CN106353445A - Method capable of detecting content of IPTG - Google Patents
Method capable of detecting content of IPTG Download PDFInfo
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- CN106353445A CN106353445A CN201610876781.XA CN201610876781A CN106353445A CN 106353445 A CN106353445 A CN 106353445A CN 201610876781 A CN201610876781 A CN 201610876781A CN 106353445 A CN106353445 A CN 106353445A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
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Abstract
The invention discloses a method capable of detecting the IPTG content. The method comprises the following steps of (1) performing sample pretreatment; (2) preparing an IPTG standard solution, performing detection by use of an ion chromatography-pulsed amperometric detection method, thereby obtaining a standard curve, wherein chromatographic conditions of the ion chromatography-pulsed amperometric detection method are as follows: a chromatographic column is a reversed phase chromatographic column, and a flowing phase is a mixed flowing phase of a buffer solution and an organic solvent; (3), performing detection on the step (1) by use of the ion chromatography-pulsed amperometric detection method, and obtaining the IPTG content. The method is capable of accurately measuring the IPTG content and achieving prominent effects.
Description
Technical field
The present invention relates to a kind of detection method of iptg content.
Background technology
Iptg, Chinese entitled isopropyl-β-d- Thiogalactopyranoside, English entitled isopropyl β-d-1-
Thiogalactopyranoside, cas:367-93-1；Its chemical formula is as follows:, it is
A kind of thio-compoundss.
Iptg is the activity inducement material of beta galactosidase, based on this characteristic, when the carrier dna(of puc series or its
He carries lacz genophore dna) with lacz deletion cells for host converted when or entered with the carrier dna of m13 phage
During row transfection, if adding x gal and iptg in plating medium, because the α of beta galactosidase is complementary, Ke Yigen
According to whether assuming white colony (or plaque) and easily pick out genetic recombinants.Additionally, it is also used as with lac
Or the induced expression thing of the expression vector of promoter such as tac uses.In prior art, iptg tries as a kind of molecular biology
Agent, is usually used in protein expression in the screening of blue white macula and the antibacterial of iptg induction etc..In technique for gene engineering, using iptg conduct
Derivant, can promote the great expression of destination protein.But iptg is expensive, toxic when usage amount is excessive, and improve
Commercial production cost.
Iptg is actually a kind of half thio compound.Due to lacking luminophore in the compound molecule of sulfur-bearing, no
Using conventional UV-visible detector detection, detection method the most frequently used at present is the hplc inspection of derivatization joint fluorescence to method
Survey method detects, such as conventional fluorescent derivatizing agent has o- phthaladehyde and n- maleimide.But this detection method wastes time and energy,
Poor selectivity, and exist and derive incomplete possibility.
Therefore, develop a kind of detection method of iptg content, to realize the Accurate Determining to iptg content in biological sample,
Obviously there is positive realistic meaning.
Content of the invention
The goal of the invention of the present invention is to provide a kind of detection method of iptg content.
To achieve the above object of the invention, the technical solution used in the present invention is: a kind of detection method of iptg content, including
Following steps:
(1) sample pre-treatments: sample is carried out ultrafiltration centrifugal treating, obtain molecular weight be less than 10000 dalton treat test sample
Product；
(2) configure iptg standard solution, detected using chromatography of ions-Pulse amperometric detection method, obtain standard curve；
The chromatographic condition of described chromatography of ions-Pulse amperometric detection method is as follows:
Chromatographic column is reversed phase chromatographic column；
Mobile phase is the mixed flow phase of buffer solution and organic solvent；Wherein, the ph value of buffer solution is 1 ~ 11；Buffer solution
Volume ratio with organic solvent is 99 ~ 80:1 ~ 20；
(3) testing sample of step (1) is detected using chromatography of ions-Pulse amperometric detection method, comparison step (2)
Standard curve, you can obtain the content of iptg；
The chromatographic condition of described chromatography of ions-Pulse amperometric detection method is identical with the chromatographic condition in step (2).
