CN110270133A - A kind of integral post plate preparation method and application based on PE sieve plate conjugation chemistry group - Google Patents
A kind of integral post plate preparation method and application based on PE sieve plate conjugation chemistry group Download PDFInfo
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- CN110270133A CN110270133A CN201910695712.2A CN201910695712A CN110270133A CN 110270133 A CN110270133 A CN 110270133A CN 201910695712 A CN201910695712 A CN 201910695712A CN 110270133 A CN110270133 A CN 110270133A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
Abstract
The invention discloses a kind of integral post plate preparation method based on PE sieve plate conjugation chemistry group, the PE sieve plate containing micropore glass bead CPG is assembled to the assembly position bottom of pass Filter column plate by S1.；It S2. will be in the seal groove of the pass Filter column plate merging negative pressure leaching device equipped with PE sieve plate, after passing through sample-adding liquid separation manipulator addition N- chloromethyl phthalimide, methylene chloride and maintaining 15 minutes, ferric trichloride-benzophenone dichloromethane solution is added and makees catalyst, it is heated to 45 DEG C of reactions and is kept for half an hour, after reaction, PE sieve plate is eluted, is drained, vacuum completes drying；S3. PE sieve plate is eluted with ethyl alcohol and methanol, is filtered, be dried in vacuo aminomethyl micropore glass bead solidifies the integral post plate of PE sieve plate.The invention also discloses integral post plates to synthesize in DNA, nucleic acid extraction and purifying, compound Solid Phase Extraction, biological sample absorption, separation, purifying and concentration, the application in chemical reaction carrier catalysis, blood purification.
Description
Technical field
The present invention relates to polyalcohol integral pole technical fields, and in particular to a kind of based on the whole of PE sieve plate conjugation chemistry group
Scapus plate preparation method and application.
Background technique
Currently, being synthesized for DNA, nucleic acid extraction and purifying, compound Solid Phase Extraction, biological sample absorption, separation, purifying
With concentration, the Related product for chemically reacting the fields such as catalytic carrier, blood purification is mostly single-column pipe, is substantially inside column tube
Using the form of two panels sieve plate folder functional form powder filler, functional form powder filler has been coupled functional type chemistry base in advance
Group, is mainly used to do DNA extraction column and SPE column, and the volume for handling sample is bigger, belongs to single tube form, is unfavorable for batch
Change, scale and miniaturization operation, the processing for micro, ultramicron and trace sample, single-column pipe are also difficult to realize.It is existing
Sample pretreatment single-column pipe, be specifically also manifested by the following aspects: 1) high production cost, use cost is also high, traditional
The production of single-column pipe all relies on manually, and artificial production cost accounts for 30% or more of entire production cost, meanwhile, function is filled out
Expect that amount of fill is more, in use, reagent consumption is big, operating cost is also high；2) functional stuffing consumption is big, functional stuffing wasteness
Also big, traditional single-column pipe, is limited, minimum 1ml column tube, the minimum 30mg of functional stuffing amount of fill in column tube by column tube volume, then
Small amount of fill is difficult control and loads precision, does not need so high amount of fill due to handling micro and trace sample, so
Lead to the waste of functional stuffing；3) processing sample size is limited, primary to test, and most multipotency handles 96 target samples simultaneously；4) place
Sample volume is managed all in 1ml or more, is unable to satisfy the pre-treatment of micro, ultramicron and trace sample, it is required matched in this way
The reagent dosage of biological sample pre-treatment is also very big, not only increases reagent consumption, increases processing cost, and place used
Managing reagent is volatile corrosive liquids poisonous and harmful to human body and environment mostly, and dosage is more, and discharging of waste liquid is more, to people
Body and environmental hazard are bigger；5) the degree of automation is not high, inefficient, and operation is extremely inconvenient, can not realize well automation,
Mass handles biological sample；6) small product size itself is with regard to big, and required matched reagent and filler matrix is also more, product sheet
The production cost of body also remains high；7) it has a single function, is unable to satisfy multi-functional, multiduty demand.
However, with biotechnology, life science, synthetic biology, biological medicine, chemical analysis, food inspection and facing
The fast development of bed diagnostics research needs to carry out DNA synthesis, core to the magnanimity biochemical composition in a large amount of biological sample
Sour extraction and purification, compound Solid Phase Extraction, biological sample absorption, separation, purifying and concentration chemically react carrier catalysis, blood
Liquid purification, to the matched instrument and equipment in the field and reagent consumptive material, more stringent requirements are proposed, such as high-throughput, ultramicron synthesis with
Detection, cost control, multi-functional, environmentally friendly etc., and reduce target sample pre-treatment to greatest extent, synthesizing and purifying reagent makes
Dosage etc..
Summary of the invention
The present invention proposes a kind of integral post plate preparation method and application based on PE sieve plate conjugation chemistry group, solves existing
There is the whole problem that column volume is big, permeability is poor in technology, a kind of preparation method of polyalcohol integral pole plate, this method base are provided
In micropore glass bead (Controlled Pore Glass, abbreviation CPG or silica gel) or the matrix immobilized PE sieve of high molecular polymer
The multiple function pipe lamella carrier of plate conjugation chemistry radical techniques prepares integral post plate, and prepared integral post plate has column bed
Small in size, the advantages that permeability is good, back pressure is low, convective mass transfer speed is fast and preparation is easy, integral post plate is greatly widened
Application range.
The technical scheme of the present invention is realized as follows:
The embodiment of the invention discloses a kind of integral post plate preparation methods based on PE sieve plate conjugation chemistry group, specific to wrap
Include following steps:
S1. it is 3.2mm by diameter, is assembled to 400ul with a thickness of the PE sieve plate containing micropore glass bead CPG of 6.25mm
The assembly position bottom of 384 pass filter plates；
S2. by the seal groove of the 384 pass filter plates merging negative pressure leaching device equipped with PE sieve plate, pass through sample-adding liquid separation
Manipulator is by 1.1mmolN- chloromethyl phthalimide, 120ul methylene chloride, and after maintaining 15min, 6ul is added
0.6mol/L ferric trichloride-benzophenone dichloromethane solution makees catalyst, is heated to 45 DEG C of reactions holdings 0.5 hour, instead
After answering, the N of 0.1mol/L hydrochloric acid, N '-dimethyl formamide solution (3*100ul), N, N '-dimethyl formamide are used respectively
(3*100ul) methylene chloride (5*100ul), methanol (2*100ul) elute PE sieve plate, drain, and vacuum completes drying；
S3. PE sieve plate is eluted with ethyl alcohol (3*100ul) and methanol (3*100ul), suction filtration, be dried in vacuo aminomethyl is micro-
Hole glass pellet (controlled pore glass, CPG) solidifies PE sieve plate, and reaction equation is shown below:
S4. the synthesis that aminomethyl micropore glass bead (controlled pore glass, CPG) solidifies PE sieve plate is completed.
2) between 3- amino -1- (4,4 '-dimethoxytrityl) -1,2-PD arm synthesis
30mmol 3- amino -1,2-PD is added in 30ml trifluoro-acetate, and reaction 12h, reaction knot is stirred at room temperature
Trifluoro-acetate is distilled off after beam, 100ml toluene is added in resultant product, and reflux water removal obtains colorless oil as product 3- trifluoro
Acetamide -1,2-PD.5mmol 3- trifluoroacetamide -1,2-PD is added in 20ml anhydrous pyridine, stirs while adding
Enter 5mmol DMT-Cl, after reaction 12h is stirred at room temperature, 2ml methanol is added and terminates reaction, solvent is distilled off, residue is added
In 50ml ethyl acetate, ethyl acetate successively is washed with saturated sodium bicarbonate solution (3*30ml) and pure water (2*30ml), is collected
Organic phase is added the stirring 2h water removal of 10g anhydrous sodium sulfate, is then filtered to remove sodium sulphate, evaporation removes ethyl acetate, obtains yellowish
The dichloromethane solution of color grease, product silica gel chromatography, 0.1% pyridine and 0~2% methanol is mobile phase progress
Gradient elution obtains light yellow oil 3- trifluoroacetamide -1- (4.4 '-dimethoxytrityl) -1,2-PD.2.5g
The methanol solution of 75ml 9mol, L ammonia is added in 3- trifluoroacetamide -1- (4.4 '-dimethoxytrityl) -1,2-PD
In, reaction is stirred at room temperature overnight, is evaporated to obtain colorless oil as product 3- amino -1- (4,4 '-dimethoxytrityl) -1,2- the third two
Alcohol, spare, reaction equation is shown below.
