echozxjjj
第2楼2006/08/15
我的缓冲液的配法是跟这个方法的:
A 0.1 mmol/L stock solution of 5-HT was prepared in 0.01 mol/L cold
perchloric acid and stored at 4C before use.
A 0.45 mmol/L of FITC stock solution was prepared with acetonitrile containing 0.8% v/v methanol and 0.5% v/v pyridine. A 45 mmol/L of FITC working solution was prepared from the above stock solution with further dilution by using the same acetonitrile mixture solution.
A 0.05 mol/L Na2HPO4 derivatization buffer solution was prepared by dissolution of 7.80 g/L of Na2HPO4 in deionized water, adjusted to pH 10.95 with 1.0 mol/L NaOH.
A 40 mmol/L sodium tetraborate and 60 mmol/L boric acid mixture buffer solution was adjusted to pH 9.0 with 1.0 mol/L NaOH and was used as the background electrolyte (BGE). The working solutions of 5-HT standards were diluted with this BGE solution.
衍生化: Ten mL 1.0 mmol/L PAs or CAs working solution or extracts was mixed with 10 mL of 45 mmol/L FITC, 200 mL 0.05 mol/L Na2HPO4 buffer, and 200 mL water, vortexed and reacted in the dark at 257C for 6 h.
进样: Samples were injected by pressure at 0.5 psi for 20 s, and separations were performed under 25 kV for 20 min with a positive
high voltage.
我的结果是,大部分时候 5-HT 都出不了峰,有两次出了(跟空白对照),但后来又重复不到.同时做了几个不同浓度的反应,1,10,100微克/L,加了同样量的FITC,只有10的那个浓度出了个峰,100 的那个浓度也有一个分叉的峰,但迁移时间推后了大概两分钟,对不上.