[go: up one dir, main page]

WO2014146147A2 - Ensemble cuve à circulation de multi-mesure pour chromatographie en phase liquide - Google Patents

Ensemble cuve à circulation de multi-mesure pour chromatographie en phase liquide Download PDF

Info

Publication number
WO2014146147A2
WO2014146147A2 PCT/US2014/038070 US2014038070W WO2014146147A2 WO 2014146147 A2 WO2014146147 A2 WO 2014146147A2 US 2014038070 W US2014038070 W US 2014038070W WO 2014146147 A2 WO2014146147 A2 WO 2014146147A2
Authority
WO
WIPO (PCT)
Prior art keywords
flow cell
light
conductivity
optical flow
cell system
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2014/038070
Other languages
English (en)
Other versions
WO2014146147A3 (fr
Inventor
Nicholas Demarco
Steven R. TJENSVOLD
Bryant J. DEAKINS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PRACTICHEM LLC
Original Assignee
PRACTICHEM LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by PRACTICHEM LLC filed Critical PRACTICHEM LLC
Priority to EP14764986.7A priority Critical patent/EP2972154A4/fr
Publication of WO2014146147A2 publication Critical patent/WO2014146147A2/fr
Publication of WO2014146147A3 publication Critical patent/WO2014146147A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/06—Illumination; Optics
    • G01N2201/066—Modifiable path; multiple paths in one sample
    • G01N2201/0668—Multiple paths; optimisable path length

