CN104841405A - Supercritical/sub-critical fluid blocking method of HPLC (High Performance Liquid Chromatography) reversed phase bonded stationary phase - Google Patents
Supercritical/sub-critical fluid blocking method of HPLC (High Performance Liquid Chromatography) reversed phase bonded stationary phase Download PDFInfo
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Abstract
The invention discloses a supercritical/sub-critical water blocking method of HPLC (High Performance Liquid Chromatography) reversed phase bonded stationary phase, which comprises the steps of using carbon dioxide supercritical fluid and/or carbon dioxide sub-critical fluid as a carrier, bring a blocking reagent and a catalyst into a supercritical/sub-critical fluid reactor loaded with silica gel reversed phase bonded stationary phase, and carrying out blocking reaction under a closed condition. According to the method, residual active silanol groups can be thoroughly closed, and lyophobic reversed phase bonded stationary phase with complete neutral charge is produced; no residual active silanol groups exchanges side effects with alkali compound cations, so that alkali compound has a very symmetrical peak shape, and the retention time and mechanism fully follow the order from low to high of hydrophobicity of the compound. The method has the advantages that the reaction condition is mild, the reversed phase bonded stationary phase has no damage, the life of a chromatographic column is long, the repeatability of production batches is very good, the use amount of a toxic organic solvent, such as methylbenzene, is minimized, the pollution to the environment is reduced to the greatest extent, and the process continuity and semi-automatization can be realized.
Description
Technical field
What the present invention relates to the inverse bonded phase of a kind of high performance liquid chromatography surpasses/subcritical fluids end blocking method.
Background technology
Take C18 as the heart that the efficient reversed-phase liquid chromatography post of representative is described to drug discovery, exploitation, method validation (validation) always, efficient reversed-phase liquid chromatography post is also extremely widely used in the field such as drug metabolism and dynamics, life science, medical treatment & health, bioanalysis detection, drugs and drug-testing, Food Safety Analysis, environmental analysis, military affairs, Homeland Security.Efficient reversed-phase liquid chromatography preparative column is also one of most important separating and purifying technology.
The exploitation of routine HPLC methods almost always from C18 as starting point, reverse-phase chromatography account for the application of more than 80%.No matter be past, present or foreseeable future, occupy dominant position in actual applications with the efficient reversed-phase liquid chromatography post that spherical Type B silica gel is material skeleton always.
Recent decades in past, countless effort is focused in the quality improving silica gel, is optimized linkage chemistry and closed-end technology.
Before two more than ten years, use organosilicon material to replace inorganic silicon material and represent an epoch-making revolution, the spherical silica gel called after Type B spherical silica gel of production as initiation material production spherical silica gel.Inorganic A type silica gel content of beary metal is very high, and Silica Surface some positions severe acidification and chelating effect etc. cause many alkali compounds rate of recovery low.Spherical Type B silica gel content of beary metal is very low, and very large degree eliminates the A type inorganic silica gel surface problem such as some positions severe acidification and chelating effect.With Type B spherical silica gel synthesizing efficient liquid chromatography stuffing, performance liquid chromatographic column product quality is caused to have qualitative leap.
Before more than ten years, first Waters company introduces the super-pressure ultra high efficiency UPLC technology in market, use the UPLC Ultra Performance Liquid Chromatography post being less than 2 μm of Type B spherical silica gels and producing, and the UHPLC Ultra Performance Liquid Chromatography post that first Advanced Material company introduces 1.7 μm and the 2.6 μm nucleocapsid silica gel type spherical silica gel productions in market represents another epoch-making revolution.UPLC, UHPLC Ultra Performance Liquid Chromatography post post effect is the three to four-fold of the efficient reversed-phase liquid chromatography post of tradition 5 μm, therefore short 5cm length UPLC is used, UHPLC Ultra Performance Liquid Chromatography post often can arrive with the same effect of the performance liquid chromatographic column of 5 μm of 25cm length, but analysis time shortens three to four-fold, substantially increase analysis speed.
On linkage chemistry, recent decades in past, except optimizing traditional C18, C8, Phenyl linkage chemistry, the most important thing is that first Supelco company introduces polarity embedded (Polar embedded) Bonded Phase in market.Polarity embedded (Polar embedded) Bonded Phase is most important product in the efficient reversed-phase liquid chromatography of C18 " satellite group ", and being that C18 and C8 Bonded Phase is most important supplements.Polarity embedded (Polar embedded) Bonded Phase originates from Supelco ABZ.The bonding method of Supelco ABZ forms a C16 acid amides with aminopropyl Bonded Phase and long-chain carboxylic acid's condensation reaction.This Bonded Phase has reasonable peak shape to alkali compounds, but is clearly sacrifice acid compound and amphoteric compound peak shape is cost, because the NH of aminopropyl Bonded Phase
2the impossible complete reaction of functional group, Bonded Phase at acidic phs surface charge is just.It should be noted that, on market, some C18 use the bonding or capping reagent production that carry positive charge.Start from ABZ, Supelco is fully recognized that the side effect of above-mentioned strategy, and therefore first use γ-aminopropylsilane synthesis C16 amide silane, be then bonded on Type B spherical silica gel, Here it is, and Supelco is famous
amide.Owing to not having patent protection, other chromatogram manufacturers imitate enthusiastically, are finally formed with acid amides or acyl ester polarity representatively embedded (Polar embedded) Bonded Phase group.The selective uniqueness of polarity embedded (Polar embedded) Bonded Phase, some are difficult being separated and may embedded in polarity (Polar embedded) Bonded Phase being well solved on C18 with C8 Bonded Phase.Under the same conditions, the qualified embedded Bonded Phase of acid amides polarity has better peak shape than corresponding C18 Bonded Phase to alkali compounds, because acid amides and alkali compounds have the interaction of polar-polar (Charger transfer), the objectively acid silanol base effect of partly shielding effect.The qualified embedded Bonded Phase of acid amides polarity can use under 100% aqueous phase condition, and much more stable than so-called aqueous phase C18.
