RU2825594C1 - Stationary phase thickness gradient chromatographic column - Google Patents

Abstract

FIELD: physics.

SUBSTANCE: invention relates to a gas chromatography device comprising a chromatographic column having a stationary phase with a positive thickness gradient. Gas chromatography device for focusing peaks of one or more target analytes includes: a chromatographic column having an inlet and an outlet, wherein a sample comprising one or more target analytes is fed into the inlet and exits the chromatographic column through the outlet, and a stationary phase deposited inside the chromatographic column, which has a positive thickness gradient, wherein the stationary phase passes from the inlet to the outlet and has a first thickness at the inlet of the chromatographic column and a second thickness at the outlet of the chromatographic column, where the second thickness is at least approximately 10% greater than the first thickness.

EFFECT: improved separation in a chromatographic column in order to improve its operational characteristics and increase the analytical capacity of gas chromatography.

28 cl, 9 dwg, 9 tbl

Description

This application claims the benefit of U.S. Provisional Application No. 62/940,038, filed November 25, 2019. This application is incorporated herein by reference in its entirety.

This invention was developed under Government Grant FA8650-17-C-9106, funded by the United States Air Force, Air Force Materiel Command. The Government has certain rights in this invention.

Field of technology

The present invention relates to gas chromatography devices comprising chromatographic columns containing a stationary phase with a positive thickness gradient to improve peak focusing.

State of the art

This section provides general information related to the present invention, which does not necessarily relate to the prior art.

Gas chromatography (GC) is an analytical technique based on the separation of compounds in the gas phase using separating columns, which allows the analysis and identification of compounds present in a target sample. GC is widely used in various industrial applications for the separation and identification of target analytes, such as volatile organic compounds or semi-volatile organic compounds. GC is particularly suitable for the analysis of complex samples containing a set of target analytes that require individual detection. GC analysis records the "peaks" of chemicals passing through a separating column. Thus, a sample containing various chemicals or target analytes is injected into the column through an injector. The column contains a material that is considered as a stationary phase. Different fractions of the sample pass through the column at different rates (due to the physical and chemical interactions of the chemicals with the material contained in the column). As target analytes elute and exit the column, the sensor can detect time-released species based on the rate at which the analytes pass through the column. These analytes can be identified and/or quantified electronically during or after detection. It would be desirable to improve the separation of the chromatographic column to improve its performance and enhance the analytical capability of the GC.

The essence of the invention

This section provides a general outline of the invention and thus does not fully disclose the scope of the invention or all of its features.

Some aspects of the present invention relate to a gas chromatography device designed to focus peaks of one or more target analytes. A chromatographic column has an inlet and an outlet, wherein a sample is fed into the inlet, including one or more target analytes, which exit the column through the outlet. A stationary phase is deposited inside the chromatographic column, which has a positive thickness gradient. The stationary phase extends from the inlet to the outlet and has a first thickness at the inlet of the chromatographic column, and has a second thickness at the outlet of the chromatographic column. The second thickness is at least approximately 10% greater than the first thickness.

According to one aspect, the first thickness is from a value greater than or equal to about 10 nm to a value less than or equal to about 10 micrometers, and the second thickness is from a value greater than or equal to about 30 nm to a value less than or equal to about 30 micrometers.

According to one aspect, the second thickness is at least approximately 100% greater than or equal to the first thickness.

According to one aspect, the second thickness is at least approximately 300% greater than or equal to the first thickness.

According to one aspect, the chromatographic column is a chromatographic column for gas microanalysis.

According to one aspect, the stationary phase comprises a siloxane polymer.

According to one other aspect, the siloxane polymer comprises at least one alkyl group or aryl group containing from 1 to 30 carbon atoms.

According to one aspect, the cross-sectional shape of the chromatographic column is selected from the group consisting of a circular, oval, rectangular and triangular shape.

The present invention also relates to a method for focusing peaks in a gas chromatography device. The method includes introducing two or more target analytes into the inlet of a chromatographic column, including a stationary phase arranged inside this chromatographic column, having a positive thickness gradient. The stationary phase extends from the inlet to the outlet and has a first thickness at the inlet of the chromatographic column, and has a second thickness at the outlet of the chromatographic column. The second thickness exceeds the first thickness by at least approximately 10%. The method further includes separating two or more target analytes in the chromatographic column. The method also includes eluting two or more target analytes from the outlet of the chromatographic column.

In one aspect, two or more target analytes are volatile organic compounds (VOCs).

According to one aspect, at least one of the two or more target analytes comprises an aromatic compound, and the resulting aromatic compound peak focusing index is greater than or equal to about 25%.

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

According to one aspect, the first thickness is from a value greater than or equal to about 10 nm to a value less than or equal to about 10 micrometers, and the second thickness is from a value greater than or equal to about 30 nm to a value less than or equal to about 30 micrometers.

According to one aspect, the second thickness is at least approximately 300% greater than or equal to the first thickness.

According to one aspect, the chromatographic column is a chromatographic column for gas microanalysis.

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

According to one aspect, the cross-sectional shape of the chromatographic column is selected from the group consisting of: round, oval, rectangular and triangular.

The present invention also relates to a method for confirming the focusing of peaks in a gas chromatography device. The method includes performing a forward operation by introducing two or more target analytes into the inlet of a chromatographic column containing a stationary phase arranged inside this chromatographic column having a positive thickness gradient. The stationary phase extends from the inlet to the outlet and has a first thickness at the inlet of the chromatographic column and a second thickness at the outlet of the chromatographic column. The second thickness is at least approximately 10% greater than the first thickness. The forward operation includes separating two or more target analytes in the chromatographic column and eluting two or more target analytes from the outlet of the chromatographic column. The method also includes performing a reverse operation by introducing two or more target analytes into the outlet of the chromatographic column containing the stationary phase. The reverse operation includes separating two or more target analytes in the chromatographic column and eluting two or more target analytes from the inlet of the chromatographic column. The method further includes comparing the chromatographic resolution obtained in the direct operation and the resolution obtained in the reverse operation, in which the peak focusing index of at least one corresponding pair of two peaks of two target analytes exceeds 5%.

The present invention also relates to a method for creating a gas chromatography device comprising a chromatographic column with a positive thickness gradient. The method includes introducing a precursor liquid into the chromatographic column. The precursor liquid includes a stationary phase precursor and a low-boiling solvent that evaporates as it moves along the chromatographic column, which increases the concentration of the stationary phase precursor as it moves along the column, thus creating a stationary phase thickness gradient. The method also includes carrying out a reaction of the stationary phase precursor or forming cross-links in the stationary phase precursor when forming a positive stationary phase thickness gradient from the inlet to the outlet, where the stationary phase has a first thickness at the inlet of the chromatographic column, and has a second thickness at the outlet of the chromatographic column. The second thickness exceeds the first thickness by at least approximately 10%.

According to one aspect, prior to introducing the precursor liquid, the inner surface of the chromatographic column is silanized.

In one aspect, silanization comprises passing a reactive silane in gaseous form through a column.

According to one aspect, a chromatographic column includes an inlet and an outlet, and introducing and reacting or cross-linking comprises: dynamically coating the inner surface of the chromatographic column by partially filling the chromatographic column with a precursor liquid, applying pressure at the inlet to force the precursor liquid along the length of the column, and applying vacuum at the outlet to evaporate the low-boiling solvent.

According to one aspect, the first thickness is from a value greater than or equal to about 10 nm to a value less than or equal to about 10 micrometers, and the second thickness is from a value greater than or equal to about 30 nm to a value less than or equal to about 30 micrometers.

According to one aspect, the second thickness is at least approximately 100% greater than or equal to the first thickness.

According to one aspect, the second thickness is at least approximately 300% greater than or equal to the first thickness.

According to one aspect, the chromatographic column is a chromatographic column for gas microanalysis.

According to one aspect, the stationary phase comprises a siloxane polymer.

According to one other aspect, the siloxane polymer comprises at least one alkyl group or aryl group containing from 1 to 30 carbon atoms.

Other areas of application of the invention will become apparent after reading the proposed description. The proposed description and specific examples are provided only to illustrate the invention and do not limit the scope of the present invention.

