Anal. Chem. 2003, 75, 2177-2180
Airborne Chemistry Coupled to Raman Spectroscopy Sabina Santesson,† Jonas Johansson,‡ Lynne S. Taylor,‡ Ia Levander,‡ Shannon Fox,§ Michael Sepaniak,§ and Staffan Nilsson*,†
Technical Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, Pharmaceutical and Analytical R&D, AstraZeneca R&D, Mo¨lndal, Sweden, and Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996
In this paper, the use of airborne chemistry (acoustically levitated drops) in combination with Raman spectroscopy is explored. We report herein the first Raman studies of crystallization processes in levitated drops and the first demonstration of surface-enhanced Raman scattering (SERS) detection in this medium. Crystallization studies on the model compounds benzamide and indomethacin resulted in the formation of two crystal modifications for each compound, suggesting that this methodology may be useful for investigation of polymorphs. SERS detection resulted in a signal enhancement of 27 000 for benzoic acid and 11 000 for rhodamine 6-G. The preliminary results presented here clearly indicate that several important applications of the combination between Raman spectroscopy and acoustic drop levitation can be expected in the future. The pharmaceutical industry has increased its efforts to shorten the development time of new pharmaceuticals. Consequently, a need for handling of small sample amounts at an increased rate has evolved. To meet this need, a number of different sample-handling approaches have been suggested. In this paper, the use of airborne chemistry in combination with Raman spectroscopy is explored. Previous research has shown that it is possible to combine optical spectroscopy and liquid drop levitation.1-4 Levitated drops, as miniaturized systems, have the benefits of diversity of application and low reagent and sample consumption.5 Raman spectroscopy in levitated drops offers a means of studying real-time processes (chemical transformations, mass transport, etc.) within the confinement of a liquid drop and provides detailed vibrational information for high selectivity or structural elucidation. For applications in pharmaceutical analysis, the miniaturized format of the drop is very advantageous since in the screening phase of * Corresponding author: Phone: +46 46 2228177. Fax: +46 46 2224525. E-mail:
[email protected]. † Lund University. ‡ AstraZeneca R&D. § University of Tennessee. (1) Preston, R. E.; Lettieri, T. R.; Semerjian, H. G. Langmuir 1985, 1, 365367. (2) Popp, J.; Hartmann, I.; Lankers, M.; Trunk, M.; Kiefer, W. Ber. BunsenGes. Phys. Chem. 1997, 101, 809-813. (3) Musick, J.; Popp, J.; Kiefer, W. J. Raman Spectrosc. 2000, 31, 217-219. (4) Davies, A. N.; Jacob, P.; Stockhaus, A.; Kuckuk, R.; Hill, W.; Hergenroder, R.; Zybin, A.; Klockow, D. Appl. Spectrosc. 2000, 54, 1831-1836. (5) Welter, E.; Neidhart, B. Fresenius J. Anal. Chem. 1997, 357, 345-350. 10.1021/ac026302w CCC: $25.00 Published on Web 04/04/2003
© 2003 American Chemical Society
a drug only very small amounts of the samples exist and at the same time a vast amount of different samples must be tested. The use of levitated drops also has the advantage of circumventing the adsorption of analytes and sample components to the walls of the handling equipment. Moreover, levitated drops often provide increased sensitivity of detection since optical interference at the walls of the sample container is avoided.5,6 Acoustically levitated drops have previously been shown to be useful in several analytical areas such as cell stimulation and inhibition,6 single-cell analysis,7 and sample preparation prior to gas chromatography5 or capillary electrophoresis/capillary electrochromatography analysis.8 There is some history of employing conventional Raman detection in levitated drops, mainly to characterize analytes in solution.1-4 One particular advantage of Raman spectroscopy is the ability to distinguish between different crystal forms (polymorphs) of the analyte.9 Although Raman spectroscopy does not provide information as