In this paper, the newly developed ion exchange phase separation method to create chitosan sub-micron particles is introduced: 1) chitosan was dissolved in a lactic acid aqueous solution. 2) the obtained chitosan solution was added stepwise in a sodium sulfate aqueous solution and cooled down to 5℃ to become slightly turbid through agglutination. 3) desalinating and deacidifying of the mixture was carried out by a dialyzing tube method. IR spectroscopy and elemental analysis indicated that the agglutination of chitosan was induced by crosslinking effect with an electrostatic interaction between sulfate anions and amino groups in the glucosamine unit although large excess of Na<sub>2</sub>SO<sub>4</sub> caused undesirable further agglutination of the resultant chitosan particles. As a result, the proper amount of Na<sub>2</sub>SO<sub>4</sub> was approximately 1.0 - 10.0 equivalent for the amino group to create the chitosan particles with a sub-micron size. In addition, we investigated an antibacterial activity test for Escherichia coli of the obtained chitosan particles. The significant antibacterial activity was observed in incubation even at neutral pH condition while the chitosan microbeads (size: ca 200 <i>∫</i>m) prepared by the conventional method and chitosan granules (size: ca 600 <i>∫</i>m) as starting materials showed almost no antibacterial activity in the same condition.
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