CN104784971A - Large-size hydrophilic organic polymer liquid monolithic chromatographic column as well as preparation and application - Google Patents
Large-size hydrophilic organic polymer liquid monolithic chromatographic column as well as preparation and application Download PDFInfo
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Abstract
The invention belongs to the technical field of chromatographic separation materials and discloses a large-size hydrophilic organic polymer liquid monolithic chromatographic column as well as preparation and an application. According to the chromatographic column, 3-[N,N-dimethyl-[2-(2-methylpropane-2-alkene acyloxy) aminopropyl] ammonio] propane-1-sulfonate is taken as a function monomer, N,N-methylene bisacrylamide is taken as a cross-linking agent, methanol is taken as a hole forming agent, azodiisobutyronitrile is taken as an initiator, and the chromatographic column is formed through in situ polymerization of the materials in a pretreated quartz capillary tube with an inner diameter larger than 200 mu m by the aid of a thermocatalytic reaction. The inner diameter of the chromatographic column is larger than that of an ordinary organic polymer monolithic chromatographic column, the sample loading amount is larger, and the required flow speed is measured in microliter, so that requirements for an instrument is lower by comparison with a nanoliter liquid phase; the column pressure of the large-size hydrophilic organic polymer liquid monolithic chromatographic column is low, and the monolithic chromatographic column is suitable for separating multiple polar compounds such as base samples, nucleoside samples, small peptide samples and other samples.
Description
Technical field
The invention belongs to chromatographic separation material technical field, be specifically related to a kind of large scale hydrophilic organic polymer liquid phase solid chromatography column and preparation and application.
Background technology
As everyone knows, liquid chromatogram and GC-MS thereof have been the extremely important parts of compartment analysis science, all have a very wide range of applications in each different field such as environment, food security, pharmacy, medical science and life sciences.Chromatographic column is the core dielectric that liquid chromatogram realizes its compartment analysis function, and therefore the development of chromatographic column directly affects the development of whole liquid chromatography technology and even each compartment analysis scientific domain.In recent years, there is the attention that high-permeability, low post pressure, mass transfer velocity are fast, new chromatographic Stationary liquid integral material that is that prepare the advantage such as simple has more and more been subject to compartment analysis scientific worker.And the exploitation of novel overall chromatographic stationary phases material also becomes one of the study hotspot in separation science field.So far, monolithic chromatogram stationary phase material has been successfully applied to the clastotypes such as reverse-phase chromatography, ion-exchange chromatography, affinity chromatography and has effectively been separated the multiple compound systems of different nature such as amino acid, polypeptide, protein, polypeptide and steroids.According to the difference of function monomer preparing integral material, solid chromatography column is mainly divided into three major types: Organic Polymer Monolithic Columns, monolithic silica column and hybrid integral post [Zou H, Huang X, Ye M, Luo Q.Monolithic stationary phases for liquid chromatography and capillary electrochromatography.J Chromatogr.A.2002; 954 (1-2): 5-32.].Wherein, because the preparation of Organic Polymer Monolithic Columns is simple, with low cost, instrument and equipment threshold is low and can select the advantages such as dissimilar functional monomer according to different compartment analysis demands, the advantage making organic polymer solid chromatography column have other two kinds of solid chromatography columns can not to compare.
Wherein, hydrophilic organic polymers solid chromatography column is the important branch of Organic Polymer Monolithic Columns, and the preparation of hydrophily solid chromatography column is the important development focus of organic polymer solid chromatography column.From nineteen ninety hydrophilic liquid phase chromatogram (Hydrophilic interaction chromatography, HILIC) [Alpert AJ.Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds.J Chromatogr.1990 after being proposed first; 499:177-96.], hydrophilic liquid phase chromatogram more and more receives the concern of compartment analysis scientific worker.About the research of this part, the people such as Boguslaw have done comprehensive and outstanding summary [Boguslaw et al.Hydrophilic interaction liquid chromatography (HILIC)-a powerful separation technique.Anal Bioanal Chem (2012) 402:231-247].
