Anal. Chem. 1998, 70, 366-372
Pump Based on Thermal Expansion of a Liquid for Delivery of a Pulse-Free Flow Particularly for Capillary Chromatography and Other Microvolume Applications Christer Ericson and Stellan Hjerte´n*
Department of Biochemistry, Biomedical Center, University of Uppsala, P.O. Box 576, S-751 23 Uppsala, Sweden
A new liquid delivery system has been designed which, without splitting, affords a constant pulse-free flow in the nanoliter to microliter range. A water-filled stainless-steel tubular spiral (or a coiled fused silica capillary) is immersed into a water bath in which temperature increments can be computer-programmed with high precision. When exposed to a (linear) temperature gradient, the enclosed water expands. The discharged water acts as a uniformly moving plunger which propels the mobile phase. Calculated flow rates obtained from a simple equation agreed with experimental values. The pump was developed because commercial piston pumps for HPLC do not perform satisfactorily at the low volumetric flows used in capillary chromatography: periodic variations in pressure and flow rate cause zone deformation and false peaks in the chromatograms. The thermal expansion pump is not based on any mechanically movable parts and is, therefore, ideal in the sense that it does not give rise to such disturbances. The potential of the pump is demonstrated by ion-exchange and hydrophobic-interaction chromatography experiments of proteins on the easyto-synthesize continuous beds with 15-µm and 320-µm inside diameters (no frits to support the beds are required). The flow rates were 10 nL/min and 5.8 µL/min when using pressure chambers of 0.11 and 11 mL, respectively. For larger volumes of the elution buffer, a greater internal volume of the pressure chamber and agitation of the enclosed liquid are required in order to achieve a rapid change in temperature. The potential of miniaturized HPLC cannot be fully utilized until two practical difficulties have been overcome: (1) simple uniform packing of columns with 10-50-µm i.d. and (2) design of a pump which delivers a pulse-free flow well below 10 µL/min at both low and high pressures without wasting the mobile phase (often harmful organic solvents) by splitting, a technique which only permits a rough estimation of the flow rate through the column. The destruction of the mobile phase often costs more than the purchase of organic solvents and is an ever-increasing environmental problem.1 In recent papers, we have shown how 366 Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
the first difficulty can be solved.2-7 The object of this paper is to demonstrate how the second difficulty can be overcome by taking advantage of the volumetric expansion of a liquid when the temperature is elevated. A pump designed according to this approach has the great advantage that it is not based on movable parts, as are piston and syringe pumps, and therefore delivers a virtually pulse-free flow. Consequently, in capillary chromatography, both zone broadening and baseline distortion can be suppressed if the thermal expansion pump is employed for elution. MATERIALS Piperazine diacrylamide (PDA), N,N,N′,N′-tetramethylethylenediamine (TEMED), ammonium persulfate (electrophoresis purity reagents), and ammonium sulfate (HPLC-grade) were from Bio-Rad (Richmond, CA). Methacrylamide (MA) and vinyltrichlorosilane were obtained from Fluka (Buchs, Switzerland). 2-Hydroxyethyl methacrylate (HEMA) was from Aldrich (Steinham, Germany), acrylic acid (AA) from Merck (Darmstadt, Germany), [3-(methacryloyloxy)propyl]trimethoxysilane (Bind-Silane A 174) from Pharmacia Biotech (Uppsala, Sweden), and N-isopropylacrylamide (IPA) from Tokyo Chemical Industries (Tokyo, Japan). Fused-silica capillaries (15-µm i.d. × 140-µm o.d. and 320-µm i.d. × 405-µm o.d) were purchased from MicroQuartz (Munich, Germany). The mobile phase was in all experiments filtrated through a filter with 0.20-µm pore size (Minisart RC 25, from Sartorius, Go¨ttingen, Germay) and then purged with nitrogen to expel dissolved oxygen. The monitoring of the sample solutes was done on-line by a modified UV detector for HPCE experiments.4 DESIGN OF THE PULSE-FREE PUMP Principle. The volume expansion of a liquid (∆V) upon elevation of temperature (∆T) is not large but is, nevertheless, (1) Wan, H. B.; Wong, M. K. J. Chromatogr. A 1996, 754, 43-47. (2) Li, Y.