CHROMATOGRAPHIC ANALYSIS WITH LOW PRESSURE DUAL GRADIENT REFOCUSING

Abstract

There is provided a system for separation of analytes in a solution. The system encompasses a cartridge or trapping column enclosing a sorbent for binding the analytes in the solution and a conduit establishing a fluid link to a valve having a holding-loop to achieve elution through the cartridge at low pressures. Prior to entry into the loop, the eluent is diluted or modified by a confluent flow stream. The valve is switchable to a position following the elution from the cartridge for emptying the holding loop through an outlet port at high pressures comparable to those required for chromatographic columns. The system may use parallel gradient formation/elution to stagger analyses so that essentially the only analytical phase that hinders a 100% duty cycle is the time required for moving the first analyte from the valve and to the detector.

Claims

1. A system for separation of analytes in a liquid sample, said system comprising: a control unit for controlling and regulating flow rate and pressure of the system; an inlet for introducing the sample into the system; a first column having a sorbent for trapping the analytes, said first column being switchable between a trapping mode and an elution mode, said trapping mode enabled when the inlet is in fluid communication with the first column, and said elution mode enabled when the analytes trapped in the first column are eluted; one or more fluid pumps connected in parallel and controlled by the control unit to establish one or more mobile phase gradients and to deliver a mobile phase at a flow rate of 300 nL/min to 50 L/min through said first column at a pressure of 1 bar to 50 bar, when the column is in elution mode, one or more holding loops wherein the mobile phase gradient(s) and the partly separated analytes eluted from the first column may be stored for further separation one or more fluid pumps connected in parallel and controlled by the control unit to deliver a make-up flow at a flow rate of 300 nL/min to 50 L/min at a pressure of 1 bar to 50 bar, said make-up flow being introduced between the first column and the holding loops, and one or more valves switchable between a low pressure position and high pressure position, wherein said low pressure position establishes a fluid connection from the first column to a holding loop, whereas said high pressure position establishes a fluid connection with a high pressure fluid pump and a conduit leading to a chromatography column, wherein the control unit is adapted to activate the high pressure fluid pump and force the gradient and analytes in said loop through the chromatography column at a flow rate of 20 nL/min to 20 L/min and a pressure of 1 bar to 3,000 bar.

2. The system of claim 1 wherein the inner diameter of the first column is at least twice as large as the inner diameter of the chromatography column.

3. The system of claim 1, wherein the chromatographic column contains C18 reversed phase material.

4. The system of claim 1, wherein the sorbent of the first column is selected from C18, C8, C4, hilic, SCX, SAX, cellulose, antibodies and derivatives thereof.

5. A system for separation of analytes in a liquid sample, said system comprising: a control unit for controlling and regulating flow rate and pressure of the system; a disposable cartridge enclosing a sorbent for binding the analytes from the liquid sample, a recipient socket for receiving the cartridge, and allowing a mobile phase to flow through the cartridge into the recipient socket, thereby eluting the analytes into the recipient socket; a conduit in fluid communication with the recipient socket, said conduit establishing a fluid link to a holding loop wherein one or more mobile phase gradient(s) and the partly separated analytes eluted from the cartridge may be stored for further separation; one or more, fluid pumps connected in parallel and controlled by the control unit to optionally establish one or more mobile phase gradients and to deliver a mobile phase at a flow rate of 300 nL/min to 50 L/min through said cartridge at a pressure of 1 bar to 50 bar, one or more, fluid pumps connected in parallel and controlled by the control unit to optionally establish one or more gradients and to deliver a make-up flow at a flow rate of 300 nL/min to 50 L/min at a pressure of 1 bar to 50 bar, said make-up flow being introduced between the recipient socket and the holding loop, and one or more valves switchable between a low pressure position and high pressure position, wherein said low pressure position establishes a fluid connection from the cartridge to the holding loop, whereas said high pressure position disconnects the fluid connection of the loop from the low pressure position and establishes a fluid connection with a high pressure fluid pump and a second conduit leading to a chromatography column, wherein the control unit is adapted to activate the high pressure fluid pump and force the gradient and analytes in said loop through the chromatography column at a flow rate of 20 nL/min to 20 L/min and a pressure of 1 bar to 3,000 bar.