Definitions

  • Detectors such as UV absorbance detectors, are measuring instruments used in liquid chromatography to detect and/or measure constituents of a liquid solution.
  • a flow cell comprises part of a detector and defines the sensor volume of the detector. The flow cell's internal cavity, through which the liquid specimen flows, exposes properties of the liquid to measuring apparatus.
  • An optical flow cell allows transmission of ultraviolet or visible light through liquid flowing therein.
  • the liquid's absorbance can be measured for the purpose of detecting the presence, and determining the concentration, of certain chemical compounds, such as species of protein, DNA or small drug molecules.
  • Absorbance is calculated using Beer-Lambert's law:
  • ⁇ is molar absorbtivity of the analyte (L mol 1 cm "1 )
  • b optical path length (cm);
  • P is the transmitted radiant flux
  • the optical flow cell is typically defined by a bore in the interior of a body, comprising a fluid inlet and fluid outlet and establishing the flow path for liquid wherein absorbance is measured.
  • the optical flow cell body typically includes a second bore, defining an optical path which allows light to pass through at least a portion of the liquid flow path. A portion of the liquid flow path is shared with the optical path and usually runs parallel to the optical path. Windows, with the aid of a sealing gasket, seal against the ends of the second bore, allowing light to pass while retaining the liquid inside the optical flow cell body.
  • the windows are often made from fused silica or another material optically transparent at the measured wavelengths, compatible with the liquid and sufficiently strong to withstand the internal flow cell cavity forces.
  • the detector body includes one or more grooves or slots on each end of the optical path where the optical path end face joins a window.
  • the groove directs fluid into one open end of the optical path and out the opposing end.
  • This combination of geometric features promotes liquid flow through the entire optical path and minimizes mixing of the currently passing liquid with liquid that had flowed through the cavity earlier.
  • a window sealing gasket made from a compliant material compatible with the liquid, like PTFE (DuPont Teflon®) or an elastomeric material like perfluoroelastomer (DuPont Kalrez®), silicone or rubber (Buna-N) is disposed between the window and the cell body as a seal, retaining the liquid in the flow cell.
  • Light absorption detectors often employ a broad spectrum light source coupled to a monochromator to select each wavelength of interest.
  • Other light absorption detectors use a broad spectrum lamp and a holographic grating to split the transmitted light into constituent wavelengths, then measure the transmitted wavelengths with a linear array of light sensitive elements such as photodiodes or charge-coupled devices (CCDs).
  • CCDs charge-coupled devices
  • Electrolytic conductivity of a liquid solution is often related to the ionic concentration of a solution. Measured in conjunction with light absorbance, conductivity provides additional information about the liquid, such as salt concentration and protein concentration. Conductivity is usually determined by measuring the resistance across two electrodes. Dilute solutions, typical of liquid chromatography, follow Kohlrauch's law: m ⁇ cation cation anion cation
  • a °° m is the molar conductivity of an electrolyte
  • Vcation and Vanion are the numbers of cations and anions per formula unit respectively;
  • X °° cat ion and X °° cat ion are the molar conductivities of the cation and anion at infinite dilution respectively.
  • Typical conductivity flow cells include two metal disks including holes in their centers through which the liquid solution flows.
  • the metal disks are separated by an insulator, preventing electrical conduction between the optical flow cell bodies other than through the liquid solution. Thereby, the disks act as electrodes for measuring the conductivity of the liquid.
  • Two conductive surfaces separated by an insulator form a capacitor.
  • the two conductivity electrodes meet this definition and, therefore, form a capacitor.
  • the capacitance of the conductivity electrodes affects the conductivity readings.
  • the present invention provides such flow cells, methods for their use, and liquid chromatography systems including such flow cells.
  • a flow cell system in one embodiment, includes first and second optical flow cell bodies, each of which includes an inlet port, an outlet port, and a fluid passage through the optical flow cell body connecting the inlet port to the outlet port.
  • a housing supports the first optical flow cell body and the second optical flow cell body, such that the first and second optical flow cell bodies are in series, with the outlet port of the first optical flow cell body adjacent to the inlet port of the second optical flow cell body.
  • the flow cells are stackable, that is, connected fluidically in series, each with its own housing.
  • a first conductive material is adjacent to the outlet port of the first optical flow cell body, positioned such that would be in contact with any fluid passing through the flow cell, a second conductive material is adjacent to the inlet port of the second optical flow cell body, positioned such that would be in contact with any fluid passing through the flow cell, and an insulator between the first and second conductive materials.
  • the optical flow cell body can be formed from a conductive material.
  • the conductive materials can be physically pressed against the flow cell bodies, or they can be attached to the optical flow cell bodies, mechanically or chemically.
  • a female part at the outlet port of the first optical flow cell body or the inlet port of the second optical flow cell body can be adapted to receive a male part of a conductive material, so that the two are physically mated.
  • the conductive material can be adhered to one or both of the optical flow cell bodies, such as by an epoxy resin or other suitable adhesive that resists degradation under the conditions of use, namely, high pressure, organic solvents, and the like.
  • the simplest conductive materials are washers, including metallic washers or washers made of or covered by conductive substances (such as conductive polymers or coatings or platings).
  • the optical flow cell bodies can include wires, metal plating, and the like.
  • a portion of the flow cell is plated with a metal, such as gold.
  • the conductivity measuring system can further include an alternating voltage source (AC source).
  • the AC source is connected, directly or indirectly, to the first and second conductive materials, so as to complete a circuit.
  • the AC source is capable of alternating at more than one frequency.
  • the presence of the two conductive electrodes with an insulator disposed between them allows one to perform conductivity measurements when a) a liquid solution is passed through the flow cell electrodes and b) the conductive materials are connected to an AC source to drive current through the materials.
  • the liquid solution completes the circuit between the conductive materials, and the current through the circuit changes as the concentration of proteins or other molecules of interest in the liquid solution changes.
  • the conductivity measurement system comprises more than two conductive material electrodes, such as 3, 4, 5, 6, or more conductive material electrodes, which, when connected to an AC source, function as reference electrodes.
  • the initial electrodes are formed by the conductive materials at the outlet port of the first optical flow cell body and the inlet port of the second optical flow cell body
  • an additional two electrodes can be formed by placing conductive materials at the inlet port of the first optical flow cell body, and the outlet port of the second optical flow cell body.
  • the AC source operates at varying frequencies depending on the measured value of conductance. By varying the AC source frequency, cell capacitive effects become apparent and separately measurable. These capacitive effects can be subtracted from the measurement to improve measurement accuracy.
  • the flow cells can be adapted for use in detecting the presence, and determining the concentration, of certain chemical compounds, such as species of protein, DNA or small drug molecules by means of absorbance and/or fluorescence.
  • the first and/or second optical flow cell bodies can include one or more paths through which light can pass, typically in a perpendicular direction to the liquid path between the inlet and outlet ports on the optical flow cell bodies.
  • the light path through the first and/or second optical flow cell bodies includes a window with an inside and an outside surface.
  • This window is formed of a material that a) transmits light at the wavelength being evaluated, and b) has suitable physical and chemical properties to withstand the conditions of use.
  • Representative materials that can be used to prepare the window(s) include fused silica (quartz), MgF, UV transparent polymers like cyclic olefin copolymers (example Topas® COC).
  • the window(s) are coated, on one or both surfaces, with an anti- reflective coating to maximize the light transmitted through the window.
  • Light from one or more suitable light sources can be passed through the window, such that it travels through the flow cell and the liquid whose absorbance is to be measured and on to a photodetector.
  • the absorbance can be computed, and where the eluent includes a protein containing one or more absorbing amino acids, the absorbance will be higher than when no protein is present.
  • Light can be passed from a single light source through one of the optical flow cell bodies, from multiple sources through one of the optical flow cell bodies, or from multiple sources through multiple optical flow cell bodies.
  • the light source(s) can be located in the housing, or can be located outside of the housing. Where the light source(s) are located outside of the housing, the light can be transmitted optionally including one or more lens elements into the housing through air, or by means of a fiber optic cable, or other fiber optic element. In this embodiment, the fiber optic element ideally resides, at least in part, within the housing.
  • the light source(s) must be capable of generating light at a wavelength that is suitable for the molecules being detected. Typically, this light is in the UV or visible range (UV/VIS). Where fluorescence is also being measured, at least one light source must be capable of generating light that stimulates fluorescence in the substance to be detected.
  • Representative light sources include, but are not limited to, lasers, light emitting diodes (LEDs), such as TOPLEDs (OSRAM Opto Semiconductor), UVTOP® (Sensor Electronic Technologies), or Nichia UV-LEDs (Nichia Corporation, Tokushima, Japan) http://www.nichia.co.jp/en/product/uvled.html) some of which provide monochromatic light, or nearly monochromatic light, and deuterium, xenon, mercury, zinc, and tungsten lamps, which provide broader spectrum light output.
  • LEDs light emitting diodes
  • TOPLEDs OSRAM Opto Semiconductor
  • UVTOP® Tinsor Electronic Technologies
  • Nichia UV-LEDs Nichia UV-LEDs (Nichia Corporation, Tokushima, Japan) http://www.nichia.co.jp/en/product/uvled.html) some of which provide monochromatic light, or nearly monochromatic light, and deuterium, x
  • the light is monochromatic light, or nearly monochromatic light, and is of a desired wavelength for the analysis, there is no need to separate or filter the light using a prism, holographic grating or bandpass filter and direct light of a desired wavelength through the optical flow cell body.
  • a prism or grating or filter it may be desirable to use a prism or grating or filter to select the desired wavelength or range of wavelengths of light and direct the resulting light through the optical flow cell body.
  • the transmitted wavelength can be varied, depending on the molecule of interest being eluted from a column and through the flow cell system.
  • the photodetectors used to measure the transmitted light rely on small currents, often measured in the nano Ampere scale. Where there is only a relatively small amount of energy being detected, even tiny levels of background noise cause the signal to noise ratio (S/N) to be detrimental to the measurement. In one embodiment, this effect is minimized by using light with a relatively high intensity. The relatively high light intensity provides significantly more signal to the detector, thus improving S/N. Liquid solutions passing through the flow cell can be negatively affected by high energy UV radiation, which can denature proteins and can precipitate on the flow cell surfaces, artificially increasing the measured absorbance.
  • this limitation is addressed by operating the light source in a high peak power, short duration pulsed mode. Between pulses, the light system cools sufficiently such that the light source lifetime is maintained at or near normal levels and the flow cell windows are not fouled.