The synthesis of arm between 3- amino -1- (4,4 '-dimethoxytrityl) -1,2-PD
3) aminomethyl micropore glass bead (controlled pore glass, CPG) solidifies the coupling of sieve plate and arm
The 384 pass filter plate of 400ul of sieve plate will be solidified equipped with CPG, is placed in the seal groove of negative pressure leaching matched with devices
It is interior, under the cooperation of sample-adding liquid separation manipulator, 7.5mg amber is added in equipped with the 384 each holes of pass filter plate for solidifying sieve plate
Amber acid anhydrides and 50ul pyridine, 8ul N- methylimidazole, 42ul tetrahydrofuran (Tetrahydrofuran THF) mixed solution,
It is shaken at room temperature overnight, the solidification sieve plate after reaction uses 50ul pyridine and 50ul tetrahydrofuran to elute respectively, obtains succinamide first
Base micropore glass bead (controlled pore glass, CPG) solidifies sieve plate, then in the 384 each holes of pass filter plate
8ul N- methylimidazole, 5ul acetic anhydride, 5ul 2, the mixed liquor of 6- lutidines and 82ul tetrahydrofuran, room temperature vibration is added
Reaction 45min is swung, respectively with 50ul tetrahydrofuran, 50ul pyridine, the elution of 100ul acetonitrile, is filtered, vacuum drying adds
2.5mmol 3- amino -1- (4,4 '-dimethoxytrityl) -1,2- propylene glycol, 3mmol I-hydroxybenzotriazole, 3mmol
N, N '-dicyclohexylcarbodiimide, 75ul pyridine mixed liquor, 20 DEG C of oscillating reactions are stayed overnight, are filtered.It is eluted again with 50ul acetonitrile
Afterwards, the mixed solution of 10ul triethylamine, 10ul water, 30ul acetonitrile is added, after shaken at room temperature reacts 45min, with 100ul acetonitrile
Elution is dried in vacuo 2h.
The mixing of 60ul 2,6- lutidines and 70ul tetrahydrofuran is added in the 384 each holes of pass filter plate again
Solution, is added formic acid and acetic anhydride mixed solution (volume ratio 2:3 adds 3 times, each 50ul) carries out formylated, room temperature reaction
1h elutes cylinder with 100ul acetonitrile, is dried in vacuo 2h, obtains target product micropore glass bead (controlled pore
Glass, CPG) matrix DNA Solid-phase organic synthesis Universal CPG carrier, reaction equation is shown below.
The connection of aminomethyl micropore glass bead (controlled pore glass, CPG) and arm
It is 20umol/g, i.e. 200nmol/ through spectrophotometry 4,4 ' dimethoxytrityl (DMT) carrying capacity
Hole.
Then the plate can be used as 200nmol, 384 hole DNA hard boards come using.
With same method, the 384 hole DNA hard boards that scale is 0.05nmol-500nmol can also be made.
Further, the PE sieve plate preparation step are as follows:
S11. the selection of raw material: the matrix fill being cured uses the micropore glass bead CPG of 80-150um partial size, high
Molecularly Imprinted Polymer curing agent uses partial size for 50um, the ultra-high molecular weight polyethylene UHMW-PE powder that molecular weight is 4,000,000；
S12. the configuration and mixing of mixed raw material: according to mass ratio, micropore glass bead CPG:UHMW-PE powder=1:1.5
Ratio prepare 25g mixed powder (i.e. 10g micropore glass bead CPG+15gUHMW-PE powder), be mixed by inversion on blending instrument
2h, it is spare；
S13. the design and manufacture of the cylindrical sieve plate curing mold of diameter 3.2mm, thickness 6.25mm: it is in side length
250.0mm, with a thickness of using slow wire feeding technique to cut out 1000 diameters equidistantly as 3.4mm on 6.5mm aluminium block, depth 6.5mm
Cylindrical hole, using the technologies retrofit such as mirror surface treatment, surface polishing, it is ensured that after mold hole precision≤0.02mm, spraying
High-temperaure coating, it is spare after Drying and cooling, the method for using the technologies retrofit such as mirror surface treatment, surface polishing make side length for
250.0mm, the upper and lower cover plates of thickness 10.0mm, after storming and capturing screw hole location with tapping machine, equally spraying high-temperaure coating, drying are cold
But spare after；
S14. mixed powder is die-filling: being 3.4mm containing 1000 diameters, depth is the aluminum mould of the cylindrical hole of 6.5mm
In tool, the micropore glass bead CPG:UHMW-PE blended powder mixed is uniformly added, with shaking platform (vibration frequency 50HZ, amplitude
3mm, time of vibration 60 seconds) to add powder vibration while vibrating flat, and upper cover plate is covered, is locked；
S15. powder curing: being heated to 220 DEG C, keeps the temperature 70min, rear by being cooled to room temperature, and takes out cylindrical solidification sieve
Plate.
Further, the 384 pass filter plate of 400ul is molded using the PP material (polypropylene material) of medical grade
Production forms the filter plate in 384 holes.
The integral post plate of the preparation method preparation of integral post plate based on PE sieve plate conjugation chemistry group is synthesized in DNA, core
Sour extraction and purification, compound Solid Phase Extraction, biological sample absorption, separation, purifying and concentration chemically react carrier catalysis, blood
Application in liquid purification.
Compared with prior art, the beneficial effects of the present invention are:
1, solve the problems, such as that high production cost, use cost are high；
Three steps of traditional Sample pretreatment single-column pipe loading method point: the first step loads a piece of lower sieve plate into void column pipe,
Then, the functional stuffing of approrpiate wts is weighed loaded in column tube；Then, on piece sieve is put into the column tube equipped with functional stuffing
Upper sieve plate is compacted with tool, is loaded, by plate.Entire production operation process is too dependent on manually, relatively complicated, and
It is limited to little power accurate weighing distribution technique, is difficult to realize automation, manually weighs and distributes substantially, causes product raw
It produces cost to remain high, method of the invention, the directly function carrier for being made of one are suitable for automation equipment and fill out automatically
Dress, intermediate link can be completed without artificial participation, and production cost substantially reduces.In addition, the function carrier after solidifying, is using
50% target sample pre-treatment reagent dosage can be at least saved in cost, save 50% or more use cost.
2, the pre-treatment of micro, denier and trace target sample is solved the problems, such as；
Traditional target sample pre-treatment column tube handles sample volume all in 1ml or more, is unable to satisfy micro, ultramicron
And the pre-treatment of trace sample, the reagent dosage of matched biological sample pre-treatment required in this way is also very big, not only increases
Reagent consumption, increases processing cost, and reagent treatment used is poisonous and harmful to human body and environment volatile mostly
Corrosive liquids, dosage is more, and discharging of waste liquid is more, bigger to human body and environmental hazard.The present invention, using unlimited proportional diluted
The mixed powder of configuration makes micropore glass bead in carrier (Controlled Pore Glass, abbreviation CPG or silica gel) or height
Molecularly Imprinted Polymer matrix content can be near minimum, has saved cost of material, improves micro, denier and trace target sample
Pre-treatment ability, and environmental protection.
3, Fillers selection hardly possible is solved the problems, such as；
Using the curing mechanism of powdered substrate and PE powder, the functional stuffing partial size of conventional target Sample pretreatment is broken
>=20um limitation becomes a reality so that≤20um even nm scaled powders matrix handles the application in product before target sample.
4, functional stuffing or carrier are solved the problems, such as to target product selectivity absorption and reserve capability；
Specific surface area is bigger, the stronger nm scaled powders matrix of adsorption capacity handles answering in product before target sample
With, significantly enhance the absorption and reserve capability of target substance, non-targeted product, impurity interference extremely strong elimination ability, make
The TIANZHU XINGNAO Capsul for obtaining biological sample is very high, and Sample pretreatment process becomes more handy.
