CN103487533A - Packing method and detecting method of gel chromatography column - Google Patents
Packing method and detecting method of gel chromatography column Download PDFInfo
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- CN103487533A CN103487533A CN201310404465.9A CN201310404465A CN103487533A CN 103487533 A CN103487533 A CN 103487533A CN 201310404465 A CN201310404465 A CN 201310404465A CN 103487533 A CN103487533 A CN 103487533A
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- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000005227 gel permeation chromatography Methods 0.000 title claims abstract description 13
- 238000012856 packing Methods 0.000 title abstract description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 68
- 230000000694 effects Effects 0.000 claims abstract description 46
- 239000011780 sodium chloride Substances 0.000 claims abstract description 34
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims abstract description 33
- 239000000243 solution Substances 0.000 claims abstract description 24
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 16
- 239000000700 radioactive tracer Substances 0.000 claims abstract description 16
- 239000008213 purified water Substances 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000012544 monitoring process Methods 0.000 claims abstract description 9
- 238000005086 pumping Methods 0.000 claims abstract description 3
- 239000000725 suspension Substances 0.000 claims description 23
- 238000013016 damping Methods 0.000 claims description 20
- 239000012530 fluid Substances 0.000 claims description 20
- 150000001447 alkali salts Chemical class 0.000 claims description 18
- 238000010828 elution Methods 0.000 claims description 18
- 229910019142 PO4 Inorganic materials 0.000 claims description 12
- 239000008366 buffered solution Substances 0.000 claims description 12
- 239000010452 phosphate Substances 0.000 claims description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 8
- 239000008363 phosphate buffer Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 abstract description 9
- 239000003513 alkali Substances 0.000 abstract description 3
- 150000003839 salts Chemical class 0.000 abstract description 3
- 239000008055 phosphate buffer solution Substances 0.000 abstract 2
- 239000000499 gel Substances 0.000 description 35
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 32
- 239000007863 gel particle Substances 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000012506 Sephacryl® Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000037656 Respiratory Sounds Diseases 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- -1 alkali salt alkali salt Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
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Abstract
The invention provides a packing method of a gel chromatography column. The packing method comprises the following steps: 1), preparing a gel suspending solution, namely, adding a 0.02-0.4mol/L NaOH solution and a phosphate buffer solution containing NaCl in gel, to form an alkali and salt mixed gel suspending solution; 2), packing, namely, adding purified water into the bottom of a column tube, and filling the gel suspending solution into the column tube; pumping the alkali and salt buffer solution in which the NaOH solution and the phosphate buffer solution containing NaCl are mixed according to the ratio of 1: (1.0-3) into the column tube, and starting the next balancing step when the level of the gel suspending solution is constant; 3), balancing, namely, balancing 1-3 times of column volume by using the buffer solution containing NaCl; and 4), column effect detecting, namely, with the NaCl as a tracer agent, monitoring the change of conductivity, so as to obtain a column effect result. Compared with the traditional packing method, the packing method provided by the invention has the packing effect same as that of the traditional method, furthermore, by adoption of the packing method provided by the invention, the packing efficiency can be greatly improved, and the disadvantages of repeat packing and large balancing volume are overcome.
Description
Technical field
The invention belongs to the chromatographic technique field, be specifically related to a kind of preparation method and detection thereof of chromatography exchange column.
Background technology
Gel chromatography claims again gel filtration, is a kind of chromatography method by the molecular size range separate substance.The method is that sample is added in the chromatographic column of gel particle abrim, then uses buffer solution elution.Large molecule can not enter in the static phase in gel particle, only stay in the mobile phase between gel particle, therefore at first flow out chromatographic column with speed faster, little molecule can freely be come in and gone out in gel particle, and form mobile equilibrium very soon between mobile phase and static phase, therefore will spend the post bed of flowing through of longer time, thereby the molecules of different sizes are separated.