Brief description of the drawings

The drawings provided in this work are given only to illustrate some embodiments of the invention and do not reflect all possible embodiments of the invention, and, thus, do not limit the scope of the present invention.

Fig. 1 shows an example of the embodiment of a system of a device for gas chromatography.

Fig. 2 is a cross-sectional view of a chromatographic column comprising a stationary phase with a positive thickness gradient to improve peak focusing, according to certain aspects of the present invention.

3A-3C illustrate peak focusing in a column containing a thickness gradient film (TGF) according to some aspects of the present invention. The film having a transition from a thinner to a thicker thickness facilitates focusing of the analyte peak as it moves along the column, as shown in Fig. 3A. Fig. 3B illustrates a setup for evaluating the performance of a column. Fig. 3C illustrates forward and reverse/inverted operation modes.

Fig. 4A-4C. Fig. 4A shows the apparatus for coating a CPGT. The column is partially coated dynamically by moving a plug of coating solution from the liquid phase, and then the mixture is ejected from the column under a pressure of 5 psi (34473.8 Pa). While ejecting the solution, a vacuum pressure of -2 psi (-13789.5 Pa) is applied to the outlet to evaporate the solvent. Fig. 4B is an SEM image of a region near the column inlet where the film thickness is 34 nm. Fig. 4C is an SEM image of a region near the column outlet where the film thickness is 241 nm.

Fig. 5A-5D. Fig. 5A is a diagram of the separation of a mixture of C ₇ -C ₁₆ alkanes in the direct operation mode. Fig. 5B is a diagram of the separation of a mixture of C ₇ -C ₁₆ alkanes under identical parameters in the reverse or inverted operation mode. Fig. 5C is a diagram of the separation of a mixture of C ₇ -C ₁₆ alkanes over the same time in the reverse mode. Fig. 5D is a diagram of the separation of a mixture of C ₇ -C ₁₆ alkanes on a column of uniform thickness.

Figure 6 shows a diagram of the differences in resolution obtained in the direct and reversed modes with identical parameters for the same time, as well as in a column with a uniform thickness during the separation of C ₇ -C ₁₆ alkanes.

Fig. 7A-7D. Fig. 7A is a diagram of the separation of a mixture of aromatic compounds in the direct mode. Fig. 7B is a diagram of the separation of a mixture of aromatic compounds under identical parameters. Fig. 7C is a diagram of the separation of a mixture of aromatic compounds over the same time in the reverse/inverted mode. Fig. 7D is a diagram of the separation of a mixture of aromatic compounds in a comparative column having a uniform thickness. Peaks 1, 2, 3, 4 and 5 correspond to benzene, toluene, ethylbenzene, ortho-xylene and 1,3-dichlorobenzene, respectively.

Figure 8 shows a diagram of the differences in resolution obtained in the forward and reverse modes with identical parameters for the same time, as well as in a column with a uniform thickness during the separation of aromatic compounds.

Fig. 9A-9C. Fig. 9A shows a diagram of the isothermal separation of C ₅ and C ₆ in the direct mode at room temperature. Fig. 9B shows a diagram of the isothermal separation of C ₅ and C ₆ at room temperature under identical parameters in the reversed/inverse mode. Fig. 9C shows a diagram of the isothermal separation of C ₅ and C ₆ at room temperature on a column of uniform thickness.

Identical parts are designated in graphic materials by identical position numbers.

Detailed description of the invention

Below, for a more detailed disclosure of the present invention, exemplary embodiments are provided, which fully disclose the scope of the invention to those skilled in the art. For a better understanding of the embodiments of the present invention, many specific details are provided, such as examples of specific compositions, components, devices and methods. Those skilled in the art should understand that it is not necessary to use specific details, that the exemplary embodiments can be implemented in various forms, and that none of the presented embodiments should be considered as limiting the scope of the invention. In some exemplary embodiments, well-known methods, well-known device designs and well-known techniques are not discussed in detail.

The terminology used herein is for the purpose of describing particular exemplary embodiments only and should not be considered limiting. As used herein, the singular forms may also refer to the plural unless the context otherwise requires. The terms "includes," "including," "comprise," and "having" have a non-limiting meaning and thus indicate the presence of the named features, elements, compositions, 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. Although the non-exhaustive term "including" should be understood as an open-ended term used to describe and disclose the various embodiments discussed herein, in some aspects this term may be considered a more limiting and restrictive term, such as "consisting of" or "consisting essentially of." Thus, in any given embodiment of the present invention, the recitation of compositions, materials, components, elements, features, integers, operations and/or process steps also specifically includes embodiments consisting of or consisting essentially of such recited compositions, materials, components, elements, features, integers, operations and/or process steps. In the case of the expression "consisting of", an alternative embodiment excludes the presence of any additional compositions, materials, components, elements, features, integers, operations and/or process steps, while in the case of the expression "consisting essentially of", any additional compositions, materials, components, elements, features, integers, operations and/or process steps that significantly affect the basic and novel characteristics are excluded from such embodiment, although any compositions, materials, components, elements, features, integers, operations and/or process steps that do not significantly affect the basic and novel characteristics may be included in this embodiment.

Any method steps, processes, and operations discussed in this work should not be considered as necessarily requiring their execution in the specific order shown or described unless the order of their execution is clearly indicated. It should also be understood that, unless otherwise indicated, additional or alternative steps may be included.

When a component, element, or layer is said to be "on," "linked to," "connected to," or "associated with" another element or layer, it may be directly on, linked to, connected to, or associated with the other component, element, or layer, or there may be intervening elements or layers. Conversely, when an element is said to be "directly on," "directly linked to," "directly connected to," or "directly associated with" another element or layer, there may be no intervening elements or layers. Other words describing interactions between elements may be similarly interpreted (e.g., "between" rather than "directly between," "near" rather than "directly adjacent," etc.). As used herein, the term "and/or" includes any and all combinations of one or more of the listed entities associated with it.

Although the terms "first," "second," "third," etc. may be used herein to describe various stages, elements, components, regions, layers, and/or regions, unless otherwise specified, such stages, elements, components, regions, layers, and/or regions should not be limited by these terms. These terms may be used only to distinguish one stage, element, component, region, layer, or region from another stage, element, component, region, layer, or region. When terms such as "first," "second," and other ordinal designations are used herein, they do not denote a sequence or order unless the context otherwise requires. Thus, the first stage, element, component, region, layer, or region discussed below may be referred to as the second stage, element, component, region, layer, or region without changing the features of a particular embodiment.

Terms relating to spatial arrangement or temporal order, such as "before," "after," "inside," "outside," "under," "below," "lower," "above," "upper," and similar terms, may be used herein for ease of reference when describing the relationship between one element or feature and another element(s) or feature(s) shown in the drawings. Terms relating to spatial arrangement or temporal order may include not only the orientations shown in the drawings, but also various orientations of the device or system during operation or maintenance.

In this document, numerical values representing approximate measurements or range limits include minor deviations from the stated values, and along with embodiments in which exact values are stated, the invention also includes embodiments in which approximate values are stated. Except for the working examples given at the end of the detailed description, all numerical values of parameters (e.g., quantities or conditions) given herein, including the appended claims, are to be understood in all cases as including the word "about", regardless of whether the numerical value is preceded by the word "about". "Approximately" indicates that the stated numerical value allows for some imprecision (with some approximation to the exact value; approximately or reasonably close to the value; approximately). If the imprecision defined by the word "about" does not exactly correspond to its ordinary meaning in the given art, then in this document "about" means at least the deviations that may arise in ordinary methods of measuring and using such parameters. For example, "approximately" may include a deviation of less than or equal to 5%, possibly less than or equal to 4%, possibly less than or equal to 3%, possibly less than or equal to 2%, optionally less than or equal to 1%, possibly less than or equal to 0.5%, and in some aspects possibly less than or equal to 0.1%.

In addition, the ranges given include all values and additionally designated ranges within the entire range, including endpoints and subranges of the ranges.

Below, examples of embodiments of the invention are considered in more detail, accompanied by drawings.