5, solve the problems, such as that functional stuffing volume is excessive in conventional target Sample pretreatment column tube；
Presently commercially available conventional target Sample pretreatment column tube, the diameter and thickness of round sieve plate folder powder filler is substantially all
In 5mm or more, and micro column tube lamella carrier of the invention, diameter and thickness can achieve 1mm hereinafter, in the world " most
It is small " target sample pre-treatment column tube lamella carrier.
6, it solves the problems, such as limited with an Experimental lots processing target sample ability；
Conventional target Sample pretreatment column tube processing sample size is limited, primary to test, and most multipotency handles 96 targets simultaneously
Sample, product of the invention, primary experiment can handle up to 384 even more than target sample, greatly improve mesh
Mark the ability of Sample pretreatment mass, scale.
7, vdiverse in function, solve multi-functional, multiduty demand；
It is not coupled micropore glass bead (the Controlled Pore of special chemical group since PE sieve plate has cured in advance
Glass, abbreviation CPG or silica gel) or high molecular polymer matrix, the carrier after this solidification be loaded onto suitable column tube and hole
After in plate, it can be needed to add the chemical group with different function according to experiment, carry out very flexible processing target sample
Predetermined substance carries out selective absorption, purifying to predetermined substance, target product and is concentrated and separates, greatly facilitates use
Person, using it becomes more flexible, more convenient.
8, reduce the discharge of pollutant, green, environmental protection；
Since matrix fill being solidificated on high molecular polymer and (powder filler is inlaid on high polymer sieve plate), institute
The solidification sieve plate of formation, reagent runner is relatively fixed, and reagent used in clear and rich function sieve plate is than isometric traditional SPE column
At least 40% is saved, so, either applied sample amount, or activation, balance, amount of reagent will be than traditional mesh used in elution cylinder
Mark Sample pretreatment link saves nearly 50% reagent.Using the present invention, while economizing on resources, before reducing target sample
The reagent of processing consumes, and discharging of waste liquid is minimized, environment is protected.
9, in addition, 96 orifice plate target sample pretreatment function carriers and 384 orifice plate target sample pretreatment function carriers
It introduces, is at home pole in target sample pre-treatment, filtering/extraction/desalination/purifying/concentrating of target product and separation industries
Big innovation solves the problems, such as mass, scale processing sample ability, so that being disposably capable of handling 96 or 384 samples
This, it is even more, the efficiency of sample process is greatly improved, enterprise production management cost is reduced, accelerates experiment process,
Have to entire target sample pre-treatment industry and greatly pushes.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 is 384 pass filter plate front views in embodiment one；
Fig. 2 is 384 pass filter plate side views in embodiment one；
Fig. 3 is 96 pass filter plate main views in embodiment seven；
Fig. 4 is 96 pass filter plate side views in embodiment seven.
Specific embodiment
The present invention can be further appreciated that by the specific embodiment of invention now given below, but they are not to this hair
Bright restriction.Some nonessential improvement and adjustment according to made by foregoing invention content for those skilled in the art,
It is considered as and is within the scope of the present invention.
Embodiment one:
Such as Fig. 1 and Fig. 2,400ul is prepared, 384 orifice plate DNA synthetic vectors integral post plates are divided into diameter 3.2mm, thickness
The cylindrical solidification sieve plate and its 384 pass filter plate of mating 400ul of 6.25mm.
1, the design, die sinking of 384 pass filter plate of 400ul and injection molding produce:
Hollow plate long 127.3mm, wide 85.0mm, high 41.46mm, plate surrounding frame width 2.5mm, plate body are uniformly equally spaced
384 holes, hole top are cuboid cavity, and side length 3.7mm, high 25.0mm, cuboid cavity lower end is gradually closed up to 3.2mm and filled
Coordination, assembly position are cylindrical cavity, diameter 3.2mm, height 6.25mm.Lower part is closed up to 1.5mm, and length is ultimately formed
For 10.0mm conical cavity to bottom end, bottom end point mouth point is using flat mouth, angle or sharp-crested design.
Injection molding machine is then used, injection molding production is carried out using the PP material (polypropylene material) of medical grade, forms the filtering of 384 holes
Plate, it is spare.
2, the cylinder of diameter 3.2mm, thickness 6.25mm solidify the preparation (by 1000 calculating of preparation) of sieve plate:
1) selection of raw material: the matrix fill being cured uses the micropore glass bead CPG of 80-150um partial size, macromolecule
Polymer curing agents use partial size for 50um, the ultra-high molecular weight polyethylene UHMW-PE powder that molecular weight is 4,000,000.
2) configuration and mixing of mixed raw material: according to mass ratio, micropore glass bead CPG:UHMW-PE powder=1:1.5
Ratio prepares 25g mixed powder (i.e. 10g micropore glass bead CPG+15gUHMW-PE powder), and 2h is mixed by inversion on blending instrument,
It is spare.
3) design and manufacture of the cylindrical sieve plate curing mold of diameter 3.2mm, thickness 6.25mm: it is in side length
250.0mm, with a thickness of using slow wire feeding technique to cut out 1000 diameters equidistantly as 3.4mm on 6.5mm aluminium block, depth 6.5mm
Cylindrical hole, using the technologies retrofit such as mirror surface treatment, surface polishing, it is ensured that after mold hole precision≤0.02mm, spraying
High-temperaure coating, it is spare after Drying and cooling.Use the technologies retrofit such as mirror surface treatment, surface polishing method make side length for
250.0mm, the upper and lower cover plates of thickness 10.0mm, after storming and capturing screw hole location with tapping machine, equally spraying high-temperaure coating, drying are cold
But spare after.
4) mixed powder is die-filling: being 3.4mm containing 1000 diameters, depth is the aluminium-making mould of the cylindrical hole of 6.5mm
It is interior, the micropore glass bead CPG:UHMW-PE blended powder mixed is uniformly added, with shaking platform (vibration frequency 50HZ, amplitude
3mm, time of vibration 60 seconds) to add powder vibration while vibrating flat, and upper cover plate is covered, is locked.
5) powder curing: being heated to 220 DEG C, keeps the temperature 70min, rear by being cooled to room temperature, and takes out cylindrical solidification sieve plate.
3, it assembles:
With limit push rod or full-automatic filling equipment by diameter 3.2mm, the cylindrical solidification sieve plate of thickness 6.25mm is assembled
To 400ul, 384 hole filters assemble position bottom.
4, the coupling of general CPG functional chemical group:
1) aminomethyl micropore glass bead (controlled pore glass, CPG) solidifies the synthesis of sieve plate:
The 384 pass filter plate of 400ul of sieve plate will be solidified equipped with CPG, is placed in the seal groove of negative pressure leaching matched with devices
It is interior, under the cooperation of sample-adding liquid separation manipulator, 1.1mmolN- chloromethane is added in equipped with the 384 pass filter plates for solidifying sieve plate
After maintaining 15min, 6ul 0.6mol/L ferric trichloride-benzophenone is added in base phthalimide, 120ul methylene chloride
Dichloromethane solution make catalyst, 45 DEG C of reaction 0.5h use the N of 0.1mol/L hydrochloric acid, N '-diformazan after reaction respectively
Base formamide solution (3*100ul), N, N '-dimethyl formamide (3*100ul) methylene chloride (5*100ul), methanol (2*
100ul) elution solidification sieve plate, is drained, and is dried in vacuo, and the sieve plate that solidifies after drying is added in the ethanol solution of 5% hydrazine of 200ul,
0.5h is reacted, is then eluted with ethyl alcohol (3*100ul), methanol (3*100ul), is filtered, be dried in vacuo to obtain aminomethyl micropore glass
Bead (controlled pore glass, CPG) solidifies sieve plate, and reaction equation is shown below.