-M.; Liao, J.-L.; Nakazato, K.; Mohammad, J.; Terenius, L.; Hjerte´n, S. Anal. Biochem. 1994, 223, 153-158. (3) Liao, J.-L.; Zhang, R.; Hjerte´n, S. J. Chromatogr. 1991, 586, 21-26. (4) Ericson, C.; Liao, J.-L.; Nakazato, K.; Hjerte´n, S. J. Chromatogr. 1997, 767, 33-41. (5) Liao, J.-L.; Li, Y.-M.; Hjerte´n, S. Anal. Biochem. 1996, 234 (1), 27-30. (6) Zeng, C.-M.; Liao, J.-L.; Nakazato, K.; Hjerte´n, S. J. Chromatogr. 1996, 753, 227-234. (7) Hjerte´n, S.; Eaker, D.; Elenbring, K.; Ericson, C.; Kubo, K.; Liao, J.-L.; Zeng, C.-M.; Lidstro¨m, P.-A.; Lindh, C.; Palm, A.; Srichiayo, T.; Valtcheva, L.; Zhang, R. Jpn J. Electrophor. 1995, 39, 105-118. S0003-2700(97)00685-9 CCC: $15.00
© 1998 American Chemical Society Published on Web 01/15/1998
Figure 1. Schematic diagram of the thermal expansion pump. S, a tubular spiral made of stainless steel (0.75-mm i.d. × 1.59-mm o.d. × 24.9 m) or a coiled fused silica capillary (320-µm i.d. × 405-µm o.d. × 1.37 m); FT, fingertight plug. The titanium rotor R can be moved up and down to increase or decrease the internal volume of the control valve. G, Teflon tubing containing the buffer gradient; Sy, syringe; SV, three-port switching valve; T, a short piece of Teflon tubing.
Figure 2. Coefficient for thermal expansion of water, RT, as a function of the temperature of water. Values for RT at different temperatures were taken from the literature.14
sufficient to be used to force the mobile phase into a column for capillary chromatography. A pump based on this principle is outlined in Figure 1. Since RT (sometimes denoted β), the cubal coefficient for thermal expansion at the temperature T, is defined as
RT )
δV 1 δT V
(1)
βp ) -
the flow rate Fexp ) δV/δt can be written
Fexp ) RTV
δT δt
where V is the volume of the liquid and δT/δt is the temperature gradient (the elevation of the temperature of the liquid per second). According to eq 2, the thermal expansion pump gives a virtually pulse-free flow because V is constant, RT changes in a monotonous mode with a change in temperature, and it is easy to generate a constant temperature gradient δT/δt. The risk of a pulsating flow is low also for the reason that the design of the pump is not based on any mechanically movable parts. Equation 2 also shows that, for a linear temperature gradient, the flow rate is constant if RT is constant. In practice, this requirement can be fulfilled, although RT varies somewhat with temperature, provided that a narrow temperature interval is chosen (see Figure 2). The fact that RT increases slowly with temperature gives rise to a small contribution to the flow rate. The magnitude of this contribution depends on the chosen temperature interval. This predictable “error” is about 1% for a temperature elevation of 1°. However, the variations in RT can be compensated for by keeping the product RTδT/δt constant, which can be achieved by a simple computer program. In this case, the flow rate will be constant within any temperature range. Influence of the Compressibility of Liquid in the Spiral on the Flow Rate. The compressibility of a liquid at temperature T and pressure P can be described by the compressibility factor βp, defined by the expression
(2)
δV 1 δP V
(3)
where V is the volume of the liquid. Using eq 4, Fcompr, the Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
367
contribution from the compressibility of the liquid in the spiral S (in Figure 1) to the flow rate, can be written as
Fcompr )
δV δP δT ) - βpV δt δT δt
(4)
Almost all chromatographic experiments are performed under such conditions that the pressure is constant during the run, i.e., during the application of the temperature gradient. Accordingly, δP/δT is zero, and, therefore, Fcompr is also zero. Thus, variations in the compressibility factor with temperature will not affect the observed flow rate, Fexp. Fcompr must be taken into account only in the few cases when the pressure changes considerably during a run, for instance, by compression of the bed, by heavy clogging of the channels or the filters, or by release of clogging material. In such cases, a constant value of Fcompr can be attained by