JP2017512310A - Chromatographic material for separating unsaturated molecules - Google Patents

Abstract

The present disclosure relates to a method of separating a subject compound, particularly an unsaturated compound of interest, from a mixture. The compounds are separated using columns having chromatographic stationary phase materials in a variety of different modes of chromatography containing a first substituent and a second substituent. The first substituent minimizes the change in retention of the compound over time under chromatographic conditions. The second substituent includes one or more aromatic hydrocarbon groups, polyaromatic hydrocarbon groups, heteroaromatic hydrocarbon groups, or polyheteroaromatic hydrocarbon groups, thereby chromatographing the compound. Retaining graphically and selectively, each group is optionally substituted with an aliphatic group.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application claims the priority of US Patent Application Publication No. 14 / 194,686, filed March 1, 2014, which is hereby incorporated by reference in its entirety. To do.

  The present disclosure relates generally to chromatographic materials for separating unsaturated molecules. The present disclosure more particularly relates to normal phase chromatography, high pressure liquid chromatography, which, in various embodiments, reduces or avoids retention drifts or changes while showing overall retention useful for the separation of unsaturated molecules. Materials for chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography and hydrophobic interaction liquid chromatography and corresponding apparatus , Kits, manufacturing methods and methods of use.

  Chromatography is a collective term for a series of experimental techniques for separating mixtures. The mixture is dissolved in the mobile phase, and the mobile phase transports the mixture within the stationary phase. Since the various components of the mixture move at different speeds, the various components are separated. Separation is based on the distribution difference between the mobile phase and the stationary phase. A slight difference in the partition coefficient of the compound results in a difference in retention on the stationary phase, thus changing the separation. Chromatography is useful for the separation of structurally related compounds such as regioisomers, chirals, diastereomers and the like. Some techniques, including SFC, are known to be particularly useful for the separation of structurally related vitamins, natural products and chemicals. However, chromatographic techniques are often insufficient to separate any structurally related compounds. For example, critical pairs of related vitamins (eg, D2 and D3, K1 and K2) are difficult to separate / split.

  Fluid or liquid chromatography packing materials can generally be classified into two types: organic materials (eg, polydivinylbenzene) and inorganic materials (eg, silica). Many organic materials are chemically stable to strongly alkaline and strongly acidic mobile phases, providing flexibility in the choice of mobile phase composition and pH. However, organic chromatographic materials can yield inefficient columns, especially with low molecular weight analytes. Many organic chromatographic materials not only lack the mechanical strength of typical chromatographic silica, but also contract and swell as the mobile phase composition changes.

  Silica is widely used in high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC) and supercritical fluid chromatography (SFC). Some applications use silica surface-modified with organic functional groups such as octadecyl (C18), octyl (C8), phenyl, amino, cyano, and the like. As stationary phases for HPLC, these packing materials can yield columns with high efficiency and no evidence of shrinkage or swelling.

  Hybrid materials can provide a solution to certain chromatographic problems experienced by silica-based packing materials. The hybrid material can provide improvements including improved high and low pH stability, mechanical stability, peak shape when used at pH 7, efficiency, retention, and desirable chromatographic selectivity.

  However, potential problems may exist with conventional hybrid materials and silica materials in other applications. One problem is insufficient peak shape for bases when used at low pH, which can adversely affect loading and peak capacity when used at low pH. Another problem is the change in retention time of acidic and basic analytes (referred to as “drift”) after the column has undergone repeated changes in the mobile phase pH (eg, repeated switching from pH 10 to 3).

  Another problem is, for example, retention drift or change in chromatographic mode with small amounts of water (eg, less than 5%, less than 1%). For example, retention drifts or changes can occur in both silica and organic-inorganic hybrid (eg, BEH Technology ™ material available from Waters Technologies Corporation, Milford, Mass.) Systems, both bonded and unbound. For the chromatographic phase, it is observed under standard SFC conditions. Other SFC stationary phases may exhibit similar retention drifts or changes.

  In various aspects and embodiments, the present disclosure provides normal phase chromatography, high pressure liquid chromatography, which reduces or avoids retention drifts or changes while showing overall retention useful for the separation of unsaturated molecules. Chromatographic materials for solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography and hydrophobic interaction liquid chromatography, and corresponding devices, kits , Manufacturing method and method of use.

  The present disclosure includes various additional advantages including, but not limited to, the ability to select / design selectivity through chemical modification selection / design.

In one embodiment, the present disclosure relates to a method for separating a compound of interest from a mixture, the method comprising providing a mixture containing the compound of interest, and applying a portion of the mixture to a chromatography system having a chromatography column. Introducing, eluting the separated compound of interest from the column, wherein the column has the following structure (i):
[X] (W) _a (Q) _b (T) _c (i)
Having a stationary phase with:
Wherein X is a chromatographic substrate containing silica, metal oxides, inorganic-organic hybrid materials, block copolymers or combinations thereof, W is selected from the group consisting of hydrogen and hydroxyl; Binds to the surface of X, Q is the first substituent that minimizes the change in analyte retention over time under low water chromatographic conditions, and T retains the analyte chromatographically Wherein T has one or more aromatic hydrocarbon groups, polyaromatic hydrocarbon groups, heterocyclic aromatic hydrocarbon groups or polyheterocyclic aromatic hydrocarbon groups, each group being Optionally substituted with an aliphatic group, and b and c are positive numbers, 0.05 ≦ (b / c) ≦ 100 and a ≧ 0.

  In some embodiments, Q is the following structure (ii):

を有し、
式中、ｎ１は１−３０の整数であり、ｎ２は１−３０の整数であり、Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４はそれぞれ独立して、水素、ヒドロキシル、フルオロ、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、低級アルキル、保護または脱保護アルコールおよび双性イオンから成る群から選択され、Ｚは、（ａ）式（Ｂ^１）_ｘ（Ｒ^５）_ｙ（Ｒ^６）_ｚＳｉ−を有し、式中、ｘが１−３の整数であり、ｙが０−２の整数であり、ｚが０−２の整数であり、ｘ＋ｙ＋ｚ＝３であり、Ｒ^５およびＲ^６がそれぞれ独立して、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、置換または非置換アリール、環式アルキル、分枝アルキル、低級アルキル、保護または脱保護アルコール、双性イオン基およびシロキサン結合から成る群から選択され、ならびにＢ^１がシロキサン結合である表面結合基または（ｂ）直接炭素−炭素結合形成を介したもしくはヘテロ原子、エステル、エーテル、チオエーテル、アミン、アミド、イミド、尿素、カーボネート、カルバメート、ヘテロ環、トリアゾールまたはウレタン結合を介した表面有機官能性ハイブリッド基への結合または（ｃ）材料の表面に共有結合していない吸着表面基のいずれかであり、Ｙは、埋め込み極性官能基、結合または脂肪族基であり、ならびにＡは、親水性末端基、官能性化基、水素、ヒドロキシル、フルオロ、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、低級アルキルおよび極性化性基から成る群から選択される。

Have
In the formula, n1 is an integer of 1-30, n2 is an integer of 1-30, and R ¹ , R ² , R ³ and R ⁴ are each independently hydrogen, hydroxyl, fluoro, methyl, ethyl, selected from the group consisting of n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, protected or deprotected alcohol and zwitterion, Z is (a) the formula (B ¹ ) _x (R ⁵ ) _Y (R ⁶ ) _z Si—, wherein x is an integer of 1-3, y is an integer of 0-2, z is an integer of 0-2, and x + y + z = 3 Each of R ⁵ and R ⁶ is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, Protection Or deprotection alcohol is selected from the group consisting of zwitterionic groups and siloxane bonds, and surface binding group or (b) direct carbon B ¹ is a siloxane bond - or heteroatom via a carbon bond formation, ester, ether , Thioethers, amines, amides, imides, ureas, carbonates, carbamates, heterocycles, triazoles or bonds to surface organofunctional hybrid groups via urethane bonds or (c) adsorbed surface groups not covalently bonded to the surface of the material Y is an embedded polar functional group, a bond or an aliphatic group, and A is a hydrophilic end group, functionalized group, hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, Isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl and polarized It is selected from the group consisting of groups.

  In some embodiments, T is the following structure (iii):

を有し、
式中、ｍ^１は１−３０の整数であり、ｍ^２は１−３０の整数であり、ｍ^３は１−３の整数であり、Ｒ^７、Ｒ^８、Ｒ^９およびＲ^１０はそれぞれ独立して、水素、ヒドロキシル、フルオロ、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、低級アルキル、保護または脱保護アルコール、双性イオン、芳香族炭化水素基およびヘテロ環芳香族炭化水素基から成る群から選択され、Ｚは、（ａ）式（Ｂ^１）_ｘ（Ｒ^５）_ｙ（Ｒ^６）_ｚＳｉ−を有し、式中、ｘが１−３の整数であり、ｙが０−２の整数であり、ｚが０−２の整数であり、ｘ＋ｙ＋ｚ＝３であり、Ｒ^５およびＲ^６がそれぞれ独立して、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、置換または非置換アリール、環式アルキル、分枝アルキル、低級アルキル、保護または脱保護アルコール、双性イオン基およびシロキサン結合から成る群から選択され、ならびにＢ^１がシロキサン結合である表面結合基、（ｂ）直接炭素−炭素結合形成を介したもしくはヘテロ原子、エステル、エーテル、チオエーテル、アミン、アミド、イミド、尿素、カーボネート、カルバメート、ヘテロ環、トリアゾールまたはウレタン結合を介した表面有機官能性ハイブリッド基への結合または（ｃ）材料の表面に共有結合していない吸着表面基であり、Ｙは、埋め込み極性官能基、結合または脂肪族基であり、Ｄは、結合、Ｎ、Ｏ、Ｓ、−（ＣＨ_２）_０−１２−Ｎ−Ｒ^１１Ｒ^１２、−（ＣＨ_２）_０−１２−Ｏ−Ｒ^１１、−（ＣＨ_２）_０−１２−Ｓ−Ｒ^１１、−（ＣＨ_２）_０−１２−Ｎ−（ＣＨ_２）_０−１２−Ｒ^１１Ｒ^１２、−（ＣＨ_２）_０−１２−Ｏ−（ＣＨ_２）_０−１２−Ｒ^１１、−（ＣＨ_２）_０−１２−Ｓ−（ＣＨ_２）_０−１２−Ｒ^１１、−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｎ−Ｒ^１１Ｒ^１２、−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｏ−Ｒ^１１、−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｓ−Ｒ^１１；−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｎ−（ＣＨ_２）_０−１２−Ｒ^１１Ｒ^１２、−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｏ−（ＣＨ_２）_０−１２−Ｒ^１１および−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｓ−（ＣＨ_２）_０−１２−Ｒ^１１から成る群から選択され、Ｒ^１１は、第１のモノ芳香族基、ポリ芳香族基、ヘテロ環芳香族基またはポリヘテロ環芳香族基であり、Ｒ^１２は、水素、脂肪族基または第２のモノ芳香族基、ポリ芳香族基、ヘテロ環芳香族基またはポリヘテロ環芳香族基であり、ここでＲ^１１およびＲ^１２は、脂肪族基によって場合により置換される。Ｑ、Ｔまたは両方の種々の画分が重合できる。

Have
In the formula, m ¹ is an integer of 1-30, m ² is an integer of 1-30, m ³ is an integer of 1-3, and R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are each independent. Hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, protected or deprotected alcohol, zwitterion, aromatic hydrocarbon group and heterocycle Selected from the group consisting of aromatic hydrocarbon groups, Z has the formula (a) formula (B ¹ ) _x (R ⁵ ) _y (R ⁶ ) _z Si—, where x is an integer of 1-3 Y is an integer of 0-2, z is an integer of 0-2, x + y + z = 3, and R ⁵ and R ⁶ are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl , Substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, protected or deprotected alcohol is selected from the group consisting of zwitterionic groups and siloxane bonds, and surface binding group B ¹ is a siloxane bond, (B) Surface organofunctional hybrid groups directly through carbon-carbon bond formation or through heteroatoms, esters, ethers, thioethers, amines, amides, imides, ureas, carbonates, carbamates, heterocycles, triazoles or urethane bonds Or (c) an adsorbing surface group that is not covalently bonded to the surface of the material, Y is an embedded polar functional group, bond or aliphatic group, and D is a bond, N, O, S, − (CH ₂ ) _0-12 —N—R ¹¹ R ¹² , — (CH ₂ ) _0-12 —O—R ¹¹ , — (CH ₂ ) _{^{0-12 -S-R 11, - (}} CH 2) 0-12 -N- (CH 2) 0-12 -R 11 R 12, - (CH 2) 0-12 -O- (CH 2) 0- _{^{12 -R 11, - (CH 2}} ) 0-12 -S- (CH 2) 0-12 -R 11, - (CH 2) 0-12 -S (O) 1-2 - (CH 2) 0- _{^{12 -N-R 11 R 12,}} - (CH 2) 0-12 -S (O) 1-2 - (CH 2) 0-12 -O-R 11, - (CH 2) 0-12 -S ( ^{_{O) 1-2 - (CH 2)}} 0-12 -S-R 11 ;-( CH 2) 0-12 -S (O) 1-2 - (CH 2) 0-12 -N- (CH 2) _{^{0-12 -R 11 R 12, - (}} CH 2) 0-12 -S (O) 1-2 - (CH 2) 0-12 -O- (CH 2) 0-12 -R 11 and _{- (CH 2) 0-12 -S (} O) 1-2 - (CH 2) 0-12 -S- selected from the group consisting of _{^{(CH 2) 0-12 -R 11,}} R 11 is first A monoaromatic group, a polyaromatic group, a heterocyclic aromatic group or a polyheterocyclic aromatic group, wherein R ¹² represents hydrogen, an aliphatic group or a second monoaromatic group, a polyaromatic group, a heterocyclic ring An aromatic group or a polyheterocyclic aromatic group, wherein R ¹¹ and R ¹² are optionally substituted with an aliphatic group. Various fractions of Q, T or both can be polymerized.

  In another embodiment, the present disclosure relates to a method for separating lipids, vitamins or polycyclic aromatic hydrocarbons from a mixture.

In another embodiment, the present disclosure provides the following structure (i):
[X] (W) _a (Q) _b (T) _c (i)
A chromatographic stationary phase having
Where X is a chromatographic substrate comprising a group of silica, metal oxides, inorganic-organic hybrid materials, block copolymers or combinations thereof, W is selected from the group consisting of hydrogen and hydroxyl, and W is Bound to the surface of X, Q is the first substituent that minimizes the variation in analyte retention over time under low water chromatographic conditions, and T retains the analyte chromatographically The second substituent, T has one or more monoaromatic, polyaromatic, heteroaromatic or polyheteroaromatic groups, each group optionally by an aliphatic group And b and c are positive numbers, 0.05 ≦ (b / c) ≦ 100 and a ≧ 0.

  In another embodiment, the disclosure provides normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography. Relates to a column, capillary column, monolithic column, microfluidic device or apparatus for chromatography or hydrophobic interaction liquid chromatography, said device or apparatus comprising a housing and a housing having at least one wall defining a chamber having an inlet and an outlet A stationary phase having the above structure, i.e., normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical Body chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction conforms to liquid chromatography housing and stationary phase.

  In another embodiment, the disclosure provides normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography. With respect to a kit for chromatography or hydrophobic interaction liquid chromatography, the kit comprises a housing having at least one wall defining a chamber having an inlet and an outlet and an upper structure disposed within the housing, ie (i) Stationary phase, normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide chromatography, hydrophilic interaction liquid chromatography Housing and stationary phase compatible with luffy or hydrophobic interaction liquid chromatography; and normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical using the housing and stationary phase Includes instructions for performing fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography.

  In another embodiment, the present disclosure is a method of preparing a stationary phase having the above structure, ie, (i), wherein a chromatography substrate is reacted with a silane coupling agent having a pendant reactive group. Reacting a second chemical agent comprising one or more aromatic hydrocarbon groups, polyaromatic hydrocarbon groups, heterocyclic aromatic hydrocarbon groups or polyheteroaromatic hydrocarbon groups with the pendant reactive groups As well as a process comprising neutralizing any remaining unreacted pendant reactive groups to form a stationary phase.

  In another embodiment, the present disclosure provides a method of preparing a stationary phase having the above structure, i.e., (i), oligomerizing a silane coupling agent having a pendant reactive group, the core surface of the oligomer Reacting with a silanized silane coupling agent, a second chemical agent comprising one or more aromatic hydrocarbon groups, polyaromatic hydrocarbon groups, heteroaromatic hydrocarbon groups or polyheteroaromatic hydrocarbon groups It relates to a process comprising reacting with the pendant reactive group and neutralizing any remaining unreacted pendant reactive group to form a stationary phase.

  In another embodiment, the disclosure provides normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography. A method for reducing or preventing retention drift in chromatography or hydrophobic interaction liquid chromatography, wherein a sample is chromatographed using a chromatography device in which a chromatographic stationary phase having the above structure (i) is placed. The present invention relates to a method comprising lithographic separation and reducing or preventing retention drift.

The present disclosure advantageously reduces or avoids retention drifts or changes while exhibiting useful overall retention, particularly for an unsaturated molecule or compound of interest. For example, in SFC, retention drifts or changes can occur in the solvent for the particles (and / or by other alcohol cosolvents) under the standard CO ₂ / MeOH mobile phase used in SFC (among other various theories). May result from alkoxylation of accessible silanols. This is a problem, as the column ages, the user will again observe the chromatographic (eg retention time) changes that can be obtained with these SFC systems when a new non-alkoxylated column is installed in the system. Because.

  In various aspects and embodiments, the present disclosure provides various solutions to such retention drifts or changes and related problems (eg, retention, peak shape, etc.) due to the selection and / or modification of chromatographic materials. To do. For example, the present invention includes special functionalization of the chromatographic core surface (eg, with specific functional groups and combinations thereof), which essentially ensures chromatographic interaction between the analyte and the chromatographic core surface. The desired interaction between the analyte and the chromatographic material is maintained.

  In other various aspects and embodiments, the present disclosure relates to chromatographic materials with significantly reduced secondary interactions (eg, unwanted interactions, non-specific adsorption) with the base particle surface. Secondary interactions between the analyte and the material surface may occur due to silanols, pendant hydrophobic groups, and polymer or hybrid backbones.

  In various aspects and embodiments, the present disclosure provides a number of advantages. For example, the present disclosure provides a stationary phase that can resolve analytes, particularly unsaturated analytes, across all classes (eg, acidic, basic and neutral) with excellent retention, peak capacity and peak shape. The peak shape of the base is less important. In various examples, the present disclosure can effectively mask silanol from an analyte of interest to produce a predictable and stable chromatographic separation. In various examples, the present disclosure can effectively eliminate retention drifts or changes due to unwanted support surface interaction with the analyte. The present disclosure can particularly effectively mask silanols of silica or silica hybrid materials. In various examples, the present disclosure can improve peak capacity and tailing across all analyte classes, but especially with respect to bases. In various examples, the present disclosure can avoid pore clogging despite bonding with oligomeric siloxane (eg, pores that greatly reduce the available surface area of the material and lead to a heterogeneous surface). Despite the accelerated silane oligomerization in this disclosure, there is no evidence of pore clogging or surface area reduction compared to polymer coatings of conventional porous silica materials that can cause Absent.).

The present disclosure provides advantages over the prior art based on its unique chemical and performance characteristics. For example, liquid separation of unsaturated compounds typically includes separation performed on a C18 bonded phase such as ACQUITY UPC ² HSS C18 SB. In such materials, the alkyl chain is a retention selector that produces high methylene / hydrophobicity selectivity but little shape / isomer selectivity. The present disclosure provides retention selectors that are capable of both methylene / hydrophobic selectivity and shape / isomer selectivity. For example, the stationary phase of the present disclosure is superior to retaining and separating fat-soluble vitamins, lipids and metabolites and providing improved shape / isomer selectivity compared to C18 bonded phases.

  The present disclosure is further illustrated by the following figures and examples, which are used for illustration only and not for limitation.

  The present disclosure is more readily understood in the context of the following drawings and detailed description. It will be appreciated by those skilled in the art that the following drawings are not necessarily to scale, emphasis instead being placed upon illustrating the inventive concepts of the invention.

