlation technique, which offers a way to identify nuclei belonging to separate spin systems. In TOCSY, cross peaks indicate protons that are coupled to each other along an unbroken chain of coupling. In our laboratory, the technique is mostly used with peptides and oligosaccharides for identifying single residues (e.g., amino acids in peptides). Each proton of an amino acid displays cross peaks with the other proton of the same amino acid. Coupling with protons of the next amino acids however does not occur because the peptide bond interrupts the spin system After identifying the amino acid residues the peptide sequence can be obtained from a subsequent 2D NOESY (nuclear Overhauser effect spectrosrnnvt eynprimp-nt which detects interactions hptwppn neicrhhnrino' a m i n o acidc
To demonstrate the suitability of the interface for structural elucidation of samples with restricted amounts, a static (solution injected directly into the detection capillary) 2D TOCSY spectrum of a protected dipeptide was accumulated (Figure 3). A1% (by weight) solution of peptide was introduced, resulting in just 10 nmol of the peptide in the detection cell (volume of —400 nL).
The signals of the protecting groups, trimethoxybenzyl (tmob) and 9-fluoromethoxycarbonyl (fmoc), are clearly visible in Figure 3. An assignment of the signals can be achieved via their chemical shift values. Hence, it follows that the signals at 7.9-7.2 and 4.3-4.1 ppm arise from the fmoc group, whereas, the signals at 6.2,4.5,3.8, and 3.3 ppm arise because of the tmob residue. The cross signals that appear between the methyl group signal at 0.9 ppm and signals at 2.2 and 5.1 ppm (shown by dotted lines) indicate the separate spin system of the valine residue whereas the glycine signal (3 6 ppm) shows no cross peaks Thus the spectrum allows the assignment of the J H NMR signals and affords confirmatory evidence for the structure of the Fmoc-ValTmob-Gly-OH dipetide CHPLC/NMR. Because the capillarycoupling setup allows the use of fully deuterated solvents, the need for solventsuppression techniques (with their associated loss of signals) is eliminated. For example, some of the signals of hop bitter acids coincide roughly with those of acetonitrile (ACN). Using undeuterated ACN, these signals are masked. The flavor ingredients of beer are complex, composed of a mixture of humulones and isohumulones. All NMR signals are necessary for full structural characterization (see Figure 4). Usually, the connectivities of the side chains can be derived from
2D COSY spectra (dotted lines in Figure 4b and 4c). Because solvent suppression is required for the spectrum in Figure 4b, important cross peaks in the 2D experiments with structural information about a side chain in question are lost. By way of contrast, when the same mixture of isohumulones was investigated by CHPLC/ NMR, using the same chromatographic conditions but with CD3CN as eluent (Figure 4c), the cross peaks in the high-field region, which were masked in the left-hand plot are now apparent allowing full structural elucidation and assignment of the hop bitter acids (14) CE/NMR. When coupling CE with NMR the separation is best performed in the detection capillary itself. The volume of the entire separation capillary is ~6 uL, and only nanoliter volumes of sample are injected. Therefore, given this small injection volume and the relatively high detection limits required for NMR, a high sample concentration is necessary. The first measurements using the saddle-type coil were performed with a detection capillary that had a cell volume of 250 nL. In spite of the rather poor sensitivity the suitability of the setup for CE/NMR on-line experiments was demonstrated with a separation of amino acids (14) The detection limit improved by enlarging the detection cell to —400 nL allowing the analyses of more demanding samples (1^—1 ft)' even stonned-
Figure 4 . S t u d i e s of n-isohumulone by separation techniques coupled to NMR. (a) Assigned structure of n-isohumulone. Stopped-flow H,H COSY spectra of n-isohumulone from (b) an HPLC separation and (c) a CHPLC separation of an isohumulone mixture. (Adapted with permission from Ref. 14.) Analytical Chemistry News & Features, May 1, 1999 3 1 9 A
Report flow 2D CE/NMR experiments became possible (15,16). For example, the contour plot of a CE/ NMR analysis of paracetamol from human urine is depicted in Figure 5a with NMR chemical shift on the horizontal axis and retention time on the vertical axis. The principal metabolites of paracetamol observed are glucuronide, sulfate, and hippurate (an endogenous compound found in human urine). CEC/NMR. On-line coupling of CEC
with NMR (14,16-18) shows even more promise than the coupling of CE or CHPLC with NMR CEC has higher sample capacity than CE, and higher separation efficiencies and shorter analysis times than CHPLC. Of special interest is the coupling of gradient CEC with NMR. Gradient CEC/NMR offers increased sample capacity as a result of sample preconcentration at the front of the column and a higher separation efficiency together with a reduction in analysis time compared with isocratic CEC/NMR (16). Figure 5b shows the analysis of the analgesic, Thomapyrin, which contains acetylsalicylic acid, caffeine, and acetaminophen, using on-line coupling of gradient CEC with NMR. The substances are eluted as sharp peaks. The ACN signal in the contour plot of the gradient CEC/NMR is shifted somewhat because of the change in solvent composition. On-column preconcentration to increase the sample's concentration and improve separation efficiency leads to more intense NMR signals.
