WO1998052034A1

WO1998052034A1 - Thermal expansion pump for pulse-free liquid flow

Info

Publication number: WO1998052034A1
Application number: PCT/US1998/010119
Authority: WO
Inventors: Stellan Hjerten; Christer Ericson
Original assignee: Bio-Rad Laboratories, Inc.
Priority date: 1997-05-15
Filing date: 1998-05-14
Publication date: 1998-11-19
Also published as: AU7577198A

Abstract

Pulse-free flow, for instance for capillary chromatography, is provided by a pump comprised of a length of heat transmissive tubing filled with a liquid that undergoes thermal expansion upon heating, in conjunction with a heating device that provides uniform heating of the tubing and its contents at a controlled rate.

Description

THERMAL EXPANSION PUMP FOR PULSE-FREE LIQUID FLOW

This invention lies in the field of liquid flows, including both micro volume flows such as those used in capillary chromatography, and larger volume flows, and addresses in particular the difficulties encountered in attempting to achieve a steady, pulse-free flow of liquid at very low flow rates.

BACKGROUND OF THE INVENTION

The use of capillaries for separations and analyses of multi-component mixtures is widely practiced in view of the advantages that capillaries offer. Among these are the ability to analyze very small sample volumes, to perform analyses in a relatively short period of time but with high reproducibility and accuracy, and to perform on-line detection, and the use of cartridges to hold the capillaries and to permit easy exchange of one separation medium or column for another, the cartridges lending themselves readily to use in automated systems for the analysis of a multitude of samples in succession.

The means by which the mixture components are separated can vary widely, reflecting the many different types and sources of mixtures that require analysis, the variety of separation media needed to separate the components, and the variety of chromatographic methods that have been developed to achieve these separations. Perhaps the most powerful group of separation techniques are those involving high performance liquid chromatography (HPLC). Included in this group are ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, molecular sieve chromatography, adsorption chromatography, and exclusion chromatography. In these techniques, the mobile phase is forced through the separation medium by the mechanical force of a pump.

When HPLC is performed with a capillary column, the performance of the pump is a particularly significant factor in the separation. As the capillary size and the flow rate through the capillary are progressively reduced, the flow pulses created by the mechanical action of the pump disrupt the detector baseline to an increasing extent, interfering with the separation results. One means of reducing this interference is by splitting the pump discharge so that only a portion of the flow enters the capillary. With this method, the pump can be run at a higher flow rate than that directed through the capillary, thereby reducing the pulsing. The disadvantages are that the flow rate through the column is not known with certainty, and the excess carrier fluid that by-passes the column is wasted.

SUMMARY OF THE INVENTION

It has now been discovered that a continuous, pulse-free and highly controlled flow can be achieved by the use of a thermal expansion pump, which includes a non-expandable (rigid-walled) vessel that is filled with a liquid that undergoes volumetric expansion upon heating, plus a heating device for heating the vessel and its contents uniformly and at a highly controlled rate. The vessel is completely filled with the liquid and fully closed except for one outlet port for discharge of the liquid, such that all volumetric expansion of the liquid is discharged through the outlet port upon heating. The vessel can be a bomb, a tank, a length of tubing, or any container that is capable of retaining and being completely filled with liquid and does not expand upon increases in the pressure of the liquid. A preferred heating device is a temperature bath under computer control monitored by a temperature sensor in the bath.

When the vessel is a length of tubing, the tubing is closed at one end and open at the other, and the temperature rise creates a discharge of the expanding liquid from the open end of the tubing at a controlled rate. Rapid equalization of temperature inside the tubing is achieved by using tubing of relatively small diameter and thin-wall construction. When the vessel is a tank or bomb, rapid equalization of temperature of the liquid is achievable by stirring, or other means of agitation of the vessel or its contents. In general, substantial equalization of temperature should be obtained within about 5 seconds, and preferably within about one second.

For capillary chromatography, the preferred vessel is a length of tubing. The discharge end of the tubing is connected to the capillary column, either directly or through an intermediate length of tubing or any enclosed reservoir that will transmit the expansion flow from the temperature-rise tubing to the capillary. In either case, sample injection will be performed at the entry end of the capillary. For systems in which the temperature-rise tubing is joined directly to the capillary, the temperature-rise tubing will contain the carrier liquid (generally a buffer solution or when desired a gradient buffer solution) that will draw the sample through the capillary. When a further length of tubing or a reservoir is interposed between the temperature-rise tubing and the capillary, the carrier liquid can be initially retained in the intervening tubing or reservoir while the liquid in the temperature- rise tubing can be any liquid that expands volumetrically upon heating. Intervening tubing is particularly useful in applications where a concentration gradient, such as a salt gradient, for example, is desired.

