Method for determining the dwell volume of a chromatography system

Abstract

The present invention describes a method for determining the dwell volume of a liquid chromatography system and a liquid chromatography system capable of determining the dwell volume of the system. The method comprises mixing a flow of a first solvent with a flow of a second solvent to form a solvent mixture. The flows of the first solvent and the second solvent are respectively reduced and increased to produce a gradient composition. The system pressure of the liquid chromatography system is measured to determine a pressure trace, which is defined as the measured system pressure as a function of time. The dwell volume of the system is determined based on the time delay determined between the gradient composition at the mixing location and the pressure trace. The method can be performed using a liquid chromatography system having a chromatographic column or a flow restrictor used to replace the chromatographic column.

Description

[0001] Related applications

[0002] This application claims the benefit of the prior filing date of U.S. Provisional Patent Application Serial No. 62 / 889,820, filed on August 21, 2019, and entitled “Method for Determining a Dwell Volume of a Chromatographic System,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present technology generally relates to chromatography systems. More specifically, the present technology relates to a method for determining the dwell volume of a liquid chromatography system based on the system pressure response to a composition gradient. Background Art

[0004] The dwell volume of a chromatography system is typically defined as the volume of the flow path extending between the location where the solvents are mixed (i.e., where the solvent gradient is formed) and the inlet (head) of the chromatography column. The solvent flow at the inlet is isocratic before the gradient begins to reach the column inlet. The gradient delay is the time difference between the start of the solvent gradient at the mixing location and the time when the solvent gradient first reaches the column inlet. The gradient delay can be determined based on knowledge of the dwell volume and solvent flow rate.

[0005] The residence volume affects the flux of gradient separation and affects the transfer of gradient method from one chromatographic system to another chromatographic system. The difference in the residence volume between the systems can cause the difference in the solvent gradient at the column. Therefore, when transferring the method between different chromatographic systems, especially when there are system differences in the residence volume and because the difference is unknown and cannot be compensated, problems may arise. For example, the method of transfer may cause the selectivity and spacing of the peaks in the chromatogram to vary. For longer gradient delays, i.e., larger residence volumes, isocratic separations can occur for a longer time before the gradient arrives at the chromatographic column. Therefore, compared with systems with small residence volumes, isocratic separations can be significant and may cause significantly different chromatograms.

[0006] A conventional technique for determining the dwell volume of a liquid chromatography system is based on measuring the dwell volume using two sources of the same solvent. The solvent supplied by one source contains other compounds that are easily sensed by a chromatographic detector. For example, for a system employing an ultraviolet (UV) light detector, a UV tracer compound can be added to the solvent at one of the solvent sources. A solvent gradient is generated by mixing the solvents supplied by the two sources. Therefore, the user needs to spend time preparing the solvent and installing the solvent source. In addition, it may be necessary to clean the flow path of the liquid chromatography system after the dwell volume measurement to prevent the system from being contaminated by foreign compounds and the system performance from deteriorating. If some system components are modified, other problems may be encountered. For example, changes in tubing and / or mixers may result in changes in the dwell volume, so the dwell volume determined earlier may no longer be accurate and may need to be remeasured. Summary of the Invention

[0007] Examples of the present disclosure include a method for determining a residence volume of a liquid chromatography system and a liquid chromatography system that can determine its own residence volume.

[0008] In one example, a method for determining the dwell volume of a liquid chromatography system includes mixing a flow of a first solvent and a flow of a second solvent at a mixing location to produce a solvent mixture in the system flow of the liquid chromatography system. During a gradient duration, the flow of the first solvent is reduced and the flow of the second solvent is increased to generate a gradient composition of the solvent mixture. The system pressure of the liquid chromatography system is measured to determine a pressure trace, which is defined as the measured system pressure as a function of time. The dwell volume of the liquid chromatography system is determined by a time delay determined between the gradient composition at the mixing location and the pressure trace.

[0009] The solvent mixture may have a viscosity that changes in response to the gradient composition. The change in viscosity may be substantially linearly proportional to the change in the solvent mixture according to the gradient composition.

[0010] The viscosity of the first solvent may be different from the viscosity of the second solvent.

[0011] The gradient composition may be a linear gradient composition or a step gradient. The gradient composition may be a non-linear gradient composition determined to provide a substantially linear pressure change for at least a portion of the pressure trace in response to the gradient composition.

[0012] The liquid chromatography system may include a chromatography column, and determining the dwell volume may include determining the product of the time delay and the flow rate of the solvent mixture and then subtracting half the volume of the chromatography column from the product.

[0013] The liquid chromatography system may include a flow restrictor configured to provide a system pressure drop for a system flow, and determining the dwell volume may include determining a product of the time delay and a flow rate of the solvent mixture.

[0014] In another example, a computer program product for determining a hold-up volume of a liquid chromatography system includes a computer-readable storage medium having computer-readable program code embodied therewith. The computer-readable program code includes:

[0015] computer readable program code configured to mix a first solvent and a second solvent at a mixing location to produce a solvent mixture in a system stream of a liquid chromatography system;

[0016] computer readable program code configured to decrease the flow of a first solvent and increase the flow of a second solvent over a gradient duration to produce a gradient composition of the solvent mixture;

[0017] computer readable program code configured to measure system pressure and determine a pressure trace based on the system pressure, the pressure trace being defined as the measured system pressure as a function of time; and

[0018] Computer readable program code configured to determine a dwell volume of a liquid chromatography system based on a time delay between a gradient composition and a pressure trace at a mixing location.

