Anal. Chem. 1998, 70, 737-742
Packed Capillary Column Solvating Gas Chromatography Using Mobile Phases That Transition from Liquid to Gas between the Column Inlet and Outlet Yufeng Shen and Milton L. Lee*
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602-5700
In this study, organic solvents at temperatures higher than their normal boiling points were used as mobile phases for packed capillary column chromatography. No restrictor or back pressure was imposed on the column outlet. As a result, a large difference in mobile-phase properties existed, and a phase transformation from liquid to gas occurred, along the column. Solvating gas chromatography (SGC) was used to characterize this chromatographic process because, near the column outlet, a gaseous mobile phase existed. Chromatographic performance characteristics including mobile-phase flow, column efficiency, mobile-phase solvating power, and solute retention were investigated using fused-silica capillary columns packed with microparticles (5- and 10-µm diameters). It was found that when the temperature was increased to values that were higher than the normal mobile-phase boiling point, the mobile-phase linear velocity increased rapidly at constant column inlet pressure. Although large differences in mobile-phase properties existed between the column inlet and outlet, high column efficiencies (reduced plate heights of less than 2) were achieved. At elevated temperature, the optimum mobile-phase linear velocity increased, and the dependence of column efficiency on linear velocity decreased. Solute retention factors also decreased with increasing temperature, even for temperatures higher than the normal boiling point of the mobile phase. Large polycyclic aromatic hydrocarbons and enantiomers were rapidly separated under SGC conditions.
power of the mobile phase facilitates this elution. In order to extend the solvating power of the mobile phase in SGC, we examined the use of organic solvents in this study. In recent years, enhanced fluidity mobile phases have been introduced in liquid chromatography (LC) to enhance the speed and efficiency of LC separations. The viscosity of mobile phases can be lowered by using elevated temperature or by adding lowviscosity fluids such as CO2.4-12 If elevated temperatures are allowed by the sample, the use of higher temperature can improve chromatographic performance including high speed,7,9-12 better column efficiency,4-6,10,12 and lower solute retention4,6-10 compared to room-temperature LC. However, the maximum temperature that can be applied is limited by the mobile-phase normal boiling point. In order to maintain the mobile phase throughout the column length as a uniform liquid (superheated liquid), a restrictor or back-pressure controller must be used.8,10 In fact, the restrictor or back-pressure controller determines the mobile-phase linear velocity and column efficiency generation rate when a specific column inlet pressure is used. More than twenty years ago, vapor carrier GC was developed for the separation of polar or low-volatility compounds.13-15 Two approaches were used in these studies. One method involved the use of liquid vapors as modifiers in gas carriers such as He and N2.13,14 The second method involved directly using liquid vapors as mobile phases, termed “steam gas-solid chromatography.”15 However, these early studies were based on conventional packed GC columns, in which large particles (60-100 mesh, 150-250 µm) were used. In contrast, when microparticle packed columns are used, the required high column inlet pressure can
Previous studies have shown that solvating gas chromatography (SGC) provides high-speed1 and high-efficiency separations2 and is even suitable for the separation of polar compounds such as free acids and amines3 when using microparticle packed capillary columns and CO2 as mobile phase. When CO2 is used as the mobile phase, the column inlet and outlet behave as supercritical fluid chromatography (SFC) and gas chromatography (GC), respectively. Elevated temperature is required to elute all but the most volatile solutes from the column, and the solvating
(4) Schmit, J. A; Henry, R. A.; Williams, R. C.; Dieckman, J. F. J. Chromatogr. Sci. 1971, 9, 645-651. (5) Knox, J. H.; Vasvari, G. J. Chromatogr. 1973, 83, 181-194. (6) Takeuchi, T.; Watanabe, T., Ishii, D. J. High Resolut. Chromatogr. Chromatogr. Commun. 1981, 4, 300-302. (7) Antia, F. D; Horva´th, Cs. J. Chromatogr. 1988, 435, 1-15. (8) Gant, J. R.; Dolan, J. W.; Snyder, L. R. J. Chromatogr. 1979, 185, 153-177. (9) Trones, R.; Iveland, A.; Greibrokk, T. J. Microcolumn Sep. 1995, 7, 505512. (10) Sheng, G.; Shen, Y.; Lee, M. L. J. Microcolumn Sep. 1997, 9, 63-72. (11) Cui, Y.; Olesik, S. Anal. Chem. 1991, 63, 1812-1819. (12) Lee, S. T.; Olesik, S. Anal. Chem. 1994, 66, 4498-4506. (13) Ackman, R. G.; Burgher, R. D. Anal. Chem. 1963, 35, 647-652. (14) Dunn, S. R.; Simenhoff, M. L.; Wesson, L. G., Jr. Anal. Chem. 1976, 48, 41-44. (15) Nonaka, A. Anal. Chem. 1972, 44, 271-276.
