Enantioselective Interactions at the Solid–Liquid Interface of an HPLC

Anal. Chem. 2008, 80, 3572–3583

Enantioselective Interactions at the Solid–Liquid Interface of an HPLC Column under Working Conditions Ronny Wirz, Davide Ferri,† and Alfons Baiker* Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, Hönggerberg, HCI, CH-8093 Zurich, Switzerland A technique is presented which allows studying the enantioselective interactions occurring at the solid–liquid interface of a chiral stationary phase (CSP) and a racemate relevant to high performance liquid chromatography (HPLC). A conventional chiral column (Chiralpak AS) was mounted on an attenuated total reflection-infrared (ATRIR) cell mimicking an HPLC setup equipped with an ATRIR detector. Racemic pantolactone (PL) was used as the selectand. This setup in combination with modulation excitation spectroscopy (MES) allows for the identification of inter- and intramolecular hydrogen bonds being crucial for enantioseparation under HPLC operation conditions. The method is based on a two step strategy. In a first step, the enantiomers are separated by the chiral column similar to a standard HPLC experiment and upon adsorption on the identical CSP deposited on the internal reflection element (IRE), they are detected by ATR-IR spectroscopy. This experiment provides a retention time for each enantiomer. From the difference in retention, a suitable frequency is calculated which is used in a second experiment where the racemate concentration is varied alternately (modulation) in a way that the pulses of (R)PL and (S)-PL exhibit a phase lag of 90° after elution through the column. This procedure allows one to gain separate information of the enantioselective selectand-CSP interaction after performing a demodulation similar to a phase sensitive detection (PSD). A further benefit of this method is the strong enhancement of the signal-to-noise ratio. The effectiveness of the method is demonstrated by investigating the observed faster decrease in retention time of the later-eluted (R)-PL, as compared to (S)-PL, when separating at higher temperatures (from 12 to 36 °C). The origin is attributed to a weakening of a specific hydrogen bond between the CdO of (R)-PL and the N–H of the CSP. High performance liquid chromatography (HPLC) is one of the most suited techniques to separate enantiomers for analytical or production-related purposes. The growing need of the pharmaceutical and life science industries for enantiopure chemicals * Corresponding author. E-mail: [email protected]. Phone: +41 44 632 31 53. † Present address: Laboratory for Solid State Chemistry and Catalysis, Empa, Ueberlandstrasse 129, CH-8600 Dübendorf, Switzerland.

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stimulates research and development of chiral stationary phases (CSP) potentially capable of separating a large variety of enantiomers. Considerable effort is made to understand the crucial processes occurring at these chiral solid–liquid interfaces, which influence the separation performance. Research and development of new CSP often include two different analytical approaches. On the one hand, the separation performance of a CSP for certain selectands is measured using HPLC experiments under particular experimental conditions (mobile phase composition, temperature, etc.). On the other hand, the origin of enantioseparation is investigated using techniques which often impose some constraints on the measuring conditions.1 An alternative strategy for gaining information on chiral interactions are computational studies of X-ray crystal structures.2,3 Experimental techniques used to elucidate enantioselective interactions between a chiral selectand and a soluble analogue of the CSP,4,5 including X-ray diffraction,6 NMR,7,8 and IR spectroscopy,9,10 have been applied. One of the few examples where the entire CSP was investigated is a FT-Raman and surface enhanced Raman spectroscopic study where a Pirkle-type CSP was explored under quasi in situ chromatographic conditions,11 the active site concentration being significantly enhanced compared to the conventional experiment. High-resolution magic-angle spinning NMR spectroscopy (HR-MAS NMR)7 and spin–spin relaxation 13C NMR8 have been reported as well to probe chiral recognition in the presence of the CSP. Attenuated total reflection infrared (ATRIR) in combination with modulation excitation spectroscopy (MES) has been proven to be a powerful technique to study enantioselective interactions occurring at the solid–liquid interface by adsorbing each enantiomer separately.9,10 (1) Yamamoto, C.; Yashima, E.; Okamoto, Y. J. Am. Chem. Soc. 2002, 124, 12583. (2) Armstrong, D. W.; Ward, T. J.; Armstrong, R. D.; Beesley, T. E. Science 1986, 232, 1132. (3) Topiol, S.; Sabio, M.; Moroz, J.; Caldwell, W. B. J. Am. Chem. Soc. 1988, 110, 8367. (4) Akasaka, K.; Gyimesi-Forrás, K.; Lämmerhofer, M.; Fujita, M.; Nobuyuki, W.; Harada, W.; Lindner, W. Chirality 2005, 17, 544. (5) Pirkle, W. H.; Pochapsky, T. C. J. Am. Chem. Soc. 1986, 108, 5627. (6) Hamilton, J. A.; Chen, L. Y. J. Am. Chem. Soc. 1988, 110, 5833. (7) Hellriegel, C.; Skogsberg, U.; Albert, K.; Lämmerhofer, M.; Maier, N. M.; Lindner, W. J. Am. Chem. Soc. 2004, 126, 3809. (8) Oguni, K.; Ito, M.; Isokawa, A.; Matsumoto, A. Chirality 1996, 8, 372. (9) Wirz, R.; Bürgi, T.; Baiker, A. Langmuir 2003, 19, 785. (10) Wirz, R.; Bürgi, T.; Lindner, W.; Baiker, A. Anal. Chem. 2004, 76, 5319. (11) Horvath, E.; Kristof, J.; Frost, R. L.; Rintoul, L.; Redey, A.; Forsling, W. J. Chromatogr., A 2000, 893, 37. 10.1021/ac702363d CCC: $40.75  2008 American Chemical Society Published on Web 04/04/2008