  • the light source is pulsed rapidly. For example, the pulse intervals may be between 0.1 and 500 milliseconds. Within each pulse, the light source will be on less than about 50% of the complete cycle, preferably less than about 25% of the cycle, and still more preferably, less than about 10% of the cycle. For example, one can have a 76 microsecond light pulse within a one millisecond cycle.
  • Pulse cycles shorter than about 0.1 milliseconds become difficult to control, resulting in signal ringing and timing inaccuracies.
  • the light pulse should not exceed around 50% of the cycle time for sufficient thermal recovery between pulses.
  • the frequency of the pulses, and the length of the cycle, can be adjusted using appropriate hardware and software controls.
  • each light source package includes more than one LED device, where each LED can be independently enabled.
  • a first light pulse from one LED can be passed through the flow cell, followed by a second light pulse through the same flow cell.
  • Both LEDs can be of the same wavelength or different wavelengths to confirm the value of the other LED or to return additional property data on the liquid samples under measurement.
  • a single light emitting device package containing more than one light LED source element within it can be used, where the light sources are substantially close to each other, such that each light source reaches the window substantially co-axially.
  • the individual light source elements can be offset from the flow cell optical axis by as much as 5° and still deliver sufficient light to the photodetector for measurement.
  • one conduit in this embodiment, the optical bore
  • time division multiplexing two or more signals are interleaved in time through the same channel, thereby minimizing the number of channels required.
  • additional flow cells are expensive and should be minimized.
  • the photodetector When the light is pulsed, it is also possible to minimize noise, improving the signal to noise ratio by synchronously gating the photodetector, so the amplifier accumulates signal in approximately the same time frame as the source light is active. Photodiodes and other photodetectors generate some electricity regardless of light falling on their active surfaces. This electricity is commonly known as "dark current" because the current flows even if the sensor's surface is dark. Accordingly, in one embodiment, the photodetector is pulsed to convert light intensity to an output current that mirrors the pulsing of the light source. In one aspect of this embodiment, the photodetector includes an amplifier, and the pulsing of the photodetector is accomplished by synchronously gating the amplifier.
  • the UV LED can be driven in a high transient pulse manner, followed by a relatively long off period for cooling and recovery.
  • This drive method makes overdriving of the LED impossible under normal conditions.
  • the microcontroller drives the LED through a pulse width modulated (PWM) output.
  • PWM pulse width modulated
  • the PWM drives a constant current circuit composed of a high slew rate operational amplifier.
  • a calibrated current sense resistor can be used to provide negative feedback to the op amp to maintain the required stable current output to minimize damage to the LED.
  • a higher amplitude pulse from the controller can be used to drive the amplifier to create a higher intensity output pulse, but with the same duration. The duration can also be modified by changing the width of the pulse.
  • the USB voltage can be boosted sufficiently to drive the LED at its required currents.
  • the circuit is current limited. Any errors from the control system that might direct the LED to remain constantly on will result in a short, high current burst of an intensity equivalent to the normal burst power, followed by a far lower average current below the LED's constant drive current damage threshold.
  • the system can include one or more lenses, filters, and/or apertures, positioned such that the lenses, filters, and/or apertures are in the path of the light emitted from one or more of the light sources, before and/or after the light passes through the first or second optical flow cell body.
  • Representative lenses include concave, convex, plano-concave, plano-convex, ball, hemispherical, collimating, and Fresnel lenses.
  • the flow cell systems can be connected to, or comprise, one or more detectors.
  • the detector is ideally a photodetector, such as a slicon photodiode, charge-coupled device (CCD), photoelectron multiplier.
  • the photodetector is external to the housing, but light is transmitted to the detector via a fiber optic element that resides, at least in part, within the housing.
  • the flow cell systems are ideally connected to, or comprise, a conductivity detector.
  • the flow cell system particularly the housing portion of the system, includes a temperature measuring device, such as a thermocouple or resistive temperature device (often called RTD, or PtlOO), although any device.
  • a temperature measuring device such as a thermocouple or resistive temperature device (often called RTD, or PtlOO), although any device.
  • the invention in another embodiment, relates to an overall liquid chromatography system that includes the flow cell systems described herein.
  • the complete liquid chromatography system typically includes one or more mobile phases, which are typically stored in one or more reservoirs, and a pump for moving the mobile phase(s).
  • the system typically also includes one or more valves.
  • the chromatography system also includes one or more chromatography columns, each of which include a suitable column packing material. Samples are injected onto the column(s) through a sample injection port. As the eluent (in this case, the mobile phase(s) passes through the end of the column, it can then pass through the flow cell systems described herein.
  • the conductivity portion of the detector system described herein can be used in a chromatography system to verify the mobile phase components have been attached to the proper fluid inlets and the control system has been configured to deliver the correct ratio of mobile phase components.
  • a chromatography system for example in biochromatography, one mobile phase component contains a substantially lower salt concentration than other mobile phase components. A transposition of this low salt component becomes apparent rapidly after the commencement of the chromatography process.
  • the conductivity portion of the detector, the absorbance portion of the detector or both the conductivity portion and the absorbance portion in concert can be used to determine the completion of a chromatographic step.
  • Certain steps such as equilibration, sample load, wash and regenerate are considered complete when the passage of additional mobile phase through the system results in effectively no additional chromatographic change.
  • the chromatographic change tends to correlate with changes in absorbance, conductivity or both absorbance and conductivity. Monitoring absorbance and conductivity during these steps and further monitoring the rate of change of these parameters often indicates completion of the step.
  • the invention relates to a method for detecting the presence or absence of protein, DNA, or another molecule of interest in an eluent by measuring the conductivity of the eluent.
  • the method involves passing a mobile phase through a liquid chromatography column comprising the flow cell systems described above.
  • the conductivity of the mobile phase as it elutes from the column (i.e., the eluent), and as it passes through the flow cell system, can be determined, and this can be used to define baseline conductivity.
  • a sample that comprises one or more molecules of interest, such as proteins or DNA can then be injected onto the liquid chromatography column.
  • a chromatographic separation is performed by pumping the eluent through the liquid chromatography column.
  • Periodic or continuous measurements of the conductivity of the eluent can be taken as the eluent passes through the optical flow cell bodies. An increase in conductance relative to the baseline conductance is indicative of the presence of a molecule of interest in the eluent.
  • the invention relates to a method for detecting the presence or absence of a protein, DNA, or other molecule of interest in an eluent, by measuring the absorbance or fluorescence of the eluent.
  • the method involves passing a mobile phase eluent through a liquid chromatography column comprising a flow cell system as described herein, to form an eluent.
  • a beam of light, at a wavelength suitable for detecting the presence of the molecule of interest in the eluent, is passed through the first and/or second optical flow cell body of the flow cell system in a direction substantially perpendicular to the direction in which the eluent flows through the optical flow cell bodies.
  • the light is then passed through the optical flow cell bodies to a photodetector, and an initial light intensity is measured.
  • a sample which includes one or more molecules of interest is then injected onto the liquid chromatography column. Chromatographic separation is performed by pumping the mobile phase through the chromatography column to form an eluent.
  • the intensity of light passed through the flow cell optical paths and directed to a photodetector can be measured, either periodically or continuously, and the light intensity can be compared to the initial light intensity. A decrease in light intensity measured by the photodetector, relative to the initial light intensity, is indicative of a protein or other compound of interest's presence in the eluent.
  • light is measured in each of the two flow cells at different wavelengths. For example, where the wavelengths are set at 260 nm and 280 nm, one can measure protein and DNA concentrations. In another embodiment, light is measured in each of the two flow cells at the same wavelength. This can help determine whether there are any distortions caused by artifacts in one of the flow cells, because in the absence of such artifacts, assuming the same light intensity, flow cell size, and flow cell composition, the two signals should be identical. In a third embodiment, in addition to having two light sources at the same light intensity, one in each flow cell, one can further have two (or more) light sources in one or both of the flow cells.
  • the system can provide pulses of light at a first wavelength in the cycle, and pulses of light at a second wavelength either within the same cycle, or in a separate cycle. It is generally preferred to allow sufficient time between pulses of light of different frequencies, so as to avoid interference by the light beams.
  • conductivity can be measured.
  • Conductivity of that specimen fluid is measured by connecting the cell bodies, or, more specifically, connecting the conductive material present on both sides of the insulator, to a power supply and applying an AC current thereto.
  • the variation in the electrical resistance of the specimen liquid can be measured, either continuously, or at desired times during a chromatographic run.
  • the conductivity is also measured in a pulsed manner rather than in a continuous manner.
  • the pulses by which conductivity is measured are timed to avoid overlapping with the light pulses, so that noise and other artifacts caused by the light signal, and detection thereof, are not inadvertently captured as part of the conductivity measurement or vice versa.
  • this flow cell system eliminates the need for a separate conductivity measuring device and adds an additional absorbance measurement at a different wavelength into a single, compact housing.
  • This compact, single-device design reduces the space that is otherwise occupied by the use of multiple, separate flow cell assemblies. It also reduces the potential for measurement error which can be introduced by the use of a series of multiple separate flow cells. This device further reduces the system's non- chromato graphic fluidic volume, reducing post-separation remixing of the eluent.
  • FIG. 1 shows a sectional view of an embodiment of a multi-measurement flow cell assembly
  • FIG. 2 shows an exploded view of an embodiment of the multi-measurement flow cell assembly
  • FIG. 3 shows an embodiment of a detector system including a multi-measurement flow cell assembly
  • FIG. 4 shows another embodiment of a multi-measurement flow cell assembly.
  • FIG. 5 shows a schematic illustration of a sample and hold circuit.
  • FIG. 6 shows an internal view of the embodiment shown in FIG. 3.
  • Flow cell assemblies comprising first and second optical flow cell bodies, each of which includes an inlet port, an outlet port, and a fluid passage through the optical flow cell body connecting the inlet port to the outlet port, are disclosed.
  • HPLC devices including the flow cell assemblies are also disclosed, as well as methods for using the flow cell assemblies.
  • a housing supports the first optical flow cell body and the second optical flow cell body, such that the first and second flow cell bodies are in series, with the outlet port of the first flow cell body adjacent to the inlet port of the second flow cell body.
  • the flow cells are stackable in series, each with its own housing.
