System and method for electroinjection using chromatography column with conductive or semiconductive stationary phase

Abstract

Systems, methods, and cartridges taught herein improve chromatographic performance in electroinjection systems featuring chromatography columns having conductive or semi-conductive stationary phases by electrically connecting a fluid junction upstream of the chromatography column to a fluid junction downstream of the chromatography column using an electrical conductor. The electrical conductor generates a voltage equipotential between a first end of the chromatography column and a second end of the chromatography column that neutralizes an electrical current flowing through the chromatography column. Accurate electroinjection current measurement can be achieved while post-column peak dispersion and repeatable retention times are achieved.

Description

Background Art

[0001] Electrospray ionization (ESI) is a technique for mass spectrometry (MS). More specifically, ESI is a "soft" ionization technique widely used to prepare gas-phase ions with low levels of fragmentation. In a typical ESI process, a high voltage is applied to a liquid to produce charged droplets, which are desolvated to form gas-phase ions. Low-flow electrospray or "nanoESI" prepares charged droplets from an emitter with a relatively small inner diameter (e.g., about 20 microns or less). The combination of low flow rate and small diameter leads to an increase in ionization efficiency (i.e., an increase in the ratio of gas-phase ions prepared per available analyte molecule in the solution) and allows a reduction in the amount of required sample. When the emitter is positioned near the mass spectrometer inlet acting as a counter electrode, the prepared gas-phase ions are sampled into an instrument for mass analysis.

[0002] ESI is often used to couple the output of a chromatography column or system to a mass spectrometer (MS). Chromatographic techniques such as liquid chromatography (LC) can be used to produce a temporal separation between molecules in solution. The eluate can be fed to an electrospray emitter to serve as the input to a mass spectrometer. Summary of the invention

[0003] The present invention provides an electrospray system. The electrospray system includes a first pre-column fluid junction for receiving a fluid sample. The electrospray system includes a chromatographic column for separating analytes in the fluid sample. The chromatographic column includes a conductive or semi-conductive stationary phase arranged between a first end and a second end of the chromatographic column. The first end is fluidically connected to the first pre-column fluid junction. The electrospray system includes a post-column fluid union having a first fluid port and a second fluid port. The first fluid port receives the analytes separated in the fluid sample from the second end of the chromatographic column. The electrospray system includes an electrospray emitter fluidically connected to the second fluid port. The electrospray system includes an electrical conductor connected to the post-column fluid union and the first pre-column fluid junction to equalize the potential between the post-column fluid union and the first pre-column fluid junction.

[0004] The present invention provides a method for reducing the electrochromatographic effect in an electrospray system. The method includes causing a fluid sample to flow from a first pre-column fluid junction to a first end of a chromatographic column. The chromatographic column includes a conductive or semi-conductive stationary phase disposed between the first end and the second end of the chromatographic column. The method includes using the chromatographic column to separate analytes in the fluid sample. The method includes outputting the separated fluid sample from the second end of the chromatographic column to a first fluid port of a post-column fluid union. The post-column fluid union has a second fluid port connected to an electrospray emitter. The method includes applying an equipotential voltage in the range of +1 kV to +10 kV or in the range of -1 kV to -10 kV at both the first pre-column fluid junction and the post-column fluid union using an electrical conductor in electrical contact with the first pre-column fluid junction and the post-column fluid union.

[0005] The present invention provides a box for inserting into an electrospray system. The box includes a first pre-column fluid junction for receiving a fluid sample. The box includes a chromatographic column for separating analytes in the fluid sample. The chromatographic column includes a conductive or semi-conductive stationary phase disposed between a first end and a second end of the chromatographic column. The first end is fluidically connected to the first pre-column fluid junction. The box includes a post-column fluid union having a first fluid port and a second fluid port. The first fluid port receives the analytes separated in the fluid sample from the second end of the chromatographic column. The box includes an electrospray emitter fluidically connected to the second fluid port. The box includes an electrical conductor connected to the post-column fluid union and the first pre-column fluid junction to equalize the potential between the post-column fluid union and the first pre-column fluid junction. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] It should be understood that the drawings are not necessarily drawn to scale, nor are the relationships between objects in the drawings necessarily drawn to scale. The drawings are intended to be depictions that clarify and understand the various embodiments of the devices, systems, and methods disclosed herein. Wherever possible, all drawings will use the same reference numerals to refer to the same or similar parts. In addition, it should be understood that the drawings are not intended to limit the scope of the present teachings in any way.

[0007] Figure 1 A conventional electrospray system comprising a chromatography column with a non-conductive stationary phase is schematically depicted.

[0008] Figure 2 Depicted is a schematic diagram of an electrospray system with a chromatography column having a conductive or semiconductive stationary phase according to some embodiments taught herein.

[0009] Figure 3 Depicted is a schematic diagram of an alternative electrospray system with a chromatography column having a conductive or semiconductive stationary phase according to some embodiments taught herein.

[0010] Figure 4 Shown is a graph of the retention time of a particular analyte in a chromatography column with a semiconducting stationary phase according to some embodiments taught herein for a series of repeated experiments under different conditions.

[0011] Figure 5 Current sensing circuits for use with embodiments of the systems, methods, and cartridges taught herein are schematically depicted.

[0012] Figure 6 A current sensing circuit including isolation suitable for use with some embodiments taught herein is schematically depicted.

[0013] Figure 7 Depicted is a routine for reducing electrochromatographic effects in an electrospray system according to various embodiments taught herein. DETAILED DESCRIPTION

[0014] The system, method and box taught herein improve the chromatographic performance in the electrospray system characterized by a chromatographic column with a stationary phase formed by a conductive or semiconductive material or substrate. In a conventional electrospray system, an electrical setting allows leakage current to pass through an eluent. Such an arrangement is not suitable for use with a chromatographic column with a conductive or semiconductive stationary phase, because the current flowing through such a conductive column causes the column to be charged over time, which subsequently produces an unfavorable interaction with the analyte during the separation of the analyte in the column. The system, method and box taught herein overcome this problem by using an electrical conductor to electrically connect the fluid confluence located upstream of the chromatographic column to the fluid union located downstream of the chromatographic column. The electrical conductor generates a high voltage equipotential between the first end of the chromatographic column and the second end of the chromatographic column, and the high voltage equipotential limits or eliminates the current flowing through the chromatographic column.

[0015] The system, method and box taught herein also enable the use of a chromatographic column with a conductive or semi-conductive stationary phase to measure the electrospray current in the system while maintaining high chromatographic performance. In conventional systems, a current monitoring circuit is connected between a power supply and an electrospray emitter in such a way that a slightly different voltage is applied to the end (i.e., inlet and outlet) of a chromatographic column. In the system, method and box taught herein, an electric conductor connecting a fluid confluence portion and a fluid union portion is connected in series with a current monitoring circuit. Therefore, there is no current flowing through a chromatographic column with a conductive or semi-conductive stationary phase, but the electrospray current can still be accurately measured.

[0016] According to some embodiments taught herein, the electrospray system, method and box taught herein realize improved chromatographic performance by including a fluid union as the only post-column fluid connection. The chromatographic column produces a temporary separation of analytes in the solution, and the analyte bands travel to the electrospray emitter. Each fluid connection that the analyte bands pass through on the way to the electrospray emitter can disturb the laminar fluid flow (e.g., flow vortex). In addition, an unswept (no flow) volume is produced at each fluid connection where the analyte diffuses (passive transport) therein. Because this is a much slower process than active transport, a portion of the analyte plug or band will lag behind the body of the plug, resulting in a "tail plug". These processes affect the quality of separation because they cause the analyte bands to disperse, or so-called "band widening". In the system, method and box taught herein, the use of an electrical conductor enables true equipotential to be achieved between the ends of the column while only a single post-column fluid connection (i.e., fluid union) is used. Thus, systems and methods according to some embodiments herein can improve chromatographic performance (e.g., reduce peak broadening and adjust retention times compared to conventional systems) by avoiding charging the conductive or semiconductive stationary phase in the chromatographic column and limiting the number of post-column fluid connections to one.

