WO2007041510A1 - Improved process for analyzing, for separating, and for isolating individual polar protic monomers and/or oligomers - Google Patents
Improved process for analyzing, for separating, and for isolating individual polar protic monomers and/or oligomers Download PDFInfo
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- WO2007041510A1 WO2007041510A1 PCT/US2006/038478 US2006038478W WO2007041510A1 WO 2007041510 A1 WO2007041510 A1 WO 2007041510A1 US 2006038478 W US2006038478 W US 2006038478W WO 2007041510 A1 WO2007041510 A1 WO 2007041510A1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/32—Bonded phase chromatography
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J20/288—Polar phases
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
- B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
Definitions
- the present invention is directed to an improved process for separating and preferably recovering individual polar protic monomers and/or oligomers, including without limitation flavan-3-ols, according to their degree of polymerization, using diol-phase liquid chromatography (LC).
- LC diol-phase liquid chromatography
- Proanthocyanidins the oligomers and polymers of flavan-3-ols, are the second most abundant natural plant phenols after lignin.
- the flavan-3-ol monomers are linked primarily through carbon-carbon linkages from the 4 position of one monomer to the 8 position of another monomer (C4 ⁇ C8) and to a lesser extent through C4 ⁇ C6 linkage(s).
- Proanthocyanidins include B-type and A-type proanthocyanidins.
- the monomers are linked via interflavan C4 ⁇ C6 and/or interflavan C4 ⁇ C8 linkages.
- Oligomers with exclusively C4 ⁇ C8 linkages are linear, while the presence of at least one C4 ⁇ C6 linkage results in a branched oligomer.
- A- type proanthocyanidins are doubly linked oligomers containing linkages at C2-O-C7 as well as at C4 ⁇ C6 or C4 ⁇ C8.
- the molecular weight of proanthocyanidins typically is expressed as degree of polymerization (DP). Individual oligomers are commonly referred to as dimers, trimers, etc.
- Procyanidins represent the largest class of proanthocyanidins. Gu et al. showed that out of 41 foods found to contain proanthocyanidins, 27 contained procyanidins. (J. Agric. and Food Chem. (2003) 51:7513). Procyanidins may include (-)-epicatechin, (+)- epicatechin, (+)-catechin and/or (-)-catechin monomeric units, as well as gallated monomers such as (-)-catechin gallate, (+)-catechin gallate, (-) epicatechin gallate and/or (+)-epicatechin gallate.
- proanthocyanidins play important roles in the color stability, astringency, and bitterness of plant foods.
- Haslam "Practical Polyphenols: From Molecular Recognition and Physiological Action” (Cambridge U. Press, 1998)).
- the notoriety of proanthocyanidins has increased due to the potential health benefits of these phenolic compounds.
- Bagchi et al. Toxicology (2000) 148:187; Foo et al., J. Natural Products (2000) 63: 1225; Steinberg et al., Am. J. Clin. Nutri. (2003) 77:1466).
- procyanidin oligomers present specific characteristics and potential benefits for use in humans and animals.
- Tempesta discloses that procyanidin oligomers having a degree of polymerization (DP) of 2-11 possess significant antiviral activity, and are useful in treating warm-blooded animals, including humans, infected with paramyxovaridae such as respiratory syncytial virus, orthomyxovaridae such as influenza A, B and C, and herpes viruses such as Herpes Simplex virus (U.S. Patent No. 5,211;944).
- paramyxovaridae such as respiratory syncytial virus
- orthomyxovaridae such as influenza A, B and C
- herpes viruses such as Herpes Simplex virus
- antineoplastic compositions comprising procyanidin oligomers having a DP of 3-11 together with a suitable earner (U.S. Patent No. 5,554,645).
- Romanczyk, Jr. et al. also disclose that procyanidin oligomers having a DP of 5-12 are useful as antioxidants (U.S. Patent No. 5,891,905).
- Schmitz et al. disclose the use of cocoa procyanidin oligomers having a DP of 2-18 together with acetyl salicylic acid as anti-platelet therapy (U.S. Patent No. 6,524,630).