Above, described organic solvent is organic solvent miscible with water, such as acetonitrile, below lower alcohol c3 and polyhydric alcohol
(as methanol, ethanol, propanol, isopropanol, ethylene glycol, glycerol, n-butyl alcohol etc.), acetone, oxolane, trifluoroacetic acid, second two
Diethylene glycol dimethyl ether, ethylenediamine, dimethylformamide (dmf) or dimethyl sulfoxide (dmso) etc..
Preferably, the filter centrifugation in described step (1) is processed as being processed using ultra-filtration centrifuge tube.Ultrafiltration herein
Centrifuge tube can adopt prior art, and it has ultrafiltration and two kinds of effects of centrifugation simultaneously, simply can efficiently process multiple lifes
Thing sample.
Preferably, in described ultra-filtration centrifuge tube, the pore diameter range of super filter tube is 0.001 ~ 0.02 micron.
Preferably, in described step (1), the molecular weight of the testing sample after sample pre-treatments is less than 3000 dongle
?.Preferably, the molecular weight of the testing sample after sample pre-treatments is less than 500 dalton.
Preferably, in described step (2), described chromatographic column be using (weak) nonbonding silica gel, cyano group, c1 (tms), c3,
C4, phenyl, c8, c18 or c6h5 as fixing phase reversed phase chromatographic column.Described chromatographic column is c18 chromatographic column.
Preferably, in described step (2), described buffer solution be ph buffer solution, described buffer solution be selected from mineral acid,
One or more of Organic Acid and Base and its esters；
Described mineral acid is hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid or carbonic acid；
Described organic acid is formic acid, acetic acid, propanoic acid, tartaric acid, oxalic acid, citric acid, malic acid, citric acid, ascorbic acid, benzene first
Acid, salicylic acid or caffeic acid；
Described alkali is sodium hydroxide or potassium hydroxide；
Described salt is sodium formate, potassium formate, sodium acetate, potassium acetate, sodium citrate, potassium citrate, sodium tartrate, tartaric acid
Potassium, sodium carbonate, potassium carbonate, sodium bicarbonate or potassium bicarbonate.
Some physical parameter (as acid-base value, dissolubility etc.) akin reagent with mentioned reagent can certainly be selected
As buffer solution.Preferably sodium acetate solution.
Preferably, in described step (2), the ph value of described buffer solution is 2 ~ 8.Preferably 3 ~ 7.More preferably 4 ~ 6.
Preferably, in described step (2), described organic solvent is acetonitrile, the lower alcohol of below c3 or polyhydric alcohol, acetone,
Oxolane, trifluoroacetic acid, glycol dimethyl ether, ethylenediamine, dimethylformamide or dimethyl sulfoxide.
The lower alcohol of described below c3 or polyhydric alcohol are methanol, ethanol, propanol, isopropanol, ethylene glycol, glycerol, positive fourth
Alcohol.
Preferably, described organic solvent is acetonitrile, methanol or acetone.More preferably acetonitrile.
Described organic solvent is to analyze pure above organic solvent.Preferably, described organic solvent is chromatographically pure organic solvent.
It is furthermore preferred that the purity of described organic solvent is 100%.
The operation principle of the present invention is as follows: the present invention carries out processing sample, rapidly and efficiently removing body using ultra-filtration centrifuge tube
Impurity in system, the testing sample solution after processing is passed through ph buffer solution anti-phase with the mixed flow phase entrance of organic solvent
Chromatographic column carries out separating, and iptg is eluted out under this condition quickly, then is detected by pulsed amperometry, thus complete
Become analysis.
Because technique scheme is used, the present invention compared with prior art has the advantage that
1. the present invention devises a kind of detection method of new iptg content, and test proves, the method for the present invention can be exactly
Measure the content of iptg, achieve significant effect；
2. the method for the present invention has the advantages that sample pre-treatments are easy, specificity is good, sensitivity is high, is suitable to popularization and application.
Brief description
Fig. 1 is the detection potential diagram measuring iptg using chromatography of ions-Pulse amperometric detection method (hpaec-pad).
Fig. 2 is the chromatogram measuring iptg using chromatography of ions-Pulse amperometric detection method (hpaec-pad).