The use of IR spectroscopy as a detection method in liquid chromatography is known since the mid-1970s,12,13 and the combination of the two techniques has been recently reviewed.14The major advantage of detection in the IR range is the sensitivity to numerous selectands due to the analysis of vibrational modes of specific functional groups, which allows the differentiation and identification of species dissolved in a medium or interacting with a solid. Although FT-IR has a potential for the elucidation of molecular interactions, severe limitations occur due to the strong energy absorption of the mobile phase. A possibility to circumvent this drawback is to make use of the short path length of the attenuated total reflectance (ATR) mode.15 For this purpose, cylindrical internal reflectance (CIRCLE) flow cells16–19 and horizontal diamond based ATR-IR cells20 were developed. Recently, the feasibility of using an ATR-IR microscope as a detection unit has been also demonstrated.21 Several coating materials like polymers,22–28 biological materials,29–31 and sol–gel films32,33 have been used to increase the detection limits of the ATR-IR technique for quantification. The coating has the advantage to increase the population density of molecules nearby the internal reflection element (IRE) used as waveguide for the infrared radiation. Here we report on a technique that facilitates the investigation of specific enantioselective interactions between a CSP and the enantiomers of a chiral selectand from experiments involving the racemate instead of adsorbing the pure enantiomers separately as described earlier.9,10 Since the pure enantiomers are not always available, it is desirable to develop an analytical technique from which identical information can be obtained using the racemate. (12) Vidrine, D. W.; Mattson, D. R. Appl. Spectrosc. 1978, 32, 502. (13) Kizer, K. L.; Mantz, A. W.; Bonar, L. C. Am. Lab. 1975, 7, 85. (14) Somsen, G. W.; Visser, T. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; John Wiley: Chichester, U.K., 2000, Vol. 12, 10837. (15) Harrick, N. J. Internal Reflection Spectroscopy; Interscience Publishers: New York, 1967. (16) Morgan, D. K.; Danielson, N. D.; Katon, J. E. Anal. Lett. 1985, 18, 1979. (17) Sperline, R. P.; Muralidharan, S.; Freiser, H. Appl. Spectrosc. 1986, 40, 1019. (18) Miller, B. E.; Danielson, N. D.; Katon, J. E. Appl. Spectrosc. 1988, 42, 401. (19) Danielson, N. D. Encyclopedia of Physical Science, 3rd ed.; Academic Press: New York, 2002; Vol. 8, pp 673. (20) Edelmann, A.; Diewok, J.; Baena, J. R.; Lendl, B. Anal. Bioanal. Chem. 2003, 376, 92. (21) Patterson, B. M.; Danielson, N. D.; Sommer, A. J. Anal. Chem. 2003, 75, 1418. (22) Heinrich, P.; Wyzgol, R.; Schrader, B.; Hatzilazaru, A.; Lübbers, D. W. Appl. Spectrosc. 1990, 44, 1641. (23) Krska, R.; Rosenberg, E.; Taga, K.; Kellner, R.; Messica, A.; Katzir, A. Appl. Phys. Lett. 1992, 61, 1778. (24) Carey, W. P.; DeGrandpre, M. D.; Jorgensen, B. S. Anal. Chem. 1989, 61, 1674. (25) Meuse, C. W.; Tomellini, S. A. Anal. Lett. 1989, 22, 2065. (26) Gobel, R.; Krska, R.; Kellner, R.; Seitz, R. W.; Tomellini, S. A. Appl. Spectrosc. 1994, 48, 678. (27) Gobel, R.; Seitz, R. W.; Tomellini, S. A.; Krska, R.; Kellner, R. Vibr. Spectrosc. 1995, 8, 141. (28) Ertan-Lamontagne, M. C.; Parthum, K. A.; Seitz, R. W.; Tomellini, S. A. Appl. Spectrosc. 1995, 48, 1539. (29) Poirier, M. A.; Lopes, T.; Singh, B. R. Appl. Spectrosc. 1994, 48, 867. (30) Taylor, G. T.; Troy, P. J.; Nullet, M.; Sharma, S. K.; Liebert, B. E.; Mower, H. F. Appl. Spectrosc. 1993, 47, 1140. (31) Kang, S. W.; Sasaki, K.; Minamitani, H. Appl. Opt. 1993, 32, 3544. (32) Han, L.; Niemczyk, T. M.; Lu, Y.; Lopez, G. P. Appl. Spectrosc. 1998, 52, 119. (33) Rivera, D.; Poston, P. E.; Uibel, R. H.; Harris, J. M. Anal. Chem. 2000, 72, 1543.

Figure 1. Schematic representation of the experimental setup. The dissolved selectands and the mobile phase were stored in two distinct reservoirs connected to an HPLC pump via a computer controlled Teflon valve. Regular switching of this valve resulted in equal pulses of the selectand solution as well as the mobile phase. The chiral column right after the HPLC pump induced a phase shift of the corresponding selectand pulses before they were detected in the ATR-IR cell by interacting with the CSP deposited on the IRE.

