Patent Description:
Chromatographic techniques are important tools for the identification and separation of complex samples. The basic principle underlying chromatographic techniques is the separation of a mixture into individual components by transporting the mixture in a moving fluid through a retentive media in a separation channel (e.g., a separation column). The moving fluid is typically referred to as the mobile phase and the retentive media is typically referred to as the stationary phase. The separation of the various constituents of the mixture is based on differential partitioning between the mobile and stationary phases. Differences in components' partition coefficient result in differential retention on the stationary phase, resulting in separation. A detector, positioned at the outlet end of the separation channel, detects each of the separated components as they exit the separation channel yielding a chromatogram.

The methods of choice for chromatographic separations have conventionally been gas chromatography (GC) and liquid chromatography (LC). One major difference between GC and LC is that the mobile phase in GC is a gas, whereas the mobile phase in LC is a liquid. Although GC is typically a sensitive method of analysis, the high temperatures required in GC make this method unsuitable for some high molecular weight biopolymers or proteins because they are denatured by heat. In addition, their low vapor pressure makes them insoluble in the gas phase. In contrast, LC does not require high temperatures and can utilize solubilizing mobile phases. LC that generally utilizes small packing particles and moderately high pressure is referred to as high-performance liquid chromatography (HPLC); whereas liquid chromatography that generally utilizes very small packing particles and high pressure is referred to as ultra-high performance liquid chromatography (UHPLC). In HPLC and UHPLC the sample is forced by a liquid at high pressure, which is the mobile phase, through a separation channel (e.g., a column) that is packed with a stationary phase, which is typically composed of irregularly or spherically shaped particles. In some embodiments, the stationary phase may be a monolithic solid.

Various materials (e.g., stainless steel, polymers such as PEEK, fused silica, etc.) have been used for column hardware (e.g., HPLC hardware or UPLC hardware) for chromatographic separation. However, each of these categories of materials exhibits deficiencies with respect to chromatographic separation of peptides and proteins in biological samples. <CIT> discloses tubing and a method for manufacture. <CIT> discloses a coating for a surface used in a device for carrying out a separation process such as affinity chromatograph, wherein said surface can comprise an alloy such as MP35N (trademark of SPS Technologies, LLC). <CIT> discloses chromatography apparatus having diffusion-bonded and surface-modified components.

Some exemplary embodiments include devices for separating a sample by chromatography and components for chromatographic separation devices. The devices or components have a wetted surface exposed to a mobile phase including the sample during chromatographic separation. The wetted surface includes an alloy material comprising nickel, cobalt, molybdenum and chromium and limited in an amount of titanium The alloy material of the wetted surface enables chromatography of samples containing peptides (e.g., histidine-containing peptides) with narrow peaks for the peptide components and without significant peak tailing of the peptide components. The alloy material is more corrosion-resistant that other materials, such as stainless steel, employed in some chromatographic separation devices. The enhanced corrosion resistance of alloys employed may increase a useful lifetime of chromatographic separation devices and components.

Devices for separating a sample by chromatography have wetted surfaces exposed to a mobile phase including the sample with one or more of the wetted surfaces including an alloy material selected to resist adsorption of proteins and peptides present in the sample. The alloy material is limited in an amount of titanium (i.e., less than <NUM> wt% titanium), e.g. less than <NUM> wt% titanium, less than <NUM> wt% titanium, less than <NUM> wt% titanium, less than <NUM> wt% titanium, less than <NUM> wt% titanium. In an embodiment, the alloy material has a composition of about <NUM> wt% cobalt, <NUM> wt% nickel, <NUM> wt% chromium, and <NUM> wt% molybdenum and is limited in an amount of titanium. In some aspects, the alloy material is included in a surface portion of a chromatography component or separation channel with a bulk portion of the chromatography component or separation channel includes a different material. In an example, the surface portion and the bulk portion are diffusion bonded to each other.

In some aspects, the components and devices having wetted surfaces including the alloy material are configured for use in microfluidic separation devices or systems (e.g., where a width or diameter of a separation channel falls in a range of <NUM> to <NUM>). In some aspects, the components and devices having wetted surfaces including the alloy material are configured for use at high pressures (e.g., at pressures in a range of <NUM> to <NUM> MPa (<NUM>,<NUM> to <NUM>,<NUM> psi)).

The invention provides a device for separating a sample by chromatography as recited by Claim <NUM>.

In some embodiments, the wall includes a surface portion including the wetted surface and a bulk portion, and the composition of the alloy material of the wetted surface is different than a composition of a material of the bulk portion. In some embodiments, the surface portion is diffusion bonded to the bulk portion. In some embodiments, most or all of the thickness of the wall may be made of the alloy.

In some embodiments, the wetted surface of the wall defines a separation channel. In some embodiments, the device further includes an electrospray tip at an outlet of the separation channel and a wetted surface of the electrospray tip includes the alloy material.

In some embodiments, the device includes two or more sheets of the alloy material, a portion of each sheet forming a portion of the wall. In some embodiments, the two or more sheets are diffusion bonded at an interface with at least a portion of the separation channel extending along the interface.

In some embodiments, the device includes two or more sheets, each sheet including a layer of the alloy material and each sheet forming a portion of the wall with the layer of alloy material of the sheet forming the wetted surface for the portion of the wall. In some embodiments, the layers of the alloy material of the two or more sheets are diffusion bonded at an interface with at least a portion of the separation channel extending along the interface.

In some embodiments, the device also includes an end fitting and wetted surfaces of the end fitting include the alloy material.

In some embodiments, the device also includes a seal ring and wetted surfaces of the seal ring include the alloy material. In some embodiments, the seal ring includes a frit and wetted surfaces of the frit include the alloy material.

In some embodiments, the device also includes a frit and wetted surfaces of the frit comprise the alloy material.

In some embodiments, the device also includes a weir and wetted surfaces of the weir comprise the alloy material.

In some embodiments, the device also includes one or more integrated valves, and wetted surfaces of the one or more integrated valves comprise the alloy material.

In some embodiments, the device also includes a distributor disk and wetted surfaces of the distributor disk include the alloy material.

In some embodiments, all surfaces of the device upstream of an outlet end of the separation channel and configured to be in contact with the mobile phase including the sample during use, excluding the stationary phase, include the alloy material.

In some embodiments, the width or diameter of the separation channel falls in a range of <NUM> to <NUM>.

In some embodiments, the device is a microfluidic device and the width or diameter of the separation channel falls in a range of <NUM> to <NUM>.

There is also described a component configured for use in a device for separating a sample by chromatography includes a body having a wetted surface exposed to a mobile phase including the sample during chromatographic separation.

In some embodiments, the wetted surface defines a separation channel through which the mobile phase including the sample flows during use. In some embodiments, a width or diameter of the separation channel is between <NUM> and <NUM>. In some embodiments, the component further includes an electrospray tip at an outlet of the separation channel, and the wetted surface of the body includes a wetted surface of the electrospray tip.

