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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

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Isolation by Ion-exchange methods

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  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

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