CN114950391A - Modified silica gel stationary phase, hydrophilic chromatographic column, and preparation method and application thereof - Google Patents
Modified silica gel stationary phase, hydrophilic chromatographic column, and preparation method and application thereof Download PDFInfo
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- CN114950391A CN114950391A CN202210523980.8A CN202210523980A CN114950391A CN 114950391 A CN114950391 A CN 114950391A CN 202210523980 A CN202210523980 A CN 202210523980A CN 114950391 A CN114950391 A CN 114950391A
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- silica gel
- stationary phase
- modified silica
- thioglycerol
- epoxy
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 title claims abstract description 75
- 230000005526 G1 to G0 transition Effects 0.000 title claims abstract description 45
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000000741 silica gel Substances 0.000 claims abstract description 33
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 33
- 238000000926 separation method Methods 0.000 claims abstract description 20
- 229920001542 oligosaccharide Polymers 0.000 claims abstract description 19
- -1 thioglycerol modified silica gel Chemical class 0.000 claims abstract description 19
- 239000004593 Epoxy Substances 0.000 claims abstract description 18
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 150000002482 oligosaccharides Chemical class 0.000 claims abstract description 11
- 229940035024 thioglycerol Drugs 0.000 claims abstract description 10
- BPSIOYPQMFLKFR-UHFFFAOYSA-N trimethoxy-[3-(oxiran-2-ylmethoxy)propyl]silane Chemical compound CO[Si](OC)(OC)CCCOCC1CO1 BPSIOYPQMFLKFR-UHFFFAOYSA-N 0.000 claims abstract description 7
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 239000005715 Fructose Substances 0.000 claims description 8
- 229930091371 Fructose Natural products 0.000 claims description 8
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 8
- LKDRXBCSQODPBY-AMVSKUEXSA-N L-(-)-Sorbose Chemical compound OCC1(O)OC[C@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-AMVSKUEXSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 8
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 7
- QWIZNVHXZXRPDR-UHFFFAOYSA-N D-melezitose Natural products O1C(CO)C(O)C(O)C(O)C1OC1C(O)C(CO)OC1(CO)OC1OC(CO)C(O)C(O)C1O QWIZNVHXZXRPDR-UHFFFAOYSA-N 0.000 claims description 7
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 7
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 claims description 7
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 7
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 7
- QWIZNVHXZXRPDR-WSCXOGSTSA-N melezitose Chemical compound O([C@@]1(O[C@@H]([C@H]([C@@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O)CO)CO)[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O QWIZNVHXZXRPDR-WSCXOGSTSA-N 0.000 claims description 7
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 6
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 6
- 229930195725 Mannitol Natural products 0.000 claims description 6
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 6
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 6
- 239000000832 lactitol Substances 0.000 claims description 6
- 235000010448 lactitol Nutrition 0.000 claims description 6
- VQHSOMBJVWLPSR-JVCRWLNRSA-N lactitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-JVCRWLNRSA-N 0.000 claims description 6
- 229960003451 lactitol Drugs 0.000 claims description 6
- 239000000845 maltitol Substances 0.000 claims description 6
- VQHSOMBJVWLPSR-WUJBLJFYSA-N maltitol Chemical compound OC[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VQHSOMBJVWLPSR-WUJBLJFYSA-N 0.000 claims description 6
- 235000010449 maltitol Nutrition 0.000 claims description 6
- 229940035436 maltitol Drugs 0.000 claims description 6
- 239000000594 mannitol Substances 0.000 claims description 6
- 235000010355 mannitol Nutrition 0.000 claims description 6
- 239000000337 buffer salt Substances 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 claims description 4
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 4
- 239000012159 carrier gas Substances 0.000 claims description 4
- 238000010828 elution Methods 0.000 claims description 4
- 229960002160 maltose Drugs 0.000 claims description 4
- 238000010907 mechanical stirring Methods 0.000 claims description 4
- 229960004793 sucrose Drugs 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 3
- 238000000105 evaporative light scattering detection Methods 0.000 claims description 3
- 229960001855 mannitol Drugs 0.000 claims description 3
