Systems and methods for electrospray using chromatographic columns with conductive or semiconductive stationary phases

Abstract

To provide systems, methods and cartridges for electrospray realizing improved chromatographic performance.SOLUTION: Systems, methods and cartridges improve chromatographic performance in electrospray systems that feature chromatographic columns having conductive or semiconductive stationary phases by electrically connecting a fluid junction located upstream of the chromatographic column to a fluid union located downstream of the chromatographic column using an electrical conductor. The electrical conductor creates a voltage equipotential between a first end of the chromatographic column and a second end of the chromatographic column that neutralizes a current flow through the chromatographic column. Accurate electrospray current measurement is enabled while post-column peak dispersion and repeatable retention time are achieved.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a system and method for electrospray using a chromatography column having a conductive or semiconductive stationary phase. [Background technology]

[0002] Electrospray ionization (ESI) is a technique used in mass spectrometry (MS). More specifically, ESI is a "soft" ionization technique that is widely used to generate gas-phase ions with low levels of fragmentation. In a typical ESI process, a high voltage is applied to a liquid to generate charged droplets, which are desolvated to form gas-phase ions. Low-flow electrospray, or "NanoESI," generates charged droplets from an emitter with a relatively small inner diameter (e.g., about 20 micrometers or less). The combination of low flow rate and small diameter results in increased ionization efficiency (i.e., an increase in the ratio of gas-phase ions generated per available analyte molecule in solution), allowing for a reduction in the amount of sample required. When this emitter is placed near the mass spectrometer inlet, which acts as a counter electrode, the generated gas-phase ions are sampled into the instrument for mass analysis.

[0003] ESI is commonly used to couple the output of a chromatography column or system to mass spectrometry (MS). Chromatographic techniques, such as liquid chromatography (LC), can be used to generate temporal separation between molecules in solution. The eluent can be fed into an electrospray emitter and serve as the input for a mass spectrometer. Summary of the Invention

[0004] Provided herein is an electrospray system. The electrospray system includes a first pre-column fluidic junction for receiving a fluid sample. The electrospray system includes a chromatography column for separating analytes in the fluid sample. The chromatography column includes a conductive or semiconductive stationary phase disposed between a first end and a second end of the chromatography column. The first end is fluidly connected to the first pre-column fluidic junction. The electrospray system includes a post-column fluidic junction having a first fluidic port and a second fluidic port. The first fluidic port receives the separated analytes in the fluid sample from the second end of the chromatography column. The electrospray system includes an electrospray emitter fluidly connected to the second fluidic port. The electrospray system includes an electrical conductor connected to the post-column fluidic junction and the first pre-column fluidic junction to equalize an electrical potential between the post-column fluidic junction and the first pre-column fluidic junction.

[0005] Provided herein is a method for reducing electrochromatographic effects in an electrospray system. The method includes flowing a fluid sample from a first pre-column fluidic junction to a first end of a chromatography column. The chromatography column includes a conductive or semiconductive stationary phase disposed between a first end and a second end of the chromatography column. The method includes separating analytes in the fluid sample using the chromatography column. The method includes outputting the separated fluid sample from a second end of the chromatography column to a first fluidic port of a post-column fluidic junction. The post-column fluidic junction has a second fluidic port connected to an electrospray emitter. The method includes applying an equipotential voltage in the range of +1 to +10 kilovolts or in the range of -1 to -10 kilovolts to both the first pre-column fluidic junction and the post-column fluidic junction using electrical conductors in electrical contact with the first pre-column fluidic junction and the post-column fluidic junction.

[0006] Provided herein is a cartridge for insertion into an electrospray system. The cartridge comprises a first pre-column fluidic junction for receiving a fluid sample. The cartridge comprises a chromatography column for separating analytes in the fluid sample. The chromatography column includes a conductive or semiconductive stationary phase disposed between a first end and a second end of the chromatography column. The first end is fluidly connected to the first pre-column fluidic junction. The cartridge comprises a post-column fluidic junction having a first fluidic port and a second fluidic port. The first fluidic port receives separated analytes in the fluid sample from the second end of the chromatography column. The cartridge comprises an electrospray emitter fluidly connected to the second fluidic port. The cartridge comprises an electrical conductor connected to the post-column fluidic junction and the first pre-column fluidic junction to equalize an electrical potential between the post-column fluidic junction and the first pre-column fluidic junction. [Brief description of the drawings]

[0007] It should be understood that the figures are not necessarily drawn to scale, and that objects within the figures are not necessarily drawn to scale in relationship to each other. The figures are representations intended to provide clarity and understanding to various embodiments of the devices, systems, and methods disclosed herein. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or similar parts. Furthermore, it should be understood that the drawings are not intended to limit the scope of the present teachings in any way. [Figure 1] FIG. 1 is a schematic diagram of a conventional electrospray system comprising a chromatography column having a non-conductive stationary phase. [Diagram 2] 1 is a schematic diagram of an electrospray system having a chromatography column with a conductive or semiconductive stationary phase according to some embodiments taught herein. [Diagram 3]FIG. 2 is a schematic diagram of an alternative electrospray system having a chromatography column with a conductive or semiconductive stationary phase according to some embodiments taught herein. [Figure 4] 1 is a graph of retention times of certain analytes in a chromatography column having a semiconducting stationary phase according to some embodiments taught herein for a series of replicate experiments under different conditions. [Diagram 5] FIG. 2 is a schematic diagram of a current sensing circuit for use with embodiments of the systems, methods, and cartridges taught herein. [Figure 6] FIG. 1 is a schematic diagram of a current sensing circuit with isolation suitable for use in some embodiments taught herein. [Figure 7] FIG. 1 illustrates a routine for reducing electrochromatographic effects in an electrospray system, according to various embodiments taught herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] The systems, methods, and cartridges taught herein improve chromatographic performance in electrospray systems featuring chromatographic columns having stationary phase(s) formed from conductive or semiconductive materials or substrates. In conventional electrospray systems, the electrical setup allows leakage current to pass through the eluent. Such configurations are not suitable for use with chromatographic columns having conductive or semiconductive stationary phases because the current flowing through such conductive columns causes the columns to charge over time, which then creates detrimental interactions with the analytes during analyte separation in the column. The systems, methods, and cartridges taught herein overcome this problem by using electrical conductors to electrically connect a fluidic junction located upstream of the chromatographic column to a fluidic coupling located downstream of the chromatographic column. The electrical conductor creates a high voltage equipotential between a first end of the chromatographic column and a second end of the chromatographic column, which limits or eliminates the current flow through the chromatographic column.

[0009] The systems, methods, and cartridges taught herein also enable measurement of electrospray current in systems using chromatographic columns with conductive or semiconductive stationary phases while maintaining high chromatographic performance. In conventional systems, a current monitoring circuit is connected between a power source and an electrospray emitter such that slightly different voltages are applied to the ends (i.e., inlet and outlet) of the chromatographic column. In the systems, methods, and cartridges taught herein, the conductors connecting the fluidic junctions and fluidic couplings are connected in series with the current monitoring circuit. Thus, although no current flows through the chromatographic column with conductive or semiconductive stationary phase, accurate measurement of the electrospray current is still possible.

[0010] According to some embodiments taught herein, the electrospray systems, methods, and cartridges taught herein allow for improved chromatographic performance by providing a fluidic junction as the only post-column fluidic connection. The chromatographic column provides a temporal separation of analytes in solution, and the analyte bands travel to the electrospray emitter. Each fluidic connection that the analyte band passes through on its way to the electrospray emitter can generate disturbances (e.g., flow vortices) to the laminar fluid flow. In addition, a non-swept (no flow) volume is created at each fluidic connection through which the analyte diffuses (passive transport). As this is a much slower process than active transport, a portion of the analyte plug or band lags behind the bulk of the plug, causing a "tail plug". These processes affect the quality of the separation, as they result in the dispersion of the analyte band, or so-called "band broadening". In the systems, methods, and cartridges taught herein, the use of electrical conductors allows for true equipotential between the ends of the columns while using only a single post-column fluidic connection (i.e., fluidic junction). Thus, the systems and methods according to some embodiments herein can improve chromatographic performance (e.g., reduce peak broadening and modulate retention times compared to conventional systems) by avoiding charging of conductive or semiconductive stationary phases in chromatographic columns and limiting the number of post-column fluidic connections to one.

