Anal. Chem. 1984, 56, 2471-2475
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Direct-Linked Analytical Scale High-Performance Liquid Chromatography/Nuclear Magnetic Resonance Spectrometry David A. Laude, Jr., and Charles L. Wilkins*
Department of Chemistry, University of California-Riverside,
Riverside, California 92521
The ablllty to perform on-line HPLC/NMR on an analytical scale Is demonstrated. An optlmlzed observatlon cell, mounted In a commerclal proton probe, achleves a resolution of less than 0.6 Hz at flow rates up to 2.0 mUmln. On-column detection limits of 50 pg are demonstrated for elght coadded scans. The ablllty to place a stainless steel LC column In the magnet bore allows dead volumes of a few mlcrollters with no loss In NMR resolutlon. With these mlnlmal dead volumes, LC/NMR reconstructions of a slxcomponent mlxture, with a tlme resolution of 4.7 8, produce chromatographic efflclencles comparable to those for conventional detectors.
the use of minimal transfer line and observation volumes. Finally, time resolution for the chromatographic run (time per data file stored) would be limited to a few seconds to provide an adequate definition of chromatographic peaks in HPLC/NMR reconstructions. The system we have developed meets these requirements and demonstrates the feasibility of on-line HPLC/NMR as a viable technique for organic mixture analysis (9). The experimental work presented here was devoted primarily to development and optimization of the system interface; optimum flow rates for maximum NMR sensitivity and resolution were determined. In addition, it was essential to minimize system dead volume. The overall system design is remarkably simple; the major advance compared to previous work is the ability to place a commercial stainless steel HPLC column directly in the NMR magnet bore without loss of NMR resolution or sensitivity. This success not only ensured complete premagnetization but also essentially eliminated transfer line volume. Once an optimum system design was obtained, systematic studies were performed to determine detection limits for on-column injections. Finally, test mixtures of up to six components were analyzed; reconstructions of the analyzed mixtures were obtained and chromatographic efficiencies were calculated and compared to those for chromatograms obtained with a UV detector.
Although the nearly universal applicability of high-performance liquid chromatography (HPLC) to the separation of organic mixtures is well recognized, the detectors most commonly employed with the technique (ultraviolet, fluorescence, refractive index) are generally nonspecific. Efforts to couple more powerful spectroscopic methods, including infrared and mass spectrometry, to HPLC have not been as successful as their GC/infrared and GC/mass spectrometry counterparts. The use of nuclear magnetic resonance spectrometry (NMR) as a detector offers the advantage of unparalleled organic structural analysis capabilities and sample requirements for HPLC and NMR which are reasonably similar. Beginning with the direct-linked HPLC/NMR experiment reported by Watanabe in 1978, preliminary work by several groups using iron magnet N M R instruments has demonstrated the feasibility of the technique (1-5). The use of high field superconducting solenoid NMR magnets provided the expected order of magnitude increase in sensitivity (6,7). More recently, the difficulties associated with the use of protonated solvents have been overcome in a successful demonstration of reverse-phase separations (7). A recent publication provides a complete review of LC/NMR work to date (8). Further progress in the development of HPLC/NMR on a more practical analytical scale has been slowed by the existence of two major problems. These are: (1)the inherent insensitivity of the NMR detector; and (2) the requirement for premagnetization of HPLC eluent before NMR detection. To date, the lack of sensitivity has necessitated either the use of preparative or semipreparative scale columns with the correspondingly larger solvent requirements or gross overloading of analytical scale columns with concurrent degradation in column efficiencies. The sample premagnetization requirement has been dealt with either by stopping the flow, resulting in long analysis times, or by use of large postcolumn magnetization regions causing large chromatographic dead volumes and associated loss in efficiencies. Within the limitations imposed by the sensitivity of the detector, an optimum set of parameters can be chosen which, if maintained, would permit successful application of HPLC/NMR on an analytical scale. On-column injection limits in the low microgram range would be required, while maintaining NMR resolution of better than 1.0 Hz in a flowing system. Maximum chromatographic efficiencies, comparable to those obtained with conventional detectors, would require 0003-2700/84/0358-247 I S 0 1.50/0