- 239000012299 nitrogen atmosphere Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000003054 catalyst Substances 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 7
- 239000000463 material Substances 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 17
- 235000013361 beverage Nutrition 0.000 description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 239000004005 microsphere Substances 0.000 description 10
- 238000004587 chromatography analysis Methods 0.000 description 8
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 239000000543 intermediate Substances 0.000 description 5
- 238000011084 recovery Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2,2'-azo-bis-isobutyronitrile Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- HQYALQRYBUJWDH-UHFFFAOYSA-N trimethoxy(propyl)silane Chemical compound CCC[Si](OC)(OC)OC HQYALQRYBUJWDH-UHFFFAOYSA-N 0.000 description 3
- OZAIFHULBGXAKX-VAWYXSNFSA-N AIBN Substances N#CC(C)(C)\N=N\C(C)(C)C#N OZAIFHULBGXAKX-VAWYXSNFSA-N 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000005452 bending Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 238000012650 click reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- GQIUQDDJKHLHTB-UHFFFAOYSA-N trichloro(ethenyl)silane Chemical compound Cl[Si](Cl)(Cl)C=C GQIUQDDJKHLHTB-UHFFFAOYSA-N 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 229920002554 vinyl polymer Polymers 0.000 description 2
- 239000005050 vinyl trichlorosilane Substances 0.000 description 2
- OXBLVCZKDOZZOJ-UHFFFAOYSA-N 2,3-Dihydrothiophene Chemical compound C1CC=CS1 OXBLVCZKDOZZOJ-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 101000860173 Myxococcus xanthus C-factor Proteins 0.000 description 1
- 229910002808 Si–O–Si Inorganic materials 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000002329 infrared spectrum Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
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- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000696 nitrogen adsorption--desorption isotherm Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000007142 ring opening reaction Methods 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 150000004756 silanes Chemical class 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000002336 sorption--desorption measurement Methods 0.000 description 1
- 238000012421 spiking Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Images
Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
Abstract
The invention provides a modified silica gel stationary phase, a hydrophilic chromatographic column, and a preparation method and application thereof. The preparation process of the modified silica gel stationary phase comprises the following steps: 3- (2, 3-epoxypropoxy) propyl trimethoxy silane reacts with silica gel to obtain epoxy silica gel, and then reacts with 1-thioglycerol to obtain thioglycerol modified silica gel. The thioglycerol modified silica gel material can be used as a hydrophilic chromatographic stationary phase and is used for good separation and accurate detection of oligosaccharide substances.
Description
Technical Field
The invention relates to the technical field of chromatographic separation, in particular to a modified silica gel stationary phase, a hydrophilic chromatographic column, and a preparation method and application thereof.
Background
Oligosaccharide compounds are commonly used additives in commercial beverages, and the detection of the content of the oligosaccharide compounds is crucial to the quality monitoring of the beverages. The oligosaccharide structure is rich in hydroxyl groups, has high polarity, and is suitable for separation and analysis by hydrophilic chromatography. However, conventional commercial hydrophilic chromatography columns cannot achieve good separation and accurate quantification of tens of oligosaccharide compounds simultaneously.
Disclosure of Invention
Based on the above, there is a need for a novel modified silica gel stationary phase, a hydrophilic chromatographic column, and a preparation method and application thereof, which can realize simultaneous separation and accurate quantification of dozens of oligosaccharides, thereby providing a new method for quality monitoring of sugar-containing or sugar-free beverages.
The invention adopts the following technical scheme:
the invention provides a modified silica gel stationary phase, which has the structural formula as follows:
the preparation method of the modified silica gel stationary phase provided by the invention comprises the following steps: reacting 3- (2, 3-epoxypropoxy) propyl trimethoxy silane with silica gel to obtain epoxy silica gel; 1-thioglycerol is adopted to react with epoxy silica gel to obtain the thioglycerol modified silica gel.
