Anal. Chem. 1997, 69, 409-415
Effect of Mobile Phase Additives in Packed-Column Subcritical and Supercritical Fluid Chromatography John A. Blackwell* and Rodger W. Stringham
Chemical Process Research and Development, Chambers Works, PRF-1 (S-1), Dupont Merck Pharmaceutical Company, Deepwater, New Jersey 08023 Jeff D. Weckwerth
Chemistry Department, Smith and Kolthoff Halls, University of Minnesota, 207 Pleasant Street S.E., Minneapolis, Minnesota 55455
The effect of mobile phase additives is investigated for a variety of compounds under subcritical and supercritical conditions using packed columns. Retention of hydrogen bond donor/acceptor analytes was found to be more dependent on the presence of mobile phase additives than weak hydrogen bond acceptor analytes. The temperature and pressure of the mobile phase are major factors in the extent of this dependence. Consequently, selectivity between homologous compounds is dependent on both the additive used and the state of the mobile phase. Efficiency is nearly always improved by the presence of mobile phase additives, more so under supercritical conditions than under subcritical conditions. These observations suggest that surface molar excesses of mobile phase additives play a large part in the resulting character of the supercritical chromatographic system. Packed-column supercritical fluid chromatography is used to an increasing extent in the pharmaceutical industry as a means for facile determination of chiral purity.1 High optimum linear velocities and mobile phase diffusivities offer the promise of fast, efficient separations. Indeed, many literature citations attest to superior separations which may be achieved using subcritical and supercritical mobile phases compared to conventional HPLC separations.2-8 Carbon dioxide alone is unacceptable as a mobile phase for most pharmaceutical compounds on packed columns. Because of its poor solvating ability, addition of polar modifiers is necessary. Recent studies indicate that various mobile phase modifiers affect retention, selectivity, and efficiency to different extents depending on the temperature and pressure of the chromatographic system.9 These studies showed that the changes in retention imparted by (1) Petersson, P.; Markides, K. E. J. Chromatogr. 1994, 666, 381-394. (2) Terfloth, G. J.; Pirkle, W. H.; Lynam, K. G.; Nicolas, E. C. J. Chromatogr. 1995, 705, 185-194. (3) Bargmann-Leyder, N.; Tambute, A.; Caude, M. Chirality 1995, 7, 311325. (4) Whatley, J. J. Chromatogr. 1995, 697, 251-255. (5) Nishikawa, Y. Anal. Sci. 1993, 9, 33-37. (6) Blum, A. M.; Lynam, K. G.; Nicolas, E. C. Chirality 1994, 6, 302-313. (7) Lynam, K. G.; Nicolas, E. C. J. Pharm. Biomed. Anal. 1993, 11, 11971206. (8) Stringham, R. W.; Lynam, K. G.; Grasso, C. C. Anal. Chem. 1994, 66, 19491954. S0003-2700(96)00888-8 CCC: $14.00
© 1997 American Chemical Society
the modifiers could be successfully modeled using linear solvation energy relationships (LSERs). LSER analyses accurately predicted retention for a wide variety of naphthalene derivatives on a cyano bonded phase under both subcritical and supercritical conditions. Although chromatographic efficiency could not be modeled using these same relationships, efficiency losses could be accounted for by the solvation parameters of each of the modifiers. Specifically, the hydrogen bond accepting ability of surface silanol groups distorted the peak shapes of hydrogen bond donor analytes. Modifiers with hydrogen bond donor character minimized these effects to a greater extent than nondonor modifiers. Conversely, the hydrogen bond donating properties of surface silanols are best attenuated by hydrogen bond accepting modifiers when hydrogen bond accepting analytes are considered. The specific nature of these effects was far greater under subcritical conditions than under supercritical conditions. Unfortunately, the effect of modifiers alone is usually insufficient to overcome the chromatographically deleterious effects of residual silanol groups for many pharmaceutical compounds. Certain functional groups, such as carboxylic acids, amines, amidines, etc., interact quite strongly with silica-based stationary phase supports, giving rise to distorted peak shapes and poor chromatographic efficiencies. Use of very strong modifiers in higher concentrations may improve efficiency, but at the cost of decreased retention and selectivity.9 A common remedy, taken from normal phase chromatography, is the use of mobile phase additives. These mobile phase components, used in relatively low concentrations, attenuate undesirable interactions between the analyte and the stationary phase. With additives being used to improve efficiency, retention may be independently controlled by the modifier. Most of the prior investigations involving the use of mobile phase additives for packed-column SFC have been of limited nature.10-14 In most cases, the chromatographic effects of a few additives are qualitatively compared for a particular type of (9) Cantrell, G. O.; Stringham, R. W.; Blackwell, J. A.; Weckwerth, J. D.; Carr, P. W. Anal. Chem. 1996, 68, 3645-3650. (10) Geiser, F. O.; Yocklovich, S. G.; Lurcott, S. M.; Guthrie, J. W.; Levy, E. J. J. Chromatogr. 1988, 459, 173-181. (11) Oudsema, J. W.; Poole, C. F. J. High Resolut. Chromatogr. 1993, 16, 130134. (12) Berger, T. A.; Deye, J. F. J. Chromatogr. 1991, 547, 377-392.
