WO2010057826A1 - Focalisation thermique dynamique de séparations chromatographiques - Google Patents

Focalisation thermique dynamique de séparations chromatographiques Download PDF

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Publication number
WO2010057826A1
WO2010057826A1 PCT/EP2009/065041 EP2009065041W WO2010057826A1 WO 2010057826 A1 WO2010057826 A1 WO 2010057826A1 EP 2009065041 W EP2009065041 W EP 2009065041W WO 2010057826 A1 WO2010057826 A1 WO 2010057826A1
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Prior art keywords
column
temperature
separation
separation column
sample
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PCT/EP2009/065041
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English (en)
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Ole Vorm
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Proxeon Biosystems A/S
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Priority to US13/128,611 priority Critical patent/US8613216B2/en
Priority to EP09752355.9A priority patent/EP2356441B1/fr
Publication of WO2010057826A1 publication Critical patent/WO2010057826A1/fr

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    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • 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
    • G01N30/06—Preparation
    • G01N30/12—Preparation by evaporation
    • G01N2030/121—Preparation by evaporation cooling; cold traps
    • G01N2030/122—Preparation by evaporation cooling; cold traps cryogenic focusing
    • 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
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G01N2030/3015—Control of physical parameters of the fluid carrier of temperature temperature gradients along 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G01N2030/303—Control of physical parameters of the fluid carrier of temperature using peltier elements
    • 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
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G01N2030/3076—Control of physical parameters of the fluid carrier of temperature using specially adapted T(t) profile

