Monitoring mobile phase composition in a liquid chromatography system using a thermal flow sensor

Abstract

To provide a method and an apparatus for monitoring a solvent composition together with a mobile phase in a fluid conduit during gradient elution in a liquid chromatography system.SOLUTION: The mobile phase of the liquid chromatography system 6 is composed of a plurality of known solvents, and the liquid chromatography system includes a thermal flow sensor 20. The mobile phase is passed by the thermal flow sensor at a known flow rate, a measurement of a transport property of the mobile phase is obtained via the thermal flow sensor, and the measurement is converted to a solvent composition using a pre-established correlation.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of liquid chromatography systems, and more particularly to the compositional analysis of the mobile phase in a liquid chromatography system using a thermal flow sensor. [Background technology]

[0002] High-performance liquid chromatography (HPLC) and ultra(H)PLC (U(H)PLC) are powerful techniques used in analytical chemistry to separate, identify, quantify, and / or purify compounds or analytes in a sample. Liquid chromatography separates samples containing various compounds in time and space by passing the sample through a chromatography column using a mobile phase. The mobile phase, consisting of a solvent or solvent mixture, carries the sample to and through the chromatography column. The chromatography column contains a stationary phase that selectively retains the sample's analytes due to their various physicochemical properties, i.e., retains each analyte to a different extent. Depending on the stationary phase, various separation principles can be used. For example, reversed-phase chromatography (separation based on hydrophobic interactions), normal-phase chromatography (separation based on hydrophilic interactions), ion-exchange chromatography (separation based on charge), and size-exclusion chromatography (separation based on molecular size) can be used, which allows analytes to elute from the column at different times (i.e., the analytes have different retention times). Regardless of the separation principle, one of the critical aspects of a liquid chromatography system is the accurate delivery of the intended mobile phase composition to obtain accurate, reliable, and reproducible results. Therefore, accurate solvent delivery is paramount to obtain high chromatographic performance.

[0003] In liquid chromatography, analytes can be eluted from a column using either isocratic or gradient elution. In isocratic elution, a single mobile phase consisting of a single solvent or a mixture of solvents in a fixed ratio is maintained throughout the chromatographic run. This results in a consistent elution strength and profile throughout the chromatographic run. In contrast, when using gradient elution, the mobile phase composition is changed over time during the chromatographic run. This change involves changing the composition (i.e., ratio) of solvents in the mobile phase according to a predetermined gradient program. Typically, the concentration of one solvent (the "weak" elution solvent) decreases while the concentration of another solvent (the "strong" elution solvent) increases. By adjusting the gradient program, analysts can achieve improved resolution, shorter analysis times, and increased sensitivity compared to isocratic elution. Because the composition of the mobile phase directly affects analyte retention times, resolution, and peak shape, ensuring the correct solvent composition at a given time and location within the system is crucial for accurate and reproducible chromatographic performance. Therefore, (real-time) monitoring of solvent composition can be used as quality control or validation to ensure proper functioning of chromatographic systems and consistency between separate chromatographic systems.

[0004] Gradient elution in a chromatography system can be achieved, for example, by using binary pumps (one pumping a solvent, e.g., water, from channel A and one pumping a different solvent, e.g., ACN, from channel B), with the speed of each individual pump being varied during the run to essentially dictate the solvent composition. However, pump speed is not the optimal measure for determining the solvent composition at any given point in the system away from the pump, because solvent composition can be affected by mixing efficiency, pump accuracy, fluid leaks, pump wear, and / or unexpected additional dwell volume within the system. Systems are designed with specific tubing configurations that have specific, predictable dwell volumes. However, systems can also contain unintended additional dwell volumes caused by improper connections of tubing, fittings, and columns, which vary from system to system and ultimately lead to unpredictable changes in retention times. The presence of additional dwell volume in tubing, fittings, and other components can result in unexpected gradient delays (i.e., the dwell volume causes a delay in gradient delivery) and inaccuracies in gradient formation due to diffusion effects. Furthermore, dwell volume also contributes to sample dispersion, where analytes disperse or diffuse along the flow path, resulting in peak broadening and reduced chromatographic resolution. Therefore, the time it takes for a solvent mixture to traverse the dwell volume can also affect the timing and shape of the gradient profile, impacting the reproducibility and accuracy of chromatographic analyses. Therefore, minimizing dwell volume is particularly important in U(H)PLC, which uses small-diameter columns and low flow rates. Furthermore, even when measures are taken to optimize solvent delivery using high-quality pumps with leak protection and minimize pulsations to maintain a constant flow rate, dissolved gases in the mobile phase can affect the accuracy of the flow rate and gradient composition, resulting in chromatographic performance variations, even with in-line degassing. Therefore, achieving accurate gradients is difficult in practice.