In some embodiments, the preparation process route of the epoxy modified silica gel is as follows: dispersing the activated silica gel in toluene containing 3- (2, 3-epoxypropoxy) propyl trimethoxy silane, performing reflux reaction for 6-10 h at 100-120 ℃ under the atmosphere of nitrogen and mechanical stirring, cooling, washing with toluene and methanol in sequence, and drying to obtain the silica gel. Preferably, the amount ratio of the activated silica gel to 3- (2, 3-glycidoxy) propyltrimethoxysilane is 2g:1 mL.
In some of these embodiments, the process for reacting 1-thioglycerol with epoxy-based silica gel is: and adding the epoxy modified silica gel into 1, 4-dioxane containing 1-thioglycerol and triethylamine, and stirring and reacting for 3-5 h at 125-140 ℃ to obtain the epoxy modified silica gel. Preferably, the amount ratio of the activated silica gel to 1-thioglycerol is 2g:1 mL.
The invention also provides application of the modified silica gel stationary phase in separation and detection of oligosaccharide substances.
The invention also provides a hydrophilic chromatographic column which is filled by adopting the modified silica gel stationary phase.
The invention also provides a method for separating oligosaccharide substances by using the modified silica gel stationary phase, which adopts a chromatographic column filled with the modified silica gel stationary phase and adopts acetonitrile (A) to NH (B) 4 Gradient elution with OAc buffer salt (concentration 5-25 mM, pH 4.0-7.5) as mobile phase (elution procedure: 0-8 min, 93% A; 8-10min, 93% A-85% A; 10-40 min, 85% A), flow rate of 0.2mL/min, column temperature 25-35 deg.C, evaporation light scattering detector drift tube temperature 85 deg.C, carrier gas (nitrogen) flow rate of 2.3mL/min as chromatographic conditionsAnd (5) carrying out separation detection.
In some of these embodiments, the saccharide substance comprises one or more of ribose, xylose, fructose, glucose, sorbose, mannitol, sucrose, maltose, maltitol, lactitol, melezitose, raffinose.
The invention has the beneficial effects that:
compared with the prior art, the novel modified silica gel stationary phase is provided for the first time, and can be prepared by reacting 3- (2, 3-epoxypropoxy) propyl trimethoxy silane with silica gel to obtain epoxy silica gel and then reversely preparing 1-thioglycerol and the epoxy silica gel. The novel modified silica gel stationary phase and the hydrophilic chromatographic column filled with the same can realize complete separation and accurate detection of 12 oligosaccharide substances simultaneously.
Drawings
FIG. 1 is a schematic diagram of a preparation process of a modified silica gel stationary phase in test example 1; (a) route for preparation of SP1 stationary phase, (b) route for preparation of SP2 stationary phase.
FIG. 2 shows the results of characterization of the raw materials, intermediates and products in the preparation of the modified silica gel stationary phase in test example 1; wherein, (a) scanning electron microscope images (interpolation: local amplification) of the silica gel microspheres, (b) nitrogen adsorption/desorption isotherms and pore size distribution curves of the silica gel microspheres, (c) infrared spectrograms of SP1, SP2 and intermediates thereof, and (d) thermogravimetric curves of SP1 and SP 2.
FIG. 3 is a chromatogram for measuring various sugar compounds in test example 2; wherein 1 represents ribose (ribose), 2 represents xylose (xylose), 3 represents fructose (fructose), 4 represents glucose (glucose), 5 represents sorbose (sorbose), 6 represents mannitol (mannitol), 7 represents sucrose (sucrose), 8 represents maltose (maltose), 9 represents maltitol (maltose), 10 represents lactitol (lactitol), 11 represents melezitose (melezitose), and 12 represents raffinose (raffinose).
FIG. 4 is a chromatogram obtained in test example 3; (a) NH (NH) 4 The concentration of OAc buffer salts; (b) pH of NH4OAc buffer salt; (c) column temperature; the chromatographic conditions and peak assignments were the same as in figure 3, except for the variables.
FIG. 5 shows a test knot obtained in test example 4Fruit; batch-to-batch reproducibility (a) and retention of Column2 relative to mobile phase flush volume (b) and Column stability (c); chromatographic conditions are as follows: (a) same as in fig. 3; (b) and (c) 93% ACN-7% NH 4 OAc (10mM, pH 6.8); the flow rate is 0.2 mL/min; the column temperature is 30 ℃; the sample injection volume is 5 mu L; the evaporative light scattering detector drift tube temperature was 85 ℃ and the carrier gas (nitrogen) flow rate was 2.3 mL/min.