Although achieve these progress, Bonded Phase capping problem there is no good solution, is to perplex the maximum problem of chromatographic field always.Trials whole up to now only obtains limited success, and Bonded Phase end-blocking almost all uses solid-liquid backflow.In fact, regardless of which type of catalyst and capping reagent used, the end-blocking under solid-liquid backflow is a blind alley.With the different pore size of the same explained hereafter of same manufacturer and the Type B spherical silica gel of different-grain diameter, the C18 Bonded Phase produced with same linkage chemistry can embody this point.In general, macro porous silica gel C18 Bonded Phase acid silanol base effect (such as
c18) corresponding mesoporous silica gel is significantly less than
c18 Bonded Phase, mesoporous silica gel
c18 Bonded Phase acid silanol base effect is significantly less than corresponding aperture silica gel
c18 Bonded Phase.In addition, in general, same aperture Type B spherical silica gel, the C18 Bonded Phase produced with same linkage chemistry, 10 μm of particle diameter Bonded Phase acid silanol base effects are significantly less than 5 μm of particle diameter Bonded Phases.5 μm of particle diameter Bonded Phases are significantly less than again 3 μm of particle diameter Bonded Phases, and 3 μm of particle diameter Bonded Phase particle diameter Bonded Phases are significantly less than again 1.7 μm of particle diameter Bonded Phases.
Summary of the invention
Research finds, some apertures that capping reagent is difficult to be diffused into silica gel under solid-liquid reflux state are inner, therefore cannot close corresponding acid silanol base, is presented as that the hangover of many alkali compounds is serious in chromatogram.Spherical silica gel is polymerized by Nano particles of silicon dioxide.Small particle diameter silica gel hole is significantly less than Large stone silica gel, and some apertures that capping reagent is difficult to be diffused into silica gel under solid-liquid reflux state are inner, therefore cannot close corresponding acid silanol base.
In order to solve the problems of the technologies described above, what the invention provides the inverse bonded phase of a kind of high performance liquid chromatography surpasses/subcritical fluids end blocking method, at utmost eliminating the acid effect of silanol base.From the viewpoint of basic science, supercritical fluid end-blocking or subcritical fluids end blocking method should be the closed-end technologies of final version, because under supercritical fluid or subcritical fluids state, diffusion coefficient is 1000 times of solid-liquid reflux state, and capping reagent fully can be diffused into all apertures inside of silica gel and at utmost close activated silica alcohol radical by chemical reaction.In theory, this mode is almost unique feasibility.
The inverse bonded phase of high performance liquid chromatography provided by the invention super/subcritical fluids end blocking method, be use Co 2 supercritical fluid and/or carbon dioxide sub critical fluid as carrier by capping reagent and catalyst strips to be equipped with the inverse bonded phase of silica gel super/sub-critical flow reactor according in, carry out end capping reaction under sealing condition.
Described in the inventive method inverse bonded cover mutually all anti-phase and containing the filler of reverse phase mechanism, include but not limited to: monofunctional group, two functional groups and trifunctional group C18, C8, phenyl, hexyl-phenyl, C5, C4, C1, PFPP, polarity embedded (Polar embedded) Bonded Phase such as acid amides or acyl ester, C
30deng.
Described high performance liquid chromatography is inverse bonded covers all silica gel or the filler containing silica gel mutually, includes but not limited to: Type B spherical silica gel, nucleocapsid silica gel, organic and inorganic hybrid silica gel.
