Systems and methods for refractive index detection

Abstract

Differential refractive index detector comprising: a flow cell body having a proximal end, a distal end, and a flow axis extending between the proximal and distal ends, the flow cell body comprising a flow cell assembly (212) consisting of individual optically transmissive elements, the flow cell assembly (212) comprising: a first assembly (214a) including a sample chamber (290) and oriented to allow sample flow parallel to the flow axis; a second assembly (214b) enclosing a reference chamber (214d) and oriented to permit a reference flow parallel to the flow axis; wherein the first assembly (214a) and the second assembly (214b) are connected and the individual optically transmissive elements provide a passage for an optical beam path that interrogates the refractive index difference between a sample located in the sample chamber (290) and a reference fluid located in the reference chamber (214d) perpendicular to the flow axis, and wherein a cross-sectional profile of at least one of the sample chamber (290) and the reference chamber (214d) perpendicular to the flow axis of the flow cell body includes at least one curved section.

Description

FIELD OF THE INVENTION

[0001] The present invention relates generally to refractive index detection and, more particularly, to systems, methods, and apparatus for measuring refractive index differences with low fluidic dispersion. BACKGROUND

[0002] The measurement of the refractive index, or RI, of a fluid, such as a gas or liquid, has widespread applications in many industrial fields. RI is a property of a fluid that characterizes its response to an externally applied electromagnetic radiation field. Different substances respond to the same radiation field to an extent that depends on the specific material, and it is this differential response that forms the basis for quantifying a given material and allowing it to be distinguished from another material. In general, the qualitative aspects of an RI measurement are less in demand than its attractiveness as a quantitative measurement tool, since many substances, such as sugar, are less suitable for other forms of analysis, such as UV absorption detection (i.e., they lack a strong UV chromophore) or fluorescence detection.RI is sometimes referred to as a universal detector because so many substances exhibit an RI response. In particular, an RI detector preceded by a separation device, such as a liquid chromatograph, will produce a response for virtually all substances. In this measurement mode, a sample containing one or more analytes is injected into a chromatographic column. The subsequent continuous flow of a clean mobile phase through the column results in a temporal separation of the individual analytes. These analytes elute, or exit the column, as individual peaks and have a characteristic volume and retention time that reflect the analyte and the column packing material. The peak is guided from the column to the RI detector, which produces a response proportional to the analyte concentration.Since each peak contains the original amount of analyte dissolved in the mobile phase, the RI response for the same injected mass will be greater the more compact or narrow the volume of the peak. Compact peaks are characteristic of minimal dispersion during transfer of the analytical peak from the column to the detector. The process by which the peak emerging from the column broadens during transfer to a downstream detector is generally referred to as post-column dispersion.

[0003] Proper management of post-column dispersion can make it possible to reduce the volumetric extent of the separation, which can lead to significant advantages in signal enhancement for many detection methods, including concentration-sensitive analyzers such as differential RI detectors. Peak volumes decrease proportionally with the cross-sectional area of ​​the column. Therefore, for two columns whose diameters differ by a factor of two, the peak volume for the column with the smaller inner diameter is expected to be four times smaller, and therefore the concentration must be four times higher for the same injected mass. There are other important advantages to moving to smaller-scale separations. For example, reduced solvent consumption is an advantage for applications that use expensive mobile phases, as is common in RI detection.

[0004] Large-scale chromatographic systems can be considered those using separation columns with internal diameters (IDs) greater than about 4 mm, smaller-scale columns with IDs in the range of about 1-4 mm, and capillary-scale systems can be considered those using columns with IDs less than about 1 mm. Chromatographic theory can predict the peak volume of a retained analyte, and it is this volume that serves as a guide to assess the effects of post-column dispersion. For the aforementioned range of columns, typical peak volumes for early-eluting analytes (k'=2) are shown in Table 1. Table 1 Säulen-ID, mm Säulenlänge, mm Partikelgröße, Mikrometer Peakvolumen (4,4%), µl Optimale Flussrate, ml / min Zeit, min, für 1 Säulenvolumen 4,6 150 3,5 180 0,56 2,94 3,0 100 1,7 44 0,49 0,95 0,5 100 1,7 1,2 0,014 0,95

[0005] In practice, system parameters such as flow rate, operating pressure, etc., are influenced by the choice of column. Relative to a large-scale separation, the same method can be performed in a small-scale system in a manner that offers advantages both in terms of time and reduced solvent consumption. Since peak volumes are smaller in smaller-scale systems, more stringent conditions apply to controlling sources of post-column dispersion. Accordingly, there is a need for differential dispersion refractometers, specifically for separations performed in small-scale systems.