The invention is useful in achieving a wide range of flow rates, but will be of particular interest in capillary HPLC where the flow rate produced is 10 μL/min (microliters per minute) or less. The invention will also be of interest in capillaries containing a continuous solid yet porous polymeric bed as a separation medium.

Further details on these and other features and advantages of the invention will be apparent from the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the coefficient of volumetric thermal expansion vs. temperature for water.

FIG. 2 is a diagram of a thermal expansion pump and capillary chromatography column utilizing the principles of the present invention. FIG. 3 is a plot of flow rate vs. temperature rise/minute for a thermal expansion pump of the present invention.

FIG. 4 is a plot of flow rate vs. pressure for a thermal expansion pump of the present invention.

FIG. 5 is a chromatogram of a protein mixture taken on a cation exchange column using a thermal expansion pump of the present invention as the carrier liquid driving force.

FIG. 6 is a chromatogram of a protein mixture taken on a smaller diameter cation exchange column, again using a thermal expansion pump of the present invention as the carrier liquid driving force.

FIG. 7 is a chromatogram of a protein mixture taken on a hydrophobic interaction column using a thermal expansion pump of the present invention as the carrier liquid driving force.

FIGS. 8a and 8b are records of pressure vs. time at two different pressures and flow rates for a conventional HPLC piston pump.

FIGS. 9a and 9b are records of pressure vs. time at four different flow rates for a thermal expansion pump of the present invention.

FIG. 10 is a diagram of a thermal expansion pump and tubular chromatography column utilizing the principles of the present invention to achieve a higher rate of flow than that of the apparatus of FIG. 2. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

This invention is primarily of interest in achieving volumetric flow rates with the range of about 0.001 μL per minute to about 100 μL per minute, although larger flow rates can also be achieved, depending on the nature of the temperature rise vessel. Preferred flow rates for certain applications, notably HPLC, are those in the range of about 0.3 μL/min to about 10 μL/min, and for extremely small capillaries, those in the range of about 0.001 μL/min to about 0.03 μL/min. Selection of the flow rate is achieved by a combination of factors, including the volumetric capacity (length and internal diameter) of the temperature-rise tube, the rate of temperature rise, and the coefficient of thermal expansion of the driving liquid retained in the temperature-rise tube.

The volumetric coefficient of thermal expansion _τ for a liquid at a given temperature T is defined as

a_τ = δV 1_ ^~δ KTT V V (1)

and the flow rate

F = — " ^~δt

can be written as

where V is the volume of the liquid and Tlbt is the rate rise of the temperature of the liquid in degrees Celsius per second. Since V is constant and _τ for typical liquids varies in a monotonic way with temperature, the result of imposing a controlled temperature rise is a pulse-free flow.

Any liquid that expands volumetrically with an increase in temperature and for which a substantially constant rate of expansion can be achieved by a continuous temperature increase can be used. Coefficients of thermal expansion and their values as a function of temperature are known and are generally available in the literature. Although the particular value of the coefficient is not critical to this invention, best results in most cases will be obtained with a liquid having a coefficient of about 1 X 10^"4 K^"1 or greater at 1 atmosphere and 20°C, preferably within the range of about 1 x 10^~4 K^"1 to about 20 X 10"⁴ K"¹ at 1 atmosphere within a temperature range of about 20°C to about 100°C. Water is one example of a liquid useful for this purpose, and its coefficient of thermal expansion as a function of temperature is shown graphically in FIG. 1. It will be noted that in the temperature range of 0-20 °C, represented by the dashed line, the slope of the coefficient vs. temperature is greater than that in the remainder of the curve, and for this reason it is preferable to operate in the region of the curve represented by the solid line. In general, it is preferable to select the operating temperature range and the rate of temperature increase such that the product of a_τ and δTVδt is substantially constant (see Equation (2)). This can be achieved by a computer program, the selection of which will be readily apparent to those skilled in the use of computers for temperature control, which is a known method of temperature control.

