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wanghf

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[求助] ph示差法

用ph示差法测定苋菜红色素的含量,我的步骤如下:
1.将苋菜烘干,磨碎,过筛,备用;
2.称取1g样品,加入25ml,40%乙醇,超声波辅助提取30min,40摄氏度,200w;
3.过滤,旋转蒸发,离心,得到提取液;
4.取1ml提取液,分别用ph1.0和ph4.5的缓冲液定容到10ml,静置20min;
5.分别在540nm(我自己扫描得到的波长)和700nm处测得吸光度值。
ph1.0的缓冲液:称取1.49gkcl,用水定容到100ml,用hcl调节ph到1.0
ph4.5的缓冲液:三水合乙酸钠2.99g,冰醋酸1.68g,加水至1L
出现的问题:加入ph1.0缓冲液的提取液颜色比ph4.5处的浅,这样测得的吸光度值前者小于后者,根据ph示差法的经验公式,两者的差值是负值
各位有碰到过这样的问题吗?求指教,非常感谢!!
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