using a computer program where the product βpδP/δT is kept constant. The total flow rate is then the algebraic sum of Fexp (eq 2) and Fcompr (eq 4). Values of βp for different liquids can be found in the literature.11 Some Practical Details. The water bath Haake L (Berlin, Germany) was connected via its thermostat Haake D8 to the computer (Scandic Products, Uppsala, Sweden), which was equipped with software for the generation of a temperature gradient in the water bath (and, thereby, in the metal spiral S in Figure 1). The software was created by Mr. Per-Axel Lidstro¨m at this institute. In our experiments, only linear gradients were used. The spiral had i.d. ) 0.75 mm, o.d. ) 1.59 mm, length ) 24.9 m, and volume ) 11.00 mL, determined by weighing. This metal spiral was exchanged for a coiled fused silica capillary (i.d. ) 320 µm, o.d. ) 405 µm, length ) 1.37 m) in a chromatography experiment on an extremely narrow column (i.d. ) 15 µm). The pressure was measured with a slightly reconstructed pressure gauge of an HPLC pump (Model 2150 from LKB Produkter, Stockholm, Sweden) (see Comparison of Pressure Traces from an HPLC Piston Pump and Those from the Thermal Expansion Pump, below). A three-port high-pressure HPLC valve (P-734) from Upchurch (Oak Harbor, WA) served as control valve to close and open ports 1 and 2. A three-port switching valve (SV) series UV from Valco (Houston, TX) was installed to create gradients in the Teflon tube G (see Formation of Small-Volume Gradients for Elution with the Thermal Expansion Pump, below). EXPERIMENTAL SECTION Measurement of the Flow Rate. By Weighing the Eluent. The flow rate was determined by weighing the delivered mobile phase periodically on a precision microscale balance (Model AE 260-S from Mettler-Toledo AG, Greifensee, Switzerland). For this purpose, the outlet end of the column was connected to a fused silica capillary (25-µm i.d.), which was inserted through a hole in the cap of a glass vial (Figure 1). The hole was only about 0.5 mm in diameter in order to prevent evaporation of the collected eluent. The upper section of the capillary was fixed in a steel tube. By Means of a Home-Made Bubble Flow Meter. A 10-cm-long transparent glass tube (1.0-mm i.d.) mounted on a horizontally aligned ruler was connected to the capillary outlet via a press-fit connector from NTK Kemi HB (Uppsala, Sweden). The inside 368
Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
walls of both the glass tube and the connector were coated with vinyltrichlorosilane to decrease the adherence of water. The time to transport the liquid meniscus between two thin lines on the glass tube was measured, and the velocity calculated was compared with that obtained by weighing. Handling of the Pump. The metal spiral S (Figure 1) was first filled with deaerated, double-distilled water via the fingertight plug FT using an HPLC pump. Port 1 was kept closed with a fingertight plug (P-550, Upchurch), and the valve SV was open at T. A small back pressure of 5-10 bar was created with the aid of the fingertight plug FT, while filling the spiral to eliminate the risk of air bubble formation. The deaeration was a precaution, since air bubbles are compressible and may, therefore, retard the initial response to the pressure induced by the thermal expansion. After the water bath was set at the starting temperature, To (usually 41 °C), the elution and equilibration buffer was sucked up into the gradient tubing G via the switching valve SV with the aid of the syringe Sy (P1 was open and P2 closed). The temperature gradient ∆T/∆t required to deliver a certain flow rate, ∆V/∆t, was obtained from diagrams similar to those in Figure 4. However, in practice, we used computer-generated diagrams with high resolution instead of the graph in Figure 4, which is presented primarily to facilitate the discussion. For an experimental determination of the flow rate, one of the two above methods was used (that based on weighing is more accurate but slower). The estimated and the experimental flow rates are compared in Figure 4. The pressure was recorded by the pressure gauge. It should be mentioned that it takes 10-20 s to establish the final pressure (and thereby the final flow rate), probably due to some slight elasticity