[0017] Accurate current-voltage curves (i.e., the current flowing through an electrospray emitter as a function of applied voltage) are important for characterizing, diagnosing, and optimizing electrospray systems. Such curves help distinguish between various electrospray schemes that primarily control the sensitivity of the instrument. In the first electrospray scheme, the electrospray voltage (V E ) is below a threshold value, resulting in the emitter not emitting a spray or dripping fluid. For example, in some embodiments, the first electrospray scheme may occur at less than 1,200 volts. When the applied electrospray voltage (V E) is sufficient to induce electrospray within a stable mandrel scheme, a second electrospray scheme occurs in which charged droplets are formed directly from filaments extending from a Taylor cone. For example, the second scheme may occur when the electrospray voltage is in the range of 1,200 volts to 2,200 volts. In the third scheme, the applied electrospray voltage results in a conical jet in which the filaments are truncated by Coulomb repulsion, thereby producing a diffuse plume of charged droplets. For example, the third scheme may occur when the electrospray voltage exceeds 2,200 volts. The three electrospray schemes are characterized by a sudden change in the slope on a graph of the spray current as a function of the applied voltage. Embodiments of the present disclosure allow for the precise determination of the spray current as a function of the applied voltage, and therefore enable the creation of such a graph. On an IV graph, the inverse of the slope (i.e., 1 / slope) represents the effective impedance for given parameters such as flow rate, tip diameter, fluid composition, etc. This knowledge is important at least for setup, spray optimization, and diagnostics, and other methods of obtaining the effective impedance (including processing images of the electrospray plume) are complex and time consuming. Thus, embodiments taught herein may enable improvement or optimization of jetting by directly measuring electrical properties without the need for complex imaging algorithms or associated techniques.

[0018] Additionally, changes in the current-voltage characteristics of the electrospray system or cartridge indicate the health of the system or cartridge. Thus, monitoring these characteristics with a controller can provide a diagnostic tool to assess the health of the system or cartridge. One of the benefits of the electrical arrangement taught in the embodiments described herein is that the improved fidelity of the current measurement readback enables direct measurement and software automation of health and operating information of the electrospray system that would otherwise have to be obtained from an optical detection device of the ESI plume.

[0019] However, conventional systems and methods for measuring electrospray current have significant disadvantages, particularly for systems with chromatographic columns having conductive or semi-conductive stationary phases. Off-the-shelf power supplies, although typically equipped with current and voltage monitor outputs, will only report the total load current. In reality, there are unknown leakage or false paths for currents generated by the conductive fluid flow itself and by conductive elements in the fluid path. In order to obtain an accurate measurement of the ESI current, other currents must be ignored or subtracted from the whole. This requires a separate independent measurement of the injection current, which is typically not provided by high voltage power supply manufacturers. Any other loads must be considered in addition, whether intentionally or unintentionally. The problem is further complicated when considering that these extremely small currents are measured in the presence of extremely high common-mode voltages (e.g., requiring an accuracy of greater than 10 nanoamperes). For the case of ±8 kilovolt power supplies, the measurement circuit typically uses a minimum common-mode rejection of approximately 100 decibels or more. In addition, tolerance matching of high voltages and high-ohmic resistors requires an accuracy better than 0.01%.

[0020] The systems, methods, and boxes taught herein enable accurate measurement of electrospray current in an electrospray system including a chromatographic column having a conductive or semiconductive stationary phase by using an electrical conductor connected in series with a current sensing circuit to generate a voltage equipotential between a pre-column fluid junction located at a first end of the chromatographic column and a post-column fluid junction located at a second end of the chromatographic column.

[0021] Figure 1 A prior art electrospray system 100 is schematically depicted, comprising a chromatography column 106 with a non-conductive stationary phase (e.g., packed polymer or glass beads). Figure 1 , Figure 2 and Figure 3 In the figure, the dotted line connection between the components represents the fluid path, and the solid line connection between the elements represents the electrical path. Prior art electrospray system 100 includes a fluid confluence 102, a chromatographic column 106 with a non-conductive stationary phase, a post-column fluid union 104, a post-column fluid confluence 122 and an electrospray emitter 114. The fluid sample is supplied to the electrospray system 100 from a liquid chromatography system 108 via the fluid confluence 102. The fluid sample travels through the chromatographic column 106 from the pre-column fluid confluence 102. The chromatographic column 106 temporarily separates the analyte in the fluid sample and outputs the separated fluid sample (that is, the fluid sample including the separated analyte). The separated fluid sample flows to the post-column fluid confluence 122, and then flows to the post-column fluid union 104, and then is emitted by the electrospray emitter 114. The power supply 110 is connected to the fluid confluence 102 and to the high side 118 of the current sensing circuit 116. The low side 120 of the current sensing circuit 116 is connected to the fluid union 104 and the post-column fluid junction 122. When a sufficient voltage is applied at the fluid union 104, the fluid at the electrospray emitter 114 is ionized and forms a stream of droplets, which 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 be ground in some cases). Thus, the flow of ionized droplets completes the circuit and produces a current that can be measured.

[0022] In some embodiments, the counter electrode 112 can float at a bias voltage referenced to ground that 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 spectrometer can serve as the counter electrode 112. In embodiments that include a small floating bias voltage, another ion optical element in the mass spectrometer can be held at ground (e.g., a c-trap), thereby allowing ions to be guided by the ion optical element held at a gradually decreasing (or increasing, depending on its polarity) voltage.

[0023] exist Figure 1In the embodiment, the current sensing circuit 116 is arranged in series with the fluid union 104 and the post-column fluid junction 122, and measures the current flowing from the power supply 110 to the fluid union 104. The conventional electrospray system 100 attempts to divert the leakage current from the measurement path of the current sensing circuit 116 by applying a common voltage to the fluid union 104 and the post-column fluid junction 122 while applying a high voltage to the fluid junction 102. In some examples, no voltage V is applied at the fluid junction 102. + . If no voltage is applied to the pre-column fluid junction 102, leakage current occurs when current flows through the conductive fluid to the grounded liquid chromatography system 108. In the present application, "leakage current" refers to current from a power source that does not travel to the electrospray emitter and the counter electrode, but instead travels along a separate path away from the emitter to ground (e.g., along a path through the fluid sample to the upstream of the chromatographic column and to the grounded liquid chromatography system 108). If the leakage current is measured by the current sensing circuit but does not travel through the electrospray emitter to ground, the leakage current may prevent accurate measurement of the electrospray current.

[0024] A voltage V is applied at the fluid confluence 102. + is not sufficient to eliminate the leakage current across the column 106 because the voltage V + Not equal to the voltage V applied to the fluid joint E This difference may be due to a non-zero voltage drop across the current sensing circuit 116 as well as other factors such as different contact resistances or material compositions at the fluid junction or fluid union. Due to the very high voltages involved, even if V + With V E A small difference between 1 and 2 can also achieve leakage currents of the order of the injection current. + , the same current leakage problem occurs, because the current will then leak to the grounded liquid chromatography system 108. However, these conventional systems include a chromatography column with a non-conductive stationary phase. A large voltage drop (up to several kilovolts) can exist across such a chromatography column 106 without affecting the performance of the chromatographic separation, because the non-conductive stationary phase works similarly whether it is in the presence or absence of current due to the voltage drop.