[0011] Accurate current-voltage curves (i.e., the current through the electrospray emitter as a function of applied voltage) are important for characterizing, diagnosing, and optimizing electrospray systems. Such curves help distinguish between the various electrospray regimes that largely govern the sensitivity of the instrument. In the first electrospray regime, the electrospray voltage (V E) is below the threshold, such that the emitter does not emit a spray or droplet fluid. For example, the first electrospray regime can occur in some embodiments at less than 1200 volts. The second electrospray regime occurs when the applied electrospray voltage (V E ) is sufficient to cause electrospray within a stable spindle regime where charged droplets form directly from a filament extending from the Taylor cone. For example, the second regime may occur when the electrospray voltage is in the range of 1200-2200 volts. In the third regime, the applied electrospray voltage results in a cone jet where severing of the filament is assisted by Coulomb repulsion, thereby generating a diffuse plume of charged droplets. For example, the third regime may occur when the electrospray voltage exceeds 2200 volts. The three electrospray regimes are characterized by an abrupt change in slope on a graph of spray current as a function of applied voltage. The embodiments of the present disclosure allow for accurate determination of spray current as a function of applied voltage, and thus, the creation of such a graph. On an I-V graph, the inverse of the slope (i.e., 1 / slope) represents the effective impedance for a given parameter such as flow rate, tip diameter, fluid composition, etc. This knowledge is important for at least set-up, spray optimization, and diagnostics; other methods for obtaining effective impedance (including processing images of the electrospray plume) are complex and time consuming. Thus, the embodiments taught herein can enable spray improvement or optimization by direct measurement of electrical properties without complex imaging algorithms or associated techniques.

[0012] Additionally, changes in the current-voltage characteristics of the electrospray system or cartridge are indicative of the health of the system or cartridge. Thus, monitoring these characteristics with a controller can provide a diagnostic tool for assessing the health of the system or cartridge. One advantage of the electrical configuration taught in the embodiments described herein is that the improved fidelity of the current measurement readback allows for direct measurement and software automation of electrospray system health and operational information that would otherwise have to be obtained from optical inspection equipment of the ESI plume.

[0013] However, conventional systems and methods for measuring electrospray current have significant drawbacks, especially for systems with chromatographic columns having conductive or semiconductive stationary phases. Off-the-shelf power supplies are most often equipped with current and voltage monitor outputs, but only report the total load current. In reality, there are unknown leakage or spurious paths of current generated by the conductive fluid flow itself and by conductive elements in the fluid path. To obtain an accurate measurement of the ESI current, other current flows must be ignored or subtracted from the total. This requires a separate and independent measurement of the spray current, which is typically not available from high voltage power supply manufacturers. Other loads, whether intentional or unintentional, must also be considered. The problem is further complicated when considering that these extremely small currents (e.g., requiring accuracy of better than 10 nanoamps) are measured in the presence of extremely high common mode voltages. For ±8 kilovolt power supplies, the measurement circuitry typically uses a minimum common mode rejection of approximately 100 decibels or more. In addition, tolerance matching of high voltage and high ohmic resistors is required with an accuracy of better than 0.01%.

[0014] The systems, methods, and cartridges taught herein enable accurate measurement of electrospray current in an electrospray system comprising a chromatography column having a conductive or semiconductive stationary phase by using electrical conductors in series with a current sensing circuit to generate an equipotential voltage between a pre-column fluidic junction located at a first end of the chromatography column and a post-column fluidic junction located at a second end of the chromatography column.

[0015] FIG. 1 shows a schematic diagram of a prior art electrospray system 100 comprising a chromatographic column 106 having a non-conductive stationary phase (e.g., packed polymer or glass beads). In FIGS. 1, 2, and 3 of this document, dashed connections between components indicate fluidic paths, and solid connections between elements indicate electrical paths. The prior art electrospray system 100 comprises a fluidic junction 102, a chromatographic column 106 having a non-conductive stationary phase, a post-column fluidic coupling 104, a post-column fluidic junction 122, and an electrospray emitter 114. A fluid sample is provided to the electrospray system 100 from a liquid chromatography system 108 via the fluidic junction 102. The fluid sample travels from the pre-column fluidic junction 102 through the chromatographic column 106. The chromatographic column 106 separates analytes in the fluid sample in time and outputs a separated fluid sample (i.e., a fluid sample containing separated analytes). The separated fluid sample flows to a post-column fluidic junction 122 and then to the post-column fluidic coupling 104 before being emitted through the electrospray emitter 114. The power supply 110 is connected to the fluidic junction 102 and to a high side 118 of a current sensing circuit 116. A low side 120 of the current sensing circuit 116 is connected to the fluidic junction 104 and to the post-column fluidic junction 122. When a sufficient voltage is applied to the fluidic junction 104, the fluid at the electrospray emitter 114 is ionized, forming a stream of droplets that are propelled from the electrospray emitter 114 to the counter electrode 112. The counter electrode 112 and the negative terminal are connected to a common ground (which may in some cases be earth ground). Thus, the stream of ionized droplets completes an electrical circuit and produces a measurable current.

[0016] In some embodiments, the counter electrode 112 can be floated at an offset voltage referenced to ground, where the offset voltage is small (less than 200 V) relative to the electrospray voltage from the power supply 110. In some embodiments, the atmosphere-to-vacuum inlet of the mass analyzer can serve as the counter electrode 112. In embodiments that include a small floating offset voltage, another ion optical element in the mass analyzer can be held at ground (e.g., a c-trap), thereby allowing ions to be guided by ion optical elements that are held at progressively lower (or higher, depending on their polarity) voltages.

[0017] 1, a current sensing circuit 116 is placed in series with the fluid coupling 104 and the post-column fluidic junction 122 and measures the current flowing from the power source 110 to the fluid coupling 104. Conventional electrospray systems 100 attempt to divert leakage current from the measurement path of the current sensing circuit 116 by applying a common voltage to the fluid coupling 104 and the post-column fluidic junction 122 while a high voltage is applied to the fluidic junction 102. In some cases, the fluidic junction 102 is coupled to a voltage V + is not applied. If no voltage is applied to the pre-column fluid junction 102, leakage current occurs when current flows through the conductive fluid stream to the grounded liquid chromatography system 108. In this application, "leakage current" refers to current from the power supply that does not travel to the electrospray emitter and counter electrode, but rather travels along a separate path away from the emitter to ground (e.g., along a path through the fluid sample, upstream of the chromatography column and to the grounded liquid chromatography system 108). Leakage current is measured by a current sensing circuit, but if it does not travel through the electrospray emitter to ground, it can prevent accurate measurement of the electrospray current.

[0018] Voltage V + is the voltage V applied to the fluid junction E Since the voltage V at the fluid junction 102 is not equal to +Application of V is insufficient to eliminate leakage current across the chromatography column 106. This difference may arise due to a non-zero voltage drop across the current sensing circuit 116 and other factors such as different contact resistances or material compositions of the fluid junctions or couplings. Due to the very high voltages involved, V + and V E Even a small difference between the voltage V and the voltage V can allow leakage currents on the order of the spray current. + If the fluid junction 102 is removed, the same problem of current leakage occurs because current leaks through the grounded liquid chromatography system 108. However, these conventional systems include chromatography columns with non-conductive stationary phases. Because non-conductive stationary phases operate similarly with or without the current through the voltage drop, a substantial voltage drop (up to several thousand volts) can exist across such a chromatography column 106 without affecting the performance of the chromatographic separation.