[0019] The liquid chromatography system may include a chromatography column and computer readable program code configured to determine a dwell volume, computer readable program code configured to determine a product of a time delay and a flow rate of a solvent mixture, and computer readable program code configured to subtract half the volume of the chromatography column from the product.

[0020] In another example, a liquid chromatography system includes a source of a first solvent, a source of a second solvent, a pump, a flow restrictor, a pressure sensor, and a processor. The mixer has a mixer inlet for receiving the first and second solvents and a mixer outlet for providing the solvent mixture. The pump has a pump inlet in fluidic communication with the mixer outlet and a pump outlet. The pump receives the solvent mixture at the pump inlet and provides the solvent mixture at system pressure in a system flow dispensed from the pump outlet. The flow restrictor has a restrictor inlet in fluidic communication with the pump outlet and a restrictor outlet. A pressure sensor is disposed in the system flow and after the pump outlet and generates a pressure signal indicating a measured system pressure. The processor is in communication with the sources of the first and second solvents and the pressure sensor. The processor is configured to generate a gradient composition in the system flow by controlling the contribution of the first solvent and the contribution of the second solvent to the mixer and to determine a pressure trace based on the pressure signal. The pressure trace is defined as the measured system pressure as a function of time. The processor is further configured to determine a time delay between the gradient composition at the mixing location and the pressure trace and to determine a dwell volume of the liquid chromatography system based on the time delay.

[0021] The liquid chromatography system may further include a gradient proportioning valve disposed between the mixer and the sources of the first solvent and the second solvent.

[0022] The processor may determine the dwell volume as the product of the time delay and the flow rate of the solvent mixture.

[0023] The flow restrictor may be a chromatography column, and determining the dwell volume comprises determining the product of the time delay and the flow rate of the solvent mixture and then subtracting half the volume of the chromatography column from the product.

[0024] In another example, a liquid chromatography system includes a source of a first solvent, a source of a second solvent, a first pump, a second pump, a mixer, a flow restrictor, a pressure sensor, and a processor. The first pump has a first pump inlet in fluidic communication with the source of the first solvent and a first pump outlet. The second pump has a second pump inlet in fluidic communication with the source of the second solvent and a second pump outlet. The mixer has first and second mixer inlets in fluidic communication with the first and second pump outlets, respectively, and a mixer outlet for providing a solvent mixture. The flow restrictor has a restrictor inlet in fluidic communication with the pump outlet and a restrictor outlet. A pressure sensor is disposed in the system flow and after the mixer outlet and generates a pressure signal indicating a measured system pressure. The processor is in communication with the first and second pumps and the pressure sensor. The processor is configured to generate a gradient composition in the system flow by controlling the contribution of the first solvent to the mixer and the contribution of the second solvent to the mixer, and to determine a pressure trace based on the pressure signal. The pressure trace is defined as the measured system pressure as a function of time. The processor is further configured to determine a time delay between the gradient composition at the mixing location and the pressure trace and to determine a dwell volume of the liquid chromatography system based on the time delay.

[0025] The processor may determine the dwell volume as the product of the time delay and the flow rate of the solvent mixture.

[0026] The flow restrictor may be a chromatography column, and determining the dwell volume may include determining the product of the time delay and the flow rate of the solvent mixture and then subtracting half the volume of the chromatography column from the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and other advantages of the present invention may be better understood by referring to the following description in conjunction with the accompanying drawings, in which the same reference numerals refer to the same elements and features in the various figures. Letters may be appended to reference numerals to distinguish similar features from those in the figures and to indicate correspondence with other features in the figures. For clarity, not every component is labeled in every drawing. The drawings are not necessarily drawn to scale, with emphasis instead on illustrating the principles of the present invention.

[0028] Figure 1 is a block diagram of an example of a liquid chromatography system that can be used to run a gradient separation.

[0029] Figure 2 is a description of how the solvent mixture travels through Figure 1 Schematic block diagram of a liquid chromatography system from the gradient proportional valve through the detector.

[0030] Figure 3 is a graphic depiction of a technique for using a UV tracer solvent to determine the residence time and residence volume of a liquid chromatography system.

[0031] Figure 4 is a graphical representation of the dependence of viscosity on the water content of a mixture of water and acetonitrile.

[0032] Figure 5 is a graphical representation of the dependence of viscosity on the water content of a mixture of water and methanol.

[0033] Figure 6 is a graphical depiction of a programmed gradient and resulting pressure trace of a liquid chromatography system used to perform a method for determining a dwell volume of a liquid chromatography system.

[0034] Figure 7A A column manager with two chromatography columns is shown, with one chromatography column optionally being included in the system flow path.

[0035] Figure 7B The volume occupied by the restrictor is shown to be negligible relative to conventional liquid chromatography systems. Figure 7A Column Manager.

[0036] Figure 8 is a graphical representation of programmed gradient composition and resulting pressure traces for an exemplary implementation of a method for determining a dwell volume for a liquid chromatography system.

[0037] Figure 9 is a graphical representation of a programmed gradient composition and resulting pressure trace for another exemplary implementation of a method for determining a dwell volume for a liquid chromatography system.

