WO2005114168A1 - Colonne mixte - Google Patents

Colonne mixte Download PDF

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Publication number
WO2005114168A1
WO2005114168A1 PCT/EP2004/051079 EP2004051079W WO2005114168A1 WO 2005114168 A1 WO2005114168 A1 WO 2005114168A1 EP 2004051079 W EP2004051079 W EP 2004051079W WO 2005114168 A1 WO2005114168 A1 WO 2005114168A1
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WO
WIPO (PCT)
Prior art keywords
column
microfluidic device
sample
outlet
inlet
Prior art date
Application number
PCT/EP2004/051079
Other languages
English (en)
Inventor
Karsten Kraiczek
Bernd Glatz
Original Assignee
Agilent Technologies, Inc.
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 Agilent Technologies, Inc. filed Critical Agilent Technologies, Inc.
Publication of WO2005114168A1 publication Critical patent/WO2005114168A1/fr

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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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/08—Preparation using an enricher
    • 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
    • G01N30/60—Construction of the column
    • G01N30/6095—Micromachined or nanomachined, e.g. micro- or nanosize
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/08—Preparation using an enricher
    • G01N2030/085—Preparation using an enricher using absorbing precolumn
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G01N2030/201—Injection using a sampling valve multiport valves, i.e. having more than two ports
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N2030/382—Flow patterns flow switching in a single column
    • 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
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G01N30/724—Nebulising, aerosol formation or ionisation
    • G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
    • 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