FIG. 6 is a representative chromatogram obtained in test example 5, the chromatographic conditions and peak assignment were the same as those in FIG. 3.
Detailed Description
The present invention is further described in detail below with reference to specific examples so that those skilled in the art can more clearly understand the present invention.
The following examples are provided only for illustrating the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by a person skilled in the art based on the specific embodiments of the present invention without any inventive step are within the scope of the present invention.
In the examples of the present invention, all the raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present invention, unless otherwise specified, all technical means used are conventional means well known to those skilled in the art.
Description of the reagents used in the experiments of the invention:
analytical grade hydrochloric acid (HCl), toluene, methanol, ethanol, 1, 4-dioxane, 2' -Azobisisobutyronitrile (AIBN), Triethylamine (TEA), ammonium acetate (NH) 4 OAc) was purchased from national chemical agents (shanghai) ltd. 1-thioglycerol and vinyltrichlorosilane are provided by Chishiai (Shanghai) chemical industry development Co., Ltd., and Bailingwei science and technology Co., Ltd., Beijing, respectively. 3- (2, 3-glycidoxy) propyltrimethoxysilane was produced by silicon New materials Co., Ltd, university of Wuhan. Ribose, xylose, fructose, glucose, sorbose, mannitol, sucrose, maltose, maltitol, lactitol, melezitose, raffinose were purchased from Allantin reagent, Inc. (Shanghai). Chromatographically pure Acetonitrile (ACN) was purchased from Sigma-Aldrich (usa).
Silica gel (S)iO 2 ) Microsphere: self-made, reference documents are: zhang SQ, Li J, Li L, Yuan X, Xu L, Shi ZG. fast section of water-soluble vitamins by hydrophilic interaction based on submicron meter flow-through silica microscopics. food Chem 2020,307: 125531.
The 12 kinds of oligose are compounded into stock solution in 20mg/mL concentration in pure water and stored at 4 deg.c.
Activating silica gel: and (3) placing the self-made silica gel microspheres under 6mol/L HCl for refluxing for 12h, then washing with water and methanol in sequence, and drying to obtain the activated silica gel.
Experimental example 1 provides 2 modified silica gel stationary phases SP1 and SP2 which can be packed to form a hydrophilic chromatographic column
(a) The preparation route of SP1 refers to Wu J, Bicker W, Lindner W.separation properties of novel and commercial polar phases in the hydraulic interaction and reversed-phase liquid chromatography mode J Sep Sci 2008,31(9):1492 + 1503, which comprises the following steps:
as shown in FIG. 1a, 3g of silica gel microspheres were ultrasonically dispersed in 30mL of toluene containing 1.5mL of vinyltrichlorosilane under a nitrogen atmosphere in a three-necked flask, heated to 110 ℃ under a nitrogen atmosphere and mechanically stirred, and reacted under reflux for 8 h. After the reaction is finished, the product is washed by toluene and methanol in sequence to obtain vinyl silica gel. The product vinyl silica gel was added to 30mL of an ethanol solution containing 1.5mL of 1-thioglycerol and 30mg of AIBN, and reacted in a 70 ℃ water bath for 12 hours with mechanical stirring. The product was washed with ethanol and dried. Thus, the thioglycerol modified silica gel stationary phase 1, abbreviated as SP1, is obtained through a thiol-ene click reaction.
(b) The preparation route of SP2 is as follows:
as shown in FIG. 1b, 3g of silica gel microspheres were ultrasonically dispersed in 30mL of toluene containing 1.5mL3- (2, 3-glycidoxy) propyltrimethoxysilane under nitrogen in a three-necked flask, heated to 110 ℃ under nitrogen and mechanically stirred and reacted at reflux for 8 h. And after the reaction is finished, sequentially washing the product by using toluene and methanol to obtain the epoxy silica gel. Epoxy silica gel was added to 30mL of 1, 4-dioxane containing 1.5mL of 1-thioglycerol and 0.45mL of triethylamine, and the mixture was refluxed at 130 ℃ for 4 hours with mechanical stirring. The product was washed with ethanol and dried. Thus, the thioglycerol modified silica gel stationary phase 2, abbreviated as SP2, is obtained through a mercapto-epoxy click reaction.