[0038] Figure 10 is a graphical representation of a programmed gradient composition and resulting pressure trace for another exemplary implementation of a method for determining a dwell volume for a liquid chromatography system.

[0039] Figure 11 is a flow chart representation of an example of a method for determining the dwell volume of a liquid chromatography system. DETAILED DESCRIPTION

[0040] References in this specification to an "example" or "implementation" indicate that a particular feature, structure, or characteristic described in connection with the example or implementation is included in at least one example or implementation of the present teachings. References to a particular example or implementation within this specification do not necessarily refer to the same example or implementation.

[0041] The present teachings will now be described in more detail with reference to the examples and specific implementations shown in the accompanying drawings. Although the present teachings have been described in conjunction with various examples, it is not intended that the present teachings be limited to such examples. In contrast, the present teachings encompass various alternatives, modifications, and equivalents, as will be understood by those skilled in the art. Those of ordinary skill in the art who can use the teachings herein will recognize additional embodiments, modifications, and examples, as well as other areas of use, within the scope of the present disclosure.

[0042] In short, the following discloses an example of a method for determining the dwell volume of a liquid chromatography system. The method includes mixing solvents to produce a gradient mobile phase. The system pressure of the liquid chromatography system is measured over time to determine a "pressure trace." The pressure trace is compared to the gradient composition (which is a function of time) at the solvent mixing location to determine a time delay. This time delay then allows the dwell volume to be calculated based on the flow rate of the solvent mixture. Advantageously, no UV tracer or other additional compounds are required. Thus, the preparation time for the dwell volume measurement is reduced and the risk of contamination by additional compounds is avoided. In addition, the liquid chromatography system does not require physical reconfiguration. For example, the method can be performed without removing the chromatographic column and rearranging the flow path with additional tubing. In addition, the reduction in operator actions used to perform the measurement reduces the possibility of human error that may lead to erroneous dwell volume determinations. In addition, the time required to perform the method and the resulting accuracy can reduce problems that may arise when transferring the method between different chromatography systems and configurations.

[0043] Figure 1 1 is a block diagram of a liquid chromatography system 10, which includes a system processor 14 (e.g., a microprocessor and controller) in communication with a user interface device 16 for receiving input parameters and displaying system information to an operator. The system processor 14 is in communication with a gradient proportional valve (GPV) 18, which is used to control the contribution of two solvents A and B contained in solvent sources (e.g., reservoirs) 22A and 22B to the solvent flow, respectively. The system processor 14 is also in communication with a pump drive module 20, which controls a system pump 24 and an injection valve 32 for injecting a sample into the solvent flow. A mixer 26 receives a solvent flow from the GPV 18 at the mixer inlet. The received solvent flow includes alternating contributions of each solvent. The mixer 26 homogenizes the composition of the solvent flow and provides a solvent mixture at the mixer outlet. The solvent mixture flows to the pump inlet of the system pump 24, where the solvent mixture is pressurized to the system pressure. The pump 24 may include a pump head that can be configured in a variety of ways. For example, the pump 24 may include a main pump head that is connected in series with an accumulator pump head.

[0044] During a separation run, sample from a sample reservoir or container 30 is injected by an injector valve 32 into the mobile phase flowing to a chromatography column 28. The column 28 is coupled to a detector 34 that provides a detector signal to the system processor 14. The detector signal is responsive to components in the sample eluting from the column 28.

[0045] During operation of the liquid chromatography system 10 with a gradient mobile phase, solvents A and B are drawn from the solvent source 22. The relative contributions of the solvents present in the mixed mobile phase depend on the discrete contributions of each solvent provided by the GPV 18. These contributions vary over time during the gradient formation according to the desired (predetermined) gradient composition of the mobile phase.

[0046] In one conventional technique for determining the dwell volume of system 10, solvent sources 22A and 22B hold solvent A and solvent B, respectively. In some implementations, solvents A and B are the same. A small amount of an organic compound that absorbs ultraviolet (UV) light (i.e., a UV tracer) is added to the solvent in solvent source 22B. System 10 then performs a gradient separation run in which the gradient contains 100% solvent A from solvent source 22A and transitions to a solvent containing 100% solvent B from solvent source 22B, which includes the UV tracer, during the gradient transition time. The gradient flowing through the system then remains at 100% solvent B before the solvent flow returns to 100% solvent A. As described in more detail below, the chromatography column is removed to implement this technique. The reason for removing the chromatography column is that the column adds volume and complicates the process by potentially retaining UV-active analytes.

[0047] Figure 2 is a schematic block diagram depicting how a solvent mixture travels through a liquid chromatography system from GPV 18 through detector 34. The system pressure is highest at the outlet of pump 24. Most of the pressure drop in the liquid chromatography system occurs across chromatography column 28. For a gradient composition to be first defined at GPV 18, there is a delay time Δt to be achieved at the inlet of column 28. This delay time Δt is defined as the time difference between the time the gradient first reaches the inlet of column 28 and the time the gradient first starts at GPV 18 (i.e., isocratic flow stops).

[0048] See also Figure 3 , shows a graphical depiction of the above technique for determining residence time and residence volume using a UV tracer solvent. The graph includes curve 40, which depicts the percentage of solvent A in the system flow at GPV 18 as a function of time. The graph includes curve 42, which depicts the percentage of solvent A at the head of column 28 as a function of time.

