HK40076216A - Chromatographic column having stationary phase thickness gradient - Google Patents

Description

Chromatographic column with stationary phase thickness gradient

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application No. 62/940,038, filed on 25/11/2019. The entire disclosure of the above-mentioned provisional application is incorporated herein by reference.

Government support

The invention is accomplished with the support of the FA8650-17-C-9106 government awarded by the air force, air force materials Committee. The government has certain rights in the invention.

Technical Field

The present disclosure relates to a gas chromatography apparatus including a chromatography column having a stationary phase with a positive thickness gradient to enhance peak focusing.

Background

This section provides background information related to the present disclosure that is not necessarily prior art.

Gas Chromatography (GC) is an analytical method for separating Gas phase compounds via a separation column that allows for analysis and identification of compounds in a target sample. GC is widely used in many industries to isolate and identify target analytes, such as volatile or semi-volatile organic compounds. GC is particularly useful for analyzing complex samples with multiple target analytes (target analytes) that need to be detected individually. GC works by observing the "peaks" of the chemicals passing through the separation column. Thus, samples with different chemicals or target analytes are introduced into the column via the sample injector. The column includes an inner material that is considered the stationary phase. Different portions of the sample pass through the column at different rates (due to the physical and chemical interaction of each chemical with the material contained in the column). As the target analyte elutes and exits from the column, the detector can distinguish between the materials that elute over time based on the rate at which the analyte passes through the column. These analytes can be electronically identified and/or quantified during or after detection. It is desirable to improve the separation within a chromatography column to improve performance and GC detection capability.

Disclosure of Invention

This section provides a general summary of the disclosure, and is not a comprehensive disclosure of its full scope or all of its features.

In certain aspects, the present disclosure relates to a gas chromatography device for peak focusing (peak focusing) of one or more target analytes. The chromatography column has an inlet and an outlet, wherein the inlet receives a sample comprising one or more target analytes exiting the column at the outlet. The stationary phase is deposited inside the chromatography column and has a positive thickness gradient. The stationary phase extends from an inlet to an outlet and has a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column. The second thickness is at least about 10% greater than the first thickness.

In one aspect, the first thickness is greater than or equal to about 10nm to less than or equal to about 10 microns and the second thickness is greater than or equal to about 30nm to less than or equal to about 30 microns.

In one aspect, the second thickness is greater than or equal to the first thickness by at least about 100%.

In one aspect, the second thickness is greater than or equal to the first thickness by at least about 300%.

In one aspect, the column is a micro gas chromatography column (microrogas chromatographic column).

In one aspect, the stationary phase comprises a siloxane polymer.

In yet another aspect, the siloxane polymer comprises at least one alkyl or aryl group comprising 1 to 30 carbon atoms.

In one aspect, the cross-sectional shape of the chromatography column is selected from the group consisting of: circular, oval, rectangular, and triangular.

The present disclosure also relates to a method of peak focusing in a gas chromatography apparatus. The method includes introducing two or more target analytes into an inlet of a chromatography column including a stationary phase having a positive thickness gradient deposited inside the chromatography column. The stationary phase extends from an inlet to an outlet and has a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column. The second thickness is at least about 10% greater than the first thickness. The method also includes separating the two or more target analytes in the chromatography column. The method also includes eluting the two or more target analytes from the outlet of the chromatography column.

In one aspect, the two or more target analytes are Volatile Organic Compounds (VOCs).

In one aspect, at least one of the two or more target analytes comprises an aromatic compound, and a total peak focusing rate (over focusing rate) for the aromatic compound is greater than or equal to about 25%.

In one aspect, at least one of the two or more target analytes comprises an alkane compound, and the total peak focusing rate for the alkane compound is greater than or equal to about 10%.

In one aspect, the first thickness is greater than or equal to about 10nm to less than or equal to about 10 microns and the second thickness is greater than or equal to about 30nm to less than or equal to about 30 microns.

In one aspect, the second thickness is greater than or equal to the first thickness by at least about 300%.

In one aspect, the chromatography column is a micro gas chromatography column.

In yet another aspect, the stationary phase comprises a siloxane polymer comprising at least one alkyl or aryl group comprising 1 to 30 carbon atoms.

In one aspect, the cross-sectional shape of the chromatography column is selected from the group consisting of: circular, oval, rectangular, and triangular.

The present disclosure also relates to a method of verifying peak focusing in a gas chromatography apparatus. The method includes performing a forward operation by introducing two or more target analytes into an inlet of a chromatography column including a stationary phase having a positive thickness gradient deposited inside the chromatography column. The stationary phase extends from an inlet to an outlet and has a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column. The second thickness is at least about 10% greater than the first thickness. The forward operation includes separating two or more target analytes in the chromatography column and eluting the two or more target analytes from an outlet of the chromatography column. The method also includes performing a reverse operation (reverse operation) by introducing the two or more target analytes to an outlet of a chromatography column, the chromatography column comprising a stationary phase. The reverse operation includes separating the two or more target analytes in the chromatography column and eluting the two or more target analytes from the inlet of the chromatography column. The method further includes comparing chromatographic resolution (chromatographic resolution) from the forward and reverse operations, wherein peak focusing rates for at least one pair of corresponding two peaks from the two target analytes are greater than 5%.

The present disclosure still further relates to a method of manufacturing a gas chromatography apparatus having a chromatography column with a positive thickness gradient. The method includes introducing a precursor liquid into a chromatography column. The precursor liquid includes a stationary phase precursor and a low boiling point solvent that volatilizes along the length of the chromatography column to increase the concentration of the stationary phase precursor as it moves along the chromatography column, thereby creating a stationary phase thickness gradient. The method also includes reacting or crosslinking the stationary phase precursor to form a positive thickness gradient stationary phase, the stationary phase extending from an inlet to an outlet and having a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column. The second thickness is at least about 10% greater than the first thickness.

In one aspect, the inner surface of the chromatography column is silanized prior to introduction of the precursor liquid.

In one aspect, silylation comprises passing the reactive silane in a gas phase through a column.

In one aspect, the chromatography column comprises an inlet and an outlet, and the introducing, and reacting or crosslinking comprises: the inner surface of the chromatography column is dynamically coated by partially filling the column with a precursor liquid, applying pressure at the inlet to force the precursor liquid down the length of the column, and applying vacuum at the outlet to evaporate the low boiling point solvent.

In one aspect, the first thickness is greater than or equal to about 10nm to less than or equal to about 10 microns and the second thickness is greater than or equal to about 30nm to less than or equal to about 30 microns.

In one aspect, the second thickness is greater than or equal to the first thickness by at least about 100%.

In one aspect, the second thickness is greater than or equal to the first thickness by at least about 300%.

In one aspect, the chromatography column is a micro gas chromatography column.

In one aspect, the stationary phase comprises a siloxane polymer.

In yet another aspect, the siloxane polymer comprises at least one alkyl or aryl group comprising 1 to 30 carbon atoms.

Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.

Drawings

The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

FIG. 1 shows an exemplary embodiment of a gas chromatography apparatus system.

Fig. 2 shows a cross-sectional view of a chromatography column having a stationary phase with a positive thickness gradient to enhance peak focusing, according to certain aspects of the present disclosure.

Fig. 3A-3C are depictions of peak focusing by a Film Thickness Gradient Column (FTGC) according to certain aspects of the present disclosure. As shown in fig. 3A, the thinner to thicker film focuses the analyte peak as the analyte travels along the column. Fig. 3B is a diagram of an arrangement for column performance evaluation. Fig. 3C is a diagram of a forward operation mode and a backward operation mode/reverse operation mode.

Fig. 4A-4C. Fig. 4A is a graphical representation of an FTGC coating setup. The column was dynamically coated by partially filling with a plug of coating solution, and then pushing the mixture away at a pressure of 5psi. With the solution pushed away, a vacuum pressure of-2 psi was applied to the outlet to evaporate the solvent. FIG. 4B is an SEM image of the region near the column inlet, and the film thickness was 34nm. FIG. 4C is an SEM image of the thickness of the film near the exit of the column at 241nm.

