Abstract
An apparatus for reaction chromatography comprising: a chromatography column, the column having a fluid outlet for an eluate flow, wherein the fluid outlet is configured with two or more fluid ports, the two or more fluid ports comprising one or more reactant ports, wherein each reactant port is for connecting a reactant flow into the eluate flow to react with the eluate flow, and one or more product ports for receiving the reacted eluate flow; one or more reactant sources in fluid communication with the one or more reactant ports to provide the reactant flow; and one or more processing units in fluid communication with the one or more product ports to process the reacted eluate flow.
Claims
1. A reaction chromatography apparatus, comprising: a chromatography column having a column outlet assembly; the column outlet assembly having a plurality of fluid ports; at least one reactant source in fluid communication with at least one of the plurality of fluid ports, wherein the reactant source is connected to the column outlet assembly and wherein no separate reaction equipment resides downstream of the chromatography column; at least one processing unit in fluid communication with at least one of the plurality of fluid ports.
2. An apparatus as claimed in claim 1 wherein the at least one processing unit is selected from the group consisting of: a detector, a fraction collector and another chromatography column.
3. An apparatus as claimed in claim 1, wherein there are two fluid ports.
4. An apparatus as claimed in claim 1 wherein at least one of the plurality of fluid ports is positioned to introduce a reactant flow only to a portion of an eluate flowing from a restricted radial region selected from a peripheral radial region of the chromatography column and a central radial region of the chromatography column, and, wherein at least one of the plurality of fluid ports is positioned to receive an at least partially reacted eluate flow.
5. An apparatus as claimed in claim 4 wherein the restricted radial region of the chromatography column is a peripheral radial region of the chromatography column.
6. An apparatus as claimed in claim 5 wherein at least one of the plurality of fluid ports receives a second portion of eluate flowing from a central radial region of the chromatography column.
7. An apparatus as claimed in claim 6 wherein the at least one of the plurality of fluid ports is in communication with a first processing unit comprising a first detector and wherein at least one of the plurality of fluid ports is in communication with a second processing unit comprising a second detector.
8. An apparatus as claimed in claim 1 wherein at least two of the plurality of fluid ports are positioned peripherally around a central axis of the chromatography column.
9. An apparatus as claimed in claim 8 having at least one of the plurality of fluid ports positioned on the central axis of the chromatography column.
10. An apparatus as claimed in claim 1 wherein the column outlet assembly comprisesa split frit having a plurality of frit sections whereby an eluate flow is segmented into a plurality of portions by the split frit.
11. An apparatus as claimed in claim 10 wherein a first frit section communicates with both a first reactant fluid port and a first product fluid port; and a second frit section communicates with both a second reactant fluid port and a second product fluid port.
12. An apparatus as claimed in claim 11 wherein the first reactant fluid port is in communication with a first reactant source and the second reactant fluid port is in communication with a second reactant source that is different from the first reactant source.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) FIG. 1 shows schematically in flow-chart form a conventional configuration of an HPLC system.
(2) FIG. 2 shows schematically an axial cross-section side view through a conventional packed chromatography column with a single sample component eluting.
(3) FIG. 3 shows the view of FIG. 2 with four different sample components eluting.
(4) FIG. 4 shows schematically an axial cross-section side view through a packed chromatography column in accordance with the invention showing the principle of a flow distributor at the outlet to enable reaction chromatography.
(5) FIG. 5A shows a perspective view of a preferred embodiment of a frit assembly for use with the invention; FIG. 5B shows the underside view of the embodiment looking in direction of arrow A; and FIG. 5C shows a side cross section view of the embodiment taken on line B-B.
(6) FIG. 5D shows a perspective view of another preferred embodiment of a frit assembly for use with the invention; and FIG. 5E shows the underside view of the embodiment.
(7) FIGS. 5F and 5G schematically show respective further embodiments of frit assemblies for use with the invention.
(8) FIGS. 5H and 5I schematically show embodiments of exit capillary arrangements at the column outlet for use with the invention.
(9) FIG. 6A shows, in exploded view, an embodiment of the invention having a split section frit and four port end fitting; and FIG. 6B shows a similar embodiment to FIG. 6A but having a single piece frit.
(10) FIG. 7 shows, in exploded view, a further embodiment of the invention having a split section frit and four port end fitting.
(11) FIG. 8A shows, in exploded view, a still further embodiment of the invention having a split section frit and seven port end fitting.
(12) FIG. 8B shows the assembled embodiment of FIG. 8A in a cut-away view.
(13) FIG. 9 shows a cut-away side view of the embodiment shown in FIG. 7, shown in assembled form and with exit plumbing attached.
(14) FIG. 10 shows schematically an embodiment for performing reaction chromatography according to the present invention.
(15) FIG. 11 shows schematically a further embodiment for performing reaction chromatography according to the present invention.
(16) FIG. 12 shows schematically another embodiment for performing reaction chromatography according to the present invention.
(17) FIG. 13 shows yet another embodiment of the invention for performing reaction chromatography, being a modification of the embodiment shown in FIG. 6.
(18) FIG. 14 shows an embodiment similar to that shown in FIG. 13 but altered to simulate a prior art method of performing reaction chromatography.
(19) FIGS. 15A-D show the chromatogram for a Comparative Example with no reaction coil present.
(20) FIGS. 16A-D show the chromatogram for an Inventive Example with no reaction coil present.
(21) FIGS. 17A-D show the chromatogram for a Comparative Example with a 99 ?L reaction coil present.
(22) FIGS. 18A-D show the chromatogram for an Inventive Example with a 99 ?L reaction coil present.
(23) FIGS. 19A-D show the chromatogram for a Comparative Example with a 200 ?L reaction coil present.
(24) FIGS. 20A-D show the chromatogram for an Inventive Example with a 200 ?L reaction coil present.
(25) FIG. 21 shows a comparison of noise levels in chromatograms for both Comparative (a) and Inventive (b) Examples.
