Analisis Asam Amino dengan Kromatografi Cairan Kinerja Tinggi Secara Derivatisasi Prakolom dan Pascakolom
Abstract
Abstract. Through an experiment process, an optimum condition of amino acid analysis using High Performance Liquid Chromatography (HPLC) with post- and pre-column derivatization methods has been found. The optimum condition of the analysis for the post-column deriyatization was obtained by using resin cation exchanger (R-SO3Na) as the column, citric acid buffer of the pH 3.0 and boric acid buffer of the pH 9.7 as the mobile phase and O-phtalaldehyde (OPA) /2-mercaptoethanoal as the derivator, In the pre-column derivatization method, Resolve 5μ spherical C18 was used as the column, solution A (methanol : THF ; H2O = 2 : 2 : 96) which contains 0.05 M Na2HPO4 and 0.05M NaOAc) and solutionB (methanol:H2O= 65 : 35) as the mobile phase, and OPA/ethantiol as the derivator. Each of both HPLC methods can separate more than 17 kinds of amino acids. The result shows that the pre-column derivatization method was more sensitive and took a shorter analysing time than the post-column derivatization method; however, secondary amino acid could not be determined by the first method.
Sari. Telah dilakukan percobaan untuk mencari kondisi optimum proses analisis asam amino dengan kromatografi cair kinerja tinggi (KCKT) secara derivatisasi prakolom dan pascakolom. Kondisi analisis optimum untuk cara derivatisasi pascakolom diperoleh dengan menggunakan resin penukar kation (R-SO3 Na+), larutan dapar sitrat pH 3,0 dan larutan dapar borat pH 9,7 sebagai fase gerak, serta O-ftalaldehida (OPA)/2-merkaptoetanol sebagai penderivatisasinya. Untuk cara derivatisasi prakolom, sebagai kolom digunakan Resolve spherical C18, sebagai fase gerak digunakan larutan A yang terdiri dari metanol : THF : air (2:2: 96) yang mengandung 0,5 M Na2 HPO4 dan larutan B yang terdiri dari metanol : air (65 :35), sedangkan sebagai zat penderivalisasi digunakan OPA/etantiol. Kedua metode KCKT ini masing-masing dapat memisahkan 17 macam campuran asam amino. Diketahui bahwa kepekaan cara derivatisasi prakolom lebih tinggi dan waktu analisisnya lebih cepat daripada cara derivatisasi pascakolom, tetapi cara prakolom tidak dapat menentukan asam amino sekunder.
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