[0025] In other conventional schemes, the pre-column fluid junction 102 and the post-column fluid junction 122 are maintained at ground potential (i.e., substantially zero volts). In such schemes, the stationary phase of the chromatographic column 106 is not subjected to current flow. However, such an arrangement is disadvantageous because it does not allow the use of the current sensing circuit 116 alone to accurately measure the electrospray current, because the current sensing circuit 116 will measure both the electrospray current and the leakage current that occurs between the post-column fluid union 104 and the post-column fluid junction 122.

[0026] Figure 1 The arrangement of the conductive or semi-conductive stationary phase causes problems in the use of chromatographic columns with conductive or semi-conductive stationary phases. Leakage current through a chromatographic column with a conductive or semi-conductive stationary phase can induce charging, which affects the flow and retention time of analytes within the column, as described below with respect to Figure 4 The performance degradation may occur even if the voltage drop across the chromatographic column is small (e.g., the voltage drop is as small as 10V). In addition, the presence of two liquid-metal interfaces (i.e., post-column fluid junction 122 and fluid union 104) creates two locations where flow disturbances may compromise the quality of analyte separation in the separated fluid sample leaving the chromatographic column because each liquid-metal interface contributes to the dispersion of the analyte bands, or so-called "band broadening".

[0027] The systems, methods and cartridges taught herein mitigate these problems of leakage current and band broadening by creating true non-zero voltage equipotentials (e.g., in the range of +1 kV and +10 kV or in the range of -1 kV and -10 kV) between the ends of a chromatographic column having a conductive or semiconductive stationary phase. In addition, because the systems taught herein reduce the number of post-column fluid junctions (e.g., fluid-metal interfaces) from two to one, band broadening effects are mitigated.

[0028] Figure 2 A schematic diagram of an electrospray system 200 with a chromatography column 302 having a conductive or semiconductive stationary phase is depicted according to some embodiments taught herein. Figure 2 In the figure, the dashed line connection between the components indicates the fluid connection, while the solid line connection between the components indicates the electrical connection. The fluid components of the electrospray system 200 include a pre-column fluid junction 206 (which can be a septum fitting or other fluid system inlet or interface in some instances), a pre-column fluid junction 204 (which can be a needle seat or fluid box inlet or interface in some instances), a pre-column fluid junction 304 (which can be an in-box fluid junction in some instances), a chromatographic column 302 having a conductive or semi-conductive stationary phase, a post-column fluid union 306, and an electrospray emitter 308. The electrospray system 200 may also include electronic components, such as a power supply 202, a current sensing circuit 210, and electrical conductors that electrically connect the components to each other. Specifically, the electrical conductor 314 connects the fluid union 306 and the fluid union 304 to equalize the potential between the fluid union 306 and the fluid union 304. By equalizing the potential (e.g., the high voltage potential V used in the electrospray emission) E ) is applied to both the fluid union portion 306 and the fluid confluence portion 304, further reducing or eliminating the possibility of current leakage and electrochemical interaction in the chromatographic column 302, while the chromatographic column 302 can float at a high potential.

[0029] The fluid sample enters the electrospray system 200 through the pre-column fluid 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 solvents, loading samples and running gradients. The fluid sample flows to the pre-column fluid junction 204 along the fluid path 218. The fluid sample then flows to the fluid junction 304 along the fluid path 220. In the embodiment where some of the components are assembled into a box form (described in more detail below), this is the point where the fluid sample has entered the box. The fluid sample flows from the fluid junction 304 along the fluid path 222 to the first end 318 of the chromatographic column 302. In the chromatographic column 302, the analyte of the fluid sample is temporarily separated in the fluid sample, so that the separated fluid sample (that is, the fluid sample including the separated analyte) is output from the second end 320 of the chromatographic column 302. For example, the chromatographic column 302 can be configured for, for example, 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 the first fluid port 310 of the fluid union 306. The fluid sample is applied with a high potential (V E ) ionization. The electrospray emitter 308 is connected to the second fluid port 312 of the fluid union 306. The ionized fluid stream is emitted from the electrospray emitter 308 as ionized droplets. For example, the electrospray emitter 308 may include a needle, and the droplets may be ejected through the distal tip of the needle. In some embodiments, the electrospray emitter 308 has an inner diameter of less than about 30 microns to achieve nanoESI. In some embodiments, the emitted ionized droplets leave the electrospray emitter 308 and travel toward the atmospheric pressure inlet to the mass spectrometer serving as the counter electrode 112. The electrospray current return is typically formed by the electrically grounded counter electrode 112, such as Figure 2 shown.

[0030] The power supply 202 is electrically connected to the high side 212 of the current sensing circuit 210 to measure the electrospray current generated by 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 may output a voltage of ±2 kV or ±8 kV. The low side 214 of the current sensing circuit 210 is connected to an electrical conductor 314, which is also connected to the fluid confluence 304 and the fluid union 306. In other words, the fluid confluence 304 and the fluid union 306 are electrically connected in parallel to each other through the electrical conductor 314, and the parallel circuit is connected in series with the current sensing circuit 210. Therefore, the electrical conductor 314 generates a true equipotential between the fluid confluence 304 and the fluid union 306 because there is no intermediate element (such as the current sensing circuit 210) between the two to generate a potential difference. Essentially, the two pre-column fluid junctions 206 and 208 form a pre-column voltage spacer that directs current around the chromatographic column to prevent current from flowing through the chromatographic column. As a result of using the pre-column voltage spacer, a small amount of voltage is dropped across the fluid path 220, while most of the voltage drop occurs along the fluid path 218. The use of the pre-column voltage spacer can be combined with Figure 1 This is in contrast to the post-column voltage spacer arrangement in conventional electrospray systems 100, 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. Due to the presence of two liquid-metal interfaces, Figure 1 The post-column voltage spacer also negatively induced band broadening.

[0031] In some embodiments, the leads of the electrical conductor 314 are directly connected to the outer conductive surface of the fluid union 306 to provide a current to the fluid flow sufficient to provide ionization therein. In some embodiments, the leads of the electrical conductor 314 are directly connected to the outer conductive surface of the fluid union 304. In some embodiments, the electrical conductor 314 may include a wire or cable and may include a shield or be a bare conductor.

[0032] Note that in some embodiments, the power supply 202 may be supplied as a combined system with the electrospray system 200. In other embodiments, the power supply 202 is not provided as part of the system, but rather the electrospray system 200 is designed to interface with a separately provided power supply 202. In some embodiments, the polarity of the power supply 202 is reversed so that the high voltage is applied to the counter electrode 112 and the return voltage lead of the power supply 202 is connected to the current sensing circuit 210.

[0033] The nanoESI plume typically carries a current between 50 nanoamperes and 500 nanoamperes. Thus, because nanoESI results in greater sensitivity and produces relatively small ion currents, these small ion currents are measured to a measurement resolution of 10 nanoamperes or better, because it is highly desirable to measure the electrospray current to a high degree of accuracy to ensure that the electrospray emitter 308 is operating in the correct electrospray regime. Because a voltage drop occurs across the current sensing circuit 210, the voltage V at the high side 212 is + and the voltage V at the low side 214 E Mismatch. Voltage V E The parallel connection is applied at the fluid union 306 and the fluid junction 304. In some embodiments, the electrical connector passes through a high voltage electrical connector 216, which can be mounted on the housing of the cassette 208, for example.

