Patent Application
20100278695
This invention was made without Federal sponsorship.
This invention relates to chromatographic separation media and to media for selectively absorbing certain chemical species, especially for analytical purposes. The media are useful in liquid chromatography, supercritical fluid chromatography and electrochromatography or electrophoresis, especially for the types of high pressure liquid chromatography and chromatography at extreme pressures of greater than 5,000 pounds per square inch (PSI), nanoflow LC, and microbore LC.
Mixtures comprising components having different chemical structures are frequently separated for analytical or preparative purposes by chromatography. An aliquot of such a mixture may be introduced into a mobile phase (which may be gas, liquid or a supercritical fluid). The mobile phase is caused to flow through a stationary phase comprising separation media. Components of the mixture may interact differently with the separation media and may be retained by it for different periods. Components may therefore elute from the separation media at different times and may be collected or detected by any means responsive to a selected property of the components of interest. As used herein, the term “sample” is directed to the solutions and mixtures which are analysed to determine one or more components of interest.
Mobile phase may be caused to flow through the separation media by virtue of its pressure (if a gas or supercritical fluid), by pumping, or by the influence of an electrical field (in electrochromatography or capillary electrophoresis). A combination of these processes may also be used.
In a related technique, a complex mixture can be separated by depositing it on separation media (typically a gel) supported on a surface, and causing the components comprised in the mixture to migrate through the media by means of an electrical field. A variety of different techniques may then be used to mark the positions of different components in the media after the field has been applied for a given time. Certain components in a complex mixture may also be selectively absorbed on separation media in order to separate them from unwanted components, and then analysed using other techniques, for example mass spectrometry or other spectroscopic techniques.
Chromatographic separation media may comprise small particles packed in a tubular column (usually made of stainless steel or fused silica) or may comprise a coating on the interior wall of a capillary tube. Some current columns comprise particles as small as 1 micron and operate at pressures as high as 15,000-100,000 psi. Although these columns generally exhibit greater separation efficiency than columns comprising larger particles, it is thought that still greater efficiency could be realised by the use of separation media more suited to the high pressures used.
In general, the efficiency of all such separation techniques is to some extent dependent on the nature of the separation media. The present invention relates to improved separation media particularly suitable for use at very high pressure of a chromatographic mobile phase, but which also may advantageously be used in a variety of other techniques to improve the separation of components in complex mixtures.
The inventors have found that, particularly in the case of chromatographic separations carried out at very high pressure, increasing the thermal conductivity of prior types of separation media, or adding material of high thermal conductivity to the separation media, improves the separation efficiency of that media. The invention therefore provides improved separation media for use in the chromatographic separation of a sample mixture. The separation media comprises first particles of a first material and second particles of a second material. The first particles are capable of at least temporarily retaining at least one component of a sample mixture. The second material is selected to have a higher thermal conductivity than said first material.
A further embodiment of the invention is directed to an apparatus for the chromatographic separation of a sample mixture comprising a tubular member having an inlet through which a fluid may enter and an outlet through which fluid may leave. The tubular member has an interior space disposed between the inlet and the outlet, wherein there is disposed in such interior space, a separation media comprising first particles of a first material and second particles of a second material. The first particles are capable of at least temporarily retaining at least one component of the sample mixture. The second material is selected to have a higher thermal conductivity than the first material.
Another embodiment of the invention is directed to apparatus for selectively absorbing at least one component of a sample mixture on separation media. The apparatus comprises means for supplying a fluid which fluid is or carries the sample mixture to the separation media. The separation media has first particles of a first material and second particles of a second material. The first particles are capable of at least temporarily retaining at least one component of the sample mixture and the second material is selected to have a higher thermal conductivity than the first material.
Preferably, the first particles of a first material comprise particles of a material conventionally used in prior types of chromatographic columns, for example silica, organic polymers, siliceous polymers, graphite. These particles are coated or uncoated. As used herein, the term “coating” refers to a layer or reaction product of the particle surface with chemical agents to bond chemically functional moieties. Such moieties may comprise alkyl, aryl, phenyl or carbamate groups, or other groups used in prior types of separation media. The first particles are porous or non-porous. The first particles are sized between 1 micron and 100 microns, but most preferably are between 1 and 10 microns. The first particles are any shape including irregular shapes, but preferably are approximately spherical.
