On-Line Coupling of Column Liquid Chromatography and Raman

The detection system is schematically depictured in Figure 1. ... To explore the potential of the direct coupling of conventional-size LC and Raman sp...
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Anal. Chem. 1999, 71, 4575-4579

On-Line Coupling of Column Liquid Chromatography and Raman Spectroscopy Using a Liquid Core Waveguide Reyer J. Dijkstra, Arjen N. Bader, Gerard Ph. Hoornweg, Udo A. Th. Brinkman, and Cees Gooijer*

Department of Analytical Chemistry and Applied Spectroscopy, Division of Chemistry, Free University Amsterdam, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands

The on-line coupling of Raman spectroscopy and conventional-size liquid chromatography (LC) by using a plastic liquid core waveguide (LCW) was explored. The LCW considered (length 30 cm, internal diameter 280 µm, internal volume 18 µL) is suitable for light guiding through aqueous LC eluents since the refractive index of the plastic perfluorinated material (denoted as Teflon AF2400) is 1.29, i.e., smaller than that of water (1.333) or methanol (1.329). Three-dimensional chromatograms showing on-the-fly Raman spectra are shown for a test mixture of 4-nitroaniline, 4-nitrophenol, 2,4-dinitrophenol, and nitrobenzene, compounds with slightly different NO2 vibrations. A limit of detection of 10 µg/mL for 4-nitroaniline, the most favorable of the test compounds, was achieved; the linearity ranged from 0.05 to 3.0 mg/ mL (six data points in duplicate, r2 ) 0.997). The improved detectability is due to the increased optical path length provided by the LCW and to the use of a largevolume-injection procedure (500-µL injections which effected a 35-fold improvement compared to standard, 10 µL, injections). The results of this preliminary study indicate that LC-Raman has potential. Raman spectroscopy is a mature laser spectroscopic technique with a high analyte identification potential and receives much attention in the recent literature from various chemical disciplines.1 Like Fourier transform infrared (FT-IR) spectroscopy, it provides detailed vibrational information, but it can be used much easier for aqueous samples and, even, biological systems. There is an obvious need to couple spectroscopic techniques such as Raman and FT-IR, as well as nuclear magnetic resonance (NMR), spectroscopy to column liquid chromatography (LC). These hyphenated systems can provide much structural information, which is complementary to that of LC-mass spectrometry (MS), which is rapidly becoming a routine technique. Unfortunately, developing on-line LC-FT-IR and LC-Raman is not a straightforward operation. Especially for reversed-phase LC, which uses aqueous organic eluents, on-line coupling with FT-IR is hardly possible because of the strong infrared absorbance of water. Such eluent interferences do not explain the lack of studies on LCRaman; in fact, aqueous eluents are quite promising since they (1) Lyon, A.; Keating, C. D.; Fox, A. P.; Baker, B. E.; He, L.; Nicewarner, S. R.; Mulvaney, D. P.; Natan, S. R. Anal. Chem. 1998, 70, 341R. 10.1021/ac9902648 CCC: $18.00 Published on Web 09/08/1999