[0034] The negative terminal of the liquid chromatography system 108, the pre-column fluid junction 206 (e.g., the system inlet), the counter electrode 112, and the power supply 202 are connected to electrical ground. It should be understood that "electrical ground" as used herein may also include potentials operating at or near electrical ground or substantially electrical ground. For example, in some instances, electrical ground may be biased up to + / -150 volts, which represents a substantially grounded source when compared to the common mode voltage of + / -2 kilovolts to 10 kilovolts taught herein.

[0035] The chromatographic column 302 includes a conductive or semiconductive material or substrate that forms and / or supports a stationary phase. For example, the stationary phase of the chromatographic column 302 may include (or be formed of): silicon, gallium arsenide, gallium nitride, indium tin oxide, n-type or p-type semiconductor material, or a doped semiconductor. In other embodiments, the stationary phase of the chromatographic column 302 may include a conductive metal such as platinum or stainless steel. In some embodiments, the stationary phase of the chromatographic column 302 includes or is supported by a conductive or semiconductive substrate disposed between the first end 318 and the second end 320 of the chromatographic column 302. In some embodiments, the chromatographic column 302 may have a 1×10 -2 Ω●cm to 1×10 2 Resistivity in the Ω●cm range.

[0036] In some embodiments, the chromatographic column 302 may include micro-replicated structural features, such as a cylinder extending from a substrate in a microfluidic flow channel. The microfluidic structural features include conductive or semi-conductive materials (such as silicon), and can be generated using micron manufacturing techniques or nano-manufacturing techniques known to those of ordinary skill in the art (including, for example, using beam lithography or electron beam lithography and deep reactive ion etching). The cylinder forms the stationary phase of the chromatographic column. In some examples, the cylinder or other microfluidic structural features are formed by a silicon substrate by etching the space around the cylinder. Examples of devices suitable for use as chromatographic columns in the 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 is composed of rectangular channels, which have been 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 a column pre-fluid confluence 304 and / or a column post fluid union 306, can also be directly integrated into a microfluidic chip. In some examples, the fluid confluence 304 may be located on a chip directly adjacent to the first end 318 of the chromatographic column 302, thereby reducing or eliminating the fluid path 222. In some examples, the fluid union 306 may be located on a chip directly adjacent to the second end 320 of the chromatographic column 302, thereby reducing or eliminating the fluid path 224. In some embodiments, the silanols in the conductive or semiconductive stationary phase of the chromatographic column 302 are chemically terminated so that the secondary (e.g., electrochemical) interaction with the analyte is reduced. Similarly, the outer layer of the conductive or semiconductive stationary phase of some examples may be chemically treated to form a layer of different materials, such as by changing silicon to silicon oxide, or may be coated with a thin layer of other materials to reduce analyte interactions. In contrast to a chromatographic column with a non-conductive stationary phase (which typically includes a non-uniform packed bed of functionalized spherical particles (e.g., fused silica particles) as a stationary phase), the bed in a conductive or semiconductive microfluidic / microfabricated chromatographic column is formed by etching a column located at a uniform distance. Compared with a packed bed column, a microfluidic chromatographic column provides several advantages. First, peak dispersion (e.g., eddy dispersion) arising from non-uniform flow paths across the column cross-section that occur in non-uniform packed bed columns is reduced in chromatographic columns with microfabricated or deep reactive ion etched features (such as pillars) due to uniform spacing between features. Second, column permeability is increased. Finally, the uniformity of microfabricated chromatographic columns results in excellent column-to-column reproducibility.

[0037] In other embodiments, the chromatography column 302 may include other conductive or semiconductive stationary phase materials, such as porous graphitic carbon beads.

[0038] The use of chromatographic columns with conductive or semiconductive stationary phases in conjunction with electrospray emitters has been limited by problems arising from the conductivity of the stationary phase. For example, doped semiconductor substrates and materials (e.g., boron-doped semiconductors) include impurities that produce holes (unoccupied electronic states), making them electron acceptors. The substrate does not conduct these holes well, resulting in the accumulation of charge. In fact, the current flowing through the chromatographic column can charge the stationary phase in the chromatographic column, which in turn interacts with the analyte during separation. Similarly, having a voltage difference between the first end and the second end of the chromatographic column can produce electrochemically induced changes in the pH of the solvent in the fluid sample, which ultimately affects the stationary phase of the chromatographic column to deteriorate the chromatographic results. In addition, the potential difference between the fluid sample and the chromatographic column or between parts of the chromatographic column can promote undesirable electrochemistry, such as oxidation or reduction of solvents or analyte species in the fluid sample. These electrical problems are amplified at high voltages (i.e., kilovolts) because leakage currents are more difficult to ignore (become less negligible) under high voltage conditions. In some conventional systems, such as Figure 1 As shown, by introducing additional post-column fluid confluence 122 between post-column fluid union 104 and the second end of chromatographic column 302 to effectively make chromatographic column grounding, these shortcomings have been alleviated. Additional post-column fluid confluence can be electrically grounded, thereby preventing current from flowing into chromatographic column. However, each liquid-metal interface downstream of the chromatographic column in the system introduces additional dispersion in the chromatographic peak (that is, the work of chromatographic column separating analytes from each other is cancelled). Therefore, the additional post-column fluid confluence 122 of the conventional system introduces additional peak dispersion. Using the electric conductor 314 as taught herein to balance the potential across the chromatographic column solves these problems, while still enabling the chromatographic column to float at high voltage and enabling the high-resolution measurement (for example, to nanoampere level) of the electrospray current performed by the current sensing circuit 210. In addition, the only post-column liquid-metal interface in the electrospray system 200 occurs at the fluid union 306, where electrospray is initiated, thereby reducing the peak dispersion in the fluid sample.

[0039] In some embodiments, fluid or electronic components may be contained in box 208. As used herein, "contained" in the box indicates that the components are at least partially installed in the box, although some elements may extend from the outer housing or shell of the box. In some embodiments, in some cases, by integrating the column and the emitter into the box form, the box form also contains atomizing gas for consistent desolvation, an integrated heater for optimal chromatography, and a memory on the board that enables use as "intelligent consumables", the use of the box form can make expert-level liquid chromatography mass spectrometry (LCMS) performance more easily available. The integrated heater can provide thermal stability and reduce the back pressure level required to achieve suitable results. In some microfluidics and / or box-based examples, the integrated heater is particularly useful for reducing back pressure, because the components of box 208 can have a lower pressure limit than the pressure limit achieved by the liquid chromatography pump of the prior art. For example, the components of box 208 (such as chromatographic columns) can have a back pressure limit of 450 bar, and the integrated heater can achieve good performance even at a lower back pressure limit. In some embodiments, box 208 can be a removable and replaceable component inserted into or extracted from the electro-injection system 200. For example, a removable or replaceable box 208 can be inserted into a receiving slot in a system housing. The insertion of the box can facilitate the fluid connection between the pre-column fluid junction 204 and the fluid path 218, and removing the removable box 208 from the electro-injection system 200 can facilitate the disconnection of the fluid connection between the pre-column fluid junction 204 and the fluid path 218. Similarly, inserting the box into the electro-injection system 200 can facilitate the low side of the current sensing circuit 210 to be electrically connected to the electrical conductor 314 through the high voltage electrical connector 216, and removing the box 208 from the system can facilitate the disengagement or disconnection of the electrical contact between the current sensing circuit 210 and the high voltage electrical connector 216. In some embodiments, the housing or shell of box 208 is formed by a non-conductive material. In some embodiments, box 208 is sealed or can be sealed to prevent the user from contacting the high voltage present inside the box 208 during operation and improve the safe handling of the box.

