WO2022198304A1 - Dispositif pour l'analyse électrophorétique d'échantillons liquides à plusieurs composants - Google Patents

Dispositif pour l'analyse électrophorétique d'échantillons liquides à plusieurs composants Download PDF

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Publication number
WO2022198304A1
WO2022198304A1 PCT/CA2022/050389 CA2022050389W WO2022198304A1 WO 2022198304 A1 WO2022198304 A1 WO 2022198304A1 CA 2022050389 W CA2022050389 W CA 2022050389W WO 2022198304 A1 WO2022198304 A1 WO 2022198304A1
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WO
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Prior art keywords
lid
capillary
vial
vials
gripper
Prior art date
Application number
PCT/CA2022/050389
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English (en)
Inventor
Maksim SLIADNEV
Original Assignee
Lumex Instruments Canada (0890278 B.C. Ltd.)
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Filing date
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Publication of WO2022198304A1 publication Critical patent/WO2022198304A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44713—Particularly adapted electric power supply

Definitions

  • the present disclosure relates to analytical chemistry in general and in particular to a capillary electrophoresis (CE) method for use in analysis of multicomponent liquid samples.
  • CE capillary electrophoresis
  • Composition of multicomponent liquid samples can be determined with separation methods.
  • electrophoretic analysis of multicomponent solutions based on high voltage electric field application to a mixture of components for their separation in a quartz capillary.
  • Capillary electrophoresis (CE) systems provide effective applications for food industry, environmental applications, pharmaceutical and chemical industries, biochemistry and forensic science.
  • Capillary electrophoretic analysis involves the injection of analyzed sample into a narrow quartz capillary that is first washed and filled with an electrolyte solution (known as a buffer solution). High voltage is applied on both ends of the capillary and mixture components of multicomponent liquid sample start migrating at different velocity, driven by the electrical field. When a migrating analyte reaches a detector (such as an optical detector based on direct or indirect ultraviolet light (UV) absorbance) located at a specified distance from capillary inlet, analytical signals related to the analyte's quantity and times of the analyte's migration are measured. Migration time is calculated as an interval between an onset of electrophoretic separation and a time point when a component is detected.
  • a detector such as an optical detector based on direct or indirect ultraviolet light (UV) absorbance
  • the detector response that is recorded when a single analyte passes through the detector zone is known as electrophoretic peak.
  • the detector response recorded as a function of time is known as an electropherogram, which can be used for qualitative identification and quantitative determination of analytes that correspond to different peaks.
  • Different components of a sample can be identified by comparing each detected component's migration time with reference migration time of analytes in a calibration (standard) solution.
  • Quantitative determination of the components in a sample can be done by comparing the detector response with reference electrophoretic peaks or electropherograms for analytes in a calibration solution.
  • Migration times depend on migration velocity of components of the buffer and the buffer velocity in the capillary. The latter depends on buffer composition and condition of capillary walls. If the previous runs contaminate the capillary walls, it affects electroosmotic flow and changes migration times.
  • Charged particle migration rate in the capillary during electrophoretic separation is equal to algebraic sum of particle electromigration rate and electroosmotic flow (EOF) rate.
  • Particle electromigration rate depends on properties of the particle (its charge, mass and structure), while EOF rate depends on capillary features.
  • EOF rate is determined by equation: where A eo f - EOF rate, z - electrokinetic surface potential, e - dielectric constant of solution, E - electric-field intensity, h - solution viscosity of buffer electrolyte.
  • Equation (1) shows that EOF rate is proportional to electrokinetic surface potential magnitude (z -potential), determined by composition and structure of the electrical double layer formed between the inner capillary surface and electrolyte. Under given conditions the equilibrium value of z-potential for clean capillary surface is maximal, while EOF rate is maximal and component migration time is minimal. For sequential sample injections the component migration time increases if the procedure of capillary washing is not carried out after the previous analysis. This is attributed to decrease of z-potential magnitude because of capillary inner surface contamination by the adsorbed admixtures, which change the electrical double layer composition. The substantial change of component migration time due to capillary contamination may cause the errors of identification of multicomponent mixture components.