[0005] UV-Vis detection is one of the most common detection methods in liquid chromatography, and in addition to detecting actual analytes, UV-Vis detectors are used to measure solvent composition to verify the compositional accuracy of pump(s) and to run step gradient profiles with mobile phase spiked with chromogenic compounds to determine the dwell volume of the system (see Non-Patent Document 1). Such tests are often performed as part of operational qualification (OQ) or quality control (QC) to ensure proper functioning of the system and further ensure that it is operating according to its intended performance and meeting quality specifications.

[0006] However, UV-Vis detectors have several drawbacks when it comes to measuring solvent composition. First, common HPLC solvents (e.g., water, MeOH, or ACN) have low absorbance in the UV spectrum, necessitating the addition of trace amounts of a strongly absorbing analyte, such as acetone, as an internal standard to measure solvent composition and determine dwell volume. Because acetone is highly volatile, its post-spike concentration in the buffer reservoir tends to change over time. Furthermore, low-flow measurements require small-volume UV-Vis sensors, but these sensor cells are expensive, prone to error, and susceptible to cell clogging. Furthermore, UV sensors cannot withstand the high pressures of the mobile phase being measured. Current UV sensors are generally limited to pressures below 130 bar (1,900 psi). Therefore, determining mobile phase composition and system dwell volume requires replacing the column with a conduit that allows the UV detector to measure the gradient profile. Finally, UV-Vis detectors drift over time, necessitating frequent calibration.

[0007] Therefore, there is a need in the art for new means and methods for determining solvent composition and / or dwell volume, especially in low flow-based and / or high pressure-based systems.

[0008] The present invention provides a novel, inexpensive and reliable method for analyzing the composition of mobile phases that can be performed as part of Operational Qualification (OQ) or Quality Control (QC) to ensure the proper functioning of a chromatographic system. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] LCGC NORTH AMERICA VOLUME 37 NUMBER 6 JUNE 2019 Summary of the Invention

[0010] Against this background, it may be considered an object of the present disclosure to enable monitoring of solvent composition in liquid chromatography systems.

[0011] One or more of these objectives may be met by the aspects of the present disclosure described below.

[0012] A first aspect of the present disclosure relates to a method of monitoring solvent composition in a liquid chromatography system, preferably during gradient elution, along with a mobile phase in a fluid conduit, wherein the mobile phase comprises or consists essentially of a plurality of known solvents, the liquid chromatography system including a flow sensor adjacent to, and preferably exposed to, the mobile phase in the fluid conduit, the method comprising: passing a mobile phase through a flow sensor at a known flow rate; - obtaining measurements of transport properties, such as thermal, electrical or viscous properties of the mobile phase via a flow sensor; - Converting the measurements to solvent composition using a pre-established correlation, e.g., a correlation curve.

[0013] The use of flow sensors adapted to measure transport properties, such as thermal, electrical, or differential pressure flow sensors, offers several advantages over traditional UV-Vis detectors, including cost, commercial availability, ability to straighten flow paths (and thus be less susceptible to clogging), ability to operate at high pressures, and no need for color additives. Furthermore, such flow sensors (especially thermal flow sensors) are stable over time and do not require frequent recalibration.

[0014] In this context, the mobile phase comprises or consists essentially of multiple known solvents, e.g., water and acetonitrile. However, it should be understood that a single solvent may contain one or more trace impurities depending on the purity of the solvent. It should also be understood that the solvent may contain additives, such as acids (e.g., TFA) or bases (e.g., NH4HCO3), commonly used in the art to adjust the pH of the mobile phase. In some embodiments, the mobile phase may comprise or consist essentially of two solvents (i.e., a binary mobile phase). In some embodiments, the mobile phase may comprise or consist essentially of three solvents (i.e., a ternary mobile phase), or in some cases, four known solvents (i.e., a quaternary mobile phase).

[0015] In this context, gradient elution should be understood as any change in the solvent composition of the mobile phase occurring during a chromatographic run, and thus may refer to, for example, a linear gradient, a step gradient, and / or a mixture thereof.

[0016] In this context, the transport properties of a mobile phase are properties that describe how the mobile phase transfers mass, energy, or momentum within the fluid. Examples of transport properties are: thermal properties, such as thermal conductivity or specific heat capacity, electrical properties, such as resistivity, capacitance, or inductance, and - Viscosity properties, such as dynamic viscosity.

[0017] Optical properties, on the other hand, describe how a material interacts with light (electromagnetic waves) and include properties such as refractive index, absorbance, reflectance, and transmittance. These properties do not describe the movement or transport of matter or energy within the material itself, but rather how the material affects light that passes through or interacts with it.

[0018] This method, commonly used in chromatography, relies on solvents that differ in at least some transport properties. For example, a commonly used solvent pair in chromatography is 0.1% formic acid in water and acetonitrile. In contrast to pure acetonitrile, which has an extremely low electrical conductivity of around 7 μS / cm, 0.1% formic acid has a relatively high electrical conductivity of approximately 154 μS / cm at 25 °C. In other words, there is a roughly 20-fold difference in resistivity between these two exemplary mobile phase solvents. As another example, the dynamic viscosity of water at 25 °C is approximately 0.89 mPa·s, while the dynamic viscosity of acetonitrile (CH3CN) at 25 °C is approximately 0.35–0.44 mPa·s—a significant difference of approximately 2-fold.

