Patent Application
20080092639
The present invention provides apparatus for controlling LC flow to optimize detection sensitivity and resolution by maintaining column pressure.
In some embodiments of the present invention, the pump (1) is of the type known to one of ordinary skill in the art. Any pump that can force a mobile phase through a separator (2) can be used.
In some embodiments of the present invention, the separator (2) is a liquid chromatography column, a high pressure liquid chromatography column, a capillary column, a nano liquid chromatography column, a reverse phase high pressure liquid chromatography column, or any subgroup thereof. The separator (2) is in fluid communication via connector (8) with the pump (1). An analyte to be separated can be present within the mobile phase within the pump (1) or can be delivered to the separator (2) via a sample injector (not shown). The separation of analyte components in the separator (2) is governed by van Deemter theory; for a column with particular dimensions and characteristics, an optimum flow rate can be predicted. Some embodiments of the present invention further comprise more than one separator (2). Separator (2) can be a stainless tube packed with fine particles (e.g., 5 μm in diameter) which have different surface characteristics (e.g., C18 chains) or any other commercially available column.
Referring to
Flow sensor (32) can be any sensor known to those skilled in the art including any commercially available sensor. The flow sensor (32) measures flow rates of the mobile phase and can be positioned as desired by the user of the system depending upon which flow rate is desired to be measured. In
In some embodiments, the apparatus further comprises a detector (4) in fluid communication via connector (10) with the flow restrictor (3). Referring to
In some embodiments, the apparatus further comprises a controller (5) in electronic communication via connectors (6), (7), (38), and (30) respectively, with the pump (1), flow restrictor (3), flow sensor (32), and optionally detector (4). In some embodiments, the controller (5) is a microprocessor and/or computer software.
According to some embodiments, the apparatus further comprises a stepper motor (31) attached to the flow restrictor (3), wherein the stepper motor (31) is optionally in electronic communication with the controller (5). In some embodiments, the stepper motor (31) is attached to the handle (not shown) of the flow restrictor (3). The stepper motor (31) activates or de-activates the flow restrictor (3). The fine adjustment of needle (11) is accomplished by the use of the stepper motor (31) which is programmed to adjust the opening (15).
The embodiments of the apparatus described above can be combined in any manner. Thus, features from one embodiment can be combined with features from any other embodiment. For example, the embodiments described above can be combined in a manner to produce an apparatus comprising: a pump (1); a separator (2) in fluid communication with the pump (1), wherein the separator (2) is a liquid chromatography column, a high pressure liquid chromatography column, a capillary column, a nano liquid chromatography column, or a reverse phase high pressure liquid chromatography column; a flow restrictor (3) in fluid communication with the separator (2), wherein the flow restrictor (3) is a needle valve comprising only one input port (12) and only one output port (13); a nanoflow sensor (32) in fluid communication with the flow restrictor; and a detector (4) in fluid communication with the flow restrictor (3), wherein the detector (4) is a mass spectrometer, a nuclear magnetic resonance detector, a radioactivity detector, an ultraviolet detector, or an electrochemical detector. Such an apparatus may further comprise a controller (5) in electronic communication with the pump (1), flow restrictor (3), flow sensor (32), and detector (4), wherein the controller (5) is a microprocessor and/or computer software. Such an apparatus may further comprise a stepper motor (31) attached to the flow restrictor (3), wherein the stepper motor (31) is in electronic communication with the controller (5).
The embodiments described above can also be combined in a manner to produce an apparatus comprising, for example: a pump (1); a separator (2) in fluid communication with the pump (1), wherein the separator (2) is a liquid chromatography column, a high pressure liquid chromatography column, a capillary column, a nano liquid chromatography column, or a reverse phase high pressure liquid chromatography column; a flow restrictor (3) in fluid communication with the separator (2), wherein the flow restrictor (3) is a needle valve comprising only one input port (12) and only one output port (13); a nanoflow sensor (32) in fluid communication with the flow restrictor; a detector (4) in fluid communication with the flow restrictor (3), wherein the detector (4) is a mass spectrometer, a nuclear magnetic resonance detector, a radioactivity detector, an ultraviolet detector, or an electrochemical detector; and a controller (5) in electronic communication with the pump (1), flow restrictor (3), flow sensor (32), and detector (4), wherein the controller (5) is a microprocessor and/or computer software. Such an apparatus may further comprise a stepper motor (31) attached to the flow restrictor (3), wherein the stepper motor (31) is in electronic communication with the controller (5).