Definitions

  • the present invention relates to a microfluidic device for sample analysis, and to a method for analyzing components of a fluid sample.
  • trapping columns have been used for accomplishing a sample enrichment.
  • a volume of fluid sample is transported through the trapping column.
  • the outlet of the trapping column is connected to an inlet of a separation column, in order to separate the various components of the sample.
  • German Patent Application DE 102 28 767 describes a microdevice for component separation in a fluid, whereby a volume of a fluid sample is controllably introduced from a sample source into a separation conduit.
  • the microfluidic device for sample analysis comprises one or more inlets adapted for supplying fluid sample and mobile phase to the microfluidic device, a column adapted for trapping a fluid sample during a sample loading phase, and for separating components of the fluid sample during a sample analysis phase, a waste outlet, and a detection unit adapted for detecting the components of the fluid sample.
  • the microfluidic device further comprises a flow path switching facility, with said flow path switching facility being operable to couple an outlet of the column with the waste outlet during the sample loading phase, and with said flow path switching facility being operable to couple the outlet of the column with the detection unit during the sample analysis phase.
  • one of the inlets is connected with the column, and the column's outlet is coupled, via the flow path switching facility, with the waste outlet.
  • the column is used as a trapping column. A sample volume is transported through the column, with sample compounds being trapped at the head of the column.
  • a sample enrichment is accomplished. Furthermore, the sample is cleaned.
  • the flow path switching facility is operated in a way that the column's outlet is connected, via the flow path switching facility, with the detection unit.
  • Mobile phase is provided to one of the inlets and transported through the column. During this sample analysis phase, the sample compounds that have been trapped at the head of the column are separated and analyzed by the detection unit.
  • a major advantage of the microfluidic device according to embodiments of the invention is that early eluting compounds can be detected.
  • the column is used as a trapping column, whereas during the sample analysis phase, the column is used as a separation column.
  • the microfluidic chip instead of two columns, the microfluidic chip only comprises one column, and accordingly, the structure of the microfluidic chip is simplified.
  • the microfluidic device comprises a flow path switching facility that is located downstream of the column's outlet.
  • the column's outlet can either be connected with the waste outlet, or with the detection unit.
  • a separation column's outlet should be directly connected with the detection unit, because any kind of switching facility located downstream of the column's outlet introduces additional dead volumes. Dead volumes, especially those located downstream of the column's outlet, cause a band broadening of the analyte and deteriorate the resolution of the obtained chromatogram.
  • the flow paths have been realized by means of a microfluidic device.
  • the flow paths and the flow path switching facility can be manufactured in a way that dead volumes are kept very small.
  • dead volumes are kept very small.
  • fittings connecting capillaries, valves, etc. are needed causing excessive band broadening, especially when using columns with nanoliter volumes.
  • no fittings of this kind are required any more, and as a consequence, the amount of dead volume can be kept small.
  • the flow path switching facility can be operated such that a sample loading flow path is set up, with said sample loading flow path providing a connection between one of the inlets, the column, and the waste outlet.
  • a sample loading flow path is set up, with said sample loading flow path providing a connection between one of the inlets, the column, and the waste outlet.
  • the flow path switching facility can be operated in a way that a sample separation flow path is established, with said sample separation flow path comprising one of the inlets, the column, and the detection unit.
  • the sample components trapped at the head of the column are successively eluted to the detection unit. There, the sample compounds can be detected.
  • fluid conduits can be implemented with a minimum of fittings. For this reason, the total amount of dead volume in the fluid conduits can be kept sufficiently small. As a consequence, band broadening is minimized, and well-resolved chromatograms are obtained.
  • the microfluidic device comprises a column of rectangular, rhomboid, semi-circular or V-shaped cross section.
  • the permeability of the column is increased.
  • the multipurpose column is used as a trapping column, and a comparatively large volume of fluid sample (e.g. in the order of ⁇ l) has to be transported through the column at a high flow rate. Therefore, a high permeability of the column is particularly advantageous during the sample loading phase.
  • the time required for loading sample into the column is reduced.
  • the flow path switching facility is implemented as a multiroute valve, with the multiroute valve being adapted for coupling the column's outlet to the waste outlet during the sample loading phase, and with the multiroute valve being adapted for connecting the column's outlet with the detection unit during the sample analysis phase.
  • the flow path switching facility is realized as a rotatable valve comprising a stator element and a rotor element, with the rotor element comprising grooves that provide a fluid communication between adjacent valve ports.
  • the microfluidic device might act as the valve's stator element, with the rotor element being rotatably mounted to the microfluidic device.
  • the one or more inlets comprise a first inlet for providing fluid sample to the microfluidic device, and a second inlet for providing mobile phase to the microfluidic device.
  • a rather large volume of fluid sample has to be transported through the column in a short period of time. For this reason, during the sample loading phase, fluid sample should be provided to the system at a high flow rate.
  • mobile phase should be provided to the second inlet at a smaller flow rate, in order to obtain a well-resolved chromatogram.
  • the first and the second inlet might e.g.
  • both the pumps may operate at an intermediate flow rate.
  • the first inlet is connected with the column during the sample loading phase, whereas the second inlet is connected with the column during the sample analysis phase.
  • the flow path switching facility might be adapted for either connecting the first inlet or the second inlet with the column.