  • a first conductive material is adjacent to the outlet port of the first optical flow cell body, positioned such that would be in contact with any fluid passing through the flow cell, a second conductive material is adjacent to the inlet port of the second optical flow cell body, positioned such that would be in contact with any fluid passing through the flow cell, and an insulator between the first and second conductive materials.
  • the flow cell assemblies allow one to reduce the number of flow cells in a liquid chromatography system. This is advantageous for several reasons. Flow cells take up space in compact liquid chromatography systems and add to the length and complexity of the flow path for the specimen solution. Further, the use of multiple flow cells adds to the number of connection points, which increases the opportunity for leaks or other failures to seal. Moreover, flow cells often increase the amount of turbulence created in the specimen solution, which can cause inaccuracies in and distortion of the conductivity and absorbance measurements. Most importantly, the additional "deal volume" created by the addition of multiple flow cells presents opportunities for previously separated compounds to remix, reversing the effects of the separation process.
  • the invention relates to a flow cell that can substantially overcome the disadvantages and problems encountered by using multiple flow cells.
  • the disclosed flow cell assembly allows for three or more property measurements to be made in a single, compact device. Conductance and the absorbance at two, three, or more distinct wavelengths, or, alternatively, duplicate absorbance measurements at a single wavelength, can all be measured simultaneously within the same detector. Further, temperature can be measured as well. Accordingly, in this embodiment, the flow cell assemblies eliminate the need for a separate conductivity measuring device and add, at least, an additional absorbance measurement at a different wavelength, or a duplicate measurement at the same wavelength, into a single, compact housing.
  • the compact, single-device design reduces the space that is otherwise occupied by multiple, separate flow cell assemblies. It also reduces the potential for measurement error which can be introduced by using a series of multiple separate flow cells.
  • the flow cell systems described herein include two or more flow cells, either within the same housing, or in separate housings.
  • the flow cells have components that enable the measurement of conductivity and/or temperature as fluids pass through the flow cells.
  • the flow cells include inlet and outlet ports, which enable them to be part of a complete chromatography system.
  • the flow cells can be part of a complete chromatography system which includes at least one reservoir including a mobile phase, a column packed with a stationary phase, a pump to pass the mobile phase through the column, an injection port to introduce a sample to be separated.
  • the flow cells described herein generally have an optical path diameter between about 1 and about 5 mm, and an optical path length of between about 0.1 and 20 mm.
  • the flow cells can be adapted for use in detecting the presence, and determining the concentration, of certain chemical compounds, such as species of protein, DNA or small drug molecules by means of absorbance and/or fluorescence.
  • the first and/or second optical flow cell bodies can include one or more paths through which light can pass, typically in a perpendicular direction to the liquid path between the inlet and outlet ports on the flow cell bodies.
  • the light path through the first and/or second flow cell bodies includes a window with an inside and an outside surface.
  • This window is formed of a material that a) transmits light at the wavelength being evaluated, and b) has suitable physical and chemical properties to withstand the conditions of use.
  • Representative materials that can be used to prepare the window(s) include fused silica (quartz), MgF, UV transparent polymers like cyclic olefin copolymers (example Topas® COC).
  • the window(s) are coated, on one or both surfaces, with an anti- reflective coating to maximize the light transmitted through the window.
  • the materials used to prepare the flow cells are, ideally, corrosion resistant, able to withstand the physical conditions of the system (i.e. pressure and forces) and preferably do not bind to proteins.
  • Titanium specifically Ti 6A1-4V
  • Other alloys composed primarily of titanium could be suitable.
  • Polymers like polyetheretherketone (PEEK) or polyvinyldisulfone or polyvinylidene fluoride (PVDF) can also be used.
  • the flow cell body can be formed from a conductive material.
  • the conductive materials can be physically pressed against the flow cell bodies, or they can be attached to the flow cell bodies, mechanically or chemically.
  • a female part at the outlet port of the first flow cell body or the inlet port of the second flow cell body can be adapted to receive a male part of a conductive material, so that the two are physically mated.
  • the conductive material can be adhered to one or both of the flow cell bodies, such as by an epoxy resin or other suitable adhesive that resists degradation under the conditions of use, namely, high pressure, organic solvents, and the like.
  • the flow cells are designed such that a light beam can pass through the transparent or translucent portion in a linear or substantially linear path, and travel to a photodetector. It is important that the path be linear or substantially linear, so as to minimize interference and maximize the signal reaching the detector.
  • a tube is inserted within the flow cell.
  • the exterior of the tube includes two (or more) spirals, and each spiral connects one of the (inlet, outlet) with the optical path of the cell.
  • the tube imparts a vortex in the fluid passing through the flow cell.
  • the flow cells comprise angled holes or spiral- forming structures (like rifling) for this same purpose.
  • Flow cells comprising a tube comprising two or more spirals, or angled holes, or spiral-forming structures, such as rifiling, are a separate embodiment of the invention, and can be used without the other features of the flow-cell systems described herein.
  • a housing supports the first optical flow cell body and the second optical flow cell body, such that the first and second optical flow cell bodies are in series, with the outlet port of the first optical flow cell body adjacent to the inlet port of the second optical flow cell body.
  • the flow cells are stackable in series, each with its own housing.
  • Light from one or more suitable light sources can be passed through the window of a flow cell body, such that it travels through the flow cell and the liquid whose absorbance is to be measured and on to a photodetector.
  • the absorbance can be computed, and where the eluent includes a protein containing one or more absorbing amino acids, the absorbance will be higher than when no protein is present.
  • Light can be passed from a single light source through one of the optical flow cell bodies, from multiple sources through one of the optical flow cell bodies, or from multiple sources through multiple optical flow cell bodies.
  • the light source(s) can be located in the housing, or can be located outside of the housing. Where the light source(s) are located outside of the housing, the light can be transmitted optionally including one or more lens elements into the housing through air, or by means of a fiber optic cable, or other fiber optic element. In this embodiment, the fiber optic element ideally resides, at least in part, within the housing.
  • the light source(s) must be capable of generating light at a wavelength that is suitable for the molecules being detected. Typically, this light is in the UV or visible range (UV/VIS). Where fluorescence is also being measured, at least one light source must be capable of generating light that stimulates fluorescence in the substance to be detected.
  • the light be monochromatic light, or, where it is not monochromatic, that it be passed through a prism, holographic grating, or bandpass filter so that light of substantially only one wavelength passes through the flow cell.
  • substantially only one wavelength is meant light with wavelength intensity following a Gaussian distribution, with around 80 or more percent of the light falling within a range of around 20 nm or less from the maximum wavelength.
  • the light is monochromatic light, or nearly monochromatic light, and is of a desired wavelength for the analysis, there is no need to separate or filter the light using a prism, holographic grating or bandpass filter and direct light of a desired wavelength through the optical flow cell body.
  • a prism or grating or filter it may be desirable to use a prism or grating or filter to select the desired wavelength or range of wavelengths of light and direct the resulting light through the optical flow cell body.
  • the transmitted wavelength can be varied, depending on the molecule of interest being eluted from a column and through the flow cell system.
  • light at a wavelength of 280 nm is absorbed by most amino acids and is preferred for measuring protein concentration, and 260 nm is preferred for measuring DNA concentration (see, for example, Sambrook and Russell (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor Laboratory Press. ISBN 978-0-87969-577-4.
  • the light is monochromatic or substantially monochromatic, and in one aspect of this embodiment, the light is monochromatic LED light.
  • the system can include one or more lenses, filters, and/or apertures, positioned such that the lenses, filters, and/or apertures are in the path of the light emitted from one or more of the light sources, before and/or after the light passes through the first or second optical flow cell body.
  • Representative lenses include concave, convex, plano-concave, plano-convex, ball, hemispherical, collimating, and Fresnel lenses.
  • Representative light sources include, but are not limited to, lasers, light emitting diodes (LEDs), such as TOPLEDs (OSRAM Opto Semiconductor), UVTOP® (Sensor Electronic Technologies), or Nichia UV-LEDs (Nichia Corporation, Japan) some of which provide monochromatic light, or nearly monochromatic light, and deuterium, xenon, mercury, zinc, and tungsten lamps, which provide broader spectrum light output.
  • LEDs light emitting diodes
  • TOPLEDs OSRAM Opto Semiconductor
  • UVTOP® Tinsor Electronic Technologies
  • Nichia UV-LEDs Nichia UV-LEDs
  • a light-emitting diode is a two-lead semiconductor light source that resembles a basic pn-junction diode, except that an LED also emits light.
  • an LED's anode lead has a voltage that is more positive than its cathode lead by at least the LED's forward voltage drop, current flows.
  • Electrons are able to recombine with holes within the device, releasing energy in the form of photons. This effect is called electroluminescence, and the color of the light (corresponding to the energy of the photon) is determined by the energy band gap of the semiconductor.
  • a single light source can include two or more LEDs, each transmitting light at a desired frequency. Where light at two more more frequencies is desired, the LEDs can be offset so that they each transmit beams of light through substantially the same path through the flow cell. Although it is most preferred that light pass directly through the flow cell to the photodetector, if the beams of light are only slightly offset, they can transmit beams down substantially the same path, such that the beams are effectively coaxial. By “substantially the same path” and “effectively coaxial” is meant that the light transmission is 95% or more of the light that would travel down the path if the path were completely straight.
  • LED lights that have a focused emission profile (i.e., which emit light with a Gaussian distribution of wavelengths centered around a desired wavelength) over using a light source that generates a broad spectrum of wavelengths that have to be passed through a filter, prism, or grating, as such can substantially simplify the flow cell systems.
  • LED lights are relatively small, so the footprint of the flow cell systems can be reduced by using LED lights.
  • UVTOP® is a series of deep ultraviolet light emitting diodes with peak emission wavelengths from 240nm to 355nm.
  • the LEDs are hermetically sealed in metal-glass transistor outline (TO) packages with a choice of UV-transparent optical windows and lenses for beam profiling. These lights can be preferred, as they can be custom tailored to emit light with a maximum emission at a desired wavelength.
  • TO metal-glass transistor outline
  • LEDs can transmit light with as narrow a wavelength distribution, or nearly so, as polychromatic light from other sources that has been passed through a prism, filter, or grating.
  • Use of LED Lights With Two or More Sources of Light can transmit light with as narrow a wavelength distribution, or nearly so, as polychromatic light from other sources that has been passed through a prism, filter, or grating.
  • each light source package includes more than one LED device, where each LED can be independently enabled.
  • a first light pulse from one LED can be passed through the flow cell, followed by a second light pulse through the same flow cell.
  • Both LEDs can be of the same wavelength or different wavelengths to confirm the value of the other LED or to return additional property data on the liquid samples under measurement.