[0049] The liquid chromatography system is operated in isocratic mode, with solvent A contributing 100% to the system flow. Subsequently, when the gradient is first formed at GPV 18 (at which time the contribution of solvent A first begins to decrease), there is a time t1. Since the liquid has a propagation time to traverse the dwell volume defined between the mixing location (i.e., the gradient formation location) and the column inlet, the time t2 when the gradient first reaches the head of column 28 is delayed by a time Δt = t2 - t1 from the time the gradient starts at GPV 18. The gradient ends at time t G1 corresponds to the moment when the solvent at GPV 18 is completely composed of solvent B. Similarly, the end of the gradient reaches the head of column 28 for the first time, t G2 The same time Δt is delayed from the moment when the end of the gradient occurs at the GPV 18 .

[0050] Generally speaking, the gradient composition at the head of column 28 is approximately the same gradient composition defined at GPV 18, but delayed by time Δt (no column in the flow path). Thus, curve 42 is similar to curve 40, but with a horizontal offset due to the time delay Δt.

[0051] The above conventional technique for determining the dwell volume places certain demands on the system operator. The operator must prepare solvent A, prepare solvent B with the required UV tracer, and run the separation. For the value on each of curves 40 and 42 that is halfway between the maximum and minimum values ​​(i.e., the 50% level), the time offset (i.e., the separation of the two curves 40 and 42) is determined. The determined time offset Δt is multiplied by the flow rate of the system flow to determine the dwell volume. Once the measurement is completed, the system is typically flushed to ensure that no UV tracer remains in the system before pre-priming the system for normal operation. This technique typically requires several hours or more, depending on the expertise of the operator performing the measurement. In addition, the technique can be affected by operator error. For example, an excess of UV tracer can cause a nonlinear response of the UV detector, resulting in inaccuracies.

[0052] In order to perform the above-mentioned measurement, the operator removes the chromatographic column 28 and replaces the part of the system flow path with a tubing with a small internal diameter. This change is performed because the volume of the chromatographic column 28 represents a major part of the entire system flow path, and therefore the measurement result of the UV detector of the gradient delay will not represent the delay occurring at the head of the column 28. In contrast, the volume of the replacement tubing only adds a small total volume (e.g., a few microliters or less) to the system flow path. Therefore, this additional tubing volume is small enough to generally make its contribution to the measured gradient delay time negligible. However, when using conventional UV tracer technology, the need to remove the chromatographic column and rearrange the flow path with additional tubing represents an additional time burden.

[0053] Although the user can leave the chromatography column 28 in place in the system with accurate knowledge of the column volume, careful attention is required to ensure that the UV tracer does not remain on the column 28, thereby causing incorrect measurements. A second concern is that any retained UV tracer may cause contamination of later separation runs.

[0054] Described below is an example of determining the residence time of a liquid chromatography system using a system pressure response (i.e., system pressure as a function of time, also referred to herein as a "pressure trace"). The system pressure trace can be measured by a pressure sensor disposed in a flow path upstream of the main system pressure drop caused by a chromatographic column (or a restrictor if no chromatographic column is present). For example, the system pressure trace can be determined by a pressure signal generated by a pressure sensor that indicates changes in system pressure over time. In some embodiments, the pressure sensor is a pressure transducer located at the pump outlet. The pressure drop on the tubing (or column, if present) is proportional to the dynamic viscosity (Poiseuille's law). Under certain conditions, the pressure trace has a slope proportional to the slope of a linear binary phase gradient. In order to achieve this, the viscosity change of the solvent mixture is substantially linear relative to the change in the solvent composition. In other words, the viscosity change of the solvent mixture is substantially linearly proportional to the change in the solvent that contributes to the total solvent flow within a limited solvent composition range. For example, for a linear decrease in solvent, the contribution of one solvent (e.g., solvent A) can result in a linear decrease in system pressure; however, such linearity is typically not present throughout the transition from 100% to 0% solvent A over the duration of the gradient.

[0055] The viscosity of a solvent mixture generally varies with the percentage of a particular solvent in the solvent mixture. For example, Figure 4 The dependence of viscosity on the presence of water in a mixture of water and acetonitrile is graphically depicted, and Figure 5 The dependence of viscosity on the presence of water in a mixture of water and methanol is graphically depicted. Each curve indicates the viscosity at a different temperature, with higher temperature curves corresponding to lower viscosities. Figure 4 For compositions between about 10% and about 40% water, the viscosity varies in a roughly linear manner and Figure 5 In the embodiment of the present invention, the viscosity varies in a generally linear manner for compositions between about 0% to about 20% and between about 70% to about 100% water.

[0056] By knowing the linear range of viscosity in a solvent mixture, the operator can program (define) the gradient composition to achieve a gradient change in the time it remains within the linear viscosity range. For example, the operator can program a mixture of acetonitrile and water that spans one of the linear ranges in a few minutes.

