Liquid chromatography system configuration

Abstract

Described is a liquid chromatography system configuration. The configuration includes a binary solvent delivery system with a first pump for a strong solvent and a second pump for a weak solvent; an injector that is placed after the first pump and before a mixing tee that is used to combine the strong and weak solvent flow; a mixer placed downstream from the mixing tee and upstream from a column, is effective in radial mixing with small internal volume.

Description

[0001] TITLE: Liquid chromatography system configuration

[0002] Cross Reference to Related Applications:

[0003] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 568,073, filed March 21, 2024, where permissible incorporated by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] The present invention relates generally to liquid chromatography, including high performance liquid chromatography ( HPLC ) and ultrahigh pressure liquid chromatography(UHPLC) , and in particular to systems and methods for large volume injection(LVI) within a liquid chromatography system.

[0006] BACKGROUND

[0007] Liquid chromatography (LC) is a technique for performing an analytical or preparative separation of a liquid sample into constituent components. During a chromatographic separation, the liquid sample is transported in a mobile phase and forced through a stationary phase that is typically provided in the form of a particle packed bed inside a column or cartridge through which the mobile phase flows. The respective compositions of the mobile phase and stationary phase are selected to cause differing components of the sample material in the column to become distributed between the mobile phase and the stationary phase to varying degrees, dependent on the respective physiochemical properties of the sample material's components. Components with higher affinity towards the stationary phase travel slower than the mobile phase, while components with weaker affinity travel more rapidly. As a result, components of differing compositions become separated from each other as the mobile phase flows through the column.

[0008] Typically, a sample to be separated is introduced into a column by first injecting a sample plug of a small volume into the mobile phase at a point upstream of the column. The mobile phase may be a mixed flow stream formed by mixing the flows of one weak solvent and one strong solvent in a solvent mixer upstream of the column. In the case of so-called binary solvent gradient, the concentration of the strong solvent in the mobile phase is increased along with time, so that strongly retained components in the sample can be eluted out of the column faster and in a reasonable period of time. Typically, the sample is injected into the column with a solvent composition that is weaker than the mobile phase.

[0009] When the sample is injected into the column with the strong solvent being at a high concentration, it may cause chromatographic band broadening relative to the injected sample volume, and thus poor chromatographic separation power. In severe cases, sample components pass through the column in the void (breakthrough). Still, in practice, there are many cases where large volume of liquid sample, sample in strong solvent or both needs to be injected and analyzed. In case that sample components have low solubility in weak solvent, sample components may be lost to any contact surface during sample preparation and analysis. If this occurs inside a sample injector, it leads to the injector carryover. Another case is for trace level analysis when concentration levels of sample components are lower than ng / mL, large injection volume is usually preferred to obtain needed sensitivity, strong sample solvent is preferred to prevent absorption loss.

[0010] When analyzing samples of biological origins, samples need to be prepared in such a way that the obtained extracts are suitable for LC separation. Typical sample preparation methods include solid phase extraction, liquid-liquid extraction and protein precipitation. Common to all sample preparation methods is that analyte of interest is in a solution with high concentration of strong solvent at a certain point of time during sample preparation. Current practices include evaporation to completely remove or partially reduce the concentration of strong solvent, reconstitution or dilution with a weak solvent. It should be advantageous to be able to directly inject the extracts in strong solvent for analysis.

[0011] In the case of two-dimensional liquid chromatography (2D-LC) for complex samples, the eluent from the first dimension and stored in a sample loop, which is usually in relatively large volume and with high concentration of strong solvent, needs to be injected for fast separation in the second dimension, and solvent mismatch is common in 2D-LC considering different chromatographic modes possibly associated with each dimension.

[0012] SUMMARY

[0013] A liquid chromatography system includes a binary solvent delivery system, an injector, a mixing tee, a mixer and a chromatographic column. The binary solvent delivery system includes a first pump that draws a strong solvent from one fluid source and a second pump that draws a weak solvent from a second fluid source. The injector is configured to receive the strong solvent flow from the first pump and to inject a sample into the strong solvent flow. The mixing tee has a first inlet connected to the injector, a second inlet connected to the second pump, and an outlet connected to the inlet of the mixer to provide the combined flow to the mixer. The mixer includes an inlet and an outlet. The inlet is configured to receive the combined fluid flow from the mixing tee and the outlet is configured to provide the mixed fluid flow to the chromatography column. The chromatography column is connected to the outlet of the mixer for performing a separation of the sample.

[0014] The injector may include a two-position six-ports valve with a sample loop. The injector may include a multiple-position valve with multiple sample loops. Furthermore, the sample loop may be a coiled tube, a packed tube or a coiled packed tube.