(26) FIG. 22 shows schematically an embodiment for performing reaction chromatography according to the present invention utilizing a split flow frit with 5 exit ports.
DETAILED DESCRIPTION OF EMBODIMENTS
(27) In order to further understand the invention, but without limiting the scope thereof, various exemplary embodiments and experiments are now described with reference to the accompanying drawings.
(28) In a conventional configuration of an HPLC system (as shown schematically in FIG. 1 in the form of a flow chart), one or more bottles of mobile phase solvent(s) 2 are delivered via tubing to a solvent delivery system 6 that employs a pump pumping at high or low pressures, or are delivered via tubing by force of gravity (low pressure only). The solvent delivery system 6 delivers desired mobile phase solvent or mixtures thereof (herein simply termed solvent) through a sample-injection port or valve 8 where a sample is introduced into the solvent flow and then into a chromatographic column 15 packed with a stationary phase or bed or provided with a monolithic stationary phase. The column is typically a circular cross section cylindrical column. The flow through the column 15 is radially dispersed over the full width of the cross-section of the column bed by a head or inlet frit 9 as well as by the column bed itself and subsequent chromatographic separation then occurs as the sample is carried by the mobile phase solvent down the length of the column. At the exit or outlet of a conventional HPLC column, the out flowing mobile phase or eluate is gathered by a second or outlet frit 11 typically held in place by an end fitting fitted to the outlet end of the column so that the entire cross section of the flow is delivered to a small exit port 40 located at the centre of the cross section of the column. That is, material from outer radial regions of the column near the wall is forced radially inwards to pass, together with material that has passed through the central radial region of the column, through the single central exit port 40. Separated components of sample are then carried by the eluate flow stream through suitable connective tubing into a detector 16, which generates a chromatographic trace.
(29) In analytical chromatography, the separated components may be either sent to waste 18 after detection, or destroyed as part of the detection. In preparative chromatography, a portion of the eluate flow is detected and that is used as the basis for collecting desired components from the flow stream using a reservoir or fraction collection device 19. The system is under the control of a Control and Data Collection System 4, e.g. a computer and associated control electronics, which in particular controls the solvent delivery system 6 and injector 8 and controls and receives data from the detector 16, as well as controlling other components. The Control and Data Collection System 4 may also process the data for output, e.g. as a chromatogram.
(30) In many instances, it is necessary to perform a post-column reaction on the eluate stream, for example to allow analysis by fluorescence, chemiluminescence, or decolorisation reaction. In such cases, a separate reactant source unit 21 downstream of the column outlet is employed to supply a reactant (reagent) into the eluate at a zero dead volume t-piece 22. The reacted sample components are then detected in the detector. This additional step, as well as the plumbing and equipment for the post column reaction, increases the time, labour and cost of detection. Typically a reaction coil 23 is employed to enhance the reaction. Broadening of the component peaks, i.e. loss of resolution may also occur as a result of the post column reaction.
(31) As illustrated in FIG. 2, which schematically shows an axial or longitudinal cross-section side view through a conventional packed chromatography column 15, a sample component applied to the head of the column from the injector via a single centre inlet port 41 accumulates there in the shape of a relatively thin, flat band 10. In three dimensions, the band 10 resembles a thin, flat disc that is bounded by the inner diameter of the column casing or wall. During separation on the column, and as the band 10 of sample is carried down the column by the mobile phase, the band begins to change shape as shown by the band at 20 and as described in more detail in the introduction above. Briefly, the centre of the band located on and around the central or longitudinal axis 17 of the column, moves faster than the perimeter of the band nearer the wall, drawing the band of material into a sort of bowl or cup shape as shown clearly by the band at 25. Furthermore, the sample near the column wall begins to spread out (broaden) and become more dilute. This phenomenon is progressive, such that it is most pronounced as the fluid leaves the column at the exit end or outlet of the column. At the end of the column 30, the bowl of material, begins to exit the centre exit hole or port 40 located on the central axis 17 after passing through a thin frit layer 11 with minimal impedance of lateral flow. To the right hand side in FIG. 2 is shown the end-on view of the end 30 of the column with the centre port 40 in the middle. The goal of the conventional arrangement is to gather the full cross section of the sample band to the centre port 40, and direct it to the detector in as sharp a peak as possible.
(32) When multiple components are applied to a packed column, their different chemical affinities for mobile and stationary phases in the column cause them to move at different rates through the column than each other. That is the basis of separation in an LC column. FIG. 3, which is analogous to FIG. 2 shows four different sample components 105, 110, 115, 120 that have been partially resolved (i.e. separated) from one another over the length of the column 15. It should be appreciated in the schematic example shown in FIG. 2B that, in the centre of the column, i.e. on the central axis 17, peaks are completely resolved. As this set of components 105, 110, 115, 120 exits the single central hole or port 40 at the centre of the chromatography column outlet, with liquid from across the full width being forced through the port, the peaks in the resulting chromatogram 135 display partial or incomplete resolution. That is, there are distinguishable peaks but they are not completely separated from each other at the early and late part of each peak, e.g. in the inter-peak region 140.
(33) In contrast to the conventional arrangement described, in preferred embodiments of the present invention, the outlet of the column is configured with a plurality of exit fluid ports in contrast to the single exit port 40 of the conventional arrangement. As described in more detail hereinafter, preferred embodiments may use an end or exit fitting (also termed end cap) on the outlet end of the column that has been modified to be unlike a conventional LC end fitting. A preferred modification is that the outlet frit and/or end fitting are designed to drain the mobile phase (eluate) from the column through multiple ports that are positioned at different points in the transverse (radial) cross section of the column when the frit and/or fitting are positioned at the outlet. In this way, the mobile phase being drained through the multiple ports emanates from different regions of the column, more particularly different radial regions of the column. This allows segregation of the different fluidic components across the diameter of the column. The flow from the multiple ports can be treated as separate portions and processed differently. By restricting the portions of eluate to limited radial regions of the column, each portion may show improvements in separation efficiency compared to the conventional case where eluate from across the whole column is gathered together and detected as one stream. In some embodiments, however, eluate from across the whole column may be reacted (and processed) as one.