[0006] A wide range of RI detectors coupled to a separation system are described in the prior art. For example, US Patent US 3,674,373 A describes a heat exchanger for a differential refractometer. It is well known that the temperature coefficient of the refractive index of most fluids is such that poor thermal control can lead to undesirable detector responses that are often larger than the signal of interest. This patent discloses a conduit with inner diameters ranging from 0.02" to 0.04" (0.51 mm to 1.02 mm) and lengths up to 12" (30.48 cm). These conduit dimensions correspond to post-column volumes of 60 to 160 µm, which are unsuitable for small-scale separations. US Patent US 3 999 856 A describes a diffractometric refractometer that measures a phase shift between a sample beam that has been passed through a reference and sample flow cell chamber.Flow cell volumes as small as 2 µl are discussed, however, such small cells generally have short mechanical path lengths, which pose limitations when attempting to measure both very small and large refractive index differences. This patent does not disclose detector volumes between the column and the flow cell or the thermal management of the sample or reference streams.

[0007] Various techniques have been described in the prior art for measuring the refractive index difference, based on a phase shift of light that has been passed through the reference and sample fluid cells and then recombined in a plane spaced from the cell. These techniques, generally referred to as interferometric methods, can be performed with small-volume flow cells, but they require small fluid pre-cell volumes and good thermal management to enable accurate RI differences.

[0008] US Patent No. 4,952,055 describes a beam-shifting technique performed on a capillary-based flow cell. While small-volume cells are feasible, a setup procedure is described that requires the alignment of the sample beam to the flow cell at an angle based on the refractive index of the cell material (glass) and the sample fluid. Consequently, measuring RI differences over a wide range of absolute RI (e.g., 1.30 to 1.60 RI units), which would be necessary in a general-purpose RI detector, would require optical realignments, which can have a negative impact on instrument performance. Other techniques, such as those employing evanescence detection (e.g., as disclosed in US Patent No. 5,311,274), can also be implemented in low-volume configurations; however, they have limited application due to their dependence on the refractive index of the light-guiding material.

[0009] US Patent Nos. 5,606,412 and 5,900,152 describe a device for modifying flow profiles in a non-circular flow cell by directing the flow substantially to the inner surfaces of the cells. The devices in these patents relate to flow cells with volumes ranging from approximately 7 to 50 µl, which are more suitable for larger-scale chromatography.

[0010] Accordingly, there is a need for robust, long-range and sensitive differential RI detectors that exhibit low dispersion.

[0011] US Patent No. 2,857,803 A relates to a differential refractometer cell. DE 18 13 397 A relates to a cuvette with two diametrically opposed windows for a beam of rays and at least one translucent, plane-parallel wall arranged obliquely to these windows. SUMMARY

[0012] The invention relates to a differential refractive index detector according to claims 1, 14 and 19. Advantageous embodiments are defined in the subclaims.

[0013] A robust, long-range, and sensitive differential RI detector exhibiting low dispersion can be provided by reducing the volume of the detector system, for example, by minimizing the length of the fluid paths within the detector, and by thermally controlling the incoming fluid flow and spatially tailored injection of the fluid into the sample chamber. Flow cells according to embodiments of the present invention are also capable of operating under high pressures.

[0014] A differential refractive index detector disclosed herein includes a flow cell body having a proximal end, a distal end, and a flow axis extending between the proximal and distal ends. The flow cell body includes a first chamber and a second chamber. In some embodiments, the first chamber may have a volume smaller than that of the second chamber. In one exemplary embodiment, either the first chamber or the second chamber may have a volume in the range of about 2 µL to about 5 µL.

[0015] The flow cell body also includes a first inflow port configured to allow fluid to flow into the first chamber, a first outflow port configured to allow fluid to flow out of the first chamber, a second inflow port configured to allow fluid to flow into the second chamber, and a second outflow port configured to allow fluid to flow out of the second chamber. At least one of the first and second inflow ports may be configured to provide fluid flow in a direction parallel to the flow axis of the flow cell body. In exemplary embodiments, at least one of the first and second inflow ports may be disposed at the proximal end of the flow cell. In exemplary embodiments, at least one of the first and second outflow ports may be disposed at the distal end of the flow cell.

[0016] The differential refractive index detector may further include an inlet conduit coupled to either the first or second inflow port. The inlet conduit may include a proximal end, a distal end, and a flow axis extending between the proximal and distal ends. In some embodiments, the inflow conduit may taper from a first diameter at the proximal end to a larger second diameter at the distal end. For example, an inner diameter of the inflow conduit may be larger than an inner diameter of the inflow conduit at the proximal end. For example, the inner diameter of the inflow conduit at the distal end may be larger than the inner diameter of the inflow conduit at the proximal end and may provide a taper angle of the inflow conduit in the range of about 8° to about 20°.