The present invention also provides methods for detecting an analyte comprising: passing a mobile phase comprising the analyte to a separator (2) at a first pump flow rate; passing the mobile phase from the separator (2) to a flow restrictor (3), wherein the separator (2) is in fluid communication with the flow restrictor (3); passing the mobile phase from the flow restrictor (3) to a detector (4) at a first restrictor flow rate, wherein the detector (4) is in fluid communication with the flow restrictor (3); and decreasing the first restrictor flow rate to a second restrictor flow rate upon reaching a first detection event. The separator (2), flow restrictor (3), and detector (4) can be any of those described herein.
In some embodiments, the mobile phase is a scintillation fluid, an organic solvent, or the like. The analyte(s) can be present in the mobile phase or can be introduced into the mobile phase by a sample injector prior to being passed to the separator (2). In addition, the analyte(s) being detected can be a single analyte or a plurality of analytes. Further, the analyte can be any molecule, compound, ion, or the like whose detection is sought.
The mobile phase comprising the analyte is passed to a separator (2) at a first pump flow rate from a pump (1). The first pump rate can be from about 0.1 ml/min to about 10 ml/min, from about 0.5 ml/min to about 5 ml/min, from about 1 ml/min to about 2 ml/min, or about 1 ml/min. As used in this context, the term “about” means±5% of the value it modifies. The mobile phase is then passed from the separator (2) to a flow restrictor (3), wherein the separator (2) is in fluid communication with the flow restrictor (3), as described above.
The mobile phase is then passed from the flow restrictor (3) to a detector (4) at a first restrictor flow rate, wherein the detector (4) is in fluid communication with the flow restrictor (3), as described above. The first restrictor flow rate can be from about 0.1 ml/min to about 10 ml/min, from about 0.5 ml/min to about 5 m/min, from about 1 ml/min to about 2 ml/min, or about 1 ml/min. As used in this context, the term “about” means±5% of the value it modifies. The second restrictor flow rate is measured by a flow sensor (32) located in an appropriate position. Flow sensors can be generally located throughout the apparatus to measure particular desired flow rates.
Upon reaching a first detection event, the first restrictor flow rate is decreased to a second restrictor flow rate. In some embodiments, the second restrictor flow rate is less than about 25%, less than about 10%, less than about 1%, less than about 0.1%, or less than about 0.01% of the first restriction flow rate. As used in this context, the term “about” means±5% of the value it modifies. In some embodiments, the first detection event is a first predetermined time, detection of the beginning of a peak from the analyte by the detector (4), or a user-determined manual adjustment. The first predetermined time can be any preset time after beginning the chromatography run desired by the user. The user-determined manual adjustment can be any point during the chromatography run at which the user desires to decrease the first restrictor flow rate. The beginning of the peak from the analyte can be determined by reaching a preset threshold (such as a particular absorbance value) or by reaching a particular selected slope of, for example, the absorbance vs. time.
According to some embodiments, decreasing the first restrictor flow rate to the second restrictor flow rate upon reaching the first detection event is affected by activation of the flow restrictor (3). In some embodiments, upon reaching the first detection event, the detector (4) signals a controller (5), which is in electronic communication with the detector (4) and flow restrictor (3), as described above, whereby the controller (5) signals the flow restrictor (3) to decrease the first restrictor flow rate to the second restrictor flow rate. The controller (5) can be any controller described above. In addition, the flow restrictor (3) can be optionally attached to a stepper motor (31), as described above. Activation of the flow restrictor (3), as used herein, does not necessarily mean complete activation whereby the first restrictor flow rate is decreased to zero.
In some embodiments, in order to provide a stable low flow rate, the opening of the flow restrictor (3) can be calibrated before use based on the pressure of pump (1) and flow rate. For example, the restrictor opening (15) can be adjusted while monitoring the target pump pressure (e.g., 100 bar) with the pump flow rate set to the target slow flow rate (e.g., 5 μl/min). The target pressure is the pump pressure under normal flow mode (e.g., 1 ml/min). When the target pressure is maintained, the setting of opening (15) is calibrated. This calibrated restrictor setting then can be used for slow flow mode. However, sometimes the opening (15) might change over time. This problem is solved by on-line adjustment of opening (15) while maintaining the target pressure with the calibrated pump slow flow rate. This method effectively enables the accurate and stable target slow flow rate under slow flow mode. The advantage of this on-line adjustment is that the pump (1) can deliver a much lower flow rate comparing to its normal flow rate range. One skilled in the art can measure the actual flow rate by adjusting the flow rate settings of pump (1) to accurately calibrate the system. For example, a pump (1) with a flow rate range of 1-5000 μl/min can be calibrated and deliver 100 nl/min flowrate by turning the pump (1) on and off in pre-determined periods of duration while maintaining the pump pressure.