But the preparation research about HILIC integral post mainly concentrates on the preparation of small size capillary column (internal diameter is less than or equal to 200), and about the rarely seen report of article prepared by larger sized HILIC integral post.But, based on the feature of integral post high-permeability, low post pressure, if can large scale be prepared and there is the integral post of Gao Zhuxiao, then greatly can reduce requirement to instrument (integral post being less than or equal to 200 microns needs microlitre/receive to rise liquid phase, and this quasi-instrument exists that price is high, Market Selection is few and stream such as easily to block at the shortcoming).But, only have at present in research about large scale capillary monolithic column: the people such as Ales prepare research [the Ales P of large volume integral post, Milos B et al.Construction of large-volume monolithic columns.Anal.Chem.2000,72,5693-5699]; The people such as Pavel prepare the methyl acrylic ester capillary monolithic column [Pavel C, Martin C et al.Methacrylate monolithic columns of 320 μm of I.D.for capillary liquid chromatography.Journal of Chromatography A.946 (2002) 99-106] of 320 micron inside diameter; The people such as A.Podgornik prepare the integral post of large column volume (8000ml) and are applied to the purifying [A.Podgornik et al.large-scale methacrylate monolithic columns:design and properties.J.Biochem.Biophys.Methods 60 (2004) 179-189] of macromolecular sample (protein, DNA etc.); The people such as Huang have done the research [Xiaojia H.et al.Octyl-type monolithic columns of 530 μm of i.d.for capillary liquid chromatography.J.chromatogr.A 1062 (2005) 183-188] of 530 micron inside diameter integral post.But, all there is a subject matter in these above-mentioned researchs: the post effect of integral post is low, and bad mechanical strength, separating effect is undesirable.Obviously, these shortcomings have impact on the further investigation and application of large scale solid chromatography column greatly.Therefore, how to prepare there is Gao Zhuxiao, the hydrophily solid chromatography column of large scale (>200 micron) of strong mechanical strength remains separation science problem in science in the urgent need to address.
Summary of the invention
In order to solve the shortcoming and defect part of above prior art, primary and foremost purpose of the present invention is the large scale hydrophilic organic polymer liquid phase solid chromatography column providing a kind of Gao Zhuxiao.
Another object of the present invention is to the preparation method that above-mentioned large scale hydrophilic organic polymer liquid phase solid chromatography column is provided.
Another object of the present invention is the application providing above-mentioned large scale hydrophilic organic polymer liquid phase solid chromatography column.
The object of the invention is achieved through the following technical solutions:
A kind of large scale hydrophilic organic polymer liquid phase solid chromatography column, described large scale hydrophilic organic polymer liquid phase solid chromatography column is with 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt (N, N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl) ammonium betaine, SPP) be function monomer, N, N-methylene-bisacrylamide (N, N-Methylenebis (acrylamide), MBA, be crosslinking agent No. CAS: 110-26-9), methyl alcohol (Methanol, be pore generating agent No. CAS: 67-56-1), azodiisobutyronitrile (Azobisisobutyronitrile, AIBN, be initator No. CAS: 78-67-1), be greater than in-situ polymerization in 200 microns of pretreated quartz capillaries by heat catalysis at internal diameter to form.
The internal diameter of described pretreated quartz capillary preferably 400 microns.
Described pretreated quartz capillary refers to through the pretreated quartz capillary of following steps: first, rinses quartz capillary 15 minutes by the NaOH solution that concentration is 1mol/L; Then, quartz capillary sealing two ends is placed on 100 DEG C of water-baths and reacts 2 hours; Then deionized water rinsing quartz capillary is used until the Acidity of Aikalinity pH=7 of eluate; Then, also nitrogen drying 4 hours are used with washed with methanol quartz capillary; After drying, methyl alcohol and 3-(isobutene acyl-oxygen) propyl trimethoxy silicane (3-(Methylacryloxy) propyl-trimethoxysilane that volume ratio is 1:1, γ-MAPs, mixed liquor fill with into quartz capillary in and guarantee in pipe bubble-free produce, sealing two ends and be placed on 60 DEG C water-baths in reaction 12 hour No. CAS: 2530-85-0); Finally respectively rinse 30 minutes with first alcohol and water respectively, nitrogen drying 12 hours, obtain through pretreated quartz capillary; By aforesaid operations, the silanol base of quartz capillary inner surface and γ-MAPs are reacted, thus γ-MAPs is bonded on silanol base.