1 shows the structure of glycidoxypropyltrimethoxysilane (GPTMS). 1 shows the structure of 1-aminoanthracene. Figure 3 shows a schematic of the reaction between an unmodified chromatographic surface and GPTMS. Figure 2 shows a schematic of the reaction between a modified chromatographic surface and 1-aminoanthracene. Figure 2 shows a schematic of a chromatographic surface crosslinked by the modifiers GPTMS and 1-aminoanthracene. FIG. 2 shows a schematic diagram of two possible synthetic routes to prepare a chromatographic stationary phase of the present disclosure. FIG. 6 shows a graph of percent retention of analyte eluted using unmodified BEH particles as stationary phases and BEH particles modified according to the present disclosure. FIG. 7A shows an exemplary lipid separation using a 1-aminoanthracene-based stationary phase as described in Example 9. FIG. 4 illustrates an exemplary lipid separation using a 1-aminoanthracene-based stationary phase as described in Example 9. FIG. FIG. 4 shows C22: 0 and C20: 0 chromatograms using a 1-aminoanthracene-based stationary phase as described in Example 9. FIG. C16: 0, C12: 0 and C8: 0 chromatograms using a 1-aminoanthracene-based stationary phase as described in Example 9 are shown. FIG. 4 shows C22: 0 and C20: 0 chromatograms using a 1-aminoanthracene-based stationary phase as described in Example 9. FIG. C16: 0, C12: 0 and C8: 0 chromatograms using a 1-aminoanthracene-based stationary phase as described in Example 9 are shown. FIG. 4 shows C24: 1 and C22: 1 chromatograms using a 1-aminoanthracene stationary phase as described in Example 9. FIG. C20: 1, C18: 1 and C14: 1 chromatograms using 1-aminoanthracene stationary phases as described in Example 9 are shown. FIG. 4 shows C24: 1 and C22: 1 chromatograms using a 1-aminoanthracene stationary phase as described in Example 9. FIG. C20: 1, C18: 1 and C14: 1 chromatograms using 1-aminoanthracene stationary phases as described in Example 9 are shown. C18: 0, C18: 1 and C18: 2 chromatograms using a 1-aminoanthracene-based stationary phase as described in Example 9 are shown. C22: 1, C22: 2 and C22: 6 chromatograms using 1-aminoanthracene stationary phases as described in Example 9 are shown. C18: 0, C18: 1 and C18: 2 chromatograms using a 1-aminoanthracene-based stationary phase as described in Example 9 are shown. C22: 1, C22: 2 and C22: 6 chromatograms using 1-aminoanthracene stationary phases as described in Example 9 are shown. Figure 6 shows chromatograms of various linolenic and eicosadienoic acids using a 1-aminoanthracene-based stationary phase as described in Example 9. Figure 6 shows chromatograms of various linolenic and eicosadienoic acids using a 1-aminoanthracene-based stationary phase as described in Example 9. 2 illustrates an exemplary lipid separation performed using a 1-aminoanthracene-based stationary phase as described in Example 9. Figure 5 shows chromatograms of various lipids using 1-aminoanthracene-based stationary phases as described in Examples 3 and 10. Figure 2 shows chromatograms of various lipids using 2-picolylamine-based stationary phases as described in Examples 3 and 10. Figure 5 shows chromatograms of various lipids using pyridine-based stationary phases as described in Examples 3 and 10. Figure 5 shows chromatograms of various lipids using 6-aminoquinoline stationary phases as described in Examples 3 and 10. Figure 2 shows chromatograms of various lipids using aniline stationary phases as described in Examples 3 and 10. Figure 5 shows chromatograms of various lipids using GPTMS-based stationary phases as described in Examples 3 and 10. Figure 4 shows chromatograms of various lipids using 4-n-octylaniline based stationary phases as described in Examples 3 and 10. C18: 0, C18: 1 and C18: 2 chromatograms using 1-aminoanthracene stationary phases as described in Examples 3 and 10 are shown. C22: 1, C22: 2 and C22: 6 chromatograms using 1-aminoanthracene stationary phases as described in Examples 3 and 10 are shown. C18: 0, C18: 1 and C18: 2 chromatograms using 2-picolylamine stationary phases as described in Examples 3 and 10 are shown. C22: 1, C22: 2 and C22: 6 chromatograms using 2-picolylamine based stationary phases as described in Examples 3 and 10 are shown. C18: 0, C18: 1 and C18: 2 chromatograms using pyridine-based stationary phases as described in Examples 3 and 10 are shown. Chromatograms of C22: 1, C22: 2 and C22: 6 using pyridine based stationary phases as described in Examples 3 and 10 are shown. C18: 0, C18: 1 and C18: 2 chromatograms using 6-aminoquinoline stationary phases as described in Examples 3 and 10 are shown. C22: 1, C22: 2 and C22: 6 chromatograms using 6-aminoquinoline stationary phases as described in Examples 3 and 10 are shown. C18: 0, C18: 1 and C18: 2 chromatograms using an aniline stationary phase as described in Examples 3 and 10 are shown. C22: 1, C22: 2 and C22: 6 chromatograms using aniline-based stationary phases as described in Examples 3 and 10 are shown. C18: 0, C18: 1 and C18: 2 chromatograms using GPTMS-based stationary phases as described in Examples 3 and 10 are shown. C22: 1, C22: 2, and C22: 6 chromatograms using GPTMS-based stationary phases as described in Examples 3 and 10 are shown. C18: 0 chromatograms using 4-n-octylaniline stationary phases as described in Examples 3 and 10 are shown. C18: 1 chromatograms using 4-n-octylaniline stationary phases as described in Examples 3 and 10 are shown. C18: 2 chromatograms using 4-n-octylaniline stationary phases as described in Examples 3 and 10 are shown. FIG. 2 shows a C22: 1 chromatogram using 4-n-octylaniline stationary phase as described in Examples 3 and 10. FIG. C22: 2 chromatograms using 4-n-octylaniline stationary phases as described in Examples 3 and 10 are shown. C22: 6 chromatograms using 4-n-octylaniline stationary phases as described in Examples 3 and 10 are shown.

  In various aspects and embodiments, the present disclosure provides normal phase chromatography, high pressure liquid chromatography, which reduces or avoids retention drifts or changes while showing overall retention useful for the separation of unsaturated molecules. Chromatographic materials for solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography and hydrophobic interaction liquid chromatography, and corresponding devices, kits , Manufacturing method and method of use. In some embodiments, the present disclosure also provides retention and separation of structurally related compounds such as critical pairs that are difficult to separate.

The present disclosure advantageously reduces or avoids retention drifts or changes while showing useful overall retention. For example, in SFC, retention drifts or changes can occur in the solvent for the particles (and / or by other alcohol cosolvents) under the standard CO ₂ / MeOH mobile phase used in SFC (among other various theories). May result from alkoxylation of accessible silanols. This is a problem, as the column ages, the user will again observe the chromatographic (eg retention time) changes that can be obtained with these SFC systems when a new non-alkoxylated column is installed in the system. Because.

  In various aspects and embodiments, the present disclosure provides for such retention drifts or changes and related problems (eg, retention, peaks) due to selective modification of chromatographic materials and / or resolution of a mixture of unsaturated compounds of interest. Various solutions).

Definitions In various aspects and embodiments, the present invention provides for mitigation or prevention of retention drifts or changes. “Retention drift” or “retention change” is an undesirable difference in elution time between chromatographic runs or experiments (eg, in run 1, peak x elutes at time y, while in run 1 + n, the peak x elutes at time z). Thus, retention drifts or changes may also cause undesirable effects including experimental noise, non-reproducibility or failure. Thus, in a broad sense, mitigation or prevention of retention drifts or changes address or counteract undesirable differences in elution times between chromatographic runs to the extent that chromatographic experiments give chromatographically acceptable results. To include.

  In some embodiments, the mitigation or prevention of retention drift or change is not absolute or constant. For example, the amount of retention drift or change that can occur while still achieving a chromatographically acceptable result is the error or variation allowed in a given experiment, sample complexity (eg, number of peaks and / or May change depending on separation. The amount of retention drift or change that can occur while still achieving chromatographically acceptable results can vary depending on the duration or reproducibility required for a given experiment (eg, reproducibility). However, if required for a relatively large number of runs, the allowed retention drift or change between runs may be small.) Thus, it should be clear that mitigation or prevention of retention drift or change does not necessarily mean that retention drift or change is completely eliminated.

  In some embodiments, the mitigation or prevention of retention drift or change can be quantified. For example, retention drifts or changes can be measured for a single peak or averaged over a set of peaks. Retention drift or change can be measured over a given period or number of runs. Retention drift or change can be measured relative to a standard value, a starting value, or between two or more given runs.

  Furthermore, retention drifts or changes can be quantified by standardized testing. For example, the% average retention change is the percent difference in the average absolute peak retention measured in the 3rd, 10th or 30th day chromatographic test from the average absolute peak retention measured in the 1st day chromatographic test. Can be calculated by calculating. On each day of the test, the column is equilibrated under one set of test conditions, followed by multiple injections of the first test mix, then equilibrated under the second set of test conditions, followed by The second test mix can be injected multiple times.

  According to this standardized test, mitigation or prevention of retention drift or change is ≦ 5% over 30 days, ≦ 4% over 30 days, ≦ 3% over 30 days, ≦ 2% over 30 days, ≦ 1% over 30 days ≦ 5% over 10 days ≦ 4% over 10 days ≦ 3% over 10 days ≦ 2% over 10 days ≦ 1% over 10 days ≦ 5% over 3 days ≦ 4% over 3 days, ≤3% over 3 days ≤2% over 3 days ≤1% over 3 days, ≤5% over 30 runs, ≤4% over 30 runs, ≤3% over 30 runs, 30 ≦ 2% over 30 runs, ≦ 1% over 30 runs, ≦ 5% over 10 runs, ≦ 4% over 10 runs, 10 ≤3% over rows, ≤2% over 10 runs, ≤1% over 10 runs, ≤5% over 3 runs, ≤4% over 3 runs, ≤3% over 3 runs Retention drifts or changes of ≦ 2% over 3 runs or ≦ 1% over 3 runs can be included.

  In other embodiments, the mitigation or prevention of retention drift or change is 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day (or run) ≦ 5.0, 4.9, 4.8, 4 7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2 .2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1, 2, 1.1, 1.0 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or It can include drift or change of .1% retention.

  “High purity” or “high purity chromatography material” includes materials prepared from high purity precursors. In certain embodiments, high purity materials have been shown to reduce metal contamination and / or chromatographic properties including, but not limited to, surface silanol acidity and surface heterogeneity.

  A “chromatographic surface” includes a surface that provides a chromatographic separation of a sample. In certain embodiments, the chromatographic surface is porous. In some embodiments, the chromatographic surface can be the surface of a particle, surface porous material or monolith. In some embodiments, the chromatographic surface is composed of one or more particles, surface porous materials or monolithic surfaces used in combination during chromatographic separation. In certain other embodiments, the chromatographic surface is non-porous.

  An “ionizable modifier” includes a functional group that retains an electron donating group or an electron withdrawing group. In some embodiments, the ionizable modifier contains one or more carboxylic acid groups, amino groups, imide groups, amide groups, pyridyl groups, imidazolyl groups, ureido groups, thionyl-ureido groups or aminosilane groups, or combinations thereof. . In other embodiments, the ionizable modifier contains a group that retains a nitrogen or phosphorus atom having a free electron unshared electron pair. In some embodiments, the ionizable modifier is covalently bonded to the material surface and has an ionizable group. In some examples, the ionizable modifier is attached to the chromatographic material by chemical modification of the surface hybrid groups.

“Hydrophobic surface groups” include surface groups on a chromatographic surface that exhibit hydrophobic properties. In certain embodiments, the hydrophobicity can be a carbon bonded phase, such as a C ₄ -C ₁₈ bonded phase. In other embodiments, the hydrophobic surface group can contain embedded polar groups such that the outer portion of the hydrophobic surface remains hydrophobic. In some examples, the hydrophobic surface group is attached to the chromatographic material by chemical modification of the surface hybrid group. In other examples, hydrophobic, C ₄ -C ₃₀ embedded polar, chiral, may be a phenylalkyl or pentafluorophenyl bonding and coating.

  A “chromatographic core” is not limited thereto, but may be silica, as defined herein, in the form of a particle, monolith or another suitable structure that forms the inner portion of the disclosed material. Includes chromatographic materials, including organic materials such as hybrid materials. In certain embodiments, the surface of the chromatographic core represents a chromatographic surface as defined herein or represents a material surrounded by a chromatographic surface as defined herein. The chromatographic surface material can be placed on the chromatographic core, bonded to the core, annealed to the core or blended with the surface of the chromatographic core so that discrete or distinct transitions can be identified. It can be bound to the chromatographic core so as to cause a step change in the material and not a discontinuous inner core surface. In certain embodiments, the chromatographic surface material may be the same as or different from the material of the chromatographic core, including but not limited to pore volume, surface area, average pore size, carbon content or It can exhibit physical or physicochemical properties that are different from the chromatographic core, including hydrolysis pH stability.

  “Hybrids” including “hybrid inorganic / organic materials” include inorganic-based structures in which the organic functional group forms an internal or “skeleton” inorganic structure as well as part of the surface of the hybrid material. The inorganic part of the hybrid material can be, for example, alumina, silica, titanium, cerium or zirconium or their oxide or ceramic material. “Hybrid” includes inorganic structures in which the organic functional group forms part of both the internal or “skeleton” inorganic structure as well as the surface of the hybrid material. As mentioned above, exemplary hybrid materials are shown in US Pat. Nos. 4,017,528, 6,528,167, 6,686,035, and 7,175,913, which Are incorporated herein by reference in their entirety.

The term “alicyclic group” includes closed ring structures of 3 or more carbon atoms. Alicyclic groups include cycloparaffins or naphthalenes that are saturated cyclic hydrocarbons, cycloolefins that are unsaturated with two or more double bonds, and cycloacetylenes that have triple bonds. These do not contain aromatic groups. Examples of cycloparaffins include cyclopropane, cyclohexane and cyclopentane. Examples of cycloolefins include cyclopentadiene cyclohexadiene and cyclooctatetraene. Alicyclic groups also include substituted alicyclic groups such as fused ring structures and alkyl substituted alicyclic groups. In the examples of alicyclic compounds, such substituents further include lower alkyl, lower alkenyl, lower alkoxy, lower alkylthio, lower alkylamino, lower alkyl carboxyl, nitro, hydroxyl, —CF ₃ , —CN, and the like. Can do.

The term “aliphatic group” includes organic compounds characterized by straight or branched chains, typically having from 1 to 24 carbon atoms. Aliphatic groups include alkyl groups, alkenyl groups, and alkynyl groups. In complex structures, the chains can be branched or bridged. In some embodiments, the aliphatic group has 2 to 24 carbon atoms, or 4 to 22 carbon atoms, or 6 to 20 carbon atoms, or 8 to 18 carbon atoms, or 10 to It can include chains having 16 carbon atoms, or 12 to 14 carbon atoms, or combinations of these numbers, such as about 6 to 12 carbon atoms or 10 to 14 carbon atoms. Alkyl groups include saturated hydrocarbons having one or more carbon atoms, including straight chain alkyl groups and branched chain alkyls. Such hydrocarbon moieties can substitute one or more carbons by, for example, a halogen group, hydroxyl group, thiol group, amino group, alkoxy group, alkylcarboxy group, alkylthio group, or nitro group. Unless otherwise specified, the term “lower aliphatic” as used herein means an aliphatic group as defined above, but having 1 to 6 carbon atoms (eg, lower alkyl, lower Alkenyl, lower alkynyl). Representative examples of such lower aliphatic groups, such as lower alkyl groups, are, for example, methyl, ethyl, n-propyl, isopropyl, 2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl, tert- Butyl, 3-thiopentyl and the like. As used herein, the term “nitro” means —NO ₂ , the term “halogen” designates —F, —Cl, —Br or —I, and the term “thiol” means SH. And the term “hydroxyl” means “—OH”. Thus, the term “alkylamino” as used herein means an alkyl group as defined above having an amino group attached thereto. Suitable alkylamino groups include groups having 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms. The term “alkylthio” refers to an alkyl group, as defined above, having a sulfhydryl group attached thereto. Suitable alkylthio groups include groups having 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms. The term “alkylcarbonyl” as used herein means an alkyl group, as defined above, having a carboxyl group attached thereto. The term “alkoxy” as used herein means an alkyl group, as defined above, having an oxygen atom attached thereto. Exemplary alkoxy groups include groups having 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms, such as methoxy, ethoxy, propoxy, tert-butoxy and the like. The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groups similar to alkyl but having at least one double or triple bond, respectively. Suitable alkenyl and alkynyl groups include groups having 2 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms.

The term “alkyl” includes saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched alkyl has 30 or fewer carbon atoms in its backbone, such as C ₁ -C ₃₀ for straight chain or C ₃ -C ₃₀ for branched chain. In certain embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone, such as C ₁ -C ₂₀ for straight chain or C ₃ -C _{20 for} branched chain, more preferably Has up to 18 carbon atoms. Similarly, preferred cycloalkyls have from 4-10 carbon atoms in their ring structure, and more preferably have 4-7 carbon atoms in their ring structure. The term “lower alkyl” denotes an alkyl group having 1 to 6 carbons in the chain and a cycloalkyl having 3 to 6 carbons in the ring structure.

  Furthermore, the term “alkyl” (including “lower alkyl”), as used throughout this disclosure, includes both “unsubstituted alkyl” and “substituted alkyl”, where “substituted alkyl” refers to a hydrocarbon backbone. And an alkyl moiety having a substituent replacing hydrogen with one or more carbons. Examples of such substituents include halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate Phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl Alkylthio, arylthio, thiocarboxylate, sulfate, sulfonate, Rufamoiru, sulfonamides, may nitro, trifluoromethyl, cyano, azido, heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be appreciated by those skilled in the art that moieties substituted with a hydrocarbon chain can be substituted per se, if appropriate. Cycloalkyls can be further substituted, for example, with the substituents described above. An “aralkyl” moiety is an alkyl substituted with an aryl having, for example, 1 to 3 separate or fused rings and 6 to about 18 carbon ring atoms, such as phenylmethyl (benzyl).

The term “amino” as used herein refers to an unsubstituted or substituted moiety of the formula —NR _a R _b , wherein R _a and R _b are each independently hydrogen, alkyl, aryl Alternatively, they are heterocyclyl, or R _a and R _b together with the nitrogen atom to which they are attached form a cyclic moiety having 3 to 8 atoms in the ring. Thus, the term “amino” includes cyclic amino moieties such as piperidinyl or pyrrolidinyl groups, unless otherwise indicated. “Amino-substituted amino group” refers to an amino group in which at least one of R _a and R _b is further substituted with an amino group.

The term “aromatic group” includes unsaturated cyclic hydrocarbons containing one or more rings. The term “monoaromatic” includes unsaturated cyclic hydrocarbons containing one ring. The term “polyaromatic” includes unsaturated cyclic hydrocarbons containing two or more rings. Aromatic groups include 5- and 6-membered monocyclic groups that can contain from 0 to 4 heteroatoms such as furan, pyrrole, pyrroline, oxazole, thiazole, imidazole, imidazoline, pyrazole, pyrazoline, pyrazolidine, isoxazole, Examples include isothiazole, benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, and thiophene. The aromatic ring is substituted at one or more ring positions by, for example, halogen, lower alkyl, lower alkenyl, lower alkoxy, lower alkylthio, lower alkylamino, lower alkyl carboxyl, nitro, hydroxyl, —CF ₃ , —CN, etc. be able to. Aromatic groups include 5- and 6-membered polycyclic groups that can contain 0 to 8 heteroatoms such as indene, indolizine, indole, isoindole, indoline, indazole, benzimidazole, benzothiazole ( benzothiazole), naphthalene, quinolidine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, quinuclidine, fluorene, carbazole, anthracene, acridine, phenazine, phenothazine, phenoxazine, etc. . Examples of the polyaromatic group include condensed aromatic groups.