Various aspects of the present invention relate to gas chromatography. As shown in Fig. 1, a simplified diagram of a gas chromatographic system 20 typically includes at least five components: (1) a carrier gas stream 20; (2) a sample inlet system 22; (3) one or more gas chromatographic columns 30; (4) a sensor 32, and (5) a data processing system 34. The carrier gas introduced as the carrier gas stream 20 (also called the mobile phase) is a highly pure and relatively inert gas such as helium, hydrogen, nitrogen, argon, or air. In a conventional system, the carrier gas 20 is passed through the column 30 simultaneously with the sample of the fluid being analyzed (the separation method). Using a sample injector 22, a given volume of a test mixture including one or more target analytes (e.g., in gaseous form) is introduced into a column 30, mixing it with a carrier gas from a carrier gas stream 20. Thus, the carrier gas stream 20 and a sample 22 (potentially containing one or more target analytes) are introduced into one or more chromatographic columns 30. The sample 22 moves through the device together with the carrier gas supplied as the carrier gas stream 20.

The target analytes of the sample 24 are separated and transported through the column 30 and then eluted therefrom. It should be noted that the eluted sample containing one or more target analytes may be eluted from the column 30 in partial fractions depending on the retention time of the corresponding target analyte, as the substances pass through the chromatographic column 30 and are separated therein. In addition, fractions of the sample exiting the column 30 may optionally be captured and reintroduced downstream.

Typically, a chromatographic column 30 performs separation because the interior surfaces of the column are coated with a material (or the interior of the column is filled with a material) that serves as a stationary phase. The term "column" broadly includes various flow channels through which fluids can flow, such as a structured flow field consisting of microelements formed in one or more substrates, or other fluid flow channels known to those skilled in the art. The stationary phase adsorbs various target analytes contained in a mixed sample to varying degrees. The difference in the degree of adsorption causes differences in retention times and, therefore, differences in the mobility of the various chemical species as they move through the column, resulting in physical separation of the target analytes in the mixed sample. In some embodiments, the chromatographic column is a gas microanalysis chromatographic column. As used herein, the term "microscale" refers to a structure having at least one dimension that is less than about 500 μm, possibly less than about 400 μm, possibly less than about 300 μm, possibly less than about 200 μm, possibly less than about 150 μm, and in some embodiments, possibly less than about 100 μm, and may also include nanoscale features. Reference herein to a microscale, a microchannel, a microfluidic channel, or a microstructure includes smaller sized structures, such as nanoscale structures. It should also be noted that while one dimension, such as a diameter, may be in the microscale range, other dimensions, such as a length, may exceed the microscale range.

The various separated components elute from the column 30 and are directed to one or more sensors 32 for analysis. Thus, one or more sensors 32 are located at the end of one or more columns 30. Thus, the sensor 32 serves to determine various chemical substances or target analytes in the sample released or eluted from the column 30 at different times. In a gas chromatographic system, the sensor 32 typically performs a destructive analysis of the eluted fractions. Typical non-limiting examples of sensors 32 include a mass spectrometer (MS) (e.g., a time-of-flight mass spectrometer (TOFMS)), a flame ionization sensor (FID), a photoionization sensor (PID), an electron capture detector (ECD), thermal conductivity detectors (TCDs) or similar sensors. The data processing system 34 is typically in communication with the sensor 32 and thus typically can store, process, and record the results of the separation analysis.

In coated capillary chromatography columns, analyte retention is achieved by interaction of the gas phase vapor with the stationary phase coated on the capillary wall. As the analytes migrate along the column, they undergo longitudinal and transverse mass transfer, which results in peak broadening, which reduces GC resolution and increases the likelihood of co-elution. Typically, proper choice of the stationary phase in the chromatography column (ensuring sufficient interaction and retention of the analyte), application of programmable temperature profiles, and splitless/splitless sample injection can improve separation and resolution in the chromatograph. In some cases, however, these methods do not achieve the desired separation. For example, in portable GCs, limited carrier gas supplies prevent splitting, and precise control of temperature programming is difficult and limited by system capacity. Furthermore, even for specialized separations (e.g., separation of highly volatile compounds in open tubular porous bed columns), complete separation of the full spectrum of target compounds can be challenging. Thus, it is desirable to create an additional way to improve the separation in columns.

Negative temperature gradient separation (NTGS) is one of the methods used to improve the performance of a column by sharpening the elution peaks. In NTGS, the column inlet is heated and a temperature gradient is created by heat exchange with the environment. Since the temperature decreases closer to the column outlet, the peak front moves slower than its tail, which leads to an overall focusing of the peaks. This effect can be optimized by adjusting different temperature profiles along the column, which provides high versatility in different conditions. However, due to the dependence of NTGS on heat exchange, the focusing varies depending on the ambient temperature, humidity, air convection velocity and thermal conductivity of the packing, which reduces reproducibility and predictability (in particular, in the case of complex temperature profiles). Sophisticated temperature control modules can be used to stabilize the temperature gradient, but this leads to an increase in the size, weight, complexity and cost of GC devices. In addition, energy losses due to this heat exchange are a significant drawback of resource-constrained systems (e.g., micro GC devices). In addition, separation of highly volatile compounds often needs to be carried out at ambient temperatures, so that no or minimal temperature gradient is created, which reduces the effect of RTGS. Thus, despite the versatility and controllability of the RTGS method, its application in some areas (e.g., in portable GC) may be limited and associated with difficulties.

The present invention relates to a new method for focusing peaks in gas chromatography. In some aspects, a gas chromatography device that ensures focusing of peaks of one or more target analytes includes a chromatographic column with an inlet and an outlet. A sample including one or more target analytes is fed to the inlet, which exits the column through an outlet. A stationary phase is placed or deposited inside the chromatographic column and has a positive thickness gradient. The stationary phase extends from the inlet to the outlet and has a first thickness at the inlet of the chromatographic column and a second thickness at the outlet of the chromatographic column. As described in more detail below, the second thickness exceeds the first thickness by at least about 10%. Thus, a positive thickness gradient of the stationary phase is a thickness gradient in which the thickness of the stationary phase film increases from the inlet toward the outlet. As the thickness of the stationary phase increases toward the outlet, the peak front moves more slowly than its tail, which generally leads to focusing of the peaks.

Fig. 2 shows an example of a chromatographic column 50 prepared in accordance with some aspects of the present invention, in which focusing of peaks of one or more target analytes is possible. In some aspects, one or more target analytes may be volatile organic compounds (VOCs) that have a relatively high vapor pressure and, therefore, are low boiling at the temperatures and pressures used in the chromatographic column 50. Volatile organic target analytes may include alkanes or aromatic compounds.

The chromatographic column 50 is limited by a wall 60. Thus, the wall 60 of the chromatographic column 50 can limit a structure having a hollow internal region 52, which can be at least partially filled with a stationary phase 64. The cross-sectional shape of the chromatographic column 50 can be selected from the group consisting of a round, oval, rectangular and triangular shape. In some aspects, the chromatographic column 50 is made of metal, silica or glass, or a polymer. The chromatographic column 50 has an inlet 70 and an outlet 72. As shown in Fig. 2, the wall 60 has a constant thickness. Thus, the first thickness of the wall 60 at the inlet 70, designated "T ₁ ", is equal to the second thickness, designated "T ₂ ", at the inlet 72.

As noted above, a mixture of one or more target compounds in the sample/carrier mobile phase interacts with the stationary phase 64 as the mixture passes through the column. Each target analyte interacts with the stationary phase 64 to a different extent or at a different rate. The analyte that interacts the least with the stationary phase 64 exits or elutes from the chromatographic column 50 first. Typically, the target analytes that interact the most with the stationary phase 64 pass through the column 50 at the slowest rate and thus elute last. Varying the characteristics of the mobile phase (carrier and sample) and the stationary phase 65 allows for the separation of different mixtures of target analytes. As shown below, according to the present invention, the stationary phase 64 has a positive thickness gradient that allows for the focusing of the peaks of one or more target analytes, and this typically means that the analyte peak is focused as the analyte passes from the inlet 70 to the outlet 72 of the chromatographic column 50.

