CN111644164B - Chromatographic material for separating unsaturated molecules - Google Patents

Chromatographic material for separating unsaturated molecules Download PDF

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Publication number
CN111644164B
CN111644164B CN202010703909.9A CN202010703909A CN111644164B CN 111644164 B CN111644164 B CN 111644164B CN 202010703909 A CN202010703909 A CN 202010703909A CN 111644164 B CN111644164 B CN 111644164B
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chromatography
chromatographic
stationary phase
vitamin
liquid chromatography
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CN202010703909.9A
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CN111644164A (en
Inventor
D·W·布劳斯米歇
J·N·费尔蔡尔德
J·F·希尔
G·伊萨奇
M·F·莫里斯
K·D·怀恩罕
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Waters Technologies Corp
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Waters Technologies Corp
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Priority claimed from US14/194,686 external-priority patent/US10092894B2/en
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Abstract

The present application relates to chromatographic materials for separating unsaturated molecules. The present disclosure relates to methods of separating related compounds, particularly unsaturated related compound(s), from mixtures. The compounds were separated using a column with chromatographic stationary phase material containing a first substituent and a second substituent for various modes of chromatography. The first substituent minimizes the change in retention of the compound over time under chromatographic conditions. The second substituent chromatographically and selectively retains the compound by incorporating one or more aromatic, polyaromatic, heterocyclic aromatic or polyheterocyclic aromatic groups, each of which is optionally substituted with an aliphatic group.

Description

Chromatographic material for separating unsaturated molecules
Cross Reference to Related Applications
The present application is a divisional application of the invention patent application with application number 201580021868.2 and name "chromatographic material for separating unsaturated molecules" of 2015, 2, 27. The present application claims priority from U.S. patent application Ser. No. 14/194,686 filed on 1/3/2014, which is incorporated herein by reference in its entirety.
Technical Field
The present disclosure relates generally to chromatographic materials for separating unsaturated molecules. The present disclosure relates more particularly in various embodiments to chromatographic materials for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, and hydrophobic interaction liquid chromatography that while exhibiting overall retention that facilitates separation of unsaturated molecules, mitigate or avoid retention drift or variation, and to corresponding devices, kits, methods of manufacture, and methods of use.
Background
Chromatography is a generic term for a set of laboratory techniques used to separate mixtures. The mixture is dissolved in the mobile phase to carry it through the stationary phase. The various components of the mixture travel at different speeds to separate them. The separation is based on differential partitioning between the mobile phase and the stationary phase. Subtle differences in partition coefficients of the compounds result in differential retention on the stationary phase, thereby altering the separation. Chromatography can be used to separate structurally related compounds such as regioisomers, chiralities, diastereomers, and the like. Some techniques, including SFC, are known to be particularly useful for separating structurally related vitamins, natural products, and chemical materials. However, chromatographic techniques are often inadequate for isolating all structurally related compounds. For example, key pairs of related vitamins (e.g., D2 and D3, K1 and K2) are difficult to isolate/resolve.
Packing materials for fluid or liquid chromatography can be broadly divided into two categories: organic materials (e.g., polydivinylbenzene) and inorganic materials (e.g., silica). Many organic materials are chemically stable to strongly basic and acidic mobile phases so that mobile phase composition and pH can be flexibly selected. However, organic chromatographic materials can produce columns with low efficiency, especially for low molecular weight analytes. Many organic chromatographic materials not only lack the mechanical strength of typical chromatographic silica, but also shrink and swell as the composition of the mobile phase 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 that has been surface derivatized 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 produce columns with high efficiency and showing no signs of shrinkage or swelling.
The hybrid material may provide a solution to certain chromatographic problems that occur when using silica-based packing materials. The hybrid materials may 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, conventional hybrid materials and silica materials can present potential problems in other applications. One problem is the poor peak shape for the base when used at low pH, which can adversely affect the load capacity and peak capacity when used at low pH. Another problem is the change in retention time of acidic and basic analytes (known as "drift") after exposing the column to repeated changes in mobile phase pH (e.g., repeated changes from pH 10 to 3).
Another problem is that drift or variation is retained, for example in chromatographic modes with small amounts of water (e.g. less than 5%, less than 1%). For example, retention drift or change was observed under standard SFC conditions for both silica-based and organic-inorganic hybrid-based chromatographic phases (bonded and unbound), such as the BEH Technology ™ material available from Waters Technologies Corporation, milford MA. Other SFC stationary phases may also exhibit similar retention drift or changes.
Disclosure of Invention
In various aspects and embodiments, the present disclosure provides chromatographic materials for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, and hydrophobic interaction liquid chromatography that while exhibiting overall retention that facilitates separation of unsaturated molecules, mitigate or avoid retention drift or variation, as well as corresponding devices, kits, methods of manufacture, and methods of use.
The present disclosure includes various additional advantages including, but not limited to, the ability to select/design selectivity through selection/design chemical modification.
In one embodiment, the present disclosure relates to a method of separating a related compound from a mixture, the method comprising providing a mixture containing the related compound, introducing a portion of the mixture into a chromatography system having a chromatography column, and eluting the separated related compound from the column, wherein the column has a stationary phase having the following structure (i):
[X](W) a (Q) b (T) c (i)
wherein X is a chromatographic substrate comprising silica, a metal oxide, an inorganic-organic hybrid material, a set of block copolymers, or a combination thereof, W is selected from hydrogen and hydroxyl groups, wherein W is bonded to the surface of X, Q is a first substituent that minimizes the change in analyte retention over time under chromatographic conditions having a low water concentration, and T is a second substituent that chromatographically retains the analyte, wherein T has one or more aromatic, polycyclic aromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups, each optionally substituted with an aliphatic group; and b and c are positive numbers, 0.05-100 (b/c), and a-0.
In some embodiments, Q has the following structure (ii):
wherein n1 is an integer of 1 to 30, n2 is an integer of 1 to 30, R 1 、R 2 、R 3 And R is 4 Each independently selected from hydrogen, hydroxy, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, protected or deprotected alcohol and a zwitterionic, Z is (a) a compound having the formula (B 1 ) x (R 5 ) y (R 6 ) z Si-, wherein x is an integer from 1 to 3, y is an integer from 0 to 2, z is an integer from 0 to 2, and x+y+z=3, R 5 And R is 6 Each independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, substituted or unsubstituted aryl, cycloalkyl, branched alkyl, lower alkyl, protected or deprotected alcohol, zwitterionic groups, and siloxane bonds, and B 1 Is a siloxane bond, or (b) is formed by a direct carbon-carbon bond or by a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or polyurethane (urethane) bond to a surface organofunctional hybrid group, or (c) is an adsorbing surface group that is not covalently attached to the surface of the material, Y is an intercalating polar functional group, bond, or aliphatic group, and a is selected from the group consisting of a hydrophilic end group, a functionalizable group, hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, and a polarizable group.
In some embodiments, T has the following structure (iii):
wherein m is 1 Is an integer of 1 to 30, m 2 Is an integer of 1 to 30, m 3 Is an integer of 1 to 3, R 7 、R 8 、R 9 And R is 10 Each independently selected from the group consisting of hydrogen, hydroxy, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, protected or deprotected alcohol, zwitterionic, aromatic hydrocarbon group and heterocyclic aromatic hydrocarbon group, Z is (a) having formula (B) 1 ) x (R 5 ) y (R 6 ) z Si-, wherein x is an integer from 1 to 3, y is an integer from 0 to 2, z is an integer from 0 to 2, and x+y+z=3, R 5 And R is 6 Each independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, substituted or unsubstituted aryl, cycloalkyl, branched alkyl, lower alkyl, protected or deprotected alcohol, zwitterionic groups, and siloxane bonds, and B 1 Is a siloxane bond, (b) is formed by a direct carbon-carbon bond or is attached to a surface organofunctional hybrid group by a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or polyurethane bond, or (c) is an adsorbed surface group that is not covalently attached to the surface of the material, Y is an intercalated polar functional group, bond, or aliphatic group, D is selected from the group consisting of 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–(CH 2 ) 0-12 –R 11 ，R 11 Is a first monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group, R 12 Is hydrogen, an aliphatic group or a second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group, wherein R 11 And R is 12 Optionally substituted with aliphatic groups. Various scores of Q, T or both may be aggregated.
In another embodiment, the disclosure relates to a method of separating lipids, vitamins, or polycyclic aromatic hydrocarbons from a mixture.
In another embodiment, the disclosure relates to a chromatographic stationary phase having the following structure (i):
[X](W) a (Q) b (T) c (i)
wherein X is a chromatographic substrate comprising silica, a metal oxide, an inorganic-organic hybrid material, a set of block copolymers, or a combination thereof, W is selected from hydrogen and hydroxyl groups, wherein W is bonded to the surface of X, Q is a first substituent that minimizes the change in analyte retention over time under chromatographic conditions having a low water concentration, and T is a second substituent that chromatographically retains the analyte, wherein T has one or more monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic, or polyheterocyclic aromatic groups, each of which is optionally substituted with an aliphatic group; and b and c are positive numbers, 0.05-100 (b/c), and a-0.
In another embodiment, the present disclosure relates to a column, capillary column, monolith, microfluidic device or apparatus for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography comprising a housing having at least one wall defining a chamber having an inlet and an outlet and a stationary phase having the structure described above, i.e. (i), disposed therein, wherein the housing and stationary phase are adapted for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography.
In another embodiment, the present disclosure relates to a kit for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography comprising a housing having at least one wall defining a chamber having an inlet and an outlet and a stationary phase disposed therein having the structure described above, i.e. (i), wherein the housing and stationary phase are suitable for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography; and instructions for performing normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography with the shell and stationary phase.
In another embodiment, the present disclosure relates to a method of preparing a stationary phase having the structure described above, i.e., (i), comprising reacting a chromatographic substrate with a silane coupling agent having a pendant reactive group, reacting a second chemical reagent comprising one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups with the pendant reactive group; and neutralizing any remaining unreacted pendant reactive groups, thereby producing the stationary phase.
In another embodiment, the present disclosure relates to a method of preparing a stationary phase having the above structure, i.e., (i), comprising oligomerizing a silane coupling agent having pendant reactive groups, reacting a core surface with the oligomerized silane coupling agent, and reacting a second chemical reagent comprising one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups with the pendant reactive groups; and neutralizing any remaining unreacted pendant reactive groups, thereby producing the stationary phase.
In another embodiment, the present disclosure relates to a method of mitigating or preventing retention drift in normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography, comprising chromatographic separation of a sample using a chromatographic device comprising a chromatographic stationary phase having the above structure, i.e., (i), disposed therein, thereby mitigating or preventing retention drift.
The present disclosure advantageously reduces or avoids retention drift or variation while exhibiting overall retention that is particularly useful for unsaturated related molecules or compounds. For example, in SFC, retention drift or variation can be attributed (in various other theories) to the standard CO used for SFC 2 Alkoxylation of the solvent-reachable silanol on the particles under MeOH mobile phase (and/or with other alcohol co-solvents). This is a problem because as the column ages, the user observes the chromatographic changes (e.g., retention time) obtained on their SFC system and again observes changes when a new non-alkoxylated column is installed on the system.
In various aspects and embodiments, the present disclosure provides various solutions to such retention drift or variation and related problems (e.g., retention, peak shape, etc.) through the selection and/or modification of chromatographic materials. For example, the present invention includes specific functionalization of the chromatographic core surface (e.g., with specific functional groups and combinations thereof), which substantially prevents chromatographic interactions between the analyte and the chromatographic core surface, which maintains the desired interactions between the analyte and the chromatographic material.
In other various aspects and embodiments, the disclosure relates to chromatographic materials having greatly reduced secondary interactions (e.g., unwanted interactions, non-specific adsorption) with the surface of matrix particles. Secondary interactions of the analyte with the surface of the material may occur due to silanol, pendant hydrophobic groups and polymeric or hybrid backbones.
In various aspects and embodiments, the present disclosure provides a number of advantages. For example, the present disclosure may provide a stationary phase that can resolve all kinds of analytes (e.g., acidic, basic, and neutral) with excellent retention, peak capacity, and peak shape, particularly unsaturated analytes, with less importance to the peak shape of the base. In various examples, the present disclosure can effectively mask silanol from relevant analytes to produce predictable and stable chromatographic separations. In various examples, the present disclosure may be effective to eliminate retention drift or variation that occurs due to unwanted interactions of the support surface with the analyte. The present disclosure may be particularly effective at masking silanol on silica or silica hybrid materials, and in various examples, the present disclosure may improve peak capacity and tailing in all analyte species, especially for alkali. In various examples, the present disclosure can avoid pore blocking, although with oligomeric siloxane bonding (e.g., compared to conventional polymeric coatings of porous silica materials, which can cause pore blocking to greatly reduce the available surface area of the material and create a non-uniform surface-although silane oligomerization is promoted in the present disclosure, there is no sign of pore blocking or surface area reduction).
The present disclosure provides advantages over the prior art based on its unique chemical and performance characteristics. For example, liquid phase separation of unsaturated compounds typically involves the formation of a C18 bonding phase, such as ACQUITY UPC 2 Separation at HSS C18 SB. On this material, the alkyl chain is a retention selectivity factor that gives high methylene/hydrophobic selectivity but very little shape/iso selectivity (retention selector). The present disclosure provides retention selection factors that enable both methylene/hydrophobic selectivity and shape/isomerism selectivity. For example, the stationary phase of the present disclosure excellently retains and separates fat-soluble vitamins, lipids, and metabolites, and provides enhanced shape/isomerism selectivity compared to the C18-bonded phase.
The present disclosure is described in more detail by the following figures and examples, which are for purposes of illustration only and not limitation.
Drawings
The present disclosure will be understood more readily from the following drawings and detailed description. It will be appreciated by those of ordinary skill in the art that the following drawings are not necessarily to scale, but are instead drawn to illustrate the inventive concepts of the present invention.
FIG. 1 shows the structures of glycidoxypropyl trimethoxysilane (GPTMS) (FIG. 1A) and 1-aminoanthracene (FIG. 1B).
Figure 2 shows a schematic of the reaction between an unmodified chromatographic surface and GPTMS.
FIG. 3 shows a schematic representation of the reaction between a modified chromatographic surface and 1-aminoanthracene.
Fig. 4 shows a schematic representation of the chromatographic surface cross-linked with the modifier GPTMS and 1-aminoanthracene.
Fig. 5 shows schematic diagrams of two possible synthetic pathways for preparing chromatographic stationary phases of the present disclosure.
Fig. 6 shows a graph of percent retention of analytes eluted using unmodified BEH particles as the stationary phase and BEH particles modified according to the present disclosure.
Fig. 7A and 7B show exemplary lipid separations using a stationary phase based on 1-aminoanthracene as described in example 9.
FIG. 8A shows chromatograms of C22:0 and C20:0 using a stationary phase based on 1-aminoanthracene as described in example 9.
FIG. 8B shows chromatograms of C16:0, C12:0 and C8:0 using a 1-aminoanthracene based stationary phase as described in example 9.
FIG. 9A shows chromatograms of C22:0 and C20:0 using a stationary phase based on 1-aminoanthracene as described in example 9.
FIG. 9B shows chromatograms of C16:0, C12:0 and C8:0 using a 1-aminoanthracene based stationary phase as described in example 9.
FIG. 10A shows chromatograms of C24:1 and C22:1 using a 1-aminoanthracene based stationary phase as described in example 9.
FIG. 10B shows chromatograms of C20:1, C18:1 and C14:1 using a 1-aminoanthracene based stationary phase as described in example 9.
FIG. 11A shows chromatograms of C24:1 and C22:1 using a 1-aminoanthracene based stationary phase as described in example 9.
FIG. 11B shows chromatograms of C20:1, C18:1 and C14:1 using a 1-aminoanthracene based stationary phase as described in example 9.
FIG. 12A shows chromatograms of C18:0, C18:1 and C18:2 using a 1-aminoanthracene based stationary phase as described in example 9.
FIG. 12B shows chromatograms of C22:1, C22:2, and C22:6 using a 1-aminoanthracene based stationary phase as described in example 9.
FIG. 13A shows chromatograms of C18:0, C18:1 and C18:2 using a 1-aminoanthracene based stationary phase as described in example 9.
FIG. 13B shows chromatograms of C22:1, C22:2 and C22:6 using a 1-aminoanthracene based stationary phase as described in example 9.
Fig. 14 and 15 show chromatograms of various linolenic and eicosadienoic acids using a stationary phase based on 1-aminoanthracene as described in example 9.
Fig. 16 shows an exemplary lipid separation achieved using a 1-aminoanthracene based stationary phase as described in example 9.
Fig. 17A shows chromatograms of various lipids using a stationary phase based on 1-aminoanthracene as described in examples 3 and 10.
Fig. 17B shows chromatograms of various lipids using a stationary phase based on 2-aminomethylpyridine as described in examples 3 and 10.
Fig. 17C shows chromatograms of various lipids using the pyridine-based stationary phases as described in examples 3 and 10.
Fig. 17D shows chromatograms of various lipids using a stationary phase based on 6-aminoquinoline as described in examples 3 and 10.
Fig. 17E shows chromatograms of various lipids using aniline-based stationary phases as described in examples 3 and 10.
Fig. 17F shows chromatograms of various lipids using GPTMS-based stationary phases as described in examples 3 and 10.
Fig. 17G shows chromatograms of various lipids using a stationary phase based on 4-n-octylaniline as described in examples 3 and 10.
FIG. 18A shows chromatograms of C18:0, C18:1 and C18:2 using stationary phases based on 1-aminoanthracene as described in examples 3 and 10.
FIG. 18B shows chromatograms of C22:1, C22:2 and C22:6 using stationary phases based on 1-aminoanthracene as described in examples 3 and 10.
FIG. 19A shows chromatograms of C18:0, C18:1 and C18:2 using stationary phases based on 2-aminomethylpyridine as described in examples 3 and 10.
FIG. 19B shows chromatograms of C22:1, C22:2 and C22:6 using stationary phases based on 2-aminomethylpyridine as described in examples 3 and 10.
FIG. 20A shows chromatograms of C18:0, C18:1 and C18:2 using pyridine-based stationary phases as described in examples 3 and 10.
FIG. 20B shows chromatograms of C22:1, C22:2 and C22:6 using pyridine-based stationary phases as described in examples 3 and 10.