In some embodiments, an amount of eluant from the separator (2) is diverted to a second separator (not shown). The eluant from the separator (2) can optionally be diluted with a second mobile phase prior to diverting the eluant to the second separator. The second mobile phase can be identical to or different from the mobile phase passing into the separator (2). In some embodiments, the second separator is a 2-dimensional high pressure liquid chromatography column. In some embodiments, diversion of an amount of eluant to the second separator is initiated by the detector (4) signaling to a controller (5), which is in electronic communication with the detector (4), separator (2), and second separator, upon reaching the first detection event.
In some embodiments, the methods further comprise decreasing the first pump flow rate of the mobile phase to the separator (2) to a second pump flow rate when the first detection event is reached. In some embodiments, a pump (1) is in fluid communication with the separator (2) and wherein upon reaching the first detection event, the detector (4) signals a controller (5), which is in electronic communication with the detector (4), flow restrictor (3), and pump (1), whereby the controller (5) signals the flow restrictor (3) to decrease the first restrictor flow rate to the second restrictor flow rate, and whereby the controller (5) also signals the pump (1) to decrease the first pump flow rate to the second pump flow rate. According to some embodiments, decreasing the first restrictor flow rate of the mobile phase containing the eluant to the second restrictor flow rate upon reaching the first detection event occurs simultaneously with, or ±0 to 5 seconds, or ±0 to 3 seconds, or ±0 to 1 second of decreasing the first pump flow rate of the mobile phase to the separator (2) to the second pump flow rate.
In some embodiments, the pressure in the separator (2) remains substantially steady after the flow restrictor (3) decreases the first restrictor flow rate to the second restrictor flow rate and the pump (1) decreases the first pump flow rate to the second pump flow rate. Pressure drop in the separator (2) depends on the flow rate of the system. Further, maintaining separator pressure preserves the separation of peaks inside the separator (2).
In some embodiments, after the first restrictor flow rate is decreased to the second restrictor flow rate and the first pump rate is optionally lowered to the second pump rate, the methods further comprise increasing the second restrictor flow rate back to about the first restrictor flow rate upon reaching a second detection event. As used in this context, the term “about” means less than 50% to greater than 200% of the first restrictor flow rate. For example, if the first restrictor flow rate is 1 ml/min, which was decreased to a second restrictor flow rate of 100 μl/min, then the flow rate could be increased to between 500 μl/min and 2 ml/min. In some embodiments, the second detection event is a second predetermined time, detection of the end of a peak from the analyte by the detector (4), or a user-determined manual adjustment. The second predetermined time can be any preset time after beginning the chromatography run desired by the user. The user-determined manual adjustment can be any point during the chromatography run at which the user desires to increase the second restrictor flow rate back to about the first restrictor flow rate. The end of the peak from the analyte can be determined by reaching a preset threshold (such as a particular absorbance value) or by reaching a particular selected slope of, for example, the absorbance vs. time.
In some embodiments, increasing the second restrictor flow rate back to about the first restrictor flow rate upon reaching the second detection event is affected by de-activation of the flow restrictor (3). In some embodiments, upon reaching the second detection event, the detector (4) signals a controller (5), which is in electronic communication with the detector (4) and flow restrictor (3), whereby the controller (5) signals the flow restrictor to increase the second restrictor flow rate back to about the first restrictor flow rate. The controller (5) can be any controller described above. In addition, the flow restrictor (3) can be optionally attached to a stepper motor (31), as described above. De-activation of the flow restrictor (3), as used herein, does not necessarily mean complete de-activation whereby the second restrictor flow rate is increased to a maximum flow rate.