- THF Tetrahydrofuran
- CNS central nervous system
- the present invention provides an improved process for separating and eluting individual polar protic monomer(s) and/or oligomer(s).
- the process comprises the steps of. (i) introducing a liquid sample containing the monomer(s) and/or oligomer(s) into a liquid chromatography (LC) column packed with a polar bonded stationary phase; (ii) separating the individual monomer(s) and/or oligomer(s), on the basis of degree of polymerization, by passing a binary mobile phase comprising an A phase consisting essentially of a polar aprotic solvent and a B phase consisting essentially of a polar protic solvent through the column; and (iii) eluting one or more individual fractions containing the monomer(s) and/or oligomer(s).
- LC liquid chromatography
- one or more of the eluted individual monomer(s) and/or oligomer(s) may be recovered for use in a food product, a medicinal food product, a nutraceutical, or a pharmaceutical product, by drying or other conventional means.
- the eluted individual monomer (s) and/or oligomer (s) may be recovered for use as standards in other separations.
- the polar protic monomer(s) and/or oligomer(s) may be proanthocyanidins, hydrolyzable tannins, oligosaccharides, oligonucleotides, peptides, acrylamides, polysorbates, polyketides, poloxamers, polyethylene glycols, polyoxyethylene alcohols or polyvinyl alcohols.
- the polar protic monomer(s) and/or oligomer(s) are proanthocyanidins, they may be proapigeninidins, proluteolinidins, protricetinidins, propelargonidins, procyanidins, prodelphinidins, proguibourtinidins, profisetinidins, prorobinetinidins, proteracacinidins and/or promelacacinidins.
- the monomer(s) are epicatechin and/or catechin
- the oligomer(s) are procyanidin oligomers thereof.
- the sample containing the monomer(s) and/or oligomer(s) is a polar, defatted cocoa extract.
- the amount of extract present in the liquid sample introduced into the LC column preferably is greater than 10 milligrams. More preferably, the amount of extract is greater than 100 milligrams. Even more preferably, the amount of extract is greater than 1 gram.
- the polar bonded stationary phase may be a diol phase, a glycerol phase, an amino phase, a cyano phase, a trimethylsilyl phase, a dimethylsilyl phase, a propyl phase, a butyl phase, a pentyl phase, a hexyl phase, a phenyl phase, a halogenated phase, or a nitro phase.
- the stationary phase is a diol phase or a glycerol phase.
- the polar aprotic solvent may be any of acetonitrile, acetone, cyclohexanone, methyl ethyl ketone, methyl tert- butyl ether, diethyl ether, dimethyl ether, methyl acetate, ethyl acetate and nitromethane
- the polar protic solvent may be any of methanol, ethanol, n-propanol, isopropanol, n-butanol and isobutanol.
- the proportion of polar aprotic solvent in the A phase may be up to 100 percent by volume, and the proportion of the polar protic solvent in the B phase may be up to 100 percent by volume.
- the remainder of the A phase and B phase may be any mineral or organic acid and/or water.
- the proportion of polar aprotic solvent in the A phase and polar protic solvent in the B phase is greater than 50 percent by volume.
- the proportion of polar aprotic solvent (A phase) and polar protic solvent (B phase) is greater than 90 percent by volume.
- the polar aprotic solvent in the A phase is about 98% acetonitrile by volume
- the polar protic solvent in the B phase is about 95% methanol by volume.
- the polar bonded stationary phase preferably has a particle size from about 3 ⁇ m to about 10 ⁇ m.
- the LC column preferably has a diameter of at least ten (10) millimeters.
- the present invention also provides a system for separating and eluting individual polar protic monomer(s) and/or oligomer(s) on the basis of degree of polymerization.
- the system comprises an LC column packed with a polar bonded stationary chromatographic phase; and a binary mobile phase comprising an A phase consisting essentially of a polar aprotic solvent and a B phase consisting essentially of a polar protic solvent for eluting one or more individual fractions containing the monomer(s) and/or oligomer(s).