The characterization results of silica gel microspheres and SP1, SP2 or intermediates are shown in FIG. 2.
As shown in FIG. 2a, the SEM image of the silica gel microspheres shows that the particle size distribution is relatively uniform, about 3-5 μm.
The nitrogen adsorption-desorption isotherm and the pore size distribution curve of the silica gel microspheres are shown in FIG. 2b, and the specific surface area of the silica gel microspheres is calculated to be 226m based on a Brunauer-Emmett-Teller model 2 The concentration of mesopores was calculated by the Barrett-Joyner-Halenda method to be around 7.4 nm.
FIG. 2c is an infrared spectrum of SP1, SP2, and intermediates thereof. 3450cm -1 The broad peak is from the stretching vibration of O-H, 1100cm -1 Super absorption of (C) and 805cm -1 The intermediate signal at (b) belongs to the stretching vibration of Si-O-Si. 965cm -1 The weak absorption of (b) originates from the epoxy group and the signal disappears in SP2 due to the ring opening process. At 2800 to 2900cm -1 Continuous micro signals are derived from C-H stretching vibration, and the signals are obvious due to the relatively high content of the epoxy silica gel and the SP 2. And 1412cm -1 The small peak at (A) is the bending vibration of C-H, which is only visible in the vinyl-modified silane and SP 1. 1650cm -1 The C ═ C signal is masked by O-H bending vibration.
As shown in FIG. 2d, the thermal weight loss ratios of SP1 and SP2 were 6.2% and 9.1% respectively at 200-800 ℃. After conversion, the coverage of 1-thioglycerol on SP1 and SP2 was 500. mu. mmol/g (2.21. mu. mmol/m), respectively 2 ) And 459. mu. mmol/g (2.03. mu. mmol/m) 2 )。
The above characterization results demonstrate the successful preparation of two stationary phases SP1, SP 2.
Experimental example 2 chromatographic Properties for the Simultaneous isolation of various oligosaccharide Compounds
The thioglycerol-modified silica gel stationary phases SP1 and SP2 prepared in Experimental example 1 were uniformly dispersed in methanol, respectively, and loaded into a stainless steel hollow column tube (250 m. times.2.1 mm id) at 7000psi using methanol as a displacement solvent to obtain a chromatography column. The Column packed with the stationary phase SP1 was named Column1 and the Column packed with the stationary phase SP2 was named Column 2.
In the hydrophilic chromatography mode, 12 kinds of oligosaccharide compounds including 6 kinds of monosaccharides (ribose, xylose, fructose, glucose, sorbitol, mannitol), 4 kinds of disaccharides (sucrose, maltose, maltitol, lactitol) and 2 kinds of trisaccharides (melezitose, raffinose) were separated using chromatography columns, Column1 and Column2, respectively, as analytes, and the separation performance thereof was evaluated.
The 12 kinds of oligose are compounded into stock solution in 20mg/mL concentration in pure water and stored at 4 deg.c. Before injection, the sample is diluted to an appropriate concentration by an initial mobile phase, and the injection volume is 5 mu L.
The chromatographic conditions are as follows: the mobile phase is ACN (A) to 10mM and NH with the pH value of 6.8 4 OAc buffer (B) flow rate 0.2 mL/min.
Gradient program:
Column1：0～6min，95％A；6～8min，95％A～90％A；8～25min，90％A。
Column2：0～8min，93％A；8～10min，93％A～85％A；10～40min，85％A。
the evaporative light scattering detector drift tube temperature was set at 85 ℃ and the carrier gas (nitrogen) flow rate was 2.3 mL/min.