Definitions

  • the present invention relates to a liquid chromatography system and a method for focusing the chromatographic separation on one, two, or more columns of a liquid chromatography system by dynamically and differentially adjusting the temperatures of different segments of the liquid transfer lines and the solid phases of the setup. More specifically, the invention primarily relates to a method for thermally focusing the eluting compounds of a pre-column and a separation column of a liquid chromatography system.
  • Detection efficiency by means of electrospray mass spectrometry is concentration dependent so the best sensitivity is achieved when samples are eluted in volumes that are as small as possible. This is done by using chromatographic columns of narrow inner diameters and low flow rates for the mobile phase. However, small column volumes provide fewer binding sites on the stationary phase such that the loading capacity is low. Overloading columns with sample results in poor resolving power, broad and asymmetric peaks and eventually deteriorating detection efficiency.
  • nano-LC which is liquid chromatography that uses a flow range of typically 50 nL/min to 500 nL/min
  • column temperature control is less common than in most other application areas and when using larger flow ranges.
  • trapping columns are used to capture samples during sample loading where such trap-columns are shorter and often of slightly wider ID than the analytical (or “separating”) columns.
  • the flow rate through pre-columns when loading and desalting samples can be much higher than when samples are applied directly onto analytical columns. This saves analysis time. Compounds eluted from the pre-column move onto the analytical column and are separated there.
  • a primary negative effect caused by the combined use of trapping columns in-line with separating columns, is that peak widths are much (typically 1.3 to 5 times) wider than when samples are loaded directly onto separating columns (entirely omitting trap-columns).
  • the chromatographic retention and resolution i.e., the separation of the different peaks in the chromatogram associated with different substances, are to some extent influenced by the system temperature.
  • the system temperature can be manipulated to optimise conditions for a specific separation task. And it is widely practised to ensure reproducible chromatographic results, by holding the temperature constant during the separation process and from analysis to analysis.
  • a thermally stabilized housing is often employed in liquid chromatography in order to keep the chromatographic bed temperature in the separation column constant. By adjusting temperatures, it is also possible to optimize the separation performance of a separation column for a given analyte and given set of mobile phases.
  • Various forms of thermally stabilized housings of the cited type are known, in which the temperature within the housing is essentially constant, such as a water bath, circulating-air heating/cooling, a heating jacket, etc.
  • the prior art includes various apparatus for thermal stabilization in liquid chromatography.
  • US 4,404,845 discloses a thermal regulator for liquid chromatographs.
  • This regulator comprises a thermostat arrangement for the mobile phase and the separation column in a liquid chromatograph, whereby the thermostat has a heat transformer through which the mobile phase passes.
  • the heat transformer comprises a heating and/or a cooling element and is positioned between a sample injection device and the inlet of the separation column.
  • the heat exchanger serves to selectively heat or cool the ambient air surrounding the separation column. Thermal regulation of the mobile phase and the separation column prevents disadvantageous temperature gradients.
  • DE8536810 discloses an oven for setting the temperature of separation columns for high pressure liquid chromatography. In order to avoid a fluctuation of the retention times due to temperature deviations, temperature regulation of the separation column is proposed.
  • the temperature of the separation column in this case is set by an oven consisting of a thermally heatable and coolable block of a metal with high thermal conductivity and with a well for accepting one or more separation columns.
  • a Peltier element is arranged on the heatable and coolable block to allow working temperatures to be set below room temperature.
  • EP438618 discloses an apparatus for thermally stabilizing a mobile phase in a liquid chromatograph.
  • This apparatus comprises a chromatographic column to which an ingoing capillary tube and an outgoing capillary tube are attached, this chromatographic column being positioned in a receptacle element that can be adjusted to a desired temperature using a temperature control unit.
  • a temperature control unit In order to avoid excessive temperature of the liquid directed to a detector through the outgoing capillary tube, which would lead to inaccurate measurement, it is proposed in this case to route the ingoing and outgoing capillary tubes parallel with one another for a predefined distance in order to achieve a heat exchange between the higher-temperature capillary tube leading to the detector and the lower-temperature ingoing capillary tube leading to the separation column.
  • TF chromatographic analysis
  • TF utilizes a steep temperature gradient between a trap column and a separating column in order to focus analytes that elute off of the trap column into a narrow band at the very beginning of or through the separating column.
  • This focusing effect is, by example, very prominent for peptides, using the same or related stationary phase materials in both columns but where the trap column is kept at, say 50 0 C, and where at least the initial part of the separating column is kept at a lower temperature, e.g. 0 0 C.
  • the separating column cooling preferably includes also a portion of the transfer tubing coming from the trap column such that mobile phase is cooled before reaching the separating column.
  • the tubing leading into the trap-column should also be temperature controlled such that buffers have reached the set trap-column temperature already before physically reaching the front end of the trap-column.
  • the cooled area of the separating column need not cover the entire length of the separating column whereas the entire length of the trap column preferably should be of uniform temperature.
  • the focusing effect increases with the size of the temperature drop.
  • the cause of the focusing effect is that analytes released at high temperatures from the pre-column at a given point (T1 ) in a mobile phase gradient will, when cooled before the separating column, strongly displace from the mobile phase to the stationary phase and be immobilized until such later point in time (T2) when the gradient has changed sufficiently to again facilitate elution.
  • T1 point in time
  • T2 later point in time
  • TF allows the matching of very large ID trap columns with narrow ID separating column without significant peak broadening effects.
  • the ID of the trap column can be e.g. 500% of the ID of the separating column while the length of the trap column may also be longer than normal without leading to detrimental peak broadening. This translates into much improved loading capacity (as this is proportional to trap column volume) of the entire system and higher loading and de-salting speeds (as this is roughly proportional to the column radius to the fourth power and only inversely proportional to the column length). In practice, for complex samples, this can provide one to two orders of magnitude higher dynamic range and/or sensitivity of nano-LC-MS experiments. With the larger diameter, pre-columns are also less likely to be blocked by particulate matter and debris contained in liquid samples and hence the overall nano-LC analysis becomes more robust than it is with the currently used, relatively narrow diameter pre-columns.
  • TF makes it possible to simultaneously achieve significantly higher loading speeds, sensitivity, robustness, and/or dynamic range than what is achieved with state-of-the-art approaches today while maintaining the same resolving power.