Described capping reagent and catalyst can use disclosed reagent in field.Such as, capping reagent includes but not limited to: 1, 1, 3, 3, 5, 5-pregnancy basic ring three silazane, octamethylcyclotetrasilazane, 1, 1, 3, 3, 5, 5, 7, 7, 9, 9-ring five dimethyl silicone polymer, 1, 1, 3, 3, 5, 5, 7, 7, 9, 9, 11, 11-dodecamethylcyclohexasiloxane, 1, 1, 3, 3, 5, 5-hexaphenyl ring three silazane, 1, 1, 3, 3, 5, 5, 7, 7-octaethyl cyclotetrasilazane, 1, 1, 3, 3, 5, 5, 7, 7, 9, 9-ten diethyl cyclopentasiloxane, 1, 1, 3, 3, 5, 5, 7, 7, 9, 9, 11, 11-ten diethyl cyclohexasiloxane, dimethyldichlorosilane, dimethyldimethoxysil,ne, trim,ethylchlorosilane, methoxytrimethylsilane, dimethyl tertiary butyl chlorosilane, normal-butyl dimethyl methoxy silane, N-butyl methyl silicon dichloride, n-butylmethyldimethoxyforane, butyl trimethoxy silane, tert-butyl group trichlorosilane, methyl trichlorosilane, MTMS etc. and their conbined usage.Catalyst includes but not limited to N, the two trimethyl silicane yl acetamide of O-, ammonia, triethylamine, 1,1,1,3,3,3-hexa methyl di silicon imide, N, N-dimethyl trimethyl silicane amine etc. and their conbined usage.
Preferably, in the supercritical fluid end blocking method of the inverse bonded phase of above-mentioned high performance liquid chromatography, the described end capping reaction time is 3 ~ 24 hours; The pressure condition of described end capping reaction is 50 ~ 600bar; Described end capping reaction temperature is 31.2 ~ 200 DEG C.
Preferably, the inverse bonded phase of above-mentioned high performance liquid chromatography super/subcritical fluids end blocking method, also comprise the step that end capping reaction terminates rear cooling, and the step of release capping reagent and catalyst after cooling.
Preferably, the inverse bonded phase of above-mentioned high performance liquid chromatography super/subcritical fluids end blocking method, after release capping reagent and catalyst, also comprise the step of cleaning end capping reaction product, and the step of dry end capping reaction product.
Preferably, the inverse bonded phase of above-mentioned high performance liquid chromatography super/subcritical fluids end blocking method in, described cleaning is by end capping reaction product acetonitrile, and the methyl alcohol formed by the volume ratio of 1:1 and water mixed liquid, acetone cleans successively.
Preferably, the inverse bonded phase of above-mentioned high performance liquid chromatography super/subcritical fluids end blocking method in, described drying condition is 80 DEG C of nitrogen protections.
Preferably, the inverse bonded phase of above-mentioned high performance liquid chromatography super/subcritical fluids end blocking method in, described high performance liquid chromatography is inverse bonded is C18, C8, phenyl, hexyl-phenyl, C5, C4, C1, PFPP, acid amides, acyl ester or C30 Bonded Phase mutually.
Described high performance liquid chromatography is inverse bonded covers all granular sizes mutually, includes but not limited to: 1.2 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4 μm, 5 μm, 6 μm, 7 μm, 10 μm, 12 μm, 15 μm, 20 μm, 40 μm, 75 μm.
Described high performance liquid chromatography is inverse bonded covers all pore sizes mutually, includes but not limited to:
and atresia.
Preferably, the inverse bonded phase of above-mentioned high performance liquid chromatography super/subcritical fluids end blocking method, described capping reagent comprises 1, 1, 3, 3, 5, 5-pregnancy basic ring three silazane, octamethylcyclotetrasilazane, 1, 1, 3, 3, 5, 5, 7, 7, 9, 9-ring five dimethyl silicone polymer, 1, 1, 3, 3, 5, 5, 7, 7, 9, 9, 11, 11-dodecamethylcyclohexasiloxane, 1, 1, 3, 3, 5, 5-hexaphenyl ring three silazane, 1, 1, 3, 3, 5, 5, 7, 7-octaethyl cyclotetrasilazane, 1, 1, 3, 3, 5, 5, 7, 7, 9, 9-ten diethyl cyclopentasiloxane, 1, 1, 3, 3, 5, 5, 7, 7, 9, 9, 11, 11-ten diethyl cyclohexasiloxane, dimethyldichlorosilane, dimethyldimethoxysil,ne, trim,ethylchlorosilane, methoxytrimethylsilane, dimethyl tertiary butyl chlorosilane, normal-butyl dimethyl methoxy silane, N-butyl methyl silicon dichloride, n-butylmethyldimethoxyforane, butyl trimethoxy silane, tert-butyl group trichlorosilane, one or more in methyl trichlorosilane and MTMS, described catalyst comprises N, one or more in O-two trimethyl silicane yl acetamide, ammonia, triethylamine, 1,1,1,3,3,3-hexa methyl di silicon imide and N, N-dimethyl trimethyl silicane amine.
High performance liquid chromatography reverse bonding phase of the present invention super/subcritical fluids end blocking method can be widely used in analyzing and/or preparing reversed-phase HPLC, UHPLC or UPLC product.Product quality is very high.
The present invention also provides a kind of performance liquid chromatographic column, the Bonded Phase of this chromatographic column adopt the inverse bonded phase of above-mentioned high performance liquid chromatography super/subcritical fluids end blocking method carries out end-blocking.