Gel chromatography is a kind of stage division that separates fast and simply of water-soluble substances, be widely used in the association areas such as biological chemistry, molecular biology and medical science, be the indispensable technological means of biomacromolecule material such as separating and purifying protein matter, biology enzyme, nucleic acid and polysaccharide, also can be used for the concentrated and desalination of sample and the aspects such as molecular mass of mensuration biomacromolecule.
Use purified water preparation gel suspension in traditional dress post technique, then gel suspension is slowly added in post, complete the dress post.In the method, because selecting purified water to carry out even glue, can not make gel particle well disperse, cause gel particle natural subsidence excessive velocities and gel bed inhomogeneous, thereby make the gel column bed crackle occur, affect the chromatography effect.Also need the pillar of often resetting simultaneously, increased working strength, work efficiency is reduced.Therefore in the urgent need to a kind of quick, high-quality dress column method, to save time, to raise the efficiency.
The post effect detects and after the dress post completes, system is detected, and survey post efficacious prescriptions method is used acetone to carry out the post effect as tracer agent and detected, but acetone is toxic reagent, and volatile, and the experiment operator tool is had a certain impact; Simultaneously, acetone easily remains in pillar, in follow-up technique, is difficult to it is removed fully, and quality and the security tool of product had a certain impact.Therefore need a kind of effect of post safely and effectively detection system, to improve quality and the security of product, avoid affecting experimenter's health simultaneously.
Summary of the invention
The objective of the invention is for the deficiencies in the prior art, propose a kind of dress column method of gel chromatography column, with alkali and salt preparation gel suspension and dress post damping fluid, by the method for phosphate buffer balance chromatographic column, filled post; The post effect detects as replace acetone to carry out a kind of method that the post effect detects with sodium chloride (NaCl).
Another object of the present invention is the detection method that proposes a kind of gel chromatography column.
The technical scheme that realizes above-mentioned purpose of the present invention is:
1, a kind of dress column method of gel chromatography column comprises step:
1) preparation gel suspension: add the alkali salt damping fluid in gel, described alkali salt damping fluid is that volume ratio is the 1:1.0-3.0 0.02-0.4mol/L NaOH solution mixed and the PBS phosphate buffered solution that contains 0.05-0.8mol/L NaCl, makes the gel suspension that gel and alkali salt damping fluid mix;
2) dress post:
A) add purified water in the column jecket bottom, then pour the gel suspension into column jecket, the 1-10% that the purified water volume is gel suspension volume;
B) load onto the column cap of chromatographic column, the gel suspension is placed under the column cap piston;
C) pump into 0.02-0.4mol/LNaOH solution and the alkali salt damping fluid that the PBS phosphate buffered solution 1:1.0-3.0 that contains 0.05-0.8mol/L NaCl mixes, when gel suspension liquid level is constant, start next equilibrium step;
3) balance: with the 1-3 times of column volume of phosphate buffer balance containing 0.05-0.8mol/LNaCl, equilibrium velocity is that 30 ± 5cm/h(damping fluid volume is column volume 1-3 times);
4) the post effect detects: with 1-4mol/L NaCl, as tracer agent, the monitoring electric conductivity value changes, and obtains post effect result.
Gel is inert carrier, and for example the sephadex of various models, comprise the gel of Sephacryl High Resolution series etc.
Wherein, in described step 1), the ratio 1:1.0-3.0 of gel and alkali salt liquid.
Wherein, described step 2) in, constant pressure while pumping into described alkali salt mixed liquor (pressure of pump) is 1 ± 0.1bar.
Particularly, described step 4) is: use 1-4mol/LNaCl as tracer agent, the 1-4% that the loading volume is column volume, with the phosphate buffer wash-out containing 0.05-0.8mol/LNaCl, loading flow velocity and elution flow rate are 30 ± 5cm/h, wash-out 1-3 times column volume (the damping fluid volume of wash-out is 1-3 times of column volume), the monitoring electric conductivity value changes, and obtains post effect result.
Wherein, the pH value of described phosphate buffered solution is 7.0-7.5.Phosphate buffered solution is prepared by conventional method, and the potassium dihydrogen phosphate and the sodium hydrogen phosphate that are generally 0.01-0.2mol/L are formulated, preferably the phosphate buffered solution of 0.01mol/L.