- the polar bonded stationary phase may be any of a diol phase, a glycerol phase, an amino phase, a cyano phase, a trimethylsilyl phase, a dimethylsilyl phase, a propyl phase, a butyl phase, a pentyl phase, a hexyl phase, a phenyl phase, a halogenated phase and a nitro phase.
- the polar bonded stationary chromatographic phase is a diol phase or a glycerol phase.
- the polar bonded stationary chromatographic phase has a particle size from about 3 ⁇ m to about 10 ⁇ m.
- the column preferably has a diameter of at least ten (10) millimeters.
- the polar aprotic solvent in the A phase may be any of acetonitrile, acetone, cyclohexanone, methyl ethyl ketone, methyl tert-butyl ether, diethyl ether, dimethyl ether, methyl acetate, ethyl acetate and nitromethane.
- the polar protic solvent in the B phase may be any of methanol, ethanol, n-propanol, isopropanol, n-butanol and isobutanol.
- Suitable polar protic monomer(s) and/or oligomer(s) for the system include proanthocyanidins, hydrolyzable tannins, oligosaccharides, oligonucleotides, peptides, acrylamides, polysorbates, polyketides, poloxamers, polyethylene glycols, polyoxyethylene alcohols and polyvinyl alcohols.
- the polar protic monomer(s) and/or oligomer(s) are proanthocyanidins, they may be proapigeninidins, proluteolinidins, protricetinidins, propelargonidins, prodelphinidins, proguibourtinidins, profisetinidins, prorobinetindins, proteracacinidins and/or promelacacinidins.
- the monomer(s) are epicatechin and/or catechin
- the oligomer(s) are procyanidin oligomers thereof.
- the proportion of polar aprotic solvent in the A phase may be up to 100% by volume, and the proportion of the polar protic solvent in the B phase may be up to 100% by volume.
- the remainder of the A phase and B phase may be any mineral or organic acid and/or water.
- the proportion of polar aprotic solvent in the A phase and polar protic solvent in the B phase is greater than 50% by volume.
- the proportion of polar aprotic solvent (A phase) and polar protic solvent (B phase) is greater than 90% by volume.
- the polar aprotic solvent in the A phase is about 98% acetonitrile by volume
- the polar protic solvent in the B phase is about 95% methanol by volume.
- the present invention also provides a process for separating and isolating xanthine(s) from polar protic monomer (s) and/or oligomer(s).
- the process comprises the steps of: (i) introducing a liquid sample containing the xanthine(s) and polar protic monomer(s) and/or oligomer(s) into a liquid chromatography (LC) column packed with a polar bonded stationary chromatographic phase; (ii) separating the xanthine(s) from the monomer(s) and/or oligomer(s) by passing an isocratic mobile phase consisting essentially of a polar aprotic solvent through the column; and (iii) eluting one or more individual fractions containing the xanthine(s).
- LC liquid chromatography
- the xanthine(s) preferably are caffeine and/or theobromine.
- the liquid sample is a polar, defatted cocoa extract.
- the eluted individual xanthine(s) may be recovered for use in a food product, a medicinal food product, a nutraceutical, or a pharmaceutical product, by drying or other conventional means.
- the eluted individual xanthine(s) also may be recovered for use as standards in other separations.
- the polar bonded stationary phase may be a diol phase, a glycerol phase, an amino phase, a cyano phase, a trimethylsilyl phase, a dimethylsilyl phase, a propyl phase, a butyl phase, a pentyl phase, a hexyl phase, a phenyl phase, a halogenated phase, or a nitro phase.
- the stationary phase is a diol phase or a glycerol phase.
- the polar aprotic solvent preferably is acetonitrile.
- the proportion of polar aprotic solvent in the isocratic mobile phase preferably is at least 90 percent by volume.
- the remainder of the mobile phase may be any mineral or organic acid and/or water.
- the polar aprotic solvent in the isocratic mobile phase is about 99% acetonitrile by volume.
- the present invention also provides a system for separating and isolating xanthine(s) from polar protic monomer(s) and/or oligomer (s).