(1) Shen, Y.; Lee, M. L. J. Chromatogr. 1997, 778, 31-42. (2) Shen, Y.; Lee, M. L. Anal. Chem. 1997, 69, 2541-2549. (3) Shen, Y.; Lee, M. L. Chromatographia 1997, 46, 587-592. S0003-2700(97)00672-0 CCC: $15.00 Published on Web 01/21/1998
© 1998 American Chemical Society
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Figure 1. Calculated relationships between Tb and P for different liquids based on the Frost-Kalkwarf-Thodos equation.23
liquefy the mobile phase, while the low pressure at the column outlet allows the existence of a gaseous mobile phase. At a specific point along the column, a phase transition from liquid to gas (vapor) occurs. Without question, the formation of vapor near the column outlet increases the mobile-phase linear velocity because of the low viscosity of the vapor. However, the large differences in densities between gases and liquids produces an extremely high mobile-phase linear velocity gradient between the column inlet and outlet (mass flow rate of the mobile phase remains constant in the column). The effect of the mobile-phase linear velocity gradient on column efficiency is of major concern in packed column chromatography when compressible mobile phases are used.16-19 Theory and experiment have shown that this effect is minimal.16,17 The effect of a phase transition along the column on column efficiency has been little studied. Our previous work indicated that when the mobile phase was changed from a supercritical fluid to a gas along the column, high column efficiencies were still achieved (reduced plate heights of smaller than 1.3).2 However, while physicochemical properties make a smooth change from supercritical fluid to gas at elevated temperatures, the situation is obviously different when changing from a liquid to a gas. The solvating power of the mobile phase increases with increasing temperature, which produces reduced retention of solutes in the column.10 However, when the column contains both superheated liquid and vapor, the mechanisms leading to solute retention in the column become more complex. In this study, the effect of mobile-phase transition from liquid to gas on chromatographic performance was investigated. Organic solvents were selected as mobile phases because aqueous mobile phases are known to damage siliceous stationary phases at elevated temperatures.20 EXPERIMENTAL SECTION Materials and Instrumentation. Spherical porous (10-µm diameter, 300-Å pores) octadecyl bonded silica (ODS) particles (16) Giddings, J. C. Anal. Chem. 1964, 36, 741-744. (17) Myers, M. N.; Giddings, J. C. Anal. Chem. 1965, 37, 1453-1457. (18) Schoenmakers, P. J.; Rothfusz, P. E.; Verhoeven, F. C. C. C. J. G. J. Chromatogr. 1987, 395, 91-110. (19) Schoenmakers, P. J.; Uunk, L. G. M. Chromatographia 1987, 24, 51-57. (20) Unger, K. K. Porous Silica; Elsevier: Amsterdam, 1979; p 12.
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Figure 2. Experimental relationship between mobile-phase linear velocity and temperature. Conditions: 260 cm × 250 µm i.d. fusedsilica capillary column packed with 10-µm (300-Å pores) ODS particles, nitromethane used as unretained marker, acetonitrile used as the mobile phase, and UV detector (254 nm). Key: (0) SGC (200 °C), ([) SGC (140 °C), (4) LC (60 °C), and (b) LC (24 °C).