EXPERIMENTAL SECTION Apparatus. Figure 1 is a block diagram representing the on-line HPLC/NMR configuration. The major components include a Varian Model 5060 ternary solvent HPLC coupled with a Nicolet 300-MHz wide bore superconducting NMR spectrometer. A commercial 5-mm proton probe was used for all experiments. The spectrometer was controlled by a Nicolet 1280 computer executing Nicolet-developed NMR software. NMR data files were stored on a 5-Mbyte Hawk dual disk drive. For all experiments the HPLC pump was placed ca. 3 m from the magnet and connections between the pump and interface were made with 1/16 in. stainless steel or Teflon tubing. A schematic diagram of the observation cell, which is mounted directly into the standard probe, is shown in Figure 2. The glass capillary tubing used in the observation region was required to obtain maximum NMR resolution. A total observe volume of 20 rL, comparable to the observe volume of a UV detector, was used for all experiments. The 0.25-mm i.d. Kel-F plug inserted in the glass tubing was necessary to limit transfer line volume. The observation cell was readily shimmed in a few minutes with Compushim, the Nicolet simplex optimization routine, applied to the 21 room-temperature shim coils. The signal used for shimming was the free induction decay (FID) of a protonated solvent flowing through the cell. NMR Parameters. The general spectral parameters for all experiments are listed in Table I. Flow Experiments. The effects of flow rate on signal amplitude were measured by flowing a 50:50 pentane-Freon-1 13 mixture through the observation region at flow rates up to 2.0 mL/min. Teflon tubing connected the pump to the cell with no LC column present. A 750-rL premagnetization region was made by coiling Teflon tubing just below the probe. The experiment was run with four coadded scans collected and stored for each flow rate. The relative signal was obtained by integrating over the free induction decay (FID) and ratioing the result to the integrated value for the static system. 0 1984 American Chemical Soclefy
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
Table I. NMR Parameters for All HPLC/NMR Experiments General Parameters for All Experiments 300.0675 4000 Hz 13 ps at 90' quadrature
SF
ffn
sw
pulse width measure mode
=
0
&0
Specific Parameters
+
n PUMP
INJECTOR
I
to waste
Teflon tubing
\ probe casing
\
I/
experiment flow
4
resolution NMR detection limits chromatographic
1 1 8
heptane 1-octene 1,4-hexadiene o-dichlorobenzene ethylbenzene naphthalene
. 5mm Kel-F sleeve 1 -_ 1.5mm glass tubing
observe regm
Kel-F plug\
' I
Teflon tubing
from column
block size
acquisition time
4K 32K 4K 4K
512 ms 4.1 s 512 ms 512 ms
Table 11. Six-Component Mixture at Various Concentrations Used in the Study of LC/NMR Reconstructions compd name
c -
acquisitions
concentration (pg per 20 p L injected) mixture 1 mixture 2 mixture 3 mixture 4 2000 2000 2000
600 600 600
3000 2000 2000
900
200 200 200 300 200 200
600 600
100 100 100
150 100 100
Chromatographic Experiments. Stainless steel tubing connected the pump to a Rheodyne injector with a 20-pL sample loop mounted directly below the magnet. The injector was connected to a stainless steel 25 cm x 4.6 mm 10 pm silica gel Alltech column with 5 cm of '/le in. X 0.007 in. stainless steel tubing. The column was connected to the in. Swagelok union at the observation cell with 5 cm of '/le in. X 0.25 mm Teflon tubing. The detection limit studies for on-column injection were performed with naphthalene and heptane as test samples. A range of sample material from 50 pg to 2 mg was injected on the column. A single stored file of eight scans for each experiment, processed with the same conditions as those for the mixture analyses, was used to determine detection limits. Table I1 lista the compound names and concentrations for three test mixtures analyzed with 100% Freon flowing at 0.5 mL/min. These data, collected over a 15-min period, were stored as eight coadded scans every 4.7 s. The range of mixture concentrations was used to determine the effects of column overload on chromatographic efficiency. The data were processed by base line correcting, application of a 0.5 Hz line smoothing, transforming, phase correcting, autoscaling, and frequency domain subtraction of an early-eluting background spectrum. LC reconstructions of the data were best performed by summation of selected integration windows for each subtracted spectrum. The windows used were 0.75 to 2.75 ppm and 6.5 to 8.5 ppm relative to Me&.