[0057] As mentioned above, conventional UV tracer techniques for determining gradient delay generally involve removing the chromatographic column and being replaced with tubing with a small internal diameter. This change is performed because the volume of the chromatographic column represents a major part of the entire system flow path, and therefore the measurement result of the UV detector of the gradient delay will not represent the delay at the head of the column. In contrast, the volume of the replacement tubing only adds a small total volume (e.g., a few microliters or less) to the system flow path. Therefore, this other tubing volume is sufficiently small that its contribution to the measured gradient delay time is generally negligible. However, when using conventional UV tracer techniques, removing the chromatographic column and rearranging the flow path with other tubing requires another time burden.

[0058] In various examples of methods for determining dwell volume described herein, the majority of the pressure drop across the liquid chromatography system occurs across the chromatography column. The presence of the chromatography column is advantageous because the pressure drop across the column ensures that the resulting pressure trace does not include a significant pressure noise component and does not require the installation of replacement tubing. Another advantage of this method is that standard mobile phase solvents, such as water, acetonitrile, and / or methanol, can be used without the need for specialized solvents (e.g., solvents with UV tracers) and without any risk of contamination.

[0059] Figure 6 is a simplified graphical depiction of a programmed gradient 50 and the resulting pressure trace 52 for a liquid chromatography system. Also shown is curve 54, which shows the programmed gradient shifted to a later time corresponding to the outlet of the chromatography column based on column volume. Curve 55 is similar to curve 54, except that curve 55 represents the programmed gradient at the same location in the system without the column. Thus, as can be seen from curves 54 and 55, half the time difference between curves 54 and 55 (corresponding to half the column volume) can be subtracted from the difference Δt between the measured pressure trace 52 and the programmed gradient 50 to determine the gradient delay t 延迟 .

[0060] More specifically, with respect to the illustrated example, the programmed gradient 50 shows a transition over four minutes from a maximum value of the first solvent (solvent A) to a minimum value of the first solvent, wherein the maximum and minimum values ​​are selected to achieve a linear response in the pressure trace 52, as described above. The pressure trace 52 decreases from the maximum pressure value until it reaches the minimum pressure value. Due to the effects of the mixer in which the solvents are mixed, some distortion of the linearity in the pressure trace 52 is apparent near the beginning and end of its transition. Due to the dwell volume and column volume, the pressure trace 52 is temporally offset by a time Δt from the programmed gradient 50. In order to properly determine the gradient delay at the head of the chromatographic column, the time offset Δt between the programmed gradient 50 and the pressure trace 52 is reduced by a time corresponding to half the column volume multiplied by the flow rate. For example, if the column has a delay time t0 associated with its full volume and flow rate, the gradient delay is Δt-t0 / 2. The column volume can be known from the product specification information. Alternatively, for some columns, other parameters of the column (such as column size and particle size) can be used to calculate the column volume. Therefore, the gradient delay at the head of the column can be easily determined using the measured value Δt and the known column volume t0.

[0061] Figure 7A A column manager 60 is shown having two chromatography columns 62A and 62B. The particular column to be used for the separation can be selected by valves 64A and 64B. The flow path for either column 62 can be used to determine the dwell volume. Figure 7B In the illustrated alternative configuration, column manager 60 may include a restrictor 66, such as a length of tubing having a negligible volume compared to the dwell volume and acting as the primary pressure drop across the chromatography system. One advantage of this alternative configuration is that the column volume need not be known. The gradient delay time is determined directly as Δt, without subtracting half the column delay time t0. In a non-limiting numerical example, the tubing has an inner diameter of 100 μm or less, and the pressure drop across the length of tubing is approximately 13.8 MPa (2,000 psi).

[0062] A method for determining the dwell volume of a liquid chromatography system was evaluated for three different configurations. The gradient was based on an aqueous mixture containing acetonitrile, wherein the percentage of acetonitrile in the mixture was limited to 60% to 90% (i.e., 40% to 10% water), so that the viscosity change was confined to an essentially linear region (see Figure 4 The method includes achieving a system pressure variation of approximately 6.9 MPa (1,000 psi) and limiting the maximum system pressure to 27.6 MPa (4,000 psi) or less depending on the variable flow rate. The parameters according to the method are selected to reduce the effects of nonlinear changes in viscosity due to pressure and temperature variations and to address solvent compressibility issues.

[0063] For each configuration, a programmed gradient of 60% acetonitrile was selected, with acetonitrile variations ranging between 10% and 30%. The initial hold volume was selected to be approximately twice the column volume, the gradient duration was selected to be at least twice the column delay time, and the final hold volume was selected to be approximately twice the column volume. Evaluations were performed at a temperature of approximately 30°C.

[0064] Figure 8 An exemplary implementation of a method for determining the residence volume of a liquid chromatography system is graphically depicted. In this example, the chromatography system is a LC-MS / MS system available from Waters Corporation, Milford, Massachusetts. UPLC H-Class System. The chromatographic column was a 2.1 mm x 75 mm column with a 2.5 μm particle size and a column volume of 0.172 mL. The gradient was an aqueous solvent mixture with a flow rate of 0.5 mL / min, transitioning from 60% acetonitrile to 75% acetonitrile over four minutes. Curve 70 depicts the programmed gradient, and curve 72 is the measured pressure trace with a maximum of 31.83 MPa (4,617 psi) and a minimum of 25.45 MPa (3,691 psi).

[0065] The measured pressure trace 72 shows Δt at a 50% (mid-range) pressure value between the minimum and maximum pressures at 0.98 minutes, corresponding to a dwell volume of 0.49 mL. After subtracting 0.086 mL, which is half the column volume, the dwell volume was determined to be 0.404 mL. This is similar to the 0.384 mL volume determined by the UV tracer method described above.