© 1999 American Chemical Society

exhibit relatively weak Raman scattering. The main problem of LC-Raman is the inherently low sensitivity of Raman spectroscopy. To date, only a few preliminary studies on LC-Raman have been published, one on micro LC and one on conventional-size LC.2,3 Cooper et al.2 explored the potential of micro LC-Raman using deuterated solvents (a viable option in view of the low solvent consumption) to increase the Raman window. They used a He/Ne laser for excitation, a fiber-optic probe attachment for guiding the excitation, and the Raman scatter light and a CCD camera for recording the spectra and achieved a limit of detection (LOD) of 75 ng for nitrobenzene (60 nL injected). Concerning the coupling of conventional-size LC and Raman spectroscopy, to our best knowledge, one preliminary study was published three years ago.3 The authors used the full power of an argon laser (1 W) for excitation and a monochannel triple monochromator for detection; for toluene an LOD of 9.6 µg (20 µL injection) was reported. It will be obvious that these results are not encouraging in terms of concentration LODs. Furthermore, the chromatograms and spectra shown in these publications suggest that in practice significantly higher analyte concentrations are required to record Raman spectra on-the-fly, the main objective of LC-Raman. To improve the analyte detectability, LC has been coupled on-line with resonance Raman spectroscopy (RRS)4 and on-line5 or atline6 with surface-enhanced (resonance) Raman spectroscopy (SER(R)S) after deposition on a substrate. The present paper deals with LC-Raman without using resonance and/or surface enhancement effects. The approach chosen to cope with the low sensitivity of Raman spectroscopy is to increase the optical path length of the detection system by using a light-guiding capillary cell, usually denoted as a liquid core waveguide (LCW). If the refractive index (RI) of the capillary cell material is lower than that of the LC eluent, total internal reflection can be achieved and the incoming light will be fully captured in the capillary. As regards the Raman scatter light, only light that (2) Cooper, S. D.; Robson, M. M.; Batchelder, D. N.; Bartle, K. D. Chromatographia 1997, 44, 257. (3) Nguyen Hong, T. D.; Jouan, M.; Nguyen Quy Dao; Bouraly, M.; Mantisi, F. J. Chromatogr., A 1996, 743, 323. (4) Chong, C. K.; Mann, C. K.; Vickers, T. J. Appl. Spectrosc. 1992, 46, 249. (5) Cabalı´n, L. M.; Rupe´rez, A.; Laserna, J. J. Anal. Chim. Acta 1996, 318, 203. (6) Somsen, G. W.; Coulter, S. K.; Gooijer, C.; Velthorst, N. H.; Brinkman, U. A. Th. Anal. Chim. Acta 1997, 349, 189.

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strikes the liquid-LCW boundary at an angle of incidence equal to or exceeding the critical angle will be captured. Until recently, no capillary cell material was available with an RI smaller than those of the solvents generally used in reversed-phase LC, such as water and methanol, which have RIs of 1.333 and 1.329, respectively (at 20 °C), while silica-based glasses have RIs larger than 1.46.7 Therefore, the suitability of the LCW approach could be demonstrated only by using high-RI liquids such as aromatic solvents, carbon disulfide, and various halogenated compounds.8 To extend the applicability of LCWs to other solvents, including water, metal-coated tubes and even uncoated borosilicate or fusedsilica tubes have been used.8 With the latter type, the protective layer is removed so that the light is guided by total reflection at the outer glasssair, rather than the liquidsglass, boundary. The results were very promising, but the loss of light characteristic of the capillary is determined by the quality and condition of its outer surface. That is, extensive cleaning prior to use and very careful handling to avoid scratching are necessary. Besides, such capillaries are extremely fragile and their applicability for routine use is, therefore, quite limited.8 Recently, a breakthrough was achieved. A new kind of plastic LCW was developed and commercialized that does not suffer from the above drawbacks. It is based on a perfluorinated polymeric material with a RI of only 1.29.7-9 Recently, Holtz et al.10 used a type of LCW to create a small-volume (∼6 nL) Raman detector, in which transverse illumination was applied and end-on detection through a transparent window at the tube terminus was used. To involve LCWs for detection purposes in LC, it is of main interest that capillaries can be obtained with an internal diameter of 280 µm or an internal volume of ∼60 µL/m. Such volumes are compatible with conventional-size LC. Our group recently used such a plastic LCW to improve the LODs of several analytes in LC with conventional absorption detection, which caused a 3050-fold enhanced detectability.11 In the present preliminary study, the suitability of these LCWs to develop LC-Raman is studied. For this purpose, nitro-substituted compounds, known for their favorable Raman response, were used as model analytes. EXPERIMENTAL SECTION LC System. The LC system consisted of an LKB model 2150 pump (Pharmacia, Uppsala, Sweden), with a laboratory-made sixport injection valve equipped with a 10- or 500-µL injection loop, a 5-µm C-18-bonded silica column (4.6 mm i.d. × 25 cm; Luma Phenomenex, Torrance, CA) and a Raman LCW detector cell. The eluent was methanol/aqueous 10 mM phosphate buffer (pH 3.0; 60/40 or 65/35, v/v) at a flow rate of 1.0 mL/min. The eluent was filtered over 0.45-µm pore size disks from Millipore (Bradford, MA) and sonicated at reduced pressure before use. Chemicals. Methanol (HPLC grade), water (HPLC grade), sodium dihydrogen phosphate, phosphoric acid, and 4-nitroaniline were obtained from Baker (Deventer, The Netherlands). 4-Nitrobenzene was from Merck (Darmstadt, Germany) and 2,4(7) Altkorn, R.; Koev, I.; Van Duyne, R. P.; Litorja, M. Appl. Opt. 1997, 36, 8992. (8) Altkorn, R.; Koev, I.; Gottlieb, A. Appl. Spectrosc. 1997, 51, 1554. (9) Waterbury, R. D.; Rao, W.; Byrne, R. H Anal. Chim. Acta 1997, 357, 99. (10) Holtz, M.; Dasgupta, P. K.; Zhang, G Anal.Chem. 1999, 71, 2934-2938. (11) Gooijer, C.; Hoornweg, G. Ph.; de Beer, T.; Bader, A.; van Iperen, D. J.; Brinkman, U. A. Th. J. Chromatogr., A 1998, 824, 1.