[0019] In other conventional schemes, the pre-column fluidic junction 102 and the post-column fluidic junction 122 are held at ground potential (i.e., substantially zero volts). In such a scheme, the stationary phase of the chromatography column 106 is not subjected to an electric current. However, such a configuration is not preferred because the current sensing circuit 116 measures both the electrospray current and the leakage current that occurs between the post-column fluidic junction 104 and the post-column fluidic junction 122, and therefore the current sensing circuit 116 cannot be used to accurately measure only the electrospray current.

[0020] The setup of FIG. 1 creates problems in the use of chromatographic columns with conductive or semiconductive stationary phases. Leakage currents through chromatographic columns with conductive or semiconductive stationary phases can induce charges that affect the flow and retention times of analytes in the column, as taught in more detail below with respect to FIG. 4. Performance degradation can occur even with small voltage drops across the chromatographic column (e.g., voltage drops on the order of 10 V). In addition, the presence of two liquid-metal interfaces (i.e., post-column liquid junction 122 and fluid coupling 104) creates two locations where flow disturbances can impair the quality of analyte separation in the separated fluid sample leaving the chromatographic column, as each liquid-metal interface contributes to the dispersion of analyte bands, or so-called "band broadening."

[0021] The systems, methods, and cartridges taught herein alleviate these problems of leakage currents and band broadening by creating a true non-zero voltage equipotential (e.g., in the range of +1 kV to +10 kV or in the range of -1 kV to -10 kV) between the ends of a chromatography column having a conductive or semiconductive stationary phase. In addition, the systems taught herein reduce the number of post-column fluidic junctions (e.g., fluid-metal interfaces) from two to one, thereby mitigating band broadening effects.

[0022] 2 shows a schematic diagram of an electrospray system 200 having a chromatography column 302 with a conductive or semiconductive stationary phase according to some embodiments taught herein. In FIG. 2, dashed connections between components indicate fluidic connections and solid connections between components indicate electrical connections. The fluidic components of the electrospray system 200 include a pre-column fluidic junction 206 (which in some cases may be a septum fitting or other fluidic system inlet or interface), a pre-column fluidic junction 204 (which in some cases may be a needle seat or fluidic cartridge inlet or interface), a pre-column fluidic junction 304 (which in some cases may be an intra-cartridge fluidic junction), a chromatography column 302 with a conductive or semiconductive stationary phase, a post-column fluidic coupling 306, and an electrospray emitter 308. The electrospray system 200 can also include electrical components such as a power source 202, a current sensing circuit 210, and electrical conductors for electrically connecting the components to each other. In particular, electrical conductor 314 connects fluid coupling 306 and fluid junction 304 to equalize the electrical potential between fluid coupling 306 and fluid junction 304. The equal electrical potential (e.g., the high voltage potential V E ) to both the fluid coupling 306 and the fluid junction 304, the possibility of current leakage and electrochemical interactions in the chromatography column 302 is further reduced or eliminated while at the same time allowing the chromatography column 302 to float at a high potential.

[0023] The fluid sample enters the electrospray system 200 through a pre-column liquid junction 206. The source of the fluid sample can be an external reservoir or an external liquid chromatography system 108. In some examples, the external liquid chromatography system 108 is responsible for delivering the solvent, loading the sample, and running the gradient. The fluid sample flows along a fluid path 218 to the pre-column fluid junction 204. The fluid sample then flows along a fluid path 220 to a fluid junction 304. In embodiments where several components are assembled into a cartridge format (described in more detail below), this is the point at which the fluid sample enters the cartridge. The fluid sample flows from the fluid junction 304 along a fluid path 222 to a first end 318 of a chromatography column 302. In the chromatography column 302, the analytes of the fluid sample are separated in time within the fluid sample such that a separated fluid sample (i.e., a fluid sample containing separated analytes) is output from a second end 320 of the chromatography column 302. For example, the chromatography column 302 can be configured, for example, for liquid chromatography (LC) to separate molecules in a liquid mobile phase. The separated fluid sample is eluted from the chromatography column and travels along the fluid path 224 to a first fluid port 310 of the fluid junction 306. The fluid sample is induced by a high potential (V E ) into the mass spectrometer. The electrospray emitter 308 is connected to a second fluid port 312 of the fluid coupling 306. The ionized fluid stream is emitted from the electrospray emitter 308 as ionized droplets. For example, the electrospray emitter 308 can comprise a needle, and the droplets can be emitted through the distal tip of the needle. In some embodiments, the electrospray emitter 308 has an inner diameter of less than about 30 micrometers to enable nanoESI. In some embodiments, the emitted ionized droplets leave the electrospray emitter 308 and travel toward an atmospheric pressure inlet to the mass spectrometer, which functions as the counter electrode 112. The electrospray current return is generally formed by the electrically grounded counter electrode 112, as shown in FIG. 2.

[0024] The power supply 202 is electrically connected to the high side 212 of the current sensing circuit 210 to measure the electrospray current resulting from the flow of ionized droplets between the electrospray emitter 308 and the counter electrode 112. In some embodiments, the power supply 202 outputs a high voltage in the range of ±1 kV to ±10 kV. For example, the power supply 202 can output a voltage of ±2 kV or ±8 kV. The low side 214 of the current sensing circuit 210 is connected to a conductor 314 that further connects to the fluid junction 304 and the fluid coupling 306. In other words, the fluid junction 304 and the fluid coupling 306 are electrically connected in parallel with each other via the conductor 314, and this parallel circuit is connected in series with the current sensing circuit 210. Thus, the conductor 314 creates a true equipotential between the fluid junction 304 and the fluid coupling 306 since there is no intermediate element (such as the current sensing circuit 210) that creates a potential difference between them. In essence, the two pre-column fluid junctions 206 and 208 form a pre-column voltage spacer that directs current around the chromatographic column to prevent it from flowing through the chromatographic column. As a result of using the pre-column voltage spacer, most of the voltage drop occurs along the fluid line 218 while a small amount of voltage drops across the fluid line 220. The use of the pre-column voltage spacer can be contrasted with the post-column voltage spacer configuration in the conventional electrospray system 100 of FIG. 1, where the improvement in electrospray current measurement is accompanied by a small voltage drop across the conductive or semiconductive stationary phase of the chromatographic column, which can lead to undesirable charging effects that affect retention times. The post-column voltage spacer of FIG. 1 also negatively induces band broadening due to the presence of two liquid-metal interfaces.

[0025] In some embodiments, the leads of the electrical conductors 314 are connected directly to an outer conductive surface of the fluid junction 306 to provide sufficient current to the fluid stream to provide ionization therein. In some embodiments, the leads of the electrical conductors 314 are connected directly to an outer conductive surface of the fluid junction 304. In some embodiments, the electrical conductors 314 may comprise a wire or cable and may comprise a shield or be bare conductors.

[0026] It should be noted that power supply 202 may be provided with electrospray system 200 as a combined system in some embodiments. In other embodiments, power supply 202 is not provided as part of the system, but rather, electrospray system 200 is designed to interface with a separately provided power supply 202. In some embodiments, the polarity of power supply 202 is reversed such that high voltage is applied to counter electrode 112 and the return voltage lead of power supply 202 is connected to current sensing circuit 210.

[0027] NanoESI plumes typically carry currents of 50-500 nanoamps. Thus, because nanoESI produces relatively small ion currents, resulting in higher sensitivity, it is highly desirable to measure the electrospray current with high precision to ensure that the electrospray emitter 308 is operating in the correct electrospray regime, and measurements are made from these small ion currents down to a measurement resolution of 10 nanoamps or better. A voltage drop occurs across the current sense circuit 210, so that the voltage V at the high side 212 + is the voltage V at the low side 214 E Does not match the voltage V E is applied in parallel to the fluid coupling 306 and the fluid junction 304. In some embodiments, the electrical connector goes through a high voltage electrical connector 216, which may be attached to the housing of the cartridge 208, for example.

[0028] The liquid chromatography system 108, the pre-column fluid junction 206 (e.g., system inlet), the counter electrode 112, and the negative terminal of the power supply 202 are connected to electrical ground. It should be understood that "electrically grounded" as used herein can also include a potential operating at or near electrical ground, or a substantial electrical ground. For example, in some instances, the electrical ground may be biased to + / - 150 volts, which represents a substantially grounded source as compared to the common mode voltages of + / - 2 to 10 kilovolts taught herein.