Compared with prior art, the present invention has following beneficial effect:
(1) use this new Method and process thoroughly can close residual active silanol, produce the hydrophobic reversed-phase Bonded Phase of complete neutral charge.
(2) the high-efficient liquid phase reverse-phase chromatographic column using this new strategy (new technology and technique) to produce or ultra high efficiency liquid phase reverse-phase chromatographic column, not residual active silanol and the side effect of alkali compounds cation exchange, therefore alkali compounds (and acid compound, neutral compound, amphoteric compound is the same) there is very symmetrical peak shape, retention time and mechanism follow compound hydrophobicity order from low to high completely.
(3) the high-efficient liquid phase reverse-phase chromatographic column that makes than prior art of alkali compounds post effect and peak shape or 1.7 μm of UPLC Ultra Performance Liquid Chromatography posts, 2.6 μm of nucleocapsid UHPLC high-efficient liquid phase reverse-phase chromatographic columns significantly improve.
(4) this new Method and process reaction condition is gentle, and inverse bonded phase not damaged, the chromatographic column life-span is long.
(5) production batch repeatability very well.
(6) high-efficient liquid phase reverse-phase chromatographic column that alkali compounds post effect and peak shape make than prior art or 1.7 μm of UPLC Ultra Performance Liquid Chromatography posts are produced in supercritical fluid and subcritical fluids enclosure method, 2.6 μm of nucleocapsid UHPLC high-efficient liquid phase reverse-phase chromatographic columns Bonded Phase in the TIC analysis of LC-MS is highly stable, No leakage.
(7) method provided by the invention guarantees Green Chemistry, reduces the use of poisonous organic solvent as toluene to greatest extent, reduces the pollution to environment to greatest extent, and can realize technique serialization and semi-automation.
Accompanying drawing explanation
Fig. 1 be the present invention super/subcritical fluids closed-end technology (left side) and solid-liquid reflux under closed-end technology (right side) compare schematic diagram;
Fig. 2 is representational inverse bonded phase pH-surface charge curve comparative result;
Fig. 3 is representational inverse bonded phase Zeta electric potential comparative result;
Fig. 4 is the results contrast detecting same sample amitriptyline, imipramine with different performance liquid chromatographic column, and wherein A is that conventional liquid-solid end-blocking is produced
c18 5 μm of 25cm × 4.6mm HPLC chromatographic columns, B is that supercritical fluid end-blocking is produced
c18 5 μm of 25cm × 4.6mm HPLC chromatographic columns, C is Supelco company
c18 5 μm of 25cm × 4.6mm HPLC chromatographic columns, D is Supelco company
c18 5 μm of 25cm × 4.6mm HPLC chromatographic columns, E is Waters company
c185 μm of 25cm × 4.6mm HPLC chromatographic column.
Fig. 5: be the chromatogram of 100A C18 5 μm of 25cm × 4.6mm HPLC column detection Prozacs that supercritical fluid end-blocking is produced.
Fig. 6 is that 5 μm of PFPP HPLC performance liquid chromatographic columns that supercritical fluid closed-end technology is produced do the result that in horse urine, aminorex is identified, aminorex LC-MS/MS MRM peak (M+1=163): 18.1 minutes, another active metabolite (M+1=162) peak: 28.0 minutes;
Fig. 7 is that subcritical fluids end blocking method closed-end technology is produced
the peptide mapping that C18 chromatographic column produces;
Fig. 8 is the peptide mapping on market above famous brand name performance liquid chromatographic column 1;
Fig. 9 is the peptide mapping on market above famous brand name performance liquid chromatographic column 2;
Figure 10 is that the 1.7 μm of UPLC produced by supercritical fluid end-blocking stablize acid amides Ultra Performance Liquid Chromatography post to the separating resulting of Chinese herbal medicine effective ingredients;
Figure 11 is the result that 2.6 μm of C18 nucleocapsid UHPLC Ultra Performance Liquid Chromatography posts that supercritical fluid closed-end technology is produced measure arginine in rat brain microdialysates and histidine.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described, can better understand the present invention and can be implemented, but illustrated embodiment is not as a limitation of the invention to make those skilled in the art.
Embodiment 1:5 μm of C18 Bonded Phase supercritical fluid end-blocking
Tradition solid-liquid backflow end blocking method: adopt Japanese Daiso company
5 μm of Type B spherical silica gels and silane reagent octadecyldimethylchlorosilane are as raw material, and use toluene is solvent, and imidazoles and pyridine are catalyst, reflux 24 hours in the nitrogen protection of refluxing toluene temperature, obtain C18 Bonded Phase after washing.Technical parameter: specific area 300m
2/ g, bond densities 3.2 μm of ol/m
2.
100g C18 Bonded Phase uses trim,ethylchlorosilane to be capping reagent, and 1,1,1,3,3,3-hexa methyl di silicon imide, N, N-dimethyl trimethyl silicane amine etc. are catalyst, reflux 24 hours in the nitrogen protection of refluxing toluene temperature.Use toluene after reaction, carrene, acetonitrile, first alcohol and water (50:50), acetone is washed successively, 80 DEG C of nitrogen protection dryings, uses after cooling down.