in the system, including the Teflon tubing G, or/and some inertia in the interface between the computer and the water bath. This delay is favorable from the point of view that abrupt increases in pressure are avoided. However, if an instant increase in pressure and flow rate is desired, the internal volume of the control valve should be diminished immediately after the start of a run by turning the rotor R clockwise (this valve arrangement can also be used with piston pumps). Formation of Small-Volume Gradients for Elution with the Thermal Expansion Pump. For cation-exchange chromatography, the mobile phase was a 48-µL linear salt gradient from 0 to 0.6 M sodium chloride in 20 mM sodium phosphate, pH 6.2, and for hydrophobic interaction chromatography a linear gradient of 48 µL from 2.4 to 0 M ammonium sulfate in 20 mM sodium phosphate, pH 6.8. Salt gradients of such small volumes were created by introducing small segments of salt solutions of different concentrations into a piece of tubing (G in Figure 1). The technique is similar to that described previously,2,3 although we increased the number of segments in the buffer tubing to achieve a still smoother transition from one concentration to the next. In order to create the gradient for cation-exchange chromatography, 16 solutions were prepared in test tubes by mixing solution A (20 mM sodium phosphate, pH 6.2) with solution B (0.6 M sodium chloride in 20 mM sodium phosphate, pH 6.2) in different proportions. The volume percentages of solution B increased gradually from 0% in test tube 1 to 100% in test tube 16. Following equilibration of the capillary columns with solution A delivered by the thermal expansion pump (or the HPLC pump), the Teflon tubing G was connected to the switching valve SV. By means of
Table 1. Composition of Monomer Solutions for Preparing Continuous Bed Capillary Columns
CIECb HICc a
PDA (g)
MA (g)
0.165 0.150
0.130
HEMA (µL)
AA (µL)
IPA (g)
16 0.100
0.14
5 M NaOH (µL)
(NH4)SO4 (g)
buffera (mL)
32
0.08 0.06
0.9 0.9
Sodium phosphate (50 mM, pH 7.0). b Cation-exchange chromatography. c Hydrophobic-interaction chromatography.
the syringe Sy, 3-µL portions from each test tube were then sucked into the gradient tubing to form a 48-µL positive salt gradient. Consequently, the equilibration buffer in test tube 1 was the last one to be sucked up into the gradient tubing. The procedure to create the gradient for hydrophobic interaction chromatography was similar to the above description, except that the order of salt concentrations in the gradient tubing G was reversed, ranging from 2.4 to 0 M ammonium sulfate in 20 mM sodium phosphate (pH 6.8), and that the column was equilibrated with the higher salt concentration (2.4 M). In both separation modes, 1 µL of a protein test mixture was sucked in, followed by 4 µL of the starting buffer. Cation exchange chromatography was also performed in a 15-µm-i.d. column. For that purpose, we used a small-volume syringe (0.5 µL) from SGE (Sydney, Australia) to create a 0.10µL linear gradient going from 0 to 0.6 M sodium chloride in 20 mM sodium phosphate, pH 6.2. The pressure chamber consisted of a coiled fused silica capillary, 320-µm i.d. × 405-µm o.d. × 1.37 m (volume, 110 µL). Therefore, for a given temperature interval, the displacement of water was 100-fold less than that delivered by the 11-mL metal spiral. Further details are given in the legend to Figure 5 B. The switching valve SV was redirected toward the capillary column, and the syringe Sy was replaced by the fingertight plug. The chromatography experiment was started by applying the desired temperature gradient in the water bath and then opening the control valve by slowly turning the rotor R upward. Preparation of Chromatographic Columns. General Considerations. The performance of the thermal expansion pump was investigated using continuous polymer bed columns for cationexchange and hydrophobic interaction chromatography. Continuous beds are synthesized from a set of water-soluble monomers and may be prepared in a beaker and then packed into a column or, as in these experiments, by polymerization directly in the capillary.2-7 The continuous beds used in this study share the properties of many other types of continuous beds described