Figure 1 schematically shows the principal experimental setup applied for this purpose. The CSP material packed in the column is also deposited on the IRE, and the selectands are admitted through the chiral column and the ATR-IR cell. The column separates the enantiomers, and the CSP coating on the IRE acts as a sensor. Although in principle any coating material can be used to increase the detection limits of the dissolved selectands, the application of the same coating material as the one packed in the column offers the possibility to study distinct enantiospecific selectands-CSP interactions under HPLC working conditions with the obvious assumption that these interactions dominate in the column as well. This is done by applying MES as previously described.34,35 Equal volumes of the selectands and the mobile phase are pulsed through the setup (column and ATR-IR cell). The two enantiomers in each selectand pulse possess different retention characteristics in the column indicated by monitoring their interaction with the coated IRE. Because of the reversibility of the adsorption process, the ATR-IR spectra are repeated after each modulation period and can be accumulated and averaged. This approach has several advantages: (i) Spectral information on the selectand-CSP interactions is obtained with a high signalto-noise ratio under working HPLC conditions. (ii) The racemate can be used instead of the pure enantiomers. (iii) The retention times of the enantiomers can be related to specific interactions between the CSP and the selectands. (iv) The effect of essential parameters like temperature or polarity of the mobile phase on the separation performance can be studied by one technique. To demonstrate the feasibility of the technique, Chiralpak AS as the CSP and the enantiomers of pantolactone as the selectands were chosen since Chiralpak AS belongs to an important class of CSPs often used in separation techniques. The reasons for the choice of this system are multiple. Racemic pantolactone can be (34) Baurecht, D.; Fringeli, U. P. Rev. Sci. Instrum. 2001, 3782. (35) Urakawa, A.; Bürgi, T.; Baiker, A. Chem. Eng. Sci. [Online early access] DOI: 10.1016/j.ces.2007.06.009.

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efficiently separated using Chiralpak AS.36 Furthermore, the pure enantiomers of pantolactone are available, and the spectra obtained by adsorption of the single enantiomers can be used as a reference to evaluate the results obtained by the proposed technique. From a spectroscopic point of view, pantolactone is ideal because it exhibits carbonyl stretch modes at higher wavenumbers than those of the CSP. Therefore, changes occurring on both the selectand and the selector can be better discriminated. SPECTRA DECONVOLUTION VIA RESPONSE DEMODULATION A detailed description of the principle of MES can be found elsewhere.34,35 In this paragraph, an overview is given that focuses on the two advantages of MES, namely, the increase of the signalto-noise ratio and the separation of two signals showing a phase shift, to study the specific enantioselective interaction between a CSP and a pair of enantiomers. A problem encountered when studying dynamic or fast phenomena is often the poor signal-to-noise ratio of the measured signals due to the limited scanning time. For reversible processes, this problem can be overcome using MES. The system is periodically perturbed by the parameter which should be investigated such as, for example, temperature, pressure, pH, or concentration. Upon periodically changing this parameter, the system is stimulated with a certain frequency and responds accordingly with an equal frequency. The response of the system can then be measured, for example, using spectroscopic techniques leading to the acquisition of periodic signals with characteristic amplitudes and phase lags with respect to the stimulation. In our case, the CSP in contact with the mobile phase is the system under investigation and is stimulated by varying the selectand concentration. The response of the system contains the information we are interested in, namely, the enantioselective interactions of the CSP and the selectands. Figure 2 illustrates the relation of the various signals with respect to the stimulation before (Figure 2a) and after (Figure 2b) the chiral column used to force two enantiomers, denoted as (S) and (R), to be adsorbed and detected on the CSP-coated IRE at different times. Before the column, the responses of the solvent front, (S), and (R) oscillate with the same frequency and are in phase with the stimulation. After the column, the oscillating response of the solvent front is slightly shifted compared to the stimulation because the front of the noninteracting solvent needs some time to pass through the column (t0). In chromatography, this time is typically used as the reference. Because of the distinct interactions of the selectands with the CSP in the column, longer retention times are observed for the two enantiomers, before these signals also start to oscillate. The more strongly bound enantiomer (for example the (R)-enantiomer) has a larger retention time than the (S)-enantiomer (t(R) > t(S)). The three oscillating responses depicted in Figure 2 show a typical phase relation with the stimulation, which is determined by the modulation period T. As it will be seen in the following, the appearance of a 90° phase lag between two pulses of (S) and (R) as depicted in Figure 2 is the key step to separate the two responses. There are several possibilities to achieve such a requirement considering that the first pulse of (R) can display a phase lag of ±90° with respect to the (n + 1)th pulse of (S). A (36) Kaida, Y.; Okamoto, Y. J. Chromatogr. 1993, 641, 267.