Fig. 5A-5D. FIG. 5A is a diagram of separation C in the forward mode of operation ₇ To C ₁₆ Figure for alkane mixtures. FIG. 5B is a diagram of separation C in the same parameter reverse or reverse mode of operation ₇ To C ₁₆ Figure for alkane mixtures. FIG. 5C is a diagram of separating C in an isochronous reverse mode ₇ To C ₁₆ Figure for alkane mixtures. FIG. 5D is a separation of C using a uniform thickness column ₇ To C ₁₆ Figure for alkane mixtures.

FIG. 6 illustrates a scheme for C ₇ To C ₁₆ The resolution difference between the forward mode of the alkane, the same parametric mode and the isochronous reverse/reverse mode, and the uniform thickness column.

Fig. 7A-7D. FIG. 7A is a diagram of separation of an aromatic mixture in forward mode. FIG. 7B is a graph of the separation of an aromatic mixture under the same parameters. Fig. 7C is a diagram of the separation of an aromatic mixture in an isochronal reverse/inverse model. Fig. 7D is a graph of separation of an aromatic mixture in a contrast column with a column of uniform thickness. Peaks 1, 2, 3, 4 and 5 correspond to benzene, toluene, ethylbenzene, ortho-xylene and 1, 3-dichlorobenzene, respectively.

Fig. 8 illustrates the resolution difference between the forward mode, the same parameter mode and the isochronous reverse mode of aromatic separation and uniform thickness columns.

Fig. 9A-9C. FIG. 9A shows a mode C in the forward mode ₅ And C ₆ Graph of room temperature isothermal separation of (1). FIG. 9B shows C in the same parameter reverse/reverse mode ₅ And C ₆ Graph of room temperature isothermal separation of (1). FIG. 9C shows a view of using a column of uniform thickness C ₅ And C ₆ Graph of room temperature isothermal separation of (1).

Corresponding reference characters indicate corresponding parts throughout the several views of the drawings.

Detailed Description

Example embodiments are provided so that this disclosure will be thorough and will fully convey the scope to those skilled in the art. Numerous specific details are set forth such as examples of specific compositions, components, devices, and methods to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well-known technologies are not described in detail.

The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" may also be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises," "comprising," "including," and "having" are open-ended and thus specify the presence of stated features, elements, components, steps, integers, operations, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. While the open-ended term "comprising" should be understood as a non-limiting term used to describe and claim the various embodiments set forth herein, in certain aspects the term may instead be understood as a more limiting and constraining term, such as "consisting of 8230; …" consisting of or "consisting essentially of 8230; \8230"; "consisting of. Thus, for any given embodiment that recites a composition, material, component, element, feature, integer, operation, and/or process step, the disclosure also specifically includes embodiments that consist of, or consist essentially of, those recited composition, material, component, element, feature, integer, operation, and/or process step. In the case of "consisting of 8230in the alternative embodiments exclude any additional compositions, materials, components, elements, features, integers, operations and/or process steps, and in the case of" consisting essentially of 8230in the alternative embodiments exclude any additional components, materials, components, elements, features, integers, operations and/or process steps that substantially affect the basic and novel characteristics from the alternative embodiments, and in the case of "consisting essentially of the alternative 8230in the alternative embodiments exclude any additional components, materials, components, elements, features, integers, operations and/or process steps that substantially affect the basic and novel characteristics from the alternative embodiments, but may include any components, materials, components, elements, features, integers, operations and/or process steps that do not substantially affect the basic and novel characteristics from the alternative embodiments.

Unless specifically identified as an order of execution, the method steps, processes, and operations described herein are not to be construed as necessarily requiring their execution in the particular order discussed or illustrated. It should also be understood that additional or alternative steps may be employed unless otherwise indicated.

When a component, element, or layer is referred to as being "on," "engaged to," "connected to," or "coupled to" another element or layer, it may be directly on, engaged, connected or coupled to the other component, element, or layer, or intervening elements or layers may be present. In contrast, when an element is referred to as being "directly on," "directly engaged to," "directly connected to" or "directly coupled to" another element or layer, there may be no intervening elements or layers present. Other words used to describe the relationship between elements should be interpreted in a similar manner (e.g., "between" and "directly between," "adjacent" and "directly adjacent," etc.). As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Although the terms first, second, third, etc. may be used herein to describe various steps, elements, components, regions, layers and/or sections, these steps, elements, components, regions, layers and/or sections should not be limited by these terms unless otherwise specified. These terms may be only used to distinguish one step, element, component, region, layer or section from another step, element, component, region, layer or section. Terms such as "first," "second," and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first step, element, component, region, layer or section discussed below could be termed a second step, element, component, region, layer or section without departing from the teachings of the example embodiments.

Spatially relative or temporally relative terms, such as "before", "after", "inside", "outside", "below", "beneath", "over", "above", and the like, may be used herein for convenience of description to describe one element or feature's relationship to another element or feature as shown. Spatially relative terms or temporally relative terms may be intended to encompass different orientations of the device or system in use or operation in addition to the orientation depicted in the figures.

Throughout this disclosure, numerical values represent approximate measurements or limits of ranges to encompass minor deviations from the given values, embodiments having approximately the stated values, and embodiments having exactly the stated values. Other than the working examples provided at the end of the detailed description, all numerical values of parameters (e.g., numerical values of quantities or conditions) in this specification (including the appended claims) are to be understood as being modified in all instances by the term "about," whether or not "about" actually appears before the numerical value. "about" means that the numerical value allows some slight imprecision (with some approach to exactness in the value; approximately or reasonably close to the value; nearly). As used herein, "about" refers to at least variations that may result from ordinary methods of measuring and using such parameters, provided that the imprecision provided by "about" is not otherwise understood in the art with such ordinary meaning. For example, "about" can include less than or equal to 5%, optionally less than or equal to 4%, optionally less than or equal to 3%, optionally less than or equal to 2%, optionally less than or equal to 1%, optionally less than or equal to 0.5%, and in certain aspects, optionally less than or equal to 0.1%.

Further, disclosure of ranges includes disclosure of all values and further divided ranges within the entire range, including the endpoints and subranges given for the ranges.

Example embodiments will now be described more fully with reference to the accompanying drawings.

In various aspects, the present teachings pertain to gas chromatography. As shown in FIG. 1, a simplified representation of a gas chromatography system 20 typically has at least five components: (1) a carrier gas source 20; (2) a sample injection system 22; (3) one or more gas chromatography columns 30; (4) a detector 32; and (5) a data processing system 34. The carrier gas introduced as carrier gas source 20 (also referred to as a mobile phase) is a high purity and relatively inert gas such as helium, hydrogen, nitrogen, argon, or air. The carrier gas 20 in conventional systems flows through the column 30 (throughout the separation process) simultaneously with the sample fluid to be tested. A sample injector (injector) 22 is introduced into the column 30 by combining a predetermined volume of a sample mixture comprising one or more target analytes to be tested (e.g., in gaseous form) with a flowing carrier gas from a carrier gas source 20. Thus, the carrier gas source 20 and the sample 22 (possibly with one or more target analytes) are introduced into one or more chromatography columns 30. Sample 22 moves through with the co-injected carrier gas from carrier gas source 20.

The target analyte species from the sample 24 is separated and transported through the column 30, thus eluting from the column. It should be noted that depending on the delay of the respective target analyte species as they pass through and are separated by the chromatography column 30, the eluted sample with the one or more target analytes may elute from the column 30 in partial fractions. In addition, the sample fraction eluted from column 30 may optionally be captured and re-injected downstream.