[0015] The mixing tee may be a simple three-way tee. The mixing tee may include mixing elements, such as a frit, a filter, a small particle packed bed or the like.

[0016] The mixer may be a mixing chamber of any shape or form, wherein, together with the mixing tee, the fluid flow through the mixer is sufficiently mixed in radial direction while the total internal volume of the mixing tee and the mixer is kept small.

[0017] The mixer may be a piece of tubing of small inner diameter and long enough to provide sufficient mixing by molecular diffusion.

[0018] The mixer may include a coiled tube, wherein radial dispersion of a fluid flow through the coiled tube is increased while axial dispersion is decreased, under laminar flow conditions. The coiled tube may be any form or shape, such as circular, rectangular or trapezoidal crosssection, spiral or helical coil form, on a flat plane or three dimensional, multiple different coils serially connected.

[0019] A mixer may be added between the first pump and the injector, and / or between the second pump and the mixing tee.

[0020] The fluid source may include a gradient proportioning valve to provide the solvent flow to the connected pump.

[0021] BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 is a block diagram of a liquid chromatography system with binary solvent configuration

[0023] FIG. 2A shows a schematic depiction of radial mixing by a mixer

[0024] FIG. 2B shows a schematic depiction of a sample plug in the mobile phase

[0025] FIG. 2C shows a schematic depiction of solvents and sample mixing with disclosed LC system FIG. 3 is a block diagram of a liquid chromatography system with disclosed configuration

[0026] FIG. 4 is a block diagram of a liquid chromatography system as in FIG.3 with a flow selection valve

[0027] FIG. 5 is a block diagram of a liquid chromatography system as in FIG.3 with a tubing coil as the mixer

[0028] FIG . 6 is a graph comparing signal traces acquired using two different mixers

[0029] FIG . 7 is a schematic representation of an injector with a sample loop in the form of a coiled tube

[0030] DESCRIPTION OF EXAMPLARY ENBODIMENTS

[0031] Reference in the specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the teaching. References to a particular embodiment within the specification do not necessarily all refer to the same embodiment.

[0032] The present teaching will now be described in detail with reference to exemplary embodiments thereof as shown in the accompanying drawings. While the present teaching is described in conjunction with various embodiments and examples, it is not intended that the present teaching be limited to such embodiments and examples. On the contrary, the present teaching encompasses various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art. For example, various embodiments described herein refer to solvents although it should be recognized that other fluids can be used. Those of ordinary skill having access to the teaching herein will recognize additional implementations, modifications, and embodiments, as well as other fields of use, which are within the scope of the present disclosure as described herein.

[0033] FIG. 1 is a block diagram of a liquid chromatography system 10 with binary solvent configuration that is commonly adopted. The liquid chromatography system 10 can be a high pressure liquid chromatography (HPLC) system, an ultrahigh pressure liquid chromatography (UHPLC) system, or the like.

[0034] The liquid chromatography system 10 includes a solvent delivery system to provide a mobile phase. The solvent delivery system includes solvent sources 11A and 11B (generally 11). Two pumps 12A and 12B are used with each pump being coupled to solvent sources 11A and 11B respectively. The solvent flows from the two pumps 12 are merged at high pressure in a fluidic tee 131 and the combined solvent flows pass through a mixer 13 that is provided downstream from the pumps 12. An injector 14 is disposed downstream from the mixer 13.

[0035] The injector 14 may be included as a feature of a sample manager or other sub system (e.g. the modulation valve interface in a 2D-LC set up) configured to inject a sample plug into the mobile phase that exits the mixer 13. The injector 14 may include an injector valve with a sample loop. The sample manager can control the injector valve and may operate in one of two states: a load state and an injection state. In the load state, the injector valve is configured to enable the sample manager to load the sample into the sample loop. In the injection state, the injector valve is configured such that the sample manager enables the sample plug in the sample loop to be injected into the continuously flowing mobile phase from the mixer 13.

[0036] The chromatographic column 15 receives the mobile phase carrying the injected sample plug, and analytes in the sample are separated and eluted from the chromatographic column 15 as elution bands. The output port of the chromatographic column 15 is fluidically coupled to a detector 16, for example, a mass spectrometer or an ultraviolet (UV) detector. From the elution bands, the detector 16 produces output signals (chromatographic peaks) from which the identity and quantity of sample components can be determined.