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Figure 2. Phase shift of response signals due to retention caused by the chiral column. (a) Before the column, all responses are in phase. (b) After the column, the response of the solvent front is slightly shifted since the solvent needs the time t0 to pass through the column. The retention times of the (S)- and (R)-enantiomers are longer (t(S) and t(R)) due to interaction with the CSP. The (R)-enantiomer develops intimate interactions with the CSP and is therefore eluted significantly later than the corresponding (S)-enantiomer. A 90° response shift for the responses of the two enantiomers is obtained by setting a proper modulation period T which can be calculated from the difference in retention times ∆t. n is the number of (S)-enantiomer pulses which are eluted before the first (R)-enantiomer pulse exits the column.

phase lag of ±90° is equal to T/4 knowing that T corresponds to 360°. Modulation periods T can be calculated from the retention time of the two enantiomers or from their difference ∆t ) t(R) t(S) if the following conditions are fulfilled (cf. Figure 2) phase lag of -90°: t(S) + nT +

T ) t(R) 4

(n ) 0, 1, ...) (1)

and phase lag of +90°: t(S) + nT -

T ) t(R) (n ) 1, 2, ...) 4 (2)

Solving for T results in T)

4∆t (4n + 1)

(n ) 0, 1, ...)

(3)

T)

4∆t (4n - 1)

(n ) 1, 2, ...)

(4)

and

In Figure 2 the phase lag of an (R) pulse relative to an (S) pulse is +90° and n ) 1. The retention time of each enantiomer is obtained in a preliminary experiment where a short pulse of the racemate is separated by the column, and spectra are continuously measured thus simulating a typical HPLC experiment.

Figure 3. Principle of spectra deconvolution via response demodulation. (a) After accumulation of several modulation periods, the time-resolved spectra can be averaged into one period where A(ν(R),t) and A(v(S),t) represent all responses originating from (R) and (S), respectively. These responses are forced to exhibit a relative phase lag of 90°. (b) The demodulation sine wave with equal frequency and phase constant φPSD as PSD PSD the corresponding response signals are determined. Here, the frequency is 1/T and φi,ph.,(R) ) 360° for A(ν(R),t) and φi,ph.,(S) ) 270° for A(ν(S),t). PSD (c) Demodulation of all response signals according to eq 5 with φi ph,(R) ) 360° results in a PSD signal with magnitude Amp(R) because responses of (R) are in phase with the demodulation sine wave. All PSD signals of (S) are silent because the responses of (S) are out of phase with the PSD demodulation sine wave (90°!). With φi,ph.,(S) ) 270°, the same parameters are determined for the other enantiomer.

After t(R), each spectrum within a period is continuously repeated after the time T, and therefore, several periods can be accumulated and averaged into one period, resulting in a considerably enhanced signal-to-noise ratio of the corresponding spectra. Figure 3a shows such time-dependent signals obtained at v(R) and v(S) which are oscillating with amplitudes Amp(R) and Amp(S) as a result of concentration modulation. The responses measured at wavenumbers v(R) and v(S) represent each signal specific of the dissolved enantiomer or the diastereomeric selectand-CSP complex. The two signals show a relative phase lag of T/4 (90°) and are superimposed in a real experiment leading to signal overlapping when v(R) and v(S) are too close. The resolution of the signals is explained in the following and is obtained after elaboration of the averaged time-resolved spectra similarly to a phase sensitive detection (PSD). This procedure is known as demodulation(eq 5):

Ak(ν, φPSD) )

2 T

∫

T

0

A(ν, t) sin(kωt + φPSD) dt

(5)

In our case, the response is measured by ATR-IR spectroscopy and therefore A(v,t) is a set of time-resolved IR spectra of sinusoidal shape (Figure 3a) for A(v(R),t) and A(v(S),t). Multiplication with a demodulation sine function of the form sin(kωt + φPSD) (Figure 3b) and integration over the whole period T results in phase-resolved spectra Ak(v,φPSD) (Figure 3c). The demodulation sine function contains the same frequency ω as the stimulation/ response or higher harmonics thereof (kω; k ) 1, 2, ...). The result of such a demodulation is that signals having other frequencies than kω in the time-resolved spectra are eliminated in the phaseresolved spectra. This is especially important for the noise of a measured signal, which consists normally of much higher frequencies than the applied modulation frequency. Therefore, it is silent after demodulation. This leads to the enhancement of the signal-to-noise ratio.

In this work, demodulation was carried out only at the fundamental frequency (k ) 1). The phase constant φPSD in the demodulation sine function can be varied from 0° to 360° with the consequence that the magnitudes of A(v(R), φPSD) and A(v(S),φPSD) range between ±Amp(R) and ±Amp(S), respectively. When φPSD iph,(R) is equal to the phase constant of the response signal A(v(R),t), they are in phase to each other as depicted in Figure 3 PSD for φiph,(R) ) 360° with the consequence that the PSD signal A(v(R),φPSD iph,(R)) becomes maximal (Amp(R)). At the same time, every response A(v(S),t) of the other enantiomer shows a 90° phase lag toward the demodulation sine wave and, therefore, is out of phase PSD PSD (φiph,(R) ) φooph,(S) ). A response signal which is out of phase with the demodulation sine wave has no PSD signal and is silent (A(v(S),φPSD ooph,(s)) ) 0). On the other hand, if demodulation in Figure PSD 3 is performed with φiph,(S) ) 270°, the response signal A(v(S),t) is in phase and a signal with magnitude Amp(S) is observed in the demodulated spectrum, whereas every signal of (R) is silent. Thus, distinct spectra of (R) and (S) can be obtained in the phasedomain, whereas their response in the time-domain is measured as a superposition with a 90° relative phase lag by performing a demodulation with the proper phase constant φPSD. Suitable phase constants are accessible by Fourier transformation (FT) of the corresponding A(v(S),t) and A(v(R),t) responses, respectively. It should be clear that the existence of at least one signal which can unambiguously be assigned to one enantiomer (A(v(R),t) or A(v(S),t)) is crucial to effectively calculate the corresponding in phase constants necessary to obtain the correct phase resolved spectra and limits the proposed technique to systems showing a nonoverlapping enantioselective signal. It should be stated once more that (R) and (S) stand for signals which originate from both the dissolved enantiomers and the corresponding enantiomer-CSP complex. In this way, spectroscopic investigations concerning Analytical Chemistry, Vol. 80, No. 10, May 15, 2008