Typically, separation is achieved within the chromatography column 30 because the inner surface of the column is coated with (or the interior of the column is packed with) a material that serves as the stationary phase. The use of the term "column" is intended to broadly include various flow paths through which fluids may flow, such as patterned flow fields from microfeatures defined in one or more substrates or other fluid flow paths as recognized by one of ordinary skill in the art. The stationary phase adsorbs different target analytes in the sample mixture to different extents. The difference in adsorption results in a difference in the delay of different chemical species as they travel down the column and in this way in a difference in mobility, thereby affecting the physical separation of the target analyte in the sample mixture. In some variations, the chromatography column is a micro-gas chromatography column. As used herein, "micro-sized" refers to structures having at least one dimension of less than about 500 μm, optionally less than about 400 μm, optionally less than about 300 μm, optionally less than about 200 μm, optionally less than about 150 μm, and in certain variations, structures having a dimension of optionally less than about 100 μm, which may also encompass nanoscale features. As used herein, reference to micro-dimensions, micro-channels, microfluidic channels, or microstructures encompasses smaller structures, such as equivalent nano-scale structures. It should also be noted that while one dimension (such as a diameter) may fall within the micro-scale range, other dimensions (such as a length) may exceed the micro-scale range.

The various separated components elute from the column 30 to one or more detectors 32 for analysis. Thus, one or more detectors 32 are located at the ends of one or more columns 30. Thus, the detector 32 is used to detect various chemical species or target analytes in the sample that emerge or elute from the column 30 at different times. Such a detector 32 is typically run in a gas chromatography system by destructive analysis of the eluted fractions. Typical non-limiting examples of the detector 32 include a Mass Spectrometer (MS) (e.g., a time-of-flight mass spectrometer (TOFMS)), a Flame Ionization Detector (FID), a photoionization detector (PID), an Electron Capture Detector (ECD), a Thermal Conductivity Detector (TCD), and the like. The data processing system 34 is also typically in communication with the detector 32 so as to be typically capable of storing, processing and recording the results of the separation test.

In wall-coated capillary chromatography columns, the retention of the analyte is made possible by the vapor interaction between the gas phase and the stationary phase coated on the capillary wall. As the analytes travel down the column, they encounter longitudinal and transverse mass transfer, which can lead to peak broadening, decrease GC resolution and increase the likelihood of co-elution. Typically, the correct choice of stationary phase within the chromatography column (to enable sufficient analyte interaction and retention), the application of temperature-programmed curves, and split/splitless sample injection enable improved chromatographic separation and resolution. However, in some cases, these methods are not sufficient to achieve the desired separation. For example, in portable GCs, the limited supply of carrier gas prevents the use of split injection, while fine control of the temperature ramp procedure is difficult and limited by system power capacity. Furthermore, even for specialized separations (e.g., separation of highly volatile compounds by porous layer open tubular columns), it can be difficult to completely separate all of the relevant compounds. Therefore, additional methods for improving column separation are desirable.

Negative Temperature Gradient Separation (NTGS) is a method used to improve column performance by sharpening the elution peaks. In NTGS, the column inlet is heated and a temperature gradient is created via heat exchange with the ambient environment. Due to the lower temperature towards the column outlet, the front of the peak travels slower than the tail, resulting in overall peak focusing. This effect can be optimized by adjusting different temperature profiles along the column, thus making a high degree of versatility under different conditions possible. However, due to the dependence of NTGS on heat exchange, the focus varies with ambient temperature, humidity, air-to-flow rate, and thermal conductivity of the fill material, thereby reducing repeatability and predictability (especially where complex temperature profiles are used). Complex thermal control modules may be used to stabilize the temperature gradient, but add additional size, weight, complexity, and cost to the GC apparatus. Furthermore, the energy loss due to the above-described heat exchange is a relevant detriment to resource-limited systems (e.g., micro-GC devices). Furthermore, the separation of highly volatile compounds generally requires near ambient temperature, so that the generation of temperature gradients can be avoided or minimized, thereby suppressing the NTGS effect. Thus, while versatile and tunable, the use of NTGS for certain applications (e.g., portable GCs) may be limited and challenging.

The present disclosure provides a new method for peak focusing during gas chromatography. In certain aspects, a gas chromatography device for peak focusing of one or more target analytes includes a chromatography column having an inlet and an outlet. The inlet receives a sample comprising one or more target analytes exiting the column at the outlet. The stationary phase is disposed and deposited within the chromatography column and has a positive thickness gradient. The stationary phase extends from an inlet to an outlet and has a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column. As will be described further below, wherein the second thickness is at least about 10% greater than the first thickness. In this way, the positive stationary phase thickness gradient is a gradient in which the stationary phase membrane thickness increases from the inlet toward the outlet. As the thickness of the stationary phase increases toward the outlet, the front of the peak travels slower than the tail, causing the overall peak to focus.

Fig. 2 shows an example of a chromatography column 50 capable of focusing peaks of one or more target analytes prepared according to certain aspects of the present disclosure. In certain aspects, the one or more target analytes may be Volatile Organic Compounds (VOCs) that have relatively high vapor pressures and thus low boiling points at the temperatures and pressures used within the chromatography column 50. The VOC target analyte may include alkanes or aromatics.

The chromatography column 50 is defined by walls 60. Thus, the walls 60 of the chromatography column 50 may define a structure having a hollow interior region 52 that may be at least partially occupied by the stationary phase 64. The cross-sectional shape of the chromatography column 50 may be selected from the group consisting of: circular, oval, rectangular, and triangular. In certain aspects, the chromatography column 50 is formed of metal, silica or glass, or a polymer. The chromatography column 50 defines an inlet 70 and an outlet 72. As shown in fig. 2, the wall 60 has a constant thickness. Thus, the wall 60 is bounded by a "T" at the inlet 70 ₁ A first thickness denoted by "T" at the outlet 72 ₂ "indicates the same second thickness.

As described above, as the mixture of one or more target compounds in the sample/carrier mobile phase and the stationary phase 64 pass through the column, the mixture interacts with the stationary phase. Each target analyte interacts with the stationary phase 64 to a different degree or at a different rate. The analyte that minimally interacts with the stationary phase 64 will first exit or elute from the column 50. Generally, the target analyte that interacts most with the stationary phase 64 travels through the column 50 at the slowest rate and thus exits last. By varying the properties of the mobile phase (carrier and sample) and stationary phase 65, different mixtures of target analytes can be separated. As will be described below, the stationary phase 64 in the context of the present technology has a positive thickness gradient that enables focusing of peaks of one or more target analytes, which generally means that analyte peaks are focused as they travel from the inlet 70 to the outlet 72 of the chromatography column 50.

In certain variations, the stationary phase 64 may comprise silicon, such as silica or a siloxane polymer. A variety of siloxane-based polymers may form the stationary phase 64. Generally, the siloxane polymer is a crosslinked polymer having a basic skeleton of silicon and oxygen, and side constituent groups (which may be the same or different), typically consisting of structural repeat units (-O-SiRR' -) _n Described, wherein R and R' may be the same or different pendant groups, and "n" may be any value greater than 2. Surface functionalization of silica or siloxane can be performed in a monomeric or polymeric reaction with different short-chain organosilanes, reacting with silanol groups. While the retention mechanism for target analytes remains unchanged, differences in surface chemistry of different stationary phases can result in changes in selectivity for different target analytes. The silicone polymer may comprise polyheterosiloxanes (polyheterosiloxanes) in which the pendant groups or repeating units may be different. Examples of suitable pendant groups can be at least one unsubstituted or substituted alkyl or aryl group containing from 1 to 30 carbon atoms, for example, including, but not limited to: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, phenyl, alkylphenyl and the like.

In various aspects, the stationary phase 64 is deposited on the inner surface 66 of the wall 60 and defines a positive thickness gradient.In certain variations, as will be described further below, the inner surface 66 may first be silanized, for example, by exposing the inner surface 66 to Hexamethyldisilazane (HMDS) vapor to form a silane-containing or silanol-containing surface coating. Then, precursors of the siloxane polymer (such as silicone resin OV-1, vinyl modified 100% dimethyl siloxane commercially available as OV-1 6001 or silicone resin OV-17 from the Ohio Valley Specialty Company, 50% phenyl siloxane-50% methyl siloxane commercially available as OV-1 6017 from the Ohio Valley Specialty Company) can be injected and reacted to form the stationary phase. Crosslinking agents (such as Dow SYLGARD) ^TM 184 reagent B (15% w/w, crosslinking agent)) may also be added together with the precursor to promote crosslinking. Notably, the stationary phase 64 is not limited to such materials, and these are provided merely as suitable examples for use in a gas chromatography column.

