离子交换色谱法分离纯化水溶性花菁染料的研究

Study on purification of water-soluble cyanine dye by anion exchange chromatography

针对水溶性不对称荧光染料及副产物不同的电荷特性,采用DEAE-52阴离子交换色谱结合葡聚糖凝胶色谱对其进行分离纯化,并以纯化后的染料作为荧光探针对抗体进行标记。结果表明,当使用含0.5 mol/L Na2SO4的0.2 mol/L PBS缓冲液(pH 7.4)进行洗脱时,DEAE-52阴离子交换色谱能够将目标染料分离,得到纯度为98.4%的产物,得率57.1%。产物经活化后用于标记抗体,标记度达到4.6,与同类别的CFTM647相当,达到了实用的要求。

In order to separate the water-soluble fluorescent dye from its by-products with different charge characteristics, DEAE-52 anion exchange chromatography and gel permeation chromatography were used. The product was labeled the antibody as a fluorescent probe. The result shows that DEAE-52 anion exchange chromatography can purify the dye efficiently when 0.2 mol/L PBS （ pH 7.4） with the addition of 0.5 mol/L Na2SO4 was used as eluent. Detecting by HPLC the purity of the product is 98.4% and yield 57.1%. After being activated, the dye was used to label the antibody. Comparing the similar dye CFTM 647, the dye＇ s D/P is 4.6. To acquire practical fluorescent probe, this method is convinced to be applicable.