In some embodiments, the component further includes one or more integrated valves, and wetted surfaces of the one or more integrated valves include the alloy material.

In some embodiments, the component is an end fitting for an inlet or an outlet of a separation column.

In some embodiments, the component is a stationary phase retaining element configured to keep a stationary phrase within a separation channel of the device. In some embodiments, the stationary phase retaining element is a frit. In some embodiments, the stationary phase retaining element is a weir structure.

In some embodiments, the body includes a surface portion including the wetted surface and a bulk portion, and a composition of the alloy material of the wetted surface is the same as a composition of a material of the bulk portion. In some embodiments, the body includes a surface portion including the wetted surface and a bulk portion, and a composition of the alloy material of wetted surface is different than a composition of a material of the body portion. In some embodiments, the surface portion is diffusion bonded to the bulk portion.

In some embodiments, the wetted surface consists of the alloy material.

There is also described a solid body configured for use as at least part of a stationary phase in a chromatographic separation device includes an alloy material as described herein.

There is also described a method of performing chromatographic separation on a sample includes providing a chromatographic separation device including a separation channel. The method also includes flowing a mobile phase carrying the sample into and through the separation channel, thereby performing chromatographic separation on the sample.

In some embodiments, the method also includes detecting components of the sample downstream of the separation channel.

In some embodiments, the sample includes proteins and the proteins in the sample are separated and detected. In some embodiments, the sample includes peptides and the peptides in the sample are separated and detected. In some embodiments, the sample comprises histidine-containing peptides and the histidine-containing peptides in the sample are separated and detected. In some embodiments, the sample includes phosphopeptides and the phosphopeptides in the sample are separated and detected.

In some embodiments, the method also includes detecting components of the sample downstream of the separation channel where the sample includes one or more of proteins or peptides, and the one or more proteins or peptides in the sample are separated and detected with a tailing factor of less than <NUM>.

There is also described a method of performing chromatographic separation includes providing a chromatographic separation device in accordance with any embodiments described herein and flowing a mobile phase carrying a sample through the chromatographic separation device, thereby separating components of the sample.

Some exemplary embodiments includes devices of components for performing enzymatic reactions that may be part of immobilized enzymatic reactor (IMER) systems. The devices configured for performing enzymatic reactions include a wall defining a chamber having an inlet and an outlet with the wall having a wetted surface exposed to a liquid sample during use. The wetted surface of the wall includes any of the alloy materials described herein. The components configured for performing enzymatic reactions include a body having a having a wetted surface exposed to a liquid sample during use with the wetted surface including any of the alloys described herein.

In some embodiments, the wall or the body comprises a surface portion including the wetted surface and a bulk portion, and the composition of the alloy material of the wetted surface is different than a composition of a material of the bulk portion. In some embodiments, the surface portion is diffusion bonded to the bulk portion. In some embodiments, the surface portion consists of the alloy material.

The alloy material is limited in an amount of titanium to <NUM> wt%. In some embodiments, the alloy material is limited in an amount of titanium to <NUM> wt%. In some embodiments, the alloy material is limited in an amount of titanium to <NUM> wt%. In some embodiments, the alloy material is limited in an amount of titanium to <NUM> wt%. In some embodiments, the alloy material is limited in an amount of titanium to less than <NUM> wt%. In some embodiments, the alloy material is limited in an amount of titanium to less than <NUM> wt%. In some embodiments, the alloy material is limited in an amount of titanium to less than <NUM> wt% titanium.

The alloy material includes both cobalt and chromium as constituents. The alloy material further includes molybdenum as a constituent.

The alloy material includes the following constituents: <NUM> wt%-<NUM> wt% cobalt; <NUM> wt%-<NUM> wt% nickel; <NUM> wt%-<NUM> wt% chromium; and <NUM> wt% -<NUM> wt% molybdenum; and limited in an amount of titanium to <NUM> wt%; and a remainder being limited to a total of <NUM> wt%. In some embodiments, the remainder is limited to a total of <NUM> wt%.

In some embodiments, the alloy material includes the following constituents: <NUM> wt%-<NUM> wt% cobalt; <NUM> wt%-<NUM> wt% nickel; <NUM> wt%-<NUM> wt% chromium; and <NUM> wt% -<NUM> wt% molybdenum; and limited in an amount of titanium to <NUM> wt%; and a remainder being limited to a total of <NUM> wt%. In some embodiments, the remainder is limited to a total of <NUM> wt%.

In some embodiments a wetted surface consists of the alloy material. In some embodiments, most of the surface area of the wetted surface is covered by the alloy material. In some embodiments, over <NUM>% of the surface area of the wetted surface is covered by the alloy material. In some embodiments, over <NUM>% of the surface area of the wetted surface is covered by the alloy material. In some embodiments, over <NUM>% of the surface area of the wetted surface is covered by the alloy material. In some embodiments, over <NUM>% of the surface area of the wetted surface is covered by the alloy material.

In some embodiments, the device or component is configured for use in ion exchange chromatography. In some embodiments, the device or component is configured for use in reversed-phase chromatography.

In some embodiments, the device or component is configured to withstand pressures used in high performance liquid chromatography. In some embodiments, the device or component is configured to withstand pressures used in ultra-high performance liquid chromatography. In some embodiments, the device or component is configured to withstand pressures of <NUM> to <NUM> MPa (<NUM>,<NUM> to <NUM>,<NUM> psi). In some embodiments, the device or component is configured to withstand pressures of <NUM> to <NUM> MPa (<NUM>,<NUM> psi to <NUM>,<NUM> psi). In some embodiments, the device or component is configured for low pressure applications.

In some embodiments, the alloy material is resistant to adsorption of proteins and resistant to adsorption of peptides. In some embodiments, the alloy material is resistant to adsorption of histidine-containing peptides. In some embodiments, the alloy material is resistant to adsorption of phosphopeptides.

Other advantages and features will become apparent from the following detailed description considered in conjunction with the accompanying drawings. It is to be understood, however, that the drawings are designed as an illustration only and not as a definition of the limits of the invention.

To assist those of skill in the art in making and using the devices and components and associated methods, reference is made to the accompanying figures, which are not necessarily to scale.

Stainless steel has been widely used as column hardware (e.g., HPLC hardware or UPLC hardware) for chromatographic separation. However, it has been found that some components of biological samples, such as peptides and proteins, often adsorb to column hardware of stainless steel and similar metals during analysis. It has been theorized that the adsorption is due to interaction between the peptides and the iron contained in the metal. The adsorption to column hardware during analysis causes disappearance of or reduced signal from these components in the chromatogram, or substantial peak broadening (see description accompanying <FIG> below).

Further, the effect of the adsorption on the chromatograph may vary for subsequent injections until the adsorption on the hardware reaches a saturation level, indicating that the column needs to be conditioned prior to analysis (see description accompanying <FIG> below). Stainless steel column hardware can be conditioned by injecting large quantities of peptide or protein prior to the analysis, which reduces the amount of adsorption of these components from samples during analysis. However, this may only be a temporary fix. Further, conditioning may waste sample and increases the time and effort required to obtain useful results.