Preferably, described NaOH solution and phosphate buffered solution volume ratio are 1:3.
A kind of detection method of gel chromatography column post effect, it is to use 1-4mol/L NaCl as tracer agent, the monitoring electric conductivity value changes, and obtains post effect result.
Described detection method is specially, with 1-4mol/L NaCl as tracer agent, the 1-4% that the loading volume is column volume, with the phosphate buffer wash-out containing 0.05-0.8mol/L NaCl, loading flow velocity and elution flow rate are 30 ± 5cm/h, wash-out 1-3 times column volume (the damping fluid volume of wash-out is 1-3 times of column volume), the monitoring electric conductivity value changes, and obtains post effect result.
Post effect computing formula: N=5.54 ［ θ r/ (W
1/2)
2(1)
Wherein, θ r is elution time, W
1/2for half-peak breadth.Be the dimensionless performance value, be accurate to 1min chronomere during measurement, be accurate to 0.1min.
Wherein, the pH value of described phosphate buffered solution is 7.0-7.5.
Useful technique effect of the present invention is:
1) using the present invention to fill column method dress post compares with tradition dress post method, the dress post effect finally obtained is the same, but can greatly improve the dress column efficiency with novel dress post method dress post, the post of dress repeatedly and the bulky defect of balance that have occurred while having overcome with tradition dress column method dress post.So the dress column method that proof the present invention proposes can replace traditional dress column method fully.
2) carrying out post effect with NaCl detects and carries out the post effect with acetone and detect and compare, the post effect result finally obtained is substantially close, all meet post effect and detect criterion of acceptability, so proof NaCl carries out method that the post effect detects and acetone, to carry out the effect that the method for post effect detection reaches be the same.
The accompanying drawing explanation
Fig. 1 fills the post process flow diagram.
Fig. 2 is that the gel chromatography column that embodiment 1 obtains is imitated figure as a result with acetone check post.
The gel chromatography column that Fig. 3 is embodiment 1 is imitated figure as a result with NaCl check post.
Embodiment
Now with following most preferred embodiment, the present invention is described, but is not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technological means used is well known to those skilled in the art.
Embodiment 1:
Gel is that GE company produces Sephacryl S-400HR..Flow process is shown in Fig. 1.
1) prepare the gel suspension: with 0.02mol/L NaOH solution and 0.01mol/LPBS(containing 0.05mol/LNaCl, pH7.2) the alkali salt damping fluid of solution 1:1 mixing carries out even glue, the ratio of gel and alkali salt damping fluid is 1:1, obtains the gel suspension
2) dress post: the purified water that adds the 1cm liquid level in the column jecket bottom, then with gel suspension dress post, the column cap of chromatographic column is installed, constant pressure is that 1.0bar pumps into 0.02mol/L NaOH solution and 0.01mol/LPBS(contains 0.05mol/L NaCl, pH7.2) the alkali salt liquid that solution 1:1 mixes, when constant voltage is constant to the gel height, the high 65cm of the gel column of preparation; Carry out next step;
3) balance: with 0.01mol/L PBS(, contain 0.05mol/L NaCl, pH7.2) 1 times of column volume of damping fluid balance, equilibrium velocity is 25cm/h;
4) the post effect detects:
A) acetone contrast: by purified water, prepare 2% acetone soln as tracer agent, the loading volume is 2% of column volume, with 0.01mol/L PBS(, contains 0.05mol/LNaCl, pH7.2) buffer solution elution, loading flow velocity and elution flow rate are 25cm/h, detect ultraviolet absorption value and change;
B) NaCl detects: with 1mol/LNaCl, as tracer agent, 0.01mol/LPBS(is containing 0.05mol/L NaCl, pH7.2) buffer solution elution, loading flow velocity and elution flow rate are 25cm/h, detect electric conductivity value and change;