The compressibility of the driving liquid is a further factor in the performance of the thermal expansion pump of this invention. The compressibility of a liquid at the temperature T and pressure P can be expressed in terms of the compressibility factor β_P , which is defined as

^{β " "} δP (³)

where V is the volume of the liquid. The flow rate F_compr, taking the compressibility factor into account, is then expressed as:

F = AT = -β V — — compr _{b t} PP ^ _T 6t <⁴>

This relation need only be considered when the pressure changes considerably during a run. This may occur as a result of compression of a bed of separation medium in the capillary, or the obstruction of channels or filters, or by the removal of obstructions. Again, a computer program can be used to maintain the product of β_p and δP/δT, and thereby the value of F_compr, constant. The total flow is then the algebraic sum of F^ and F compr' For relatively large-volume flows, the vessel can be a tank, bomb or other non- tubular vessel, with a volumetric capacity of 100 mL or greater, preferably from about 100 mL to about 10 L. A stirrer such as a magnetic stirrer can be used to achieve rapid equalization of the temperature. For relatively small-volume flows, the vessel can be a length of tubing, with inner diameter of abot 1.0 cm or less and volumetric capacity of 100 mL or less, and preferably and inner diameter within the range of about 0.01 mm to about 1.0 cm and a volumetric capacity within the range of about 0.01 mL to about 100 mL.

In the case of capillary chromatography, the driving liquid can be the same liquid entering the capillary where chromatographic separation is being conducted, but it is preferred that an intermediate reservoir or length of tubing separate the temperature rise vessel from the capillary, such that the driving liquid does not enter the capillary. In this way, the capillary is isolated from the temperature rise in the driving liquid. When an intermediate reservoir or length of tubing are thus used, the driving liquid can be one that is either miscible or partially or totally immiscible with the carrier liquid passing into the capillary. When the two liquids form separate phases, the intermediate reservoir or length of tubing must be configured or arranged such that only the carrier liquid enters the capillary.

The temperature rise vessel can be of any material that will retain the thermal expansoin liquid and that will not expand to any substantial degree in response to an increase in pressure of the liquid retained inside that is likely to be encountered during the operation of the pump (t^'.e. , excluding increases in pressure caused by vaporization). For a temperature rise vessel that is a length of tubing, the temperature rise tube when in operation is closed at one end so that volumetric expansion will drive the expanding liquid from the other end only. Metals such as stainless steel as well as other materials such as silica capillaries can be used for the temperature rise tube. The inner diameter of the tubing can vary, and will be selected according to the needs of the system, as indicated above. The volumetric capacity of the tubing can likewise vary, depending on the needs of the system as indicated above. Preferred ranges are given above, and further preferred ranges are a volumetric capacity of from about 0.03 mL to about 30 mL and an inner diameter of from about 0J mm to about 3.0 mm. The wall thickness of the tubing is also a factor, and will be selected accordingly. In most applications, the wall thickness will range from about 10 microns to about 1,000 microns.

The rate of temperature rise can vary as well, and will be selected according to the desired flow rate. In most cases, the temperature rise vessel and the driving liquid will be selected such that an appropriate rate of temperature rise will be from about 0.05 to about 20 degrees Celsius per minute, and preferably from about 0.1 to about 5 degrees Celsius per minute.

The temperature rise is achieved by any conventional means that is susceptible to a high level of control and rates within the ranges cited above. For tubing, immersion of the tubing in a thermally controlled liquid bath is a preferred method, particularly a temperature bath whose temperature is continually monitored and adjusted by computer. For vessels such as tanks and bombs, the temperature can be controlled by a heating coil in the tank or bomb interior, either electrically heated or having a heat-transfer fluid passing through the interior of the coil. In general, however, any heating device that will provide controlled heating at the desired rate can be used.

While the thermal expansion pump of this invention is of use in any application where a pulse-free flow of liquid at a highly controlled yet very slow rate is needed, the invention is of particular interest in capillary chromatography. The pump is useful with any kind of chromatographic bed, including those in which the bed is a continuous solid porous bed that has been formed in the capillary itself. An example of one such bed is that formed from a polymerization reaction mixture containing one or more water-soluble polymerizable compounds such as vinyl, allyl, acrylic and methacrylic compounds, and a crosslinking agent, and ammonium sulfate, in amounts such that:

(a) the sum of the weight percents of the monomers and crosslinking agents relative to the aqueous polymerization reaction mixture is within the range of about 10% to about 40%; (b) the mole fraction of crosslinking agent relative to all monomers present in the polymerization reaction mixture is within the range of about 0.3 to about 0.4; and