Figure 3. Experimental relationship between mobile-phase linear velocity and column inlet pressure in SGC and LC. Conditions, the same as in Figure 2. Key: (]) 200 and (0) 400 atm column inlet pressure.
were purchased from Phenomenex (Torrance, CA), and spherical porous (5-µm diameter, 200-Å pores) ODS particles were purchased from YMC (Wilmington, NC). Fused-silica capillary tubing was purchased from Polymicro Technologies (Phoenix, AZ). Column connections were made using PEEK tubing and zero dead volume unions (Valco Instruments, Houston, TX). The preparation of packed capillary columns was carried out using a Lee Scientific model 600 SFC instrument (Dionex, Salt Lake Division, Salt Lake City, UT). SFC-grade CO2 (Scott Specialty Gases, Plumsteadville, PA) was used for the slurry packing of packed capillary columns. For SGC experiments, a µLC-500 microflow pump (Isco, Lincoln, NE) was used to deliver the mobile phase. A Lee Scientific model 600 SFC oven was used to control the temperature of the column. A model 203 UV/visible detector (Linear Scientific Instruments, Reno, NV) was used for detection. Preparation of Packed Capillary Columns. A CO2 slurry packing method was modified to prepare long packed capillary columns.2,21 The procedure is outlined as follows. One end of (21) Malik, A.; Li, W.; Lee, M. L. J. Microcolumn Sep. 1993, 5, 361-367.
Table 1. Relationship between Total Column Efficiency (N, Plates) and Mobile-Phase Linear Velocity (u, cm s-1) under LC and SGC Conditionsa
Figure 4. Experimental relationship between reduced plate height and mobile-phase linear velocity in SGC, HTLC, and RTLC. Conditions: benzene used as test solute; other conditions and notations are the same as in Figure 3.
Figure 5. Experimental relationship between optimum mobile-phase linear velocity and temperature. Conditions are the same as in Figure 4.
the fused-silica capillary column was connected to a zero dead volume union using PEEK tubing to position a steel screen (2µm pores, Valco) to support the particles, and the other end of the union was left open to the atmosphere. The open end of the column was connected to a stainless steel vessel, into which microparticles were introduced. Packing was carried out at an initial pressure of 40 atm and at room temperature. The particles were packed into the column by gradually increasing the pressure while the column was submerged in an ultrasonic bath. At ∼120 atm, when the column was fully packed, it was conditioned at room temperature and ∼200 atm in the ultrasonic bath for 10 min. Finally, the column was allowed to depressurize slowly overnight. SGC and LC Experiments. A manual liquid injector (Valco) with a rotor volume of 0.06 µL was used for introduction of samples. The pump outlet was connected to the valve injector through a 50 cm × 0.1 cm i.d. preheated stainless steel tube in the oven. A tee was connected to the injector valve by using a 10 cm × 125 µm i.d. steel tube. A 15 cm × 9 µm i.d. fused-silica capillary was used as a split line, providing a split ratio of >100. The separation columns were connected to the tee using PEEK tubing. A 10 cm × 100 µm i.d. fused-silica capillary was used to connect the separation column and the UV detector. A detection window was made on this connection tubing, and the distance
LC (24 °C)
LC (60 °C)
u
N
u
N
SGC (140 °C) u
N
SGC (200 °C) u
N
0.07 0.08 0.10 0.11 1.12 1.14 0.15 0.16 0.17 0.18 0.19 0.20
132 000 144 000 144 000 142 000 136 000 127 000 113 000 109 000 104 000 99 000 97 000 94 000
0.11 0.12 0.14 0.15 0.18 0.19 0.20 0.21 0.22 0.24 0.25 0.26
146 000 148 000 144 000 136 000 127 000 123 000 113 000 109 000 108 000 102 000 98 000 97 000
0.20 0.21 0.22 0.23 0.26 0.29 0.32 0.36 0.38 0.40 0.42 0.44
125 000 141 000 147 000 146 000 137 000 125 000 120 000 115 000 111 000 108 000 104 000 102 000
0.34 0.34 0.37 0.40 0.43 0.46 0.49 0.52 0.55 0.57 0.60 0.61
114 000 125 000 137 000 144 000 137 000 134 000 130 000 126 000 124 000 121 000 120 000 118 000
a Conditions: benzene test solute; other conditions are the same as in Figure 2.