(34) Various exemplary embodiments of the invention are now described.
(35) A first preferred feature of the invention is that the frit at the outlet is modified to separate and segregate mobile phase arriving from the centre of the column cross section from mobile phase arriving from the region surrounding the centre (i.e. from the perimeter region). Thus, the flow of eluate is split by the frit into a portion which has travelled through the centre of the column and a portion which has travelled through the perimeter region. A second preferred feature is that the centre and perimeter flows are then taken off into different exit ports in a flow distributor (e.g. steel end fitting or cap) that is typically fitted (e.g. screwed) to the end of the column. It is possible in some embodiments to use such a distributor without the split-frit which splits the eluate since the flow distributor with multiple ports may alone perform splitting of the eluate flow into the different portions, e.g. where the surface of the flow distributor which faces the frit lies close to the surface of the frit, preferably contacting the surface of the frit.
(36) FIG. 4 illustrates schematically the principle of a flow distributor at the outlet of the column 15, which is a packed column, e.g. for HPLC. FIG. 4 shows a schematic longitudinal cross sectional side view of the column similar to FIGS. 2 and 3. To the right hand side of the longitudinal cross section side view is shown an end-on view of the column outlet (i.e. an end-on view of the flow distributor 172 on the end of the column). The flow distributor comprises a centre outlet port 175, positioned similarly to the single centre port 40 of the conventional arrangement that can receive and transmit eluate flowing from the central radial region of the cross section of the column (i.e. a region located on the central axis 17 of the column). The flow distributor further comprises six peripheral ports 180 located equally and symmetrically spaced around the central port 175 that can receive and transmit eluate that is flowing in the perimeter region. Any of the peripheral ports, e.g. half of them (in this case three), could be used as reactant ports. That is, reactant may be flowed from a reactant source (not shown) through one or more of the peripheral ports into the column outlet to encounter the eluate flowing in the peripheral region and react therewith. This reaction chromatography is described in more detail below.
(37) The right hand side of FIG. 4 illustrates the better resolved peaks in a resultant chromatogram (trace 195 showing baseline resolution of the peaks, 200) which arises from detection of only the eluate from the central port 175. The chromatogram 195 shows an improvement in resolving power compared to the chromatogram 135 obtained using the conventional column arrangement as shown in FIG. 3 where all the eluate from across the full cross section of the column is gathered together and detected. The eluate from the one or more peripheral ports 180 collectively forms one portion of eluate that is not processed with the eluate from the central port 175. For example, in one embodiment in which eluate from the central port 175 is detected using a detector, the eluate from the peripheral ports 180 may instead be reacted, as hereafter described in more detail, and detected using another, separate detector and/or separately collected and/or sent to the inlet of the same or another column for a further chromatographic separation in order to better resolve the components, optionally after being re-concentrated before such further chromatographic separation. The peripheral eluate also exhibits better resolved peaks than in a conventional arrangement as shown in FIG. 3. Without being in any way limiting on the scope of the invention, this is believed to be due to the fact that a portion of eluate taken from a restricted radial region has a smaller axial spread of sample than eluate taken from across the full width of the column.
(38) There are different possible ways to design an outlet frit 11 suitable for the purpose of segregating flows of mobile phase from the outlet of the column. FIG. 5A shows a perspective view of one preferred embodiment of a frit assembly 220, with FIG. 5B showing an underside view of the embodiment looking in direction of arrow A and FIG. 5C showing a side cross section view taken on line B-B. In the embodiment shown, the frit assembly is assembled from sections of frit, i.e. a central, circular frit disc 235 and surrounding annular concentric frit ring 245 both made of porous material conventionally used as frit material, e.g. steel, which are separated from each other by a solid, non-porous flow barrier in the form of concentric ring 240, e.g. made of polymer such as PEEK. The non-porous intervening ring 240 prevents lateral flow of eluate between the two frit sections 235 and 245, thus keeping the central and peripheral eluate flows separate. The width of the non-porous ring 240 is lower than the width of the frit sections in this case, thereby to reduce drag on separated components in the eluate flow. The disc and rings 235, 240, 245 are fixed together and held inside a ring shaped, profiled outer fitting 250 also made of solid, non-porous material, e.g. PEEK, which acts as a fitting to the end of the column as described below.
(39) The aforesaid parts 235, 240, 245, 250 are thus assembled so that they fit together to form the assembly 220 which acts as a frit cap, wherein the outer ring 250 is dimensioned and profiled with an extended peripheral edge 224 which fits over the end of the column so that the under-side 222 of the frit cap is push-fitted (i.e. friction fitted) over the end of the column. Frit assemblies for use with the invention, like the assembly 220, typically may be push-fitted as shown or screwed onto the column end, but are preferably applied by push or friction fit. The over-side of the frit cap 225 in use is in contact with the steel end fitting (described in more detail hereafter) once the end fitting is screwed onto the end of the column. The non-porous intervening ring 240 protrudes slightly above the face or edge of the frit sections 235 and 245 on the downstream side, as evident in the protruding edge 241 of the non-porous ring 240 shown in the side view of FIG. 5C. The protruding edge 241 of the non-porous ring 240 can seal against the flow distributor, i.e. the underside of the flow distributor, to thereby separate zones of eluate adjacent each frit section. In this embodiment, the peripheral zone of eluate (adjacent outer frit section 245) is thus separated from central zone of eluate (adjacent central frit section 235). In a similar way, the non-porous, outer fitting or ring 250 also protrudes slightly above the face or edge of the frit sections 235 and 245 on the downstream side, which enables it to seal the against the flow distributor as well (to form an outer seal).
