Anal. Chem. 2000, 72, 1253-1262
High-Temperature Ultrafast Liquid Chromatography Bingwen Yan,† Jianhong Zhao,† James S. Brown,‡ John Blackwell,§ and Peter W. Carr*.†
Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minnesota 55455, Department of Chemical Engineering, Georgia Institute of Technology, 778 Atlantic Drive, Atlanta, Georgia 30332, and Dupont Pharmaceuticals Company, Chemical Process Research and Development, Chambers Works, PRF-1, Deepwater, New Jersey 08023
A novel liquid chromatographic system which enables high temperature ultrafast liquid chromatography (HTUFLC) has been designed through the careful consideration of heat transfer, band broadening, and pressure drop. Studies of the effect of linear velocity on the HETP show that column efficiency at high velocity, especially of wellretained solutes, dramatically improves at higher temperatures. At 150 °C, at a flow rate of 15 mL/min with a 5 cm by 4.6 mm (i.d.) column packed with 3 µm polystyrenecoated zirconia porous particles, long chain alkylphenones were completely resolved, and the analysis time could be decreased by a factor of 50 compared to that at room temperature (25 °C) at a conventional flow rate (4 mL/ min). In addition, using pure water as the mobile phase, five phenols were separated in less than 30 s. In high performance liquid chromatography (HPLC), most separations are optimized by first selecting the most appropriate type of stationary and mobile phase.1 Subsequently, resolution is optimized by adjusting the volume fraction of strong solvent or pH. Until recently temperature has been neglected as a variable during HPLC method development.2 This is in large part due to the fact that the mobile phase composition is easily adjusted and has a greater effect on selectivity than do small changes in temperature.3 Large changes in temperature have only recently been investigated, as most silica-based phases are thermally unstable and can only be used at temperatures only marginally higher than ambient.3,4 This allows for a maximum increase in temperature of only 30-40 °C above room temperature. This corresponds to an absolute temperature change of less than 12%. On the basis of the seminal contribution of Horvath and Antia,5 it should be clear that analysis time in HPLC can be considerably improved by using much higher temperatures. Recently, a number of thermally stable silica-based phases were developed for the chromatographic separation of large molecules at elevated temperature.2,5,6 It is well-known that temperature has †
University of Minnesota. Georgia Institute of Technology. § Dupont Pharmaceutical Co. (1) Snyder, L. R.; Glajch, J. L.; Kirkland, J. J. Practical HPLC Method Development, 2nd ed.; John Wiley & Sons: New York, 1997. (2) Chen, H.; Horvath, C. J. Chromatogr. 1995, 705, 3-20. (3) Kikta, E. J.; Grushka, E. Anal. Chem. 1976, 48, 1098-1104. (4) Colin, H.; Diez-masa, J. C.; Guiochon, G. J. Chromatogr. 1978, 167, 41-65. (5) Antia, F. D.; Horvath, C. J. Chromatogr. 1988, 435, 1-15. ‡
10.1021/ac991008y CCC: $19.00 Published on Web 02/19/2000
© 2000 American Chemical Society
a greater effect on the retention, efficiency, and selectivity of large solutes than it has on small solutes.2,5,6 Hancock et al.7,8 studied the selectivity of peptides and proteins on “sterically protected” C8 and C18 columns by varying mobile phase composition and temperature. They found that the use of high temperature (85 °C) can greatly improve selectivity. Horvath et al.2,9,10 used pellicular stationary phases to separate proteins at 120 °C. Over the past few years, high stability zirconia-based stationary phases, such as polybutadiene-11-13 and polystyrene-coated14 zirconia RPLC supports, have been developed in this laboratory. A few studies of the efficiency, selectivity, and thermodynamic properties of polybutadiene-coated zirconia (PBD-ZrO2) at high temperature have appeared. Separation times on the order of seconds were achieved.11-14 High-temperature ultrafast liquid chromatography (HTUFLC) requires precise temperature control, the ability to generate a high flow rate, and, most importantly, a stationary phase stable enough to withstand the harsh conditions. Conventional HPLC systems are limited to temperatures of about 80 °C and flow rates of about 5 mL/min. To design a high-quality HTUFLC system, it is absolutely necessary to preheat the mobile phase before it enters the column to avoid unacceptable peak broadening.2,10,15 This requires the estimation or measurement of the system parameters affecting heat transfer. It is well-known that at high temperature thermostating only the column and not the eluent is inadequate.16 A significant difference (>5 °C) in temperature between the incoming eluent and the column greatly degrades column efficiency and causes severe band broadening and peak splitting.15,17 This is probably due to radial temperature gradients and therefore radial retention and viscosity gradients in the column. It is also possible that it results from viscous fingering.18,19 If the entering (6) Ooms, B. LC-GC International 1996, 9, 574-585. (7) Hancock, W. S.; Chloupek, R. C.; Kirkland, J. J.; Snyder, L. R. J. Chromatogr. 1994, 686, 31-43. (8) Chloupek, R. C.; Hancock, W. S.; Marchylo, B. A.; Kirkland, J. J.; Boyes, B. E.; Snyder, L. R. J. Chromatogr. 1994, 686, 45-59. (9) Chen, M. H.; Horvath, C. J. Chromatogr. 1997, 788, 51-61. (10) Chen, H.; Horvath, C. Anal. Methods Instrum. 1993, 1, 213-222. (11) Li, J. W.; Carr, P. W. Anal. Chem. 1997, 69, 837-43. (12) Li, J. W.; Carr, P. W. Anal. Chem. 1997, 69, 2193-2201. (13) Li, J. W.; Hu, Y.; Carr, P. W. Anal. Chem. 1997, 69, 3884-8. (14) Zhao, J.; Carr, P. W. Anal. Chem. 1999, 71, 5217-5224. (15) Poppe, H.; Kraak, J. C. J. Chromatogr. 1983, 282, 399-412. (16) Schrenker, H. J. Chromatogr. 1981, 213, 243. (17) Rozing, G. R.; Goetz, H. J. Chromatogr. 1989, 476, 3-19. (18) Castells, R. C.; Castells, C. B.; Castells, M. A. J. Chromatogr. 1997, 775, 73-79.