Analytical Chemistry, Vol. 69, No. 3, February 1, 1997 409
compound under a limited set of conditions. While this information is very useful for that particular type of separation, it does not provide information as to which mobile phase additive is most appropriate for a particular type of separation under different temperature and pressure conditions. This hit-or-miss approach to additive choice precludes the selection of novel mobile phase additives, chosen with a specific function in mind based on the compound’s chemical properties. The scope of this study is to determine the relative effects of a number of various mobile phase additives in a carbon dioxidemethanol mobile phase system. Prior studies indicate that methanol is a very strong mobile phase modifier and is a good first choice for the elution of polar pharmaceutical compounds under most temperature and pressure conditions. Mobile phase additives are compared under both subcritical and supercritical conditions for a wide variety of naphthalene derivatives, specifically chosen to represent the varied types of functional groups expected for many pharmaceutical compounds. Various additives are chosen to span a large range of solvation parameters to determine whether specific effects may be correlated to particular solvation properties of the additives. The effect of these mobile phase additives on retention, selectivity, and efficiency will be demonstrated. The effect of various additives on chiral selectivity and resolution will be briefly examined. EXPERIMENTAL SECTION Chromatographic System. The chromatographic system used in this study was a Gilson SF3 system (Gilson, Inc. Middleton, WI). Carbon dioxide mobile phase was pumped with a Gilson Model 308 pump with a thermostated head. Methanol modifier containing 0.13 M additive was pumped with a Gilson Model 306 pump. Mixing took place in a Gilson Model 811C dynamic mixer with a 1.5 mL mixing chamber. Fixed loop injections (5 µL) were made using a Gilson Model 231XL sampling injector. Column thermostating was accomplished using a Gilson Model 831 temperature regulator. Detection was accomplished at 210 nm using a Gilson Model 117 variable wavelength UV detector with a 7 µL high-pressure flow cell. Column backpressure was maintained using a Gilson Model 821 pressure regulator. Retention times, peak widths at half-height, resolution factors, and tailing factors were determined using Unipoint software obtained from Gilson. Column. A Zorbax SB-Cyano column (250 mm long; 4.6 mm diameter; 5 µm particles) was used for the achiral studies and was obtained from Mac-Mod (Chadds Ford, PA). A Chirex 3012 column (150 mm long; 4.6 mm diameter; 5 µm particles) with the (R)-phenylglycine-3,5-dinitroaniline selector was used for the chiral separations and was obtained from Phenomenex (Torrance, CA). Chromatographic Conditions. Naphthalene derivatives were chromatographed using carbon dioxide modified with 10 vol % methanol containing 0.13 M additive. Total flow through the system was 1.0 mL/min (liquid flow). Column temperature was maintained at 25 °C (subcritical for all mobile phase mixtures) or 80 °C (supercritical for all mobile phase mixtures).15 Backpressure was maintained at 150 bar. Inlet pressure varied among the various mobile phase compositions between 155 and 160 bar. When additives were changed, the mobile phase containing the (13) Berger, T. A.; Deye, J. F. J. Chromatogr. Sci. 1991, 29, 310-317. (14) Berger, T. A.; Deye, J. F. J. Chromatogr. Sci. 1991, 29, 26-30. (15) Chueh, P. L.; Prausnitz, J. M. AIChE J. 1967, 13, 1107-1113.