Aminomethyl micropore glass bead (controlled pore glass, CPG) solidifies the synthesis of sieve plate
2) between 3- amino -1- (4,4 '-dimethoxytrityl) -1,2-PD arm synthesis
30mmol 3- amino -1,2-PD is added in 30ml trifluoro-acetate, and reaction 12h, reaction knot is stirred at room temperature
Trifluoro-acetate is distilled off after beam, 100ml toluene is added in resultant product, and reflux water removal obtains colorless oil as product 3- trifluoro
Acetamide -1,2-PD.5mmol 3- trifluoroacetamide -1,2-PD is added in 20ml anhydrous pyridine, stirs while adding
Enter 5mmol DMT-Cl, after reaction 12h is stirred at room temperature, 2ml methanol is added and terminates reaction, solvent is distilled off, residue is added
In 50ml ethyl acetate, ethyl acetate successively is washed with saturated sodium bicarbonate solution (3*30ml) and pure water (2*30ml), is collected
Organic phase is added the stirring 2h water removal of 10g anhydrous sodium sulfate, is then filtered to remove sodium sulphate, evaporation removes ethyl acetate, obtains yellowish
The dichloromethane solution of color grease, product silica gel chromatography, 0.1% pyridine and 0~2% methanol is mobile phase progress
Gradient elution obtains light yellow oil 3- trifluoroacetamide -1- (4.4 '-dimethoxytrityl) -1,2-PD.2.5g
The methanol solution of 75ml 9mol, L ammonia is added in 3- trifluoroacetamide -1- (4.4 '-dimethoxytrityl) -1,2-PD
In, reaction is stirred at room temperature overnight, is evaporated to obtain colorless oil as product 3- amino -1- (4,4 '-dimethoxytrityl) -1,2- the third two
Alcohol, spare, reaction equation is shown below:
The synthesis of arm between 3- amino -1- (4,4 '-dimethoxytrityl) -1,2-PD
3) aminomethyl micropore glass bead (controlled pore glass, CPG) solidifies the coupling of sieve plate and arm:
The 384 pass filter plate of 400ul of sieve plate will be solidified equipped with CPG, is placed in the seal groove of negative pressure leaching matched with devices
It is interior, under the cooperation of sample-adding liquid separation manipulator, 7.5mg amber is added in equipped with the 384 each holes of pass filter plate for solidifying sieve plate
Amber acid anhydrides and 50ul pyridine, 8ul N- methylimidazole, 42ul tetrahydrofuran (Tetrahydrofuran THF) mixed solution,
It is shaken at room temperature overnight, the solidification sieve plate after reaction uses 50ul pyridine and 50ul tetrahydrofuran to elute respectively, obtains succinamide first
Base micropore glass bead (controlled pore glass, CPG) solidifies sieve plate, then in the 384 each holes of pass filter plate
8ul N- methylimidazole, 5ul acetic anhydride, 5ul 2, the mixed liquor of 6- lutidines and 82ul tetrahydrofuran, room temperature vibration is added
Reaction 45min is swung, respectively with 50ul tetrahydrofuran, 50ul pyridine, the elution of 100ul acetonitrile, is filtered, vacuum drying adds
2.5mmol 3- amino -1- (4,4 '-dimethoxytrityl) -1,2- propylene glycol, 3mmol I-hydroxybenzotriazole, 3mmol
N, N '-dicyclohexylcarbodiimide, 75ul pyridine mixed liquor, 20 DEG C of oscillating reactions are stayed overnight, are filtered.It is eluted again with 50ul acetonitrile
Afterwards, the mixed solution of 10ul triethylamine, 10ul water, 30ul acetonitrile is added, after shaken at room temperature reacts 45min, with 100ul acetonitrile
Elution is dried in vacuo 2h.
The mixing of 60ul 2,6- lutidines and 70ul tetrahydrofuran is added in the 384 each holes of pass filter plate again
Solution, is added formic acid and acetic anhydride mixed solution (volume ratio 2:3 adds 3 times, each 50ul) carries out formylated, room temperature reaction
1h elutes cylinder with 100ul acetonitrile, is dried in vacuo 2h, obtains target product micropore glass bead (controlled pore
Glass, CPG) matrix DNA Solid-phase organic synthesis Universal CPG carrier, reaction equation is shown below.
The connection of aminomethyl micropore glass bead (controlled pore glass, CPG) and arm
It is 20umol/g, i.e. 200nmol/ through spectrophotometry 4,4 ' dimethoxytrityl (DMT) carrying capacity
Hole.
Then the plate can be used as 200nmol, 384 hole DNA hard boards come using.
With same method, the 384 hole DNA hard boards that scale is 0.05nmol-500nmol can also be made.
Compared with prior art, the beneficial effects of the present invention are:
1, solve the problems, such as that high production cost, use cost are high；
Three steps of traditional Sample pretreatment single-column pipe loading method point: the first step loads a piece of lower sieve plate into void column pipe,
Then, the functional stuffing of approrpiate wts is weighed loaded in column tube；Then, on piece sieve is put into the column tube equipped with functional stuffing
Upper sieve plate is compacted with tool, is loaded, by plate.Entire production operation process is too dependent on manually, relatively complicated, and
It is limited to little power accurate weighing distribution technique, is difficult to realize automation, manually weighs and distributes substantially, causes product raw
It produces cost to remain high, method of the invention, the directly function carrier for being made of one are suitable for automation equipment and fill out automatically
Dress, intermediate link can be completed without artificial participation, and production cost substantially reduces.In addition, the function carrier after solidifying, is using
50% target sample pre-treatment reagent dosage can be at least saved in cost, save 50% or more use cost.
2, the pre-treatment of micro, denier and trace target sample is solved the problems, such as；
Traditional target sample pre-treatment column tube handles sample volume all in 1ml or more, is unable to satisfy micro, ultramicron
And the pre-treatment of trace sample, the reagent dosage of matched biological sample pre-treatment required in this way is also very big, not only increases
Reagent consumption, increases processing cost, and reagent treatment used is poisonous and harmful to human body and environment volatile mostly
Corrosive liquids, dosage is more, and discharging of waste liquid is more, bigger to human body and environmental hazard.The present invention, using unlimited proportional diluted
The mixed powder of configuration makes micropore glass bead in carrier (Controlled Pore Glass, abbreviation CPG or silica gel) or height
Molecularly Imprinted Polymer matrix content can be near minimum, has saved cost of material, improves micro, denier and trace target sample
Pre-treatment ability, and environmental protection.
3, Fillers selection hardly possible is solved the problems, such as；
Using the curing mechanism of powdered substrate and PE powder, the functional stuffing partial size of conventional target Sample pretreatment is broken
>=20um limitation becomes a reality so that≤20um even nm scaled powders matrix handles the application in product before target sample.
4, functional stuffing or carrier are solved the problems, such as to target product selectivity absorption and reserve capability；
Specific surface area is bigger, the stronger nm scaled powders matrix of adsorption capacity handles answering in product before target sample
With, significantly enhance the absorption and reserve capability of target substance, non-targeted product, impurity interference extremely strong elimination ability, make
The TIANZHU XINGNAO Capsul for obtaining biological sample is very high, and Sample pretreatment process becomes more handy.
5, solve the problems, such as that functional stuffing volume is excessive in conventional target Sample pretreatment column tube；
Presently commercially available conventional target Sample pretreatment column tube, the diameter and thickness of round sieve plate folder powder filler is substantially all
In 5mm or more, and micro column tube lamella carrier of the invention, diameter and thickness can achieve 1mm hereinafter, in the world " most
It is small " target sample pre-treatment column tube lamella carrier.
6, it solves the problems, such as limited with an Experimental lots processing target sample ability
Conventional target Sample pretreatment column tube processing sample size is limited, primary to test, and most multipotency handles 96 targets simultaneously
Sample, product of the invention, primary experiment can handle up to 384 even more than target sample, greatly improve mesh
Mark the ability of Sample pretreatment mass, scale.
7, vdiverse in function, solve multi-functional, multiduty demand；
It is not coupled micropore glass bead (the Controlled Pore of special chemical group since PE sieve plate has cured in advance
Glass, abbreviation CPG or silica gel) or high molecular polymer matrix, the carrier after this solidification be loaded onto suitable column tube and hole
After in plate, it can be needed to add the chemical group with different function according to experiment, carry out very flexible processing target sample
Predetermined substance carries out selective absorption, purifying to predetermined substance, target product and is concentrated and separates, greatly facilitates use
Person, using it becomes more flexible, more convenient.