[0040] Placing the fluid junction 304 and the fluid union 306 within the box 208 can advantageously provide additional electrical shielding of these components to ensure that voltage equipotential conditions are maintained and to improve safety by preventing users from making physical contact with high voltages. Even small resistivity differences along the electrical connections due to differences in the condition of the cables, electrical contacts, fluid unions and junctions, and the surface state of the fluid unions or junctions (such as the amount of oxidation on the surface) can produce measurable leakage currents that can affect chromatographic performance. Placing the fluid junction 304 and the fluid union 306 within the box 208 can prevent subsequent movement of these fluid parts when a pressurized fluid sample is passed through them, thereby ensuring improved electrical contact between the electrical conductor 314 and these components.

[0041] Figure 2 The current sensing circuit 210 is depicted as being located outside of the box 208. However, it is also contemplated that the current sensing circuit 210 may be located within the box 208. In such embodiments, the current sensing circuit 210 is placed in series between the high voltage electrical connector 216 and the electrical conductor 314. For example, the power source 202 and the high side 212 of the current sensing circuit 210 may be connected through the high voltage electrical connector 216 and the low side 214 of the current sensing circuit 210 may be directly connected to the electrical conductor 314 without an intermediate high voltage electrical connector 216.

[0042] In some embodiments, the column pre-fluid junction 204 may have an inner diameter in the range of 50 microns to 500 microns. In an exemplary embodiment, the column pre-fluid junction 204 may have an inner diameter of 280 microns. In some embodiments, the fluid junction 304 may include one or more conductive materials. For example, the fluid junction 304 may include a noble metal or stainless steel. In some embodiments, the fluid junction 304 or the fluid union 306 may include a multi-port pipe fixture or fittings known to those skilled in the art. In some embodiments, the fluid junction 304 and the fluid union 306 are the same parts in terms of material composition, shape, or both material composition and shape. In actual implementation, different electrical connections and fluid connections may produce different resistivities. By using the same material or parts of the same shape for the fluid junction 304 and the fluid union 306, the difference in contact resistance can be reduced. Undesirable ground currents may appear in the system such as through the shell. In some embodiments, the shell is formed by a non-conductive material.

[0043] In some embodiments, the pre-column fluid junction 204 and the fluid junction 304 can be merged into a combined fluid junction. In such embodiments, fluid path 220 is eliminated and the combined fluid junction is maintained at the transmitter voltage V E Down.

[0044] In some embodiments, the fluid union 306 may include one or more conductive materials. For example, the fluid union 306 may include stainless steel. Figure 2 and Figure 3The depicted fluid union 306 is a dual-port device (i.e., the fluid union 306 includes a first fluid port 310 and a second fluid port 312). However, in the LC-MS procedure, it may be desirable to add certain chemicals to the mobile phase or introduce certain chemicals into the separated fluid sample to affect analyte ionization in order to improve the analyte signal after the separated fluid sample leaves the chromatographic column but before the electrospray emission. Alternatively, some additives can be used to suppress undesirable signals or selectively enhance the signal of a specific compound in the mixture. Therefore, in some configurations, the fluid union 306 may include additional fluid ports (e.g., a third fluid port, a fourth fluid port, etc.) to receive additional fluid streams (e.g., a second fluid, a third fluid, etc.), which are mixed in the fluid union 306 with the fluid sample received from the chromatographic column 302. In various embodiments, the additional fluid streams may each be derived from a separate reservoir or from an additional fluid column such as an additional chromatographic column. In the embodiment where the additional fluid port of the fluid union receives additional fluid stream from the additional chromatographic column, the electrical conductor 314 can extend to be connected to the additional fluid junction upstream of the additional chromatographic column. Therefore, equipotential can be established between the fluid union 306, the fluid junction 304 and these additional fluid unions. In some embodiments, the additional fluid stream can include a post-column additive. Post-column additives (PCA) can be used to affect the electrospray ionization process in various ways, such as promoting desolvation, changing charge state distribution or other known methods, and they can be solvents or other chemical moieties.

[0045] In various embodiments taught herein, fluid paths 218, 220, 222, 224 are formed by flexible or rigid tubing. In some embodiments, fluid paths 218, 220, 222, 224 may include paths or capillaries formed at least in part by fused silica or plastic / polymer materials. Fluid paths 218, 220, 222, 224 may include capillaries with inner coatings such as polyetheretherketone (PEEK). In some embodiments, fluid path 218 may have an inner diameter in the range of 10 microns to 50 microns or in the range of 20 microns to 40 microns. In some embodiments, fluid path 218 may have an inner diameter of 30 microns. Fluid path 218 may have a length in the range of 10 cm to 50 cm or in the range of 20 cm to 40 cm. In some embodiments, fluid path 218 may have a length of 35 cm. In some embodiments, fluid path 220 may have an inner diameter in the range of 10 microns to 50 microns or in the range of 20 microns to 40 microns. In some embodiments, the fluid path 220 may have an inner diameter of 30 microns. The fluid path 220 may have a length in the range of 10 mm to 50 mm, or in the range of 20 mm to 40 mm, or in the range of 50 mm to 100 mm. In some embodiments, the fluid path 220 may have a length of 35 mm.

[0046] In some embodiments, fluid path 222 may have an inner diameter in the range of 5 microns to 50 microns. In some embodiments, fluid path 222 may have an inner diameter of 5 microns, 10 microns, 20 microns, 25 microns, 30 microns, or 40 microns. Fluid path 222 may have a length in the range of 10mm to 50mm, or in the range of 20mm to 40mm, or in the range of 50mm to 100mm. In some embodiments, fluid path 222 may have a length of 35mm. In some embodiments, fluid path 224 may have an inner diameter in the range of 5 microns to 50 microns. In some embodiments, fluid path 224 may have an inner diameter in the range of 5 microns, 10 microns, 20 microns, 25 microns, 30 microns, or 40 microns. Fluid path 224 may have a length in the range of 10mm to 50mm, or in the range of 20mm to 40mm, or in the range of 50mm to 100mm. In some embodiments, fluid path 224 may have a length of 35mm. Fluid path 224 may 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 microns or 10 microns, while fluid paths 218, 220 have an inner diameter of 30 microns.

[0047] In some embodiments, box (more specifically, same chromatographic column and electrospray emitter) can be used for hundreds of injections. However, a given chromatographic column tends to have a longer service life than a given electrospray emitter. Therefore, in some embodiments, the first fluid port 310, the second fluid port 312 of the fluid union 306 or both the first fluid port and the second fluid port are configured to enable the disconnection and reconnection of the electrospray emitter attached thereto or to realize fluid path. In such embodiments, the electrospray emitter (or the fluid union 306) can be removed from box 208 and replaced by a new electrospray emitter 308 or fluid union 306. The ability of this exchange emitter makes it possible to make full use of the life of the column. In some embodiments, fluid path 222 and fluid path 224 are configured to be disconnected and reconnected from the first end 318 and the second end 320 of the chromatographic column 302, respectively. By having a fluid path that can be removed, the chromatographic column 106 can be removed from the box and replaced.