  The term “aryl” refers to 5- and 6-membered monocyclic aromatic groups that may contain from 0 to 4 heteroatoms, such as unsubstituted or substituted benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole , Pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl and the like. Aromatic rings can be substituted at one or more ring positions with such substituents, for example as described above for alkyl groups. Suitable aryl groups include unsubstituted and substituted phenyl groups. The term “aryloxy” as used herein means an aryl group, as defined above, having an oxygen atom attached thereto. The term “aralkoxy” as used herein means an aralkyl group, as defined above, having an oxygen atom attached thereto. Suitable aralkoxy groups have 1 to 3 separate or fused rings and 6 to about 18 carbon ring atoms, for example O-benzyl.

  The term “ceramic precursor” is intended to include any compound that results in the formation of a ceramic material.

  The term “chiral moiety” is intended to include any functional group that allows chiral or stereoselective synthesis. Chiral moieties include, but are not limited to, substituents having at least one chiral center, natural and unnatural amino acids, peptides and proteins, derivatized cellulose, macrocyclic antibiotics, cyclodextrins, crown ethers As well as metal complexes.

  The term “embedded polar functional group” is a functional group that provides an integrated polar moiety so that the interaction with a basic sample is reduced by shielding unreacted silanol groups on the silica surface. Embedded polar functional groups include, but are not limited to, carbonates, amides, ureas, ethers (eg, —O— between carbon-containing groups) as disclosed in US Pat. No. 5,374,755. , Thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocyclic, triazole or carbamate functional groups and chiral moieties.

  The term “chromatography-enhancing pore shape” is disclosed herein, for example, which has been found to improve the chromatographic resolution of a material, as distinguished from other chromatographic media in the art. Includes the shape of the pore structure of the material. For example, a shape can be formed, selected or structured, for example, to determine whether the chromatographic resolution of a material is “improved” compared to a shape known in the art or used conventionally Various characteristics and / or factors can be used. Examples of these factors include high separation efficiency, longer column lifetime, and high mass transfer characteristics (such as demonstrated by reduced band broadening and good peak shape). These characteristics can be measured or observed using techniques recognized in the art. For example, the chromatographically enhanced pore shape of the porous inorganic / organic hybrid material of the present invention is distinguished from prior art materials by the absence of an “ink bottle” or “shell” pore shape or morphology, Both “ink bottles” or “shell” pore shapes or configurations are undesirable because, for example, they reduce mass transfer rates and result in lower efficiencies.

Chromatographically enhanced pore shapes are found in hybrid materials containing only a small population of micropores. A small population of micropores is obtained with a hybrid material when the total pores with a diameter of less than about 34 mm is less than about 110 m ² / g relative to the specific surface area of the material. Such a hybrid material with a low micropore surface area (MSA) improves chromatography, including high separation efficiency and good mass transfer properties (eg as evidenced by reduced band broadening and good peak shape) Bring. Micropore surface area (MSA) is defined as the surface area of pores having a diameter of 34 mm or less, as determined by multipoint nitrogen sorption analysis from the isotherm adsorption leg using the BJH method. As used herein, the acronyms “MSA” and “MPA” are used interchangeably to indicate “micropore surface area”.

  The term “functionalizable group” includes organic functional groups that impart certain chromatographic functional groups to the chromatographic stationary phase.

The term “heterocyclic group” includes closed ring structures in which one or more of the atoms in the ring is an element other than carbon, such as nitrogen, sulfur or oxygen. Heterocyclic groups can be saturated or unsaturated, and heterocyclic groups such as pyrrole and furan can have aromatic character, i.e., "heteroaromatic groups". These include one or more ring structures. A heterocyclic group containing two or more ring structures is a “polyheterocyclic aromatic group”. These groups can have fused ring structures such as quinoline and isoquinoline. Other examples of heterocyclic groups include pyridine and purine. Heterocyclic groups are also substituted with one or more constituent atoms by, for example, halogen, lower alkyl, lower alkenyl, lower alkoxy, lower alkylthio, lower alkylamino, lower alkylcarboxyl, nitro, hydroxyl, —CF ₃ , —CN, etc. can do. Suitable heteroaromatic and heteroalicyclic groups are generally 1 to 3 separate or fused rings having 3 to about 8 ring members per ring and one or more N, O or S atoms. For example, coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl.

  The term “metal oxide precursor” is intended to include any compound that contains a metal and results in the formation of a metal oxide such as alumina, silica, titanium oxide, zirconium oxide.

The term “monolith” is intended to include a collection of individual particles packed in a bed configuration in which the shape and morphology of the individual particles are maintained. The particles are advantageously filled with a material that binds the particles. Any number of bonding materials well known in the art, such as linear or cross-linked polymers of divinylbenzene, methacrylate, urethane, alkene, alkyne, amine, amide, isocyanate or epoxy groups, as well as organoalkoxysilanes, tetraalkoxysilanes, polyorganoalkoxysiloxanes Polyethoxysiloxane condensation reactants, as well as ceramic precursors can be used. In certain embodiments, the term “monolith” refers to a hybrid monolith made by other methods, such as the hybrid monolith detailed in US Pat. No. 7,250,214; 0-99 mole percent silica (eg, SiO 2). ₂ ) a hybrid monolith prepared from the condensation of one or more monomers containing; a hybrid monolith prepared from coalesced porous inorganic / organic particles; a hybrid monolith with chromatographically enhanced pore shape; Hybrid monolith with no pore shape; hybrid monolith with regular pore structure; hybrid monolith with aperiodic pore structure; hybrid monolith with amorphous or amorphous molecular regularity; with crystalline domain or region Hybrid model Squirrel; including hybrid monoliths and a variety of different aspect ratios; hybrid monolith with a variety of different macropores characteristics and mesopores characteristics. In certain embodiments, the term “monolith” refers to an inorganic monolith such as G.I. Guiochon / J. Chromatogr. A 1168 (2007) 101-168 is also included.

  The term “nanoparticle” refers to a fine particle / particle that can be crystalline or non-crystalline, having at least one dimension of less than about 100 nm, eg, a diameter or particle thickness of less than about 100 nm (0.1 mm). Granules, or fine components of powder / nano powder. Nanoparticles have properties that are often superior to these, which are different from those of conventional bulk materials, including, for example, higher strength, hardness, ductility, sinterability, and higher reactivity. Substantial scientific research has continued to characterize nanomaterials, and small amounts of nanomaterials (primarily as nanosized powders) have been achieved by multiple methods including colloidal precipitation, mechanical grinding and gas phase nucleation and growth. The material is synthesized. An extensive review documents recent developments in nanophase materials, which are incorporated herein by reference: Gleiter, H. et al. (1989) “Nano-crystalline materials”, Prog. Mater. Sci. 33: 223-315 and Siegel, R.M. W. (1993) "Synthesis and properties of nano-phase materials", Mater. Sci. Eng. A168: 189-197. In certain embodiments, the nanoparticles comprise the following silicon carbide, aluminum, diamond, cerium, carbon black, carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium, gadolinium, iron, nickel, samarium, silicon, silver, Contains oxides or nitrides of titanium, zinc, boron and mixtures thereof. In certain embodiments, the nanoparticles of the present disclosure comprise diamond, zirconium oxide (amorphous, monoclinic, tetragonal and cubic forms), titanium oxide (amorphous form, anatase form, brookite form and Rutile form), aluminum (amorphous form, alpha form and gamma form) and boronitride (cubic form). In certain embodiments, the nanoparticles of the present disclosure are selected from nanodiamonds, silicon carbide, titanium dioxide (anatase form), cubic boronitrides, and any combination thereof. Further, in certain embodiments, the nanoparticles can be crystalline or amorphous. In certain embodiments, the nanoparticles are 100 mm or less in diameter, such as 50 mm or less, such as 20 mm or less in diameter.

  Further, it should be understood that nanoparticles characterized as being dispersed within the composites of the present disclosure are those described as externally added nanoparticles. This is a composition with significant similarity to nanoparticles, i.e. putative nanoparticles that can be formed in situ, that can include those aggregates in which macromolecular structures such as particles have been generated internally In contrast to

  The term “substantially irregular” indicates no pore regularity based on X-ray powder diffraction analysis. Specifically, “substantially irregular” is defined by the absence of a peak in the diffraction angle corresponding to a d value (or d interval) of at least 1 nm in the X-ray diffraction pattern.

  “Surface modifiers” typically include organic functional groups that impart certain chromatographic functional groups to the chromatographic stationary phase. Porous inorganic / organic hybrid materials have both organic and silanol groups that can be further substituted or derivatized with a surface modifier.

  The term “surface modified” is used herein to describe a composite material of the present disclosure having both organic and silanol groups that can be further substituted or derivatized with a surface modifier. . “Surface modifiers” comprise (usually) organic functional groups that impart certain chromatographic functional groups to the chromatographic stationary phase. Surface modifiers as disclosed herein bind to the substrate, for example, through derivatization or coating, and subsequent cross-linking, imparting the surface modifier's chemical properties to the substrate. In one embodiment, the organic groups of the hybrid material react to form an organic covalent bond with the surface modifier. Modifiers include, but are not limited to, nucleophilic reactions, electrophilic reactions, cycloaddition reactions, free radical reactions, carbene reactions, nitrene reactions and carbocation reactions in organic and polymer chemistry. Organic covalent bonds to the organic groups of the material can be formed through several well-known mechanisms. Organic covalent bonds include, but are not limited to, the formation of covalent bonds between common elements of organic chemistry, including hydrogen, boron, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur and halogen. Then it is defined. Furthermore, carbon-silicon bonds and carbon-oxygen-silicon bonds are defined as organic covalent bonds, while silicon-oxygen-silicon bonds are not defined as organic covalent bonds. Various synthetic transformations are well known in the literature, see, for example, March, J. et al. See Advanced Organic Chemistry, 3rd Edition, Wiley, New York, 1985.

  The chromatographic materials of the present disclosure can include those comprising a group of core materials of silica core materials, metal oxide core materials, inorganic-organic hybrid materials, or block copolymers thereof. The core material can be a high purity chromatographic core composition as discussed herein. Similarly, the chromatographic core material can be a normal, eg non-pure version / analog / congener, of the high purity material discussed herein.

  Examples of suitable core materials include, but are not limited to, conventional chromatographic silica materials, metal oxide materials, inorganic-organic hybrid materials or a group of these block copolymers, ceramics, silicon oxides, imides Examples include silicon nitride, silicon nitride, aluminum silicon nitride, diimide silicon, and silicon oxynitride. Further examples of suitable core materials (for use with or without modification) include US Patent Application Publication Nos. 2009/0127177, 2007/0135304, 2009/0209722, 2007/0215547, 2007/0141325. No. 2011/0049056, 2012/0055860 and 2012/0273404, and WO 2008/103423, which are incorporated herein by reference in their entirety.

  The chromatography core material can be in the form of discrete particles or can be a monolith. The chromatographic core material can be any porous material, can be commercially available, or known methods such as US Pat. Nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913, which are hereby incorporated by reference in their entirety. In some embodiments, the chromatography core material can be a non-porous core.

The composition of the chromatographic surface material and the chromatographic core material can be changed by one skilled in the art to provide improved chromatographic selectivity, improved column chemical stability, improved column efficiency and / or improved mechanical strength. Can do. Similarly, the composition of the surrounding material results in a change in hydrophilic / lipophilic balance (HLB), surface charge (eg isoelectric point or silanol pK _a ) and / or surface functionality to improve chromatographic separation. Further, in some embodiments, the composition of the chromatographic material can also provide surface functionality that can be utilized for further surface modification.

  The ionizable groups and hydrophobic surface groups of the chromatographic material of the present disclosure can be prepared using known methods. Some of the ionizable modifier reagents are commercially available. For example, silanes having aminoalkyltrialkoxysilane, methylaminoalkyltrialkoxysilane and pyridylalkyltrialkoxysilane are commercially available. Other silanes such as chloropropylalkyltrichlorosilane and chloropropylalkyltrialkoxysilane are also commercially available. These can be coupled and reacted with imidazole to produce an imidazolylalkylsilyl surface species, or can be coupled and reacted with pyridine to produce a pyridylalkylsilyl surface species. Although not limited thereto, sulfopropyltrisilanol, carboxyethylsilanetriol, 2- (carbomethoxy) ethylmethyldichlorosilane, 2- (carbomethoxy) ethyltrichlorosilane, 2- (carbomethoxy) ethyltrimethoxy Silane, n- (trimethoxysilylpropyl) ethylenediamine, triacetic acid, (2-diethylphosphatoethyl) triethoxysilane, 2- (chlorosulfonylphenyl) ethyltrichlorosilane and 2- (chlorosulfonylphenyl) ethyltrimethoxysilane. Other acidic modifiers are also commercially available.

  It is known to those skilled in the art to synthesize these types of silanes using general synthetic protocols including Grignard reactions and hydrosilylation. The product can be purified by chromatography, recrystallization or distillation.

  Other additives such as isocyanates are also commercially available or can be synthesized by one skilled in the art. A common isocyanate formation protocol is the reaction of a primary amine with a reagent known as phosgene or triphosgene.

In one aspect, the present disclosure relates to a method of separating a compound of interest from a mixture, the method comprising: (a) providing a mixture containing the compound of interest; (b) a chromatography system having a chromatography column. Introducing a portion of the mixture, (c) eluting the separated target compound from the column, wherein the column has the following structure (i):
[X] (W) _a (Q) _b (T) _c (i)
Having a stationary phase having the formula:
X is a chromatographic substrate containing silica, metal oxides, inorganic-organic hybrid materials, group of block copolymers or combinations thereof;
W is selected from the group consisting of hydrogen and hydroxyl, W binds to the surface of X,
Q is the first substituent that minimizes the change in retention of the analyte over time under chromatographic conditions with a low moisture concentration;
T is a second substituent that chromatographically retains the analyte, and T has one or more monoaromatic, polyaromatic, heteroaromatic or polyheteroaromatic groups Each group is optionally substituted by an aliphatic group, and b and c are positive numbers, 0.05 ≦ (b / c) ≦ 100 and a ≧ 0.

  In various embodiments, the selectivity of the chromatographic material can be controlled or influenced by the choice of Q and / or T, the density of Q and / or T on the surface, or a combination thereof. Can do. In some embodiments, both Q and T play a role in retention of the compound of interest. In other embodiments, T selectively retains different compounds of interest.

  In various embodiments, the present disclosure provides binding chromatographic materials that can bind a ligand. The high density coverage achieved by the bonding method of the present invention may be 2 to 3 times higher than the silane bonding chemistry currently practiced with conventional SFC materials. By combining the high Q and / or T coverage with other properties, the interaction between the surface silanol and the analyte can be prevented.

  In various embodiments, the present disclosure provides a high density of bonded phases that increase the retention of retention caused by analyte interactions with surface silanols or other secondary retention mechanisms. Drift or change is prevented. The elimination of these secondary and trace selectivities greatly improves peak shape and column peak capacity, especially for basic analytes.

  In various embodiments, the present disclosure provides for the use of a two-component system based on coupling chemistry that results in uniform coverage of mixed surface functional groups. Unlike mixed particle beds, where two separate particles with different surface chemistries are mixed, this material is uniform throughout and has predictable surface properties. Columns packed with such materials are less likely to be chromatographically unstable due to insufficient particle mixing or particle type separation during column packing.

  In various embodiments, the present disclosure provides column packing that is facilitated by the use of a single particle slurry.

  In various embodiments, the present disclosure provides a chromatographic packing material with improved selectivity for acidic, neutral and basic analytes in a single column without the use of mixed particle or multiparticulate beds. .

  In various embodiments, the present disclosure provides a ratio of components at the particle surface that can be easily manipulated to alter the selectivity of the support and provide a wide choice of chromatographic separations.

  In various embodiments, the present disclosure provides for the crosslinked membrane of the silane surface modifier to be formed by either polymerization of surface reactive groups or addition of a crosslinking agent, either before, during or after the addition of the selective ligand. Provides the binding chemistry that is formed.

  In certain other embodiments, the chromatographic material of the present disclosure is non-porous. In another embodiment, the chromatographic material of the present disclosure is hydrolytically stable at a pH of about 1 to about 14, a pH of about 10 to about 14, or a pH of about 1 to about 5.

  In another aspect, the present disclosure provides a material as described herein, wherein the chromatographic material further comprises a nanoparticle dispersed within the chromatographic surface or a mixture of more than one nanoparticle.

  In certain embodiments, the nanoparticles are present at less than 20% by weight of the nanocomposite, less than 10% by weight of the nanocomposite or less than 5% by weight of the nanocomposite.

  In other embodiments, the nanoparticles are crystalline or amorphous and are silicon carbide, aluminum, diamond, cerium, carbon black, carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium, gadolinium, iron, It can be nickel, samarium, silicon, silver, titanium, zinc, boron, oxides thereof or nitrides thereof. In certain embodiments, the nanoparticle is a material that includes one or more moieties selected from the group consisting of nanodiamond, silicon carbide, titanium dioxide, and cubic-boronitride. In other embodiments, the nanoparticles can be 200 nm or less in diameter, 100 nm or less in diameter, 50 nm or less in diameter, or 20 nm or less in diameter.

  In one or more embodiments, Q is:

によって表され、
式中：
ｎ^１は、１−３０の整数であり；
ｎ^２は、１−３０の整数であり；
Ｒ^１、Ｒ^２、Ｒ^３およびＲ^４はそれぞれ独立して、水素、ヒドロキシル、フルオロ、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、低級アルキル、保護または脱保護アルコールおよび双性イオンから成る群から選択され；
Ｚは、
（ａ）式（Ｂ^１）_ｘ（Ｒ^５）_ｙ（Ｒ^６）_ｚＳｉ−を有する表面結合基であって、式中、ｘが１−３の整数であり、ｙが０−２の整数であり、ｚが０−２の整数であり、およびｘ＋ｙ＋ｚ＝３であり；Ｒ^５およびＲ^６がそれぞれ独立してメチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、置換または非置換アリール、環式アルキル、分枝アルキル、低級アルキル、保護または脱保護アルコール、双性イオン基およびシロキサン結合から成る群から選択され；ならびにＢ^１がシロキサン結合である、表面結合基；
（ｂ）直接炭素−炭素結合形成を介したまたはヘテロ原子、エステル、エーテル、チオエーテル、アミン、アミド、イミド、尿素、カーボネート、カルバメート、ヘテロ環、トリアゾールもしくはウレタン結合を介した表面有機官能性ハイブリッド基への結合；または
（ｃ）材料の表面に共有結合していない、吸着された表面基であり；
Ｙは、埋め込み極性官能基であり；ならびに
Ａは、親水性末端基、官能性化基、水素、ヒドロキシル、フルオロ、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、低級アルキルおよび極性化性基から成る群から選択される。

Represented by
In the formula:
n ¹ is an integer from 1-30;
n ² is an integer from 1-30;
R ¹ , R ² , R ³ and R ⁴ are each independently hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, protected or deprotected Selected from the group consisting of alcohol and zwitterions;
Z is
(A) a surface binding group having the formula (B ¹ ) _x (R ⁵ ) _y (R ⁶ ) _z Si—, wherein x is an integer of 1-3 and y is an integer of 0-2 And z is an integer from 0-2 and x + y + z = 3; R ⁵ and R ⁶ are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, A surface binding group selected from the group consisting of substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, protected or deprotected alcohol, zwitterionic group and siloxane bond; and B ¹ is a siloxane bond;
(B) Surface organofunctional hybrid groups directly through carbon-carbon bond formation or through heteroatoms, esters, ethers, thioethers, amines, amides, imides, ureas, carbonates, carbamates, heterocycles, triazoles or urethane bonds Or (c) an adsorbed surface group that is not covalently bonded to the surface of the material;
Y is an embedded polar functional group; and A is a hydrophilic end group, functionalized group, hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, Selected from the group consisting of lower alkyl and polarizable groups.