In some embodiments, the methods further comprise increasing the second pump flow rate of the mobile phase to the separator (2) back to about the first pump flow rate. As used in this context, the term “about” means less than 50% to greater than 200% of the first pump rate. For example, if the first pump flow rate is 1 ml/min, which was decreased to a second pump flow rate of 100 μl/min, then the flow rate could be increased to between 500 μl/min and 2 ml/min. In some embodiments, a pump (1) is in fluid communication with the separator (2) and comprises the mobile phase for passing into the separator (2), and upon reaching the second detection event, the detector (4) signals a controller (5), which is in electronic communication with the detector (4), flow restrictor (3), and pump (1), whereby the controller (5) signals the flow restrictor (3) to increase the second flow restrictor flow rate back to about the first restrictor flow rate, and whereby the controller (5) also signals the pump (1) to increase the second pump flow rate back to about the first pump flow rate.
According to some embodiments, increasing the second restrictor flow rate back to about the first restrictor flow rate upon reaching the second detection event occurs simultaneously with, or ±0 to 5 seconds, or ±0 to 3 seconds, or ±0 to 1 second of increasing the second pump flow rate of the mobile phase to the separator (2) back to about the first pump flow rate.
The effects of the methods described herein are demonstrated in
The embodiments of the methods described above can be combined in any manner. Thus, features from one embodiment can be combined with features from any other embodiment. For example, the embodiments described above can be combined in a manner to produce methods for detecting an analyte comprising: passing a mobile phase comprising the analyte to a separator (2) at a first pump flow rate, wherein the separator (2) is a liquid chromatography column, a high pressure liquid chromatography column, a capillary column, a nano liquid chromatography column, or a reverse phase high pressure liquid chromatography column; passing the mobile phase from the separator (2) to a flow restrictor (3), wherein the separator (2) is in fluid communication with the flow restrictor (3), and wherein the flow restrictor (3) is a needle valve; passing the mobile phase from the flow restrictor (3) to a detector (4) at a first restrictor flow rate, wherein the detector (4) is in fluid communication with the flow restrictor (3), and wherein the detector (4) is a mass spectrometer, a nuclear magnetic resonance detector, a radioactivity detector, an ultraviolet detector, or an electrochemical detector; decreasing the first restrictor flow rate to a second restrictor flow rate upon reaching a first detection event, wherein the second restriction flow rate is less than about 25% of the first restriction flow rate; decreasing the first pump flow rate of the mobile phase to the separator (2) to a second pump flow rate; increasing the second restrictor flow rate back to about the first restrictor flow rate upon reaching a second detection event; and increasing the second pump flow rate of the mobile phase to the separator (2) back to about the first pump flow rate.
The embodiments described above can be combined in a manner to produce methods for detecting an analyte comprising: passing a mobile phase comprising the analyte to a separator (2) at a first pump flow rate, wherein the separator (2) is a liquid chromatography column, a high pressure liquid chromatography column, a capillary column, a nano liquid chromatography column, or a reverse phase high pressure liquid chromatography column; passing the mobile phase from the separator (2) to a flow restrictor (3), wherein the separator (2) is in fluid communication with the flow restrictor (3), and wherein the flow restrictor (3) is a needle valve; passing the mobile phase from the flow restrictor (3) to a detector (4) at a first restrictor flow rate, wherein the detector (4) is in fluid communication with the flow restrictor (3), and wherein the detector (4) is a mass spectrometer, a nuclear magnetic resonance detector, a radioactivity detector, an ultraviolet detector, or an electrochemical detector; upon reaching a first detection event, the detector (4) signals a controller (5), which is in electronic communication with the detector (4) and flow restrictor (3), whereby the controller (5) signals the flow restrictor (3) to decrease the first restrictor flow rate to the second restrictor flow rate, wherein the second restriction flow rate is less than about 10% of the first restriction flow rate, and also upon reaching the first detection event, the detector (4) simultaneously signals the controller (5), which is also in electronic communication with a pump (1), which is in fluid communication with the separator (2), whereby the controller (5) also signals the pump (1) to decrease the first pump flow rate to the second pump flow rate, wherein the first detection event is a first predetermined time, detection of the beginning of a peak from the analyte by the detector, or a user determined manual adjustment, and wherein the controller (5) is a microprocessor and/or computer software; upon reaching a second detection event, the detector (4) signals the controller (5) whereby the controller (5) signals the flow restrictor (3) to increase the second restrictor flow rate back to about the first restrictor flow rate, and whereby the controller (5) signals the pump (1) to increase the second pump flow rate back to about the first pump flow rate, wherein the second detection event is a second predetermined time, detection of the end of a peak from the analyte by the detector (4), or a user determined manual adjustment.
It should be understood that the embodiments and drawings described herein are only some examples of the apparatus and methods described herein and are not to be construed as limiting the invention in any manner. In addition, various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