In some embodiments, the stationary phase 64 may include silicon, such as in the form of silica or a siloxane polymer. The stationary phase 64 may be prepared from various siloxane-based polymers. Typically, the siloxane polymer is a cross-linked polymer whose backbone consists of silicon and oxygen, having pendant groups that may be the same or different, which is typically described by the structural repeating unit (-O-SiRR'-) _n , where R and R' may be the same or different pendant groups, and "n" may have any value greater than 2. The surface of silica or siloxane can be functionalized by reacting monomers or polymers with various short-chain organic silanes, resulting in reactions of silanol groups. In this case, the mechanism of retention of target analytes remains the same, but differences in the surface chemistry of the different stationary phases create differences in selectivity with respect to various target analytes. Siloxane polymers may include polyheterosiloxanes that may contain various pendant groups or repeating units. An example of suitable pendant groups may be at least one unsubstituted or substituted alkyl group or aryl group comprising from 1 to 30 carbon atoms, examples of which include, but are not limited to: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, phenyl, alkylphenyl and the like groups.

In various aspects, the stationary phase 64 is supported on the inner surface 66 of the wall 60 and has a positive thickness gradient. In some embodiments, as described in more detail below, the inner surface 66 can first be silanized, such as by exposing the inner surface 66 to hexamethyldisilazane (HMDS) vapor, resulting in a surface coating comprising a silane or silanol. A siloxane polymer precursor, such as Silicone OV-1, a vinyl-modified 100% dimethyl silicone commercially available from Ohio Valley Specialty Company under the designation OV-1 6001, or Silicone OV-17, a vinyl-modified 50% phenyl silicone and 50% methyl silicone commercially available from Ohio Valley Specialty Company under the designation OV-1 6017, can then be introduced and reacted to form the stationary phase. Along with the precursor, a crosslinking agent such as Dow SYLGARD™ 184 Reagent B (15% by weight, crosslinking agent) may also be added. It should be noted that the choice of stationary phase material 64 is not limited to the listed materials, which are given here only as suitable examples of materials for the manufacture of a gas chromatographic column.

As shown, the stationary phase 64 extends along the inner surface 66 of the wall 60 from the inlet 70 to the inlet 72. Thus, the stationary phase 64 forms a continuous coating or film on the inner surface 66. At the inlet 70, the stationary phase 64 has a first thickness, designated "t ₁ ". At the outlet 72, the stationary phase 64 also has a second thickness, designated "t ₂ ". The second thickness (t2) is at least approximately 10% greater than the first thickness (t ₁ ). Thus, the stationary phase 64 has a positive thickness gradient along the length of the chromatographic column 50. As a result, the internal region 52 has a first diameter "d ₁ ", determined by the first thickness (t ₁ ) of the stationary phase 64 at the inlet 70. The internal region 52 has a second diameter "d ₂ ", determined by the second thickness (t ₂ ) of the stationary phase 64 at the outlet 72. The first diameter (d ₁ ) is greater than the second diameter (d ₁ ). Thus, the stationary phase 64 located in the chromatographic column 50 has a positive thickness gradient.

In some aspects, the thickness gradient gradually increases along the length of the chromatographic column 50 from a first thickness (t ₁ ) to a second thickness (t2). The change in thickness of the stationary phase 64 may be constant or vary along the length of the chromatographic column 50, but the thickness increases from the inlet 70 to the outlet 72.

In some embodiments, the second thickness (t ₂ ) is at least about 50% greater than the first thickness (t ₁ ), possibly at least about 100% greater than the first thickness (t ₁ ), possibly at least about 150% greater than the first thickness (t ₁ ), possibly at least about 200% greater than the first thickness (t ₁ ), possibly at least about 250% greater than the first thickness (t ₁ ), and in some embodiments, the second thickness (t ₂ ) is possibly at least about 300% greater than the first thickness (t ₁ ). The first thickness (t ₁ ) can optionally be from a value greater than or equal to about 10 nm to a value less than or equal to about 10 micrometers, and the second thickness (12) can be from a value greater than or equal to about 30 nm to a value less than or equal to about 30 micrometers. In one example, the first thickness (t ₁ ) is approximately 30 nm and the second thickness (t ₁ ) is approximately 30 micrometers.

3A-3C illustrate peak focusing by using a film with a positive thickness gradient according to the present invention, performed in a column containing a film with a thickness gradient (FGT) according to some aspects of the present invention. As can be seen from Fig. 3A, a peak 82 of a certain analyte 80 becomes more and more focused as the analyte 80 moves along a column 84 containing a stationary phase 86 with a positive thickness gradient, passing from an inlet 88 to an outlet 90 of the column 84. Thus, the film of the stationary phase 86, the thickness of which changes from thinner to thicker (in the direction from the inlet 88 to the outlet 90), focuses the analyte peak 82 as the analyte moves along the column in the direction of the shaped arrow, as shown in Fig. 3A. Fig. 3B illustrates a setup for evaluating the performance of a column. Column 84 is placed in fluid communication with injector 94 of benchtop GC 96 Agilent 6890 equipped with flame ionization detector (FID) 98. In Fig. 3C, forward (top) and reverse/inverted (bottom) operating modes are shown. In other words, sample containing at least two target analytes is introduced in forward mode (top) through inlet 88, after which it moves toward outlet 90 of chromatographic column 84 in the direction of the arrow. In reverse or inverted mode (bottom), sample containing at least two target analytes is introduced at outlet 90 of chromatographic column 84, after which it moves toward inlet 88 in the direction of the arrow.

In some aspects, the present invention relates to a method for focusing peaks in a gas chromatography device. The method may include introducing two or more target analytes at the inlet of a chromatographic column, including a stationary phase arranged within the chromatographic column, having a positive thickness gradient. The stationary phase extends from the inlet to the outlet and has a first thickness at the inlet of the chromatographic column, and has a second thickness at the outlet of the chromatographic column. The second thickness is at least about 10% greater than the first thickness. The stationary phase may be any stationary phase described above. The method includes separating two or more target analytes in the chromatographic column. Additionally, the two or more target analytes are then eluted from the outlet of the chromatographic column.

In some aspects, two or more target analytes are volatile organic compounds (VOCs). According to one aspect, at least one of the two or more target analytes comprises an aromatic compound, and the resulting aromatic compound peak focusing index is greater than or equal to about 25%, such as may be greater than or equal to about 26%, optionally greater than or equal to about 27%, and in some embodiments, possibly greater than or equal to about 28%.

According to another aspect, at least one of the two or more target analytes comprises an alkane, and the resulting alkane peak focusing index is greater than or equal to about 10%, and in some aspects, possibly greater than or equal to about 11%.

According to other aspects, the present invention relates to a method for confirming the focusing of peaks in a gas chromatography device. The method includes performing a direct operation by introducing two or more target analytes at the inlet of a chromatographic column including a stationary phase deposited inside this chromatographic column having a positive thickness gradient. The stationary phase extends from the inlet to the outlet and has a first thickness at the inlet of the chromatographic column and a second thickness at the outlet of the chromatographic column, where the second thickness exceeds the first thickness by at least about 10%. The stationary phase can have any of the structures and compositions described above. The direct operation includes separating two or more target analytes in the chromatographic column and then eluting the two or more target analytes from the outlet of the chromatographic column. Then, an inverse operation is performed, including introducing two or more target analytes at the outlet of the chromatographic column including the stationary phase. The inverse operation further includes separating two or more target analytes in the chromatographic column and eluting the two or more target analytes at the outlet of the chromatographic column. The method further comprises comparing the chromatographic resolution values of the direct operation and the reverse operation, where the peak focusing index of at least one corresponding pair of two peaks of two target analytes exceeds 5% and is possibly greater than or equal to approximately 10%.

The present invention also relates to a method for creating a gas chromatography device comprising a chromatographic column with a positive thickness gradient. The method comprises introducing a precursor liquid into the chromatographic column. The precursor liquid comprises a stationary phase precursor and a low-boiling solvent that evaporates as it moves along the chromatographic column, so that the concentration of the stationary phase precursor increases as it moves along the column, which results in the creation of a stationary phase thickness gradient. The method also comprises a reaction of the stationary phase precursor or the formation of cross-links in the stationary phase precursor, which results in obtaining a positive stationary phase thickness gradient along the length from the inlet to the outlet, so that the stationary phase has a first thickness at the inlet of the chromatographic column, and has a second thickness at the outlet of the chromatographic column. The second thickness exceeds the first thickness by at least approximately 10%.

