Screening Metal Binding Selectivities of Macrocycle Mixtures by HPLC

binding selectivities in binary and complex mixtures by electrospray ionization mass spectrometry. Esther M. Tristani , George R. Dubay , Alvin L...
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Anal. Chem. 2001, 73, 384-390

Screening Metal Binding Selectivities of Macrocycle Mixtures by HPLC-ESI-MS and Postcolumn Reactions Esther C. Kempen and Jennifer S. Brodbelt*

Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas 78712 Richard A. Bartsch

Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061 Michael T. Blanda and Dustin B. Farmer

Department of Chemistry, Southwest Texas State University, San Marcos, Texas 78666

A rapid method for the screening of metal binding selectivities of host compounds in mixtures is presented. This method involves the separation of mixtures of hosts by HPLC, followed by postcolumn complexation with one or more metals, then analysis by mass spectrometry. The intensities of the host-guest complexes in the mass spectra correlate with the binding selectivities of the hosts. The method was applied to a series of lariat ethers that were synthesized as ion-selective reagents for ion-selective electrodes. The compounds most selective for Na+ vs Li+ and K+ were identified. Additionally, a mixture of substituted calixarenes was screened for alkali-metal-binding selectivity. These compounds were determined to be selective for Cs+ over Rb+, K+, and Na+. Exploiting the principles of molecular recognition1-3 has become one of the most powerful and versatile ways of designing new molecules for specific purposes, such as for the development of ion extraction agents, ion sensors, or enzyme mimics. The creation of new hosts relies as much on novel synthetic design strategies as it does on suitable analytical methods for characterization of the binding selectivities. Conventional methods for determining binding selectivities,4,5 such as potentiometry, spectrophotometry, and NMR titrimetry, have been the most popular methods but do not offer the greatest sensitivity or versatility for * Corresponding author: Phone: 512-471-0028. Fax: 512-471-8696. E-mail: [email protected]. (1) Molecular Recognition: Chemical and Biochemical Problems; Roberts, S. M., Ed.; Royal Society of Chemistry: Cambridge, 1992. (2) Principles of Molecular Recognition; Buckingham, A. D., Legon, A. C., Roberts, S. M., Eds.; Backie Academic: Glasgow, 1992. (3) Comprehensive Supramolecular Chemistry; Gokel, G. W., Ed.; Elsevier Science: New York, 1996; Vol. 1. (4) Martell, A. E.; Hancock, R. D. Metal Complexes in Aqueous Solutions; Plenum Press: New York, 1996; Chapter 7. (5) Tsukube, H.; Furuta, H.; Odani, A.; Takeda, Y.; Kudo, Y.; Inoue, Y.; Liu, Y.; Sakamoto, H.; Kimura, K. In Comprehensive Supramolecular Chemistry, Davies, J. E. D., Ripmeester, J. A., Eds.; Elsevier: New York, 1996; Vol. 8, Chapter 10.