[0029] The chromatography column 302 comprises a conductive or semiconductive material or substrate that forms and / or supports a stationary phase. For example, the stationary phase of the chromatography column 302 may include (or may be formed from) silicon, gallium arsenide, gallium nitride, indium tin oxide, n-type or p-type semiconductor materials, or doped semiconductors. In other embodiments, the stationary phase of the chromatography column 302 may include a conductive metal, such as platinum or stainless steel. In some embodiments, the stationary phase of the chromatography column 302 includes or is supported by a conductive or semiconductive substrate disposed between a first end 318 and a second end 320 of the chromatography column 302. In some embodiments, the chromatography column 302 may include a stationary phase of 1×10 -2 Ω cm~1×10 2 They can have resistivities in the Ω·cm range.

[0030] In some embodiments, the chromatography column 302 can include microfabricated structural features, such as pillars, that extend from a substrate within the microfluidic flow channel. The microfluidic structural features can include conductive or semiconductive materials, such as silicon, and can be produced using micro- or nanofabrication techniques known to those skilled in the art, including, for example, using photo- or e-beam lithography and deep reactive ion etching. The pillars form the stationary phase of the chromatography column. In some examples, the pillars or other microfluidic structural features are formed from a silicon substrate by etching voids surrounding the pillars. Examples of devices suitable for use as chromatography columns in embodiments taught herein are described in U.S. Patent Application Publication No. 2016 / 0001199, the entire contents of which are incorporated herein by reference. In some embodiments, the microfluidic column consists of a rectangular channel etched into a microfluidic chip (e.g., a semiconductor chip or substrate) using deep reactive etching techniques. Certain features of the electrospray system 300, such as the pre-column fluidic junction 304 and / or the post-column fluidic junction 306, can also be integrated directly onto the microfluidic chip. In some cases, the fluidic junction 304 can be located on-chip directly adjacent to the first end 318 of the chromatography column 302, thus reducing or eliminating the fluidic path 222. In some examples, the fluidic coupling 306 can be located on-chip directly adjacent to the second end 320 of the chromatography column 302, thus reducing or eliminating the fluidic path 224. In some embodiments, the silanols in the conductive or semiconductive stationary phase of the chromatography column 302 are chemically end-capped to reduce secondary (e.g., electrochemical) interactions with analytes. Similarly, the outer layer of the conductive or semiconductive stationary phase of some examples can be chemically treated to form a layer of a different material, such as by converting silicon to silicon oxide, or can be coated with a thin layer of another material to reduce analyte interactions.In contrast to chromatography columns with non-conductive stationary phases, which usually contain a heterogeneous packed bed of functionalized spherical particles (e.g., fused silica particles) as the stationary phase, the bed in conductive or semi-conductive microfluidic / microfabricated chromatography columns is formed by etched pillars spaced at uniform distances. Compared to packed bed columns, microfluidic chromatography columns offer several advantages. First, peak dispersion (e.g., vortex dispersion), which arises from a non-uniform flow path across the column cross section in heterogeneous packed bed columns, is reduced in chromatography columns with microfabricated or deep reactive ion etched features such as pillars due to the uniform spacing between features. Second, column permeability is increased. Finally, the homogeneity of microfabricated chromatography columns results in excellent column-to-column reproducibility.

[0031] In other embodiments, the chromatography column 302 can include other conductive or semi-conductive stationary phase materials, such as porous graphitic carbon beads.

[0032] The use of chromatography columns having conductive or semiconductive stationary phases with electrospray emitters has been limited by problems arising due to the electrical conductivity of the stationary phase. For example, doped semiconductor substrates and materials (e.g., boron-doped semiconductors) contain impurities that generate holes (unoccupied electronic states) making them electron acceptors. The substrate does not conduct these holes well, resulting in the accumulation of charge. Indeed, the current passing through the chromatography column can cause the charging of the stationary phase in the chromatography column, which in turn interacts with the analytes during separation. Similarly, having a voltage difference between a first end and a second end of the chromatography column can generate electrochemically induced changes in the pH of the solvent in the fluid sample, which ultimately affects the stationary phase of the chromatography column to deteriorate the chromatographic results. Furthermore, the potential difference between the fluid sample and the chromatography column, or between portions of the chromatography column, can promote undesirable electrochemistry, such as oxidation or reduction of the solvent or analyte species in the fluid sample. These electrical problems are magnified at high voltages (i.e., kilovolts) because leakage currents become more difficult (and cannot be) ignored under high voltage conditions. In some conventional systems, these drawbacks have been mitigated by effectively grounding the chromatographic column by introducing an additional post-column fluidic junction 122 between the post-column fluidic coupling 104 and the second end of the chromatographic column 302, as shown in FIG. 1. The additional post-column fluidic junction can be electrically grounded, thus preventing the flow of electrical current to the chromatographic column. However, each liquid-metal interface in the system downstream of the chromatographic column introduces additional dispersion to the chromatographic peaks (i.e., undoing the work of the chromatographic column to separate the analytes from one another). Thus, the additional post-column fluidic junction 122 in conventional systems introduces additional peak dispersion.The use of electrical conductors 314 as taught herein to balance the potential across the chromatography column solves these problems while still allowing for floating of the chromatography column at high voltages and enabling high resolution measurement of the electrospray current (e.g., down to the nanoamp level) by current sensing circuitry 210. Furthermore, the only post-column liquid-metal interface in electrospray system 200 occurs at fluid junction 306 where electrospray is initiated, thus reducing peak dispersion in the fluid sample.

[0033] In some embodiments, the fluidic or electrical components may be housed within the cartridge 208. As used herein, "housed" within the cartridge indicates that the components are at least partially mounted within the cartridge, although some elements may extend from the outer housing or casing of the cartridge. In some embodiments, the use of a cartridge format may make it easier to achieve expert-level liquid chromatography mass spectrometry (LCMS) performance by integrating the column and emitter into a cartridge format that also includes, in some cases, a nebulizing gas for consistent desolvation, an integrated heater for optimal chromatography, and on-board memory to enable use as a "smart consumable." The integrated heater can provide thermal stability and reduce the level of backpressure required to achieve adequate results. In some microfluidic and / or cartridge-based implementations, the integrated heater is particularly useful for reducing backpressure, since the cartridge 208 components may have lower pressure limits than would be achieved by state-of-the-art liquid chromatography pumps. For example, the cartridge 208 components, such as the chromatography column, may have a backpressure limit of 450 bar, and the integrated heater allows for good performance even at the lower backpressure limit. The cartridge 208, in some embodiments, may be a removable and replaceable component that is inserted into or withdrawn from the electrospray system 200. For example, the removable or replaceable cartridge 208 may be inserted into a receiving slot in the system housing. Insertion of the cartridge may facilitate a fluid connection between the pre-column fluidic junction 204 and the fluid pathway 218, while removal of the removable cartridge 208 from the electrospray system 200 may facilitate severing the fluid connection between the pre-column fluidic junction 204 and the fluid pathway 218.Similarly, insertion of the cartridge into the electrospray system 200 can facilitate electrical connection to the low side conductor 314 of the current sense circuit 210 via the high voltage electrical connector 216, and removal of the cartridge 208 from the system can facilitate disengagement or cutting of electrical contact between the current sense circuit 210 and the high voltage electrical connector 216. In some embodiments, the housing or casing of the cartridge 208 is formed from a non-conductive material. In some embodiments, the cartridge 208 is sealed or sealable to prevent user contact with the high voltages present within the cartridge 208 during operation and to improve safe handling of the cartridge.