In some aspects, prior to introducing the precursor liquid, the interior surface of the chromatography column is silanated. Silanating may include passing a reactive silane in gaseous form through the column. In one example, the reactive silane may be hexamethyldisilazane (HMDS) vapor that forms a silane-containing surface coating on the interior surface of the column. In some embodiments, the reactive silane may be passed through the chromatography column multiple times.

A siloxane polymer precursor such as Silicone OV-1, a vinyl-modified 100% dimethyl silicone available from Ohio Valley Specialty Company as OV-1 6001, or Silicone OV-17, a vinyl-modified 50% phenyl silicone and 50% methyl silicone available from Ohio Valley Specialty Company as OV-1 6017, may be added and reacted to form the stationary phase. A cross-linking agent such as Dow SYLGARD™ 184 Reagent B (15 wt.% cross-linking agent) may also be added with the precursor. It should be noted that the choice of stationary phase material is not limited to the listed materials, which are provided herein merely as suitable examples of materials for the manufacture of a gas chromatographic column.

The chromatographic column includes an inlet and an outlet, and the introduction and reaction or cross-linking may involve dynamic coating of the interior surface of the chromatographic column. This may be accomplished by partially filling the chromatographic column with a precursor liquid, applying pressure at the inlet to force the precursor liquid down the length of the column, and applying a vacuum at the outlet to evaporate the low boiling solvent. For example, a plug of precursor liquid may be forced through the column by applying a pressure of 5 psi (34473.8 Pa) at the inlet. A vacuum pressure of 2 psi (-13789.5 Pa) may be applied at the outlet to evaporate the low boiling solvent.

Examples

Gas chromatography (GC) columns with a stationary phase thickness gradient allow focusing of analyte peaks and improve separation resolution. Theoretical analysis and modeling show that focusing occurs at a positive thickness gradient, i.e. at a stationary phase thickness that increases along the column. Peak focusing was confirmed experimentally by coating a 5 m long capillary column with a film thickness ranging from 34 nm at the column inlet to 241 nm at the column outlet. The column was tested with C ₅ to C ₁₆ alkanes and aromatics in the forward (from smaller to larger thickness) and reverse (from larger to smaller thickness) modes, and the results were compared with those obtained in a column with a uniform coating thickness of 131 nm.

Comparison of the resolution values obtained in the direct mode and in a uniform column thickness shows that the overall focusing gain (i.e. increase in peak capacity) is 11.7% for alkanes and 28.2% for aromatics.

The focusing effect was also demonstrated for isothermal separations of highly volatile compounds at room temperature and for temperature programmed separations with different rates of temperature increase. In all cases, the peak capacities in direct separation modes were higher than in other modes, indicating that the positive thickness gradient facilitates the focusing of analyte peaks. Thus, the present thickness gradient technique can be widely applied to various stationary phases and column types as a general method for improving GC separation performance.

Experimental setup A column containing a film thickness gradient (FTG) was mounted on an Agilent 6890 GC stage equipped with a flame ionization detector (FID, see Fig. 3B). Ultrahigh purity helium was used as a carrier gas. To evaluate the peak focusing effect, analytes were injected from the end with a thinner coating (forward mode, i.e., moving from a thinner to a thicker film) or from the end with a thicker coating (reverse mode, i.e., moving from a thicker to a thinner film), as shown in Fig. 3C. For comparison, a column with a uniform coating thickness (the film thickness is equal to the average thickness) was also studied on the same setup. All experiments were carried out at a programmed temperature and constant pressure. The methods of programming the temperature and injection pressure are presented in Table 1.

Materials

C ₅ -C ₁₆ hydrocarbons, benzene, toluene, ethylbenzene, ortho-xylene, 1,3-dichlorobenzene, nitrobenzene, and dichloromethane of analytical grade were purchased from Sigma-Aldrich (St. Louis, MO). Vinyl-modified OV-1 (P/N 6001) and OV-17 (P/N 6017) were purchased from Ohio Valley Specialty Company (Marietta, OH). Dow SYLGARD™ 184 Reagent B was purchased from Ellsworth Adhesive (Germantown, WI). Deactivated fused silica tubing (cat. no. 10010, 250 μm i.d.) and an RTX-5 column (cat. no. 10205, cut to 5 m length, 250 μm i.d., and 0.1 μm film thickness) were purchased from Restek (Bellefonte, PA). A DB-1MS column (cat. no. 122-0162, cut to 5 m length, 250 μm i.d., and 0.25 μm film thickness) was purchased from Agilent (Santa Clara, CA). All materials were used as received without further purification or modification.

Column Coating Reagents OV-1 (75% w/w), OV-17 (10% w/w) and Dow SYLGARD™ 184 Reagent B (15% w/w, crosslinking agent) were dissolved in dichloromethane to obtain a 2% w/w coating solution (phenyl content in the stationary phase 5%). As shown in Fig. 4A, a 5 m long capillary column 100 (internal diameter 250 μm) was silanized by 8 repeated injections of hexamethyldisilazane (HMDS) vapor before coating. Then, 80 μl of coating solution 102 were injected into the capillary 100 through the column inlet 104 using a syringe pump. To move the coating solution 102 toward the outlet 108, a positive pressure 106 of 5 psi (34473.8 Pa) was applied at the inlet 104. At the outlet 108, a negative vacuum pressure 110 of -2 psi (-13789.5 Pa) was applied through a 1 m long blank column 112 (internal diameter 250 μm), which ensured a constant plug velocity of the coating solution. During coating, a small volume of low-boiling dichloromethane evaporates under vacuum, causing the concentration of the coating solution to gradually increase and hence the film thickness to increase as the plug 102 of the coating solution moves from the inlet 104 of the column to the outlet 108. After coating, dry air is continuously passed through the column 100 for 2 hours, followed by a cross-linking reaction at 80°C for another 2 hours and subsequent deactivation with HMDS. The column 100 is then maintained at 230°C for 3 hours under a helium flow of 0.5 ml/min. In the same manner, the column is coated to a uniform film thickness using a 1 wt% coating solution (of the same composition as the solutions used above but at a higher dilution) and applying a positive pressure of 5 psi (34473.8 Pa) at the inlet to force the coating solution toward the outlet (without applying a vacuum).

Simulation conditions

The separation of a C ₈ -C ₁₅ mixture was simulated in the forward and reverse modes, as well as in a column with a uniform thickness equivalent to the average film thickness gradient (the separation conditions are presented in Table 1). The film thickness in the 5 m column varied from 34 nm to 241 nm (from the inlet to the outlet in the forward mode and vice versa in the reverse mode). It should be noted that to calculate the retention factor k(x, t), knowledge of the distribution coefficient K(t) (Eq. (2)) is required, which is calculated from the known values. The simulated retention times and full width at half maximum peak height (FWHM) are presented in Table 2, and the resolution values are presented in Table 3.

Uniform Coating Control Column A Restek RTX-5 column was used as a control for the separation of C ₇ -C ₁₆ alkanes in forward and reverse modes, with no expected differences in separation. The separation conditions are presented in Table 1. The p-values for retention time and FWHM (from 5 runs) were calculated using a paired Student's t-test and converting the resulting t-score to a p-value. Significance was set at p = 0.05; no significant differences were observed between forward and reverse modes for all analyte peaks. Similarly, no significant differences were observed between forward and reverse modes when a 5 m Agilent DB-1MS column was used to separate C ₇ -C ₁₅ alkanes (data not shown).

Characterization of the Stationary Phase To determine the thickness of the stationary phase, KPGT was frozen in liquid nitrogen and then several pieces were broken off. Scanning electron microscope (SEM) images were taken near the column inlet (thinner film) and near the column outlet (thicker film). Figures 4(B) and (C) show that the film thickness increases from 34 nm to 241 nm along the column from inlet to outlet, and the gradient is approximately 41 nm/m. A column with uniform film thickness was also examined at the inlet and outlet, and it was found that the film thickness was 131 nm at both ends of the column.