FIG. 21A shows chromatograms of C18:0, C18:1 and C18:2 using stationary phases based on 6-aminoquinoline as described in examples 3 and 10.
FIG. 21B shows chromatograms of C22:1, C22:2 and C22:6 using 6-aminoquinoline based stationary phases as described in examples 3 and 10.
FIG. 22A shows chromatograms of C18:0, C18:1 and C18:2 using aniline based stationary phases as described in examples 3 and 10.
FIG. 22B shows chromatograms of C22:1, C22:2 and C22:6 using aniline based stationary phases as described in examples 3 and 10.
FIG. 23A shows chromatograms of C18:0, C18:1 and C18:2 using GPTMS based stationary phases as described in examples 3 and 10.
FIG. 23B shows chromatograms of C22:1, C22:2 and C22:6 using GPTMS based stationary phases as described in examples 3 and 10.
FIGS. 24A-24F show chromatograms of C18:0, C18:1, C18:2, C22:1, C22:2 and C22:6 using stationary phases based on 4-n-octylaniline as described in examples 3 and 10.
Detailed Description
In various aspects and embodiments, the present disclosure provides chromatographic materials for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, and hydrophobic interaction liquid chromatography that while exhibiting overall retention that facilitates separation of unsaturated molecules, mitigate or avoid retention drift or variation, as well as corresponding devices, kits, methods of manufacture, and methods of use. In some embodiments, the present disclosure also provides for the retention and isolation of structurally related compounds that are difficult to separate, such as key pairs.
The present disclosure advantageously mitigates or avoids retention drift or variation while exhibiting useful overall retention. For example, in SFC, retention drift or variation can be attributed (in various other theories) to the standard CO used for SFC 2 Alkoxylation of the solvent-reachable silanol on the particles under MeOH mobile phase (and/or with other alcohol co-solvents). This is a problem because as the column ages, the user observes the chromatographic changes (e.g., retention time) obtained on their SFC system and again observes changes when a new non-alkoxylated column is installed on the system.
In various aspects and embodiments, the present disclosure provides various solutions to such retention drift or variation and related problems (e.g., retention, peak shape, etc.) by selective modification of chromatographic materials and/or resolution of mixtures of unsaturated related compounds.
Definition of the definition
In various aspects and embodiments, the invention is used to mitigate or prevent retention drift or variation. "retention drift" or "retention change" may include undesirable elution time differences between chromatographic runs or experiments (e.g., in run 1, peak x elutes at time y, but in run 1+n, peak x elutes at time z). Thus, retention drift or variation can cause undesirable effects, including experimental noise, irreproducibility, or failure. Thus, in a broad sense, reducing or preventing retention drift or variation includes resolving or counteracting undesirable elution time differences between chromatographic runs to the extent that the chromatographic experiment provides chromatographic acceptable results.
In some embodiments, the mitigation or prevention of retention drift or change is not an absolute value or a constant value. For example, the amount of retention drift or variation that can occur while still achieving a chromatographically acceptable result can vary depending on the error or variance that is acceptable in a given experiment, the complexity of the sample (e.g., the number of peaks and/or separations). The amount of retention drift or change that can occur while still achieving a chromatographically acceptable result can vary depending on the duration of a given experiment or the desired reproducibility (e.g., the allowable retention drift or change between runs can be smaller if reproducibility over a greater number of runs is desired). Thus, it should be clear that reducing or preventing the retention drift or variation does not necessarily mean absolutely eliminating the retention drift or variation.
In some embodiments, retention drift or variation may be quantitatively reduced or prevented. For example, drift or variation may be retained for a single peak measurement, or an average value of a set of peaks may be derived. The reserve drift or change can be measured over a given period or number of runs. The reserve drift or change may be measured relative to a standard value, a starting value, or between two or more given runs.
Furthermore, the retention drift or change can be quantified by standardized tests. For example, the average% retention change can be calculated by taking the percentage difference of the average absolute peak retention measured by the day 3, 10, or 30 chromatographic test from the average absolute peak retention measured at day 1 chromatographic test. For each day of the test, the column may be equilibrated under one set of test conditions followed by multiple injections of the first test mixture, then equilibrated under a second set of conditions followed by multiple injections of the second test mixture.
According to this standardized test, the test results, reducing or preventing retention drift or change may include retention drift or change of 30 days-5%, 30 days-4%, 30 days-3%, 30 days-2%, 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 times-5%, 30 times-4%, 30 times-3%, 30 times-2%, 30 times-1%, 10 times-5%, 10 times-4%, 10 times-3%, 10 times-2%, 10 times-1%, 3 times-4%, 3 times-3%, 3 times-2%, or 3 times-1%.
In the case of the use of the present invention in the case of a further embodiment, reducing or preventing a retention drift or change may comprise a change in the retention of 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). Ltoreq.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, 1.1, 9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.0, 1.8, 1.0, 0.0, 3.0, 0.0, 0.0.0, 0.0.
"high purity" or "high purity chromatographic material" includes materials made from high purity precursors. In certain aspects, the high purity material has reduced metal contamination and/or non-reduced chromatographic properties, including, but not limited to, the acidity of the surface silanol and the surface inhomogeneity.
"chromatographic surface" includes surfaces that provide chromatographic separation of a sample. In certain aspects, the chromatographic surface is porous. In some aspects, the chromatographic surface may be a surface of a particle, a surface porous material, or a monolith. In certain aspects, the chromatographic surface is comprised of the surface of one or more particles, surface porous materials, or monoliths used in combination in a chromatographic separation process. In other aspects, the chromatographic surface is pore-free.
"ionizable modifier" includes functional groups with electron donating or electron withdrawing groups. In certain aspects, the ionizable modifier contains one or more carboxylic acid groups, amino groups, imino groups, amido groups, pyridyl groups, imidazolyl groups, ureido groups, thionyl-ureido groups, or aminosilane groups, or a combination thereof. In other aspects, the ionizable modifier contains a group with a nitrogen or phosphorus atom that has a free electron lone pair. In certain aspects, the ionizable modifier is covalently attached to the surface of the material and has an ionizable group. In some cases, it is attached to the chromatographic material by chemical modification of the surface hybridization groups.
"hydrophobic surface groups" include surface groups that exhibit hydrophobicity on chromatographic surfaces. In certain aspects, the hydrophobic group may be a nitrogen-bonded phase, such as C 4 To C 18 And a bonding phase. In other aspects, the hydrophobic surface groups may contain embedded polar groups to keep the exterior of the hydrophobic surface hydrophobic. In some cases, it is attached to the chromatographic material by chemical modification of the surface hybridization groups. In other cases, the hydrophobic group may be C 4 -C 30 Embedded polar, chiral, phenylalkyl or pentafluorophenyl bonded coatings.
"chromatography core" includes chromatographic materials forming the interior of the materials of the present disclosure, including but not limited to organic materials as defined herein, such as silica or hybrid materials, in the form of particles, monoliths, or another suitable structure. In certain aspects, 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 may be disposed on or bonded or annealed to the chromatographic core in a manner that recognizes discrete or distinct transitions or may be bonded to the chromatographic core in a manner that blends with the surface of the chromatographic core to cause the material to fade and without a discrete core surface. In certain embodiments, the chromatographic surface material may be the same as or different from the material of the chromatographic core and may exhibit different physical or physicochemical properties from the chromatographic core, including, but not limited to, pore volume, surface area, average pore diameter, carbon content, or hydrolytic pH stability.
"hybrid" includes "hybrid inorganic/organic materials," including inorganic-based structures in which organic functional groups are incorporated into internal or "backbone" inorganic structures as well as hybrid material surfaces. The inorganic portion of the hybrid material may be, for example, alumina, silica, titanium, cerium or zirconium or oxides thereof, or a ceramic material. "hybridization" includes inorganic-based structures in which organic functional groups are incorporated into internal or "backbone" inorganic structures as well as the surface of the hybrid material. Exemplary hybrid materials, as described above, are shown in U.S. Pat. nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913, the contents of which are incorporated herein by reference in their entirety.
The term "cycloaliphatic radical" includes closed-loop structures having three or more carbon atoms. Cycloaliphatic groups include cycloalkanes or cycloalkanes (which are saturated cyclic hydrocarbons), cycloalkenes (which are unsaturated, have two or more double bonds), and cycloalkynes having a triple bond. They do not include aromatic groups. Examples of cycloalkanes include cyclopropane, cyclohexane and cyclopentane. Examples of cycloolefins include cyclopentadiene, cyclohexadiene, and cyclooctatetraene. Cycloaliphatic radicals also include fused ring structures and substituted cycloaliphatic radicals, such as alkyl-substituted cycloaliphatic radicals. In the case of cycloaliphatic radicals, such substituents may further include lower alkyl, lower alkenyl, lower alkoxy, lower alkylthio, lower alkylamino, lower alkylcarboxy, nitro, hydroxy, -CF 3 -CN, etc.
The term "aliphatic radical" includes organic compounds characterized by a straight or branched chain, typically having from 1 to 24 carbon atoms. Aliphatic groups include alkyl, alkenyl, and alkynyl groups. In complex structures, the chain may be branched or crosslinked. In some embodiments, the aliphatic group may comprise a chain having 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 16 carbon atoms, or 12 to 14 carbon atoms, or any combination 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 alkyl groups. Such hydrocarbon moieties may be substituted on one or more carbons with, for example, halogen, hydroxy, thiol, amino, alkoxy, alkylcarboxyl, alkylthio, or nitro. As used herein, unless the carbon number is otherwise specified, "lower aliphatic "refers to an aliphatic group as defined above having 1 to 6 carbon atoms (e.g., lower alkyl, lower alkenyl, lower alkynyl). Representative of such lower aliphatic groups, e.g., lower alkyl, are methyl, ethyl, n-propyl, isopropyl, 2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl, tert-butyl, 3-thiopentyl, and the like. The term "nitro" as used herein refers to-NO 2 The method comprises the steps of carrying out a first treatment on the surface of the The term "halogen" refers to-F, -Cl, -Br or-I; the term "thiol" refers to SH; and the term "hydroxy" refers to-OH. Thus, the term "alkylamino" as used herein refers to an alkyl group as defined above having an amino group attached thereto. Suitable alkylamino groups include groups having from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms. The term "alkylthio" refers to an alkyl group as defined above having a mercapto group attached thereto. Suitable alkylthio groups include groups having from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms. The term "alkylcarboxy" as used herein refers to an alkyl group as defined above having a carboxy group attached thereto. The term "alkoxy" as used herein refers to an alkyl group as defined above having an oxygen atom attached thereto. Representative alkoxy groups include groups having from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms, such as methoxy, ethoxy, propoxy, t-butoxy, and the like. The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups similar to alkyl groups but containing 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, branched alkyl, cycloalkyl (alicyclic), alkyl substituted cycloalkyl, and cycloalkyl substituted alkyl. In certain embodiments, the linear or branched alkyl groups have 30 or fewer carbon atoms in their backbone, e.g., C for linear chains 1 -C 30 Or C for branched chains 3 -C 30 . In certain embodiments, the linear or branched alkyl groups have 20 or fewer carbon atoms in their backbone, e.g., C for linear chains 1 -C 20 Or C for branched chains 3 -C 20 More preferably 18 or less. Likewise, cycloalkyl groups having 4 to 10 carbon atoms in their ring structure are preferred, with 4 to 7 carbon atoms in the ring structure being more preferred. The term "lower alkyl" refers to alkyl groups having 1 to 6 carbons in the chain and cycloalkyl groups having 3 to 6 carbons in the ring structure.
Furthermore, the term "alkyl" (including "lower alkyl") as used throughout this disclosure includes "unsubstituted alkyl" and "substituted alkyl" which refers to an alkyl moiety having a substituent on one or more carbons of the hydrocarbon backbone that replaces hydrogen. Such substituents may include, for example, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, sulfanylcarbonyl, alkoxy, phosphate, phosphonato, phosphinato (phosphinato), cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), amido (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonate (sulfonato), sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those skilled in the art will appreciate that moieties substituted on the hydrocarbon chain may themselves be substituted, if appropriate. Cycloalkyl groups may be further substituted, for example, with the substituents described above. An "aralkyl" moiety is an alkyl group substituted with, for example, an aryl group having 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 the formula-NR a R b Wherein R is an unsubstituted or substituted moiety of a And R is b Each independently is hydrogen, alkyl, aryl or heterocyclyl, or R a And R is b Together with the nitrogen atom to which they are attached, form a cyclic moiety having 3 to 8 atoms in the ring. Thus, unless otherwise indicated, the term "amino" includes cyclic amino moieties such as piperidinyl or pyrrolidinyl. "amino substituted AmmoniaThe radical "means where R a And R is b An amino group further substituted with an amino group.
The term "aromatic radical" includes unsaturated cyclic hydrocarbons containing one or more rings. The term "monocyclic aromatic" includes unsaturated cyclic hydrocarbons containing one ring. The term "polycyclic aromatic" includes unsaturated cyclic hydrocarbons containing two or more rings. Aromatic groups include 5-and 6-membered monocyclic groups which may include 0 to 4 heteroatoms such as furan, pyrrole, pyrroline, oxazole, thiazole, imidazole, imidazoline, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, benzene, pyridine, pyridazine, pyrimidine, pyrazine, triazine, thiophene and the like. The aromatic ring may be substituted at one or more ring positions by, for example, halogen, lower alkyl, lower alkenyl, lower alkoxy, lower alkylthio, lower alkylamino, lower alkylcarboxy, nitro, hydroxy, -CF 3 -CN, etc. Aromatic groups include 5-and 6-membered polycyclic groups which may include 0 to 8 heteroatoms, such as indene, indolizine, indole, isoindole, indoline, indazole, benzimidazole, benzothiazole, naphthalene, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1, 8-naphthyridine, quinuclidine, fluorene, carbazole, anthracene, acridine, phenazine, phenothiazine, phenoxazine, pyrene, and the like. Polycyclic aryl groups include fused aryl groups.
The term "aryl" includes 5-and 6-membered monocyclic aryl groups, which may include 0 to 4 heteroatoms, such as unsubstituted or substituted benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine and the like. Aryl also includes polycyclic fused aryl groups such as naphthyl, quinolinyl, indolyl, and the like. The aromatic ring may be substituted at one or more ring positions with substituents such as described above for alkyl. Suitable aryl groups include unsubstituted and substituted phenyl groups. The term "aryloxy" as used herein refers to an aryl group as defined above having an oxygen atom attached thereto. The term "aralkoxy" as used herein refers to 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, such as 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 capable of 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 celluloses, macrocyclic antibiotics, cyclodextrins, crown ethers, and metal complexes.
The term "embedded polar functional group" is a functional group that provides an overall polar moiety such that interaction with the basic sample is reduced due to unreacted silanol groups on the surface of the shielding silica. The embedded polar functional groups include, but are not limited to, carbonate, amide, urea, ether (e.g., -O- "between carbon-containing groups), thioether, thionyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocycle, triazole functional groups or carbamate functional groups, and chiral moieties as disclosed in U.S. patent No. 5,374,755.
The term "chromatographic enhancing pore geometry" includes the geometry of the pore structure of the materials of the present disclosure, which has been found to enhance the chromatographic separation capacity of the materials, e.g., as compared to other chromatographic media in the art. For example, the geometry may be formed, selected, or constructed, and various properties and/or factors may be used to determine whether the chromatographic separation capacity of the material has been "enhanced," e.g., compared to geometries known or conventionally used in the art. Examples of such factors include high separation efficiency, longer column life, and high mass transfer properties (as evidenced by, for example, reduced band broadening and good peak shape). These properties can be measured or observed using techniques recognized in the art. For example, the chromatograph-enhanced pore geometry of the present porous inorganic/organic hybrid material differs from prior art materials by the absence of "ink bottle" or "shell shape" pore geometry or morphology (neither of which is desirable because they, for example, reduce mass transfer rates to cause lower efficiencies).
Chromatographic enhanced pore geometry is found in hybrid materials containing only a small number of micropores. A small number of micropores are achieved in the hybrid material when all pores of diameter of about <34 a constitute less than about 110 square meters per gram of the specific surface area of the material. Hybrid materials with such low Micropore Surface Area (MSA) provide chromatographic enhancement, including high separation efficiency and good mass transfer properties (as demonstrated by, for example, reduced band broadening and good peak shape). Micropore Surface Area (MSA) is defined as the surface area in pores having a diameter less than or equal to 34 a as measured by multipoint nitrogen adsorption analysis from the adsorption branches of an isotherm using the BJH method. The acronyms "MSA" and "MP" are used interchangeably herein to refer to "micropore surface area".
The term "functional group" includes organic functional groups that impart a specific chromatographic function to a chromatographic stationary phase.
The term "heterocyclic group" includes closed ring structures in which one or more atoms in the ring is a non-carbon element, such as nitrogen, sulfur, or oxygen. The heterocyclic groups may be saturated or unsaturated, and heterocyclic groups such as pyrrole and furan may be of aromatic nature, i.e. "heteroaryl". They comprise one or more ring structures. A heterocyclic group having two or more ring structures is a "polyheterocyclic aryl". These groups may have condensed ring structures such as quinoline and isoquinoline. Other examples of heterocyclic groups include pyridine and purine. Heterocyclic groups may also be substituted at one or more of the constituent atoms by, for example, halogen, lower alkyl, lower alkenyl, lower alkoxy, lower alkylthio, lower alkylamino, lower alkylcarboxy, nitro, hydroxy, -CF 3 -CN, etc. Suitable heteroaromatic and heteroalicyclic groups typically have 1 to 3 separate or fused rings, 3 to about 8 members each and one or more N, O or S atoms, for example coumarin, quinoline, pyridine, pyrazine, pyrimidine, furan, pyrrole, thiophene, thiazole, oxazole, imidazole, indole, benzofuran, benzothiazole, tetrahydrofuran, tetrahydropyran, piperidine, morpholine and pyrrolidinyl groups.
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, titania, zirconia.