previously:2,3,8,9 for instance, a resolution that is independent of flow rate or even increases with an increase in flow rate. Activation of the Capillary Wall with Methacryloyl Groups. In order to bind the continuous bed covalently, the fused silica wall was treated in the following way. By means of a water aspirator, the capillary was washed with toluene and acetone and was then treated for 30 min with 0.2 M sodium hydroxide, followed by 0.2 M hydrochloric acid for 30 min, and a final flushing with distilled water. The capillary wall was then activated with a solution of [3-(methacryloyloxy)propyl]trimethoxysilane in acetone (30% v/v).10 (8) Hjerte´n, S.; Nakazato, K.; Mohammad, J.; Eaker, D. Chromatographia 1993, 37, 287-294. (9) Hjerte´n, S.; Li, Y.-M.; Liao, J.-L.; Mohammad, J.; Nakazato, K.; Pettersson, G. Nature (London) 1992, 356, 810-811. (10) Hjerte´n, S. J. Chromatogr. 1985, 347, 191-198.
The methoxy groups in this compound react with the silanol groups at the surface of the silica wall to form Si-O-Si-C bonds, leaving free methacryloyl groups for reaction with the monomers forming the continuous bed. Consequently, during the course of the subsequent polymerization, the continuous bed becomes attached covalently to the capillary wall. Preparation of the Continuous Bed in situ in the Capillary. Table 1 shows the composition of the monomer solutions used to synthesize continuous beds for hydrophobic-interaction and cationexchange chromatography. Each monomer solution was degassed with a stream of nitrogen and supplemented with 10 µL of 10% (w/v) ammonium persulfate and 10 µL of 5% (v/v) TEMED aqueous solutions before being sucked into the methacryloylactivated column. The polymerization proceeded for 24 h with both ends of the capillary plugged with desiccator grease. A window for on-line detection was made after the polymer bed was prepared (according to a method described in ref 4). RESULTS AND DISCUSSION Relation between Flow Rate and Pressure. The flow rate was measured by determining the mass of the buffer leaving the capillary, as outlined above. An ideal pump produces a volumetric flow which is independent of column back-pressure; i.e., the programmed flow rate should be the same for all columns and all compositions of the mobile phase. For two columns with different flow resistances, the pressure across the bed must, therefore, be different, and for each column the flow rate should be proportional to the pressure. Two capillary columns were employed to investigate whether the pump described herein fulfills these requirements, one giving 12 bar and the other 66 bar at a flow rate of 10 µL/min, and, as shown in Figure 3, both gave rise to a linear relationship between flow rate and pressure. The larger scattering of points at lower flow rates originates from the difficulties in determining accurately small changes in the masses of the effluent fractions. Calculated and Measured Flow Rates. From eq 2 and from literature values of RT,14 the flow rate Fexp ()∆V/∆t) of the water forced out of the spiral S was calculated for different temperature gradients. A plot is shown in Figure 4. The full line represents the calculated flow rates, and the dotted line corresponds to flow rates determined by weighing the displaced water. The close agreement between calculated and measured rates indicates that the heat transfer was efficient, that the seals were tight, and that the stainless steel tube and pieces of Teflon tubings were sufficiently inelastic. Table 2 is a complement to Figure 4. Observe that RT varies relatively strongly with temperature in the range 0-20 °C (the dotted line in Figure 2). This temperature (11) Lange’s Handbook of Chemistry, 12th ed.; Dean, J. A., Ed.; McGraw-Hill Book Co.: New York, 1978.
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Table 2. Measured Flow Rate and Flow Rate Calculated from Eq 2
Figure 3. Flow rate as a function of pressure for two capillary columns with different flow resistance. Column A (320-µm i.d. × 6 cm) gave a back pressure of 12 bar at 10 µL/min and column B (320µm i.d. × 26 cm) 66 bar at the same flow rate. Buffers: 20 mM sodium phosphate, pH 6.2. The compositions of the beds were different, leading to different back pressures for a given flow rate.