8, reduce the discharge of pollutant, green, environmental protection；
Since matrix fill being solidificated on high molecular polymer and (powder filler is inlaid on high polymer sieve plate), institute
The solidification sieve plate of formation, reagent runner is relatively fixed, and reagent used in clear and rich function sieve plate is than isometric traditional SPE column
At least 40% is saved, so, either applied sample amount, or activation, balance, amount of reagent will be than traditional mesh used in elution cylinder
Mark Sample pretreatment link saves nearly 50% reagent.Using the present invention, while economizing on resources, before reducing target sample
The reagent of processing consumes, and discharging of waste liquid is minimized, environment is protected.
9, in addition, 96 orifice plate target sample pretreatment function carriers and 384 orifice plate target sample pretreatment function carriers
It introduces, is at home pole in target sample pre-treatment, filtering/extraction/desalination/purifying/concentrating of target product and separation industries
Big innovation solves the problems, such as mass, scale processing sample ability, so that being disposably capable of handling 96 or 384 samples
This, it is even more, the efficiency of sample process is greatly improved, enterprise production management cost is reduced, accelerates experiment process,
Have to entire target sample pre-treatment industry and greatly pushes.
Embodiment two:
600ul is prepared, DNA (nucleic acid) is small to mention integral post, silica Silica content 0.25mg, DNA extracted amount 0-
5ug, is divided into diameter 2.25mm, and the cylindrical solidification sieve plate of thickness 1.0mm and its matched 600ul are centrifuged tubing void column pipe.
1, the design, die sinking and injection molding that 600ul is centrifuged tubing void column pipe produce:
600ul centrifugation tubing void column pipe size of adopting international standards opens injection mold, expects (poly- third with the PP of medical grade
Alkene material) carry out injection molding production.
2, the cylinder of diameter 2.25mm, thickness 1.0mm solidify the preparation (by 1000 calculating of preparation) of sieve plate:
1) raw material selects: using silicon dioxide powder Silica and the molecular weight of 300nm partial size for 4,000,000, partial size is
The UHMW-PE powder of 25um, according to volume ratio, silicon dioxide powder Silica:UHMW-PE powder=1:1.5 ratio prepares mixing
Powder is mixed by inversion 2h on blending instrument, spare.
3) design and manufacture of the cylindrical sieve plate curing mold of diameter 2.25mm, thickness 1.0mm: it is in side length
250.0mm, with a thickness of using slow wire feeding technique to cut out 1000 diameters equidistantly as 2.4mm on 1.2mm aluminium block, depth 1.2mm
Cylindrical hole, pass through the technologies retrofit such as mirror surface treatment, surface polishing, it is ensured that after mold hole precision≤0.02mm, spraying
High-temperaure coating, it is spare after Drying and cooling.Use the technologies retrofit such as mirror surface treatment, surface polishing method make side length for
250.0mm, the upper and lower cover plates of thickness 10.0mm, after storming and capturing screw hole location with tapping machine, equally spraying high-temperaure coating, drying are cold
But spare after.
4) mixed powder is die-filling: being 2.4mm containing 1000 diameters, depth is the aluminium-making mould of the cylindrical hole of 1.2mm
It is interior, the silicon dioxide powder Silica mixed and UHMW-PE blended powder are uniformly added, with shaking platform (vibration frequency 50HZ, amplitude
3mm, time of vibration 60 seconds) to add powder vibration while vibrating flat, and upper cover plate is covered, is locked.
5) mixed powder solidifies: being heated to 220 DEG C, is cooled to room temperature again after keeping the temperature 60min, takes out cylindrical solidification sieve
Plate.
3, it assembles:
With limit push rod or full-automatic filling equipment by diameter 2.25mm, the cylindrical solidification sieve plate of thickness 1.0mm is assembled
Tubing void column bottom of the tube is centrifuged to 600ul.
4, the coupling of hydroxy-functional chemical group:
It will be centrifuged tubing column tube equipped with the 600ul for solidifying sieve plate, is placed in the sealing hole of negative pressure leaching matched with devices, is adding
Under the cooperation of sample liquid separation manipulator, using the method for piranha, gradually added in three times in equipped with the single-column pipe for solidifying sieve plate
The volume ratio of 100ul is the concentrated sulfuric acid: non-bonded silica gel is activated in 85 DEG C of reaction 0.5h in the piranha solution of H2O2=2:1
In after our hydroxyl groups that need, be heated to 80 degree after cleaning, keep the temperature 5 hours, drying, wherein silicon dioxide powder is even
Connection hydroxyl, which becomes Silica functional stuffing, can do nucleic acid extraction or solid-phase extraction column plate, structure after overactivation are as follows:
(note: piranha solution is the concentrated sulfuric acid and dioxygen aqueous mixtures of heat, for removing all organic matters, while can
So that hydroxylating occurs for the surface of most of material.The elemental oxygen that H2SO4+H2O2 → H3O++HSO4-+O is generated is extremely strong
Oxidant.)
The integral post can be used as 600ul, DNA (nucleic acid) is small mention column come using.
With same method, the scale that can also make is up to DNA (nucleic acid) extraction column of g grades of 2ml-300ml.
Embodiment three:
1ml, C18 18 alkyl silica gel integral post are prepared, C18 powder filler content 130mg is divided into diameter 5.8mm, thick
Spend the cylindrical function sieve plate and its mating 1ml syringe class void column pipe of 30mm.
1, the design, die sinking of 1ml syringe class void column pipe and injection molding produce:
1ml syringe class void column pipe size of adopting international standards opens injection mold, expects (polypropylene with the PP of medical grade
Material) carry out injection molding production.
2, the cylinder of diameter 5.8mm, thickness 30.0mm solidify the preparation (by 100 calculating of preparation) of sieve plate:
1) selection of raw material: the matrix fill being cured uses the silica white of 40-70um partial size, high molecular polymer solidification
Agent uses partial size for 100um, the ultra-high molecular weight polyethylene UHMW-PE powder that molecular weight is 3,000,000.
2) configuration and mixing of mixed raw material: according to mass ratio, silica white: UHMW-PE powder=1:1.5 ratio is prepared
33g mixed powder (i.e. 13.2g silica white+19.8gUHMW-PE powder), is mixed by inversion 2h on blending instrument, spare.
3) design and manufacture of the cylindrical sieve plate curing mold of diameter 5.8mm, thickness 30.0mm: it is in side length
150.0mm, with a thickness of using slow wire feeding technique to cut out 100 diameters equidistantly as 6.0mm on 30.5mm aluminium block, depth 30.5mm
Cylindrical hole, pass through the technologies retrofit such as mirror surface treatment, surface polishing, it is ensured that sprayed after mold hole precision≤0.02mm resistance to
High temperature coating, it is spare after Drying and cooling.Use the technologies retrofit such as mirror surface treatment, surface polishing method make side length for
150.0mm, the upper and lower cover plates of thickness 10.0mm or so after storming and capturing screw hole location with tapping machine, equally spray high-temperaure coating, dry
It is dry and cold rear spare.
4) mixed powder is die-filling: containing 100 6.0mm, in the cylindrical aluminium-making mould of thickness 30.5mm, is uniformly adding
Add the silica white mixed and UHMW-PE blended powder, with shaking platform (vibration frequency 50HZ, amplitude 3mm, time of vibration 60 seconds) one
The vibration of addition powder is flat on one side for side vibration, covers upper cover plate, locks.
5) mixed powder solidifies: being heated to 220 DEG C, passes through after heat preservation 40min and be cooled to room temperature, takes out cylindrical solidification and sieve
Plate.
3, it assembles:
With limit push rod or full-automatic filling equipment by diameter 5.8mm, the cylindrical solidification sieve plate of thickness 30.0mm is assembled
To 1ml syringe class void column bottom of the tube.
