Abstract
A nano-flow fractionator apparatus, comprises: one or more sources of mobile phase solvent; a source of auxiliary solvent; a sample injection valve; a chromatographic column having an inner diameter of less than or equal to 75 micro-meters, a column inlet end and a column outlet end; a solvent fraction delivery line comprising: an inlet end that is configured to receive eluate that is emitted from the column outlet end and an outlet end that is configured to dispense the eluate to each of a plurality of sample fraction containers; a fluid junction configured to receive the eluate that is emitted from the column outlet end and to receive a flow of the auxiliary solvent that is delivered from the source of auxiliary solvent and to deliver the eluate and the flow of auxiliary solvent to the solvent fraction delivery line.
Claims
1. A nano-flow fractionator apparatus, comprising: one or more sources of mobile phase solvent; a source of auxiliary solvent; a sample injection valve; a chromatographic column having an inner diameter of less than 75 micro-meters (m) and having a column inlet end and a column outlet end; a first fluidic line coupled between the one or more sources of mobile phase solvent and an inlet port of the sample injection valve; a second fluidic line coupled between an outlet port of the sample injection valve and the chromatographic column; a solvent fraction delivery line comprising: an inlet end that is configured to receive eluate that is emitted from the column outlet end; and an outlet end that is configured to dispense the eluate to each of a plurality of sample fraction containers; and a fluid junction configured to receive the eluate that is emitted from the column outlet end and to receive a flow of the auxiliary solvent that is delivered from the source of auxiliary solvent and to deliver the eluate and the flow of auxiliary solvent to the solvent fraction delivery line, wherein the solvent fraction delivery line is configured to outlet the flow of the auxiliary solvent as a sheath flow that at least partially surrounds the eluate emerging from the outlet end of the solvent fraction delivery line.
2. A nano-flow fractionator apparatus as recited in claim 1, further comprising a robotic arm configured to move the outlet end of the solvent fraction delivery line to a receiving portion of each of the sample fraction containers.
3. A nano-flow fractionator apparatus as recited in claim 1, further comprising a moveable sample table upon which the plurality of sample fraction containers is supported, the moveable table configured to position a receiving portion of each of the sample fraction containers at the outlet end of the solvent fraction delivery line.
4. (canceled)
5. A nano-flow fractionator apparatus as recited in claim 1, wherein the fluid junction comprises a penetration of an auxiliary transfer line through a sleeve of the solvent fraction delivery line and into a cylindrical annular conduit of the of the solvent fraction delivery line.
6. (canceled)
7. A nano-flow fractionator apparatus as recited in claim 1, wherein: the source of auxiliary solvent comprises a fluid pump; the one or more sources of mobile phase solvent comprise at least one container of mobile phase solvent; and the fluid pump of the source of auxiliary solvent is configured to draw the auxiliary solvent from the at least one container of mobile phase solvent.
8. A nano-flow fractionator apparatus as recited in claim 1, wherein the source of auxiliary solvent comprises: a container of the auxiliary solvent; a switching valve comprising an inlet port that is configured to draw the auxiliary solvent from the container of the auxiliary solvent; and a syringe pump fluidically coupled to another port of the switching valve.
9. A nano-flow fractionator apparatus as recited in claim 1, wherein the source of auxiliary solvent comprises: a container of the auxiliary solvent; and a source of pressurized gas or air that is fluidically coupled to the container of the auxiliary solvent.
10. A nano-flow fractionator apparatus as recited in claim 1, wherein the one or more sources of mobile phase solvents comprise one or more pumps and the one or more pumps are configured to pump the sample and the mobile phase solvents through the chromatographic column at a flow rate in the range of 100 nanoliters (nL) per minute to 2 L per minute.
11-20. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The above noted and various other aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings, not necessarily drawn to scale, in which:
[0022] FIG. 1A is a schematic depiction of fraction pooling within multiple wells of a multi-well collection plate during the course of a chromatographic separation;
[0023] FIG. 1B is a schematic depiction of a known system for nano-chromatographic separation of a sample into various fractions with fraction pooling;
[0024] FIG. 2A is a schematic depiction of a first chromatographic nano-flow fractionator in accordance with the present teachings;
[0025] FIG. 2B is a schematic depiction of a second chromatographic nano-flow fractionator in accordance with the present teachings;
[0026] FIG. 2C is a schematic depiction of a third chromatographic nano-flow fractionator in accordance with the present teachings; and
[0027] FIG. 3 is a schematic depiction of the end of a fraction delivery tube in accordance with the present teachings.