The term "monolith" is intended to include a collection of individual particles packed in the form of a bed, wherein the shape and morphology of the individual particles are maintained. The particles are advantageously packed with a material that binds the particles together. Any bonding material known in the art may be used, such as divinylbenzene, methacrylate, urethane, alkene, alkyne, amine, amide, isocyanate or epoxy based linear or crosslinked polymers, as well as condensation reactions of organoalkoxysilanes, tetraalkoxysilanes, polyorganoalkoxysiloxanes, polyethoxysilanes, and ceramic precursors. In certain embodiments, the term "monolith" also includes hybrid monoliths made by other methods, such as the hybrid monoliths detailed in U.S. patent No. 7,250,214; from one or more of the compounds containing 0-99 mol% of silicon dioxide (e.g. SiO 2 ) A hybrid monolith made from the condensation of monomers of (a); a hybrid monolith made of agglomerated porous inorganic/organic particles; a hybrid monolith having a chromatographic-enhanced pore geometry; a hybrid monolith without chromatographic enhancing pore geometry; a hybrid monolith having an ordered pore structure; a hybrid monolith having a non-periodic pore structure; a hybrid monolith having amorphous or amorphous molecular ordering; a hybrid monolith having crystalline domains or regions; a hybrid monolith having a variety of macroporous and mesoporous properties; and various different aspect ratio hybridization monoliths. In certain embodiments, the term "monolith" also includes inorganic monoliths, such as G.Guiochon-J. Chromatogr. A1168 (2007) those described in 101-168.
The term "nanoparticle" is a microscopic member of a microscopic particle/particle or powder/nanopowder having at least one dimension less than about 100 nanometers, such as a diameter or particle thickness less than about 100 nanometers (0.1 millimeter), which may be crystalline or amorphous. Nanoparticles have properties that are different from and generally superior to conventional bulk materials, including, for example, greater strength, hardness, ductility, sinterability, and greater reactivity, among others. Much scientific research continues to be devoted to determining the properties of nanomaterials, with small amounts having been synthesized by many methods (primarily as nanoscale powders Last), including colloidal precipitation, mechanical grinding, and vapor nucleation and growth. A number of reviews have recorded the recent developments in nanophase materials and are incorporated herein by reference: gleiter, H. (1989) "Nano-crystalline materials," A. RTM., B.H.) "Prog. Mater. Sci.33:223-315 and Siegel, R.W. (1993) "Synthesis and properties of nano-phase materials," J.P.) "Mater. Sci. Eng. A168:189-197. In certain embodiments, the nanoparticle comprises an oxide or nitride of: silicon carbide, aluminum, diamond, cerium, carbon black, carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium, gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc, boron, and mixtures thereof. In certain embodiments, the nanoparticles of the present disclosure are selected from diamond, zirconia (amorphous, monoclinic, tetragonal, and cubic forms), titania (amorphous, anatase, brookite, and rutile forms), aluminum (amorphous, alpha, and gamma forms), and boron nitride (cubic forms). In particular embodiments, the nanoparticles of the present disclosure are selected from nanodiamond, silicon carbide, titanium dioxide (anatase form), cubic boron nitride, and any combinations thereof. Furthermore, in certain embodiments, the nanoparticle may be crystalline or amorphous. In certain embodiments, the nanoparticle is less than or equal to 100 millimeters in diameter, such as less than or equal to 50 millimeters in diameter, such as less than or equal to 20 millimeters in diameter.
Furthermore, nanoparticles featuring dispersion within the composite of the present disclosure are intended to describe exogenously added nanoparticles. This is different from nanoparticles that can be formed in situ or from formations (formations) that have significant similarity to putative (push) nanoparticles, where, for example, macromolecular structures such as particles may comprise endogenously generated aggregates of these.
The term "substantially disordered" refers to the lack of pore order based on x-ray powder diffraction analysis. In particular, "substantially disordered" is determined by the lack of peaks in the x-ray diffraction pattern at diffraction angles corresponding to d-values (or d-spacing) of at least 1 nm.
"surface modifying agents" generally include organic functional groups that impart specific chromatographic functions to a chromatographic stationary phase. The porous inorganic/organic hybrid material has organic groups and silanol groups that may be additionally substituted or derivatized with surface modifying agents.
The word "surface modified" is used herein to describe a composite of the present disclosure having organic groups and silanol groups that may be additionally substituted or derivatized with a surface modifying agent. "surface modifying agents" include organic functional groups that (generally) impart a specific chromatographic function to a chromatographic stationary phase. The surface modifying agents as disclosed herein are bonded to the substrate, for example, by derivatization or coating and subsequent crosslinking, to impart chemical properties to the substrate. In one embodiment, the organic groups of the hybrid material react to form organic covalent bonds with the surface modifying agent. The modifier may form an organic covalent bond with the organic group of the material by a number of mechanisms well known in organic and polymer chemistry including, but not limited to, nucleophilic, electrophilic, cycloaddition, free radical, carbene, nitrene, and carbocationic reactions. Organic covalent bonds are defined as those involving covalent bond formation between common elements of organic chemistry including, but not limited to, hydrogen, boron, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur, and halogens. Furthermore, carbon-silicon and carbon-oxygen-silicon bonds are defined as organic covalent bonds, whereas silicon-oxygen-silicon bonds are not defined as organic covalent bonds. Various synthetic transformations are well known in the literature, see e.g. March, J. Advanced Organic Chemistry, 3 rd edition, Wiley, New York, 1985。
Chromatographic materials of the present disclosure may include those comprising a silica core, a metal oxide core, an inorganic-organic hybrid material, or a set of block copolymer cores. The core material may be a high purity chromatographic core composition as discussed herein. Similarly, the chromatographic core may be in the normal (e.g., non-high purity) form/analog/homolog of the high purity materials 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 block copolymers thereof, ceramics, silica, iminosilicon nitride, silicon aluminum nitride, diimine silicon and silicon oxynitride. Further examples of suitable core materials (modified or unmodified for use) are described in U.S. publication nos. 2009/0127777, 2007/01355304, 2009/0209722, 2007/0215547, 2007/0141325, 2011/0049056, 2012/0055860 and 2012/0273404, and international publication No. WO2008/103423, which are incorporated herein by reference in their entirety.
The chromatographic core may be in the form of discrete particles or may be a monolith. The chromatographic core may be any porous material and is commercially available or may be manufactured by known methods such as those described in, for example, U.S. patent nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913, which are incorporated herein by reference in their entirety. In some embodiments, the chromatographic core may be a pore-free core.
One of ordinary skill in the art can vary the composition of the chromatographic surface material and the chromatographic core material to provide enhanced chromatographic selectivity, enhanced column chemical stability, enhanced column efficiency, and/or enhanced mechanical strength. Similarly, the composition of the surrounding material provides a hydrophilic/lipophilic balance (HLB), surface charge (e.g., isoelectric point or silanol pK a ) And/or a change in surface functionality to enhance chromatographic separation. In addition, in some embodiments, the composition of the chromatographic material may also provide surface functionality that may be used for further surface modification.
The ionizable groups and hydrophobic surface groups of the chromatographic materials of the present disclosure can be prepared using known methods. Some ionizable modifiers are commercially available. For example, silanes, including aminoalkyl trialkoxysilanes, methylaminoalkyl trialkoxysilanes, and pyridylalkyl trialkoxysilanes are commercially available. Other silanes, such as chloropropyl alkyl trichlorosilane and chloropropyl alkyl trialkoxysilane, are also commercially available. These may be bonded and reacted with imidazole to produce an imidazolylalkylsilyl surface species, or with pyridine to produce a pyridylalkylsilyl surface species. Other acidic modifiers are also commercially available, including, but not limited to, sulfopropyltrisilanol, carboxyethylsilanetriol, 2- (carbomethoxy) ethylmethyldichlorosilane, 2- (carbomethoxy) ethyltrichlorosilane, 2- (carbomethoxy) ethyltrimethoxysilane, n- (trimethoxysilylpropyl) ethylenediamine, triacetic acid, (2-diethylphosphorylethyl) triethoxysilane, 2- (chlorosulfonylphenyl) ethyltrichlorosilane, and 2- (chlorosulfonylphenyl) ethyltrimethoxysilane.
The synthesis of these types of silanes using common synthetic procedures, including grignard reactions and hydrosilylation, is known to those skilled in the art. The product may be purified by chromatography, recrystallisation 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 procedure is the reaction of a primary amine with phosgene or a reagent known as triphosgene.
In one aspect, the present disclosure relates to a method of separating a related compound from a mixture, the method comprising (a) providing a mixture containing the related compound; (b) Introducing a portion of the mixture into a chromatography system having a chromatography column; and (c) eluting the separated related compounds from the column; wherein the column has a stationary phase having the following structure (i):
[X](W) a (Q) b (T) c (i)
wherein:
x is a chromatographic substrate comprising silica, metal oxide, inorganic-organic hybrid material, a set of block copolymers, or a combination thereof;
w is selected from hydrogen and hydroxy, wherein W is bonded to the surface of X;
q is a first substituent that minimizes variation in analyte retention over time under chromatographic conditions with low water concentration;
t is a second substituent chromatographically retaining the analyte, wherein T has one or more monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic, or polyheterocyclic aromatic groups, each optionally substituted with an aliphatic group; and is also provided with
b and c are positive numbers, 0.05-100 (b/c), and a-0.
In various embodiments, the selectivity of the chromatographic material may be controlled or affected by the choice of Q and/or T, the density of Q and/or T on the surface, or a combination thereof. In some embodiments, Q and T play a role in retaining the relevant compounds. In other embodiments, T selectively retains each of the different related compounds.
In various embodiments, the present disclosure provides a bonded chromatographic material to which a ligand may be attached. The high density coverage achieved by the bonding method of the present invention can be 2 to 3X higher than current silane bonding chemistries in conventional SFC materials. The combination of high coverage and other properties of Q and/or T may prevent interaction of surface silanols with the analyte.
In various embodiments, the present disclosure proposes a high density of the binding phase that enhances retention and prevents retention drift or change caused by analyte interactions with surface silanol or other secondary retention mechanisms. The elimination of these secondary and secondary selective components greatly improves peak shape and column peak capacity, especially for alkaline analytes.
In various embodiments, the present disclosure proposes that the use of a two-component system based on coupling chemistry results in uniform coverage of mixed surface functional groups. Unlike a mixed particle bed, in which two separate particles having different surface chemistries are mixed, such materials have uniform and predictable surface characteristics throughout. Columns packed with such materials are less prone to chromatographic instability due to poor particle mixing or particle type segregation during column packing.
In various embodiments, the present disclosure provides for simplified column packing due to the use of a single particle slurry.
In various embodiments, the present disclosure provides chromatographic packing materials with improved selectivity for acidic, neutral, and basic analytes in a single column without the use of mixed or multiparticulate-based beds.
In various embodiments, the present disclosure proposes that the ratio of components on the particle surface can be easily controlled to alter the selectivity of the support, providing a wide range of chromatographic separation options.
In various embodiments, the present disclosure provides bonding chemistry to form crosslinked films of silane surface modifiers by polymerization of surface reactive groups or by adding a crosslinking agent before or after the addition of selective ligands.
In other embodiments, the chromatographic materials of the present disclosure are non-porous. In another embodiment, the chromatographic material of the present disclosure is at a pH of about 1 to about 14; at a pH of about 10 to about 14; or hydrolytically stable at 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 nanoparticles or a mixture of more than one nanoparticle dispersed within the chromatographic surface.
In certain embodiments, the nanoparticle is present at < 20% by weight of the nanocomposite, < 10% by weight of the nanocomposite, or < 5% by weight of the nanocomposite.
In other embodiments, the nanoparticle is crystalline or amorphous and may be silicon carbide, aluminum, diamond, cerium, carbon black, carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium, gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc, boron, oxides thereof, or nitrides thereof. In certain embodiments, the nanoparticle is a substance comprising one or more moieties selected from the group consisting of nanodiamond, silicon carbide, titanium dioxide, and cubic boron nitride. In other embodiments, the nanoparticle may be less than or equal to 200 nanometers in diameter, less than or equal to 100 nanometers in diameter, less than or equal to 50 nanometers in diameter, or less than or equal to 20 nanometers in diameter.
In one or more embodiments, Q is represented by the formula:
wherein:
n 1 is an integer of 1 to 30;
n 2 is an integer of 1 to 30;
R 1 、R 2 、R 3 and R is 4 Each independently selected from the group consisting of hydrogen, hydroxy, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, lower alkyl, protected or deprotected alcohol and a zwitterionic;
Z is
(a) Tool withHaving formula (B) 1 ) x (R 5 ) y (R 6 ) z Si-wherein x is an integer from 1 to 3, y is an integer from 0 to 2, z is an integer from 0 to 2, and x+y+z=3; r is R 5 And R is 6 Each independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, substituted or unsubstituted aryl, cycloalkyl, branched alkyl, lower alkyl, protected or deprotected alcohol, zwitterionic groups, and siloxane bonds; and B is 1 Is a siloxane bond;
(b) Attachment to the surface organofunctional hybrid group through direct carbon-carbon bond formation or through heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or polyurethane bonds; or (b)
(c) An adsorption surface group not covalently attached to the surface of the material;
y is an intercalating polar functional group; and is also provided with
A is selected from the group consisting of hydrophilic end groups, functionalizable groups, hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, lower alkyl, and polarizable groups.
In one or more embodiments, T is represented by the formula:
wherein the method comprises the steps of
m 1 Is an integer of 1 to 30;
m 2 is an integer of 1 to 30;
m 3 is an integer of 1 to 3;
R 7 、R 8 、R 9 and R is 10 Each independently selected from the group consisting of hydrogen, hydroxy, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, lower alkyl, protected or deprotected alcohol, zwitterion, aromatic hydrocarbon group, and heterocyclic aromatic hydrocarbon group;
Z is
(a) Has the following structure(B 1 ) x (R 5 ) y (R 6 ) z Si-wherein x is an integer from 1 to 3, y is an integer from 0 to 2, z is an integer from 0 to 2, and x+y+z=3; r is R 5 And R is 6 Each independently selected from methyl, ethyl, n-propyl, isopropyl,n-Ding (n-Ding) Radical, isobutyl, tert-butylSubstituted or unsubstituted aryl, cycloalkyl, branched alkyl, lower alkyl, protected or deprotected alcohol, zwitterionic group, and siloxane bond; and B is 1 Is a siloxane bond;
(b) Attachment to the surface organofunctional hybrid group through direct carbon-carbon bond formation or through heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or polyurethane bonds; or (b)
(c) An adsorption surface group not covalently attached to the surface of the material;
y is an intercalating polar functional group;
d is selected from the group consisting of 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–(CH 2 ) 0-12 –R 11 ，
R 11 is a first monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group; and is also provided with
R 12 Is hydrogen, an aliphatic group or a second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group, wherein R 11 And R is 12 Optionally substituted with one or more groups selected from aliphatic groups, halogen, hydroxy, thiol, amino, alkoxy, alkylcarboxy, alkylthio, and nitro.
In some embodiments, R 11 Or R is 12 The first or second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group of (c) may be a polycyclic aromatic or polyheterocyclic aromatic hydrocarbon having at least 2 aromatic rings. R is R 11 Or R is 12 The first or second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group of (c) may also be a polycyclic aromatic or polyheterocyclic aromatic hydrocarbon having at least 3 aromatic rings. R is R 11 Or R is 12 The first or second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group of (c) may also be a polycyclic aromatic or polyheterocyclic aromatic hydrocarbon having at least 4 aromatic rings.
In one embodiment, R 11 Or R is 12 The first or second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polycyclic aromatic group of (a) is selected from furan, pyrrole, pyrroline, oxazole, thiazole, imidazole, imidazoline, pyrazole, pyrazoline, pyrazolidine, isoxazole, isothiazole, benzene, pyridine,pyridazines, pyrimidines, pyrazines, triazines, thiophenes, indenes, indolizines, indoles, isoindoles, indolines, indazoles, benzimidazoles, benzothiazoles, naphthalenes, quinolizines, quinolines, isoquinolines, cinnolines, phthalazines, quinazolines, quinoxalines, 1, 8-naphthyridines, quinuclidines, fluorenes, carbazoles, anthracenes, acridines, phenazines, phenothiazines, phenoxazines, pyrenes and derivatives thereof, wherein the group is unsubstituted or optionally substituted with an aliphatic group.
In other embodiments, R 11 Or R is 12 The first or second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic radical of (C) may be substituted by at least one C 1 -C 24 Aliphatic group substitution. In particular, the radical may be substituted by at least one C 2 -C 22 Aliphatic group, a C 3 -C 20 Aliphatic group, a C 4 -C 18 Aliphatic group, a C 5 -C 16 Aliphatic group, a C 6 -C 14 Aliphatic group, a C 7 -C 12 Aliphatic group, a C 8 -C 10 Aliphatic groups or aliphatic groups of any combination of the foregoing carbon lengths, e.g. C 8 -C 18 Aliphatic groups or groups of various other sizes as described in this disclosure.
R11 or R12 may be an aminoanthracene (e.g., 1-aminoanthracene, 2-aminoanthracene, or 9-aminoanthracene) or a methylamino anthracene (e.g., 1-methylamino anthracene, 2-methylamino anthracene, or 9-methylamino anthracene). The aminoanthracene or methylaminoanthracene may be substituted on the ring structure with another aliphatic group, such as lower alkyl. In some embodiments, the amino-anthracene or methylamino-anthracene may have the formula (X) -amino- (Y) -alkyl-anthracene or (X) -methylamino- (Y) -alkyl-anthracene, where X is 1, 2, or 9 and Y is 1-10, representing a carbon position on the anthracene (e.g., 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-anthracene; 1-methylamino-1-methyl-anthracene; 1-methylamino-2-methyl-anthracene; 1-methylamino-3-methyl-anthracene; 1-methylamino-4-methyl-anthracene; 1-methylamino-5-methyl-anthracene; 1-methylamino-6-methyl-anthracene; 1-amino-8-methyl-anthracene; 1-amino-9-methyl-anthracene; 1-amino-10-methyl-anthracene) Base-anthracene, etc.).
In other embodiments, the amino-anthracene or methylaminoanthracene may have the formula (X) -amino- (Y) -alkyl- (Z) -alkyl-anthracene or (X) -methylamino- (Y) -alkyl- (Z) -alkyl-anthracene, where X is 1, 2, or 9 and Y and Z are 1-10, representing carbon positions on the anthracene, provided that Y and Z are not identical (e.g., 1-amino-1-methyl-2-methyl-anthracene, 1-amino-1-methyl-3-methyl-anthracene, 1-amino-1-methyl-4-methyl-anthracene, 1-amino-1-methyl-5-methyl-anthracene, 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 may be disubstituted on the ring structure with a second polar group, such as an amine (e.g., 1-amino, 4-N, N-dimethylaminoanthracene).
