题目内容

某公司技术人员需要利用离子交换层析法分离氨基酸,实验过程如下。1.试剂与材料苯乙烯磺酸钠型树脂(强酸1×8,100-200目);无水乙醇;2mol/L盐酸溶液;2mol/LNaOH溶液;1mol/LNaOH溶液。天冬氨酸的pI为2.97,组氨酸的pI为7.59,赖氨酸的pI为9.74。标准氨基酸溶液:天冬氨酸、赖氨酸和组氨酸均配制成2mg/mL的0.1mol/L的盐酸溶液。混合氨基酸溶液:将3种标准氨基酸溶液按1∶2.5∶10的比例混合。洗脱液1:0.45mol/L、pH( )柠檬酸缓冲液;洗脱液2:0.2 mol/L、pH( )Tris-HCl缓冲液;洗脱液3:0.01mol/L pH( ) NaOH缓冲液2. 器材25cm×1cm层析管, ( )、 ( ),分光光度计。3. 操作方法 (1)( )将干的强酸型树脂用蒸馏水浸泡过夜,使之充分溶胀。用4倍体积的乙醇浸泡2小时,倾去清液,洗至中性。再用2mol/L 氢氧化钠处理,做法同上;然后用2mol/L HCl溶液浸泡2小时,倾去清液,洗至中性。最后用欲使用缓冲液浸泡。(2)( )取直径1cm,长度25cm的层析柱。将柱垂直至于铁架上。自顶部注入上述经处理的树脂悬浮液,关闭层析柱出口,待树脂沉降后,放出过量溶液,再加入一些树脂,至树脂沉降至 距离顶端2~3cm的高度即可。(3)( )用pH5.8的柠檬酸缓冲液冲洗平衡交换柱。调节流速为0.5mL/min,流出液达到床体积的4倍时即( ) 为平衡完成的标志,然后可以上样。(4)( )由柱上端旋转加入氨基酸的混合液0.25~0.5mL,同时开始收集流出液。当样品液面靠近床面时,即刻加入0.5mL柠檬酸缓冲液冲洗加样品处。待缓冲液面靠近床面时,再加入0.5mL缓冲液。如此重复两次,然后,将进水口连通洗脱瓶,层析柱出水口与( )相连。(5)洗脱用pH5.8的柠檬酸缓冲液洗脱,并用试管收集洗脱液,每管收集1mL,共收集30~40管。然后换( )洗脱,并用试管收集洗脱液,每管收集1mL,共收集30~40管;最后换 ( )洗脱,并用试管收集洗脱液,每管收集1mL,共收集30~40管;4、实验结果与分析根据氨基酸的解离性质(天冬氨酸pI2.97,组氨酸pI7.59,赖氨酸pI9.74),分析该实验条件下氨基酸从层析柱洗脱下来的顺序( )。

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