DETAILED DESCRIPTION
[0028] The following description is presented to enable any person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the described embodiments will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. Thus, the present invention is not intended to be limited to the embodiments and examples shown but is to be accorded the widest possible scope in accordance with the features and principles shown and described. To fully appreciate the features of the present invention in greater detail, please refer to FIGS. 1A, 1B, 2A-2C and 3, in which like reference numbers refer to like elements, in conjunction with the following discussion. It should be noted that all instances of the word line when used in reference to the movement of or in conjunction with a fluid (e.g., delivery line. transfer line, outlet line, etc.) are to be understood as referring, in a general sense, to a conduit that transfers fluid from one location to another, such as a tube or pipe or tubing or the like, without implication of or restriction to any particular material or materials, properties, size, shape, length, or form.
[0029] FIG. 2A is a schematic depiction of a chromatography nanoflow fractionator system 100 in accordance with the present teachings. In similarity to the system 1 that is illustrated in FIG. 1B, the system 100 comprises a first solvent delivery sub-system 2a, a second solvent delivery sub-system 2b, an injector 7, a chromatographic column 12 that receives a sample portion dissolved in a mobile phase from the injector 7 and a gradient valve 6 that is fluidically coupled between the injector 7 and the two solvent delivery sub-systems 2a, 2b. Unlike the known system 1, however, the column outlet line 18 of the system 100 does not deliver eluate fractions to a multiport distribution valve but, instead delivers the eluate fractions to a fluid junction 113 that combines the eluate with an auxiliary flow of an additional solvent 103 from an auxiliary solvent source 102. As depicted in FIG. 2A, the auxiliary solvent source 102 comprises an additional pump 104 and an additional switching valve 105. An auxiliary transfer line 119 delivers the auxiliary flow of an additional solvent to the fluid junction 113 at which it combines with the eluate from the chromatographic column 12 as transferred by column outlet line 18. The additional pump 104 is a low-pressure pump and, thus, the flow of additional solvent through the auxiliary transfer line 119 occurs at approximately atmospheric pressure. This is in contrast to the high-pressure flow (e.g., generally 100-350 bars pressure) within the fluidic lines that couple the first and second solvent delivery sub-systems 2a, 2b to the gradient valve 6 and to the multiport injection valve 10 and within the outlet tubing 16 that couples the multiport injection valve 10 to the nano-flow chromatographic column 12. The post column lines (outlet line 18 and a fraction delivery line 114) may comprise silica or PEEK tubing that is of small diameter (approximately 10 m inner diameter) to avoid diffusion. The post-column lines and the transfer line carry little to no back pressure (less than 150 bars) between the outlet end of the column 12 and the collection plate 15, depending on the lengths and inner diameters of these lines. In any event the pressure within the post-column lines and the transfer lines is significantly less than in the pre-column line 16.
[0030] In the system 100 (FIG. 2A), the combined eluate fractions and auxiliary solvent flow are transferred from the fluid junction 113 to a series of fraction collection containers (exemplified, in this depiction, by multi-well plate 15) by the single fraction delivery line 114. As will be described in greater detail below, operation of the system 100 causes the various eluate fractions to be delivered to the different fraction containers in cyclical sequential fashion as described above. However, because the system comprises only a single fraction delivery line 114 (as opposed to the multiple fraction delivery lines of the system 1), the receiving containers are changed by means of mechanical relative movement between the containers and the outlet end of the fraction delivery line 114. In the system 100 illustrated in FIG. 2A, this relative movement is effected by repositioning of the outlet end of the fraction delivery line 114 (assumed to comprise a flexible tubing) by a robotic arm structure 117 that is mechanically coupled to the fraction delivery line 114. Alternatively, the fraction delivery line 114 may remain stationary while the containers are repositioned between transfer of separate fractions. In the system shown in FIG. 2A, this repositioning may be carried out by lateral movement of a motorized moveable support table (not specifically illustrated) that supports the multi-well plate 15. An electronic controller 80 comprises electronic connections to the valves, pumps and robotic arm (and/or motorized moveable support table) and comprises program instructions that synchronize the operations of the pumps with the configurations of the valves for the purposes of providing appropriate mobile phase compositions to the nano-flow column, of controlling the injection of sample into the injection valve and for controlling the positioning of the fraction delivery line relative to the multiple fraction collection containers, either by control of the robotic arm or of a motorized moveable support table. If sample portions are provided by an autosampler apparatus (not specifically shown in FIG. 2A, then the controller 80 may also comprise a communication link to the autosampler apparatus that causes synchronization of the operation of the injector with the delivery of sample portions to the injector by the autosampler apparatus.