Figure 6. Relationship between solute retention factor and temperature in SGC and LC. Conditions: 400 atm column inlet pressure; (b) and ([) retention factors for 5,6,11,12-tetraphenylnaphthacene and 9,10-diphenylanthracene in acetonitrile mobile phase, respectively. Other conditions are the same as in Figure 2.
between the window and the column outlet was ∼1 cm. The UV detector was used at a wavelength of 254 nm with a time constant of 1 s. In order to obtain stable detection, the eluent was cooled at 24 °C in the 100 µm i.d. fused-silica connecting tubing between the column outlet and the detection window with circulating water. When using acetonitrile as the mobile phase and ODS particles as packing material, nitromethane was used as an unretained marker.22 All organic solvents used in the SGC experiments were HPLC grade (Fisher Scientific, Fair Lawn, NJ). RESULTS AND DISCUSSION SGC Using Mobile Phases That Transition from Liquid to Gas in the Column. Liquid vaporization is determined by the boiling point (Tb), which is affected by pressure (P). Little data are available for providing details about the relationships between Tb and P of organic solvents. The Frost-Kalkwarf(22) Engelhardt, H.; Mu ¨ ller, H.; Dreyer, B. Chromatographia 1984, 19, 240245.
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Figure 7. Fast SGC separations of large PAHs using various mobile phases. Conditions: 55 cm × 250 µm i.d. fused-silica capillary column packed with 5-µm polymeric ODS particles (200-Å pores), 300-atm column inlet pressure, and temperature program from 65 to 250 °C at 25 °C min-1. Mobile phases: (A) acetonitrile, (B) hexane, and (C) methanol. Other conditions are the same as in Figure 2.
Thodos vapor pressure equation23 was used to carry out approximate calculations in this study. Figure 1 shows the calculated results for acetonitrile, methanol, hexane, and methylene chloride. The critical temperatures of methylene chloride, methanol, acetonitrile, and hexane are 510, 513, 546, and 508 K,24 respectively, and the highest point in each curve in Figure 1 is the critical point of the solvent. For a solvent having high Tb, the applied pressure produces a significant influence on Tb. For example, a pressure of only 25 atm can increase the boiling point of acetonitrile to 200 °C. In this study, the column outlet was connected to a 10 cm × 100 µm i.d. capillary tube, on which a window was made for UV detection. The pressure drop produced by this tube was examined, and it was found that a pressure drop of 0.7 atm was produced when using a large flow rate of 200 µL min-1 of acetonitrile at room temperature. The actual flow rate through the column was less than 20 µL min-1 in this study, and the pressure drop produced by the connection tube to the detector could be neglected. The column outlet was opened to ambient (1 atm). Since pressure has a significant effect on the solvent boiling point, the pressure profile of the mobile phase along the column
is of critical importance in SGC. Currently, no accurate method for calculation of this profile for SGC is available, and a gross estimation must be made. For example, at 200 °C and at a column inlet pressure from 100 to 400 atm, 1/5-1/20 of the total column length contains acetonitrile vapor and the rest contains superheated liquid if the pressure changes linearly along the column length. Therefore, both liquid and gaseous mobile phases exist in the column. This chromatography can also be called SGC because solvating gaseous vapors exit the column. When the temperature is higher than the critical point of the mobile phase, the situation is properly considered to be SGC where the mobile phase change is from supercritical fluid to gas.1,2 Mobile-Phase Flow. At a specific column inlet pressure, the mobile-phase flow is determined by the mobile-phase viscosity (η). The value of η for liquid decreases with increasing temperature.25 While gas viscosity increases with temperature, the magnitude of these gaseous viscosities can be neglected compared to typical liquid viscosities.26 In SGC, because of the decreased viscosity of the superheated liquid coupled with the fact that part of the column length contains gaseous mobile phase, the mobilephase flow encounters much less resistance at elevated temperature.
