The present invention relates to a capillary assembly suitable for connecting various components of an analytical measuring device, for example a liquid chromatograph or a capillary electrophoresis device, to each other.
In a liquid chromatographic (LC) system, connecting capillaries, as well as columns made from capillaries, are often used.
In a liquid chromatographic system, the LC column is located between an injector and an LC detector to separate one or more constituents of interest from the various interferences in a sample to permit detection of these constituents of interest by an LC detector.
Capillary LC is a micro-version of traditional liquid chromatography and its popularity has grown rapidly during the past decades. Capillary LC columns have extremely low solvent consumption and require low volumes of samples for analysis. NanoLC is the name given to further miniaturization of chromatography, where flow-rates are typically below 1,000 nL/min and column diameters are typically around 75 μm (inner diam.). Analogous to traditional liquid chromatography, nano-LC and capillary-LC also consists of a micro-pump, a capillary column, a detector, and a data processing device. The capillary column is important to the system because it is where the separation process occurs.
A capillary LC column is manufactured by packing a capillary column with silica media, such as bonded silica particles, also referred to as packing material. Different types of materials, such as fused silica glass, stainless steel, and high-tensile polymers, have been used for capillary columns. Due to their unique features, fused silica glass capillaries are the most common for preparation of capillary LC columns. Fused silica capillary columns have inner diameters of less than 1 mm and, typically, less than 0.25 mm. They are strong and can withstand high packing pressure. It is easy to control their column dimensions during manufacturing, and the columns do not deform during packing. Further, the wall of a fused silica capillary is smooth, which is very desirable for packing.
Although fused silica capillaries have some unsurpassed advantages, they do have certain limitations. The most significant limitation stems from the brittle and fragile nature of the glass material from which they are made. The fragile nature of a thin, fused silica capillary makes packing, shipping, and handling difficult. A layer of polyimide is generally coated on the outside of the fused silica capillary for protection. However, if the polyimide layer has incurred even a small scratch during production or handling, it will lose its effect and the capillary can break with just a gentle touch.
To avoid damage to the packed capillary LC column, a shielding layer of stainless steel is sometimes provided for protection. Although the currently available steel shield do prevent the capillaries from breaking, they are rigid and thus require long connecting capillaries to install the capillary column between the injector and the detector of an LC system. This generates unnecessary extra column dead volume which degrades separation efficiency. Moreover, a separate assembly process is required in addition to the packing process, which will add extra cost to capillary LC column production.
When connecting a fused silica glass column firmly to another component, a sleeve is often needed to tighten and secure an end-fitting on the end of the capillary column. During the packing process, one end of the capillary is typically enclosed with an end-fitting assembly and the other end is connected to a slurry reservoir. A flexible sleeve is employed in the end-fitting assembly during packing because sufficient tightening is required to enclose the end for high pressure packing. The sleeve facilitates tightening and compensates for the size of the capillary, which is too narrow for the end-fitting. The packing pressure can force the end-fitting assembly open if there is insufficient tightening, while too much tightening can damage the capillary.
One particular use of HPLC is in the field of proteomics, i.e. the study of the entire protein complement of a cell or tissue sample where proteolytic fragments of proteins (e.g. peptides) are separated by HPLC prior to detection by mass spectrometry. Since the samples being analyzed in proteomics experiments are typically very complex and available in only very low quantities, it is frequently a challenge to obtain sufficient sensitivity and analysis speed. Sensitivity is optimized by reducing the flow rate of the mobile phase in combination with use of nano-bore columns (i.e. columns of narrow inner diameter).
Whereas the use of nano-bore columns is required in order to optimize the analytical sensitivity, it does however cause a range of complications inasmuch as it is difficult to connect tubing of narrow inner diameter in a fail-safe manner; tubing with the required bore-size can only be manufactured in a certain small selection of fragile materials; and the narrow diameters used require a very high chromatographic pressure in order to force liquid through the tubing at the required flow rate.
WO2009/147001 A1 discloses an integrated separation column having various fittings. For example FIG. 1 of that document shows an embodiment, where the integrated column (including fittings and electrospray needle) is embedded in a plastic material. Meanwhile there is no disclosure of sleeves that can readily be connected with other means, such as pumps, valves, analytical devices etc. The fittings used in WO2009/147001 comprise sheaths (or tubes) on which the ferrules can be placed and tightened; such sheaths or metal tubes are commonly used in analytic chemistry. However, in WO2009/147001 A1 the entire assembly comprising column, sheaths and ferrules, is covered with plastic material, and hence the sheaths cannot be connected to other means without removing the plastic material covering the ferrules. Since the very aim of the invention described in WO2009/147001 A1 is to provide an integrated separation column including fittings, where the consumer has no access to the fittings (being covered by a plastic material), WO2009/147001A1 does not provide a generic capillary.
