Investigation of Experimental Conditions for Analyzing ε-Polylysine with HPLC-UV-MS

碩士 === 國立高雄大學 === 應用化學系碩士班 === 107 === Preliminary goal of this research is to develop an effective HPLC method for analyzing ε-polylysine (ε-PL) from various suppliers. After the method was developed, we found that the robustness of the method is very poor and can easily misinterpret experimental r...

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Bibliographic Details
Main Authors: HUANG, YI-CHUN, 黃怡郡
Other Authors: HO, YEUNG-HAW
Format: Others
Language:zh-TW
Published: 2019
Online Access:http://ndltd.ncl.edu.tw/handle/k7xt6d
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Summary:碩士 === 國立高雄大學 === 應用化學系碩士班 === 107 === Preliminary goal of this research is to develop an effective HPLC method for analyzing ε-polylysine (ε-PL) from various suppliers. After the method was developed, we found that the robustness of the method is very poor and can easily misinterpret experimental results. Therefore, we further investigated the factor that cause the poor robustness of the HPLC method. Trifluoroacetic acid (TFA) is an essential mobile phase modifier for analyzing ε-PL with various degree of polymerization. Using formic acid and acetic acid as the modifier cannot resolve ε-PL with various degree of polymerization. TFA acted as an ion-pair reagent. Number and strength of multi-interactions between TFA and ε-PL will affect the conformation and the net charge of ε-PL---TFAs adduct resulted in different retention time. ε-PLs coeluted before the retention time of mobile phase while MeOH/0.1% TFA=50/50 was used as mobile phase. ε-PLs were retained in column while MeOH/0.1% TFA=20/80 was used as mobile phase. Accidentally, we noticed ε-PL from two different vendors (L and S) had different retention time under same experimental conditions. We speculated that matrix effect was the cause of disagreement. We studied the effect of several sample pretreatment on separation of ε-PLs. The pretreatments included (1) adjusted pH of L and S to same pH; (2) heated up L and S then cool down them to room temperatures; (3) heated up L and S then added EDTA into the solutions and cool down the solution to room temperatures; (4) diluted L and S with various organic solvents. The experimental results showed: (1) the matrix in sample did affect the retention time of ε-PL in column; (2) the mobile phase cannot overcame the matrix effect originated from sample L and S. In conclusion, results indicated ε-PL exhibited molten globule conformation properties in L and extended conformation properties in S.