6. The system of claim 5, wherein the inner diameter of the cartridge is at least twice as large as the inner diameter of the chromatography column.

7. The system of claim 5, wherein the cartridge is selected from: a. a pipette tip with chromatographic resin embedded and fixated therein; b. a length of tube, wherein its inner and/or outer surfaces may be cylindrical, conical, spherical or any combination thereof; and c. a planar disc.

8. The system of claim 5, wherein the chromatographic column contains C18 reversed phase material.

9. The system of claim 5, wherein the sorbent of the cartridge is selected from C18, C8, C4, hilic, SCX, SAX, cellulose, antibodies and derivatives thereof.

10. The system according to claim 1, wherein the control unit establishes a flow through the first column or cartridge and the make-up flow each below 10 mL/min while the flow through the chromatography column during separation is below 5 mL/min.

11. The system according to 10, wherein the control unit computes and subsequently is lead to establish flows from the individual mobile phase pumps that are non-linear over time such that the resulting mobile phase gradient eluted from the holding loop be a piecewise linear mobile phase gradient.

12. The system of claim 1, wherein the control unit is adapted to establish a flow through the first column or cartridge, and the make-up flow each above 50 L/min and below 10 mL/min while the flow through the chromatography column during separation is below one fifth of the flow through the first column or cartridge.

13. The system of claim 1, wherein the control unit is adapted to establish flows from the one or more fluid pumps in the range of less than 10 L/min, more preferably less than 5 L/min, and most preferably less than 2 L/min at a pressure of 1 bar to 50 bar.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The above, as well as other advantages of the present invention, will become readily apparent to those skilled in the art from the following when considered in the light of the accompanying drawings.

[0047] FIG. 1 shows a configuration enabling low-pressure gradient formation through a trap column (partial elution possible), post trap dilution of eluent, into storage loop, and a high pressure elution through an analytical column.

[0048] FIG. 2 shows isocratic elution through a disposable cartridge and subsequent gradient formation before entering storage loop.

[0049] FIG. 3 shows low pressure gradient through cartridge and subsequent dilution before entering storage loop.

[0050] FIG. 4 shows Low pressure gradient through cartridge and subsequent gradient modification before entering into storage loop (or trapping column).

[0051] FIG. 5 shows an algorithmic computation of flows needed for forming the desired gradient.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The above, as well as other advantages of the present invention will become readily apparent to those skilled in the art from the following detailed description of preferred embodiments when considered in the light of the accompanying drawings. The present invention relates to the following aspects: [0053] 1. Low pressure gradient formation and low pressure elution through a first column (which may be an ordinary trapping column or a disposable sorbent cartridge) followed by low pressure gradient modification (dilution, mostly) before positioning into a holding loop, also at low pressure. Gradient with partly separated analytes are then flushed from loop under high pressure for further separation on separation column. [0054] 2. Algorithmic computation of pump flow required in the first binary gradient relative to the gradient modification flow in order to obtain a (piece-wise) linear resulting gradient in the holding loop.