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many applications. For example, the NMR method is not ultrasensitive, thus requiring adequate amounts (often in the 0.1 mM range with over 0.5 mL required) of host and guest molecules for complete characterization. The potentiometric methods suffer from limited solvent compatibility, generally requiring the use of nonorganic solvent systems. In recent years, electrospray ionization-mass spectrometry6 (ESI-MS) has emerged as a new method for measuring binding selectivities.7-17 In this method, the intensities of complexes observed in the mass spectra obtained upon electrospray ionization of solutions containing various host and guest species correlate with the distribution of host-guest complexes existing in solution. This method has been successfully used to determine the alkali-metal-binding selectivities of crown ethers,7,8 lariat ethers,12 and calixarenes9,10 in solutions ranging from aqueous to organic. Moreover, binding selectivities can be obtained using as little as a few nanomoles of hosts. A new mass spectrometric strategy for rapid evaluation of binding selectivities of hosts present in mixtures reported herein involves uniting the separation and screening steps via postcolumn (6) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom Rev. 1990, 9, 37. (7) Blair, S.; Kempen, E. C.; Brodbelt, J. S. J. Am. Soc. Mass Spectrom. 1998, 9, 1049-1059. (8) Brodbelt, J. S.; Kempen, E.; Reyzer, M. Structural Chemistry, 1999, 10, 213-219. (9) Goolsby, B.; Hall, B. J.; Brodbelt, J. S.; Adou, E.; Blanda, M. Int. J. Mass Spectrom. 1999, 193, 197-204. (10) Blanda, M. T.; Farmer, D. B.; Brodbelt, J. S.; Goolsby, B. J. Am. Chem. Soc. 2000, 122, 1486-1491. (11) Blair, S. M.; Brodbelt, J. S.; Marchand, A. P.; Kumar, K. A.; Chong, H.-S. Anal. Chem. 2000, 72, 2433-2445. (12) Kempen, E. C.; Brodbelt, J. S.; Bartsch, R. A.; Jang, Y.; Kim, J. S. Anal. Chem. 1999, 71, 5493-5500. (13) Reyzer, M. L.; Brodbelt,J. S.; Marchand, A. P.; Chen, Z.; Huang, Z.; Namboothiri, I. N. N. Int. J. Mass Spectrom. 2000, accepted. (14) Blair, S.; Brodbelt, J.; Marchand, A.; Chong, H.-S.; Alidhodzic, S. J. Am. Soc. Mass Spectrom. 2000, 11, 884-89. (15) Wang, K.; Gokel, G. W.; J. Org. Chem. 1996, 61, 4693-4697. (16) Young, D.-S.; Hung, H.-Y.; Liu, L. K. J. Mass Spectrom. 1997, 32, 432437. (17) Young, D.-S.; Hung, H.-Y.; Liu, L. K. Rapid Commun. Mass Spectrom. 1997, 11, 769-773. 10.1021/ac0010476 CCC: $20.00

© 2001 American Chemical Society Published on Web 12/15/2000

the strategy was optimized on a commercial HPLC-ESI-ion trap instrument that mixed the host and guest solutions via a tee. An array of control experiments used to validate the postcolumn screening method and optimization of the experimental conditions required for the determination of binding selectivities is also presented.

Figure 1. Dibenzo-16-crown-5 lariat ethers and bis-bridged calix[6]arenes.

reactions after HPLC. Postcolumn reactions have had great general utility for increased sensitivity of detection after HPLC in nonmass spectrometric applications.18 Postcolumn reactions have had limited use for HPLC-MS applications.19-22 For instance, Kohler et al. used postcolumn addition of metal chlorides for the analysis of carbohydrates and achieved improved detection sensitivities.19 Karlsson added alkali metal ions to the sheath flow of an ESI interface to ionize cyclodextrins and oligosaccharides by LC-ESI-MS.20 Shen et al. used a mixture of a transition metal and an auxiliary chelating agent like 2,2-bipyridine to form complexes with quinolone antibiotics with enhanced sensitivity.21 More recently, Creaser and co-workers used the postcolumn addition of copper to aid in the characterization of peptides,22 and Browner et al. developed a novel method for increasing the sensitivity of detection of organoselenium compounds based on postcolumn complexation with 18-crown-6.23 In the present study, the host compounds were separated by HPLC prior to exposure to the guests of interest in an online postcolumn reaction. Two mixtures of synthetic hosts (Figure 1) are analyzed with the HPLC-MS method in the present report. We had previously developed and validated the use of ESI-MS to evaluate the alkali-metal-binding selectivities of individual synthetic hosts; therefore, the framework for extending the method to screen mixtures was in place.7-14 The studies were initially undertaken using a home-built HPLC-ESI-ion trap system that mixed the host and guest solutions by a sheath flow device, then (18) Brinkman, U. A. T.; Frei, R. W.; Lingeman, H. J. Chromatogr. 1989, 492, 251-298. (19) Kohler, M.; Leary, J. Anal. Chem. 1995, 67, 3501. (20) Karlsson, K. E. J. Chromatogr. A 1998, 794, 359. (21) Shen, J.; Brodbelt, J. S. Rapid Commun. Mass Spectrom. 1999, 13, 13811389. (22) Creaser, C. S.; Lill, J. R.; Bonner, P. L. R.; Hill, S. C.; Rees, R. C. Analyst, 2000, 125, 599. (23) Shou, W. Z.; Woznichak, M. M.; May, S. W.; Browner, R. F. Anal. Chem. 2000, 72, 3266-3271.

