Evaluation of Temperature Effects on Selectivity in RPLC Separations

chlorophenols, temperature changes did significantly af- fect selectivity. We feel that simple mixtures with one dominant retention mechanismse.g., so...
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Anal. Chem. 1997, 69, 2202-2206

Evaluation of Temperature Effects on Selectivity in RPLC Separations Using Polybutadiene-Coated Zirconia Jianwei Li† and Peter W. Carr*

Department of Chemistry, Kolthoff and Smith Halls, University of Minnesota, 207 Pleasant Street, SE, Minneapolis, Minnesota 55455

The effect of temperature on selectivity in RPLC method development has been evaluated on polybutadiene-coated zirconia. We find that the influence of temperature on selectivity depends strongly on solute type. For solutes of similar structure such as polyaromatic hydrocarbons, temperature has almost no effect on selectivity; however, for solutes with very different functional groups such as chlorophenols, temperature changes did significantly affect selectivity. We feel that simple mixtures with one dominant retention mechanismse.g., solvophobic retentionswill not be helped appreciably by adjusting temperature. However, in complex mixtures with polar and ionizable solutes, optimization by varying the temperature may well be fruitful. In a previous paper, we characterized the effect of temperature on selectivity, efficiency, and stability of polybutadiene (PBD)coated zirconia phases.1 We found, depending on the type of analyte, that temperature can be a very important variable that can be adjusted to improve the separation selectivity. For example, we saw no change in elution sequence of alkylbenzenes as temperature was increased. In contrast, for a set of tricyclic antidepressants, there were very substantial changes in elution order for these more polar, partially protonated species. This conclusion is consistent with the result obtained by Hancock et al., who used temperature as an effective separation variable in the separation of peptides and proteins on conventional bonded phases.2,3 We demonstrated that the PBD-coated zirconia phase is stable at a temperature of 100 °C over at least 7000 column volumes. The exceptional thermal stability of PBD-coated zirconia allows the addition of another dimension, temperature, to method development in RPLC. In addition, the use of elevated temperatures in RPLC usually shortens the analysis time, resulting in savings in time and organic solvent. For low molecular weight species (MW < 1000), it is usually the case that enthalpy changes are larger for later eluting bands than for earlier peaks. This is invariably the case for a homologous series.4-9 However, as pointed out by Melander et al.,4 particularly for solutes of similar structure, the enthalpy change is often linearly related to the entropy of retention. Thus, the

aforementioned relationship between retention and enthalpy change can be expressed quantitatively as2

log(ki′) ) A(∆Hi) + B

(1)

where ki′ denotes the capacity factor for a series of solutes at some temperature T, and A and B are solute-independent constants which vary with all the experimental conditions, including temperature. If a set of solutes, such as a homologous series, follows eq 1 exactly, no major changes in selectivity will occur as the temperature is varied, because the difference in enthalpies for a pair of bands will be the same. All k′ values will eventually become the same at the so-called compensation or isokinetic temperature.4 This is why we often see a monotonic decrease in the selectivity of all pairs of bands as temperature approaches the usually superambient isokinetic temperature in RPLC. To take advantage of temperature to improve selectivity, we rely on the failure of eq 1, so that chromatographic peaks move at different rates as the temperature is varied. The failure of eq 1 is commonly associated with the existence of more than one type of interaction or separation mechanism and with certain specific chemical effects.2 These chemical effects include, for example, retention on more than one stationary phase site (e.g., cations retained on ionized silanols in addition to solvophobic retention on the bonded phase), ionization of sample molecules (e.g., AH and A-, BH+ and B), and other secondary equilibria such as ion pairing. Moreover, eq 1 often fails in situations where the separation mechanism is based on the shape selectivity,10 chiral recognition,11-17 complexation,18 protein interactions,19-21 or macrocycle mediation.22 The selectivity effects mentioned above will be more pronounced for large molecules such as peptides and proteins than

† Current address: 3M Pharmaceuticals, 3M Center, Building 270-4S-02, St. Paul, MN 55455-1000. (1) Li, J.; Carr, P. W. Anal. Chem. 1997, 69, 2193-2201. (2) Hancock, W. S.; Chloupek, R. C.; Kirkland, J. J.; Snyder, L. R. J. Chromatogr. 1994, 686, 31-43. (3) Chloupek, R. C.; Hancock, W. S.; Marchylo, B. A.; Kirkland, J. J.; Boyes, B. E.; Snyder, L. R. J. Chromatogr. 1994, 686, 45-59.