[0055] FIG. 1 shows the schematics of a simple implementation of the present invention as applied to normal reversed phase chromatography with a multi-use trapping column [1] positioned across a 6-port two-position valve [2] such that a sample may be loaded from an autosampler (not shown) into an injection port [3]; when the sample of analytes dissolved in liquid passes through the trapping column, analytes will be captured on the column while the solvent flows through the column to the waste port [4](the valve must be in the other position, i.e. the one not shown here). When the valve is switched (the depicted position), a mobile phase gradient from two low pressure pumps [5 &6] is formed at a mixing Tee [7] and the liquid moves through the trapping column and sequentially elutes all analytes as a result of the gradually increasing eluting strength of the mobile phase gradient. After the trapping column, a second fluid stream, from a third pump [8] is added at a second mixing Tee [9] upon which the now diluted gradient (containing the partly separated analytes) is placed in a holding loop [10] that is positioned across a second sheer valve [11]. All these liquid handling steps can occur at relatively low pressure, e.g. around 1 to 20 bar (although for some analyses it may be advantageous to use up to 50 bar) even if a high flow-rate of, say up to 50 L/min is used. When the second sheer valve switches position, the low pressure fluidics components get disconnected while the holding loop is now in-line with a high pressure fluid source [12] on one side and a separation column [13] on the other side. The modified gradient is now forced by the high pressure pump out of the loop, through the separation column to a detector. At the column head, each analyte will bind to the stationary phase until a later point in time where the gradient has reached the same elution strength that eluted the analyte from the trapping column material (provided the stationary phase is equivalent in the two columns). As the low pressure components are disconnected, the elution may happen at any back pressure that the valve and pump can withstand. This same functionality may also be achieved by using 3 needle valves instead of one sheer valve. Needle valves can currently withstand up to 40,000 psi on a routine basis while such pressures may be delivered by e.g. pneumatic pumps. The present invention therefore also provides for a very powerful means of loading ultra-high pressure columns well above the pressure ranges currently provided by commercial systems.

[0056] As a concrete implementation example, the trapping column may contain C18 reverse phase chromatography material in a conduit that is e.g. 10 mm long and 300 m inner diameter. A gradient flow that elutes the sample may be formed from water and acetonitrile at e.g. 20 L/min while maintaining low back pressure. The added mobile phase at the second Tee may be water at an initial flow rate of 20 L/min and dropping to zero at the end of the gradient; this approximately reduces the acetonitrile composition of the original gradient to around half initially while it ends at the same level as the original gradient. The holding loop may be a 7 m long piece of fused silica tubing with an inner diameter of 100 m where the narrow diameter is chosen because it reduces diffusion of the gradient during the time it stays in the loop. The separation column may be a 20 cm long conduit with an inner diameter of 150 m and containing C18 reversed phase material. Elution of the partly separated analytes and the gradient from the holding loop could for instance happen at a rate of 1 L/min and the elution through the separation column would therefore take more than 20 times longer than the elution from the trap column.

[0057] FIG. 2 shows a setup also in accord with the present invention; in this setup an isocratic flow from a source [87] elutes peptides from a disposable sorbent cartridge [85] while a make-up fluid from another source [88] is added, again at a connection [83] just after the recipient socket [84]. Initially the flow rate of the make-up flow is high but it is gradually reduced such that the liquid composition within the holding loop [81] de-facto becomes the desired gradient. The analytes of all species will be in the most diluted fluid volume at the distal point of the holding loop (closest to the separation column [91]). Once the analytes and the gradient are in the holding loop, the valve [86] will switch and thereby place the loop in-line between a high-pressure liquid delivery source [89] (e.g. a syringe pump) on the one side, and a conduit [90] leading to a separation column [91] on the other side. As the gradient with analytes reaches the head of the separation column, all analyte species adsorbs to the stationary phase material (if the mobile phase has been sufficiently diluted) until such later point in the gradient where each analyte species is released and the thus focused species are propagating through the column towards the detector.

[0058] As shown, the selected high pressure valve is a 2-position 10-port valve which accommodates the use of two holding loops in parallel. This allows the next sample and gradient to be loaded into one holding loop while the previous sample and gradient is still being analysed. Since the low pressure steps (of eluting with a first gradient through the disposable sorbent cartridge and adding make-up fluid and positioning the resulting gradient in a holding loop) are so fast relative to most elutions through the separation column, then the effect of using two holding loops in parallel is that the next sample is already loaded and ready as soon as the previous sample is finished. Further, the gradient is positioned at the very end of the holding loop, meaning that the dwell time is exceedingly small and the net effect of pre-loading and small dwell time is that this system can have near 100% duty cycle.