R11 or R12 may be naphthylamine (e.g., 1-naphthylamine or 2-naphthylamine) or methylnaphthylamine (e.g., 1-methylnaphthylamine or 2-methylnaphthylamine). The naphthylamine or methylnaphthylamine may be substituted with another aliphatic group, such as lower alkyl. In some embodiments, the naphthylamine or methylnaphthylamine may 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 (e.g., 1-amino-1-methyl-naphthalene; 1-amino-2-methyl-naphthalene; 1-amino-3-methyl-naphthalene; 1-amino-4-methyl-naphthalene; 1-amino-5-methyl-naphthalene; 1-amino-6-methyl-naphthalene; 1-amino-7-methyl-naphthalene; 1-amino-8-methyl-naphthalene; 9 and 10 positions may not be substituted; 1-methylamino-1-methyl-naphthalene; 1-methylamino-2-methyl-naphthalene; 1-methylamino-3-methyl-naphthalene; 1-methylamino-4-methyl-naphthalene; 1-methylamino-5-methyl-naphthalene; 1-methylamino-6-methyl-naphthalene; 1-methylamino-7-methyl-naphthalene; 1-amino-7-methyl-naphthalene; 1-methyl-8-methyl-naphthalene, etc.).
In other embodiments, the naphthylamine or methylnaphthylamine may have the formula (X ') -amino- (Y ') -alkyl- (Z ') -alkyl-naphthalene or (X ') -methylamino- (Y ') -alkyl- (Z ') -alkyl-naphthalene, wherein X ' is 1 or 2 and Y ' and Z ' are 1-10, representing carbon positions on naphthalene, provided that Y ' and Z ' are different (e.g., 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-amino-1-methyl-8-methyl-naphthalene; and the like).
R11 or R12 may be an aminophenol (e.g., 1-aminophenol, 2-aminophenol, 3-aminophenol, 4-aminophenol or 9-aminophenol) or a methylamino-phenanthrene (e.g., 1-methylamino-phenanthrene, 2-methylamino-phenanthrene, 3-methylamino-phenanthrene, 4-methylamino-phenanthrene or 9-methylamino-phenanthrene). The aminophenol or methylamino phenanthrene may be substituted on the ring structure with another aliphatic group, for example lower alkyl. In some embodiments, the amino phenanthrene or methylaminophenanthrene can have 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 a carbon position on the phenanthrene (e.g., 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 aminophenol or methylamino phenanthrene can have 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 (excluding the conjugated carbon), representing a carbon position on the phenanthrene, provided that Y and Z are not the same (e.g., 1-amino-1-methyl-2-methyl-phenanthrene; etc.).
Similarly, R11 or R12 may be an aminopyrene (e.g., 1-aminopyrene, 2-aminopyrene, 3-aminopyrene, 4-aminopyrene, or 5-aminopyrene) or a methylaminopyrene (e.g., 1-methylaminopyrene, 2-methylaminopyrene, 3-methylaminopyrene, 4-methylaminopyrene, or 5-methylaminopyrene). The aminopyrene and methylaminopyrene may be substituted similarly to the aminoanthracene and methylaminoanthracene.
Similarly, R11 or R12 may be amino chrysene (e.g., 1-amino chrysene, 2-amino chrysene, 3-amino chrysene, 4-amino chrysene, 5-amino chrysene, or 6-amino chrysene) or methylamino chrysene (e.g., 1-methylamino chrysene, 2-methylamino chrysene, 3-methylamino chrysene, 4-methylamino chrysene, 5-methylamino chrysene, or 6-methylamino chrysene). The amino group chrysene and the methylamino group chrysene may be substituted similarly to the amino anthracene and the methylamino anthracene.
In some embodiments, T is represented by one of the following structures:
wherein each E is independently a bond or lower alkyl optionally substituted with hydroxy, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, lower alkyl, a protected or deprotected alcohol, a zwitterionic, an aromatic or heterocyclic aromatic group, and wherein Z, Y, R 9 、m 1 、m 2 And D is as defined above.
In other embodiments, T is represented by one of the following structures:
/>
therein Z, Y, R 9 、m 1 、m 2 And D is as defined above.
In some embodiments, b and c are positive numbers, the ratio is 0.05.ltoreq.b/c.ltoreq.100, and a.gtoreq.0. In some embodiments, Q and T are different, while in other embodiments, Q and T are the same. Q may include two or more different portions, and T may include two or more different portions. 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%.
In one or more embodiments, Q is non-polar. In some embodiments, Q comprises a borate or nitro functionality. In some embodiments, Q is represented by one of the following formulas:
Wherein Z may comprise a compound having formula (B 1 ) x (R 5 ) y (R 6 ) z Si-, wherein x is an integer from 1 to 3, y is an integer from 0 to 2, z is an integer from 0 to 2, and x+y+z=3. R is R 5 And R is 6 Each independently represents methyl ethyl group,n-Ding (n-Ding) Radical, isobutyl, tert-butyl、Different speciesPropyl, tertiary hexyl, substituted or unsubstituted aryl, cycloalkyl, branched alkyl, lower alkyl, protected or deprotected alcohol or zwitterionic group, and B 1 May represent a siloxane bond.
In another embodiment, Z is a linkage to the surface organofunctional hybrid group through direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or polyurethane bond. In yet another embodiment, Z is an adsorbing surface group that is not covalently attached to the surface of the material.
In some embodiments, T is represented by one of the following formulas:
/>
/>
wherein Z may comprise a compound having formula (B 1 ) x (R 5 ) y (R 6 ) z Si-, wherein x is an integer from 1 to 3, y is an integer from 0 to 2, z is an integer from 0 to 2, and x+y+z=3. R is R 5 And R is 6 Can independently represent methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, substituted or unsubstituted aryl, cycloalkyl, branched alkyl, lower alkyl, protected or deprotected alcohol, zwitterionic group and siloxane bond at each occurrence, and B 1 May represent a siloxane bond. In some embodiments, Z is a linkage to the surface organofunctional hybrid group through direct carbon-carbon bond formation or through a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or polyurethane bond. In some embodiments, Z is an adsorbing surface group that is not covalently attached to the surface of the material.
In some embodiments, R 11 Or R is 12 The first or second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group of (c) may be converted into a cyclic olefin. For example, pyridine groups may be converted to cyclic olefins in solution under certain conditions. In some cases, the cyclic olefin retains sufficient unsaturation to retain and isolate the structurally related compound(s) from the mixture. In certain instances, stationary phases of the present disclosure containing cyclic olefins can retain, separate, and resolve key pairs associated with vitamins (e.g., D2 and D3, K1 and K2).
The Q and T substituents may also be polymerized. The Q and T substituents may be polymerized onto each itself, e.g., Q-Q, T-T, or with each other, e.g., Q-T. As shown in fig. 4, polymerization may occur at the surface level between siloxane groups and/or between hydrocarbon substituents. The polymerization between substituents may produce a crosslinked surface coating or a second coating on the surface coating, such as a second layer of substituents that may also be polymerized or unpolymerized or a mixture of both. The degree of polymerization of the Q and T substituents can vary. For example, a first fraction of Q may be bonded to X, and a second fraction of Q may be aggregated. Likewise, a first fraction of T may be bonded to X, and a second fraction of T may be polymerized. In another embodiment, a first fraction of Q may be bonded to X, a second fraction of Q may be polymerized, a third fraction of T may be bonded to X, and a fourth fraction of T may be polymerized. The polymeric moieties of Q and T may self-polymerize or mutually polymerize.
In one or more embodiments of any of the above aspects, X is a high purity chromatographic material having a core surface that is alkoxylated by a chromatographic mobile phase under chromatographic conditions. X may be a chromatographic material having a core surface alkoxylated by a chromatographic mobile phase under chromatographic conditions. In some embodiments, the functional group comprising Q is a diol. The functional group comprising T may be an amine, an ether, a thioether, or a combination thereof. T may include a chiral functional group suitable for chiral separation, Q may include a chiral functional group suitable for chiral separation, or T and Q may both include a chiral functional group suitable for chiral separation.
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 aggregate surface coverage is greater than 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 micromoles per square meter.
In some embodiments of the above aspects, the chromatographic stationary phase exhibits a retention drift or change of 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 3 days, 5% over 3 days, 4% over 3 days, 3% over 3 days, 1% over 3 days, 5% over 30 times, 4% over 30 times, 3% over 30 times, 2% over 30 times, 1% over 10 times, 5% over 10 times, 4% over 10 times, 3% over 10 times, 2% over 10 times, 1% over 3 times, 4% over 3% times, 2% over 3 times, or 1% over 3 times.
In some embodiments, the core material consists essentially of a silica material. Optionally, 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 may optionally be in the form of a multiparticulate, monolith or surface porous material. In some embodiments, the stationary phase material does not have a chromatographic enhancing pore geometry, while in other embodiments, the stationary phase material has a chromatographic enhancing pore geometry. The stationary phase material may be in the form of a spherical material, a non-spherical material (e.g., including loops, polyhedrons). In certain embodiments, the stationary phase material has a highly spherical core morphology, a rod-shaped core morphology, a bent rod-shaped core morphology, a doughnut-shaped core morphology; or dumbbell-shaped core morphology. In certain embodiments, the stationary phase material has a mixture of highly spherical, rod-shaped, bent rod-shaped, loop-shaped, or dumbbell-shaped morphologies.
In some embodiments, the stationary phase material has a surface area of about 25 to 1100 square meters per gram, about 150 to 750 square meters per gram, or about 300 to 500 square meters per gram. In some embodiments, the stationary phase material has a pore volume of about 0.2 to 2.0 cc/g or about 0.7 to 1.5 cc/g. In some embodiments, the stationary phase material has a micropore surface area of less than about 105 square meters per gram, less than about 80 square meters per gram, or less than about 50 square meters per gram. The stationary phase material may have an average pore size of about 20 to 1500 a, about 50 to 1000 a, about 60 to 750 a, or about 65 to 200 a. In some embodiments, the multiparticulates 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 has an aggregate surface coverage of ≡2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, or 5 micromoles per square meter; or X comprises a non-silica core or a silica-organic hybrid core, c=0, and Q has an aggregate surface coverage of ≡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 or 5 micromoles per square meter; or b > 0, c > 0, and Q has an aggregate surface coverage of ≡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 or 5 micromoles per square meter.
In other embodiments, Q has an aggregate surface coverage of ≡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 or 2.0 micromoles per square meter. In other embodiments, T has an aggregate surface coverage of ≡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 or 3.5 micromoles per square meter. T may also have an aggregate surface coverage of about 0.1 to about 4.0 micromoles per square meter, or about 0.2 to about 3.9 micromoles per square meter, or about 0.3 to about 3.8 micromoles per square meter, or about 0.4 to about 3.7 micromoles per square meter, or about 0.5 to about 3.6 micromoles per square meter, or about 1.0 to about 3.5 micromoles per square meter, or about 1.2 to about 3.0 micromoles per square meter, or any combination of values, such as about 3.0 to about 4.0 micromoles per square meter.
In other embodiments, the total combined coverage of Q and T is greater than or equal to 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 micromoles per square meter.
The chromatographic stationary phase can be used for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography.
The chromatographic stationary phase may include radially-adjusted pores, non-radially-adjusted pores, ordered pores, non-ordered pores, monodisperse pores, non-monodisperse pores, smooth surfaces, roughened surfaces, or a combination thereof. In one or more embodiments, T has one ionizable group, T has more than one ionizable group, T has two or more ionizable groups with the same pKa, or T has two or more ionizable groups with different pKa.
In another embodiment, the disclosure relates to a chromatographic stationary phase having the following structure (i):
[X](W) a (Q) b (T) c (i)
wherein X is a chromatographic substrate comprising silica, metal oxide, inorganic-organic hybrid material, a set of block copolymers, or a combination thereof; w is selected from hydrogen and hydroxy, wherein W is bonded to the surface of X; q is a first substituent that minimizes variation in analyte retention over time under chromatographic conditions with low water concentration; t is a second substituent chromatographically retaining the analyte, wherein T has one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups, each optionally substituted with an aliphatic group; and b and c are positive numbers, 0.05-100 (b/c), and a-0.
In another embodiment, the present disclosure relates to a column, capillary column, microfluidic device or apparatus for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography comprising a housing having at least one wall defining a chamber having an inlet and an outlet and a stationary phase as described in the present disclosure disposed therein, wherein the housing and stationary phase are suitable for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography.
In another embodiment, the present disclosure relates to a kit for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography comprising a housing having at least one wall defining a chamber having an inlet and an outlet and a stationary phase as described in the present disclosure disposed therein, wherein the housing and stationary phase are suitable for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography; and instructions for performing normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography with the shell and stationary phase.
The kit may comprise 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 disclosure disposed therein. The device may have a preformed frit (frit), a frit generated from interconnect material, or a frit-free device. The shell and stationary phase are suitable for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography or a combination thereof. In addition, instructions for performing normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography, or a combination thereof, with the housing and stationary phase may be included.
Accordingly, the kits of the present disclosure may be used to practice the methods of the invention described herein. In addition, the kits of the invention can be used to analyze a variety of different samples and sample types, including those described below.
In one or more embodiments, the present invention contemplates kits containing aspects of the present disclosure to reduce or mitigate the effects of retention drift or variation. For example, a kit may contain a chromatographic column packed with a stationary phase medium of the present disclosure. In some embodiments, the packed column may be used directly in a standard chromatographic system (e.g., a commercially available chromatographic system such as a Waters Acquity chromatographic system). The kit may further comprise instructions for use. In addition, the kit may further contain a stock sample for calibrating the instrument and/or confirming the substantial absence of pure analyte retention drift or change. The kit may include any or all of the above components (e.g., stationary phase, packed column, or chromatographic apparatus) to mitigate the effects of retention drift or variation.
In another embodiment, the present disclosure relates to a method of reducing or preventing retention drift in normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography, comprising chromatographic separation of a sample using a chromatographic device comprising a chromatographic stationary phase as described in the present disclosure, thereby reducing or preventing retention drift.
In one or more embodiments, reducing or preventing retention drift or change includes retention drift or change of 30 days-5%, 30 days-4%, 30 days-3%, 30 days-2%, 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 days-5%, 30 times-4%, 30 times-3%, 30 times-2%, 30 times-1%, 10 times-5%, 10 times-4%, 10 times-3%, 10 times-2%, 10 times-1%, 3 times-5%, 3 times-4%, 3 times-3%, 3 times-2%, or 3 times-1%. 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 modifying a chromatography core as used herein is understood to include functionalizing the chromatography core with, for example, polar silanes or other functional groups, thereby reducing or avoiding retention drift or variation. For example, functionalization may substantially prevent chromatographic interactions between the analyte and the chromatographic core (e.g., effective elimination of chromatographic effects of core surface silanols and/or alkoxylated silanols). In some cases, functionalization (e.g., using nonpolar groups) can reduce the retention of the column. Thus, in various embodiments, functionalization of the chromatographic core can include the use of hydrophilic, polar, ionizable, and/or charged functional groups that interact with the analyte chromatogram to maintain or achieve a useful overall retention of the chromatogram. Such end-capping groups may be introduced, for example, by standard bonding chemistry.
In some embodiments, functionalization provides a permanent bond. Therefore, it is important to select a functionalization suitable for this chromatographic phase. In preferred embodiments, the chromatographic material has chromatographically desirable properties (e.g., overall retention). It is therefore important in some embodiments to select functionalization with properties that mimic the desirable (e.g., overall retention) properties of conventional chromatographic materials.
In various embodiments, the chemical nature of the functional groups may be selected to achieve a desired effect. For example, one or more hydrophilic, polar, ionizable, and/or charged functional groups may be used to achieve a desired interaction with the analyte (e.g., a chromatographically acceptable retention) and/or mobile phase (e.g., rejection of alcohol that may alkoxylate the surface of the chromatographic core). Likewise, the end group size and/or spacing may be selected to mask the core surface and/or to effect chiral separation.
Similarly, the concentration of functionalization can be varied. In some embodiments, larger and/or stronger-interacting functional groups may reduce or avoid retention drift or change at lower concentrations (e.g., as compared to smaller functional groups). In other embodiments, coverage may be adjusted for a desired property. For example, non-polar functional groups may be used at a lower coverage than polar functional groups (e.g., to maintain a desired retention). In various embodiments, functionalization may use one or more polar or non-polar end groups or a combination thereof. In some embodiments, the surface area of the chromatographic medium is increased or decreased to compensate for decreased or increased retention due to the change in polarity of the functional groups.
In another embodiment, the present disclosure relates to a method of preparing a stationary phase as described in the present disclosure, comprising reacting a chromatographic substrate with a silane coupling agent having pendant reactive groups; reacting a second chemical reagent comprising one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups with the pendant reactive groups; and neutralizing any remaining unreacted pendant reactive groups, thereby producing the stationary phase.
In another embodiment, the present disclosure relates to a method of preparing a stationary phase as described in the present disclosure, comprising oligomerizing a silane coupling agent having pendant reactive groups; reacting the core surface with an oligomeric silane coupling agent; reacting a second chemical reagent comprising one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups with the pendant reactive groups; and neutralizing any remaining unreacted pendant reactive groups, thereby producing the stationary phase.
In one or more embodiments, Q is derived from an agent having one of the following structures:
。
in some embodiments, Y comprises one of the following structures:
/>
wherein R is associated with the Y group 6 And R is 7 Each group is independently an aliphatic group. In some embodiments, the Y group may also be a bond or an aliphatic group.
The above-described methods can be used to make any material as described herein (e.g., chromatographic stationary phase materials). For example, the methods of the present disclosure can include a method of reacting a chromatographic stationary phase (e.g., silica particles) with a chemical reagent (e.g., any of the above reagents as described herein) to chemically modify the surface of the stationary phase to mitigate the effects of retention drift or change.
The present disclosure includes various devices (e.g., chromatographic columns, capillaries, and microfluidic devices and systems for their use) comprising the chromatographic materials described herein. Although some illustrative examples are discussed below, one of ordinary skill in the art will appreciate that the present disclosure contemplates many different embodiments, including but not limited to chromatography columns, devices, methods of use, or kits.