The separation results are shown in FIG. 3. The proportion of ACN in the mobile phase of Column1 was generally higher than that of Column2, with the initial proportion being even as high as 95%, which has approached the upper limit of the hydrophilic chromatography, and should have a strong retention probability, but the experimental results show that the first four pairs of analytes could not be completely separated, in particular melezitose and raffinose, with their corresponding peaks completely coinciding. In Column2, 12 oligosaccharides were completely separated, and the degree of separation of glucose and sorbose, which was the closest, was 1.51, and thus it was concluded that Column2 packed with SP2 had better hydrophilic chromatographic separation performance.
Test example 3 examination of the influence of buffer salt concentration, pH, and column temperature on the separation Effect
Referring to test example 2, this experiment further investigated the effect of varying chromatographic conditions on the separation of oligosaccharide compounds when SP2 was used as a hydrophilic chromatographic stationary phase, and the results are shown statistically in FIG. 4.
As can be seen from FIG. 4, at NH 4 Under the conditions that the concentration of the OAc buffer solution is 5-25 mM, the pH value is 4.0-7.5 and the Column temperature is 25-35 ℃, 12 oligosaccharide compounds are kept and slightly changed, but good separation can be obtained, which shows that the small-range adjustment of chromatographic conditions hardly affects the separation of the oligosaccharide compounds on Column2 (using SP2 as a stationary phase), and shows that the application range of the chromatographic stationary phase SP2 is wide.
Test example 4 batch-to-batch reproducibility and column stability investigation
The batch reproducibility under the same synthetic route was investigated with reference to the chromatographic detection conditions of test example 2, and 3 batches of the synthesized thioglycerol-modified silica gel stationary phase SP2 were respectively used to fill the chromatographic columns and were named Column2-1, Column2-2 and Column 2-3.
The results are shown in FIG. 5 a. It can be seen that the elution order and retention of each analyte is relatively stable, with RSD% of each analyte retention factor ranging from 1.42% to 3.33%. The degrees of separation of glucose and sorbose in Column2-1, Column2-2 and Column2-3 were 1.51, 1.67 and 1.57, respectively. RSD% < 6% for the degree of separation and RSD% < 10% for the theoretical plate number were within acceptable ranges.
Passing through mobile phase 93% ACN-7% NH 4 After continuous washing of OAc (10mM, pH 6.8), the retention factors and theoretical plate numbers of the 4 monosaccharides on Column2 (using SP2 as the stationary phase) were recorded to evaluate Column stability.
As shown in FIGS. 5b and 5c, the RSD% of retention factors of xylose, fructose, glucose and sorbose were 1.18%, 1.90%, 2.03% and 2.80%, respectively, and the RSD% of theoretical plate number was 5.44%, 5.73%, 5.86% and 4.42%, respectively, with a small and reasonable fluctuation range.
Test results show that Column2 filled with the thioglycerol modified silica gel stationary phase SP2 has good batch-to-batch reproducibility and stability.
Test example 5 quantitative analysis of sugar in beverage
The experiment establishes a low-sugar beverage quality monitoring method, which is essentially to develop a quantitative detection method for oligosaccharide substances in the beverage by utilizing Column2 (filled with a thioglycerol modified silica gel stationary phase SP2), carry out methodology verification under the chromatographic condition of the experimental example 2, then respectively obtain 13 commercial beverages, carry out filtration, ultrasonic exhaust and proper dilution pretreatment, and carry out actual sample detection under the same test conditions of the experimental example 2. The chromatograms of 3 representative beverages are shown in figure 6.
The linearity, detection Limit (LOD) and quantitation Limit (LOQ) statistics are given in the following table:
it can be seen that the peak areas are well linear with the concentration of each sugar, and the relative numbers of each analyte in the corresponding linear range are not lower than 0.9921. The detection Limit (LOD) and the quantification Limit (LOQ) are respectively set at 5-50 mu g/mL based on the signal-to-noise ratio (S/N) of 3 and 10 -1 And 10 to 100. mu.g/mL -1 Within the range, meets the beverage detection requirements.