[0066] After the gradient expires at 7.0 minutes, the solvent composition is held constant until 10.0 minutes when the programmed gradient composition returns to 60% acetonitrile in a step change. As can be seen, the second Δt can be determined by the rise in pressure trace 72. In this example, the second Δt is 0.90 minutes. Therefore, the method is actually performed twice, wherein in the first case, the water flow decreases and the acetonitrile flow increases, and in the second case, the acetonitrile flow decreases and the water flow increases. In other words, the flow of the second solvent decreases while the flow of the first solvent increases. The second value of Δt for increasing the pressure ramp should be similar to the first value obtained by decreasing the pressure ramp and therefore serves as a check on the first value. Alternatively, the two values ​​can be averaged to determine the dwell volume, although it is generally preferred to use Δt determined for a linear gradient rather than a step change in composition because linear gradient techniques are generally more accurate.

[0067] Figure 9Another exemplary implementation of the method is depicted. In this example, the chromatography system is an Alliance HPLC system, and the chromatography column is a 3.0 mm x 150 mm column with a particle size of 2.5 μm and a column volume of 0.7 mL. The gradient is an aqueous solvent mixture with a flow rate of 0.3 mL / min that transitions from 60% acetonitrile to 85% acetonitrile over four minutes. Curve 74 depicts the programmed gradient, and curve 76 is the measured pressure trace with a maximum of 20.48 MPa (2,970 psi) and a minimum of 11.49 MPa (1,667 psi).

[0068] The measured pressure trace 76 exhibits a Δt of 4.82 minutes at a mid-range pressure value corresponding to a dwell volume of 1.446 mL. (The measured pressure trace 76 also provides a second Δt of 4.87 minutes based on the step change back to the initial solvent composition.) After subtracting 0.35 mL, which is half the column volume, the dwell volume is determined to be 1.096 mL. This is similar to the 1.14 mL volume determined by the UV tracer method described above.

[0069] Figure 10 Another exemplary embodiment of the method is depicted. In this example, the chromatography system is a chromatographic system available from Waters Corporation, Milford, Massachusetts. Arc system. The chromatography column was a 4.6 mm x 250 mm column with a 5 μm particle size and a column volume of 2.32 mL. The gradient was an aqueous solvent mixture that transitioned from 60% acetonitrile to 90% acetonitrile over ten minutes at a flow rate of 1.0 mL / min. Curve 78 depicts the programmed gradient, and curve 80 is the measured pressure trace with a maximum of 15.81 MPa (2,293 psi) and a minimum of 9.45 MPa (1,370 psi).

[0070] The measured pressure trace 80 exhibits a first Δt of 1.16 minutes at a mid-range pressure value corresponding to a dwell volume of 1.99 mL. The second Δt, determined from the rise in pressure trace 80 resulting from the reverse linear gradient starting at 22.0 minutes, is also 1.16 minutes. After subtracting 1.16 mL, which is half the column volume, the dwell volume is determined to be 0.83 mL. This is similar to the 0.81 mL volume determined by the UV tracer method described above.

[0071] In the above examples, a linear gradient is used to generate the pressure trace. In an alternative embodiment of the method, the linear gradient is replaced by a step change in the solvent mixture composition. In other words, the solvent mixture composition suddenly changes from one value to a different value. The two compositions preferably correspond to the linear region of viscosity as a function of composition ( Figure 4 and Figure 5 ). The resulting pressure trace includes faster transitions between maxima and minima; however, the principles associated with measuring time delays and column volume adjustments remain the same.

[0072] In another alternative implementation of this method, the programmed gradient can be intentionally selected to be a nonlinear gradient. The nonlinear gradient can be defined to compensate for the nonlinearity of viscosity changes, so that the pressure trace can have improved linearity. Thus, this intentional "distortion" in the linearity of the gradient composition change enables operation over a wider range of viscosity values, thereby providing a linear pressure response and enabling the offset between the programmed gradient and the measured pressure trace to be easily determined for dwell volume estimation.

[0073] In some embodiments, the present invention provides the liquid chromatography system of the present invention.Although the above example of description is for the liquid chromatography system that wherein solvent is mixed under lower (for example, atmospheric pressure) pressure, in other specific implementations of the method, the liquid chromatography system with a separate pump (high pressure gradient generation) for every solvent can be used.In this type of specific implementation, the solvent delivered from the outlet of each pump is provided to the corresponding inlet of blender, and blender provides high pressure solvent mixture to injector valve and chromatographic column subsequently.The flow velocity of pump is timely controlled, to maintain the constant solvent mixture flow velocity of the solvent composition with variation.Measure system pressure at the position of blender outlet or downstream, and perform the calculation of gradient delay and dwell volume as described above for low pressure mixing example.