As shown, the stationary phase 64 extends along the inner surface 66 of the wall 60 from an inlet 70 to an outlet 72. The stationary phase 64 thus forms a continuous coating or film on the inner surface 66. The stationary phase 64 has a structure represented by "t" at the inlet 70 ₁ "first thickness. The stationary phase 64 also defines a flow path designated by "t" at the outlet 72 ₂ "second thickness. A second thickness (t) ₂ ) Is greater than the first thickness (t) ₁ ) At least about 10% greater. Thus, the stationary phase 64 defines a positive gradient thickness along the length of the chromatography column 50. In this manner, the interior region 52 has a first thickness "t" at the inlet 70 from the stationary phase 64 ₁ "defined first diameter (d) ₁ ). The inner region 52 has a second thickness "t" at the outlet 72 from the stationary phase 64 ₂ "defined second diameter (d) ₂ ). A first diameter (d) ₁ ) Greater than the second diameter (d) ₂ ). In this manner, the stationary phase 64 defines a positive thickness gradient within the chromatographic column 50.

In certain aspects, the thickness gradient is at a first thickness (t) ₁ ) And a second thickness (t) ₂ ) Gradually increasing along the length of the column 50. The thickness variation of the stationary phase 64 may be constant or vary along the length of the chromatography column 50, but the thickness increases from the inlet 70 to the outlet 72.

In certain variations, the second thickness (t) ₂ ) Is greater than the first thickness (t) ₁ ) At least about 50% greater, optionally than the first thickness (t) ₁ ) At least about 100% greater, optionally than the first thickness (t) ₁ ) At least about 150% greater, optionally than the first thickness (t) ₁ ) At least about 200% greater, optionally than the first thickness (t) ₁ ) At least about 250% greater, and in certain variations, optionally a second thickness (t) ₂ ) Is greater than the first thickness (t) ₁ ) At least about 300% greater. A first thickness (t) ₁ ) Optionally, can be greater than or equal to about 10nm to less than or equal to about 10 microns, and a second thickness (t) ₂ ) From greater than or equal to about 30nm to less than or equal to about 30 microns. In one variant, the first thickness (t) ₁ ) About 30nm, second thickness (t) ₂ ) About 30 microns.

Fig. 3A-3C are depictions of peak focusing by employing a positive film thickness gradient, also referred to herein as a Film Thickness Gradient Column (FTGC), in accordance with certain aspects of the present disclosure. As shown in fig. 3A, for a given analyte 80, the peak 82 becomes increasingly focused as the analyte 80 travels down the column 84 having a stationary phase 86 with a positive thickness gradient extending from an inlet 88 to an outlet 90 of the column 84. As shown in fig. 3A, the stationary phase 86 film thickness is from thinner to thicker (in the direction from inlet 88 to outlet 90) in such a way as to focus the analyte peak 82 as the analyte travels along the column in the direction of the block arrow. Fig. 3B is a diagram of an arrangement for column performance evaluation. The column 84 is mounted in fluid communication with an injector 94 of an Agilent 6890 bench GC 96 equipped with a Flame Ionization Detector (FID) 98. Fig. 3C is an illustration of a forward (top) mode of operation and a reverse/reverse (bottom) mode of operation. In other words, a sample containing at least two target analytes is introduced from the inlet 88 in a forward mode (top) and travels in the direction of the block arrow towards the outlet 90 of the chromatography column 84. In the reverse or reverse mode (bottom), a sample containing at least two target analytes is introduced to the outlet 90 of the chromatographic column 84 and then travels in the direction of the block arrow towards the inlet 88.

In certain aspects, the present disclosure contemplates a method of performing peak focusing in a gas chromatography apparatus. The method can include introducing two or more target analytes into an inlet of a chromatography column including a stationary phase deposited within the chromatography column and having a positive thickness gradient. The stationary phase extends from an inlet to an outlet and has a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column. The second thickness is at least about 10% greater than the first thickness. The stationary phase may be any of those described previously. The method includes separating two or more target analytes in a chromatography column. In addition, the two or more target analytes are then eluted from the outlet of the chromatography column.

In certain aspects, the two or more target analytes are Volatile Organic Compounds (VOCs). In one aspect, at least one of the two or more target analytes comprises an aromatic compound, and the total peak focusing rate for the aromatic compound is greater than or equal to about 25%, for example, can be greater than or equal to about 26%, optionally greater than or equal to about 27%, and in certain variations, optionally greater than or equal to about 28%.

In another aspect, at least one of the two or more target analytes comprises an alkane compound, and the total peak focusing rate for the alkane compound is greater than or equal to about 10%, and in certain variations, optionally greater than or equal to about 11%.

In yet another aspect, the present disclosure contemplates a method of verifying peak focusing in a gas chromatography apparatus. The method includes performing a forward operation by introducing two or more target analytes into an inlet of a chromatography column including a stationary phase having a positive thickness gradient deposited within the chromatography column. The stationary phase extends from an inlet to an outlet and has a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column, wherein the second thickness is at least about 10% greater than the first thickness. The stationary phase may have any of the designs and compositions described previously. Forward operation involves separating two or more target analytes in a chromatography column followed by elution of the two or more target analytes from an outlet of the chromatography column. The reverse operation is then performed by introducing two or more target analytes to an outlet in a chromatography column, the chromatography column comprising a stationary phase. The reverse operation also includes separating the two or more target analytes in the chromatography column and eluting the two or more target analytes from the inlet of the chromatography column. The method further comprises comparing chromatographic resolutions from the forward and reverse operations, wherein the peak focus ratio of at least one pair of corresponding two peaks from the two target analytes is greater than 5%, and optionally greater than or equal to about 10%.

The present disclosure also contemplates a method of manufacturing a gas chromatography apparatus having a chromatography column with a positive thickness gradient. The method comprises introducing a precursor liquid into a chromatography column. The precursor liquid includes a stationary phase precursor and a low boiling point solvent that volatilizes along the length of the chromatography column, thereby increasing the concentration of the stationary phase precursor as it moves along the column and thus creating a stationary phase thickness gradient. The method also includes reacting or crosslinking the stationary phase precursor to form a positive thickness gradient stationary phase extending from an inlet to an outlet and having a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column. The second thickness is at least about 10% greater than the first thickness.

In certain aspects, the inner surface of the chromatography column is silanized prior to introducing the precursor liquid. Silanization may include passing the reactive silane in the gas phase through a column. In one embodiment, the reactive silane may be Hexamethyldisilazane (HMDS) vapor, which HMDS vapor forms a silane-containing surface coating on the interior surface. In certain variations, the reactive silane may be passed through the chromatography column multiple times.

Then, precursors of siloxane polymers (such as silicone resin OV-1, vinyl modified 100% dimethylsiloxane commercially available as OV-1 6001 or silicone resin OV-17 from ohio valley specialty, vinyl modified 50% phenylsiloxane-50% methylsiloxane commercially available as OV-1 6017 from ohio valley specialty) can be injected and reacted to form the stationary phase. Crosslinking agents (such as Dow SYLGARD) ^TM 184 reagent B (15% w/w, crosslinking agent)) may also be added together with the precursor to promote crosslinking. Obviously, the stationary phaseSuch materials are not limited and these are provided only as suitable examples of gas chromatography columns.

The chromatography column includes an inlet and an outlet, and introducing and reacting (or cross-linking) may include dynamically coating an inner surface of the chromatography column. This may be accomplished by partially filling the chromatography column with the precursor liquid, applying pressure at the inlet to force the precursor liquid down the length of the column, and applying vacuum at the outlet to evaporate the low boiling point solvent. For example, a plug of precursor liquid may be pushed through the column by applying a pressure of 5psi at the inlet. A vacuum pressure of-2 psi may be applied to the outlet to evaporate the low boiling point solvent.