[0019] Thus, the transport properties observed for a mobile phase of known solvent at a known flow rate and, in some cases, temperature can be used to derive the solvent composition (i.e., the solvent composition that should result in the observed transport properties).

[0020] In a preferred embodiment, the flow sensor is a thermal flow sensor including a heat-generating element adjacent to, and preferably exposed to, the mobile phase in the fluid conduit, and the method includes the step of introducing a known amount of thermal energy into the mobile phase via the heat-generating element, the transport property being a thermal property such as thermal conductivity or specific heat capacity, and measurements being obtained in response to the known amount of thermal energy introduced by the heat-generating element into the mobile phase via the thermal flow sensor.

[0021] Thermal flow sensors offer several advantages over traditional UV-Vis detectors, including cost, commercial availability, ability to straighten flow paths (and therefore less likely to clog), ability to operate at high pressures, and no need for color additives. Furthermore, thermal flow sensors are stable over time and do not require frequent recalibration. Furthermore, thermal flow sensors are more sensitive than electrochemical sensors, and, at least when used to measure mobile phase viscosity, are often more compact, rugged, and stable than differential pressure flow sensors.

[0022] Additionally or alternatively, the plurality of known solvents in the mobile phase preferably comprises two known solvents (i.e., a binary mobile phase, e.g., water and acetonitrile), but may also comprise three known solvents (i.e., a ternary mobile phase), or even four known solvents (i.e., a quaternary mobile phase). Each of the plurality of known solvents has a distinct thermal property, such as a distinct specific heat capacity or a distinct thermal conductivity. During gradient elution, the composition of the mobile phase can be changed only with respect to two of the known solvents. Thus, in the case of a ternary mobile phase, one of the known solvents can be maintained at a constant proportion of the mobile phase during gradient elution. Similarly, in the case of a quaternary mobile phase, two of the known solvents can be maintained at a constant proportion of the mobile phase during mobile phase gradient elution.

[0023] Additionally or alternatively, a flow sensor can be positioned in the fluid conduit downstream of the mixer and upstream of, preferably adjacent to, the chromatography column. Most preferably, the flow sensor is positioned immediately upstream of and adjacent to, the chromatography column to accurately determine the dwell volume of the chromatography system and further determine the solvent composition at the column inlet, thereby accounting for diffusion effects that arise due to dwell / dead volume and affect solvent composition. Alternatively, but less preferably, the flow sensor can be positioned downstream of the chromatography column.

[0024] The disclosed method can be used in the operational qualification (OQ) or quality control (QC) of any analytical (or preparative) liquid chromatography system, and thus can be used with a variety of chromatography column diameters and at a variety of flow rates and pressures, for example, in HLPC, UPLC, UHPLC, capillary LC, or nanoflow LC systems.

[0025] Additionally or alternatively, the known flow rate can be a substantially constant flow rate. Alternatively, the known flow rate can involve a known flow rate gradient. As another example, the known flow rate can be known but not constant, and the method can involve compensating for the known, non-constant flow rate via a compensation algorithm. For example, keeping the flow rate through a flow sensor located immediately upstream of a chromatography column constant facilitates the conversion of flow rate measurements to solvent composition because it minimizes the number of parameters required in the correlation table (or algorithm). However, it is also possible to convert varying flow rates to solvent composition measurements, as long as the flow rates are known and mapped a priori. Thus, while the present disclosure teaches the use of a constant flow rate for the conversion to composition, in more complex cases, varying flow rates may be used for the same purpose.

[0026] Additionally or alternatively, the known flow rate can be in the range of 1 nL / min to 20 mL / min, preferably 1 nL / min to 2 mL / min, more preferably 5 nL / min to 200 μL / min, and even more preferably 10 nL / min to 100 μL / min. It would be advantageous if this method utilizing a flow sensor could be applied to monitor solvent composition at such low flow rates. Additionally or alternatively, the known flow rate can be up to 20 mL / min, preferably up to 2 mL / min, or more preferably up to 100 μL / min. Additionally or alternatively, the known flow rate can be at least 1 nL / min, preferably at least 5 nL / min, or more preferably at least 10 nL / min.

[0027] The disclosed methods are particularly useful in LC systems operating at high pressures where the mobile phase pressure precludes the use of UV detectors, which typically can only withstand pressures of about 10-35 bar (for standard UV detectors) or about 100-140 bar (for high-pressure UV detectors). Thus, UV detectors cannot withstand the pressures upstream of the chromatography column, which often range from 200 to 1,200 bar, depending on the LC system.