Analytical Chemistry, Vol. 72, No. 6, March 15, 2000 1253
mobile phase temperature is significantly lower than the column temperature, the fluid temperature will be higher near the wall than it is at the center of the column. The higher temperature near the wall will result in lower retention and viscosity which, in turn, will result in an increased solute zone velocity near the tube wall. The differences in the solute velocities at the center and near the wall of the column cause peak broadening and splitting. We also need to limit the problems caused by the rather considerable length of tubing needed to bring the mobile phase up to the column temperature. These include the extracolumn peak broadening20 and increased system back pressure21 (see below). One of the important putative benefits of the use of high temperature is ultrafast separation. Traditional HPLC methods often require from 5 min to 15 or 20 min for even simple analyses. While instrumentation and control are more challenging, the potential advantages of HTUFLC are as follows: for most solutes, analysis time can be reduced at elevated temperature because the enthalpy of solute transfer from the mobile to the stationary phase is favorable in RPLC. As a result, retention decreases as temperature is increased, and many separations can be completed in less than 30 s at high temperature and high flow rate.22,23 In addition, as temperature is increased, the viscosity of the eluent decreases and thus the system back pressure is greatly reduced.4 These advantages of HTUFLC expand the range of applicability of traditional HPLC to areas requiring fast analysis and high sample throughput, including process control, clinical chemistry, and online monitoring of chemical reactions.24 Another important benefit of HTUFLC is that water can sometimes be used as the sole eluent, thereby mitigating many problems, including the toxicity, flammability, and cost related to the use of organic solvents. Smith et al.25,26 used water as the sole eluent at 200 °C to separate phenols and barbiturates on a polystyrene-divinylbenzene polymeric phase. Ingelse27 also studied the separation of alcohols at high temperature using water as the eluent and a flame ionization detection in conjunction with conventional octdecylsilane-silica and carbon columns. Here we present the details of a novel design of HTUFLC system, in which the extent of heat transfer was calculated a priori and the effect of extracolumn tubing on efficiency was minimized. We also evaluate the effect of temperature on the chromatographic efficiency and selectivity for a series of alkylphenones. Finally, an ultrafast separation of some phenolic pollutants was achieved at high temperature using pure water as the mobile phase. EXPERIMENTAL SECTION Reagents. All reagents were purchased and were reagent grade or better. The mobile phase contained HPLC grade acetonitrile (ACN) from Mallinckrodt (Paris, KY). Deionized water (19) Broyles, B. S.; Shalliker, R. A.; Cherrak, D. E.; Guiochon, G. J. Chromatogr. 1998, 822, 173-187. (20) Martin, M.; Eon, C.; Guiochon, G. J. Chromatogr. 1975, 108, 229-241. (21) Mortimer, R. G. Physical Chemistry; The Benjamin/Cummings Publishing Co. Inc. Redwood City, CA, 1993. (22) Olson, K. C.; Gehant, R. L. Biotechnol. Prog 1992, 8, 562. (23) Mant, C.; Hodges, R. HPLC of Peptide and Proteins: Analysis and Conformation; CRC Press: Boca Raton, FL, 1990. (24) Paliwell, S. K.; Nadler, T. K.; Regnier, F. E. TIBTECH 1993, 11, 95. (25) Smith, R. M.; Burgess, R. J. J. Chromatogr. A 1997, 785, 49-55. (26) Smith, R. M.; Burgess, R. J. Anal. Commun. 1996, 33, 327-329. (27) Ingelse, B. A.; Janssen, H. G.; Cramers, C. A. J. High Resol. Chromatogr. 1998, 21, 613-616.
1254 Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
Figure 1. The chromatographic system: (1) solvent reservoir, (2) pumps, (3) injector, (4) “T”, (5) stainless steel tubing, (6) heating bath, (7) heat exchanger, (8) low dead volume “T”, (9) in-line filter, (10) analytical column, (11) cooling bath, (12) cooling tubing, (13) UV detector, (14) data system, and (15) back pressure adjuster.
was filtered through a 0.45 µm filter (Gelman Science Inc., Ann Arbor, MI) and then boiled to remove carbon dioxide. All solvents were filtered with a 0.45 µm filtration disk and sonicated to remove air bubbles before use. Alkylphenones (acetophenone, octanophenone, decanophenone, dodecanophenone, and tetradecanophenone) and phenolic compounds (phenol, 2-methylphenol, 4-ethylphenol, 2,4.6-trimethylphenol, and 4-tert-butylphenol) used in this study were purchased from Aldrich (Milwaukee, WI). Deuterium oxide (D2O) (CIL Cambridge Isotope Laboratories, Woburn, MA) was used to determine the dead volume. HTUFLC System. The chromatographic system used here is shown in Figure 1. It consists of three Altex pumps (Model 110A, Altex Scientific, Inc.), a valve injector, a homemade column thermostat, a HP1090 UV detector, and a HP-3394 data system (Hewlett-Packard, Wilmington, DE). The system was designed to be thermostated in such a fashion as to minimize band broadening. The eluent is delivered from the reservoir by two paths using three pumps (see Figure 1). One path (the high flow path) is operated at high flow rate (>10 mL/min) and the eluent (not the sample) is passed through a single large heat exchanger made from 2.5 m of wide inner diameter stainless tubing [1/16 in. (o.d.) × 0.03 in. (i.d.), Alltech Associates Inc., Deerfield, IL] with a 5 cm short section of narrow-bore tubing [1/16 in. (o.d.) × 0.004 in. (i.d.)] at the end. The sample delivery path (the low flow path) is typically operated at low flow rate (1 mL/min). The eluent is passed through the injector and a small heat exchanger made from a 10 cm length of very narrow-bore stainless tubing [1/16 in. (o.d.) × 0.004 in. (i.d.)]. The two paths are combined at a very low dead volume “ T ” (U-428, Upchurch Scientific, Inc.) and the eluent then goes through a high pressure in-line filter (Model 7335, Alltech Associates Inc., Deerfield, IL). The analytical column and the above heat exchangers were placed in a silicone oil bath that was thermostated to (1 °C. Since the UV detector cannot tolerate high temperature, the hot eluent coming from the column outlet must be cooled by use of an ice bath with a 20 cm length of narrow-bore tubing [1/16 in. (o.d.) × 0.004 in. (i.d.)]. The mobile phase was then passed through a UV detector equipped with a back pressure regulator at its outlet. The back pressure is needed to prevent the mobile phase from boiling at higher temperatures. Usually 20 bar back pressure is sufficient to prevent mobile phase boiling. As the flow rate was changed, the back pressure regulator was adjusted appropriately. Note that the entire HTUFLC system must be leakfree for obvious safety reasons.