Another approach to address adsorption of components of biological samples during analysis is to passivate surfaces of the hardware that will contact with the sample during analysis. It has been theorized that the adsorption is due to interaction between components, such as peptides, and the iron contained in the stainless steel or other metal used for column hardware. In one approach, stainless steel hardware may be exposed to an acid that preferentially dissolves iron, resulting in iron-deficient and chromium-rich material at the surface of the stainless steel hardware and presumably decreasing adsorption. However, this passivation may only be a temporary solution. For example, the iron-deficient surface may be damaged (e.g., scratched by the stationary phase) during use, exposing the underlying iron-rich stainless steel. Further, passivation of narrow bore tubes, such as tubes with an inner diameter of <NUM> or less, is difficult to perform reproducibly and inexpensively in a manufacturing environment.

Alternatively, non-metal materials could be employed for the surfaces of column hardware to avoid problems of adsorption of biological sample components on metal surfaces. Polymers such as polyether ether ketone (PEEK) could be used for column hardware instead of metals such as stainless steel, except that polymer materials do not have sufficient mechanical strength to be used at the high pressures required for HPLC and UHPLC. Alternatively, stainless steel columns could be sleeved or coated with polymers such as PEEK or polytetrafluoroethylene (e.g., TEFLON from DuPont). While this approach would increase the mechanical strength of the columns, it would be difficult to manufacture coated or sleeved columns with small diameters, particularly those having a diameter of less than <NUM>. Further, polymeric materials may deleteriously adsorb proteins and peptides by hydrophobic interaction when using highly aqueous mobile phases such as those employed in size-exclusion and ion-exchange chromatography. Alternatively, fused silica could be used instead of stainless steel for the column; however, it is more difficult to manufacture columns from fused silica than from stainless steel. Additionally, the silanols on the fused silica surface can interact with proteins and peptides by ion exchange.

The embodiments of the invention address or avoid problems arising from the use of conventional column materials such as stainless steel, polymers such as PEEK, and silica for chromatographic column components when performing chromatography on biological samples including peptides (e.g., histidine-containing peptides) and proteins. There are described not only devices for separating a sample by chromatography, but also components configured for use in a device for separating a sample by chromatography, and methods for performing chromatographic separation on a sample. In device and component embodiments, the device or component includes a wetted surface exposed to a mobile phase including the sample during chromatographic separation with the wetted surface of the wall including an alloy material. The alloy material includes nickel, cobalt, molybdenum and chromium and is limited in an amount of titanium. The alloy is resistant to adsorption of proteins and resistant to the adsorption of peptides (e.g., histidine-containing peptides).

<FIG> depict a device in the form of a chromatographic column assembly <NUM> for separating a sample by chromatography, in accordance with an embodiment. Device <NUM> includes a wall <NUM> having a wetted surface <NUM> that is exposed to a mobile phase including the sample during chromatographic separation. The wetted surface <NUM> of the wall includes an alloy material. In some embodiments, the wetted surface of the wall defines a separation channel <NUM>, as shown.

The alloy material is limited in an amount of titanium to <NUM> wt%. In some embodiments, the alloy material is limited in an amount of titanium to <NUM> wt%. In some embodiments, the alloy material is limited in an amount of titanium to <NUM> wt%.

The alloy includes both cobalt and chromium as constituents. The alloy material further includes molybdenum as a constituent.

The alloy material includes the following constituents: <NUM> wt%-<NUM> wt% cobalt; <NUM> wt%-<NUM> wt% nickel; <NUM> wt%-<NUM> wt% chromium; and <NUM> wt% -<NUM> wt% molybdenum; limited in an amount of titanium to <NUM> wt%; and a remainder being limited to a total of <NUM> wt%. In some embodiments, the reminder is limited to a total of <NUM> wt%.

In some embodiments, the alloy material includes the following constituents: <NUM> wt%-<NUM> wt% cobalt; <NUM> wt%-<NUM> wt% nickel; <NUM> wt%-<NUM> wt% chromium; and <NUM> wt% -<NUM> wt% molybdenum; limited in an amount of titanium to <NUM> wt%; and a remainder being limited to a total of <NUM> wt%. In some embodiments, the reminder is limited to a total of <NUM> wt%.

For example, in some embodiments, the alloy may be MP35N LT a trademark of SPS Technologies, LLC of Jenkintown, PA, which is comprised of approximately <NUM> wt% cobalt, <NUM> wt% nickel, <NUM> wt% chromium, and <NUM> wt% molybdenum with less than <NUM> wt% titanium. This alloy is known to have strong mechanical properties and can withstand high pressures, such as those used in UHPLC.

The alloy material is resistant to adsorption of proteins and resistant to adsorption of peptides. In some embodiments the alloy material is resistant to adsorption of histidine-containing peptides. In some embodiments, the alloy material is resistant to adsorption of phosphopeptides.

The inventors have determined that chromatographic separation components employing a nickel-cobalt alloy that is limited in an amount of titanium to less than <NUM> wt% on wetted surfaces exhibit superior performance with respect to resistance to adsorption of biological sample components (e.g., proteins and peptides) as compared to stainless steel components when performing chromatographic separation of biological samples. Further, the inventors determined that decreasing the amount of titanium in the alloy improved the quality of the chromatographic separation. In particular, the inventors determined that a separation column with wetted surfaces of MP35N LT alloy did not show significant peak broadening or peak tailing during analysis of histidine-containing peptides. Further, the inventors determined that a separation column with wetted surfaces of MP35N LT did not require conditioning of the column and showed consistent peak width over successive injections during analysis of peptide-containing samples. The impressive performance of a nickel-cobalt alloy in chromatographic separation of samples with peptides and proteins was unexpected, as nickel ions and cobalt ions are known to chelate certain types of peptides or proteins by metal chelation interaction chromatography, which would lead to the expectation that nickel and cobalt in an alloy would lead to greater interaction with peptides or proteins. Experimental results comparing the performance of stainless steel, fused silica, and MP35N LT alloy chromatographic separation components during separation of samples with biological components are detailed below in the examples section with respect to <FIG>.

Turning again to the device <NUM> of <FIG>, in some embodiments, most or all of the thickness of the wall <NUM> may be made of the alloy. For example, <FIG> shows a detail view of the wall <NUM>, in which the most or all of the thickness of the wall <NUM> is made of the alloy material (e.g., MP35N LT), in accordance with some embodiments.