EXPERIMENTAL SECTION HPLC-MS. HPLC of the lariat ether compounds was performed on a Waters 2690 Separations Module equipped with a 3.5-µm Waters Symmetry 2.1 × 50 mm C18 column and using an isocratic mobile phase of 70% MeOH, 25% H2O, and 5% MeCN at a flow rate of 50 µL/min. A mixture (5 µL) containing 1.0 mM of each lariat ether was injected onto the column. The HPLC effluent was split 50/50 before the postcolumn reaction mixture of alkali metal chlorides (LiCl, NaCl, and KCl at 1.0 mM each in methanol) was added at a flow rate of 2 µL/min. The postcolumn reaction took place using an apparatus previously characterized in detail.21 This mixing device consists of a set of concentric tubes in which the HPLC effluent flows through the central tube and the postcolumn reagent flows through the outer tube, creating a sheath flow-type device. These two solutions mix at the exit of the device. The mass spectrometric detector used for this portion of the study was a Finnigan ion trap mass spectrometer operated in the mass selective instability mode with modified ITD electronics to allow axial modulation. The electrospray interface was based on a design developed by the ion trap group at Oak Ridge National Laboratory which involved differentially pumped regions containing ion-focusing lenses,24 with no heated capillary. For the mixture of calixarenes, a Finnigan LCQ-Duo mass spectrometer was used. For each run, a 5-µL injection of a solution of 5.4 × 10-4 g/mL of the calixarene mixture in acetonitrile was used. HPLC of these compounds was performed with a 3.5-µm Waters Symmetry 2.1 mm × 50 mm C8 column using an isocratic mobile phase of 90% CH3OH/10% CHCl3 at a flow rate of 100 µL/ min. No splitting of the effluent was performed prior to mass spectrometric detection. The postcolumn reagent mixture (alkali metal chlorides CsCl, RbCl, KCl, and NaCl at 1.5 × 10-4 M each in methanol) was introduced at 10 µL/min through a tee union upstream from the electrospray source. A tee was used for this work, as opposed to the sheath flow apparatus used for the lariat ether compounds, because the tee gave superior mixing and reproducibility. Compounds. The lariat ether compounds were provided by Prof. Richard Bartsch at Texas Tech University (Figure 1).25-27 The combinatorially synthesized mixture of bis-bridged calix[6]arenes was provided by Prof. Michael Blanda at Southwest Texas University (Figure 1). The library was constructed by reacting 0.25 mmol of each of the non-bis-bridged 37,40-dialkylated calix[6]arene precursors (refer to Figure 1) with dibromomethyl-mxylene in the presence of sodium hydride and tetrahydrofuran. The resulting calixarene compounds were obtained as a mixture, (24) Van Berkel, G. J.; Glish, G. L.; McLuckey, S. A. Anal. Chem. 1990, 62, 1284-1289. (25) Bartsch, R. A.; Heo, G. S.; Kang, S. I.; Liu, Y.; Strzelbicki, J.; Bills, L. J. J. Org. Chem. 1993, 48, 4864. (26) Ohki, A.; Lu, J. P.; Bartsch, R. A. Anal. Chem. 1994, 66, 651-654. (27) Ohki, A.; Lu, J. P.; Huang, X.; Bartsch, R. A. Anal. Chem. 1994, 66, 43324336.