Supercritical fluid end blocking method of the present invention: 100g C18 Bonded Phase is placed into S and I of Supercritical fluid device centre position shown in SC-method on the left of Fig. 1, excessive (capping reagent and C
18the molar concentration rate of Bonded Phase silanol base is 2:1) trim,ethylchlorosilane and 1,1,1,3,3,3-hexa methyl di silicon imide, N, N-dimethyl trimethyl silicane amine is placed into Fig. 1 Supercritical fluid device centre position 2 as capping reagent and catalyst.First the temperature of reaction system rises to 31.2 DEG C, and pressure rises to 73.8bar, this time CO
2become supercritical fluid.The temperature of reaction system rises to 100 DEG C in 10 minutes, and pressure rises to 414bar, and supercritical fluid end-blocking continues 3 ~ 8 hours.After reaction, use fresh CO
2supercritical fluid Rapid Cleaning 20 ~ 30 minutes, cooling, decompression.Further use acetonitrile, the mixed liquor of first alcohol and water (volume ratio 50:50), acetone is washed successively, 80 DEG C of nitrogen protection dryings, uses, be labeled as Novel C18 after cooling down.
Embodiment 2: surface charge titration
Surface charge titration, Zeta electric potential is the most important Surface Characterization technology of colloid and two, interface science field.Titration method: 10 grams of chromatogram Bonded Phases are placed on constant-current titration indoor; with 10mM sodium chloride (80%) and methyl alcohol (20%) as liquid phase; by 0.1M hydrochloric acid adjusted to ph to 3.0; pH8 is titrated to 0.1M NaOH under nitrogen protection from pH value 3.0; use again 0.1M hydrochloric acid back titration to pH3.0, be titrated to pH8.0 with 0.1MNaOH again.
Native state potential titration data is scaled pH-surface charge curve after being delivered to computer.
PH-surface charge curve can clearly reveal the surface charge of the reverse-phase chromatography product on much market as new aspect test platform very much.
Type B spherical silica gel itself has 8 μm of ol/m
2silanol base, be only wherein that activity can be dissociated less than half silanol base.Under acidic pH, only a small amount of very active silanol base can dissociate.PH value increases, and increasing silanol base dissociates.On pH-surface charge density curve, Silica Surface always carries negative electrical charge.PH value increases, and Silica Surface negative charge density is larger.In theory, inverse bonded and end-blocking will cause pH3 ~ 8 Silica Surface negative charge density to become zero completely.From practical application angle, the very active silanol base that can dissociate under acidic pH is by complete bonding or close all right.
The pH-surface charge curve of C18 Bonded Phase, Supelco company Discovery HS C18 and Supelco company SupelcoilABZ Plus tri-kinds of inverse bonded phases that the present embodiment is prepared embodiment 1 supercritical fluid end blocking method compares, and the results are shown in Figure 2.
Supercritical fluid end-blocking C18 Bonded Phase in Novel C18 and embodiment 1 in figure, surface charge density is zero, proves that the silanol base that can dissociate is by complete bonding or close.C18 Bonded Phase prepared by corresponding solid-liquid backflow end blocking method is equal to Supelco company of U.S. Discovery HS C18.
Supelco company of U.S. Discovery HS C18 still has many active silanol bases, is presented as that the hangover of many alkali compounds is serious in chromatogram.
Supelco company of U.S. Supelcoil ABZ Plus aminopropyl Bonded Phase synthesis, NH
2the impossible complete reaction of functional group, thus under acidic pH, Silica Surface electric charge is just.PH value increases, and waits negative electrical charge that the silanol base dissociated carries more than NH
2during the positive charge that functional group carries, Bonded Phase surface charge density becomes negative.Therefore the Bonded Phase that Supelco ABZ Plus and the similar principle of some other use are produced is both sexes Bonded Phases, is not classical anti-phase.Be presented as in chromatogram that many alkali compounds are due to positive charge-positive charge repulsive interaction peak shape improvement, acid compound is peak shape hangover under acidic pH, under neutral pH conditions, do not have enough reservations owing to dissociating.
Embodiment 3:Zeta electromotive force
PH-surface charge density curve itself is difficult to the isoelectric point (i.e.p) determining Bonded Phase.Adjoint is that Zeta electric potential measures.The Zeta electric potential of Supelco HS C18 and Supelco ABZ Plus as shown in Figure 3.
The isoelectric point of Supelco HS C18 is at pH2.7, and the isoelectric point of silica gel itself is similar.SupelcoABZ Plus is owing to there being residual NH
2functional group, isoelectric point shifts to pH4.4.
Because supercritical fluid end-blocking at utmost encloses activated silica alcohol radical, Novel C18 surface charge density is almost zero, and Zeta electric potential measures also cannot observe surface charge.
Embodiment 4: chromatogram is tested
Supercritical fluid end-blocking at utmost encloses activated silica alcohol radical, causes performance liquid chromatographic column product quality to have the enhancing of essence.To produce with supercritical fluid end-blocking of the present invention
on C18, C8 and market, similar aperture product (famous brand name performance liquid chromatographic column) compares.