[0074] Figure 11 1 is a flow chart representation of a general example of a method 100 for determining the hold-up volume of a liquid chromatography system. The method 100 includes mixing (110) a flow of a first solvent and a flow of a second solvent at a mixing location (e.g., a GPV) to produce a solvent mixture in the system flow. During a gradient duration, the flow of the first solvent decreases and the flow of the second solvent increases (120) to generate a gradient composition of the solvent mixture. Although in some embodiments, the gradient composition is a linear gradient, in alternative embodiments, the gradient is a nonlinear gradient or a step gradient. The system pressure of the liquid chromatography system is measured (130) to determine a pressure trace, which is defined as the measured system pressure as a function of time. In some embodiments, the system pressure is measured at the output of a system pump. In alternative embodiments, the system pressure can be measured at different locations upstream of a chromatography column or a system flow restrictor (if a chromatography column is not present). After determining the pressure trace, the hold-up volume of the liquid chromatography system is determined (140) using a time delay between the gradient composition at the mixing location and the pressure trace.

[0075] Method 100 can be performed in an automated manner, and if the chromatographic column is in a system flow, the method takes column volume into account. In some specific implementations, the column volume is calculated using the predetermined information of the column (e.g., column size, particle size, etc.). For example, a lookup table can store the predetermined information of a plurality of columns. The column volume is then calculated by retrieving the stored information in the lookup table associated with the column used and using the retrieved information to calculate the column volume, thereby determining the column volume of a specific column. Alternatively, a lookup table can store previously determined values ​​of the column volumes of different column types, and the column volume of the column in use can be directly determined by retrieving from the lookup table.

[0076] The method and its variations can be implemented in software (including firmware, resident software, microcode, etc.) or as a combination of software and hardware. In addition, the method can be implemented in the form of a computer program product embodied in one or more computer-readable media having computer-readable program code embodied thereon.

[0077] Any combination of one or more computer-readable storage media may be utilized. The computer-readable medium may be a computer-readable signal medium or a computer-readable storage medium. The computer-readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, device, or apparatus, or any suitable combination of the foregoing. More specific examples (not an exhaustive list) of computer-readable storage media would include the following: an electrical connection with one or more wires, a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disk read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing. The computer-readable storage medium may be any tangible medium that may contain or store a program for use by or in conjunction with an instruction execution system, device, or apparatus.

[0078] A computer-readable signal medium may include a propagated data signal having computer-readable program code embodied thereon, for example, in baseband or as part of a carrier wave. Such a propagated signal may take any of a variety of forms, including but not limited to electromagnetic, optical, or any suitable combination thereof. A computer-readable signal medium may be any computer-readable medium that is not a computer-readable storage medium and that can communicate, propagate, or transport a program for use by or in conjunction with an instruction execution system, apparatus, or device. The program code embodied on a computer-readable medium may be transmitted using any suitable medium, including but not limited to wireless, wireline, fiber optic cable, RF, etc., or any suitable combination of the foregoing.

[0079] The computer program code for performing the operation of various aspects of the present invention can be written in any combination of one or more programming languages, and the programming language comprises object-oriented programming language and conventional process programming language.Program code can be executed completely on the user's computer, partly on the user's computer, as an independent software package, partly on the user's computer and partly on a remote computer or completely on a remote computer or server.In the latter case, the remote computer can be connected to the user's computer by any type of network (comprising local area network (LAN) or wide area network (WAN)), or can be connected to an external computer (such as by using the Internet of an Internet service provider).

[0080] These computer program instructions may be provided to a processor of a general-purpose computer, a special-purpose computer, or other programmable data processing device to produce a machine, so that the instructions executed by the processor of the computer or other programmable data processing device form a means for implementing the functions / actions of the various methods described. Figure 1 The computer program instructions may also be stored in a computer-readable medium that can instruct a computer, other programmable data processing device, or other apparatus to operate in a specific manner, such that the instructions stored in the computer-readable medium produce an article of manufacture that includes instructions for implementing the functions / actions of the method. The computer program instructions may also be loaded onto a computer, other programmable data processing device, or other apparatus to cause a series of operational steps to be executed on the computer, other programmable device, or other apparatus to produce a computer-implemented process, such that the instructions executed on the computer or other programmable device provide a process for implementing the functions / actions.

[0081] In the example of the method for determining the residence volume of a liquid chromatography system described above, various advantages are achieved. No additional compounds, such as UV tracers, are utilized, thereby reducing operator preparation time and eliminating the risk of cross-contamination by compounds intended for later separation. In addition, the liquid chromatography system does not require physical reconfiguration. For example, the method can be performed without removing the chromatographic column and rearranging the flow path with additional tubing. Advantageously, the method allows for efficient determination of the residence volume of the chromatography system, thereby reducing operator interaction and therefore reducing the possibility of human error. In addition, the time required for the method and the resulting accuracy can reduce the difficulties encountered when transferring the method between different chromatography systems and configurations.

[0082] While the present invention has been shown and described with reference to particular embodiments, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the scope of the invention as recited in the following claims.

Claims

1. A method for determining the dwell volume of a liquid chromatography system, comprising: mixing the stream of the first solvent and the stream of the second solvent at a mixing location to produce a solvent mixture in a system stream of the liquid chromatography system; decreasing the flow of the first solvent and increasing the flow of the second solvent over a gradient duration to produce a gradient composition of the solvent mixture; measuring a system pressure of the liquid chromatography system to determine a pressure trace, the pressure trace being defined as the measured system pressure as a function of time; as well as A dwell volume of the liquid chromatography system is determined based on a time delay determined between the gradient composition and the pressure trace at the mixing location.