[0048] Figure 3 An electrospray system 300 with a chromatography column 302 having a conductive or semiconductive stationary phase according to some embodiments taught herein is depicted. The electrospray system 300 includes a chromatography column 302 having a first end 318 and a second end 320, a post-column fluid union 306, a pre-column fluid junction 304, and an electrospray emitter 308. The post-column fluid union 306 and the pre-column fluid junction 304 are connected by an electrical conductor 314. The electrical connection of the fluid union 306 to the fluid junction 304 via the electrical conductor 314 generates a true equipotential between the first end 318 and the second end 320 of the chromatography column 302.

[0049] In some embodiments, the electrospray system 300 may include a housing that shields and protects the components in the housing. In some embodiments, the housing may be shaped as a box that can be inserted into a larger system in a removable manner. The housing may include an inlet port 316 for fluid that is connected to the fluid junction 304. The housing may also include a high voltage electrical connector 322 mounted thereon or passed therethrough. Power supply 202 may be used to apply voltage to the high voltage electrical connector 322. The fluid sample is supplied to the inlet port 316 from a fluid source. The fluid sample flows through the main body of the inlet port 316 and the fluid junction 304 along fluid path 222 to reach the first end 318 of the chromatographic column 302. In the chromatographic column 302, the analyte of the fluid sample is temporarily separated in the fluid sample, so that the separated fluid sample (that is, the fluid sample including the separated analyte) is output from the second end 320 of the chromatographic column 302. The separated fluid sample travels to the first fluid port 310 of the fluid union 306 along the fluid path 224. The fluid sample is applied with a high potential (V +) ionization. The electrospray emitter 308 is connected to the second fluid port 312 of the fluid union 306. The ionized fluid stream is emitted from the electrospray emitter 308 as ionized droplets. For example, the electrospray emitter 308 may include a needle, and the droplets may be ejected through the distal tip of the needle. In some embodiments, the electrospray emitter 308 has an inner diameter of less than about 30 microns to achieve nanoESI. In some embodiments, the emitted ionized droplets leave the electrospray emitter 308 and travel toward the atmospheric pressure inlet to the mass spectrometer serving as the counter electrode 112. The electrospray current return is typically formed by the electrically grounded counter electrode 112, such as Figure 3 shown.

[0050] In the absence of a current sensing circuit, the electric injection system 300 can receive a high voltage V from the power supply 202. + And the high voltage V is transmitted through the electrical conductor + Directly distributed to the fluid confluence portion 304 and the fluid union portion 306. In other embodiments, Figure 2 , Figure 5 and / or Figure 6 A current sensing circuit similar to the current sensing circuit described above may be placed in the circuit between the power source 202 and the high voltage electrical connector 322. Note that in some embodiments, the power source 202 may be supplied as a combined system with the electric injection system 300. In other embodiments, the electric injection system 300 is designed to interface with a separately provided power source 202.

[0051] Figure 4 A graph showing the retention time of a particular analyte (specifically, a peptide with 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 repeated experiments under different conditions. These experiments demonstrate the effect on the analyte charge retention time due to non-isopotential voltage conditions on a chromatography column having a conductive or semiconductive stationary phase. Note that, for example, Figure 1 In the electrospray system 100 of FIG. 1 , a voltage difference (or voltage drop) of tens of volts across the chromatographic column can easily occur due to the high voltage (e.g., up to 8 kV) applied to the emitter. For example, for a current sensing resistor with a resistance of 10 MΩ and an electrospray current of 5 μA, a difference of 50 V across the current sensing circuit 116 and thereby across the Figure 1 The chromatographic column 106 of the electrospray system 100 is shown.

[0052] Under the first condition 402, the chromatographic column is grounded (i.e., the chromatographic column is isolated from the high voltage applied to the electrospray emitter by a grounded fluid junction placed between the emitter and the column). Under the second condition 404, the chromatographic column 302 is floated under a high voltage (i.e., a voltage sufficient to induce electrospray ionization of the fluid sample), wherein there is an isoelectric condition between the first end and the second end of the chromatographic column, as taught in various embodiments taught herein. As described above, the system taught herein can achieve floating of the chromatographic column without using a second fluid junction downstream of the chromatographic column, which induces peak dispersion. Across both the first condition 402 and the second condition 404, the average retention time is relatively stable and does not evolve or change with repeated experiments.

[0053] Under the third condition 406, the fourth condition 408, and the fifth condition 410, the voltage differences of +1.6V, +9.6V, and +19.2V are respectively located at the fluid confluence / combination portion (e.g., Figure 1 The fluid confluence 102 and the fluid union 104 of the system are intentionally induced, as would occur in a conventional system such as the electrospray system 100. Figure 4 As shown, the stability of analyte retention times decreases significantly over repeated experiments due to charge accumulation in the semiconducting stationary phase of the chromatographic column. Therefore, experimental results from early runs in an experimental series can no longer be reliably compared with experimental results from later runs in the experimental series.

[0054] After the fifth condition 410 is applied to the chromatographic column 302, the experimental condition returns to the second condition 404, wherein the chromatographic column 302 is floated at a high voltage, wherein equal voltages are applied to the first end 318 and the second end 320 (e.g., as occurs in the electrospray system 300, wherein equal voltages are applied to the fluid confluence 304 and the fluid union 306). It can be seen that retention time repeatability recovers with some early experimental runs to return to stable retention times as further experimental runs proceed. Finally, the chromatographic column 302 is subjected to the sixth condition 412, wherein an intentional voltage difference of -19.2 V is applied between the first end 318 and the second end 320 of the chromatographic column 302. Again, retention times become unstable and vary between experiments when the accumulated charge within the semiconductive chromatographic column 302 begins to affect the analyte retention times within the chromatographic column.

[0055] Table 1 below gives the Figure 4 The coefficient of variation measured for each of the experimental conditions plotted. As these values ​​show, the experimental variation is significantly reduced under conditions where the first and second ends of the chromatographic column are at equal potential.

[0056]

[0057] Figure 5 An example current sensing circuit 210 for use with embodiments of the systems, methods, and boxes taught herein is depicted. The current sensing circuit 210 may include a current sensing resistor 510 disposed between a high side 212 and a low side 214 of the current sensing circuit 210. In some embodiments, the current sensing circuit 210 consists only of the current sensing resistor 510, a first electrical lead connecting the high side 212 of the current sensing circuit 210 to a voltmeter, and a second electrical lead connecting the low side 214 of the current sensing circuit 210 to the voltmeter. In such an arrangement, the voltmeter can measure high voltages with high accuracy. To reduce the cost of the current sensing circuit 210, additional electronic components (such as Figure 5 Additional electronic components (as shown) can be used to attenuate the voltage levels so that data acquisition electronics compatible with modern computing equipment can be used.

[0058] exist Figure 5 In the illustrated embodiment, the current sensing circuit 210 includes a current sensing 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 sensing resistor 510 is severely attenuated via a precision voltage divider (502, 504) to meet typical data acquisition and computer voltage potentials. In one embodiment, the precision voltage dividers 502, 504 operate at a step-down ratio of 1,000:1. The voltage obtained at the output of the voltage divider 502, 504 can be subtracted using a differential amplifier 506. The difference obtained at the output 514 of the current sensing circuit 210 provides a direct proportional measurement of the ESI injection current. In various embodiments, the resistance of the current sensing resistor 510 can be in the range of 10kΩ to 10GΩ, in the range of 1MΩ to 1GΩ, or in the range of 10MΩ to 100MΩ.

[0059] Optionally, the controller 508 may be coupled to the output 514 of the current sensing circuit 210. The controller 508 may be configured to receive the output current directly from the output 514 or a digitized representation of the output 514. In some embodiments, the controller 508 determines the state of the electrospray emitter based on the measured current. Specifically, the state of the electrospray emitter includes an operating mode within a specific electrospray scheme.