[0037] The mixer 13 can be configured to passively mix the combined solvent flow from the mixing tee 131, as depicted in Fig. 2A. The weak solvent 133 and the strong solvent 132 enter the mixer 13 and can be mixed effectively in Fig. 2D radial direction (cross-sectional) that is perpendicular to the flow direction 130, and exit the mixer 13 as a fully mixed mobile phase 134. However, in the axial flow direction 130, a large mixer volume is required to provide a substantial reduction or a near complete elimination of the periodic flow fluctuation (manifested as baseline noise of the detector 16 signal output) resulting from the pumps 12.

[0038] The separation power of a LC system 10 as depicted in FIG.l is determined by the column 15. The narrower the elution bands or zones (in time or volume, also referred to as chromatographic peaks) exiting the column 15, the higher the separation performance. For example, UHPLC utilizes sub 2-pm particle packed LC column to achieve chromatographic peak widths in time less than 1 second. However, there are many constraints that must be satisfied in order to achieve the highest separation power that a LC column can afford. The injection volume is one of them. Even smaller injection volume is necessary for UHPLC to further reduce axial band broadening, compared to HPLC.

[0039] FIG. 2B shows a schematic depiction of a sample plug 135 surrounded by the mobile phase 134 in the sample loop of the injector 14. The sample plug 135 should have a significant axial dimension (physical length or volume) considering a reasonable injection volume and the small internal diameter of the sample loop of the injector 14. It is easy for people skilled in the art to understand that a huge mixer volume relative to the sample volume may be needed to effectively mix the sample plug 135 with significant amount of the mobile phase 135. The imaginary mixer 113 is not present in a typical LC system as depicted in FIG.l because the mixer volume should be prohibitive. Due to that there is no effective mixing after the injector and before the column, large volume injection with or without solvent mismatch suffers from severe loss of separation power. There are a few literatures in which small inline mixers were used to mitigate issues with large volume injection to limited degrees.

[0040] With a typical LC system as depicted in FIG.1, an approach called on-column focusing is utilized to inject as much sample volume as possible, by which a sample solvent composition is weak enough to cause high affinity (also called retention) towards the stationary phase in a LC column, to achieve chromatographic peak widths close to the optimum. In practice, a sample may contain many components, some are retained very weakly, necessitating weak injection solvent composition, while some are retained too strongly, it will take too long for them to elute from the column in a weak mobile phase, which is not changed during a LC separation (the isocratic mode). With gradient elution mode, the concentration of the strong solvent in the mobile phase keeps increasing with time, and strongly retained components in the sample can be eluted from the column faster in a reasonable time. Another benefit of gradient elution mode is that the elution band keeps being narrowed during the gradient (the compression effect).

[0041] With gradient elution mode, there is one factor, called dwell volume (or gradient delay volume), which is critical to fast separation. In FIG. 1, the dwell volume 120 is a system volume from the mixing point 131 at which the mobile phase solvents are mixed until they reach the head of the column 15, including the mixing tee 131, the mixer 13, the injector 14 and multiple connecting conduits or tubing 101. The gradient needs to flow through the dwell volume to reach the head of the column to take effect. Before that, the mobile phase stays the same as the initial composition, thus being an isocratic step.

[0042] To address the foregoing problems, in whole or in part, and / or other problems that may have been observed by persons skilled in the art, the present disclosure provides methods, processes, systems, apparatus, instruments, and / or devices, as described by way of example in implementations set forth below.

[0043] According to one embodiment, an improved LC system is depicted in FIG. 3. All components in a LC system depicted by FIG. 3 are present in a LC system depicted by FIG .1. The major differences between the two include: 1) the injector 14 is positioned between the pump 12A and the mixing tee 131, the pump 12A is used to deliver the strong solvent 132 through the injector 14; 2) a mixer 13', different from the mixer 13 in FIG. 1 (explanation below), is positioned directly before the column 15 and after the mixing tee 131. Apparently, the dwell volume 120' does not include the internal volume (e.g. the sample loop) of the injector 14. The sample loop in the injector 14 may be as large as needed to hold large volume of sample to be injected, without contributing to the dwell volume 120'. The traditional mixers 13 can still be placed between pump 12A and the injector 14 as well as between pump 12B and the mixing point 131, if needed and dependent on pump design. However, the mixers' volumes should not be included in the dwell volume 120'. These mixers may have as large internal volume as needed to eliminate the flow noise from the pumps 12 completely.

[0044] FIG. 2C shows a schematic depiction of mixing solvents and sample in a LC system with disclosed configuration. The sample is injected into the strong solvent flow 132 first, and then the strong solvent flow 132 and the weak solvent flow 133 are combined in the mixing tee 131 before entering the mixer 13'. Similar to FIG. 2A, a small mixer volume is needed for effective radial mixing since the axial mixing as in FIG. 2B is not critical or needed (least axial dispersion is preferred). Large volume injection can be achieved provided that the initial mobile phase composition is weak enough to achieve on-column focusing, even larger volume can be injected with gradient elution due to the compression effect. One drawback is that it will take too long for injection to complete if the strong solvent flow rate is too low.