4, the coupling of octadecyl functional chemical group:
By equipped with the 1ml syringe class column tube for solidifying sieve plate, it is placed in the sealing hole of negative pressure leaching matched with devices, is being loaded
Under the cooperation of liquid separation manipulator, 0.2ml dehydrated alcohol is successively added in equipped with the single-column pipe for solidifying sieve plate, 0.3ml is added and goes
Ionized water adds 0.4ml octadecyl trimethoxysilane (C18) liquid, keeps flow velocity 0.2d/min, is guaranteeing that cylinder is clear
In the case where profit, step more than three times is operated repeatedly, after reaction, is drained the reagent inside cylinder, is added 1ml ammonium hydroxide,
Flow velocity 0.2d/min is kept, repeats above step twice, it is after reaction, multiple with ethanol washing, it is dry in vacuum oven
Processing, silicon dioxide powder, which is coupled octadecyl, becomes C18 functional stuffing, can do C18 solid-phase extraction column, structure are as follows:
The integral post can be used as 1ml, C18 18 alkyl silica gel column come using.
With same method, the scale that can also make is up to the C18 18 alkyl silica gel integral post of g grades of 2ml-300ml.
Example IV:
3ml, WCX (cation exchange) integral post are prepared, polystyrene PS powder filler content 267mg is divided into diameter
The cylindrical function sieve plate of 9.0mm, thickness 30mm and its mating 3ml syringe class void column pipe.
1, the design, die sinking of 3ml syringe class void column pipe and injection molding produce:
3ml syringe class void column pipe size of adopting international standards opens injection mold, expects (polypropylene with the PP of medical grade
Material) carry out injection molding production.
2, the cylinder of diameter 9.0mm, thickness 30.0mm solidify the preparation (by 100 calculating of preparation) of sieve plate:
1) selection of raw material: the matrix fill being cured uses the polystyrene PS powder of 40-80um partial size, polyphosphazene polymer
Closing object curing agent uses partial size for 100um, the ultra-high molecular weight polyethylene UHMW-PE powder that molecular weight is 3,000,000.
2) configuration and mixing of mixed raw material: according to mass ratio, polystyrene PS powder: UHMW-PE powder=1:2 ratio
Example prepares 80g mixed powder (i.e. 26.7g polystyrene PS powder+53.3gUHMW-PE powder), and 2h is mixed by inversion on blending instrument,
It is spare.
3) design and manufacture of the cylindrical sieve plate curing mold of diameter 9.0mm, thickness 30.0mm: it is in side length
200.0mm, with a thickness of using slow wire feeding technique to cut out 100 diameters equidistantly as 9.25mm on 30.5mm aluminium block, depth is
The cylindrical hole of 30.5mm is passing through the technologies retrofit such as mirror surface treatment, surface polishing, it is ensured that after mold hole precision≤0.02mm
High-temperaure coating is sprayed, it is spare after Drying and cooling.Side is made using the method for the technologies retrofit such as mirror surface treatment, surface polishing
A length of 200.0mm, the upper and lower cover plates of thickness 10.0mm or so, after storming and capturing screw hole location with tapping machine, the same high temperature resistant that sprays is applied
Layer, it is spare after Drying and cooling.
4) mixed powder is die-filling: containing 100 9.25mm, in the cylindrical aluminium-making mould of thickness 30.5mm, uniformly
The polystyrene PS powder mixed and UHMW-PE blended powder are added, with shaking platform (vibration frequency 50HZ, amplitude 3mm, vibration
60 seconds time) to add powder vibration while vibrating flat, and upper cover plate is covered, is locked.
5) mixed powder solidifies: being heated to 160 DEG C, leads to after heat preservation 50min and be cooled to room temperature, takes out cylindrical solidification and sieve
Plate.
3, it assembles:
With limit push rod or full-automatic filling equipment by diameter 9.0mm, the cylindrical solidification sieve plate of thickness 30.0mm is assembled
To 3ml syringe class void column bottom of the tube.
4, the coupling of carboxyl functionality chemical group:
1) polystyrene PS solidifies the acetylation of sieve plate:
By equipped with the 3ml syringe class column tube for solidifying sieve plate, it is placed in the sealing hole of negative pressure leaching matched with devices, is being loaded
Under the cooperation of liquid separation manipulator, 0.6ml methylene chloride (DCM), 0.6ml are added in order in equipped with the single-column pipe for solidifying sieve plate
Acetylation reagent (Reg) adds the catalyst (Cat) of 1ml, reacts 20min under normal temperature and pressure reversely in the case where logical nitrogen,
After reaction, successively clean suction filtration with THF, cryosel sour (3%), distilled water, until filtrate with silver nitrate solution detection without chlorine from
It is again with methanol diafiltration 5 times, spare after vacuum drying until son.
2) the polystyrene PS after acetylation solidifies the carboxylated of sieve plate:
The 3ml syringe class column tube of sieve plate will be solidified equipped with the polystyrene PS after acetylation, is placed in negative pressure leaching device
In matched sealing hole, under the cooperation of sample-adding liquid separation manipulator, the dichloro of 1ml is added in equipped with the single-column pipe for solidifying sieve plate
Methane (DCM) keeps flow velocity 0.2d/min, in the case where guaranteeing that cylinder is clear and rich, operates step more than three times, reaction knot repeatedly
Bromine water, KOH, water are prepared into bromine water aqueous slkali at 0 DEG C by Shu Hou；This solution is added in column tube, flow velocity 0.2d/ is kept
Min operates step more than three times in the case where guaranteeing that cylinder is clear and rich repeatedly, after reaction, uses the dilute of suitable volumes respectively
Hydrochloric acid, distilled water, ethyl alcohol, methanol are sufficiently washed, are filtered, after vacuum drying.
Its reaction equation is shown below:
The integral post can be used as 3ml, WCX (cation exchange) integral post come using.
With same method, the WCX (cation exchange) that the scale that can also make is up to g grades of 1ml-300ml is whole
Column.
Embodiment five:
Prepare 6ml, SCX phenylbenzimidazole sulfonic acid strong cation exchange integral post, non-bonded silica gel Silica powder filler content
720mg/ branch is divided into diameter 13.0mm, the cylindrical function sieve plate of thickness 30mm and its mating 6ml syringe class void column pipe.
1, the design, die sinking of 6ml syringe class void column pipe and injection molding produce:
6ml syringe class void column pipe size of adopting international standards opens injection mold, expects (polypropylene with the PP of medical grade
Material) carry out injection molding production.
2, the cylinder of diameter 13.0mm, thickness 30.0mm solidify the preparation (by 100 calculating of preparation) of sieve plate:
1) selection of raw material: the matrix fill being cured uses the non-bonded silica gel Silica powder of 40-80um partial size,
High molecular polymer curing agent uses partial size for 100um, the ultra-high molecular weight polyethylene UHMW-PE powder that molecular weight is 3,000,000.
2) configuration and mixing of mixed raw material: according to mass ratio, non-bonded silica gel Silica powder: powder=1 UHMW-PE:
1.5 ratio prepares 180g mixed powder (i.e. the non-bonded silica gel Silica powder+108gUHMW-PE powder of 72g), in blending instrument
On be mixed by inversion 2h, it is spare.
3) design and manufacture of the cylindrical sieve plate curing mold of diameter 13.0mm, thickness 30.0mm: it is in side length
250.0mm, with a thickness of using slow wire feeding technique to cut out 100 diameters equidistantly as 13.3mm on 30.5mm aluminium block, depth is
The cylindrical hole of 30.5mm is passing through the technologies retrofit such as mirror surface treatment, surface polishing, it is ensured that after mold hole precision≤0.02mm
High-temperaure coating is sprayed, it is spare after Drying and cooling.Side is made using the method for the technologies retrofit such as mirror surface treatment, surface polishing
A length of 250.0mm, the upper and lower cover plates of thickness 10.0mm or so, after storming and capturing screw hole location with tapping machine, the same high temperature resistant that sprays is applied
Layer, it is spare after Drying and cooling.
4) mixed powder is die-filling: containing 100 13.3mm, in the cylindrical aluminium-making mould of thickness 30.5mm, uniformly
The non-bonded silica gel Silica powder mixed and UHMW-PE blended powder are added, with shaking platform (vibration frequency 50HZ, amplitude
3mm, time of vibration 60 seconds) to add powder vibration while vibrating flat, and upper cover plate is covered, is locked.