RESULTS AND DISCUSSION It can be readily demonstrated that NMR spectra in flowing systems show both an increase in signal amplitude and a decrease in NMR resolution. These empirical observations are readily explained by the equations 1 - -1 -(1) Tlobsd TIetatic
+-
where Tlobsd and TZobsd are the spin-lattice and spin-spin relaxation time in the flowing system and r is the lifetime of
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1984
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0 0
1'
'i Y
tl z
11
1
0' .I
0.00
I
0: 2s
0: so
d.7~
I:OC
FLOW RATE
1.'
zs
(ML/MIN)
;.so
1:7s
Figure 3. The effect of flow rate on slgnal intensity for the 20-pL flow cell. Complete premagnetization is achieved with a 75GpL precell coil. Integration of the FID for a 50:50 pentane-Freon mixture provided slgnal intensity.
a nucleus in the observation cell (IO). For example, in the 20-pL cell a t 0.5 mL/min, r is about 2.4 s. The equations require that at faster flow rates (smaller T ) , both TIobsd and TZobsd are smaller. A smaller apparent TIoMresults from introduction of magnetized nuclei into the observed region while the saturated nuclei, which have yet to return to equilibrium, are swept from the cell. This suggests that in a flowing system, signals can be enhanced because the delay time in a static system of greater than 3T1 for nuclei to relax is unnecessary. This phenomenon is demonstrated in Figure 3 where the optimum signal amplitude vs. flow rate is determined for the 20-pL LC/NMR cell. From the plot, an optimum flow rate for signal enhancement of 0.6 mL/min is obtained. However, even over a wide range of flow rates up to about 2.0 mL/min, the typical range for LC flow rates, the sensitivity is better than that for a static system. It should be pointed out that at very fast flow rates, greater than 2 mL/min for the LC/NMR cell, the signal drops below that for the static system because nuclei leave the cell too fast to be observed. The shorter apparent TZobsd in a flowing system results because not all nuclei remain in the observation cell for the entire data acquisition time. Since TZoMis inversely related to the line width 0
AWllz = L Tzobsd
00
0.25
i.00
(3)
there is a broadening of the flowing NMR signal. This fact, coupled with the difficulties in spinnillg an LC/NMR cell, suggests that LC/NMR spectra might have inadequate NMR resolution. Figure 4 is a plot, with Figure 5 containing selected spectra, of the results for the experiment on resolution as a function of flow rate for the 2 0 - ~ LLC/NMR cell. Although the expected decrease in resolution with increasing flow rate is exhibited, the superior design of the cell still permits an NMR resolution of 0.54 Hz for ODCB resonances at 2 mL/min. The resolution obtained, even at high flow rates, certainly exceeds the requirements needed for high-resolution spectra of LC/ NMR eluents. The static system resolution of 0.18 Hz is
0.50
FL%
1.16
RATE1.oiML/MIN)
1.50
1.75
1.00
Flgure 4. The effect of flow rate on resolution is demonstrated for 20% ODCB in Freon. R1
RES = 0 . 4 4 HZ
R1
RES = 0.54 HZ
Figure 5. 'H NMR spectra of ODCB with the NMR resolution determined at various flow rates. Spectrum A is at a flow rate of 0.5 mLlmin, and spectrum B Is at a flow rate of 2.0 mL/mln.