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new additive was allowed to equilibrate for at least 4 h to ensure that the old additive was washed from the system. After this equilibration period, the baseline was constant at the lowest integrator attenuation, and replicate injections of probe analytes produced identical retention times. Chiral analytes were chromatographed using carbon dioxide modified with 25 vol % methanol containing 1 vol % additive. Flow was maintained at 1.5 mL/min at 20 or 70 °C (subcritical for all mobile phase mixtures). Backpressure was maintained at 200 bar, while inlet pressure varied between 210 and 215 bar. Chemicals. Various 1-substituted naphthalene derivatives, 1,10′-bi-2-naphthol, tri-n-butyl phosphate, trimethyl phosphate, acetamide, formic acid, and n-hexylamine were obtained from Aldrich (Milwaukee, WI) and were reagent grade or better. Dansylated amino acid enantiomers and (1-naphthyl)ethylamine enantiomers were obtained from Sigma (St. Louis, MO). Spectroscopic or HPLC grade trifluoroacetic acid, acetic acid, triethylamine, and triethanolamine were obtained from J. T. Baker (Phillipsburg, NJ). Water was obtained from a Milli-Q water system (Millipore Corp. Bedford, MA). Methanol was from EM Science (Gibbstown, NJ). SFC grade carbon dioxide (without helium headspace) was obtained from Scott Specialty Gases (Plumsteadville, PA). Samples. Solutions of the 1-substituted naphthalene derivatives were prepared in methanol to a final concentration of 100 µg/mL. Note that 1-naphthylamine is a known carcinogen and should be handled with extreme caution. Chiral analytes were dissolved in methanol to a final concentration of 1 mg/mL. LSER Regressions. Retention data were regressed against experimentally determined9,16 solvation parameters according to the relationship:
∑β
log k ′ ) SP0 + l log L87 + sπ2H + b
H 2
∑R
+a
H
2
+
rR2 (1) where SP0 is the regression intercept, log L87 is the gas-to-apolane partition coefficient at 200 °C, π2H is the analyte polarizability/ dipolarity, β2H is the analyte hydrogen bond basicity, R2H is the analyte hydrogen bond acidity, and R2 is the analyte excess molar refraction. Each term was systematically evaluated as to its statistical significance with both partial and full data sets. Experimentally, it was determined that the gas-to-apolane partition coefficient contribution was statistically insignificant. Final regressions were performed with the following resulting equation:
∑β
log k ′ ) SP0 + sπ2H + b
H
2
∑R
+a
H
2
+ rR2
(2)
RESULTS AND DISCUSSION The chromatographic properties of 10 mobile phase additives were evaluated with respect to retention, selectivity, and efficiency using a system with a carbon dioxide-based mobile phase. Ten volume percent of methanol was used both as a mobile phase modifier and as a carrier for the mobile phase additive under subcritical and supercritical conditions. The relevant properties of these mobile phase additives are given in Table 1. The choice (16) Weckwerth, J. D. Ph.D. Thesis, University of Minnesota, 1996. (17) Abraham, M. H. Chem. Soc. Rev. 1993, 73-83. (18) Kamlet, M. J.; Abboud, J. M.; Abraham, M. H.; Taft, R. W. J. Org. Chem. 1983, 48, 2877-2887. (19) Reid, R. C.; Prausnitz, J. M.; Poling, B. E. The Properties of Gases and Liquids, 4th ed.; McGraw-Hill: New York, 1987; Appendix A.
Table 1. Mobile Phase Additive Propertiesa
Table 2. Analyte Solvation Parameters derivative (-R)a
π2H b
ΣR2H c
Σβ2H d
R2e
log L87 f
log L16
π*
R
β
R2
dipole (D)
probe no.