[0017] The differential refractive index detector may also include an outflow conduit coupled to either the first or second outflow port. The outflow conduit may include a proximal end, a distal end, and a flow axis extending between the proximal and distal ends. In some embodiments, the outflow conduit may be tapered from a first diameter at the proximal end to a smaller second diameter at the distal end. For example, an inner diameter of the outflow conduit at the proximal end may be larger than an inner diameter of the outflow conduit at the distal end. For example, the inner diameter of the outflow conduit at the proximal end is larger than the inner diameter of the outflow conduit at the distal end and may provide an outflow conduit taper angle in the range of about 8° to about 20°.

[0018] The flow cell of the refractive index detector can be formed from various materials. For example, at least a portion of the flow cell body can be formed from clear quartz.

[0019] Also disclosed is a differential refractive index detector comprising a flow cell body having a proximal end, a distal end, a flow axis extending between the proximal end and the distal end, and a first chamber and a second chamber. Each of the first chamber and the second chamber may have an interior surface extending substantially parallel to the flow axis of the flow cell body. According to an exemplary embodiment, the flow cell body may comprise a first unit and a second unit, wherein the first unit defines the first chamber and the second unit defines the second chamber. The flow cell body may also comprise at least one window configured to prevent fluid communication between the first chamber and the second chamber, wherein the first window is configured to transmit light between the first chamber and the second chamber.For example, the at least one window may be formed of clear quartz.

[0020] According to some embodiments, at least a portion of the inner surface of either the first or second chamber may be shaped to minimize sharp corners extending along the flow axis. According to the invention, a cross-sectional profile of either the first chamber or the second chamber includes at least one curved portion perpendicular to the flow axis of the flow cell body.

[0021] The flow cell body may also include a first inflow port configured to allow fluid to flow into the first chamber, a first outflow port configured to allow fluid to flow out of the first chamber, a second inflow port configured to allow fluid to flow into the second chamber, and a second outflow port configured to allow fluid to flow out of the second chamber. At least one of the first and second inflow ports may be configured to provide fluid flow in a direction parallel to the flow axis of the flow cell body. According to exemplary embodiments, one of the first and second inflow ports may be disposed at the proximal end of the flow cell. In some embodiments, at least one of the first and second outflow ports may be disposed at the distal end of the flow cell.

[0022] The differential refractive index detector may also include an inflow conduit coupled to either the first inflow port or the second inflow port. The inflow conduit may have a proximal end, a distal end, and a flow axis extending between the proximal end and the distal end. In some embodiments, the inflow conduit may taper from a first diameter at the proximal end to a larger second diameter at the distal end. For example, the inner diameter of the inflow conduit at the distal end may be larger than an inner diameter of the inflow conduit at the proximal end. For example, the inner diameter of the inflow conduit at the distal end may be larger than the inner diameter of the inflow conduit at the proximal end and may provide an inflow conduit taper angle in the range of about 8° to about 20°.

[0023] The differential refractive index detector may also include an outflow conduit coupled to either the first or second outflow opening. The outflow conduit may have a proximal end, a distal end, and a flow axis extending between the proximal end and the distal end. In some embodiments, the outflow conduit may taper from a first diameter at the proximal end to a smaller second diameter at the distal end. For example, an inner diameter of the outflow conduit at the proximal end may be larger than an inner diameter of the outflow conduit at the distal end. For example, the inner diameter of the outflow conduit at the proximal end may be larger than the inner diameter of the outflow conduit at the distal end and may provide an outflow conduit taper angle in the range of about 8° to about 20°. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The invention will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings. Fig. 1 shows a schematic representation of a differential RI detector coupled to a separation system according to an embodiment of the present invention; Fig. Figure 2 A shows a refractometer flow cell with a prismatic sample and reference chambers; Fig. Figure 2B shows a cross-sectional view of the flow cell of Fig. 2A along line aa; Fig. 3 A shows a refractometer flow cell assembly according to an embodiment of the present invention; Fig. Figure 3B is a cross-sectional view of the flow cell of the Fig. 3 A along line bb; Fig. Figure 3C is a cross-sectional view of the flow cell of Fig. 3 A along line cc; Fig. Figure 4 is an exemplary dispersion versus flow rate plot for two differential RI detectors; Fig. Figure 5A shows a refractometer flow cell assembly designed to withstand high working pressures according to an embodiment of the present invention; Fig. Figure 5B is a cross-sectional view of the flow cell of Fig. 5A along line AA; Fig. Figure 6A shows a reference chamber of the flow cell arrangement of Fig. 5A according to an embodiment of the present invention; Fig. Figure 6B is a sectional view of the reference chamber of Fig. 6A along line BB; Fig. Figure 6C is a sectional view of the reference chamber of Fig. 6A along line CC; Fig. 6 D shows a window of the reference chamber of Fig. 6A according to an embodiment of the present invention; Fig. Figure 7A shows the sample chamber of the flow cell array of Fig. 5A according to an embodiment of the present invention; and Fig. Figure 7B is a sectional view of the sample chamber of Fig. 7A along line DD. DETAILED DESCRIPTION

[0025] Exemplary embodiments are described below to provide a thorough understanding of the principles, structure, function, manufacture, and uses of the devices and methods disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings.