[0060] In some embodiments, the controller 508 includes a user interface 512 that is configured to display or otherwise alert a user or operator of the determined electrospray scheme. In other embodiments, the controller 508 may interface with an external user interface. In some embodiments, the controller 508 may determine the health of the cartridge 208, for example, by tracking and identifying changes in the output current relative to the applied voltage over time. If the change is significant (e.g., exceeds a threshold limit), the controller 508 may indicate to the user that the cartridge 208 or a portion of the cartridge 208 (such as a chromatography column 302 or an electrospray emitter 308) should be replaced.

[0061] exist Figure 5 In the current sensing circuit 210, the current measurement is obtained with high accuracy using relatively inexpensive electronics without an isolation amplifier. One difficulty with direct high-side measurement techniques (i.e., measurement of the electrospray current at 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 severely attenuated in order to protect downstream electronics. Another solution used to overcome these difficulties is to protect downstream electronics by isolating or electrically floating the large common-mode supply voltage and sensing circuits. The measured quantity can be transferred to downstream electronics across the isolation barrier using modulation and demodulation techniques.

[0062] Figure 6 An alternative current sensing circuit 210 is schematically depicted as an isolation amplifier circuit suitable for use with some embodiments taught herein. The isolation amplifier circuit can electrically float a large input voltage. In this embodiment, the current sensing circuit 210 includes a current sensing resistor 510. The high side 212 and the low side 214 of the current sensing 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 sensing circuit 210, which can be optionally connected to a controller 508, as described above with respect to Figure 5 described. Figure 6 The current sensing circuit 210 described in also benefits from using an isolated power supply 202 for the floating amplifier and a modulation and demodulation means across an isolation barrier (not shown).Modulation and demodulation may introduce system noise.

[0063] Other embodiments of current sensing circuits and current monitoring circuits suitable for use with embodiments of the systems, methods, and boxes taught herein are described in U.S. Patent Application Publication No. 2023 / 0030920 to Lindseth et al., the entire contents of which are incorporated herein by reference.

[0064] Figure 7 A routine 700 for reducing electrochromatographic effects in an electrospray system according to various embodiments taught herein is depicted. In box 702, the routine 700 causes a fluid sample to flow from a first pre-column fluid junction 304 to a first end 318 of a chromatographic column 302. The chromatographic column 302 includes a conductive or semi-conductive stationary phase disposed between the first end 318 and the second end 320 of the chromatographic column. In box 704, the routine 700 uses the chromatographic column 302 to separate analytes within the fluid sample. In box 706, the routine 700 outputs the separated fluid sample from the second end of the chromatographic column to a first fluid port 310 of a post-column fluid union 306. The fluid union 306 has a second fluid port 312 connected to an electrospray emitter 308. In box 708, the routine 700 applies an equipotential voltage in the range of +1 kV to +10 kV or in the range of -1 kV to -10 kV at both the first pre-column fluid junction 304 and the post-column fluid junction 306 using an electrical conductor 314 in electrical contact with the first pre-column fluid junction and the post-column fluid junction. In optional box 710, the routine 700 applies a first voltage to the high side 212 of the current sensing circuit 210 using the power supply 202. In optional box 712, the routine 700 transfers the equipotential voltage from the low side 214 of the current sensing circuit 210 to the electrical conductor 314.

[0065] Many of the embodiments taught herein are described in a replaceable ESI cassette (e.g., Figure 2 The present invention is described in the context of a cartridge 208 in the system. However, the techniques taught herein may also be implemented in other systems that do not include the cartridge. In such embodiments without the cartridge, the system still advantageously provides the speed and simplicity of replacing the electrospray emitter (e.g., by disconnecting the needle from the fluid union) without disturbing or otherwise disassembling the remaining fluid and electrical connections in the system.

[0066] Although the present system, method and box and the associated advantages have been described in detail, it should be understood that various changes, substitutions and modifications may be made 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 specific embodiments of the processes, machines, manufactures, material compositions, modes, methods and steps described in the specification. As will be readily understood by those of ordinary skill in the art from the present disclosure, according to the present disclosure, currently existing or later to be developed processes, machines, products, material compositions, components, methods or steps that perform substantially the same functions as the corresponding embodiments taught herein or achieve substantially the same results as the corresponding embodiments taught may be utilized. Therefore, the appended claims are intended to include such processes, machines, manufactures, material compositions, modes, methods or steps within their scope.

[0067] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described in any way.

[0068] In the detailed description of various embodiments, for the purpose of explanation, many specific details are set forth to provide a thorough understanding of the disclosed embodiments. However, it will be understood by those skilled in the art that these various embodiments may be practiced with or without these specific details. In other examples, structures and devices are shown in block diagram form. In addition, it will be readily appreciated by those skilled in the art that the specific order of presenting and executing methods is exemplary, and it is contemplated that the order may be varied (unless otherwise explicitly stated) and still remain within the spirit and scope of the various embodiments disclosed herein.

[0069] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and internet web pages, are expressly incorporated by reference in their entirety for any purpose. Unless otherwise described, 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.

[0070] It should 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 there are slight and insubstantial deviations within the scope of the present teachings. In this application, unless otherwise specifically provided, the use of the singular includes the plural. In addition, the use of "comprising", "including" and "containing" is not intended to be limiting. It should be understood that both the foregoing general description and the following detailed description are merely exemplary and illustrative, and are not limitations of the present teachings.

[0071] As used herein, "a" or "an" may also mean "at least one" or "one or more." In addition, the use of "or" is inclusive, such that the phrase "A or B" is true when "A" is true, "B" is true, or both "A" and "B" are true. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0072] "System" describes a set of real or abstract components comprising a whole, in which each component interacts or relates to at least one other component within the whole.

[0073] The advantages and features of the present disclosure are further described by the following examples:

[0074] Embodiment 1. An electrospray system, the electrospray system comprising: a first pre-column fluid junction for receiving a fluid sample; a chromatographic column for separating analytes in the fluid sample, the chromatographic column comprising a conductive or semi-conductive stationary phase arranged between a first end and a second end of the chromatographic column, the first end being fluidically connected to the first pre-column fluid junction; a post-column fluid junction having a first fluid port and a second fluid port, the first fluid port receiving the analytes separated from the fluid sample from the second end of the chromatographic column; an electrospray emitter fluidically connected to the second fluid port; and an electrical conductor connected to the post-column fluid junction and the first pre-column fluid junction to equalize the potential between the post-column fluid junction and the first pre-column fluid junction.

[0075] Embodiment 2. The electric injection system according to embodiment 1 further comprises 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.

[0076] Embodiment 3. The electronic injection system according to embodiment 2 further comprising a power supply electrically connected to the high side of the current sensing circuit.

[0077] Embodiment 4. According to the electrospray system of embodiment 3, the electrospray system also includes a second pre-column fluid junction for transporting the fluid sample to the first pre-column fluid junction, wherein the power supply is electrically connected to supply a high voltage to the second pre-column fluid junction.

[0078] Embodiment 5. The electrospray system according to embodiment 1, wherein the post-column fluid junction and the first pre-column fluid junction are electrically connected in parallel with each other and in series with a current sensing circuit.

[0079] Embodiment 6. The electrospray system of embodiment 1, wherein the fluid path connecting the second end of the chromatography column and the post-column fluid union has an inner diameter of 30 microns or less to reduce peak broadening.