additive trifluoroacetic acid acetic acid formic acid water triethylamine triethanolamine n-hexylamine acetamide trimethyl phosphate tri-n-butyl phosphate
na 1.750 na 0.260 3.040 na 3.655 na na na
0.50b 0.65 0.60 0.45 0.15 na 0.35 1.30 1.10 0.90
na 0.61 0.75 0.82 0.00 na 0.16 0.54 0.00 0.00
na 0.44 0.38 0.35 0.79 na 0.61 0.68 1.00 1.21
na 0.265 0.300 0.000 0.101 na 0.197 0.460 0.113 -0.100
2.3d 1.7d 1.4d 1.8c 0.7d 3.6e na 3.8d na 3.1e
1 2 3 4 5 6 7 8
Weak Hydrogen Bond Acceptor Analytes -H 0.85 0.00 0.22 1.38 -CH3 0.87 0.00 0.22 1.44 -CH2CH3 0.87 0.00 0.23 1.43 -C6H5 1.08 0.00 0.30 1.91 -F 0.82 0.00 0.18 1.32 -Cl 0.92 0.00 0.15 1.54 -Br 0.97 0.00 0.17 1.67 -I 1.04 0.00 0.20 1.84
0.332 0.477 0.567 1.060 0.329 0.559 0.670 0.804
9 10 11 12 13 14 15 16
Strong Hydrogen Bond Acceptor Analytes -NO2 1.29 0.00 0.36 1.35 -CN 1.25 0.00 0.41 1.19 -CH2CN 1.44 0.00 0.53 1.43 -(CO)H 1.19 0.00 0.47 1.47 -OCH3 0.99 0.00 0.37 1.70 -OCH2CH3 0.96 0.00 0.40 1.63 -O(CO)CH3 1.25 0.00 0.62 1.13 -N(CH3)2 0.93 0.00 0.49 1.57
0.756 0.633 0.832 0.662 0.618 0.687 0.703 0.674
17 18 19 20 21 22 23
Hydrogen Bond Donor/Acceptor Analytes -OH 1.20 0.67 0.38 1.19 -CH2OH 1.19 0.27 0.64 1.64 -CH2CH2OH 1.21 0.23 0.72 1.67 -COOH 1.27 0.52 0.48 1.20 -CH2COOH 1.35 0.54 0.40g 1.30 -NH2 1.23 0.19 0.49 1.74 -CH2NH2 1.10 0.00 0.80 1.95
0.515 0.688 0.798 0.731 0.838 0.660 0.754
a
na indicates data not available. Values from ref 17 except where noted. b Bulk value from ref 18. c Values from ref 19. d Values from ref e 20. Values from ref 21.
of additives spans the range of typical acidic and basic additives, as well as some included solely to determine whether significant solvation properties alone, without Brønsted acidity or basicity, are sufficient to produce desirable chromatographic effects. The analytes used in this study consist of a wide variety of R-substituted naphthalene derivatives. These compounds have been well characterized as to their solvation properties.16 Many of these analytes have been used previously to characterize the effects of mobile phase modifiers in a similar system.9 The relevant solvation parameters for these analytes are given in Table 2. Analytes are nearly evenly distributed between weak hydrogen bond acceptor analytes, strong hydrogen bond acceptor analytes, and hydrogen bond acceptor/donor analytes in order to account for the many types of compounds anticipated in pharmaceutical analyses. Efficiency Effects. Mobile phase additives are used primarily in normal and reversed phase chromatography to improve peak shape and increase efficiency.22 Figure 1 shows the reduced plate heights obtained for 1-naphthoic acid and 1-naphthylamine for the various additives under subcritical and supercritical conditions. These solutes represent the extremes of acidity and basicity for the probe set and are representative of the effects observed for other analytes. The mobile phase additives are ranked with respect to their increasing reduced plate heights under supercritical conditions. All additives improved efficiency under supercritical conditions for 1-naphthoic acid. This is likely due to the preferential adsorption of mobile phase components at the chromatographic interface.23 From these observations, it appears that the additives adsorb to a greater extent than the methanol modifier. Under supercritical conditions, surface molar excesses of mobile phase additives act to change the chemical nature of the stationary phase surface. Any additive that may sterically block access to the surface silanol groups will act to improve efficiency. Under subcritical conditions, where this effect is not present, neutral additives do nothing to improve efficiency. Only Brønsted acidic and basic additives, which may have higher adsorption energies at the silanol sites, are effective at improving efficiency. (20) Lide, D. R. CRC Handbook of Chemistry and Physics, 75th ed.; CRC Press: Boca Raton, FL, 1994. (21) Dean, J. A. Lange’s Handbook of Chemistry, 13th ed.; McGraw-Hill: New York, 1985. (22) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography, 2nd ed.; J. Wiley and Sons: New York, 1979. (23) Strubinger, J. R.; Song, H.; Parcher, J. F. Anal. Chem. 1991, 63, 104-108.
a
R
b
Solute dipolarity/polarizability, see ref 16. c Solute hydrogen bond acidity, see ref 16. d Solute hydrogen bond basicity, see ref 16. e Solute excess molar refraction, see ref 16. f Gas-to-apolane partition coefficient at 200 °C, see ref 16. g Estimated value.