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Figure 1. Schematic representation of the experimental setup. Key: L1-L5, lenses; OF, optical fiber; HBF, holographic band-pass filter; LC, effluent from liquid chromatograph; LCW, liquid core waveguide; NF, notch filter; SP, spectrograph; OMA, optical multichannel array. (Inset) FT, fingertight; TH, termination head; QW, quartz window.

dinitroaniline from Fluka (Buchs, Switzerland); 4-nitrophenol was synthesized in-house. Detection System. The detection system is schematically depictured in Figure 1. The light of a Spectra Physics (Mountain View, CA) argon ion laser Series 2000, with a wavelength of 514.5 nm, was focused by lens L1 (f, 50 mm) on a quartz optical fiber with a core of 600 µm (Ensign Bickford Optics, Avon, CT), transported over 15 m and focused by lens L2 (f, 30 mm) on a holographic band-pass filter for 514.5 nm (Kaiser Optical Systems, Ann Arbor, MI), and coupled into the LCW by a lens L3 (f, 100 mm). The Raman scattering was collected in the “forward mode”. It was projected by lens L4 (f, 50 mm) onto a holographic super notch filter for 514.5 nm (HSNF-514-xx, Kaiser Optical Systems) to get rid of the laser light and subsequently focused by lens L5 (f, 100 mm) onto a single-stage spectrograph (Monospec 18, Scientific Measurement Systems, Grand Junction, CO). This monochromator had a focal length of 156 mm and a grating of 1200 lines/mm, which resulted in a spectroscopic resolution of 0.5 nm. The spectrum was recorded, with an exposure time of 1 s and a 2-fold accumulation, or 200 ms and a 10-fold accumulation, by a cooled EG&G Princeton Applied Research intensified linear photodiode array detector and an EG&G OMA III series model 1460 optical multichannel analyzer (Princeton, NJ). The LCW used in this study was purchased from Ocean Optics (Dundin, FL). It consists of Teflon AF2400 (code, LPC-1) and has diameters of 530 µm o.d. and 280 µm i.d. Both ends of the LCW were glued to a standard (Valco) chromatographic fingertight. Interfacing of the LCW with the laser light was achieved by using two laboratory-made poly(ethylene terephthalate) termination heads (Figure 1, see inset). In each of these, two holes were drilled and provided with a Valco thread. A tiny eluent chamber was formed between these holes, which was sealed by a quartz window. The effective length of the LCW in this setup was 30 cm. RESULTS AND DISCUSSION To explore the potential of the direct coupling of conventionalsize LC and Raman spectroscopy, an experimental setup was

Figure 2. (A) 3D chromatogram (240-840-s window) of a mixture of 1.4 mg/mL 4-nitroaniline (peak 1; 298 s), 1.8 mg/mL 4-nitrophenol (peak 2; 404 s), 2.4 mg/mL 2,4-dinitrophenol (peak 3; 484 s), and 2.8 mg/mL nitrobenzene (peak 4; 680 s); (B) background-subtracted 3D chromatogram. Eluent: methanol/aqueous 10 mM phosphate buffer (pH 3.0; 60/40, v/v). Injection solvent: methanol/water (40/60, v/v). Exposure time: 1 s and a 2-fold accumulation.