detailed as with X-ray diffraction, it is a much faster and convenient technique and is also an excellent tool for in situ measurements. It is fast enough for monitoring the rapid kinetics of solid-state transitions. The phenomenon of polymorphism is of critical interest to the pharmaceutical industry. There is currently considerable interest in low-consumption methods that facilitate rapid evaluation of polymorphism tendency. The promising combination of Raman spectroscopy and crystallization in levitated drops should help to meet this analytical challenge. The utility of conventional Raman spectroscopy can be limited, particularly for dilute systems, by a general lack of sensitivity. However, enormous improvements in sensitivity have been realized via surface-enhanced Raman scattering (SERS), to our knowledge an approach not previously explored for levitated drops. Fleischmann and co-workers’ initial discovery of a large SERS effect demonstrated Raman enhancement of up to 108 for molecules adsorbed on or near roughened metallic surfaces.10 Recent reports have cited enhancements with values greater than 1012, thus making possible single-molecule detection with SERS.11,12 (6) Santesson, S.; Andersson, M.; Degerman, E.; Johansson, T.; Nilsson, J.; Nilsson, S. Anal. Chem. 2000, 72, 3412-3418. (7) Santesson, S.; Degerman, E.; Johansson, T.; Nilsson, J.; Nilsson, S. Am. Lab. 2001, 33, 13. (8) Petersson, M.; Nilsson, J.; Wallman, L.; Laurell, T.; Johansson, J.; Nilsson, S. J. Chromatogr., B 1998, 714, 39-46. (9) Deeley, C. M.; Spragg, R. A.; Thelfall, T. L. Appl. Spectrosc. 1991, 47A, 1217-1223. (10) Fleischmann, M.; Hendra, P. J.; Mcquilla, A. J. Chem. Phys. Lett. 1974, 26, 163-166.
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Figure 1. Schematic of instrumental setup for Raman spectroscopy in acoustically levitated drops. Inset: (a) levitated drop, (b) ultrasonic transducer, (c) ultrasonic reflector, (d) dispenser droplet trajectory, and (e) dispenser.
While a wide variety of metallic nanostructures have been employed in SERS experiments,13 most of these structures are not amenable to use in liquid media or cannot be readily introduced into levitated drops. However, colloidal solutions of silver and gold are readily prepared and can be used in levitated drops. Nanoparticle size, degree of aggregation, and surface activation are factors that influence the observed SERS activity of these colloidal media. When properly prepared, however, extremely high sensitivity is possible for liquid-phase or dried nanoparticles of silver and gold.4,14 The combination of in situ Raman/SERS probing with levitated drop sample manipulation could well provide a unique approach to studying interesting chemical systems, including those of pharmaceutical and biological significance. We report herein the first Raman studies of crystallization processes in levitated drops and the first demonstration of SERS detection in this medium. EXPERIMENTAL SECTION Chemicals. Model compounds benzamide, indomethacin, benzoic acid, and rhodamine 6-G were purchased from Sigma Aldrich or Merck. A concentrated stock solution for each compound was prepared and diluted to produce standard solutions ranging from 10-2 to 10-6 M. Benzoic acid was dissolved in methanol, rhodamine 6-G in double-distilled water (Elgastat Maxima, Elga, England), benzamide in ethanol, and indomethacin in ethanol and 1-butanol. Silver colloid solutions were prepared using a modification of the original citrate reduction method by Lee and Miesel.15 All glassware was cleaned in 2 M HNO3 (Merck) and rinsed thoroughly with double-distilled water before use. An 18-mg sample of AgNO3 (Scharlau) was dissolved in 100 mL of double(11) Nie, S. M.; Emery, S. R. Science 1997, 275, 1102-1106. (12) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667-1670. (13) Vo-Dinh, T. TrAC, Trends Anal. Chem. 1998, 17, 557-582. (14) Krug, J. T.; Wang, G. D.; Emory, S. R.; Nie, S. M. J. Am. Chem. Soc. 1999, 121, 9208-9214. (15) Lee, P. C.; Miesel, D. J. Phys. Chem. 1982, 86, 3391-3395.