  • the flow path switching facility is implemented as a multiroute valve comprising at least two different positions.
  • the valve's first position corresponds to the sample loading phase.
  • the multiroute valve is switched to its second position, which corresponds to the sample analysis phase.
  • the second inlet is connected to the column, and the column's outlet is coupled with the detection unit.
  • a mobile phase gradient is supplied to the column, the sample's components are separated, and components that show up at the column's outlet are detected by the detection unit.
  • the flow path switching facility only comprises one multi-route valve adapted for switching between the sample loading flow path and the sample analysis flow path.
  • the flow path switching facility additionally comprises an input selection valve adapted for coupling either the first inlet or the second inlet with the column.
  • the flow rate of the pump connected to the common inlet is set to a large value during the sample loading phase, whereas during the sample analysis phase, the pump's flow rate is reduced.
  • a mobile phase gradient is applied to the column during the sample analysis phase.
  • the elution strength of the mobile phase is continuously increased during the sample analysis phase.
  • the various components of the sample that have been trapped at the head of the column are successively flushed to the detection unit.
  • the mobile phase is a mixture of water and an organic solvent like e.g. acetonitrile.
  • the mobile phase gradient is generated by continuously increasing the amount of organic solvent.
  • the column is a liquid chromatography column that is filled with packing material. The retention time of a certain compound is determined by the compound's interactions with the mobile phase and the stationary phase.
  • the microfluidic device comprises a microchannel that provides a connection between the flow path switching facility and the detection unit. Especially in the part of the flow path behind the column's outlet, the amount of dead volume has to be kept small, because otherwise, the bands of sample compounds obtained at the column's outlet will broaden, and resolution will be lost.
  • the preferred length of the microchannel ranges from 5 mm to 50 mm, and the preferred width is between 2 ⁇ m and 30 ⁇ m.
  • the detection unit comprises a mass spectroscopic detection unit, with the various sample compounds being detected according to their respective molecular weights.
  • the microfluidic device is equipped with an electrospray device.
  • the electrospray device might e.g. comprise an electrospray nozzle adapted for vaporizing the sample compounds obtained at the column's outlet.
  • the electrospray device might e.g. comprise a power supply adapted for ionizing the fluid sample's components.
  • the obtained ions are mass spectroscopically analyzed according to their molecular weights.
  • An electrospray device of the above-described type is well-suited for being integrated on a microfluidic device.
  • the detection unit is implemented as a fluorescence detection unit, whereby sample compounds that have been fluorescently labeled by attaching a fluorescent marker tag are detected by analyzing the emitted fluorescence light as a function of time.
  • interfering moieties such as e.g. salts
  • interfering moieties are washed to the waste outlet during the sample loading phase.
  • salts are flushed to the detection unit.
  • the microfluidic device is implemented as an integrated microfluidic chip.
  • the interior volume of the flow paths between the various elements is reduced. Furthermore, the amount of dead volume is reduced, and the resolution of the obtained chromatogram is improved.
  • the microfluidic device is realized as a multilayer structure comprising two, three or more layers of polymeric material.
  • a polymeric material like e.g. polyimide or polyetheretherketone (PEEK) is utilized.
  • the various layers of the microfluidic device are formed using a suitable microstructuring technique that provides the required accuracy. Then, the microstructured polymeric films are assembled in a subsequent bonding process.
  • microstructuring polymeric films with an accuracy in the micron or even sub-micron range.
  • These techniques comprise laser ablation, hot embossing, microinjection molding, wet etching, dry etching, plasma etching, and several photolithographic procedures, as e.g. known from semiconductor manufacturing processes.
  • the multilayer structure comprises a top layer, an intermediate layer and a bottom layer.
  • the column is part of the intermediate layer, and for this reason, the height of the column corresponds to the thickness of the intermediate layer.
  • the thickness of the polymeric layers ranges from 10 ⁇ m to 300 ⁇ m.
  • Fig. 1 shows a first embodiment of a microfluidic chip device according to the present invention
  • Fig. 2 shows two positions of a multi-route valve that correspond to a sample loading phase and to a sample analysis phase, respectively;
  • Fig. 3 shows how the top layer, the medium layer and the bottom layer of the polymeric chip device are manufactured
  • Fig. 4 is a cross section of the polymeric chip device along the line A-A';
  • Fig. 5 shows an electrospray nozzle at the tip of the polymeric chip device
  • Fig. 6 shows a cross section of a fitting used in prior art solutions
  • Fig. 7 is a chromatogram of BSA
  • Fig. 8 shows another embodiment of a microfluidic chip device according to the present invention.
  • Fig. 1 shows a first embodiment of a microfluidic chip device adapted for separating and analyzing molecular compounds of a fluid sample.
  • the microfluidic chip 1 which is shown from above, comprises a separation column 2 that is filled with packing material.
  • the microfluidic chip 1 further comprises a 2-position/6-port multi-route valve 3, whereby port D of the multi-route valve 3 is connected with the inlet of the separation column 2, and whereby the outlet of the separation column 2 is coupled with port A of the multi-route valve 3.
  • the compounds of the fluid sample have been separated, they are detected, e.g. by means of mass spectroscopy.
  • a microchannel 5 couples port B of the multi-route valve 3 with an electrospray nozzle 4 at the tip of the microfluidic chip.
  • the electrospray nozzle 4 is adapted for vaporizing the components of the fluid sample, which are then provided to a mass spectroscopy unit.
  • the microfluidic chip 1 comprises separate inlets for sample and mobile phase.
  • a sample inlet 6 is coupled with port E, and a mobile phase inlet 7 is coupled with port C of the multi -route valve 3.
  • Port F of the multi-route valve 3 is connected with a waste outlet 8.
  • the microfluidic chip 1 can be realized as a multilayer structure comprising two or more microstructured polymeric films. In order to provide for a precise alignment of the various layers relative to each other, each of the layers comprises several positioning holes 9.