A challenging probe of hydrogen bond basicity, 1-naphthylmethylamine, was included in these studies as a model Brønsted basic analyte. Only a mobile phase containing n-hexylamine as an additive was successful in eluting this primary amine. For 1-naphthylamine, some additives decreased efficiency relative to a mobile phase without any additive, although only acetamide showed any significant loss of efficiency under supercritical conditions. Under subcritical conditions, only trifluoroacetic acid showed any significant loss of efficiency relative to mobile phase void of additives. Retention Effects. The effect an additive can have on retention is highly dependent on the functionality of the analyte. Analytes with functional groups that can interact with strong stationary phase adsorption sites, such as residual silanol groups, will show much larger differences in retention than other types of analytes. The effectiveness with which the mobile phase additive disrupts this strong interaction will determine the extent to which retention is altered. Therefore, additives should be able to be ranked as to their relative effectiveness toward disrupting these various specific molecular interactions. A relatively unambiguous method for determining the relative strengths of specific molecular interactions in a chromatographic system is through the use of linear solvation energy relationships or solvatochromic analysis.24 Earlier studies have shown this technique to be quite useful for determining the relative properties of various mobile phase modifiers for the same type of system as (24) Carr, P. W. Microchem. J. 1993, 48, 4-28.
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411
Figure 1. Reduced plate heights for (a) 1-naphthoic acid and (b) 1-naphthylamine under (0) subcritical and (O) supercritical conditions. Additives: TEOH, triethanolamine; HexNH2, n-hexylamine; TEA, triethylamine; AcAm, acetamide; TMP, trimethyl phosphate; TBP, tri-n-butyl phosphate; HOAc, acetic acid; MeOH, no additive; H2O, water; HCOOH, formic acid; and TFA, trifluoroacetic acid.
Figure 2. Effect of various mobile phase additives on retention for (a) subcritical (25 °C/150 bar) and (b) supercritical conditions (80 °C/150 bar). Analytes are (O) 1-phenylnaphthalene and (0) 1-naphthoic acid. Additives as abbreviated in Figure 1.
employed here.9 In this study, the main difference is that many more polar analytes are included in order to gain a greater understanding of how highly polar functional groups react to the presence of various mobile phase additives. It is exactly these types of “difficult” analytes for which additives are generally used. Figure 2 confirms that, under subcritical conditions, weak hydrogen bond acceptor analytes show relatively little difference in retention when various mobile phase additives are used. Strong hydrogen bond acceptor and donor/acceptor analytes show more 412
Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
significant differences in retention which are additive dependent. Under subcritical conditions, mobile phase additives are dispersed between the stationary and mobile phases, as determined by the additive’s partition coefficient for this system. Many additives which can interact with residual silanol groups will selectively partition to these sites and attenuate increased retention via specific interactions. This interaction can be more selective when additives are used, since a wider range of solvation parameters is available for use among additives than among chromatographically
Figure 3. LSER regression coefficients for analyte retention under (9) subcritical and (b) supercritical conditions. Error bars denote variability in regression coefficients. Coefficients are given for (a) stationary phase hydrogen bond basicity (R), (b) stationary phase dipolarity/polarizability (s), (c) stationary phase hydrogen bond acidity (β), and (d) stationary phase excess molar refraction (r). Additives as abbreviated in Figure 1.