Test compounds is tricyclics imipramine, amitriptyline.
Mobile phase is pH7 phosphate buffer 70%+ methyl alcohol 30% (volume).
It should be noted that, use 5 μm of C1825cm × 4.6mm HPLC performance liquid chromatographic columns prepared by traditional solid-liquid backflow end blocking method in embodiment 1, imipramine, amitriptyline tailing factor are respectively 2.0 and 2.3 (the results are shown in Figure 4A).Use 5 μm of C1825cm × 4.6mm HPLC performance liquid chromatographic columns prepared by supercritical fluid end-blocking liquid backflow end blocking method, imipramine, amitriptyline tailing factor are respectively 1.08 and 1.09 (the results are shown in Figure 4B).Supelco
c18 and
c18, Waters company
c18 comparative result is shown in Fig. 4 C, 4D and 4E.
Embodiment 5: the comparison of alkali compounds chromatogram test result data
With supercritical fluid end-blocking produce
the comparative result of the alkali compounds chromatogram test test data of C18 and famous brand name product is in table 1:
Table 1
With supercritical fluid end-blocking produce
c18 is in the anti-phase test of standard, and post effect 26000, toluene peak trails 1.03.Reversed-phase HPLC column is at pH7 phosphate buffer: when doing the test of alkali compounds chromatogram in methanol as mobile phase (70:30), and the hangover of amitriptyline, imipramine and Prozac is 1.03,1.08 and 1.09 respectively, without chromatographic peak conditions of streaking.Post effect is 19000,19000 and 16000 respectively.
Use the 100A C185um25cmx4.6mm HPLC column that supercritical fluid closed-end technology is produced, mobile phase is pH7 phosphate buffer 70%+30% methyl alcohol, and Prozac HPLC chromatogram is shown in Fig. 5.
Supelco company
c18 and
c18, Waters company
c18, Restek company DB C18 amitriptyline, imipramine and Prozac have obvious chromatographic peak hangover, and post effect is on the low side.
These two tests clearly illustrate that uses supercritical fluid end-blocking to produce
c18 has remarkable superiority.
Embodiment 6: stability test
With supercritical fluid end-blocking produce
c18, dress up two 5cm × 4.6mm HPLC chromatographic columns, first do anti-phase test, then do the test of alkali compounds chromatogram at acid ph value mobile phase (30% methyl alcohol+0.1% trifluoracetic acid) and pH neutral mobile phase (pH7 phosphate buffer 70%+ methyl alcohol 30%) respectively.Then continue to use acid ph value mobile phase or pH neutral mobile phase (pH7 phosphate buffer 70%+ methyl alcohol 30%) to do stability test.At set intervals, these two HPLC chromatographic columns do anti-phase test and the test of alkali compounds chromatogram again.Test result sees the following form.
Table 2
Table 3
Above-mentioned data show, after 8400 empty bed volumes, all compound post effects, retention time and peak shape do not change, and prove to use supercritical fluid end-blocking to produce
c
18stability is very good.
Embodiment 7: Purification and Characterization aminorex (aminorex)
Aminorex (aminorex) belongs to first kind illegal stimulants, a kind of parasiticidal veterinary drug, and the aminorex produced in the livestock bodies such as horse, pig, ox once allowed 150 show jumping competition person's suspension.
The PFPP chromatographic column that supercritical fluid end-blocking of the present invention is produced, plays vital effect at the evaluation program of aminorex.
5 liters of horse urine concentrate and partial purification through multistep flash chromatography post, obtain about 10g and slightly extract sample.Use reversed-phase HPLC or LC-MS/MS can not confirm that aminorex exists.This is because aminorex LC-MS/MS MRM peak (M+1=163) may be the isotope contribution of another active metabolite (M+1=162), in reversed-phased high performace liquid chromatographic, two peaks can not be separated.But in 5 μm of PFPP HPLC chromatographic columns that supercritical fluid closed-end technology is produced, the difference of two peak retention times reaches ten minutes (see Fig. 5), aminorex LC-MS/MS MRM peak (M+1=163): 18.1 minutes, another active metabolite (M+1=162) peak: 28.0 minutes.See Fig. 6.
Chromatographic column: PFPP5 μm of 25cm × 4.6mm HPLC
HPLC gradient:
Step table:
Embodiment 8:
c18 Bonded Phase subcritical fluids end-blocking
Macro porous silica gel is under high pressure frangible, therefore sometimes needs to use subcritical fluids end blocking method.
Adopt Japanese Daiso company
5 μm of Type B spherical silica gels and silane reagent octadecyldimethylchlorosilane are as raw material, and use toluene is solvent, and imidazoles and pyridine are catalyst, reflux 12 hours in the nitrogen protection of refluxing toluene temperature, obtain C18 Bonded Phase after washing.Technical parameter: specific area 100m
2/ g, bond densities 3.4 μm of ol/m
2.