[0026] Those of ordinary skill in the art will understand that the devices and methods described herein and illustrated in the accompanying drawings are merely non-limiting embodiments, and that the scope of the present invention is defined solely by the claims. The features illustrated or described in connection with an exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present invention.

[0027] Fig. 1 illustrates an exemplary separation system including a differential RI detector. The separation system includes a separation unit 1. The separation unit 1 may include various modules. For example, the separation unit 1 may include modules such as a sample organizer, a pump for pumping fluid(s), a sample injector, a separation column, a column manager, and a central control unit, such as a computer on which software for controlling the entire separation is installed. The separation system may also include an RI detector 20. The RI detector 20 may include various fluidic and thermal conditioning or control devices, which are discussed in more detail below. The RI detector 20 may also include an optical detection system configured to quantify the difference in the refractive indices of the fluids contained in the separation chambers of a flow cell 12.As will be discussed in more detail below, the detector 20 has two primary modes of operation, referred to herein as a cleaning mode and a normal mode.

[0028] When operating in cleaning mode, the separation unit 1 delivers a reference fluid along a path 2 to a detector inlet 3a. In cleaning mode, a valve in the drain unit 30 is closed. The valve forces the flow along the path between the detector inlet 3a and the flow cell inlet 10a, through the sample chamber 14a of the flow cell 12 and then into the reference chamber 14b of the flow cell 12, then to exit the reference chamber along the lines 17b and 18a, and finally to enter the drain unit 24, from which the fluid can be diverted to either a waste container or a recycling container. After a complete cleaning operation in cleaning mode, the fluid composition within the chambers 14a, 14b of the flow cell 12 is the same. During this period, i.e.If the fluid composition of the flow cell chambers 14a, 14b is identical, the optical detection system can be used to acquire a calibration signal. The calibration data can include a detector output signal and can be stored in a digital memory location.

[0029] During normal mode operation, a test sample is injected onto the column within test unit 1, and separation of this sample into one or more analytes begins as the reference fluid is passed through the column at a constant flow rate. The eluent from the column is fed to the RI detector 20 via fluid line 2. According to exemplary embodiments, fluid line 2 can be sized to minimize dispersion between the column outlet and the connection at detector inlet 3a, wherein the detector can also be of the low-dispersion type. According to some embodiments, the temperature of the interior of detector 20 can be controlled by a master controller (not shown). In normal mode, a valve in drain unit 30 is open, resulting in an active fluid path along line 4b and via connection 3b into drain unit 30.The drain unit 30 can be configured to divert the flow to either a waste receptacle or a recycling receptacle. In normal mode, there is no flow along line 17a into the reference chamber 14b or any leakage from the reference chamber 14b along line 18a. However, a hydraulic connection still exists between these flow paths, which can prevent undesirable detector responses that may result from pressure changes associated with the separation unit 1, for example, by maintaining the two chambers of the flow cell 12 at the same pressure. During normal mode operation, the detector output signal is recorded.The detector output signal is processed to remove the calibration signal recorded during the purge mode and is scaled using known factors to obtain a detector output value representing the refractive index difference between the analyte contained in sample chamber 14a and the reference fluid contained in chamber 14b of flow cell 12. The analyte concentration can be related to the refractive index difference via a separate calibration step. The plot of the detector output signal versus time forms a chromatogram.

[0030] As mentioned above, the temperature of the interior of the detector 20 can be controlled, for example, by a master temperature controller. According to some embodiments, the incoming test fluid can be thermally conditioned. For example, a first internal thermal conditioning module 5 can adjust the temperature of the incoming fluid in line 4a to the temperature of the fluid flow exiting through line 4b. According to exemplary embodiments, the thermal conditioning module 5 can be a counterflow heat exchanger. The fluidic volume of line 4a in the first internal conditioning module 5 can be reduced to a sufficiently small value while maintaining high efficiency so that its contribution to the overall peak dispersion can be minimized for a wide range of flow rates and fluid viscosities.According to some embodiments, the detector 20 may include a second thermal conditioning unit 9. The second thermal conditioning unit 9 may be a heat sink whose absolute temperature is slightly regulated by a master heating control unit 9. The flow cell 12 and the fluidic inlet and outlet ports 10a, 10b, 16a, 16b may be attached to the second thermal conditioning unit 9 by standard mechanical means, for example, by screws, press fits, silencing, soldering, etc. Further thermal shielding may be provided by a housing that provides further isolation from other components, such as circuit boards, etc., inside the detector 20.