The second particles comprise the second material that has a higher thermal conductivity than the material of which the first particles are comprised. Preferably, the second material is a metal; such as gold, silver, aluminium, copper, tungsten, and molybdenum; carbon allotropes, especially various forms of diamond; and ceramics, such as alumina, aluminium nitride, titanium carbide, silicon carbide, or zirconium oxide.
Preferably, the second material has a thermal conductivity greater than 0.5 W·cm−1·° K−1, and, most preferably, greater than 1 W·cm−1·° K−1. Preferably, the thermal conductivity of the second material is approximately tenfold greater than that of the first material.
Preferably, the first and second particles are approximately the same size. The first particles are normally sized as the particles that might be used to prepare prior types of separation media comprising only the first particles.
Preferably, approximately 1% and 25% of separation media comprise the second particles. The remainder of the separation media is the first particles. A more preferred range is between 5% and 20% of the separation media is the second particles; and, more preferred, about 10%.
Preferably, the first particles are responsible for the at least temporary retention of components of a sample by the separation media. The second particles are primarily added to increase the thermal conductivity of the substrate to a value greater than that of the first particles alone. However, those skilled in the art will recognise that the second particles also interact with components of the sample and for at least some of those components to be at least temporarily retained by the second particles.
The inventors have found that by the use of separation media as described, the performance of high-pressure liquid chromatography in particular can be significantly improved. For example, the separation between two chromatographic peaks which are poorly resolved using separation media comprising only the first particles can be significantly improved using separation media comprising high thermal conductivity second particles in addition to the first particles, using otherwise similar chromatographic conditions.
Without wishing to be bound by theory, the inventors believe that the use of separation media according to the invention may reduce the detrimental effect of frictional heating that is thought to take place at very high pressure. It is speculated that the use of separation media having a higher thermal conductivity than that of the first particles alone can reduce the thermal gradients in the mobile phase that are probably caused by this heating and in so doing increase the resolution of the separation.
Other features and advantages of the present invention will be apparent to those skilled in the art upon reading the detailed description and viewing the Figures that follow.
Embodiments of the invention will now be described in detail with reference to the figures, in which:
Embodiments of the present invention will be described in detail as to preferred devices, articles of manufacture and methods featuring a separation media. As used herein, unless the context requires otherwise, the term “column” refers to all separation devices having a conduit in which a separation media is placed, including column cartridges and capillaries. Those skilled in the art will recognise that the preferred embodiments of the present invention are capable of modification and alteration and that the present description should not be perceived as limiting.
Turning now to
Separation media provided by the invention has utility for chromatographic or electrophoretic separation of components of a sample mixture, or for the selective absorption of one or more components of a mixture on a substrate, typically prior to analysis of the absorbed components by another analytical technique. Preferably, the first particles are particles conventionally used for chromatographic separations or the selective absorption of different chemical species. For example, the first material from which the first particles are made may be chosen from the following list:
The above list is by way of example only and it is within the scope of the invention to provide first material comprising other materials capable of retaining or selectively absorbing components of a mixture.
The first particles have a diameter sized between 1 and 100 μm. For chromatographic embodiments, for example reverse-phase liquid chromatography, the first particles are between 1 and 10 μm. For extreme pressure applications, the first particles are sized less than 2 μm. The particles can be any shape including irregular shapes. Preferably, the particles are approximately spherical. The first particles are porous or non-porous, and, preferably, are porous.
The second material, from which the second particles are made, is selected to have a higher thermal conductivity than the first material from which the first particles are made. Typically, the second material may have a thermal conductivity in the range 0.1-50 W·cm−1·° K−1. The thermal conductivity of the second material is, preferably, selected to be ten-fold greater than the thermal conductivity of the material of the first particle. A preferred range is 1-10 W·cm−1·° K−1, to 1-50 W·cm−1·° K−1.
The second material is selected to have as little chromatographic effect. The addition of a second material type may influence peak shape unless the second material is relatively inert under the conditions used for the separation or absorption. The potential detrimental effect on peak shape is addressed by maintaining the concentration of the second particle as low as possible while taking advantage of the thermal conductivity.