Polystyrene-Coated Zirconia (PS-ZrO2) Stationary Phase. Bare zirconia (ZrO2) was made in this lab using the PICA synthesis method28 (Batch PICA-7). The particle size is 2.5 µm; the specific surface area is 30.7 m2/g (by nitrogen sorptometry). The PS-ZrO2 stationary phase was made in this laboratory. Its preparation and characterization have been reported elsewhere.14 The carbon content of the PS-ZrO2 used in this study was 2% (w/w). We packed these PS-ZrO2 particles into a 50 mm × 4.6 mm (i.d.) stainless steel column (Alltech Associates Inc., Deerfield, IL) using the upward stirred slurry method. 2-Propanol was used as the pushing solvent and the slurry solvent was 50/50 (v/v) 2-propanol/tetrahydrofuran. The packing process has not been fully optimized as yet. Chromatographic Conditions. Mixtures of alkylphenones were separated using different mobile phase compositions [40%, 35%, 30% and 25% ACN (v/v)], at different temperatures (40, 80, 120, and 150 °C), and at different flow rates (4, 8, 10, and 15 mL/ min). The solute concentrations were all 5 mg/mL. The volume injected was typically 2 µL, and the UV detector wavelength was set to 254 nm. The separation of the phenolic compounds was carried out using pure water as the mobile phase at 120 °C. To determine the effect of temperature on column efficiency, plots of plate height (H) vs interstitial linear velocity (ue) at four different temperatures (25, 80, 120, 150 °C) were analyzed. The mobile phase compositions (see Table 5) were adjusted at each temperature to keep the retention factors (k′) in nearly the same range (between 0 and 10). Computation of the Plate Height and Linear Velocity. The plate number was calculated as
N ) 6.28
(tR - tex)2 WAR/AH2 - Wex2
(1)
where tR is the retention time of the solute, and WAR/AH is the area-to-height based peak width for the solute as reported by the data system. Wex and tex are the extracolumn contributions to the peak width and retention time, respectively. Wex and tex were obtained from measurements of the peak produced by acetone and D2O without the column in place and were read from a highspeed chart recorder, respectively. The interstitial linear velocity (cm/s) was computed as
ue )
F 60eπRc2
(2)
where F is the flow rate (mL/min), e is the external porosity (taken as 0.38 for our column), and Rc is the column radius (0.23 cm). Finally, we fitted the data to the Knox equation using Sigma plot (Jandel Scientific Inc., San Rafael, CA) to obtain the A, B, and C coefficients. A program written in Excel (Microsoft 95) was used to calculate the temperature as a function of axial position in the cooling system in order to estimate the average viscosity and diffusion coefficient (see below).
Figure 2. Schematic of one-dimensional heat flow into the mobile phase through a hollow cylinder immersed in a hot fluid.
and thermally stable stationary phase. As described in the introduction, PS-ZrO2 fulfills these requirements. Homemade columns packed with PS-ZrO2 possess excellent thermal stability, good efficiency, and uniformity.14 Good temperature control is necessary and can be achieved by the use of thermostated oil bath. Control of temperature gradients due to the thermal resistance to heat transfer is also necessary and can be achieved by the proper combination of system variables and plumbing configuration. Poppe et al.15 and others29 studied heating effects on peak broadening; their results15 show that column efficiency in a liquid bath is much better than in an air bath because heat transfer through a liquid is much better than through air. The need for a liquid bath instead of an air bath is particularly severe at high flow rates. Even the simplest calculations show that much longer equilibration tubes are needed in an air bath. Whenever there is a significant temperature difference (5 °C) between the incoming mobile phase and the column wall, peak broadening results. To minimize the temperature difference and prevent peak broadening in the column, the mobile phase must be preheated from room temperature to a temperature close to that of the column wall. To solve this problem, we carefully estimated the heat transfer rate between the flowing eluent and the silicone oil bath and designed our system accordingly. As shown in Figure 2, the tubing in the heating bath is modeled as a hollow cylinder with an inner radius, r1; outer radius, r2; and length, L. Assuming that the heat capacity (Cp) is relatively independent of temperature, the heat input rate required to raise the temperature of flowing mobile phase from the inlet temperature (Tin) to the outlet temperature (Tout) is
q˘ ) m˘ Cp(Tout - Tin)
(3)
RESULTS AND DISCUSSION Heat Transfer of the Chromatographic System. The practical application of HTUFLC requires a mechanically, chemically,
m˘ is the mass flow rate of mobile phase and Cp is the mean specific heat of the fluid. In a differential element of length dx (see Figure
(28) Sun, L.; Annen, M.; Lorenzano-Porras, C. F.; Carr, P. W.; McCormick, A. J. Colloid Interface Sci. 1994, 163, 464-73.
(29) McCown, S. M.; Southern, D.; Morrison, B. E.; Garteiz, D. J. Chromatogr. 1986, 483-492.
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1255
2), the thermal resistance (Rth) is given by the following equation30
()
r2 r1 1 1 + + Rth ) 2πr1h1 dx 2πκw dx 2πr2h2 dx ln
(4)
where h1 and h2 are the convective heat transfer coefficients between the mobile phase and the tube inner wall, and between the oil bath and the tube outer wall, respectively. κw is the thermal conductivity of the stainless steel tubing wall. The first term in eq 4 represents the overall average resistance to convective heat transfer from the inner wall of the tubing to the bulk mobile phase, the second term is the resistance to conductive heat transfer through the tube wall, and the third term is the resistance to convective heat transfer from the oil bath to the outer wall of the tubing. For a constant heat flux through the tube wall and under laminar flow, the inner convection coefficient, h1, can be calculated as follows:30
h1 ) 2.182
κm r1
(5)
where κm is the thermal conduction coefficient of the mobile phase. Substituting eq 5 into eq 4, we obtain:
()
r2 r1 1 1 Rth ) + + 4.634πκm dx 2πκw dx 2πr2h2 dx ln
(6)
Note that under the conditions of laminar flow, the inner radius of the tubing, r1, appears only in the second term, which represents the resistance to heat transfer through the tube wall. As long as the tube wall is thin, and the conduction coefficient of the tube wall, κw, is large, the thermal resistance to heat transfer through the tube wall will be small compared to the two convective resistances. In this case, as shown in Table 1, the length of the tubing required to achieve thermal equilibrium will be almost independent of the tubing inner radius (r1). The differential heat flow rate, dq˘ , along a very short length of the flow system can be written as an equation similar to that of Ohm’s law in electric-circuit theory,
q˘ x - q˘ x+dx ) m˘ Cp (Tx - Tx+dx) )
T x - TB Rth
(7)
where Tx and TB represent the temperatures at the axial position x in the mobile phase and the silicone oil bath, respectively. If, we define R′th as
R′th ) Rth dx and dT ) Tx+dx - Tx, then (30) Holman, J. P. Heat Transfer, 7th ed.; McGraw-Hill: New York, 1990.