The theoretical basis for peak focusing is given below. Effective speed, analyte in section x (distance from the column inlet) at a given time t is determined by the expression:

Where represents the velocity of the mobile phase, and - retention factor:

distribution coefficient determined from the expression

Where - universal gas constant, and represents the time-dependent temperature of the column at the site represents the change in Gibbs free energy associated with the transition of the analyte from the stationary to the mobile phase, which can be calculated from the change in enthalpy and entropy analyte:

The phase relation β is determined by the expression

Where - the internal diameter of the column and the film thickness, respectively.

Thus, Eq. (2) can be expressed as follows:

where A is a constant for a given column. The retention factor over the length of the column, can be written as follows:

Equation (7) shows that with a partial increase in the retention factor, at a distance along the length of the column, two parameters contribute: a negative temperature gradient, given by the first variable, and a positive film thickness gradient, given by the second variable. Retention factor gradient is related to the velocity gradient by equation (1); thus, both the negative temperature gradient and the positive film thickness gradient result in a velocity difference between the front and tail of the analyte band, which contributes to band focusing (i.e., the spatial distribution of the analyte is affected by the spatially varying velocity gradient). At the outlet during elution, the band appears as a time-varying peak, which may be narrower than the corresponding peak of the unfocused band. In other words, peak focusing (an observed quantity) is the result of band focusing within the column. The equivalence of these two gradients can be expressed as follows:

For peak focusing, the film thickness gradient has several advantages over the traditional temperature gradient. First, the film thickness gradient is independent of the column temperature, which allows for peak focusing of analytes of any volatile compounds at any operating temperature. In particular, peaks of highly volatile compounds are difficult to focus in negative temperature gradient (NTG) separations, but this can be done using the technique of the invention (e.g., using a chromatographic device and a FTIG method). Second, while the temperature gradient can be varied by using a heater and conventional conditions (such as heater placement, heat dissipation, column size/weight, column channel location, ambient temperature, and air flow), the film thickness gradient is always constant and allows for more reliable and reproducible GC operations (due to less exposure to environmental influences). Finally, FTIG according to some aspects of the present invention can be used without additional ancillary equipment (such as heaters or coolers required for FTIG), which significantly reduces the complexity of the device for further integration. However, despite these advantages, in some respects, film thickness gradient separation may be less versatile than ROTG, since here the gradient is fixed, while the temperature gradient can be changed by changing the heat source and/or removing heat. In addition, increasing film thickness towards the column outlet may slow down the mass transfer, which may distort the peak focusing effect. The mass transfer effect was investigated in the simulations below.

In this simulation, the temperature gradient (i.e., RTTG) was not taken into account, and only the effect of the film thickness gradient was analyzed. The time-dependent peak concentration of the analyte moving along the column is determined by solving the convection-diffusion equation for the transition state:

Where defined in Eq. (1). Effective diffusion can be calculated from the values of local dispersion D and retention factor

Where - film thickness, and - diffusion constant of the mobile phase. It should be noted that D includes mass transfer/diffusion in both longitudinal and transverse directions. The value can be expressed as follows:

Where is the diffusion constant (which depends on the molar masses and atomic and structural diffusion volumes of the analytes and mobile phase molecules), and is the diffusion constant of the stationary phase. Local pressure determined based on pressures at the input and output, respectively:

where L is the length of the column. The velocity in the mobile phase is is described by the formula

where is viscosity is given as a function of the reference viscosity at temperature and the indicator depending on the type of gas:

It should be noted that in Eq. (12) the temperature is given in the form in accordance with the assumption that at a given time t the temperature remains the same along the entire length of the column. Equation (9) can be solved by applying a finite difference model to a discrete time instant (t) and radius vectors (i)

Where - the simulated distance and time step. Combining these equations yields:

The solution to Eq. (19) is the time-dependent movement of the analyte peak along the column.

To model Eq. (19), it is necessary to establish some boundary conditions. First, at time t=0, the peak of the injected analyte has the shape of a Gaussian peak, i.e.:

where σ is the initial dispersion. It should be noted that the initial peak at time t = 0 is at x = 3σ. After the initial introduction through the column inlet, no additional analytes are introduced into the column:

At the column outlet, the final concentration on the grid is approximately equal to the concentration on the left-hand side (since it cannot be calculated using equation (16)), i.e.:

The peak retention times and full widths at half maximum (FWHM) can be determined at the column outlet (i.e., at x = L) because the spatially varying concentration given by Eq. (19) is used to construct a two-dimensional concentration matrix in which both position and time vary. For example, the separation of C ₈ -C ₁₅ compounds can be simulated 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. As a control experiment, simulations were also performed in a column with uniform film thickness (at a thickness equivalent to the average value for the film thickness in the forward/reverse mode). The temperature was increased from 40 °C to 240 °C at a rate of 30 °C/min. The injection pressure was set to 3.45 psi (23787 Pa) (the outlet pressure was equal to the ambient pressure, i.e. 1 atm (1.013⋅10 ⁵ Pa)). The film thickness in the 5 m column was varied from 34 nm to 241 nm (from inlet to outlet in the forward mode and vice versa in the reverse mode). Monitoring the concentration along the second axis (i.e. time) allows determining the time-varying concentration vector corresponding to the signal received from the outlet sensor. The maximum value (varying with time) corresponds to the elution/retention time, and the FWHM can be determined from the time at which the concentration is half the peak value. Additionally, the resolution (R) between adjacent peaks can be calculated using the following formula:

Where - the retention times of the two peaks, and - corresponding to the PSHPM.

The peak retention times and full width at half maximum (FWHM) measured at the column outlet (i.e. at x=L) are presented in Table 1. Table 1 presents the programmed temperature and injection pressure profiles used in the simulation of the uniform film thickness control experiment, the separation of _C7 - _C16 alkanes, the separation of aromatic compounds, and the separation of highly volatile alkanes ( _C5 and _C6 ).

Table 2 presents the simulated retention times (RT) and full widths at half maximum (FWHM) for the _C8 - _C15 separation in the forward and reverse modes. The RT and FWHM values for a uniform coating thickness are also presented for comparison. The temperature was ramped up from 40°C at 30°C/min and the injection pressure was 3.45 psi (~23,787 Pa). The column length was 5 m. All values are presented in minutes. Additional analysis is provided in Table 3.

Table 3 presents the values of resolution (R) between adjacent peaks obtained in the simulation for C ₈ -C ₁₅ mixtures in the forward and reverse modes and in a column with a uniform coating thickness. The resolution values obtained in the forward mode exceed the resolution values obtained in the reverse mode and in a column with a uniform coating thickness. The difference in resolutions was determined using the formula

From the data presented in Table 1, it is evident that the analyte peaks in the forward and reverse modes emerge from the column at different times; this is because the separation conditions for a given analyte are different in the two modes. In the forward mode, the analyte first interacts with the thinner film at low temperatures and reaches the thicker film at high temperatures, while in the reverse mode, the opposite occurs. As a result, the retention times of the analytes in the two modes are different, and the full widths at half maximum (FWHM) of the peaks cannot be compared directly when evaluating column performance. Instead, the resolution R between the two peaks, calculated using Equation 23, is used to analyze the separation performance (see Table 2).

From the data presented in Table 2, it is evident that, compared to the reverse mode, the resolution between adjacent peaks is higher in the forward mode, i.e., during the same time interval, more peaks can be obtained in the forward mode than in the reverse mode. The resolution values in the column with uniform film thickness exceed the resolution values in the reverse mode, but are always lower than the resolution values in the forward mode, which thus proves the focusing of the peaks in the forward mode. Further analysis of the resolution values of adjacent peaks shows that the difference in resolution values between the forward and reverse modes (i.e. ) decreases as the retention time of the analyte increases. This may be due to slower mass transfer (i.e., lateral diffusion), which is more pronounced for heavier compounds, in the region of the thicker film. In the forward mode, this effect results in peak broadening for less volatile compounds closer to the column outlet, reducing the focusing provided by the column. In contrast, in the reverse mode, the thinner film at the outlet provides less broadening, which reduces defocusing due to the reverse gradient. Thus, for lower volatility compounds, the resolution difference between the forward and reverse modes is reduced.