The spiking recovery test was performed over 3 days, with 12 sugar compounds added at 3 concentration levels in beverage 7 (pulsed) to assess matrix interference. The results are shown in the following table:
the recovery rate of each oligosaccharide is 89.54% -113.15%. The recovery rate RSD% of three repeated tests at each concentration level is less than 10%, the recovery rate of three concentration levels is the result of continuous three-day measurement, and the RSD% is also less than 10%, which indicates that the intra-day precision and the inter-day precision are good.
The results of the 13 commercial beverage tests are shown in the following table:
as can be seen from the statistical results, fructose, glucose and sucrose are common sugar additives in commercial beverages, and have wide sources and low cost. Maltitol, a representative sugar alcohol, has gradually replaced the conventional sugar to be used in diet drinks. Except the beverage 1, the detection content of all beverages is consistent with the content of the labels, and the accuracy advantage of the method for detecting the oligosaccharide content in the beverages by using the hydrophilic chromatography with the thioglycerol modified silica gel SP2 as a stationary phase is further verified.
The methodological verification and the actual sample detection result show that the hydrophilic chromatography taking the thioglycerol modified silica gel SP2 as the stationary phase is used for separating and detecting oligosaccharide compounds in the beverage, and has the advantages of complete separation, high accuracy, good precision, good reproducibility and strong matrix interference resistance.
It should be noted that the above examples are only for further illustration and description of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment, and is not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The modified silica gel stationary phase is characterized in that the structural formula of the modified silica gel stationary phase is as follows:
2. a method for preparing a modified silica gel stationary phase according to claim 1, comprising the steps of:
reacting 3- (2, 3-epoxypropoxy) propyl trimethoxy silane with silica gel to obtain epoxy silica gel;
1-thioglycerol is adopted to react with epoxy silica gel to obtain the thioglycerol modified silica gel.
3. The method for preparing the modified silica gel stationary phase according to claim 2, wherein the preparation process route of the epoxy-based silica gel is as follows: dispersing the activated silica gel in a toluene solution containing 3- (2, 3-epoxypropoxy) propyl trimethoxy silane, performing reflux reaction for 6-10 h at 100-120 ℃ under the condition of nitrogen atmosphere and mechanical stirring, cooling, washing with toluene and methanol in sequence, and drying to obtain the silica gel.
4. The method for preparing modified silica gel stationary phase according to claim 3, wherein the amount ratio of activated silica gel to 3- (2, 3-epoxypropoxy) propyltrimethoxysilane is 2g:1 mL.
5. The method for preparing the modified silica gel stationary phase according to any one of claims 2 to 4, wherein the process for reacting the 1-thioglycerol with the epoxy silica gel comprises the following steps: and adding the epoxy silica gel into 1, 4-dioxane containing 1-thioglycerol and triethylamine, and stirring and reacting for 3-5 h at 125-140 ℃ to obtain the catalyst.
6. The method for preparing a modified silica gel stationary phase according to claim 5, wherein the amount ratio of the activated silica gel to 1-thioglycerol is 2g:1 mL.
7. Use of the modified silica gel stationary phase of claim 1 for separation and detection of oligomeric species.
8. A hydrophilic chromatographic column packed with the modified silica gel stationary phase of claim 1.
9. A method of using the modified silica gel of claim 1 to immobilize and separate oligomeric species, characterized in thatA chromatographic column packed with the modified silica gel stationary phase according to claim 1, wherein the chromatographic column is packed with (A) acetonitrile to (B) NH having a concentration of 5 to 25mM and a pH of 4.0 to 7.5 4 And performing gradient elution by using OAc buffer salt as a mobile phase, wherein the gradient elution program is as follows: 0-8 min, 93% A; 8-10min, 93-85% of A; 10-40 min, 85% A; and performing separation detection by using the conditions of a flow rate of 0.2mL/min, a column temperature of 25-35 ℃, an evaporative light scattering detector drift tube temperature of 85 ℃ and a carrier gas flow rate of 2.3mL/min as chromatographic conditions.
10. The method of claim 9, wherein the oligosaccharide species comprises one or more of ribose, xylose, fructose, glucose, sorbose, mannitol, sucrose, maltose, maltitol, lactitol, melezitose, raffinose.
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