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and the exact quantity of each compound in the mixture was unknown. RESULTS AND DISCUSSION The general approach was to use the HPLC to provide a rudimentary separation of each complex synthetic mixture, then to use the postcolumn reaction method to mix each eluting host with a series of guests (in this case, alkali metal ions), followed by ESI-MS to determine the relative binding selectivities on the basis of the mass spectral intensities of the relevant complexes. Numerous steps were needed to validate this approach, including the comparison of the results that were obtained upon mixing the host and guest solutions using the postcolumn reaction method to those results obtained for premixed solutions containing well-defined amounts of hosts and guests (i.e., compositions created to mimic the predicted concentrations of components in the postcolumn mixing experiments). Determinations of the optimal flow rates needed for adequate mixing and the appropriate metal ion concentrations for optimum selectivity information were also undertaken. Alkali-metal-binding selectivities for each host in the postcolumn reaction experiments were derived from the ion chromatograms by plotting the selected ion chromatograms for each metal complex of interest, integrating the total peak areas, and converting to percentages. Only isocratic chromatographic methods were used to provide a constant solvent environment for the selectivity measurements which are known to be solvent dependent. Validation of the Postcolumn Selectivity Method Using the Sheath Flow Apparatus. The first steps entailed characterization of the sheath flow mixing device to compare the distributions of host-guest complexes obtained upon mixing the separate host and guest solutions with the distributions obtained for equivalent premixed host-guest solutions. Dibenzo-18-crown-6, a well-studied host with known binding constants for Na+ and K+,28 was used for these studies. Two solutions were prepared: the host solution consisted of 1.0 × 10-4 M dibenzo-18-crown-6 in methanol and the guest solution consisted of 1.0 × 10-3 M each NaCl and KCl in methanol. In one experiment, the host solution was introduced at a flow rate of 25 µL/min through the central tube of the mixing apparatus (the tube through which the HPLC effluent would normally flow) and the guest solution was introduced through the outer tube (through which the postcolumn reagent would normally flow) at a flow rate of 2 µL/min. The ESI mass spectrum obtained for the resulting solution obtained by mixing the two solutions with the sheath flow mixing device is shown in Figure 2A. In the second experiment, 25 µL of the host solution was mixed directly with 2 µL of the guest solution, yielding a net concentration of 9.3 × 10-5 M of dibenzo-18-crown-6 and 7.4 × 10-5 M of each metal. The ESI mass spectrum obtained for this premixed solution is shown in Figure 2B. The theoretical equilibrium ratio of the concentrations of (dibenzo-18-crown-6 + K)+ to (dibenzo-18crown-6 + Na)+ complexes for this solution (calculated on the basis of log KK+ ) 5.0 and log KNa+ ) 4.37)28 is 2.1 (for a solution containing 9.3 × 10-5 M dibenzo-18-crown-6 and 7.4 × 10-5 M each of NaCl and KCl). The experimental ratio of the intensities of the (dibenzo-18-crown-6 + K)+ vs (dibenzo-18-crown-6 + Na)+ (28) Gokel, G., Crown Ethers and Cryptands; The Royal Society of Chemistry: Cambridge, 1991; p 74.

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Figure 2. Comparison of alkali metal ion selectivity of dibenzo-18crown-6 via (A) postcolumn reaction mode with sheath flow mixing vs (B) premixed mode. Host solution ) 1.0 × 10-4 M dibenzo-18crown-6 in methanol; guest solution ) 1.0 × 10-3 M each of NaCl and KCl in methanol. A: Host solution at 25 µL/min and guest solution at 2 µL/min mixing via sheath flow. B: Solution containing 25 µL host solution and 2 µL guest solution.

complexes obtained using the premixed solution mixture is 2.4 ( 0.2 (Figure 2B), while the ratio obtained using the sheath flow apparatus to mix the two solutions is 2.8 ( 0.3 (Figure 2A). The mass spectra that were obtained for both the premixed and sheath flow-mixed solutions indicate the preference of dibenzo-18-crown-6 for binding K+ over Na+, in agreement with the theoretical equilibrium prediction. The fact that the ratios of K+ to Na+ complexes differ somewhat for the premixed and sheath flowmixed solutions suggests that the final composition of the solution created by the sheath flow device may not exactly duplicate the mixture obtained upon premixing of the host and guest solutions, but the qualitative agreement suggests that screening of binding selectivities of hosts is possible. A similar experiment was undertaken using the same concentrations of dibenzo-18-crown-6 and metals as above but with the 70% CH3OH, 25% H2O, 5% CH3CN solvent system used as the mobile phase for the subsequent HPLC experiments involving the lariat ethers. These experiments were aimed at confirming that self-consistent selectivity ratios could be obtained for other solvent systems when comparing the premixed and sheath flow-mixed solutions. The ratio of the intensities of the (dibenzo-18-crown-6 + K)+ vs (dibenzo-18-crown-6 + Na)+ complexes for this solvent system obtained using the sheath flow apparatus to mix the two solutions is 3.9 ( 0.4, and the ratio for the premixed solution is 3.4 ( 0.2. These results confirm the agreement between the two methods of mixing, thus supporting the use of a postcolumn mixing apparatus to undertake binding selectivity experiments. Because binding constants for the dibenzo-18-crown-6/alkali metal ion complexes in this ternary HPLC solvent mixture are not known, comparison of the experimental distribution of (dibenzo18-crown-6 + Na+) and (dibenzo-18-crown-6 + Na+) complexes with the theoretical equilibrium distribution was not possible. Determination of Binding Selectivities of Lariat Ethers by HPLC-ESI-MS. The affinities of the sym-dibenzo-16-crown-5 lariat ether compounds, 1-7, for alkali metal ions (Li+, Na+, and K+) have been previously studied in our lab12 by using the electrospray ionization selectivity method pioneered by this

