This invention relates generally to method and apparatus for the precise measurement of the flowrate of pressure-driven liquids and more particularly, for precisely measuring fluid flow rate, thereby providing for accurate control of the flowrate and composition, of liquids injected into high pressure liquid chromatography systems.
In liquid chromatography, chemical separations may be performed by flowing a fluid (the mobile phase) past an immobilized material (the stationary phase) inside a liquid chromatography (LC) column. By injecting a sample consisting of multiple components into one end of the LC column, allowing them to be separated into distinct bands as the sample flows through the LC column, and detecting those bands near the exit end of the LC column this technique is used for chemical analysis of mixtures. In those systems, the separation is governed by the dynamic partitioning of the analyte between the mobile phase and the stationary phase. Control of the separation may be achieved by adjusting the flowrate as well as the composition of the mobile phase or the stationary phase or both to influence analyte partitioning.
High-performance liquid chromatography (HPLC) is an established analytical technique that relies on high-pressure means, that can be mechanical pumps (generally a gear- or cam-driven pump capable of generating pressures in excess of 5,000 psi), to drive a fluid sample through a specially prepared column. The HPLC separation material or stationary phase is typically a thick bed packed with fine particles. HPLC columns can also be packed with special polymers or resins. Regardless of the column packing used, the HPLC column presents a very large resistance to flow, hence the need for high pressures to drive the sample being analyzed through the system.
Conventional HPLC systems typically employ separation columns of about 3-5 mm in diameter and flow rates xcx9c3-5 mL/min. However, miniaturization of the separation column (microbore columns) offers several advantages, including improved efficiency, mass detection sensitivity, low solvent consumption, small sample quantity, and easier coupling to a detector such as mass spectrometers and flame-based detectors and several analytical methods using miniaturized or capillary columns have been developed for micro-HPLC. These columns generally have inside diameters of 1 mm or less.
Gradient elution is a process that has been developed for HPLC and micro-HPLC wherein the mobile phase composition is varied during separation for separating a wide variety of complex samples. The gradient elution approach is useful when the components of the mixture have a range of properties and no single mobile phase composition is appropriate for separating all of them. In HPLC, the creation of the solvent gradient is typically accomplished by using two or more means for generating high-pressure to deliver two different fluids into a small mixing chamber. The mixed liquid is then forced into the HPLC column. The composition of the mobile phase is controlled and varied by adjusting the relative flowrate from the individual pumps to achieve gradient elution.
Low volume flowrates of varying composition are required in capillary-based separation techniques when a single mobile phase is insufficient to separate all of the chemical components of a sample. Conventional approaches of providing a variable composition mobile phase at low flowrates are expensive to implement, slow to respond to external control, and are generally unreliable in composition for flow rates of less than 1 xcexcL/min. Although the miniaturization of separation columns offers the above-mentioned advantages, accurately and consistently delivering a xcexcL/min gradient flow into a capillary column (e.g., 10-100 xcexcm i. d.) packed with micrometer-size particles poses a difficult problem.
Low volume flowrates (typically in the range of nL/min to tens of nL/min) are also required for liquid chromatography/mass spectrometry (LC/MS) systems, where the output of a liquid chromatography column is injected into the input of a mass spectrometer.
Further, there is, as yet, no convenient means for handling fluctuations in the output of the high pressure pumps conventionally used in HPLC. These fluctuations can affect the flowrate of the mobile phase which, in turn, can have a deleterious effect on the analysis. Conventional means of correcting for such operational fluctuations, such as a servo loop cannot be used because the heavy damping required makes the system time response too slow to use servo control.
What is required is a system that will provide pressure driven flows at precisely measured and thus accurately controlled flowrates, wherein the flowrate can be in the range of mL/min to nL/min. Further, the system must be compatible with microbore columns and the desire for small sample quantity, low solvent consumption, improved efficiency, the ability to run samples in parallel, and field portability. In addition, it is desirable that the flowrate measuring system provide a signal that can be used for a servo loop or feedback control system for a high pressure pumping system.
It is an object of this invention to provide an apparatus for accurate measurement of the flowrate of liquids in high pressure systems.
It is a further object is to provide an apparatus for accurate measurement of the flowrate of liquids in high pressure liquid chromatography (HPLC) systems.
It is yet another object of this invention to provide for accurate measurement of liquid flowrates in micro-HPLC systems at flowrates in the range of mL/min to nL/min.
A further object is to provide for accurate control of fluid flowrate and composition of the mobile phase in gradient elution chromatography systems.
Yet another object of the invention is to provide a system of feedback control to eliminate fluctuations that can occur during the operation of high pressure generating means.
Another object is to provide a substrate having a microchannel system disposed thereon comprising at least one electrokinetic pump and associated flowmeter in combination with a chromatography column.
Another object of the invention is to provide a method for accurately measuring and controlling the flowrate of liquids in a high pressure system.
These and other objects of the present invention will become apparent from the following description and accompanying drawings.
The invention involves measuring the pressure drop through a porous bed of material disposed at the outlet of a high pressure pumping means and by the use of Darcy""s Law, determining the flowrate of the pumped liquid. The pressure drop through the porous bed of material not only provides an accurate determination of the fluid flowrate but also provides for controlling the fluid flowrate. Further, the pressure drop through the porous bed of material can provide an error signal that can be used as input to a servo loop to control the pressure supplied by the high pressure pumping means and thereby eliminate fluctuations in fluid flowrate. The system provides superior performance, including faster response and higher reliability at very low flowrates (xcx9cnL/min), and the ability to produce arbitrary gradient profiles, and can be used for a variety of applications.
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