In some embodiments, the wall <NUM> includes a surface portion including the wetted surface and a bulk portion, and the composition of the alloy material of the wetted surface is different than a composition of a material of the bulk portion. For example, <FIG> shows a detail view, in accordance with another embodiment, in which the wall <NUM> includes a surface portion <NUM> and a bulk portion <NUM>. The wetted surface <NUM> is disposed on the surface portion <NUM> of the alloy material (e.g., MP35N LT) and the bulk portion <NUM> has a different composition (e.g., stainless steel). The material of the bulk portion <NUM> may be selected for desirable mechanical properties or chemical properties (e.g., strength or corrosion resistance) and/or for issues relating to cost (e.g., the material of the bulk portion may be less expensive than the alloy material). Other materials that could be employed for the bulk portion include, but are not limited to stainless steel, titanium, aluminum, carbon fiber composites, PEEK, polyolefins, ceramics, etc. In some embodiments, the surface portion <NUM> is deposited on the bulk portion <NUM>. In some embodiments, the surface portion <NUM> is diffusion bonded to the bulk portion <NUM>. In some embodiments, the surface portion <NUM> is welded to the bulk portion <NUM>. In some embodiments, the bulk portion <NUM> is a sleeve around and in contact with the surface portion <NUM>.

In some embodiments the wetted surface <NUM> consists of the alloy material. In some embodiments, most of the surface area of the wetted surface is covered by the alloy material. In some embodiments, over <NUM>% of the surface area of the wetted surface is covered by the alloy material. In some embodiments, over <NUM>% of the surface area of the wetted surface is covered by the alloy material. In some embodiments, over <NUM>% of the surface area of the wetted surface is covered by the alloy material. In some embodiments, over <NUM>% of the surface area of the wetted surface is covered by the alloy material.

In some embodiments, the wetted surface <NUM> of the wall <NUM> defines a separation channel <NUM>, as shown. In some embodiments, the alloy is employed in the wall <NUM> of the separation channel <NUM> or in at least a surface portion <NUM> of the wall <NUM> of the separation channel <NUM>. In some embodiments, the alloy is also employed in one or more wetted surfaces of one or more additional components of the device <NUM>.

For example, in some embodiments, the device <NUM> also includes an end fitting <NUM> and wetted surfaces of the end fitting comprise the alloy material (see detail view of <FIG>). In some embodiments the device <NUM> also includes a frit <NUM> and wetted surfaces <NUM> of the frit <NUM> comprise the alloy material (see <FIG>). In some embodiments, the device <NUM> further includes a housing <NUM> that includes the frit <NUM>. The detail view of <FIG> shows the housing <NUM> and the frit <NUM> as part of the column assembly device <NUM>. <FIG> show different views of the housing <NUM>.

In some embodiments, a device also includes a seal ring <NUM> and a wetted surface <NUM> of the seal ring includes the alloy material. <FIG> depicts a cross-sectional view of an end of a column assembly <NUM>' that includes a wall <NUM>', a wetted surface <NUM>', an end fitting <NUM>', a frit <NUM>' and a seal ring <NUM> with wetted surface <NUM>. <FIG> depict different views of the seal ring <NUM>.

In some embodiments, the device <NUM> also includes a distributor disk <NUM> and wetted surfaces <NUM> of the distributor disk <NUM> comprise the alloy (see <FIG> shows the distributor disk <NUM> and a housing assembly <NUM> with an associated filter or frit <NUM>. In some embodiments, a housing assembly <NUM> including the distributor disk <NUM> and associated filter or frit <NUM> is located upstream and downstream of the wall of the separation column.

The chromatographic separation assembly would normally include a stationary phase (not shown) within the separation channel <NUM>. In some embodiments of the device, all surfaces of the device <NUM> upstream of and outlet end of the separation channel <NUM> and configured to be in contact with the mobile phase including the sample during use, excluding the stationary phase, comprise the alloy material. For example in some embodiments, wetted surfaces of the wall <NUM>, the end fittings <NUM> and the frit <NUM> include the alloy material.

Embodiments include components for use in a device for separating a sample by chromatography. The component has a body having a wetted surface exposed to a mobile phase including the sample during chromatographic separation, The wetted surface includes an alloy material as described above with respect to the device embodiments. For example, in some embodiments the component is the wall <NUM> having a wetted surface <NUM> that defines a chromatographic separation channel <NUM>. In some embodiments, the component is an end fitting <NUM>. In some embodiments, the component is a frit <NUM> and the wetted surface is a wetted surface of the frit <NUM>. In some embodiments, the component is a stationary phase retaining element configured to keep a stationary phase within a separation channel of the device. In some embodiments, the stationary phase retaining element is a frit <NUM>. In some embodiments, the frit additionally or alternatively filters solid particulates upstream of a chromatographic separation channel. In some embodiments, the frit is upstream of a chromatographic separation channel and may be used to keep particulates off of a separate column inlet frit directly upstream of the chromatographic separation channel.

Embodiments are not limited to chromatographic separation devices with cylindrical columns. One of ordinary skill in the art will appreciate that embodiments of chromatographic separation devices may employ other geometries for separation channels. For example, device <NUM> shown in <FIG> has a planar chromatographic chip geometry. Device <NUM> may alternately be described as a component configured for use in a device for separating a sample by chromatography. <FIG> is an exploded view of the layers of the device <NUM>. The device <NUM> includes a first layer <NUM> (e.g., a top plate) and a second layer <NUM> (e.g., a bottom plate). The second layer <NUM> includes grooves <NUM> and <NUM>, which may be formed by electrochemical micromachining (EMM), also known as electroetching or electrochemical micromachining through a photomask, or milling, that form portions of a chromatographic separation channel. The first layer <NUM> includes holes <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> and slot <NUM>, and the second layer 10b includes holes <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and slot <NUM>, which may be made by micro electrical discharge machining (micro-EDM), wire EDM, mechanical drilling, and/or laser drilling. Holes <NUM> and <NUM> are used as fluidic access ports or vias. Holes <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> are used to attach a fitting at an exterior edge of the device <NUM> to provide fluidic access on the edge or side <NUM> of the device <NUM>. Holes <NUM>, <NUM> and slots <NUM>, <NUM> are used for alignment of the first layer <NUM> and the second layer <NUM>.

The first layer <NUM> is joined to the second layer <NUM> to form device <NUM>. In some embodiments, the first layer <NUM> is joined to the second layer <NUM> by diffusion bonding or other suitable techniques, such as clamping with a gasket seal. <FIG> schematically depicts a cross-sectional view of the device taken through line A-A of <FIG> after the first layer <NUM> and the second layer <NUM> have been joined together. <FIG> and <FIG> are not to scale and some relative dimensions are changed for ease of illustration. As shown in <FIG>, after the joining of the first layer <NUM> and the second layer <NUM>, the groove <NUM> and a surface of the first layer <NUM> form a channel <NUM> capable of holding fluids hermetically under high hydraulic pressures. Similarly, groove <NUM> and the first layer <NUM> form another portion of channel <NUM> when the first layer <NUM> and the second layer <NUM> are joined. In some embodiments, a width w of the channel <NUM> is in a range of <NUM>-<NUM>.

The first layer <NUM> and the second layer <NUM> together form a wall of the device with wetted surface <NUM>. Alternatively, the first layer <NUM>, the second layer <NUM> may be described as together forming a body having a wetted surface <NUM>. The wetted surface <NUM> includes the alloy that includes the following constituents: nickel; and cobalt and/or chromium; and limited in an amount of titanium to <NUM> wt%. All of the features and variations of the alloy described above with respect to device <NUM> are also applicable to device <NUM>.