Table 1 below lists some materials that may be considered for use as the second material of the invention, together with their approximate thermal conductivities. The list is not limiting, however, and other materials may be suitable.
These thermal conductivities may be compared with the values for some commonly used chromatographic separation materials that might be used for the first particles, listed in Table 2 below:
It can be seen from Tables 1 and 2 that the addition of second particles selected from table 1 to those listed in table 2 can markedly increase the thermal conductivity of the resulting media, especially when the second material has a very high thermal conductivity, such as diamond, gold, or certain forms of graphite.
Embodiments of separation media according to the invention suitable for chromatographic separations comprise a mixture of first and second particles comprising, for example, 1%-25% by volume of the second particles and the remainder substantially comprising the first particles. Preferably, the second particles comprise between 5% and 20% of the separation media. A concentration of second particles of approximately 10% of the separation media is a reasonable compromise with respect to the advantages of reducing thermal gradients and providing an appropriate well defined peak.
A preferred separation media has first particles of a hybrid siliceous polymer and second particles of gold, diamond, or graphite. The hybrid siliceous particles may be a methylpolyethoxysilane (structure I below) or a polyethoxysilane, (structure II below). Structure I:
The open valences above are terminal hydrogen, or alkylene, alkynylene, or arylene groups or are bonded to further subgroups of the structures depicted. As used above, x and y are integers from 1 to infinity.
Alternatively, they may comprise any of the polymers described in U.S. Pat. No. 6,686,035, the contents of which are herein incorporated by reference. These polymers may be represented by the formulae SiO2/[R2pR4qSiOt]n and SiO2/[R6(R2rSiOt)m]n, where R2 and R4 are independently C1-C18 aliphatic or aromatic moieties, R6 is a substituted or unsubstituted C1-C18 alkylene, alkynylene, or arylene moiety bridging two or more silicon atoms. The following also applies:
These siliceous particles are coated or uncoated. As used herein, the term “coated” refers to having the surface bonded to one or more functional groups. These functional groups are selected for the chromatographic separation to be carried out. For example, their surfaces may be modified with hydrocarbon groups, including alkyl C8 or C18 groups, phenyl, aryl, or carbamate groups. Methods of preparing and coating these siliceous polymeric particles are disclosed in the above-referenced U.S. Pat. No. 6,886,035.
Particles of structure I and sizes between 5 μm and 25 μm are available in the form of “XTerra™” chromatographic columns from Waters Corporation, Milford, Mass. Particles of structure II having a size of 1.7 μm are also available in the form of “ACQUITY UPLC®” or “XBridge™” chromatographic columns from Waters Corporation. Both types of particles are available with a variety of organofunctional coatings.
Siliceous polymer separation media according to the invention is made by mixing the polymer particles described above with similar-sized particles selected from Table 2 above. Gold, graphite and diamond, as a material for second particles, is preferred. The media may comprise between 1 and 25% of the second particles. However, about 10% gold or diamond has been found to give good results.
Diamond particles suitable for use in separation media according to the invention comprise natural diamond or high-pressure synthetic diamond manufactured by chemical vapour deposition (CVD) induced by RF, microwave, electron cyclotron resonance (ECR), etc, or by ion sputtering processes. Suitable diamond particles are commercially available, for example from Element Six, Netherlands.
Turning now to
Turning now to
The sample injection device 14 is used to introduce a sample from a reservoir 27 into the flow of mobile phase from the pump 13. Constituents of a mobile phase (which may comprise a mixture of several solvents and/or additives) are stored in a reservoir system 16. The pump 13 is provided to pump the mobile phase through the column 4, and may be capable of providing to the column 4 a mobile phase whose composition varies with time, for example to allow gradient elution to be carried out. Eluent from the column 4 is received by a detector or collector system 15.
The HPLC system 111 may be used for either analytical purposes or preparative purposes. If used for analytical purposes, detector system 15 may comprise a detector responsive to at least one of the components comprised in a sample mixture to be analysed. Suitable detectors include UV absorption detectors, evaporative light scattering detectors, refractive index detectors, fluorescence detectors, electrochemical detectors, or mass spectrometric detectors. If the apparatus is to be used to prepare samples of constituents comprised in a sample mixture, detector system 15 may comprise a non-destructive detector such as a UV absorption detector, and a number of vessels or a sample plate comprising a number of wells, each vessel or well for receiving a particular constituent as it elutes from the column 4.