In some embodiments, the present disclosure provides a column or device for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography, or a combination thereof. The column or device comprises a housing having at least one wall defining a chamber having an inlet and an outlet, and a stationary phase disposed therein according to any embodiment of the disclosure. The device may have a preformed frit, a frit generated from interconnect material, or a frit-free device. The shell and stationary phase are suitable for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography or a combination thereof.
Accordingly, the devices of the present disclosure may contain (e.g., be filled with) the materials of the present disclosure (e.g., chromatographic stationary phases, such as chemically modified stationary phases suitable for reducing or mitigating retention drift or change). Furthermore, the apparatus of the present disclosure may be used to perform the methods of the present disclosure as described herein.
In one embodiment, the present disclosure is in the form of a packed column. The column may be packed with a stationary phase (e.g., chromatographic material) as described herein. Such columns may be used to perform different types of chromatography (e.g., normal phase chromatography, supercritical fluid chromatography, carbon dioxide-based chromatography, hydrophobic interaction liquid chromatography, hydrophilic interaction liquid chromatography, subcritical fluid chromatography, high pressure liquid chromatography, and solvated gas chromatography) while mitigating or avoiding retention drift or variation.
The column can be used in combination with existing chromatographic platforms, such as commercially available chromatographic systems, including Waters Alliance HPLC systems, waters acquisition systems, or Waters UPC systems 2 The system is used in combination. The columns of the present disclosure can be used for many different mass throughputs (e.g., analytical scale chromatography, preparative scale chromatography) while mitigating the effects of retention drift or variation. Likewise, the device and system can be used in capillaries and microfluidics The present disclosure is embodied in (e.g., commercially available and known to those of ordinary skill in the art). The choice of columns, capillaries and microfluidic devices and related systems will be readily understood by those of ordinary skill in the art.
In various embodiments, materials according to the present disclosure may be used in micro-diameter columns used on SFC, HPLC, and/or UHPLC systems. In various embodiments, materials according to the present disclosure may be used for fast balancing columns, long life columns, and SFCs with water stable columns.
The present disclosure may be used to retain, isolate and/or analyze a number of different compounds from a number of different samples from a number of different fields, such as from clinical chemistry, medicine, veterinary medicine, forensic chemistry, pharmacology, food industry, occupational safety, and environmental pollution. The plurality of samples include, but are not limited to, small organic molecules, proteins, nucleic acids, lipids, fatty acids, carbohydrates, polymers, and the like. Similarly, the present disclosure may be used to isolate small molecules, polar small molecules, analytes for pharmaceutical products, biomolecules, antibodies, polymers and oligomers, sugars, glycan assays, petrochemical assays, lipid assays, peptides, phosphopeptides, oligonucleotides, DNA, RNA, polar acids, polycyclic aromatic hydrocarbons, food assays, chemical assays, biological assays, drugs of abuse, forensics, pesticides, agrochemicals, biomimetics, formulations.
Analytes that can be separated by chromatography of the present disclosure can include essentially any related molecule, including, for example, small organic molecules, lipids, peptides, nucleic acids, synthetic polymers.
Clinical chemistry target analytes can include any molecule present in an organism (e.g., human, animal, fungal, bacterial, viral, etc.). For example, clinical chemistry target analytes include, but are not limited to, proteins, metabolites, biomarkers, and drugs.
Human and veterinary target analytes may include any molecule useful for diagnosing, preventing or treating a disease or condition in a subject. For example, human and veterinary target analytes include, but are not limited to, disease markers, preventive or therapeutic agents.
Forensic chemical target analytes may include any molecules present in a sample taken from a crime scene, such as a sample taken from a victim's body (e.g., a tissue or body fluid sample, hair, blood, semen, urine, etc.). For example, clinical chemistry target analytes include, but are not limited to, toxicants, drugs and their metabolites, biomarkers and identification compounds.
The pharmacological target analyte may include any molecule that is a drug or a metabolite thereof or that may be useful in the design, synthesis and monitoring of a drug. For example, pharmacological analytes of interest include, but are not limited to, prophylactic and/or therapeutic agents, prodrugs, intermediates and metabolites thereof. Pharmacological analysis may include bioequivalence tests, for example, associated with approval, manufacture, and monitoring of a generic name drug.
Food industry and agricultural target analytes may include any molecules related to monitoring the safety of food, beverages, and/or other food industry/agricultural products. Examples of target analytes from the food industry field include, but are not limited to, pathogen markers, allergens (e.g., gluten and nut proteins), and mycotoxins.
Analytes of interest may include polypeptides (e.g., 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, carboxyglutamic acid polymers), peptides, proteins, glycoproteins, lipoproteins; peptide-nucleic acid; hormones (such as peptide hormones (e.g., TRH and vasopressin), synthetic and industrial polypeptides.
In some embodiments, the related 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 lipids" as used herein refers to the constituent of fats and oils that do not contain double bonds at the acyl chain sites 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 a constituent of fats and oils that contain one or more sites of unsaturation in the molecule. These may be present in the fatty acid part of the molecule, as in triglycerides, phospholipids and glycolipids, or in the alkyl chain of the molecule, as in carotenoids, hydrocarbons and fat-soluble vitamins. The lipid is selected from saturated and unsaturated fatty acids, phospholipids, glycerolipids, glycerophospholipids, lysophosphoglycerides (lysophosphoglycerides), sphingolipids, sterol lipids, pregnenolone lipids, glycolipids, carotenoids, waxes and polyketides.
In another embodiment, the present disclosure may be used to retain, separate and analyze polycyclic aromatic hydrocarbons and related compounds. Polycyclic Aromatic Hydrocarbons (PAHs) constitute a class of non-functionalized aromatic compounds consisting of fused aromatic rings. Approximately 2000 compounds are classified as PAHs. PAHs and their derivatives are widely found in the environment due to combustion processes, such as fossil fuel combustion. They bind strongly to soil organic matter (humic acid) and their degradation rate in soil and other environmental areas (environmental compartment) is generally slow. In addition, PAHs reaching the waterway rapidly migrate into the sediment.
PAHs are also formed during domestic and industrial combustion processes such as extraction of vegetable oils, herbs, spices and other food materials, smoking and grilling of food materials and the like. PAHs are toxic carcinogenic compounds. Their presence and control is increasingly important in the field of food safety and health and safety regulations. Several effects of PAHs are enzyme induction, immunosuppression, teratogenicity, and promotion of tumors.
In particular embodiments, the lipid may be a saturated or unsaturated fatty acid, monoacylglyceride, diacylglyceride, triacylglyceride, phospholipid or steroid. Triglycerides (TG, triacylglycerols, TAGs or triacylglycerides) are esters derived from glycerol and three fatty acids. As blood lipids, they facilitate the bi-directional transfer of fat and blood glucose from the liver. There are many triglycerides: depending on the oil source, some are highly unsaturated and some are lower. Triglycerides are the major components of vegetable oils (usually more unsaturated) and animal fats (usually more saturated). Triglycerides are the main component of human skin oils.
Steroids are a class of organic compounds that contain a characteristic arrangement of four cycloalkane rings that are interconnected. Examples of steroids include dietary fat cholesterol, the sex hormones estradiol and testosterone, and the anti-inflammatory agent dexamethasone. The steroid nucleus consists of 17 carbon atoms bonded together in the form of four fused rings: three cyclohexane rings and one cyclopentane ring. Steroids are altered by the functional groups attached to the tetracyclic nucleus and the oxidation state of these rings. Sterols are a special form of steroid having a hydroxyl group in position-3 and a skeleton derived from cholestane.
Hundreds or thousands of different sterols are found in plants, animals and fungi. All steroids are made in cells from sterols lanosterol (animals and fungi) or from cycloartenol (plants). Both lanosterol and cycloartenol are derived from cyclization of the triterpene squalene.
In other embodiments, the related compound may be a fat-soluble vitamin selected from vitamin C, vitamin B, or derivatives or combinations thereof. Fat-soluble vitamins are relevant because they are difficult to dissolve, often requiring strong organic solvents and separation via RPLC. Typically, the fat-soluble vitamins are separated by means of a C18 silane (ODS) or other alkyl bonding phase using SFC conditions. These processes typically use very weak CO-solvents and higher percentages of CO in the mobile phase 2 . One of the advantages of the present disclosure is the modification of the stationary phase with pi-electron rich selectivity factor (selector), e.g., 1-aminoanthracene. Coupling pi-electron rich selectivity factors to the stationary phase maximizes the selective chromatographic separation between two key pairs, such as vitamins D2 and D3, or K1 and K2.
Another advantage of the present disclosure is that the stationary phase has a selectivity factor with a relatively high pKa such that no acid or basic additives are required to achieve the separation. In one embodiment, the present disclosure relates to a method of using a mobile phase of an acid-free additive and/or a basic additive. In other embodiments, the present disclosure relates to methods of using mobile phases having 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 acid additives and/or basic additives. The use of only carbon dioxide modified with methanol as a co-solvent can result in a rapid general process. For example, the separation of the above-described vitamin key pair is achieved using a stationary phase of the present disclosure having, for example, 1-aminoanthracene as a selection factor. In one embodiment, the resolution level between vitamin key pairs is higher than current methods (resolution per same column length; sub-2 [ mu ] m particles and 50 mm column length are used).
The present disclosure is useful for retaining, isolating and analyzing vitamin C and related compounds. Vitamin C [ 2-oxo-L-threo-hexenoic acid-1, 4-lactone2,3-enediol (2-oxo-L-threo-hexono-1, 4-lactone2, 3-enediol) ] or L-ascorbic acid is a water-soluble vitamin and an essential nutrient for human body. Which is necessary for collagen formation required for normal growth and development and tissue repair in all parts of the body. Vitamin C also acts as an antioxidant blocking damage caused by free radicals and directly reducing toxic chemicals and contaminants.
Since vitamin C is not produced in humans, it is mainly obtained from dietary sources such as fruits and vegetables. Dietary vitamin C deficiency may cause vitamin C deficiency. Severe vitamin C deficiency, also known as "scurvy", results in the formation of liver spots, spongiform gums (sponges) and mucosal bleeding or even death on the skin.
Vitamin C is now used not only as a dietary supplement, but also as an adjunct therapy for some viral infections and advanced cancers. The recommended daily intake of vitamin C for adult humans to prevent deficiency is 75, 75 mg for females and 90 mg for males, both with an upper tolerance limit of 2,000 mg. For therapeutic use in detoxification and cancer treatment, vitamin C is administered intravenously at much higher doses. Although vitamin C toxicity is less clinically common, relatively high oral intake doses may cause gastric discomfort and diarrhea. Determination of vitamin C blood concentration has been developed and used by patients and physicians to assess nutritional status or optimize therapeutic dosages. Measurement of these compounds is a useful indicator of vitamin C nutritional status and efficacy of certain vitamin C analogs.
In another embodiment, the present disclosure may be used to retain, isolate and analyze vitamin B and related compounds. B vitamins are a class of water-soluble vitamins that play an important role in cellular metabolism. The B vitamins were once considered to be single vitamins, simply referred to as vitamin B. Later studies showed that they are chemically different vitamins that often coexist in the same food product. In general, supplements containing all 8 weight are referred to as vitamin B complex. The B vitamin supplements alone are represented by the vitamin's private name (e.g., B1, B2, B3, etc.). The list of B vitamins includes: vitamin B1 (thiamine), vitamin B2 (nuclear), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal or pyridoxamine or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid), vitamin B12 (various cobalamins; commonly referred to as cyanocobalamin in vitamin supplements).
The vitamin B compounds act differently. For example, thiamine plays a central role in the generation of energy from carbohydrates. It is involved in RNA and DNA production and in neural function. The active form is a coenzyme called thiamine pyrophosphate (TPP), which is involved in the metabolic conversion of pyruvate to acetyl CoA (CoA). The nuclear element participates in energy production for electron transport chains, citric acid circulation, and fatty acid catabolism (β -oxidation). Nicotinic acid consists of two structures: niacin and niacinamide. Nicotinic acid has two coenzyme forms: nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NADP). Both play an important role in energy transfer reactions in the metabolism of glucose, fat and alcohols. NAD carries hydrogen and their electrons during metabolic reactions, including the path from the citric acid cycle to the electron transport chain. NADP is a coenzyme in lipid and nucleic acid synthesis.
Pantothenic acid is involved in the oxidation of fatty acids and carbohydrates. Coenzyme a, which can be synthesized from pantothenic acid, is involved in the synthesis of amino acids, fatty acids, ketones, cholesterol, phospholipids, steroid hormones, neurotransmitters (e.g., acetylcholine) and antibodies. Pyridoxine is usually stored in the body in the form of pyridoxal 5' -phosphate (PLP), which is a coenzyme for vitamin B6. Pyridoxine is involved in the metabolism of amino acids and lipids; synthesis of neurotransmitters and hemoglobin, and production of niacin (vitamin B3). Pyridoxine also plays an important role in gluconeogenesis. Biotin plays a key role in the metabolism of lipids, proteins and carbohydrates. It is a key coenzyme for four carboxylase enzymes: acetyl-coa carboxylase involved in the synthesis of fatty acids from acetate; propionyl-coa carboxylase involved in gluconeogenesis; beta-methylcrotonyl-coa carboxylase involved in leucine metabolism; and pyruvate-CoA carboxylase involved in the metabolism of energy, amino acids and cholesterol.
Folic acid acts as a coenzyme in the form of Tetrahydrofolate (THF), which is involved in the transfer of single carbon units in the metabolism of nucleic acids and amino acids. THF is involved in pyrimidine nucleotide synthesis and is therefore required for normal cell division, especially during gestation and infancy, which are rapid growth phases. Folic acid also contributes to erythropoiesis (production of red blood cells). Vitamin B12 is involved in cellular metabolism of carbohydrates, proteins and lipids. Which is necessary for the production of blood cells in bone marrow, nerve sheath and proteins. Vitamin B12 acts as a coenzyme in the intermediary metabolism of the reaction of methionine synthase with mecobalamin and the reaction of methylmalonyl-coa mutase with adenosylcobalamin.
The effect of any deficiency of these vitamins is different. Vitamin B1 thiamine deficiency causes beriberi. Symptoms of this disease of the nervous system include weight loss, mood disorders, wernike encephalopathy (impaired sensory perception), weakness and limb pain, irregular heart cycles and oedema (swelling of body tissues). Heart failure and death may occur in severe cases. Chronic thiamine deficiency can also cause colza-koff syndrome, irreversible dementia characterized by amnesia and compensatory deficiency.
Vitamin B2 nuclear yellow deficiency causes riboflavin deficiency. Symptoms may include lip lesions (chapped lips), high sensitivity to sunlight, keratitis, glossitis (tongue inflammation), seborrheic dermatitis or pseudosyphilis (particularly affecting the scrotum or labia majora and the oral cavity), pharyngitis (sore throat), congestion, and edema of the throat and oral mucosa.
Vitamin B3 niacin deficiency, together with tryptophan deficiency, causes pellagra. Symptoms include aggression, dermatitis, insomnia, frailty, confusion, and diarrhea. In severe cases, brown skin disease may cause dementia and death (3 (+1) Ds: dermatitis, diarrhea, dementia and death).
Vitamin B5 pantothenate deficiency can cause acne and paresthesia, although this is unusual. Vitamin B6 pyridoxine deficiency may cause microcytic anemia (because phosphopyridoxine is a cofactor for heme synthesis), depression, dermatitis, hypertension (hypertension), water retention and elevated homocysteine levels. Vitamin B7 biotin deficiency does not usually cause symptoms in adults, but can cause growth disorders and neurological disorders in infants. Even if dietary biotin intake is normal, multiple carboxylase deficiency (congenital metabolic abnormality) can cause biotin deficiency.
Vitamin B9 folate deficiency causes megaloblastic anemia and elevated homocysteine levels. The lack of a pregnant woman can cause birth defects. Supplementation is usually recommended during pregnancy. Studies have shown that folic acid may also slow down the potential effect of age on the brain. Vitamin B12 cobalamin deficiency causes megaloblastic anemia, elevated homocysteine, peripheral neuropathy, memory loss and other cognitive deficits. It is most likely to occur in the elderly, as intestinal absorption decays with age; autoimmune disease pernicious anemia is another common cause. It also causes symptoms of mania and psychosis. In rare extreme cases, paralysis can result. Measurements of these compounds are useful indicators of vitamin B nutritional status and efficacy of certain vitamin B analogues.
In other embodiments, the related compound may be a fat-soluble vitamin selected from vitamin D, vitamin a, vitamin K, vitamin E, beta carotene, or derivatives or combinations thereof. The present disclosure is useful for retaining, isolating and analyzing vitamin D and related compounds. Vitamin D is an essential nutrient with important physiological roles in the positive regulation of calcium homeostasis. Vitamin D can be synthesized de novo in the skin by exposure to sunlight or it can be absorbed from the diet. Vitamin D has two forms; vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D3 is a form synthesized by animals from the head. It is also a common supplement added to dairy products and certain foods produced in the united states. Both dietary and intrinsically synthesized vitamin D3 must be metabolically activated to produce bioactive metabolites. In humans, the initial step of vitamin D3 activation occurs mainly in the liver and involves hydroxylation to form the intermediate metabolite 25-hydroxycholecalciferol (calcitol), which is enzymatically hydroxylated at the 25 position. Calcitonin is the major form of vitamin D3 in the circulation. The circulating calcitonin is then converted by the kidneys to form 1, 25-dihydroxyvitamin D3 (calcitriol), which is generally considered the most biologically active vitamin D3 metabolite. Vitamin D2 is derived from fungal and plant sources. Many over-the-counter dietary supplements contain ergocalciferol (vitamin D2) rather than cholecalciferol (vitamin D3). Drisdol (the only highly potent prescription form of vitamin D available in the United states) is formulated with ergocalciferol. Vitamin D2 undergoes a metabolic activation pathway in humans similar to that of vitamin D3 to form the metabolites calcitriol and calcitriol. Vitamin D2 and vitamin D3 have long been considered bioequivalent in humans, but recent reports indicate that these two forms of vitamin D differ in terms of bioactivity and bioavailability. Measurement of these compounds is a useful indicator of vitamin D nutritional status and efficacy of certain vitamin D analogues.