developed that provides much operational freedom and does not require the use of a Raman microscope including a triplemonochromator facility (Figure 1). An optical fiber (OF) of ∼15-m length was used to connect the argon ion laser and the LC instrument. The light exiting from the OF was filtered by a holographic band-pass filter to remove nonlasing emission from the laser as well as Raman scatter produced by the optical fiber and subsequently focused, in the best way possible, on the core of the LCW. There is, of course, a significant loss of laser light since the internal diameter of the OF (600 µm) is substantially larger than that of the LCW (280 µm). The power of the laser light finally entering the LCW is ∼40 mW. The laser light exiting from the LCW is removed by a high-quality notch filter, which allows the recording of Raman spectra in the forward mode without the need of an expensive triple monochromator (characterized by excellent stray light rejection, but with a poor light throughput).12 We used an LCW of 30-cm length, with an internal volume of 18 µL, which is sufficiently small to prevent undue band broadening of the LC peaks by the detector cell. Four test compounds were used, all of which contained a nitro substituent, which usually leads to a high Raman response. Preliminary experiments were performed using a standard injection volume of 10 µL. In a mixture of 4-nitroaniline, 4-nitrophenol, 2,4-dinitrophenol, and nitrobenzene at a level of 8 mg/mL, both 4-nitroaniline and 4-nitrophenol gave a good response, but 2,4-dinitrophenol and nitrobenzene could not be detected. To enhance the analyte detectability in terms of

concentration units, a rather large sample volume (500 µL) was injected, and so-called on-column focusing was used, a robust and well-established procedure, frequently involved in trace analyses of aqueous samples.13 This implies that the analytes are dissolved in a noneluting solvent containing, typically, at least 20% less organic modifier than the eluent. The analytes will then be trapped on the top of the LC column and are desorbed only when the LC run is started after the sample introduction. Under these conditions, injection volumes of 500 µL do not cause serious band broadening. Figure 2A shows the 3D chromatogram of a 1-3 mg/mL mixture of 4-nitroaniline, 4-nitrophenol, 2,4-dinitrophenol, and nitrobenzene thus obtained. The separation of the analytes is satisfactory, but the peaks are rather broad, which indicates that for the present sample the percentage methanol in the injection solvent is too high. Improved peak shapes can be obtained by using less modifier (see below), but then the on-column focusing approach can only be used over a limited analyte concentration range because of solubility problems of the test compounds. As was expected, the two strong vibration bands of methanol at 1023 and 1452 cm-1 dominate the 3D chromatogram, while the Raman bands of water cannot be seen. To improve the quality of the 3D chromatogram, a background-subtraction routine was written; the background was set equal to the average of, in this case, the final 50 spectra of the chromatogram covering the retention window from 740 to 840 s. Figure 2B shows the background-subtracted 3D chromatogram. The Raman spectra of the four test compounds

(12) Asher, S. A, Anal. Chem. 1993, 65, 59A.

(13) Ling, B. L.; Baeyens, W.; Dewaele, C. J. Microcolumn Sep. 1992, 4, 17.

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Figure 3. Raman spectra of a mixture of 0.5 mg/mL (A) 4-nitroaniline, (B) 4-nitrophenol, (C) 2,4-dinitrophenol, and (D) nitrobenzene; (‚‚‚) background spectrum of methanol. Eluent: methanol/aqueous 10 mM phosphate buffer (pH 3.0; 65/35, v/v). Injection solvent: methanol/water (25/75, v/v). Exposure time: 10-fold accumulation of 200 ms.