(c) the amount concentration of ammonium sulfate in the polymerization reaction mixture is within the range of about 1.8 to about 4.0 M. Examples of polymerizable compounds are vinyl acetate, vinyl propylamine, acrylic acid, butyl aery late, acrylamide, methacrylamide, glycidyl methacrylate, glycidyl acrylate, methylene-bis-acrylamide, and piperazine diacrylamide. For ion exchange columns, charged compounds, due to the inclusion of functional groups, will be included. Examples of functional groups for anion exchangers are quaternary ammonium groups with either three or four alkyl substitutions on the nitrogen atom, the alkyl groups being primarily methyl or ethyl, and in some cases themselves substituted, for example with hydroxyl groups. Examples of functional groups for cation exchangers are sulfonic acid groups and carboxylic acid groups, joined either directly to the resin or through linkages. Examples of functional groups for reversed phase chromatography are octylacrylate and octadecylacrylate. The crosslinking agent(s) will be selected in accordance with the monomers. For polyacrylamides and polymers of other forms of acrylic acid, examples of suitable crosslinking agents are bisacrylamides, diacrylates, and a wide range of terminal dienes. Specific examples are dihydroxyethylenebisacrylamide, diallyltartardiamide, triallyl citric triamide, ethylene diacrylate, bisacrylylcystamine, N,N'-methylenebis- acrylamide and piperazine diacrylamide. Appropriately derivatized, the column can be used for anion exchange, cation exchange, hydrophobic interaction, affinity, or reversed phase chromatography.

The following examples are offered as non-limiting illustration of the invention. EXAMPLES

Apparatus for Examples 1 through 3

A thermal expansion pump in accordance with this invention was constructed as shown in FIG. 2, where it is shown with a capillary column whose flow rate is controlled by the pump. Components of the pump 11 are a water bath 12 with built-in heater and thermostat, a computer 13 regulating the temperature of the water bath based on the thermostat potential, a spiral stainless steel tube 14 24.9 m in length, with inner diameter 0.75 mm and outer diameter 1.59 mm and an internal volume of 11.00 mL, and a pressure gauge 15. In experiments where a capillary of extremely small (15-micron) diameter was used, the stainless steel tube was replaced with a coiled fused silica capillary of length 1.37 m, inner diameter 320 microns, and outer diameter 405 microns. A control valve 16 consisting of a three-port high-pressure HPLC valve was connected to the pump discharge line, the valve containing two open-and-close ports 17, 18 and a titanium rotor 19 to increase or decrease the internal volume of the control valve. A Teflon tube 20 used in the formation of salt gradients joined the control valve output to a three-port switching valve 21. The switching valve led to a capillary column 22, a UV detector 23, and a precision micro-scale balance 24. Also shown in the drawing is a syringe 25 for drawing fluids through the system, as explained below.

The inner diameter of the gradient tubing 20 was selected in accordance with the inner diameter of the capillary column 22: for a 320 μm capillary column, gradient tubing with an inner diameter of 250 μm was used; for a 15 μm capillary column, gradient tubing with an inner diameter of 25 μm was used.

The micro-scale balance was Model AE 260-S from Mettler-Toledo AG (Greifensee, Switzerland), and was used for obtaining measurements of the flow rate through the capillary column 22. The balance weighed the column eluent collected in a glass vial 26, that was fed by a fused silica capillary (25 micron inner diameter) leading from the outlet end of the capillary column and passing into the vial through a hole in the cap of the vial. The hole was 0.5 mm in diameter to prevent evaporation of the column eluent. As an alternative to the micro-scale balance, a bubble flow meter was constructed by mounting a 10-cm long transparent glass tube (1.0 mm inner diameter) to a horizontal ruler and joining the tube to the capillary outlet by a press-fit connector. Both the glass tube and the connector were coated with vinyltrichlorosilane to lower the affinity of the tube and connector to water. Flow rates were determined by measuring the time required for the meniscus to travel between two tbin lines on the tube. Determination of Temperature Rise/Flow Rate Relation

Measurements of flow rate delivered by the thermal expansion pump as a function of the rate of temperature rise were compared with the calculated relation to determine the accuracy of the system. To perform the. measurements;: the stainless steel spiral tube 14 was first filled with deaerated, double-distilled water by an HPLC pump, keeping the syringe port 17 closed and the switch valve 21 open to the input line 31. A small back pressure of 5-10 bar was created while filling the spiral tube, thereby eliminating the risk of air bubble formation. The water bath was set at a starting temperature T₀ (usually between 40°C and 45°C), and the control valve 16 was turned to open the syringe port 17 and close the pump discharge port 18. With the switching valve 21 unchanged to provide a flow passage between the gradient tubing 20 and the input line 31, elution or equilibration buffer was drawn backward up the input line 31 through the switching valve 21 and into the gradient tubing 20, with the aid of the syringe 25. The switching valve 21 was then turned to connect the thermal pump 11 to the capillary column 22, and the controlled temperature rise at selected rates was begun, while pressure was recorded on the pressure gauge 15.