- the system comprises a liquid chromatography column packed with a polar bonded stationary chromatographic phase; and an isocratic mobile phase consisting essentially of a polar aprotic solvent for eluting one or more individual fractions containing the xanthine(s).
- the polar bonded stationary phase may be any of a diol phase, a glycerol phase, an amino phase, a cyano phase, a trimethylsilyl phase, a dimethylsilyl phase, a propyl phase, a butyl phase, a pentyl phase, a hexyl phase, a phenyl phase, a halogenated phase and a nitro phase.
- the stationary phase is a diol phase or a glycerol phase.
- the stationary phase has a particle size from about 3 ⁇ m to about 10 ⁇ m.
- the column preferably has a diameter of at least ten (10) millimeters.
- the polar aprotic solvent preferably is acetonitrile.
- the proportion of polar aprotic solvent in the isocratic mobile phase preferably is greater than 90% by volume.
- the remainder may be any mineral or organic acid and/or water.
- the polar aprotic solvent in the isocratic mobile phase is about 99% by volume acetonitrile.
- the present invention also provides a process for separating and eluting xanthine(s) and individual polar protic monomer(s) and/or oligomer(s).
- the process comprises the steps of: (i) introducing a liquid sample containing the xanthine(s) and polar protic monomer(s) and/or oligomer(s) into a liquid chromatography column packed with a polar bonded stationary chromatographic phase; (ii) separating the xanthine(s) from the monomer(s) and/or oligomer(s) by passing an isocratic mobile phase consisting essentially of a polar aprotic solvent through the column, and eluting one or more individual fractions containing the xanthine(s); (iii) subsequently separating the individual monomer(s) and/or oligomer(s), on the basis of degree of polymerization, by passing a binary mobile phase comprising an A phase consisting essentially of a
- the eluted xanthine(s) and/or one or more of the eluted individual monomer(s) and/or oligomer(s) may be recovered for use in a food product, a medicinal food product, a nutraceutical, or a pharmaceutical product, by drying or other conventional means.
- the eluted xanthine(s) and/or eluted individual monomer(s) and/or oligomer(s) also may be recovered for use as standards in other separations.
- the xanthine(s) preferably are caffeine and/or theobromine.
- the monomer(s) and/or oligomer(s) may be proanthocyanidins, hydrolyzable tannins, oligosaccharides, oligonucleotides, peptides, acrylamides, polysorbates, polyketides, poloxamers, polyethylene glycols, polyoxyethylene alcohols and polyvinyl alcohols.
- the polar bonded stationary phase may be a diol phase, a glycerol phase, an amino phase, a cyano phase, a trimethylsilyl phase, a dimethylsilyl phase, a propyl phase, a butyl phase, a pentyl phase, a hexyl phase, a phenyl phase, a halogenated phase, or a nitro phase.
- Figure 1 Structure of monomeric flavan-3-ols and procyanidin oligomers linked through carbon-carbon C4— >C8 and C4 ⁇ C6 linkage.
- Figure 4 HPLC chromatograms of procyanidins, using Lichrosphere Silica (bottom) and Develosil Diol (top) as stationary phase.
- Figure 8 LC trace chromatogram of preparative system for separating caffeine and theobromine from CP extract.
- the present invention discloses an improved process for separating and isolating polar protic monomer(s) and/or oligomer(s) on the basis of degree of polymerization, using a diol-based LC system having a binary mobile phase to elute one or more individual fractions containing individual polar protic monomer(s) and/or oligomer(s), as well as a system for same.
- Figure 1 shows the structure of monomeric flavan-3-ols and procyanidin oligomers linked through C4 ⁇ C8 and C4 ⁇ C6 linkage.
- the improved processes and system of the present invention use a diol- based stationary phase LC column and binary mobile phase comprising an A phase of acetonitrile and acetic acid (CH 3 CNiHOAc) in a 98:2 (v/v) mix, and a B phase of methanol, water and acetic acid (CH 3 OHiH 2 OiHOAc) in a 95:3:2 (v/v/v) mix.
