1 / 9

Isolation by Ion-exchange methods

Isolation by Ion-exchange methods. Theory & Application. 2009 11 17 Kang MinSeok & Chun JeaMoo. Theory of Ion exchange Chromatography. Porous (DVB% low). Porous ( macropore in particle). High-Porous / Macroeticular (DVB% high). Gel. I. Starting condition . II. Adsorption

tallis
Download Presentation

Isolation by Ion-exchange methods

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Isolation by Ion-exchange methods Theory &Application 2009 11 17 KangMinSeok& Chun JeaMoo

  2. Theory of Ion exchange Chromatography Porous (DVB% low) Porous (macropore in particle) High-Porous / Macroeticular (DVB% high) Gel

  3. I. Starting condition II. Adsorption of sample III. Starting desorption IV. End of desorption V. Regeneration + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + Practical Procedure Ions and ions of the sample are in competition with each other.

  4. 5.1 Anionic Compounds 5.1.1 Cephamycin A and B 1. Acidify the fermentation broth and filter. Ambelite XAD-2 2. Pass filtrate through XAD-2 resin and elute with 60% aqueous MeOH. Desalting 3. Concentrate eluateand adjust pH to 3.5 with aqueous NH4OH. 4. Dilute with H20 and pass through Amberlite IRA-68 (Cl) resin column. Ambelite IRA-68 5. Elute with 1M NaNO3 in 0.1M NaOAc(pH 7.5), collecting fractions. Weak anion exchange 6. Bioassay and combine active fractions adjust to pH 3.0 and desalt on XAD-2 as under 2 above. 7. Concentrate eluate, adjust to pH 4.0 lyophilize. DEAE-sephadex A-25 8. Dissolve in 0.5M NH4Br-0.05M AcOHbuffer. Weak anion exchange 9. Chromatograph on DEAE-SephadexA-25. 10. Bioassay fractions; pool active fractions. 11. Pass each pool through XAD-2 resin and elute with 90% aqueous MeOH.

  5. 5.1 Anionic Compounds 5.1.2 Zaragozic acids Amberlyst A-21 1. Extract whole fermentation broth with EtOAc at pH 2. • 2. Adsorbed onto Amberlyst A-21 (acetate cycle) Weak anion exchange Removal acidic Materials • 3. Elute with 3% NH4Cl in 90% aqueous MeOH Diaion HP-20 • 4. Desalt eluate with Diaion HP-20 Desalting

  6. 5.2 Cationic Compounds 5.2.1 Palau’amine • 1. Lypophilize sponge and extract with MeOH. Cellex CM (Na+) 2. Evaporate solvent and triturate with water. Weak Cation exchanger • 3. Pass through Cellex CM (Na cycle) resin column. Sephasex LH-20 • 4. Elute with step gradient NaCl; bioassay fractions. • 5. Lypophilize and desalt by trituration with EtOH. Removal contaminants • 6. Chromatograph ethanol soluble material on • Sephadex LH-20, eluting with MeOH.

  7. 5.2 Cationic Compounds 5.2.2 Gualamycin Dowex-50W 1. Filter broth; pass through charcoal column. 2. Wash with water; elute with step gradient of MeOH. 3. Bioassay fractions; pool active fractions. 4. Pass through Dowex-50W(Hþ cycle) resin column. 5. Wash with water; elute with 2.8% aqueous ammonia. 6. Bioassay fractions; lypophilize and redissolve in water. 7. Pass through CM-Sephadex (Na+) resin column. 8. Elute with step gradient of aqueous NaCl. 9. Bioassay: desalt. Weak anion exchange Strong cation exchange CM-Sephadex Weak Cation exchange

  8. 5.2 Cationic Compounds 5.2.2 Paromomycins Amberlite IRC-50 1. Adjust broth to pH 3.0; filter. 2. Adjust filtrate to pH 7.0 with NaOH. 3. Pass through Amberlite IRC-50 (NH4+) resin column. 4. Wash with water; elute with 0.5M aqueous NH4OH. 5. Bioassay eluate fractions; concentrate active fractions in vacuo; adjust to pH 7.0. 6. Pass through Amberlite CG-50 (NH3) resin column. 7. Wash with water; elute with step gradient aqueous NH4OH (0.05–0.3M). 8. Bioassay fractions; evaporate Weak anion exchange Weak cation exchange Amberlite CG-50 Weak Cation exchange

  9. Thank you for your attention

More Related