A stable pressure and flow rate were achieved within 10-20 seconds. The need for this equilibration time can be attributed to elasticity in the system, including the gradient tubing 20, a lag time in the interface between the computer 13 and the water bath 12, or both. While this affords the advantage of eliminating abrupt increases in pressure, a faster rate of increase of the pressure and flow rate can be obtained by adjusting the rotor 19 to lower the internal volume in the control valve 16.

The rate of temperature increase Δ77Δt required to deliver a particular flow rate AV/Δt using the stainless steel tube was calculated by Equation (2) above, and compared to actual flow rates measured by the micro-scale balance with an eluent temperature of 25°C (density 0.997 g/cm³). A temperature rise of from 41°C to 52°C was performed at rates ranging from 0.28°C/min to 2.20°C/min, in each case displacing a total volume of 52.4 μL. The value of α_τ used in Equation (2) was 4.237 x 10^"4 KJ, which is the mean of _τ values from the literature (Handbook of Chemistry and Physics, 68th ed., Robert C. Weast, ed., CRC Press, Inc., 1985) at twelve one-degree temperature increments over the range. The experimental temperature rise/flow rate relation is compared to the calculated temperature rise/flow rate relation in Table I, where each value is the mean of two weighings: TABLE I

Temperature Rise/Flow Rate Relation —

Measured vs. Calculated

Δ77Δt AVI At measured AV/At calculated

(°C/min) (μL/min) (μL/min)

0.28 1.19 1.309

0.37 1.64 1.745

0.55 2.54 2.618

0.69 3.14 3.272

1.10 5.04 5.236

1.57 7.31 7.480

2.20 10.32 10.472

The values in this table are plotted in FIG. 3, where the dashed line represents the measured data and the solid line represents the calculated relation. The closeness of the dashed and solid lines demonstrates a close correlation between the calculated and measured rates, confirming the predictability and reproducibility of the thermal pump.

Determination of Variation of Flow Rate With Column Back Pressure

To establish that the thermal expansion pump of FIG. 2 produces a flow rate that is proportional to the pressure drop across the column, two columns were prepared, one producing a back pressure of 12 bar to achieve a flow rate of 10 μL/min, and the other producing a back pressure of 66 bar to achieve the same flow rate. The bed dimensions were 320 μm internal diameter, 6 cm length, and 320 μm internal diameter, 26 cm length, respectively. The compositions of the beds differed.

The thermal expansion pump (with the stainless steel coil) was used to deliver a 20 mM sodium phosphate buffer, pH 6.2, to the columns over a range of flow rates, and back pressures were measured for each flow rate. The results are plotted in FIG. 4, where the "A" curve, with diamond-shaped points, represents the lower back-pressure column, and the "B" curve, with circular points, represents the higher back-pressure column. The plot shows that both columns gave a linear relation between the flow rate and the back pressure. The larger scattering of points at lower flow rates reflects the relatively small differences in the masses of the effluent fractions and the difficulties in making accurate measurements of these differences.

Preparation of Continuous Bed Separation Media in Column

Continuous bed separation media were prepared in a fused silica capillary column 140 mm in length (110 mm effective length), with inner diameter 320 μm and outer diameter 405 μm with an external polyimide coating, by the following procedures.

Before adding the monomer solution, a short section of the polyimide coating was burned off of the capillary surface to serve as a window for on-line detection. The capillary interior was then washed with toluene and then acetone, then treated for 30 minutes with 0.2 M NaOH, followed by 30 minutes with 0.2 M HCl, and finally rinsed with distilled water. The capillary wall was then activated with 3-methacryloyloxypropyl trimethoxysilane in acetone (30% by volume) to place methacryloyl acid residues on the internal capillary wall.