In another embodiment, the present disclosure may be used to retain, isolate, and analyze vitamin a and related compounds. Vitamin a is a class of unsaturated nutritional organic compounds that includes retinol, retinal, retinoic acid and several provitamin a carotenoids, of which β -carotene is the most important. Vitamin a has multiple functions: it is important for growth and development, maintenance of the immune system and good vision. The retina of the eye requires vitamin a in the form of retinaldehyde which is combined with the protein opsin to form rhodopsin light absorbing molecules which are necessary for low light vision (scotopic vision) and colour vision. Vitamin a also plays a very different role as an irreversible oxidized form of retinol (known as retinoic acid), which is a hormone-like growth factor important for epithelial and other cells.
In foods of animal origin, the major form of vitamin a is the ester, mainly retinyl palmitate, which is converted to retinol (chemically an alcohol) in the small intestine. The retinol form serves as a storage form for the vitamin and can be converted to and from its visually active aldehyde form, retinaldehyde. The relevant acids (retinoic acid), metabolites irreversibly synthesized by vitamin a, are only partially vitamin a active and do not play a role in the visual cycle in the retina. Retinoic acid is used for growth and cell differentiation.
All forms of vitamin a have a β -ionone ring to which an isoprenoid chain is attached, known as retinyl. Both of these structural features are essential for vitamin activity. The orange element of carrot-beta-carotene-can be presented as two linked retinyl groups that are utilized in vivo to contribute to vitamin a levels. Alpha-carotene and gamma-carotene also have a mono-retinyl group, which imparts some of their vitamin activity.
Vitamin a exists in food in two main forms: (i) Retinol, a form of vitamin a that is absorbed when an animal food source is consumed, is a yellow fat-soluble substance. The vitamin is present in the tissue as a retinyl ester due to the instability of the pure alcohol form. It is also commercially produced and administered in the form of an ester, such as retinol acetate or palmitate. (ii) Carotenes alpha-carotene, beta-carotene, gamma-carotene; and lutein β -cryptoxanthin (all of which contain a β -ionone ring), but no other carotenoids, acting as provitamins a in herbivores and omnivores, possess an enzyme (15-15' -dioxygenase) that cleaves β -carotene in the intestinal mucosa and converts it to retinol. In general, carnivores are poor converters of ionone-containing carotenoids, and pure carnivores such as cats and ferrets lack 15-15' -dioxygenase and are unable to convert any of the carotenoids to retinoids (so that none of the carotenoids are in the form of vitamin a for these species). Measurements of these compounds are useful indicators of vitamin a nutritional status and the efficacy of certain vitamin a analogs.
In another embodiment, the present disclosure may be used to retain, isolate and analyze vitamin K and related compounds. Vitamin K is a structurally similar class of fat-soluble vitamins required for human post-translational modification of certain proteins required for blood clotting and in metabolic pathways in bone and other tissues. They are 2-methyl-1, 4-naphthoquinone (3-) derivatives. Such vitamins include two natural vitamins: vitamin K1 and vitamin K2.
Vitamin K1, also known as phylloquinone, phytonmadione or phytomenadione, is synthesized by plants and is present in the highest amounts in green leaf vegetables because it is directly involved in photosynthesis. It can be considered to be a "plant form" of vitamin K. It is active in animals and can exert the classical function of vitamin K in animals including its activity in the production of thromboplastin. The animal may also convert it to vitamin K2.
Vitamin K2, the major storage form in animals, has several subtypes, which differ in isoprenoid chain length. These vitamin K2 homologs are known as menaquinones and are characterized by the number of isoprenoid residues in their side chains. Menaquinones are abbreviated MK-n, where M represents menaquinones, K represents vitamin K, and n represents the isoprenoid side chain residue number. For example, menaquinone-4 (abbreviated MK-4) has four isoprene residues in its side chain. Menaquinone-4 (also referred to as menatetrenone by its four isoprene residues) is the most common type of vitamin K2 in animal products, because MK-4 is usually synthesized from vitamin K1 in certain animal tissues (arterial wall, pancreas and testis) by replacing the chlorophyllin tail with an unsaturated geranylgeranyl tail containing four isoprene units, thereby producing menaquinone-4. Such homologs of vitamin K2 may have a different enzymatic function than vitamin K1.
Bacteria in the colon (large intestine) can also convert K1 to vitamin K2. In addition, bacteria typically lengthen the isoprenoid side chain of vitamin K2 to produce a range of vitamin K2 forms, most particularly MK-7 to MK-11 homologs of vitamin K2. All forms of K2 except MK-4 can only be produced by bacteria, which utilize these forms in anaerobic respiration. MK-7 and other bacterially derived forms of vitamin K2 exhibit vitamin K activity in animals, but the additional utility of MK-7 over MK-4, if any, is unclear and still under investigation.
Three synthetic types of vitamin K are known: vitamins K3, K4 and K5. Although natural K1 and all K2 homologs have proven nontoxic, synthetic form K3 (menaquinone) has been shown to be toxic. K4 and K5 are also non-toxic. The measurement of these compounds is a useful indicator of vitamin K nutritional status and the efficacy of certain vitamin K analogues.
In another embodiment, the present disclosure may be used to retain, isolate and analyze vitamin E and related compounds. Vitamin E refers to a class of eight fat-soluble compounds including tocopherols and tocotrienols. Among the many different forms of vitamin E, gamma-tocopherol is most common in north american diets. Gamma-tocopherol can be present in corn oil, soybean oil, margarine and sauces. The most bioactive form of alpha-tocopherol, vitamin E, is the second most common form of vitamin E in the diet. This variant is most abundant in wheat germ oil, sunflower oil and safflower oil. As a fat-soluble antioxidant, it terminates the production of reactive oxygen species formed upon oxidation of fat. Amounts exceeding 1,000 milligrams (1,500 IU) per day are referred to as overvitamin E, as they may increase the risk of bleeding problems and vitamin K deficiency. Measurement of these compounds is a useful indicator of vitamin E nutritional status and efficacy of certain vitamin E analogs.
Generally, a sample is a composition that includes at least one analyte of interest (e.g., an analyte of the type or kind disclosed above, along with a matrix). The sample may comprise a solid, a liquid, a gas, a mixture, a material (e.g., having an intermediate consistency such as an extract, a cell, a tissue, an organism), or a combination thereof. In various embodiments, the sample is a body sample, an environmental sample, a food sample, a synthetic sample, an extract (e.g., obtained by an isolation technique), or a combination thereof.
The body sample may comprise any sample derived from the body of an individual. In this regard, the individual may be an animal, such as a mammal, e.g., a human. Other exemplary individuals include mice, rats, guinea pigs, rabbits, cats, dogs, goats, sheep, pigs, cows, or horses. The individual may be a patient, e.g., an individual having a disease or suspected of having a disease. The body sample may be a body fluid or tissue, for example extracted for scientific or medical testing, for example for studying or diagnosing a disease (e.g. by detecting and/or identifying the presence of a pathogen or biomarker). The body sample may also include cells, such as pathogens or cells (e.g., tumor cells) of an individual body sample. Such body samples may be obtained by known methods, including tissue biopsies (e.g., drill biopsies) and by extracting blood, bronchial aspirates, sputum, urine, stool, or other body fluids. Exemplary body samples include body fluids, whole blood, plasma, serum, cord blood (particularly blood obtained by percutaneous cord blood sampling (PUBS), cerebral Spinal Fluid (CSF), saliva, amniotic fluid, breast milk, secretions, pustules, urine, feces, meconium, skin, nails, hair, umbilicus, gastric contents, placenta, bone marrow, peripheral Blood Lymphocytes (PBLs), and solid organ tissue extracts.
Environmental samples may include any sample derived from an environment, such as a natural environment (e.g., sea, soil, air, and plant systems) or an artificial environment (e.g., canal, tunnel, building). Exemplary environmental samples include water (e.g., drinking water, river water, surface water, groundwater, drinking water, sewage, effluent water, wastewater, or leachate), soil, air, sediment, biological systems (e.g., soil organisms), plant systems, animal systems (e.g., fish), and soil bodies (e.g., cuttings).
Food samples may include any sample derived from food (including beverages). Such food samples may be used for a variety of purposes including, for example, (1) checking whether food is safe; (2) Checking whether the food contains harmful contaminants or whether the food does not contain harmful contaminants when the food is eaten (sample is left); (3) Checking whether the food contains only allowed additives (e.g. compliance with regulations); (4) Check if it contains the correct level of mandatory ingredients (e.g. if the statement on the food label is correct); or (5) analyzing the amount of nutrients contained in the food. Exemplary food samples include edible products of animal, vegetable or synthetic origin (e.g., milk, bread, eggs or meat), cereals, beverages and portions thereof, such as leave-on. The food sample may also include fruits, vegetables, beans, nuts, oilseeds, cereals, tea, coffee, herbal extracts, cocoa, hops, herbs, spices, sugar plants, meat, fat, kidneys, liver, viscera, milk, eggs, honey, fish and beverages.
Synthetic samples may include any sample derived from an industrial process. The industrial process may be a biological industrial process (e.g., a process using biological material containing genetic information and capable of self-replication or replication in a biological system, such as a fermentation process using transfected cells) or a non-biological industrial process (e.g., chemical synthesis or degradation of a compound, such as a drug). The synthetic samples can be used to check and monitor the progress of an industrial process, to determine the yield of a desired product and/or to measure the amount of byproducts and/or raw materials.
Examples
Material
Unless otherwise indicated, all reagents were used as received. Those skilled in the art will recognize that the following supplies and equivalents of the suppliers exist, and therefore the following suppliers should not be considered limiting.
Characterization technique
Those skilled in the art will recognize that there are equivalents to the following instruments and suppliers, and thus the following instruments should not be considered limiting.
The% C values were measured by combustion analysis (CE-440 elemental analyzer; exeter Analytical inc., north chemmford, MA) or by coulomb carbon analyzer (modules CM5300, CM5014, UIC inc., joliet, IL). The bromine and chlorine content was determined by flask burning followed by ion chromatography (Atlantic Microlab, norcross, GA). Using multipoint N 2 Adsorption (Micromeritics ASAP 2400;Micromeritics Instruments Inc), norcross, GA) measures the Specific Surface Area (SSA), specific Pore Volume (SPV) and average pore size (APD) of these materials. SSA is calculated using BET method, SPV is P/P 0 >A single point value of 0.98 was measured,and APD was calculated from the desorption branch of the isotherm using the BJH method. As a subtraction of pores from Specific Surface Area (SSA)<34. Cumulative adsorption pore size data for a Micropore Surface Area (MSA) was determined. The median mesoporous diameter (MMPD) and Mesoporous Pore Volume (MPV) were measured by mercury porosimetry (Micromeritics AutoPore II 9220 or AutoPore IV, micromeritics, norcross, GA). Skeletal density was measured using Micromeritics AccuPyc 1330 Helium Pycnometer (V2.04N, norcross, GA). Particle size was measured using a Beckman Coulter Multisizer analyzer (30 μm pore size, 70,000 counts; miami, FL). Particle diameter (dp) was measured as 50% cumulative diameter of volume-based particle size distribution 50 ). The distribution width is measured as 90% cumulative volume diameter divided by 10% cumulative volume diameter (referred to as the 90/10 ratio). The viscosity of these materials was determined using a Brookfield digital viscometer Model DV-II (Middleboro, mass.). The pH measurements were performed with an Oakton pH100 series instrument (Cole-Palmer, vernon Hills, illinois) and calibrated at ambient temperature using Orion (Thermo Electron, beverly, MA) pH buffer standards just prior to use. Titration was performed using a Metrohm 716 DMS Titrino auto-titrator (Metrohm, herssau, switzerland) and reported as milliequivalents per gram (mequiv/g). By titration of OH released upon addition of sodium thiosulfate - The level of epoxide coverage was determined. Using Bruker Instruments Avance-300 spectrometer (7 mm double broadband probe) to obtain multi-core 13 C, 29 Si) CP-MAS NMR spectra. The spin speed is typically 5.0-6.5 kHz and the cyclic delay is 5 seconds. And the cross-polarization contact time was 6 milliseconds. Using external standard adamantane 13 cCP-MAS NMR, [ delta ] 38.55) and hexamethylcyclotrisiloxane ] 29 Si CP-MAS NMR, delta-9.62) reported relative to tetramethylsilane recording 13 C and C 29 Si CP-MAS NMR spectral shifts. The population (population) of different silicon environments was evaluated by spectral deconvolution using DMFit software. [ Massiot, d.; fayon, f.; capron, M.; king, i.; le Calv, s.; alonso, b.; durand, j. -o; bujoli, b.; gan, z.; hoatson, G.Magn.Reson. Chem.2002, 40, 70-76]。
Example 1 epoxide layer for stationary phaseIs prepared from
In a typical reaction, the hybridized porous particles were dispersed in a solution of glycidoxypropyl trimethoxysilane/methanol (0.25 mL/g) (GLYMO, aldrich, milwaukee, wis.) in 20 mM acetate buffer (pH 5.5, prepared using acetic acid and sodium acetate, J.T. Baker,5 mL/g dilution) which had been premixed for 60 minutes at 70 ℃. The mixture was kept at 70℃for 20 hours. The reaction was then cooled, the product was filtered and washed successively with water and methanol (j.t. Baker). The product was then dried under reduced pressure at 80℃for 16 hours. The specific particles used are shown in table 1.
Specific base particles used in Table 1
Entries Material
B1 Hybrid organosilica (3.8 μm, 90 Ǻ APD, 1.3 cc/g TPV) 1
B2 Hybrid organosilica (3.8 μm, 115 Ǻ APD, 1.3 cc/g TPV) 1
B3 Hybrid organosilica (2.3 [ mu ] m, 115 Ǻ APD, 1.3 cc/g TPV) 1
B4 Hybrid organosilica (1.7 μm, 143 Ǻ APD, 0.73 cc/g TPV)
B5 Hybrid organosilica (1.7 μm, 106 Ǻ APD, 1.25 cc/g TPV)
1 As described in US7919177, US 7223473, US6686035 and WO 2011084506.
The reaction data are presented in table 2. Specific changes to this general procedure include: 1) material 1E was prepared using a 6 hour reaction time, 2) material 1F was prepared using 100 mM acetate buffer, 3) material 1G was prepared using 50℃for 20 hours, 4) material 1H was prepared using 50℃pre-mix 50℃hold temperature. The total surface coverage of 3.90-6.0 micromoles per square meter was determined from the difference in% C of particles before and after surface modification as measured by elemental analysis. By passing through 13 Analysis of these materials by C CP-MAS NMR spectroscopy indicated the presence of a mixture of epoxy groups and glycol groups for these materials.
TABLE 2 initial layer coverage of stationary phase
1 This refers to the aggregate coverage from the bonded GPTMS silane-coverage from unhydrolyzed epoxide + coverage from hydrolyzed epoxide (as diol)
2 As determined by titration.
Example 2 preparation of stationary phase with diol functionality
In a typical reaction, the hybridized porous particles were dispersed in a solution of glycidoxypropyl trimethoxysilane/methanol (0.25 mL/g) (GLYMO, aldrich, milwaukee, wis.) in 20 mM acetate buffer (pH 5.5, prepared using acetic acid and sodium acetate, J.T. Baker,5 mL/g dilution) which had been premixed for 60 minutes at 70 ℃. The mixture was maintained at 70℃for 20 hours. The reaction was then cooled, the product was filtered and washed successively with water and methanol (j.t. Baker).The material was then refluxed in 0.1M acetic acid solution (5 mL/g diluted, j.t. Baker) for 20 hours at 70 ℃. The reaction was then cooled, the product was filtered and washed successively with water and methanol (j.t. Baker). The product was then dried under reduced pressure at 80℃for 16 hours. The reaction data are presented in table 3. Surface coverage of 0.93-6.0 micromoles per square meter was determined from the difference in% C of particles before and after surface modification as measured by elemental analysis. By passing through 13 Analysis of these materials by ccp-MAS NMR spectroscopy showed no measurable amount of epoxy groups remained, and only diol groups were present.
TABLE 3 initial layer coverage of stationary phase
。
Example 3 preparation of stationary phase with Mixed functionality
In standard experiments, 10 grams of the material made above was dispersed in a solvent such as, but not limited to, water, isopropanol, or dioxane. The amount of nucleophile exceeding the epoxide coverage determined for the material made above was added and the mixture was heated to 70 ℃ for 16 hours. Table 4 provides a list of specific nucleophiles used. After the reaction, the particles were washed successively with water and 0.5M acetic acid, and the material was then stirred in 0.1M acetic acid solution (5 mL/g dilution, j.t. Baker) for 20 hours at 70 ℃. The reaction was then cooled, the product was filtered and washed successively with water and methanol (j.t. Baker). The product was then dried under reduced pressure at 80℃for 16 hours. The reaction data are presented in table 5. The nucleophile surface concentration was determined from the difference in% C,% N or% S of the particles before and after surface modification by elemental analysis, and was in the range of 0.2 to 2.3 micromoles per square meter. By passing through 13 Analysis of these materials by ccp-MAS NMR spectroscopy indicated that no measurable amount of epoxy groups remained.
Table 4 list of specific nucleophiles used
Entries Nucleophile
N1 1-aminoanthracene
N2 4-n-octylaniline
N3 6-aminoquinolines
N4 Aniline
N5 1-naphthylamine
N6 8-aminoquinolines
N7 2-aminoanthracene
N8 Benzyl amines
N9 2-Aminomethylpyridine
N10 Pyridine compound
N11 N-octadecylamine
N12 Diethylamine
TABLE 5
。
Example 4 further characterization of stationary phase
The general procedure for particle bonding/functionalization detailed in examples 1-3 was applied to modify the surface silanol groups of different porous materials. Including monoliths, spheres, particles, surface porous and irregular materials, which are silica, hybrid inorganic/organic materials, hybrid inorganic/organic surface layers on hybrid inorganic/organic, silica, titania, alumina, zirconia, polymeric or carbon materials, and silica surface layers on hybrid inorganic/organic, silica, titania, alumina, zirconia or polymeric or carbon materials. Also included are stationary phase materials in the form of spherical materials, non-spherical materials (e.g., including loops, polyhedra); a core form having a high degree of sphericity, a rod-shaped core form, a bent rod-shaped core form, a ring-shaped core form; or a dumbbell-shaped core-form stationary phase material; and stationary phase materials having a mixture of highly spherical, rod-shaped, bent rod-shaped, loop-shaped or dumbbell-shaped morphology. Exemplary hybrid materials are shown in U.S. Pat. Nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913, and International publication No. WO2008/103423, the contents of which are incorporated herein by reference in their entirety. Surface porous particles include those described in U.S. publication nos. 2013/0110205, 2007/0189944, and 2010/061367, the contents of which are incorporated herein by reference in their entirety. The particle size of the spherical, granular or irregular material may be 5-500 microns; more preferably 15-100 microns; more preferably 20-80 microns; more preferably 40-60 microns. APDs of these materials can be 30 to 2,000 a; more preferably 40 to 200 a; more preferably 50 to 150 a. SSA for these materials can range from 20 to 1000 square meters per gram; more preferably from 90 to 800 square meters per gram; more preferably 150 to 600 square meters per gram; more preferably 300 to 550 square meters per gram. The TPV of these materials may be 0.3 to 1.5 cc/g; more preferably 0.5 to 1.4 cc/g; more preferably 0.7 to 1.3 cc/g. The large pore diameter of the monolith may vary from 0.1 to 30 microns, more preferably from 0.5 to 25 microns, more preferably from 1 to 20 microns.