[0080] Embodiment 7. An electrospray system according to embodiment 1, wherein the chromatographic column, the post-column fluid junction, the electrospray emitter, the electrical conductor and the first pre-column fluid junction are contained in a replaceable box.

[0081] Embodiment 8. An electric injection system according to embodiment 7, wherein the box includes a high voltage electrical connector, which is electrically connected to the electrical conductor or to a current sensing circuit connected in series with the electrical conductor.

[0082] Embodiment 9. An electric injection system according to embodiment 8, wherein the current sensing circuit is housed in the replaceable box.

[0083] Embodiment 10. The electrospray system of embodiment 7, wherein the replaceable cartridge comprises a housing that electrically shields the first pre-column fluid junction and the post-column fluid union.

[0084] Embodiment 11. An electrospray system according to Embodiment 1, wherein the length of the fluid path between the first pre-column fluid junction and the chromatographic column and the length of the fluid path between the chromatographic column and the post-column fluid junction are both less than 100 mm.

[0085] Embodiment 12. The electric injection system according to embodiment 1 further comprises a third pre-column fluid junction electrically connected to ground, wherein the second pre-column fluid junction and the third pre-column fluid junction form a pre-column voltage spacer.

[0086] Example 13. A method for reducing the electrochromatographic effect in an electrospray system, the method comprising: allowing a fluid sample to flow from a first pre-column fluid junction to a first end of a chromatographic column, the chromatographic column comprising a conductive or semi-conductive stationary phase disposed between the first end and the second end of the chromatographic column; using the chromatographic column to separate analytes in the fluid sample; outputting the separated fluid sample from the second end of the chromatographic column to a first fluid port of a post-column fluid union, the post-column fluid union having a second fluid port connected to an electrospray emitter; and applying an equipotential voltage in the range of +1 kV to +10 kV or in the range of -1 kV to -10 kV at both the first pre-column fluid junction and the post-column fluid union using an electrical conductor electrically contacting the first pre-column fluid junction and the post-column fluid union.

[0087] Embodiment 14. The method of embodiment 13, further comprising: applying a first voltage to a high side of a current sensing circuit using a power supply; and transferring the equipotential voltage from a low side of the current sensing circuit to the electrical conductor.

[0088] Embodiment 15. The method according to embodiment 14, wherein the post-column fluid junction and the first pre-column fluid junction are electrically connected in parallel with each other and in series with the current sensing circuit.

[0089] Embodiment 16. A method according to embodiment 13, wherein outputting the separated fluid sample from the second end of the chromatographic column to the first port of the post-column fluid union includes passing the separated fluid sample through a fluid path having an inner diameter of 30 microns or less to reduce peak broadening.

[0090] Embodiment 17. The method of Embodiment 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.

[0091] Example 18. A method according to Example 13, wherein the chromatographic column, the post-column fluid junction, the electrospray emitter, the electrical conductor and the first pre-column fluid junction are contained in a replaceable box, and the method also includes: connecting the second pre-column fluid junction of the box to the source of the fluid sample; and allowing the fluid sample to flow from the second pre-column fluid junction to the first pre-column fluid junction.

[0092] Embodiment 19. The method of Embodiment 18, wherein the box includes a high voltage electrical connector electrically connected to the electrical conductor, the method further comprising: electrically connecting a power source to the high voltage electrical connector.

[0093] Embodiment 20. The method of Embodiment 14, wherein the first voltage is greater than the equipotential voltage.

[0094] Embodiment 21. A box for inserting into an electrospray system, the box comprising: a first pre-column fluid junction for receiving a fluid sample; a chromatographic column for separating analytes in the fluid sample, the chromatographic column comprising a conductive or semi-conductive stationary phase arranged between a first end and a second end of the chromatographic column, the first end being fluidically connected to the first pre-column fluid junction; a post-column fluid union having a first fluid port and a second fluid port, the first fluid port receiving the analytes separated from the fluid sample from the second end of the chromatographic column; an electrospray emitter fluidically connected to the second fluid port; and an electrical conductor connected to the post-column fluid union and the first pre-column fluid junction to equalize the potential between the post-column fluid union and the first pre-column fluid junction.

Claims

1. An electric injection system, the electric injection system comprising: a first pre-column fluid junction for receiving a fluid sample; a chromatographic column for separating analytes in the fluid sample, the chromatographic column comprising a conductive or semi-conductive stationary phase disposed between a first end and a second end of the chromatographic column, the first end being fluidically connected to the first pre-column fluid junction; a post-column fluid junction having a first fluid port and a second fluid port, the first fluid port receiving the separated analytes in the fluid sample from the second end of the chromatographic column; an electrospray emitter fluidly connected to the second fluid port; and An electrical conductor connected to the post-column fluid junction and the first pre-column fluid junction to equalize the potential between the post-column fluid junction and the first pre-column fluid junction. 2 . The electronic injection 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 electronic injection 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 fluid junction for delivering the fluid sample to the first pre-column fluid junction, wherein the power supply is electrically connected to supply a high voltage to the second pre-column fluid junction.

5. The electric injection system of claim 1, wherein the post-column fluid union section and the first pre-column fluid confluence section are electrically connected in parallel to each other and are connected in series with a current sensing circuit.

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

7. The electrospray system of claim 1, wherein the chromatographic column, the post-column fluid junction, the electrospray emitter, the electrical conductor, and the first pre-column fluid junction are housed in a replaceable cartridge.

8. An electric injection system according to claim 7, wherein the box includes a high voltage electrical connector, which is electrically connected to the electrical conductor or to a current sensing circuit connected in series with the electrical conductor.

9. An electric injection system as claimed in claim 8, wherein the current sensing circuit is housed in the replaceable box.

10. The electrospray system of claim 7, wherein the replaceable cartridge comprises a housing that electrically shields the first pre-column fluid junction and the post-column fluid union.

11. The electrospray system according to claim 1, wherein the length of the fluid path between the first pre-column fluid junction and the chromatographic column and the length of the fluid path between the chromatographic column and the post-column fluid junction are both less than 100 mm.

12. The electric injection system of claim 1, 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. A method for reducing electrochromatographic effects in an electrospray system, the method comprising: Allowing a fluid sample to flow from a first pre-column fluid junction to a first end of a chromatographic column, the chromatographic column comprising a conductive or semi-conductive stationary phase disposed between the first end and a second end of the chromatographic column; separating analytes in the fluid sample using the chromatographic column; outputting the separated fluid sample from the second end of the chromatography column to a first fluid port of a post-column fluid union having a second fluid port connected to an electrospray emitter; and An equipotential voltage in the range of +1 kV to +10 kV or in the range of -1 kV to -10 kV is applied at 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. The method according to claim 13, further comprising: applying a first voltage to a high side of a current sensing circuit using a power supply; as well as The equipotential voltage is transferred from a low side of the current sensing circuit to the electrical conductor. 15 . The method of claim 14 , wherein the post-column fluid junction and the first pre-column fluid junction are electrically connected in parallel to each other and in series with the current sensing circuit.

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

17. 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 method of claim 13, wherein the chromatography column, the post-column fluid junction, the electrospray emitter, the electrical conductor, and the first pre-column fluid junction are contained in a replaceable cartridge, the method further comprising: connecting a second pre-column fluid junction of the cartridge to a source of the fluid sample; as well as The fluid sample is made to flow from the second pre-column fluid junction to the first pre-column fluid junction.

19. The method of claim 18, wherein the box includes a high voltage electrical connector electrically connected to the electrical conductor, the method further comprising: A power source is electrically connected to the high voltage electrical connector.

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

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

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