- a phase of acetonitrile and acetic acid CH 3 CNiHOAc
- B phase of methanol, water and acetic acid CH 3 OHiH 2 OiHOAc
- Figure 4 compares the chromatograms obtained using a diol stationery phase (top) and a silica stationery phase (bottom).
- the diol stationary phase provided for greater separation of individual oligomers, particularly for greater separation of oligomers having a DP of 7 or higher.
- the improved processes also are more useful than current processes since they can be used with a wider range of solvents (including water) and can be used in laboratories equipped only with a binary LC pump.
- Another benefit is the capability to analyze other compounds (i.e., other flavenoids, caffeine, and theobromine) in addition to procyanidins in a single LC run, especially in conjunction with mass spectrometry (LC-MS).
- LC-MS mass spectrometry
- any polar protic monomer(s) and/or oligomer(s) including without limitation other proanthocyanidins (e.g., proapigeninidins, proluteolinidins, protricetinidins, propelargonidins, prodelphinidins, proguibourtinidins, profisetinidins, prorobinetindins, proteracacinidins and promelacacinidins), hydrolyzable tannins, oligosaccharides, oligonucleotides, peptides, acrylamides, polysorbates, polyketides, poloxamers, polyethylene glycols, polyoxyethylene alcohols and polyvinyl alcohols.
- proanthocyanidins e.g., proapigeninidins, proluteolinidins, protricetinidins, propelargonidins, prodelphinidins, proguibourtinidins, profisetinidins
- polar protic monomer(s) and/or oligomer(s) include peanut skins, cinnamon, blueberries, apples, sorghum, hawthorne, cranberries, and grapes.
- the disclosed processes and system are suitable for separating sugar polymers such as maltosaccharides, cyclodextrins, N- acetylchitooligosaccharides, and pridylamino sugar chains.
- CP extract A cocoa polyphenol (CP) extract was prepared via a multi-step process aimed at minimizing degradation of the cocoa procyanidins. Cocoa beans were harvested, washed free of pulp, and dried. Under ambient conditions the dried beans were expeller-pressed to remove cocoa butter. The expeller cake was then ground and extracted with ethanol:water (70:30 v/v). Solids were removed by centrifugation. The extract liquid was evaporated under reduced pressure to remove the ethanol and finally spray dried.
- CP cocoa polyphenol
- NP-HPLC-MS Normal-phase high performance liquid chromatography mass spectrometry
- the column used was Develosil Diol (250 x 4.6 mm ID., 5 ⁇ particle size) purchased from Phenomenex (Torrance, California).
- the binary mobile phase consisted of (A) acetonitrile: acetic acid (98:2, v/v) and (B) methanol:water:acetic acid (95:3:2, v/v/v). Separations were effected by a linear gradient (of the mobile phase) at 30° C with an 0.8 mL/min flow rate as follows: 0-35 minutes, 0-40% B; 35-40 min, 40% B isocratic; 40-45 min, 40-0% B, followed by a 5 minute re-equilibrate time.
- Conditions for analysis in the positive ion mode included introduction of 0.05M NaCl at a flow rate of 0.05 mL/min to assist ionization, a capillary voltage of 3.5 kV, a fragmentor voltage of 100 V, a nebulizing pressure of 25 psig, and a drying gas temperature of 350 0 C.
- Conditions for analysis in the negative ion mode included 1.5 M NH 4 OH as a buffering agent at a flow rate of 0.09 mL/min for 29 minutes, and then at 0.05 mL/min.
- Capillary voltage was 3 kV
- fragmentor voltage was 75 V
- nebulizing pressure was 25 psig
- drying gas temperature was 350 0 C.
- the chromatographic mobile phase consisted of methylene chloride (CH 2 Cl 2 ), methanol (CH 3 OH), and acetic acid:water (1:1) (HOAc:H 2 O).
- Starting mobile phase conditions were 82% CH 2 Cl 2 , 14% CH 3 OH and 4% (HOAc:H 2 O). Subsequently CH 3 OH was ramped to
- Figure 2 shows the chromatogram of an unfermented, defatted cacao seed extract.