  • electroosmotic flow markers (hereinafter referred to as EOF markers) have been used. Components with zero or low electrophoretic mobility (electromigration rate) in the buffer are added to a sample. The migration time measured for these EOF markers allows for an estimation of the electroosmotic flow rate and computation of corrected migration times of the detected analytes.
  • EOF markers substantially increases the cost of analysis.
  • the EOF marker method is not versatile, because different types of buffer solution require different EOF markers.
  • Another disadvantage of the EOF marker method is that it is not useful for negative electroosmotic flow rate, since the EOF markers would not reach detection zone at all.
  • the value of the streaming potential can be determined by measuring the potential difference between the capillary ends at a certain pressure difference between the capillary ends.
  • Streaming potential magnitude is directly related with electroosmotic flow rate value A eo f ⁇ Streaming potential U s , measured during capillary washing with electrolyte solution by a means for measuring potential difference between capillary ends at definite pressure difference P between the said capillary ends, is proportional to electrokinetic surface potential (z- potential) in a similar way as electroosmotic flow rate A eo f, ⁇ eR
  • the streaming potential can be measured during preparation when the capillary is washed with electrolyte solution to monitor the condition of the capillary walls.
  • an autosampler is used to automatically introduce samples into the CE system.
  • Use of an autosampler also increases the reproducibility of multicomponent solutions analyses.
  • Reproducibility of CE analysis is based on both stable condition of capillary walls and stable composition of buffers and samples.
  • Evaporation of buffer solution or change of capillary walls condition can change migration times.
  • Evaporation of samples can change concentrations.
  • Contamination of samples or buffer can pollute samples and change properties of the buffer which may affect reproducibility.
  • each electrophoretic analysis may last for 15 minutes, so it may take several hours to analyze 50 or more samples. During this time, liquid multicomponent samples can evaporate, causing a change of sample and buffer composition.
  • most known CE electrophoretic devices work with opened vials. This can cause evaporation of volatile components from samples or from the buffer.
  • CE electrophoretic devices have an autosampler capable of operating with vials sealed with a film or membrane that is pierced by a special needle containing the capillary and the electrode. Keeping vials sealed prevents evaporation, however the needle piercing the membrane can transfer contaminants (e.g. dust particles) from an external surface of the membrane into the vial.
  • these systems do not control changes of migration times due to changes of capillary walls conditions. Therefore, in these devices the objective of solution stability is not fully achieved as the stability is affected not only by evaporation but by cross-contamination as well.
  • FIG. 1 is a schematic of a CE device comprising an autosampler and lid removing means.
  • FIG. 2 is a schematic of the CE device with a lid orientation means and the lid removing means.
  • FIG. 3 is a vial holder.
  • FIG. 4 is a vial holder mounted in a carousel.
  • FIG. 5 is a side view of the lid removing means in an initial position, with a lid gripper driver moving a kinematic chain to start operation.
  • FIG. 6 is a side view of the lid removing means of FIG. 5 with a lid-catching hook pulling the edge of a sample vial lid upwards.
  • FIG. 7 is a side view of the lid removing means of FIG. 5 in a terminal position, when the lid is removed from the vial.
  • FIG. 8 is a schematic of an embodiment of a CE device.
  • FIG. 9 is a schematic of the CE device of FIG. 8 with measuring electrodes and means for moving vials and for disconnecting of measurement circuit.
  • the present disclosure provides an improved CE device with increased or improved sample throughput and reproducibility of electrophoretic analyses of multicomponent liquid samples by reducing the risks of contamination and evaporation.
  • the examples and embodiments described herein may be implemented in an electrophoretic multicomponent analysis device such as that described and shown in United States Patent No. 8,298,393.