Described large scale hydrophilic organic polymer liquid phase solid chromatography column has continuous print loose structure (SPP-co-MBA); It is by the monomer 3-[N of end strips hydrophilic sulfonic acid group, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt (SPP) sequentially connect crosslinking agent N, N-methylene-bisacrylamide (MBA), 3-(isobutene acyl-oxygen) propyl trimethoxy silicane (γ-MAPs) and quartz capillary inwall silanol base form.
The preparation method of above-mentioned large scale hydrophilic organic polymer liquid phase solid chromatography column, comprise following operating procedure: by monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt, crosslinking agent N, N-methylene-bisacrylamide, pore generating agent methyl alcohol and initator azodiisobutyronitrile mix, after ultrasonic degas, pour into internal diameter and be greater than 200 microns in pretreated quartz capillary column, then by quartz capillary sealing two ends, water-bath reaction 12 hours is put into; After completion of the reaction, the impurity in removing quartz capillary and unreacted reactant, obtain large scale internal diameter hydrophilic organic polymer liquid phase monolithic chromatographic column.
It is react 6 ~ 24 hours within the scope of 50 ~ 65 DEG C that described reaction refers in temperature; Preferably react 12 hours under temperature is 60 DEG C of conditions.
The mass ratio of described monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt and crosslinking agent N, N-methylene-bisacrylamide is in 80:20 to 90:10 scope; The preferred 87:13 of mass ratio of monomer and crosslinking agent.
The quality of described initator azodiisobutyronitrile is monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt and crosslinking agent N, N-methylene-bisacrylamide gross mass 0.5% ~ 1%; The quality of preferred initator is 1% of monomer and crosslinking agent gross mass.
Described monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt and the quality sum of crosslinking agent N, N-methylene-bisacrylamide and the mass ratio of pore generating agent methyl alcohol be in 25:75 to 45:55 scope; The quality sum of preferred monomers and crosslinking agent and the mass ratio of pore generating agent are 32.5:67.5.
Impurity in described removing quartz capillary and unreacted reactant operate according to following steps: be connected with high-pressure pump one end of quartz capillary, rinse with organic solvent; Described organic solvent is at least one in methyl alcohol, acetonitrile and acetone.
The application of above-mentioned large scale hydrophilic organic polymer liquid phase solid chromatography column in the compartment analysis research of polar substances; Described polar substances is base, ucleosides and small peptide.
Preparation principle of the present invention is: have good post effect and separating power for making large scale hydrophilic organic polymer liquid phase solid chromatography column of the present invention, monomer selects 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt, higher amphipathic ability is possessed than monomer involved in prior art, make prepared integral post have larger hydrophilic interaction interval, and larger hydrophilic interaction interval ensure that the separating power stronger to polar substances; Select single pore generating agent system (methyl alcohol), make whole polymerization system simpler, simplify preparation technology, from probability, more easily polymerization produces the polyalcohol integral pole bed of single uniformly continous, and the polyalcohol integral pole bed of single uniformly continous is the prerequisite that integral post has Gao Zhuxiao.
Preparation and the method tool of large scale hydrophilic organic polymer liquid phase solid chromatography column of the present invention have the following advantages and beneficial effect:
(1) hydrophilic organic polymer liquid phase monolithic chromatogram column internal diameter of the present invention can reach 400 microns, has good mechanical strength, not easily occurs rupturing and the problem of subsiding, can resistance to higher pressure (>20MPa); In prior art seldom report prepare internal diameter be greater than 200 microns, Gao Zhuxiao, the Organic Polymer Monolithic Columns of high mechanical properties.