Examples

Stationary phase thickness gradient Gas Chromatography (GC) columns can focus analyte peaks and improve separation resolution. Theoretical analysis and simulations indicate that focusing via a positive thickness gradient (i.e., stationary phase thickness) increases along the column. Peak focusing was experimentally verified by coating a 5m long capillary column with a film thickness that varied from 34nm at the inlet of the column to 241nm at the outlet of the column. The column uses C ₅ To C ₁₆ The alkane and aromatic compounds of (a) were analyzed in forward (thin to thick) and reverse (thick to thin) modes and compared to a column of uniform thickness having a thickness of 131nm.

A comparison of the forward mode with the resolution of the uniform thickness column shows that the total focusing rate (i.e., improvement in peak capacity) for the alkane is 11.7% and the total focusing rate for the aromatic is 28.2%.

Focusing effects were also demonstrated for isothermal room temperature separations of highly volatile compounds and temperature programmed separations with different ramp rates. In all cases, the peak capacity from forward mode separation was higher than the peak capacity from the other modes, indicating that a positive thickness gradient was able to focus the analyte peak. Therefore, as a general method for improving the separation performance of GC, this thickness gradient technique can be widely applied to various stationary phases and column types.

Test setup

The FTGC was mounted in an Agilent 6890 bench-top GC equipped with a flame ionization detector (FID, see FIG. 3B). Ultra-high purity helium gas was used as the carrier gas. As shown in fig. 3C, the evaluation of the peak focusing effect is performed with the analyte injected from the thinner coated end (forward mode, i.e., traveling from the thinner film to the thinner film) or the thicker coated end (reverse mode, i.e., traveling from the thicker film to the thinner film). Uniform thickness columns (film thickness same as average thickness) were also evaluated using the same settings for comparison. All experiments were performed using a constant pressure ramping procedure. The temperature programmed method and indenter are provided in table 1.

Material

Analytical Standard grade C ₅ To C ₁₆ Benzene, toluene, ethylbenzene, o-xylene, 1, 3-dichlorobenzene, nitrobenzene and methylene chloride were purchased from Sigma-Aldrich (Sigma-Aldrich) (st louis, missouri). Vinyl-modified OV-1 (P/N6001) and OV-17 (P/N6017) were purchased from Ohio valley specialty Inc. (Mary Tower, ohio). Ceramic SYLGARD ^TM 184 reagent B was purchased from ellsvershot binder (Ellsworth Adhesive) (hitman, wi). A deactivated fused silica tube (P/N10010, 250 μm ID) and RTX-5column (P/N10205 length 5m, 250 μm ID, 0.1 μm film thickness) were purchased from Restek (Ruitacon, pa.). DB-1MS columns (P/N122-0162, length of 5m, internal diameter of 250 μm, film thickness of 0.25 μm) were purchased from Agilent (Santa Clara, calif.). All materials were used as purchased without further purification or modification.

Chromatography column coating

OV-1 (75% w/w), OV-17 (10% w/w) and the Dow SYLGARD ^TM 184 reagent B (15% w/w, crosslinker) was dissolved in dichloromethane to yield a 2% (w/w) coating solution (effectively 5% phenyl stationary phase). A 5m long capillary column (250 μm i.d.) was silanized prior to coating by 8 repeated injections of Hexamethyldisilazane (HMDS) vapor. Subsequently, 80 μ L of the coating solution was loaded into the capillary 100 from the column inlet via a syringe pump (syringe pump) (fig. 4A). A positive pressure of 5-psi was applied from the inlet to drive the coating solution to the outlet. Passing a negative 2-psi vacuum pressure 110A 1m virtual column (250 μm i.d.) was applied to the outlet, which ensured a constant coating plug velocity. During coating, a small amount of low boiling point dichloromethane evaporated rapidly under vacuum, and as the coating solution plug moved from the inlet to the outlet of the column, the coating solution concentration gradually increased, and thus the film thickness increased. After coating, dry air was continuously passed through the column for 2 hours, followed by additional crosslinking at 80 ℃ for 2 hours, followed by deactivation with HMDS. The column was then aged at 230 ℃ for 3 hours at a flow of 0.5mL/min of helium. Using the same method, a column with a uniform thickness membrane was coated with 1% (w/w) of a coating solution (same composition as above, but diluted) and a positive pressure of 5-psi was applied from the inlet to force the coating solution to the outlet (no vacuum applied).

Simulation setup

C ₈ To C ₁₅ The simulations of the separations were performed in forward and reverse modes and using uniform thicknesses equivalent to the average gradient film thickness (separation conditions in table 1). For a 5m column, the film thickness varied from 34nm to 241nm (forward mode from entrance to exit, reverse mode from exit to entrance). It is worth noting that the calculation of the retention factor K (x, t) requires assigning the value of the coefficient K (t) (eq. (2)), which is based on the reference [22 ]]The values in (1) are estimated (see section S3-Simulation parameters and results in supplementary data (Simulation parameters and results in Supporting Information)). Table 2 provides the simulated retention times and FWHM, and table 3 provides the resolution.

Uniform thickness control

Restek RTX-5 columns were used for separation of C7 to C16 alkanes in forward and reverse mode as controls, and no difference in separation was expected. The isolation conditions are provided in table 1. P-values for retention time and FWHM (over 5 runs) were calculated using paired "student" T-tests and the resulting T-score was converted to a p-value. Significant at p = 0.05; no significant difference between the forward and reverse modes was observed for any analyte peak (see section S4-Control experiment using a Restek RTX-5column in supplementary data (Control experiment using an a Restek RTX-5column in Supporting Information))). Likewise, for C ₇ -C ₁₅ In other words, there was no significant difference between the forward and reverse modes when using a 5m long agilent DB-1MS column (data not shown).

Stationary phase characterization

To characterize the thickness of the stationary phase, FTGC was first frozen in liquid nitrogen and then several pieces were cut. Scanning Electron Microscope (SEM) images were taken near the column inlet (thinner membrane) and outlet (thicker membrane). FIGS. 2 (B) and (C) show an increase in film thickness from 34nm to 241nm with a gradient of about 41nm/m from the inlet to the outlet. Uniform thickness columns were also characterized at the inlet and outlet, with the film thickness at both ends of the column being 131nm.

The theoretical explanation for peak focusing is as follows:

effective velocity u of analyte at position x (distance from the inlet of the column) _eff (x, t) and a given time t are started by:

wherein u is _M (x, t) is the velocity of the mobile phase, and k (x, t) is the retention factor:

the distribution coefficient K (t) is defined as

Where R is the universal gas constant and T (x, T) is the time-dependent column temperature at position x. Δ G is the Gibbs free energy (Gibbs free energy) change associated with an analyte moving from a stationary phase to a mobile phase and can be calculated from the change in enthalpy of the analyte (Δ H) and the change in entropy of the analyte (Δ S)

ΔG＝ΔH-TΔS。(4)

The phase ratio beta is defined by

Wherein d is _i And d _f (x) Respectively the column inside diameter and the film thickness. Therefore, equation (2) can be expressed as

Where A is a constant for a given column. The retention factors δ k (x, t) varying along the column can be written (see derivation in supplementary data)

Equation (7) shows that the slight increase in retention factor δ k/k along the column at distance δ x contributes two fold: a negative temperature gradient given by the first term and a positive film thickness gradient given by the second term. The retention factor gradient (δ k/k) is related to the velocity gradient in equation (1); thus, both negative temperature gradients and positive film thickness gradients result in a velocity difference between the leading and trailing portions of the ribbon, allowing the ribbon to focus (e.g., the spatial distribution of the analyte experiences a spatially varying velocity gradient). At the exit, the band is observed as a time-varying peak during elution, which may be narrower than the corresponding peak from the unfocused band. In other words, peak focusing (observable) exists as a result of in-column band focusing. The equivalence of these two gradients can be expressed as

Film thickness gradients have several advantages over conventional temperature gradient-based peak focusing. First, the film thickness gradient is independent of column temperature, which allows for focusing of any volatile analyte at any operating temperature. Especially high volatile compounds that are difficult to focus with NTGS, can be accomplished according to the techniques of the present invention (e.g., with FTGC chromatography devices and processes). Second, while the temperature gradient may vary with heater and ambient conditions (such as heater arrangement, heat dissipation, column size/weight, column channel arrangement, and ambient temperature and gas flow), the film thickness gradient is always constant and allows for more reliable and repeatable GC operation (less susceptible to the environment). Finally, FTGCs according to certain aspects of the present disclosure may be used without additional accessories (such as heaters or coolers required by NTGS), which significantly reduces device complexity for future integration. However, despite these advantages, in some aspects, separation based on film thickness gradients may not be as versatile as NTGS because the gradient is fixed and the temperature gradient can be adjusted by varying the heat source and/or drain (drain). Furthermore, increasing film thickness towards the exit of the column may result in slow mass transfer, which may counteract the peak focusing effect. The mass transfer effect was examined in the following simulation.