Test findings:
A) see Fig. 2 uv detection method, in figure, horizontal ordinate is the time, the min of unit; Ordinate is the 280nm ultraviolet absorption value.2% acetone: post effect (N)=13708, As=1.06 meets the post effect and detects the criterion of acceptability requirement;
B) see Fig. 3 conductance detection: horizontal ordinate is the time, the min of unit; Ordinate is electric conductivity value, the mS/cm of unit, and 1mol/L NaCl: post effect (N)=13421, As=1.10 meets the post effect and detects the criterion of acceptability requirement
Embodiment 2
1) prepare the gel suspension: with 0.4mol/LNaOH solution and 0.01mol/LPBS(containing 0.8mol/LNaCl, pH7.4) solution 1:3 mixing carries out even glue; Gel and alkali salt alkali salt damping fluid liquid proportional 1:3; Obtain the gel suspension
2) dress post: the purified water that adds the 2cm liquid level in the column jecket bottom, the gel suspension of packing into, load onto column cap, then pump into 0.4mol/L NaOH solution and 0.01mol/L PBS(containing 0.8mol/L NaCl, pH7.4) the alkali salt damping fluid that solution 1:3 mixes prepares gel column, constant pressure is that 1.0bar is extremely highly constant, the high 70cm of the gel column of preparation;
3) balance: with 0.01mol/L PBS(, contain 0.8mol/LNaCl, pH7.4) 3 times of column volumes of damping fluid balance, equilibrium velocity is 35cm/h;
4) the post effect detects:
A) acetone contrast: by purified water, prepare 2% acetone soln as tracer agent, the loading volume is 4% of column volume, with 0.01mol/LPBS(, contains 0.8mol/LNaCl, pH7.4) buffer solution elution, loading flow velocity and elution flow rate are 35cm/h, detect ultraviolet absorption value and change;
B) NaCl detects: with 4mol/LNaCl, as tracer agent, 0.01mol/LPBS(is containing 0.8mol/L NaCl, pH7.4) buffer solution elution, loading flow velocity and elution flow rate are 35cm/h, detect conductivity value and change;
Test findings:
A) 2% acetone: post effect (N)=10071, As=1.28 meets the post effect and detects the criterion of acceptability requirement;
B) 4mol/L NaCl: post effect (N)=10341, As=1.32 meets the post effect and detects the criterion of acceptability requirement.
Embodiment 3
1) prepare the gel suspension: with 0.2mol/LNaOH solution and 0.01mol/LPBS(containing 0.3mol/L NaCl, pH7.3) solution 1:1.5 mixing carries out even glue, gel and alkali salt damping fluid ratio 1:1.5; Obtain the gel suspension;
2) dress post: the purified water that adds the 4cm liquid level in the column jecket bottom, the gel suspension of packing into, load onto column cap, pump into 0.2mol/L NaOH solution and 0.01mol/LPBS(containing 0.3mol/L NaCl, pH7.3) the alkali salt liquid that solution 1:1.5 mixes prepares gel column, constant pressure is that 1.0bar is extremely highly constant, the high 72cm of the gel column of preparation;
3) balance: with 0.01mol/L PBS(, contain 0.3mol/LNaCl, pH7.3) 1.5 times of column volumes of damping fluid balance, equilibrium velocity is 34cm/h;
4) the post effect detects:
A) acetone contrast: by purified water, prepare 2% acetone soln as tracer agent, the loading volume is 2% of column volume, with 0.01mol/LPBS(, contains 0.3mol/L NaCl, pH7.2) buffer solution elution, loading flow velocity and elution flow rate are 34cm/h, detect ultraviolet absorption value and change;
B) NaCl detects: with 2mol/L NaCl, as tracer agent, 0.01mol/LPBS(is containing 0.3mol/L NaCl, pH7.3) buffer solution elution, loading flow velocity and elution flow rate are 34cm/h, detect electric conductivity value and change;
Test findings:
A) 2% acetone: post effect (N)=10071, As=1.28 meets the post effect and detects the criterion of acceptability requirement;
B) 2mol/L NaCl: post effect (N)=10341, As=1.32 meets the post effect and detects the criterion of acceptability requirement.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvement and replacement, these improvement and replacement also should be considered as protection scope of the present invention.