  • the microfluidic chip 1 represents the stator of the 2-position/6-port multi-route valve 3.
  • the multi-route valve 3 further comprises a rotor element that is located adjacent to the bottom surface of the microfluidic chip 1.
  • the rotor element comprises three grooves 10, with each of the grooves 10 providing a fluid connection between two adjacent ports of the multi-route valve 3.
  • Fig.2Aand Fig.2B give a more detailed view of the multi-route valve's first and second position and of the resulting flow paths. When the valve is set to the first position, which is shown in Fig. 2A, sample can be loaded into the separation column.
  • the sample inlet 6 is connected, via one of the rotor's grooves 10, with the inlet of the separation column 2.
  • the outlet of the separation column 2 is coupled, via another groove 10, with the waste outlet 8.
  • the sample inlet 6 might e.g. be coupled with a first pump, with the first pump being operative to pump a fluid sample through the separation column 2 to the waste outlet 8.
  • the fluid sample might e.g. be an aqueous solution of different sample compounds. As the elution strength of the aqueous solution is rather low, the sample compounds of interest are trapped at the separation column's head 12.
  • fluid sample might e.g.
  • the separation column 2 can be transported through the separation column 2 for a few minutes at a rate of several ⁇ l/min, in order to accomplish an enrichment of the sample compounds at the separation column's head 12.
  • Interfering moieties such as e.g. salts, which might obstruct further detection, are washed to the waste outlet 8.
  • the sample can be cleaned.
  • the mobile phase inlet 7 is connected, via another groove 10 and via the microchannel 5, with the electrospray nozzle 4.
  • the mobile phase inlet 7 might be coupled with a second pump that is adapted for providing the mobile phase at a rate of approximately 50-400 nl/min.
  • a constant flow of mobile phase through the electrospray nozzle 4 is kept up during the sample loading phase, and the liquid flow through the electrospray nozzle 4 is not disrupted.
  • the mobile phase is a mixture of water and an organic solvent like e.g. acetonitrile.
  • the multi-route valve 3 is switched to the second position shown in Fig. 2B.
  • the sample inlet 6 is connected, via one of the grooves 10, with the waste outlet 8.
  • the mobile phase inlet 7 is coupled, via another groove 10, with the inlet of the separation column 2, and the separation column's outlet is connected, via one of the grooves 10 and via the microchannel 5, with the electrospray device.
  • the separation column 2 might e.g. be implemented as a liquid chromatography column. However, the separation column 2 might be adapted to any separation technique like e.g. size exclusion chromatography, ion chromatography, capillary electrophoresis, isoelectric focusing, or electrophoretic focusing via a field gradient.
  • the chromatography column might e.g. be packed with a stationary phase. Typically, a packing material with a surface area of 10-500 m 2 /g is used. For example, silica gel beads of approximately 5 ⁇ m diameter can be used as a packing material.
  • the velocity at which a particular sample component travels through the separation column 2 depends on the component's partition between mobile phase and stationary phase. Furthermore, in order to separate the different molecular species of the sample, a mobile phase gradient with a continuously increasing amount of organic solvent might be supplied to the separation column 2. By continuously increasing the elution strength, the compounds of the sample trapped at the column's head 12 are separated according to one of molecular weight, size, polarity, charge, hydrophobicity, etc. For example, in reverse phase liquid chromatography, where the stationary phase offers a hydrophobic surface and the mobile phase is usually a mixture of water and organic solvent, the least hydrophobic component moves through the chromatography bed first, followed by other components, in order of increasing hydrophobicity.
  • the separation col umn 2 has to be reconditioned. Then, the multi-route valve 3 can be switched back to the sample loading position shown in Fig.2A, and another fluid sample can be loaded into the separation column 2.
  • a microfluidic chip of the type shown in Fig. 1 can be realized as a multilayer structure comprising a multitude of microstructured polymeric films.
  • Fig. 3 shows how these polymeric films can be manufactured.
  • the microfluidic chip shown in Fig. 1 might e.g. be composed of a top layer 13, a medium layer 14, and a bottom layer 15.
  • the three layers can be made of one single polymeric material.
  • the three layers 13, 14, 15 can be cut out of a single sheet 16 of polymeric film.
  • a solvent-resistant polymer like e.g. polyimide or polyetheretherketone (PEEK) is used, with the thickness of the polymeric film ranging from 10 ⁇ m to 300 ⁇ m.
  • the upper layer 13 After the upper layer 13 has been microstructured, it comprises a sample inlet 17, a mobile phase inlet 18, and a waste outlet 19.
  • the upper layer 13 further comprises positioning holes 20 for aligning the upper layer 13 relative to the other layers.
  • the medium layer 14 comprises holes 21, 22, 23 for connecting the sample inlet 17, the mobile phase inlet 18 and the waste outlet 19 with the valve's rotor. Besides that, a channel 24 is cut out of the polymeric film. Later on, the channel 24 will be filled with packing material, in order to form a separation column. The channel 24 connects a hole 26 with a hole 25, whereby the holes 25, 26 provide a connection to the valve's rotor.
  • the length of the channel 24 might be between 1 cm and 1 m, further preferably between 5 cm and 25 cm.
  • the channel 24 might have a width between 10 ⁇ m and several hundred ⁇ m.
  • a microchannel 29 having a width between 2 ⁇ m and 30 ⁇ m is cut out of the polymeric film.
  • the microchannel 29 is tapered at the tip of the microfluidic chip.
  • the medium layer 14 further comprises positioning holes 30 for aligning the layer 14 relative to the other layers.
  • the bottom layer 15 comprises six holes 31 to 36 that are adapted for providing a connection between the corresponding holes of the medium layer 14 and the valve's rotor element, which is pressed towards the bottom layer 15 from below.
  • the bottom layer 15 further comprises positioning holes 37 for aligning the bottom layer 15 relative to the other layers.
  • microstructuring the sheet 16 of polymeric film with the required accuracy.
  • these techniques comprise molding, e.g. microinjection molding, hot embossing, laser ablation, various etching techniques like e.g. dry etching, wet etching, plasma etching, reactive ion etching (RIE), plasma etching, etc.