[0031] The person skilled in the art will recognize that a post-column peak shape of an analyte is typically determined by the dispersive properties of the fluid path from the separation unit through the detector, for example from 1 to 15a in Fig. 1. Lines and connections with low dispersion, for example line 2 and detector inlet 3a in Fig. 1, are well known and readily available. Low-dispersion tubing can be characterized by having small lumen diameters and smooth surfaces. Exemplary low-dispersion connectors include commercially available zero-dead-volume connectors.

[0032] The dispersive properties of the fluid path from the separation unit through the detector are also influenced by the length and volume of the different sections of the fluid path. On the fluid path from 3a to 10b in Fig. 1 and for a volume of the line 4a of less than about 20 µl, for example, the efficiency of the heat exchanger 5 can be maintained at a very high level, for example, by more than 80% for flow rates greater than 2 ml / min. The efficiency of the heat exchanger 5 can also be maintained for flow rates greater than 2 ml / min, for volumes of the line 4a of about 10 µl to about 2 µl. The volume of the line 7 within the thermal conditioning unit 9 can also be reduced to less than about 10 µl, and in some embodiments to less than about 5 µl, and the volume can still provide the level of thermal stabilization required for low noise.For example, a differential RI detector employing the low volume thermal conditioning units described herein can achieve noise levels comparable to an RI detector with a thermal conditioning volume more than 10 times larger. In conjunction with the reduced conduit volumes, the flow pattern into chamber 14a can also be spatially adjusted, for example, by internally tapering junction 10a, as further demonstrated below.

[0033] The flow through a pipe is influenced by several factors, including the shape of the pipe. For example, Fig. 2 A and Fig. 2B shows a refractometer flow cell 12 comprising axial openings. However, axial openings may be insufficient to minimize any effects of disturbances on flow profiles in fluid chambers having rectangular or triangular cross-sections, such as in the Fig. 2A and Fig. 2B is shown.

[0034] Fluid orifices having a conical profile, and in some embodiments coupled with sufficiently small conduit volumes leading into the RI cell, can reduce dispersion in a mage so that differential RI detection can be performed in small-scale separations. Fig. Figure 3A shows a flow cell according to an embodiment of the present invention. The sectional view of Fig. 3B shows various connection details.

[0035] In the exemplary flow cell of the Fig. 3A and Fig. 3B, a sample flows into an RI cell 12 through a low-volume, small-bore conduit 7 before reaching the sample chamber 14a. When the fluid from conduit 7 reaches the chamber 14a, energy loss occurs due to a sudden expansion of the cross-sectional area of ​​the sample chamber 14a. Such changes in cross-section can lead to undesirable effects, such as flow reversal (vortexes), resulting from very low fluid velocities along the inner corners of the chamber. To mitigate energy losses due to this sudden change in hydraulic diameter between the conduit 7 and the prism chamber 14a, a taper (cone) with a suitable angle is provided. The purpose of the taper is to allow a gradual change in velocity and minimize energy loss when the fluid reaches the prism chamber 14a.The cone angle may be chosen to optimize the transition in velocity, i.e., to promote a smooth and gradual change in velocity. For example, the total cone angle may range from about 8° to about 20°. In some embodiments, the cone angle may range from about 8° to about 10° or about 10° to about 20°. For example, the cone angle may be about 8°, about 10°, or about 20°. In an exemplary embodiment, the hydraulic diameter of the outlet of the conical section may be chosen to coincide with the largest diameter of an inner circle defined by the boundaries of the prism chamber 14a, as represented by D(h) in FIG. Fig. 3C shown.

[0036] In exemplary embodiments, the transition from the line 7 into the sample chamber 14a may be provided by a connection 11 having a connecting bore 10a, as shown in Fig. 3B. As mentioned above, tapering the connecting bore 10a can reduce the fluid velocity and the resulting dispersion. Accordingly, the transition of the conduit 17a into the reference chamber 14b can be provided by a connecting bore 16a in the connection 11, which can also be tapered, as described above. Similarly, the transition of the sample chamber 14a and the reference chamber 14b can be provided by a connection 13 having connecting bores 10b and 16b, which can also be tapered. In other embodiments, the connecting bores 10a, 10b, 16a, 16b can be non-tapered. For example, the connecting bores 16a, 16b can have a straight bore that matches the inner diameter of the fluid lines 17a and 17b.