5) mixed powder solidifies: 220 DEG C are heated to, 50min is kept the temperature, it is rear by being cooled to room temperature, and take out cylindrical solidification
Sieve plate.
3, it assembles:
With limit push rod or full-automatic filling equipment by diameter 13.0mm, the cylindrical solidification sieve plate of thickness 30.0mm is assembled
To 6ml syringe class void column bottom of the tube.
4, the coupling of phenylbenzimidazole sulfonic acid strong cation functional chemical group:
1) non-bonded silica gel Silica solidifies the pretreatment of sieve plate:
By equipped with the 6ml syringe class column tube for solidifying sieve plate, it is placed in the sealing hole of negative pressure leaching matched with devices, is being loaded
Under the cooperation of liquid separation manipulator, the Loprazolam water that 4ml mass fraction is 5% is added in equipped with the single-column pipe for solidifying sieve plate
Solution keeps flow velocity 0.2d/min, in the case where guaranteeing that cylinder is clear and rich, operates step more than three times repeatedly, after reaction,
The reagent inside cylinder is drained, is washed with distilled water repeatedly, until filtrate is neutrality；It filters, in 80 DEG C of vacuum ovens at drying
After reason, then it is placed in drier and saves backup.
2) non-bonded silica gel Silica solidifies the surface chemical modification of sieve plate:
The 6ml syringe class column tube of sieve plate will be solidified equipped with pretreated non-bonded silica gel Silica, is placed in negative pressure pumping
In the sealing hole for filtering matched with devices, under the cooperation of sample-adding liquid separation manipulator, 2ml mixed solvent is successively added in single-column pipe
In (volume ratio of ethyl alcohol and water be 1:1), the KH-570 of 1ml is added, the isothermal reaction 0.5h at 50 DEG C after suction filtration, is repeated
Above step 2 times, after reaction, with 4ml ethanolic extraction 3 times, to remove the KH-570 for not participating in reaction, then it is vacuum dried
Afterwards, spare.
3) the modified surface bond for solidifying sieve plate:
Equipped with 1ml styrene, 2ml toluene are added in modified solidification sieve plate, drawing for styrene quality 2% is added
Agent AIBN is sent out, 80 DEG C of isothermal reactions under protection of argon gas keep flow velocity 0.2d/min, in the case where guaranteeing that cylinder is clear and rich, instead
Step drains the reagent inside cylinder, again with toluene extracting, to remove physical absorption after reaction more than three times for multiple operation
It is spare after dry in the polymer of surface.
4) sulfonation for solidifying sieve plate after being bonded:
The concentrated sulfuric acid of 4ml 98% is added in equipped with the column tube for solidifying sieve plate after bonding, in 85 DEG C of sulfonation 0.5h, weight
It operating 2 times, after suction filtration, is first washed with the dilute sulfuric acid of 10ml50% again, then suction filtration is washed with deionized water to neutrality, after suction filtration,
After vacuum drying.
The integral post can be used as 6ml, and SCX phenylbenzimidazole sulfonic acid strong cat ion exchange column is come using silicon dioxide powder is coupled benzene
Base sulfonic acid becomes SCX functional stuffing, can do SCX strong cation exchange solid-phase extraction column, structure are as follows:
With same method, the SCX phenylbenzimidazole sulfonic acid strong cation that the scale that can also make is up to g grades of 2ml-300ml is handed over
Change column.
Embodiment six:
Prepare 12ml, NH2 (amino) integral post is divided into diameter 15.8mm, the cylindrical solidification sieve plate of thickness 30.0mm and
Its mating 12ml syringe class void column pipe.
1, the design, die sinking of 12ml syringe class void column pipe and injection molding produce:
12ml syringe class void column pipe size of adopting international standards opens injection mold, expects (polypropylene with the PP of medical grade
Material) carry out injection molding production.
2, the cylinder of diameter 15.8mm, thickness 30.0mm solidify the preparation of sieve plate (by 100 calculating of preparation)
1) selection of raw material: the matrix fill being cured uses the silica white of 30-80um partial size, high molecular polymer solidification
Agent uses partial size for 120um, the ultra-high molecular weight polyethylene UHMW-PE powder that molecular weight is 3,000,000.
2) configuration and mixing of mixed raw material: according to mass ratio, silicon powder: it is mixed that UHMW-PE powder=1:2 ratio prepares 261g
It closes powder (i.e. 87g silicon powder+174gUHMW-PE powder), 2h is mixed by inversion on blending instrument, it is spare.
3) design and manufacture of the cylindrical sieve plate curing mold of diameter 15.8mm, thickness 30.0mm: it is in side length
250.0mm, with a thickness of using slow wire feeding technique to cut out 100 diameters equidistantly as 16.0mm on 30.5mm aluminium block, depth is
The cylindrical hole of 30.5mm is passing through the technologies retrofit such as mirror surface treatment, surface polishing, it is ensured that after mold hole precision≤0.02mm
High-temperaure coating is sprayed, it is spare after Drying and cooling.Side is made using the method for the technologies retrofit such as mirror surface treatment, surface polishing
A length of 250.0mm, the upper and lower cover plates of thickness 10.0mm or so, after storming and capturing screw hole location with tapping machine, the same high temperature resistant that sprays is applied
Layer, it is spare after Drying and cooling.
4) mixed powder is die-filling: containing 100 16.0mm, in the cylindrical aluminium-making mould of thickness 30.5mm, uniformly
The silica white mixed and UHMW-PE blended powder are added, with shaking platform (vibration frequency 50HZ, amplitude 3mm, time of vibration 60 seconds)
It is flat that powder vibration is added while vibrating, and is covered upper cover plate, is locked.
5) mixed powder solidifies: 200 DEG C are heated to, 70min is kept the temperature, it is rear by being cooled to room temperature, and take out cylindrical solidification
Sieve plate.
3, it assembles:
With limit push rod or full-automatic filling equipment by diameter 15.8mm, the cylindrical solidification sieve plate of thickness 30.0mm is assembled
To 12ml syringe class void column bottom of the tube.
4, the coupling of amino functional chemical group:
By equipped with the 12ml syringe class column tube for solidifying sieve plate, it is placed in the sealing hole of negative pressure leaching matched with devices, is adding
Under the cooperation of sample liquid separation manipulator, 6ml isopropanol is added in equipped with the single-column pipe for solidifying sieve plate, keeps flow velocity 0.2d/min,
In the case where guaranteeing that cylinder is clear and rich, step more than three times is operated repeatedly, adds 1mlAPETS, guarantees flow velocity 0.2d/min,
Repeat above step twice, after reaction, cleaning is drained, and is placed in a vacuum drying oven drying process, and silicon powder is coupled ammonia
Propyl becomes NH2 functional stuffing, can do NH2 solid-phase extraction column, structure are as follows:
The integral post can be used as 12ml, NH2 (amino) column come using.
With same method, NH2 (amino) integral post of 1ml, 3ml, 6ml can also be made.
Embodiment seven:
Such as Fig. 3 and Fig. 4,1.5ml is prepared, MAX (mixed type anion exchange) integral post plate is divided into diameter 7.8mm, thick
Spend the cylindrical solidification sieve plate and its 96 pass filter plate of mating 1.5ml of 8.0mm.
1, the design, die sinking of 96 pass filter plate of 1.5ml and injection molding produce:
The 96 pass high 52mm of filter plate hollow plate of 1.5ml, is derived by 96 void column pipes, and orifice plate top outer diameter is side length
The cuboid cavity of 8.1mm, height 14mm, cavity lower part close up to form cylindrical assembly position, and cylinder assembly position diameter is
7.8mm is highly 14mm, assembles position or less and uses Luer-style Interface design, orifice plate bottom end point mouth point uses flat mouth or sharp-crested
Design.Moreover, the edge of 96 hole hollow plates is provided with sealed frame, two sides are provided with location hole, the location hole side two.
It is spare using mould steel production mold after mold design is good.
Injection molding machine is then used, injection molding production is carried out using the PP material (polypropylene material) of medical grade, forms the filtering of 96 holes
Plate, it is spare.