Figure 4. Measured (]) and calculated (O, eq 2) flow rates plotted against the temperature gradient. V ) 11.00 mL. Values of RT were taken from the literature.14
interval should, therefore, be avoided to accomplish a flow rate as constant as possible at a constant linear temperature gradient (eq 2). However, as mentioned earlier, any temperature interval can be chosen, provided that the computer program is designed to keep the product RTδT/δt constant. Capillary Chromatography on Continuous Beds Using the Thermal Expansion Pump. Continuous polymer beds were derivatized in two ways for capillary chromatography: with isopropyl groups for hydrophobic-interaction chromatography6 and with propionic acid groups for cation-exchange chromatography.2,12 These columns were used to test the performance of the pulse-free thermal expansion pump (Figure 5). Observe the great similarities in the appearances of the chromatograms in experiments 5A and 5B despite the large differences in the diameters of the beds. The samples, consisting of proteins, were injected (12) Hjerte´n, S.; Liao, J.-L.; Zhang, R. J. Chromatogr. 1989, 473, 273-275.
370 Analytical Chemistry, Vol. 70, No. 2, January 15, 1998
∆V/∆t (µL/min)
∆T/∆t (°C/min)a
measuredb
calculatedc
0.275 0.367 0.550 0.687 1.100 1.571 2.200
1.19 1.64 2.54 3.14 5.04 7.31 10.32
1.31 1.74 2.62 3.27 5.24 7.48 10.47
a Temperature range 41-52 °C, which corresponds to a total displacement of 52.4 µL. b Flow rate determined by weighing the eluent at 25 °C (F ) 0.997 g/cm3). The values are the mean of two weighings. c Flow rate calculated from eq 2: ∆V/∆t ) R V∆T/∆t, where ∆T/∆t T is the linear computerized temperature gradient and RT (the mean value for the temperature interval 41-52 °C) ) (RT1 + RT2 + ... RTn)/n. For n ) 12, RT ) 4.327 × 10-4 K-1. Values of RT at different temperatures were taken from ref 14.
as described in the section, Formation of Small-Volume Gradients for Elution with the Thermal Expansion Pump. The flow rate was determined with a calibrated bubble meter (see Measurement of the Flow Rate, in the Experimental Section) the accuracy of which was double-checked by weighing the eluent fractions. Further details are given in the legend to Figure 5. When only the salt gradient prepared from the 16 buffer segments was monitored at very high UV sensitivity (205 nm, 0.0005 AU), a fairly even baseline, without “steps”, was observed, which indicates that the mixing of two adjacent buffer segments was highly efficient, owing largely to the hydrodynamic, parabolic deformation of the segments combined with radial diffusion. Since the smoothness of the gradient also depends on the inner diameter of the gradient tubing G (Figure 1), a few bore sizes were tested before we settled on 250-µm i.d for the 320-µm columns. For cation-exchange chromatography in the 15-µm column, tubing G was exchanged for a fused-silica capillary of 25-µm i.d. Other bores may be required when the separation conditions are altered (e.g., changed volume of the salt segments or other dimensions of the column). It should be stressed that all chromatographic steps could be completed within roughly the same time, independently of whether the thermal pump or a conventional HPLC piston pump was used. Comparison of Pressure Traces from an HPLC Piston Pump and Those from the Thermal Expansion Pump. A recorder was connected to the voltage output of the pressure meter of a modified HPLC pump. The built-in piezoelectric pressure transducer was, in this way, utilized as a pressure gauge. It provided an immediate and sensitive registration of pressure fluctuations smaller than 0.1 bar. With this arrangement, pressure traces from the HPLC piston pump and the thermal expansion pump were recorded. Both types of pumps were tested using the same set of continuous bed columns and the same pressure gauge. The output signal from the HPLC pump itself was not utilized. As shown in the diagrams in Figure 6, the pressure traces obtained from the thermal expansion pump were almost free from noise and fluctuations and were considerably more stable than those from the HPLC piston pump. Even at 50 µL/min (50 bar), which is a substantial flow rate in capillary chromatography, the pressure pulses were negligible. The very steady performance