Glossary
2D: two dimensional b: receiver band width B;. .nduced magnetic field B0: magnetic field strength CE: capillary electrophoresis CEC: capillary electrochromatography CHPLC: capillary HPLC COSY: correlation spectroscopy EOF: electroosomotic flow ESI-MS: electrospray ionization mass spectrometry /: coil filling factor (= V^/VJ I: spin quantum number n: noise figure N: number of nuclei in the detection volume Q: qualiiy factor of the resonating circuit T: ttmperature TOCSY: total correlation spectroscopy Vc: volume of the coil Vs: detection volume y: magnetogyric ratio
Conclusions and expectations
Numerous obstacles to coupling capillary separation methods with NMR have been overcome. The main problem of sensitivity has been sufficiently solved so that samples in the range of 100-500 pmol can be analyzed. This still requires overloading the chromatographic and electrophoretic columns, especially in the case of opentubular capillary CE. Electrochromatography, as a hybrid of reversed-phase HPLC and CE tolerates higher sample capacities and provides sharper elution profiles. This is especially true of gradient CEC which has proved to be very efficient Therefore CEC/NMR has great potential
Figure 5. (a) CE/NMR electropherogram of paracetamol: (1) glucuronide, (2) sulfate, and (3) hippurate. (Adapted w i t h permssion from Ref. 18.) (b) Gradient CEC/NMR chromatogram of the separation of Thomapyrin ( 1 0 % solution in MeOD, injection volume of - 5 0 0 nL): (1) acetylsalicylic acid, (2) caffeine, and (3) acetaminophen. (Adapted w i t h permission from Ref. 16.) 320 A
Many innovations are conceivable and certainly will be the topic of ongoing investigations. It is evident that reducing the diameter of the saddle-type rf coil will lead to better sensitivity. However, new coil arrangements and new spectrometer designs are also being pursued. The practical results already being presented demonstrate the enormous potential of capillary separation techniques coupled with NMR. These approaches can be expected to play a key role in the separation identification and structure elucidation of complicated natural and synthetic mixtures.
Analytical Chemistry News & Features, May 1, 1999
The authors wish to thank J. Nicholson, J. .indon, and their working group at Birkbeck College (U.K.) for a gift of paracetamol metabolites; Klaus Albert and Bruker GmbH (Germany) for their long-term cooperation in thefieldof HPLC/NMR Ii-Hong Tseng, W. Schaal, and E. Grom for their technical assistance; G. J. Nichollon for reading the manuscript; and the Deutsche Forschungsgemeinschaft Graduiertenkolleg "Analytische Chemie" for financial support. We are grateful lor a photograph of the detection unit provided by Bruker Physik (Rheinstetten, Germany). References
(1) Cole, R. B., Ed. Electrospray Ionization Mass Spectrometry; Wiley & Sons: New York, 1997. (2) Bayer, E.; Rentel, C; Gfrorer, P. Angew. Chem..,n press, ,999. (3) Ernst, R. R; Bodenhausen, G; Wokaun, A. Principles of Nuclear Magnetic Resonance in One and Two Dimensions; Oxford University Press: Oxford, U.K., 1991. (4) Watanabe, N.; Niki, E. Proc.Jpn. Acad., Ser. B 1978, 54,194-99. (5) Bayer, E.; Albertt K; Nieder, M.; Grom, E.; Keller, T./. Chromatogr. 1979,186,497-507. (6) Lindon, J. C; Nicholson, J. K; Sidelmann, U. G; Wilson, I. D. DrugMetab. Rev. 1997,29, 705-46. (7) Albert, K.; Bayer, E. Anal. Methods InI strum. 1995,2, 302-14. (8) Albert, K./. Chromatogr., A 1995, 703, 123-47. (9) Wu, N.; Peck, T. L.; Webb, A. G; Magin, R. L.. Sweedler, J. V./. Am. Chem. Soc. 1994,116, 7929-30.