[0028] Additionally or alternatively, the pressure of the mobile phase (upstream of the chromatography column) may be at least 200 bar, suitably at least 300 bar, preferably at least 400 bar, more preferably at least 500 bar, even more preferably at least 700 bar, or most preferably at least 1000 bar. It would be advantageous if this method, which utilises a flow sensor as opposed to a UV-VIS sensor, could be applied to such high pressure liquid chromatography processes.

[0029] Additionally or alternatively, the flow sensor may include a pipe section, the inlet and outlet ends of which may form part of a fluid conduit. The fluid volume of the pipe section between the inlet and outlet ends may be up to 1 μL, preferably up to 150 nL, or more preferably up to 100 nL. This has the advantage of limiting the dwell volume added to the liquid chromatography system.

[0030] Additionally or alternatively, the flow sensor may include at least one first sensing element and at least one second sensing element exposed to the mobile phase downstream of the first sensing element.

[0031] Additionally, if the flow sensor is a thermal flow sensor, a heating element can be disposed between the first sensing element and the second sensing element. The first and second sensing elements can be disposed symmetrically on either side of the heating element. Additionally, the measurement is the temperature difference between the first and second sensing elements, which can be used to derive thermal properties of the mobile phase, such as thermal conductivity or specific heat capacity.

[0032] Additionally or alternatively, the fluid conduit may include a measurement volume defined from the first sensing element to the second sensing element, the measurement volume being at most 50 nL, preferably at most 25 nL, or more preferably at most 10 nL. For example, the measurement volume may be defined as the volume from a cross section of the fluid conduit at the first sensing element to a cross section of the fluid conduit at the second sensing element.

[0033] Additionally or alternatively, the mobile phase may comprise laminar flow at the flow sensor, preferably with a Reynolds number of up to 2300. The liquid chromatography system may include one or more flow conditioning elements disposed upstream of the flow sensor. For example, a first flow conditioning element may be configured to condition the mobile phase for laminar flow at the flow sensor, and / or a second flow conditioning element may be configured to mix the mobile phase to ensure adequate mixing at the flow sensor.

[0034] Additionally or alternatively, the thermal flow sensor may preferably be a calorimetric thermal flow sensor. However, in alternative embodiments, a hot wire thermal flow sensor, a hot film thermal flow sensor, or a time-of-flight thermal flow sensor may be utilized. A time-of-flight thermal flow sensor includes a heating element that generates a heat pulse and a temperature sensing element located a known distance from the heater. The solvent composition can then be determined from the propagation time required for the heat pulse to reach the temperature sensing element at a known flow rate. A hot wire or hot film flow sensor includes a first heating element exposed to the flow. The effect of the flow on the power consumption or temperature of the hot wire / hot film is proportional to the solvent composition of the mobile phase at a known flow rate. Similar to a hot wire / hot film flow sensor, a calorimetric flow sensor also has a heating element exposed to the flow. Furthermore, a calorimetric flow sensor includes two temperature sensors: a first sensing element upstream of the heating element and a second sensing element downstream of the heating element. The temperature sensors are arranged symmetrically with respect to the heating element. In the absence of flow, the heating element ideally creates a symmetrical heat distribution around itself, so the temperature sensors should measure the same temperature. However, flow disrupts the heat distribution, resulting in the temperature of the downstream second sensing element being higher than the upstream first sensing element. Therefore, at a known flow rate, the temperature difference ΔT between the two temperature sensing elements is a function of the heat capacity or thermal conductivity of the solvent and, therefore, the solvent composition of the mobile phase.

[0035] Additionally or alternatively, the known amount of energy can be a pulse of energy or a continuous power.

[0036] Additionally or alternatively, the method may further comprise the steps of: - determining the composition error by comparing the obtained solvent composition with the expected solvent composition.

[0037] In a first embodiment, the method may be performed as part of an operational qualification (OQ) prior to starting up a liquid chromatography process, and - initiating a liquid chromatography process in accordance with a determination that the composition error is below a predetermined threshold; - sending an alert to the liquid chromatography system in accordance with a determination that the composition error is above a predetermined threshold.

[0038] This first embodiment of the method may further include removing the flow sensor from the fluid conduit and resealing the fluid conduit before initiating the liquid chromatography process.

[0039] In a second embodiment, the method is carried out during a liquid chromatography process, and further - continuing the liquid chromatography process in accordance with a determination that the compositional error is less than or equal to a predetermined threshold; - sending an alert to the liquid chromatography system in accordance with a determination that the composition error is above a predetermined threshold.

[0040] This second embodiment of the method may further include performing the method continuously to monitor the solvent composition in the liquid chromatography system in real time.

[0041] Additionally or alternatively, this method may further comprise a third embodiment that combines the first embodiment (i.e., performing a start-up check before starting the liquid chromatography process) with the second embodiment (i.e., monitoring the solvent composition during the liquid chromatography process), in which the flow sensor remains attached during the liquid chromatography process.