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preferential interactions of the CSP and the selectands can be performed without the need of having the pure enantiomers available. EXPERIMENTAL METHODS Materials. The chiral selectands (S)-pantolactone ((S)-PL, Fluka, ∼99% purum) and (R)-pantolactone ((R)-PL, Fluka, g99% puriss) were used as received. Mesitylene (MSY, Fluka, ∼99% puriss) was used as the internal standard in all experiments and showed no significant interaction with the CSP. Chiralpak AS particles (20 µm particle size) kindly provided by Chiral Technologies-Europe were used as the CSP. A Chiralpak AS column was used (Chiral Technologies; 4.6 cm i.d., 240 mm length, and 20 µm particle size) for separation. 2-Propanol 20 vol % (Fluka, >99.8% puriss) in cyclohexane (Acros, >99%) was used as the mobile phase. The internal reflection element (IRE) for the ATR-IR experiments was a ZnSe prism (52 mm × 20 mm × 2 mm, 45°, Crystran Ldt.). With the assumption of refractive indices of 1.4 for the wet CSP film and 2.4 for ZnSe, a penetration depth dp of 1.7 µm (0.9 µm) at 1000 cm-1 (1800 cm-1) was calculated. Film Preparation. Chiralpak AS particles (6 mg) were stirred in approximately 5 mL of CH2Cl2 (J. T. Baker) for 2 h. Approximately 1.5 mL of the solution was then dropped onto the IRE on an area corresponding to 151 mm2. The solution was allowed to evaporate, and the resulting film (t(R)) until (R)-PL in the first pulse passed the ATR cell. The experimental parameters are listed in Table 1 for each temperature. The corresponding demodulated ATR-IR spectra of each enantiomer adsorbed on the selector were obtained at the particular temperature by a response separation via demodulation and offer the possibility to relate the temperature-dependent separation performance with specific selectand-CSP interactions. Theoretical calculation using Gaussian0339 were performed on a strongly simplified model system of (R)-PL bound to one single side branch of the CSP as well as the single (R)-PL and the side branch, respectively, to explain and support the behavior of the observed ATR-IR signals mainly originating from chemical groups involved in hydrogen bonding. Therefore, the B3LYP method using a 6-31 gdp basis set was applied where the structures were first optimized before a normal-mode analysis was performed. To support the observations made in the ATR-IR experiments concerning separation of pantolactone by Chiralpak AS chiral

stationary phase at different temperatures, similar experiments were performed on a standard HPLC setup. Separation was performed at 12-36 °C in steps of 3 °C. Before the experiment was started, the system was equilibrated for 2 h at the corresponding temperature by flowing the mobile phase. The experiment was repeated once as a check. The values for selectand concentration as well as the injection volume were different compared to the ATR-IR experiments and matched the necessary HPLC conditions. Nevertheless, a similar behavior was observed for the retention of (S)- and (R)-PL when the temperature was increased. RESULTS AND DISCUSSION Method Evaluation. The collection of IR spectra after admittance of a 120 s pulse of the racemate solution resulted in wavenumber dependent chromatograms. Figure 4 shows the chromatograms obtained for such an experiment at three different wavenumbers. The CdO stretching vibration of dissolved pantolactone is expected at 1782 cm-1; therefore, the corresponding chromatogram (Figure 4a) contains the contribution from both enantiomers. The maximum of the (S)-PL signal was detected 1065 s after the measurement was started whereas that of (R)-PL after 1532 s. Note, that the correct assignment of the absolute configuration was possible due to experiments performed with only one enantiomer. The difference in retention time of the two enantiomers is therefore ∆t ) 467 s. A signal at 1716 cm-1 was only observed for (R)-PL when adsorbed on the CSP, and the corresponding chromatogram is depicted in Figure 4b. The signal is assigned to a CdO stretching vibration of one of the carbamate side chains of the CSP hydrogen bonded to the sOH group of (R)-PL.9 The internal standard mesitylene (MSY) was not interacting with the CSP, and its prominent signal at 1607 cm-1 (quadrant mode of the aromatic system) could be used to determine t0 (651 s), the time at which the solvent front reached the spectroscopic cell. The quality of the obtained chromatograms in terms of retention, peak width, and peak resolution is not optimal compared to the ones achieved by a standard HPLC setup (comparison depicted in Figure 7). However, these factors are sufficient for the purpose of using modulation excitation spectroscopy (MES). In principle a better chromatogram would be obtained by a shorter pulse at higher selectand concentration leading to a smaller peak width. However, in the following modulation experiment as discussed above, the pulse width is given by the calculated modulation period T. For the same reason, the selectand concentration should be kept as low as possible to minimize overloading of the column during a modulation experiment or to use a (39) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar; S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M. ; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J.; HratchianH. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004.