useful modifiers. Under supercritical conditions, aggregation of mobile phase additives at the chromatographic interface alters the chemical nature of the surface. This is reflected by the significant additive-dependent change in retention for the weak hydrogen bond acceptor analyte, as well as the hydrogen bond donor/acceptor analyte in Figure 2. To explore the effect of specific chemical interactions on these analytes, a detailed LSER analysis was performed for each of the additive-containing mobile phases according to eq 2. The results of these regressions are shown in Figure 3. For both subcritical and supercritical conditions, dipolarity/polarizability and hydrogen bond donating ability, denoted by the s and a terms, respectively, are the dominant characteristics that determine analyte retention. This is anticipated due to the nature of the cyano stationary phase used in these studies. Aliphatic cyano groups are very good hydrogen bond acceptors which possess very high dipole moments. In the regression analysis, analyte hydrogen bond accepting ability is a highly variable but less dominant factor, likely due to the presence of acidic residual silanol groups. Regression intercepts are nearly uniform, indicating that most of the chemical information regarding retention is accounted for in the solvatochromic terms used in these regressions.24 The R values shown in Figure 3a indicate that most of the mobile phase additives follow a similar trend, regardless of
whether the mobile phase is subcritical or supercritical. The notable exception is the significant increase in the R terms when basic additives are used under supercritical conditions. Since preferential adsorption of additives is anticipated under supercritical conditions, it is not surprising that Brønsted basic additives increase the effective hydrogen bond basicity of the stationary phase. Surprisingly, the trialkyl phosphate additives do not show similar effects despite their very high hydrogen bond basicity values (Table 1). Inspection of the other solvatochromic coefficients in Figure 3b-d reveals some interesting trends. All of the coefficients, with the exception of the relatively invariable molar excess refraction terms, are larger under supercritical conditions than for subcritical conditions. These results are consistent with earlier studies of mobile phase modifiers, where selectivity was almost always greater under supercritical conditions than under subcritical conditions.9 Selectivity Effects. The effect that each additive has on selectivity was evaluated further. In Figure 4, the capacity factor for each analyte obtained when using an additive in the mobile phase was divided by the capacity factor obtained in a system without an additive. This ratio was then plotted for each of the derivatives in each of the additive-containing systems. Probe derivative numbers are consistent with Table 2. Figure 4a shows that, under subcritical conditions, most of the derivatives show little or no selectivity differences when chromatographed with various additives. The derivatives that do show significant differences in selectivity are the hydrogen bond donor/acceptor derivatives. These highly polar analytes are especially prone to changes induced by silanol interactions. Each additive shows different selectivities due to varying degrees of competitive blocking of the silanol interactions. Figure 4b shows a somewhat similar effect when considering the hydrogen bond donor/acceptor derivatives. What is striking is the high degree of selectivity between additives for the other derivatives which is independent of the probe molecule. The trifluoroacetic acid-containing mobile phase shows significantly increased relative retention compared to the acetamide-containing mobile phase. This observation is consistent with the formation of different surface chemistries with additive-dependent properties by virtue of the surface aggregation of additive at the chromatographic interface. Chiral Selectivity. Many chiral pharmaceutical compounds possess one or more hydrogen bond donor/acceptor functional groups. These functional groups are essential for the chiral recognition process for most molecules. Mobile phase additives may alter chiral selectivity through their effect on these specific interactions. This was briefly investigated by chromatographing a variety of chiral analytes with various mobile phase additives at two different temperatures. High methanol concentrations were necessary in order to elute these polar compounds from the silicabased chiral stationary phase. Both operating temperatures are subcritical, due to the large increase in critical parameters with high modifier concentrations,15 but represent a reasonable temperature range for practical separations. Table 3 shows that the mobile phase additive has a large and variable effect on chiral selectivity. Addition of additives has a detrimental effect on the selectivity factor obtained for dansylated leucine for both temperatures. Some additives improve selectivity for dansylated methionine, (1-naphthyl)ethylamine, and 1,1′-bi-2Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
413
Figure 4. Normalized retention for the naphthalene derivatives with various mobile phase additives under (a) subcritical and (b) supercritical conditions. Probe numbers for each naphthalene derivative are obtained from Table 2. Additives: (9) trifluoroacetic acid, (0) acetic acid, ([) formic acid, (]) water, (2) acetamide, (4) triethylamine, (b) n-hexylamine, (O) triethanolamine, (×) trimethyl phosphate, and (*) tri-n-butyl phosphate. Table 3. Selectivity Factors for Chiral Analytes Using Various Additives none dans-DL-Leu dans-DL-Met (R/S)-(1-naphthyl)ethylamine 1,1′-bi-2-naphthol
1.24 1.00 1.07
dans-DL-Leu dans-DL-Met (R/S)-(1-naphthyl)ethylamine 1,1′-bi-2-naphthol
1.15 1.10 0.98
1.55
1.17
TFA
HOAc
H2O
TEA
C6NH2
1.17 1.00 1.06
1.20 1.15 1.02
1.16 1.12 1.01
1.51
1.48
1.30
1.25
70 °C; 200 bar 1.04 1.14 1.04 1.00 1.02 0.98
1.10 1.12 0.98
1.12 1.09 0.96
1.10 1.08 0.96
1.16
1.12
1.11
20 °C; 200 bar 1.09 1.22 1.08 1.00 1.05 1.03 1.62
1.18
1.17
naphthol, while others decrease selectivity. Interestingly, the elution order is reversed for (1-naphthyl)ethylamine at higher temperatures for all but the trifluoroacetic acid-containing mobile phase. This suggests that these operating temperatures are close to the isoelution temperature for this system.25 Table 4 shows that additives also have large, variable effects (25) Stringham, R. W.; Blackwell, J. A. Anal. Chem. 1996, 68, 2179-2185.