[0059] FIG. 3 shows a further implementation in accord with the present invention; in this setup two pumps [101 and 102] are used to deliver a gradient through the cartridge thereby eluting different analytes at different times of the gradient, all the while, an isocratic make-up fluid is again added from a third pump [103] downstream of the cartridge such that it modifies the gradient as it elutes from the cartridge. As an example, this could be a gradient that runs through a C18 sorbent containing cartridge and starts out at 2% organic phase in water and gradually increases to 40% organic phase (e.g. acetonitrile) while the make-up fluid may be pure water that initially is added at an equal flow rate as the gradient and where the flow rate is gradually reduced over the course of the gradient such that the gradient entering into the holding loop (and containing the semi-separated analytes) is now a gradient that runs from 1% to 35%. Following a valve switch, this gradient is then flowed through a C18 sorbent containing separation column but since each analyte species is present at a point that contains less organic solvent than the composition that eluted it from the C18 of the cartridge, then each analyte will bind temporarily onto the separation column until a little later in the gradient where they again would elute rapidly. The gradient modification from the make-up fluid is thus used to make each species re-sorb and focus onto the separation column.

[0060] While the setup in FIG. 3 has one more syringe pump than the setup in FIG. 2 and therefore is more complex, the setup in FIG. 3 is easier to optimise in terms of diluting the gradient such that analytes are re-focused across the entire chromatogram.

[0061] FIG. 4 shows a further modification also in accord with the present invention where two pumps [101 and 102] are used to deliver a gradient through the disposable sorbent cartridge thereby eluting different analytes at different times of the gradient, all the while, downstream of the cartridge, a make-up fluid is added from two pumps [103 and 104] such that the make up fluid may also be ramped as a gradient such that this make-up gradient modifies the original gradient that elutes from the cartridge. This provides a very high degree of flexibility to control the resulting gradient that enters into a holding loop (or runs through a trapping column as the case may be).

[0062] One advantage that is obtained from having two syringe pumps available for the make-up flow, is that the entire gradient including a separation column rinsing step with very high eluting strength can be formed and loaded into each holding loop. With the setup in FIG. 3, a rinsing step would require that the high eluting strength liquid be delivered by the high pressure pump whereas the setup in FIG. 4, can use any liquid, including water, in the high pressure pump in order to push the gradient through the separation column. In the schematic shown in FIG. 4, the liquid of the high pressure pump only has one function, namely to push the gradient out of the holding loop. This is easier to control than if the high pressure pump solvent also was used for rinsing steps.

Pump Control Aspects of the Present Invention

[0063] The mobile phase output of each of the 3 or 4 pumps involved in generating the first gradient and providing the make-up flow needs to be controlled and synchronized carefully. Difficulties in generating linear, binary gradients from three solvent flows arise from the fact that the volume between the two mixing points (V.sub.tt) is always greater than zero. While a change in flow-rate from any of the solvents has an instantaneous effect on the collective output flow rate immediately downstream of the second mixing Tee, any changes in composition will be delayed owing to the volume between the first and second mixing Tees.

[0064] As schematically represented in FIG. 5, two solvent streams [201 and 202] are mixed in a first mixing tee [203], followed by confluent addition of a third solvent stream [204] in a second mixing tee [205] before forming a collective output. This figure exemplifies mixing of solvents A and B prior to the disposable cartridge, and then addition of a dilutive solvent C after the cartridge. The two mixing points are then separated by a volume V.sub.tt, where the relationship between the volume V.sub.tt and the functions for flows of A and B (F.sub.A and F.sub.B) can be described by the following integral over time:

.sub.x=t.sub.d.sup.x=t(F.sub.A(x)+F.sub.B(x))dx=V.sub.tt

allowing us to determine a point in time, t.sub.d, where the actual mixing of solvent A and B took place.

[0065] That is, if the flow of A and B is described by continuous or stepwise continuous functions and the volume V.sub.tt is known, we can solve the integral and solve for t.sub.d for any given time, t (symbolically or numerically). Knowing t.sub.d as a function of t allow us to calculate the composition of the solvent mix A/B entering the second mixing point (MixTee2).