Example 5 the stationary phase shows minimal analyte retention change over time under chromatographic conditions
The mean% retention change was calculated by taking the percentage difference of the mean absolute peak retention measured by the day 3, 10 or 30 chromatography test to the mean absolute peak retention measured at day 1 chromatography test. For each day of the test, the column was equilibrated for 20 minutes under Mix1 test conditions followed by three injections of Mix1, then equilibrated for 10 minutes under Mix2 test conditions followed by three injections of Mix2. The conditions are shown in table 6. The results are shown in tables 7 and 8.
The% less retention was calculated by taking the percentage difference between the 1 st balance absolute peak retention measured for Mix1 and Mix2 and the 1 st balance absolute peak retention measured for Mix1 and Mix2 on example 1A.
TABLE 6 chromatographic test conditions for measuring retention variation
Cosolvent Mix1 5% methanol
Sample Mix1 3-Benzoylpyridine (0.1 mg/mL)
Cosolvent Mix2 10% methanol
Sample Mix2 Caffeine, thymine, papaverine, prednisolone, sulfanilamide (0.2 mg/mL each)
Column size 2.1 x 150 mm
Flow rate 1.0 mL/min
Column temperature 50℃
Back pressure 1800 psi
Detector for detecting a target object Acquisy PDA with SFC Flow Cell
Detector arrangement 254 nm 40 spec/sec
Weak washing needle Isopropyl alcohol
Injection of 1.0 Mu L (2.0 mu L cycle, PLUNO injection mode)
Instrument for measuring and controlling the intensity of light UPC²
Software for providing a plurality of applications Empower
TABLE 7 retention change over time of materials from example 2
It was shown that this test was not performed on this material.
TABLE 8 retention change over time from the comparative materials
。
It was shown that this test was not performed on this material.
EXAMPLE 6 organic-inorganic hybrid particles with glycidoxypropyl trimethoxysilane (GPTMS) and 1-aminoanthracene Subsurface functionalization
The structures of GPTMS and 1-aminoanthracene are shown in FIG. 1. Fig. 1A shows the structure of GPTMS (silane surface modifier). Portion 105 shows the surface reactive groups of GPTMS (trialkoxysilane) and portion 110 shows the reactive groups (epoxide). FIG. 1B shows the selective ligand (1-aminoanthracene).
The GPTMS was first pre-formed by pre-incubation at 70 ℃ in 20 mM sodium acetate buffer pH 5.0. During the incubation process, small oligomers of hydrolyzed silane are formed. After a suitable pre-incubation period, the particles to be modified are added in the form of a dry powder. The oligomer and any remaining monomers react with the material surface to produce a high surface coverage of the silane modifier covalently attached to the material surface as shown in fig. 2.
FIG. 2 shows the reaction of a silane coupling agent with the surface of an organic-inorganic hybrid material. For simplicity, silane (205) is depicted as a monomer. The preformed oligomeric silane may be coupled in the same manner as the surface reactive groups (210). In some embodiments, a portion of the silane that is not attached to the surface may also be coupled to the coating (e.g., cross-polymerized). Under reaction conditions, methanol (215) is eliminated to produce surface modified particles (220).
After the silane has reacted with the chromatographic material, by reaction with MilliQ H 2 O washing removes excess reagent and buffer salts and transfers the material to an organic solvent (e.g., 1, 4-dioxane) and adds 1-aminoanthracene. The amino group of 1-aminoanthracene is coupled to the surface through a pendant epoxide group of GPTMS. The proportion of the converted epoxy groups can be controlled by limiting the amount of 1-aminoanthracene added. The coupling material was then washed into 0.5M acetic acid and the unreacted epoxide groups were hydrolyzed to the corresponding diols as shown in fig. 3.
The resulting 1-aminoanthracene/diol surface contains a uniform distribution of 1-aminoanthracene groups and provides excellent selectivity while the diol shields the surface silanol from analyte interactions. The multicomponent surface is superior to either the diol surface alone or the 1-aminoanthracene surface alone.
FIG. 3 shows the reaction of surface-modified particles (305) with selective ligands (310). Reaction conditions (315) are given and include treatment with isopropanol and 0.5M acetic acid at 70 ℃. As a result stationary phase particles (320) with a multicomponent surface for chromatographic separation are obtained.
Alternatively, the multicomponent surface may be produced under reaction conditions (which simultaneously bond to the substrate surface, partially react the pendant reactive groups to form inert pendant groups and also cause limited polymerization between the pendant reactive groups on adjacent silane coupling agent molecules) using a silane coupling agent having the pendant reactive groups as the bonding phase under conditions to produce a polymeric surface.
Similarly, the multicomponent surface can be fabricated by covalent bonding to interact with the analyte under conditions that produce the polymeric surface to affect the retained second chemical agent by introducing charged, uncharged, polar, nonpolar, lipophilic or hydrophilic properties into the chromatographic phase. Alternatively, under certain conditions, the epoxy groups of GPTMS may react with the hydroxyl groups of adjacent silanes to form ether bridges that crosslink GPTMS on the surface. Such crosslinking may provide stability to the bonding phase and may also enhance silanol protection. The presence of such crosslinks is consistent with NMR analysis of these materials. One embodiment of the type of crosslinked surface is shown in fig. 4. Fig. 4 also shows the cross-linked silane groups in the coating, wherein the silane is not attached to the surface. This structure demonstrates the formation of ether bridges by polymerization of the surface epoxide.
Example 7
Fig. 5 shows two possible synthetic pathways for preparing the chromatographic stationary phases of the present disclosure. As shown in scheme (500), unmodified BEH particles (505) may be chemically modified in at least two different ways. Accordingly, as an option, a chemical modifier is first prepared by reacting GPTMS with 1-aminoanthracene (510). Reagent 510 is then reacted with the BEH particles (505) to produce a functionalized chromatographic surface (515).
Alternatively, fig. 5 shows a different synthetic pathway. In this embodiment, particles 505 react directly with GPTMS to produce a GPTMS-modified surface (520). Surface 520 may then be reacted with 1-aminoanthracene (525) to produce a functionalized chromatographic surface (515).
In a preferred embodiment, the second reaction pathway comprising first reacting the particles 505 with GPTMS followed by functionalization with 1-aminoanthracene (525) performs better than the first reaction pathway comprising reacting the particles 505 with the preformed chemical modifier 510.
Example 8 GPTMS binding on organic-inorganic hybrid materials reduces retention drift or variation
As shown in fig. 6, treatment of the Bridged Ethylene Hybrid (BEH) stationary phase with glycidoxypropyl trimethoxysilane (GPTMS) followed by epoxide ring opening reaction to produce diol can significantly mitigate the retention drift effect. Graph 600 shows a graph of the% original retention of unfunctionalized 3 [ mu ] m BEH particles (605) compared to glycol functionalized 3 [ mu ] m BEH particles (610). The% original retention is given as a function of time, the number of days is given on the x-axis. The results demonstrate that in at least some preferred embodiments, functionalization of the chromatographic surface with GPTMS and subsequent epoxide ring opening to produce diol can mitigate retention drift. The results also show that GPTMS coating alone solves the problem of retention drift and also provides significant retention.
Example 9 separation of related Compounds Using 1-aminoanthracene-based stationary phases
Using simple chromatographic conditions, the methods and stationary phases of the present disclosure provide for the separation of related compounds, such as lipids and fat-soluble vitamins, that are superior to current prior art methods. They also provide different selectivities, which facilitate the resolution of different classes of compounds.
Using ACQUITY UPC 2 A system provided with: aconvergencechromatographybinarysolventmanager(ccBSM),asamplemanager(SM),aconvergencechromatographymanager(CCM),acolumnmanager(CM-a)andaphotodiodearray(pda)wereused. The system and separation conditions are provided as follows:
the first condition is that the flow rate is gradient, 3-20% methanol/CO in 2 min 2 The method comprises the steps of carrying out a first treatment on the surface of the 20% methanol/CO 2 for 0.5 min; 20-3% methanol/CO in 0.5 min 2 . ABPR set 2175 psi. Column temperature 40 ℃. Detection 235 nm, compensation 400-500 nm (320 nm for vitamin A, compensation 400-500 nm, if desired). Column dimensions 3.0 x 50 mm. Stationary phase 1-aminoanthracene modified diol as provided in example 7, 2.5 μm.
Second condition is that the flow rate is 2.0 mL/min. Gradient is 3-8% methanol/CO in 3 min 2 (# 8 curve); 8-35% methanol/CO in 0.5 min 2 (# 1 curve); 35% methanol/CO 2 1 minute; 35-3% methanol/CO in 0.5 min 2 (# 1 curve). ABPR 1800 psi. Column temperature 50 ℃. Sample GLC85 lipid standards (Nu-chek Prep, elysian, MN, USA), 1 g/L in CHCl 3 In stock solution, 20X was diluted with 1:1 heptane/IPA. Column dimensions 3.0x50 mm. Stationary phase 1-aminoanthracene modified diol as provided in example 7, 2.5 μm.
An ACQUITY SQD2 mass spectrometer was also used, 3.46kV capillary; a 25V cone; a 350 ℃ source; 500 L/hr desolventizing gas; 10 L/hr cone gas; LM Res 11.8; HM Res 15.1; ion energy of 0.2 provides peak detection and structural determination.
Figures 7-16 show lipid separations achieved using the methods and stationary phases of the present disclosure. The results of these separations demonstrate that the 1-aminoanthracene coupling is excellent in retaining and separating fat-soluble vitamins, lipids and metabolites. The 1-aminoanthracene coupling enhances shape/isomerism selectivity compared to the C18 bonding phase.
These results are surprising because mixing hydrophobic and hydrophilic groups on the same ligand is not only challenging, but is also an atypical motif (motif) for the stationary phase. Typically pi-electron rich ligands are used for very specialized separations and to provide slightly different selectivities than the C18 binding phase. Found in stationary phases based on 1-aminoanthracene and e.g. ACQUITY UPC 2 There is a very different selectivity between HSS C18 SB (used for comparison purposes in this disclosure).
In the case of fat-soluble vitamins, as their name suggests, these molecules are nonpolar and generally have few ionizable groups. In some embodiments, the stationary phase may contain residual amines that do not enhance the separation of the nonpolar compounds. In some embodiments, these groups may control the surface pH of the ligand. The presence of these groups does not indicate the possibility of isolation of fat-soluble vitamins. Typically, in the C18 bonding phase, e.g. ACQUITY UPC 2 Vitamin, lipid and metabolite separation was performed on HSS C18 SB. On this material, the alkyl chain is a retention selectivity factor that gives high methylene/hydrophobic selectivity but little shape/iso-selectivity. Materials of the present disclosure, such as those containing 1-aminoanthracene, retain and isolate lipid-soluble vitamins, lipids, and metabolites excellently. These materials also enhance shape/isomerism selectivity compared to the C18 bonding phase.
Similar conclusions were drawn when used with lipids, i.e. 1-aminoanthracene coupling was better than ACQUITY UPC 2 HSS C18 SB. When using the optimization method, more lipid peaks were resolved on the 1-aminoanthracene coupling prototype. The observed increased peak number correlates with the carbon chain length of the fatty acid and the selectivity of the saturation. 1-aminoanthracene prototype resolution "Fast and Simple Free Fatty Acids Analysis Using UPC 2 The problem noted in/MS "(Library Number: APTNT134753626; part Number 720004763 en), which states"Reversed phase chromatography separates lipids according to chain length and unsaturation. The problem is that In the dual nature of the reverse phase separation (reduced retention time of double bonds in fatty acyl chains)And fatty acyl chain length improves retention Time) may hinder the analysis of the actual sample; the fraction of components is typically so large that it is difficult to identify "due to co-elution". The stationary phase of the present disclosure brings more retention of unsaturated fatty acid chains (more double bonds bring longer retention time) and longer chain length. The separation is based on an improved retention of carbon number and double bond number, which is an excellent improvement compared to the alkyl-bonded stationary phase.
Those skilled in the art will appreciate that the present disclosure may be used to develop and regulate methods in other fields of application: fine chemicals/materials (OLEDs, agrochemicals, dyes/organic dyes, conformational polymers, polymer additives and surfactants), foods and environments (pesticides, glycerides, edible oils, tobacco, food adulterants), pharmaceutical/life sciences (lipid profile, natural products, DMPK/bioassays, impurity profile, medicinal chemistry) and forensics/research (opiates, drugs of abuse, steroids, fatty acids, antidepressants, gunpowder components, explosives).
In addition, these materials and methods can be used as part of a multi-dimensional experiment (2D-SFC, SFC-LC, etc.). Other materials that combine with these include the Argentation (silver impregnated material) and existing lipid methods such as those on CSH brand columns.
Example 10 separation of related compounds using various stationary phases of the present disclosure
Other chromatographic materials were prepared according to the previous examples using different selection factors. Lipid analysis was performed on these additional stationary phases. The test material included a stationary phase using the following selection factors: 1-aminoanthracene, 2-aminomethylpyridine, pyridine, 6-aminoquinoline, aniline, diol and 4-n-octylaniline.
General chromatographic conditions were provided as follows: sample: GLC85 lipid (Nu-chek Prep), 1 g/L in CHCl 3 In (a) and (b); dilute 20x with 1:1 IPA: heptane. The system comprises: UPC2 w/SQD 2, ESI-ionization. Mobile phase 97.5/2.5 MeOH/H2O w/0.1M NH3 make-up stream. 3.0x50mm column. The sample contains C 4 -C 24 A mixture of 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 these stationary phases, profiles and carbon bond number based each lipid. In general, the stationary phase containing 1-aminoanthracene and 4-n-octylaniline shows sufficient resolution of the mixture, based on the number of double bonds, and an increase in retention. The majority of lipid peaks in the samples could not be resolved using stationary phases of 2-aminomethylpyridine, pyridine, 6-aminoquinoline and aniline coupling and diols. Table 9 provides a summary of the performance of these stationary phases.
Table 9 properties of various stationary phases of the present disclosure
。
Table 9 provides a summary of the results. The retention window was measured as the time difference between the first and last elution peak of the lipid mixture. It is desirable to maximize this value because it represents a separation space. Material 3A provides the highest retention window value, 200% of HSS C18 SB (comparative). Peak capacity is measured as the retention window divided by the average peak width of all peaks eluting in that retention window. Material 3E provides a significant improvement over HSS C18 SB, with a peak capacity of 20% greater. The retention values of C18:0, C18:1, C22:1 and C22:6 represent the retention times of lipids having carbon chain lengths of 18 and 22, respectively, and 0, 1 and 6 double bonds in the chain, respectively. Materials 3E and HSS C18 SB showed reduced retention with increasing number of double bonds, while all other materials showed increased retention, and the (retention) range of all C18 and C22 lipids in the mixture was calculated by subtracting the retention time of the lipid C18 or C22 with the highest level of unsaturation (most double bonds) from the retention time of the saturated lipid C18 or C22, respectively. Negative values in these columns represent a decrease in retention based on the number of double bonds present in the chain. The positive C18 and C22 range values for the materials in this list represent unique selectivities compared to HSS C18 SB. The total Double Bond (DB) range is the total 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, which is embodied in material 3A. In some embodiments, the materials of the present disclosure provide DB values of greater than 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 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 1.5 under normal or conventional chromatographic conditions.
It was observed that N-octylaniline, HSS C18 SB (comparison column), 1-aminoanthracene and anilino stationary phases provided the best peak capacities. Peak capacity is the retention window divided by the average peak width at 13.4% (4 sigma width). Pyridine, 6-aminoquinoline, 2-aminomethylpyridine and 1-aminoanthracene provide the best C18 and C22 ranges. C18 ranges are the time differences between C18:2 and C18:0. Negative values indicate that C18:2 eluted before C18:0. The same applies to the C22 range. 1-aminoanthracene, pyridine, 6-aminoquinoline and 2-aminomethylpyridine provide the best total double bond range. C18:0 has 18 carbons and 0 double bond; c18:1 has 18 carbons and 1 double bond; etc. 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 the various stationary phases of the present disclosure
Two key pairs, vitamins K1 and K2 and vitamins D2 and D3, were tested using the stationary phase of the present disclosure to determine whether these materials improved the resolution of these pairs. Chromatographic conditions are provided in table 10. Table 11 provides a summary of the results. For each test material, the resolution of two key pair vitamins K1 and K2 and vitamins D2 and D3 were measured, respectively. The materials 3J, 3H and 3P significantly improved the resolution of the key pair compared to HSS C18 SB (comparison), especially for vitamins K1 and K2. The percentage improvement was calculated as follows:
。
The smaller particle size of materials 3J, 3H and 3P is expected to further improve the resolution of the key pair. For example, it is expected that decreasing the particle size of material 3P would increase the observed resolution by another 44%. It is believed that the conjugated portions of materials 3J, 3H and 3P enhance the interaction between the chromatographic surface and vitamins to promote unique selectivity compared to HSS C18 SB.
In some embodiments, the materials of the present disclosure provide Rs values of vitamin D3/D4 of greater than 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 under normal or conventional chromatographic conditions. In some embodiments, the materials of the present disclosure provide Rs values of vitamin K1/K2 of greater than 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5 under normal or conventional chromatographic conditions.