Focusing of peaks of alkane mixture. The peak focusing capabilities of the CPGT were assessed by separating a mixture of _C7 - _C16 alkanes. 0.025 μl of liquid was used for injection at a flow split ratio of 5:1. The same separation conditions were created in the direct mode, in a column with a uniform film thickness, and in the reverse mode (which were designated as "identical parameters of the reverse mode", see Table 1 - alkane mixtures). Examples of chromatograms are shown in Figs. 5A - 5D.

It is evident from Fig. 6 that in the forward mode, in the uniform film thickness column and at identical parameters in the reverse mode, the analyte peaks are eluted after different time intervals, which is consistent with the modeling data (Table 2). This is due to the fact that the separation conditions of a particular analyte in the two specified modes are different, and they also differ from the conditions in the uniform film thickness column. In the forward mode, the analyte first interacts with the thinner film at low temperatures and then reaches the thicker film at high temperatures; in the reverse mode, everything happens vice versa. In the uniform film thickness column, the analytes interact with the uniform film thickness at all temperatures. As a result, the retention times of the analytes in the two specified modes and in the uniform film thickness column are different, and it is not possible to make a direct comparison of the full width at half maximum (FWHM) of the peaks when evaluating the performance of the column. In contrast, the resolution values between adjacent peaks (e.g., C ₇ and C ₈ , C ₈ and C ₉ , etc.) are used to analyze separation characteristics. The resolution values for identical parameters in the reverse mode and in a column with uniform film thickness are subtracted from the corresponding resolution values in the forward mode; the resolution differences between all pairs of adjacent peaks (averaged over 5 experiments) are plotted in Fig. 6. The p-values for the difference in resolution were calculated using the paired Student's t-test (over 5 experiments in forward and reverse mode with identical parameters and in a column with uniform film thickness) and converting the resulting T-score into a p-value (see Table 3). Significance was taken at the p=0.05 level, indicating a much higher resolution between all pairs of adjacent peaks in forward mode compared to reverse mode with identical parameters. This is consistent with the modeling data (Table 3) and is also confirmed by the above-mentioned resolution values in forward mode, meaning that more peaks are obtained in forward mode during the same time interval than in reverse mode. The uniform column resolution values are lower than the direct mode resolution values up to the C ₁₀ /C ₁₁ mixture, but for the C ₁₅ /C ₁₆ pairs the uniform column resolution is higher. A general analysis of the performance characteristics is given below.

Table 4 presents the p-values obtained in comparing the separation of _C7 - _C16 alkanes in the forward mode, in a uniform film thickness column, in the reverse mode under identical parameters (IP), and in the reverse mode for the same time (RT). Significance was accepted at the p-value level of 0.05. All p-values were significant in the comparison of the forward and reverse IP modes, while the p-values obtained in the separation of _C7 -C13 were significant for the reverse RT mode. The resolution values in the forward mode significantly exceeded the resolution values in the uniform film thickness column up to the _C10 / _C11 mixture, while the resolution in the uniform film thickness column was significantly higher for the _C15 / _C16 mixture.

To further analyze the discrepancy in retention times, a second series of chromatograms was obtained at a lower rate of temperature increase in the reverse mode, which was reduced so that C ₁₆ (the last of the eluting analytes) was eluted at the same time as in the forward mode (this is designated as "reverse mode for equal time", see Table 1 - separation conditions, chromatograms in Figs. 5A - 5D, resolution differences in Fig. 6, Table 4 - p values). In this case, again in the forward mode, the resolution in separating alkane pairs in the C ₇ -C ₁₃ range (results obtained in 5 experiments) was significantly higher, however, in the reverse mode for equal time, the resolution in separating C ₁₃ -C ₁₆ was close to the resolution in the forward mode. Although in the forward mode not all local resolution values (i.e. between adjacent pairs of alkanes) exceed the corresponding resolution values in the reverse mode for an equal time (or separation in a column with a uniform film thickness), in the forward mode a significantly higher peak capacity (PC), defined as the sum of all resolution values (Table 5), is achieved compared to all other modes. Analysis of the focusing index (FI), defined as

shows that the overall focusing performance in the forward mode compared to a column with uniform film thickness, the reverse modes at identical parameters, and the reverse mode for equal time are approximately 11.7%, 26.8%, and 29.8%, respectively.

Table 5. Figures 5A-5D show the peak capacities, overall resolution values, p values, and focusing indices obtained when comparing the separation of _C7 - _C16 alkanes in the direct mode with the separation in the reverse modes with identical parameters (IP and equal time (ET)) and in a column with a uniform film thickness. Significance was taken at the p=0.05 level. The peak capacity in the direct mode significantly exceeds the peak capacity in all other modes.

Peak Focusing in Aromatic Mixture Analysis Peak focusing in KPGT was also analyzed using the example of separation of aromatic mixture containing benzene (B), toluene (T), ethylbenzene (E), ortho-xylene (X) and 1,3-dichlorobenzene (DCB). The liquid mixture in an amount of 0.025 μL was injected at a split ratio of 5:1 (the separation conditions are given in Table 1 - aromatic mixture). Examples of chromatograms are shown in Figs. 7A-7D, and the resolution differences are shown in Fig. 8. The p values for the local resolution difference (calculated for 5 experiments) are presented in Table 6. Table 7 presents the peak capacities, p values and focusing indices, which demonstrate that the peak capacity in the direct mode is significantly larger than the peak capacity in all other modes. Thus, whether the separation parameters are kept constant (and the analytes elute faster in the reverse mode with identical parameters) or are varied so that the last compound is eluted at the same time (in the forward and reverse modes for the same time), the separation in the forward mode is always better than in any of the reverse modes. The separation in the forward mode is also better than in a column with a uniform film thickness, as confirmed by the focusing index of 28.2% (Table 7). Thus, overall, it is shown that the use of the forward mode (i.e. a positive film thickness gradient) allows for an improvement in the peak capacity of the separation.

Table 6 presents the p-values obtained when comparing the separation of aromatic compounds in the forward mode with their separation in a column with uniform film thickness, in the reverse mode with identical parameters (IP), and in the reverse mode for equal time (RT). The elution order and reductions are shown in Figs. 7A-7D. Significance was taken at the p-value of 0.05. All p-values indicate a significant improvement in resolution in the forward mode.

The peak capacities, p-values and focusing indices obtained when comparing the separation of aromatic compounds in the direct mode with their separation in the reverse modes with identical parameters (IP) and equal time (RT) and in a column with a uniform film thickness are presented in Table 7 and in Figs. 7A - 7D. Significance was taken at the level of p = 0.05. The separation in the direct mode is significantly superior to the separation in all other modes.

Effect of rising temperature

To determine the effect of the rate of temperature increase on peak focusing, a mixture of C ₇ -C ₁₀ compounds was separated at four different rates of temperature increase (0, 10, 20, and 30 °C/min, ramping from 60 °C without delay) in the forward mode, in the reverse mode under identical parameters, and on a uniform film thickness column. The pressure was 3.45 psi (23787 Pa) (2.7 mL/min at 60 °C) and the split ratio was 15:1 for all separations (0.025 μL injection of liquid mixture). Methane was injected to determine the escape time of the unretained component and was found to be 0.36 min at all rates of temperature increase. The resolution and focusing values for each temperature profile are presented in Table 8 (values are averages of 5 experiments). In the forward mode, at higher rates of temperature increase, the analytes are exposed to relatively high temperatures by the time they reach the thicker stationary phase closer to the column outlet. Thus, the analyte is retained for a shorter time in the thicker film, resulting in reduced peak broadening. In the reverse mode, the analytes first interact with the thicker stationary phase at lower temperatures and then migrate to the thinner stationary phase at higher temperatures. The peak broadening due to the thinner stationary phases is already small; thus, in the reverse mode, the overall reduction in peak broadening due to the temperature increase is lower. Thus, the focusing efficiency increases with increasing rate of temperature increase, up to 61.9% for the forward versus reverse mode comparison and up to 68.1% for the forward versus uniform column mode comparison, at a temperature increase rate of 30 °C/min.