  • the device of the present disclosure includes an autosampler, means for immersing the ends of a capillary into an electrolyte vial and a sample vial, means for generating electrolyte flow, means for applying voltage across the capillary ends, a detector connected to the capillary, and a control and signal processing system.
  • the autosampler holds vials sealed with removable lids and comprises a lid-removing means for opening vials to access multicomponent liquid samples by removing the lids from the vials. This reduces evaporation prior to analysis. Since the lids are not pierced, the risk of contamination from piercing needles is avoided.
  • the removable lids are asymmetric, said lids have a tip on one side of their edge.
  • the autosampler comprises lid orientation elements to position the vial lids in a predefined position relative to the lid-removing means.
  • the lid-removing means includes a lid gripper and a lid gripper driver, while the lid gripper comprises a lid-catching hook and a supporting heel while the lid gripper driver comprises a tilting means (e.g. a pivoting joint or a cam mechanism) allowing the lid gripper to tilt around the supporting heel so that the lid-catching hook pulls an edge of the lid upwards whereas the supporting heel contacting with another part of the lid prevents the vial to be pulled from the autosampler.
  • a tilting means e.g. a pivoting joint or a cam mechanism
  • the autosampler comprises a lid position sensor that detects if the lid was removed to prevent damaging the capillary and the electrode.
  • the lid removal means removes the lids from the vial such that potential contaminants (e.g. dust particles) on the external lid surface drop outside of the vial to further reduce the risk of contamination. Removed lids are dropped into a collecting container.
  • the CE device can include a system for monitoring the capillary walls for degradation or contamination.
  • the monitoring system includes means for generating electrolyte flow in the capillary that builds up and maintains a preset differential pressure between the capillary ends during rinsing, and streaming potential measurement means that make electrical contact with the capillary ends, thus forming a closed electric measurement circuit with the electrolyte in the capillary so that the potential difference between the capillary ends can be measured.
  • FIG. 1 schematically shows the CE device.
  • Device control and data processing may be executed by an external computer 1, which is connected with the device through a communication port.
  • controllers 2 provide diagnostics and control of the device. All units are functioning mainly to provide the reliable work of the main device unit - a capillary cassette 3.
  • the device autosampler comprises two carousels, an external carousel 4 and an internal carousel 5. These carousels hold vials for both inlet 6 and outlet 7 ends of the capillary. Conventional polymer vials can be used in both carousels.
  • Lid-removing means 9 is integrated into the autosampler to remove lids before the analysis.
  • FIG. 2 schematically shows the layout of the means in the autosampler.
  • the present invention enables to use the conventional polymer vials in both carousels of the autosampler, such as Eppendorf-type polypropylene vials, sealed with the proper lids, which altogether reduces time for the sample preparation and analysis.
  • FIGS. 2-4 shows a way to mount vials into the carousel by means of a vial holder 46.
  • the vial is inserted into the vial holder 46.
  • the top of the vial holder 46 is shaped with protruding flanges 48 that limit the placement of the vial to the proper orientation.
  • the lid typically has a protruding lip 49 to facilitate lid removal.
  • the lip 49 of the vial lid 50 is positioned between the flanges, as can be seen in FIG 4.
  • the exterior of the vial holder 46 is contoured so it can only fit into the carousel one way, to reduce the likelihood of operator error. In the example of FIG.
  • the vial holder 46 has a cross-sectional profile that can generally be described as a truncated circle, and fits into a corresponding aperture in the carousel so that a portion of the vial holder 46 protrudes from the top of the carousel.
  • a lid-removing means 13 is integrated into the autosampler 11 to open vials and access multicomponent liquid samples just prior to the analysis.
  • the autosampler comprises container 15 for collecting the lids removed.
  • the autosampler also comprises the lid orientation elements 12 to keep the vials lids in a predefined position relative to the lid-removing means and a system for lid position control 14 and 14' for the external 4 and internal 5 carousels.
  • the lid orientation elements comprise the vial holder 46 and a limiter 47.
  • FIG. 3 depicts a non-limiting example of the vial holder 46.
  • the vial holder 46 is shaped to receive a vial and is further provided with a flange which restricts the orientation of the vial within the vial holder 46 to a predetermined position.