Means may also be provided to minimize the temperature gradient along the axis of the tubular member 6, which may otherwise develop due to the flow of mobile phase through the separation media 5. An insulated jacket 30 comprising a heater and one or more temperature sensors may be disposed around the tubular member 6. A temperature controller 31 may also be provided to control the power supplied to the heater in response to a signal from the one or more temperature sensors so that the temperature of the tubular member 6 is maintained approximately constant along at least a substantial portion of its length. In the alternative, in the event the components being separated are heat sensitive, the heater may be replaced by one or more cooling devices (for example, Peltier effect devices) and the temperature controller 31 adjusted to maintain the temperature of the tubular member 6 below ambient.
Alternative methods of minimizing the temperature gradient along the tubular member 6, comprise, by way of example, without limitation, immersion of tubular member 6 in a temperature-controlled water bath, or subjecting the tubular member 6 to a flow of heated (or cooled) air from a fan.
HPLC systems 111 for carrying out a high-resolution analytical separations are sold by several vendors, such as the ALLIANCE® and ACQUITY UPLC® systems available form Waters Corporation (Milford. MA). HPLC systems 111 for preparative uses are sold by several vendors such as the Delta 600 fluid handling unit also available from Waters Corporation.
A suitable column 19 for use in the apparatus of
One embodiment of the present invention features a capillary columns similar to capillary column 19, shown in
An embodiment of the invention for selectively absorbing one or more constituents of a sample mixture is illustrated in
The separation media is of any of the forms described, however, for this discussion the separation media will be described as being chemically modified in order to provide selective absorption of a desired chemical species. For example, linker moieties are provided in a manner known in the art to attach antibodies to the surface. The antibodies have specific affinity to selected haptens, often proteins, so that particular proteins can be retained on the coating through an immunological interaction. This embodiment can be used to extract specific chemical species or even specific molecules from a complex mixture for analytical or preparative purposes. The higher thermal conductivity of coatings allow for distribution and dissipation of thermal energy. The plates 26 are used for matrix laser desorption ionization (MALDI) processes.
In the alternative, the coating 27 is a gel, for example a polyacrylamide gel, allowing the apparatus to be used for gel electrophoresis. In such an embodiment, the first particles of the invention may comprise polyacrylamide to which second particles of gold or diamond are added to increase the thermal conductivity of the gel.
Substrate 26 of the
Embodiments of the invention according to
In the alternative, solid substrates 26 according to the invention are be produced in granular or powder form with chemically modified surfaces that allow the grains to selectively adsorb specific types of chemical species. These embodiments may be used to extract species that are selectively absorbed on them from complex mixtures.
Four 50 mm long chromatographic columns, listed in Table 3 below, were packed using a downward slurry packing method with mixtures of “ACQUITY UPLC®” 1.7 μm BEH particles (structure II above, and available from Waters corporation, Milford, Mass.) and various proportions of non-porous, irregularly shaped 1.5-2.5 μm diamond particles.
For the following experiments, a mobile phase comprising 65% acetonitile, 35% water was used. A test sample comprising 9.6 μg/ml thiourea, 0.77 mg/ml toluene, 96 μg/ml naphthalene, 384 μg/ml acenaphthene, 500 μg/ml benzene, 0.1 μl/ml heptanophenone and 3 μl/ml amylbenzene, was used to obtain the results listed below. A sample loop of 2 μl volume was employed, and detection was by a UV absorbance detector tuned to 254 nm. A Waters corporation “ACQUITY UPLC®” pump system was used.
Data relevant to the performance of the four columns is listed in Table 4. The figures in table 4 relate to the heptanophenone component in the test sample. Data relating to “ambient” operation was obtained with the external surface of the columns in contact with ambient air, whereas the “adiabatic” data was obtained with the columns insulated from ambient air by layers of glass fibre tape. The retention factors listed have been corrected for extra-column contributions.
This application claims priority from U.S. Provisional Patent Application No. 60/916,611, filed May 8, 2007. The contents of these applications are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US08/62737 | 5/6/2008 | WO | 00 | 2/25/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60916611 | May 2007 | US |