  • TF generally alleviates the peak broadening caused by diffusion and turbulence in large void volumes in-between the trap and the separating columns.
  • void-volume issues are frequently the result of imperfect connections between columns, fittings, and tubing where such imperfections cause "dead-volumes" (that are unswept) and perturb the fluid flow in a manner that causes peak broadening.
  • TF essentially only dead volumes and perturbations lying after the separating column will lead to peak broadening, thus the technique is more forgiving than current approaches where there slightest imperfection can lead to large peak broadening effects and therefore reduced sensitivity.
  • the cooled separating column can bind certain analytes and impurities (such as detergents and polymers) so strongly that these remain immobilized on the column throughout the entire gradient, even during its latter part which is usually devised to help clean the separating column prior to next sample being analyzed.
  • This will lead to accumulation of analytes and impurities on the column such that the column may be rendered useless or performing badly after only few sample injections.
  • it is an advantage to raise the temperature of the separating column to at least the same temperature as that of the trap column towards the very last part of the gradient, such that every molecular species that is transferred from the trap to the separating column will also be washed out of the separating column for each analysis cycle.
  • DTF When including these temperature changes over time, TF should be referred to as DTF which has the just mentioned significant advantages over STF.
  • Focusi ng effects have previously been ach ieved and is common ly used i n chromatography by carefully matching different stationary phases in trap and separating columns. This approach is often cumbersome and normally only helps focus few select compounds and not a broader group of compounds. In addition, unwanted negative effects such as specificity switches (resulting in split peaks) may be observed.
  • TF is simpler to deploy and control and focuses over broad ranges of compounds simultaneously. It is however possible to achieve even further optimized separation effects by coupling the use of TF with the matching of different trap and separating column materials.
  • the temperature of the separating column may be raised to at least the same temperature as that of the trap column towards the very last part of the gradient, such that every molecular species that is transferred from the trap to the separating column will also be washed out of the separating column for each analysis cycle.
  • the pre-column is operated at a lower temperature than the analytical (separation) column. As a result, the break-through of early eluting substances can be reduced or prevented.
  • the present invention may further increase the capacity of the pre-column by using an ID of the pre-column that is from 100% to 500%, preferably from 120% to 350%, more preferably from 130% to 200%, and most preferably from 150% to 170% of the ID of the separating column while the length of the trap column is from 1 % to 300%, preferably from 10% to 150%, more preferably from 60% to 120%, and most preferably from 70% to 100% of the length of the separating column.
  • an ID of the pre-column that is from 100% to 500%, preferably from 120% to 350%, more preferably from 130% to 200%, and most preferably from 150% to 170% of the ID of the separating column while the length of the trap column is from 1 % to 300%, preferably from 10% to 150%, more preferably from 60% to 120%, and most preferably from 70% to 100% of the length of the separating column.
  • the present invention moreover, provides a liquid chromatography system with a pre- column for enriching a sample; a separation column with a fluid-transfer link to the pre- column; a first heat exchanger device arranged on the pre-column and enabling independent control of the pre-column temperature; a second heat exchanger device arranged on the first between 5% and 20%, preferably 30%, more preferably 40%, and most preferably at least 50% of the length of the separation column enabling independent control of the temperature of the first part of the separation column; and occasionally a third heat exchanger device arranged on the rest of the separation column enabling independent control of the temperature of this part of the separation column.
  • the present invention provides, in an advantageous manner, a physical and electrical de-coupling of the heat exchangers of the pre- and separation columns of a liquid chromatography system, so that two colu mns, namely the pre-column and the separation column as well as the first part of the separation column, can be operated at different temperatures, resulting in the ability to optimize the selectivity and/or separation efficiency.
  • Figs. 1A to 1 C show a first embodiment of the cooling/heating devices required for a liquid chromatography system in accordance with the present invention.
  • Fig 1A shows the thermo-element used for heating and cooling of the separation column, in this case, a Peltier element in contact with an elongated block of aluminium that has a groove which precisely fits around the column (0 360 ⁇ m). The aluminium piece is surrounded by an insulator layer made of plastic and foam in order to minimize the amount of energy exchanged with the ambient air (please note the lid is shown in exploded view).
  • Fig 1 B similarly shows the thermoelement used for heating and cooling of the pre-column.
  • thermo-element This is also an aluminium block with grooves cut for a precise fit to the pre-column.
  • connection fitting and some of the tubing upstream of the pre-column i.e. towards the LC system
  • the thermo-element is enclosed in the thermo-element to ensure that the liquid (and sample) has reached the desired temperature by the time it reaches the front edge of the pre-column.
  • the cooling and heating events are more time critical for the pre-column than for the analytical column and also the system must be able to cool/heat pre-columns of significantly wider diameter (than the separation columns have) and therefore this thermo-element was made with two larger Peltier elements and a substantially larger heat sink.
  • Fig 1 C shows both thermo-elements relative to oneanother where the pre- and separation- columns have been connected by a piece of transfer tubing, preferably of low inner diameter, in this case 25 ⁇ m ID.
  • Fig. 2 compares chromatograms taken with the chromatography system of the present invention without (top pane) and with (bottom pane) thermal focusing. Both chromatograms were recorded with otherwise identical experimental parameters.
  • the sample loaded was a tryptic digest of BSA (bovine serum albumin, Mw 66 kDal) and the ion trace is displayed for two values of mass-to-charge ratios, namely the peptides eluting to give rise to ions of m/z 722.6 and 582.5, resp..
  • the separation column used had an inner diameter of 75 ⁇ m and a length of 8 cm and hence its volume was approximately 1 12 nl_.
  • the pre-column and an artificially introduced dead-volume immediately before the separation column were approximately 2050 nl_, i.e. a volume that is around 1 8 times larger than that of the separation column .
  • the upper chromatogram shows the envelope of the peaks eluting under normal running conditions, that is without thermal focusing turned on. Both of the two peaks are around 40 seconds wide at the intensity level of 5% above baseline. Also both peaks exhibit pronounced asymmetry.
  • the lower chromatogram shows the same experiment albeit with thermal focusing turned on. The two peaks have now been focused such that they have become significantly more symmetrical and the peak widths at 5% above base line has dropped to around 12 seconds. As a result of the narrower peaks, the signal intensity is almost 3 times higher and the general signal-to-noise-ration of the entire chromatogram has improved.
  • Figs. 1A to 1 C show a first embodiment of the cooling/heating devices required for a liquid chromatography system in accordance with the present invention.