There is a need, therefore, for a means to facilitate the use of fragile column materials.
It is therefore an object of the present invention to provide a device that can protect the capillary during packing and handling, and alleviate the other shortcomings of the fragile fused silica capillary.
It is another object of the present invention to provide a capillary assembly for analytical measurement technology which has a small and substantially constant inner diameter, a smooth inner wall, and which can be equipped with end fitting in an easy way, and which does not have the previously mentioned disadvantages.
The present invention solves the above problems by providing a means to facilitate the use of fragile column materials. This involves reinforcing the fragile tubing by the addition of steel or PEEK sleeves and/or embedding the fragile tubing in an injection-molded resin such that the fragile tubing is not exposed directly to operator handling and manipulation. Further functional improvement is obtained by including additional components inside the resin. Thereby a versatile and robust capillary assembly is achieved.
As mentioned above, the capillary assembly according to the present invention makes use of sleeves, preferably steel or PEEK-sleeves, provided only in the end regions of the capillary. In the regions of the capillary where there are no sleeves the capillary is coated by a flexible plasticlayer, which is in direct contact with the capillary. In that way an additional protection against scratches is achieved.
In a preferred embodiment of the invention the glass capillary is a fused silica glass capillary; however, other materials can also be used, for example boro-silicate glass and thin-walled polymer and metal tubing.
The present invention is based on a method by which a capillary column, such as a silica glass capillary column, or connecting capillary tubing along with the accompanying end sleeves for connection via a fitting with adjacent liquid conduits is embedded in a polymer matrix.
In accordance herewith, the present invention is directed to a method for producing a capillary assembly, preferably a fused silica assembly, said method comprising:
The present invention also provides a capillary assembly comprising:
It is important to stress that molded plastic coating exclusively coats the capillary and a part of the sleeves, and not e.g. ferrules or other fittings as in WO2009/147001 A1. Accordingly, the capillary assembly of the present invention can be readily disconnected from the means it is connected to, which is in contrast to the integrated device in WO2009/147001 A1, where the connection is limited to the specific fitting protruding from the coating material.
Plastifying the part may be achieved in various ways, preferably by heating the plastic material beyond the softening temperature for bringing it in its softening range and making it soft. In a preferred embodiment the entire column and fittings are surrounded by the plastic material. The molding part may be a pre-formed part adapted to the shape of the silica capillary and of the forming tool.
The forming of the molding part may be achieved by closing the forming tool and exerting pressure on the pre-formed part. Alternatively, this is achieved by closing the forming tool and heating the forming tool together with the plastic material.
In preferred embodiments of the present invention, the forming of the molded part may be achieved by injecting molten plastic material into a mold wherein the capillary with sleeves are located and allowing the molten plastic to embed these parts and cool off and harden to become solid. Alternatively, the molded part may be shaped by exerting pressure on the plastic material caused by the thermal expansion of the plastic material by heating the closed forming tool comprising the plastic material, alternatively by exerting pressure on the plastic material by closing the forming tool, or actively cooling down the plastic material and/or the forming tool. Still another alternative embodiment may be achieved by mixing chemicals that subsequently polymerize inside a mold thereby embedding the capillary with sleeves and other related components.
Preferably, the plastic materials of the present invention are thermoplastic hotmelts based on polyamide or polyurethane, such as those marketed under the tradename MacroMelt (Henkel Kommanditgesellschaft). These include at least one room-temperature-flowable polymerizable compound in combination with a polymeric matrix present in an amount sufficient to render the composition non-flowable at temperatures of at least about 49° C. The polymerizable compound or composition may be selected from a wide group of materials including anaerobics, epoxies, acrylics, polyurethanes, olefinic compounds and combinations thereof.
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According to the present invention, devices and techniques for HPLC applications are provided. Merely by way of example, the invention has been applied to a high pressure liquid chromatography process. But it would be recognized that the invention has a much broader range of applicability.
Embodiments may comprise one or more of the following: a part surrounding an HPLC column with end fittings that are plastified and molded within a forming tool for forming or for shaping the form of the integrated column and for fixing sleeves (and eventually end-fittings). The molding part comprises a plastic material. Advantageously, this technique enables sealing and positioning of sleeves and column. Advantageously, the forming tool can form the column to a desired shape with a good dimensional stability and a high reproducibility. Additionally, close tolerances can be held or maintained, for example, by exactly adjusting the process parameters like the temperature and the detention time within the forming tool.