(4) Melander, W. R.; Campbell, D. E.; Horva´th, Cs. J. Chromatogr. 1978, 158, 215-225. (5) Chmielowiec, J.; Sawatzky, B. J. Chromatogr. Sci. 1979, 17, 245-252. (6) Vigh, G.; Varga-Puchony, Z. V. J. Chromatogr. 1980, 196, 1-9. (7) Kikta, E. J.; Grushka, E. Anal. Chem. 1976, 48, 1098-1104. (8) Colin, H.; Diez-masa, J. C.; Guiochon, G.; Czajkowska, T.; Miedziak, I. J. Chromatogr. 1978, 167, 41-65. (9) Robbat, A.; Liu, T. Y. J. Chromatogr. 1990, 513, 117-135. (10) Sander, L. C.; Wise, S. A. Anal. Chem. 1989, 61, 1749-1754. (11) Pirkle, W. H.; Burke, J. A. J. Chromatogr. 1991, 557, 173-185. (12) Takagi, T.; Suzuki, T. J. Chromatogr. 1992, 625, 163-168. (13) Labl, M.; Fang, S.; Hruby, K. J. J. Chromatogr. 1991, 586, 145-148. (14) Henderson, D. E.; Mello, J. A. J. Chromatogr. 1990, 499, 79-88. (15) Sander, L. C.; Craft, N. E. Anal. Chem. 1990, 62, 1545-1547. (16) Sentell, K. B.; Henderson, A. N. Anal. Chim. Acta 1991, 246, 136-149. (17) Paesen, K.; Goetz, J. F. J. Chromatogr. 1978, 157, 185-196. (18) Smith, R. G.; Drake, P. A.; Lamb, J. D. J. Chromatogr. 1991, 546, 139-149.

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small solutes for two reasons. First, the enthalpies of large solutes are greater than those of small solutes. Second, interactions between large solutes and the stationary phase often involve multiple, complex retention mechanisms.19-21,23 In this work, we evaluated the effect of temperature on selectivity in RPLC by varying both the mobile phase composition and the temperature. Several different kinds of samples were separated, including samples of similar structure (polyaromatic hydrocarbons, amino acids, nucleosides) and samples with different functional groups, such as chlorophenols. EXPERIMENTAL SECTION PBD-Coated Zirconia Particles and Columns. The PBDcoated zirconia particles used in this study are the same as those described in the previous studies.1,24 Thus, the preparation, physical characterization, and packing of PBD-coated zirconia particles have been fully detailed elsewhere.1 The particle size and pore diameter were about 2.5 µm and 200 Å, respectively. The phase contained 5.6% carbon (w/w), except for the separation of nucleosides, where a 2.7% carbon load phase was used. The column dimensions were 100 mm × 4.6 mm i.d. Reagents. All reagents were obtained from commercial sources and were reagent grade or better, unless noted below. The organic solvent used for liquid chromatography was ChromAR HPLC grade acetonitrile (Mallinckrodt Chemical Co., Paris, KY). DI water was filtered through a 0.45 µm filter (Gelman Sciences Inc., Ann Arbor, MI) and then boiled to remove carbon dioxide before use. All solvents were filtered a second time with a 0.45 µm filtration disk. Chemicals used included chlorobenzene, acetophenone, ethyl benzoate, benzyl cyanide, 1,4-dimethylnitrobenzene, N-ethylaniline, 4-ethylphenol, benzamide, benzophenone, biphenyl, naphthalene, anisole, phenol, 4-amino-2,6-dichlorophenol, 4-chlorophenol, 4-chloro-3-methylphenol, 2,4,5-trimethylphenol, 2,4,6-trichlorophenol, 2,4,5-trichlorophenol, and pentachlorophenol. They were all purchased from Aldrich (Aldrich Chemical Co., Milwaukee, WI). The phenylthiohydantoin amino acids used in this study included leucine, proline, phenylalanine, tyrosine, tryptophan, threonine, glutamine, arginine, histidine, and methionine (Sigma Chemical Co., St. Louis, MO). The peptides used included Asp-Phe (Asp ) aspartate, Phe ) phenylalanine), Asp-Asp, Asp-Asp-Asp, and Asp-Asp-Asp-Asp (Sigma). Standard HPLC samples of nucleosides and polyaromatic hydrocarbons were obtained from Supelco (Supelco, Inc., Bellefonte, PA). Ammonium fluoride and phosphate were used as strong Lewis bases (and also as buffers, see below) to block the Lewis acid sites on the zirconia surface so that the Lewis acid-base interactions between the solutes and the zirconia surface can be minimized.25 Ammonium carbonate, tris(hydroxymethyl)aminomethane (TRIS), potassium phosphate (diabasic), and potassium phosphate (monobasic) were used as mobile phase buffers. Hexanesulfonate and tetrabutylammonium perchlorate were used as ion-pairing reagents. Phosphoric acid and sodium hydroxide were used to adjust the mobile phase pH. All were from Aldrich. (19) Chen, H.; Horva´th, Cs. J. Chromatogr. 1995, 705, 3-20. (20) Chen, H.; Horva´th, Cs. Anal. Methods Instrum. 1993, 1 (4), 213-222. (21) Kalghatgi, K.; Horva´th, Cs. J. Chromatogr. 1988, 443, 343-353. (22) Kura, G.; Kitamura, E.; Baba, Y. J. Chromatogr. 1993, 628, 241-246. (23) Natia, F. D.; Horva´th, Cs. J. Chromatogr. 1988, 435, 1-15. (24) Li, J.; Carr, P. W. Anal. Chem. 1996, 68, 2857-2868. (25) Nawrochi, J.; Rigney, M. P.; McCormick, A.; Carr, P. W. J. Chromatogr. 1993, 657, 229-282.