Continuous beds were prepared in the activated capillary for either cation exchange chromatography or hydrophobic interaction chromatography, using monomer solutions with the components and amounts shown in Table II:

TABLE II

Compositions of Monomer Solutions for Continuous Bed Capillary Columns

Amounts Used To Prepare Beds For:

Cation Hydrophobic

Exchange Interaction

Component Chromatography Chromatography

Piperazine Diacrylamide (g) 0.165 0.150

Methacrylamide (g) 0 130 —

2-Hydroxyethyl

— 0.100 Methacrylate (μL)

Acrylic Acid (μL) 16 —

Isopropyl Alcohol (g) — 0.15

5M NaOH (μL) 32 —

(NH₄)₂SO₄ (g) 0.08 0.06

Sodium Phosphate, 50 mM,

1.0 1.0 pH 7.0 (mL)

Each monomer solution was degassed with a stream of nitrogen and supplemented with 10 μL of 10% ammonium persulfate and 10 μL of 5% aqueous TEMED (N,N,N,N- tetramethylethylenediamine) -.oiution befor : being aspirated into the activated column. Polymerization proceeded for 24 hours.

EXAMPLE 1

This example illustrates the use of the thermal expansion pump of the present invention as the driving force for cation exchange chromatography, using a linear salt gradient.

A 48-μL linear salt gradient extending from 0 to 0.6 M NaCl in 20 mM sodium phosphate, pH 6.2 was prepared as follows. Sixteen (16) solutions were prepared in test tubes by mixing solution A (20 mM sodium phosphate, pH 6.2) with solution B (0.6 M NaCl in 20 mM Na^PO,,, pH 6.2) in different proportions, the volume percentage of solution B increasing in equal increments from 0% in the first test tube to 100% in the sixteenth test tube. Using the system shown in FIG. 1, the capillary column 22 was equilibrated with solution A delivered by the thermal expansion pump 11, with the switching valve 21 in position to direct the flow from the pump into the column. The switching valve was then turned to permit fluid to be drawn backward into the gradient tubing 20 through the input line 31, and by means of the syringe 25, 3-μL portions from each of the sixteen test tubes in succession, beginning with the sixteenth test tube (100% solution B) and ending with the first (100% solution A) were then drawn into the gradient tubing 20 to form the 48-μL positive salt gradient in the tubing.

To determine whether a smooth salt gradient had been prepared in this manner, a test run was made through the capillary with no sample having been added, and with monitoring at very high UV sensitivity (205 nm, 0.0005 absorption units (AU)). The detector output was an approximately even baseline without stepwise changes. This indicated an efficient mixing of adjacent buffer mixtures to form a smooth gradient, due to the hydrodynamic parabolic deformation of the interfaces between the segments combined with radial diffusion.

To illustrate a protein separation, the syringe 25 was used to draw a 1-μL aliquot of a protein test mixture into the tubing 20 (through the input line 31, with the switching valve 21 connecting the input line 31 to the gradient tubing 20), followed by 4 μL of the starting buffer (i.e., an additional 4 μL of the last of the sixteen buffers to be drawn into the tubing). The switching valve 21 was then redirected toward the capillary column 22, and the syringe 25 was replaced with a finger-tight plug. The chromatography experiment was then begun by starting the selected temperature rise in the water bath 12 and opening the control valve 16 by slowly turning the rotor 19 upwards. The protein mixture consisted of 0.4 mg/mL each of myoglobin (horse), cytochrome C, and lysozyme, plus 1.2 mg/mL of ribonuclease.

The coil in the thermal pump was the stainless steel coil described above, and the pump was operated with a programmed linear temperature gradient of 1.25°C/min, starting at 41 °C, and a constant pressure of 30 bar (measured), producing a flow rate of 5.8 μL/min. The capillary column was as described above, with 1 10 mm effective length and 320 μm inner diameter. Detection was performed at 280 nm wavelength. The resulting chromatogram is shown in FIG. 5, whei peak 1 represents myoglobin, peak 2 represents ribonuclease, peak 3 represents cytochrome C, and peak 4 represents lysozyme. A second cation exchange chromatography run was performed on the same protein mixture, but with a smaller capillary column and a smaller coil in the thermal expansion pump. The column was a fused silica capillary 140 mm in length (1 10 mm effective length), with inner diameter 15 μm and outer diameter 140 μm, treated and packed with a continuous bed in the same manner as the larger capillary. The pressure coil in the thermal expansion pump was the coiled fused silica capillary 1.37 m in length with an internal diameter of 320 μm. This coil produced a volume change that was 100-fold less than that of the stainless steel tube. The pump was operated with a programmed linear temperature gradient of 0.25°C/min, starting at 41°C, and a constant pressure of 28 bar (measured), producing a flow rate of 10 nL/min without splitting of the mobile phase. The sample volume was 0.01 μL. The resulting chromatogram is shown in FIG. 6. The similarity in appearance between the chromatograms of FIGS. 5 and 6 is significant. Both show a clean separation of the individual proteins and high resolution for each peak.