  • a limiter 47 prevents the improperly installed vial from entering the work area.
  • the limiter 47 may be optical or mechanical.
  • An optical limiter may comprise a light emitter and light receiver that detects a presence of a light beam. The optical limiter will throw a signal to the controllers 2 when the beam is interrupted by the lid tip.
  • a mechanical limiter will throw a signal to the controllers 2 if the tip touches a special stop provided on the mechanical limiter. Consequently, the system for lid position control will throw an error and stop the carousel if the lid 50 is oriented incorrectly.
  • FIG. 5 shows the initial position of the lid removing means. This position as a standby mode of said means.
  • the lid-removing means includes a stationary part and a movable part.
  • the stationary part comprises a lid gripper driver 16, a standby mode sensor 17, a pivot ring 18, and a spring 19.
  • the movable part of the lid removing means comprises a drive mechanism, a linkage 21 and a lid gripper 22.
  • the drive mechanism is affixed on a driver axis.
  • the lid gripper driver 16 is a mechanism that pivots around the axis 27.
  • the lid gripper 22 is biased by the spring 19 against the pivot axis 27 of the linkage.
  • the lid gripper 22 comprises a lid-catching hook 28 and a supporting heel 29.
  • the lid gripper driver 16 comprises tilting means allowing the lid gripper 22 to tilt around the supporting heel 29.
  • the lid-catching hook 28 pulls a lid edge 32 upwards whereas the supporting heel 29 contacting with another part of the lid prevents a vial 30 to be pulled from a mounting set 31.
  • the lid gripper driver 16 and the pivot ring 18 are affixed on the stationary part.
  • the vial 30 is positioned in the mounting set 31 in the way to keep the lid edge 32 in a predefined position relative to the lid-removing means.
  • the capillary should be washed after each analysis (FIG. 8 and FIG. 9).
  • the required cleaning efficiency of capillary walls from adsorbed admixtures can be determined by those skilled in the art through an empirical choice of compositions of washing solution and washing time. Cleaning efficiency is determined after analysis based on the migration time reproducibility for analytes.
  • FIG. 8 illustrates an apparatus for electrophoretic analysis of multicomponent solutions.
  • the apparatus contains a capillary 33, placed into a means for capillary installation 34, vials for electrolyte 35 and 35', vials for analyzed samples 36, and a means for moving said vials 37, which may immerse capillary ends to the vials.
  • a means for generating the electrolyte flow through capillary 38 is connected with the vial 35, where the input end of the capillary 33 is located (in FIG. 8 and FIG. 9).
  • a means for applying voltage between ends of capillary 39 is electrically connected with the vials 35, 35', where ends of capillary 33 are located.
  • a means for streaming potential measurements 41 is made with possibility of electric connection with the ends of the capillary 33 during washing.
  • the device contains a detector 40, which may be connected to the capillary 33.
  • a control and signal processing system 42 is electrically connected to the detector 40, the means for applying voltage 39, as well as the means for creation of electrolyte flow through capillary 38, and the means for streaming potential measurement 41.
  • the means for moving vials 37 is a device for moving vials with solutions, placing selected vial in position at the selected capillary end (operating position of vial).
  • the means for moving vials 37 thus positions the vials in what may be referred to as their operating position, as shown in FIGS. 6 and 7.
  • the means for capillary installation 34 permits immersion of the ends of the capillary 33 into the vials 35 and 35' in operating position.
  • the vial interior is sealed from ambient air but is connected with the means for creation of electrolyte flow through capillary 38.
  • the means for generation of electrolyte flow through capillary 38 is a device for creating and maintaining a defined pressure difference between the ends of the capillary 33 submerged in the vials 35, 35'.
  • the means for applying voltage 39 is equipped with electrodes 43, installed so to submerge their ends to the vials 35 and 35', when in operating position.
  • the means for streaming potential measurement 41 includes a means for pressure differential measurement between the ends of capillary 44.
  • the means for streaming potential measurements 41, electrolyte in vials 35 and 35', and the capillary 33 filled with electrolyte form an electric measurement circuit.
  • the means for streaming potential measurements 41 also includes a means for opening the streaming potential measurements circuit 45, which can be used to prevent high voltage effects on the means for streaming potential measurement 41.
