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

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

3. + + + + + + + + + + + + + + + + V. Regeneration V. Regeneration + + + + + + + + + + II. Adsorption of sample II. Adsorption of sample + + + + + + + + - - + + + + + + + + + + + + + + + + I. Starting condition I. Starting condition + + + + + + + + - - III. Starting desorption III. Starting desorption + + + + + + + + + + + + + + + + + + + + + + + + + + IV. End of desorption IV. End of desorption + + + + + + + + + + + + + + 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. 2. Pass filtrate through XAD-2 resin and elute with 60% aqueous MeOH. 3. Concentrate eluate and adjust pH to 3.5 with aqueous NH4OH. 4. Dilute with H20 and pass through Amberlite IRA-68 (Cl) resin column. 5. Elute with 1M NaNO3 in 0.1M NaOAc (pH 7.5), collecting fractions. 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. 8. Dissolve in 0.5M NH4Br-0.05M AcOH buffer. 9. Chromatograph on DEAE-Sephadex A-25. 10. Bioassay fractions; pool active fractions. 11. Pass each pool through XAD-2 resin and elute with 90% aqueous MeOH. Ambelite XAD-2 Ambelite IRA-68 DEAE-sephadex A-25 Desalting Weak anion exchange

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

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

7. 5.2 Cationic Compounds 5.2.2 Gualamycin 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. Dowex-50W CM-Sephadex Weak anion exchange Weak Cation exchange Strong cation exchange

8. 5.2 Cationic Compounds 5.2.2 Paromomycins 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 Amberlite IRC-50 Amberlite CG-50 Weak anion exchange Weak Cation exchange Weak cation exchange

9. Thank you for your attention