(2) internal diameter of hydrophilic organic polymer liquid phase solid chromatography column of the present invention is larger than general organic polymer solid chromatography column, and volume containing the sample is larger, and required flow rate is micro updating thus to the requirement of instrument lower (rising liquid phase compared to receiving); And large scale hydrophilic organic polymer liquid phase solid chromatography column post forces down, be applicable to being separated numerous polar compound, as samples such as ucleosides, base class, small peptides, and show outstanding separating power and separative efficiency;
(3) hydrophilic organic polymer liquid phase solid chromatography column of the present invention has very high post effect (column length 15 centimetres, best plate height is 6.99 microns, the number of plates 143,000 every meter), be obviously better than the post effect of existing bibliographical information and business post of the same type;
(4) preparation technology of hydrophilic organic polymer liquid phase solid chromatography column of the present invention is simple, and cost is low.
Accompanying drawing explanation
Fig. 1 and Fig. 2 is the scanning electron microscope (SEM) photograph that embodiment 1 prepares gained SPP-co-MBA large scale hydrophilic organic polymer liquid phase solid chromatography column.
Fig. 3 is the retention behavior figure that embodiment 1 prepares gained SPP-co-MBA large scale hydrophilic organic polymer liquid phase solid chromatography column.
Fig. 4 is the chromatogram that embodiment 1 prepares that the degree sample separation such as gained SPP-co-MBA large scale hydrophilic organic polymer liquid phase solid chromatography column embody post effect.
Fig. 5 is the curve map of plate height with change of line speed that embodiment 1 prepares that gained SPP-co-MBA large scale hydrophilic organic polymer liquid phase solid chromatography column embodies post effect.
Fig. 6 is that embodiment 1 prepares gained SPP-co-MBA large scale hydrophilic organic polymer liquid phase solid chromatography column separation base and nucleosides chromatogram.
Fig. 7 is the chromatogram that embodiment 1 prepares that gained SPP-co-MBA large scale hydrophilic organic polymer liquid phase solid chromatography column is separated little peptide sample.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1 ~ 6 use through pretreated quartz capillary (400 micron inside diameter) be prepare by following operating procedure: rinse quartz capillary 15min by the NaOH solution of 1mol/L, then quartz capillary sealing two ends be placed on 100 DEG C of water-baths and react 2 hours; Then, with deionized water rinsing quartz capillary until eluate Acidity of Aikalinity pH=7; Then nitrogen drying is used 4 hours with washed with methanol quartz capillary; After drying, be that the methyl alcohol of 1:1 and 3-(isobutene acyl-oxygen) propyl trimethoxy silicane (γ-MAPs) mixed liquor pour in quartz capillary volume ratio, sealing two ends and to be placed in 60 DEG C of water-baths reaction 12 hours; Finally respectively rinse 30 minutes with first alcohol and water respectively, nitrogen drying 12 hours, obtains through pretreated quartz capillary.
Embodiment 1
By monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt 0.0870g, crosslinking agent N, N-methylene-bisacrylamide 0.0132g, pore generating agent methyl alcohol 0.2100g and initator azodiisobutyronitrile 0.0020g, be mixed with reaction mixture, ultrasonic degas pours into after 5 minutes in pretreated quartz capillary, then by quartz capillary sealing two ends, 60 DEG C of water-bath reactions 12 hours are put into; After completion of the reaction quartz capillary is connected with high-pressure pump, rinse out impurity and unreacted reactant, obtain the hydrophilic organic polymer liquid phase monolithic chromatographic column of SPP-co-MBA large scale (400 micron inside diameter), in post, the electron scanning Electronic Speculum result of organic polymer as depicted in figs. 1 and 2.