(40) FIGS. 5D and 5E show views of a similar frit assembly construction to that shown in FIGS. 5A, B and C except that the frit disc 235 and rings 240 and 245 are of different relative areas thereby splitting the eluate flow into portions from central and peripheral regions of different relative transverse area. As examples, the ratio of the area of the outer frit section 245 (245) to the area of the central frit disc 235 (235) may vary, e.g. from 90%:10% to 50%:50% but typically from 80%:20% to 50%:50% with ratios outside this range also possible. As examples the aforesaid ratio may be about 70%:30% or 75%:25%. Preferably, the said ratio is about 2:1. The ratio of the areas of the frit sections may be a means to vary the ratio of the respective volumes of the split portions of eluate flow. In some embodiments, it may be possible to use a central frit disc 235 (235) that has a different, e.g. lower, density compared to the frit sections 245 (245).
(41) FIGS. 5F and 5G show further frit designs which could be used in the present invention to split the eluate flow at the column outlet, in which a central porous frit disc 535 is surrounded by a plurality of porous frit discs 550, each of the frit discs 535 and 550 being located in a non-porous body 540, which thereby prevents lateral flow of eluate between the two frit sections 535 and 550. Other arrangements similar to those in FIGS. 5F and 5G could have more or less than the six peripheral frit discs 550 shown. The relative areas of the central and peripheral frit discs 535 and 550 may be varied, with examples of different relative areas being shown in FIGS. 5F and 5G.
(42) In certain other embodiments, as shown in FIGS. 5H and 5I, the column outlet 580 may be provided with a plurality of exit capillaries 585, 590 to channel the eluate flow in separate portions out of the end of the column, wherein a radially central capillary 585 is arranged to channel the first portion of the flow from the central region of the column and ten capillaries 590 peripherally arranged around the central capillary 585 are provided to direct the second portion of the flow. Thus, the first and second portions are channeled in separate capillaries and thus are split from each other. Preferably, in such embodiments, frits 592 are provided in the ends of the capillaries as shown in FIG. 5I. The total number of capillaries may be varied, e.g. 5, 6, 7, 8, 9, 10, 11, or 12 or more capillaries.
(43) FIG. 6 shows, in exploded view, an embodiment of the invention, which could be used, for example, with a standard analytical HPLC steel column 270 of 4.6 mm internal diameter. The embodiment in FIG. 6A uses a split frit assembly, 278, 279, and a flow distributor in the form of a steel end fitting 280 having multiple flow channels therein with corresponding fluid ports 281, 282. The parts (inset 285) of the split section frit 278 (corresponding to parts 235, 240 and 245 in FIGS. 5A-E) are assembled and placed at the column outlet. The frit parts 285 comprise a central frit disc section 255 held in a non-porous intermediate PEEK ring 260, with ring 260 in turn held in an outer peripheral ring frit section 265. Over the frit parts 285, there is a fitting 279, i.e. an outer cap made of PEEK, which acts as a flow adapter and serves to align the respective separated flow paths through the split frit 278 with respective fluid ports 281, 282 of the end fitting 280 as described later. The fitting 279 comprises a main body 271 which closely fits over the end 268 of the column (by friction fit) and encloses the frit parts 285 of the split section frit 278 at the column end. In an alternative arrangement, instead of friction fit, the fitting 279 could fit to the end of the column by a screw connection (i.e. an internal thread inside the main body 271 of the fitting 279 could screw onto an external thread on the end 268 of the column).
(44) The fitting 279 has a radially central aperture 272 which is aligned with the central frit section 255 of the split frit to thereby allow passage through the aperture 272 of eluate emanating from the central radial region of the column which has passed through the central frit section. The fitting 279 also has a plurality of, preferably equally spaced, peripheral apertures 273 (in this case 5 apertures) which are positioned peripherally around the central aperture 272 and lie in an annular recess in the end face 277 of the adapter. It will be appreciated that the apertures 273 could be circular or they could be slits or of some other shape. The peripheral apertures 273 in this case are radially aligned with the peripheral frit section 265 of the split frit. The apertures 273 thereby allow passage through of eluate emanating from the peripheral radial region of the column that has passed through the peripheral frit section 265. The peripheral eluate collected through apertures 273 in this way is communicated into the annular recess in which the apertures 273 lie in the end face 277 of the fitting 279.
(45) The steel end-fitting or end-cap 280 is screwed, either hand tightened or tightened with the aid of a tool if necessary, onto the external screw thread 275 on the outside of the column end. The end-fitting 280 provides one or more fluid ports, in this case three fluid ports, 281, for fluid communication with mobile phase exiting the column from the peripheral radial regions of the column (i.e. via peripheral frit section 265 and peripheral apertures 273) and a central fluid port 282 for communication with mobile phase exiting the column from the central radial region of the column (i.e. via central frit section 255 and aperture 272).
(46) When assembled, the underside of the tightened end fitting 280 (not visible in the figure) contacts the end face of fitting 279. The annular peripheral recess described in the end of fitting 279 containing apertures 273 is aligned with and in fluid communication with the peripheral ports 281 so that the peripheral flow of eluate through the peripheral apertures 273 is thereby in fluid communication with the peripheral ports 281 in the end fitting 280. The annular nature of the recess means that rotational alignment of the peripheral ports 281 with the peripheral apertures 273 is not required. Eluate in communication with peripheral ports 281 emanates from the same peripheral radial region and thus is preferably processed together as one portion of eluate, e.g. reacted and detected. Most typically, a reacted eluate flow from the peripheral ports 281 will be processed as one portion. However, it will be appreciated that flow from two or more peripheral ports could be processed separately. Indeed, the eluate flow from each peripheral port could be processed separately. One or more of the peripheral ports are preferably utilised as reactant ports. That is, reactant from a reactant source (not shown) is introduced into the column outlet through one or more of the peripheral fluid ports 281.
(47) The central aperture 272 in the fitting 279 on the other hand is in fluid communication with the central port 282 of the end fitting. The eluate from central port 282 of the end fitting thus emanates from the central radial region of the column and is processed differently to the eluate from the peripheral ports. The eluate from central port 282 may flow unreacted to a separate detector for example. In another case, the central port could be closed or capped so that only peripheral ports 281 are used as reactant and product ports.