[0045] FIG. 4 shows an embodiment according to the disclosure that includes a flow selection valve 17 after the pump 12. The flow selection valve can be any kind of two position valve with at least four ports, two for inlets and two for outlets. Two inlet ports are connected to pump 12A and 12B respectively. One outlet port is connected to injector 14 and one outlet port is connected to the mixing tee 131. By switching valve 17, either the strong solvent from pump 12A or the weak solvent from pump 12B can be connected to the injector. With the weak solvent flows through the injector, large volume of sample in weak solvent can be injected for some applications. FIG. 5 shows an embodiment according to the disclosure that uses a piece of helically coiled tube as the mixer 13". It is theoretically and experimentally shown that in coiled tubes, the centrifugal forces present in fluid layers in a laminar flow regime contribute to increased radial mixing across the inner diameter of the tube while, at the same time, reducing the axial mixing along the tube. The centrifugal forces are generated by the curvature of the coiled tube. As the radius of the coil is decreased, the radial dispersion increases and the axial dispersion decreases. In general, smaller curvature radius leads to greater radial mixing with a certain flow rate and the inner diameter of the tube. The flow properties of coiled tubes match well with the requirements of the mixer 13": small mixing volume, effective radial mixing and least axial dispersion. Small mixing volume, thus small dwell volume, is critical to mobile phase gradient fidelity, especially for fast gradient separation.

[0046] FIG .6 is a graph 100 of detector signal traces for two mixers, including the mixer 13" in the form of a coiled tube of FIG . 3, according to embodiments of the present disclosure. In this graph, the upper traces 101 corresponds to signal outputs with a straight tube (with same length and internal diameter as the coiled mixer) used as the mixer, while the lower traces 102 corresponds to the coiled mixer of the present disclosure. It can be seen in this graph that the traces 102, which corresponds to the coiled mixer of this disclosure, has narrow and symmetric chromatographic peaks and no peak shape distortion, compared to the traces 101 of the straight tube mixer. In this embodiment, the top traces 101 corresponding to the straight tube mixer are much wider and badly deformed. The coiled mixer is formed by winding the 1 / 16" PEEK tubing (0.01" id and 30" long) around a rod of 0.360" outer diameter. The internal volume of the tubing is calculated as 38.6 pL, plus the internal volume of the mixing tee 131 of 0.57 pL, resulting in a dwell volume less than 40 pL. The flow rate of the mobile phase (mixture of methanol as the strong solvent and water as the weak solvent ) used is 0.4 mL / min, with a 50 mm by 2 mm C18 column. The injection volume is 50 pL and the injection solvent is 100% acetonitrile. The signal traces are acquired by a tandem mass spectrometry detector in multiple reaction monitoring mode.

[0047] FIG .7 is a schematic representation of an injector 14 with a sample loop in the form of a coiled tube. As shown, a two-position six-port valve 141 in loading mode has a sample loop 142 in the form of a coiled tube that is connected to its two ports 143 and 144. Besides the coiled sample loop 142 for its potential reduction in axial dispersion, the injection valve 141, all fluidic connections to it, and its operation is common to any LC system. However, according to the embodiment depicted in FIG. 3, only strong solvent 134 flows through the injection valve. In general, this should lead to less carryover by the injection valve 141 considering high solubility of sample components in the strong solvent 134. Additionally, as shown in FIG. 5, since a large loop volume can be used without contributing to the dwell volume 120', it is possible to bracket the sample plug 135 with strong solvent 134 on both sides and position the sample plug away from the injection valve 141 in load position. When the rotator of the injection valve turns to the injection position, there are no sample components inside the internal volume (including ports and grooves) of the injection valve, leading to no absorption loss of sample components to any contact surface and total elimination of potential carryover.

[0048] The disclosed LC system configurations capable of injecting large volume of sample in strong solvent make it possible to simplify sample preparation procedure. In the case of solid phase extraction, an elution solvent that is usually strong is used; the eluted sample needs to be fully evaporated and then reconstituted in a weak solution. In the case of protein precipitation, an organic solvent that is usually a strong solvent is used to precipitate the proteins in the biological samples; the supernatant is either diluted with a weak solvent or fully evaporated and then reconstituted in a weak solution. With the disclosed configuration, for both cases, a large volume of the eluted sample or the supernatant can be injected to obtain the required sensitivity, eliminating the laborious, problematic and environment unfriendly procedures, such as supernatant dilution or solvent evaporation. By this way, sample preparation is simplified and becomes easy to automate.

