On-line derivatization fluorescence detection coupled 2-D HPLC for amino acid enantiomers analysis from aspartame thermal hydrolysis in Coca Cola Zero

• Main Authors: Shing-Chen Wu, 吳幸真
• Other Authors: Cheanyeh Cheng
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/xx89y7

碩士 === 中原大學 === 化學研究所 === 98 === A two-dimensional high-performance liquid chromatography (2-D HPLC) system was developed by combining an ultraviolet detector coupled reversed-phase chromatography and a fluorescence detector coupled ligand-exchange chromatography (LEC) with column-switching technique to study the thermal hydrolysis of aspartame in Coca-Cola Zero. Aspartame was quantified by the matrix matched external standard calibration curve method. The linear concentration range of the external standard calibration curve was of 0~50 μg mL-1 and possessed a linear corrlelation coefficient of 0.9984 which gave a limit of detection (LOD) of 1.3 μg mL-1. The amino acid enantiomers produced from the thermal hydrolysis of aspartame was separated by ligand-exchange column and detected by on-line postcolumn fluorescence derivatization fluorescence detection. The quantitative analysis was made by the matrix matched internal standard calibration method and 4 μg mL-1 D-leucine was used as the internal standard. The linear concentration range of the four internal standard calibration curves for the four amino acid enantiomers (D- and L-aspartic acid and D- and L-phenylalanine was all in the range of 0~10 μg mL-1 and their linear correlation coefficients were in the range of 0.9988-0.9997. The LODs obtained from the four standard calibration curves of D- and L-aspartic acid and D- and L-phenylalanine were all 0.2 μg mL-1. However, the LOD for D- and L-aspartic acid and D- and L-phenylalanine with ultraviolet detection was 2.5 and 2.6 μg mL-1 , respectively. Therefore, the sensitivity of flurorescence detection for amino acid enantiomers was 12-13 folds better than that of the ultraviolet detection. The thermal hydrolysis of aspartame in Coca-Cola Zero was performed with a special designed mricro-reactor and at four different temperatures (37, 60, 90 and 120℃) for one day or more than one day. The yield was 2.1%～19.3% for D,L-aspartic acid and 2.5%～21.3% for D,L-phenylalanine acid, respectively. The thermal hydrolysis of aspartame was also performed by microwave heating and tested at three different powers (320 W, 560 W, and 800 W) for 1 or 3 minutes. Only L-aspartic acid was produced with yields of 2.1% ~ 2.3%. The analysis accuracy tested by the spike experiments were 90%~99%. The relative standard deviations (RSDs) of repeated sample measurements were all smaller than 6.7%. Therefore, the developed 2D-HPLC not only can simultaneously determine the dipeptide aspartame but also can determine its four thermal hydrolysis amino acid enantiomer products with high sensitivity, accuracy, and precision.