  • a single light emitting device package containing more than one light LED source element within it can be used, where the light sources are substantially close to each other, such that each light source reaches the window substantially co-axially.
  • the individual light source elements can be offset from the flow cell optical axis by as much as 5° and still deliver sufficient light to the photodetector for measurement.
  • one conduit in this embodiment, the optical bore
  • time division multiplexing two or more signals are interleaved in time through the same channel, thereby minimizing the number of channels required.
  • additional flow cells are expensive and should be minimized.
  • time-division multiplexing is used primarily for digital signals, but may be applied in analog multiplexing, in which two or more signals are transferred appearing simultaneously as sub-channels in one communication channel, but are physically taking turns on the channel.
  • a light source comprising more than one LED light, with each LED transmitting light at a different wavelength, allows one to transmit light through a flow cell at more than one wavelength, using pulses of light at each desired wavelength.
  • the light source(s) can be located in the housing, or can be located outside of the housing. Where the light source(s) are located outside of the housing, the light can be transmitted optionally including one or more lens elements into the housing through air, or by means of a fiber optic cable, or other fiber optic element. In this embodiment, the fiber optic element ideally resides, at least in part, within the housing.
  • the light source(s) must be capable of generating light at a wavelength that is suitable for the molecules being detected. Typically, this light is in the UV or UV/VIS range. Where fluorescence is also being measured, at least one light source must be capable of generating light that elicits fluorescence in the substance to be detected.
  • the photodetectors used to measure the transmitted light rely on small currents, often measured in the nano Ampere scale. Where there is only a relatively small amount of energy being detected, even tiny levels of background noise cause the signal to noise ratio (S/N) to be detrimental to the measurement. In one embodiment, this effect is minimized by using light with a relatively high intensity. The relatively high light intensity provides significantly more signal to the detector, thus improving S/N. Liquid solutions passing through the flow cell can be negatively affected by high energy UV radiation, which can denature proteins and can precipitate on the flow cell surfaces, artificially increasing the measured absorbance.
  • this limitation is addressed by operating the light source in a high peak power, short duration pulsed mode. Between pulses, the light system cools sufficiently such that the light source lifetime is maintained at or near normal levels and the flow cell windows are not fouled.
  • the light source is pulsed rapidly. For example, the pulse intervals may be between 0.1 and 500 milliseconds. Within each pulse, the light source will be on less than about 50% of the complete cycle, preferably less than about 25% of the cycle, and still more preferably, less than about 10% of the cycle. For example, one can have a 76 microsecond light pulse within a one millisecond cycle.
  • Pulse cycles shorter than about 0.1 milliseconds become difficult to control, resulting in signal ringing and timing inaccuracies.
  • the light pulse should not exceed around 50% of the cycle time for sufficient thermal recovery between pulses.
  • the frequency of the pulses, and the length of the cycle, can be adjusted using appropriate hardware and software controls.
  • light from one or more of the suitable light sources described can be passed through the window(s) of the flow cells, such that the light travels through the flow cell(s), and the liquid whose absorbance is to be measured, and on to a photodetector.
  • the absorbance of the light can be measured, and where the eluent includes a protein containing one or more absorbing amino acids, the absorbance will be higher than when no protein is present.
  • Light can be passed from a single light source through one of the optical flow cell bodies, from multiple sources through one of the optical flow cell bodies, or from multiple sources through both of the optical flow cell bodies.
  • the light passes on to a photodetector.
  • the photodetector can be, for example, a quantum dot photoconductor or a photodiode, each of which can handle wavelengths in the visible and infrared spectral regions.
  • a photodiode is an active component that converts light into an electrical voltage (photovoltaic effect) or photocurrent.
  • the p-n junction in the silicon semiconductor generally serves as the physical basis for this process, though a structure rather than a p-n junction can be used to increase the speed of response.
  • photons with sufficient energy are absorbed by the detector, this results in the formation of charge carriers (electron-hole pairs), which are separated in the space-charge region and thus generate the photocurrent.
  • Photodiodes may contain optical filters, built-in lenses, and may have large or small surface areas.
  • the photodetector When the light is pulsed, it is also possible to minimize noise, improving the signal to noise ratio by synchronously gating the photodetector, so the amplifier accumulates signal in approximately the same time frame as the source light is active. Photodiodes and other photodetectors generate some electricity regardless of light falling on their active surfaces. This electricity is commonly known as "dark current" because the current flows even if the sensor's surface is dark. Accordingly, in one embodiment, the photodetector is pulsed to convert light intensity to an output current that mirrors the pulsing of the light source. In one aspect of this embodiment, the photodetector includes an amplifier, and the pulsing of the photodetector is accomplished by synchronously gating the amplifier.
  • the photodiode measuring circuit depends on precision microsecond timing to provide a highly accurate measurement.
  • the control system includes a microcontroller.
  • the microcontroller may be busy with another task for a few microseconds, resulting in measurement start or duration delays. This error in timing would be detrimental to the light measurement, reducing the accuracy of the absorbance measurement.
  • the same gating signal used to enable the LED serves a second purpose; connecting the photodiode to a storage capacitor.
  • This capacitor forms a simple, inexpensive and effective sample and hold circuit, storing the photodiode energy for later measurement. The energy remains in the capacitor until the microcontroller instructs the analog to digital converter to measure the energy by measuring the voltage across the capacitor.
  • FIG. 5 A schematic illustration of a representative circuit is shown in Figure 5.
  • the stream of pulses is composed of a series of digital on (high) and off (low) voltages.
  • this series of changing voltages is generated by pulse width modulation (PWM) circuitry and instructional code (firmware and software).
  • PWM pulse width modulation
  • a pulse width modulated output series oscillates between digital on and off under supervisory control.
  • the period of the pulse is the time between rising transitions (off to on).
  • the width is the duration of each on transition.
  • the period of the PWM may be 1000 microseconds.
  • the pulse width might be 76 microseconds.
  • a LED and photodiode amplifier could be gated by the on portion of the PWM cycle, with the LED and photodiode amplifier being active for the 76 millisecond portion of the pwm and being off for the 924 millisecond portion of the PWM cycle.
  • the circuit can be configured to make the LED and photodiode amplifier active during the off portion of the PWM cycle and inactive during the on portion of the PWM cycle.
  • the PWM circuitry may be incorporated into the controlling and computing element, for example a Freescale Kinetis K20 microcontroller, an Atmel ATMEGA or ATTiny microcontroller) or the PWM circuitry can be constructed from individual logic elements.
  • the stream of pulses may be created using a resistor- capacitor circuit (RC) or an inductor-resistor-capacitor circuit (LCR).
  • RC resistor- capacitor circuit
  • LCR inductor-resistor-capacitor circuit
  • Further embodiments could use commercially available integrated circuits constructed primarily to provide timing functions. Examples include the generic "555 timer IC" family.
  • the LCR and R-C circuits suffer from tolerance and temperature induced inaccuracies.
  • the 555 timer is less susceptible to these inaccuracies but is not as easily adjusted as the purpose-built PWM circuitry.
  • a computer processor output pin may be toggled directly by the processor's instruction code using the internal or external timing standard incorporated within the processor. Codes monitor the elapsed time and toggle the output pin between high and low at precisely the proper time. This timing and toggling can be accomplished with interrupts and interrupt service routines, whereby instructional code monitors the processor's clock and calls a subroutine whenever the processor clock reports a preprogrammed elapsed time. The subroutine instructs the processor to toggle the output pin. The subroutine may also set the next interrupt service routine to continue the toggling process indefinitely.
  • light is measured in each of the two flow cells at different wavelengths. For example, where the wavelengths are set at 260 nm and 280 nm, one can measure protein and DNA concentrations. In another embodiment, light is measured in each of the two flow cells at the same wavelength. This can help determine whether there are any distortions caused by artifacts in one of the flow cells, because in the absence of such artifacts, assuming the same light intensity, flow cell size, and flow cell composition, the two signals should be identical. In a third embodiment, in addition to having two light sources at the same light intensity, one in each flow cell, one can further have two (or more) light sources in one or both of the flow cells.
  • the system can provide pulses of light at a first wavelength in the cycle, and pulses of light at a second wavelength either within the same cycle, or in a separate cycle. It is generally preferred to allow sufficient time between pulses of light of different frequencies, so as to avoid interference by the light beams.
  • the flow cell systems described herein further comprise components that enable the conductivity of the mobile phase to be measured as it passes through the flow cell.
  • a conductivity cell is a device that includes electrodes which sense the electrical conductivity of a substance, which, in an HPLC instrument, is the mobile phase plus any compounds of interest that have eluted through the column and are present in the mobile phase.
  • Common configurations include two electrode cells and four electrode conductivity cells.
  • the main trait that differentiates each type is the conductivity cell constant (K). Electrode geometry, size, the distance between each, and the pattern of the electrical field present determine the cell constant. Generally, the constant is higher for cells with small electrodes that are spaced far apart, and is lower for cells with larger electrodes spaced closer together.
  • the cell constant and the conductance of the material have to be multiplied.
  • the fringe field effect must also be factored into the equation, which is simplified by also performing a measurement of a solution in which the electrical conductivity is known.
  • a first conductive material is adjacent to the outlet port of the first optical flow cell body, positioned such that would be in contact with any fluid passing through the flow cell
  • a second conductive material is adjacent to the inlet port of the second optical flow cell body, positioned such that would be in contact with any fluid passing through the flow cell, and an insulator between the first and second conductive materials.
  • conductivity can be measured.
  • Conductivity of that specimen fluid is measured by connecting the cell bodies, or, more specifically, connecting the conductive material present on both sides of the insulator, to a power supply and applying an AC current thereto.
  • the variation in the electrical resistance of the specimen liquid can be measured, either continuously, or at desired times during a chromatographic run.
  • the conductivity is also measured in a pulsed manner rather than in a continuous manner.
  • the pulses by which conductivity is measured are timed to avoid overlapping with the light pulses, so that noise and other artifacts caused by the light signal, and detection thereof, are not inadvertently captured as part of the conductivity measurement or vice versa.
  • the conductive materials can be physically pressed against the optical flow cell bodies, or they can be attached to the optical flow cell bodies, mechanically or chemically.
  • a female part at the outlet port of the first optical flow cell body or the inlet port of the optical second flow cell body can be adapted to receive a male part of a conductive material, so that the two are physically mated.