- the labels 2-14 indicate the degree of polymerization of procyanidins in the peaks.
- Gu et al. the flavan-3-ol monomeric and oligomeric composition of unfermented cacao consists exclusively of (-)-epicatechin and (+)-catechin. See J. Agric. and Food Chem. (2003) 51:7513. In agreement with the chromatographic separation described herein, Gu et al.
- the bonded diol phase showed similar retention characteristics only for the procyanidin monomers, i.e., (-)-epicatechin and (+)-catechin.
- the bonded diol phase showed stronger retention characteristics for the dimer through the decamer with retention increasing with degree of polymerization.
- the retention time for the decamer fraction was about 50% longer than that observed for the Lichrosphere Silica phase under identical conditions. Consistent with the stronger retention characteristics of the diol phase, an increase in speciation was also observed. This was apparent throughout the entire chromatogram and even impacted the monomer region at 10 minutes.
- the diol phase yielded almost baseline resolution of (-)-epicatechin and (+)-catechin while the silica phase showed significant co-elution.
- One of the major benefits of the diol stationary phase process described herein is the ability to scale up the process with a smaller concern for safety issues and disposal costs. Due to solvent choice and the relative simplicity of the binary mobile phase gradient used for the analytical scale, the process was able to be transferred to a semi- preparative (250 x 21.5 mm) and then to the preparative (300 x 50 mm) scale HPLC system with alterations in only the gradient and flow-rate.
- Suitable stationary phases include, but are not limited to, a glycerol phase, an amino phase (preferably a propyl amine phase), a cyano phase (preferably a cyanopropyl phase), a trimethylsilyl phase, a dimethylsilyl phase, a propyl phase, a butyl phase, a pentyl phase, a hexyl phase, a phenyl phase, a halogenated phase, or a nitro phase.
- the disclosed particle size is 5 ⁇ m, it will be understood by those of ordinary skill in the art that, particularly with respect to a diol stationary phase, any commercially available particle size may be used for the separation.
- the commercially available diol particle sizes include 3, 5 and 10 ⁇ m.
- the binary mobile phase may be a combination of any polar aprotic solvent (A phase) followed by any polar protic solvent (B phase).
- Suitable polar aprotic solvents for the (A) phase include, in addition to acetonitrile, acetone, cyclohexanone, methyl ethyl ketone, methyl tert-butyl ether, diethyl ether, dimethyl ether, methyl acetate, ethyl acetate, and nitromethane.
- Suitable polar protic solvents for the (JB) phase include, in addition to methanol, ethanol, n-propanol, isopropanol, n-butanol and isobutanol. It will be understood that the proportion of polar aprotic solvent in the A phase and polar protic solvent in the B phase may be between 0 and 100% by volume, preferably is greater than 50% by volume, and even more preferably is greater than 90% by volume. The remainder of the A phase and B phase may be any mineral or organic acid (e.g., acetic acid) and/or water.
- acetic acid e.g., acetic acid
- the improved processes and system of the present invention for separating polar protic oligomers based on degree of polymerization on an analytical scale and for separating and isolating the oligomers on a preparative scale provide several advantages over existing approaches.
- the polar bonded stationary chromatographic phase is more robust than silica, and can tolerate a wider range of solvents.
- the long term adsorption properties of silica are compromised in the presence of water, whereas with the polar bonded stationary chromatographic phase, such as a diol or glycerol stationary phase, this is not a concern.
- the diol phase When directly compared to the silica phase, the diol phase was shown to reduce or eliminate surface adsorption, thereby providing for increased speciation of individual oligomers, including especially oligomers with a DP of at least 10.
- a binary mobile phase is used, rather than the typical tertiary or quaternary phases, thereby making the process readily adaptable to researchers lacking sophisticated quaternary HPLC pumps.
- the solvents used in the disclosed process are less dangerous and more environmentally friendly than those currently employed, vis., the carcinogen/mutagen methylene chloride and the CNS depressant tetrahydrofuran (THF).