(23) Reid, R. C.; Prausnitz, J. M.; Sherwood, T. K. The Properties of Gases and Liquids, 3rd ed.; McGraw Hill Book Co.: New York, 1977; p 188. (24) CRC Handbook of Chemistry and Physics, 77th ed.; CRC Press: Boca Raton, FL, 1996; pp 6-57.
(25) Giddings, J. C. Unified Separation Science; John Wiley & Sons: New York, 1991; Chapter 4. (26) Vargaftik, N. B. Handbook of Physical Properties of Liquids and Gases; Hemisphere Publishing Co., New York, 1983.
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Figure 8. Fast SGC separation of enantiomers. Conditions: 95 cm × 250 µm i.d. fused-silica capillary column packed with 5-µm silica particles (80-Å pores), 0.5% (w/w) permethylated β-cyclodextrin in methanol mobile phase, 380 atm column inlet pressure, and 140 °C. Other conditions are the same as in Figure 2.
Figure 2 shows the experimental relationship between column inlet pressure (Pi) and resultant mobile-phase linear velocity (u) at various temperatures. Since compressible mobile phases are formed in SGC experiments, the measured mobile-phase linear velocity is an average of all values along the column length. An excellent linear relationship was observed between u and Pi (linear regression coefficient larger than 0.999). Although liquid vapor was formed near the column outlet when the temperature was higher than the normal boiling point of the mobile phase, a smooth change in mobile-phase linear velocity was found at each specific column inlet pressure. Figure 3 shows experimental results of u as a function of 1/T at column inlet pressures of 200 and 400 atm. A rapid increase in mobile-phase linear velocity is obtained with increasing temperature. This results from the fact that both the decrease in liquid viscosity and the increase in the percentage of column length that contains gaseous mobile phase are gradual, smooth processes. Column Efficiency. When using mobile phases that change from liquid to gas in the column, there is a large mobile-phase linear velocity gradient as well as phase change along the column. It has been experimentally demonstrated that high column efficiency can be obtained in SGC, even when a large mobilephase gradient exists along the column as the mobile phase is changed from a supercritical fluid to a gas.2 Since the change from supercritical fluid to gas is usually smooth at elevated temperature, the influence of the phase change on the column efficiency is small and can be neglected. In this study, the mobile
phase was changed from liquid to gas, and the effect of this change on column efficiency was experimentally examined. Figure 4 shows the experimental relationship between h and u under SGC (140 and 200 °C) and LC (24 and 60 °C) conditions using acetonitrile as mobile phase. The minimum reduced plate height (hmin) was slightly increased from 1.7 to 1.8 when the temperature was increased from 60 to 200 °C. This indicates that neither the large mobile-phase linear velocity gradient nor the phase change from superheated liquid to gas along the column has a significant influence on column efficiency in SGC. Results from a previous study showed that when the same packed capillary column was used in LC or SFC, no significant difference in hmin existed; however, an obvious increase in hmin was found when the column was used under GC conditions.27 When packed columns containing microparticles are combined with solvating liquids or supercritical fluids, SGC can provide high efficiency. The column efficiency generation rate is influenced significantly by the diffusion of solutes in the mobile phase. The diffusion coefficient of the solute in the mobile phase (Dm) depends on temperature. Increasing the temperature can increase the diffusion coefficient of a substance in a liquid,28 which improves the plates/time. The optimal mobile-phase linear velocities (uopt) in Figure 4 were 0.085, 0.124, 0.234, and 0.398 cm s-1 at 24, 60, 140, and 200 °C, respectively. These data show that an increase in uopt exists with increasing temperature (Figure 5). It was found that the rate of decrease in total column efficiency (N) in SGC became smaller compared to LC when u was larger than uopt. This can be expected because, at high u, N is greatly affected by the mass-transfer resistance of solutes in the mobile phase. Table 1 shows the experimental relationships between N and u at various temperatures. More than 100 000 total plates were obtained when using a mobile-phase linear velocity of greater than 0.6 cm s-1 in SGC at 200 °C, while a mobile-phase linear velocity of less than 0.25 cm s-1 was required for LC at 60 °C to achieve this efficiency. Increasing