Figure 1. Separation of polyaromatic hydrocarbons. Chromatographic conditions: mobile phase (premixed), 50% ACN + 50% H2O (20 mM NH4F); solute concentration, 0.1-2 mg/mL; injection volume, 1 µL; flow rate, 1.0 mL/min; detection, UV 254 nm. Solutes: 1, naphthalene; 2, acenaphthylene; 3, acenaphthene; 4, fluorene; 5, phenanthrene; 6, anthracene; 7, fluoranthene; 8, pyrene; 9, benz[a]anthracene; 10, chrysene; 11, benzo[b]fluoranthene; 12, benzo[k]fluoranthene; 13, benzo[a]pyrene; 14, dibenz[a,h]anthracene; 15, benzo[ghi]perylene; 16, indeno[1,2,3-cd]pyrene.

Chromatographic Apparatus. All chromatographic experiments were carried out on a fully automated Hewlett Packard 1090 liquid chromatograph with binary gradient pumps, an autosampler, a temperature controller, a UV detector, and a computer-based chemstation (Hewlett Packard S.A., Wilmington, DE). A pressure regulator was installed at the outlet of the detector to control the pressure stability and to minimize air bubbles. Chromatographic Conditions. The chromatographic conditions are given in the figure legend for each separation. The mobile phase composition was controlled by a binary pumping system. Eluent A was usually 50% ACN and 50% water (premixed and degassed), while eluent B was either 100% water or 100% ACN, depending on the ACN composition needed for a separation. Buffers for each separation were included in the two eluents. This setup of the mobile phase allowed us to reduce the air bubbles generated by mixing ACN and water, particularly at high temperatures. The mobile phase usually included a strong Lewis base (to interact with the Lewis acid sites on zirconia’s surface25) and a buffer to control pH. RESULTS AND DISCUSSION Figure 1 shows the separation of a sample of 16 polyaromatic hydrocarbons (PAH). These solutes are all nonpolar (hydrophobic), nonionizable species; thus, we expect that there will be only one retention mechanism for these solutes. Previously we showed that PBD-coated zirconia does not have the solute shape selectivAnalytical Chemistry, Vol. 69, No. 11, June 1, 1997

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Figure 2. Separation of amino acids. Chromatographic conditions: mobile phase, 15% ACN + 85% H2O (20 mM NH4F + 50 mM (NH4)2CO3, pH ) 7); solute concentration, 0.5 mg/mL; injection volume, 1 µL; flow rate, 1 mL/min; detection, 254 nm. Solutes (PTH-labeled amino acids): 1, threonine; 2, glutamine; 3, methionine; 4, histidine; 5, tyrosine; 6, proline; 7, leucine; 8, phenylalanine; 9, arginine; 10, tryptophan.

