CN103487533B - 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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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 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 by the chromatography method of 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 with speed faster, first flow out chromatographic column, little molecule can freely be come in and gone out in gel particle, and between mobile phase and static phase, form mobile equilibrium very soon, therefore will spend the post bed of flowing through of longer time, thereby make the molecules of different sizes be able to separation.
Gel chromatography is a kind of quick and simple separated stage division 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.
In traditional dress post technique, use purified water preparation gel suspension, then gel suspension is slowly added in post, complete 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 gel column bed occur crackle, affect 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.
It is after dress post completes, system to be detected that post effect detects, and survey post efficacious prescriptions method is used acetone to carry out post effect as tracer agent and detected, but acetone is toxic reagent, and volatile, and experiment operator tool is had a certain impact; Meanwhile, acetone easily remains in pillar, is difficult to it to remove completely in follow-up technique, and the quality of product and security tool are 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 object 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, fill post; Post effect detects as replace acetone to carry out a kind of method that 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 dress column method for gel chromatography column, comprises step:
1) preparation gel suspension: add 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 mixing 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) in column jecket bottom, add purified water, then pour gel suspension into column jecket, purified water volume is the 1-10% of gel suspension volume;
B) load onto the column cap of chromatographic column, gel suspension is placed under 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) post effect detects: with 1-4mol/L NaCl, as tracer agent, monitoring electric conductivity value changes, and obtains post effect result.
Gel is inert carrier, and for example the sephadex of various models, comprises 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, loading volume is the 1-4% of 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), 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 detection method for gel chromatography column post effect, it is to use 1-4mol/L NaCl as tracer agent, monitoring electric conductivity value changes, and obtains post effect result.
Described detection method is specially, with 1-4mol/L NaCl as tracer agent, loading volume is the 1-4% of 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), 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 dimensionless performance value, during measurement, be accurate to 1min chronomere, 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 obtaining is the same, but with novel dress post method dress post, can greatly improve dress column efficiency, the post of dress repeatedly and the bulky defect of balance that while having overcome with tradition dress column method dress post, have occurred.So the dress column method that proof the present invention proposes can replace traditional dress column method completely.
2) with NaCl, carrying out post effect detects and carries out post effect with acetone and detect and compare, the post effect result finally obtaining is substantially close, all meet post effect and detect criterion of acceptability, so proof NaCl carries out method that post effect detects and acetone, to carry out the effect that the method for post effect detection reaches be the same.
Accompanying drawing explanation
Fig. 1 fills post process flow diagram.
Fig. 2 is the acetone check post effect result figure for gel chromatography column that embodiment 1 obtains.
Fig. 3 is the NaCl check post effect result figure for gel chromatography column of embodiment 1.
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 WeiGE company produces Sephacryl S-400HR..Flow process is shown in Fig. 1.
1) prepare 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 gel suspension
2) dress post: the purified water that adds 1cm liquid level in 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 gel height, the high 65cm of gel column of preparation; Carry out next step;
3) balance: contain 0.05mol/L NaCl, pH7.2 with 0.01mol/L PBS() 1 times of column volume of damping fluid balance, equilibrium velocity is 25cm/h;
4) post effect detects:
A) acetone contrast: prepare 2% acetone soln as tracer agent by purified water, 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 280nm ultraviolet absorption value.2% acetone: post effect (N)=13708, As=1.06 meets post effect and detects 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, 1mol/L NaCl: post effect (N)=13421, As=1.10 meets post effect and detects criterion of acceptability requirement
Embodiment 2
1) prepare 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 gel suspension
2) dress post: the purified water that adds 2cm liquid level in column jecket bottom, pack gel suspension 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 is prepared gel column, constant pressure is that 1.0bar is extremely highly constant, the high 70cm of gel column of preparation;
3) balance: contain 0.8mol/LNaCl, pH7.4 with 0.01mol/L PBS() 3 times of column volumes of damping fluid balance, equilibrium velocity is 35cm/h;
4) post effect detects:
A) acetone contrast: prepare 2% acetone soln as tracer agent by purified water, 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 post effect and detects criterion of acceptability requirement;
B) 4mol/L NaCl: post effect (N)=10341, As=1.32 meets post effect and detects criterion of acceptability requirement.
Embodiment 3
1) prepare 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 gel suspension;
2) dress post: the purified water that adds 4cm liquid level in column jecket bottom, pack gel suspension 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 is prepared gel column, constant pressure is that 1.0bar is extremely highly constant, the high 72cm of gel column of preparation;
3) balance: contain 0.3mol/LNaCl, pH7.3 with 0.01mol/L PBS() 1.5 times of column volumes of damping fluid balance, equilibrium velocity is 34cm/h;
4) post effect detects:
A) acetone contrast: prepare 2% acetone soln as tracer agent by purified water, 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 post effect and detects criterion of acceptability requirement;
B) 2mol/L NaCl: post effect (N)=10341, As=1.32 meets post effect and detects 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, do not departing under the prerequisite of 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 (6)
1. a dress column method for gel chromatography column, is characterized in that, comprises step:
1) preparation gel suspension: add 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 mixing 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) in column jecket bottom, add purified water, then pour gel suspension into column jecket, purified water volume is the 1-10% of 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/L NaCl, equilibrium velocity is 30 ± 5cm/h;
4) post effect detects: with 1-4mol/L NaCl, as tracer agent, monitoring electric conductivity value changes, and obtains post effect result.
2. dress column method according to claim 1, is characterized in that, described step 1) in, the ratio of gel and alkali salt damping fluid 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 damping fluid, constant pressure is 1 ± 0.1bar.
4. dress column method according to claim 1, it is characterized in that, described step 4) be: use 1-4mol/LNaCl as tracer agent, loading volume is the 1-4% of 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, 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 the arbitrary described dress column method of claim 1-4, it is characterized in that, described NaOH solution and phosphate buffered solution volume ratio are 1:3.0.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1586699A (en) * | 2004-07-16 | 2005-03-02 | 大连大学 | Gel column mounting method |
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| CN1586699A (en) * | 2004-07-16 | 2005-03-02 | 大连大学 | Gel column mounting method |
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| Lars Hagel.Gel-Filtration Chromatography.《Current Protocols in Protein Science Gel-Filtration Chromatography》.1998, * |
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