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distilled water. The suspension was brought to boil, and 2 mL of a 1% solution of sodium citrate (Sigma Aldrich) was added. The solution was kept boiling for ∼1 h while stirring. Instrumentation. An instrumental setup was developed to allow Raman spectroscopy in acoustically levitated drops as shown in Figure 1. A HoloProbe Raman spectrometer equipped with a charge-coupled device detector (Kaiser Optical Inc.) was used for these studies. A 250-mW diode laser at 785 nm was used for excitation. The laser beam was focused at a point at ∼65 mm from the probe, which was fixed on a microtranslation stage. The focus was set at a distance from the drop of ∼5 mm in order to avoid signal fluctuation due to drop movements in and out of the focus and to reduce solvent evaporation due to high laser irradiation powers. The laser power was estimated to 50-100 mW at the sample. The Raman probe head was kept in a fixed position to the ultrasonic levitator APOS BA10 (Dantec Dynamics, Erlangen, Germany). The acoustic levitator generates a standing wave with equally spaced nodes and antinodes by multiple reflections between an ultrasonic radiator and a solid reflector and operates with a frequency of 100 kHz.5,6,8 Sample drops of volume 20-100 nL were positioned in the levitator using an in-house-developed flow-through dispenser6,7 or a Hamilton syringe. Adjustments in focus on the drop were made by moving the levitator. Procedure. (a) Crystallization. Reference Raman spectra were first collected from the substances in powder form. The powder was positioned on a metal plate inserted in the levitator at a position corresponding to the node in the levitator where the levitated drop was later positioned. Drops of ∼100 nL of the standard solutions were then positioned in that same node of the levitator and Raman spectra gathered during the evaporation of the drop. The collection of spectra was stopped when the drop was completely evaporated and no further changes in the spectra could be observed. The exposure time was 3 s per spectrum. (b) SERS. Reference Raman spectra were first gathered from the stock solution for each compound in levitated drops (without
Figure 2. Raman spectra of indomethacin: (A) Reference R-indomethacin; (B) from ethanol and (C) from 1-butanol.
silver colloid). These spectra were collected with an exposure time of 3 or 10 s. Standard solutions of a volume of 120 µL were then mixed with 1200 µL of colloid solution. Spectra were gathered, typically with an exposure time of 1 s, as long as the drop stayed stable in the levitator. As soon as the drop had evaporated, a new drop was generated and new spectra were collected. Enhancements were calculated as a ratio of the response factors of the SERS signal and the conventional Raman signal. RESULTS AND DISCUSSION The combination of sample levitation and Raman spectroscopy was utilized for crystallization studies of two different samples known to exhibit polymorphism. The first system studied, the antiinflammatory drug indomethacin, can exist as a number of different solid-state modifications depending on the conditions of crystallization.16 Common forms include the stable γ polymorph and the metastable R polymorph. In addition, a number of solvated crystals have been described, and the amorphous form has been characterized. Raman spectra of several of these forms have been published.17
Figure 2 shows Raman spectra obtained from crystallization experiments with indomethacin. When indomethacin was dissolved in ethanol, the spectrum obtained from the drop following solvent evaporation was the same as that of the R polymorph, as can clearly be seen from the figure. This crystal form was found to be stable over the time scale of the experiment (data not shown). However, when 1-butanol was used as the solvent, a hitherto unknown Raman spectrum was obtained. Although it is known that indomethacin has a tendency to form solvates, no 1-butanol solvate has been reported. Moreover, crystallization from solution invariably produces the R and γ forms, with the exception of crystallization from warm methanol, whereby a third nonsolvated polymorph can be obtained.16 The Raman spectrum obtained in our experiments does not correspond to any of these forms (the possibility of an amorphous form has also been eliminated). It can be supposed that either a new solvate or a new polymorph has been produced by the unique conditions found in the levitated drop. Benzamide was the first organic substance for which polymorphism was detected.18 The metastable polymorph is usually extremely difficult to detect due to rapid transformation to the stable form, particularly when crystallized from solvent rather than the melt.19 In our crystallization experiments with benzamide, ethanol was used as the solvent. Three different experimental outcomes were observed. In one case, the stable crystal form was observed to form immediately during evaporation. In a second experiment, results from which are shown in Figure 3, the metastable form was briefly observed before converting to the stable form. Such behavior is consistent with Ostwalds rule of stages whereby the metastable form is expected to crystallize before the stable form. In the third instance, the metastable form crystallized and was stable over the duration of the experiment (∼3 min). This behavior is most likely due to differences in experimental parameters between the experiments. The resultant solid-state form depends on the evaporation rate and hence on the size of the drop, which was not exactly the same in all
Figure 3. Raman spectra from a levitated drop of benzamide in ethanol illustrating the formation of metastable form and transition to stable form.