Figure 3. HPLC total ion chromatogram of lariat ethers using postcolumn complexation. Table 1. Percentages of Dibenzo-16-crown-5 Lariat Ether Complexes Obtained from HPLC Postcolumn Reaction Modea area %

lariat ether

R1

R2

1 2 3 4 5 6 7

H n-C3H7 H n-C3H7 H n-C3H7 n-C3H7

OMe OMe OCH2COOH OCH2COOH OCH2CON(Me)2 OCH2CON(Me)2 OCH2CONH2

(LE + Li)+ (LE + Na)+ (LE + K)+ 14 11 4 7 3 6 5

63 59 64 77 75 77 83

23 30 32 17 22 17 12

a The average standard deviation is ( 12% of the listed number. HPLC conditions: 3.5 µm Waters Symmetry C18 2.1 × 50 mm column. Mobile phase: 70% MeOH, 25% H2O, and 5% MeCN at 50 µL/min with a 50/50 split before reaction. Postcolumn reagent mixture: 1.0 × 10-3 M LiCl, NaCl, and KCl, each in MeOH at a flow rate of 2 µL/min.

group.7-15 In our previous study of the lariat ethers, the intensities of the lariat ether/metal ion complexes in the ESI-mass spectra were examined for solutions containing a single lariat ether with an array of metals in order to determine metal ion selectivities of the lariat ethers. A mixture of lariat ethers was thus used for initial evaluation of the HPLC postcolumn screening method. The HPLC chromatogram obtained for the separation of seven lariat ethers using an isocratic mobile phase of 70% CH3OH, 25% H2O, and 5% CH3CN is shown in Figure 3. The metal solution (1.0 × 10-3 M each metal in methanol) was introduced via the sheath flow at 2 µL/min. The peak areas for each lariat ether 1-alkali metal ion complex were integrated to give the mass spectral percentages of the lariat ether 1-alkali metal ion complexes. The distribution of complexes obtained in this way is summarized in Table 1 for all of the lariat ethers in the mixture. Compounds 4-7 show enhanced sodium selectivity over the other lariat ethers. In general, Li+ complexes are not prominent species. Another experiment was undertaken to confirm that alkali metal selectivities that were obtained by using the postcolumn mixing mode were similar to those that were obtained for premixed solutions containing a lariat ether and the metal salts in a 5% MeCN, 24% H2O, and 71% MeOH solution, a composition that corresponds to the solvent environment obtained by mixing