  • the conductive material can be adhered to one or both of the optical flow cell bodies, such as by an epoxy resin or other suitable adhesive that resists degradation under the conditions of use, namely, high pressure, organic solvents, and the like.
  • the simplest conductive materials are washers, including metallic washers or washers made of or covered by conductive substances (such as conductive polymers or coatings or platings).
  • the optical flow cell bodies can include wires, metal plating, and the like.
  • a portion of the flow cell is plated with a metal, such as gold.
  • the conductivity measuring system can further include an alternating voltage source (AC source).
  • the AC source is connected, directly or indirectly, to the first and second conductive materials, so as to complete a circuit.
  • the AC source is capable of alternating at more than one frequency.
  • the presence of the two conductive electrodes with an insulator disposed between them allows one to perform conductivity measurements when a) a liquid solution is passed through the flow cell electrodes and b) the conductive materials are connected to an AC source to drive current through the materials.
  • the liquid solution completes the circuit between the conductive materials, and the current through the circuit changes as the concentration of proteins or other molecules of interest in the liquid solution changes.
  • the flow cell systems described herein comprise two or more electrodes, made from a conductive material compatible with the fluids to be measured. Often, the same materials used in the flow cell can be used (examples of which include titanium Ti 6A1-4V and similar alloys).
  • the electrode is produced from a material which forms an oxide that is minimally insulating, so an oxide coating, if and when formed, does not interfere with the conductivity measurement.
  • Platinum (Pt) and gold (Au) electrodes are minimally reactive but expensive, but the electrodes can be plated with Pt or Au to enhance conductivity.
  • the insulator separating the conductivity electrodes is made from an essentially non-conductive, physically rigid material compatible with the liquid to be measured.
  • Representative insulators include fluorinated polymers, such as PTFE (DuPont Teflon®), ETFE (DuPont Tefzel®), FEP, PVDF, PES and PEEK.
  • a system of op-amps and switches creates an alternating current (AC) square wave to excite the conductivity electrodes.
  • the AC reversals minimize galvanic transfer of metal from one electrode to the other.
  • the conducted current is measured indirectly by measuring the voltage drop across a current sense resistor. This voltage is amplified and converted to a digital signal using an analog to digital converter system.
  • the conductivity measurement system comprises more than two conductive material electrodes, such as 3, 4, 5, 6, or more conductive material electrodes, which, when connected to an AC source, function as reference electrodes.
  • the initial electrodes are formed by the conductive materials at the outlet port of the first flow cell body and the inlet port of the second flow cell body
  • an additional two electrodes can be formed by placing conductive materials at the inlet port of the first flow cell body, and the outlet port of the second flow cell body.
  • an additional two electrodes can be formed by placing conductive materials at the inlet port of the first optical flow cell body, and the outlet port of the second optical flow cell body.
  • the AC source operates at varying frequencies depending on the measured value of conductance. By varying the AC source frequency, cell capacitive effects become apparent and separately measurable. These capacitive effects can be subtracted from the measurement to improve measurement accuracy.
  • the electrodes, flow cells, and insulator used to measure conductivity are just some of the components used to measure conductivity. In addition, it is also useful to provide a transmitter and controller for signal conditioning, as well as a connecting cable.
  • a circuit creates the AC voltage delivered to the electrodes.
  • the circuit consists of an alternating pulse train that switches from an inverting amplifier to a non-inverting amplifier. Alternately, the pulse train can switch from a low gain amplifier to a high gain amplifier, creating an alternating output.
  • the pulse train can be created by a computer processor, a R-C or LCR network or a 555 timer, or PWM or directly from a programmed computer processor manipulating one or more output pins to create a pulse train.
  • ADC analog to digital converter
  • the sensing section's digital representation of the measured voltage is read multiple times (between 2 and 100,000, for example) before the computer processor delivers a final value.
  • the final value is an average of the multiple readings to smooth the effect of a few aberrant readings within a measurement cycle.
  • the measured conductance varies with the liquid's temperature.
  • the value which is a digital value, is typically corrected for temperature effects using the following calculations:
  • C25 is the measured conductivity at 25° C
  • a is the temperature coefficient of the calibration solution in °C
  • C t is the temperature at which the conductivity measurement is desired.
  • the flow cell system particularly the housing portion of the system, includes a temperature measuring device, such as a thermocouple or resistive temperature device (often called RTD, or PtlOO), although any device.
  • a temperature measuring device such as a thermocouple or resistive temperature device (often called RTD, or PtlOO), although any device.
  • One way to determine the concentration of biomolecules in the mobile phase as it passes through a flow cell is to measure the UV transmission of the mobile phase, and compare the transmission to a set of dilution-based standard curves, or to use an internal standard.
  • HPLC-diode array detectors including those with features such as multiple wavelength monitoring, are capable of maintaining a high degree of response reproducibility over extended periods of time. Because of this high degree of reproducibility, it has been suggested that detector response factors, rather than dilution based standard curves, can also be used to measure concentrations of proteins and pharmaceuticals. Eberlein, "Quantitation of proteins using HPLC-detector response rather than standard curve comparison,” J Pharm Biomed Anal, Sep;13(10): 1263-71 (1995).
  • the flow cell systems described herein enable one to measure UV transmission, using relatively small flow cells, and relatively simplified optics and/or electronics, that is as accurate as measurements taken using conventional flow cells with relatively larger footprints and more complicated optics and/or electronics.
  • a sample to be analyzed is introduced into a flow cell.
  • the flow cell is irradiated with excitation light; light of a specific wavelength.
  • the amount of fluorescence emitted from the excited sample is measured by a photodetector to determine the concentration or amount of each of the components of the sample.
  • excitation radiation from an excitation source having a first wavelength, passes through excitation optics.
  • the excitation optics cause the excitation radiation to excite the sample.
  • fluorescent proteins in the sample emit radiation which has a wavelength that is different from the excitation wavelength.
  • Collection optics then collect the emission from the sample.
  • the device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned.
  • Conductivity detection is used to detect inorganic and organic ionic species in the mobile phase. All ionic species are electrically conducting, so conductometric detection is a universal detection technique.
  • Conductivity the reciprocal of electrical resistivity, is the ability of a solution to conduct electricity. Conductivity is results from the mobility of dissociated anions and cations in a solution. In low to moderate concentrations, conductivity can be predicted. In high concentrations, physical effects contribute to the electron mobility.
  • Conductivity is temperature dependent. Unless stated otherwise, conductivity references standard temperature (25 °C).
  • a chloride salt like potassium chloride (KC1) or sodium chloride (NaCl) in deionized water can be used to create standardized solutions. iVlo!ar mass
  • DAC digital-to-analog converter
  • a reasonable range for this voltage is a 100: 1 ratio from 5 Vp-p to 50 mVp-p.
  • a 20- bit DAC provides 262K: 1 resolution assuming two resolution bits are wasted as noise.
  • the capacitance of the cell affects conductivity.
  • the separated electrodes naturally form a capacitor.
  • a charge is stored on this unintentional capacitor. This charge artificially increases the apparent conductivity.
  • Increasing the alternation [frequency as the apparent conductivity increases reduces the capacitive error because the unintentional capacitor has less time to charge to a value affecting the measurement.
  • the YSI 3100 laboratory conductivity instrument (YSI Incorporated, Yellow Spring, OH), uses the frequencies shown in the following table through the measuring range, and these frequencies can also be used in the devices described herein:
  • the invention in another embodiment, relates to an overall liquid chromatography system that includes the flow cell systems described herein.
  • the complete liquid chromatography system typically includes one or more mobile phases, which are typically stored in one or more reservoirs, and a pump for moving the mobile phase(s).
  • the system typically also includes one or more valves.
  • the chromatography system also includes one or more chromatography columns, each of which include a suitable column packing material. Samples are injected onto the column(s) through a sample injection port. As the eluent (in this case, the mobile phase(s) passes through the end of the column, it can then pass through the flow cell systems described herein.
  • the eluent liquid exiting the chromatography column is evaluated in one or more measuring instruments known to those in the art as Detectors.
  • Detectors In addition to including the windows through which light can pass, and conductive materials that can serve as electrodes when a current is applied, it is also important, for example, to be able to use information regarding the absorbance, fluorescence, or conductivity to determine when a molecule of interest has eluted from a column.
  • the flow cell systems can be connected to, or comprise, one or more detectors.
  • the detector is ideally a photodetector, such as a photodiode, charge-coupled device (CCD), photoelectron multiplier.
  • the photodetector is external to the housing, but light is transmitted to the detector via a fiber optic element that resides, at least in part, within the housing.
  • the flow cell systems are ideally connected to, or comprise, a conductivity detector.
  • the systems comprise:
  • an analysis column provided downstream of the sample injection portion on the analysis flow passage to separate a sample injected from the sample injection portion into individual components.
  • the detectors are provided downstream of the analysis column on the analysis flow passage to detect each of the components separated by the analysis column.
  • the invention relates to a method for detecting the presence or absence of protein, DNA, or another molecule of interest in an eluent by measuring the conductivity of the eluent.
  • the method involves passing a mobile phase through a liquid chromatography column comprising the flow cell systems described above.
  • the conductivity of the mobile phase as it elutes from the column (i.e., the eluent) as it passes through the flow cell system can be determined, and this can be used to define baseline conductivity.
  • a sample that comprises one or more molecules of interest, such as proteins or DNA can then be injected onto the liquid chromatography column.
  • a chromatographic separation is performed by pumping the eluent through the liquid chromatography column.
  • Periodic or continuous measurements of the conductivity of the eluent can be taken as the eluent passes through the flow cell bodies. An increase in conductance relative to the baseline conductance is indicative of the presence of a molecule of interest in the eluent.
  • the invention relates to a method for detecting the presence or absence of a protein, DNA, or other molecule of interest in an eluent, by measuring the absorbance or fluorescence of the eluent.
  • the method involves passing a mobile phase eluent through a liquid chromatography column comprising a flow cell system as described herein, to form an eluent.
  • a beam of light at a wavelength suitable for detecting the presence of the molecule of interest in the eluent, is passed through the first and/or second optical flow cell body of the flow cell system in a direction substantially perpendicular to the direction in which the eluent flows through the optical flow cell bodies.
  • the light is then passed through the flow cell bodies to a photodetector, and an initial light intensity is measured.