  In one or more embodiments, T is:

によって表され、
式中：
ｍ^１は、１−３０の整数であり；
ｍ^２は、１−３０の整数であり；
ｍ^３は、１−３の整数であり；
Ｒ^７、Ｒ^８、Ｒ^９およびＲ^１０はそれぞれ独立して、水素、ヒドロキシル、フルオロ、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、低級アルキル、保護または脱保護アルコール、双性イオン、芳香族炭化水素基およびヘテロ環芳香族炭化水素基から成る群から選択され；
Ｚは、
（ａ）式（Ｂ^１）_ｘ（Ｒ^５）_ｙ（Ｒ^６）_ｚＳｉ−を有する表面結合基であって、式中、ｘが１−３の整数であり、ｙが０−２の整数であり、ｚが０−２の整数であり、ｘ＋ｙ＋ｚ＝３であり；Ｒ^５およびＲ^６がそれぞれ独立してメチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、置換または非置換アリール、環式アルキル、分枝アルキル、低級アルキル、保護または脱保護アルコール、双性イオン基およびシロキサン結合から成る群から選択され；ならびにＢ^１がシロキサン結合である、表面結合基；
（ｂ）直接炭素−炭素結合形成を介したまたはヘテロ原子、エステル、エーテル、チオエーテル、アミン、アミド、イミド、尿素、カーボネート、カルバメート、ヘテロ環、トリアゾールもしくはウレタン結合を介した表面有機官能性ハイブリッド基への結合；または
（ｃ）材料の表面に共有結合していない、吸着された表面基であり；
Ｙは、埋め込み極性官能基であり；
Ｄは、結合、Ｎ、Ｏ、Ｓ、
−（ＣＨ_２）_０−１２−Ｎ−Ｒ^１１Ｒ^１２、
−（ＣＨ_２）_０−１２−Ｏ−Ｒ^１１、
−（ＣＨ_２）_０−１２−Ｓ−Ｒ^１１、
−（ＣＨ_２）_０−１２−Ｎ−（ＣＨ_２）_０−１２−Ｒ^１１Ｒ^１２、
−（ＣＨ_２）_０−１２−Ｏ−（ＣＨ_２）_０−１２−Ｒ^１１、
−（ＣＨ_２）_０−１２−Ｓ−（ＣＨ_２）_０−１２−Ｒ^１１、
−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｎ−Ｒ^１１Ｒ^１２、
−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｏ−Ｒ^１１、
−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｓ−Ｒ^１１；
−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｎ−（ＣＨ_２）_０−１２−Ｒ^１１Ｒ^１２、
−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｏ−（ＣＨ_２）_０−１２−Ｒ^１１および
−（ＣＨ_２）_０−１２−Ｓ（Ｏ）_１−２−（ＣＨ_２）_０−１２−Ｓ−（ＣＨ_２）_０−１２−Ｒ^１１から成る群から選択され；
Ｒ^１１は、第１のモノ芳香族基、ポリ芳香族基、ヘテロ環芳香族基またはポリヘテロ環芳香族基であり；ならびに
Ｒ^１２は、水素、脂肪族基または第２のモノ芳香族基、ポリ芳香族基、ヘテロ環芳香族基もしくはポリヘテロ環芳香族基であり、式中、Ｒ^１１およびＲ^１２は、脂肪族基、ハロゲン、ヒドロキシル基、チオール基、アミノ基、アルコキシル基、アルキルカルボキシ基、アルキルチオ基およびニトロ基から成る群から選択される１個以上の基によって場合により置換される。

Represented by
In the formula:
m ¹ is an integer of 1-30;
m ² is an integer from 1-30;
m ³ is an integer of 1-3;
R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are each independently hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, protected or deprotected Selected from the group consisting of alcohols, zwitterions, aromatic hydrocarbon groups and heterocyclic aromatic hydrocarbon groups;
Z is
(A) a surface binding group having the formula (B ¹ ) _x (R ⁵ ) _y (R ⁶ ) _z Si—, wherein x is an integer of 1-3 and y is an integer of 0-2 And z is an integer of 0-2 and x + y + z = 3; R ⁵ and R ⁶ are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, substituted Or a surface binding group selected from the group consisting of unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, protected or deprotected alcohol, zwitterionic group and siloxane bond; and B ¹ is a siloxane bond;
(B) Surface organofunctional hybrid groups directly through carbon-carbon bond formation or through heteroatoms, esters, ethers, thioethers, amines, amides, imides, ureas, carbonates, carbamates, heterocycles, triazoles or urethane bonds Or (c) an adsorbed surface group that is not covalently bonded to the surface of the material;
Y is an embedded polar functional group;
D is a bond, N, O, S,
^{_{- (CH 2) 0-12 -N-}} R 11 R 12,
^{_{- (CH 2) 0-12 -O-}} R 11,
^{_{- (CH 2) 0-12 -S-}} R 11,
_{- (CH 2) 0-12 -N- (} CH 2) 0-12 -R 11 R 12,
_{- (CH 2) 0-12 -O- (} CH 2) 0-12 -R 11,
_{- (CH 2) 0-12 -S- (} CH 2) 0-12 -R 11,
_{- (CH 2) 0-12 -S (} O) 1-2 - (CH 2) 0-12 -N-R 11 R 12,
_{- (CH 2) 0-12 -S (} O) 1-2 - (CH 2) 0-12 -O-R 11,
_{- (CH 2) 0-12 -S (} O) 1-2 - (CH 2) 0-12 -S-R 11;
_{- (CH 2) 0-12 -S (} O) 1-2 - (CH 2) 0-12 -N- (CH 2) 0-12 -R 11 R 12,
_{- (CH 2) 0-12 -S (} O) 1-2 - (CH 2) 0-12 -O- (CH 2) 0-12 -R 11 and _{- (CH 2) 0-12 -S (} O _{) 1-2 - (CH 2) 0-12} -S- ( selected from the group consisting of _{CH 2)} 0-12 ^{-R 11;}
R ¹¹ is first mono-aromatic group, polyaromatic group, a heterocyclic aromatic group or polyheterocyclic aromatic group; and R ¹² is hydrogen, an aliphatic group or the second mono-aromatic group, A polyaromatic group, a heteroaromatic group or a polyheteroaromatic group, wherein R ¹¹ and R ¹² are an aliphatic group, halogen, hydroxyl group, thiol group, amino group, alkoxyl group, alkylcarboxy group Optionally substituted with one or more groups selected from the group consisting of alkylthio groups and nitro groups.

In some embodiments, the first or second monoaromatic group, polyaromatic group, heteroaromatic group, or polyheteroaromatic group of R ¹¹ and R ¹² has at least two aromatic rings. It can be a polyaromatic or polyheterocyclic aromatic hydrocarbon. The first or second monoaromatic group, polyaromatic group, heterocyclic aromatic group or polyheteroaromatic group of R ¹¹ and R ^{12 is} a polyaromatic or polyheterocyclic aromatic group having at least three aromatic rings. It can also be a group hydrocarbon. The first or second monoaromatic group, polyaromatic group, heterocyclic aromatic group or polyheteroaromatic group of R ¹¹ and R ^{12 is} a polyaromatic or polyheterocyclic aromatic group having at least four aromatic rings. It can also be a group hydrocarbon.

In one embodiment, the first or second monoaromatic group, polyaromatic group, heterocyclic aromatic group or polyheteroaromatic group of R ¹¹ or R ¹² is furan, pyrrole, pyrroline, oxazole, thiazole, Imidazole, imidazoline, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, thiophene, indene, indolizine, indole, isoindole, indoline, indazole, benzimidazole, benzothiazole ( benzthiazole), naphthalene, quinolidine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, quinuclidine, fluorene, carbazole Anthracene, acridine, phenazine (phanazine), it is selected phenothiazine, phenoxazine, from the group consisting of pyrene and its derivatives, wherein said group is substituted by an aliphatic group by is unsubstituted or.

In other embodiments, the first or second monoaromatic group, polyaromatic group, heteroaromatic group, or polyheteroaromatic group of R ¹¹ or R ¹² is at least one C ₁ -C ₂₄ fatty acid. It can be substituted by a group. In particular, said group, at least one _C 2 _{-C 22} aliphatic group, one _C 3 _{-C 20} aliphatic group, one _C 4 _{-C 18} aliphatic group, one _C 5 -C ₁₆ aliphatic groups, 1 C ₆ -C ₁₄ aliphatic group, 1 C ₇ -C ₁₂ aliphatic group, 1 C ₈ -C ₁₀ aliphatic group or one of the above-mentioned carbon lengths Can be substituted by a combination of aliphatic groups such as C ₈ -C ₁₈ aliphatic groups or other various sized groups as described in this disclosure.

R ¹¹ or R ¹² is aminoanthracene (eg 1-aminoanthracene, 2-aminoanthracene or 9-aminoanthracene) or methylaminoanthracene (eg 1-methylaminoanthracene, 2-methylaminoanthracene or 9-methylaminoanthracene) Can be. Aminoanthracene or methylaminoanthracene can be substituted on the ring structure by one or more aliphatic groups, such as lower alkyl. In some embodiments, the aminoanthracene or methylaminoanthracene can have the formula (X) -amino- (Y) -alkyl-anthracene or (X) -methylamino- (Y) -alkyl-anthracene, Wherein X is 1, 2 or 9, and Y is 1-10 representing the carbon position on the anthracene (eg 1-amino-1-methyl-anthracene; 1-amino-2-methyl-anthracene 1-amino-3-methyl-anthracene; 1-amino-4-methyl-anthracene; 1-amino-5-methyl-anthracene; 1-amino-6-methyl-anthracene; 1-amino-7-methyl-anthracene 1-amino-8-methyl-anthracene; 1-amino-9-methyl-anthracene; 1-amino-10-methyl-a 1-methylamino-1-methyl-anthracene; 1-methylamino-3-methyl-anthracene; 1-methylamino-4-methyl-anthracene; 1-methylamino 1-methylamino-6-methyl-anthracene; 1-methylamino-7-methyl-anthracene; 1-methylamino-8-methyl-anthracene; 1-methylamino-9-methyl-anthracene And 1-methylamino-10-methyl-anthracene).

  In other embodiments, the aminoanthracene or methylaminoanthracene is of the formula (X) -amino- (Y) -alkyl- (Z) -alkyl-anthracene or (X) -methylamino- (Y) -alkyl- (Z ) -Alkyl-anthracene, wherein X is 1, 2 or 9, Y and Z are 1-10 representing the carbon position on the anthracene, provided that Y and Z are not the same No (eg 1-amino-1-methyl-2-methyl-anthracene; 1-amino-1-methyl-3-methyl-anthracene; 1-amino-1-methyl-4-methyl-anthracene; 1-amino-1 1-amino-1-methyl-6-methyl-anthracene; 1-amino-1-methyl-7-methyl-anthracene 1-amino-1-methyl-8-methyl - anthracene; 1-amino-1-methyl-9-methyl - anthracene; 1-amino-1-methyl-10-methyl - anthracene, etc.). The aminoanthracene or methylaminoanthracene can be disubstituted on the ring structure with a second polar group, such as an amine (eg 1-amino, 4-N, N-dimethylaminoanthracene).

R ¹¹ or R ¹² can be naphthylamine (eg 1-naphthylamine or 2-naphthylamine) or methylnaphthylamine (eg 1-methylnaphthylamine or 2-methylnaphthylamine). Naphthylamine or methylnaphthylamine can be substituted by one or more aliphatic groups such as lower alkyl. In some embodiments, the naphthylamine or methylnaphthylamine can have the formula (X ′)-amino- (Y ′)-alkyl-naphthalene or (X ′)-methylamino- (Y ′)-alkyl-naphthalene. Wherein X ′ is 1 or 2, and Y ′ is 1-8 representing a carbon position on naphthalene (eg 1-amino-1-methyl-naphthalene; 1-amino-2-methyl- 1-amino-3-methyl-naphthalene; 1-amino-4-methyl-naphthalene; 1-amino-5-methyl-naphthalene; 1-amino-6-methyl-naphthalene; 1-amino-7-methyl- 1-amino-8-methyl-naphthalene; positions 9 and 10 cannot be substituted; 1-methylamino-1-methyl-naphthalene; 1-methylamino-2- 1-methylamino-3-methyl-naphthalene; 1-methylamino-4-methyl-naphthalene; 1-methylamino-5-methyl-naphthalene; 1-methylamino-6-methyl-naphthalene; Methylamino-7-methyl-naphthalene; 1-methylamino-8-methyl-naphthalene and the like).

  In other embodiments, the naphthylamine or methylnaphthylamine has the formula (X ′)-amino- (Y ′)-alkyl- (Z ′)-alkyl-naphthalene or (X ′)-methylamino- (Y ′)-alkyl. -(Z ')-alkyl-naphthalene, wherein X' is 1 or 2, Y 'and Z' are 1-10 representing the carbon position on naphthalene, provided that Y ' And Z ′ are not the same (eg 1-amino-1-methyl-2-methyl-naphthalene; 1-amino-1-methyl-3-methyl-naphthalene; 1-amino-1-methyl-4-methyl-naphthalene 1-amino-1-methyl-5-methyl-naphthalene; 1-amino-1-methyl-6-methyl-naphthalene; 1-amino-1-methyl-7-methyl-naphthalene; -1-methyl-8-methyl - naphthalene, etc.).

R ¹¹ or R ¹² is aminophenanthrene (eg 1-aminophenanthrene, 2-aminophenanthrene, 3-aminophenanthrene, 4-aminophenanthrene or 9-aminophenanthrene) or methylaminophenanthrene (eg 1-methylaminophenanthrene, 2-aminophenanthrene, Methylaminophenanthrene, 3-methylaminophenanthrene, 4-methylaminophenanthrene or 9-methylaminophenanthrene). Aminophenanthrene or methylaminophenanthrene can be substituted on the ring structure by one or more aliphatic groups, such as lower alkyl. In some embodiments, the aminophenanthrene or methylaminophenanthrene has the formula (X ″)-amino- (Y ″)-alkyl-phenanthrene or (X ″)-methylamino- (Y ″)-alkyl-phenanthrene. Wherein X is 1, 2, 3, 4 or 9 and Y is 1-10 representing the carbon position on the phenanthrene (eg 1-amino-1-methyl-phenanthrene; 1-amino 2-methyl-phenanthrene; 1-methylamino-1-methyl-phenanthrene; 1-methylamino-2-methyl-phenanthrene, etc.).

  In other embodiments, the aminophenanthrene or methylaminophenanthrene is of the formula (X ″)-amino- (Y ″)-alkyl- (Z ″)-alkyl-phenanthrene or (X ″)-methylamino- (Y ″). -Alkyl- (Z ″)-alkyl-phenanthrene, wherein X is 1, 2, 3, 4 or 9 and Y and Z are 1-10 representing the carbon position on the phenanthrene Yes (bonding carbon is not counted), but Y and Z are not the same (eg 1-amino-1-methyl-2-methyl-phenanthrene, etc.).

Similarly, R ¹¹ or R ¹² is aminopyrene (eg 1-aminopyrene, 2-aminopyrene, 3-aminopyrene, 4-aminopyrene or 5-aminopyrene) or methylaminopyrene (eg 1-methylaminopyrene, 2-methylaminopyrene). , 3-methylaminopyrene, 4-methylaminopyrene or 5-methylaminopyrene). Aminopyrene and methylaminopyrene can be substituted similarly to aminoanthracene and methylaminoanthracene.

Similarly, R ¹¹ or R ¹² is an aminochrysene (eg 1-aminochrysene, 2-aminochrysene, 3-aminochrysene, 4-aminochrysene, 5-aminochrysene or 6-aminochrysene) or methylaminochrysene (eg 1-methylaminochrysene, 2-methylaminochrysene, 3-methylaminochrysene, 4-methylaminochrysene, 5-methylaminochrysene or 6-methylaminochrysene). Aminochrysene and methylaminochrysene can be substituted similarly to aminoanthracene and methylaminoanthracene.

  In some embodiments, T is the structure:

の１つによって示され、
式中、各Ｅは独立して、結合またはヒドロキシル、フルオロ、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、低級アルキル、保護または脱保護アルコール、双性イオン、芳香族炭化水素基もしくはヘテロ環芳香族炭化水素基によって場合により置換することができる低級アルキルであり、Ｚ、Ｙ、Ｒ^９、ｍ^１、ｍ^２およびＤは、上で定義した通りである。

Indicated by one of the
Wherein each E is independently a bond or hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, protected or deprotected alcohol, zwitterion, aromatic Lower alkyl which can be optionally substituted by an aromatic hydrocarbon group or a heterocyclic aromatic hydrocarbon group, and Z, Y, R ⁹ , m ¹ , m ² and D are as defined above.

  In another embodiment, T is the structure:

の１つによって示され、
式中、Ｚ、Ｙ、Ｒ^９、ｍ^１、ｍ^２およびＤは、上で定義した通りである。

Indicated by one of the
In the formula, Z, Y, R ⁹ , m ¹ , m ² and D are as defined above.

  In some embodiments, b and c are positive numbers and the ratios 0.05 ≦ (b / c) ≦ 100 and a ≧ 0. In some embodiments, Q and T are different, but in other embodiments Q and T are the same. Q can include two or more different parts, and T can include two or more different parts. In some embodiments, the first, second, third, fourth and fifth fractions are each independently about 0-100, 1-99, 5-95, 10-90, 20-80. 30-70, 40-60, 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% is there.

  In one or more embodiments, Q is nonpolar. In some embodiments, Q comprises a borate or nitro functional group. In some embodiments, Q is

の１つによって表され、
式中、Ｚは、式（Ｂ^１）_ｘ（Ｒ^５）_ｙ（Ｒ^６）_ｚＳｉ−を有する表面結合基を含むことができ、ｘは１−３の整数であり、ｙは０−２の整数であり、ｚは０−２の整数であり、ｘ＋ｙ＋ｚ＝３である。Ｒ^５およびＲ^６の各出現は独立して、メチル、エチル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、イソプロピル、テキシル、置換または非置換アリール、環式アルキル、分枝アルキル、低級アルキル、保護または脱保護アルコールまたは双性イオン基を表すことができ、Ｂ^１はシロキサン結合を表すことができる。

Represented by one of the following:
Wherein Z can include a surface binding group having the formula (B ¹ ) _x (R ⁵ ) _y (R ⁶ ) _z Si—, where x is an integer of 1-3 and y is 0-2. Z is an integer of 0-2, and x + y + z = 3. Each occurrence of R ⁵ and R ⁶ is independently methyl, ethyl, n-butyl, isobutyl, tert-butyl, isopropyl, texyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, protected or A deprotected alcohol or zwitterionic group can be represented and B ¹ can represent a siloxane bond.

  In another embodiment, Z is directly via carbon-carbon bond formation or via a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole or urethane bond. Bond to surface organofunctional hybrid group. In yet another embodiment, Z is an adsorbed surface group that is not covalently bonded to the surface of the material.

  In some embodiments, T is

の１つによって表され、
式中、Ｚは、式（Ｂ^１）_ｘ（Ｒ^５）_ｙ（Ｒ^６）_ｚＳｉ−を有する表面結合基を含むことができ、ｘは１−３の整数であり、ｙは０−２の整数であり、ｚは０−２の整数であり、ｘ＋ｙ＋ｚ＝３である。Ｒ^５およびＲ^６の各出現は独立して、メチル、エチル、ｎ−プロピル、イソプロピル、ｎ−ブチル、イソブチル、ｔｅｒｔ−ブチル、置換または非置換アリール、環式アルキル、分枝アルキル、低級アルキル、保護または脱保護アルコール、双性イオン基およびシロキサン結合を表すことができ、Ｂ^１はシロキサン結合を表すことができる。幾つかの実施形態において、Ｚは、直接炭素−炭素結合形成を介したまたはヘテロ原子、エステル、エーテル、チオエーテル、アミン、アミド、イミド、尿素、カーボネート、カルバメート、ヘテロ環、トリアゾールもしくはウレタン結合を介した表面有機官能性ハイブリッド基への結合である。幾つかの実施形態において、Ｚは、材料の表面に共有結合していない、吸着された表面基である。

Represented by one of the following:
Wherein Z can include a surface binding group having the formula (B ¹ ) _x (R ⁵ ) _y (R ⁶ ) _z Si—, where x is an integer of 1-3 and y is 0-2. Z is an integer of 0-2, and x + y + z = 3. Each occurrence of R ⁵ and R ⁶ is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, Protected or deprotected alcohols, zwitterionic groups and siloxane bonds can be represented, and B ¹ can represent siloxane bonds. In some embodiments, Z is through direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or urethane bond. Bond to the surface organofunctional hybrid group. In some embodiments, Z is an adsorbed surface group that is not covalently bonded to the surface of the material.