Table 8 presents the values of resolution (R), peak capacities (PC), and focusing indices obtained in the separation of _C7 - _C10 compounds in the direct mode, reverse modes, and in a column with uniform film thickness at different rates of temperature increase. In all separation experiments, the initial temperature was 60°C and the carrier gas injection pressure was 3.45 psi (2.7 mL/min at 60°C). A volume of 0.025 μL of the liquid mixture was injected at a split ratio of 15:1.

Focusing in the Separation of Highly Volatile Compounds In contrast to the negative temperature gradient (NTG) separation, the thickness gradient column (FTGC) separation allows focusing of peaks at low temperatures (provided that such peaks are retained at these temperatures for a sufficient time), at which it is difficult to establish a temperature gradient. To confirm this fact, isothermal separations at room temperature (Table 1) of compounds _C5 and _C6 (Figs. 9A–9C) were carried out. The resolution values, p values, and focusing indices are presented in Table 9 (the values are averaged over 5 experiments). A focusing index of 40.2% was obtained with an average resolution in the direct mode of 2.97 and a resolution in the uniform film thickness column of 2.12. It should be noted that in the ROTG mode it is difficult to achieve the same peak focusing effect for highly volatile compounds, since only small temperature gradients can be created at low operating temperatures.

Table 9 presents the resolution values, p values, and focusing indices for the separation of _C5 and _C6 at room temperature (26°C) in the forward mode, reverse mode, and in a column with uniform film thickness. Significance was taken at the p=0.05 level.

This paper describes a technique for the preparation and characterization of a column with a thickness gradient of the stationary phase, which allows for peak focusing. The experimental results are discussed in combination with theoretical analysis and modeling and confirm the presence of improved characteristics of the separation of various compounds in the direct mode, including the focused separation of highly volatile compounds at room temperature. The advantages of the CGT compared to the ROTG are: wider ranges of operating temperatures and compound volatility, simple operation without auxiliary equipment, less dependence on environmental conditions and greater compactness. The technique of using a thickness gradient of the stationary phase can be easily adapted to various branches of GC and can be applied to stationary phases prepared from any materials of any thickness, provided that a gradient can be created. In addition, this technique is applicable to both conventional capillary columns of circular cross section and microcolumns of rectangular cross section.

The above disclosure of embodiments of the invention is given for the purpose of their illustration and description. It is not exhaustive and does not limit the invention. Individual elements or features of a particular embodiment are usually not limited to this particular embodiment, but, where possible, are interchangeable and can be included in another selected embodiment, even if this is not specifically indicated and described. This can also be implemented in various ways. Such changes should not be considered as departures from the subject of the invention, and all such modifications are included in the scope of the invention.

Claims (42)

1. A gas chromatographic device designed to focus peaks of one or more target analytes, which comprises: a chromatographic column having an inlet and an outlet, wherein a sample comprising one or more target analytes is fed into the inlet and exits the chromatographic column through the outlet, and a stationary phase deposited within a chromatographic column that has 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 chromatographic column and a second thickness at the outlet of the chromatographic column, wherein the second thickness is at least approximately 10% greater than the first thickness. 2. The gas chromatography device of claim 1, wherein the first thickness is from a value greater than or equal to about 10 nm to a value less than or equal to about 10 micrometers, and the second thickness is from a value greater than or equal to about 30 nm to a value less than or equal to about 30 micrometers. 3. The gas chromatography device of claim 1, wherein the second thickness is at least approximately 100% greater than or equal to the first thickness. 4. The gas chromatography device of claim 1, wherein the second thickness is at least about 300% greater than or equal to the first thickness. 5. A device for gas chromatography according to claim 1, wherein the chromatographic column is a chromatographic column for gas microanalysis. 6. A gas chromatography device according to claim 1, wherein the stationary phase comprises a siloxane polymer. 7. The gas chromatography device according to claim 6, wherein the siloxane polymer contains at least one alkyl group or aryl group containing from 1 to 30 carbon atoms. 8. A device for gas chromatography according to claim 1, wherein the cross-sectional shape of the chromatographic column is selected from the group consisting of: round, oval, rectangular and triangular. 9. A method for focusing peaks in a gas chromatography device, comprising: introducing two or more target analytes into the inlet of a chromatographic column comprising a stationary phase deposited within the chromatographic column 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 chromatographic column and a second thickness at the outlet of the chromatographic column, wherein the second thickness is at least approximately 10% greater than the first thickness; separation of two or more target analytes in a chromatographic column; and elution of two or more target analytes from the output of a chromatographic column. 10. The method of claim 9, wherein 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 the resulting aromatic compound peak focusing index 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, and the resulting alkane peak focusing index is greater than or equal to about 10%. 13. The method of claim 9, wherein the first thickness is from a value greater than or equal to about 10 nm to a value less than or equal to about 10 micrometers, and the second thickness is from a value greater than or equal to about 30 nm to a value less than or equal to about 30 micrometers. 14. The method of claim 9, wherein the second thickness is at least approximately 300% greater than or equal to the first thickness. 15. The method according to claim 9, wherein the chromatographic column is a chromatographic column for gas microanalysis. 16. The method according to claim 9, wherein the stationary phase comprises a siloxane polymer containing at least one alkyl group or aryl group containing from 1 to 30 carbon atoms. 17. The method according to claim 9, wherein the cross-sectional shape of the chromatographic column is selected from the group consisting of: round, oval, rectangular and triangular. 18. A method for confirming peak focusing in a gas chromatographic device, wherein the method comprises: performing a direct operation by introducing two or more target analytes into the inlet of a chromatographic column comprising a stationary phase deposited within the chromatographic column having a positive thickness gradient, wherein the stationary phase extends from the inlet to the outlet of the chromatographic column and has a first thickness at the inlet of the chromatographic column and a second thickness at the outlet, wherein the second thickness is at least approximately 10% greater than the first thickness; separation of two or more target analytes in a chromatographic column: elution of two or more target analytes from the output of a chromatographic column; performing the reverse operation by introducing two or more target analytes into the outlet of a chromatographic column containing a stationary phase; separation of two or more target analytes in a chromatographic column; elution of two or more target analytes from the inlet of a chromatographic column and a comparison of the chromatographic resolution values of the direct operation and the reverse operation, in which the peak focusing index of at least one corresponding pair of two peaks of two target analytes exceeds 5%. 19. A method for creating a device for gas chromatography, including a chromatographic column with a positive thickness gradient, wherein the method includes: introducing a precursor liquid into a chromatographic column, wherein the precursor liquid comprises a stationary phase precursor and a low boiling solvent that evaporates as it moves along the length of the chromatographic column, resulting in an increase in the concentration of the stationary phase precursor as the precursor liquid moves along the chromatographic column, and reacting the stationary phase precursor or cross-linking the stationary phase precursor to form a positive thickness gradient of the stationary phase extending from the inlet to the outlet and having a first thickness at the inlet of the chromatographic column and a second thickness at the outlet of the chromatographic column, wherein the second thickness is at least approximately 10% greater than the first thickness. 20. The method according to claim 19, wherein before introducing the precursor liquid, the inner surface of the chromatographic column is silanized. 21. The method of claim 20, wherein silanization comprises passing a reactive silane in gaseous form through a chromatographic column. 22. The method of claim 19, wherein the chromatographic column includes an inlet and an outlet, and introducing and carrying out the reaction or forming cross-links includes dynamically coating the inner surface of the chromatographic column by partially filling the chromatographic column with a precursor liquid, applying pressure at the inlet to force the precursor liquid along the length of the chromatographic column, and applying a vacuum at the outlet to evaporate the low-boiling solvent. 23. The method of claim 19, wherein the first thickness is from a value greater than or equal to about 10 nm to a value less than or equal to about 10 micrometers, and the second thickness is from a value greater than or equal to about 30 nm to a value less than or equal to about 30 micrometers. 24. The method of claim 19, wherein the second thickness is at least approximately 100% greater than or equal to the first thickness. 25. The method of claim 19, wherein the second thickness is at least approximately 300% greater than or equal to the first thickness. 26. The method according to claim 19, wherein the chromatographic column is a chromatographic column for gas microanalysis. 27. The method according to claim 19, wherein the stationary phase with a positive thickness gradient comprises a siloxane polymer. 28. The method of claim 27, wherein the siloxane polymer contains at least one alkyl group or aryl group containing from 1 to 30 carbon atoms.