Embodiment 2
By monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt 0.0851g, crosslinking agent N, N-methylene-bisacrylamide 0.0150g, pore generating agent methyl alcohol 0.2089g and initator azodiisobutyronitrile 0.0033g, be mixed with reaction mixture, pour into after ultrasonic degas in pretreated quartz capillary, then by quartz capillary sealing two ends, put into 60 DEG C of water-bath reactions 12 hours, after completion of the reaction quartz capillary is connected with high-pressure pump, rinse removing unreacted reactant, obtain SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column.
Embodiment 3
By monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt 0.0900g, crosslinking agent N, N-methylene-bisacrylamide 0.0104g, pore generating agent methyl alcohol 0.2119g initator azodiisobutyronitrile 0.0035g, be mixed with reaction mixture, pour into after ultrasonic degas in pretreated quartz capillary, then by quartz capillary sealing two ends, put into 60 DEG C of water-bath reactions 12 hours, after completion of the reaction quartz capillary is connected with high-pressure pump, rinse removing unreacted reactant, obtain SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column.
Embodiment 4
By monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt 0.1014g, crosslinking agent N, N-methylene-bisacrylamide 0.0214g, pore generating agent methyl alcohol 0.2866g and initator azodiisobutyronitrile 0.0042g, be mixed with reaction mixture, pour into after ultrasonic degas in pretreated quartz capillary, then by quartz capillary sealing two ends, 60 DEG C of water-bath reactions 12 hours are put into; After completion of the reaction quartz capillary is connected with high-pressure pump, rinses removing unreacted reactant, obtain SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column.
Embodiment 5
By monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt 0.0839g, crosslinking agent N, N-methylene-bisacrylamide 0.0178g, pore generating agent methyl alcohol 0.2112g and initator azodiisobutyronitrile 0.0036g, be mixed with reaction mixture, pour into after ultrasonic degas in pretreated quartz capillary, then by quartz capillary sealing two ends, 60 DEG C of water-bath reactions 12 hours are put into; After completion of the reaction quartz capillary is connected with high-pressure pump, rinses removing unreacted reactant, obtain SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column.
Embodiment 6
By monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt 0.0826g, crosslinking agent N, N-methylene-bisacrylamide 0.0174g, pore generating agent methyl alcohol 0.1866g and initator azodiisobutyronitrile 0.0026g, be mixed with reaction mixture, pour into after ultrasonic degas in pretreated quartz capillary, then by quartz capillary sealing two ends, 60 DEG C of water-bath reactions 12 hours are put into; After completion of the reaction quartz capillary is connected with high-pressure pump, rinses removing unreacted reactant, obtain SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column.
Embodiment 1 prepares the separating property test of gained SPP-co-MBA large scale hydrophilic organic polymer liquid phase solid chromatography column:
(1) liquid phase retention behavior:
By SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column of embodiment 1 gained, acetonitrile and water are mobile phase, toluene and thiocarbamide are test compounds, and test their retention behaviors under the following conditions, result as shown in Figure 3.
Test condition:
As seen from Figure 3: under low acetonitrile concentration, (5%, v/v) two curves have no crossing, hydrophily interacts and still exists, large between aqueous favoring interaction region.Compared to document [Jiang Z, Smith NW, Ferguson PD, Taylor MR.Hydrophilic interaction chromatography using methacrylate-based monolithic capillary column for the separation of polar analytes.Anal Chem.2007; 79 (3): 1243-50.], obviously increase between aqueous favoring interaction region.
(2) the degree sample separation test such as:
SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column is separated the standard sample of 3 opposed polarities:
By SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column of embodiment 1 gained, isocratic elution, the separating effect of n-compound in integral post of test 3 opposed polarities, and investigate and weigh the post effect of this integral post with this, result is as shown in Figure 4.
Test condition:
As seen from Figure 4: above 3 biased samples utilize SPP-co-MBA large scale of the present invention (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column to be separated, good separating effect can be reached, can find out prepared by the present invention chromatographic column and have very high post effect (theoretical cam curve of compound thiourea be 143,000 every meter) from the peak shape of chromatogram and peak symmetry.