ity24 exhibited by conventional polymeric alkylsilane bonded phases.26 The sole difference in terms of retention between these solutes is their relative hydrophobicity. Figure 1 shows that the analysis time can be decreased from about 70 min at 30 °C to about 20 min at 90 °C. Even though there is a significant decrease in analysis time, the overall quality of the resolution at high temperature is only slightly poorer (by about 10%) than that at 30 °C, and the resolution of these solutes at 90 °C is still quite acceptable. We note that the 9/10 and 11/12 pairs are structural isomers, differing only in their shapes (see Figure 1). Thus, it is not easy to resolve them on the PBD phase due to the lack of shape selectivity.24 The detector response at 90 °C relative to that at 30 °C is significantly improved (by about 3-fold) due to the reduced retention times and sharper peaks. This is evidently important in trace analysis. Figure 2 shows a separation of a set of PTH amino acids. We expect this sample to be more complicated than the PAH mixture because each solute has both hydrophobic and polar groups. Fluoride is present in the mobile phase to serve as a strong Lewis base, and ammonium carbonate was used as both a buffer and a weak Lewis base. As indicated in Figure 2, the selectivity decreases as the temperature is increased from 40 to 100 °C. This is very obvious for solute groups 1/2/3 and 7/8/10. These peaks are fully resolved at 40 °C and are merged at 100 °C. However, solutes 9 and 10 (arginine and tryptophan) reverse their elution sequence. Figure 3 demonstrates the separation of a set of simple aromatics. The purpose of this separation is to investigate the (26) Sander, L. C.; Wise, S. A. J. Chromatogr. 1993, 656, 335-351.

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Figure 3. Separation of a general aromatic mixture. Chromatographic conditions: mobile phase, gradient (20 mM NH4F and 50 mM TRIS, pH ) 9); solute concentration, 1.5 mg/mL; injection volume, 1 µL; flow rate, 1.0 mL/min; detection, 254 nm. Solutes: 1, benzamide; 2, acetophenone; 3, benzyl cyanide; 4, 4-ethylphenol; 5, N-ethylaniline; 6, anisole; 7, ethyl benzoate; 8, 1,4-dimethylnitrobenzene; 9, benzophenone; 10, naphthalene; 11, biphenyl. Gradient conditions: at 40 °C, 0-6 min, 20% ACN, 6-25 min, 20-50% ACN; at 70 °C, 0-4 min, 20% ACN, 4-15 min, 20-50% ACN; at 100 °C, 0-3 min, 20% ACN, 3-10 min, 20-50% ACN. The dashed-box region was “zoomed in” for clarity on the right.

selectivity change with temperature for a simple set of organic solutes with different functional groups. It is typical because the solutes differ only by a single polar functional group. Note that we use a gradient to decrease the analysis time and that each gradient step was scaled for the relevant temperature. The analysis time decreases from 20 min at 40 °C to less than 10 min at 100 °C without loss in resolution. It is also evident that, even for this very simple but typical sample, temperature has a very important effect on selectivity. This can be seen for solutes 3, 4, and 6. Notice the change in elution sequence for solute pairs 2/3, 2/4, and 5/6. This separation clearly indicates that increased temperature can not only shorten analysis time without sacrificing resolution but can also change the selectivity when two solutes differ by only a single polar functional group. Figure 4 shows the separation of a set of 12 nucleosides via an ion pair technique. At a pH of 3, all solutes are protonated, and hexanesulfonate forms ion pairs with the solutes. In addition, the ion-pairing reagent increases the retention of the nucleosides because they are very polar. All peaks are resolved at 40 °C; however, we did not identify the peaks. It is important to note that, in this separation, temperature did not decrease the retention time, but it did decrease the selectivity. This means that the enthalpy changes for the solutes are very small, although the relative differences in enthalpy are significant.

Figure 4. Separation of nucleosides. Chromatographic conditions: mobile phase, 75% ACN + 25% H2O (25 mM KH2PO4 + 20 mM hexanesulfonate, titrated to pH ) 3 with H3PO4); solute concentration, 20-100 µg/mL; injection volume, 2 µL; flow rate, 0.75 mL/min; detection, 260 nm. Solutes: pseudouridine, cytidine, 3-methylcytidine, uridine, 1-methyladenosine, 2-thiocytidine, 5-methylcyidine, 7-methylguanosine, 2′-O-methylcytidine, inosine, guanosine, ribothymidine.

Figure 5. Separation of chlorophenols. Chromatographic conditions: mobile phase, 32% ACN + 68% H2O (20 mM NH4F, pH ) 5); solute concentration, 0.5 mg/mL; injection volume, 1 µL; flow rate, 1 mL/min; detection, 254 nm. Solutes: 1, phenol; 2, 4-amino-2,6dichlorophenol; 3, 4-chlorophenol; 4, 4-chloro-3-methylphenol; 5, 2,4,5-trimethylphenol; 6, 2,4,6-trichlorophenol; 7, 2,4,5-trichlorophenol; 8, pentachlorophenol.