- THF tetrahydrofuran
- Figure 8 shows an LC trace generated by the preparative HPLC system. Peak 1 is theobromine, peak 2 is caffeine, peak three is the monomer (flavanol) and peak 4 is oligomers with DP>1. Holding initial mobile phase conditions for 15 minutes (i.e., 0% B phase, 100% A phase), theobromine, caffeine and monomer were eluted first. Adjusting the mobile phase to high polarity allowed for quick recovery of CP oligomers (DP>1).
- Trace (a) is the fluorescence detection of the CP extract.
- Trace (b) is the UV detection of the same material. It is known that xanthines do not fluoresce substantially and CPs do not display strong signals under UV detection. Using both detection modes thus gives a more comprehensive picture.
- Trace (c) shows the UV trace of the xanthines removed from the CP extract. Although the analysis of individual fractions of theobromine and caffeine are not displayed, they can be separated and physically isolated. Additionally, the monomer can be separated and isolated from the xanthines. See trace (d).
- Trace (e) shows the UV trace of the CP extract after removal of the monomer and xanthines. As is evident, the sample contains only oligomers of DP >2.
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EP06825350A EP1945326A1 (en) | 2005-10-03 | 2006-10-03 | Improved process for analyzing, for separating, and for isolating individual polar protic monomers and/or oligomers |
CA002623593A CA2623593A1 (en) | 2005-10-03 | 2006-10-03 | Improved process for analyzing, for separating, and for isolating individual polar protic monomers and/or oligomers |
AU2006299573A AU2006299573A1 (en) | 2005-10-03 | 2006-10-03 | Improved process for analyzing, for separating, and for isolating individual polar protic monomers and/or oligomers |
JP2008534601A JP2009510476A (en) | 2005-10-03 | 2006-10-03 | Improved method for analyzing, separating and isolating individual polar protic monomers and / or oligomers |
IL190467A IL190467A0 (en) | 2005-10-03 | 2008-03-27 | Improved process for analyzing, for separating, and for isolating individual polar protic monomers and/or oligomers |
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US11/242,210 US7566401B2 (en) | 2005-10-03 | 2005-10-03 | Process for separating and isolating xanthines, individual polar protic monomers, and polar protic oligomers |
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EP (1) | EP1945326A1 (en) |
JP (1) | JP2009510476A (en) |
CN (1) | CN101355995A (en) |
AU (1) | AU2006299573A1 (en) |
CA (1) | CA2623593A1 (en) |
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Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
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US7566401B2 (en) * | 2005-10-03 | 2009-07-28 | Kelm Mark A | Process for separating and isolating xanthines, individual polar protic monomers, and polar protic oligomers |
US8568798B2 (en) | 2009-04-03 | 2013-10-29 | Dianaplantsciences, S.A.S. | Production and extraction of procyanidins from plant cell cultures |
BRPI0904246A2 (en) * | 2009-08-14 | 2015-06-02 | Univ Minas Gerais | Extractive-analytical method for determination of tannins in plant inputs and / or products |
US8932468B2 (en) * | 2010-01-12 | 2015-01-13 | The United States Of America As Represented By The Secretary Of The Army | Separation of enzymatically synthesized polyepicatechin via high performance liquid chromatography |
US8431176B2 (en) * | 2010-03-08 | 2013-04-30 | The United States Of America As Represented By The Secretary Of The Army | Liquid chromatographic fraction of enzymatically polymerized flavonoid as an antioxidant |
CN103237457A (en) | 2010-10-04 | 2013-08-07 | 戴安娜植物科学有限公司 | Production and extraction of procyanidins from plant cells cultures |
CN103175904B (en) * | 2011-12-20 | 2015-07-08 | 天津天狮生物发展有限公司 | Method for determining procyanidine in healthcare food |