actually comparable to that expected for spinning systems; this is explained because the narrow 1.5-mm diameter of the capillary tubing allows better shimming than wider bore 5-mm NMR tubes. Perhaps the most significant improvement in the LC/NMR interface is the successful placement of a commercial stainless steel column directly in the magnet bore. Ideally, spectra obtained with the column in the magnet bore would have signal-to-noise and resolution values comparable to those obtained with the column outside the bore. This was readily demonstrated by flowing a 5050 pentane-Freon-113 mixture and positioning the column up the bore while shimming. Even with the LC column inserted completely, a resolution of 0.64 Hz was obtained compared with resolution of 0.67 Hz for the column outside the magnet. Signal to noise ratios were also the same. The importance of placing the column in the
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
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ETHYLBENZENE I N FLOW CELL IMfCRCGRiMS) - - --- -Figure 6. A study of system sensitivity for the 20-pL flow cell in the
commercial proton probe demonstrates detection limits of a few micrograms of ethylbenzene. The data are obtained for single scans at a flow rate of 0.5 pLlmin. magnet bore should be recognized. As a result of this approach, minimal postcolumn volumes (a few microliters) are achieved, opening the possibility of obtaining chromatographic efficiencies matching those for more conventional analytical-scale LC systems. As high field superconducting magnets have become available, detection limits for proton NMR have been extended to the low microgram range, concurrently extending the capabilities of LC/NMR to the analytical scale. A systematic study of detection limits for the 20-pL LC/NMR cell was undertaken as part of the present research. Figure 6 is a plot of S I N vs. concentration of ethylbenzene for these experiments. In each case, a single scan was taken a t a flow rate of 0.5 mL/min. The plot suggests that as little as 2 pg in the observation cell can be detected with a SIN of 2:l for one scan. It is important to note that these detection limits, although useful in determining the ultimate system capabilities, are not equivalent to those for an amount injected onto an LC column; the resulting band broadening would obviously require a larger amount of material be injected on the column to obtain comparable SIN. To study detection limits for on-column injection, naphthalene and heptane were chosen as typical sample compounds and minimum injection limits estimated. These limits could certainly be artifically lowered by choosing favorable compounds with large ratios of protons per mole of compound at each NMR resonance. As Figure 7 indicates, spectra with excellent resolution and acceptable S I N are obtained even when as little as 50 pg is injected. Note that each of these spectra results from a single stored file containing eight coadded scans collected over 4.7 s. Coadding spectra over the entire chromatographic peak would have allowed even lower injection limits to be reached. Reconstructionsand Chromatographic Efficiency. A further requirement of analytical scale HPLC/NMR is the capability of providing chromatographic reconstructions. Although reconstructions of preparative scale experiments have been demonstrated (5),more commonly the chromatogram is provided by linking in series before the NMR an additional detector. This experimental design has the disadvantage of requiring large transfer volumes which degrade
AI
HEPTANE
81
NRPHTHRLENE
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Flgure 7. 'H NMR spectra for heptane and naphthalene, each at 50 fig injected on the column. The spectra are for eight coadded scans in a system flowing at 0.5 mL/min.
the chromatography at the NMR detector. The use of ultraviolet detectors has the further disadvantage of detecting only a subset of the compounds that are NMR observable. Several problems arise in aquiring an HPLC/NMR reconstruction comparable to the traces from the conventional analog detectors. In a flowing system large numbers of scans cannot be coadded across the chromatographic peak; detection limits are consequently much higher than with a static system. If a large cell volume is used to increase residence times for longer observation of the eluent (7), not only are there an insufficient number of data files collected to define the peak but chromatographic efficiencies are much lower. If larger quantities of material are injected on the column, gross column overload will lead to a dramatic drop in efficiencies. Results presented earlier suggest a compromise of conflicting parameters which would make possible analytical scale reconstructions equivalent to those for analog detectors. The flow cell used in the present work has a total transfer and observe volume of less than 30 pL which is comparable to volumes for a UV detector. Detection limits for injection of 50 pg of material were demonstrated; at these levels column overload is not a problem. Importantly, the 50-pg spectra were acquired with only eight scans and a time resolution of 4.7 s. Since the typical analytical scale peak elutes over 20 to 60 s, an