Figure 5. Capillary chromatographic experiments based on the continuous beds and the thermal expansion pump. (A) Cation-exchange chromatography of proteins on a continuous polymer bed derivatized with propionoic acid residues. Capillary column: 140 mm (effective length, 110 mm) × 320-µm i.d. × 405-µm o.d. Mobile phase: 48-µL linear gradient from 0 to 0.6 M sodium chloride in 20 mM sodium phosphate, pH 6.2. Flow rate (measured): 5.8 µL/min without splitting of the mobile phase. Programmed linear temperature gradient: 1.25 °C/min. Temperature interval: 41-52 °C. Constant pressure: 30 bar (measured). The pressure chamber was a stainless steel tube (0.75-mm i.d. × 1.59-mm o.d. × 24.9 m; volume, 11.00 mL; wall thickness, 0.84 mm) coiled into a spiral. On-tube detection was at wavelength 280 nm. Sample: 1 µL of a protein mixture, 0.4 mg/mL of each protein except for ribonuclease A, which had a 3-fold higher concentration. 1, myoglobin (horse); 2, ribonuclease A; 3, cytochrome c; 4, lysozyme. (B) Cation-exchange chromatography on a continuous polymer bed derivatized with propionic acid residues. Capillary column: 140 mm (effective length, 110 mm) × 15-µm i.d. × 140-µm o.d. Mobile phase: 0.10-µL linear gradient from 0 to 0.6 M sodium chloride in 20 mM sodium phosphate, pH 6.2. Flow rate (calculated): 10 nL/min without splitting of the mobile phase. Programmed linear temperature gradient: 0.25 °C/min. Temperature interval: 41-49 °C. Constant pressure: 28 bar (measured). Sample volume: 10 nL. The pressure chamber was a fused-silica capillary, 320-µm i.d. × 405-µm o.d. × 1.37 m (volume, 110 µL). ∆V for this capillary is 100-fold less than that of the spiral of 11 mL (S in Figure 1) used in all the other experiments. Other conditions are as in (A). (C) Hydrophobic-interaction chromatography of proteins on a continuous polymer bed derivatized with isopropyl groups. Capillary column: 130 mm (effective length, 100 mm) × 320-µm i.d. × 405-µm o.d. Mobile phase: 48-µL linear gradient from 2.4 to 0 M ammonium sulfate in 20 mM sodium phosphate, pH 6.8. Flow rate (measured): 5.8 µL/min without splitting of the mobile phase. Programmed linear temperature gradient: 1.25 °C/min. Temperature interval: 41-52 °C. Constant pressure: 24 bar (measured). On-tube detection was at wavelength 280 nm. Sample: 1 µL of a protein mixture, 0.4 mg/mL of each protein except for cytochrome c (0.7 mg/mL). 1, cytochrome c; 2, myoglobin (horse); 3, ribonuclease A; 4, lysozyme; 5, R-chymotrypsinogen. The same pressure chamber as in (A) was used.
at 0.01-1 µL/min is a prerequisite for efficient separations on very narrow bore columns (for instance, 15-µm i.d., as used in the experiment presented in Figure 5B). A set of traces at other pressures was similar to those in Figure 6 for the two pumping systems. A minor reduction of the baseline pulses generated by the HPLC pump could be achieved by running the pump at a higher flow rate in combination with a splitter column (to produce the required flow rate through the separation column). However, such an arrangement is wasteful of solvents and makes the pump unusable for measuring the pressure over the column and the rate of the flow through the column. Prior to the experiments, the HPLC pump was fitted with new seals and bearings and was tuned for optimum performance. The pressure diagrams obtained were at least as stable as standards given in the handbook for the HPLC pump.13 The two pistons in the drive unit of the HPLC pump are propelled by a microprocessor-controlled dc motor via a gear (13) LKB Produkter AB, Bromma, Sweden, Instruction Manual to LKB 2150 HPLC pump, 1985. (14) Handbook of Chemistry and Physics, 68th ed.; Weast, R. C., Ed.; CRC Press, Inc.: Boca Raton, FL, 1987.