[0034] Locating the fluidic junctions 304 and 306 within the cartridge 208 advantageously provides additional electrical shielding of these components to ensure that voltage equipotential conditions are maintained and improve safety by preventing users from coming into physical contact with high voltages. Even small resistivity differences along the electrical connections due to differences in the condition of the electrical cables, electrical contacts, fluidic junctions and junctions, as well as the fluidic junction or junction surface conditions, such as the amount of oxidation on the surfaces, can generate measurable leakage currents that can affect chromatographic performance. Locating the fluidic junctions 304 and 306 within the cartridge 208 prevents subsequent movement of these fluidic components as the pressurized fluid sample is pumped through them, thus ensuring improved electrical contact between the electrical conductors 314 and these components.

[0035] 2 depicts the current sense circuit 210 as being external to the cartridge 208. However, it is contemplated that the current sense circuit 210 may be disposed within the cartridge 208. In such an embodiment, the current sense circuit 210 is placed in series between the high voltage electrical connector 216 and the electrical conductor 314. For example, the high side 212 of the power source 202 and the current sense circuit 210 may be connected via the high voltage electrical connector 216, and the low side 214 of the current sense circuit 210 may be connected directly to the electrical conductor 314 without the intervening high voltage electrical connector 216.

[0036] The pre-column fluid junction 204 can have an inner diameter ranging from 50 microns to 500 microns in some embodiments. In an exemplary embodiment, the pre-column fluid junction 204 can have an inner diameter of 280 microns. In some embodiments, the fluid junction 304 can include one or more conductive materials. For example, the fluid junction 304 can include a precious metal or stainless steel. In some embodiments, the fluid junction 304 or the fluid coupling 306 can include a multi-port plumbing fixture or fitting known to those skilled in the art. In some embodiments, the fluid junction 304 and the fluid coupling 306 are identical parts in terms of material composition, shape, or both material composition and shape. In practical implementation, different electrical and fluid connections can result in different resistivities. By using identical materials or identically shaped parts for the fluid junction 304 and the fluid coupling 306, differences in contact resistance can be reduced. Undesirable ground currents can occur in the system, such as through the housing. In some embodiments, the housing is formed from a non-conductive material.

[0037] In some embodiments, the pre-column fluidic junction 204 and the fluidic junction 304 can be merged into a combined fluidic junction. In such embodiments, the fluidic path 220 is eliminated and the combined fluidic junction is connected to the emitter voltage V E is held in

[0038] In some embodiments, the fluid coupling 306 can include one or more conductive materials. For example, the fluid coupling 306 can include stainless steel. The fluid coupling 306 as shown in FIG. 2 and FIG. 3 is a two-port device (i.e., the fluid coupling 306 includes a first fluid port 310 and a second fluid port 312). However, in LC-MS procedures, it may be desirable to add certain chemicals to the mobile phase or introduce certain chemicals to the separated fluid sample after it has exited the chromatography column but before the electrospray emission to improve the analyte signal, so as to affect analyte ionization. Alternatively, some additives may be used to suppress undesired signals or selectively enhance the signals of certain compounds in the mixture. Thus, in some configurations, the fluid coupling 306 can include additional fluid ports (e.g., a third fluid port, a fourth fluid port, etc.) for receiving additional fluid streams (e.g., a second fluid, a third fluid, etc.) that mix in the fluid coupling 306 with the fluid sample received from the chromatography column 302. In various embodiments, the additional fluid streams may each be provided from a separate reservoir or from an additional fluid column, such as an additional chromatography column. In embodiments where the additional fluid streams are received at additional fluid ports of the fluidic junction from the additional chromatography column, the electrical conductor 314 may be extended to connect to additional fluidic junctions upstream of the additional chromatography column. Thus, an equipotential may be established between the fluidic junction 306, the fluidic junction 304, and these additional fluidic junctions. The additional fluid streams may include post-column additives in some embodiments. Post-column additives (PCAs) may be used in various ways, for example, to promote desolvation, change charge state distribution, or affect the electrospray ionization process in other known ways, and may be solvents or other chemical moieties.

[0039] In various embodiments taught herein, the fluid pathways 218, 220, 222, 224 are formed from flexible or rigid tubing. In some embodiments, the fluid pathways 218, 220, 222, 224 can include pathways or capillaries formed at least in part from fused silica or plastic / polymeric materials. The fluid pathways 218, 220, 222, 224 can include capillaries having an internal coating such as polyetheretherketone (PEEK). In some embodiments, the fluid pathways 218 can have an inner diameter in the range of 10-50 microns, or in the range of 20-40 microns. The fluid pathways 218 can have an inner diameter of 30 microns, in some embodiments. The fluid pathways 218 can have a length in the range of 10-50 cm, or in the range of 20-40 cm. The fluid pathways 218 can have a length of 35 cm, in some embodiments. In some embodiments, the fluid pathways 220 can have an inner diameter in the range of 10-50 microns, or in the range of 20-40 microns. The fluid path 220, in some embodiments, can have an inner diameter of 30 microns. The fluid path 220 can have a length in the range of 10-50 mm, or in the range of 20-40 mm, or in the range of 50-100 mm. The fluid path 220, in some embodiments, can have a length of 35 mm.

[0040] In some embodiments, the fluid path 222 can have an inner diameter in the range of 5-50 microns. The fluid path 222 can have an inner diameter of 5 microns, 10 microns, 20 microns, 25 microns, 30 microns, or 40 microns, in some embodiments. The fluid path 222 can have a length in the range of 10-50 mm, or in the range of 20-40 mm, or in the range of 50-100 mm. The fluid path 222 can have a length of 35 mm, in some embodiments. In some embodiments, the fluid path 224 can have an inner diameter in the range of 5-50 microns. The fluid path 224 can have an inner diameter in the range of 5 microns, 10 microns, 20 microns, 25 microns, 30 microns, or 40 microns, in some embodiments. The fluid path 224 can have a length in the range of 10-50 mm, or in the range of 20-40 mm, or in the range of 50-100 mm. The fluid path 224 can have a length of 35 mm, in some embodiments. Fluid path 224 can have reduced inner and outer diameters compared to other fluid paths in the system to help reduce peak broadening. In one example, fluid path 224 has an inner diameter of 5 or 10 microns and fluid paths 218, 220 have an inner diameter of 30 microns.

[0041] In some embodiments, the cartridge (more specifically, the same chromatography column and electrospray emitter) can be used for hundreds of injections. However, a given chromatography column tends to have a longer useful life than a given electrospray emitter. Thus, in some embodiments, the first fluid port 310, the second fluid port 312, or both the first and second fluid ports of the fluid coupling 306 are configured to allow for disconnection and reconnection of the fluid path or electrospray emitter attached thereto. In such embodiments, the electrospray emitter (or the fluid coupling 306) can be removed from the cartridge 208 and replaced with a new electrospray emitter 308 or fluid coupling 306. This ability to replace the emitter allows for the full life of the column to be used. In some embodiments, the fluid paths 222 and 224 are configured to be disconnected and reconnected from the first end 318 and the second end 320 of the chromatography column 302, respectively. By having a removable fluid path, the chromatography column 106 can be removed from the cartridge and replaced.

[0042] 3 shows an electrospray system 300 having a chromatography column 302 having a conductive or semiconductive stationary phase according to some embodiments taught herein. The electrospray system 300 comprises a chromatography column 302 having a first end 318 and a second end 320, a post-column fluidic coupling 306, a pre-column fluidic junction 304, and an electrospray emitter 308. The post-column fluidic coupling 306 and the pre-column fluidic junction 304 are connected by electrical conductors 314. The electrical connection of the fluidic coupling 306 to the fluidic junction 304 by the electrical conductors 314 creates a true equipotential between the first end 318 and the second end 320 of the chromatography column 302.