2, the cylinder of diameter 7.8mm, thickness 8.0mm solidify the preparation (by 100 calculating of preparation) of sieve plate:
1) selection of raw material: the matrix fill being cured is poly- using the polystyrene-divinylbenzene height of 20-80um partial size
Object (PS/DVB), high molecular polymer curing agent use partial size for 25um, the low form super high molecular weight that molecular weight is 5,000,000
Polyethylene UHMW-PE powder.
2) configuration and mixing of mixed raw material: according to mass ratio, PS/DVB:UHMW-PE powder=1:2 ratio prepares 15g
Mixed powder (i.e. 5gPS/DVB+10gUHMW-PE powder), is mixed by inversion 2h on blending instrument, spare.
3) design and manufacture of the cylindrical sieve plate curing mold of diameter 7.8mm, thickness 8.0mm: it is in side length
150.0mm, with a thickness of using slow wire feeding technique to cut out 100 diameters equidistantly as 8.0mm on 8.5mm aluminium block, depth is 8.5mm's
Cylindrical hole is passing through the technologies retrofit such as mirror surface treatment, surface polishing, it is ensured that spray resistance to height after mold hole precision≤0.02mm
Warm coating, it is spare after Drying and cooling.Use the technologies retrofit such as mirror surface treatment, surface polishing method make side length for
150.0mm, the upper and lower cover plates of thickness 10.0mm or so after storming and capturing screw hole location with tapping machine, equally spray high-temperaure coating, dry
It is dry and cold rear spare.
4) mixed powder is die-filling: containing 100 8.0mm, in the cylindrical aluminium-making mould of thickness 8.5mm, is uniformly adding
Add the PS/DVB mixed and UHMW-PE blended powder, with shaking platform (vibration frequency 50HZ, amplitude 3mm, time of vibration 60 seconds) one
The vibration of addition powder is flat on one side for side vibration, covers upper cover plate, locks.
5) powder curing: being heated to 135 DEG C, keeps the temperature 40min, rear by being cooled to room temperature, and takes out cylindrical solidification sieve plate.
3, it assembles:
With limit push rod or full-automatic filling equipment by diameter 7.8mm, the cylindrical solidification sieve plate of thickness 8.0mm is assembled to
The 96 pass filter plate bottom 1.5ml.
4, the coupling of quaternary ammonium group functional chemical group:
1) PS/DVB solidifies the sulfonation of sieve plate:
The 96 pass filter plate of 1.5ml of sieve plate will be solidified equipped with PS/DVB, is placed in the seal groove of negative pressure leaching matched with devices
It is interior, under the cooperation of sample-adding liquid separation manipulator, 1ml1-, 2 two chloroethenes are added in equipped with the 96 pass filter plates for solidifying sieve plate
Alkane keeps flow velocity 0.5d/min, and vacuum is drained, and the 1ml concentrated sulfuric acid is added, and keeps flow velocity 0.2d/min, at 85 DEG C, reaction
0.5h is cleaned with acetone after reaction and is removed 1-, 2 dichloroethanes, filtrate is washed with deionized finally as neutrality, then
96 hole filters are placed in a vacuum drying oven spare after being dried.
2) PS/DVB after sulfonation solidifies the coupling quaternary ammonium group functional chemical group of sieve plate with 1,3- dibromopropane and four
Methyl-diethyl-amine is raw material, using acetonitrile as solvent.Monomer concentration is 2mol/L, reacts 36h in 70 DEG C of stirred in water bath.Layering removes
Acetonitrile solvent is removed, is dissolved in water, is filtered to remove insoluble substance, and be diluted to weak solution.The PS/DVB for being added to sulfonation is solid
Change in sieve plate, 70 DEG C of water-bath 30min.It is neutral with deionized water filtration washing to filtrate, then 96 hole filters is placed in
After being dried in vacuum oven.
3) polystyrene-divinylbenzene high polymer coupling quaternary ammonium salt becomes MAX functional stuffing, can do MAX mixed type
Anion-exchange solid phase extraction column plate, structure are as follows:
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (4)
1. a kind of integral post plate preparation method based on PE sieve plate conjugation chemistry group, which is characterized in that specifically include following step
It is rapid:
S1. it is 3.2mm by the diameter containing micropore glass bead CPG, is assembled to 400ul 384 with a thickness of the PE sieve plate of 6.25mm
The assembly position bottom of pass filter plate；
S2. by the seal groove of the 384 pass filter plates merging negative pressure leaching device equipped with PE sieve plate, pass through sample-adding liquid separation machinery
Hand is by 1.1mmolN- chloromethyl phthalimide, 120ul methylene chloride, and after maintaining 15min, 6ul0.6mol/L tri- is added
Iron chloride-benzophenone dichloromethane solution makees catalyst, is heated to 45 DEG C of reactions and is kept for 0.5 hour, after reaction, is divided
Not Yong 0.1mol/L hydrochloric acid N, N '-dimethyl formamide solution (3*100ul), N, N '-dimethyl formamide (3*100ul) two
Chloromethanes (5*100ul), methanol (2*100ul) elute PE sieve plate, drain, and vacuum completes drying；
S3. PE sieve plate is eluted with ethyl alcohol (3*100ul) and methanol (3*100ul), filters, is dried in vacuo to obtain aminomethyl micropore glass
Glass bead (controlled pore glass, CPG) solidifies PE sieve plate, and reaction equation is shown below:
S4. the synthesis that aminomethyl micropore glass bead (controlled pore glass, CPG) solidifies PE sieve plate is completed.
2. the integral post plate preparation method according to claim 1 based on PE sieve plate conjugation chemistry group, which is characterized in that
In step sl, the PE sieve plate preparation step are as follows:
S11. the selection of raw material: the matrix fill being cured uses the micropore glass bead CPG of 80-150um partial size, polyphosphazene polymer
Closing object curing agent uses partial size for 50um, the ultra-high molecular weight polyethylene UHMW-PE powder that molecular weight is 4,000,000；
S12. the configuration and mixing of mixed raw material: according to mass ratio, micropore glass bead CPG:UHMW-PE powder=1:1.5 ratio
Example prepares 25g mixed powder (i.e. 10g micropore glass bead CPG+15gUHMW-PE powder), and 2h is mixed by inversion on blending instrument, standby
With；
S13. the design and manufacture of the cylindrical sieve plate curing mold of diameter 3.2mm, thickness 6.25mm: being 250.0mm in side length,
With a thickness of using slow wire feeding technique to cut out 1000 diameters equidistantly as 3.4mm on 6.5mm aluminium block, depth is the cylindrical hole of 6.5mm,
Using the technologies retrofit such as mirror surface treatment, surface polishing, it is ensured that after mold hole precision≤0.02mm, spraying high temperature resistant is applied
Layer, it is spare after Drying and cooling, use the method production side length of the technologies retrofit such as mirror surface treatment, surface polishing for 250.0mm,
The upper and lower cover plates of thickness 10.0mm after storming and capturing screw hole location with tapping machine, equally sprays high-temperaure coating, Drying and cooling standby
With；
S14. mixed powder is die-filling: it is being 3.4mm containing 1000 diameters, depth is in the aluminium-making mould of the cylindrical hole of 6.5mm,
Uniformly add the micropore glass bead CPG:UHMW-PE blended powder mixed, with shaking platform (vibration frequency 50HZ, amplitude 3mm,
Time of vibration 60 seconds) to add powder vibration while vibrating flat, and upper cover plate is covered, is locked；
S15. powder curing: being heated to 220 DEG C, keeps the temperature 70min, rear by being cooled to room temperature, and takes out cylindrical solidification sieve plate.
3. the integral post plate preparation method according to claim 1 based on PE sieve plate conjugation chemistry group, which is characterized in that
The 384 pass filter plate of 400ul carries out injection molding production using the PP material (polypropylene material) of medical grade, forms the mistake in 384 holes
Filter plate.
4. entirety prepared by the preparation method of the integral post plate according to claim 1 based on PE sieve plate conjugation chemistry group
Column plate is synthesized in DNA, nucleic acid extraction and purifying, compound Solid Phase Extraction, biological sample absorption, separation, purifying and concentration, chemistry
Application in reaction carriers catalysis, blood purification.
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