US9506897B2 (en) | 2012-11-16 | 2016-11-29 | Agilent Technologies, Inc. | Methods and compositions for improved ion-exchange chromatography |
JP2017067677A (en) * | 2015-10-01 | 2017-04-06 | 住友ベークライト株式会社 | Method for preparing purified substance of label sugar |
TW201733605A (en) * | 2016-02-29 | 2017-10-01 | 明治股份有限公司 | Cocoa-derived water extract, beverage containing the same, method for producing cocoa extract, and method for extracting polyphenols |
CN111830147A (en) * | 2020-06-09 | 2020-10-27 | 贵州茅台酒股份有限公司 | Method for detecting concentration of tannin in sorghum |
CN111999398B (en) * | 2020-07-09 | 2022-03-08 | 武汉迈特维尔生物科技有限公司 | Liquid chromatography-mass spectrometry analysis method for caffeine and metabolites thereof |
CN111830168B (en) * | 2020-07-23 | 2022-07-22 | 吉林医药学院 | LC-HR-MS/MS quantitative analysis method of poloxamer |
CN113740447A (en) * | 2021-07-05 | 2021-12-03 | 贵州茅台酒股份有限公司 | Identification and analysis method of key color generation substances of sorghum seed coats |
Citations (2)
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US5670673A (en) * | 1990-11-02 | 1997-09-23 | University Of Florida | Method for the isolation and purification of taxol and its natural analogues |
US20050065341A1 (en) * | 2003-08-25 | 2005-03-24 | Guoquan Wang | Substituted 8-heteroaryl xanthines |
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US6210679B1 (en) | 1999-01-07 | 2001-04-03 | Hauser, Inc. | Method for isolation of caffeine-free catechins from green tea |
US6627232B1 (en) | 2000-06-09 | 2003-09-30 | Mars Incorporated | Method for extracting cocoa procyanidins |
US20030100082A1 (en) | 2001-10-31 | 2003-05-29 | Mary Ann Lila | Methods for isolation of proanthocyanidins from flavonoid-producing cell culture |
WO2003080852A1 (en) | 2002-03-26 | 2003-10-02 | Lek Pharmaceutical And Chemical Company D.D. | Process for the preparation of a desired erythropoietin glyco-isoform profile |
US20050006534A1 (en) * | 2002-12-20 | 2005-01-13 | D. Kay Shillings | Organizer for medical tubes and cables |
WO2005042569A1 (en) | 2003-10-24 | 2005-05-12 | Amgen, Inc. | Process for purifying proteins in a hydrophobic interaction chromatography flow-through fraction |
US7485735B2 (en) * | 2005-10-03 | 2009-02-03 | Mars, Inc. | Process for selectively extracting procyanidins |
US7566401B2 (en) * | 2005-10-03 | 2009-07-28 | Kelm Mark A | Process for separating and isolating xanthines, individual polar protic monomers, and polar protic oligomers |
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- 2006-10-03 AU AU2006299573A patent/AU2006299573A1/en not_active Abandoned
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Patent Citations (2)
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US5670673A (en) * | 1990-11-02 | 1997-09-23 | University Of Florida | Method for the isolation and purification of taxol and its natural analogues |
US20050065341A1 (en) * | 2003-08-25 | 2005-03-24 | Guoquan Wang | Substituted 8-heteroaryl xanthines |
Non-Patent Citations (1)
Title |
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GU L. ET AL.: "Fractionation of Polymer Procyanidins from Lowbush Blueberry and Quantification of Procyanidins in Selected Foods with an Optimized Normal-Phase HPLC-MS Fluorescent Detection Method", J. AGRIC. FOOD CHEM., vol. 50, 2002, pages 4852 - 4860, XP003011367 * |
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Publication number | Publication date |
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US20100016536A1 (en) | 2010-01-21 |
CA2623593A1 (en) | 2007-04-12 |
CN101355995A (en) | 2009-01-28 |
IL190467A0 (en) | 2008-11-03 |
AU2006299573A1 (en) | 2007-04-12 |
WO2007041448A1 (en) | 2007-04-12 |
US7566401B2 (en) | 2009-07-28 |
JP2009510476A (en) | 2009-03-12 |
US20070075020A1 (en) | 2007-04-05 |
EP1945326A1 (en) | 2008-07-23 |
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