(48) The fluid ports 281, 282 have internally threaded surfaces (female threads) to accept a screw fitting for plumbing to a reactant source, detector etc. as described below. Alternatively, it is possible to arrange the ports to have a male thread, e.g. male thread on a protruding portion, onto which is screwed a fitting for plumbing.
(49) The embodiment in FIG. 6B is largely the same as shown in FIG. 6A but instead of the split section frit 278 it uses a frit assembly that has a single frit piece 278 inside the fitting 279, wherein the fitting 279 divides the flow at the downstream side of the frit piece by means of its apertures 272 and 273.
(50) FIG. 7 illustrates an exploded view of an example of a column 293, having at its outlet an externally threaded end portion 293, on which is screwed end fitting 289, which has an internal threaded surface for this connection. A split frit assembly 290, having frit parts as shown (see exploded view inset), is push-fitted over a portion 299 of the column end having a smaller external diameter than the threaded portion 293. The split frit assembly 290 is held in placed once the end fitting 289 is screwed on the end of the column. The split frit assembly 290 in this embodiment comprises a central frit disc 295 and peripheral frit ring 294 which are separated from each other by non-porous PEEK ring 296 as a flow barrier. The split frit sections are held in outer PEEK ring fitting 297 which functions as a push fitting cap to fit over the end 299 of the column. The fitting 297 is further secured to the column end with annular steel band 298 which grips the circumference of the PEEK fitting 297.
(51) In the FIG. 7 embodiment, the fitting 297 is different to the fitting 279 acting as flow adapter in FIG. 6. In this example, the underside of end fitting 289 seals against the non-porous parts (i.e. parts 296, 297) of the frit assembly 290 to thereby keep fluidly separate the portions of eluate flowing from the frit sections 295 and 294. The end fitting 289 has one central fluid port 292 at its centre and three peripheral fluid ports 291 surrounding it, although it should be understood that the present invention contemplates any number of peripheral ports 291, e.g. one or more peripheral ports 291. Preferred examples may have from 3 to 10 peripheral exit ports, particularly 3, 4, 5, 6, 7 or 8 peripheral ports. End fittings with 3 and 6 peripheral ports are good examples. Furthermore, the present invention contemplates any number of central fluid ports (i.e. those ports that communicate with a flow of eluate from a central radial region), e.g. one or more central ports. Preferably, however, there is one central port as in the embodiment shown. The end fitting central port 292 lies radially in the central region of the column and is radially aligned with the central frit section 295 and thereby communicates with the portion of eluate flow through the central frit section. The three peripheral ports 291 lie radially in the peripheral region of the column and are radially aligned with the peripheral frit ring 294 and thereby communicate with the portion of eluate flowing through the peripheral frit ring. The fluid ports 291, 292 have internally threaded surfaces to accept a screw fitting for plumbing as described below.
(52) FIG. 8A illustrates an exploded view of an alternative frit assembly 303 and end-fitting 305 generally similar to that shown in FIG. 7. In this example though, the end-fitting 305 has six peripheral fluid ports 302 around one central port 301. The assembled frit assembly 303 and end-fitting 305 are shown in FIG. 8B, which has been cut away to show the relationship of the assembled parts inside.
(53) FIG. 9 illustrates a cut-away side view of the same four-port end fitting shown in FIG. 7, but shown assembled on the column 293 together with fluid plumbing attached. It can be seen more clearly from this view that the fluid ports 291 and 292 comprise respective channels 291 and 292 running through the end fitting 289 from its inner surface which is in fluid communication with the frit assembly 290 to its outer surface.
(54) In one embodiment, an exit plumbing tube 331, of standard type for HPLC, is fitted to the centre fluid port 292 by means of male screw fitting 341. Plumbing tubes 332, again of standard type, are fitted to the peripheral ports 291 by screw fittings 342 of the same type as screw fitting 341. The screw fittings 341 and 342 are screwed into the channels 292 and 291 of the ports 292 and 291 respectively, which channels carry an internal (female) screw thread for this purpose, and the plumbing tubes 331, 332 are thereby compression fitted. It will be appreciated that in other designs the plumbing could be fitted to the ports by means of a female screw fitting which fits to a male port on the end fitting.
(55) The end fitting 289 is tightly screw fitted on the end of the column 310 so that inner surface or underside of the end fitting 289 contacts the end face of the frit assembly 290 and thereby the respective central and peripheral portions of eluate passing through the frit are in separate fluid communication with the respective central and peripheral ports 292 and 291 of the end fitting. The underside of end fitting 289 seals against the non-porous parts (i.e. parts 296, 297) of the frit assembly 290 to thereby keep fluidly separate the portions of eluate flowing from the frit sections 295 and 294 into the respective ports 292 and 291.
(56) It will be appreciated that many further embodiments of the present invention may be devised to provide a segmented flow at the column outlet.
(57) It can be seen from the foregoing description that the present invention has the great advantage that it can be practiced using existing conventional packed chromatography columns. By segmenting the eluate flow, e.g. by providing an end fitting having multiple ports, eluate from different regions of the column can be processed differently. The present invention may therefore, for example, be practiced using a conventional HPLC system wherein one portion of the eluate flow is reacted and directed to a detector and another portion is sent processed in another way without being reacted. Even when the eluate is reacted and processed as ne portion only, the invention is applicable to conventional columns by virtue of requiring only a modified end fitting (flow distributor) and preferably split frit.
(58) The present invention may also be practiced in which only one eluate flow leaves the column and is reacted. Such an embodiment represents a simple implementation of the invention, i.e. with a single eluate flow being reacted. In other words, there is no need in some embodiments to have a split flow of eluate, with different portions of eluate flow being either reacted or not reacted or reacted differently. This may be achieved for example using apparatus described above with reference to the Figures and blocking or capping, for example, the central port so that only a single portion of eluate in the peripheral region is reacted by supplying reactant via at least one peripheral port and extracting reacted eluate product via at least one other peripheral port. If such reactions on single portions only are required, then such means as described above for segmenting the eluate at the outlet (e.g. a split frit) may not be necessary. Moreover, in such embodiments, the flow distributor may be configured with only a required number (one or more) of reactant ports to enable reactant flow into the outlet and a required number (one or more) of product ports to receive and transmit product but without any other ports than these.