In some embodiments, the first layer <NUM> and the second layer <NUM> each include a surface portion and a bulk portion as depicted in <FIG>. The first layer <NUM> includes a surface portion <NUM> including the alloy and a bulk portion <NUM> of a different material (e.g., stainless steel) and the second layer <NUM> includes a surface portion <NUM> including the alloy and a bulk portion <NUM> of a different material (e.g., stainless steel). The bulk portion and the surface portion may be joined together by any suitable techniques, which include, but are not limited to, diffusion bonding, clamping, overmolding, etc..

After fabrication and bonding or joining of the first layer and the second layer, the channel <NUM> is normally packed with a stationary phase (e.g., micrometer sized particles). The device <NUM> may include one or more stationary phase retaining elements at the end or ends of a separation column portion of the channel <NUM> to prevent the stationary phase from flowing out of the channel <NUM>. In some embodiments, the stationary phase retaining element is a frit, which may be formed by sintering the stationary phase particles together in some portion of the separation column or by immobilizing some of the stationary phase particles using some other suitable method. In some embodiments, the stationary phase retaining element is a weir formed in the device <NUM> by narrowing a portion of the groove <NUM> formed in the second layer <NUM> thereby narrowing the corresponding portion of the resulting channel. For example, <FIG> schematically depict a weir included in device <NUM>, in accordance with some embodiments. Groove <NUM> narrows at weir <NUM> forming a narrowed groove <NUM> and corresponding narrowed channel <NUM> downstream of the weir. The weir <NUM> has a wetted surface <NUM>. As another example, <FIG> schematically depict an alternative geometry of a weir included in device <NUM>, in accordance with some embodiments. In <FIG>, groove <NUM> narrows and becomes shallower at weir <NUM> and then widens and deepens to form two channels <NUM>, <NUM> respectively, connected by a narrower and shallower neck <NUM>. The weir <NUM> has a wetted surface <NUM>. In some embodiments, a plurality of weirs may be formed at the end of a channel. In some embodiments, a wetted surface or wetted surfaces of one or more stationary phase retaining elements include the alloy. For example, a wetted surface <NUM> of weir <NUM> or a wetted surface of weir <NUM> includes the alloy in some embodiments.

In some embodiments, the device <NUM> includes an electrospray tip. For example, <FIG> and 9D show an embodiment of an end <NUM>' of the device having an integrated electrospray tip <NUM>. The electrospray tip <NUM> is formed by cutting the end <NUM> of the device <NUM> (e.g., using EMM and/or EDM) to form the tip geometry. Channel <NUM> narrows at weir <NUM> to become narrowed channel <NUM>, which exits the device <NUM> at the electrospray tip <NUM>. In some embodiments, a wetted surface of the electrospray tip includes the alloy.

In some embodiments, the device <NUM> further includes one or more integrated valves (not shown), and wetted surfaces of the one or more integrated valves include the alloy material.

In some embodiments, a device may include three layers employing slots to define a channel. For example, <FIG> schematically depict a device <NUM> having a first layer <NUM>, second layer <NUM>, and a third layer <NUM>. Device <NUM> may alternately be described as a component configured for use in a device for separating a sample by chromatography. The first layer <NUM> has similar holes and slots as those described above with respect to first layer <NUM> of device <NUM>. The second layer <NUM> has similar holes and slots as those described above with respect to second layer <NUM> of device <NUM>; however, instead of grooves, the second layer <NUM> has slots <NUM> and <NUM>. The third layer <NUM> has holes and slots for alignment similar to those described above with respect to second layer <NUM> of device <NUM>. First layer <NUM>, second layer <NUM> and third layer <NUM> are joined together. The cross-sectional view in <FIG> depicts how surfaces of the first layer <NUM> and the third layer <NUM> and the slot <NUM> of the second layer <NUM> form a hermetically sealed channel <NUM>. When joined together, the first layer <NUM>, the second layer <NUM> and the third layer <NUM> can be described as forming a wall of the device <NUM>. Alternatively, the first layer <NUM>, the second layer <NUM> and the third layer <NUM> can be described as forming a body of the device <NUM>. Surfaces of the slot <NUM> and surfaces of the first layer <NUM> and third layer <NUM> form the wetted surfaces <NUM> of the wall or body, which include the alloy.

In some embodiments, the first layer <NUM> and the third layer <NUM> each include a surface portion and a bulk portion as depicted in <FIG>. The first layer <NUM> includes a surface portion <NUM> including the alloy and a bulk portion <NUM> of a different material (e.g., stainless steel) and the third layer <NUM> includes a surface portion <NUM> including the alloy and a bulk portion <NUM> of a different material (e.g., stainless steel). The bulk portion and the surface portion may be joined together by any suitable techniques, which include, but are not limited to, diffusion bonding, clamping, overmolding, etc..

Additional information regarding planar geometry chromatographic separation devices and manufacturing methods for such devices appears in <CIT> entitled "Chromatography Apparatus Having Diffusion-Bonded And Surface-Modified Components,". In some embodiments, the separation device may be part of a microfluidic cartridge.

In some embodiments, the device is configured for low pressure applications. For example, the device could be configured for use in solid phase extraction. In another embodiment, the device is configured for use in supercritical fluid chromatography (SFC). In another embodiment, the device is configured for use in gas chromatography. Low pressure applications, as used herein, refers to applications in which the pressure in the device falls within a range of atmospheric pressure to <NUM>. 89MPa (<NUM> psi).

In some embodiments, the device is configured to withstand pressures used in high performance liquid chromatography. As used herein, high performance liquid chromatography refers to liquid chromatography in which the mobile phase is subjected to pressures of between <NUM> to <NUM> MPa (<NUM>,<NUM> and <NUM>,<NUM> psi).

In some embodiments, the device is configured to withstand pressures used in ultra-high performance liquid chromatography. As used herein, ultra high performance liquid chromatography refers to liquid chromatography in which the mobile phase is subjected to pressures of greater than <NUM> MPa (<NUM>,<NUM> psi).

In some embodiments, the device is configured to withstand pressures of <NUM> to <NUM> MPa (<NUM>,<NUM> to <NUM>,<NUM> psi) during use. In some embodiments, the device is configured to withstand pressures of <NUM> to <NUM> MPa (<NUM>,<NUM> psi to <NUM>,<NUM> psi) during use. In some embodiments, the device is configured to withstand pressures of <NUM> to <NUM> MPa (<NUM>,<NUM> to <NUM>,<NUM> psi) during use. In some embodiments, the device is configured to withstand pressures of <NUM> to <NUM> MPa (<NUM>,<NUM> psi to <NUM>,<NUM> psi) during use.