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Figure 5. Time-resolved responses of the signals at 1782 and 1716 cm-1 obtained from three distinct but similar modulation experiments of (a,d) racemic PL, (b,e) (R)-PL, and (c,f) (S)-PL at 12 °C. The signal at 1782 cm-1 (νCdO) represents both enantiomers in solution. The signal at 1716 cm-1 is characteristic of the (R)-PL-CSP complex. With a modulation period T ) 623 s, the responses of the two enantiomers are expected to have a 90° phase shift (T/4 ) 156 s).

Figure 4. ATR-IR spectra and chromatograms of the “one-pulse” experiment. MSY, (S)-PL, and (R)-PL were separated by a Chiralpak AS column, and successive ATR spectra were measured after the column using the IRE covered with the same CSP material as a sensor. (a-c) Chromatograms measured following the intensity of the signals at 1782 cm-1 (νCdO, PL), 1716 cm-1 (νCdO, (R)-PL), and 1607 cm-1 (MSY, t0 ) 644 s). (d) ATR-IR spectrum measured after 1064 s when (S)-PL reached the spectroscopic cell. (e) ATR-IR spectrum measured after 1531 s when (R)-PL reached the spectroscopic cell. The temperature was 12 °C.

preparative instead of an analytical column. Figure 4d shows the ATR-IR spectrum measured after 1064 s when (S)-PL was detected, whereas the corresponding spectrum of (R)-PL measured after 1531 s is depicted in Figure 4e. Although some difference is already clear from these spectra, their poor signalto-noise ratio hardly allows the unambiguous identification and interpretation of signals or signal shifts of specific functional groups. From the spectra shown in Figure 4, only the retention time of the selectands can be reliably obtained from the largest signals corresponding to the modes of carbonyl groups in the region above 1700 cm-1. Employing a chiral column for separation and ATR-IR spectroscopy combined with MES allows the identification of enantiomerCSP interactions from high-quality spectra. Therefore, continuous pulses of the racemate were performed, resulting in a mutual shift of the enantiomer pulses induced by the column when detected on the IRE. This approach renders the use of neat enantiomers to study specific selectand-CSP interactions superfluous. 3578

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Parts a and d of Figure 5 show the transient responses of the signals at 1782 and 1716 cm-1 of a modulation experiment where the concentration of racemic PL was modulated from 0 to 3 mM. As mentioned, the signal at 1782 cm-1 belongs to both enantiomers, whereas the signal at 1716 cm-1 indicates a distinct interaction between (R)-PL and the CSP. Because of the presence of the chiral column, the response to (R)-PL should be observed with a +90° phase shift compared to the response to (S)-PL, when a modulation period T ) 623 s is applied as calculated from eq 4 (∆t ) 467 s, n ) 1). A 90° phase shift is optimal for the deconvolution of spectra using response demodulation and corresponds to a shift of 156 s (T/4). The experimental shift qualitatively fits well with the predicted 156 s as demonstrated in Figure 5a. Quantitative values for the experimentally observed shift can hardly be given because no distinct signal could be clearly assigned to the (S)-PL adsorbed on the CSP which could have been compared with the signal observed at 1716 cm-1. The reliability of the technique, however, could be tested by carrying out two similar modulation experiments, where the concentration of each enantiomer was periodically changed from 0 to 1.5 mM. The kinetics of the signals at 1782 and 1716 cm-1 is shown in parts c and f of Figure 5 for (R)-PL and in parts ca and f of Figure 5 for (S)-PL. Additionally, an unambiguous assignment of the elution order of the enantiomers can be obtained from this experiment. The response measured at 1716 cm-1 was only observed for (R)-PL (Figure 5e) since it corresponds to the response shown in Figure 5b. By comparison of the responses measured at 1782 cm-1 (Figure 5b,c), a shift of 107° was calculated corresponding to 185 s (as explained in the following paragraphs). Figure 5 reveals that the response obtained after modulation of the racemate is approximately a superposition of phase and

Figure 6. Phase-resolved spectra demodulated with a φPSD of 224° (solid) and 134° (dashed) for three similar modulation experiments. The concentration of (a) racemic PL, (b) (R)-PL, and (c) (S)-PL were modulated at 12 °C with a period T ) 623 s, and the corresponding time-resolved spectra were transformed into phase-resolved spectra. With φPSD ) 224°, the demodulation sine wave is in phase with the time-dependent signal measured at 1716 cm-1 (sensitive to (R)-PL only!). The corresponding out of phase spectrum is obtained with φPSD ) 134°. The characteristic MSY signal is marked by O and the uncompensated signals of 2-propanol by /.

amplitude of the responses obtained in the experiment with each enantiomer showing a phase shift of approximately 90°. To obtain the two demodulated spectra each showing the characteristic features of the interaction of a single enantiomer with the CSP, the appropriate in phase constants had to be determined first. These phase constants can be calculated by performing a fast Fourier transformation (FFT) of a response signal originating from an interaction that can be exclusively assigned to one enantiomer. For this system, only the signal at 1716 cm-1 originating from the (R)-PL-CSP complex is not affected by any other signals. Therefore, it can be used to PSD determine the phase constant φiph providing the in phase spectrum of the (R)-PL-CSP complex according to eq 5. Since no such response could be specifically assigned to (S)-PL due to the absence of such a signal, the corresponding phase-resolved spectrum was derived using the phase constant that was out of phase with the response at 1716 cm-1. Figure 6a shows the