For this simulation, the temperature gradient (i.e., NTGS) was not considered; only the film thickness gradient was analyzed. The time-dependent concentration c of the analyte peak traveling along the column is determined by solving the transient convection-diffusion equation

Wherein u is _eff (x, t) is given in equation (1). Effective diffusion coefficient u _eff (x, t) can be calculated from the local diffusion D and the retention factor k (x, t)

d _f As a film thickness, and D _M As the mobile phase diffusion constant. Notably, D includes longitudinal and lateral mass transfer/diffusion. D _M (x, t) may be represented as

Diffusion constant D _C (depending on the molar weight of the analyte and mobile phase molecules, and the atomic and structural diffusion volumes) and D _S As the stationary phase diffusion constant. The partial pressure p (x) being defined by the inlet and outlet pressures p _in And p _out Determining

L is the length of the column. u. of _M Is the velocity in the mobile phase, which is given by

The viscosity eta is provided as ₀ Index α associated with gas type _n Lower reference viscosity eta ₀ Function of (c):

in equation (12), it is worth noting that the temperature T (x, T) is provided as T (T) assuming that the temperature along the column remains constant for a given time T. Equation (9) can be solved by applying a finite difference model to the discrete time (t) and position (i) vectors

Δ x and Δ t are analog distances and time steps. Combine these to obtain

The solution of equation (19) yields a time-dependent movement of the analyte peak along the column.

By modeling equation (19), several boundary conditions must be set. First, at t =0, the injection peak has a gaussian peak shape, i.e.

Where σ is the initial diffusion. Notably, the initial peak at time t =0 is located at x =3 σ. At the column inlet, after the initial injection, no additional analyte was injected into the column:

C(0,t)＝0。(21)

at the column outlet, the final mesh concentration is approximately the same as the left mesh concentration (since it cannot be calculated by equation (16)), i.e.

C(L,t)＝C(L-Δx,t)。(22)

By observing, using equation (19), the peak retention time and full width at half maximum (FWHM), spatially varying concentrations can be measured at the column outlet (i.e., x = L) to construct a two-dimensional concentration matrix that varies with position and time ₈ To C ₁₅ Separation of compounds can be achieved with positive (i.e., from thin to thick, or "forward" mode) and negative (i.e., from thick to thin, or "reverse" mode) film thickness gradients. Simulations of uniform thickness (thickness equal to average forward/reverse mode film thickness) films were also performed as controls. The temperature was increased from 40 ℃ to 240 ℃ at a rate of 30 ℃/min. Pressure head set at 3.45psi (outlet set to ambient)Pressure, i.e., 1 atmosphere). For a 5m column, the film thickness varied from 34nm to 241nm (forward mode from entrance to exit, reverse mode from exit to entrance). By observing the concentration along a second dimension (i.e., in time), a vector of concentration changes over time can be obtained, which corresponds to the signal obtained from the detector at the outlet. The maximum (time-varying) corresponds to the elution/retention time, and the FWHM can be measured by observing the concentration at half the time of the peak. The resolution (R) between adjacent peaks can be calculated additionally using a formula

Wherein t is ₁ And t ₂ Is the retention time of two peaks, and w ₁ And w ₂ Is the corresponding FWHM.

Peak retention time and full width at half maximum (FWHM) can be measured at the column outlet (i.e., x = L) and are provided in table 1. Table 1 shows alkane C for simulation, uniform thickness control ₇ To C ₁₆ Separation of aromatic compounds and highly volatile alkanes (C) ₅ And C ₆ ) The separated temperature programmed profile and head pressure.

TABLE 1

Table 2 shows the data for C in forward and reverse modes ₈ To C ₁₅ The simulated Retention Time (RT) and full width at half maximum (FWHM). RT and FWHM for uniform coating thickness are also provided for reference. The temperature was ramped up from 40 ℃ at a rate of 30 ℃/min with a head pressure of 3.45psi. The column length was 5m. All values are provided in minutes. Additional analyses are provided in table 3.

TABLE 2

	RT fwd	FWHM fwd	RT uni	FWHM uni	RT bkwd	FWHM bkwd
							C 8	0.460	0.0382	0.449	0.0519	0.439	0.0684
C 9	0.731	0.0473	0.714	0.0704	0.697	0.0995
							C 10	1.157	0.0574	1.136	0.0885	1.113	0.1296
C 11	1.639	0.0656	1.615	0.1005	1.560	0.1487
							C 12	2.106	0.0713	2.082	0.1073	2.055	0.1584
C 13	2.591	0.0763	2.568	0.1120	2.541	0.1640
							C 14	3.050	0.0807	3.027	0.1158	3.001	0.1679
C 15	3.460	0.0842	3.437	0.1186	3.411	0.1708

Table 3 shows the use for C in forward and reverse modes and in uniform thickness ₈ To C ₁₅ The simulated resolution (R) between adjacent peaks. The forward mode resolution is greater than both the reverse mode and the uniform thickness resolution. The difference in resolution is defined as R _diff ＝R _fwd -R _bkwd 。

TABLE 3

Table 1 shows the analyte peaks eluting at different times in forward and reverse mode; this is because the separation conditions for a given analyte are different between the two modes. In the forward mode, the analyte is first exposed to a thinner film at low temperature and then reaches a thicker film at high temperature, which is quite the opposite of what the analyte experiences in the reverse mode. Thus, in evaluating column performance, the retention times for the analytes in these two modes are different and the full width at half maximum (FWHM) cannot be directly compared. Instead, resolution R was used between the two peaks to analyze the separation performance (see table 2), which is given by equation 23.

Table 2 shows that the forward mode yields higher resolution between adjacent peaks when compared to the reverse mode, which means that the forward mode may contain more peaks than the reverse mode within the same time interval. The uniform thickness resolution is greater than the reverse mode resolution, but always less than the forward mode resolution, thus indicating that peak focusing is achieved in the forward mode. Further analysis of adjacent peak resolutions indicates a difference in resolution (i.e., R) between the forward mode and the reverse mode _{Difference value} ＝R _{Forward direction} -R _{Reverse direction} ) Decreases with increasing analyte retention. This may be due to slower mass transport (i.e., lateral diffusion) in thicker film regions — this is more pronounced for heavier compounds. In the forward mode, this effect causes the lower volatility compounds to be wider closer to the column outlet, thereby counteracting the focusing provided by the column. Conversely, in the reverse mode, a thinner film at the exit results in less broadening, which counteracts the defocusing from the reverse gradient. Thus, the difference in resolution between the forward mode and the reverse mode is reduced for lower volatility compounds.

The alkane mixture was subjected to peak focusing. Peak focusing capability of FTGC by separation C ₇ To C ₁₆ Alkane mixtures were evaluated. 0.025 μ L of liquid was used for injection at a split ratio of 5. The same separation conditions were used for the forward mode, the uniform thickness column and the reverse mode (denoted "same parameter reverse mode", see table 1-alkane mixture). Example chromatograms are shown in fig. 5A-5D.