In some embodiments, a width or diameter of a separation channel of a device or component falls in a range of <NUM> to <NUM>. In some embodiments, a width or diameter of a separation channel of a device or component falls in a range of <NUM> to <NUM>. For a prep column, a width or diameter may be as large as <NUM>. In some embodiments, such as a microfluidic device or a microfluidic component, a width or diameter of a separation column may be in the range of <NUM> to <NUM>.

The use of the alloy for a wetted surface of a device or component is particularly beneficial in a microfluidic system, as opposed to using surface-treated stainless steel for wetted surfaces because of the difficulties associated with accomplishing effective surface treatment in microfluidic systems.

The use of the alloy for a wetted surface of a device or component is beneficial, as compared to using surface treated fused silica because it is easier to manufacture separation columns from metals such as the alloy than from silica, and because the silanols of the fused silica wetted surface can interact with proteins and peptides by ion exchange.

Solely for illustrative purposes, use of devices taught herein will be described with reference to device <NUM> described above with respect to <FIG> In use, a chromatographic separation device <NUM> including a separation channel <NUM> is provided. The separation channel <NUM> has wetted surfaces <NUM> including an alloy material. The alloy material includes the following constituents nickel; and cobalt and/or chromium; and limited in an amount of titanium to <NUM> wt%. Other features and aspects of the alloy material in accordance with various embodiments are described above. A mobile phase is flowed carrying the sample into and through the separation channel <NUM>, thereby performing chromatographic separation on the sample. In some embodiments, the method further includes detecting components of the sample. In some embodiments, the sample includes proteins and the proteins in the sample are separated and detected. In some embodiments, the sample includes peptides and the peptides in the sample are separated and detected. In some embodiments, the sample includes histidine-containing peptides and the histidine-containing peptides in the sample are separated and detected. In some embodiments, the sample includes phosphopeptides and the phosphopeptides in the sample are separated and detected. In some embodiments, the sample includes one or more of peptides or proteins and the one or more proteins or peptides in the sample are separated and detected with a U. Pharmacopeia (USP) tailing factor of less than <NUM>. The USP tailing factor is calculated as the ratio of the width of a peak to <NUM> times the width of the front of the peak both measured at <NUM>% of the height of the peak as indicated in the formula for tailing factor below: <MAT> where T is the tailing factor f<NUM>% is the width of the front of the peak at <NUM>% of maximum peak height and t<NUM>% is the width of the tail of the peak at <NUM>% of maximum peak height.

There could be also provided a variety of separation devices having a stationary phase including an alloy material described herein. In various embodiments, separation devices with a stationary phase including an alloy material described herein include, for example, chromatographic columns; thin layer plates; filtration membranes; sample cleanup devices and microtiter plates; packings for HPLC columns; solid phase extraction (SPE) devices; ion-exchange chromatography devices; magnetic beads; affinity chromatographic and SPE sorbents; sequestering reagents; solid supports for combinatorial chemistry; solid supports for oligosaccharide, polypeptides, and/or oligonucleotide synthesis; solid supported biological assays; capillary biological assay devices for mass spectrometry; templates for controlled large pore polymer films; capillary chromatography devices; electrokinetic pump packing materials; packing materials for microfluidic devices; polymer additives; catalysis supports; and packings materials for microchip separation devices.

In some embodiments, alloy materials as described herein can be packed into preparatory, microbore, capillary, and microfluidic devices. In some embodiments, a solid stationary phase in a device includes the alloy materials as described herein. In some embodiments, surfaces of a solid stationary phase in a device include the alloy materials described herein. In some embodiments, both the solid phase and a wetted surface of a wall of the device include an alloy or alloys as described herein.

Embodiments can be used for all modes of chromatography. Separation modes include but are not limited to reversed phase, normal phase, size exclusion, ion exchange, affinity, hydrophobic interaction and hydrophilic interaction. In addition, the alloys described herein can be employed in sample preparation devices for all the above modes.

Some embodiments include an immobilized enzymatic reactor (IMER). In some embodiments, the IMER includes a wall defining a chamber having an inlet and an outlet and a solid stationary phase covalently linked to an enzyme within the chamber. In use, a liquid sample including a polymer and an analyte flows into the chamber through the inlet, interacts with the immobilized enzyme and flows out of the chamber through the outlet. In some embodiments, the solid stationary phase of the IMER includes an alloy described herein. In some embodiments a wetted surface of the wall of the chamber (i.e., a surface that is in contact with the liquid sample during use) includes an alloy described herein. In some embodiments, both a wetted surface of the wall of the chamber and the solid stationary phase of the IMER include one or more of the alloys described herein. In some embodiments, the IMER device includes additional components or fittings and wetted surfaces of one or more of the additional components or fittings include one or more alloys described herein. In some embodiments, the IMERs are suitable for operation under pressures in the range of about <NUM> to <NUM> MPa (<NUM>,<NUM> to <NUM>,<NUM> psi). In some embodiments, the IMERs are suitable for operation under pressures in the range of <NUM> to <NUM> MPa (<NUM>,<NUM> to <NUM>,<NUM> psi). Additional details regarding IMER devices and systems are provided in <CIT> and entitled "Immobilized Enzymatic Reactor,". In some embodiments, the chamber of the IMER may have a configuration similar to that of chromatographic separation devices described herein. One of ordinary skill in the art in view of the present disclosure will appreciate that the drawings and description herein regarding devices, columns and components for chromatographic separation also applies, in large part to IMERs. For example, a structure described herein as a separation column or a separation channel may also or alternatively be viewed and described as a chamber in which a stationary phase can be disposed for an IMER.

Enzymes that could be immobilized on the stationary phase in an IMER include, but are not limited to: pepsin, protease, cellulose, lipase, amylase, glucoamylase, glucose isomerase, xylanase, phtase, arabinanase, polygalacturonase, hydrolase, chymosin, urease, pectinase, beta-gluconase, ligase, glycosidase, polymerase, phosphatase, kinase, ceramidase. In certain embodiments, the enzyme is trypsin, PNGase F, pepsin, chymotrypsin, peptidase, bromelain, papain, IdeS, or IdeZ, or mixtures thereof.

In some embodiments, the stationary phase includes immobilized affinity reagents, which include, but are not limited to: Protein G, Lambda, Kappa, Protein Y, Protein L, aptamers, affimers, amyloids, lectins, or activated resins for user generated affinity phases such as streptavidin and epoxy. The target molecules of the immobilized affinity reagents include, but are not limited to: proteins, IgG, IgM, insulin, peptides, small molecules, toxins, afflatoxins, mycotoxins, citrinin, deoxynivalenol, vomitoxin, fumonisin, ochratoxin, zearalenone, fusarium, or mixtures thereof. In some embodiments, the immobilized affinity immobilized affinity reagents or the target molecules of immobilized affinity reagents are incorporated into a technology platform such as the Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) affinity workflow from SISCAPA Assay Technologies Inc. (see e.g., <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; and <CIT>).

In some embodiments, the stationary phase is modified to include one or more immobilized affinity and immobilized enzyme materials.