[0042] A second aspect of the present disclosure relates to a method of obtaining a pre-established correlation for use in a method according to the first aspect, said method comprising: - passing a mobile phase containing a predetermined solvent composition of a plurality of known solvents through a thermal flow sensor at a known flow rate; - introducing a known amount of heat energy into the mobile phase via a heat generating element of a thermal flow sensor; - obtaining a measurement of the dissipation of thermal energy from the heat-generating element via a thermal flow sensor; - preferably repeating the foregoing steps for one or more additional predetermined solvent composition(s); - Correlating the measurement(s) with the predetermined solvent composition(s) to obtain a pre-established correlation.

[0043] It is also envisioned that by measuring solvent composition and flow rate pairs and establishing a correlation matrix, the thermal flow sensor analysis can derive the solvent composition at different, known, constant flow rates.

[0044] A third aspect of the present disclosure relates to a chromatography system including a thermal flow sensor for monitoring the solvent composition of a binary mobile phase, the thermal flow sensor being in fluid communication with a chromatography column and a sample loop during elution, the thermal flow sensor being positioned upstream of, and preferably adjacent to, the chromatography column. [Brief explanation of the drawings]

[0045] Embodiments of the present invention are described in more detail below with reference to the accompanying figures, which illustrate one way of implementing the invention and are not to be construed as limiting other possible embodiments falling within the scope of the appended claim set. [Figure 1] 1 shows a simplified liquid chromatography system including a thermal flow sensor for use in a method according to a first aspect of the present disclosure. [Figure 2] 1 illustrates the operating principle of a thermal flow sensor for use in the method according to the first aspect of the present disclosure. [Figure 3A] 10 shows exemplary results of a correlation sequence to obtain a pre-established correlation for use in a method according to a first aspect of the present disclosure. [Figure 3B]1 shows an exemplary correlation curve for use in the method according to the first aspect of the present disclosure. [Figure 4] The operating principles of an electrical flow sensor and a differential pressure flow sensor are shown. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention relates to the discovery that thermal flow sensors can be used in the compositional analysis of mobile phases in liquid chromatography systems. This discovery enables the use of thermal flow sensors in operational qualification (OQ) or quality control (QC) to ensure that liquid chromatography systems are operating according to their intended performance and meet quality specifications. Flow sensors are commonly employed in HPLC systems to measure the flow rates of each individual solvent prior to mixing the mobile phases in the chromatography system, allowing for real-time validation of HPLC system performance.

[0047] To measure solvent composition, a thermal flow sensor can be placed in the HPLC system 6 between the mixer and the chromatography column to measure the composition of the mobile phase. Preferably, however, the thermal flow sensor is located adjacent to the inlet of the chromatography column to account for dwell volume in the system. FIG. 1 shows a simplified embodiment of the present invention in a typical setup of an HPLC system 6 with binary pumps 7a and 7b drawing solvent from two flasks 8a and 8b (i.e., from channels A and B) through a filter (not shown). The pumps are conveniently monitored and controlled individually using flow sensors (not shown) in a feedback loop to ensure constant flow rates. The solvent passes through the mixer 10 and is delivered to the chromatography column 16 via the sample injector 13. A thermal flow sensor 20 is positioned between the sample injector 13 and the chromatography column 16. In one preferred embodiment, the thermal flow sensor is positioned adjacent to the inlet of the chromatography column 16.

[0048] In this context, it should be understood that FIG. 1 illustrates only one HPLC setup to illustrate the preferred placement of the thermal flow sensor.

[0049] It will therefore be apparent that the present invention may be used in any setup that uses, for example, a quaternary pump instead of a binary pump, or a cartridge or trapping column before the sample loop, as described, for example, in WO 2018 / 059639 A1 and WO 2018 / 059640. Regardless of the setup of the chromatography system, a thermal flow sensor is preferably positioned close to the inlet of the chromatography column, for example, to measure the mobile phase composition in real time.

[0050] Flow sensors in HPLC systems can include piston-type flow sensors, pressure-type flow sensors, or thermal-type flow sensors. Thermal-type flow sensors measure the flow rate of a fluid (liquid or gas) using the principle of heat transfer. They rely on the ability of fluid flow to change the thermal profile around the sensor.