The molding part can be realized as a pre-formed part, wherein the shape of the pre-formed part is adapted to the shape of the column/capillart and sleeves/fittings and of the forming tool. The pre-formed molding part can be plastified by heating the plastic material above or beyond the softening temperature and bringing it in its softening range for making it soft and pliable. Advantageously, the plastified plastic material can be evenly formed to the outer surfaces of the column and fittings. This enables a homogenous force distribution across the surfaces. Besides this, the mechanical stress after forming can be reduced.
In embodiments, the pre-formed molding part can comprise two or more component parts, wherein said component parts are joined to each other.
Most advantageously, the molding part can be realized by injecting molten plastic material into a mold and allowing this to cool to such temperature where the plastic forms a stable solid which may be flexible or entirely rigid depending on the chosen chemical composition of the plastic material.
Compared to standard HPLC designs, then UHPLCs (ultra-high pressure range HPLCs) are designed to generate the higher backing pressure by using e.g. stronger motors on pumps and stronger valves and composites inside valves and other active components. While these components can be made with due care and consideration from present materials, the currently most limiting element has been the tubing that carries the solvent at pressures above 5,000. For low-flow chromatography systems, i.e. flow rates below 5 mL/min, the outer diameter of the standard LC tubing is usually one of three standard sizes: 360 μm, 1/32″, and 1/16″. The inner diameters tend to range from 5 μm to 300 μm, but any size combination of 360 μm OD and more than 200 μm ID will have very thin wall thickness and will be too fragile for normal use and handling.
The material used for LC tubing is typically one of: steel (316), fused silica glass, or
PEEK. Newer types of tubing combine two of these materials in order to obtain select advantages associated with each of the materials. Unfortunately, whether materials are used separately or mixed, each existing type of tubing on the market has severe disadvantages that hinder their robust use in nano-flow LC at ultra high pressures. For instance:
Therefore, whereas multiple types of tubing for capillary and nano-flow chromatography exists, none of the existing materials or material combinations presents satisfactory solutions in terms of mechanical and physical robustness, chemical inertness, or selection of inner diameter.
The present invention describes methodology and apparatus that provides greatly improved capillary tubing and column products. In a preferred implementation the new tubing is an assembled product that contains an inner core of fused silica glass tubing that is coated with poly-imide as most commonly used. The desired length of tubing is cut from a reel of tubing and each end is covered (i.e. sleeved) with a concentric polymer tube or steel tube that has a tight fit to the inner tube. That is, the OD of the fused silica tubing is few micrometers smaller than the ID of the sleeve. Then the portion of the fused silica tubing, that is not covered by the sleeves, is embedded in a polymer resin by injection molding (in a mold) that will subsequently harden to form a protective outer layer around the fused silica. The resin may also cover parts of the sleeves at one or both ends and it may also be advantageous to include additional components inside the resin embedded volume in order to provide additional functionality of the complete assembly.
This inner diameter of the fused silica tubing can be obtained in many dimensions while the outer diameter tends to conform to one of few standard sizes. In a preferred implementation the fused silica tubing is approximately 360 μm OD, a size for which sleeves are readily available. These sleeves often have an outer diameter of approximately 1/32″ or 1/16″ which again is a standard size for connectors and fittings used in the field of chromatography. Sleeves can be made of per-fluoro-polymers, steel, or PEEK in a preferred implementation. Normal lengths of sleeves range from around 2 cm to 5 cm.
Resin for injection molding may be of many chemical compositions. In a preferred implementation, a hotmelt resin based on poly-urethane (MacroMelt from Henkel) was used to give a robust but somewhat flexible material that binds well to the poly-imide layer of the fused silica tubing and also binds to the outer surface of the sleeves.
For resin embedded fused silica tubing when made according to the descriptions herein, we have found several advantages over the current state of the art, including:
When embedded in resin, fused silica is readily pressure proof up to about 20,000 psi when the inner diameter of the glass is less than 150 μm. The poly-imide layer cannot be scratched owing to the protective resin layer hence the assembly is robust even when handled and flexed while under pressure.
Sleeves and ferrules can be firmly coordinated relative to the liquid transfer conduit such that it facilitates leak proof assembly with fittings and other active components of an HPLC system.
Number | Date | Country | Kind |
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PA 2011 00288 | Apr 2011 | DK | national |
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/DK2012/050120 | 4/11/2012 | WO | 00 | 11/20/2013 |
Number | Date | Country | |
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61474344 | Apr 2011 | US |