In some embodiments, a column for a separation device or for an IMER has an inner diameter of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> ID, or a diameter there between. In some embodiments, a column for a separation device or for an IMER has an inner diameter falling in a range of <NUM>-<NUM>.

In some embodiments, a column for a separation device or for an IMER has a length of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> <NUM>, <NUM>, <NUM> length, or a length falling there between.

In some embodiments, a component for a separation device or for an IMER is compatible with or configured to be used with <NUM> ID column hardware, <NUM> ID column hardware, <NUM> ID column hardware, <NUM> I D column hardware, <NUM> ID column hardware, <NUM> ID column hardware, <NUM> ID column hardware, <NUM>-<NUM> ID column hardware, planar geometry chromatographic separation devices, diffusion-bonded separation devices, or microfluidic cartridges,.

Incorporating the alloy materials described herein into devices may improve the lifetimes of the devices due to the improved corrosion resistance of the alloy material as compared with other materials normally used on surfaces of such devices. Embodiments of devices that have a stationary phase including an alloy as described herein may exhibit improved lifetimes. Embodiments of devices that have one or more wetted surfaces that include an alloy as described herein may exhibit improved lifetimes.

Experimental data showing the improved corrosion resistance of chromatographic hardware made of the alloys disclosed herein as compared with the corrosion resistance of stainless steel is presented below in the examples section.

Some embodiments provide a kit including a device or one or more components of a device as described herein, and instructions for use. In one embodiment, the instructions are for use with a separation device, e.g., chromatographic columns, thin layer plates, filtration membranes, sample cleanup devices, solid phase extraction device, microfluidic device, and microtiter plates. In one embodiment, the instructions are for use with an immobilized enzymatic reactor device.

The inventors performed chromatographic separation on samples including peptides to compare the performance of conventional stainless steel and silica separation columns to example separation columns.

<FIG> includes chromatograms of a tryptic digest of a sample of enolase analyzed with a conventional stainless steel separation column. Chromatogram <NUM> shows significant peak broadening for an extracted mass <NUM> of peptide <NUM> in the sample. Chromatogram <NUM> shows significant peak broadening for an extracted mass <NUM> of peptide <NUM> in the sample. Chromatogram <NUM> shows even more significant peak broadening for an extracted mass <NUM> of peptide <NUM> in the sample. Chromatogram <NUM> of the total ion current shows the broadened peaks <NUM>, <NUM>, <NUM> for peptide <NUM>, peptide <NUM> and peptide <NUM> respectively. The peaks for peptides <NUM>, <NUM> and <NUM> show relatively low signal to noise ratio in addition to significant broadening. These chromatograms illustrate a problem with the use of a stainless steel column for samples including peptides. When a sample including enolase is analyzed on the stainless steel column, some peptides such as histidine-containing peptides do not appear in the chromatogram or have extremely broad peak shape, often observed with significant peak tailing. The peptides are tryptic digests of enolase, which are included in the MASSPREP Enolase Digest with Phosphopeptides Mix available from Waters Corporation of Milford, MA. Peptide <NUM> is T3 (sequence WLTGPQLADLYHSLMK), Peptide <NUM> is T44 (sequence AAQDSFAAGWGVMVSHR) and Peptide <NUM> is T51-<NUM> (sequence IEEELGDNAVFAGENFHHGDKL). Further information regarding the peptides are available in the publication "<NPL>.

For comparison, <FIG> includes chromatograms of a tryptic digest of the same sample of enolase analyzed with a column constructed with fused silica tubing, specifically the NANOEASE column from Waters Corporation of Milford, Massachusetts. In contrast to chromatogram <NUM> from the stainless steel column, chromatogram <NUM> shows a narrow peak and high signal to noise ratio for the extracted mass <NUM> of peptide <NUM> in the sample. In contrast to chromatogram <NUM> from the stainless steel column, chromatogram <NUM> shows a narrow peak and high signal to noise ratio for the extracted mass <NUM> of peptide <NUM> in the sample. In contrast to chromatogram <NUM> from the stainless steel column, chromatogram <NUM> shows a narrow peak and high signal to noise ratio for the extracted mass <NUM> of peptide <NUM> in the sample. Chromatogram <NUM> shows the total ion current and the excellent signal to noise ratio of peaks <NUM>, <NUM>, <NUM> for peptide <NUM>, peptide <NUM> and peptide <NUM> respectively. The contrast between the chromatograms in <FIG> and <FIG> illustrates how poor the stainless steel column performed for separation of these three peptides.

Another problem with the stainless steel column hardware is that for some peptides, it takes several injections to obtain acceptable peak shape, indicating that the column needs to be conditioned for the analysis. Graph <NUM> in <FIG> shows how the peak width of a peptide significantly changes over <NUM> injections in a stainless steel column, demonstrating that the stainless steel column requires substantial conditioning to obtain consistent results. Such conditioning wastes sample and experimental time. In contrast, graph <NUM> of <FIG> demonstrates how the peak width for the peptide is consistent from injections <NUM> through <NUM> indicating that the fused silica column does not need significant conditioning.

Although the fused silica column performed well for these three peptides, fused silica may be an undesirable material for many applications due to the difficulty in making devices and components from fused silica as compared with making devices and components from a metal material.

Some surface modifications can be made in a stainless steel separation column for better performance with peptide containing samples such as surface passivation of wetted surfaces or coatings that can be applied to wetted surfaces (e.g., creating an iron-deficient and chromium-rich surface or coating with polymers); however, such surface modifications and coatings can fail if the surface or coating is damaged during use. Further, such surface treatments and coatings are difficult to accomplish in microscale devices. Embodiments employing the alloys described herein avoid the need for surface treatments or coatings on wetted surfaces thereby reducing complexity in manufacturing.

An example separation column was constructed using a nickel-cobalt alloy material, specifically, MP35N LT, for the column hardware. This alloy has approximately <NUM> wt% cobalt, <NUM> wt% nickel, <NUM> wt% chromium, and <NUM> wt% molybdenum with less than <NUM> wt% titanium. The example separation column was used for separation and detection of the enolase peptide sample, with the results shown in <FIG> and <FIG>.

In contrast to chromatogram <NUM> for the stainless steel column, chromatogram <NUM> for the example column shows a narrow peak and high signal to noise ratio for the extracted mass <NUM> of peptide <NUM> in the sample. In contrast to chromatogram <NUM> for the stainless steel column, chromatogram <NUM> for the example column shows a narrow peak and high signal to noise ratio for the extracted mass <NUM> of peptide <NUM> in the sample. In contrast to chromatogram <NUM> for the stainless steel column, chromatogram <NUM> for the example column shows a narrow peak and high signal to noise ratio for the extracted mass <NUM> of peptide <NUM> in the sample. Chromatogram <NUM> for the example column shows the total ion current and the excellent signal to noise ratio for peaks <NUM>, <NUM>, <NUM> for peptide <NUM>, peptide <NUM> and peptide <NUM>, respectively. The contrast between the graphs in <FIG> and <FIG> demonstrates the superior performance of the example column as compared to the stainless steel column for separation and detection of these three peptides. Peptides whose peaks were broadened or missing in the stainless steel column data are present, narrow, and have good signal to noise ratio in the example column data. Comparison of <FIG> for the example column and <FIG> for the fused silica column shows that the example column performed at least as well as fused silica for separation and detection of the three peptides.