[0051] FIG. 2 illustrates the operating principle of a recommended thermal flow sensor 20. The thermal flow sensor 20 includes a heating element 21, a first temperature sensor 22 positioned upstream of the heating element 21, and a second temperature sensor 23 positioned downstream of the heating element 21. The role of the heating element 21 is to dissipate thermal energy into the mobile phase 11 by passing an electric current through the heating element via resistive heating, delivering a known amount of energy. As the mobile phase 11 flows through the heating element 21, heat is transferred from the heating element 21 to the mobile phase 11 via convection at a known flow rate. The first and second temperature sensors 22, 23 detect a heat distribution 24 (or thermal profile) by measuring the temperature of the liquid before and after passing through the heating element 21. In the absence of flow, the heat distribution 24 remains symmetrical, and the temperatures of both sensors 22, 23 remain equal (if the sensors 22, 23 are positioned symmetrically around the heating element 21). As the mobile phase flows across the sensors 22, 23, this equilibrium is disrupted. As illustrated in Figure 2, fluid flow transports heat from the heating element 21 toward the downstream temperature sensor 23, distorting the heat distribution 24. This shifts the heat distribution 24, resulting in a temperature difference between the two sensors 22, 23. This temperature difference can be correlated to flow rate because the heat transfer rate is proportional to the fluid velocity if the thermal properties of the fluid remain constant. As fluid flow rate increases, more heat is transported away from the heating element, resulting in a more significant temperature difference between the sensors, and vice versa. Thermal flow sensors are widely used in measuring flow rates in HPLC pumps.

[0052] The inventors have found that a thermal flow sensor can be used in a method for the compositional analysis of a solvent mixture based on the realization that the heat distribution 24 around the heating element 21 is affected not only by the flow rate but also by the thermal properties of the mobile phase, such as the thermal conductivity or specific heat capacity, which are determined by the specific composition of the mobile phase (the ratio and type of solvents in the mobile phase).

[0053] For example, if the thermal conductivity of the mobile phase changes over time during gradient elution, the temperature difference between the first temperature sensor 22 located upstream and the second temperature sensor 23 located downstream will also change because the mobile phase with lower thermal conductivity will transfer heat at a lower rate than the mobile phase with higher thermal conductivity. Similarly, in this case, if the flow rate is held constant, a mobile phase with lower specific heat capacity will result in a higher temperature difference than a mobile phase with higher specific heat capacity. The inventors have discovered that the use of conventional thermal flow sensors can provide an accurate, reliable, and inexpensive means for measuring the composition of the mobile phase. This can be useful, for example, in operational qualification (OQ) or quality control (QC) to verify the composition accuracy of the pump(s) and ensure proper system function. It can also be used, for example, for real-time monitoring of solvent composition during gradient elution according to a specified gradient program, provided a constant flow rate is maintained.

[0054] Thus, the monitoring method involves introducing a known amount of thermal energy into the mobile phase 11 via a heat generating element 21, obtaining a measurement of the dissipation of the thermal energy from the heat generating element 21 via a thermal flow sensor 20, and converting the measurement to a solvent composition using a pre-established correlation, thereby obtaining the solvent composition of the mobile phase.

[0055] In both the first and second embodiments, the method further comprises: - determining a composition error by comparing the obtained solvent composition with an expected solvent composition.

[0056] In a first embodiment, the method may be performed as part of an operational qualification (OQ) or quality control (QC) prior to starting up a liquid chromatography process, and - initiating a liquid chromatography process in accordance with a determination that the composition error is below a predetermined threshold; - sending an alert to the liquid chromatography system in accordance with a determination that the composition error is above a predetermined threshold.

[0057] This alert may indicate excessive dwell volume, a leak, a pump miscalibration, or other causes. If an alert is received by a liquid chromatography system, the system or operator may decide to refrain from starting the liquid chromatography process. The operator may then inspect the system to identify the cause of the alert.

[0058] This first embodiment of the method may further include removing the thermal flow sensor from the fluid conduit and resealing the fluid conduit before starting the liquid chromatography process.

[0059] In a second embodiment, the method is carried out during a liquid chromatography process, and further - continuing the liquid chromatography process in accordance with a determination that the compositional error is less than or equal to a predetermined threshold; - sending an alert to the liquid chromatography system in accordance with a determination that the composition error is above a predetermined threshold.

[0060] This alert may indicate excessive dwell volume, a leak, a pump miscalibration, or other causes. If an alert is received by the liquid chromatography system, the system or operator may decide to stop the liquid chromatography process. The operator may then inspect the system to identify the cause of the alert.

[0061] This second embodiment of the method may further include performing the method continuously to monitor the solvent composition in the liquid chromatography system in real time.

[0062] A third embodiment of this method is also envisioned combining the first embodiment, i.e., performing this method as part of an operational qualification (OQ) or quality control (QC) before starting a liquid chromatography process, with the second embodiment, i.e., monitoring the solvent composition during a liquid chromatography process. In the third embodiment, the thermal flow sensor remains installed during the liquid chromatography process.

[0063] Therefore, the method of the present invention can be used to provide reliable information about the composition of the mobile phase of a chromatography system, allowing the operator of the chromatography system to check in a simple and easy way whether the system is working as expected before starting the chromatography process, thereby allowing the operator to monitor the system performance in real time.