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experiments. Thus, it is likely that a smaller drop would favor the metastable form. Furthermore, the presence of impurities originating from the injection device, which act as a seed for one particular solid-state form, cannot be ruled out. Further experiments are clearly necessary to determine the parameters controlling crystallization. Further experimentation was conducted using silver colloid in the levitated drops, to initially demonstrate the ability to use SERS with levitated drops and its advantages over the use of conventional Raman in this medium. No attempts at optimization were made, but the experiments were performed solely to investigate the possibilities and potential problems of silver colloid SERS in levitated drops. In Figure 4, benzoic acid (A) shows a large augmentation of the SERS signal (a) over the nonenhanced spectrum (b). This is illustrated even more clearly when it is noted that the concentration of benzoic acid for the SERS spectrum was 110 times less than with the conventional Raman, and the laser exposure was only 1/10 the time. Using the peak at 1004 cm-1 as a basis of calculation, the signal enhancement was estimated to be 27 000. Similarly, rhodamine 6-G (B) shows a signal enhancement of ∼11 000 for the SERS (a) over the conventional Raman spectrum (b). Subtle differences between the conventional Raman and SERS spectra such as seen in Figure 4 are seen but are quite common. Additionally, the SERS spectra for these compounds are very similar to those obtained in our laboratory for samples not in levitated drops. Optimization of analyte and silver colloid concentrations is likely to improve the enhancement factor further. Although not incorporated into these preliminary experiments, it has been demonstrated that certain additives to the colloid solution such as NaCl can improve SERS enhancements by initiating partial aggregation of silver colloids and by other mechanisms.20 Thus, further augmentation of the SERS enhancement and improvement in S/N may be achieved by adjusting the environment in the drop (e.g., adding electrolytes, changing the solvent, altering the colloidal density) or by altering irradiation conditions (e.g., changing the wavelength or intensity). Such studies may be conducted readily with the levitated drop approach. The preliminary results presented here indicate that crystallization of organic substances in levitated drops, coupled with Raman spectroscopy, offers a number of advantages to conventional techniques when crystallization behavior is explored. The rapid analysis time afforded by the Raman probe would appear to be conducive to the detection of short-lived metastable forms. In addition, only a small amount of sample is required, an advantage in some industries, such as pharmaceutical research, where new chemicals are produced in small quantities early in the research process. Our studies show that, by varying the solvent system, different crystal modifications can be produced, as in conventional crystallization, but with substantially reduced analysis time and sample consumption. In addition, the unusual conditions (no container, ultrasonic field) may result in crystal forms not produced during conventional crystallization, thus (16) (17) (18) (19)
Borka, L. Acta Pharm. Suecica 1974, 11, 295-303. Taylor, L. S.; Zografi, G. Pharm. Res. 1997, 14, 1691-1698. Wohler, F.; Liebig, J. Ann. Pharm. 1832, 3. Bernstein, J. Polymorphism in molecular crystals; Oxford University Press: Oxford, U.K., 2002; p 251. (20) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R.; Feld, M. Chem. Rev. 1999, 99, 2957-2975.
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Figure 4. (A) Conventional Raman and SERS spectra of benzoic acid; 3.6 µM and 1-s exposure time for the enhanced (a) vs 0.41 mM and an exposure time of 10 s for the nonenhanced (b). Inset: The scale of the nonenhanced spectrum (b) is magnified 20 times. (B) Conventional Raman and SERS spectra of rhodamine 6-G; 9.0 µM and 3-s exposure time for the enhanced (a) vs 1.0 mM and an exposure time of 10 s for the nonenhanced (b). Inset: The scale of the nonenhanced spectrum (b) is magnified 20 times.
providing extended information about assembly processes in molecular crystals. Finally, the ability to use SERS as a sensitive detection and characterization method in levitated drops has been demonstrated, and its improvements over conventional Raman illustrated. Optimization of the enhancement factors could yield even better results in the future. ACKNOWLEDGMENT This research was supported in part by the Swedish Research Council (VR), the Crafoord Foundation, the R. W. Johnson Research Institute, and the U.S. Department of Energy (DOE), Basic Energy Sciences under Grant DE-FG02-02ER15331 with the University of Tennessee. Received for review November 11, 2002. Accepted March 3, 2003. AC026302W