In some embodiments, the first or second monoaromatic group, polyaromatic group, heterocyclic aromatic group or polyheteroaromatic group of R ¹¹ or R ¹² can be converted to a cycloolefin. For example, a pyridine group can be converted to a cycloolefin in a solution under certain conditions. In certain circumstances, the cycloolefin retains sufficient unsaturation to retain and separate the structurally related compound of interest from the mixture. In certain circumstances, stationary phases of the present disclosure containing cycloolefins can retain, separate and split critical pairs associated with vitamins (eg, D2 and D3, K1 and K2).

  Q and T substituents can also be polymerized. The Q and T substituents can each be polymerized themselves, for example QQ, TT or can be polymerized to each other, for example QT. As shown in FIG. 4, polymerization can occur at the surface level between siloxane groups, between hydrocarbon substituents, or both. Polymerization between the substituents produces a cross-linked surface coating or a second coating on the surface coating, such as a second layer of substituents that can be polymerized or not polymerized, or a mixture of both. The degree of polymerization of the Q and T substituents can be varied. For example, the first fraction of Q can bind to X and the second fraction of Q can polymerize. Similarly, the first fraction of T can bind to X and the second fraction of T can polymerize. In another embodiment, the first fraction of Q can bind to X, the second fraction of Q can polymerize, and the third fraction of T can bind to X. And the fourth fraction of T can be polymerized. The polymerized portions of Q and T can polymerize to themselves or to each other.

  In one or more embodiments of any of the above aspects, X is a high purity chromatographic material having a core surface that undergoes alkoxylation by a chromatographic mobile phase under chromatographic conditions. X can be a chromatographic material having a core surface that undergoes alkoxylation by chromatographic mobile phase under chromatographic conditions. In some embodiments, the functional group comprising Q is a diol. The functional group comprising T can be an amine, ether, thioether or a combination thereof. T can contain a chiral functional group suitable for chiral separation, Q can contain a chiral functional group suitable for chiral separation, or both T and Q can contain a chiral functional group suitable for chiral separation Can do.

In one or more embodiments of the above aspect, the ratio b / c is about 0.05-75, 0.05-50, 0.1, 0.2, 0.3, 0.4, 0.5. , 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80 or 90. In some embodiments, the surface of X does not include silica and b = 0 or c = 0. In some embodiments, the total surface coverage is about 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2. 9, 3.0, 3.1, 3.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 5, 6, 7, or 8 μmol / m ² Over.

  In some embodiments of the above aspect, the chromatography stationary phase is ≦ 5% over 30 days, ≦ 4% over 30 days, ≦ 3% over 30 days, ≦ 2% over 30 days, ≦ 1 over 30 days % For 10 days ≤ 5%, 10 days ≤ 4%, 10 days ≤ 3%, 10 days ≤ 2%, 10 days ≤ 1%, 3 days ≤ 5%, 3 days ≤ 4% ≤3% over 3 days ≤2% over 3 days ≤1% over 3 days, ≤5% over 30 runs, ≤4% over 30 runs, ≤3% over 30 runs, 30 ≦ 2% over 30 runs, ≦ 1% over 30 runs, ≦ 5% over 10 runs, ≦ 4% over 10 runs, 10 runs Over ≤3%, over 10 runs ≤2%, over 10 runs ≤1%, over 3 runs ≤5%, over 3 runs ≤4%, over 3 runs ≤3% It shows a drift or change in retention of ≦ 2% over 3 runs or ≦ 1% over 3 runs.

  In some embodiments, the core material consists essentially of a silica material. In some cases, the core material consists essentially of an organic-inorganic hybrid material or a surface porous material. In one or more embodiments, the core material consists essentially of an inorganic material having a hybrid surface layer, a hybrid material having an inorganic surface layer, a surrounding hybrid layer, or a hybrid material having a different hybrid surface layer. The stationary phase material can optionally be in the form of a plurality of particles, a monolith or a surface porous material. In some embodiments, the stationary phase material does not have a chromatographically enhanced pore shape, while in other embodiments, the stationary phase material has a chromatographically enhanced pore shape. The stationary phase material can be in the form of a spherical material, a non-spherical material (eg including toroids, polyhedra). In certain embodiments, it has a stationary phase material, a highly spherical core form, a bar shaped core form, a curved rod shaped core form, a toroid shaped core form or a dumbbell shaped core form. In certain embodiments, the stationary phase material has a mixture in the form of highly spherical, rod-shaped, curved rod-shaped, toroid-shaped, or dumbbell-shaped.

In some embodiments, the stationary phase material has a surface area of about 25 to 1100 m ² / g, about 150 to 750 m ² / g, or about 300 to 500 m ² / g. In some embodiments, the stationary phase material has a pore volume of about 0.2 to 2.0 cm ³ / g or about 0.7 to 1.5 cm ³ / g. In some embodiments, the stationary phase material has a micropore surface area of less than about 105 m ² / g, less than about 80 m ² / g, or less than about 50 m ² / g. The stationary phase material can have an average pore size of about 20 to 1500 inches, about 50 to 1000 inches, about 60 to 750 inches or about 65 to 200 inches. In some embodiments, the plurality of particles have a size of about 0.2 to 100 microns, about 0.5 to 10 microns, or about 1.5 to 5 microns.

In one or more embodiments, X comprises a silica core, c = 0, and Q is ≧ 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5. Has a total surface coverage of 4.0, 4.5 or 5 μmol / m ² , or X comprises a non-silica or silica-organic hybrid core, c = 0, Q is ≧ 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1. Having a total surface coverage of 8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5 μmol / m ² , or b> 0, c> 0 and Q is ≧ 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, It has a total surface coverage of .5,4.0,4.5 or 5 [mu] mol / ^{m 2.}

In other embodiments, Q is ≧ 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, It has a total surface coverage of 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 μmol / m ² . In other embodiments, T is ≧ 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, It has a total surface coverage of 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0 or 3.5 μmol / m ² . T from the from about 0.1 to about 4.0μmol / ^{m 2} or about 0.2 to about 3.9μmol / ^{m 2} or about 0.3 to about 3.8μmol / ^{m 2} or about 0.4 to about 3. 7 μmol / m ² or about 0.5 to about 3.6 μmol / m ² or about 1.0 to about 3.5 μmol / m ² or about 1.2 to about 3.0 μmol / m ² or any combination of values; For example, it also has a total surface coverage of about 3.0 to about 4.0 μmol / m ² .

In other embodiments, the total coverage of Q and T is ≧ 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1 .3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 5.5, 6.0 or 6.5 μmol / m ² .

  Chromatographic stationary phase can be normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide chromatography, hydrophilic interaction liquid chromatography or hydrophobic It can be adapted for interactive liquid chromatography.

  Chromatographic stationary phase may include radial tuned pores, non-radial tuned pores, regular pores, irregular pores, monodisperse pores, non-monodisperse pores, smooth surfaces, rough surfaces or combinations thereof it can. In one or more embodiments, T has one ionic group, T has more than one ionic group, and T has two or more ionic groups of the same pKa. Or T has two or more ionic groups of different pKa.

In another embodiment, the present disclosure provides the following structure (i):
[X] (W) _a (Q) _b (T) _c (i)
A chromatographic stationary phase having
Where X is a chromatographic substrate comprising a group of silica, metal oxides, inorganic-organic hybrid materials, block copolymers or combinations thereof, W is selected from the group consisting of hydrogen and hydroxyl, and W is Q is the first substituent that binds to the surface of X and Q minimizes the change in retention of the analyte over time under chromatographic conditions with a low moisture concentration, and T is chromatographically the analyte. A second substituent to be held, wherein T has one or more aromatic groups, polyaromatic groups, heterocyclic aromatic groups or polyheteroaromatic hydrocarbon groups, each group represented by an aliphatic group Optionally substituted, and b and c are positive numbers, 0.05 ≦ (b / c) ≦ 100 and a ≧ 0.

  In another embodiment, the disclosure provides normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography. Relates to a column, capillary column, microfluidic device or apparatus for chromatography or hydrophobic interaction liquid chromatography, said device or apparatus comprising a housing having at least one wall defining a chamber having an inlet and an outlet, and in the housing A stationary phase as described in the present disclosure, comprising normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography , Containing carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction conforms to liquid chromatography housing and stationary phase.

  In another embodiment, the disclosure provides normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography. A kit for graphic or hydrophobic interaction liquid chromatography, the kit comprising a housing having at least one wall defining a chamber having an inlet and an outlet, as described in the present disclosure disposed within the housing The stationary phase is normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide chromatography, hydrophilic Suitable for interaction liquid chromatography or hydrophobic interaction liquid chromatography; and normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography using the housing and stationary phase, Includes instructions for performing critical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography.

  The kit can include a housing having at least one wall defining a chamber having an inlet and an outlet, and a stationary phase according to any embodiment of the present disclosure disposed within the housing. The device can have a preformed frit, a frit produced by an interconnect material, or a frit-free device. Housing and stationary phase can be normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide chromatography, hydrophilic interaction liquid chromatography or hydrophobicity It can be adapted for interactive liquid chromatography or a combination thereof. In addition, normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide chromatography, hydrophilic interaction liquid chromatography using the housing and stationary phase Alternatively, instructions for performing hydrophobic interaction liquid chromatography or a combination thereof can be included.

  Thus, the kits of the present disclosure can be used to carry out the methods of the invention described herein. In addition, the kits of the present invention can be used to analyze a wide variety of different samples and sample types, including those described below.

  In one or more embodiments, the present invention can contemplate kits containing aspects of the present disclosure to reduce or alleviate the effects of retention drift or change. For example, the kit can contain a chromatography column packed with a stationary phase medium of the present disclosure. In some embodiments, packed columns can be used directly in standard chromatography systems (eg, commercially available chromatography systems such as the Waters Acquity® chromatography system). The kit can further contain instructions for use. In addition, the kit may further contain a pure analyte stock sample for instrument calibration and / or to confirm that there is substantially no retention drift or change. The kit can include any of the components described above (eg, stationary phase, packed column or chromatography device) to reduce retention drift or changes.

  In another embodiment, the disclosure provides normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography. A method of reducing or preventing retention drift in chromatography or hydrophobic interaction liquid chromatography, wherein a sample is chromatographically separated using a chromatography device comprising a chromatographic stationary phase as described in this disclosure. Relates to a method comprising reducing or preventing retention drift.

  In one or more embodiments, the mitigation or prevention of retention drift or change is ≦ 5% over 30 days, ≦ 4% over 30 days, ≦ 3% over 30 days, ≦ 2% over 30 days, over 30 days ≤1%, 10 days ≤5%, 10 days ≤4%, 10 days ≤3%, 10 days ≤2%, 10 days ≤1%, 3 days ≤5%, 3 days ≤ 4%, 3 days ≤ 3%, 3 days ≤ 2%, 3 days ≤ 1%, 30 runs ≤ 5%, 30 runs ≤ 4%, 30 runs ≤ 3% ≦ 2% over 30 runs, ≦ 1% over 30 runs, ≦ 5% over 10 runs, ≦ 4% over 10 runs, 10 ≦ 3% over 10 runs ≦ 2% over 10 runs ≦ 1% over 10 runs ≦ 5% over 3 runs ≦ 4% over 3 runs ≦ 4% over 3 runs ≦ 3 %, Hold drift or change ≦ 2% over 3 runs or ≦ 1% over 3 runs. In some embodiments, reducing or preventing retention drift or change comprises substantially eliminating the effect of alkoxylation and / or dealkoxylation of the chromatographic material on retention.

  The concept of chemically modified chromatographic core material, as used herein, is understood to include functionalizing the chromatographic core with, for example, polar silanes or other functional groups to reduce or avoid retention drifts or changes. Is done. For example, functionalization can essentially interfere with the chromatographic interaction between the analyte and the chromatographic core (eg, effectively eliminates the chromatographic effects of core surface silanol and / or alkoxylated silanol). In some cases, functionalization (eg, using non-polar groups) can reduce column retention. Thus, in various embodiments, the functionalization of the chromatographic core material is a hydrophilic group, polar group that interacts chromatographically with the analyte to preserve or achieve chromatographically useful overall retention. Use of ionizable groups and / or charged functional groups. Such end-capping groups can be introduced, for example, by standard coupling chemistry.

  In some embodiments, the functionalization provides a permanent bond. It is therefore important to select an appropriate functionalization for the chromatographic phase. In preferred embodiments, the chromatographic material has properties that are chromatographically desirable (eg, overall retention). Thus, in some embodiments, it is important to select a functionalization that has properties that can mimic desirable (eg, overall retention) properties of conventional chromatographic materials.

  In various embodiments, the chemical properties of the functional group can be selected to achieve the desired effect. For example, with one or more hydrophilic groups, polar groups, ionizable groups and / or charged functional groups, the desired interaction with the analyte (eg chromatographically acceptable retention) and / or mobile phase Desired interactions (eg, elimination of alcohols that can alkoxylate the chromatographic core surface). Similarly, the size and / or conformation of the end cap can be selected to mask the core surface and / or perform chiral separation.

  Similarly, the concentration of functionalization can be varied. In some embodiments, larger and / or stronger interacting functional groups can reduce or avoid retention drifts or changes at lower concentrations (eg, compared to smaller functional groups). In other embodiments, the coverage can be adjusted to the desired characteristics. For example, nonpolar functional groups can be used at a lower coverage than polar functional groups (eg, to maintain desirable retention). In various embodiments, the functionalization can use one or more polar or non-polar end caps or combinations thereof. In some embodiments, the surface area of the chromatography media is increased or decreased to compensate for the decrease or increase in retention due to the altered polarity of the functional group.

  In another embodiment, the present disclosure is a method of preparing a stationary phase as described in the present disclosure, wherein a chromatography substrate is reacted with a silane coupling agent having a pendant reactive group, Reacting the second chemical agent containing the above aromatic hydrocarbon group, polyaromatic hydrocarbon group, heteroaromatic hydrocarbon group or polyheteroaromatic hydrocarbon group with the pendant reactive group, and remaining The method comprises neutralizing any unreacted pendant reactive groups to produce a stationary phase.

  In another embodiment, the present disclosure is a method of preparing a stationary phase as described in the present disclosure, comprising oligomerizing a silane coupling agent having a pendant reactive group, the core surface of the oligomerized silane cup Reacting with a ring agent, reacting a second chemical agent comprising one or more aromatic groups, polyaromatic groups, heteroaromatic groups or polyheteroaromatic hydrocarbon groups with the pendant reactive groups. As well as a process comprising neutralizing any remaining unreacted pendant reactive groups to form a stationary phase.

  In one or more embodiments, Q is the structure:

の１つ（ｏｎｅ ｏｒ ｔｈｅ）を有する試薬に由来する。

From one or the other.

  In some embodiments, Y is the following structure:

の１つを含む。

One of these.

In the formula, each of the R ⁶ group and the R ⁷ group bonded to the Y group is an aliphatic group. In certain embodiments, the Y group may be a bond or an aliphatic group.

  The above method can be used to make any of the materials as described herein (eg, chromatographic stationary phase materials). For example, the disclosed method can react a chromatographic stationary phase (eg, silica particles) with a chemical reagent (eg, any of the above-described reagents as described herein) to chemically modify the surface of the stationary phase. And a method of mitigating the effects of retention drift or change.

  The present disclosure includes various apparatuses (eg, chromatography columns, capillaries and microfluidic devices and systems for their use) that include the chromatographic materials described herein. Although several illustrative examples are discussed below, one of ordinary skill in the art will recognize that the present disclosure is not limited to this, but includes a number of different implementations, including chromatography columns, devices, methods of use or kits. Understand that the form can be considered.

  In some embodiments, the present disclosure includes normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interactive liquids. A column or apparatus for chromatography or hydrophobic interaction liquid chromatography or a combination thereof is provided. The column or apparatus includes a housing having at least one wall defining a chamber having an inlet and an outlet, and a stationary phase according to any embodiment of the present disclosure disposed within the housing. The device can have a preformed frit, a frit produced by an interconnect material, or a frit-free device. Housing and stationary phase can be normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide chromatography, hydrophilic interaction liquid chromatography or hydrophobicity It can be adapted for interactive liquid chromatography or a combination thereof.

  Accordingly, an apparatus of the present disclosure contains a material of the present disclosure (eg, a chromatographic stationary phase such as a chemically modified stationary phase adapted to reduce or mitigate retention drift or change). )can do. Furthermore, the apparatus of the present invention can be used to carry out the method of the present invention as described herein.

  In one embodiment, the present disclosure is in the form of a packed column. The column can be packed with a stationary phase (eg, chromatographic material) as described herein. Such columns are capable of different types of chromatography (eg normal phase chromatography, supercritical fluid chromatography, carbon dioxide chromatography, hydrophobic interaction liquid chromatography, while reducing or avoiding retention drifts or changes, Hydrophilic interaction liquid chromatography, subcritical fluid chromatography, high pressure liquid chromatography and solvation gas chromatography) can be used.

Column, Waters Alliance (R) HPLC system, including Waters Acquity (R) system or Waters ^UPC 2 (R) system, can be used in combination with existing chromatographic platforms such as the commercially available chromatographic systems. The columns of the present disclosure can be used for a number of different mass throughputs (eg, analytical scale chromatography, preparative scale chromatography) while mitigating the effects of retention drift or change. Similarly, the present disclosure can be embodied in capillaries and microfluidic devices and systems for their use (eg, commercially available and known to those skilled in the art). The selection of columns, capillaries and microfluidic devices and related systems can be readily understood by those skilled in the art.

  In various embodiments, materials according to the present disclosure can have application in microbore columns for use in SFC, HPLC and / or UHPLC systems. In various embodiments, materials according to the present disclosure may have application in SFCs having fast equilibration columns, long life columns, water stable columns.

  Using this disclosure, compounds of multiple different subjects from many different samples from many different fields such as clinical chemistry, medicine, veterinary medicine, forensic chemistry, pharmacology, food industry, occupational safety and environmental contamination Can be retained, separated and / or analyzed. Samples include, but are not limited to, small organic molecules, proteins, nucleic acids, lipids, fatty acids, carbohydrates, polymers, and the like. Similarly, the disclosure includes small molecules, polar small molecules, analytes for use in pharmaceuticals, biomolecules, antibodies, polymers and oligomers, saccharides, glycan analysis, petrochemical analysis, lipid analysis, peptides, phosphopeptides, oligonucleotides , DNA, RNA, polar acids, polyaromatic hydrocarbons, food analysis, chemical analysis, bioanalysis, drug abuse, forensic medicine, pesticides, pesticides, biosimilars, can be used for separation of formulations.

  Analytes suitable for chromatographic separation according to the present disclosure can include essentially any molecule of interest, including, for example, small organic molecules, lipids, peptides, nucleic acids, synthetic polymers.

  The target analyte of clinical chemistry can include any molecule present in an organism (eg, human body, animal body, fungi, bacteria, virus, etc.). For example, clinical chemistry target analytes include, but are not limited to, proteins, metabolites, biomarkers, and drugs.

  Human and veterinary target analytes can include any molecule that can be used in the diagnosis, prevention or treatment of a disease or condition of interest. For example, human and veterinary target analytes include, but are not limited to, disease markers, prophylactic or therapeutic agents.

  Forensic chemistry target analytes include any molecule present in a sample taken from a crime scene, such as a sample from a victim's body (eg, tissue or fluid sample, hair, blood, semen, urine, etc.) Can be mentioned. For example, clinical chemistry target analytes include, but are not limited to, toxicants, drugs and their metabolites, biomarkers and identifying compounds.

  A target analyte in pharmacology can include any molecule that is a pharmaceutical product or metabolite thereof or that can be used for drug design, synthesis, and monitoring. For example, pharmacological target analytes include, but are not limited to, prophylactic and / or therapeutic agents, prodrugs, intermediates and metabolites thereof. Pharmacological analysis can include bioequivalence testing, for example in connection with generic drug approval, manufacturing and monitoring.

  Food industry and agricultural target analytes can include any molecule associated with the monitoring of food, beverage and / or other food industry / agricultural product safety. Examples of target analytes from the field of the food industry include, but are not limited to, pathogen markers, allergens (eg, gluten and nut proteins) and mycotoxins.

  Target analytes include polypeptides (eg, naturally and / or non-naturally occurring amino acids such as Gly, Ala, Val, Leu, Ile, Pro, Phe, Trp, Cys, Met, Ser, Thr, Tyr, His, Lys, Arg, Asp, Glu, Asn, Gln, selenocysteine, ornithine, citrulline, hydroxyproline, methyllysine, carboxyglutamate polymer), peptide, protein, glycoprotein, lipoprotein; peptide-nucleic acid; hormone (eg peptide hormone ( Mention may be made, for example, of TRH and vasopressin) as well as synthetic and industrial polypeptides.