2. The method of claim 1, wherein the solvent mixture has a viscosity and wherein the viscosity varies in response to the gradient composition.

3. The method of claim 2, wherein the change in viscosity is linearly proportional to the change in the solvent mixture according to the gradient composition. The method of claim 1 , wherein the viscosity of the first solvent is different from the viscosity of the second solvent. The method according to claim 1 , wherein the gradient composition is a linear gradient composition. The method of claim 1 , wherein the gradient composition is a step gradient.

7. The method of claim 1 , wherein the liquid chromatography system comprises a chromatography column and wherein determining the dwell volume comprises: determining the product of the time delay and the flow rate of the solvent mixture; as well as Half the volume of the chromatography column is subtracted from the product.

8. The method of claim 1, wherein the liquid chromatography system comprises a flow restrictor configured to provide a system pressure drop for the system flow, and wherein determining the dwell volume comprises determining the product of the time delay and the flow rate of the solvent mixture. 9 . The method of claim 1 , wherein the gradient composition is a non-linear gradient composition determined to provide a linear pressure change for at least a portion of the pressure trace in response to the gradient composition.

10. A computer program product for determining a dwell volume of a liquid chromatography system, the computer program product comprising: A computer-readable storage medium having computer-readable program code embodied therewith, the computer-readable program code being executable by a computer of a liquid chromatography system, the computer-readable program code comprising: computer readable program code configured to mix a first solvent and a second solvent at a mixing location to produce a solvent mixture in a system flow of the liquid chromatography system; computer readable program code configured to decrease the flow of the first solvent and increase the flow of the second solvent over a gradient duration to produce a gradient composition of the solvent mixture; computer readable program code configured to measure system pressure and determine a pressure trace from the system pressure, the pressure trace being defined as the measured system pressure as a function of time; and Computer readable program code configured to determine a dwell volume of the liquid chromatography system based on a time delay between the gradient composition and the pressure trace at the mixing location.

11. The computer program product of claim 10, wherein the liquid chromatography system comprises a chromatography column, and wherein the computer readable program code configured to determine a dwell volume comprises: computer readable program code configured to determine a product of the time delay and the flow rate of the solvent mixture; as well as Computer readable program code configured to subtract half the volume of the chromatography column from the product.

12. A liquid chromatography system comprising: a source of a first solvent and a source of a second solvent; a mixer having a mixer inlet for receiving the first solvent and the second solvent and a mixer outlet for providing a solvent mixture; a pump having a pump inlet in fluid communication with the mixer outlet and having a pump outlet, the pump receiving the solvent mixture at the pump inlet and providing the solvent mixture at system pressure in a system stream dispensed from the pump outlet; a flow restrictor having a restrictor inlet in fluid communication with the pump outlet and having a restrictor outlet; a pressure sensor disposed in the system flow after the pump outlet and generating a pressure signal indicative of a measured system pressure; as well as a processor in communication with the sources of the first and second solvents and with the pressure sensor, the processor configured to: generating a gradient composition in the system flow by controlling a contribution of the first solvent to the mixer and a contribution of the second solvent to the mixer; determining a pressure trace from the pressure signal, the pressure trace being defined as measured system pressure as a function of time; determining a time delay between the gradient composition at the mixing location and the pressure trace; as well as The dwell volume of the liquid chromatography system is determined based on the time delay.

13. The liquid chromatography system of claim 12, further comprising a gradient proportioning valve disposed between the mixer and the sources of the first solvent and the second solvent.

14. The liquid chromatography system of claim 12, wherein the processor determines the dwell volume as the product of the time delay and the flow rate of the solvent mixture.

15. The liquid chromatography system of claim 12, wherein the flow restrictor is a chromatography column and wherein the determining of the dwell volume comprises: determining the product of the time delay and the flow rate of the solvent mixture; as well as Half the volume of the chromatography column is subtracted from the product.

16. A liquid chromatography system, comprising: a source of a first solvent and a source of a second solvent; a first pump having a first pump inlet in fluid communication with the source of the first solvent and having a first pump outlet; a second pump having a second pump inlet in fluid communication with the source of the second solvent and having a second pump outlet; a mixer having a first mixer inlet and a second mixer inlet in fluid communication with the first pump outlet and the second pump outlet, respectively, the mixer having a mixer outlet for providing a solvent mixture; a flow restrictor having a restrictor inlet in fluid communication with the pump outlet and having a restrictor outlet; a pressure sensor disposed in the system flow after the mixer outlet and generating a pressure signal indicative of a measured system pressure; as well as a processor in communication with the first and second pumps and the pressure sensor, the processor configured to: generating a gradient composition in the system flow by controlling a contribution of the first solvent to the mixer and a contribution of the second solvent to the mixer; determining a pressure trace from the pressure signal, the pressure trace being defined as the measured system pressure as a function of time; determining a time delay between the gradient composition at the mixing location and the pressure trace; as well as The dwell volume of the liquid chromatography system is determined based on the time delay.

17. The liquid chromatography system of claim 16, wherein the processor determines the dwell volume as the product of the time delay and the flow rate of the solvent mixture.

18. The liquid chromatography system of claim 16, wherein the flow restrictor is a chromatography column, and wherein determining the dwell volume comprises: determining the product of the time delay and the flow rate of the solvent mixture; as well as Half the volume of the chromatography column is subtracted from the product.

Images