[0064] 2, the thermal flow sensor 20 includes a pipe segment 25 having an inlet end 26 and an outlet end 27 that define a fluid volume of approximately 100 nL. The measurement volume between a first cross section 28 of the fluid conduit at the first sensing element 22 and a second cross section 29 of the fluid conduit at the second sensing element 23 is approximately 10 nL. Therefore, the thermal flow sensor 20 has a negligible effect on the dead volume of the system. The pipe segment 25 (and thus the thermal flow sensor 20) can be removed from the liquid chromatography system 6 if desired.

[0065] For example, the thermal flow sensor 20 can be included as part of a periodic (e.g., daily, weekly, monthly) operational qualification (OQ) or quality control (QC) process. Alternatively, the thermal flow sensor 20 can remain in place to perform continuous (e.g., real-time) or intermittent monitoring of the liquid chromatography process.

[0066] This method relies on establishing a relationship between the observed measurements of the thermal flow sensor and the resulting solvent composition. Such a relationship can be established by first performing a correlation sequence, whereby a set of known liquid compositions is sequentially passed through the thermal flow sensor 20 while the measurements of the thermal flow sensor 20, e.g., the temperature difference ΔT, are recorded as a function of time t. Only after the measurements of the current known liquid composition have settled is the next known liquid composition passed through the thermal flow sensor 20. The result is illustrated in FIG. 3A. In this example, a first known liquid composition (comprising 100% solvent A and 0% solvent B) is passed through the thermal flow sensor 20, after which the temperature difference is measured to have a first value ΔT1. Next, a second known liquid composition (comprising 67% solvent A and 33% solvent B) is passed through the thermal flow sensor 20, after which the temperature difference is measured to have a second value ΔT2. A third known liquid composition (containing 33% solvent A and 67% solvent B) yields a third value ΔT3, and a fourth known liquid composition (containing 0% solvent A and 100% solvent B) yields a fourth value ΔT4. From these results, a rough correlation between the thermal flow sensor measurements and liquid composition can be established. The above example was established by running four different liquid compositions with 33% step changes (i.e., 100%, 67%, 33%, and 0% solvent A). A finer correlation can be established by running smaller step changes. Figure 3B illustrates the relationship between the temperature difference measured by the thermal flow sensor and the percentage of solvent A (also in a binary solvent composition containing solvent B) established with a 2% step change. Alternatively, a known linear gradient of solvent composition can be passed through the thermal flow sensor to establish a correlation.

[0067] It is also envisioned that by measuring solvent composition and flow rate pairs and establishing a correlation matrix, the thermal flow sensor may be able to derive solvent composition at different, known, constant flow rates.

[0068] Referring to FIG. 4, which illustrates an alternative embodiment of a flow sensor, the flow sensor 20 may alternatively be implemented as either an electrical flow sensor 20' or a differential pressure flow sensor 20''.

[0069] The electrical flow sensor 20' relies on measuring changes in the electrical properties of the mobile phase and includes first and second sensing elements 22, 23 in the form of electrodes electrically connected to the mobile phase. For example, the electrical flow sensor 20 can measure changes in the resistivity of the mobile phase. A voltage difference is applied between the electrodes. The current conducted between the electrodes through the mobile phase can be derived using Ohm's law (R = U / I), where R is the resistance, U is the (known) potential difference, and I is the measured current. A commonly used solvent pair in chromatography is water and 0.1% formic acid in acetonitrile. In contrast to pure acetonitrile, which has a very low conductivity of 7 μS / cm, 0.1% formic acid has a relatively high conductivity of approximately 154 μS / cm at 25°C. In other words, there is a resistivity difference of approximately 20 times between these two exemplary mobile phase solvents. With known flow rates and temperatures, it is possible to establish a correlation that converts the measured current between the two electrodes 22, 23 into the solvent composition of the mobile phase. It should be noted that, although less preferred, other electrical properties, such as the capacitance and inductance of the mobile phase, can be measured and the solvent composition can be derived in a similar manner.

[0070] The differential pressure flow sensor 20″ relies on measuring the change in viscosity of the mobile phase with changes in solvent composition. Viscosity is determined by the Poiseuille equation (μ=(π*r 4 The dynamic viscosity of a conduit can be determined by measuring the pressure drop across the conduit at known flow rates and temperatures using the equation (8*ΔP / (8*L*Q)), where μ is the dynamic viscosity, r is the radius of the conduit, ΔP is the pressure difference between the pressure-sensing elements, L is the length of the conduit between the pressure-sensing elements, and Q is the known volumetric flow rate. The dynamic viscosity of water at 25°C is approximately 0.89 mPa·s, while the dynamic viscosity of acetonitrile (CH3CN) at 25°C is approximately 0.35-0.44 mPa·s—a significant difference of about two-fold. With known flow rates and temperatures, it is possible to create a conversion table that converts the measured pressure difference across the conduit to the solvent composition of the mobile phase. [Explanation of symbols]