(59) Using apparatus as described above with reference to the Figures, various implementations of the present invention may be achieved.
(60) A reaction chromatography embodiment is shown schematically in FIG. 10. A column fitted at its outlet end with a flow distributor 405, which may be one of the embodiments shown in FIGS. 6, 7 and 9, is shown end-on. The flow distributor is preferably in the form of an end fitting that is fitted to the end of the chromatography column as previously described. Thus, the embodiment uses a segmented parallel flow column, such as described herein, for reaction chromatography. A single central port fluid port 410 and three peripheral fluid ports 412 are shown. A first one of the peripheral fluid ports 412 is closed with a cap 414. A second one of the peripheral ports 412 is connected to a reactant source 420 which comprises a reservoir of reactant and a pump to supply a stream of reactant 422 into the second peripheral port 412. The second peripheral port 412 is thus a reactant port. The reactant mixes with eluate eluting from the peripheral region of the column that has passed through the outer frit section and the mixture of reactant and eluate from the column exits the flow distributor via a third one of the peripheral ports 412 as a reacted eluate flow 430. The third peripheral port 412 is thus a product port. Reaction occurs between one or more components in the eluate and the reactant. The reaction may begin in the flow distributor and may continue downstream of the distributor. Thus, herein, the term reacted eluate does not necessarily mean fully reacted eluate but only that at least mixing of reactant and eluate has taken place and reaction has at least begun to occur or will occur downstream. An optional reaction coil 440 may be supplied for the reaction to proceed downstream of the flow distributor. This may be useful where the reaction is slow but may not be required where the reaction is sufficiently fast.
(61) The reactant, for example, may be DPPH solution for reaction with antioxidant components in the eluate. This reaction results in decolorisation of the DPPH solution, which can be detected, e.g. at 500 nm by a UV detector. The reaction product is detected downstream at a detector 450, which, for example, could be the mentioned UV detector.
(62) The reactions can be timed to occur when they are required, e.g. to correspond to the appearance at the outlet of a target component. Since chromatographic migration of components through the column is predictable, the application of the reactant into the outlet at a particular time can be carried out. Thus, the reactant can be timed to flow into the outlet when it is required, e.g. to correspond to the appearance at the outlet of a target component. This procedure is performed whenever a target component elutes.
(63) It will be appreciated that in other embodiments, the peripheral fluid port 412 closed with cap 414 could instead not be fitted with a cap but be connected with the outflow from the other peripheral fluid port that transmits the reacted product to the detector.
(64) In addition to the reactant port and product port, eluate flows from the column and flow distributor via the central port 410. This central port is aligned with the central frit section and receives eluate flow from the centre of the column bed. Since this central port and the central portion of eluate flow that it communicates with are not in fluid communication with any of the other ports or the peripheral portion of eluate that is reacted, the central eluate flow 465 through the central port remains unreacted. This central, unreacted eluate 465 is detected as usual at a detector 460, which may be a typical HPLC type detector such as UV detector.
(65) A split frit, in particular an annular frit arrangement, is used with the reaction chromatography apparatus of FIG. 10 so that it enables the eluate flow to be split into the two portions (central and peripheral) and enables differences between the unreacted, native eluate and the reacted eluate to be observed and so changes in the system to be gauged. A suitable annular frit arrangement is shown schematically in FIG. 10 that comprises a central frit section 470, an annular non-porous flow barrier 472 and an annular outer frit section 474. The central frit section 470 and thus the central portion of eluate flowing therethrough is in fluid communication with the central fluid port 410 but is fluidly separated by the flow barrier 472 from the peripheral eluate flow. The annular frit section 474 and thus the peripheral portion of eluate flowing therethrough is in fluid communication with the outer peripheral fluid ports 412. With the eluate portions divided and fluidly separated in this way, one portion (peripheral in this case) can be reacted as described above whilst the other portion (central in this case) passes unreacted to a processing unit (detector).
(66) A similar reaction chromatography embodiment to that of FIG. 10 is shown schematically in FIG. 11. The arrangement is mostly the same but in this case the first peripheral port 412 is also a reactant port so that there are two reactant ports in all. The first peripheral port (reactant port) in this case can flow a second reactant stream 428 into the outlet from a second reactant source 426. The peripheral eluate portion in this case mixes with both the first and second reactants and the combination exits as product stream 430 via the third peripheral port 412 as before. A reaction coil is not shown in this embodiment as this is merely optional for the invention.
(67) FIG. 12 shows schematically an embodiment of the invention for performing two separate reactions simultaneously (multiplexed reactions). In the design shown, a total of five fluid ports are provided in the flow distributor 505, comprising four peripheral ports 512 and one central port 510. Once again an annular frit is provided but in this embodiment, the outer annular frit section 474 is split into two further sections 474a and 474b, such that each further section resembles an approximately semi-circular shape. The two outer frit sections 474a and 474b are fluidly separated from each other (and from the central frit section 470) by the flow barrier 472. This enables two separate reactions to be performed due to the absence of cross-flow in reagents through the frit. One reaction may be performed on the eluate flowing through one frit peripheral section 474a while another reaction may be performed on the eluate flowing through the other peripheral frit section 474b. Also, the two fluidly separated peripheral portions of eluate are separated from a central portion of eluate flowing through central frit section 470.