Fig. 6 shows the analyte peaks eluting at different times in forward mode and reverse mode of the same parameters for a uniform thickness column, consistent with the simulation (table 2). This is because the separation conditions for a given analyte are different between the two modes, which in turn are different from a uniform thickness column. In the forward mode, the analyte is first exposed to a thinner film at low temperature and then reaches a thicker film at high temperature, which is quite the opposite of what the analyte experiences in the reverse mode. In a uniform thickness column, the analyte experiences the same film thickness at all temperatures. Thus, in evaluating the column performance, this is whereIn both modes and in the homogeneous column, the retention times for the analytes are different and the full width at half maximum (FWHM) cannot be directly compared. Conversely, the resolution between adjacent peaks (e.g., C) ₇ And C ₈ 、C ₈ And C ₉ Etc.) to analyze separation performance. The resolution of the same parameter reverse mode and the resolution of the uniform column are subtracted from the corresponding resolution in the forward mode; resolution difference (i.e., R) between all adjacent pairs of peaks (average of 5 runs) _{Forward direction} -R _{Same parameter reverse direction} Or R _{Forward direction} -R _Uniformity ) Is plotted in fig. 6. P-values for resolution differences were calculated using paired "student" T-tests (5 runs in forward and reverse mode with the same parameters and homogeneous columns) and the resulting T-scores were converted to p-values (see table 3). Significance was seen at p =0.05, indicating that the forward mode had significantly higher resolution between all pairs of adjacent peaks compared to the reverse mode of the same parameters. This is confirmed by the simulation (table 3), which also shows a higher resolution in the forward mode, which means that the forward mode may contain more peaks than the reverse mode in the same time interval. Uniform thickness pillar resolution below and up to C ₁₀ /C ₁₁ Forward mode resolution of, but for C ₁₅ /C ₁ For the sake of clarity, the uniformity resolution is higher. The overall performance analysis is provided below.

Table 4 shows the results for C ₇ To C ₁₆ P-values between the forward mode of alkane separation and uniform thickness, the reverse mode with the same parameters (IP), and the reverse mode with Equal Time (ET). Significance was observed at p = 0.05. All p values are significant between the forward mode and the IP reverse mode, whereas for the ET reverse mode, C is significant ₇ To C ₁₃ The p-value of (a) is significant. Significance of forward mode resolution up to C ₁₀ /C ₁₁ All above the uniform thickness resolution, and for C ₁₅ /C ₁₆ In other words, the resolution of uniform thickness is more significant.

TABLE 4

To further account for the difference between retention times, a second set of chromatograms was obtained by reducing the reverse mode ramp rate to ensure C ₁₆ (the last analyte eluted) eluted simultaneously with the forward mode (this is denoted as "isochronous reverse mode", see table 1-separation conditions, fig. 5A-5D chromatograms, fig. 6-resolution differences, table 4-p values). Likewise, the forward mode is for C ₇ And C ₁₃ The alkane pair between provides significantly higher resolution (results obtained from 5 runs), but for C ₁₃ To C ₁₆ Similar to the isochronous reverse mode is performed. Although the forward mode is not superior to the isochronous reverse mode (or uniform thickness column) for all local resolutions (i.e., between adjacent alkane pairs), the forward mode has a significantly higher Peak Capacity (PC), defined as the sum of all resolutions, than all other modes (table 5). Focus ratio analysis, defined as

This indicates that the forward mode indicates total focusing rates of about 11.7%, 26.8% and 29.8%, respectively, when compared to uniform thickness columns, the same parameter reverse mode and the isochronous reverse mode.

TABLE 5

Forward peak capacity	49.34±0.841
		Reverse peak capacity (IP)	38.90±0.831
p value	1.73e-4
		Rate of focusing (IP)	26.84％
Converse peak capacity (ET)	38.02±2.400
		p value (ET)	4.62e-4
Rate of focusing (ET)	29.76％
		Uniform peak capacity	44.18±0.483
p value (Uniform)	1.63e-4
		Rate of focusing (uniformity)	11.67％

Table 5. For C in FIGS. 5A-5D ₇ To C ₁₆ For alkane separation, forward mode, reverse mode with the same parameters (IP), reverse mode with isochronous (ET), and peak capacity, total resolution, p-value, and focusing rate with uniform thickness. Significance was observed at p = 0.05. The peak capacity in the forward mode is significantly better than the peak capacity of all other modes.

Peak focusing on aromatic mixtures

FTGC peak focusing also analyzed the separation of aromatic mixtures containing benzene, toluene, ethylbenzene, ortho-xylene, and 1, 3-dichlorobenzene. 0.025 μ L of the mixed liquid was injected at a split ratio of 5. Example chromatograms are shown in fig. 7A-7D, and resolution differences are shown in fig. 8. Local resolution difference p values (calculated from 5 runs) are provided in table 6. The peak capacity, p-value and focus rate are provided in table 7, showing that the forward mode has a significantly higher peak capacity than all other modes. Thus, whether the separation parameters are kept constant (and the analyte elutes faster in reverse mode with the same parameters) or are varied to ensure simultaneous elution of the final compounds (in forward mode and isochronous reverse mode), the separation performance in forward mode is always better than either reverse mode. The forward mode was also superior to the uniform thickness column, indicating a focusing rate of 28.2% (table 7). Thus, overall, the forward mode (i.e., positive film thickness gradient) demonstrates the ability to increase the separation peak capacity.

Table 6 shows the p-values between the forward mode and the uniform thickness, the same parameter (IP) reverse mode, and the Equal Time (ET) reverse mode for aromatics separation. For elution order and abbreviations, see fig. 7A-7D. Significance was noted at p = 0.05. All p-values show a significantly improved resolution in forward mode.

TABLE 6

p value	p value (IP)	p value (ET)	p value (Uniform)
				B/T	3.08e-7	5.56e-7	1.83e-5
T/E	3.84e-7	9.28e-7	4.20e-6
				E/X	9.32e-6	8.28e-5	0.012
X/DCB	5.95e-6	2.06e-5	2.28e-5

Table 7 shows the peak capacity, p-value and focusing rate between the forward mode, the reverse mode with the same parameters (IP) and the isochronous (ET) reverse mode for aromatic separation in fig. 7A-7D, and uniform thickness. Significance was observed at p = 0.05. The separation of the forward mode is significantly better than the separation of all other modes.

TABLE 7

Forward peak capacity	13.47±0.089
		Reverse peak capacity (IP)	9.59±0.060
p value	1.60e-7
		Rate of focusing (IP)	40.35％
Converse peak capacity (ET)	9.85±0.093
		p value (ET)	1.56e-6
Focusing rate (ET)	36.73％
		Uniform peak capacity	10.50±0.146
p value (Uniform)	1.13e-5
		Rate of focusing (uniformity)	28.22％

Effect of temperature rise

To demonstrate how the ramp rate affects peak focusing, C with four different ramp rates (0, 10, 20, and 30 ℃/min, ramp from 60 ℃ without hold) was performed in the forward mode, the same parameter reverse mode, and using a uniform thickness column ₇ To C ₁₀ Separation of (4). The pressure was 3.45psi (2.7 mL/min at 60 ℃) and all separated fractions were in a ratio of 15. The blank time was measured by methane injection and all ramp rates were found to be 0.36 minutes. The resolution and focus rate for each temperature profile (values provided as an average of 5 runs) are provided in table 8). In the forward mode, at higher ramp rates, the analyte will encounter a relatively higher temperature when it reaches the thicker stationary phase closer to the column outlet. Thus, the analyte spends less time in the thicker film, also reducing peak broadening. In the reverse mode, the analyte first encounters a thicker stationary phase at a lower temperature and then flows toward a thinner stationary phase at a higher temperature. Peak broadening from low thickness stationary phases is already low; therefore, in the reverse mode, the peak broadening due to the temperature increase is generally reduced. Therefore, the focusing rate increases with an increase in the temperature rising rate, and the focusing rate is as high as 61.9% compared to the forward and reverse modes and 68.1% compared to the forward mode and the uniform thickness at a rate of 30 ℃/min.

Table 8 shows the resolution (R), peak Capacity (PC) and focus rate for the forward mode, reverse mode and uniform thickness for the C7 to C10 separations at different ramp rates. The initial temperature for all separations was 60 ℃ and the carrier gas head pressure was 3.45psi (2.7 mL/mi at 60 ℃). 0.025 μ L of the mixed liquid was injected using a split ratio of 15.