[0031] FIG. 2B is a schematic depiction of a second chromatographic nano-flow fractionator system 130 in accordance with the present teachings. The system 130 depicted in FIG. 2B is similar, in most respects, to the system 100 depicted in FIG. 2A except that the flow of additional solvent within the auxiliary transfer line 119 is propelled by a pressurized or flow-regulated gas source 133 instead of by a dedicated pump within the auxiliary solvent source 102. Although the gas source 133 is schematically indicated as a gas cylinder in FIG. 2B, the gas source 133 may comprise any alternative gas source, such as an air compressor. The gas source 133 may be located remotely from the location of the system 130 in which case the flow of gas may be fluidically coupled to the auxiliary solvent source 102 via existing infrastructure (e.g., house gas). As previously described, the flow of additional solvent through the auxiliary transfer line 119 occurs at approximately atmospheric pressure.
[0032] FIG. 2C is a schematic depiction of a second chromatographic nano-flow fractionator system 140 in accordance with the present teachings. The system 140 depicted in FIG. 2C is similar, in most respects, to the system 100 depicted in FIG. 2A except that the flow of additional solvent within the auxiliary transfer line 119 is provided from one or more existing solvent reservoirs (e.g., from either the mobile phase A reservoir of the first solvent delivery sub-system 2a, the mobile phase B reservoir of the second solvent delivery sub-system 2b, or a combination of the two reservoirs) via auxiliary solvent supply line 145 as shown. The flow of the auxiliary solvent supply line 145 is at approximately atmospheric pressure, in contrast to the high-pressure flow of mobile phases within the fluid lines that couple the first and second solvent delivery sub-systems 2a, 2b to the gradient valve 6 and to the multiport injection valve 10.
[0033] In all of the herein-described chromatographic nano-flow fractionator systems, the fractionated sample fluid flowing within the column outlet line 18 and the fraction delivery line 114 is, after having exited the nano-flow chromatographic column 12, at approximately atmospheric pressure. Likewise, the auxiliary fluid that flows within the auxiliary transfer line 119 is similarly at near atmospheric pressure. The fractionated sample fluid and the auxiliary fluid mix at or downstream from the fluid junction 113 with the flow rate of the auxiliary fluid being from two-times to five-times greater than the flow rate of the fractionated sample fluid. The additional flow of the auxiliary fluid overcomes the surface tension at the end of fraction delivery line 114 that would otherwise delay or prevent dispensing of the fractionated sample fluid into the containers 15.
[0034] In many instances, the fluid junction 113 may comprise a simple tee-junction. Alternatively, however, the fluid junction together with the fraction delivery line may comprise a specialized structure, as illustrated in FIG. 3, that causes the emerging auxiliary fluid to exit the end of the fraction delivery line as a sheath flow that coaxially surrounds the emerging flow of the fraction delivery line. FIG. 3 illustrates such a specialized structure in which the fraction delivery line 114 comprises two fluid conduits, the first of which is an extension of the column outlet line 18 that carries the fractionated sample fluid 149a and the second of which is an annular cylindrical gap 148 that surrounds the extension of the column outlet line 18. The annular cylindrical gap 148 is disposed between the extension of the column outlet line 18 and an outer sleeve tube 147. The auxiliary transfer line 119 passes through the outer sleeve tube 147 at the fluid junction 113 at a point upstream from the outlet end 146 of the fraction delivery line 114, at which point the auxiliary fluid 149b is delivered into the annular cylindrical gap 148. The resulting co-axial flow of the two fluids through a portion of the fraction delivery line 114 causes the auxiliary fluid to exit the fraction delivery line in the form of a sheath flow that co-axially surrounds any droplets of sample fluid that may form at the outlet end 146 and that urges said droplets to detach from the tip.
[0035] An improved chromatograph fractionator for general and multidimensional nano-flow chromatography has been disclosed. The discussion included in this application is intended to serve as a basic description. The present invention is not intended to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Any patents, patent applications, patent application publications or other literature mentioned herein are hereby incorporated by reference herein in their respective entirety as if fully set forth herein, except that, in the event of any conflict between the incorporated reference and the present specification, the language of the present specification will control.