  • the means for opening streaming potential measurement electric circuit includes measurement electrodes 46 (FIG. 9), electrically connected with the means for pressure differential measurement 44 and with the means for streaming potential measurement 41, and which are inserted into the vials 35 and 35'.
  • the vials 36 and 36' with analyzed samples are installed in operating position and the vials 35 and 35' with measurement electrodes 46 are taken out of operating position, and the measurement electric circuit appeared open.
  • Separation of the injected multicomponent solutions is done by applying high voltage between the ends of the capillary 33.
  • High voltage originates from a high voltage power supply unit, which is connected with two electrodes, immersed in the inlet and outlet vials.
  • the lid-removing means 13 open the vial and throw the lid into a collecting container (FIG. 2). This reduces the risk of cross-contamination between samples.
  • FIG. 6. The operation of the lid-removing means of the invention is shown in FIG. 6. and FIG. 7.
  • the autosampler lifts the vial 30 to the operation area of the lid removing means and the lid removing means starts the operation process from a standby mode.
  • the lid gripper driver 16 with the lever 25 affixed on it operates the tilting means, e.g. a pivoting joint or a cam mechanism, which executes reciprocating cycles. This causes the bearing 24 to slide in the slot 23.
  • the tilting means e.g. a pivoting joint or a cam mechanism
  • the movable part moves so that lid catching hook moves down and then left to catch the edge of the lid 32 (FIG. 6). After that, the movable part continues forward motion left and causes the lid gripper to slide on the lid. In the meantime, the supporting heel 29 contacts with another part of the lid.
  • the lid gripper 22 tilts around the axis 27 and changes its angular position relative to the stationary vial.
  • the lid-catching hook 28 pulls an edge of the lid 32 upwards (FIG. 7) and pulls it up to fully open the vial.
  • the lid gripper 22 drops the lid into a collecting container.
  • Said collecting container can hold lids from several analysis cycles and can be emptied anytime.
  • the said lid-removing means removes lids outwards to the container so that no possible contaminants from the lid reach the vial during opening process (FIG. 7).
  • the system for lid position control 14 may repeat the opening procedure.
  • the external computer may display an error message to operator and continue the analysis for the next sample.
  • Capillary washing (FIG. 8 and FIG. 9) is carried out as follows: the selected vial with solution is installed in operating position, where the end of capillary 33 is submerged into washing solution of the selected vial, and internal chamber of the selected vial is sealed from ambient air and pneumatically connected with the means for generating electrolyte flow through capillary 38, mean increasing pressure in the selected vial, displacing washing solution through capillary 33 to a collecting vial (not shown in the figure).
  • a streaming potential magnitude is determined.
  • vials 35 and 35' with buffer electrolyte are installed in operating position so that input and output ends of capillary 33 are immersed into the buffer electrolyte.
  • Electrodes 43 ends are also inserted into the vials with the buffer electrolyte.
  • a means for applying voltage 39 is switched off the electrodes by means for opening streaming potential measurements circuit 45, being part of the streaming potential measurement means.
  • the streaming potential measurement means 41 closes measurement electric circuit, which includes the electrodes 43 and buffer electrolyte in both the vials 35, 35' and the capillary 33.
  • the flow generation means 38 provides zero flow through capillary by creation of zero pressure difference between the ends of capillary.
  • the streaming potential measurement apparatus measures background potential difference between the ends of the capillary 33.
  • the means for flow generation 38 then increases pressure difference between the ends of capillary from zero to a selected value, providing an increasing flow rate of buffer electrolyte through the capillary 33.
  • the means for flow generation 38 maintains the mentioned selected pressure difference between capillary ends and provides the selected flow rate of buffer electrolyte through the capillary 33.
  • a steady flow rate corresponds to a steady (working) potential difference between the capillary ends.
  • the streaming potential measurement means 41 measures the magnitude of working potential difference between the ends of the capillary 33, maintaining the selected pressure difference between them. High reproducibility of the analysis can be achieved even with a large number of samples and during a long analysis cycle. The risk of evaporation or cross-contamination of samples is reduced, and, and degradation of the capillary walls can be detected.