demodulated spectra obtained from the modulation of the racemate where sin(ωt + φPSD) (eq 5) is in phase (solid) and out of phase (dashed) with respect to the signal at 1716 cm-1. The phase constant used to extract the in phase spectrum from the set of PSD -1 phase-resolved spectra obtained by demodulation was φiph,1716cm ) 224°. Consequently, the demodulated spectrum exhibiting (S)PSD PL-CSP interactions is obtained with φooph,1716cm -1 ) 134° PSD (∆φ ) 90°). These two spectra are the designated result of a response separation experiment where the racemate concentration was modulated. The two spectra in Figure 6a show a strong enhancement of the signal-to-noise ratio compared to the spectra shown in parts d and e of Figure 4, demonstrating the dramatic gain in signalto-noise ratio. Note that even signals with 10-4 absorbance units can be resolved below 1700 cm-1. Parts b and c of Figure 6 display the result after demodulation using the same phase constant as applied in Figure 6a (φPSD ) 224° and 134°) of the experiments, in which the concentration of, respectively, (R)-PL and (S)-PL was modulated. These spectra are compared with those shown in Figure 6a to evaluate the theory of spectra deconvolution via a response demodulation. When in fact a shift of 90° between the responses of (R)-PL and (S)-PL could be introduced as qualitatively observed in Figure 5a, the obtained demodulated spectra in the case of Figure 6b contain information of the enantiomer (R)-PL in contact with the CSP (φPSD ) 224°) and an almost silent spectrum (φPSD ) 134°). The opposite is expected for Figure 6c, where the (S)-PL-CSP spectrum (baseline spectrum) was obtained by demodulation with φPSD ) 134° (φPSD ) 224°). The striking resemblance of the demodulated spectra in Figure 6b (φPSD ) 224°) and Figure 6c (φPSD ) 134°) with those shown in Figure 6a, for the experiment with the racemate, clearly demonstrates the feasibility of the method. In all phase-resolved spectra, the signal at 1606 cm-1 belongs to MSY and has an intensity varying within the modulus of its modulation amplitude in the time domain because the response of MSY is generally not out of phase with the sine wave used for demodulation. The additional signals (1500–1300, 1100, and 953 cm-1) observed in the “silent” spectra in parts b and c of Figure 6 originate from incomplete compensation of the mobile phase and in particular of 2-propanol. It should be noted that some discrepancy was encountered when determining the in phase constants φPSD iph,1782cm-1 for each single enantiomer experiment that are accessible by FFT of the responses shown in Figure 5b ((R)-PL) and Figure 5c ((S)-PL). The PSD PSD -1 ) 232° for (R)-PL and φ values of φiph,1782cm iph,1782cm-1 ) 125° for (S)-PL differ somewhat from 224° and 134°, respectively. The origin of this mismatch may be the mutual interference between pulses of the two enantiomers flowing through the column and competing for adsorption sites. This would lead to a faster elution of both enantiomers in the case where the racemate concentration was modulated. Most likely, the mismatch is attributed to an inaccurate determination of the corresponding retention times because of the rather broad chromatogram peaks (Figure 4a). As a consequence, a modulation period is then used based on eq 4 which leads to a phase shift of the response of each enantiomer of not exactly 90°. Whereas the discrepancy is caused by the nature of the experiment in the first case, the signals in the demodulated spectra become smaller than the desired amplitudes Analytical Chemistry, Vol. 80, No. 10, May 15, 2008

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and are underestimated in the second case due to a reading error. It is, however, a clear advantage of the technique that a small error in the determination of retention times and therefore the modulation period T has only a very minor influence on the resulting intensity A(v,φPSD) in a certain range of φPSD. Since A(v,φPSD) is a function of φPSD in the form of a sine wave (see eq PSD 5), φPSD can differ from φiph by ±18° (±31 s for T ) 623 s) and PSD the resulting A(v,φ ) still reaches 95% of the maximum PSD amplitude A(v,φiph ). Therefore, the signal intensities in the demodulated spectra of Figure 6a as well as the corresponding spectra depicted in parts b and c of Figure 6 lie within an error of less than 5% compared to the intensities which would be obtained by demodulation with the phase constants φPSD iph,1782cm-1 ) 232° ((R)PSD -1 ) 125° ((S)-PL). PL) and φiph,1782cm Another factor influencing the correct value of the intensity in the demodulated spectra is the complete removal of each enantiomer from the CSP within a half period of the modulation experiment. Since the intensity of a signal in the demodulated in phase spectrum reflects the change of the corresponding signal within a modulation period, this signal becomes smaller when part of the selectands remain adsorbed on the CSP. A test for complete removal of the selectands within a modulation period was performed by continuously measuring ATR-IR spectra immediately after concentration modulation was stopped and the mobile phase was flown. In this way, spectra of the last two to three pulses and finally the baseline as the reference were obtained. The corresponding spectra can be found in the Supporting Information. In general, it can be summarized that high-quality ATR-IR spectra of each enantiomer interacting with the CSP can be obtained according to the explained procedure although the racemate was supplied to the setup. Application of the Method and Interpretation of IR Signals. To give an example of a possible application of the proposed technique, separation of PL by Chiralpak AS was studied at different temperatures starting from 12 to 36 °C in steps of 3 °C. At each temperature, first an experiment was performed where one selectand pulse (120 s) was admitted through the setup. Figure 7a shows the corresponding chromatograms which were obtained by following the ATR-IR signal located at 1782 cm-1. It is obvious from Figure 7a that the chromatogram peak of the faster eluted (S)-PL is slightly affected by increasing the temperature from 12 to 36 °C. The peak maximum at 12 °C was determined at around 1065 s and dropped to 909 s at 36 °C (∆156 s). The later eluted (R)-PL peak dropped from 1532 (12 °C) to 1170 s (36 °C) (∆362 s) suggesting that the interaction between the CSP and (R)-PL is more sensitive toward temperature than that of its corresponding enantiomer. At the same time, the retention for mesitylene as the internal standard did not show any dependency on increasing the temperature but remained constant within a range of 27 s as quoted in Table 2. Retention factors k(R)/(S) ) t(R)/(S)/t0 and selectivity factors R )k(R)/k(S) were calculated at each temperature. However, compared to chromatograms obtained by a standard HPLC setup (Figure 7b), these results suffer from the conditions necessary for the ATR-IR spectroscopic measurements. The signal-to-noise ratio of the obtained chromatogram peaks is rather poor. Reasons can be found in the much 3580

