Differential refractometer for gradient chromatography

The present disclosure describes a differential refractometer for gradient chromatography. In an exemplary embodiment, the differential refractometer includes a solvent delay volume, an eluent flow meter coupled to an eluent inlet of a sample cell, a solvent flow regulator coupled to an outlet of the solvent delay volume and coupled to a solvent inlet of a reference cell, an instrument controller configured to receive the eluent flow rate from the eluent flow meter, configured to receive the solvent flow rate from the solvent flow regulator, configured to receive a flow rate ratio from a flow rate ratio data source, wherein the flow rate ratio indicates a ratio of the eluent flow rate to the solvent flow rate, and an optical bench configured to measure a difference between a refractive index of the eluent present in the sample cell and a refractive index of the solvent present in the reference cell.

BACKGROUND

The present disclosure relates to differential refractometry, and more specifically, to a differential refractometer for gradient chromatography.

SUMMARY

The present disclosure describes a differential refractometer for gradient chromatography. In an exemplary embodiment, the differential refractometer includes (1) a solvent delay volume configured to be coupled to an outlet of a chromatography pump, (2) an eluent flow meter configured to be coupled to an outlet of a chromatography column and coupled to an eluent inlet of a sample cell, where the eluent flow meter is configured to measure an eluent flow rate of an eluent flowing through the sample cell, (3) a solvent flow regulator coupled to an outlet of the solvent delay volume and coupled to a solvent inlet of a reference cell, where the solvent flow regulator is configured to measure and to regulate a solvent flow rate of a solvent flowing through the reference cell, (4) an instrument controller configured to receive the eluent flow rate from the eluent flow meter, configured to receive the solvent flow rate from the solvent flow regulator, configured to receive a flow rate ratio from a flow rate ratio data source, wherein the flow rate ratio indicates a ratio of the eluent flow rate to the solvent flow rate, and configured to transmit a flow command to the solvent flow regulator to achieve the flow rate ratio, and (5) an optical bench configured to measure, in response to receiving from the instrument controller a signal indicating that the flow rate ratio has been achieved, a difference between a refractive index of the eluent present in the sample cell and a refractive index of the solvent present in the reference cell.

DETAILED DESCRIPTION

The present disclosure describes a differential refractometer for gradient chromatography. In an exemplary embodiment, the differential refractometer includes (1) a solvent delay volume configured to be coupled to an outlet of a chromatography pump, (2) an eluent flow meter configured to be coupled to an outlet of a chromatography column and coupled to an eluent inlet of a sample cell, where the eluent flow meter is configured to measure an eluent flow rate of an eluent flowing through the sample cell, (3) a solvent flow regulator coupled to an outlet of the solvent delay volume and coupled to a solvent inlet of a reference cell, where the solvent flow regulator is configured to measure and to regulate a solvent flow rate of a solvent flowing through the reference cell, (4) an instrument controller configured to receive the eluent flow rate from the eluent flow meter, configured to receive the solvent flow rate from the solvent flow regulator, configured to receive a flow rate ratio from a flow rate ratio data source, wherein the flow rate ratio indicates a ratio of the eluent flow rate to the solvent flow rate, and configured to transmit a flow command to the solvent flow regulator to achieve the flow rate ratio, and (5) an optical bench configured to measure, in response to receiving from the instrument controller a signal indicating that the flow rate ratio has been achieved, a difference between a refractive index of the eluent present in the sample cell and a refractive index of the solvent present in the reference cell. In an embodiment, the flow rate ratio is a ratio of a volume of a void in the chromatography column to a volume of a void in the solvent delay column. In an embodiment, the flow rate ratio is a ratio of a sum of a volume of a void in the chromatography column, a volume of tubing coupled to the chromatography column, and a volume of the eluent flow meter, to a sum of a volume of a void in the solvent delay column, a volume of tubing coupled to the solvent delay column, and a volume of the solvent flow regulator.

In an exemplary embodiment, the differential refractometer includes (1) a solvent delay volume configured to be coupled to an outlet of a chromatography pump, (2) an eluent flow meter configured to be coupled to an outlet of a chromatography column and coupled to an eluent inlet of a sample cell, where the eluent flow meter is configured to measure an eluent flow rate of an eluent flowing through the sample cell, (3) a solvent flow regulator coupled to an outlet of the solvent delay volume and coupled to a solvent inlet of a reference cell, where the solvent flow regulator is configured to measure and to regulate a solvent flow rate of a solvent flowing through the reference cell, and (4) an instrument controller configured to receive the eluent flow rate from the eluent flow meter, configured to receive the solvent flow rate from the solvent flow regulator, configured to receive a flow rate ratio from a flow rate ratio data source, wherein the flow rate ratio indicates a ratio of the eluent flow rate to the solvent flow rate, and configured to transmit a flow command to the solvent flow regulator to achieve the flow rate ratio. In a further embodiment, the differential refractometer further includes an optical bench configured to measure, in response to receiving from the instrument controller a signal indicating that the flow rate ratio has been achieved, a difference between a refractive index of the eluent present in the sample cell and a refractive index of the solvent present in the reference cell. In an embodiment, the flow rate ratio is a ratio of a volume of a void in the chromatography column to a volume of a void in the solvent delay column. In an embodiment, the flow rate ratio is a ratio of a sum of a volume of a void in the chromatography column, a volume of tubing coupled to the chromatography column, and a volume of the eluent flow meter, to a sum of a volume of a void in the solvent delay column, a volume of tubing coupled to the solvent delay column, and a volume of the solvent flow regulator.