  In some embodiments, the subject compound is a saturated or unsaturated lipid, vitamin, or polycyclic aromatic hydrocarbon. The term “saturated” as used herein refers to a component that does not contain a double bond at the acyl site of the molecule. The term “saturated lipid” as used herein refers to fat and oil components that do not contain a double bond at the acyl chain site of the molecule. The term “unsaturated” as used herein refers to a component that contains one or more sites of unsaturation in the molecule. The term “unsaturated lipid” as used herein refers to the components of fats and oils that contain one or more sites of unsaturation in the molecule. They can occur in the fatty acid part of molecules such as triglycerides, phospholipids and glycolipids, or in the alkyl chains in the molecule, for example in carotenoids, hydrocarbons and fat-soluble vitamins. The lipid can be selected from the group consisting of saturated and unsaturated fatty acids, phospholipids, glycerolipids, glycerophospholipids, lysophosphoglycerolipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, carotenoids, waxes and polyketides.

  In another embodiment, the present disclosure can be used to retain, separate and resolve polycyclic aromatic hydrocarbons and related compounds. Polycyclic aromatic hydrocarbons (PAH) form a family of non-functionalized aromatic compounds composed of fused aromatic rings. About 2000 compounds are classified as PAH. PAH and its derivatives are widespread in the environment as a result of combustion processes such as fossil fuel combustion. They bind strongly to soil organic matter (humic acids) and their degradation rate in the soil and other environmental compartments is usually low. Furthermore, the PAH that reaches the water stream migrates rapidly into the sediment.

  PAH is also produced during home and industrial combustion processes such as extraction of vegetable oils, herbs, spices and other food ingredients, smoked food ingredients and grilled foods. PAH is a toxic and carcinogenic compound. Controlling the presence and level of PAH has become increasingly important in the context of food safety and health and safety regulations. Some of the effects of PAH are enzyme induction, immunosuppression, teratogenicity and tumor promotion.

  In certain embodiments, the lipid can be a saturated or unsaturated fatty acid, monoacyl glyceride, diacyl glyceride, triacyl glyceride, phospholipid or steroid. Triglycerides (TG, triacylglycerol, TAG or triacylglycerides) are esters derived from glycerol and three fatty acids. As blood lipids, they can help bi-directional movement of adipocyte lipids and blood glucose from the liver. There are many triglycerides depending on the oil source, some are highly unsaturated and some are less unsaturated. Triglycerides are the major constituents of vegetable oils (usually more unsaturated) and animal fats (usually more saturated). Triglycerides are the main component of human skin oil.

  Steroids are a type of organic compound that contains a characteristic arrangement of four cycloalkane rings joined together. Examples of steroids include lipid cholesterol, the sex hormones estradiol and testosterone, and the anti-inflammatory drug dexamethasone. The core of the steroid is composed of 17 carbon atoms bonded together in the form of four fused rings, ie, three cyclohexane rings and one cyclopentane ring. Steroids vary by the functional group attached to the four ring cores and by the oxidation state of the ring. Sterols are a special form of steroids with a hydroxyl group at the 3 position and a skeleton derived from cholestane.

  Hundreds of characteristic steroids have been found in plants, animals and fungi. All steroids are produced in cells from either the sterols lanosterol (animals and fungi) or cycloartenol (plants). Both lanosterol and cycloartenol are derived from cyclization of the triterpene squalene.

In other embodiments, the subject compound can be a fat-soluble vitamin selected from the group consisting of vitamin C, vitamin B, or derivatives or combinations thereof. Fat soluble vitamins are interesting because they are difficult to dissolve, require strong organic solvents and are often separated by RPLC. Typically, fat soluble vitamins are separated by use of C18 silane (ODS) and other alkyl bonded phases using SFC conditions. These methods often use a very weak co-solvent and a very high percentage of CO ₂ in the mobile phase. One advantage of the present disclosure is that the stationary phase is modified by a π-electron rich selector, such as 1-aminoanthracene. Coupling a π-electron rich selector to the stationary phase maximizes chromatographic selectivity between two critical pairs such as vitamins D2 and D3 or K1 and K2.

  Another advantage of the present disclosure is that the stationary phase has a selector with a relatively high pKa so that no acid or basic additive is required to perform the separation. In one embodiment, the present disclosure relates to a method that utilizes a mobile phase that does not include an acid additive, a basic additive, or both. In other embodiments, the present disclosure is less than 5.0%, or 4.0%, or 3.0%, or 2.0%, 1.0%, or 0.5%, or 0.2 %, Or 0.1%, or 0.05% of the process using a mobile phase containing acid additive, basic additive or both. When only carbon dioxide modified with methanol is used as a co-solvent, a high-speed and versatile method can be achieved. For example, separation of the critical pair of vitamins described above is performed using the stationary phase of the present disclosure having, for example, 1-aminoanthracene as a selector. In one embodiment, the level of splitting between critical pairs of vitamins is higher than current methods (splitting by the same column length, using particles less than 2 μm and a 50 mm column length).

  The present disclosure can be used to retain, separate and split vitamin C and related compounds. Vitamin C [2-oxo-L-threo-hexono-1,4-lactone 2,3-enediol], or L-ascorbic acid, is an essential nutrient for water-soluble vitamins and humans. Vitamin C is essential for the formation of collagen that is necessary for normal growth and development and tissue repair in all parts of the body. Vitamin C also functions as an antioxidant that prevents damage caused by free radicals and directly reduces toxic chemicals and contaminants.

  Since humans do not produce vitamin C in the body, they are mainly obtained from dietary sources such as fruits and vegetables. A deficiency in dietary vitamin C can cause vitamin C deficiency. Severe vitamin C deficiency, also known as “scurvy”, leads to the production of melasma on the skin, bleeding from the spongy gingiva and mucous membranes, or even death.

  Currently, vitamin C is used not only as a dietary supplement, but also as an adjunct therapy for some viral infections and end-stage cancer. The recommended daily intake of vitamin C for adults to prevent deficiency is 75 mg for women, 90 mg for men, and the upper limit of tolerance for both is 2,000 mg. For therapeutic applications in detoxification and cancer treatment, vitamin C is administered intravenously at much higher doses. Although vitamin C toxicity is rare clinically, ingestion of relatively high doses can lead to stomach upset and diarrhea. Vitamin C blood concentration assays have been developed and used by patients and physicians to assess nutritional status or to optimize therapeutic dosage. Measurement of these compounds is a useful indicator of vitamin C nutritional status and the efficacy of certain vitamin C analogs.

  In another embodiment, the present disclosure can be used to retain, separate and split vitamin B and related compounds. B vitamins are a group of water-soluble vitamins that play an important role in cell metabolism. B vitamins were once considered as a single vitamin and were simply called vitamin B. Later studies have shown that these are chemically distinct vitamins that often coexist in the same food. In general, dietary supplements containing all eight are called vitamin B complexes. Individual B vitamin supplements are referred to by the specific name of each vitamin (eg, B1, B2, B3, etc.). List of B vitamins include vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal or pyridoxamine or pyridoxine hydrochloride), vitamins B7 (biotin), vitamin B9 (folic acid), vitamin B12 (various cobalamins; usually cyanocobalamin in vitamin supplements).

  The role of vitamin B compounds varies. For example, thiamine plays a central role in energy generation from carbohydrates. Thiamine is involved in RNA and DNA production and neural function. The active form of thiamine is a coenzyme called thiamine pyrophosphate (TPP), which plays a role in the conversion of pyruvate to acetyl coenzyme A (CoA) in metabolism. Riboflavin is involved in the production of energy for the electron transport system, the citrate cycle and fatty acid catabolism (beta oxidation). Niacin is composed of two structures: nicotinic acid and nicotinamide. Niacin has two coenzyme forms: nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Both play an important role in energy transfer reactions in glucose, fat and alcohol metabolism. NAD carries hydrogen and its electrons during metabolic reactions including the pathway from the citrate cycle to the electron transport system. NADP is a coenzyme in lipid and nucleic acid synthesis.

  Pantothenic acid is involved in the oxidation of fatty acids and carbohydrates. Coenzyme A is synthesized from pantothenic acid and is involved in the synthesis of amino acids, fatty acids, ketones, cholesterol, phospholipids, steroid hormones, neurotransmitters (eg acetylcholine) and antibodies. Pyridoxine is normally stored in the body as pyridoxal 5'-phosphate (PLP), a coenzyme form of vitamin B6. Pyridoxine is involved in amino acid and lipid metabolism; neurotransmitter and hemoglobin synthesis and nicotinic acid (vitamin B3) production. Pyridoxine also plays an important role in gluconeogenesis. Biotin plays an important role in lipid, protein and carbohydrate metabolism. Biotin has four types of carboxylases: acetyl CoA carboxylase involved in fatty acid synthesis from acetate; propionyl CoA carboxylase involved in gluconeogenesis; β-methylcrotonyl Coa carboxylase involved in leucine metabolism; and energy, amino acid And an important coenzyme of pyruvate CoA carboxylase involved in cholesterol metabolism.

  Folic acid acts as a coenzyme in the form of tetrahydrofolate (THF) involved in the transmission of single carbon units in the metabolism of nucleic acids and amino acids. Since THF is involved in pyrimidine nucleotide synthesis, it is required, among other things, for normal cell division during pregnancy and childhood, a period of rapid growth. Folate also assists in erythropoiesis, the production of red blood cells. Vitamin B12 is involved in the cellular metabolism of carbohydrates, proteins and lipids. Vitamin B12 is essential for the production of blood cells, nerve sheaths and proteins in the bone marrow. Vitamin B12 functions as a coenzyme in intermediary metabolism for the methionine synthase reaction with methylcobalamin and the methylmalonyl CoA mutase reaction with adenosylcobalamin.

  The effects of any deficiency of these vitamins vary. Vitamin B1 thiamine deficiency causes beriberi. Symptoms of this nervous system disease include weight loss, emotional disorders, Wernicke encephalopathy (sensory cognitive impairment), weakness and pain in the extremities, irregular heartbeats and edema (swelling of body tissue). In advanced cases, heart failure and death may occur. Chronic thiamine deficiency can also cause Korsakov syndrome, an irreversible dementia characterized by amnesia and compensatory narrative.

  Vitamin B2 riboflavin deficiency causes riboflavin deficiency. Symptoms include cheilosis (cracking of the lips), severe sunlight sensitivity, stomatitis, glossitis (lingual inflammation), seborrheic dermatitis or pseudo-syphilis (especially scrotum or labia) And pharyngitis (sore throat), hyperemia and edema of the pharynx and oral mucosa.

  Vitamin B3 niacin deficiency, together with tryptophan deficiency, causes pellagra. Symptoms include aggression, dermatitis, insomnia, weakness, mental confusion and diarrhea. In advanced cases, pellagra can lead to dementia and death (3 (+1) D: dermatitis, diarrhea, dementia and death.

  Vitamin B5 pantothenic acid deficiency is uncommon but can result in acne and sensory abnormalities. Vitamin B6 pyridoxine deficiency is associated with microcytic anemia (because pyridoxyl phosphate is a cofactor for heme synthesis), depression, dermatitis, high blood pressure (hypertension), water retention and increased levels of homocysteine. It can be connected. Vitamin B7 biotin deficiency typically does not cause symptoms in adults, but can lead to infant growth and neuropathy. Complex carboxylase deficiency is an inborn error of metabolism and can lead to biotin deficiency even if dietary biotin intake is normal.

  Vitamin B9 folate deficiency results in macrocytic anemia and elevated levels of homocysteine. Deficiency in pregnant women can lead to birth defects. Supplementation during pregnancy is often recommended. Researchers have shown that folic acid can also delay the insidious effects of age on the brain. Vitamin B12 cobalamin deficiency results in macrocytic anemia, elevated homocysteine, peripheral neuropathy, memory loss and other cognitive impairments. This deficiency is most likely to occur in the elderly because absorption through the gastrointestinal tract decreases with age. Pernicious anemia, an autoimmune disease, is another common cause. This deficiency can also cause symptoms of mania and psychosis. In rare and extreme cases, paralysis may occur. Measurement of these compounds is a useful indicator of vitamin B nutritional status and the efficacy of certain vitamin B analogs.

  In other embodiments, the subject compound can be a fat-soluble vitamin selected from the group consisting of vitamin D, vitamin A, vitamin K, vitamin E, beta carotene, or derivatives or combinations thereof. The present disclosure can be used to retain, separate and split vitamin D and related compounds. Vitamin D is an essential nutrient that has an important physiological role in the positive regulation of calcium homeostasis. Vitamin D can be generated de novo in the skin by exposure to sunlight or can be absorbed from the diet. There are two forms of vitamin D; vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D3 is a form de novo synthesized by animals. Vitamin D3 is also a common dietary supplement added to dairy products and certain foods produced in the United States. Dietary vitamin D3 and endogenously synthesized vitamin D3 need to undergo metabolic activation to produce bioactive metabolites. In humans, the first step of vitamin D3 activation takes place primarily in the liver and forms the intermediate metabolite 25-hydroxycholecalciferol (calciferdiol), which is enzymatically hydroxylated at position 25. For hydroxylation. Calcifediol is the main form of vitamin D3 in the circulation. Circulating calciferdiol is then converted by the kidney to form 1,25-dihydroxyvitamin D3 (calcitriol), which is generally considered the metabolite of vitamin D3 with the highest biological activity. Vitamin D2 is derived from fungal and plant sources. Many commercially available dietary supplements contain ergocalciferol (vitamin D2) rather than cholecalciferol (vitamin D3). Dorisdole is the only high-titer formulation of vitamin D available in the United States and is formulated with ergocalciferol. Vitamin D2 forms the metabolites calciferdiol and calcitriol via a metabolic activation pathway similar to vitamin D3 in humans. Vitamin D2 and vitamin D3 have long been considered bioequivalent in humans, but recent reports indicate differences in the bioactivity and bioavailability of these two forms of vitamin D. It has been suggested that this is possible. Measurement of these compounds is a useful indicator of vitamin D nutritional status and the efficacy of certain vitamin D analogs.

  In another embodiment, the present disclosure can be used to retain, separate and split vitamin A and related compounds. Vitamin A is a group of unsaturated nutritive organic compounds including retinol, retinal, retinoic acid and multiple provitamin A carotenoids, of which beta-carotene is the most important. Vitamin A has multiple functions. Vitamin A is important for growth and development, maintenance of the immune system and good visibility. Vitamin A is required by the retina of the eye in the form of retinal, which combines with the protein opsin to form the light-absorbing molecule rhodopsin, which is required for both low light (dark vision) and color vision. . Vitamin A functions in a very different role as an irreversible oxidized form of retinol, known as retinoic acid, an important hormone-like growth factor for epithelium and other cells.

  In animal source foods, the major form of vitamin A is an ester, primarily retinyl palmitate, which is converted to retinol (chemically alcohol) in the small intestine. The retinol form serves as a storage form of vitamins and can be converted to and from its visually active aldehyde form, retinal. The related acid (retinoic acid), a metabolite that can be irreversibly synthesized from vitamin A, has only partial vitamin A activity and does not function in the retina because of the visual cycle. Retinoic acid is used for growth and cell differentiation.

  All forms of vitamin A have a beta-ionone ring with an isoprenoid chain attached, called a retinyl group. Both structural features are essential for vitamin activity. Beta carotene, the orange pigment of carrot, can be expressed as two linked retinyl groups that are used to contribute to vitamin A levels in the body. Alpha carotene and gamma carotene also have one retinyl group that gives them some vitamin activity.

  Vitamin A can be found in food in two main forms: (i) Retinol is a form of vitamin A that is absorbed when eating animal food sources and is a yellow fat-soluble substance. . Since the pure alcohol form is unstable, the vitamin is found in the form of retinyl esters in tissues. Retinol is also produced commercially and administered as an ester such as retinyl acetate or palmitate. (Ii) carotene alpha carotene, beta carotene, gamma carotene; and xanthophyll beta cryptoxanthin (all of which contain a beta ionone ring), except for other carotenoids, cleave beta carotene in the small intestinal mucosa and retinol it. It functions as provitamin A in herbivores and omnivorous animals that have an enzyme (15-15′-dioxygenase) that converts to. In general, carnivores do not fully convert ionone-containing carotenoids, and pure carnivores such as cats and ferrets lack 15-15′-dioxygenase and neither carotenoid can be converted to retinal (these ) Does not produce any carotenoids in the form of vitamin A.) Measurement of these compounds is a useful indicator of vitamin A nutritional status and the efficacy of certain vitamin A analogs.

  In another embodiment, the present disclosure can be used to retain, separate and split vitamin K and related compounds. Vitamin K is a group of structurally similar fat-soluble vitamins required by the human body for the post-translational modification of certain proteins required for blood clotting and in metabolic pathways in bone and other tissues. These are 2-methyl-1,4-naphthoquinone (3-) derivatives. This group of vitamins contains two natural vitamers: vitamin K1 and vitamin K2.

  Vitamin K1, also known as phylloquinone, phytomenadione or phytonadione, is synthesized by plants and most commonly found in green leafy vegetables because it is directly involved in photosynthesis. Vitamin K1 can be considered as the “plant form” of vitamin K. It is active in animals and can perform the classic functions of vitamin K in animals, including the activity of vitamin K in the production of blood clotting proteins. Animals can also convert vitamin K1 to vitamin K2.

  Vitamin K2 is the main storage form in animals and has multiple subtypes with different isoprenoid chain lengths. These vitamin K2 homologs are called menaquinones and are characterized by the number of isoprenoid residues in their side chains. Menaquinone is shortened to MK-n, M represents menaquinone, K represents vitamin K, and n represents the number of isoprenoid side chain residues. For example, menaquinone-4 (abbreviated as MK-4) has 4 isoprene residues in its side chain. Menaquinone-4 (also known as menatetrenone from its four isoprene residues) is the most common type of vitamin K2 in animal products, vitamin K1 in certain animal tissues (arterial wall, pancreas and testis) This is because MK-4 is usually synthesized by replacing the phytyl tail with an unsaturated geranylgeranyl tail containing 4 isoprene units, thereby yielding menaquinone-4. This homologue of vitamin K2 may have an enzymatic function distinct from that of vitamin K1.

  Bacteria in the colon (colon) can also convert K1 to vitamin K2. In addition, bacteria typically extend the isoprenoid side chain of vitamin K2 to produce a series of vitamin K2 forms, most notably the MK-11 homologue from MK-7 of vitamin K2. All forms of K2, except MK-4, can only be produced by bacteria that use these forms in anaerobic respiration. Although other bacterially derived forms of MK-7 and vitamin K2 show vitamin K activity in animals, the additional utility of MK-7 over MK-4 is unknown, if any, and is currently under investigation It is a problem.

  Three synthetic types of vitamin K, vitamins K3, K4 and K5, are known. Although the natural K1 homolog and all K2 homologs have been found to be non-toxic, the synthetic form K3 (menadione) has shown toxicity. K4 and K5 are also non-toxic. Measurement of these compounds is a useful indicator of vitamin K nutritional status and the efficacy of certain vitamin K analogs.

  In another embodiment, the present disclosure can be used to retain, separate and split vitamin E and related compounds. Vitamin E represents a group of eight fat-soluble compounds including tocopherol and tocotrienol. Of many different forms of vitamin E, γ-tocopherol is the most common in the North American diet. γ-Tocopherol can be found in corn oil, soybean oil, margarine and dressings. α-Tocopherol is the most biologically active form of vitamin E and the second most common form of vitamin E in the diet. This variant can be found most abundantly in wheat germ oil, sunflower oil and safflower oil. α-Tocopherol, as a fat-soluble antioxidant, stops the production of reactive oxygen species that are formed when fat is oxidized. An amount over 1,000 mg (1,500 IU) per day is called hypervitamin E because it can increase the risk of bleeding problems and vitamin K deficiency. Measurement of these compounds is a useful indicator of vitamin E nutritional status and the efficacy of certain vitamin E analogs.

