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目的采用柱前衍生化反相高效液相荧光色谱法拆分肌肽的对映体。方法采用邻苯二甲醛和N-乙酰-L-半胱氨酸为柱前手性衍生化试剂,反相液相色谱法分离肌肽的光学异构体。考察衍生化反应条件对肌肽衍生化反应、不同色谱条件下对肌肽光学异构体分离的影响。通过光学异构体分离过程中的热力学参数的计算,探讨光学异构体分离过程的驱动力。结果邻苯二甲醛和N-乙酰-L-半胱氨酸的摩尔比为1∶2时,以无水乙醇与硼酸缓冲液(pH9.8)为溶剂溶解,作为衍生化试剂,于室温下避光反应5 min,肌肽转化成异吲哚产物;在柱温35℃时,肌肽光学异构体的衍生化产物分离因子(α)和分离度(R)分别为1.533、3.947。其光学异构体-衍生产物的拆分过程主要是一个焓驱动的过程。结论所用方法简单快速、灵敏度高、重复性好,适用于肌肽的手性拆分。
Objective To separate enantiomers of carnosine by precolumn derivatization reversed-phase high performance liquid chromatography. Methods Ophthalmicdehyde and N-acetyl-L-cysteine were used as pre-column chiral derivatization reagents. The optical isomers of carnosine were separated by reversed-phase liquid chromatography. The effects of derivatization conditions on the derivatization of carnosine and the separation of carnosine optical isomers under different chromatographic conditions were investigated. Through the calculation of the thermodynamic parameters during the separation of optical isomers, the driving force of the optical isomers separation process is discussed. Results When the molar ratio of o-phthalaldehyde to N-acetyl-L-cysteine was 1: 2, it was dissolved in absolute ethanol and boric acid buffer (pH 9.8) as a solvent to derivatize at room temperature The carnosine was converted to isoindole after 5 min of dark reaction. The derivatization products (α) and resolution (R) of carnosine optical isomers were 1.533 and 3.947 at 35 ℃ respectively. The resolution process of its optical isomer-derivative is mainly an enthalpy-driven process. Conclusion The method is simple, rapid, sensitive and reproducible. It is suitable for the chiral separation of carnosine.