[0043] In some embodiments, the electrospray system 300 can include a housing that shields and protects the components within the housing. In some embodiments, the housing can be molded as a cartridge that can be removably inserted into a larger system. The housing can include an inlet port 316 for fluid connected to the fluid junction 304. The housing can also include a high voltage electrical connector 322 attached to or passing through the housing. A voltage can be applied to the high voltage electrical connector 322 using a power source 202. A fluid sample is provided to the inlet port 316 from a fluid source. The fluid sample passes through the inlet port 316 and the body of the fluid junction 304 along a fluid path 222 to a first end 318 of the chromatography column 302. In the chromatography column 302, the analytes of the fluid sample are separated in time within the fluid sample such that a separated fluid sample (i.e., a fluid sample containing separated analytes) is output from a second end 320 of the chromatography column 302. The separated fluid sample travels along a fluid path 224 to a first fluid port 310 of the fluid junction 306. The fluid sample is coupled to the fluid junction 306 via a high potential (V + ) into the mass spectrometer. The electrospray emitter 308 is connected to a second fluid port 312 of the fluid coupling 306. The ionized fluid stream is emitted from the electrospray emitter 308 as ionized droplets. For example, the electrospray emitter 308 can comprise a needle, and the droplets can be emitted through the distal tip of the needle. In some embodiments, the electrospray emitter 308 has an inner diameter of less than about 30 micrometers to enable nanoESI. In some embodiments, the emitted ionized droplets leave the electrospray emitter 308 and travel toward an atmospheric pressure inlet to the mass spectrometer, which functions as the counter electrode 112. The electrospray current return is generally formed by the electrically grounded counter electrode 112, as shown in FIG. 3.

[0044] In the absence of a current sensing circuit, the electrospray system 300 receives a high voltage V + Receives high voltage V + can be distributed directly to fluid junction 304 and fluid coupling 306 via electrical conductors. In other embodiments, a current sensing circuit similar to that described in connection with Figures 2, 5, and / or 6 can be in circuit between power source 202 and high voltage electrical connector 322. Note that power source 202 can be supplied with electrospray system 300 as a combined system in some embodiments. In other embodiments, electrospray system 300 is designed to interface with a separately provided power source 202.

[0045] FIG. 4 shows a graph of retention time of a particular analyte (specifically, a peptide having the amino acid sequence GLILVGGYGTR) in a chromatography column 302 having a semiconductive stationary phase according to some embodiments taught herein for a series of replicate experiments under different conditions. These experiments demonstrate the effect of charging due to non-equipotential voltage conditions on a chromatography column having a conductive or semiconductive stationary phase on analyte retention time. Note that because high voltages (e.g., up to 8 kV) are applied to the emitter, for example, in the electrospray system 100 of FIG. 1, a voltage difference (or drop) of tens of volts across the chromatography column can easily occur. For example, for a current sensing resistor with a resistance of 10 MΩ and an electrospray current of 5 μA, a 50 V difference occurs across the current sensing circuit 116 and, therefore, across the chromatography column 106 of the electrospray system 100 shown in FIG. 1.

[0046] Under first condition 402, the chromatography column is grounded (i.e., the chromatography column is insulated from the high voltage applied to the electrospray emitter by a grounded fluid junction placed between the emitter and the column). Under second condition 404, the chromatography column 302 is floated at a high voltage (i.e., a voltage sufficient to induce electrospray ionization of the fluid sample) at equipotential conditions between the first and second ends of the chromatography column as taught in various embodiments taught herein. As discussed above, the systems taught herein can achieve floating of the chromatography column without the use of a second fluid junction downstream of the chromatography column, which would cause peak dispersion. Over both first condition 402 and second condition 404, the average retention time is relatively stable and does not evolve or change over replicate experiments.

[0047] Under the third condition 406, the fourth condition 408, and the fifth condition 410, voltage differences of +1.6V, +9.6V, and +19.2V are intentionally induced between the fluid junctions / couplings (e.g., fluid junction 102 and fluid junction 104 in the system of FIG. 1) located at the first end 318 and the second end 320 of the chromatography column 302, respectively, as occurs in a conventional system such as the electrospray system 100. As can be seen in FIG. 4, the stability of the analyte retention times is significantly degraded over replicate experiments due to charge accumulation in the semiconductive stationary phase of the chromatography column. As a result, experimental results from early runs in an experimental series can no longer be reliably compared to experimental results from later runs in the experimental series.

[0048] After applying the fifth condition 410 to the chromatography column 302, the experimental conditions are returned to the second condition 404 and the chromatography column 302 is floated at high voltage with equal voltages applied to the first end 318 and the second end 320 (e.g., as occurs in the electrospray system 300 where equal voltages are applied to the fluidic junction 304 and the fluidic coupling 306). It can be seen that retention time reproducibility is restored over several initial experimental runs and returns to stable retention times as further experimental runs are performed. Finally, the chromatography column 302 is subjected to a sixth condition 412 where an intentional voltage difference of -19.2 V is applied between the first end 318 and the second end 320 of the chromatography column 302. Again, the retention times become unstable and change from experiment to experiment as the accumulated charge in the semiconductor chromatography column 302 begins to affect analyte retention within the chromatography column.

[0049] Table 1 below shows the coefficients of variation measured for each of the experimental conditions shown in Figure 4. As shown by these values, the experimental variation is significantly reduced under conditions where the first and second ends of the chromatographic column are at equal potentials.

[0050] [Table 1]

[0051] FIG. 5 illustrates an exemplary current sense circuit 210 for use with embodiments of the systems, methods, and cartridges taught herein. The current sense circuit 210 can include a current sense resistor 510 disposed between the high side 212 and the low side 214 of the current sense circuit 210. In some embodiments, the current sense circuit 210 consists only of the current sense resistor 510, a first electrical lead connecting the high side 212 of the current sense circuit 210 to a voltmeter, and a second electrical lead connecting the low side 214 of the current sense circuit 210 to a voltmeter. Such a configuration allows the voltmeter to measure high voltages with high accuracy. To reduce the cost of the current sense circuit 210, additional electronic elements such as those shown in FIG. 5 can be used to attenuate the voltage level, allowing the use of data acquisition electronics compatible with modern computing devices.

[0052] In the embodiment shown in FIG. 5, the current sense circuit 210 comprises a current sense resistor 510, a first voltage divider 502, a second voltage divider 504, and a differential amplifier 506. The voltage between the high side 212 and the low side 214 of the current sense resistor 510 is significantly attenuated via a precision voltage divider (502, 504) to match typical data collection and computer voltage potentials. In one embodiment, the precision voltage dividers 502, 504 operate at a reduction ratio of 1000:1. The voltages obtained at the outputs of the voltage dividers 502, 504 can be subtracted using a differential amplifier 506. The difference obtained at the output 514 of the current sense circuit 210 provides a directly proportional measurement of the ESI spray current. In various embodiments, the resistance of the current sense resistor 510 can be in the range of 10 kΩ to 10 GΩ, in the range of 1 MΩ to 1 GΩ, or in the range of 10 MΩ to 100 MΩ.

[0053] Optionally, controller 508 can be coupled to output 514 of current sense circuit 210. Controller 508 may be configured to receive the output current directly from output 514 or a digitized representation of output 514. In some embodiments, controller 508 determines a state of the electrospray emitter based on the measured current flow. In particular, the state of the electrospray emitter includes an operating mode within a particular electrospray regime.

[0054] In some embodiments, the controller 508 includes a user interface 512 configured to display or otherwise alert a user or operator of the determined electrospray regime. In other embodiments, the controller 508 can interface with an external user interface. In some embodiments, the controller 508 can determine the health of the cartridge 208, for example, by tracking and identifying changes in the output current versus the applied voltage over time. If the changes are significant (e.g., exceed a threshold limit), the controller 508 can indicate to a user that the cartridge 208, or a portion of the cartridge 208, such as the chromatography column 302 or the electrospray emitter 308, should be replaced.

[0055] In the current sensing circuit 210 of FIG. 5, current measurements are obtained with high accuracy using relatively inexpensive electronics without an isolation amplifier. One difficulty with direct high-side measurement techniques (i.e., measuring the electrospray current on the high-voltage side of the circuit, as opposed to low-side measurement of the return current from the counter electrode 112 to the power supply 202) is measuring the current to nanoampere resolution in the presence of very high common-mode voltages (e.g., + / - 8 kilovolts). Such high common-mode voltages are then heavily attenuated to protect the downstream electronics. Another solution to overcome these difficulties is by isolating or electrically floating the large common-mode power supply voltages and the sensing circuit to protect the downstream electronics. The measured quantities may be converted across an isolation barrier to the downstream electronics using modulation and demodulation techniques.