Specific embodiment
With reference to embodiment, the invention will be further described:
Embodiment one
Referring to shown in Fig. 1 ~ 2, a kind of detection method of iptg content, comprise the steps:
(1) sample pre-treatments: take appropriate sample super filter tube to be centrifuged 20min, take filtrate, filtrate is with mobile phase a(Sodium Acetate Trihydrate
Solution) 100 times of dilution, obtain the testing sample that molecular weight is less than or equal to 3000 dalton；
(2) configure iptg standard solution, detected using chromatography of ions-Pulse amperometric detection method, obtain standard curve；
The chromatographic condition of described chromatography of ions-Pulse amperometric detection method is as follows:
C18 chromatographic column (acclaimò(c18,5 microns, 4.6 150mm), dionex)；
Mobile phase is the mixing buffer solution of mobile phase a and mobile phase b, and wherein, the Sodium Acetate Trihydrate for 4 ~ 6 is molten for ph value for mobile phase a
Liquid, before use through 0.45 micron of membrane filtration, degassing, it is stored in n2；Mobile phase b is 100% acetonitrile；Column temperature: 25 DEG C, on pressure
Limit: 3000psi, flow velocity: 1.0ml/min, sampling volume: 50 microlitres, elution requirement: a:b=90:10, time 20min；
(3) testing sample of step (1) is detected using chromatography of ions-Pulse amperometric detection method, comparison step (2)
Standard curve, processing data, you can obtain the content of iptg；
The chromatographic condition of described chromatography of ions-Pulse amperometric detection method is identical with the chromatographic condition in step (2).
Fig. 1 is the detection potential diagram measuring iptg using chromatography of ions-Pulse amperometric detection method (hpaec-pad)；Fig. 2 is
Measure the chromatogram of iptg using chromatography of ions-Pulse amperometric detection method (hpaec-pad).As can be seen from the figure adopt this work
Make system, iptg can quick appearance, sensitivity is high, and peak type is symmetrical, and does not interfere with.
Above-described embodiment is only the preferred embodiment of the present invention it is impossible to limit the scope of protection of the invention according to this, this
The change of any unsubstantiality that the technical staff in field is done on the basis of the present invention and replacement belong to the present invention and are wanted
Seek the scope of protection.
Claims (10)
1. a kind of detection method of iptg content is it is characterised in that comprise the steps:
(1) sample pre-treatments: sample is carried out ultrafiltration centrifugal treating, obtain molecular weight be less than 10000 dalton treat test sample
Product；
(2) configure iptg standard solution, detected using chromatography of ions-Pulse amperometric detection method, obtain standard curve；
The chromatographic condition of described chromatography of ions-Pulse amperometric detection method is as follows:
Chromatographic column reversed phase chromatographic column；
Mobile phase is the mixed flow phase of buffer solution and organic solvent；Wherein, the ph value of buffer solution is 1 ~ 11；Buffer solution
Volume ratio with organic solvent is 99 ~ 80:1 ~ 20；
(3) testing sample of step (1) is detected using chromatography of ions-Pulse amperometric detection method, comparison step (2)
Standard curve, you can obtain the content of iptg；
The chromatographic condition of described chromatography of ions-Pulse amperometric detection method is identical with the chromatographic condition in step (2).
2. iptg content according to claim 1 detection method it is characterised in that: the filtration in described step (1) from
The heart is processed as being processed using ultra-filtration centrifuge tube.
3. iptg content according to claim 2 detection method it is characterised in that: super filter tube in described ultra-filtration centrifuge tube
Pore diameter range be 0.001 ~ 0.02 micron.
4. iptg content according to claim 1 detection method it is characterised in that: in described step (1), through sample
The molecular weight of the testing sample after pre-treatment is less than 3000 dalton.
5. iptg content according to claim 1 detection method it is characterised in that: in described step (2), described chromatograph
Post be using (weak) nonbonding silica gel, cyano group, c1 (tms), c3, c4, phenyl, c8, c18 or c6h5 as fixing phase anti-phase color
Spectrum post.