  In general, a sample is a composition comprising at least one target analyte (eg, the class or type of analyte disclosed above together with a matrix). Samples can include solids, liquids, gases, mixtures, materials (eg, intermediate consistency of extracts, cells, tissues, organisms, etc.) or combinations thereof. In various embodiments, the sample is a body sample, an environmental sample, a food sample, a synthetic sample, an extract (eg, obtained by a separation technique) or a combination thereof.

  As the body sample, any sample derived from the body of an individual can be mentioned. In this context, the individual can be an animal, eg a mammal, eg a human. Other examples of individuals include mice, rats, guinea pigs, rabbits, cats, dogs, goats, sheep, pigs, cows or horses. The individual can be a patient, eg, an individual who is suffering from or suspected of having a disease. A body sample is a bodily fluid or body tissue collected for the purpose of chemical or medical testing, such as for testing or diagnosing disease (eg, by detecting and / or identifying the presence of a pathogen or biomarker) be able to. The body sample can also include cells, for example, pathogens or cells (eg, tumor cells) of an individual's body sample. Such body samples can be obtained by known methods including tissue biopsy (eg punch biopsy) and by collecting blood, bronchial aspirate, saliva, urine, feces or other body fluids. Exemplary body samples include body fluid, whole blood, plasma, serum, umbilical cord blood (especially blood obtained by percutaneous umbilical cord blood collection (PUBS)), cerebrospinal fluid (CSF), saliva, amniotic fluid, breast milk, secretion Examples include fluid, purulent fluid, urine, feces, meconium, skin, nails, hair, navel, stomach contents, placenta, bone marrow, peripheral blood lymphocytes (PBL) and solid organ tissue extracts.

  An environmental sample can include any sample derived from an environment such as a natural environment (eg, sea, soil, air and flora) or an artificial environment (eg, canals, tunnels, buildings). Exemplary environmental samples include water (drinking water, river water, surface water, ground water, drinking water, sewage, drainage, wastewater or leachate), soil, air, sediment, biota (eg, soil biota), Examples include flora, fauna (eg fish) and soil blocks (eg excavated material).

  The food sample can include any sample derived from food (including beverages). Such food samples are, for example, (1) to test whether the food is safe; (2) whether the food contains harmful pollutants at the time of eating (storage sample) or food (3) To check whether food contains only approved additives (eg compliance with laws and regulations); (4) Food is an essential ingredient Used for a variety of purposes including: to test whether it contains the correct level of (eg, whether the label on the food is correct); or (5) to analyze the amount of nutrients the food contains can do. Exemplary food samples include animal, plant or synthetic source edible products (eg milk, bread, eggs or meat), meals, beverages and portions thereof, eg stored samples. Food samples include fruits, vegetables, beans, nuts, oil seeds, oil fruits, cereals, tea, coffee, herbal exudates, cocoa, hops, herbs, spices, sugar plants, meat, fat, kidneys, liver, Mention may also be made of offal, milk, eggs, honey, fish and beverages.

  As a synthetic sample, any sample derived from an industrial process can be mentioned. An industrial process is a biological industrial process (eg, a process that uses genetic material that contains genetic information and can be regenerated itself or regenerated in a biological system such as a fermentation process using transfected cells) or non-biological. It can be an industrial process (eg chemical synthesis or degradation of compounds such as pharmaceuticals). Synthetic samples can be used to inspect and monitor the progress of industrial processes to determine the yield of the desired product and / or to measure the amount of by-products and / or starting materials.

Materials All reagents were used as received unless otherwise stated. Those skilled in the art will appreciate that the following suppliers and supplier equivalents exist and, therefore, the suppliers described below should not be construed as limiting.

Characterization Techniques Those skilled in the art will appreciate that the following equipment and supplier equivalents exist and, therefore, the equipment described below should not be construed as limiting.

C% values are determined by combustion analysis (CE-440 elemental analyzer; Exeter Analytical Inc., North Chelmsford, Mass.) Or a coulometric carbon analyzer (modules CM5300, CM5014, UIC Inc., Joliet, Illinois). It was measured. Bromine and chlorine content was measured by flask combustion followed by ion chromatography (Atlantic Microlab, Norcross, GA). The specific surface area (SSA), specific pore volume (SPV) and average pore size (APD) of these materials were measured using a multi-point N ₂ adsorption method (Micromeritics ASAP2400; Micromeritics Instruments Inc., Norcross, GA). . SSA was calculated using the BET method, SPV was a single point value determined for P / P ₀ > 0.98, and APD was calculated from the desorption leg of the isotherm using the BJH method. Micropore surface area (MSA) was determined as cumulative adsorption pore size data for pores less than 34 mm subtracted from specific surface area (SSA). Median mesopore diameter (MMPD) and mesopore pore volume (MPV) were measured by Mercury porosimetry (Micromeritics AutoPore II 9220 or AutoPore IV, Micromeritics, Norcross, Ga.). 04N, Norcross, GA). The particle size was measured using a Beckman Coulter Multisizer 3 analyzer (30 μm aperture, 70,000 counts; Miami, FL). The particle size (dp ₅₀ ) was measured as the 50% cumulative diameter of the volume-based particle size distribution. The width of the distribution was measured as 90% cumulative volume diameter divided by 10% cumulative volume diameter (meaning 90/10 ratio). The viscosities of these materials were measured using a Brookfield Digital Viscometer Model DV-II (Middleboro, Mass.). The pH measurements were made with an Oakton pH 100 series meter (Cole-Palmer, Vernon Hills, Ill.) and calibrated with the Orion (Thermo Electron, Beverly, Mass.) pH buffer standard at ambient temperature just prior to use. Titration is performed using a Metrohm 716 DMS Titrino automatic titrator (Metrohm, Helisau, Switzerland) and is reported as milliequivalents per gram (mequiv / g). Epoxide coverage levels were determined by titrating OH ⁻ released upon addition of sodium thiosulfate. Multinuclear ( ¹³ C, ²⁹ Si) CP-MAS NMR spectra were obtained using a Bruker Instruments Avance-300 spectrometer (7 mm dual broad band probe). The spin speed was typically 5.0-6.5 kHz, the latency was 5 seconds, and the cross-polarization contact time was 6 milliseconds. The reported ¹³ C and ²⁹ Si CP-MAS NMR spectral shifts were compared to external standard adamantane ( ¹³ C CP-MAS NMR, δ 38.55) and hexamethylcyclotrisiloxane ( ²⁹ Si CP-MAS NMR, δ-9.62). Was recorded in comparison with tetramethylsilane. Populations of different silicon environments were evaluated by spectral deconvolution using DMFit software [Massiot, D. et al. Fayon, F .; Capron, M .; King, I .; Le Calve, S .; Alonso, B .; Durand, J .; -O. Bujoli, B .; Gan, Z .; Hotson, G .; Magn. Reson. Chem. 2002, 40, 70-76].

Example 1 Preparation of Epoxide Layer for Stationary Phase In a typical reaction, hybrid porous particles were premixed at 70 ° C. for 60 minutes using 20 mM acetate buffer (pH 5.5, acetic acid and sodium acetate). Prepared, dispersed in a solution of glycidoxypropyltrimethoxysilane / methanol (0.25 mL / g) (GLYMO, Aldrich, Milwaukee, Wis.) According to JT Baker, 5 mL / g dilution). The mixture was held at 70 ° C. for 20 hours. The reaction was then cooled and the product was filtered and washed successively with water and methanol (JT Baker). The product was then dried at 80 ° C. under reduced pressure for 16 hours. The specific particles used are shown in Table 1.

The reaction data is listed in Table 2. Specific changes to this general procedure include: 1) Material 1E was prepared using a reaction time of 6 hours, 2) Material 1F was prepared using 100 mM acetate buffer, 3) Material 1G was prepared using 20 hours holding at 50 ° C. 4) Material 1H was prepared using 50 ° C. premix and 50 ° C. holding. The total surface coverage of 3.90-6.0 μmol / m ² was determined by the difference in particle C% before and after surface modification measured by elemental analysis. Analysis of these materials by ¹³ C CP-MAS NMR spectroscopy shows that a mixture of epoxy and diol groups exists for these materials.

Example 2 Preparation of Stationary Phase Having Diol Functional Groups In a typical reaction, hybrid porous particles were premixed at 70 ° C. for 60 minutes using 20 mM acetate buffer (pH 5.5, acetic acid and sodium acetate). And glycidoxypropyltrimethoxysilane / methanol solution (0.25 mL / g) (GLYMO, Aldrich, Milwaukee, Wis.) According to JT Baker, 5 mL / g dilution). The mixture was held at 70 ° C. for 20 hours. The reaction was then cooled and the product was filtered and washed successively with water and methanol (J. TBaker). The material was then refluxed in a 0.1 M acetic acid solution (5 mL / g dilution, JT Baker) at 70 ° C. for 20 hours. The reaction was then cooled and the product was filtered and washed successively with water and methanol (JT Baker). The product was then dried at 80 ° C. under reduced pressure for 16 hours. The reaction data is listed in Table 3. The surface coverage of 0.93-6.0 μmol / m ² was determined by the difference in particle C% before and after surface modification measured by elemental analysis. Analysis of these materials by ¹³ C CP-MAS NMR spectroscopy shows that no measurable amount of epoxy groups remains and only diol groups are present for these materials.

Example 3 Preparation of Stationary Phase with Mixed Functionality In a standard experiment, 10 g of the material prepared above was dispersed in a solvent such as, but not limited to, water, isopropanol or dioxane. An amount of nucleophile exceeding the epoxide coverage determined for the material prepared above was added and the mixture was heated at 70 ° C. for 16 hours. Table 4 shows specific nucleophiles used. After the reaction, the particles were washed successively with water and 0.5 M acetic acid, and then the material was stirred in a 0.1 M acetic acid solution (5 mL / g dilution, JT Baker) at 70 ° C. for 20 hours. The reaction was then cooled and the product was filtered and washed successively with water and methanol (JT Baker). The product was then dried at 80 ° C. under reduced pressure for 16 hours. Reaction data is listed in Table 5. The nucleophile surface concentration of 0.2-2.3 μmol / m ² was determined by the difference in particle C%, N% or S% before and after surface modification measured by elemental analysis. Analysis of these materials by ¹³ C CP-MAS NMR spectroscopy shows that no measurable amount of epoxide groups remain.

Example 4 Further Characterization of Stationary Phase The general procedure for particle binding / functionalization detailed in Examples 1-3 is applied to modify the surface silanol groups of various porous materials. This includes silica, hybrid inorganic / organic materials, hybrid inorganic / organic, silica, titania, alumina, zirconia, hybrid inorganic / organic surface layers on polymer or carbon materials, and hybrid inorganic / organic, silica, titania, alumina, Monolithic, spherical, granular, surface porous and irregular materials that are silica surface layers on zirconia or polymer or carbon materials are included. A stationary phase material in the form of a spherical material, a non-spherical material (eg including a toroid, polyhedron); Also included are stationary phase materials having a mixture of highly spherical, rod, curved, toroidal or dumbbell shaped forms. Examples of hybrid materials are shown in U.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913, and WO 2008/103423, The contents of which are hereby incorporated by reference in their entirety. Surface porous particles include those described in US Patent Application Publication Nos. 2013/0112605, 2007/0189944, and 2010/061367, the contents of which are hereby incorporated by reference in their entirety. It has been. The particle size of the spherical, granular or irregular material can vary from 5-500 μm, more preferably 15-100 μm, more preferably 20-80 μm, more preferably 40-60 μm. The APD of these materials can vary from 30 to 2000 mm, more preferably from 40 to 200 mm, more preferably from 50 to 150 mm. The SSA of these materials can vary from 20 to 1000 m ² / g, more preferably from 90 to 800 m ² / g, more preferably from 150 to 600 m ² / g, more preferably from 300 to 550 m ² / g. The TPV of these materials may vary from 0.3 to 1.5 cm ³ / g, more preferably from 0.5 to 1.4 cm ³ / g, more preferably from 0.7 to 1.3 cm ³ / g. it can. The macropore diameter of the monolithic material can vary from 0.1 to 30 μm, more preferably from 0.5 to 25 μm, more preferably from 1 to 20 μm.

[Example 5] The stationary phase shows the minimum analyte retention change over time under chromatographic conditions. The average retention change% is the third and tenth day from the absolute peak retention average measured in the first day chromatography test. Calculations were made by determining the percent difference in absolute peak retention averages determined from eye or day 30 chromatographic tests. For each test day, the column was equilibrated for 20 minutes under Mix1 test conditions, followed by 3 injections of Mix1, then equilibration for 10 minutes under Mix2 test conditions, followed by 3 injections of Mix2. The conditions are shown in Table 6. The results are shown in Tables 7 and 8.

  The% retention reduction was calculated by determining the percent difference of the first day absolute peak retention average measured for Mix1 and Mix2 from the first day absolute peak retention average measured for Mix1 and Mix2 in Example 1A.

Example 6 Surface Functionalization of Organic-Inorganic Hybrid Particles with Glycidoxypropyltrimethoxysilane (GPTMS) and 1-aminoanthracene The structures of GPTMS and 1-aminoanthracene are shown in FIG. FIG. 1A shows the structure of GPTMS, which is a silane surface modifier. Part 105 represents a surface reactive group of GPTMS (trialkoxysilane), and part 110 represents a reactive group (epoxide). FIG. 1B shows a selective ligand (1-aminoanthracene).

  GPTMS is first pre-formed with oligomers by preincubation at 70 ° C. in 20 mM sodium acetate buffer pH 5.0. During the incubation, small oligomers of hydrolyzed silane are formed. After an appropriate preincubation period, the modified particles are added as a dry powder. The oligomer and any residual monomer react with the material surface, resulting in a high surface coverage of the silane modifier covalently bonded to the material surface as shown in FIG.

  FIG. 2 shows the reaction between the silane coupling agent and the organic-inorganic hybrid material surface. Silane (205) is depicted as a monomer for simplicity. The preformed oligomeric silane can be coupled in the same manner as the surface reactive group (210). In some embodiments, portions of the silane that are not bound to the surface can also be coupled to the coating (eg, cross-polymerization). Under the reaction conditions, methanol is lost (215) to obtain surface modified particles (220).

After the silane has reacted with the chromatographic material, excess reagents and buffer salts are removed by washing with MilliQ H ₂ O, the material is transferred into an organic solvent (eg, 1,4-dioxane), and 1-amino Add anthracene. The amino group of 1-aminoanthracene is coupled to the surface via the pendant epoxide group of GPTMS. The portion of the epoxy group converted can be adjusted by limiting the amount of 1-aminoanthracene added. The coupled material is then washed into 0.5M acetic acid and the unreacted epoxide groups are hydrolyzed to the corresponding diol as shown in FIG.

  The resulting 1-aminoanthracene / diol surface has a uniformly distributed 1-aminoanthracene group and provides excellent selectivity, but the diol shields the surface silanol from interaction with the analyte. Multi-component surfaces are superior to diol-only surfaces or 1-aminoanthracene-only surfaces.

  FIG. 3 shows the reaction between the surface modified particles (305) and the selective ligand (310). Reaction conditions (315) are given, including treatment with isopropanol and 0.5M acetic acid at 70 ° C. The result is a stationary phase particle with a multicomponent surface for chromatographic separation (320).

  Alternatively, the multi-component surface can be bonded simultaneously to the substrate surface, causing the pendant reactive groups to partially react to form inert pendant groups and limited between pendant reactive groups on adjacent silane coupling agent molecules. By using a silane coupling agent with a pendant reactive group as a binder phase under reaction conditions that result in polymerisation, it can be produced under conditions that produce a polymerized surface.

  Similarly, a multi-component surface can interact with an analyte that affects retention by introducing charged, uncharged, polar, non-polar, lipophilic or hydrophilic properties into the chromatographic phase. Can be produced under conditions that give rise to a polymerized surface. Alternatively, under certain conditions, the epoxy group of GPTMS can react with the hydroxyl group of an adjacent silane to form an ether bridge, which crosslinks GPTMS on the surface. Such cross-linking can provide stability to the binder phase and can also improve silanol shielding. The presence of such crosslinks is consistent with NMR analysis of these materials. An embodiment of the type of cross-linked surface is shown in FIG. FIG. 4 also shows cross-linked silane groups in a coating where no silane is bonded to the surface. Such a structure demonstrates the formation of ether bridges by polymerization of surface epoxides.

[Example 7]
FIG. 5 shows two possible synthetic routes to prepare the chromatographic stationary phase of the present disclosure. As shown in scheme (500), unmodified BEH particles (505) can be chemically modified in at least two different ways. Thus, one option is to first prepare the chemical modifier (510) by reacting GPTMS with 1-aminoanthracene. Reagent 510 then reacts with BEH particles (505) to yield a functionalized chromatography surface (515).

  Alternatively, FIG. 5 shows a different synthesis route. In this embodiment, particles 505 react directly with GPTMS to produce a GPTMS modified surface (520). Surface 520 can then react with 1-aminoanthracene (525) to yield a functionalized chromatography surface (515).

  In a preferred embodiment, a second reaction pathway comprising first reacting particle 505 with GPTMS followed by functionalization with 1-aminoanthracene (525) is a chemical modifier 510 that has previously formed particle 505. In preference to the first reaction path comprising reacting with.

Example 8 GPTMS binding on organic-inorganic hybrid mitigates retention drift or change Treatment of cross-linked ethylene hybrid (BEH) stationary phase with glycidoxypropyltrimethoxysilane (GPTMS) as shown in FIG. The subsequent epoxide ring-opening reaction to give the next diol can significantly reduce the effect of retention drift. Graph 600 shows a plot of the original% retention of unfunctionalized 3 μm BEH particles (605) compared to diol functionalized 3 μm BEH particles (610). The original% retention is shown as a function of time in days on the X axis. The results show that in at least some preferred embodiments, functionalization of the chromatographic surface with GPTMS and epoxide ring opening to give the next diol can reduce retention drift. The results also show that the GPTMS coating alone addresses the retention drift problem and also provides significant retention.

Example 9 Separation of compounds of interest using 1-aminoanthracene stationary phase The disclosed method and stationary phase uses simple chromatographic conditions, compared to current state of the art methods, The methods and stationary phases of the present disclosure that provide excellent separation of the subject compounds, such as lipids and fat-soluble vitamins, also provide different selectivity, which is advantageous for partitioning different classes of compounds.

ACQUITY UPC with Convergence Chromatography Binary Solvent Manager (ccBSM), Sample Manager (SM), Convergence Chromatography Manager (CCM), Column Manager (CM-A) and Photodiode Array (PDA) ^Two systems were used. The system and separation conditions are shown below:
First condition: flow rate: Gradient: in CO ₂ 2 min 3-20% methanol; 0.5 min CO ₂ in 20-3% methanol; 0.5 min with 20% methanol in CO2. ABPR setting: 2175 psi. Column temperature: 40 ° C. Detection: 235 nm correction. 400-500 nm (320 nm correction 400-500 nm as necessary for vitamin A). Column dimensions: 3.0 × 50 mm. Stationary phase: 1-aminoanthracene modified diol, 2.5 μm, as given in Example 7.

Second condition: flow rate: 2.0 mL / min. Gradient: 3 minutes in a CO ₂ in 3-8% methanol (# 8 curve); CO 0.5 min ₂ in 8-35% methanol (# 1 curve); CO ₂ 1 min at 35% methanol in; CO ₂ Medium with 35-3% methanol for 0.5 min (# 1 curve). ABPR: 1800 psi. Column temperature: 50 ° C. Sample: GLC85 lipid standard (Nu-chek Prep, Elysian, MN, USA), diluted 20-fold with 1: 1 heptane: IPA. 1 g / L in CHCl ₃ stock. Column dimensions: 3.0 × 50 mm. Stationary phase: 1-aminoanthracene modified diol, 2.5 μm, as given in Example 7.

  Peak detection and structure determination were also performed using the ACQUITY SQD2 mass spectrometer: 3.46 kV capillary; 25 V cone; 350 ° C. source; 500 L / hr, desolvated gas; 10 L / hr, cone gas; LM Res 11.8; Obtained using -0.2 ion energy.

  Figures 7-16 illustrate lipid separations obtained using the methods and stationary phases of the present disclosure. The results of these separations indicate that 1-aminoanthracene coupling is exceptionally good at retaining and separating fat-soluble vitamins, lipids and metabolites. 1-aminoanthracene coupling increases shape / isomer selectivity compared to C18 bonded phase.