Definitions

Particle

A particle may be a constituent of a liquid sample aliquot. Such particles may be molecules of varying types and sizes, nanoparticles, virus like particles, liposomes, emulsions, bacteria, and colloids. These particles may range in size on the order of nanometer to microns.

Analysis of Macromolecular or Particle Species in Solution

The analysis of macromolecular or particle species in solution may be achieved by preparing a sample in an appropriate solvent and then injecting an aliquot thereof into a separation system such as a liquid chromatography (LC) column or field flow fractionation (FFF) channel where the different species of particles contained within the sample are separated into their various constituencies. Once separated, generally based on size, mass, or column affinity, the samples may be subjected to analysis by means of light scattering, refractive index, ultraviolet absorption, electrophoretic mobility, and viscometric response.

Concentration Detector

Differential Refractive Index Detector

A differential refractive index detector (dRI), or differential refractometer, or refractive index detector (RI or RID), is a detector that measures the refractive index of an analyte relative to the solvent. They are often used as detectors for high-performance liquid chromatography and size exclusion chromatography. dRIs are considered to be universal detectors because they can detect anything with a refractive index different from the solvent, but they have low sensitivity. When light leaves one material and enters another it bends, or refracts. The refractive index of a material is a measure of how much light bends when it enters.

A differential refractive index detector contain a flow cell with the following two parts: one for the sample; and one for the reference solvent. The dRI measures the refractive index of both components. When only solvent is passing through the sample component, the measured refractive index of both components is the same, but when an analyte passes through the flow cell, the two measured refractive indices are different. The difference appears as a peak in the chromatogram. Differential refractive index detectors are often used for the analysis of polymer samples in size exclusion chromatography. A dRI could output a concentration detector signal value corresponding to a concentration value of a sample.

dRI instruments, which measure small changes in refractive index of a solution, are highly desirable for measuring analyte concentration in solutions (e.g., in a chromatographic or FFF separation). dRI instruments are commonly used for isocratic chromatography, where the solvent is constant, and therefore has a constant refractive index, throughout the separation. Since dRI measurement does not require the analyte to contain chromophores or fluorophores, dRI measurement is suitable for a very wide range of molecular or macromolecular analytes including dissolved salts, polysaccharides, synthetic polymers, proteins, nucleic acids, lipids and more. dRI measurement offers a very wide range of concentration measurements, from ng/mL to g/mL.

dRI measurement involves measuring the difference in refractive index between (a) the solution containing solvent and analyte, located in the sample cell, and (b) the solvent alone located in the reference cell. The solution in the sample cell is usually changing (e.g., as eluent flows from a chromatographic separation system or another source of sample solution, the quantity of dissolved analyte changes). The solvent in the reference cell is not changed in the course of the measurement. Hence if, in the sample cell, the solvent is constant and only the analyte concentration changes, as in an isocratic separation, dRI provides a signal proportional to the analyte concentration.

Gradient Chromatography

Gradient chromatography involves mixing two solutions to provide a series of mobile phases that enhance macromolecular separation. Typically the different mixtures have different refractive indices, leading to large changes in dRI signals, which leads to error in analyte concentration measurement or even obscuration of the analyte dRI signal. Commonly, macromolecules such as proteins or polysaccharides are separated by gradient chromatography.

Current Technologies

Current technologies, as depicted inFIG.1A,FIG.1B,FIG.1C, andFIG.1D, depict how, if the solvent in the sample cell is also changing (e.g., as in a gradient chromatography such as ion-exchange chromatography (changing salt concentration) or reverse-phase chromatography (changing ratio between polar and non-polar solvent)), dRI provides a signal proportional to both the analyte concentration and changing solvent composition, which is not useful for determining analyte concentration. Thus, there is a need a differential refractometer for gradient chromatography.

Referring toFIG.2, in an exemplary embodiment, the differential refractometer includes (1) a solvent delay volume210configured to be coupled to an outlet of a chromatography pump202, (2) an eluent flow meter220configured to be coupled to an outlet of a chromatography column204and coupled to an eluent inlet of a sample cell230, where eluent flow meter220is configured to measure an eluent flow rate of an eluent flowing through sample cell230, (3) a solvent flow regulator240coupled to an outlet of solvent delay volume210and coupled to a solvent inlet of a reference cell250, where solvent flow regulator240is configured to measure and to regulate a solvent flow rate of a solvent flowing through reference cell250, (4) an instrument controller260configured to receive the eluent flow rate from eluent flow meter220, configured to receive the solvent flow rate from solvent flow regulator240, configured to receive a flow rate ratio from a flow rate ratio data source206, wherein the flow rate ratio indicates a ratio of the eluent flow rate to the solvent flow rate, and configured to transmit a flow command to solvent flow regulator240to achieve the flow rate ratio, and (5) an optical bench270configured to measure, in response to receiving from instrument controller260a signal indicating that the flow rate ratio has been achieved, a difference between a refractive index of the eluent present in sample cell230and a refractive index of the solvent present in reference cell250.