[0056] FIG. 6 illustrates a schematic of an alternative current sense circuit 210 as an isolation amplifier circuit suitable for use in some embodiments taught herein. An isolation amplifier circuit allows large input voltages to float electrically. In this embodiment, the current sense circuit 210 comprises a current sense resistor 510. The high side 212 and the low side 214 of the current sense circuit 210 are electrically coupled to a first buffer 602 and a second buffer 604, respectively. The outputs from the first buffer 602 and the second buffer 604 are connected to a differential amplifier 606. The output of the differential amplifier 606 flows to an isolation amplifier 608. The output of the isolation amplifier 608 forms the output 514 of the current sense circuit 210, which can optionally be connected to a controller 508 as described above with respect to FIG. 5. The current sense circuit 210 described in FIG. 6 also benefits from the use of an isolated power supply 202 for the floating amplifier, as well as a means of modulation and demodulation across the isolation barrier (not shown). Modulation and demodulation can introduce systematic noise.

[0057] Other embodiments of current sensing and monitoring circuits suitable for use with embodiments of the systems, methods, and cartridges taught herein are described in U.S. Patent Application Publication No. 2023 / 0030920 by Lindseth et al., the entire contents of which are incorporated herein by reference.

[0058] 7 illustrates a routine 700 for reducing electrochromatographic effects in an electrospray system according to various embodiments taught herein. In block 702, the routine 700 flows a fluid sample from a first pre-column fluidic junction 304 to a first end 318 of a chromatography column 302. The chromatography column 302 includes a conductive or semiconductive stationary phase disposed between the first end 318 and a second end 320 of the chromatography column. In block 704, the routine 700 separates analytes in the fluid sample using the chromatography column 302. In block 706, the routine 700 outputs the separated fluid sample from the second end of the chromatography column to a first fluidic port 310 of a post-column fluidic junction 306. The fluidic junction 306 has a second fluidic port 312 connected to an electrospray emitter 308. At block 708, the routine 700 applies an equipotential voltage in the range of +1 to +10 kilovolts or −1 to −10 kilovolts to both the first pre-column fluid junction 304 and the post-column fluid coupling 306 using an electrical conductor 314 in electrical contact with the first pre-column fluid junction and the post-column fluid coupling. At optional block 710, the routine 700 applies a first voltage to the high side 212 of the current sense circuit 210 using the power supply 202. At optional block 712, the routine 700 transmits the equipotential voltage from the low side 214 of the current sense circuit 210 to the electrical conductor 314.

[0059] Many of the embodiments taught herein relate to replaceable ESI cartridges (e.g., cartridge 208 in FIG. 2). However, the techniques taught herein may also be implemented in other systems that do not include a cartridge. In such embodiments without a cartridge, the system still advantageously provides speed and simplicity in replacing an electrospray emitter (e.g., by disconnecting the needle from the fluid coupling) without disturbing or otherwise disassembling the remaining fluidic and electrical connections in the system.

[0060] Although the present system, method and cartridge and associated advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, compositions, means, methods and steps described herein. As one skilled in the art will readily appreciate from this disclosure, any currently existing or later developed process, machine, manufacture, composition, means, method or step that performs substantially the same function or achieves substantially the same result as the corresponding embodiments taught herein may be utilized in accordance with the present disclosure. It is therefore intended that the appended claims include within their scope such processes, machines, manufacture, compositions, means, methods or steps.

[0061] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

[0062] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments may be practiced without or with these specific details. In other instances, structures and devices are shown in block diagram form. Moreover, those skilled in the art will readily appreciate that the specific order in which the methods are presented and performed is illustrative (unless expressly stated otherwise), and it is contemplated that the order may be altered and still remain within the spirit and scope of the various embodiments disclosed herein.

[0063] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, are expressly incorporated by reference in their entirety for any purpose. Unless otherwise explained, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the various embodiments taught herein belong.

[0064] It will be understood that there is an implicit "about" before the specific temperatures, concentrations, times, pressures, flow rates, cross-sectional areas, etc. discussed in the present teachings, so that very small insignificant deviations are within the scope of the present teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Similarly, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing", "include", "includes", and "including" is not intended to be limiting. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present teachings.

[0065] As used herein, "a" or "an" may refer to "at least one" or "one or more." Additionally, the use of "or" is inclusive, such that a phrase "A or B" is applicable when "A" is applicable, when "B" is applicable, or when both "A" and "B" are applicable. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0066] A "system" refers to a set of components, whether real or abstract, in which each component interacts with or comprises a whole in relation to at least one other component within the whole.

[0067] The advantages and features of the present disclosure can be further illustrated by the following examples.

[0068] Example 1. An electrospray system comprising: a first pre-column fluidic junction for receiving a fluid sample; a chromatography column for separating analytes in the fluid sample, the chromatography column including a conductive or semiconductive stationary phase disposed between a first end and a second end of the chromatography column, the first end being fluidly connected to the first pre-column fluidic junction; a post-column fluidic junction having a first fluidic port and a second fluidic port, the first fluidic port receiving separated analytes in the fluid sample from the second end of the chromatography column; an electrospray emitter fluidly connected to the second fluidic port; and an electrical conductor connected to the post-column fluidic junction and the first pre-column fluidic junction to equalize the electrical potential between the post-column fluidic junction and the first pre-column fluidic junction.

[0069] Example 2. The electrospray system of Example 1, further comprising a current sensing circuit having a high side and a low side, the low side of the current sensing circuit being electrically connected to the electrical conductor.

[0070] Example 3. The electrospray system of Example 2, further comprising a power supply electrically connected to the high side of the current sensing circuit.

[0071] Example 4. An electrospray system as described in Example 3, further comprising a second pre-column fluidic junction for delivering a fluid sample to the first pre-column fluidic junction, and a power source electrically connected to supply high voltage to the second pre-column fluidic junction.

[0072] Example 5. The electrospray system of Example 1, wherein the post-column fluidic junction and the first pre-column fluidic junction are electrically connected in parallel with each other and in series with a current sensing circuit.

[0073] Example 6. An electrospray system as described in Example 1, wherein the fluid pathway connecting the second end of the chromatography column and the post-column fluidic junction has an inner diameter of 30 micrometers or less to reduce peak broadening.

[0074] Example 7. The electrospray system described in Example 1, wherein the chromatography column, the post-column fluidic coupling, the electrospray emitter, the electrical conductor, and the first pre-column fluidic junction are contained within a replaceable cartridge.

[0075] Example 8. The electrospray system of Example 7, wherein the cartridge comprises a high voltage electrical connector electrically connected to the electrical conductor or to a current sensing circuit in series with the electrical conductor.

[0076] Example 9. An electrospray system as described in Example 8, wherein the current sensing circuitry is housed within a replaceable cartridge.

[0077] Example 10. The electrospray system of Example 7, wherein the replaceable cartridge comprises a housing that electrically isolates the first pre-column fluidic junction and the post-column fluidic coupling.

[0078] Example 11. An electrospray system as described in Example 1, wherein the length of the fluid path between the first pre-column fluid junction and the chromatography column, and the length of the fluid path between the chromatography column and the post-column fluid junction are each less than 100 mm.

[0079] Example 12. The electrospray system of Example 1, further comprising a third pre-column fluidic junction electrically connected to ground, the second pre-column fluidic junction and the third pre-column fluidic junction forming a pre-column voltage spacer.

[0080] Example 13. A method for reducing electrochromatographic effects in an electrospray system, comprising: flowing a fluid sample from a first pre-column fluidic junction to a first end of a chromatography column, the chromatography column comprising a conductive or semi-conductive stationary phase disposed between the first end and a second end of the chromatography column; separating analytes in the fluid sample using the chromatography column; outputting the separated fluid sample from the second end of the chromatography column to a first fluid port of a post-column fluidic junction, the post-column fluidic junction having a second fluid port connected to an electrospray emitter; and applying an equipotential voltage in the range of +1 to +10 kilovolts or -1 to -10 kilovolts at both the first pre-column fluidic junction and the post-column fluidic junction using electrical conductors in electrical contact with the first pre-column fluidic junction and the post-column fluidic junction.