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Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
(8)
2π dx dT )Tx - TB m˘ CpR′th
(9)
Integration is straightforward
(
ln
)
Tin - TB 2πL ) Tout - TB m˘ CpR′th
(10)
On the basis of eq 10, if we know the temperatures of the silicone oil (TB) and the inlet (Tin) and outlet (Tout) of the eluent heating tube, then the required length (L) of tubing can be calculated for different flow rates and tubing diameters (see Table 1). Here water is assumed to be the fluid being heated to the outlet temperature (Tout). Note that the temperature of the mobile phase approaches the desired set point asymptotically. Table 1 shows that very long tubes are needed to heat the mobile phase from 25 °C to a temperature 5 °C below the oil bath temperature. The higher the flow rate, the longer the tube needed. Even when 0.004 in. diameter tube is used, the tubing length is rather long. Initially it appeared to us that high back pressure and especially the peak broadening associated with the use of the long tubing would make it difficult to achieve ultrafast chromatography at very high flow rates and high temperatures. We overcame these problems by configuring the system so that most of the mobile phase was heated before it entered the column by using the two-path system described in the Experimental Section. Although this new design results in sample dilution by the flow ratio, it does minimize the precolumn band broadening induced by the lengthy tubing needed to thermally equilibrate the mobile phase to the desired temperature. More importantly it allows us to test the basic concepts of this work and especially allows us to examine the effect of temperature on the column efficiency. Equation 10 was also used to calculate temperature as a function of axial position in the cooling system. Figure 3 shows the results. If the column effluent is at 150 °C, then at a flow rate of 15 mL/min, 20 cm of tubing is needed to allow the eluent to cool to 40 °C. We also use Figure 3 to estimate the average axial viscosity and solute diffusion coefficient at any temperature and flow rate in the cooling system (see below). To study the effect of flow ratio of the two pathways on the peak shape at high temperature and high flow rate, we maintained the total flow rate at 13 mL/min at 150 °C and changed the flow ratio. The inlet temperature at the “T” of the mobile phase from the low flow path can be calculated via eq 10. The temperature of the mobile phase from the high flow path is 150 °C since the associated tubing is long enough to fully preheat the mobile phase from 25 to 150 °C. Table 2 shows the temperature computed at the “T” at different flow ratios. It shows that the peak broadening, especially for well-retained solutes, will result when the temperature difference between the column inlet and the mobile phase exceeds about 1.4 °C.15 Figure 4 shows a series of chromatograms at different flow rate ratios. We can see that the flow ratio affects the peak shape, especially of the well-retained solutes. This clearly shows that a significant difference in temperature between the incoming mobile phase and the column can cause severe band broadening and peak splitting. There is no peak broadening at a
Table 1. Computed Effect of Flow Rate and Tubing Diameter on the Minimum Length of Tubing Needed To Achieve Thermal Equilibriuma
tubing inner diameter (in.) 0.004 0.007 0.010
required tubing length (cm) at various flow rates (mL/min) 5 10 15 34 34 33
68 67 66
103 101 99
a Based on eq 10. The parameters are as follows. Temperatures: heating bath, 200 °C, inlet and outlet of fluid in the tubing, 25 and 195 °C, respectively. The fluid properties are as follows: mean specific heat of water, Cp ) 4.323 J/g °C; dynamic viscosity, 0.1 cP; density, 0.928 g/mL. Thermal conductivity of stainless tubing, κw ) 16.3 W/m °C. Equations for the convective heat transfer coefficients can be found in the work of Holman.27 The inner convection coefficient, h1, varies with tubing inner diameter but is independent of flow rate: h1 (0.004 in.) ) 25 772 W/m2 °C, h1 (0.007 in.) ) 14 727 W/m2 °C, h1 (0.010 in.) ) 10 309 W/m2 °C. h1 is assumed to be in the laminar region even at high Reynold’s number. The external convection coefficient, h2, was held constant at a value of 1442 W/m2 °C.27
Table 2. Computed Temperature of the Column Eluenta as a Function of Flow Rate Ratio flow rate (mL/min)
temp (°C)
F1
F2
T1b
T3c
temp difference (°C)d
1 2 3 4 5
12 11 10 9 8
149 141 128 116 106
150 149 145 140 133
0.1 1 5 10 17
a Results are computed by assuming the system described in the Experimental Section. The heating bath temperature is 150 °C. b This is the temperature of the fluid carrying the sample at the entrance to the mixing “T”. It is computed from eq 10 using the flow rate F1. c This is the computed temperature at the exit of the mixing “T” in Figure 1. The fluid at flow F2 is assumed to be at 150 °C and the fluid at flow F1 is at T1. d This is the computed difference in temperature between the column set point (150 °C) and T3, the temperature of the combined fluid flows into the column.
Figure 3. Calculated column effluent temperature vs axial position. All conditions are described in the Experimental Section. The cooling bath is assumed to be at 0 °C.
solute flow rate of 1 mL/min because the temperature difference between fluid and column is small (see Table 2). In all subsequent work we choose 1 mL/min as the upper limit for the flow rate in the sample delivery path. Extracolumn Band Broadening. Band broadening takes place not only in the column but also in the injector, connecting tubing, and the detector.20 The total variance (σtotal2) of a chromatographic peak is the sum of all possible band broadening processes:
σtotal2 ) σc2 + σinj2 + σt2 + σd2
(11)
σc2, σinj2, σt2, and σd2 represent the variance of the column, injection tubing, and detector, respectively. Dispersion in the injector and detector are to a first approximation independent of flow rate. The peak variance in volume units due to the injector depends on the volume injected and the type of injector, and the variance from the detector depends on the volume of the detector cell and the detectors response time. Band broadening in the connecting tubing depends on the flow rate, the tubing length and its inner
Figure 4. Chromatograms showing the effect of flow ratio on the peak shape. The solutes are alkylphenones. Experimental conditions: mobile phase is 20% ACN (v/v) and total flow rate is 13 mL/ min at 150 °C. F1 and F2 are the flow rates in the low and high flow paths, respectively.