EXAMPLE 2

This example illustrates the use of the thermal expansion pump of the present invention as the driving force for hydrophobic interaction chromatography, again using a linear salt gradient although a decreasing gradient rather than an increasing gradient. The gradient mobile phase extended from 2.4 to 0 M (NH₄)₂SO₄ in 20 mM

Na_jPO,,, pH 6.8, and was prepared in a manner analogous to that of the gradient mobile phase of Example 1, using the 2.4 M (NH₄)₂SO₄ solution in one test tube, the solution lacking (NH₄)₂SO₄ in another, and fourteen graduated mixtures of the two in intermediate test tubes, and loading small aliquots from each tube in succession into the gradient tubing 20. The separation was then performed on a 1-μL protein mixture consisting of 0.07 mg/mL cytochrome C and 0.4 μL each of myoglobin (horse), ribonuclease, lysozyme, and α-chymotrypsinogen, using the hydrophobic interaction separation medium described above. The capillary column had an effective length of 100 mm and an inner diameter of 320 μm. The coil in the thermal pump was the stainless steel coil described above, and the pump was operated with a programmed linear temperature gradient of

1.25°C/min, starting at 41°C, and a constant pressure of 24 bar (measured), producing a flow rate of 5.8 μL/min. Detection was performed at 280 nm wavelength.

The resulting chromatogram is shown in FIG. 7, where peak 1 represents cytochrome C, peak 2 represents myoglobin, peak 3 represents ribonuclease, peak 4 represents lysozyme, and peak 5 represents α-chymotrypsinogen. Complete separation and high resolution were achieved.

EXAMPLE 3

This example compares the pressure vs. time performance of the thermal expansion pump of the present invention with a convention HPLC piston pump. The HPLC piston pump used in this comparison was Model No. 2150 obtained from LKB, Stockholm, Sweden. To achieve the low volumetric flow required for capillary chromatography, a splitting capillary was utilized (i.e., the pump discharge was divided between two capillaries arranged in parallel). Since this arrangement permitted the pump to operate at a flow rate higher than that delivered to the individual capillaries, the splitting capillary provided the system with a further advantage by rendering the disturbances caused by the reciprocating motion of the pistons less pronounced than they would otherwise have been. For the thermal expansion pump, the stainless steel coil described above was used.

Two continuous bed columns were used. The columns had different compositions and therefore different flow resistances. The bed heights were 120 mm and the inner diameters 320 μm. Back pressures of 6 and 10 bar were used, producing flow rates through the capillary of 50 μL/min and 10 μL/min, respectively. The thermal expansion pump was tested at the same flow rates as well as flow rates of 1 μL/min (at 1 bar back pressure) and 10 nL/min (at 28 bar back pressure), flow rates too low for the HPLC piston pump, plus 50 μL/min at 50 bar back pressure.

The pressure traces are shown in FIGS. 8a and 8b for the HPLC piston pump and FIGS. 9a and 9b for the thermal expansion pump. These traces indicate that the pressure traces from the thermal expansion pump were almost free from noise and fluctuations at all flow rates and back pressures, and considerably more stable than those from the HPLC piston pump. Even at 50μL/min (50 bar), which is a high flow rate for capillary chromatography, the pressure pulses were negligible. The steady pressure observed at flow rates of 1 μL/min and 10 nL/min is a prerequisite for efficient separations on very narrow bore columns.

EXAMPLE 4

FIG. 10 illustrates a variation on the apparatus of FIG. 2, designed to produce a higher- volume flow while still using the principles of the present invention. The liquid 40 whose expansion causes the flow in the thermal expansion pump in this example is retained in an insulated tank 41 that is closed except for one discharge port 42. With the tank completely filled with liquid, all thermal expansion of the liquid in the tank causes flow through the discharge port. The temperature of the liquid in the tank, and the rate of temperature rise, is controlled by a heating coil 43, in combination with a magnetic stirrer bar 44. Circulating inside the heating coil 43 is a heat transfer liquid, whose temperature is controlled by an external temperature control bath 45, similar to the temperature control bath 12 of FIG. 2. The temperature of the temperature control bath 45 is controlled by a built-in thermostat and a computer 46 similar to the computer 13 of FIG. 2. A circulation pump 47 causes the heat transfer liquid to circulate between the coil 43 in the thermal expansion liquid tank 42 and the coil 48 in the temperature control bath 45.