[0081] Example 14. The method of example 13, further comprising applying a first voltage to a high side of the current sensing circuit using a power source, and transmitting an equipotential voltage to the conductor from a low side of the current sensing circuit.

[0082] Example 15. The method of example 14, wherein the post-column fluidic coupling and the first pre-column fluidic junction are electrically connected in parallel with each other and in series with a current sensing circuit.

[0083] Example 16. The method of example 13, wherein outputting the separated fluid sample from the second end of the chromatography column to the first port of the post-column fluid coupling comprises flowing the separated fluid sample through a fluid path having an inner diameter of 30 micrometers or less to reduce peak broadening.

[0084] Example 17. The method of example 13, wherein flowing the fluid sample from the first pre-column fluid junction to the first end of the chromatography column comprises flowing the fluid sample through a fluid path having a length of less than 100 mm.

[0085] Example 18. The method of Example 13, wherein the chromatography column, the post-column fluidic junction, the electrospray emitter, the electrical conductor, and the first pre-column fluidic junction are contained within a replaceable cartridge, and the method further comprises connecting the second pre-column fluidic junction of the cartridge to a source of a fluid sample, and flowing the fluid sample from the second pre-column fluidic junction to the first pre-column fluidic junction.

[0086] Example 19. The method of example 18, wherein the cartridge comprises a high voltage electrical connector electrically connected to the electrical conductor, and the method further comprises electrically connecting a power source to the high voltage electrical connector.

[0087] Example 20. The method of example 14, wherein the first voltage is greater than the equipotential voltage.

[0088] Example 21. A cartridge for insertion into an electrospray system, comprising: a first pre-column fluidic junction for receiving a fluid sample; a chromatography column for separating analytes in the fluid sample, the chromatography column comprising a conductive or semi-conductive stationary phase disposed between a first end and a second end of the chromatography column, the first end being fluidly connected to the first pre-column fluidic junction; a post-column fluidic junction having a first fluidic port and a second fluidic port, the first fluidic port receiving separated analytes in the fluid sample from the second end of the chromatography column; an electrospray emitter fluidly connected to the second fluidic port; and an electrical conductor connected to the post-column fluidic junction and the first pre-column fluidic junction to equalize the electrical potential between the post-column fluidic junction and the first pre-column fluidic junction.

Claims

1. 1. An electrospray system comprising: a first pre-column fluid junction for receiving the fluid sample; a chromatography column for separating analytes in the fluid sample, the chromatography column including a conductive or semiconductive stationary phase disposed between a first end and a second end of the chromatography column, the first end being fluidly connected to the first pre-column fluidic junction; a post-column fluidic connection having a first fluidic port and a second fluidic port, the first fluidic port receiving separated analytes in the fluid sample from the second end of the chromatography column; an electrospray emitter fluidly connected to the second fluid port; an electrical conductor connected to the post-column fluid junction and the first pre-column fluid junction to equalize the electrical potential between the post-column fluid junction and the first pre-column fluid junction;

2. 10. The electrospray system of claim 1, further comprising a current sensing circuit having a high side and a low side, the low side of the current sensing circuit being electrically connected to the electrical conductor.

3. The electrospray system of claim 2 , further comprising a power supply electrically connected to the high side of the current sensing circuit.

4. The electrospray system of claim 3, further comprising a second pre-column fluidic junction for delivering the fluid sample to the first pre-column fluidic junction, the power source being electrically connected to supply a high voltage to the second pre-column fluidic junction.

5. 2. The electrospray system of claim 1, wherein the post-column fluid coupling and the first pre-column fluid junction are electrically connected in parallel with each other and in series with a current sensing circuit.

6. 10. The electrospray system of claim 1, wherein a fluid path connecting the second end of the chromatography column and the post-column fluid junction has an inner diameter of 30 micrometers or less to reduce peak broadening.

7. 10. The electrospray system of claim 1, wherein the chromatography column, the post-column fluidic coupling, the electrospray emitter, the electrical conductor, and the first pre-column fluidic junction are contained within a replaceable cartridge.

8. 8. The electrospray system of claim 7, wherein the cartridge comprises a high voltage electrical connector electrically connected to the electrical conductor or to a current sensing circuit in series with the electrical conductor.

9. The electrospray system of claim 8 , wherein the current sensing circuitry is contained within the replaceable cartridge.

10. 8. The electrospray system of claim 7, wherein the replaceable cartridge comprises a housing that electrically isolates the first pre-column fluidic junction and the post-column fluidic coupling.

11. 2. The electrospray system of claim 1, wherein the length of the fluid path between the first pre-column fluid junction and the chromatography column, and the length of the fluid path between the chromatography column and the post-column fluid junction are each less than 100 mm.

12. 5. The electrospray system of claim 4, further comprising a third pre-column fluid junction electrically connected to ground, the second pre-column fluid junction and the third pre-column fluid junction forming a pre-column voltage spacer.

13. 1. A method for reducing electrochromatographic effects in an electrospray system, comprising: flowing a fluid sample from a first pre-column fluid junction to a first end of a chromatography column, the chromatography column including a conductive or semi-conductive stationary phase disposed between the first end and a second end of the chromatography column; separating analytes in the fluid sample using the chromatography column; outputting the separated fluid sample from the second end of the chromatography column to a first fluid port of a post-column fluid coupling, the post-column fluid coupling having a second fluid port connected to an electrospray emitter; applying an equipotential voltage in the range of +1 to +10 kilovolts or in the range of -1 to -10 kilovolts to both the first pre-column fluid junction and the post-column fluid junction using an electrical conductor in electrical contact with the first pre-column fluid junction and the post-column fluid junction.

14. applying a first voltage to a high side of a current sense circuit using a power supply; 14. The method of claim 13, further comprising transmitting the equipotential voltage from a low side of the current sense circuit to the electrical conductor.

15. 15. The method of claim 14, wherein the post-column fluid coupling and the first pre-column fluid junction are electrically connected in parallel with each other and in series with the current sensing circuit.

16. 14. The method of claim 13, wherein outputting the separated fluid sample from the second end of the chromatography column to a first port of the post-column fluid coupling comprises flowing the separated fluid sample through a fluid path having an inner diameter of 30 micrometers or less to reduce peak broadening.

17. 14. The method of claim 13, wherein flowing the fluid sample from the first pre-column fluid junction to the first end of the chromatography column comprises flowing the fluid sample through a fluid path having a length of less than 100 mm.

18. The chromatography column, the post-column fluidic junction, the electrospray emitter, the electrical conductor, and the first pre-column fluidic junction are contained within a replaceable cartridge, and the method comprises: connecting a second pre-column fluidic junction of the cartridge to a source of the fluid sample; 14. The method of claim 13, further comprising flowing the fluid sample from the second pre-column fluid junction to the first pre-column fluid junction.

19. the cartridge comprising a high voltage electrical connector electrically connected to the electrical conductor, the method comprising:

20. The method of claim 18, further comprising electrically connecting a power source to the high voltage electrical connector.

20. The method of claim 14 , wherein the first voltage is greater than the equipotential voltage.

21. 1. A cartridge for insertion into an electrospray system, comprising: a first pre-column fluid junction for receiving the fluid sample; a chromatography column for separating analytes in the fluid sample, the chromatography column including a conductive or semiconductive stationary phase disposed between a first end and a second end of the chromatography column, the first end being fluidly connected to the first pre-column fluidic junction; a post-column fluidic connection having a first fluidic port and a second fluidic port, the first fluidic port receiving separated analytes in the fluid sample from the second end of the chromatography column; an electrospray emitter fluidly connected to the second fluid port; a conductor connected to the post-column fluid junction and to the first pre-column fluid junction to equalize an electrical potential between the post-column fluid junction and the first pre-column fluid junction.

Images