now show up more distinctly. Interestingly, in some spectra negative peaks are observed at 1023 and 1452 cm-1, i.e., at 372 and 484 s. They are not caused by the injection plug, which elutes much earlier and is not included in the figure. At 372 s no positive peaks are observed at all, while at 484 s 2,4-dinitrophenol is eluting. The reduction of the methanol peak intensities cannot readily be explained. One might speculate that at 372 and 484 s species are eluting (with the species at 372 s having no Raman response in the 840-2005-cm-1 region) that change the RI of the medium in the LCW and, thus, the response for the methanol Raman peaks. The response linearity was tested for 4-nitroaniline. The 3D chromatograms were integrated at 1300 cm-1. There was a good 4578

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linearity over the range 0.050-3.0 mg/mL (six data points in duplicate; r2 ) 0.997). Of course, the LODs that can ultimately be achieved in LCRaman are strongly dependent on the sharpness of the chromatographic peaks. To improve the peak shape, samples containing less organic modifier, viz. 25%, were used. Under these conditions, a factor 35 in sensitivity was gained by using a large-volumeinjection (500 µL) instead of the standard injection volume (10 µL). Figure 3 shows the Raman spectra obtained for a mixture containing 0.5 mg/mL of each compound. From this figure, the LODs were estimated as 10 µg/mL for 4-nitroaniline, 50 µg/mL for 4-nitrophenol, 250 µg/mL for 2,4-dinitrophenol, and 500 µg/

nitrobenzene (1346 cm-1); its concentration is too close to the LOD. The chromatograms of Figure 4 underline the potential of LC-Raman for identification purposes, although substantial improvements will have to be realized to make the method suitable for relevant applications.

Figure 4. Chromatograms recorded at (A) 1300, (B) 1311, and (C) 1331 cm-1, extracted from the 3D chromatogram of Figure 3. Injection of a mixture of 0.5 mg/mL (1) 4-nitroaniline, (2), 4-nitrophenol, (3) 2,4-dinitrophenol, and (not visible) nitrobenzene. Eluent: methanol/ aqueous 10 mM phosphate buffer (pH 3.0; 65/35, v/v). Injection solvent: methanol/water (25/75, v/v). Exposure time: 10-fold accumulation of 200 ms.

mL for nitrobenzene. These concentration LODs are better than reported for earlier LC-Raman studies where, at least milligram per milliliter levels were required. 2,3 In the spectra of Figure 3, the vibration band of the NO2 substituent can easily be recognized in the 1300-1350-cm-1 region. Interestingly, the four analytes can be distinguished by the wavenumber shift of this band. Figure 4 shows chromatograms recorded at 1300, 1311, and 1331 cm-1, which were extracted from the same LC run as for Figure 3. The selected wavenumbers correspond with the NO2 vibration bands of 4-nitroaniline, 4-nitrophenol, and 2,4-dinitrophenol, respectively. No LC peak was observed at the wavenumber of the NO2 vibration band of

CONCLUSIONS The results obtained in this preliminary study are quite encouraging with the LOD of 10 µg/mL for 4-nitroaniline, the most favorable of the test compounds, indicating that LC-Raman apparently has perspective (although it should not be forgotten that nitro compounds have favorable Raman responses). It should be noted that the improved detectability (which cannot easily be compared with the few literature data available) is due to the use of the LCW as well as to the large-volume-injection procedure (which effected a 35-fold improvement of the LODs). To further improve detectability the laser powersin the present setup only 40 mWswill have to be increased; 10-fold higher powers are readily available. Second, the use of longer LCWs should be considered. For the present LCW of 280 µm i.d., the length should not exceed 30 cm since otherwise considerable additional band broadening of the LC peaks will occur. If waveguides with smaller inner diameters would be available, much longer cells would become feasible (e.g. for an i.d. of 100 µm, a length of 250 cm). Third, LCWs with a much smaller inner diameter will have a much smaller detector cell volume, which is especially relevant in micro LC, and the use of deuterated solvents will become a viable option. For the test compounds considered here, the use of deuterated methanol would significantly increase the Raman window and therefore suppress the interferences caused by the eluent.2 Finally, next to conventional LC-Raman, we shall attempt to further develop LC-RRS, which also can benefit from introducing the LCW technology. ACKNOWLEDGMENT We greatly acknowledge Mr. Tjipke de Beer for his advice.

Received for review March 9, 1999. Accepted July 15, 1999. AC9902648

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