The volume of thermal expansion liquid 41 in the tank 41 is considerably greater than the volume of the thermal expansion liquid in the coil 14 of FIG. 2. The same increase in temperature therefore produces a greater increase in volume although the proportional increase relative to the total volume of thermal expansion liquid in both cases will the same, assuming equal coefficients of thermal expansion. The higher volumetric flow rate in FIG. 10 can be used for any application where a pulse-free flow is required; one example is the higher diameter chromatographic separation column 49 shown in the Figure. Detection of separated components is achieved by a conventional detector 50.

The foregoing is offered primarily for purposes of illustration. It will be readily apparent to those skilled in the art that modifications and substitutions in terms of the materials, procedures and other parameters of the system may be introduced without departing from the spirit and scope of the invention.

Claims

WE CLAIM:

1. A method for creating a continuous flow of liquid, said method comprising: (a) completely filling a vessel with liquid, said vessel being of substantially non-expandable material, with means for rapid equalization of temperature of liquid contained therein, to substantially equalize temperature changes therein within about 5 seconds; and (b) while said vessel is filled with said liquid and closed except for one outlet port, raising the temperature of said liquid within said vessel at a controlled rate of temperature rise ranging from about 0.05┬░C per minute to about 20┬░C per minute, causing said liquid to be discharged from said outlet port due to thermal expansion of said liquid, said rate of temperature rise being selected to achieve a selected rate of liquid discharge through said outlet port.

2. A method in accordance with claim 1 in which said controlled rate of temperature rise of step (b) is obtained by external application of heat to said vessel.

3. A method in accordance with claim 1 in which:: step (a) comprises filling a length of tubing having first and second ends with said liquid, said tubing being formed of heat-transmissive material and having a volumetric capacity of from about 0.01 mL to about 100 mL and an inner diameter of from about 0.01 mm to about 1.0 cm; and step (b) comprises raising the temperature of said tubing at a controlled rate of temperature rise ranging from about 0.05┬░C per minute to about 20┬░C per minute with said first end of said tubing closed, causing said liquid to be discharged from said second end due to thermal expansion of said liquid, said rate of temperature rise being selected to achieve a selected rate of liquid discharge.

4. A method in accordance with claim 3 in which said tubing is immersed in a controlled-temperature bath and (b) is performed by raising the temperature of said bath at said controlled rate.

5. A method in accordance with claim 3 in which said liquid has a coefficient of thermal expansion within the range of about 1 x 10^"4 K^"1 to about 20 x 10"⁴ K^"1 at 1 atmosphere within a temperature range of about 20┬░C to about 100┬░C.

6. A method for separating a mixture of species in a liquid sample into components by capillary chromatography by passing a carrier liquid containing said sample through a capillary at a controlled volumetric flow rate, said method comprising: (a) joining an inlet end of said capillary to an enclosed reservoir filled with said carrier liquid, said enclosed reservoir fed by a length of tubing of heat- transmissive material with a volumetric capacity of from about 0.01 mL to about 100 mL and an inner diameter of from about 0.01 mm to about 1.0 cm, said tubing filled with a thermally expandable liquid; (b) injecting said liquid sample into said inlet end of said capillary; and (c) raising the temperature of said tubing at a controlled rate of temperature rise ranging from about 0.05┬░C per minute to about 20┬░C per minute with said first end of said tubing closed, causing said thermally expandable liquid to be discharged into said reservoir by thermal expansion of said thermally expandable liquid and thereby driving said carrier liquid from said reservoir through said capillary, said rate of temperature rise being selected to produce said controlled volumetric flow rate of carrier liquid.

7. A method in accordance with claim 6 in which said tubing is immersed in a controlled-temperature bath and (b) is performed by raising the temperature of said bath at said controlled rate.

8. A method in accordance with claim 6 in which said liquid has a coefficient of thermal expansion within the range of about 1 x 10^"4 K^"1 to about 20 x 10^"4 K^"1 at 1 atmosphere within a temperature range of about 20┬░C to about 100┬░C.

9. A method in accordance with claim 6 in which said reservoir is a length of connecting tubing having an inner diameter ranging from about 0.3 to about 3.0 times the inner diameter of said capillary.

10. A method in accordance with claim 9 further comprising filling said connecting tubing with said carrier liquid with a salt gradient along the length of said connecting tubing prior to (c).