[00001]${\mathrm{Composition}}_{\mathrm{ABMix}.Math..Math.2}\left(t\right)={\mathrm{Composition}}_{\mathrm{ABMix}.Math..Math.1}\left(\mathrm{td}\left(t\right)\right)=\frac{F_B\left(\mathrm{td}\left(t\right)\right)}{F_A\left(\mathrm{td}\left(t\right)\right)+F_B\left(\mathrm{td}\left(t\right)\right)}$

[0066] Combined with a function for flow C, F.sub.C, the composition leaving the second mixing point can be described:

[00002]${\mathrm{Composition}}_{\mathrm{ABCMix}.Math..Math.2}\left(t\right)=\frac{\frac{F_B\left(\mathrm{td}\left(t\right)\right)}{F_A\left(\mathrm{td}\left(t\right)\right)+F_B\left(\mathrm{td}\left(t\right)\right)}.Math.\left(F_A\left(t\right)+F_B\left(t\right)\right)}{F_A\left(t\right)+F_B\left(t\right)+F_C\left(t\right)}$

[0067] Placing some simple restraints on the system allows us to find solutions to the above equations and generate the desired gradient (e.g. a linear gradient).

[0068] As a first solution:

[0069] The combined flow is at any t set to a constant: F.sub.out, and F.sub.C is set to a first order polynomial: F.sub.C(t)=.sub.C t+.sub.C. This allow us to first find t.sub.d(t) by substituting F.sub.A F.sub.B with F.sub.outF.sub.C:

.sub.t.sub.d.sup.t(F.sub.A(x)+F.sub.B(x))dx=V.sub.tt=V.sub.t.sub.d.sup.t(F.sub.outF.sub.C(t))dx

[0070] The relevant solution if .sub.C0 is:

[00003]$t_d=\frac{-\sqrt{\begin{array}{c}{}_c^2.Math.t^2+2.Math.{}_c.Math.{}_c.Math.t+2.Math.{}_c.Math..Math.F_{\mathrm{out}}.Math.t+\\ 2.Math.{}_c.Math.\mathrm{Vtt}+{}_c^2-2.Math.{}_c.Math..Math.F_{\mathrm{out}}+F_{\mathrm{out}}^2\end{array}}-{}_c+F_{\mathrm{out}}}{{}_c}$

[0071] The composition can be simplified to:

[00004]${\mathrm{Composition}}_{\mathrm{ABCMix}.Math..Math.2}\left(t\right)=C_o\left(t\right)=\frac{\frac{F_B\left(\mathrm{td}\left(t\right)\right)}{F_{\mathrm{out}}-F_C\left(\mathrm{td}\left(t\right)\right)}.Math.\left(F_{\mathrm{out}}-F_C\left(t\right)\right)}{F_{\mathrm{out}}}$

[0072] By defining F.sub.out, V.sub.tt and the C flow function (by defining the .sub.C and .sub.C), as well as the (linear) gradient, timetables for A, B and C may be found. The above equation solved for F.sub.B yields:

[00005]$F_B\left(\mathrm{td}\left(t\right)\right)=\frac{C_o\left(t\right).Math..Math.F_{\mathrm{out}}\left(F_{\mathrm{out}}-F_C\left(\mathrm{td}\left(t\right)\right)\right)}{\left(F_{\mathrm{out}}-F_C\left(t\right)\right)}$

[0073] As F.sub.C(t) is defined, F.sub.A(td(t)) is also known. F.sub.A(td(t))=F.sub.outF.sub.C(td(t))F.sub.B(td(t)). Timetables for the flows are thus found in the form of {F.sub.A(td(t)), F.sub.B(td(t)), F.sub.C(td(t))}

[0074] E.g. a total flow of 10 uL/min and a composition of 4% B at time 0 will dictate flow-rates for A, B, C for an earlier t.sub.d (e.g. 50 seconds earlier depending on how C is defined), which is the first timepoint in the timetable of A, B and C.

REFERENCES

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