Claims (9)
1. the dress column method of a gel chromatography column, is characterized in that, comprises step:
1) preparation gel suspension: add the alkali salt damping fluid in gel, described alkali salt damping fluid is that volume ratio is the 1:1.0-3.0 0.02-0.4mol/L NaOH solution mixed and the PBS phosphate buffered solution that contains 0.05-0.8mol/L NaCl, makes the gel suspension that gel and alkali salt damping fluid mix;
2) dress post:
A) add purified water in the column jecket bottom, then pour the gel suspension into column jecket, the 1-10% that the purified water volume is gel suspension volume;
B) load onto the column cap of chromatographic column;
C) pump into 0.02-0.4mol/L NaOH solution and the alkali salt damping fluid that the PBS phosphate buffered solution 1:1.0-3.0 that contains 0.05-0.8mol/L NaCl mixes, when gel suspension liquid level is constant, start next equilibrium step;
3) balance: with the 1-3 times of column volume of phosphate buffer balance containing 0.05-0.8mol/LNaCl, equilibrium velocity is 30 ± 5cm/h;
4) the post effect detects: with 1-4mol/LNaCl, as tracer agent, the monitoring electric conductivity value changes, and obtains post effect result.
2. dress column method according to claim 1, is characterized in that, in described step 1), the ratio of gel and alkali salt liquid is 1:1.0-3.0.
3. dress column method according to claim 1, is characterized in that, described step 2) in, while pumping into described alkali salt mixed liquor, constant pressure is 1 ± 0.1bar.
4. dress column method according to claim 1, it is characterized in that, described step 4) is: use 1-4mol/LNaCl as tracer agent, the 1-4% that the loading volume is column volume, with the phosphate buffer wash-out containing 0.05-0.8mol/LNaCl, loading flow velocity and elution flow rate are 30 ± 5cm/h, wash-out 1-3 times column volume, the monitoring electric conductivity value changes, and obtains post effect result.
5. according to the arbitrary described dress column method of claim 1-4, it is characterized in that, the pH value of described phosphate buffered solution is 7.0-7.5.
6. according to arbitrary the filled column method of claim 1-4, it is characterized in that, described NaOH solution and phosphate buffered solution volume ratio are 1:3.0.
7. the detection method of a gel chromatography column post effect, is characterized in that, with 1-4mol/LNaCl, as tracer agent, the monitoring electric conductivity value changes, and obtains post effect result.
8. detection method according to claim 7, it is characterized in that, with 1-4mol/LNaCl as tracer agent, the 1-4% that the loading volume is column volume, with the phosphate buffer wash-out containing 0.05-0.8mol/L NaCl, loading flow velocity and elution flow rate are 30 ± 5cm/h, wash-out 1-3 times column volume, the monitoring electric conductivity value changes, and obtains post effect result.
9. detection method according to claim 8, is characterized in that, the pH value of described phosphate buffered solution is 7.0-7.5.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1586699A (en) * | 2004-07-16 | 2005-03-02 | 大连大学 | Gel column mounting method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1586699A (en) * | 2004-07-16 | 2005-03-02 | 大连大学 | Gel column mounting method |
Non-Patent Citations (5)
| Title |
|---|
| GE HEALTHCARE: "《Application note 28-9372-07 AA Column effciency testing》", 31 January 2010 * |
| LARS HAGEL: "《Current Protocols in Protein Science Gel-Filtration Chromatography》", 31 December 1998 * |
| YOSHIO KATO ET AL.: "Packing of Toyopearl columns for gel filtration III. Semi-constant-pressure packing", 《JOURNAI OF CHROMATOGRAPHY》 * |
| 卫秀英 等: "葡聚糖凝胶层析—蛋白质脱盐实验的技术改进", 《河南职技士院学报》 * |
| 胡晓倩 等: "凝胶过滤分离蛋白质实验条件的研究", 《中国生化药物杂志》 * |
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