6. iptg content according to claim 1 detection method it is characterised in that: in described step (2), described buffering
Solution is ph buffer solution, and described buffer solution is selected from one or more of mineral acid, Organic Acid and Base and its esters；
Described mineral acid is hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid or carbonic acid；
Described organic acid is formic acid, acetic acid, propanoic acid, tartaric acid, oxalic acid, citric acid, malic acid, citric acid, ascorbic acid, benzene first
Acid, salicylic acid or caffeic acid；
Described alkali is sodium hydroxide or potassium hydroxide；
Described salt is sodium formate, potassium formate, sodium acetate, potassium acetate, sodium citrate, potassium citrate, sodium tartrate, tartaric acid
Potassium, sodium carbonate, potassium carbonate, sodium bicarbonate or potassium bicarbonate.
7. iptg content according to claim 1 detection method it is characterised in that: in described step (2), described buffering
The ph value of solution is 2 ~ 8.
8. iptg content according to claim 1 detection method it is characterised in that: in described step (2), described organic
Solvent is acetonitrile, the lower alcohol of below c3 or polyhydric alcohol, acetone, oxolane, trifluoroacetic acid, glycol dimethyl ether, ethylenediamine,
Dimethylformamide or dimethyl sulfoxide.
9. iptg content according to claim 1 detection method it is characterised in that: in described step (2), described organic
Solvent is chromatographically pure organic solvent.
10. iptg content according to claim 1 detection method it is characterised in that: in described step (2), described slow
Rushing solution and the volume ratio of organic solvent is 95 ~ 85:5 ~ 15.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201610876781.XA CN106353445A (en) | 2016-09-30 | 2016-09-30 | Method capable of detecting content of IPTG |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610876781.XA CN106353445A (en) | 2016-09-30 | 2016-09-30 | Method capable of detecting content of IPTG |
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| CN201610876781.XA Pending CN106353445A (en) | 2016-09-30 | 2016-09-30 | Method capable of detecting content of IPTG |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107356462A (en) * | 2017-06-20 | 2017-11-17 | 齐鲁制药有限公司 | The method for eliminating the double meglumine baterial endotoxin test disturbing factors of Fosaprepitant |
| CN115144482A (en) * | 2022-02-25 | 2022-10-04 | 南京汉欣医药科技有限公司 | Method for quantitatively detecting content of isopropyl-beta-D-thiogalactoside in polypeptide or protein |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003830A (en) * | 2006-12-31 | 2007-07-25 | 华东师范大学 | Method for counting colibacillus in water body rapidly |
| CN101437837A (en) * | 2006-03-11 | 2009-05-20 | 瑞诺弗有限责任公司 | Protein folding |
| CN102994601A (en) * | 2012-12-13 | 2013-03-27 | 山东大学 | Method for preparing collagen small peptide by utilizing marine collagenase MCP-01 |
| CN103172730A (en) * | 2013-03-18 | 2013-06-26 | 昆明理工大学 | Bufo melanostictus schneider cystatin as well gene and application thereof |
-
2016
- 2016-09-30 CN CN201610876781.XA patent/CN106353445A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101437837A (en) * | 2006-03-11 | 2009-05-20 | 瑞诺弗有限责任公司 | Protein folding |
| CN101003830A (en) * | 2006-12-31 | 2007-07-25 | 华东师范大学 | Method for counting colibacillus in water body rapidly |
| CN102994601A (en) * | 2012-12-13 | 2013-03-27 | 山东大学 | Method for preparing collagen small peptide by utilizing marine collagenase MCP-01 |
| CN103172730A (en) * | 2013-03-18 | 2013-06-26 | 昆明理工大学 | Bufo melanostictus schneider cystatin as well gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| ALFRED FERNA´NDEZ: "Development and Validation of a Liquid Chromatography-Mass Spectrometry Assay for the Quantitation of IPTG in E. Coli Fed-Batch Cultures", 《ANAL. CHEM.》 * |
| SWATI J. MODI等: "Determination of thio-based additives for biopharmaceuticals by pulsed electrochemical detection following HPLC", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107356462A (en) * | 2017-06-20 | 2017-11-17 | 齐鲁制药有限公司 | The method for eliminating the double meglumine baterial endotoxin test disturbing factors of Fosaprepitant |
| CN115144482A (en) * | 2022-02-25 | 2022-10-04 | 南京汉欣医药科技有限公司 | Method for quantitatively detecting content of isopropyl-beta-D-thiogalactoside in polypeptide or protein |
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