These results are surprising, mixing hydrophobic and hydrophilic groups with the same ligand is not only difficult, but also an unusual theme for the stationary phase. In many cases, ligands rich in π electrons are used for very specific separations and are used to give slightly different selectivity from C18 bonded phases. It has been found that there is a very different selectivity between 1-aminoanthracene-based stationary phases and for example ACQUITY UPC ² HSS C18 SB (used for comparison purposes in this disclosure).

In the case of fat-soluble vitamins, as the name suggests, these molecules are nonpolar and often have few ionizable groups. In some embodiments, the stationary phase can contain residual amines that cannot improve the separation of non-polar compounds. In some embodiments, these groups can control the surface pH of the ligand. The presence of these groups does not point out that it is possible to separate fat-soluble vitamins. Usually, the separation of vitamins, lipids and metabolites is carried out on a C18 bonded phase, eg ACQUITY UPC ² HSS C18 SB. In such materials, the alkyl chain is a retention selector that produces high methylene / hydrophobicity selectivity but little shape / isomer selectivity. The materials of the present disclosure, such as those containing 1-aminoanthracene, are exceptionally good at retaining and separating fat-soluble vitamins, lipids and metabolites. These materials also improve shape / isomer selectivity compared to C18 bonded phases.

Similar results were obtained with the lipid treatment that the 1-aminoanthracene coupling was superior to ACQUITY UPC ² HSS C18 SB. When using the optimized method, more lipid peaks are resolved in the prototype with 1-aminoanthracene coupled. The increase in the number of observed peaks is related to the selectivity of the fatty acid carbon chain length and saturation. The 1-aminoanthracene prototype addresses the problem indicated by “Fast and Simple Free Fatty Acids Analysis UPC ² / MS” (Library Number: APTNT 13453626; Part Number 720004763en). Separation of lipids by both chain length and degree of unsaturation.The problem is the two-sided nature of the reverse run separation process (double bonds in fatty acyl chains reduce retention time and fatty acyl chain length is retention time) ”) Will interfere with the analysis of the actual sample; the number of components is often too high, making identification difficult due to co-elution.” The stationary phase of the present disclosure results in increased retention for unsaturated fatty acid chains (more double bonds result in longer retention times) and longer chain lengths. Separation is based on the number of carbon atoms and the number of double bonds to increase retention, which is a significant improvement over alkyl bonded stationary phases.

  Those skilled in the art can use the present disclosure to make fine chemicals / materials (OLEDs, pesticides, dyes / organic dyes, conformational polymers, polymer additives and surfactants), foods and the environment (insecticides, glycerides, edible oils, Tobacco, food contamination, pharmaceutical / life science (lipid profiling, natural products, DMPK / biological analysis, impurity profiling, medicinal chemistry) and forensic science / research (opiates, drugs of abuse, steroids, fatty acids, antidepressants, Understand that methods of explosives, explosives) can be developed and adjusted in other application areas.

  In addition, these materials and methods can be used as part of a multidimensional experiment (2D-SFC, SFC-LC, etc.). Other materials used in combination with these include agate silver (silver impregnated material) and existing lipid methods such as those in CSH brand columns.

Example 10 Separation of compounds of interest using various stationary phases of this disclosure Additional chromatographic materials were prepared using another selector according to the previous example. Lipid analysis was performed on these additional stationary phases. Test materials include stationary phases using the following selectors: 1-aminoanthracene, 2-picolylamine, pyridine, 6-aminoquinoline, aniline, diol and 4-n-octylaniline.

General chromatographic conditions are given as follows: Sample: GLC85 lipid (Nu-chek Prep), 1 g / L in CHCl ₃ , 1: 1 IPA: heptane diluted 20-fold. System: UPC2, w / SQD2, ESI-ionization. Mobile phase: 97.5 / 2.5 MeOH / H 2 O, w / 0.1 M NH 3 makeup flow. 3.0 x 50 mm column. The samples contained a mixture of C ₄ -C ₂₄ lipids (m / z: 87; 115.1; 143.1; 171.1; 185.1; 199.2; 213.2; 227.2. 241.2; 255.2; 269.2; 283.3; 311.3; 339.3; 225.2; 239.2; 253.2; 267.2; 277.2; 279.2; 281; .2; 303.2; 305.2; 307.3; 309.3; 327.2; 335.3; 337.3; 365.3).

  Figures 17-24 show lipid separation using both these stationary phases, both on the basis of the number of carbon bonds and on individual lipids. In general, stationary phases containing 1-aminoanthracene and 4-n-octylaniline show sufficient degradation of the mixture and increased retention based on the number of double bonds. Stationary phases using 2-picolylamine, pyridine, 6-aminoquinoline and aniline couplings and diols cannot resolve most lipid peaks in the sample. Table 9 shows a summary of the performance of these stationary phases.

  Table 9 shows a summary of the results. The retention window is measured as the time difference between the first and last elution peak of the lipid mixture. Since this value represents the separation space, it is desirable to maximize it. Material 3A gives a maximum retention window value that is 200% greater than HSS C18 SB (comparative). The peak capacity is measured as the retention window divided by the average peak width of all peaks eluted within the retention window. Material 3E provides a significant improvement in peak capacity by 20% over HSS C18 SB. Retention values of C18: 0, C18: 1, C22: 1 and C22: 6 represent the retention times of lipids with 18 and 22 carbon lengths, respectively, 0, 1 and 6 double bonds in the chain, respectively. Material 3E and HSS C18 SB show a decrease in retention as the number of double bonds increases, while all others show an increase in retention. The (retention) range of all C18 and C22 lipids in the mixture is from the retention time of each saturated lipid C18 or C22 to the retention time of the lipid with the highest level of unsaturation (most double bonds), C18 or C22, respectively. Calculate by subtracting The negative value of either of these columns represents a decrease in retention based on the number of double bonds present in the chain. The positive C18 and C22 range values for this list of materials show unique selectivity compared to HSS C18 SB. The full double bond (DB) range is the full range of C18 and C22 lipid species, calculated from the absolute value of the sum of the C18 and C22 ranges. It is desirable to maximize this value shown for material 3A. In some embodiments, the material of the present disclosure is 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, under normal or normal chromatographic conditions. 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4 or 1. Gives more than 5 DBs.

  It can be seen that the best peak capacity is provided by N-octyl-aniline, HSS C18 SB (comparative column), 1-aminoanthracene and aniline based stationary phase. Peak capacity is the retention window divided by the average peak width (4σ width) at 13.4%. The best C18 and C22 ranges are given by pyridine, 6-aminoquinoline, 2-picolylamine and 1-aminoanthracene. The C18 range is the time difference between C18: 2 and C18: 0. A negative number indicates that C18: 2 eluted before C18: 0. The same applies to the C22 range. The best full double bond range is also given by 1-aminoanthracene, pyridine, 6-aminoquinoline and 2-picolylamine. C18: 0 has 18 carbons and 0 double bonds, C18: 1 has 18 carbons and 1 double bond, and so on. The total DB (double bond) range is the difference between the maximum and minimum retention times of the C18 and C22 series.

Example 11 Separation of vitamins D and K using various stationary phases of the present disclosure Two critical pairs, vitamins K1 and K2 and vitamins D2 and D3, were tested using the stationary phases of the present disclosure. It was determined whether these materials improved the splitting of these pairs. Chromatographic conditions are shown in Table 10. Table 11 shows a summary of the results. For each material tested, the splitting of two critical pairs, vitamins K1 and K2 and vitamins D2 and D3, respectively, was measured. Materials 3J, 3H and 3P significantly improve the split of critical pairs, especially vitamins K1 and K2, compared to HSS C18 SB (comparison). Calculate the percent improvement as follows:

  It is expected that the splitting of critical pairs will increase further as the particle sizes of materials 3J, 3H and 3P are smaller. For example, when the particle size of material 3P is reduced, the observed split is expected to increase by another 44%. The conjugate portion of the materials 3J, 3H and 3P is believed to increase the interaction of the chromatographic surface with vitamins that exert their own selectivity compared to HSS C18 SB.

  In some embodiments, the materials of the present disclosure are 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 under normal or normal chromatographic conditions. Or give an Rs value for vitamin D3 / D4 of greater than 4.5. In some embodiments, the materials of the present disclosure are 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 under normal or normal chromatographic conditions. Or give an Rs value of vitamin K1 / K2 greater than 4.5.

  Unless otherwise noted, all techniques, including the use of kits and reagents, can be performed according to manufacturer information, methods known in the art.

  The recitation of value ranges herein is merely intended to serve as a simple way of referring to each distinct value included within the range. Unless otherwise indicated, each distinct value is incorporated herein as if it were individually listed. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer specifications and instructions) is hereby incorporated by reference in its entirety.

  This specification should be understood to disclose and encompass all possible permutations and combinations of the described aspects, embodiments and examples, unless the context clearly indicates otherwise. Those skilled in the art will appreciate that the invention can be practiced other than as summarized and described in the aspects, embodiments, and examples presented for purposes of illustration and not limitation.

Claims (30)

A method for separating a target compound from a mixture comprising:
(A) providing a mixture containing the target compound;
(B) introducing a portion of the mixture into a chromatography system having a chromatography column; and (c) eluting the separated target compound from the column;
The column has the following structure (i):
[X] (W) _a (Q) _b (T) _c (i)
Having a stationary phase having the formula:
X is a chromatographic substrate containing silica, metal oxide, inorganic-organic hybrid material, group of block copolymers or combinations thereof,
W is selected from the group consisting of hydrogen and hydroxyl, W binds to the surface of X,
Q is the first substituent that minimizes the change in analyte retention over time under chromatographic conditions with low moisture concentration;
T is a second substituent that chromatographically retains the analyte, and T has one or more monoaromatic, polyaromatic, heteroaromatic or polyheteroaromatic groups. Wherein each group is optionally substituted with an aliphatic group, and b and c are positive numbers, 0.05 ≦ (b / c) ≦ 100 and a ≧ 0.   2. The method of claim 1, wherein the first fraction of Q is coupled to X and the second fraction of Q is polymerized.   2. The method of claim 1, wherein the first fraction of T is coupled to X and the second fraction of T is polymerized.   A first fraction of Q is coupled to X, a second fraction of Q is polymerized, a third fraction of T is coupled to X, and a fourth fraction of T is polymerized. Item 2. The method according to Item 1.   The method of claim 1, wherein a portion of the second fraction of Q and a portion of the fourth fraction of T are polymerized with each other. Q is the following structure (ii):

In the formula:
n ¹ is an integer of 1-30;
n ² is an integer of 1-30;
R ¹ , R ² , R ³ and R ⁴ are each independently hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, protected or deprotected Selected from the group consisting of alcohol and zwitterions;
Z is
a) Formula (B ¹ ) _x (R ⁵ ) _y (R ⁶ ) _z A surface binding group having Si—, where x is an integer of 1-3, y is an integer of 0-2, z Is an integer from 0-2 and x + y + z = 3;
R ⁵ and R ⁶ are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, protected or Selected from the group consisting of deprotected alcohols, zwitterionic groups and siloxane bonds; and a surface binding group wherein B ¹ is a siloxane bond;
b) to surface organofunctional hybrid groups directly through carbon-carbon bond formation or through heteroatoms, esters, ethers, thioethers, amines, amides, imides, ureas, carbonates, carbamates, heterocycles, triazoles or urethane bonds. C) adsorbed surface groups that are not covalently bonded to the surface of the material;
Y is an embedded polar functional group, a bond or an aliphatic group; and A is a hydrophilic end group, a functionalized group, hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 2. The process of claim 1 selected from the group consisting of tert-butyl, lower alkyl and polarizable groups. T is the following structure (iii):

In the formula:
m ¹ is an integer of 1-30;
m ² is an integer of 1-30;
m ³ is an integer of 1-3;
R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are each independently hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, protected or deprotected Selected from the group consisting of alcohols, zwitterions, aromatic hydrocarbon groups and heterocyclic aromatic hydrocarbon groups;
Z is
a) Formula (B ¹ ) _x (R ⁵ ) _y (R ⁶ ) _z A surface binding group having Si—, where x is an integer of 1-3, y is an integer of 0-2, z Is an integer from 0-2 and x + y + z = 3;
R ⁵ and R ⁶ are each independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, substituted or unsubstituted aryl, cyclic alkyl, branched alkyl, lower alkyl, protected or Selected from the group consisting of deprotected alcohols, zwitterionic groups and siloxane bonds; and a surface binding group wherein B ¹ is a siloxane bond;
b) to surface organofunctional hybrid groups directly through carbon-carbon bond formation or through heteroatoms, esters, ethers, thioethers, amines, amides, imides, ureas, carbonates, carbamates, heterocycles, triazoles or urethane bonds. C) adsorbed surface groups that are not covalently bonded to the surface of the material;
Y is an embedded polar functional group, bond or aliphatic group;
D is a bond, N, O, S,
^{_{- (CH 2) 0-12 -N-}} R 11 R 12,
^{_{- (CH 2) 0-12 -O-}} R 11,
^{_{- (CH 2) 0-12 -S-}} R 11,
_{- (CH 2) 0-12 -N- (} CH 2) 0-12 -R 11 R 12,
_{- (CH 2) 0-12 -O- (} CH 2) 0-12 -R 11,
_{- (CH 2) 0-12 -S- (} CH 2) 0-12 -R 11,
_{- (CH 2) 0-12 -S (} O) 1-2 - (CH 2) 0-12 -N-R 11 R 12,
_{- (CH 2) 0-12 -S (} O) 1-2 - (CH 2) 0-12 -O-R 11,
_{- (CH 2) 0-12 -S (} O) 1--2 - (CH 2) 0-12 -S-R 11;
_{- (CH 2) 0-12 -S (} O) 1-2 - (CH 2) 0-12 -N- (CH 2) 0-12 -R 11 R 12,
_{- (CH 2) 0-12 -S (} O) 1-2 - (CH 2) 0-12 -O- (CH 2) 0-12 -R 11 and _{- (CH 2) 0-12 -S (} O _{) 1--2 - (CH 2) 0-12} -S- ( selected from the group consisting of _{CH 2)} 0-12 ^{-R 11;}
R ¹¹ is a first monoaromatic group, polyaromatic group, heterocyclic aromatic group or polyheterocyclic aromatic group; and R ¹² is hydrogen, an aliphatic group or second monoaromatic group, polyaromatic The method of claim 1, wherein the group is an aromatic group, a heterocyclic aromatic group, or a polyheterocyclic aromatic group, and R ¹¹ and R ¹² are optionally substituted by an aliphatic group.   The method according to claim 1, wherein the target compound is a lipid, a vitamin, or a polycyclic aromatic hydrocarbon.   9. The method of claim 8, wherein the lipid is a saturated or unsaturated fatty acid, monoacyl glyceride, diacyl glyceride, triacyl glyceride, phospholipid, sphingolipid or steroid.   9. The method of claim 8, wherein the vitamin is a water soluble vitamin selected from the group consisting of vitamin C, vitamin B, or derivatives or combinations thereof.   9. The method of claim 8, wherein the vitamin is a fat soluble vitamin selected from the group consisting of vitamin A, vitamin D, vitamin K, vitamin E, beta carotene, or derivatives or combinations thereof. A polyaromatic in which at least the first or second monoaromatic group, polyaromatic group, heteroaromatic group or polyheteroaromatic group of R ¹¹ or R ¹² has at least two aromatic rings, or The method according to claim 7, which is a polyheterocyclic aromatic. A polyaromatic group in which at least the first or second monoaromatic group, polyaromatic group, heteroaromatic group or polyheteroaromatic group of R ¹¹ or R ¹² has at least three aromatic rings, or The method according to claim 7, which is a polyheterocyclic aromatic. A polyaromatic in which at least the first or second monoaromatic group, polyaromatic group, heteroaromatic group or polyheteroaromatic group of R ¹¹ or R ¹² has at least four aromatic rings, or The method according to claim 7, which is a polyheterocyclic aromatic hydrocarbon. At least the first or second monoaromatic group, polyaromatic group, heterocyclic aromatic group or polyheteroaromatic group of R ¹¹ or R ¹² is represented by at least one C ₄ -C ₂₄ aliphatic group. 8. The method of claim 7, wherein the method is substituted. The first monoaromatic group, polyaromatic group, heteroaromatic group or polyheteroaromatic group of R ¹¹ or R ¹² is furan, pyrrole, pyrroline, oxazole, thiazole, imidazole, imidazoline, pyrazole, pyrazoline, Pyrazolidine, isoxazole, isothiazole, benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, thiophene, indene, indolizine, indole, isoindole, indoline, indazole, benzimidazole, benzothiazole, naphthalene, Quinolidine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, quinuclidine, fluorene, carbazole, Selected from the group consisting of anthracene, acridine, phenazine, phenothiazine, phenoxazine, pyrene, phenanthrene, chrysene and derivatives thereof, wherein said group is unsubstituted or optionally substituted by an aliphatic group The method according to claim 7.   The method according to claim 6, wherein the functional group contained in Q is a diol. Q is the following structure:

The method of claim 17 represented by: T has the following structure:

The method of claim 7 represented by one of the following:   The method of claim 1, wherein the surface of X undergoes alkoxylation with a chromatographic mobile phase under chromatographic conditions.   21. The method of claim 20, wherein a substantial portion of the surface of X is not subject to alkoxylation by a chromatographic mobile phase under chromatographic conditions.   The method of claim 1, wherein the surface of X is silica free and b = 0 or c = 0.   The method of claim 1, wherein the chromatographic stationary phase is compatible with supercritical fluid chromatography.   The method of claim 1, wherein the chromatographic stationary phase is compatible with carbon dioxide-based chromatography. The following structure (i):
[X] (W) _a (Q) _b (T) _c (i)
A chromatographic stationary phase having
Wherein X is a chromatographic substrate containing silica, a metal oxide, an inorganic-organic hybrid material, a group of block copolymers, or a combination thereof,
W is selected from the group consisting of hydrogen and hydroxyl, W binds to the surface of X,
Q is the first substituent that minimizes the change in retention of the analyte over time under chromatographic conditions with a low moisture concentration;
T is a second substituent that chromatographically retains the analyte, and T represents one or more aromatic groups, polyaromatic groups, heterocyclic aromatic groups, or polyheteroaromatic hydrocarbon groups. Each group is optionally substituted with an aliphatic group,
And a chromatographic stationary phase wherein b and c are positive numbers and 0.05 ≦ (b / c) ≦ 100 and a ≧ 0. For normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography A column, capillary column, microfluidic device or apparatus
26. A housing having at least one wall defining a chamber having an inlet and an outlet, and a stationary phase disposed within the housing, wherein the housing and stationary phase are normal phase chromatography, high pressure liquid chromatography. Column, capillary column, suitable for chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, Microfluidic device or apparatus. For normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography Of the kit,
26. A housing having at least one wall defining a chamber having an inlet and an outlet, and a stationary phase disposed within the housing, wherein the housing and stationary phase are normal phase chromatography, high pressure liquid chromatography. Compatible with chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography, and the housing and fixation Phase normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide chromatography, hydrophilic interaction A kit comprising instructions for performing liquid chromatography or hydrophobic interaction liquid chromatography. Reacting the chromatography substrate with a silane coupling agent having pendant reactive groups;
Reacting said pendant reactive group with a second chemical agent comprising one or more aromatic hydrocarbon groups, polyaromatic hydrocarbon groups, heteroaromatic hydrocarbon groups or polyheteroaromatic hydrocarbon groups; 26. A method of preparing a stationary phase according to claim 25, comprising neutralizing any remaining unreacted pendant reactive groups to produce the stationary phase of claim 25. Oligomerizing a silane coupling agent having pendant reactive groups;
Reacting the core surface with the oligomerized silane coupling agent;
Reacting said pendant reactive group with a second chemical agent comprising one or more aromatic hydrocarbon groups, polyaromatic hydrocarbon groups, heteroaromatic hydrocarbon groups or polyheteroaromatic hydrocarbon groups; 26. A method of preparing a stationary phase according to claim 25, comprising neutralizing any remaining unreacted pendant reactive groups to produce the stationary phase of claim 25. In normal phase chromatography, high pressure liquid chromatography, solvation gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography A method of reducing or preventing retention drift,
26. A method comprising chromatographically separating a sample using a chromatography device comprising a chromatographic stationary phase according to claim 25 to reduce or prevent retention drift.

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