diameter, the temperature of the eluent, and the difference in temperature between the tubing wall and the center of the tube (see below). The key point is to keep the three geometrical parameters at a minimum so that peak dispersion in the tubing is minimal. When the flow rate is very high, σinj2 and σd2 will be negligible. Here we only discuss the effect of the connecting tubing on extracolumn broadening. As discussed above, in HTUFLC a substantial amount of tubing is needed to heat the eluent before the column and then cool it before the detector. According to the Golay equation,31 we assume that the broadening that takes place in the connecting tubing can Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
1257
be described by the equation 2
H)
r1 u 2Dm + u 24Dm
(12)
σt2 ) (πrl2)2LH
(13)
where L is the length of the tubing. Assuming a nearly Gaussian peak:
( ) πr12LF 24Dm
(14)
A number of correlations are available for estimating diffusion coefficients.32-35 The solute diffusion coefficients in this study were calculated by the Wilke-Chang method,36
D (cm /s) ) 7.40 × 10-8 2
xψBMWBT ηVA0.6
(15)
where A and B denote the solute and mobile phase, respectively. VA is the molar volume (mL/mol) of the liquid solute at its normal boiling point and can be calculated by a group contribution approach.33 η is the mobile phase viscosity (cP), T is the temperature (K), ψB is the solvent association factor, and MWB is the molecular weight of the mobile phase (g/mol). (31) Golay, M. J. E., Gas Chromatography; Butterworth: London, 1958. (32) Scheibel, E. G. Ind. Eng. Chem 1954, 1954, 2007-2008. (33) Reddy, K. A.; Doraiswamy, L. K. Ind. Eng. Chem. Fundam 1967, 6, 77. (34) Lusis, M. A.; Ratchiff, G. A. Can. J. Chem. Eng. 1968, 46. (35) Hayduk, W.; Landie, H. AICHE J. 1974, 20, 611-615. (36) Wilke, C. R.; Chang, P. AICHE J. 1955, 1, 264-270.
1258
w1/2 (µL) at various F (mL/min) 5 10 15
tubing inner diameter (in.)
where H is the plate height, r1 is the inner radius of the tubing, Dm is the diffusion coefficient of the sample in the mobile phase, and u is the average linear velocity of the eluent. At any practical linear velocity in LC and especially at high flow rate, the second term is totally dominant; thus, we will neglect the first term. We caution that eq 12 is based on a number of assumptionssan isothermal, incompressible, Newtonian fluid in fully developed laminar flowsthat are keys to its validity.31 It is very significant that the eluent in the thermal equilibrium tubing is not in thermal equilibrium in either the axial or radial directions. This means that the diffusion coefficient in eq 12 is not independent of axial position. This problem is addressed below in some detail. Additionally, the radial temperature gradient introduces a very complex effect; namely, it generates a radial viscosity gradient. This means that in the heating section the fluid near the wall has a lower viscosity than the fluid in the center of the tube. In turn, this means that the linear velocity near the wall will be commensurately higher than it otherwise would be, thereby counteracting the Aris-Taylor dispersion phenomenon which broadens the zone. This is a very complex effect which we consider to be beyond the scope of the present work. The variance in volume units due to the tubing can be obtained from the following equation
w1/22 ) 5.54
Table 3. Computed Effect of Flow Rate and Tubing Diameter on Estimates of the Extracolumn Broadeninga in the Heating and Cooling Tubing
Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
0.004 0.007 0.01
10 (6)b 30 (17) 61 (35)
13 (8) 39 (23) 70 (47)
15 (9) 45 (27) 94 (54)
a For 40 cm of tubing, 20 cm of which is in the heating bath and 20 cm is in the cooling bath. The mobile phase is 25% ACN (v/v). Acetophenone was used as solute. Based on eq 16, in the heating bath, the parameters are Lt ) 20 cm, Dm ) 5.9 × 10-5 cm2/s at 150 °C obtained by eq 15;33 (33); in the cooling bath, Lt ) 20 cm, Dm was obtained by eq 17, so Dave is 2.6 × 10-5 cm2/s at 5 mL/min, 2.9 × 10-5 cm2/s at 10 mL/min, and 3.2 × 10-5 cm2/s at 15 mL/min. b The number in parentheses is the estimated peak half-width in the cooling system.
The viscosity of acetonitrile-water mixtures at different temperatures was estimated from the following equation, developed by Grunberg and Nissan37 and modified by Chen and Horvath10
[(
ηφ,T ) exp φ -3.476 +
726 1566 + (1 - φ) -5.414 + + T T 929 φ(1 - φ) -1.762 + (16) T
)
(
)
)]
(
where φ is the volume fraction of acetonitrile in the mobile phase and T is the temperature (K). The viscosity at any temperature and flow rate can be estimated from eq 16 and Figure 3 (see Table 3). We calculated the theoretical extracolumn peak broadening of the system based on a total of 40 cm of narrow-bore tubing [0.004 in. (i.d.)], where 20 cm of tubing is used in the heater and 20 cm is used in the cooling system. Because only a small fraction of the total extracolumn broadening takes place in the narrow, 20 cm tube from point a to b in Figure 1, we simply assumed a temperature of 150 °C and estimated a diffusion coefficient of 5.9 × 10-5 cm2/s based on eq 15. Broadening in this section is small because the flow rate is low (1 mL/min). We assume a temperature of 150 °C from the “T” to the column based on the calculations in Table 2. To accurately estimate the broadening in the postcolumn cooling system, we need to estimate the average diffusion coefficient in the tubing. We do so by use of the following equation and the rectangle rule for numerical integration:
Dave )
1 Tin - Tout
∫ D(T) dT ≈ ∑ T
D(T)∆T in - Tout
(17)
D(T) can be estimated from eqs 15 and 16 and Figure 3. Tin is the temperature of the eluent exiting the column (150 °C). Tout is the temperature of the fluid exiting the cooling system estimated by eq 10. D(T) and Tout change with the flow rate and temperature. The results are shown in Table 3. The average diffusion coefficient is then used in eq 14 to compute the tubing contribution to the peak half-width. (37) Grunberg, L.; Nissan, A. H. Nature 1930, 125, 309.