(3) chromatography column tower plate height is tested with change of line speed:
SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column is separated the plate height H of the standard sample of 3 opposed polarities and the graph of relation of linear velocity u:
By SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column of embodiment 1 gained, isocratic elution under different in flow rate, the separating effect of n-compound under different in flow rate in integral post of test 2 opposed polarities and the post effect parameter of correspondence, result as shown in Figure 5.
Test condition:
As seen from Figure 5: SPP-co-MBA large scale of the present invention (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column all has very high post effect on separation opposed polarity compound, and under optimum flow rate condition, plate height is close to 7 microns.
(4) base and the test of nucleosides separating effect:
SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column is separated base and Nucleoside samples:
By SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column of embodiment 1 gained, gradient elution, test a series of base and the separating effect of Nucleoside samples in integral post, result as shown in Figure 6.
Test condition:
Sample: thymidine, uracil, adenine, adenosine, uridine, cytimidine, inosine, cytidine, guanosine;
Mobile phase: 0min 93% acetonitrile and 7% water (v/v)
19min 85% acetonitrile and 15% water (v/v)
20min 93% acetonitrile and 7% water (v/v)
Flow velocity: 5ul/min
Determined wavelength: 254nm
As seen from Figure 6: above 9 biased samples utilize the integral post prepared by the present invention [SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column] to be separated, and can reach good separating effect.
(5) little peptide sample separation measure of merit:
SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column is separated small peptide biased sample:
By SPP-co-MBA large scale (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column of embodiment 1 gained, gradient elution, test the separating effect of a series of little peptides in this integral post, result as shown in Figure 7.
Test condition:
Sample: Tyr-Phe, Gly-Phe, Gly-Val, Ala-Tyr, Ala-Gly, Gly-Gly, L-carnosine, Gly-Gly-Gly;
Mobile phase: solvent orange 2 A: 10% acetonitrile and 90% water (containing 5mM ammonium formate, pH=3.0)
Solvent B:90% acetonitrile and 10% water (containing 5mM ammonium formate, pH=3.0)
0min 90% solvent B and 10% solvent orange 2 A
20min 65% solvent B and 35% solvent orange 2 A
Flow velocity: 5ul/min
Determined wavelength: 214nm
As seen from Figure 7: above 8 little peptide biased samples utilize SPP-co-MBA large scale of the present invention (400 micron inside diameter) hydrophilic organic polymer liquid phase monolithic chromatographic column to be separated, and can reach good separating effect.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a large scale hydrophilic organic polymer liquid phase solid chromatography column, it is characterized in that: described chromatographic column is with 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt is function monomer, N, N-methylene-bisacrylamide is crosslinking agent, methyl alcohol is pore generating agent, and azodiisobutyronitrile is initator, is greater than in-situ polymerization in 200 microns of pretreated quartz capillaries forms by heat catalysis at internal diameter.
2. a kind of large scale hydrophilic organic polymer liquid phase solid chromatography column according to claim 1, is characterized in that: the internal diameter of described quartz capillary is 400 microns.
3. a kind of large scale hydrophilic organic polymer liquid phase solid chromatography column according to claim 1 and 2, it is characterized in that: described pretreated quartz capillary refers to through the pretreated quartz capillary of following steps: first, rinse quartz capillary 15 minutes by the NaOH solution that concentration is 1mol/L; Then, quartz capillary sealing two ends is placed on 100 DEG C of water-baths and reacts 2 hours; Then deionized water rinsing quartz capillary is used until the Acidity of Aikalinity pH=7 of eluate; Then, nitrogen drying 4 hours are used with after washed with methanol quartz capillary; After drying, be that the methyl alcohol of 1:1 and 3-(isobutene acyl-oxygen) propyl trimethoxy silicane mixed liquor to be filled with in quartz capillary and to guarantee that Guan Zhongwu produces bubble volume ratio, sealing two ends and to be placed in 60 DEG C of water-baths reaction 12 hours; Finally respectively rinse 30 minutes with first alcohol and water respectively, nitrogen drying 12 hours, obtains through pretreated quartz capillary.