the temperature decreased the time needed to obtain high efficiency. The rate of loss in column efficiency, (∂N/∂u)T, with increasing mobile-phase linear velocity was analyzed using the last five points for each condition in Table 1. Values of -81 799, -152 850, -269 111, and -382 110 plates s cm-1 were obtained for 200 (SGC), 140 (SGC), 60 (LC), and 24 °C (LC), respectively. Column efficiency losses resulting from an increase in linear velocity were reduced more than 4 times when the temperature was increased from 24 to 200 °C. Although the column efficiency generation rate was improved when using mobile phases that were changed from liquids to gases in the column, the plates per unit time were still less than achieved using mobile phases that were changed from supercritical fluids to gases using the same packed capillary column.2 Solute Retention. Increasing the temperature influences the interactions between solute, mobile phase, and stationary phase in chromatography. It has been shown that when the temperature is increased in LC, the retention factor decreases.10 However, when liquid mobile phases vaporize, the retention behavior of solutes need to be examined. In this study, large polycyclic aromatic hydrocarbons (PAHs) were used as test solutes. (27) Shen, Y.; Lee, M. L. Chromatographia 1997, 46, 537-544. (28) Wilke, C. R.; Chang, P. Am. Inst. Chem. Eng. J. 1955, 1, 264-270.
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Figure 6 shows experimental relationships between k and T for 9,10-diphenylanthracene and 5,6,11,12-tetraphenylnaphthacene. Increasing the temperature decreased the retention factors of the solutes even at temperatures higher than the normal boiling point of the mobile phase. An approximately linear relationship between Ln k and 1/T was observed, and this relationship remained at temperatures higher than the normal boiling point of the mobile phase. The solvating power of a superheated organic solvent and its vapor can be used to carry out fast separations of large molecules. Figure 7 shows fast SGC separations of large PAHs using various organic solvents as mobile phases. Hexane provided the best column efficiency and peak shapes for the test solutes. A poor dibenzo[fg,qr]pentacene peak was obtained when methanol was used as mobile phase. At room temperature, the test solutes were eluted from the column in approximately 50 min when neat acetonitrile, methanol, or hexane was used as mobile phase. Methylene chloride was also examined, and it was found that the strong solvating power of the mobile phase made it difficult to separate the test solutes because they were only slightly retained, even at room temperature. The high solvating power of superheated liquid and vapor mobile phases allows the use of additives to achieve special separations. Figure 8 shows a fast SGC separation of enantiomers using permethylated β-cyclodextrin as an additive in methanol. An excellent separation was achieved. Because of the high solvating power of superheated liquid-tovapor mobile phases, it is hard to use them for the separation of small molecules. By using highly retaining and selective station-
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ary phases and by diluting the mobile phase with low-polarity solvents such as carbon dioxide or methyl chloride, this problem can be greatly reduced. Column Stability and Detection. An upper temperature of 240 °C was applied when acetonitrile or hexane was used as mobile phase and 150 °C for methanol mobile phase. No column efficiency and separation losses were observed for more than six weeks of SGC experiments. Baseline noise problems were encountered when UV detection was used in this study. Low temperatures can be used to liquefy the vapor for favorable detection. We examined the effect of UV detector temperature on the detector noise. When the temperature was lower than the normal boiling point of the mobile phase, a satisfactory baseline was obtained. At the boiling point, the noise became significant. When the column temperature was lower than 220 °C, the circulating cooling water controlled the temperature below the normal boiling points of the mobile phases tested. However, when high temperature (e.g., higher than ∼250 °C) and large mobile-phase linear velocity (e.g., larger than ∼1 cm s-1) were used, a desirable baseline could not be obtained. The UV detector is not really suitable for SGC when liquid-to-vapor mobile phases are used. Other detection methods such as thermal conductivity should be evaluated.
Received for review June 26, 1997. Accepted November 17, 1997. AC970672G