adequate number of data files to define the peak are available. Table I1 describes the six-component mixture and the various concentrations used to determine the effect of overload on chromatographic efficiencies. In addition, a UV trace was obtained for a sample injection well within column capacity. Figure 8 contains representative reconstructions of the chromatographic runs. As expected, mixture B, with injections of 600 pg per component, visibly degraded the resolution. Reconstructions for mixture A, at 100 pg injected, provided much improved efficiencies; little tailing is observed and all but the first two peaks are easily base line resolved. Although the UV detector trace demonstrated optimum chromatographic resolution at detector limits several orders of magnitude less than possible with the NMR detector, the tre-
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
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A) A
10
4
6
8
0 PPM
2
B
1
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(rnin.1 Fbure 8. Flgure A is the UV trace at 100 ng per component. Figures B and C are reconstructions of HPLC/NMR experiments for mixtures B and D, respectively, listed In Table I. Time resolutions of 4.7 s are obtained as 89 files are collected over ca. 7 min. time
Table 111. Effects of Column Overload on Chromatographic Efficiencies for Various Concentrations of the Six-Component Mixture detector
uv
NMR NMR NMR NMR
amt injectedb
efficiencies (plates/m)" peak 4 peak 5 peak 6
100 ng 100 pg 200 pg 600 pg 2000 pg
32000 28000 22000 19000
26000 18000
24000 11000
32000 28000 33000 10000 6 000
"Efficiencies (plates/m) are calculated with the equation: N = 5.54(t,/Aw,12)2(100/L,,I (cm)). bValues are for approximate amounts injected per component in the six-component mixture. Actual amounts are listed in Table 11. mendous advantage of the nearly universal nature of the NMR detector is evident; Figure 9 presents complete NMR spectra of three components from mixture C which are not UV active and would be undetected. Table I11 provides a more quantitative evaluation of column capacity effects on efficiencies. Clearly, the efficiencies for the 2-mg and 600-pg injections are severely degraded relative to those for the UV trace. However, near the lower limits of NMR detection the experiments a t 200 and 100 pg injected provided much higher efficiencies which approach those for the UV detector. Unfortunately, a difficulty in providing accurate quantitative date for HPLC/NMR efficiencies arises from sampling limitations; the discrete data, collected at 4.7-s intervals, does not completely define the peak shape and, in particular, produces shorter peak heights. With these shorter peak heights, the calculated efficiencies for the HPLC/NMR reconstructions me necessarily smaller. Despite this problem, Table I11 and Figure 8 both clearly show that injections below 200 pg provide adequate reconstructions with minimal decrease in efficiencies resulting from column overload.
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Flgure 0. 'H NMR spectra of various compounds from mixture c . Each spectrum Is eight coadded scans at 0.5 mLlmin. Spectrum A is 200 pg of heptane, spectrum B is 200 pg of l,Chexadlene, and spectrum C is 200 pg of 1-octene injected on the column.
CONCLUSION The feasibility of analytical scale HPLC/NMR has been demonstrated for mixture analysis. However, at least an order of magnitude increase in sensitivity, bringing detection limit8 to the submicrogram level, is required to make the technique applicable to trace analysis. The increased sensitivity is most easily achieved by optimizing probe design for capillary size volumes; work is under way in development of the new probes. Equally important to the successful advance of HPLC/NMR is application to reverse-phase separations; preliminary work with various solvent suppression techniques is promising. LITERATURE CITED ( I ) Watanabe, N.; Nlkl, E. R o c . Jpn. Acad., Ser. B 1978, 54, 194. (2) . . Sayer. E.; Albert. K.: NLeder. M.: Grom, E.: Keller, T. J. Chromatour. 1979, 186, 497. (3) Baver, E.; Albert, K.; Nleder, M.; Grom. E.; Zhu, An. Fmsenlus' 2. Anal. Chem. 1980, 304, 111. (4) Buddrus, J.; Herzog, H. Org. Magn. Reson. 1980, 13, 153. (5) Haw, J. R.; Glass, T. E.; Hausler, D. W.; Motell, E.; Dorn, H. C. Anal. Chem. 1980, 52, 1135. (6) Haw. J. F.; Glass, T. E.; Dorn, H. C. Anal. Chem. W81, 53, 2327. (7) Bayer, E.; Albert, K.; Nleder, M.; Grom, E.: Wolff, 0.:Rlndilsbacher, M. Anal. Chem. 1982, 54, 1747. (8) Dorn, H. C. Anal. Chem. 1984, 56, 747A. (9) Laude, D. A.; Brown, R. S.;Wllklns, C. L. 1984 Pittsburgh Conference and Exposition; March 5, 1984; Paper No. 107. (IO) Zhernovol, A. I.: Latyshev, 0. D. "NMR In a Flowing Liquid": Consultants Bureau: New York. 1965.
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RECEIVEDfor review March 26,1983. Accepted June 18,1984. The support of the National Science Foundation under Grant CHE-82-08073is gratefully acknowledged, as is partial support from the donors of the Petroleum Research Fund, administered by the American Chemical Society. Support for purchase of the wide bore NMR spectrometer under an NSF Department Research Instrument Grant (CHE-8203497) is gratefully acknowledged.