Table 3 shows the theoretical estimates for peak half-width due to the heating and cooling sections at different flow rates and different tubing diameters using acetophenone as the test solute and 25% ACN (v/v) as the eluent. It is evident that wide-bore tubing (0.01 in.) causes more severe band broadening than does narrow-bore tubing (0.004 in.). Clearly narrow-bore tubing must be used when designing a system for fast LC. However, the use of narrow-bore tubing significantly increases the back pressure, which seriously contravenes our desire to achieve the fastest possible separation. The calculation shows that more broadening takes place in the cooling system than in the heating section. Limitations to High Flow Rate. The viscosities of the mobile phases used in RPLC decrease very significantly as temperature is increased (see eq 16). For example, for a temperature change from 25 to 200 °C, eq 16 predicts that the viscosity of acetonitrile decreases 3-fold and that of water decreases 8-fold. Consequently, the system pressure drop decreases markedly and high flow rates are attainable within the pressure limits of conventional pumps. Therefore, at high temperature it should be possible to achieve much faster separations by use of high flow rates. However, as the flow rate is increased, back pressure will increase correspondingly and thus the real challenge in doing ultrafast chromatography is to overcome the back pressure created by the high flow rates. The back pressure of the packed column can be estimated by combining Darcy’s equation for the velocity of the mobile phase in a laminar flow system and the Kozeny-Carman equation for the specific column permeability:38
∆Pc ) 180η
(
)( )( )
(1 - e)2 Lc 3
e
dp
2
F πRc2
(18)
The pressure drop (∆Pc, dyn/cm2 ) is related to viscosity (η, g/cm s), external packing porosity (e, typically 0.38), column length (Lc, cm), particle diameter (dp, cm), flow rate (F, cm3/s), and the inner radius of the column (Rc, cm). For a given column, the length, radius, and particle size are fixed while the flow rate and viscosity are the only variables. In addition to the back pressure caused by the column, the narrow-bore external tubing in the system will add rather significantly to the back pressure. In particular, the HTUFLC system contains a significant length of tubing in the heating and cooling portions. If we assume that the mobile phase is incompressible and the flow is laminar, according to Poiseuille’s equation,21
∆P )
8LηF πrl4
(19)
While this equation is strictly correct only for isothermal, incompressible, fully developed laminar flow, it probably provides a reasonable estimate of the pressure drop in the 0.004 in. diameter tubing for Reynold’s numbers up to 15 00039 (Figure 5). The back pressure (∆Pt, dyn/cm2) of the tubing depends on the length (L, cm) and radius (r1, cm) of the tubing, the viscosity (η, (38) Poole, C. F.; Schuett, S. A. Contemporary Practice of Chromatography; Elsevier: Amsterdam, 1984. (39) Geankoplis, C. J. Transport Processes and Unit Operation; Allyn and Bacon: Englewood Cliffs, NJ, 1983.
Figure 5. The theoretical and experimental pressure drops at different temperatures. Lines are based on the theoretically calculated values of the back pressure resulting from the tubing and column according to eqs 18 and 19. Symbols b, 2, 1, and 9 indicate experiment data at 25, 80, 120, and 150 °C. For details see Table 4.
P), and the flow rate (F, cm3/s) of the mobile phase. From eq 19, we can see that the pressure drop is proportional to the reciprocal of the fourth power of tubing radius at fixed flow rate, mobile phase composition, and length of tubing. This implies that use of the narrow-bore tubing could produce very high back pressure. However, as discussed earlier, we cannot use wide-bore tubing, since it will cause severe band broadening, or shorter tubing, since the fluid will not be thermally equilibrated. We evaluated the theoretical and experimental back pressure of the system including the 20 cm of tubing used in the heat exchanger and the 20 cm of tubing in the cooling system; in addition, we used a 5 cm long PS-ZrO2 column. Since there is a gradient in temperature in the cooling system, we averaged the viscosity by means of the following equation:
ηave )
1 Tin - Tout
η∆T ∫ η(T) dT ≈ T∑- T in
(20)
out
ηTx can be estimated from eq 16 and Figure 3. As shown in Figure 5, we observe reasonable agreement between the estimated and experimental pressure drop of the system in 25% ACN (v/v). Furthermore, Table 4 shows that at 25 °C the maximum achievable flow rate is only 3.5 mL/min (the pressure drop being 379 bar) while at 150 °C the flow rate can be increased to 14 mL/min with a total back pressure of 360 bar. We point out that at 150 °C and a flow of 14 mL/min, 30% of the pressure drop results from the tubing. Clearly, most of the pressure is due to the column. We will show theoretically and experimentally that this HTUFLC system can be operated at flow rates up to 15 mL/min and that it is suitable for ultrafast separations. Effect of Temperature on Column Efficiency. In liquid chromatography the dependence of plate height (H) on interstitial linear velocity (ue) is usually empirically described by the Knox equation. Although in preliminary studies we found that the Knox equation usually but not invariably gave better fits than the van Deemter equation, the trends in the fitting coefficients (A, B, and C; see below) with temperature were more consistent; conseAnalytical Chemistry, Vol. 72, No. 6, March 15, 2000
1259
Table 4. The Calculated and Measured Pressure Drop of the Systema at Different Temperatures and Flow Rates ∆P (bar) tubingb
column temp (°C)
F (mL/min)
calcd
measured
calcd
measured
25 80 120 150
3.5 7 10 14
287 262 242 255
259 322 332 251
92 103 101 105
83 95 93 99
a The system is 40 cm narrow-bore tubing (0.004 in.) and a 5 × 0.46 cm column with 2.5 µm particles. The viscosity of the mobile phase (25% ACN (v/v)) was calculated from eq 16 to be 0.82, 0.37, 0.24, and 0.18 cP at 25, 80, 120, and 150 °C, respectively. b For 40 cm tubing, 20 cm tubing being in the heating bath and 20 cm in the cooling bath. The average viscosity of the cooling system was estimated by eq 20 at 25, 80, 120, and 150 °C to be 0.82, 0.62, 0.45, 0.37 cP, respectively, and the pressure drop is calculated by eq 19.
Figure 6. Plate height vs linear velocity at various temperatures for weakly retained solutes; experimental conditions are given in Table 5: 4, 25 °C (acetophenone, k′ ) 0.50); 3, 80 °C (acetophenone, k′ ) 0.26); 0, 120 °C (acetophenone, k′ ) 0.15); O, 150 °C (acetophenone, k′ ) 0.10).