  • etching techniques like e.g. dry etching, wet etching, plasma etching, reactive ion etching (RIE), plasma etching, etc.
  • RIE reactive ion etching
  • a patterning mask may be generated on the polymeric film's surface.
  • photolithographic techniques might be used for generating a patterning mask.
  • Laser ablation is an important technique for microstructuring a polymeric film.
  • short pulses of intense ultraviolet light are absorbed in a thin surface layer of material.
  • Preferred pulse energies are greater than about 100 millijoules per square centimeter and pulse durations are shorter than about one microsecond.
  • the intense ultraviolet light photo- dissociates the chemical bonds in the substrate surface.
  • the absorbed ultraviolet energy is concentrated in such a small volume of material that it rapidly heats the dissociated fragments and ejects them away from the substrate surface. Because these processes occur so quickly, there is no time for heat to propagate to the surrounding material. As a result, the surrounding region is not melted or otherwise damaged, and the parameter of ablated features can replicate the shape of the incident optical beam with precision on the scale of about one micron or less.
  • a first possibility for bonding the polymeric films is to use a thermal lamination process, whereby the multilayer structure is subjected to heat.
  • a direct bonding process might be employed.
  • the adhesion between adjacent polymeric layers can be improved by subjecting the respective surfaces to some kind of surface treatment before bringing the surfaces into contact with one another.
  • the surfaces of the polymeric films might be irradiated with UV light, in order to break up some of the chemical bonds in the vicinity of the surface.
  • Athird possibility is to laminate the polymeric layers with an adhesive film before bringing the layers into contact with each other.
  • the microfluidic chip device might be composed of two layers instead of three. In this case, for implementing the separation channel and the microchannel, grooves are formed in either one of the top layer and the bottom layer, or in both the top and the bottom layer. Further alternatively, the microfluidic chip device might be composed of more than three layers of polymeric material. For example, the microfluidic chip device might comprise four, five or even more layers of polymeric material.
  • Fig.4 shows a cross section of the microfluidic chip along the line A-A', which is depicted in Fig. 1.
  • the multilayer structure comprises a top layer 38, a medium layer 39 and a bottom layer 40.
  • the thickness of each one of the layers amounts to 50 ⁇ m, and hence, the height of the separation channel 41 is equal to 50 ⁇ m as well.
  • the permeability of the separation channel is higher than the permeability of a cylindrical capillary with the same cross section.
  • the separation channel might have a semi-circular, a rhomboid, or a V-shaped cross section. The increased permeability of the separation channel allows to perform sample loading at a large flow rate.
  • Fig. 5 shows the tip of the microfluidic chip.
  • the microfluidic chip consists of a top layer 42, a medium layer 43, and a bottom layer 44.
  • the medium layer 43 comprises a microchannel 45, with the tapered end of the microchannel 45 forming an electrospray nozzle 46.
  • the diameter of the electrospray nozzle's outlet is approximately equal to 10 ⁇ m.
  • the ionized sample molecules that are ejected from the electrospray nozzle 46 are transported by means of a strong electric field to a mass spectroscopy unit for further analysis. In order to get rid of the solvent molecules, a jet of nitrogen might be directed towards the outlet of the electrospray nozzle 46.
  • Fig. 7 shows a chromatography spectrum that has been acquired using a microfluidic chip of the type shown in Fig. 1. The results correspond to a sample of 5 fm BSA (Bovine Serum Albumine).
  • BSA Bovine Serum Albumine
  • two nanopumps have been connected to the sample inlet 6 and to the mobile phase inlet 7, respectively.
  • a microfluidic chip according to embodiments of the present invention allows to acquire well-resolved chromatograms.
  • One major reason for the good resolution is that, due to the integrated approach, the total amount of dead volume in the separation flow path can be kept very small. Dead volume, which might e.g. be due to variations of the flow path's inner diameter, causes sample dispersion. As a consequence, band broadening occurs, and the resolution of the obtained chromatograms is deteriorated.
  • FIG. 6 shows a cross section of a fitting 47 that can be used for connecting two capillaries 48, 49.
  • the capillaries 48, 49 are inserted into the fitting 47, and the screws 50, 51 are tightened.
  • the ferules 52, 53 are pressed against the narrowing of the fitting 47.
  • Each fitting introduces additional dead volume to the flow path. Especially in the region 54, additional dead volume is generated.
  • Fig. 8 shows another embodiment of the present invention.
  • the microfluidic chip 55 comprises a separation column 56, which is filled with packing material, and which is connected between port D and port A of the 2-position/6-port multi-route valve 57.
  • the rotor of the multi-route valve 57 comprises three grooves 58 that provide a fluid communication between adjacent ports of the multi-route valve 3.
  • a microchannel 59 connects port B of the multi-route valve 57 with an electrospray nozzle 60 at the tip of the microfluidic chip 55.
  • An inlet 61 adapted for providing both sample and mobile phase is connected to port D of the multi-route valve 57.
  • the microfluidic chip 55 is preferably realized as a multilayer structure, with each of the layers comprising positioning holes 63 for aligning the different layers relative to one another.
  • the multi-route valve 57 is set to a first position, which is shown in Fig. 8.
  • the inlet 61 is connected with the inlet of the separation column 56, and the outlet of the separation column 56 is connected, via one of the grooves 58, with the waste outlet 62.
  • fluid sample is transported through the separation column 56 and trapped at the head of the column.
  • the multi- route valve 57 is rotated to its second position.
  • the inlet 61 remains connected with the inlet of the separation column 56, but now, the outlet of the separation column 56 is connected, via one of the grooves 58, to the microchannel 59 and to the electrospray nozzle 60.
  • a mobile phase gradient with an increasing amount of organic solvent is provided to the separation column 56.
  • the various components of the sample are separated in dependence on their respective interactions with the mobile phase and the stationary phase.
  • the electrospray unit is adapted for detecting the different components of the sample, and a chromatogram like the one shown in Fig. 6 is obtained.