Figure 5 shows the separation of a set of phenolic compounds. It contains eight solutes. This separation clearly demonstrates the potential importance of temperature as an effective variable for optimizing a separation. At a low temperature (40 °C), peaks 4 and 5 are merged, and peak 8 elutes last. As the temperature is increased to 70 °C, peaks 4 and 5 are baseline resolved, and peak 8 moves close to peak 6. As temperature is further increased to 100 °C, the selectivity of peaks 4 and 5 is further improved, and peak 8 is quite narrow and now precedes peak 6. Figure 5 also shows that the analysis time can be reduced from about 11 min at 40 °C to about 4 min at 100 °C with a significant improvement in selectivity. Solutes 1 and 2 are not separated at all temperatures because their k′ values are very small (close to column holdup time). Peak 8 is very broad relative to other peaks at 40 °C, indicating that it is probably retained by more than one retention mechanism. In fact, all chlorophenols interact with zirconia’s surface by Lewis acid-base processes.27 However, pentachlorophenol appears to be excessively broad, even when fluoride is added to the mobile phase to block Lewis interaction. We do not fully understand this phenomenon but point out that, unlike the other phenols, pentachlorophenol is more acidic and ionized (pKa ≈ 4.5) at the mobile phase pH. To separate solutes 1 and 2, the mobile phase was further adjusted at 100 °C, and Figure 6 shows that all peaks are now well separated. Peak 8 is shifted back to the last position, and it is broad, due, most likely, to multiple retention mechanisms. As a final example, Figure 7 shows the separation of a set of aspartate peptides via an ion-pairing technique. The mobile phase

Figure 6. Separation of chlorophenols. Chromatographic conditions: mobile phase, 15% ACN + 85% H2O (20 mM NH4F, pH ) 5); solute concentration, 0.5 mg/mL; injection volume, 1 µL; flow rate, 1 mL/min; detection, 254 nm. Solutes: same as in Figure 5.

(27) Li, J.; Carr, P. W. Anal. Chim. Acta 1996, 334, 239-250.

was controlled at pH 7 so that the carboxylic groups of the solutes remain fully dissociated. Tetrabutylammonium forms ion pairs with the carboxylic groups, and perchlorate forms ion pairs with the positive functional groups. These ion-pairing reagents, as indicated earlier, not only decrease the electronic interactions between the solutes and the negatively charged surface (due to the surface bound phosphate ions) but also increase the absolute retention of these solutes. It is an interesting separation because, at the low temperature of 40 °C, the analysis time is about 6 min. However, as temperature is increased, the analysis time increases to 8 min. Solutes 1 and 2 are essentially independent of temperature; however, solutes 3 and 4 shift significantly to longer retention times. This indicates that the enthalpy changes for these Analytical Chemistry, Vol. 69, No. 11, June 1, 1997

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Finally, we emphasize that the column with 5.6% (w/w) carbon was used to develop all separations except the separation of the nucleosides. Throughout this work, we saw no change in retention or peak shape on this column due to aging. CONCLUSIONS We have separated various mixtures of low molecular weight species on PBD-coated zirconia by varying both the mobile phase composition and temperature. We find that the effect of temperature on the selectivity depends on the type of solute. For solutes with similar structure (or functional groups) in a sample such as polyaromatic hydrocarbons, temperature caused almost no effect on or slightly decreased the selectivity; however, for solutes with very different functional groups such as chlorophenols, temperature variation can be a very effective way to change the selectivity. The complexity of the molecular mixture is not a sure guide as to the effect of temperature on selectivity, as we did not see any improvement in the separation of a set of amino acids with temperature. Even though we have not used high molecular weight solutes in this study, temperature is expected to have an even more pronounced effect on their selectivity in RPLC. We also expect that, as more functional groups are added to a solute, the retention mechanism will become more complex, leading to a significant change in selectivity upon change in temperature. Figure 7. Separation of aspartate peptides. Chromatographic conditions: mobile phase, 64% ACN + 36% H2O (50 mM K2HPO4 and 50 mM (C4H9)4NClO4, titrated to pH ) 7 with H3PO4); solute concentration, 0.6 mg/mL; injection volume, 2 µL; flow rate, 0.75 mL/ min; detection, 220 nm. Solutes: 1, Asp-Phe; 2, Asp-Asp; 3, AspAsp-Asp; 4, Asp-Asp-Asp-Asp.

two solutes are positive. It is noted that the mechanism of the separation is ion-pairing, and temperature often has a more complicated effect on retention and selectivity.28 (28) Ooma, B. LC‚GC 1996, 4, 306-324.

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ACKNOWLEDGMENT The authors acknowledge the financial support by Grant GM 45988-05 from the National Institutes of Health. Received for review August 20, 1996. Accepted March 3, 1997.X AC9608681 X

Abstract published in Advance ACS Abstracts, May 1, 1997.