414 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
Table 4. Resolution Factors for Chrial Analytes Using Various Additives none dans-DL-Leu dans-DL-Met (R/S)-(1-naphthyl)ethylamine 1,1′-bi-2-naphthol
2.89 0.00 0.27
dans-DL-Leu dans-DL-Met (R/S)-(1-naphthyl)ethylamine 1,1′-bi-2-naphthol
2.39 1.80 0.18
6.78
2.70
TFA
HOAc
H2O
TEA
C6NH2
1.93 0.00 0.31
2.84 2.11 0.20
2.04 1.71 0.08
6.47
6.35
4.07
3.36
70 °C; 200 bar 0.45 2.37 0.47 0.00 0.33 0.10
1.82 2.22 0.14
2.24 1.47 0.45
1.92 1.39 0.48
2.44
1.91
1.56
20 °C; 200 bar 1.28 3.08 1.15 0.00 0.24 0.17 7.55
2.73
2.62
on chiral resolution, where the effects of selectivity and efficiency are combined. In some cases, efficiency is improved by the addition of modifiers, such as the case for dansylated methionine at 20 °C using triethylamine. In other cases, resolution is decreased, as in the case of dansylated leucine with trifluoroacetic acid. A confounding factor with these comparisons, however, is that retention has not been normalized to correct for its effect on resolution.26 Regardless, the large changes in resolution obtained
here indicate that additives may be used effectively to enhance chiral selectivity. These preliminary results warrant further investigation. It should be noted that the low resolution factors obtained for (1-naphthyl)ethylamine reflect the close proximity to the isoelution temperature. From the variable resolution and selectivity factors, it is evident that additives also have an effect on the isoelution temperature. Other Factors. In addition to the effects on retention, selectivity, and efficiency, other practical factors must be considered in order to make best use of mobile phase additives. During the course of these studies, it was observed that commercial supplies of certain mobile phase additives precluded the use of low ultraviolet wavelengths for detection. Among these, the basic additives triethylamine and triethanolamine proved most troublesome. Detection at 210 nm was not possible with these additives, although reagent grade n-hexylamine proved quite satisfactory at low wavelengths when a basic additive was desired. The issue of system peaks should also be considered when choosing a mobile phase additive. UV absorbing additives produce one or more negative system peaks which may interfere with analyte quantitation, especially under supercritical conditions where surface concentrations are quite high. Among the best additives, with respect to minimizing this effect, are water, n-hexylamine, and the trialkyl phosphates. Triethanolamine produced two very significant system peaks, which may prove too troublesome for most applications.
CONCLUSIONS Mobile phase additives are quite effective at attenuating the effect of deleterious silanol effects and changing the overall nature of the stationary phase. Efficiency is significantly affected by the choice of mobile phase additive and the temperature of the system. Any additive improves efficiency under supercritical conditions, whereas only Brønsted acidic and basic additives improve efficiency under subcritical conditions. Retention and selectivity may also be affected by the mobile phase additive, especially for compounds containing functional groups that are strong hydrogen bond donor/acceptors. The choice of operating conditions, subcritical or supercritical, largely determines the extent of these additional effects. For chiral separations, selectivity and resolution are significantly and variably affected by the presence of mobile phase additives. Despite the very low concentrations normally used in separations, the effects can be profound. ACKNOWLEDGMENT The authors thank Dr. Peter Carr for many useful discussions. SUPPORTING INFORMATION AVAILABLE Listing of subcritical and supercritical retention data and solvatochromic regression coefficients (5 pages). Ordering information is given on any current masthead page. Received for review September 5, 1996. November 18, 1996.X
Accepted
AC9608883 (26) Stringham, R. W. Chirality 1996, 8, 249-257.
X
Abstract published in Advance ACS Abstracts, January 1, 1997.
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