摘要
建立了一条液相色谱法两步纯化tBoc固相合成的戈那瑞林粗品的路线。第一步采用离子交换色谱进行初步分离,考察了CM 25 Sepharose Fast Flow填料和CMC填料对分离的影响;选用了CM 25 Sepharose Fast Flow填料,先用pH值6.0、50 mmol.L-1NaAc缓冲溶液平衡,再上样,然后用pH值6.0、50 mmol.L-1NaAc、500 mmol.L-1氯化钠缓冲溶液洗脱,此步除去了粗品中的大部分非极性杂质和部分极性杂质。第二步用制备型反相高效液相色谱(C18色谱柱)进行纯化,考察了不同流动相组成磷酸三乙胺(TEAP)和三氟乙酸(TFA)对分离纯化的影响;选用了TFA体系,流动相A为0.1%TFA、5%乙腈水溶液;流动相B为0.1%TFA、80%乙腈水溶液,梯度洗脱。终产物纯度可达98.5%以上,总收率为72%。
In this paper,a purification route using the liquid chromatography was established on gonadorelin synthesized by the solid phase peptide synthesis（SPPS） method using tBoc group for protecting α-amino acid.Two steps were included in the purification route.The first step was the ion exchange chromatography（IEC）.Two matrixes including CM 25 Sepharose Fast Flow and CMC were screened in order to optimize the separation.CM 25 Sepharose Fast Flow was determined to be used for its higher recovery rate and higher resolution.The medium was first equilibrated with pH value 6.0,50 mmol·L^-1NaAc buffer and eluted with pH value 6.0,50 mmol·L^-1NaAc,500 mmol·L^-1NaCl buffer.After this step,majority of nonpolar impurities and part of polar impurities were removed.The second step was reversed phase high performance liquid preparative chromatography（RP-HPLC） on C18 column.Several eluent systems containing TEAP or TFA were screened in order to optimize the separation.The system containing TFA was decided to be used for its higher recovery rate and higher resolution.Acetonitrile gradient elution was used.Mobile phase composition A was 0.1% TFA,5% acetonitrile in water;Mobile phase composition B was 0.1% TFA,80% acetonitrile in water.Acetonitrile gradient elution was used.Then the final product exceeded 98.5% of purity.The total yield was 72%.Those operation conditions during the isolation and purification process were also studied and optimized in this thesis.
出处
《化学与生物工程》
CAS
2010年第9期88-91,共4页
Chemistry & Bioengineering
关键词
戈那瑞林
固相多肽合成
离子交换色谱
反相高效液相色谱
纯化
gonadorelin
solid phase peptide synthesis（SPPS）
ion exchange chromatography（IEC）
reversed phase high performance liquid chromatography（RP-HPLC）
purification