the HPLC effluent (i.e., 70% CH3OH, 25% H2O, and 5% CH3CN at 50 µL/min) with the postcolumn metal solution (metals in 100% methanol at 2 µL/min). An example of the resulting mass spectrum for this premixed solution is shown in Figure 4 for lariat ether 5. The lariat ether is most selective for Na+ in this premixed solution, and the distribution of complexes is similar to that reported in Table 1 for the postcolumn mixing mode. Validation of the Postcolumn Selectivity Method Using the Mixing Tee. On the basis of the promising results obtained with the home-built HPLC-MS system, the method was implemented on a commercial LC-MS instrument (Finnigan LCQ-Duo) equipped with a mixing tee for addition of the postcolumn reagent mixture. The sheath flow apparatus available with the ESI interface underwent clogging over time, leading to an unstable signal and nonreproducible results, and causing carryover for subsequent experiments. Thus, a mixing tee was used as a more robust postcolumn mixing device. The host mixture of interest for this facet of the study consisted of an array of calixarenes (Figure 1). Validation experiments similar to those described previously for the sheath flow mixing device were also undertaken using the mixing tee. The control experiments were likewise performed using dibenzo-18-crown-6 as the host and Na+ and K+ as the guests. First, the distribution of complexes obtained for a premixed hostguest solution and a solution obtained by mixing the host solution and the guest solution via the tee were compared. In this case, the host solution consisted of 1.5 × 10-5 M dibenzo-18-crown-6 in methanol, and the guest solution consisted of 1.5 × 10-4 M each NaCl and KCl in methanol. For the mixing tee experiment, the host solution was introduced at a flow rate of 100 µL/min through one side of the mixing tee, and the guest solution was admitted through the other side of the tee at a flow rate of 10 µL/min. Again, this experiment was designed to mimic the mixing conditions that were used for the analysis of a mixture of calixarene compounds but with a host for which binding constants are known. In comparison, the ESI mass spectrum was also obtained for a premixed solution containing 100 µL of the host solution and 10 µL of the guest solution (yielding 1.4 × 10-5 M dibenzo-18-crown-6 and 1.4 × 10-5 M each metal). The experimental ratio of intensities of the (dibenzo-18-crown-6 + K)+ vs (dibenzo-18-crown-6 + Na)+ complexes obtained for the premixed solution mixture is 3.5 ( 0.2, and the ratio of the intensities obtained for the mixture obtained using the tee apparatus is 4.0 ( 0.4. The theoretical distribution of (dibenzo-18-crown-6 + K)+ vs (dibenzo-18-crown-6 + Na)+ complexes for this solution (calculated on the basis of log KK+ ) 5.0 and log KNa+ ) 4.37)28 is 3.0 (for a solution containing 1.4 × 10-5 M dibenzo-18-crown-6 and 1.4 × 10-5 M each metal). The preference for the complexation of K+ over Na+ by dibenzo-18-crown-6 is observed in both types of ESI-MS experiments, which is in agreement with the theoretical equilibrium prediction and supports the use of the mixing tee for screening binding selectivities of hosts. Optimization of Postcolumn Flow Rate and Metal Ion Concentration. Selection of the best flow rate and concentration of the postcolumn metal solution in order to maximize the attainment of equilibrium for the host-guest complexation merited examination of a range of conditions. Optimization of the flow rate of the postcolumn metal solution was undertaken using Analytical Chemistry, Vol. 73, No. 2, January 15, 2001

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Figure 4. Alkali metal ion selectivity of lariat ether 5 in the HPLC solvent system. 1.5 × 10-4 M each of lariat ether 5, LiCl, NaCl, and KCl in 5% MeCN/24% H2O/71% MeOH. Table 2. Effect of Metal Ion Concentration on the Binding Selectivity of Dibenzo-18-crown-6a

Figure 5. Effect of flow rate of metal ion solution on the binding selectivity of dibenzo-18-crown-6. Dibenzo-18-crown-6 in methanol solution (1.5 × 10-5 M) was introduced at a constant flow rate of 100 µL/min. The metal solution containing 1.5 × 10-4 M each of NaCl and KCl in methanol was introduced via the tee at flow rates varying from 2 to 25 µL/min. The theoretical equilibrium distribution is the ratio of K+ to Na+ complexes that was obtained on the basis of the expected composition of the solutions and the binding constants of dibenzo-18-crown-6 (log kK+ ) 5.0, log kNa+ ) 4.37).28

dibenzo-18-crown-6 at 1.5 × 10-5 M in methanol infused at 100 µL/min (i.e., the typical flow rate used in the HPLC experiments) and a concentration of 1.5 × 10-4 M for the metal ion solution in methanol introduced via the tee. The flow rate of the metal solution was varied from 2 to 25 µL/min, and the intensities of the (dibenzo-18-crown-6 + K)+ and (dibenzo-18-crown-6 + Na)+ complexes were monitored. The results are plotted in Figure 5 as the ratio of the intensity of the (dibenzo-18-crown-6 + K)+ complex to the intensity of the (dibenzo-18-crown-6 + Na)+ complex as a function of the flow rate of the metal ion solution. Also plotted in Figure 5 is the theoretical equilibrium ratio of K+ to Na+ complexes calculated on the basis of the predicted composition of the mixed solutions (assuming complete mixing of the dibenzo-18-crown-6 and metal solutions) and the known binding constants of dibenzo-18-crown-6 with both K+ and Na+.28 The theoretical ratio increases with the flow rate of the metal solution because the overall concentration of the metals increases relative to that of dibenzo-18-crown-6, thus increasing the selectivity for K+ over Na+. The experimentally obtained ratios also increase with flow rate, with the ratios at the lowest flow rates having substantially lower values than the theoretical ratios and the experimental ratios at the highest flow rates having large standard deviations. Incomplete mixing occurs when the metal solution flow rate is less than 10 µL/min; thus, a true equilibrium of the two solutions is not obtained (Figure 5). Perhaps the large disparity between the flow 388 Analytical Chemistry, Vol. 73, No. 2, January 15, 2001