[0037] The conduits 7, 17a, 15a, and 17b may have an internal profile of elliptical or circular shape. In exemplary embodiments, the internal diameter of the fluid conduits, for example, fluid conduits 7, 15a, 17a, and / or 17b, may be less than or equal to approximately 0.011" (0.28 mm). For example, the internal diameter of these fluid conduits may be less than or equal to approximately 0.005" (0.13 mm) or in the range of approximately 0.005" (0.13 mm) to approximately 0.011" (0.28 mm). In exemplary embodiments, the flow cell chamber volume, i.e., the volume of the sample chamber or the reference chamber, may be less than or equal to approximately 5 µl. For example, the flow cell chamber volume may be in the range of approximately 2 µl to approximately 5 µl or less than or equal to approximately 2 µl.In one embodiment, the cross-sections of the sample chamber and reference chamber flow cells may be right-angled triangles having equal side lengths of about 0.80 mm, and the flow cell chambers may have a length of about 4.0 mm, resulting in flow cell chambers having an internal volume of about 1.3 µl. For such a flow cell, the largest diameter of an internal circle is defined by the boundaries of the sample chamber 14a or the reference chamber 14b and would be as shown in FIG. Fig. 3C, approximately 0.468 mm (0.0184").

[0038] Standardized fasteners, such as welding or soldering, can be used to attach the metal lines to the conical sections, such as line 7 to conical section 10a. The cell 12 can be sealed with any connection arrangement using compliant seals made of materials such as Teflon or PEEK. The seals can be provided in the form of flat plates with suitable openings or as O-rings to allow the unobstructed flow of fluid into and out of the respective chamber.

[0039] Fig. Figure 4 is a plot of dispersion for a range of flow rates with respect to a standard differential RI detector intended for large-scale separations and a reduced dispersion detector according to embodiments of the present invention. As shown in Fig. As shown in Figure 4, the dispersion of the standard detector for the optimal flow rate range for small- and large-scale separations according to Table 1 is at least 4 times greater than that of the reduced dispersion detector according to the present invention. The approximately 3-fold speed benefit of switching to a small column shown in Table 1 can be achieved without any reduction in chromatographic efficiency when using reduced dispersion detectors according to the present invention. Since many chromatographic methods are not sample-bound, the reduced dispersion can also be utilized during loading, where virtually the same mass is on the column as for the large system, and which should result in an increase in peak height of approximately 4 times. Such an increase provides multiple advantages, such as lower limits of detection.

[0040] The differential refractometer flow cell 12 of Fig. 3 can be manufactured in a conventional manner as an all-glass assembly, for example, from optically clear fused silica. However, an all-glass assembly can limit the operating pressures acting on the cell to less than about 100 psi (6.89 bar). Beyond this pressure limit, various elements of the flow cell are prone to fracture. In exemplary embodiments of the present invention, the flow cells can be constructed from discrete optically transparent elements incorporated into a mechanical framework, so that the overall pressure resistance is significantly higher. Fig. Figures 5A to 7B illustrate elements of such an arrangement. The flow cell arrangement of the Fig. 5A to 7B provide low dispersion and withstand high pressures, for example, higher than about 100 psi (6.89 bar). The flow cell arrangement of the Fig. 5A to 7B, which is described in more detail below, works equally well for large and small scale separations and is particularly advantageous for small scale separations.

[0041] The flow cell arrangement of Fig. 5A is similar to the one in Fig. 3A, and the differences result from the configuration of the flow cell 212. Sample flow enters the differential RI cell through line 207, whose volume diameter 250 is selected according to desired dispersion properties, as discussed above. The dimensions of the effluent line 215a from this chamber are selected accordingly. As discussed above, the inflow line 217a and the effluent line 217b of the reference chamber are generally not critical with respect to the dispersion of the analysis peak. The fluid lines 217a and 217b are in the Fig. 5A, the fluid lines 217a and 217b are shown entering the chamber perpendicular to the sample lines 207 and 215a. In other embodiments, the fluid lines 217a and 217b may enter the flow cell parallel to the sample lines 207 and 215a.