(68) Each frit section 474a and 474b is in fluid communication with both a reactant port and a product port. The frit section 474a is in communication with a first peripheral port 512 which is a reactant port fed with a first reactant from a first pumped reactant source 520a. The frit section 474a is furthermore in communication with another peripheral port 512 which is a first product port for receiving the product of the mixed first reactant and eluate and sending the product stream (via optional reaction coil shown) to a first detector 550a. The other frit section 474b is in communication with a further peripheral port 512 which is a reactant port fed with a second reactant from a second pumped reactant source 520b. The other frit section 474b is furthermore in communication with yet another peripheral port 512 which is a second product port for receiving the product of the mixed second reactant and eluate and sending the product stream (via optional reaction coil shown) to a second detector 550b. The two different product streams may optionally exit through a loop. The product streams may be independently detected. As mere examples of different reactants, the first reactant could be DPPH reagent for antioxidants and the second reagent could be Xanthine oxidase used as part of a biodetector, e.g. in anti arthritic studies.
(69) As in the previous embodiment, in addition to the peripheral reactant ports and product ports, eluate flows from the column and flow distributor via the central port 510. This central port is aligned with the central frit section 474 and receives eluate flow from the centre of the column bed. Since this central port and the central portion of eluate flow that it communicates with are not in fluid communication with any of the other ports or the peripheral portion of eluate that is reacted, the central eluate flow through the central port 510 remains unreacted. This central, unreacted eluate is independently detected as usual at a detector 560, which may be a typical UV detector.
(70) The invention may have application in the following situations, for example: synthesizing compounds on a small scale e.g. nano-scale; natural product screening; protein digestions, optionally where digests could flow directly to a mass spectrometer or to a 2.sup.nd dimension chromatography column; catalytic processes, e.g. in the food or pharmaceutical industries.
EXAMPLES
(71) Details of experiments and results are given below to further illustrate the invention by way of examples.
Inventive Example
(72) Reaction chromatography according to the present invention was performed using a segmented parallel flow column with end fitting as shown in FIG. 6. Modifications were made at the fluid ports of the end fitting as shown schematically in FIG. 13. The mobile phase was flowed through the column 600 from its inlet to outlet by means of upstream pump 1 (not shown). At the end of the column was fitted a four port, parallel segmented flow end fitting 600, as described above (and as shown in FIG. 6). An annular segmented frit and flow directing cap, as described hereinabove, were utilised to segment the eluate flow at the outlet. Two of the ports 610 were sealed with plugs (not shown), being the central port and one of the peripheral ports. One of the peripheral ports 612 constituted a reactant port and delivered a flow of reactant into the column outlet from a reactant source 614 that was supplied by a pump 2. The final one of the peripheral ports 622 constituted a product port and carried a flow of reacted eluate product away from the column outlet to a UV detector 624 and from there to a waste reservoir 630. The location of a reaction coil (when used) is shown by reference 640. The eluate exiting the column in this way can undergo efficient reaction with the reactant introduced into the peripheral zone of the end fitting.
Comparative Example
(73) For comparison to the example embodying the present invention shown in FIG. 13, a conventional reaction chromatography configuration was constructed in which the same column and four port end fitting as shown in FIG. 13 was used but with the alterations shown in FIG. 14. In the comparative case, all three peripheral ports 710 were plugged and only the central port 712 was open to direct the eluate into a zero dead volume t-piece 725 located separate from and downstream of the column. In this case, reactant was delivered into the t-piece 725 from a reactant source 714 that was supplied by a pump 2. The product was carried away from the t-piece 725 to a UV detector 724 and from there to a waste reservoir 730. The location of a reaction coil (when used) is shown by reference 740.
(74) To test the two set-ups, reaction chromatography was performed for the analysis of coffee with simultaneous DPPH and UV detection. Espresso coffee was analysed using HPLC with DPPH (1,1-Diphenyl-2-picryl-hydrazyl) and UV detection.
(75) Espresso coffee was used as the sample, as it is known to contain numerous antioxidants. Reaction between the DPPH radical and an antioxidant results in decolorisation of the DPPH solution. This decolorisation can be detected at ?500 nm using a UV detector. The DPPH reaction is relatively slow, therefore most conventional DPPH detection processes employ reaction coils (commonly 800 uL in volume), resulting in substantial band broadening and leading to poor peak shape and resolution.
(76) The tests illustrated that post column reagents can be mixed with eluting sample within the end fitting of the HPLC column (Inventive Example) and a reaction occurs that can be monitored. The resulting chromatograms were compared with the chromatograms obtained using the Comparative Example set-up of FIG. 14 and the results are described below.
Experimental Details
(77) Sample Preparation:
(78) 30 mL of Ristretto espresso coffee was freshly prepared using a Delonghi espresso machine. The coffee was filtered using a 0.45 um PVDF syringe filter and allowed to cool to room temperature prior to analysis.
(79) Preparation of DPPH Reagent:
(80) A solution of 0.05 mgmL.sup.?1 DPPH in methanol was accurately prepared using volumetric glassware.
(81) Chromatographic Column:
(82) All experiments were conducted using a HYPERSIL GOLD?, Gold reversed phase column (100?4.6 mm) fitted with a four-way parallel segmented flow end fitting as shown in FIGS. 13 and 14.
(83) Chromatographic Conditions:
(84) Ristretto coffee (20 mL injection volume) was analysed using a SHIMADZU? analytical HPLC system which comprised a SHIMADZU? LC-20ADvp quaternary pump, SHIMADZU? SIL-10ADvp auto injector, SHIMADZU? SPD-M10Avp PDA detector and a Degassex model DG-440 inline degasser unit.
(85) Separation was achieved using a water/methanol: gradient starting from 5% (methanol) and finishing at 100% methanol. The gradient rate was 2%/min. The mobile phase flow rate was 1.0 mLmin.sup.?1.
(86) Chromatograms were extracted at 275 nm for UV detection and 500 nm for the DPPH reaction using simultaneous dual wavelength detection.
(87) DPPH was added to the flow stream using a SHIMADZU? LC-19ADvp quarternary pump at flow rates as described below.
