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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- Prior art keywords
- acid
- detection method
- iptg
- content according
- organic solvent
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Links
- 238000000034 method Methods 0.000 title abstract description 11
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 title abstract description 8
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 238000004082 amperometric method Methods 0.000 claims abstract description 19
- 239000003960 organic solvent Substances 0.000 claims abstract description 19
- 239000007853 buffer solution Substances 0.000 claims abstract description 16
- 239000012086 standard solution Substances 0.000 claims abstract description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 18
- 238000004587 chromatography analysis Methods 0.000 claims description 16
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 11
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
- 238000000108 ultra-filtration Methods 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000002203 pretreatment Methods 0.000 claims description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 6
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 239000000243 solution Substances 0.000 claims description 6
- 235000017281 sodium acetate Nutrition 0.000 claims description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
- 235000015165 citric acid Nutrition 0.000 claims description 4
- 229910052500 inorganic mineral Inorganic materials 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- 239000011707 mineral Substances 0.000 claims description 4
- 235000010755 mineral Nutrition 0.000 claims description 4
- 150000007524 organic acids Chemical class 0.000 claims description 4
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 claims description 4
- 150000005846 sugar alcohols Polymers 0.000 claims description 4
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 claims description 4
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 3
- 239000002585 base Substances 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 claims description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 claims description 2
- BPHVHMBNGQRCNN-UHFFFAOYSA-N 2,3-dihydroxybutanedioic acid;potassium Chemical compound [K].OC(=O)C(O)C(O)C(O)=O BPHVHMBNGQRCNN-UHFFFAOYSA-N 0.000 claims description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 2
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 claims description 2
- XTHFKEDIFFGKHM-UHFFFAOYSA-N Dimethoxyethane Chemical compound COCCOC XTHFKEDIFFGKHM-UHFFFAOYSA-N 0.000 claims description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 239000004280 Sodium formate Substances 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 2
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 claims description 2
- 235000011054 acetic acid Nutrition 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 235000010323 ascorbic acid Nutrition 0.000 claims description 2
- 239000011668 ascorbic acid Substances 0.000 claims description 2
- 229960005070 ascorbic acid Drugs 0.000 claims description 2
- 229940074360 caffeic acid Drugs 0.000 claims description 2
- 235000004883 caffeic acid Nutrition 0.000 claims description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims description 2
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 claims description 2
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- 239000001630 malic acid Substances 0.000 claims description 2
- 235000011090 malic acid Nutrition 0.000 claims description 2
- DUWWHGPELOTTOE-UHFFFAOYSA-N n-(5-chloro-2,4-dimethoxyphenyl)-3-oxobutanamide Chemical compound COC1=CC(OC)=C(NC(=O)CC(C)=O)C=C1Cl DUWWHGPELOTTOE-UHFFFAOYSA-N 0.000 claims description 2
- 229910017604 nitric acid Inorganic materials 0.000 claims description 2
- 150000007530 organic bases Chemical class 0.000 claims description 2
- 235000006408 oxalic acid Nutrition 0.000 claims description 2
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 235000011056 potassium acetate Nutrition 0.000 claims description 2
- 239000011736 potassium bicarbonate Substances 0.000 claims description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 2
- 235000015497 potassium bicarbonate Nutrition 0.000 claims description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 2
- 235000011181 potassium carbonates Nutrition 0.000 claims description 2
- 239000001508 potassium citrate Substances 0.000 claims description 2
- 229960002635 potassium citrate Drugs 0.000 claims description 2
- QEEAPRPFLLJWCF-UHFFFAOYSA-K potassium citrate (anhydrous) Chemical compound [K+].[K+].[K+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O QEEAPRPFLLJWCF-UHFFFAOYSA-K 0.000 claims description 2
- 235000011082 potassium citrates Nutrition 0.000 claims description 2
- WFIZEGIEIOHZCP-UHFFFAOYSA-M potassium formate Chemical compound [K+].[O-]C=O WFIZEGIEIOHZCP-UHFFFAOYSA-M 0.000 claims description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
- 235000019260 propionic acid Nutrition 0.000 claims description 2
- 229960004889 salicylic acid Drugs 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 239000000741 silica gel Substances 0.000 claims description 2
- 229910002027 silica gel Inorganic materials 0.000 claims description 2
- HELHAJAZNSDZJO-OLXYHTOASA-L sodium L-tartrate Chemical compound [Na+].[Na+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O HELHAJAZNSDZJO-OLXYHTOASA-L 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 235000017550 sodium carbonate Nutrition 0.000 claims description 2
- 239000001509 sodium citrate Substances 0.000 claims description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 2
- 235000011083 sodium citrates Nutrition 0.000 claims description 2
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical group [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 claims description 2
- 235000019254 sodium formate Nutrition 0.000 claims description 2
- 239000001433 sodium tartrate Substances 0.000 claims description 2
- 229960002167 sodium tartrate Drugs 0.000 claims description 2
- 235000011004 sodium tartrates Nutrition 0.000 claims description 2
- 239000001117 sulphuric acid Substances 0.000 claims description 2
- 235000011149 sulphuric acid Nutrition 0.000 claims description 2
- 239000011975 tartaric acid Substances 0.000 claims description 2
- 235000002906 tartaric acid Nutrition 0.000 claims description 2
- 230000003139 buffering effect Effects 0.000 claims 2
- 238000001914 filtration Methods 0.000 claims 1
- 238000001228 spectrum Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 4
- 239000000523 sample Substances 0.000 description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- BDKLKNJTMLIAFE-UHFFFAOYSA-N 2-(3-fluorophenyl)-1,3-oxazole-4-carbaldehyde Chemical compound FC1=CC=CC(C=2OC=C(C=O)N=2)=C1 BDKLKNJTMLIAFE-UHFFFAOYSA-N 0.000 description 2
- 102000005936 beta-Galactosidase Human genes 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 229940087562 sodium acetate trihydrate Drugs 0.000 description 2
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 1
- 206010025421 Macule Diseases 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- SBZXBUIDTXKZTM-UHFFFAOYSA-N diglyme Chemical compound COCCOCCOC SBZXBUIDTXKZTM-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004730 pulsed amperometry Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- -1 thio compound Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
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.
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