  • a sample which includes one or more molecules of interest is then injected onto the liquid chromatography column. Chromatographic separation is performed by pumping the mobile phase through the chromatography column to form an eluent.
  • the intensity of light passed through the flow cell optical paths and directed to a photodetector can be measured, either periodically or continuously, and the light intensity can be compared to the initial light intensity. A decrease in light intensity measured by the photodetector, relative to the initial light intensity, is indicative of a protein or other compound of interest's presence in the eluent.
  • the conductivity portion of the detector system described herein can be used in a chromatography system to verify the mobile phase components have been attached to the proper fluid inlets and the control system has been configured to deliver the correct ratio of mobile phase components.
  • a chromatography system for example in biochromatography, one mobile phase component contains a substantially lower salt concentration than other mobile phase components. A transposition of this low salt component becomes apparent rapidly after the commencement of the chromatography process.
  • the flow cells described herein can be used in a method for verifying the mobile phase components in a chromatography system during gradient elution, wherein one of the mobile phase components is a salt solution.
  • the method involves: a) performing gradient elution on a chromatography column using a mobile phase comprising at least two mobile phase components, at least one of which is a salt solution,
  • each of the mobile phase components is introduced onto the chromatography column through a fluid inlet, the conductivity of the mobile phase varies with the concentration of the salt solution in the mobile phase in a repeatable and predictable manner, and the salt concentration of the mobile phase is adjusted based on a pre-determined schedule, using a control system configured to deliver the correct ratio of the mobile phase components,
  • the user performing the gradient elution can verify that the mobile phase components have been attached to the proper fluid inlets, and/or that the control system has been configured to deliver the correct ratio of mobile phase components.
  • the conductivity can be measured using a flow cell as described herein.
  • the method can be performed on any chromatography system that includes a conductivity detector.
  • the conductivity portion of the detector, the absorbance portion of the detector or both the conductivity portion and the absorbance portion in concert can be used to determine the completion of a chromatographic step.
  • Certain steps such as equilibration, sample load, wash and regenerate are considered complete when the passage of additional mobile phase through the system results in effectively no additional chromatographic change.
  • the chromatographic change tends to correlate with changes in absorbance, conductivity or both absorbance and conductivity. Monitoring absorbance and conductivity during these steps and further monitoring the rate of change of these parameters therefore indicates completion of the step.
  • the flow cells described herein can be used in a method for determining whether a chromatographic step has been completed.
  • Representative steps to be analyzed for completeness include equilibration, sample loading, column washing, and column regeneration. The method involves:
  • the equilibrating, loading, washing, or renegerating step is determined to be complete.
  • the conductivity and/or absorbance can be measured using a flow cell as described herein.
  • the method can be performed on any chromatography system including a conductivity and/or absorbance detector.
  • FIG 1 shows one example of a flow cell assembly as described herein, which includes two flow cells within a non-conductive housing (40). Fluid flows into a first flow cell through a fluid inlet port (10). Each flow cell body (30), which acts as a conductivity detector, is held in place in the housing (40) using retaining rings (20). Light passes into each flow cell body (30) through a quartz window (50), which window is held in place using a window retainer (60). The light passes through the windows through pre-determined optical paths (70). Between each flow cell is a pair of conductive washers (80), with a non- conductive ledge (90) between the washers.
  • FIG 2 is an exploded view of a flow cell assembly without a housing, showing two flow cell bodies (30), functioning as conductivity electrodes, each with an optical path (70) through which light can pass, one with a fluid inlet port (10), and the other with a fluid outlet port (120).
  • a conductive washer (80) is attached to each flow cell body (30), and a conductivity insulator (100) is present between the two flow cell bodies (30).
  • a shielded wire 110) is connected to the two washers (80).
  • FIG 3 is a view of the flow cell assembly showing light passing through the flow cell bodies onto photodiodes, and showing a controller for the flow cell assembly.
  • Two flow cell bodies (5) and (6) are secured in a housing (11) using flow cell stack nuts (8) and (9). Light from LED emitters (1) and (2) flows through the two flow cell bodies (5) and (6) through optical axes (12) and (13). After the light flows through the flow cell bodies, it impinges on photodiodes (3) and (4).
  • the two flow cell bodies (5) and (6) are separated by a conductivity insulator (7), and connected by a shielded wire. Conductance is measured as solutions pass through the flow cells. The measurement of conductivity and UV absorbance is controlled by a controller (10).
  • FIG 6 is an internal view of the embodiment shown in FIG 3.
  • the flow cell stack nuts (8) and (9) hold together the two flow cell bodies (5) and (6), and a conductivity insulator (7) between the two flow cell bodies.
  • the two flow cell bodies each include a window (50) through which UV light can be transmitted.
  • one embodiment of the flow cell assembly includes two flow cells pressed together in a single housing with an insulator, such as a fluoropolymer disc, between them.
  • each flow cell acts as an electrode on either side of the insulator because the insulator prevents electrical conduction between the cell bodies other than through the liquid specimen.
  • the detector assembly provides a continuous path, or hollow bore, whereby a specimen fluid travels directly through two consecutively aligned flow cells.
  • conductivity of that specimen fluid is measured by connecting the cell bodies to a power supply and applying an AC current thereto such that the variation in electrical resistance of the specimen liquid can be measured.
  • each optical flow cell body provides an optical path that allows light to travel from a light emitter positioned on one side of the flow cell to a detector positioned on the opposite side of the optical flow cell body.
  • the optical path runs through the center of the flow cell and perpendicular to its length.
  • windows may be secured into pockets on either side of the optical path and held in place by, for example, press fit aluminum rings.
  • the light emitters on each of the optical flow cell bodies are configured to emit different wavelengths of light enabling the device to detect the presence of two different materials.
  • one emitter may be configured to emit 260 nanometer wavelength light to detect DNA while the other emits 280 nanometer wavelength light to detect proteins.
  • Each of the two flow cells has a fluid inlet port on one end and an outlet port at the opposing end.
  • the flow cells are aligned in series, such that the outlet end of the first flow cell aligns with the inlet end of the second flow cell.
  • the first and second optical flow cell bodies may be secured together by a detector housing.
  • the light emitters and detectors may also be secured to or by the detector housing.
  • the detector housing is preferably made of a material that absorbs light so that ambient light does not additively interfere with the transmitted light.
  • a material that is non-conductive which isolates the flow cells and enables them to act as electrodes for conductivity measurement.
  • the detector housing may be comprised of acetal, such as Delrin made by DuPont.
  • the optical flow cell bodies may be secured to the detector housing using numerous means or materials.
  • the two flow cells and insulator may be compressed together in the detector housing using two retaining rings, one placed at the inlet end of the first optical flow cell body, and the other placed at the outlet end of the second optical flow cell body.
  • the retaining rings may be threaded and may screw into the interior edge of the detector housing.
  • the retaining rings can be bolted or glued, or fastened to the detector housing using other fastening means known in the art.
  • each optical flow cell body can have a lip or a flange designed to be bolted or otherwise fastened to the detector housing, thereby eliminating the retaining ring all together.
  • the optical flow cell bodies may be formed to have a shoulder that abuts the ledge in the detector housing.
  • the first optical flow cell body has a shoulder near its outlet port while the second optical flow cell body has a shoulder near its inlet port such that the optical flow cell bodies can be inserted into the detector housing and be compressed against the ledge, or towards the ledge against the insulator separating the two optical flow cell bodies.
  • the optical flow cell bodies are adapted to be electrodes for measuring the conductivity of the liquid.
  • Each of the optical flow cell bodies are connected, e.g. via a conductive wire, to a conductivity analyzer.
  • the conductivity analyzer may be part of the controller for the detection system. As demonstrated in FIG. 3, the controller may manage the light emission and detection devices in both the first and second optical flow cell bodies, as well as the conductivity measurement system.
  • the flow cells also have a conductivity measurement location where the flow cell attaches to the conductivity analyzer circuit. Detection and measurement of the current passing through the liquid from one optical flow cell body to the other may be detected in any number of ways.
  • the outlet end of the first optical flow cell body and the inlet end of the second optical flow cell body are each fitted with a conductive washer.
  • the optical flow cell bodies are in contact with conductive wave washers soldered to Wires. Specifically, a conductive wave washer is positioned against each flow cell between the shoulder of the flow cell and the ledge of the detector housing.
  • the wave washer is comprised of conductive material, such as copper or gold-plated copper, which contacts the flow cell housing and allows the conductivity reading between the cells to be measured.
  • the wave washer does not necessarily contact the flow cell body around its entire circumference, and may only contact the optical flow cell body in one location or a few locations.
  • the wave washer provides the benefit of having a variable width that can fill the space between the detector housing and the shoulder of the flow cell, allowing for added tolerance in the detector assembly and allowing the system to fit tightly and easily together.
  • the conductive washer may be a standard, flat washer.
  • the assembly may not include a washer at all, and the contact point for conductivity measurement can be a node, hook, clamp, or other contact means by which current can travel between the flow cell bodies and wires or probes leading to the conductivity analyzer circuit.
  • the contact point can be a conductive ring, node, or disk embedded at a point in the detector housing that comes in contact with the respective flow cell body.
  • the detector housing may be fitted with a pin and spring assembly that provides a conductive contact with one or more of the flow cell bodies.
  • the flow cell assembly includes two spring (140) and pin (150) assemblies, one for each optical flow cell body (30).
  • the spring and pin assemblies are fitted into two bores (160), in the side of the detector housing (40), one aligned with each flow cell.
  • the spring and pin assemblies can alternatively reside in slots or holes in the side of the detector housing (130), or can be fastened to the detector housing by other means.
  • the detector housing supports each spring and pin assembly, while also permitting the assemblies to move or slide within the housing as required for the pin to make contact with and accommodate the optical flow cell bodies. Specifically, when each flow cell is inserted into the housing during assembly, the pins come in contact with the outer edge of the optical flow cell bodies.
  • the optical flow cell body exerts force against the pin, compressing the spring, which is permitted to move within the detector housing.
  • the pin is allowed to adjust to optical flow cell bodies of varying widths, which allows for tolerance within the assembly.
  • the pins (150) can slide in slots or grooves (130) in the optical flow cell bodies (30).
  • the slots or grooves (130) can be shaped to engage the pin (150) in its fully extended position, and to compress the pin (150) as the optical flow cell body (30) is inserted into the housing (40).