[0042] As in Fig. 5B, the flow cell prism assembly 212 comprises two parts defined by discrete assemblies 214a and 214b. Assembly 214a includes the sample chamber 214d. Assembly 214b includes the reference chamber 214d. These assemblies may be connected together in a known manner, such as by screws, clamps, or other fastening means (not shown). The main connection between assembly 214a and assembly 214b includes an O-ring seal formed by O-ring 282 and a corresponding groove 281 in assembly 214b. Optically transparent windows 260a, 260b, 260c provide a passage for an optical beam that interrogates the refractive index difference between the sample and the reference fluid. The windows 260a, 260b, 260c also confine the respective fluids within each chamber by allowing flow through the corresponding inlet and outlet conduits.The optical window 260c prevents the two fluids from directly communicating, while the windows 260a and 260b prevent leakage from each chamber of the cell 212. The windows 260a and 260b can be sealingly connected to their respective assemblies by edge seals 280 or foil-like seals 282 disposed between the respective window and its receiving surface 284.

[0043] In some embodiments, the flow cell 212 may include bore features 250a and 250b selected according to the optical element coupled to the flow cell. For example, the dimensions of the bore features 250a and 250b may be used to accommodate apertures that define a precise size of the optical beam passing through the flow cell 212. In other examples, the bore features 250a and 250b may accommodate clamps or other retention mechanisms to hold the windows 260a and 260b in their respective recesses.

[0044] In exemplary embodiments, the sample chamber 290 may be formed by a circular or elliptical bore provided through the longitudinal axis of the assembly 214a. After machining to accommodate the windows 260a and 260c, the circular bore 290 may provide a chamber profile in which sharp corners are substantially eliminated. According to the invention, a cross-sectional profile of the sample chamber 290, when viewed perpendicular to the flow axis of the flow cell body, includes at least one curved section. The resulting chamber profile provides several advantages, including smoother flow profiles leading to improved dispersion characteristics. In some cases, a portion of the bore 290 is not interrogated by the optical beam. Nevertheless, the improved flow profile may produce an overall reduction in peak dispersion.In some embodiments, the dimensions of the reference chamber may be enlarged relative to the dimensions of the sample chamber. In some embodiments, the cross-sectional profile of the reference chamber in the reference assembly 214b may be elliptical or circular. In exemplary embodiments, the flow cell chamber volume, i.e., the volume of the sample chamber or the reference chamber, may be less than or equal to about 5 µl. For example, the flow cell chamber volume may be in the range of about 2 µl to about 5 µl, or it may be less than or equal to about 2 µl.

[0045] The Fig. Figures 6A through 6D illustrate the reference assembly 214b in greater detail, showing features associated with various surfaces. The fluid conduits 217a and 217b may be attached to the assembly 214b using silencing or other techniques.

[0046] For example, the distal ends 216a and 216b of the fluid lines 217a and 217b can be inserted into corresponding recesses in the eye surface of the assembly 214b. The window 260b can be in a space 251b, as in Fig. 6B. The Fig. The length L, width W and thickness t of the window 260b illustrated in Figure 6D can be selected according to known design formulas based on desired pressure values ​​for the cell, window material and seal loads. Fig. 7A and Fig. 7B illustrate the sample assembly 214a in greater detail and show features associated with various surfaces. For clarity, the lead 207 is removed from the Fig. 7A omitted. Fig. Figure 7B illustrates a sectional view along the line DD in Fig. 7A. The window 260a may be in a room 251a, shown in Fig. 7B, as shown in Fig.7B, the window 260c can be accommodated in the space 286.