TABLE 10 chromatographic conditions
Cosolvent Methanol
Gradient of 3-20% cosolvent in 2 min
Sample of Vitamins A, vitamin A acetate, E, vitamin E acetate, K1, K2, D3 (0.2 mg/mL each)
Column size 3.0 x 50 mm
Flow rate 1.2 mL/min
Column temperature 40℃
Back pressure 2175 psi
Detector for detecting a target object Acquisy PDA with SFC Flow Cell
Detector arrangement 235 nm 40 spec/sec (320 nm for vitamin A)
Weak washing needle Isopropyl alcohol
Injection of 0.5 Mu L (2.0 mu L cycle, PLUNO injection mode)
Instrument for measuring and controlling the intensity of light UPC²
Software for providing a plurality of applications Empower
Table 11 performance of various stationary phases of the present disclosure in vitamin D and K separations
Material Vitamin k Rs Vitamin D Rs % improvement of vitamin D Rs (compared to HSS C18 SB)
HSS C18SB 0.23 0.74 0
2F 2.09 0.26 -64
3L 3.40 0.46 -38
3O 2.20 0.22 -71
3N 0.30 0.32 -57
3F 2.77 0.43 -42
3I 3.86 0.67 -9
3G 3.54 0.54 -27
3M 2.65 / /
3E 2.01 0.00 -100
3J 3.49 0.86 16
3H 3.97 0.79 7
3P 3.81 0.99 34
Unless otherwise indicated, all techniques, including the use of kits and reagents, may be performed according to manufacturer's information, methods known in the art.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated, each individual numerical value is incorporated into the specification as if it were individually recited. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, and specifications) is incorporated by reference in its entirety.
The description should be understood as disclosing and including all possible permutations and combinations of the described aspects, embodiments and examples unless the context indicates otherwise. Those of ordinary skill in the art will appreciate that the present invention can be practiced by other than the summarized and described aspects, embodiments, and examples, which are presented for purposes of illustration and not of limitation.
The application can comprise the following technical scheme:
1. a method of separating a related compound from a mixture, the method comprising:
(a) Providing a mixture comprising the related compound;
(b) Introducing a portion of the mixture into a chromatography system having a chromatography column; and
(c) Eluting the separated related compounds from the column;
wherein the column has a stationary phase having the following structure (i):
[X](W) a (Q) b (T) c (i)
wherein:
x is a chromatographic substrate comprising silica, metal oxide, inorganic-organic hybrid material, a set of block copolymers, or a combination thereof;
w is selected from hydrogen and hydroxy, wherein W is bonded to the surface of X;
q is a first substituent that minimizes variation in analyte retention over time under chromatographic conditions with low water concentration;
t is a second substituent chromatographically retaining the analyte, wherein T has one or more monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic, or polyheterocyclic aromatic groups, each optionally substituted with an aliphatic group; and is also provided with
b and c are positive numbers, 0.05-100 (b/c), and a-0.
2. The method of scheme 1, wherein a first fraction of Q is bonded to X and a second fraction of Q is polymerized.
3. The method of scheme 1, wherein a first fraction of T is bonded to X and a second fraction of T is polymerized.
4. The method of scheme 1, wherein a first score of Q is bonded to X, a second score of Q is polymerized, a third score of T is bonded to X, and a fourth score of T is polymerized.
5. The method of scheme 1, wherein a portion of Q of the second fraction and a portion of T of the fourth fraction are aggregated with each other.
6. The method of scheme 1 wherein Q has the following structure (ii):
wherein:
n 1 is an integer of 1 to 30;
n 2 is an integer of 1 to 30;
R 1 、R 2 、R 3 and R is 4 Each independently selected from the group consisting of hydrogen, hydroxy, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, lower alkyl, protected or deprotected alcohol and a zwitterionic;
z is
a) Having the formula (B) 1 ) x (R 5 ) y (R 6 ) z Si-wherein x is an integer from 1 to 3, y is an integer from 0 to 2, z is an integer from 0 to 2, and x+y+z=3;
R 5 and R is 6 Each independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, substituted or unsubstituted aryl, cycloalkyl, branched alkyl, lower alkyl, protected or deprotected alcohol, zwitterionic groups, and siloxane bonds; and is also provided with
B 1 Is a siloxane bond;
b) Attachment to the surface organofunctional hybrid group through direct carbon-carbon bond formation or through heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or polyurethane bonds; or (b)
c) An adsorption surface group not covalently attached to the surface of the material;
Y is an intercalating polar functional group, bond or aliphatic group; and is also provided with
A is selected from the group consisting of hydrophilic end groups, functionalizable groups, hydrogen, hydroxyl, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, lower alkyl, and polarizable groups.
7. The method of scheme 1, wherein T has the following structure (iii):
wherein the method comprises the steps of
m 1 Is an integer of 1 to 30;
m 2 is an integer of 1 to 30;
m 3 is an integer of 1 to 3;
R 7 、R 8 、R 9 and R is 10 Each independently selected from the group consisting of hydrogen, hydroxy, fluoro, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, lower alkyl, protected or deprotected alcohol, zwitterion, aromatic hydrocarbon group, and heterocyclic aromatic hydrocarbon group;
z is
a) Having the formula (B) 1 ) x (R 5 ) y (R 6 ) z Si-wherein x is an integer from 1 to 3, y is an integer from 0 to 2, z is an integer from 0 to 2, and x+y+z=3;
R 5 and R is 6 Each independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, substituted or unsubstituted aryl, cycloalkyl, branched alkyl, lower alkyl, protected or deprotected alcohol, zwitterionic groups, and siloxane bonds; and is also provided with
B 1 Is a siloxane bond;
b) Attachment to the surface organofunctional hybrid group through direct carbon-carbon bond formation or through heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole, or polyurethane bonds; or (b)
c) An adsorption surface group not covalently attached to the surface of the material;
y is an intercalating polar functional group, bond or aliphatic group;
d is selected from the group consisting of 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–(CH 2 ) 0-12 –R 11 ；
R 11 is a first monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group; and is also provided with
R 12 Is hydrogen, aliphatic or a second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or poly-cyclicHeterocyclic aromatic groups, wherein R 11 And R is 12 Optionally substituted with aliphatic groups.
8. The method of scheme 1, wherein the related compound is a lipid, a vitamin, or a polycyclic aromatic hydrocarbon.
9. The method of scheme 8, wherein said lipid is a saturated or unsaturated fatty acid, monoacylglyceride, diacylglyceride, triacylglyceride, phospholipid, sphingolipid, or steroid.
10. The method of claim 8, wherein the vitamin is a water-soluble vitamin selected from vitamin C, vitamin B, or derivatives or combinations thereof.
11. The method of claim 8, wherein the vitamin is a fat-soluble cellulose selected from the group consisting of vitamin a, vitamin D, vitamin K, vitamin E, beta-carotene, or derivatives or combinations thereof.
12. The method of scheme 7, wherein R 11 Or R is 12 At least the first or second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group of (c) is a polycyclic aromatic or polyheterocyclic aromatic hydrocarbon having at least 2 aromatic rings.
13. The method of scheme 7, wherein R 11 Or R is 12 At least the first or second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group of (c) is a polycyclic aromatic or polyheterocyclic aromatic hydrocarbon having at least 3 aromatic rings.
14. The method of scheme 7, wherein R 11 Or R is 12 At least the first or second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polyheterocyclic aromatic group of (c) is a polycyclic aromatic or polyheterocyclic aromatic hydrocarbon having at least 4 aromatic rings.
15. The method of scheme 7, wherein R 11 Or R is 12 At least one of the first or second monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polycyclic aromatic groups of (C) 4 -C 24 Aliphatic group substitution.
16. The method of scheme 7, wherein R 11 Or R is 12 The first monocyclic aromatic, polycyclic aromatic, heterocyclic aromatic or polycyclic aromatic group of (a) is selected from furan, pyrrole, pyrroline, oxazole, thiazole, imidazole, imidazoline, pyrazole, pyrazoline, pyrazolidine,isoxazoles, isothiazoles, benzenes, pyridines, pyridazines, pyrimidines, pyrazines, triazines, thiophenes, indenes, indolizines, indoles, isoindoles, indolines, indazoles, benzimidazoles, benzothiazoles, naphthalenes, quinolizines, quinolines, isoquinolines, cinnolines, phthalazines, quinazolines, quinoxalines, 1, 8-naphthyridines, quinuclidines, fluorenes, carbazoles, anthracenes, acridines, phenazines, phenothiazines, phenoxazines, pyrenes, phenanthrenes, chrysene and derivatives thereof, wherein the groups are unsubstituted or optionally substituted with aliphatic groups.
17. The method of scheme 6 wherein Q comprises a functional group that is a diol.
18. The method of scheme 17 wherein Q is represented by the following structure:
。
19. the method of scheme 7, wherein T is represented by one of the following structures:
。
20. the method of scheme 1, wherein the surface of X is alkoxylated under chromatographic conditions by a chromatographic mobile phase.
21. The method of scheme 20, wherein a majority of the surface of X is not alkoxylated by the chromatographic mobile phase under chromatographic conditions.
22. The method of scheme 1, wherein the surface of X does not comprise silica, and b=0 or c=0.
23. The method of scheme 1, wherein the chromatographic stationary phase is suitable for supercritical fluid chromatography.
24. The method of scheme 1, wherein the chromatographic stationary phase is suitable for carbon dioxide-based chromatography.
25. A chromatographic stationary phase having the following structure (i):
[X](W) a (Q) b (T) c (i)
wherein:
x is a chromatographic substrate comprising silica, metal oxide, inorganic-organic hybrid material, a set of block copolymers, or a combination thereof;
w is selected from hydrogen and hydroxy, wherein W is bonded to the surface of X;
q is a first substituent that minimizes variation in analyte retention over time under chromatographic conditions with low water concentration;
t is a second substituent chromatographically retaining the analyte, wherein T has one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups, each optionally substituted with an aliphatic group; and is also provided with
b and c are positive numbers, 0.05-100 (b/c), and a-0.
26. A column, capillary column, microfluidic device or apparatus for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography comprising:
a housing having at least one wall defining a chamber having an inlet and an outlet, and a stationary phase according to scheme 25 disposed therein, wherein the housing and stationary phase are suitable for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography.
27. A kit for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography comprising:
a housing having at least one wall defining a chamber having an inlet and an outlet, and a stationary phase according to scheme 25 disposed therein, wherein the housing and stationary phase are suitable for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography; and
Instructions for performing normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography with the shell and stationary phase.
28. A method of preparing a stationary phase according to scheme 25, comprising:
reacting the chromatographic substrate with a silane coupling agent having pendant reactive groups;
reacting a second chemical reagent comprising one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups with the pendant reactive groups; and
neutralizing any remaining unreacted pendant reactive groups,
thereby producing a stationary phase according to scheme 25.
29. A method of preparing a stationary phase according to scheme 25, comprising:
oligomerizing a silane coupling agent having pendant reactive groups;
reacting the core surface with an oligomeric silane coupling agent;
reacting a second chemical reagent comprising one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups with the pendant reactive groups; and
neutralizing any remaining unreacted pendant reactive groups,
thereby producing a stationary phase according to scheme 25.
30. A method of mitigating or preventing retention drift in normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography, comprising:
the sample is chromatographically separated using a chromatographic apparatus comprising a chromatographic stationary phase according to scheme 25, thereby mitigating or preventing retention drift.

Claims (30)

1. A chromatographic stationary phase having the following structure (i):
[X](W) a (Q) b (T) c (i)
wherein:
x is a chromatographic substrate comprising a surface and comprising silica, a metal oxide, an inorganic-organic hybrid material, a set of block copolymers, or a combination thereof;
w is selected from hydrogen and hydroxy, wherein W is bonded to the surface of X;
q is:
t is represented by one of the following structures:
a is zero or a positive number;
b is a positive number;
c is a positive number;
wherein the b/c ratio is greater than or equal to 0.05 and less than or equal to 100; and
z is independently selected from:
(a) Having the formula (B) 1 ) x (R 5 ) y (R 6 ) z Si-wherein x is an integer from 1 to 3, y is an integer from 0 to 2, z is an integer from 0 to 2, and x+y+z=3; r is R 5 And R is 6 Each independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, substituted or unsubstituted aryl, cycloalkyl, branched alkyl, lower alkyl, protected or deprotected alcohol, zwitterionic groups, and siloxane bonds; and B is 1 Is a siloxane bond;
(b) Attachment to the surface organofunctional hybrid group via a direct carbon-carbon bond or via a heteroatom, ester, ether, thioether, amine, amide, imide, urea, carbonate, carbamate, heterocycle, triazole or urethane linkage; or (b)
(c) There are no adsorbed surface groups covalently attached to the surface of X.
2. The stationary phase of claim 1, wherein the surface of X is alkoxylated by a chromatographic mobile phase under chromatographic conditions.
3. The stationary phase of claim 2, wherein a majority of the surface of X is not alkoxylated by chromatographic mobile phase under chromatographic conditions.
4. The stationary phase of claim 1, wherein the chromatographic stationary phase is suitable for supercritical fluid chromatography.
5. The stationary phase of claim 1, wherein the chromatographic stationary phase is suitable for carbon dioxide-based chromatography.
6. The stationary phase of claim 1, wherein a first fraction of Q is bonded to X and a second fraction of Q is polymerized.
7. The stationary phase of claim 1, wherein a first fraction of T is bonded to X and a second fraction of T is polymerized.
8. The stationary phase of claim 1, wherein a first fraction of Q is bonded to X, a second fraction of Q is polymerized, a third fraction of T is bonded to X, and a fourth fraction of T is polymerized.
9. The stationary phase of claim 1, wherein a portion of Q of the second fraction and a portion of T of the fourth fraction are polymerized with each other.
10. A column, capillary column, microfluidic device or apparatus for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography comprising:
a housing having at least one wall defining a chamber having an inlet and an outlet, and the stationary phase of claim 1 disposed therein, wherein the housing and stationary phase are suitable for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography.
11. A kit for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography comprising:
A housing having at least one wall defining a chamber having an inlet and an outlet, and the stationary phase of claim 1 disposed therein, wherein the housing and stationary phase are adapted for normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography; and
instructions for performing normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide based chromatography, hydrophilic interaction liquid chromatography or hydrophobic interaction liquid chromatography with the shell and stationary phase.
12. A method of preparing the stationary phase of claim 1, the method comprising:
reacting the chromatographic substrate with a silane coupling agent having pendant reactive groups;
reacting a second chemical reagent selected from the group consisting of 1-aminoanthracene, 2-aminoanthracene, and 9-aminoanthracene with the pendant reactive group; and
neutralizing any remaining unreacted pendant reactive groups;
thereby producing a stationary phase according to claim 1.
13. A method of preparing the stationary phase of claim 1, comprising:
oligomerizing a silane coupling agent having pendant reactive groups;
reacting the core surface with an oligomeric silane coupling agent;
reacting a second chemical reagent selected from the group consisting of 1-aminoanthracene, 2-aminoanthracene, and 9-aminoanthracene with the pendant reactive group; and
neutralizing any remaining unreacted pendant reactive groups;
thereby producing a stationary phase according to claim 1.
14. A method of mitigating or preventing retention drift in normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography, comprising:
chromatographic separation of a sample using a chromatographic apparatus comprising a chromatographic stationary phase according to claim 1, thereby reducing or preventing retention drift.
15. A method of separating a related compound from a mixture, the method comprising:
(a) Providing a mixture comprising the related compound;
(b) Introducing a portion of the mixture into a chromatography system having a chromatography column; and
(c) Eluting the separated related compounds from the chromatographic column;
Wherein the chromatographic column has a chromatographic stationary phase according to claim 1, wherein Q and T polymerize such that the chromatographic stationary phase comprises:
or mixtures thereof.
16. The method of claim 15, wherein the related compound is a lipid, a vitamin, or a polycyclic aromatic hydrocarbon.
17. The method of claim 16, wherein the lipid is a saturated or unsaturated fatty acid, a monoacylglyceride, a diacylglyceride, a triacylglyceride, a phospholipid, a sphingolipid, or a steroid.
18. The method of claim 16, wherein the vitamin is a water-soluble vitamin selected from vitamin C, vitamin B, or derivatives or combinations thereof.
19. The method of claim 16, wherein the vitamin is a fat-soluble cellulose selected from vitamin a, vitamin D, vitamin K, vitamin E, beta carotene, or derivatives or combinations thereof.
20. The method of claim 15, wherein the chromatographic stationary phase is suitable for supercritical fluid chromatography.
21. The method of claim 15, wherein the chromatographic stationary phase is suitable for carbon dioxide-based chromatography.
22. A method of mitigating or preventing retention drift in normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography, comprising:
Chromatographic separation of the mixture using a chromatographic system comprising the stationary phase of claim 15, thereby reducing or preventing retention drift.
23. A method of separating a related compound from a mixture, the method comprising:
(a) Providing a mixture comprising the related compound;
(b) Introducing a portion of the mixture into a chromatography system having a chromatography column; and
(c) Eluting the separated related compounds from the chromatographic column;
wherein the chromatographic column has a chromatographic stationary phase according to claim 1, wherein Q and T polymerize such that the chromatographic stationary phase comprises:
or mixtures thereof.
24. The method of claim 23, wherein the related compound is a lipid, a vitamin, or a polycyclic aromatic hydrocarbon.
25. The method of claim 24, wherein the lipid is a saturated or unsaturated fatty acid, a monoacylglyceride, a diacylglyceride, a triacylglyceride, a phospholipid, a sphingolipid, or a steroid.
26. The method of claim 24, wherein the vitamin is a water-soluble vitamin selected from vitamin C, vitamin B, or derivatives or combinations thereof.
27. The method of claim 24, wherein the vitamin is a fat-soluble cellulose selected from vitamin a, vitamin D, vitamin K, vitamin E, beta carotene, or derivatives or combinations thereof.
28. The method of claim 23, wherein the chromatographic stationary phase is suitable for supercritical fluid chromatography.
29. The method of claim 23, wherein the chromatographic stationary phase is suitable for carbon dioxide-based chromatography.
30. A method of mitigating or preventing retention drift in normal phase chromatography, high pressure liquid chromatography, solvated gas chromatography, supercritical fluid chromatography, subcritical fluid chromatography, carbon dioxide-based chromatography, hydrophilic interaction liquid chromatography, or hydrophobic interaction liquid chromatography, comprising:
chromatographic separation of the mixture using a chromatographic system comprising the chromatographic stationary phase of claim 23, thereby reducing or preventing retention drift.
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