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Figure 7. Chromatograms of PL obtained at different temperatures. In the ATR-IR experiments, the signal measured at 1782 cm-1 is depicted in part a, whereas in the standard HPLC experiments chromatograms were sampled with a UV detector at a wavelength of 220 nm (b). The same conditions (chiral column, temperatures, and flow) were used for both setups. The selectand concentrations as well as the injection volume were different. Separation of PL performed with both setups showed a faster decrease of retention for (R)-PL than for (S)-PL when increasing the temperature from 12 to 36 °C. In part a, the retention of (S)-PL dropped from 1065 to 909 s (for (R)-PL from 1532 to 1170 s). In part b, (S)-PL ((R)-PL) was eluted after 702 s (966 s) at 12 °C and 594 s (738 s) at 36 °C. The retention time of mesitylene was not influenced by temperature for both series of experiments. Table 2. Retention- and Selectivity Factors Obtained for the ATR-IR and the Standard HPLC Experimentsa temp [°C]

t0 (MSY) [s]

12 15 18 21 24 27 30 33 36

651 661 662 647 651 635 654 645 640

ATR experiment k(S)-PL k(R)-PL R 0.65 0.58 0.59 0.56 0.52 0.50 0.48 0.42 0.41

1.38 1.22 1.18 1.13 1.05 0.99 0.92 0.84 0.78

2.11 2.10 2.00 2.00 2.04 1.98 1.93 1.97 1.90

HPLC experiment k(S)-PL k(R)-PL R 0.82 0.79 0.75 0.72 0.69 0.66 0.63 0.60 0.58

1.51 1.43 1.34 1.28 1.21 1.14 1.08 1.03 0.96

1.83 1.82 1.80 1.78 1.77 1.74 1.72 1.70 1.66

a The values were calculated using the retention time of MSY as reference. The flow was set to 0.6 mL/min. The mobile phase was 20 vol % 2-propanol in cyclohexane. A selectand pulse (3 mM in PL and 5 mM MSY) of 1.2 mL was separated in the case of the ATR-IR setup and 10 µL (7.5 mM in PL and 5 mM MSY) in the case of the standard HPLC setup.

higher “injection volume” (120 s pulse at 0.6 mL/min) applied with the ATR-IR setup compared with the 10 µL which is used in standard HPLC. In the following modulation experiments, however, the injection volume is given by the applied modulation period T. The column may, therefore, be overloaded, especially at higher temperatures. However, the column has mainly the task to introduce a phase shift for the two enantiomer responses in the detection using ATR-IR. Therefore, retention times may be inaccurate but reflect the correct trends

Figure 8. Effect of temperature on distinct ATR-IR signals. (a) Spectrum of the dry CSP coating at room temperature (RT). (b) ATR spectrum of (S)-PL dissolved in the mobile phase at RT. (c) In phase spectra of (c) (S)-PL and (d) (R)-PL adsorbed on the CSP at 12 (bold) to 36 °C in steps of 6 °C. The two corresponding spectra of adsorbed (S)- and (R)-PL measured at a certain temperature were obtained from the same modulation experiment. The characteristic MSY signal is marked by O and the uncompensated signals of 2-propanol and cyclohexane by /.

for this system as observed in Figure 7b. Here, the (R)-PL peak showed a stronger influence (dropped from 966 to 738 s) upon a temperature increase compared to the (S)-PL peak (702 to 594 s). The difference in retention time obtained from the ATR-IR experiment was used to calculate modulation periods which were then applied in a second experiment performed at each temperature as described in the previous paragraph. The experimental parameters of each modulation experiment as well as the corresponding phase constants used to deconvolute the spectral information of each enantiomer being adsorbed on the CSP film can be found in Table 1. The corresponding phase-resolved spectra are depicted for the sake of clarity in steps of 6 °C from 12 °C (bold) to 36 °C in Figure 8c for the (S)-PL and in Figure 8d for (R)-PL. As reference, ATR-IR spectra of the dry selector film (Figure 8a) as well as the dissolved selectand (Figure 8b) measured at room temperature are presented. The carbonyl stretching vibrations of PL were observed at higher energies (>1750 cm-1) than those of the selector (