Claims

[1] Differential refractive index detector comprising: a flow cell body having a proximal end, a distal end, and a flow axis extending between the proximal and distal ends, the flow cell body comprising a flow cell assembly (212) comprised of individual optically transmissive elements, the flow cell assembly (212) comprising: a first assembly (214a) enclosing a sample chamber (290) and oriented to allow sample flow parallel to the flow axis; a second assembly (214b) enclosing a reference chamber (214d) and oriented to permit a reference flow parallel to the flow axis; wherein the first assembly (214a) and the second assembly (214b) are connected, and the individual optically transmissive elements provide a passage for an optical beam path that interrogates the refractive index difference between a sample located in the sample chamber (290) and a reference fluid located in the reference chamber (214d) perpendicular to the flow axis, and wherein a cross-sectional profile of at least one of the sample chamber (290) and the reference chamber (214d) perpendicular to the flow axis of the flow cell body includes at least one curved section. [2] A differential refractive index detector according to claim 1, wherein at least one of the sample chamber (290) and the reference chamber (214d) is formed by at least one of a circular bore and an elliptical bore provided through a long axis of the flow cell body. [3] A differential refractive index detector according to claim 1, further comprising: a first window (260c) which fluidically separates the sample chamber (290) from the reference chamber (214d). [4] A differential refractive index detector according to claim 3, further comprising: a second window (260a) which prevents fluid from escaping from the sample chamber (290) to the outside of the flow cell body. [5] A differential refractive index detector according to claim 4, further comprising: a third window (260b) which prevents fluid from escaping from the reference chamber (214d) to the outside of the flow cell body. [6] The differential refractive index detector of claim 1, wherein the first assembly (214a) including the sample chamber (290) and the second assembly (214b) including the reference chamber (214d) are connected by at least one of the following elements: a fastener; a screw; and a clamp. [7] A differential refractive index detector according to claim 1, further comprising: a main connection connecting the first assembly (214a) including the sample chamber (290) and the second assembly (214b) including the reference chamber (214d), the main connection including an O-ring seal (282) and a corresponding groove (281). [8] A differential refractive index detector according to claim 4, wherein the second window (260a) is sealed from the sample chamber (290) by at least one selected from an edge seal (282) and a seal (281) arranged between the second window (260a) and a receiving surface (284). [9] A differential refractive index detector according to claim 5, wherein the third window (260b) is sealed from the reference chamber (214d) by at least one selected from an edge seal (282) and a seal (281) disposed between the third window (260b) and a receiving surface (284). [10] A differential refractive index detector according to claim 1, further comprising: at least one counterbore receiving at least one of a clamp and a retention mechanism for retaining at least one of a second window (260a) and a third window (260b) in a recess of at least one of the first assembly (214a) and the second assembly (214b). [11] A differential refractive index detector according to claim 1, wherein sharp corners of at least one of the sample chamber (290) and the reference chamber (214d) have been eliminated. [12] The differential refractive index detector of claim 1, wherein a volume of at least one of the sample chamber (290) and the reference chamber (214d) is at least one of: less than or equal to about 5 µl; in a range of about 2 µl to 5 µl; and less than or equal to about 2 µl. [13] A differential refractive index detector according to claim 5, wherein at least one of: the second window (260a) is received in a cavity of the first assembly (214a) enclosing the sample chamber (290); and the third window (260b) is received in a cavity of the second arrangement (214b) which encloses the reference chamber (214d). [14] Differential refractive index detector comprising: a flow cell body having a proximal end, a distal end, and a flow axis extending between the proximal and distal ends, the flow cell body comprising a flow cell assembly (212) comprised of individual optically transmissive elements enclosing a sample chamber (290) and a reference chamber (214d), the individual optically transmissive elements providing a passage for an optical beam path that samples the refractive index difference between a sample located in the sample chamber (290) and a reference fluid located in the reference chamber (214d) perpendicular to the flow axis, and a cross-sectional profile of at least one of the sample chamber (290) and the reference chamber (214d) perpendicular to the flow axis of the flow cell body includes at least one curved portion. [15] A differential refractive index detector according to claim 14, wherein at least one of the sample chamber (290) and the reference chamber (214d) is formed by at least one of a circular bore and an elliptical bore provided through a long axis of the flow cell body. [16] A differential refractive index detector according to claim 14, wherein sharp corners of at least one of the sample chamber (290) and the reference chamber (214d) have been eliminated. [17] A differential refractive index detector according to claim 14, wherein the flow cell assembly (212) further comprises: a first assembly (214a) enclosing the sample chamber (290); a second arrangement (214b) enclosing the reference chamber (214d); and wherein the first arrangement (214a) and the second arrangement (214b) are connected. [18] A differential refractive index detector according to claim 17, wherein the first assembly (214a) including the sample chamber (290) and the second assembly (214b) including the reference chamber (214d) are connected by at least one of the following elements: a fastener; a screw; and a clamp. [19] Differential refractive index detector comprising: a flow cell body having a proximal end, a distal end, and a flow axis extending between the proximal and distal ends, the flow cell body comprising a flow cell assembly (212) comprised of individual optically transmissive elements, the flow cell assembly (212) including: a sample chamber (290); a reference chamber (214d); a first window (260c) fluidically separating the sample chamber (290) from the reference chamber (214d); a second window (260a) which prevents fluid from escaping from the sample chamber (290) outside the flow cell body; a third window (260b) preventing fluid from escaping from the reference chamber (214d) outside the flow cell body; wherein the flow cell assembly (212) comprises: a first assembly (214a) including the sample chamber (290) and a second window (260a) and oriented to allow sample flow parallel to the flow axis; a second arrangement (214b) enclosing the reference chamber (214d) and the third window (260b) and oriented to enable a reference flow parallel to the flow axis; wherein the first assembly (214a) and the second assembly (214b) are connected by a fastening element and a cross-sectional profile of at least one of the sample chamber (290) and the reference chamber (214d) perpendicular to the flow axis includes at least one curved section, and the individual optically transmissive elements provide a passage for an optical beam path that interrogates the refractive index difference between a sample located in the sample chamber (290) and a reference fluid located in the reference chamber (214d) perpendicular to the flow axis.

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