Figure 7. Plate height vs linear velocity at various temperatures for moderately retained solutes; experimental conditions are given in Table 5: 4, 25 °C (octanophenone, k′ ) 3.87); 3, 80 °C (decanophenone, k′ ) 3.15); 0, 120 °C (decanophenone, k′ ) 5.70); O, 150 °C (decanophenone, k′ ) 1.65).
Figure 8. Plate height vs linear velocity at various temperatures for well-retained solutes; experimental conditions are given in Table 5: 4, 25 °C (decanophenone, k′ ) 12.2); 3, 80 °C (dodecanophenone, k′ ) 7.39); 0, 120 °C (tetradecanophenone, k′ ) 12.3); O, 150 °C (tetradecanophenone, k′ ) 7.00).
quently, we used the van Deemeter equation to fit the data:40
H ) A + B/ue + Cue
(21)
The A term reflects band broadening from eddy dispersion and is directly related to the quality of the column packing. The B term accounts for longitudinal (axial) diffusion while the C term represents the resistance to mass transfer in the stagnant mobile phase and stationary phase.41 Both B and C explicitly depend on the solute retention factor.41,42 Figures 6-8 show plots of the plate height versus ue at all temperatures studied. The fitting results are summarized in Table 5. We also give an estimate of the solute’s diffusion coefficient from eqs 15 and 16 for reference. Our most important observation is that, for all solutes at all k′ values, the C coefficient decreases 4-fold to nearly 9-fold as the temperature is increased. It is much (40) Scott, R. P. W., Liquid Chromatography Column Theory; John Wiley & Sons: New York, 1990. (41) Horvath, C.; Lin, H. J. Chromatogr. 1978, 149, 43-70. (42) Knox, J. H.; Scott, H. J. Chromatogr. 1983, 282, 297-313.
1260 Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
smaller at 150 °C than at 25 °C. The decrease in the C coefficient is to some extent due to the decrease in the solute’s retention factor at high temperature. However, most of the decrease is due to the increase in the solute’s diffusion coefficient. If we hold k′ nearly constant, for example, by considering octanophenone (k′ ) 3.87 at 25 °C) compared to decanophenone (k′ ) 3.15 at 80 °C), dodecanophenone (k′ ) 7.39 at 80 °C) to tetradecanophenone (k′ ) 7.00 at 150 °C), and decanophenone (k′ ) 12.2 at 25 °C) to tetradecanophenone (k′ ) 12.3 at 120 °C), we see a marked decrease in the C coefficient as the temperature is raised. The more retained is the solute, the more significant is the reduction in C. This indicates that resistance to interphase mass transfer is greatly reduced as the column temperature is increased. The improvement in mass transfer at the high temperatures may be due to either the increased rate of diffusion in the stagnant mobile phase or in the stationary phase. We conclude that because diffusion coefficients increase as the temperature is increased and the C term in the van Deemter equation depends on the inverse of the diffusion coefficient, most of the improvement in the C term
Table 5. Effect of Temperature on Column Dynamics van Deemter equation coefficientsa
experimental conditions T (°C)
mobile phase (% ACN (v/v))
solute
Dm × (cm2/s)b
k′
A × 103 cm
B × 104 (cm2/s)
C × 104 (cm2/s)
uopt (cm/s)
25 90 120 150 25 80 120 150 25 80 120 150
40 40 30 25 40 40 30 25 40 40 30 25
acetophenone acetophenone acetophenone acetophenone octanophenone decanophenone decanophenone decanophenone decanophenone dodecanophenone tetradecanophenone tetradecanophenone
0.10 0.25 0.41 0.59 0.08 0.15 0.25 0.36 0.06 0.14 0.22 0.31
0.50 0.26 0.15 0.10 3.87 3.15 5.70 1.65 12.2 7.39 12.3 7.00
1.0 ( 0.03 1.1 ( 0.06 1.0 ( 0.08 1.1 ( 0.02 1.1 ( 0.04 0.90 ( 0.05 0.91 ( 0.03 1.0 ( 0.05 1.0 ( 0.06 0.93 ( 0.04 1.0 ( 0.05 1.1 ( 0.02
0.28 ( 0.03 0.58 ( 0.11 1.4 ( 0.22 1.5 ( 0.05 0.18 ( 0.03 0.6 ( 0.09 1.2 ( 0.08 1.3 ( 0.08 0.23 ( 0.05 0.63 ( 0.07 1.0 ( 0.12 1.3 ( 0.07
1.2 ( 0.06 0.46 ( 0.05 0.37 ( 0.04 0.27 ( 0.01 1.4 ( 0.06 0.80 ( 0.03 0.44 ( 0.01 0.31 ( 0.03 1.9 ( 0.09 0.77 ( 0.03 0.38 ( 0.02 0.20 ( 0.08
0.2 0.4 0.7 0.8 0.1 0.3 0.6 0.7 0.1 0.3 0.6 0.8
104
a The standard deviation of the coefficients are also given. b Estimated solute diffusion coefficient in the indicated mobile phase at the temperature of the calculation based on eqs 15 and 16.
results from enhanced diffusion and not from the decrease in k′ as the temperature is increased. As shown in Table 5, the B coefficients at 150 °C are much greater than are those at 25 °C. Fundamental chromatographic theories4,43 state that the B term should be about 1.5-2Dm. Thus B must increase as the column temperature is increased. In fact, the B terms observed here at 25 °C are close to those one would estimate on the basis of the Wilke-Chang equation. For example, eq 15 with 40% acetonitrile at 25 °C gives a Dm value of 7.8 × 10-6 cm2/s for octanophenone, as compared to the measured B values of 1.8 × 10-5 cm2/s, which is acceptably close to 1.5Dm. Using an estimate of the viscosity of 25% acetonitrile at 150 °C based on eq 15 yields an estimate of Dm of 3.6 × 10-5 cm2/s for decanophenone. This is somewhat larger than the increase we see in the B term (1.3 × 10-4 cm2/s). The B term is only significant in the low velocity region and its consequences are nearly negligible at high operating velocities. In contrast to the B and C coefficients, the A coefficient seems to be relatively constant and independent of retention factor and temperature. We expect that the A term is dictated more by the bed structure and hydrodynamic dispersion and should be relatively independent of temperature. The work of Berthod44,45 serves as a caution against detailed interpretation of the fitting coefficients. The range in linear velocities encompassed by the present study is too narrow for the accurate measurement of A, B and C, so we feel it best not to discuss them in detail. We must point out that the column does not appear to be well-packed since the A term is about 3-4 times the particle diameter. Even though the extra column broadening does affect the column efficiency, we still observe some important trends in Figures 6-8. We see that as temperature is increased, the plate height improves in the high-velocity region but worsens at very low velocities. We also note that an increase in optimum velocity as temperature is increased. This is consistent with an increase in the analyte’s diffusion coefficient. At this point we conclude that it is beneficial to operate a liquid chromatograph at both high temperature and very high flow rates to improve efficiency, shorten analysis time, and optimize resolution. (43) Deemter, J. J. V.; Zuiderweg, F. J.; Klinkenberg, A. Chem. Eng. Sci. 1956, 5, 271-276. (44) Berthod, A. J. Liquid Chromatogr. 1989, 12, 1169-1185. (45) Berthod, A. J. Liquid Chromatogr. 1989, 12, 1187-1201.