conc metal soln (M), each metal

theor equilib distribb complexes K+/Na+

experimental distribc complexes K+/Na+

3.75 × 10-5 M 7.5 × 10-5 M 1.5 × 10-4 M 3.0 × 10-4 M

2.5 2.7 3.0 3.3

0.3 1.4 2.0 2.2

a Host solution was composed of 1.5 × 10-5 M dibenzo-18-crown-6 in methanol at a flow rate of 100 µL/min. Metal solutions consisted of equal quantities of NaCl and KCl and were introduced through one branch of the tee apparatus at a flow rate of 10 µL/min. b Theoretical equilibrium distribution is the ratio of K+ to Na+ complexes obtained on the basis of the expected composition of the solutions and the binding constants of dibenzo-18-crown-6 (log kk+ ) 5.0, log kNa+ ) 4.37).28 c Experimental distribution is the ratio of peak intensities corresponding to the K+ and Na+ complexes.

rates of the host and guest solutions prevents uniform and efficient mixing. This effect does not occur in cases where the metal solution flow rate is at least 10 µL/min. When the metal ion solution is added at flow rates >10 µL/min, the reproducibility of the measurements decreases, as shown by the large error bars in Figure 5. This effect may be due to the difficulty in desolvating the dibenzo-18-crown-6/metal complexes in the interface region of the mass spectrometer due to an excess of solvent, thus allowing survival of solvated complexes that leads to a reduction of the ion signal. This solvation issue generally affects the sodium complexes, exaggerating the ratio of K+ to Na+ complexes. Due to this desolvation problem, the lowest flow that yielded acceptable results was used for the HPLC experiments involving the calixarenes (i.e., 10 µL/min). Optimization of the concentration of the metal ion solution was performed using dibenzo-18-crown-6 (1.5 × 10-5 M) as the model host in methanol, and K+ and Na+ as the guests at a range of concentrations varying from 3.75 × 10-5 M to 3.0 × 10-4 M (for each metal ion) in methanol. It was expected that a larger concentration of metals would promote a faster approach to equilibrium. The dibenzo-18-crown-6 solution was introduced at a flow rate of 100 µL/min, and the metal solution was introduced at a flow rate of 10 µL/min for this evaluation. Here again, the ratio of the intensities of the (dibenzo-18-crown-6 + K)+ to (dibenzo-18-crown-6 + Na)+ complexes was compared to the theoretical equilibrium ratio of the K+ to Na+ complexes (Table 2). The theoretical equilibrium ratio was calculated on the basis of the expected composition of the mixed solution and the known binding constants of dibenzo-18-crown-6 with K+ and Na+. As the concentration of the metal solution increases, the observed distribution of (dibenzo-18-crown-6 + K)+ to (dibenzo-

Figure 6. HPLC selected ion chromatograms of calix[6]arenes (8-13)/Cs+ complexes.

18-crown-6 + Na)+ complexes increases. When the metal ions are added at low concentrations, a reversal of the expected K+/ Na+ selectivity is observed (i.e., the Na+ complexes are more intense than the K+ complexes, contrary to the expected preference for K+). This reversal occurs because the metal ion concentration is so low relative to the host concentration that many host molecules remain unbound in solution. These unbound host molecules bind with contaminants that are present in the ESI assembly (in this case, ubiquitous sodium), thus giving unusually high sodium complexes and yielding unreliable results. Although the agreement of the ESI-MS results with the theoretical equilibrium ratios improves at greater metal ion concentrations, a loss of signal occurs at very high metal concentrations (i.e., 3.0 × 10-4 M, each metal) because the metal salts precipitate onto the orifice of the mass spectrometer, clogging the orifice. Thus, the best choice for the concentration of the metal ion solution when undertaking experiments involving continuous infusion of the metal solution is 1.5 × 10-4 M of each metal. In summary, both the flow rate and the concentration of the metal solution require careful optimization to ensure the closest approach to equilibrium for the host-guest complexation. Typically, the best strategy would involve maximizing the concentrations of the metals up to the limit of causing clogging of the ESI interface and using a moderate flow rate to prevent introduction of excess solvent into the interface while maintaining good mixing conditions. The observed metal binding selectivities are also dependent on the solvent composition of the mobile phase, but this effect is largely related to the intrinsic solubilities of the hosts and would be a considerable influence on even conventional measurements of binding selectivities (i.e., those methods that do not involve an on-line LCMS mode). Moreover, the use of gradients for the chromatographic separation should be avoided, because a variable solvent environment would significantly alter the binding selectivities observed within a group of hosts. Determination of Binding Selectivities of Bis-Bridged Calix[6]Arenes by HPLC-ESI-MS. After optimization of the postcolumn reaction conditions, the binding selectivities of a