(10) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V.Anal. Chetn. 1 9 9 4 , 66, 3849-57. (11) Behnke, B.; Schlotterbeck, G.; Tallarek, U.; Strohschein, S.; Tseng, L.-H.; Keller, T.; Albert, A.; Bayer, E.Ana.. Chetn. 1996, 68,1110-15. (12) Schlotterbeck, G.; Tseng, L.-H.; Haendel, A; Braumann, U.; Albert, K.Anal. Chem. 1997, 69,1421-25. (13) Albert, K.; Schlotterbeck, G.; Tseng, L.-H.; Braumann, V.J. Chrotnatogr.,A 1996, 750, 303-09. (14) Pusecker, K.; Schewitz, J.; Gfrorer, P.; Tseng, L.-H., Albert, K.; Bayer, E. Anal. Chem. 1998, 70, 3280-85. (15) Schewitz, J.; Pusecker, K.; Gfrorer, P.; Gotz, U.; Tseng, L.-H.; Albert, K.; Bayer, E.; Chromatographic!, accepted, 1999. (16) Gfrorer, P.; Schewitz, J.; Pusecker, K.; Tseng, L.-H.; Albert, K.; Bayer, E. Electrophoresis, 1999,20, 3-8. (17) Pusecker, K., et al. Anal. Comni. 1998, 35, 213-15. (18) Schewitz, J., et al. Analyst 1998,123, 2835-37. (19) Altria, K. D., Ed. Capillary Electrophoresis Guidebook: Principles, Operation, and Applications; Humana Press: Totowa, NJ, 1996.
(20) Heller, C, Ed. Analysis of Nucleic Acids by Capillary Electrophoresis; Vieweg & Son: Braunschweig, Germany, 1997. (21) Issaq, H. J. Electrophoresis 1997,18, 2438-52. (22) Tsuda, T , Ed. Electric Field Applications in Chromatography, Industrial and Chemical Processes; VCH Verlagsgesellschaft: Weinheim, Germany, 1995. (23) Robson, M. M.; Cikalo, M. G.; Myers, P.; Euerby, M. R.; Bartle, K. D.J. Microcolumn Sep. 1997, 9, 357-72. (24) Colon, L. A.; Reynolds, K. J.; Alicea-Maldonado, R.; Fermier, A. M. Electrophoresii 1997,18, 2162-74. (25) Verheij, E. R.; Tjaden, U. R.; Niessen, W. M. A.; van der Greef, J./. Chromatogr. 1991,554, 339-49. (26) Schmeer, K.; Behnke, B.; Bayer, E.Anal. Chem. 1995, 67, 3656-58. (27) Huang, P.; Wu, J.-T; Lubman, D. M. Anal. Chem. 1998, 70, 3003-08. (28) Webb, A. G. Prog. Nucl. Magn. Reson. Spectrosc. 1997,31,1-42. (29) Olson, D. L; Peck, T L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science 1995, 270,1967-70.
Petra Gfrorer is a graduate student whose research focuses on CEC and CHPLC and their coupling with ESI-MS, coordination ion spray MS, and NMR. Jens Schewitz is a graduate student whose research focuses on coupling CE with ESI-MS and NMR, and developing a new solid-phase extraction method for the isolation of nucleotides. Klaus Pusecker's graduate research focused on 2D-NMR, the analysis ofhop bitters, and the coupling of miniaturized separation techniques with NMR. He is currently a postdoctoral associate at Novartis Pharma (Switzerland). Ernst Bayer is a retired professor of chemistry and the current head of the Research Centerfor Peptide and Nucleic Acid Chemistry. He focuses his research on natural compounds peptides and antisense oligonucleotides and on the coupling ofseparation methods to ESI-MS coordination ion spray MS and NMR Address correspondence to Bayer at the Research Center for Peptide and Nucleic Acid Chemistrv UniDprsity ofTiJhinffpn AufdprMnrppwztplle 18 D 72076 Tubingen Germany (ernst hayer® nwi-tiiiehivioevi dp)
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REFERENCES
K. Imai, Y. Watanabe, Anal. Cliim. Acta., 130, 377 (1981),T. Toyo'oka, T. Suzuki, Y. Saito, S. Uzu, K. Imai, Analyst, 114, 413 (1989) S. Uzu, S. Kanda, K. Imai, K. Nakashima, S. Akiyama, Analyst, 115, 1477 (1990). T. Toyo'oka, M. Ishibashi, Y. Yakeda. K. Nakashima. S. Akiyama, S. Uzu, K. Imai./. Chromatogr,. 588, 61 (1991) T. Toyo'oka, M. Ishibashi. T. Terao, Analyst. 117. 727 (1992). Tokyo Kasci Kogyo. Ipn. Pat. Appl., JP 4-361656. Tokyo Kasei Kogyo, Jpn. Pat. Appl., JP 5-354873
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