mechanism and two cam wheels. Cam wheels are rotating disks with irregular profiles, attached to a revolving shaft that forwards a reciprocating motion to the pistons. The positive cyclic pressure peaks observed for the HPLC pump (Figure 6) were attributed to the cam profile. To reduce these pulses, this and many other piston pumps are designed with automatic compressibility compensation. However, this additional function does not give a notable damping at the low or moderate pressures used in the experiments described in this investigation. The thermal expansion pump was designed primarily for use in chromatography columns of 320-µm i.d. or smaller and for elution volumes up to 200 µL. If larger volumes of discharged liquid are required, the water used in the experiments described herein can be exchanged for a liquid having a larger thermal expansion coefficient, or the volume of the pressure chamber can easily be increased. In the latter approach, for instance, a metal cylinder equipped with a device for efficient (magnetic) stirring can be an alternative to the long narrow-bore metal tube (S in Figure 1). Attempts to use a stainless-steel tube of larger inner (15) Schneiderheize, J. M.; Hogan, B. L. Anal. Chem. 1996, 68, 3758-3762.
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water bath should be sufficiently low to allow a rapid temperature change. Stainless steel is an appropriate material for the pressure chamber due to its inertness and rigidity. However, a tube made from a metal with higher thermal conductivity may be preferable.
A
B
Figure 6. Pressure traces from an HPLC piston pump (A) and the thermal expansion micropump (B). Columns: A continuous bed column (I) with the dimentions 320-µm i.d. × 120 mm gave a back pressure of 10 bar at 10 µL/min. Another column (II) with the same dimensions but with a different gel composition afforded 50 µL/min at 6 bar. Both pumps were tested on both columns using these sets of pressure and flow rate. The thermal expansion pump was also tested on column I at a flow rate of 1 µL/min (1 bar) and on column III (15-µm i.d. × 110 mm) at 10 nL/min (28 bar) (the piston pump could not deliver such low flow rates). A comparison between the pressure traces A and B shows that a thermal expansion pump delivers a virtually constant flow, whereas the flow from the piston pump is pulsating.
diameter (4.6 mm) was, as expected, unsuccessful due to slow heat response. Mixing inside the expanding liquid must be carried out without creation of gas bubbles. The volume of the
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CONCLUSION The pulse-free thermal expansion pump described herein can deliver extremely low volumetric flows (below 0.01 µL/min). It can, furthermore, generate extremely high pressures. The limit is determined by the mechanical strength of the pressure chamber, connecting units, the chromatographic tube, etc. Using the thermal expansion pump for capillary chromatography, the consumption of analytes and buffers can be decreased, which is particularly important when the sample is expensive or available only in small amounts and when the mobile phase contains harmful organic solvents, such as acetonitrile, as in reversed-phase chromatography. The conventional method using standard pumps in combination with splitting does not reduce the total volume of the mobile phase leaving the pump. In addition, the method does not permit accurate determination of the flow rate through the column. There are no technical hindrances to automate the pump and adapt it to the various applications where pulse-free, low flow rates are required. Using two pumps and appropriate valves, gradients can be created in the same way as with conventional HPLC piston pumps. The pressure chamber only needs to be cooled down between runs, not opened and refilled. Thus, the thermal expansion pump has the potential to become an alternative also to motor-driven syringe displacement pumps or appropriate syringes for accurate microinjections. It may also be utilized for medical applications, for instance, for in vivo microdialysis sampling.15 ACKNOWLEDGMENT This study has been supported financially by the Swedish Natural Science Research Council and the Swedish Research Council for Engineering Sciences. Received for review July 1, 1997. Accepted October 30, 1997.X AC970685U X
Abstract published in Advance ACS Abstracts, December 15, 1997.
