论文总字数：18288字

摘 要

氨基酸是生命物质基础-蛋白质的基本构成，是生命体能量的重要来源。它作为一种有机化合物，主要包含了氨基和羧基等基团。它在各种生物功能中都有着及其重要的作用，与生命体的活动息息相通。氨基酸可以用作食品添加剂、保健品、表面活性剂、化妆品等等。氨基酸的分析在许多领域都有着重要意义，比如蛋白质化学、生物化学、临床医学、食品科学、化学工程等。氨基酸的分析方法自1958年起，就不断出现新的创新，从最开始的阳离子交换色谱分离-柱后茚三酮衍生化法到近年来被提出的液相色谱质谱联用(LC/MS)法等，但是这些方法大多存在分析时间长、过程繁或者就是仪器专用性强、费用高等缺陷。而高效液相色谱法是至今为止分析氨基酸最为广泛的技术，本实验采用的邻苯二甲醛(OPA)柱前衍生反相高效液相色谱法则具有衍生时间快、灵敏度高、基本无副产物、色谱基线稳定等优点。所以要建立OPA柱前衍生反相高效液相色谱法，让检测限更低、灵敏度更高、速度更快。

本实验过程中，用高效液相色谱，柱前衍生法，对18种氨基酸进行检测，因为衍生剂的选型对检测的影响较大，本实验选用邻苯二甲醛。采用C₁₈色谱柱，流动相A为甲醇、乙腈和水体积比为45：45：10的混合溶液，流动相B为10 mmol/L PH=7.25磷酸二氢钾溶液，柱温为40 ℃，进样大小为10 μL；荧光检测器λex=350，λem=450，梯度洗脱并调整流动相流速，分析氨基酸。

结果表明本实验操作时间短、无干扰条件、操作简便、灵敏度较高、准确度高，可以用于氨基酸的检测。

关键词：氨基酸分析 高效液相色谱 OPA 荧光检测器

Analysis of Amino Acid

Abstract

Amino acid is the basic material of life - the basic composition of protein, an organic compound composed of a carboxyl group and an amino group. It is very important to various biological functions and is related to life activities closely. Application of amino acids in many food additives, health products, surfactants, cosmetics, etc. The analysis of amino acids has important implications in many fields, such as protein chemistry, biochemistry, clinical medicine, food science, and chemical engineering. Amino Acid Analysis Methods Since 1958, new innovations have emerged, starting with the first cation exchange chromatography-column ninhydrin derivatization method and the proposed liquid chromatography-mass spectrometry (LC/MS). Law, etc., but most of these methods have long analysis time, complicated process or high specificity and high cost. So far, the most widely used technique for analyzing amino acids is still high performance liquid chromatography.. The OPA pre-column RP-HPLC method used in this experiment has the advantages of fast derivation time, high sensitivity, almost no by-products, and stable chromatographic baseline. Therefore, it is necessary to establish an OPA pre-staged reversed-phase HPLC, then lower the detection limit, higher the level of sensitivity and higher the speed.

In this experiment, o-phthalaldehyde (OPA) was used as a pre-column derivatization agent, and 18 amino acids were detected by a reversed-phase high-performance liquid phase method using a fluorescence detector as a detector. InertSustain C18 column (5 μm, 4.6mm×250mm), mobile phase A is V methanol: V acetonitrile: V water = 45:45:10, mobile phase B is 10mmol / l PH=7.25 potassium dihydrogen phosphate, the column temperature is 40 ° C, inject 10 microliters into the injection vial; the fluorescence detector λex=350, λem=450, the gradient eluted and the mobile phase flow rate was adjusted to analyze the amino acids.

The results show that the experiment has short operation time, no interference condition, simple operation, high sensitivity and high accuracy, and can be used for amino acid detection.

Key Words:Amino acids analysis OPA HPLC Fluorescence detector

目录

摘 要 I

Abstract II

目录 i

第一章 文献综述 1

1.1氨基酸 1

1.2氨基酸的分析的意义 2

1.3氨基酸的分析方法 2

1.3.1色谱法 2

1.3.2化学法 4

1.3.3电化学分析法 4

1.3.4毛细管电泳法 4

1.3.5分光光度法 4

1.4本论文构想及主要研究内容 5

第二章 实验部分 6

2.1原料和设备 6

2.1.1化学试剂 6

2.1.2设备 7

2.2氨基酸分子量的测量 8

2.2.1仪器和试剂 8

2.2.2色谱条件 8

2.2.3样品制备 8

2.2.4测量 8

2.3氨基酸混合溶液的测量 8

2.3.1仪器和试剂 8

2.3.2色谱分析条件 9

2.3.3样品制备 9

2.3.4OPA衍生试剂的配置 9

2.3.5氨基酸的衍生 9

第三章 结果与讨论 11

3.1分子量测量结果 11

3.2流动相流速的选择 12

3.3泵A浓度的选择 12

3.4洗脱的确定 12

3.5衍生剂用量对衍生反应的影响 12

3.6柱温箱温度的选择 13

3.7流动相PH对分离的影响 13

3.8氨基酸样品的检测 15

3.9氨基酸样品的定性和定量分析 16

第四章 结论与展望 18

参考文献 19

致谢 22

第一章 文献综述

1.1氨基酸

氨基酸是蛋白质的基本构成，一种由羧基和氨基组成的有机化合物。它在许多生物功能中都有着及其重要的作用，与生命体的活动息息相通^[1]。

请支付后下载全文，论文总字数：18288字