[0071] 6 Chromatography Systems 7a,7b Binary Pump 8a, 8b Flasks 9a,9b Filter 10 Mixer 11 Mobile phase 16 Chromatography Columns 20 Thermal flow sensor 21 Heating Elements 22 First temperature sensor 23 Second temperature sensor 24 Heat distribution 25 pipe sections 26 Inlet end 27 Outlet end 28 First Section 29 Second Cross Section

Claims

1. 1. A method of monitoring solvent composition in a liquid chromatography system (6) with a mobile phase (11) in a fluid conduit, the mobile phase comprising or consisting essentially of a plurality of known solvents, the liquid chromatography system including a flow sensor, preferably a thermal flow sensor, adjacent the mobile phase in the fluid conduit, the method comprising: passing the mobile phase through the flow sensor at a known flow rate; obtaining measurements of transport properties, such as thermal, electrical or viscous properties, of the mobile phase via the flow sensor; converting said measurements to solvent composition using a pre-established correlation; A method comprising:

2. 2. The method of claim 1, wherein the flow sensor is a thermal flow sensor (20) including a heat-generating element (21) adjacent to the mobile phase in the fluid conduit, the method comprising the step of introducing a known amount of thermal energy into the mobile phase via the heat-generating element (21), the transport property being a thermal property such as thermal conductivity or specific heat capacity, and the measurement value being obtained in response to the known amount of thermal energy introduced by the heat-generating element into the mobile phase via the thermal flow sensor.

3. 2. The method of claim 1, wherein the flow sensor is positioned in the fluid conduit upstream of, preferably adjacent to, a chromatography column (16) of the liquid chromatography system.

4. 4. The method of any one of claims 1 to 3, wherein the known flow rate is in the range of 1 nL / min to 20 mL / min, preferably 1 nL / min to 2 mL / min, more preferably 5 nL / min to 200 μL / min, and even more preferably 10 nL / min to 100 μL / min.

5. The method according to any one of claims 1 to 3, wherein the pressure of the mobile phase at the flow sensor is at least 200 bar, suitably at least 300 bar, preferably at least 500 bar, or more preferably at least 1000 bar.

6. 4. The method of any one of claims 1 to 3, wherein the flow sensor comprises a pipe section (25), an inlet end (26) and an outlet end (27) of the pipe section forming part of the fluid conduit, and wherein a fluid volume of the pipe section between the inlet end and the outlet end is at most 1 μL, preferably at most 150 nL, or more preferably at most 100 nL.

7. The flow sensor includes at least one first sensing element (22), at least one second sensing element (23) disposed downstream of the first sensing element, and The method according to any one of claims 1 to 3, comprising:

8. 8. The method of claim 7, wherein the fluid conduit includes a measurement volume defined from the first sensing element to the second sensing element, the measurement volume being at most 50 nL, preferably at most 25 nL, or more preferably at most 10 nL.

9. 9. The method of claim 1, further comprising determining a composition error by comparing the obtained solvent composition with an expected solvent composition.

10. The method is performed as part of an operational qualification (OQ) or quality control (QC) prior to starting up a liquid chromatography process, and the method further comprises: initiating the liquid chromatography process in accordance with a determination that the compositional error is less than or equal to a predetermined threshold; sending an alert to the liquid chromatography system in response to a determination that the compositional error is above a predetermined threshold; 10. The method of claim 9, comprising:

11. The method of claim 10 , wherein the steps of removing the flow sensor from the fluid conduit and resealing the fluid conduit are performed before starting the liquid chromatography process.

12. The method is carried out during a liquid chromatography process and further comprises: continuing the liquid chromatography process in accordance with a determination that the compositional error is less than or equal to a predetermined threshold; sending an alert to the liquid chromatography system in response to a determination that the compositional error is above a predetermined threshold; 10. The method of claim 9, comprising:

13. 13. The method of claim 12, wherein the method is performed continuously to monitor the solvent composition in the liquid chromatography system in real time.

14. A method for obtaining a pre-established correlation for use in the method of any one of claims 1 to 13, said method comprising: passing a mobile phase (11) comprising a predetermined solvent composition of a plurality of known solvents, such as a first known solvent and a second known solvent, at a known flow rate through a flow sensor (20), preferably a thermal flow sensor; Preferably, introducing a known amount of thermal energy into the mobile phase via a heat generating element (21) of the thermal flow sensor; obtaining a measurement of a transport property of the heat generating element via the flow sensor, preferably by measuring the dissipation of thermal energy from the heat generating element; Preferably, repeating the foregoing steps for one or more additional predetermined solvent compositions; correlating said measured value(s) with said predetermined solvent composition(s) to obtain said pre-established correlation; A method comprising:

15. A chromatography system comprising a flow sensor, preferably a thermal flow sensor, for monitoring the solvent composition of a mobile phase, said flow sensor being fluidly connected to a chromatography column and a sample loop during elution, said flow sensor being positioned upstream of, preferably adjacent to, said chromatography column.

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