In an embodiment, the flow rate ratio is a ratio of a volume of a void in chromatography column204to a volume of a void in solvent delay column210. In an embodiment, the flow rate ratio is a ratio of a sum of a volume of a void in chromatography column204, a volume of tubing coupled to chromatography column204, and a volume of eluent flow meter220, to a sum of a volume of a void in solvent delay column210, a volume of tubing coupled to solvent delay column210, and a volume of solvent flow regulator240.

FIG.3Adepicts a differential refractometer in accordance with an embodiment.FIG.3Bdepicts a sample cell of a differential refractometer in accordance with an embodiment.

In an exemplary embodiment, instrument controller260is a standalone computer system, such as computer system500shown inFIG.5, a network of distributed computers, where at least some of the computers are computer systems such as computer system500shown inFIG.5, or a cloud computing node server, such as computer system500shown inFIG.5. In an embodiment, instrument controller260is a computer system500as shown inFIG.5, that executes a differential refractometer for gradient chromatography script or computer software application that carries out the operations of at least a method carried out by instrument controller260. In an embodiment, instrument controller260is a computer system/server512as shown inFIG.5, that executes a differential refractometer for gradient chromatography script or computer software application that carries out the operations of at least a method carried out by instrument controller260. In an embodiment, instrument controller260is a processing unit516as shown inFIG.5, that executes a differential refractometer for gradient chromatography script or computer software application that carries out the operations of at least a method carried out by instrument controller260.

For example, reference cell250is provided with a changing solvent, matched to the changing solvent in sample cell230by splitting the flow emerging from the solvent mixing device, mixer203, prior to its entering the sample loop, sending one portion to reference cell250and the remainder to continue within the chromatography system passing through the sample loop and separation column, chromatography column204, before entering the dRI instrument's sample cell230. In a further example, the portion of solvent destined for the dRI instrument's reference cell250passes through a delay volume, solvent delay volume210, thereby inducing a timing delay equivalent to the delay experienced, upon passage through the chromatographic column, chromatography column204, by the portion of solvent destined for sample cell230. In this manner, for example, at each point in time, the same solvent composition would be present in sample cell230and reference cell250, and the differential RI would be proportional only to the analyte concentration. In addition, for example, in order to maintain correct and constant flow rates through the chromatographic column, chromatography column204, and the delay column, solvent delay column210, a flow meter, eluent flow meter220, is placed in-line with the chromatography flow path, and a flow regulator, solvent flow regulator240, is placed in-line with the solvent delay flow path. For example, if solvent delay column210were to contain one tenth of the volume of chromatography column204, the flow rate through solvent delay column210is maintained at one tenth of the flow rate though chromatography column204.

Example

For example,FIG.4AandFIG.4Bdepict how well the disclosed differential refractometer determines analyte concentration.

Computer System

In an exemplary embodiment, the computer system is a computer system500as shown inFIG.5. Computer system500is only one example of a computer system and is not intended to suggest any limitation as to the scope of use or functionality of embodiments of the present invention. Regardless, computer system500is capable of being implemented to perform and/or performing any of the functionality/operations of the present invention.

As shown inFIG.5, computer system/server512in computer system500is shown in the form of a general-purpose computing device. The components of computer system/server512may include, but are not limited to, one or more processors or processing units516, a system memory528, and a bus518that couples various system components including system memory528to processor516.

Computer system/server512typically includes a variety of computer system readable media. Such media may be any available media that is accessible by computer system/server512, and includes both volatile and non-volatile media, removable and non-removable media.

System memory528can include computer system readable media in the form of volatile memory, such as random access memory (RAM)530and/or cache memory532.

Program/utility540, having a set (at least one) of program modules542, may be stored in memory528by way of example, and not limitation. Exemplary program modules542may include an operating system, one or more application programs, other program modules, and program data. Each of the operating system, one or more application programs, other program modules, and program data or some combination thereof, may include an implementation of a networking environment. Program modules542generally carry out the functions and/or methodologies of embodiments of the present invention.

Computer system/server512may also communicate with one or more external devices514such as a keyboard, a pointing device, a display524, one or more devices that enable a user to interact with computer system/server512, and/or any devices (e.g., network card, modem, etc.) that enable computer system/server512to communicate with one or more other computing devices. Such communication can occur via Input/Output (I/O) interfaces522. Still yet, computer system/server512can communicate with one or more networks such as a local area network (LAN), a general wide area network (WAN), and/or a public network (e.g., the Internet) via network adapter520. As depicted, network adapter520communicates with the other components of computer system/server512via bus518. It should be understood that although not shown, other hardware and/or software components could be used in conjunction with computer system/server512. Examples, include, but are not limited to: microcode, device drivers, redundant processing units, external disk drive arrays, RAID systems, tape drives, and data archival storage systems.

Computer Program Product