Table 6. Effect of Temperature on Slope of ln k′ vs Carbon Number for Alkylphenonesa temp (°C)
Rc (slope)b
∆G°CH2 (kcal/mol)c
rd
25 80 120 150
0.557 ( 0.007 0.457 ( 0.010 0.412 ( 0.007 0.378 ( 0.009
-330 ( 4 -321 ( 7 -322 ( 7 -318 ( 6
0.9994 0.9995 0.9988 0.9993
a All data are based on PS-ZrO at mobile phase composition of 2 40% ACN (v/v). b Slope of ln k′ vs carbon number. c ∆G°CH2 denotes the free energy of transfer of a methylene unit from the mobile phase to the PS stationary phase. It is obtained as -RT ln Rc. d Correlation coefficient of ln k′ vs carbon number.
Thermodynamic Studies. In RPLC, the relationship between the retention factor and the carbon number in a homologous series of solutes can be expressed quantitatively as4,14,46,47
ln k′ ) ln k0 + (ln Rc)nC
(22)
where nC is the number of the methylene groups, ln k0′ is the absolute retention of the parent or the functional group that defines the homologue series, and Rc represents the hydrophobic selectivity, which is related to the free energy of transfer per methylene group (∆G°CH2 ) -RT ln Rc; R is the gas constant, T is temperature (K)) from the mobile phase to the stationary phase. The slopes and ∆G°CH2 are given in Table 6. To determine effect of the temperature on the selectivity of the separation, the methylene group selectivity at different temperatures can be expressed by
[
]
∆G°CH2(T0) ∆G°CH2(T) RT ) exp RT0 RT0 RT
(23)
where T0 is the reference temperature and RT is the selectivity at the reference temperature. If we substitute the ∆G°CH2(150) and ∆G°CH2(25) into eq 23, the selectivity at 150 °C will decrease by 16% as compare to 25 °C. However, the slight decease in selectivity (46) Karger, L. K.; Gant, J. R.; A., H.; Weiner, P. H. J. Chromatogr. 1976, 128, 65-78. (47) Vigh, G.; Varga-Puchony, Z. J. Chromatogr. 1980, 196, 1-9.
Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
1261
Figure 9. Chromatograms showing the effect of temperature on the separation of alkylphenones. Experimental conditions: mobile phase A, 30% ACN (v/v) and flow rate is 4 mL/min at 25 °C; mobile phase B, 25% ACN (v/v) and flow rate is 15 mL/min at 150 °C. Peaks: 1, acetophenone; 2, octanophenone; 3, decanophenone; 4, dodecanophenone; 5, tetradecanophenone.
is not significant and can be readily reclaimed by adjusting the solvent strength.48 We need to point out that, in general, selectivity depends not only on temperature and mobile phase but also on the solutes and type of stationary phases.14 Application of High-Temperature Ultrafast Separation. To demonstrate the speed that can be achieved at high temperature and high flow rate, we show, in Figure 9, a simple separation of alkylphenones. At 25 °C, we can perform the separation at a maximum flow rate of only 4 mL/min due to pressure limitations. On the other hand, at 150 °C, we can achieve a flow rate of 15 mL/min and at the same time the concentration of organic modifier can be reduced to maintain good resolution. Most excitingly, the analysis time is remarkably decreased from 20 min at 25 °C to 24 s at 150 °C without any practically significant loss in resolution. This supports our perspective that HTUFLC is powerful and can be used to achieve very fast separations in a few tens of seconds. As discussed above, the eluent viscosity and the back pressure decrease at high temperature so that high flow rate can be achieved. At the same time, the concentration of organic modifier can be decreased to optimize resolution. Thus it is sometimes quite practical to perform ultrafast separations with water as the eluent at high temperatures.25,27 The use of purely aqueous mobile phases in HPLC can sometimes overcome many of the problems related to the use of organic solvents, including their toxicity, flammability, and high cost. We believe that with high temperature fully aqueous mobile phase RPLC will be used more routinely soon. As shown in Figure 10, the separation of environmentally hazardous, phenolic compounds on the PS-ZrO2 column can be (48) Li, J. W.; Carr, P. W. Anal. Chem. 1996, 68, 2857-68.
1262 Analytical Chemistry, Vol. 72, No. 6, March 15, 2000
Figure 10. Chromatograms showing the separation of phenols on PS-ZrO2 at 120 °C. Experimental conditions: mobile phase, 100% water and flow rate is 12 mL/min at 120 °C. Peaks: 1, phenol; 2, 2-methylphenol; 3, 4-ethylphenol; 4, 2,4,6-trimethylphenol; and 5, 4-tert-butylphenol.
accomplished in less than 30 s in pure water at a temperature of 120 °C and a flow rate of 12 mL/min. CONCLUSIONS (1) The pressure drop in the narrow-bore tubing contributes significantly to the overall pressure drop in the system. Nonetheless, the column produces most of the back pressure. The practical maximum flow rate at 150 °C is 15 mL/min for a maximum overall pressure drop of 350 bar. (2) Column efficiency at high flow rate is significantly improved by increasing the column temperature. (3) The separation of alkyphenones shows that HTUFLC is a powerful approach to achieving separations that are as much as 50 times faster than can be achieved at room temperature. (4) The use of high temperatures can greatly reduce the use of organic solvents. The ultrafast separation of phenolic compounds at 120 °C was achieved using pure water as the mobile phase. (5) The use of an air bath to heat the system at high flow rates entails very long thermal equilibration tubing and concommitantly introduces excess bond broadening. ACKNOWLEDGMENT The authors acknowledge the financial support of the National Institute of Health. Received for review August 31, 1999. Accepted December 30, 1999. AC991008Y