Subcritical fluids end blocking method of the present invention: 100g C18 Bonded Phase is placed into super/subcritical fluids equipment centre position S and I shown in SC-method on the left of Fig. 1, excessive (molar concentration rate of capping reagent and C18 Bonded Phase silanol base is 2:1) trim,ethylchlorosilane and 1,1,1,3,3,3-hexa methyl di silicon imide, N, N-dimethyl trimethyl silicane amine is placed into Fig. 1 subcritical fluids equipment centre position 2 as capping reagent and catalyst.First the temperature of reaction system rises to 31.2 DEG C, and pressure rises to 60bar, this time CO
2become subcritical fluids.The temperature of reaction system rises to 100 DEG C in 10 minutes, and pressure is stabilized in 60bar, this time CO
2be stabilized in subcritical fluids state, subcritical fluids end-blocking continues 3 ~ 8 hours.After reaction, use fresh CO
2subcritical fluids Rapid Cleaning 20 ~ 30 minutes, cooling, decompression.Further use acetonitrile, the mixed liquor of first alcohol and water (volume ratio 50:50), acetone is washed successively, 80 DEG C of nitrogen protection dryings, uses, be labeled as after cooling down
c18.
Embodiment 9: peptide mapping resolution ratio
Peptide mapping is protein chemistry analytical method the most classical and the most effective.
c18 chromatographic column uses single function individual layer high density bonding, then carries out end-blocking by subcritical fluids end blocking method of the present invention, and therefore all polypeptide can both obtain good reservation and symmetrical peak shape.Result peptide mapping resolution ratio is the highest, thus obtains protein sequence to greatest extent, the information of structure and purity.
the peptide mapping that C18 chromatographic column produces obtains the peak more than famous brand name performance liquid chromatographic column on market.
Chromatography post:
c
185 μm of 15cm × 4.6mm;
Mobile phase A: 95% water: 5% acetonitrile: 0.1% trifluoracetic acid;
Mobile phase B: 50% water: 50% acetonitrile: 0.1% trifluoracetic acid;
Sampling volume: 50 μ L.UV detect: 215nm. temperature: 30 DEG C.
Step table:
Sample: carboxymethyl hemoglobin tryptic digestion liquid (50mM carbonic hydroammonium).
Carboxymethyl hemoglobin tryptic digestion liquid exists
the peptide peak (Fig. 7) having 71 to differentiate in peptide mapping above C18 chromatographic column.
The peptide peak (Fig. 8) having 61 to differentiate in the peptide mapping of carboxymethyl hemoglobin tryptic digestion liquid commercially above famous brand name performance liquid chromatographic column 1.
The peptide peak (Fig. 9) having 68 to differentiate in the peptide mapping of carboxymethyl hemoglobin tryptic digestion liquid commercially above famous brand name performance liquid chromatographic column 2.
Embodiment 10:1.7 μm of UPLC Ultra Performance Liquid Chromatography post
The 1.7 μm of UPLC produced by supercritical fluid end-blocking stablize the separation that acid amides Ultra Performance Liquid Chromatography post is used successfully to Chinese herbal medicine effective ingredients.Separating resulting is shown in Figure 10, and linear gradient is from 100% aqueous phase, and in upper figure, there is symmetrical peak shape at each peak.
Embodiment 11:2.6 μm of UHPLC nucleocapsid Ultra Performance Liquid Chromatography post
The 2.6 μm of C18 nucleocapsid UHPLC Ultra Performance Liquid Chromatography posts produced by supercritical fluid end-blocking are used successfully to arginine in Accurate Determining rat brain microdialysate and histidine, the results are shown in Figure 11.These two basic amino acid Dansyl Derivatives famous brand name 2.6 μm of UHPLC nucleocapsid Ultra Performance Liquid Chromatography post peaks on staple market seriously trail not even not out.
Test condition:
Sample: the arginine in rat brain microdialysate and histidine dansyl Cl derivative
Mobile phase A: Milli-Q ultra-pure water-0.1% formic acid
Mobile phase B: acetonitrile-0.1% formic acid
Flow velocity: 0.3mL/min
Temperature: 25 DEG C
Sample size: 10 μ L
Instrument: Perkin Elmer200 micro pump
ABI API4000 tandem mass spectrometer
Leap Technologies automatic sampler
Linear gradient:
0 minute → 0.5 minute 70%A+30%B
Within 0.5 minute → 3.5 minutes, linear gradient is to 10%A+90%B
Within 3.5 minutes → 4.0 minutes, linear gradient is to 70%A+30%B
4.0 minutes → 4.5 minutes 70%A+30%B.
The above embodiment is only that protection scope of the present invention is not limited thereto in order to absolutely prove the preferred embodiment that the present invention lifts.The equivalent alternative or conversion that those skilled in the art do on basis of the present invention, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
Claims (10)
1. the inverse bonded phase of high performance liquid chromatography super/subcritical fluids end blocking method, it is characterized in that, use Co 2 supercritical fluid and/or carbon dioxide sub critical fluid as carrier by capping reagent and catalyst strips to be equipped with the inverse bonded phase of silica gel super/sub-critical flow reactor according in, carry out end capping reaction under sealing condition.