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Abstract

Cette invention concerne un dispositif microfluidique pour analyse d'échantillons. Ce dispositif microfluidique comprend une ou plusieurs entrées (6, 7) conçues pour fournir un échantillon de fluide et une phase mobile audit dispositif, une colonne (2) prévue pour piéger un échantillon de fluide pendant la phase de chargement d'échantillons et pour séparer les composants de l'échantillon de fluide pendant la phase d'analyse d'échantillon; une sortie pour déchets (8) et une unité de détection des composants de l'échantillon de fluide. Le dispositif microfluidique comprend en outre une unité de commutation de chemins d'écoulement (3) pouvant relier une sortie de la colonne soit avec la sortie pour déchets pendant la phase de chargement d'échantillons, soit avec l'unité de détection pendant la phase d'analyse.
PCT/EP2004/051079 2004-05-22 2004-06-09 Colonne mixte WO2005114168A1 (fr)

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WO2016128316A1 (fr) * 2015-02-09 2016-08-18 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Moyen et procédés de réduction au minimum de volume balayé et de volume mort dans des applications chromatographiques

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EP0708331A1 (fr) * 1994-10-19 1996-04-24 Hewlett-Packard Company Dispositif miniaturisé et complètement intégré pour l'analyse et manoeuvre des échantillons liquides
EP1338894A2 (fr) * 2002-02-26 2003-08-27 Agilent Technologies, Inc. Dispositif générateur microfluide de gradient d'une phase mobile

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EP0708331A1 (fr) * 1994-10-19 1996-04-24 Hewlett-Packard Company Dispositif miniaturisé et complètement intégré pour l'analyse et manoeuvre des échantillons liquides
EP1338894A2 (fr) * 2002-02-26 2003-08-27 Agilent Technologies, Inc. Dispositif générateur microfluide de gradient d'une phase mobile

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016128316A1 (fr) * 2015-02-09 2016-08-18 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Moyen et procédés de réduction au minimum de volume balayé et de volume mort dans des applications chromatographiques
CN107209155A (zh) * 2015-02-09 2017-09-26 马克斯-普朗克科学促进学会 用于最小化色谱应用中扫掠和死体积的装置和方法

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