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目的响应面法优化检测己二酸二肼(adipate hydrazine,ADH)衍化后破伤风类毒素(tetanus toxoid,TT)纯度的排阻型高效液相(SEC-HPLC)法。方法 SEC-HPLC层析柱Superdex 200 increase 3.2/300流动相为:20 mmol/L Na_2CO_3/NaHCO_3,150 mmol/L NaCl,pH 9.0,柱温25℃,单针检测时间60 min。采用响应面法及中心复合表面设计模型(Central Composite Face-centred,CCF)对层析柱的流速、进样量、样品浓度进行优化,通过分离度、峰对称性、峰理论塔板数、单针进样时间等评价指标,确定最优色谱参数,并在最优色谱参数下连续进样100针,验证其分离效果。结果色谱柱的最优色谱条件为:流速50μl/min,进样量5μl,样品浓度1.0 mg/ml,连续进样100针各响应量均满足优化目标。结论成功采用响应面法优化了检测ADH衍化后TT纯度的SEC-HPLC法,且优化后的方法具有良好的稳定性。
OBJECTIVE To determine the purity of tetanus toxoid (TT) derived from adipate hydrazine (ADH) by surface-modified high performance liquid chromatography (HPLC). Methods SEC-HPLC column Superdex 200 increase 3.2 / 300 mobile phase was: 20 mmol / L Na 2 CO 3 / NaHCO 3, 150 mmol / L NaCl, pH 9.0, column temperature 25 ℃, single needle detection time 60 min. The response surface method and Central Composite Face-centred (CCF) were used to optimize the chromatographic column flow rate, injection volume and sample concentration. The separation, peak symmetry, peak theoretical plate number, Needle injection time and other evaluation indicators to determine the optimal chromatographic parameters, and in the optimal chromatographic parameters continuous injection of 100 needles to verify the separation effect. Results The optimal chromatographic conditions were: flow rate 50μl / min, injection volume 5μl, sample concentration 1.0 mg / ml, continuous injection of 100-pin response to meet the optimization objectives. Conclusion The response surface method was successfully used to optimize the SEC purity of TT after derivatization with ADH, and the optimized method has good stability.