Landscapes

  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Optical Measuring Cells (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

L'invention concerne un détecteur permettant de détecter les constituants d'un liquide destiné à être utilisé dans une chromatographie en phase liquide. Le détecteur comprend un premier corps de cuve à circulation optique et un second corps de cuve à circulation optique, chacun étant doté d'un canal le traversant qui permet le passage d'un liquide depuis un orifice d'entrée jusqu'à un orifice de sortie. Les premier et second corps de cuve à circulation optique sont agencés en série de sorte que le liquide sortant de l'orifice de sortie du premier corps de cuve à circulation optique entre dans l'orifice d'entrée du second corps de cuve à circulation optique. Un isolant est disposé entre le premier corps de cuve à circulation optique et le second corps de cuve à circulation optique, lequel isolant est conçu pour isoler électriquement le premier corps de cuve à circulation optique du second corps de cuve à circulation optique tout en permettant au liquide de passer du premier corps de cuve à circulation optique au second corps de cuve à circulation optique. Le premier corps de cuve à circulation optique est conçu pour faciliter la mesure de l'absorption par le liquide d'une première longueur d'onde de lumière et le second corps de cuve à circulation optique est conçu pour faciliter la mesure de l'absorption par le liquide d'une seconde longueur d'onde de lumière. Les premier et second corps de cuve à circulation optique sont en outre conçus pour tenir lieu d'électrodes afin de mesurer la conductivité du liquide.
PCT/US2014/038070 2013-03-15 2014-05-15 Ensemble cuve à circulation de multi-mesure pour chromatographie en phase liquide Ceased WO2014146147A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP14764986.7A EP2972154A4 (fr) 2013-03-15 2014-05-15 Ensemble cuve à circulation de multi-mesure pour chromatographie en phase liquide

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US201361794081P 2013-03-15 2013-03-15
US61/794,081 2013-03-15
US201361824219P 2013-05-16 2013-05-16
US61/824,219 2013-05-16
US201461953284P 2014-03-14 2014-03-14
US61/953,284 2014-03-14

Publications (2)

Publication Number Publication Date
WO2014146147A2 true WO2014146147A2 (fr) 2014-09-18
WO2014146147A3 WO2014146147A3 (fr) 2015-01-08

Family

ID=51538624

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/038070 Ceased WO2014146147A2 (fr) 2013-03-15 2014-05-15 Ensemble cuve à circulation de multi-mesure pour chromatographie en phase liquide

Country Status (2)

Country Link
EP (1) EP2972154A4 (fr)
WO (1) WO2014146147A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017536550A (ja) * 2014-11-25 2017-12-07 ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ 吸光度測定装置への蛍光検出機能の統合
WO2021094009A1 (fr) * 2019-11-14 2021-05-20 World Precision Instruments Germany Gmbh Système de cuve à circulation pour analyse de fluide optique et système de bioréacteur

Family Cites Families (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5914054U (ja) * 1982-07-19 1984-01-27 東ソー株式会社 液体クロマトグラフ用フロ−セル
EP0143901B1 (fr) * 1983-11-23 1988-06-29 Bacharach, Inc. Détecteur multifonctionnel
US6307204B1 (en) * 1997-11-14 2001-10-23 Jasco Corporation UV detector for liquid chromatography
EP1208189B1 (fr) * 1999-05-28 2004-10-06 Cepheid Dispositif et procede d'analyse d'ehantillons liquides
US6755074B2 (en) * 2001-02-27 2004-06-29 Isco, Inc. Liquid chromatographic method and system
GB2388189B (en) * 2002-04-29 2006-01-11 Robert Jeffrey Geddes Carr Optical detection and analysis of particles
AU2002952602A0 (en) * 2002-11-12 2002-11-28 Varian Australia Pty Ltd Flow through cell for optical spectroscopy
US7355706B2 (en) * 2003-04-04 2008-04-08 Hach Ultra Analytics, Inc. Particle detection system implemented with an immersed optical system
US7125492B2 (en) * 2003-07-17 2006-10-24 Agilent Technologies, Inc. Additives for reversed-phase HPLC mobile phases
US7184141B2 (en) * 2004-03-23 2007-02-27 Lockheed Martin Corporation Optical flow cell for tribological systems
US7209223B1 (en) * 2004-11-15 2007-04-24 Luna Innovations Incorporated Optical device for measuring optical properties of a sample and method relating thereto
GB0513128D0 (en) * 2005-06-27 2005-08-03 Ojk Consulting Ltd An optical arrangement for a flow cytometer
JP4817442B2 (ja) * 2006-07-31 2011-11-16 シスメックス株式会社 粒子分析装置用光学系、及びそれを用いた粒子分析装置
US7804594B2 (en) * 2006-12-29 2010-09-28 Abbott Laboratories, Inc. Method and apparatus for rapidly counting and identifying biological particles in a flow stream
JP2008256530A (ja) * 2007-04-05 2008-10-23 Shimadzu Corp 蛍光検出器及びその蛍光検出器を備えた液体クロマトグラフ
US20110141465A1 (en) * 2008-05-22 2011-06-16 Waters Technologies Corporation Light-Guiding Flow Cells And Analytical Devices Using The Same
US8293101B2 (en) * 2009-03-13 2012-10-23 Terrasep, Llc Methods and apparatus for centrifugal liquid chromatography
US20120184722A1 (en) * 2009-10-08 2012-07-19 Victor Samper Chromatography components

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of EP2972154A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017536550A (ja) * 2014-11-25 2017-12-07 ジーイー・ヘルスケア・バイオサイエンス・アクチボラグ 吸光度測定装置への蛍光検出機能の統合
WO2021094009A1 (fr) * 2019-11-14 2021-05-20 World Precision Instruments Germany Gmbh Système de cuve à circulation pour analyse de fluide optique et système de bioréacteur
US12174125B2 (en) 2019-11-14 2024-12-24 World Precision Instruments Germany Gmbh Flow cell system for optical fluid analysis and bioreactor system

Also Published As

Publication number Publication date
EP2972154A4 (fr) 2016-11-16
EP2972154A2 (fr) 2016-01-20
WO2014146147A3 (fr) 2015-01-08

Similar Documents

Publication Publication Date Title
US9194848B2 (en) Multi-measurement flow cell assembly for liquid chromatography
JP6726233B2 (ja) 真空紫外吸収分光方法
US11513006B2 (en) Systems and methods for an absorbance detector with optical reference
EP3142765B1 (fr) Détecteur d'absorption d'uv à base de del miniature de faible puissance avec limites de détection faibles pour chromatographie liquide capillaire
US7319522B2 (en) Systems and methods for in situ spectroscopic measurements
US20150153272A1 (en) Methods and apparatus for measuring the light absorbance of a substance in a solution
JP6380665B2 (ja) 光学測定装置
US11422117B2 (en) Combined UV/Vis-absorption and conductivity flow cell for liquid chromatography
US9664610B2 (en) Systems for fluid analysis using electromagnetic energy that is reflected a number of times through a fluid contained within a reflective chamber
CN103884671A (zh) 一种不分光红外(ndir)co2气体传感器
EP2972154A2 (fr) Ensemble cuve à circulation de multi-mesure pour chromatographie en phase liquide
CN104931636A (zh) 基于ccd的紫外毛细管柱上检测仪
US20180335408A1 (en) Combined fluorescence and absorption detector for on-column detection after capillary separation techniques
EP1754045A1 (fr) SYSTEMES ET PROCEDES DE REALISATION DE MESURES SPECTROSCOPIQUES <I>IN SITU</I>
CN221260800U (zh) 一种红外气体传感器
CN101871923B (zh) 液相测量装置
Chouhan Novel Detection Methods and Gas-Free Hydroxide Eluent Generator for Capillary Liquid/Ion Chromatography
CN120801192A (zh) 基于深紫外发光二极管的毛细管尺度柱上检测器

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14764986

Country of ref document: EP

Kind code of ref document: A2

REEP Request for entry into the european phase

Ref document number: 2014764986

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2014764986

Country of ref document: EP

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14764986

Country of ref document: EP

Kind code of ref document: A2