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Abstract

L'invention concerne un dispositif d'électrophorèse capillaire pour l'analyse électrophorétique de solutions à plusieurs composants, comprenant un capillaire, un échantillonneur automatique avec des moyens de positionnement des extrémités du capillaire dans des flacons d'électrolyte et de solutions d'échantillon, des moyens de génération d'un flux d'électrolyte, des moyens d'application d'une tension aux extrémités du capillaire, un détecteur relié au capillaire, et un système de commande et de traitement du signal, les flacons pour les solutions d'échantillon étant scellés par des couvercles amovibles. L'échantillonneur automatique comprend un moyen de retrait du couvercle avec un dispositif de préhension de couvercle doté d'un crochet d'accrochage de couvercle et d'un talon d'appui, et un dispositif d'entraînement du dispositif de préhension de couvercle qui fait basculer le dispositif de préhension de couvercle autour du talon d'appui. Le crochet d'accrochage du couvercle tire un bord du couvercle amovible vers le haut tandis que le talon d'appui empêche le flacon d'être délogé.
PCT/CA2022/050389 2021-03-25 2022-03-15 Dispositif pour l'analyse électrophorétique d'échantillons liquides à plusieurs composants WO2022198304A1 (fr)

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CA3113278 2021-03-25
CA3113278A CA3113278A1 (fr) 2021-03-25 2021-03-25 Dispositif d'analyse electrophoretique d'echantillons de liquide multicomposants

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4927265A (en) * 1988-04-29 1990-05-22 501 Microphoretic Systems, Inc. Detector for fluorescence and absorption spectroscopy
US6086736A (en) * 1996-08-02 2000-07-11 Texas Tech University Electromigration injection from a microreservoir-electrode in capillary separation systems
US20070111329A1 (en) * 2003-11-07 2007-05-17 Guzman Norberto A Electrophoresis apparatus having at least one auxiliary buffer passage
WO2021038112A2 (fr) * 2019-08-30 2021-03-04 Tallinn University Of Technology Appareil et procédé pour la détermination de substances interdites
US20210246188A1 (en) * 2005-12-20 2021-08-12 Bristol-Myers Squibb Company Compositions and methods for producing a composition

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4927265A (en) * 1988-04-29 1990-05-22 501 Microphoretic Systems, Inc. Detector for fluorescence and absorption spectroscopy
US6086736A (en) * 1996-08-02 2000-07-11 Texas Tech University Electromigration injection from a microreservoir-electrode in capillary separation systems
US20070111329A1 (en) * 2003-11-07 2007-05-17 Guzman Norberto A Electrophoresis apparatus having at least one auxiliary buffer passage
US20210246188A1 (en) * 2005-12-20 2021-08-12 Bristol-Myers Squibb Company Compositions and methods for producing a composition
WO2021038112A2 (fr) * 2019-08-30 2021-03-04 Tallinn University Of Technology Appareil et procédé pour la détermination de substances interdites

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XIE, H.-Y. ET AL.: "In-line coupling headspace liquid-phase microextraction with capillary electrophoresis", JOURNAL OF CHROMATOGRAPHY A, vol. 1217, no. 8, 2010, pages 1203 - 1207, XP026877344 *

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