[0049] The disclosed LC system configurations may be beneficial for 2D-LC in that : 1) large sample volume can be injected into the first dimension to improve overall sensitivity; 2) the modulation interface including an injector can hold large volume of eluent from the first dimension without loss; 3) completely decoupling the first and second dimensions allows various chromatographic separation modes to be used in each dimension free from solvent incompatibility or mismatch; 4) small dwell volume allows fast gradient separation particularly for the second dimension; 5) a simple and flexible system configuration for easy method development and optimization.

[0050] In FIG. 5, a helically coiled tube is used; however, it will be recognized that in some implementations, the coiled tube may be replaced by other forms of a channel, in terms of curvature and cross section. For example, the coiled tube may be defined on a plane of a microchip or be an internal channel in a substrate.

[0051] While various examples have been shown and described, the description is intended to be exemplary, rather than limiting and it should be understood by those of ordinary skill in the art that various changes in form and detail may be made therein without departing from the scope of the invention as recited in the accompanying claims.

Claims

CLAIMSWhat is claimed is:

1. A liquid chromatography system comprising: a. a binary solvent delivery system including a first pump that draws a strong solvent from one fluid source and a second pump that draws a second weak solvent from a second fluid source; b. an injector configured to receive the strong solvent flow from the first pump and to inject a liquid sample into the strong solvent flow; c. a mixing tee having a first inlet connected to the injector, a second inlet connected to the second pump, and an outlet connected to the inlet of a mixer to provide the combined flow to the mixer; d. a mixed fluid flow from the outlet of the mixer; and e. a chromatography column connected to the outlet of the mixer, wherein the chromatography column provides separation the liquid sample.

2. The liquid chromatography system of claim 1 wherein the injector has a two-position six- port valve with a sample loop.

3. The liquid chromatography system of claim 2 wherein the sample loop is a coiled tube, a packed tube or a coiled packed tube.

4. The liquid chromatography system of claim 2 wherein the injector has a multipleposition valve with multiple sample loops.

5. The liquid chromatography system of claim 1 wherein the mixing tee is a simple three- way tee.

6. The liquid chromatography system of claim 1 wherein the mixing tee includes a frit element, a filter element, or a small particle packed bed element.

7. The liquid chromatography system of claim 1 having a mixer with a mixing chamber having any shape couple with the mixing tee to sufficiently mixes the fluid flow in a radial direction while maintaining a total internal volume in the mixing tee and the mixer small.

8. The liquid chromatography system of claim 7 wherein the mixing chamber includes a piece of small inner diameter tubing which is long enough to provide sufficient mixing by molecular diffusion.

9. The liquid chromatography system of claim 7 wherein the mixing chamber contains a coiled tube for radial dispersion of the fluid flow while decreasing axial dispersion under laminar flow conditions.

10. The liquid chromatography system of claim 9 wherein the coiled tube has a curved flow path in a form that is circular, rectangular, trapezoidal cross-section, spiral or helical on a flat plane or having three dimensional multiple different coils serially connected.

11. The liquid chromatography system of claim 1 further having the mixer between the first pump and the injector.

12. The liquid chromatography system of claim 1 further having the mixer between the second pump and the mixing tee.

13. The liquid chromatography system of claim 1 further comprising a two-position solvent selection valve to guide either the strong solvent or the weak solvent to the injector.

14. The liquid chromatography system of claim 1 having the fluid source further comprising a gradient proportioning valve to provide the solvent flow to the connected pump.

15. A method for large liquid sample volume injection comprising: a. directing a first strong solvent flow stream and a second weak solvent flow stream to a low volume mixing tee; b. mixing the two solvent flow streams within the low volume mixing tee to create a combined solvent stream; c. guiding the combined solvent stream through a low volume mixer to achieve efficient radial mixing and obtain an initial mobile phase composition; d. injecting a large volume of the liquid sample into the first strong solvent flow stream before the low volume mixing tee; and e. starting a gradient elution program, wherein injection of the large liquid sample volume is completed is completely loaded.

16. The method for large liquid sample volume injection of claim 15 wherein the solvent in the liquid sample dissolved is less than or equally as strong as the strong solvent of a mobile phase.

17. The method for large liquid sample volume injection of claim 16 wherein the initial mobile phase composition results in sufficiently high retention for an analyte of interest.

18. The method for large liquid sample volume injection of claim 15 wherein the liquid sample volume is loaded in a time period equal to the liquid sample volume divided by the flow rate of the first strong solvent.

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