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Separation medium with iminodisuccinic acid as ligand and preparation method and application of separation medium

An iminodisuccinic acid and separation medium technology, which is applied in the field of chromatographic separation, can solve the problems such as no chromatographic separation medium, and achieve the effects of improving the loss problem, good repeatability, and improving separation and adsorption capacity.

Active Publication Date: 2017-01-18
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the application of IDS in the field of chromatographic separation media

Method used

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  • Separation medium with iminodisuccinic acid as ligand and preparation method and application of separation medium
  • Separation medium with iminodisuccinic acid as ligand and preparation method and application of separation medium
  • Separation medium with iminodisuccinic acid as ligand and preparation method and application of separation medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The main raw materials used in this example: macroporous silica gel (7μm, pore size 30nm), γ-glycidyloxypropyltrimethoxysilane, FeCl 3 ,CuSO 4 ,NiCl 2 ,ZnSO 4 ,CoCl 2 and Ca(NO 3 ) 2 For commercially available goods.

[0055] Preparation of silica gel hard matrix separation medium with IDS as ligand:

[0056] Put 10g of silica gel into a beaker filled with 50mL of 1.0mol / L HCl, and vibrate ultrasonically for 2-3min. Filter and wash with water until neutral, i.e. no chlorine ions can be detected, put the wet silica gel into a container containing 100mL 1.0mol / L HNO 3 Heating to reflux in the beaker for 1h. Cool the solution, filter it with No. 4 sand core glass dry pan, wash it with water until neutral, and dry it at 90-100°C for 4 hours before use.

[0057] Weigh 2g of acid-treated dry silica gel and place it in a flask, add 40mL of 0.1mol / L NaAc-HAc buffer solution (pH 4.0), sonicate for 2min, add 2mL of γ-GLDP dropwise, continue to react at 90°C for 2h, after...

Embodiment 2

[0060] Using the separation medium prepared in Example 1, the samples of silica gel and IDS-silica gel media were prepared by KBr pellet method, and infrared (IR) characterization was performed.

[0061] Infrared data of silica gel, FT-IR (ν / cm -1 ): 3421 (vs, v O-H ),3149(s,v H2O ). Infrared data of IDS-silica gel, FT-IR (ν / cm -1 ): 3469(s,ν O-H ), 2931, 2869 (s, C-H), 1992, 1876, 1728cm -1 (Stretching vibration of C=O), 1631cm -1 (m,d H-C-H ),1574(s,ν C＝O ),1472cm -1 (s, v c-c ),1409cm -1 (vs,ν C＝O ),1257cm -1 (s, v C-N ),1191(s,C-O-C),1075(vs,ν Si-O ),817(s,δ Si-C ).

[0062] see figure 1 , it can be seen from the infrared data that in the infrared data of silica gel and IDS-silica gel, respectively, at 3421cm -1 and 3469cm -1 There are silanol stretching vibration absorption peaks. In addition, the infrared data of IDS-silica gel also shows that the absorption peak of OH- stretching vibration is a characteristic peak. 2931cm -1 and 2869cm -1 CH 2 Th...

Embodiment 3

[0064] Using the separation medium prepared in Example 1, the mixture of bovine serum albumin (BSA), ribonuclease (RNase), and lysozyme (Lys) was separated by ion exchange chromatography.

[0065] Chromatographic column: 50×4.6mm stainless steel column; stationary phase: IDS-silica gel; mobile phase: A (equilibrium liquid), 20mmol / L PB (pH 6.0), mobile phase B (eluent): A+0.5mol / L NaCl (pH 6.0); mobile phase flow rate: 1.0mL / min; linear gradient 20min, 100%A-100%B, 100%B extended for 10min. Standard protein: 1.BSA; 2.RNase; 3.Lys.

[0066] Several acidic and basic proteins were effectively separated on IDS-silica gel column. And the order of the peaks flows out in the order of increasing isoelectric points: that is, BSA (pI=4.9) flows out first without retention, and RNase (pI=7.8) and Lys (pI=11.0-11.4) flow out sequentially. see figure 2 , the results showed that the obtained IDS-silica gel media had typical cation exchange properties.

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Abstract

The invention discloses a separation medium with iminodisuccinic acid (IDS) as ligand and a preparation method and application of the separation medium. As the ligand, the IDS is bonded to a chromatographic filler through a spacer arm, so as to prepare a novel chromatographic medium with ion exchange chromatography, high-strength metal-chelating chromatographic properties and a dual separation function. When metal ions are not immobilized, the ion exchange properties of the separation medium can be rivaled with a conventional IDA medium; when the metal ions are immobilized, the metal-chelating chromatographic properties of the separation medium are far superior to those of a commercial IDA column, and are comparable to those of an EDTA column, and defects of a conventional common metal-chelating immobilized phase filler with IDA as the ligand are overcome. Meanwhile, a good basis is made for solving the problem of metal ion loss on an IMAC column in later periods. The separation medium has great values for expanding the application range and the application prospects of the IMAC column, and increasing the market benefits of the IMAC filler.

Description

technical field [0001] The invention belongs to the technical field of chromatographic separation, and in particular relates to a separation medium with iminodisuccinic acid as a ligand, a preparation method and application thereof. Background technique [0002] Since the concept of metal chelate affinity chromatography (MCAC) was proposed in 1975, ion exchangers with aminocarboxyl complexing agents as ligands have been widely used in the field of chromatographic separation and purification. For this type of ligand, the bare column that does not fix metal ions can be used as a cation exchanger to separate some positively charged components, and compared with general carboxylic acid cation exchangers, the pH range of chromatographic work can be reduced from 4.0 to 2.0-3.0. On the other hand, using the coordination of N and O atoms in the aminocarboxyl group, the metal in the solvent can be removed. This type of ion exchanger has been widely used in many fields such as food, ...

Claims

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Application Information

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IPC IPC(8): B01J20/283B01J20/285B01J20/286B01J20/30B01J39/17C08B37/12C08F257/00C08F8/32C08G12/40
CPCB01J20/283B01J20/285B01J20/286B01J39/16B01J2220/4812B01J2220/52C08B37/0039C08F8/32C08G12/40C08F257/00
Inventor 李蓉陈斌张宁李晨马晓迅
Owner NORTHWEST UNIV
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