Penteado José Carlo Pires, Rigobello-Masini Marilda, Liria Cleber Wanderlei, Miranda M Terêsa Machini, Masini Jorge Cesar
Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.
J Sep Sci. 2009 Aug;32(15-16):2827-34. doi: 10.1002/jssc.200800741.
This paper describes the development and application of an RP HPLC method using a C(18) monolithic stationary phase for the separation and quantification of extra- and intracellular amino acids in a batch cultivation of the marine alga Tetraselmis gracilis. Fluorimetric detection was made after separation of the o-phthaldialdehyde 2-mercaptoethanol (OPA-2MCE) derivatives using a binary gradient elution. Separation of 19 amino acids was achieved with resolution >1.5 in about 39 min at a flow rate of 1.5 mL/min. RSD of analyses in seawater medium ranged from 0.36% for Orn (0.50 micromol/L) to 12% for Ile (0.10 micromol/L). The main constituents of the intracellular dissolved free amino acids (DFAAs) in the exponential growth phase were arginine (Arg), asparagine (Asn), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), glutamine (Gln), and leucine (Leu). The major amino acids excreted to the media were valine (Val), Ala, Ser, and Gly. The monolithic phase facilitates the analysis by shortening the separation time and saving solvents and instrumentation costs (indeed conventional HPLC instrumentation can be used, running at lower pressures than those ones used with packed particle columns).
本文描述了一种使用C(18)整体固定相的反相高效液相色谱（RP HPLC）方法的开发与应用，该方法用于在海洋藻类纤细四爿藻的分批培养中分离和定量细胞外和细胞内氨基酸。使用二元梯度洗脱分离邻苯二甲醛-2-巯基乙醇（OPA-2MCE）衍生物后进行荧光检测。在流速为1.5 mL/min的条件下，约39分钟内实现了19种氨基酸的分离，分离度>1.5。在海水培养基中的分析相对标准偏差（RSD）范围为：鸟氨酸（Orn，0.50 μmol/L）为0.36%至异亮氨酸（Ile，0.10 μmol/L）为12%。指数生长期细胞内溶解游离氨基酸（DFAAs）的主要成分是精氨酸（Arg）、天冬酰胺（Asn）、丙氨酸（Ala）、天冬氨酸（Asp）、谷氨酸（Glu）、丝氨酸（Ser）、甘氨酸（Gly）、谷氨酰胺（Gln）和亮氨酸（Leu）。分泌到培养基中的主要氨基酸是缬氨酸（Val）、Ala、Ser和Gly。整体固定相通过缩短分离时间、节省溶剂和仪器成本促进了分析（实际上可以使用传统的HPLC仪器，运行压力低于填充颗粒柱所用的压力）。