2. the inverse bonded phase of high performance liquid chromatography according to claim 1 super/subcritical fluids end blocking method, it is characterized in that, the described end capping reaction time is 3 ~ 24 hours; The pressure condition of described end capping reaction is 50 ~ 600bar; The temperature of described end capping reaction is 31.2 ~ 200 DEG C.
3. the inverse bonded phase of high performance liquid chromatography according to claim 1 super/subcritical fluids end blocking method, is characterized in that, also comprise the step that end capping reaction terminates rear cooling, and the step of release capping reagent and catalyst after cooling.
4. the inverse bonded phase of high performance liquid chromatography according to claim 3 super/subcritical fluids end blocking method, is characterized in that, after release capping reagent and catalyst, also comprises the step of cleaning end capping reaction product, and the step of dry end capping reaction product.
5. the inverse bonded phase of high performance liquid chromatography according to claim 4 super/subcritical fluids end blocking method, it is characterized in that, described cleaning is by end capping reaction product acetonitrile, and the methyl alcohol formed by the volume ratio of 1:1 and water mixed liquid, acetone cleans successively.
6. the inverse bonded phase of high performance liquid chromatography according to claim 4 super/subcritical fluids end blocking method, it is characterized in that, described drying condition is 80 DEG C of nitrogen protections.
7. according to the arbitrary described inverse bonded phase of high performance liquid chromatography of claim 1 ~ 6 super/subcritical fluids end blocking method, it is characterized in that, described high performance liquid chromatography is inverse bonded is C18, C8, phenyl, hexyl-phenyl, C5, C4, C1, PFPP, acid amides, acyl ester or C30 Bonded Phase mutually.
8. according to the arbitrary described inverse bonded phase of high performance liquid chromatography of claim 1 ~ 6 super/subcritical fluids end blocking method, it is characterized in that, described capping reagent comprises 1,1,3,3,5,5-pregnancy basic ring three silazane, octamethylcyclotetrasilazane, 1,1,3,3,5,5,7,7,9,9-ring five dimethyl silicone polymer, 1,1,3,3,5,5,7,7,9,9,11,11-dodecamethylcyclohexasiloxane, 1,1,3,3,5,5-hexaphenyl ring three silazane, 1,1,3,3,5,5,7,7-octaethyl cyclotetrasilazane, 1,1,3,3,5,5,7,7,9,9-ten diethyl cyclopentasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11-ten diethyl cyclohexasiloxane, dimethyldichlorosilane, dimethyldimethoxysil,ne, trim,ethylchlorosilane, methoxytrimethylsilane, dimethyl tertiary butyl chlorosilane, normal-butyl dimethyl methoxy silane, N-butyl methyl silicon dichloride, n-butylmethyldimethoxyforane, butyl trimethoxy silane, tert-butyl group trichlorosilane, one or more in methyl trichlorosilane and MTMS, described catalyst comprises N, one or more in O-two trimethyl silicane yl acetamide, ammonia, triethylamine, 1,1,1,3,3,3-hexa methyl di silicon imide and N, N-dimethyl trimethyl silicane amine.
9. the arbitrary described inverse bonded phase of high performance liquid chromatography of claim 1 ~ 8 super/subcritical fluids end blocking method is in the application analyzed and/or prepare in reversed-phase HPLC, UHPLC or UPLC product.
10. a performance liquid chromatographic column, is characterized in that, the inverse bonded arbitrary described method of claim 1 ~ 8 that adopts mutually of this chromatographic column carries out end-blocking.
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| CN101479830A (en) * | 2006-06-27 | 2009-07-08 | 朗姆研究公司 | Repairing and restoring strength of etch-damaged low-K dielectric materials |
| CN102091607A (en) * | 2010-12-27 | 2011-06-15 | 天津博纳艾杰尔科技有限公司 | Multi-layer structure high-purity bonded silica gel chromatographic filler and preparation method thereof |
| WO2013134100A1 (en) * | 2012-03-08 | 2013-09-12 | Waters Technologies Corporation | Back pressure regulation |
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| CN101479830A (en) * | 2006-06-27 | 2009-07-08 | 朗姆研究公司 | Repairing and restoring strength of etch-damaged low-K dielectric materials |
| CN102091607A (en) * | 2010-12-27 | 2011-06-15 | 天津博纳艾杰尔科技有限公司 | Multi-layer structure high-purity bonded silica gel chromatographic filler and preparation method thereof |
| WO2013134100A1 (en) * | 2012-03-08 | 2013-09-12 | Waters Technologies Corporation | Back pressure regulation |
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| CN109475838A (en) * | 2016-06-03 | 2019-03-15 | 安捷伦科技有限公司 | Functionalized particle with modified phase |
| CN109475838B (en) * | 2016-06-03 | 2022-05-03 | 安捷伦科技有限公司 | Functionalized particles with modified phase |
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