4. a kind of large scale hydrophilic organic polymer liquid phase solid chromatography column according to claim 3, is characterized in that: described large scale hydrophilic organic polymer liquid phase solid chromatography column has continuous print loose structure; It is by the monomer 3-[N of end strips hydrophilic sulfonic acid group, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt sequentially connects crosslinking agent N, and the silanol base of N-methylene-bisacrylamide, 3-(isobutene acyl-oxygen) propyl trimethoxy silicane and quartz capillary inner surface is formed.
5. the preparation method of a kind of large scale hydrophilic organic polymer liquid phase solid chromatography column described in any one of Claims 1 to 4, it is characterized in that comprising following operating procedure: by monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt, crosslinking agent N, N-methylene-bisacrylamide, pore generating agent methyl alcohol and the mixing of initator azodiisobutyronitrile, after ultrasonic degas, pour into internal diameter and be greater than 200 microns in pretreated quartz capillary column, then by quartz capillary sealing two ends, put into water-bath reaction 12 hours, after completion of the reaction, the impurity in removing quartz capillary and unreacted reactant, obtain large scale internal diameter hydrophilic organic polymer liquid phase monolithic chromatographic column.
6. the preparation method of a kind of large scale hydrophilic organic polymer liquid phase solid chromatography column according to claim 5, is characterized in that: it is react 6 ~ 24 hours within the scope of 50 ~ 65 DEG C that described reaction refers in temperature.
7. the preparation method of a kind of large scale hydrophilic organic polymer liquid phase solid chromatography column according to claim 5, it is characterized in that: described monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] mass ratio of propane-1-acid inner salt and crosslinking agent N, N-methylene-bisacrylamide is in 80:20 to 90:10 scope; The quality of described initator azodiisobutyronitrile is monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt and crosslinking agent N, N-methylene-bisacrylamide gross mass 0.5% ~ 1%; Described monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt and the quality sum of crosslinking agent N, N-methylene-bisacrylamide and the mass ratio of pore generating agent methyl alcohol be in 25:75 to 45:55 scope.
8. the preparation method of a kind of large scale hydrophilic organic polymer liquid phase solid chromatography column according to claim 7, it is characterized in that: described monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] mass ratio of propane-1-acid inner salt and crosslinking agent N, N-methylene-bisacrylamide is 87:13; The quality of described initator azodiisobutyronitrile is 1% of monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt and crosslinking agent N, N-methylene-bisacrylamide gross mass; Described monomer 3-[N, N-dimethyl-[2-(2-methyl-prop-2-alkene acyloxy) aminocarbonyl propyl] ammonium] propane-1-acid inner salt and the quality sum of crosslinking agent N, N-methylene-bisacrylamide and the mass ratio of pore generating agent methyl alcohol be 32.5:67.5.
9. the preparation method of a kind of large scale hydrophilic organic polymer liquid phase solid chromatography column according to claim 5, it is characterized in that: the impurity in described removing quartz capillary and unreacted reactant operate according to following steps: be connected with high-pressure pump one end of quartz capillary, rinse with organic solvent; Described organic solvent is at least one in methyl alcohol, acetonitrile and acetone.
10. the application of a kind of large scale hydrophilic organic polymer liquid phase solid chromatography column in base class, ucleosides and small peptide sample separation described in any one of Claims 1 to 4.
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| CN105233810A (en) * | 2015-09-30 | 2016-01-13 | 暨南大学 | Bionic double-chain phospholipid film monolithic column, and making method and application thereof |
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| CN105233810A (en) * | 2015-09-30 | 2016-01-13 | 暨南大学 | Bionic double-chain phospholipid film monolithic column, and making method and application thereof |
| CN105233810B (en) * | 2015-09-30 | 2017-07-04 | 暨南大学 | A kind of bionical dichain phospholipids film integral post and preparation method and application |
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