  • Fig 1A shows the thermo-element used for heating and cooling of the separation column, in this case, a Peltier element in contact with an elongated block of aluminium that has a groove which precisely fits around the column (0 360 ⁇ m). The aluminium piece is surrounded by an insulator layer made of plastic and foam in order to minimize the amount of energy exchanged with the ambient air (please note the lid is shown in exploded view).
  • Fig 1 B similarly shows the thermoelement used for heating and cooling of the pre-column.
  • Fig. 2 compares chromatograms taken with the chromatography system of the present invention without (top pane) and with (bottom pane) thermal focusing. Both chromatograms were recorded with otherwise identical experimental parameters.
  • the sample loaded was a tryptic digest of BSA (bovine serum albumin, Mw 66 kDal) and the ion trace is displayed for two values of mass-to-charge ratios, namely the peptides eluting to give rise to ions of m/z 722.6 and 582.5, resp..
  • the separation column used had an inner diameter of 75 ⁇ m and a length of 8 cm and hence its volume was approximately 1 12 nl_.
  • Figure 3 shows a schematic representation of the temperatures of the pre-column and the separation-column as a function of time during a chromatographic analysis that uses DTF.
  • the two temperature extrema need not be the same or even near the same for the two columns as the figure might imply. Neither axis is drawn to scale.
  • the separation-column temperature must be held at "high" for longer time than for the pre-column. But for most analyses, the separation column temperature is only raised for a relatively short period of the overall cycle.
  • the invention has been implemented and tested for the use in nano-LC-MS.
  • the implementation deploys Peltier cooling/heating elements with aluminum heat sink and fan. Peltier elements heat small aluminum bars that are covered with isolating foam. Temperature sensors and computer controlled feedback regulation is used to regulate temperatures within margins of less than one degree from the set-point except during the rapid transitions between alternating temperatures in the DTF cycle. Synchronization with the LC analysis cycle (specifically, the sample loading and de-salting and gradient formation) ensures that DTF benefits are optimized (see also fig 3).
  • Figs. 1A-C show a liquid chromatography system in which a pre-column for enriching the sample and a separation column are arranged (and which may include collective or discrete housings (not shown)).
  • heat exchangers and possibly their housings
  • First heat exchanger is associated with the pre-column
  • second and third heat exchangers are associated with the separation column.
  • the sample is loaded onto the pre-column e.g. by means of an LC pump via an inlet transfer tube.
  • the outlet of the separation column is connected to a detector (not shown).
  • Figs. 1 The arrangement shown in Figs. 1 permits three different temperature environments to be realized.
  • heat exchangers are connected to a control unit that enables independent control of the pre-column temperature and/or the separation column temperatures.
  • the pre-column and thereby the sample contained therein is heated to a second temperature at least 10 0 C higher and typically 40 0 C higher than the operating temperature of the separation column.
  • a second temperature at least 10 0 C higher and typically 40 0 C higher than the operating temperature of the separation column.
  • the experiment used an in-house packed pre-column of inner diameter 150 ⁇ m and length of around 1 cm, packed with C18 reverse phase material (5 ⁇ m bead size, ReproSil PurA, 120A pore size, Manufacturer: Dr. Maisch, Ammerbuch-Entringen, Germany). This was connected to a 10 cm long piece of empty tubing of ID 500 ⁇ m, which corresponds to a volume of 2 ⁇ l_, in order to demonstrate the effect a dead- volume or a larger pre-column has. Following this empty tubing came an in-house packed separation column of inner diameter 75 ⁇ m and length of around 8 cm, packed with C18 reverse phase material (3 ⁇ m bead size, ReproSil PurA, 120A pore size, Manufacturer: Dr. Maisch, Ammerbuch-Entringen, Germany).
  • both pre-column and separation column were at ambient temperature of around 23 degrees centigrade whereas the experiment with DTF maintained a temperature of the pre-column of 40 degrees centigrade and a separation column temperature of 0 degrees centigrade during the gradient elution.
  • the first, second, and third heat exchanger devices are thermally decoupled, but still arranged in the same column housing, without a partition between the heat exchanger devices.
  • the first, second, and third heat exchanger devices can be heated or cooled independently of each other with equal or with different temperatures.
  • a feature contributing to the thermal decoupling of the first and second heat exchanger devices is that there is no circulation of air in the column housing so that the first and second heat exchanger devices, due to their spacing and orientation, can hardly exchange heat, even if they are arranged in the same column housing.
  • the heat exchanger devices in a preferred embodiment of the invention are characterized by a material having good heat conducting properties, by thermally well integrated fluid capillaries for pre-thermostating of the solvent/sample mixture, and by ribs for receiving the column and for heat transfer thereto.
  • the heat exchanger devices can be heated and cooled in a controlled way.
  • the heat transport for thermal control of the separation column is accomplished via the solvent in the tubing prior to the column, via the contact between the separation column and the heat exchanger and via heat radiation and convection between the ribs supporting the separation column.
  • the described solution has the advantage that different temperature settings of the first and second heat exchanger devices are possible without negative thermalinfluences between these devices, even if they are arranged in the same column housing. It is an advantage of the controlled sample pre-thermostating that radial and longitudinal temperature differences of the solvent are kept as small as possible, since the temperature of the separation column and of the solvent flowing into it are substantially equal, thus avoiding any heat exchange of the solvent inside the separation column, which would have a negative effect on the chromatographic performance.
  • the heat exchanger capillaries in the first and second heat exchanger devices have different lengths over which heat is exchanged, so that a wide range of solvent flow rates can be covered.
  • the inlet and outlet connections of the heat exchangers are arranged such that the shortest possible connection to the separation column is achieved, thus ensuring a minimized dead volume.
  • the present invention has been described above and implented with reverse phase chromatography, C18 stationary phase material, and aquous/organic solvent as the mobile phases.
  • the principle of temperature affecting the distribution of analytes between the mobile and stationary phases applies also in other separation techniques.
  • An d h en ce the present invention is applicable to other modes of chromatography, e.g. normal phase LC, low pressure chromatography, ion exchange chromatography and more.
  • the invention applies to multi-dimensional separation techniques, i.e. hyphenated techniques that seperate analytes by means of two or more different physico-chemical characteristics. An especially prominent example of this would be 2-dimensional LC with strong cation exchange (SCX) separation in the first dimension followed by reverse phase separation in the second dimension.
  • SCX strong cation exchange
  • the present invention has been described and implemented using a pre-column (also called a trap-column) and a separation column (also called an analytical column) but it should apply equally well to one column separated into different segments only by the temperature control of said segments.
  • a pre-column also called a trap-column
  • a separation column also called an analytical column
  • the present invention has been described and implemented using just one pre-column segment and one separating column segment but it should apply equally well to multiple cooling/heating segments that are positioned sequentially along one long column or a number of columns.