(88) Results
(89) Experiments for both the Inventive Example and Comparative Example were conducted using 99 uL, 200 uL reaction coils and no coil (that is, eluent and reagent enter the detector through minimum tubing after mixing) using a DPPH:eluent flow rate ratio of 1.5:1. DPPH chromatograms and UV chromatograms are shown in the Figures now described for the Inventive Example and Comparative Example.
(90) ResultsNo Reaction Coil
(91) FIGS. 15A-D show the chromatogram for the Comparative Example with no reaction coil present (in each figure the upper trace is the UV chromatogram and the lower trace is the DPPH chromatogram). FIG. 15A shows the full chromatogram; FIG. 15B shows the zoomed in (intensity) full chromatogram; FIG. 15C shows the zoomed in 0-5 min region; and FIG. 15D shows the zoomed in 10 to 18 min region.
(92) FIGS. 16A-D show the chromatogram for the Inventive Example with no reaction coil present (in each figure the upper trace is the UV chromatogram and the lower trace is the DPPH chromatogram). FIG. 16A shows the full chromatogram; FIG. 16B shows the zoomed in (intensity) full chromatogram; FIG. 16C shows the zoomed in 0-5 min region; and FIG. 16D shows the zoomed in 10 to 18 min region.
(93) Results99 ?L Reaction Coil
(94) FIGS. 17A-D show the chromatogram for the Comparative Example with a 99 ?L reaction coil present (in each figure the upper trace is the UV chromatogram and the lower trace is the DPPH chromatogram). FIG. 17A shows the full chromatogram; FIG. 17B shows the zoomed in (intensity) full chromatogram; FIG. 17C shows the zoomed in 0-5 min region; and FIG. 17D shows the zoomed in 10 to 18 min region.
(95) FIGS. 18A-D show the chromatogram for the Inventive Example with a 99 ?L reaction coil present (in each figure the upper trace is the UV chromatogram and the lower trace is the DPPH chromatogram). FIG. 18A shows the full chromatogram; FIG. 18B shows the zoomed in (intensity) full chromatogram; FIG. 18C shows the zoomed in 0-5 min region; and FIG. 18D shows the zoomed in 10 to 18 min region.
(96) Results200 ?L Reaction Coil
(97) FIGS. 19A-D show the chromatogram for the Comparative Example with a 200 ?L reaction coil present (in each figure the upper trace is the UV chromatogram and the lower trace is the DPPH chromatogram). FIG. 19A shows the full chromatogram; FIG. 19B shows the zoomed in (intensity) full chromatogram; FIG. 19C shows the zoomed in 0-5 min region; and FIG. 19D shows the zoomed in 10 to 18 min region.
(98) FIGS. 20A-D show the chromatogram for the Inventive Example with a 200 ?L reaction coil present (in each figure the upper trace is the UV chromatogram and the lower trace is the DPPH chromatogram). FIG. 20A shows the full chromatogram; FIG. 20B shows the zoomed in (intensity) full chromatogram; FIG. 20C shows the zoomed in 0-5 min region; and FIG. 20D shows the zoomed in 10 to 18 min region.
(99) The Inventive Example enabled the DPPH to be readily decolourised. From the results it is evident that although the DPPH detection in the Comparative Example with post-column reaction is slightly more sensitive than the corresponding Inventive Example, the Inventive Example has significantly lower noise, giving an overall signal-to-noise advantage in that case. This is more clearly shown in FIG. 21 by comparing the results for both Comparative Examples (a) and Inventive Examples (b) and zooming in on the 13 to 14 min region. It is also evident from FIG. 21 that the noise in the Inventive Example remains approximately constant irrespective of reaction coil volume, which is not true for the Comparative Example. Thus, with the Inventive Example, it was possible to conduct simultaneous DPPH and UV analysis with substantially less noise and comparable sensitivity to the conventional post column reaction configuration of the Comparative Example.
(100) One of the further benefits of the present invention is the ability to perform multiplex reactions and/or detection (i.e. simultaneous reactions and/or detection using multiple detectors). As shown schematically in FIG. 22 the end fitting could be modified, and preferably used with a segmented frit as described herein (see e.g. FIG. 12), so that one half of the fitting 760 reacts one portion of eluate at the outlet by flowing in a reactant, e.g. derivatising agent 1, through port 764. The combined eluate and derivatising agent product mixture then exits through port 766 to a flow cell and detection. At the same time, the other half of the fitting 762 reacts another portion of eluate at the outlet by flowing in a different reactant, e.g. derivatising agent 2, through port 768. This second combined eluate and derivatising agent product mixture then exits through port 770 to a second flow cell and detection. A third portion of eluate flow 772 may also be detected separately, which preferably has not undergone a reaction and so can be used to gauge changes compared to the reacted flows.
(101) A large range of possible uses of the present invention include: the selective digestion of proteins/peptides with enzymes, e.g. such as trypsin; the screening of biologically active molecules; selective protein isolation; the derivatisation of compounds to facilitate detection, e.g. fluorescence detection; on-column stereoselective synthesis of compounds; chemiluminescence; and nanoscale synthesis.
(102) As used herein, including in the claims, unless the context indicates otherwise, singular forms of the terms herein are to be construed as including the plural form and vice versa. For instance, unless the context indicates otherwise, a singular reference, such as a or an means one or more.
(103) Throughout the description and claims of this specification, the words comprise, including, having and contain and variations of the words, for example comprising and comprises etc, mean including but not limited to, and are not intended to (and do not) exclude other components.
(104) It will be appreciated that variations to the foregoing embodiments of the invention can be made while still falling within the scope of the invention. Each feature disclosed in this specification, unless stated otherwise, may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
(105) The use of any and all examples, or exemplary language (for instance, such as, for example, e.g. and like language) provided herein, is intended merely to better illustrate the invention and does not indicate a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
(106) Any steps described in this specification may be performed in any order or simultaneously unless stated or the context requires otherwise.
(107) All of the features disclosed in this specification may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. In particular, the preferred features of the invention are applicable to all aspects of the invention and may be used in any combination. Likewise, features described in non-essential combinations may be used separately (not in combination).