Table 3. Percentages of Bis-Bridged Calix[6]Arene (CA) Complexes Obtained from HPLC Postcolumn Reaction Modea

a Average standard deviation is ( 5% of the listed number. HPLC: 3.5 µm Waters Symmetry C8 2.1 × 50 mm column. Mobile phase: 90% MeOH and 10% CHCl3 at 100 µL/min, no split. Postcolumn reagent: 1.0 × 10-4 M NaCl, KCl, RbCl, and CsCl each in methanol, at 10 µL/min.

mixture of calixarenes prepared by a combinatorial method were screened. The separation of the calixarene mixture entailed using a mobile phase composition of 90% CH3OH/10% CHCl3 at 100 µL/ min with the addition of the postcolumn mixture of metals (1.0 × 10-4 M each) at 10 µL/min in methanol via the tee. The selected ion chromatograms for the Cs+ complexes that were obtained upon HPLC separation followed by postcolumn complexation of the calixarene compounds 8-13 are shown in Figure 6. The percentages of each calixarene complex with Cs+, Rb+, K+, and Na+, obtained by integration of the selected ion chromatograms, are given in Table 3. Compounds 8-13 are selective for the larger Cs+ ion over the Rb+, K+, and Na+ ions (>82% of all complexes with each calixarene contain Cs+). This binding preference is due to the large cavity size of the calix[6]arene macrocycles, as well as the potential for greater cation-Π interactions of Cs+ with the aromatic groups of the ligands.29 Although there are slight differences in selectivities of the different compounds, they are (29) Ma, J. C.; Dougherty, D. A. Chem. Rev. 1997, 97, 1303-1324.

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quite small; in fact, they are within experimental error. The differences in the various substituents cause little modification in the metal selectivities of these compounds because the cavity size created by the base calixarene structure is highly size-selective for Cs+. CONCLUSIONS HPLC coupled with online postcolumn complexation and ESIMS is a promising method for the rapid screening of metal binding selectivities of mixtures of hosts. Both sheath flow and tee options were evaluated for mixing the eluting hosts with the guests, and the tee method proved to be more robust and reproducible and was not prone to clogging. The experimental parameters that influence the effective mixing of the hosts and guests, including the flow rate and concentration of the guest mixture, require careful optimization to ensure a reasonable approach to equilibrium for the host-guest complexation reactions. Off-line isolation of compounds is not required for screening of the binding selectivities, and any compounds that coelute may still be differentiated by their mass spectra. This method also allows for the inclusion of potential binding interferences in the postcolumn mixture of guests, such as other alkali metal ions or ammonium ions, to determine the binding selectivities for targeted guests vs

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interfering guests. Moreover, the postcolumn reaction mixtures can be tailored to any type and number of guest ions. The major limitations of the online LC-ESI-MS method are the dependence on the optimization of the postcolumn complexation conditions, a process that, because of its dynamic nature, makes it difficult to ensure attainment of equilibrium, and the influence of the specific mobile phase composition on binding selectivities. Finally, only isocratic chromatographic methods should be avoided to provide a constant solvent environment when screening the selectivities of groups of hosts. ACKNOWLEDGMENT J.S.B. gratefully acknowledges financial support from the National Science Foundation (CHE-9820755), the Robert A. Welch Foundation (Grant no. F1155), and the Texas Advanced Technology Program (003658-0206). M.T.B. acknowledges financial support from the Robert A. Welch Foundation (Grant no. AI-1443)

Received for review November 1, 2000. AC0010476

September

1,

2000.

Accepted