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Abstract

L'invention concerne un procédé pour focaliser thermiquement un analyte sur une précolonne pour une chromatographie en phase liquide, le procédé comprenant l'enrichissement de l'échantillon sur ladite précolonne à une température inférieure à la température de fonctionnement d'une colonne de séparation, l'injection de l'échantillon sur la colonne de séparation par chauffage de la précolonne jusqu'à une température supérieure à la température de la colonne de séparation, et l'élution de l'analyte à partir de la colonne de séparation. Éventuellement, l'échantillon peut être soumis à un gradient de température sur la colonne de séparation, ou la colonne de séparation peut être chauffée pour supprimer un échantillon non élué après achèvement de la séparation.
PCT/EP2009/065041 2008-11-20 2009-11-12 Focalisation thermique dynamique de séparations chromatographiques WO2010057826A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US13/128,611 US8613216B2 (en) 2008-11-20 2009-11-12 Dynamic thermal focusing of chromatographic separations
EP09752355.9A EP2356441B1 (fr) 2008-11-20 2009-11-12 Focalisation thermique dynamique de séparations chromatographiques

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US11632308P 2008-11-20 2008-11-20
US61/116,323 2008-11-20

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CN110376323A (zh) * 2019-06-26 2019-10-25 北京中医药大学东直门医院 一种毛细管色谱柱的加热装置及加热方法

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