<FIG> includes a graph <NUM> of peptide peak width for subsequent sample injections in the example column. As shown, there was very little peak width variation for the second to tenth sample injections demonstrating that the example column did not need conditioning, unlike the stainless steel column. Thus, use of the example column would save sample and time by avoiding the need for column conditioning, relative to use of the stainless steel column.

As noted above, nickel ions and cobalt ions are known to chelate certain types of peptides or proteins by metal chelation interaction chromatography, which would indicate that a nickel-cobalt alloy may not be desirable for reducing interactions with peptides and proteins.

The alloys described herein are also expected to perform well in applications such as ion-exchange separations, size exclusion chromatography (SEC), and other applications where high buffer and salt concentrations are commonly used. To counteract the poor performance of stainless steel, columns for these classes of separations commonly employ materials such as glass and PEEK, both of which can limit the design pressure of the columns and/or significantly affect the cost and complexity of the column design.

In the process of developing the invention, the inventors also explored nickel-cobalt alloys having higher levels of titanium. For these experiments described below, a sample of tubing material was placed in line with a separation column and was exposed to the pressure gradient and to the sample during sample separation. The reference tubing material was fused silica and the tubing material being evaluated was MP35N, a trademarked alloy of SPS Technologies, LLC of Jenkintown, PA, which includes <NUM> wt% cobalt, <NUM> wt% nickel, <NUM> wt% chromium, <NUM> wt% molybdenum, and about <NUM> wt% titanium <FIG> includes LC/MS chromatograms for a sample including enolase peptides for both the fused silica tubing (chromatogram <NUM>) and for the MP35N tubing (chromatogram <NUM>). As shown, a phosphopeptide that was clearly present in the chromatogram <NUM> produced with the system including the fused silica tube was absent from the chromatogram <NUM> produced with the system including the MP35N tube as indicated by box <NUM>. Thus, the inventors determined that the adsorption of peptides is sensitive to titanium levels in the alloy and that titanium levels in the nickel-cobalt alloy should be less than <NUM> wt% titanium.

An immobilized enzymatic reactor is prepared by packing a stationary phase of porous particles having immobilized pepsin on the surface of the particles in UPLC column hardware (<NUM> × <NUM>) made from MP35N. The packed MP35N column was used with an ultra performance liquid chromatography system, specifically, the NANOACQUITY ULTRAPERFORMANCE LC system from Waters Corp. of Milford, MA. Further details regarding preparing and using an IMER with a column made from a different material appear in <CIT> and entitled "Immobilized Enzymatic Reactor,". The packed MP35N column withstands elevated pressures, exhibits good digestive performance, and exhibits decreased adsorptive losses of peptides that are generated in the on-line digestion as compared with traditional stainless steel hardware.

Having low or minimal corrosion, rusting, or metals leaching from the chromatographic hardware is very important in several types of separations, including (but not limited to): ion-exchange chromatography, ion-chromatography, Size Exclusion Chromatography, Reversed-Phase Chromatographic, Hydrophobic Interaction Chromatography, Hydrophilic interaction chromatography, Gel Permeation chromatography, Normal-Phase Chromatography, Chiral Chromatography, Supercritical Fluid Chromatography, and Subcritical Fluid Chromatography. The avoidance of corrosion in chromatographic hardware is especially important in ion-exchange chromatography. To avoid corrosion when exposed to mobile phases that are high or low pH, and contain elevated salt, many commercial suppliers of ion-exchange columns use non-metallic hardware, such as plastic columns and in particular PEEK chromatographic hardware.

It should be noted that plastic or PEEK chromatographic hardware is not suitable for ultra performance liquid chromatography. It is difficult to efficiently pack chromatographic media under the required pressures for ultra performance liquid chromatography when the chromatographic hardware includes PEEK or is plastic lined steel, and the use conditions for ultra performance liquid chromatography are above the normal operating range for plastic or PEEK chromatographic hardware.

As such there is a need for mechanically strong, pressure tolerant chromatographic hardware with a lower propensity toward corrosion than stainless steel, that can be used for ultra performance liquid chromatography.

To compare corrosion resistance, stainless steel and nickel-cobalt alloy material (specifically, MP35N LT) chromatographic column hardware tubes (<NUM> × <NUM>) were separately filled with an acid (<NUM> <NUM>,<NUM>-dimethylpiperazine buffer with <NUM> NaCl and <NUM>% sodium azide, adjusted to pH <NUM> using dilute hydrochloric acid), capped at either end with plastic fittings, and stored at <NUM> for one week. As explained above, MP35N LT is a low titanium grade (less than <NUM> wt% Ti) of MP35N. Following this acid exposure, the tubes were emptied into plastic vials. The two samples along with a control acid solution were analyzed for metals content (ICP-MS, VHG Labs, Manchester NH, uncertainty estimated at +/-<NUM>%). The results of these studies are shown in the table below.

Increased levels of metals present in the acid solution stored in the tubes of stainless steel and MP35N LT as compared with the control acid solution evidences a reaction between acid and the metal surface indicating corrosion occurred. As shown in the table, the acid stored in the MP35N LT column had a reduced overall metal concentration as compared to the acid stored in the stainless steel column. As such, one can conclude that the MP35N LT had reduced corrosion with acid exposure when compared with traditional stainless steel chromatographic columns. This corrosion resistance indicates that low titanium nickel-cobalt alloys, such as MP35N LT, which are mechanically strong enough to be used for ultra performance liquid chromatography, may be particularly useful for a variety of chromatographic separations, including (but not limited to): ion-exchange chromatography, ion-chromatography, Size Exclusion Chromatography, Reversed-Phase Chromatographic, Hydrophobic Interaction Chromatography, Hydrophilic interaction chromatography, Gel Permeation chromatography, Normal-Phase Chromatography, Chiral Chromatography, Supercritical Fluid Chromatography, and Subcritical Fluid Chromatography. The use of MP35N LT chromatographic hardware is especially suited for the use in ion-exchange chromatography, and ultra performance ion-exchange liquid chromatography.

Claim 1:
A device for separating a sample by chromatography, the device comprising:
a wall (<NUM>) having a wetted surface (<NUM>) exposed to a mobile phase including the sample during chromatographic separation, wherein the wetted surface (<NUM>) of the wall includes an alloy material comprising the following constituents:
<NUM> wt%-<NUM> wt% cobalt;
<NUM> wt%-<NUM> wt% nickel;
<NUM> wt%-<NUM> wt% chromium; and
<NUM> wt%-<NUM> wt% molybdenum; and
a remainder being limited to a total of <NUM> wt% and limited
in an amount of titanium to <NUM> wt%.