TABLE 8

Focusing for highly volatile compounds

Unlike NTGS, FTGC gradients are able to focus peaks at low temperatures where temperature gradients are difficult to generate (as long as these peaks are reasonably retained at these temperatures). To prove this, C is performed ₅ And C ₆ Room temperature isothermal separation (table 1) (fig. 9A-9C). The resolution, p-value and focus rate (average of 5 runs) are provided in table 9. The focusing rate reaches 40.2%, the average forward mode resolution is 2.97, and the uniform thickness resolution is 2.12. It is noteworthy for NTGS that the same peak focusing effect is difficult to achieve with highly volatile compounds because only small temperature gradients can be generated at low operating temperatures.

Table 9 shows C for room temperature (26 ℃ C.) ₅ And C ₆ Resolution, p-value and focus rate of separated forward mode, reverse mode and uniform thickness. Significance was observed at p = 0.05.

TABLE 9

Forward resolution	2.97±0.140
		Inverse resolution	1.85±0.055
p value (reverse)	1.17e-4
		Focusing rate (reverse)	60.4％
Uniform resolution	2.12±0.049
		p value (Uniform)	4.87e-4
Rate of focusing (uniformity)	40.2％

The development and evaluation of stationary phase thickness gradient column technology capable of achieving peak focusing is described in detail herein. The experimental results, confirmed by theoretical analysis and simulation, show an increase in the separation performance of various compounds in the forward mode, including focused separation of highly volatile compounds at room temperature. Compared with NTGS, FTGC has the advantages of large applicable temperature and compound volatility range, simple operation without auxiliary equipment, small dependence on environmental conditions and good compactness. This stationary phase thickness gradient technique can be readily applied to a wide range of GC applications and can be used for stationary phases of any material or thickness as long as a gradient can be generated. Furthermore, it is applicable to both capillary columns of regular circular cross-section and micro-columns of rectangular cross-section. The foregoing description of the embodiments has been presented for purposes of illustration and description. This description is not intended to be exhaustive or to limit the disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but, where applicable, are interchangeable and can be used in a selected embodiment, even if not specifically shown or described. The same parts may also be varied in many ways. Such variations are not to be regarded as a departure from the disclosure, and all such modifications are intended to be included within the scope of the disclosure.

Claims (28)

1. A gas chromatography apparatus for peak focusing of one or more target analytes, comprising:

a chromatography column having an inlet and an outlet, wherein the inlet receives a sample comprising one or more target analytes exiting the chromatography column at the outlet; and

a stationary phase deposited within the chromatography column and having a positive thickness gradient, wherein the stationary phase extends from the inlet to the outlet and has a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column, wherein the second thickness is at least about 10% greater than the first thickness.

2. The gas chromatography apparatus of claim 1, wherein the first thickness is greater than or equal to about 10nm to less than or equal to about 10 microns and the second thickness is greater than or equal to about 30nm to less than or equal to about 30 microns.

3. The gas chromatography apparatus of claim 1, wherein the second thickness is greater than or equal to the first thickness by at least about 100%.

4. The gas chromatography apparatus of claim 1, wherein the second thickness is greater than or equal to the first thickness by at least about 300%.

5. The gas chromatography apparatus of claim 1, wherein the chromatography column is a micro-gas chromatography column.

6. The gas chromatography apparatus of claim 1, wherein the stationary phase comprises a siloxane polymer.

7. The gas chromatography apparatus of claim 6, wherein the siloxane polymer comprises at least one alkyl or aryl group comprising 1 to 30 carbon atoms.

8. The gas chromatography apparatus of claim 1, wherein the cross-sectional shape of the chromatography column is selected from the group consisting of: circular, oval, rectangular, and triangular.

9. A method of peak focusing in a gas chromatography apparatus, the method comprising:

introducing two or more target analytes into an inlet of a chromatography column, the chromatography column comprising: a stationary phase deposited inside the chromatography column and having a positive thickness gradient, wherein the stationary phase extends from the inlet to an outlet and has a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column, wherein the second thickness is at least about 10% greater than the first thickness;

separating the two or more target analytes in the chromatography column; and

eluting the two or more target analytes from the outlet of the chromatography column.

10. The method of claim 9, wherein the two or more target analytes are Volatile Organic Compounds (VOCs).

11. The method of claim 9, wherein at least one of the two or more target analytes comprises an aromatic compound and a total peak focusing rate for the aromatic compound is greater than or equal to about 25%.

12. The method of claim 9, wherein at least one of the two or more target analytes comprises an alkane compound and a total peak focus rate for the alkane compound is greater than or equal to about 10%.

13. The method of claim 9, wherein the first thickness is greater than or equal to about 10nm to less than or equal to about 10 microns and the second thickness is greater than or equal to about 30nm to less than or equal to about 30 microns.

14. The method of claim 9, wherein the second thickness is greater than or equal to the first thickness by at least about 300%.

15. The method of claim 9, wherein the chromatography column is a micro-gas chromatography column.

16. The method of claim 9, wherein the stationary phase comprises a siloxane polymer comprising at least one alkyl or aryl group comprising 1 to 30 carbon atoms.

17. The method of claim 9, wherein the cross-sectional shape of the chromatography column is selected from the group consisting of: circular, oval, rectangular, and triangular.

18. A method of verifying peak focusing in a gas chromatography apparatus, the method comprising:

forward operation is performed by introducing two or more target analytes into an inlet of a chromatography column comprising: a stationary phase deposited inside the chromatography column and having a positive thickness gradient, wherein the stationary phase extends from the inlet to an outlet of the chromatography column and has a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column, wherein the second thickness is at least about 10% greater than the first thickness;

separating the two or more target analytes in the chromatography column;

eluting the two or more target analytes from the outlet of the chromatography column;

performing a reverse operation by introducing the two or more target analytes into the outlet of the chromatography column, the chromatography column comprising a stationary phase;

separating the two or more target analytes in the chromatography column;

eluting the two or more target analytes from the inlet of the chromatography column; and

comparing chromatographic resolutions from the forward and reverse operations, wherein a peak focus ratio of at least one pair of corresponding two peaks from two target analytes is greater than 5%.

19. A method of manufacturing a gas chromatography apparatus having a chromatography column with a positive thickness gradient, the method comprising:

introducing a precursor liquid into the chromatography column, wherein the precursor liquid comprises a stationary phase precursor and a low boiling point solvent that volatilizes along the length of the chromatography column to increase the concentration of the stationary phase precursor as the stationary phase precursor moves along the chromatography column; and

reacting or crosslinking the stationary phase precursor to form the positive thickness gradient stationary phase extending from the inlet to the outlet and having a first thickness at the inlet of the chromatography column and a second thickness at the outlet of the chromatography column, wherein the second thickness is at least about 10% greater than the first thickness.

20. The method of claim 19, wherein the inner surface of the chromatography column is silanized prior to introducing the precursor liquid.

21. The method of claim 20, wherein the silanization comprises passing reactive silane in a gas phase through the chromatography column.

22. The method of claim 19, wherein the chromatography column comprises an inlet and an outlet, and the introducing, and reacting or crosslinking comprises: dynamically coating the inner surface of the chromatography column by partially filling the chromatography column with the precursor liquid, applying pressure at the inlet to force the precursor liquid down the length of the chromatography column, and applying vacuum at the outlet to evaporate the low boiling point solvent.

23. The method of claim 19, wherein the first thickness is greater than or equal to about 10nm to less than or equal to about 10 microns and the second thickness is greater than or equal to about 30nm to less than or equal to about 30 microns.

24. The method of claim 19, wherein the second thickness is at least about 100% greater than or equal to the first thickness.

25. The method of claim 19, wherein the second thickness is greater than or equal to the first thickness by at least about 300%.

26. The method of claim 19, wherein the chromatography column is a micro-gas chromatography column.

27. The method of claim 19, wherein the positive thickness gradient stationary phase comprises a siloxane polymer.

28. The method of claim 27, wherein the siloxane polymer comprises at least one alkyl or aryl group comprising 1 to 30 carbon atoms.