饲料中氨基酸的HPLC检测方法研究

发布时间：2018-06-06 23:51

本文选题：饲料 + 氨基酸　；参考：《河北农业大学》2015年硕士论文

【摘要】：饲料中的氨基酸是畜禽的重要营养物质,动物对蛋白质的需求实际上是对氨基酸的需求。饲料中含有的氨基酸种类和含量是判定饲料质量高低的重要指标。因此,饲料中氨基酸的准确分析、测定十分重要。但氨基酸分析误差较大,可能是由于不同的水解条件和不同的分析方法所造成的。氨基酸的分析测定是一个复杂的过程,所以需要建立一套完善的饲料中氨基酸的检测方法。蛋白质的氨基酸分析分成三大步骤,第一,把蛋白质彻底的水解为游离的氨基酸混合物;第二,用色谱的方法把混合的氨基酸分离成单个的氨基酸;第三,测定每个氨基酸的含量。目前,采用常规的酸水解法测定蛋白质氨基酸的含量,因为色氨酸在酸水解过程中几乎都被破坏,无法与其他的氨基酸一起测定,所以只能单独测定。本研究分为两大部分,一部分是柱前衍生高效液相色谱法测定饲料中的氨基酸;另一部分是高效液相色谱法测定饲料中的色氨酸。具体内容如下:本试验建立了以2,4-二硝基氯苯为柱前衍生剂的高效液相色谱法测定饲料中的氨基酸。通过对前处理条件中酸解剂、酸样比、盐酸浓度、水解温度、水解时间、通氮气、微波水解时间、微波水解温度、微波水解与常规水解的比较,衍生条件中衍生剂用量、衍生反应时间、衍生反应温度、衍生反应缓冲液、衍生反应缓冲液p H?色谱条件中色谱柱、缓冲液、缓冲液p H?缓冲液浓度、三乙胺加入量、洗脱程序、流速、色谱柱温度、有机洗脱溶剂、检测波长进行研宄,建立了19种氨基酸的标准曲线,其相关系数范围为0.9901?0.9993;检出限为0.69?5.70mg/L;加标回收率为91.9%?110.7%。结果表明,本方法操作简单,无干扰,具有较好的准确度和较高的灵敏度,能够满足饲料中氨基酸的定量分析。本试验还建立了高效液相色谱法测定饲料中的色氨酸。通过对前处理条件中碱解剂种类、碱解剂浓度、碱解剂用量、碱解时间、碱解温度、通氮气,色谱条件中缓冲液、流动相p H?有机洗脱溶剂、流动相组成、流动相流速、色谱柱温度、检测波长的研宄,建立了色氨酸的标准曲线,其相关系数为0.9998;检出限为O.llmg/L;加标回收率为93.5%。结果表明,本方法操作简单,无干扰,具有较好的准确度和较高的灵敏度,能够满足饲料中氨基酸的定量分析。
[Abstract]:Amino acids in feed are important nutrients for livestock and poultry. The kinds and contents of amino acids in feed are important indexes to judge the quality of feed. Therefore, the accurate analysis and determination of amino acids in feed is very important. However, the error of amino acid analysis may be due to different hydrolysis conditions and different analytical methods. The analysis and determination of amino acids is a complex process, so it is necessary to establish a complete method for the determination of amino acids in feed. The amino acid analysis of proteins is divided into three steps: first, the complete hydrolysis of proteins into free amino acid mixtures; second, separation of mixed amino acids into single amino acids by chromatographic method; third, The content of each amino acid was determined. At present, the conventional acid hydrolysis method is used to determine the content of protein amino acids. Because tryptophan is destroyed in the process of acid hydrolysis, it can not be determined together with other amino acids, so it can only be determined separately. This study is divided into two parts, one is the determination of amino acids in feed by precolumn derivatization HPLC, and the other is the determination of tryptophan in feed by HPLC. The main contents are as follows: the determination of amino acids in feed by high performance liquid chromatography (HPLC) with 2o 4-dinitrochlorobenzene as precolumn derivative was established. By comparing the acid hydrolysis agent, acid sample ratio, hydrochloric acid concentration, hydrolysis temperature, hydrolysis time, nitrogen gas, microwave hydrolysis time, microwave hydrolysis temperature, microwave hydrolysis and conventional hydrolysis, the amount of derivative in the derivatization condition was compared. Derivatization reaction time, reaction temperature, buffer solution pH? In chromatographic conditions, the column, buffer solution pH? Buffer concentration, triethylamine addition, elution procedure, flow rate, chromatographic column temperature, organic elution solvent, detection wavelength were studied, and the standard curves of 19 amino acids were established. The range of correlation coefficient was 0.9901g / L 0.9993, the detection limit was 0.69 ~ 5.70 mg / L, and the recovery rate was 91.9% ~ 110.7. The results showed that the method was simple, without interference, had better accuracy and sensitivity, and could be used for quantitative analysis of amino acids in feed. The determination of tryptophan in feed by HPLC was also established. In this paper, the kinds of alkali hydrolysates, the concentration of alkali hydrolysates, the amount of alkali hydrolysates, the time of alkali hydrolysis, the temperature of alkali hydrolysis, nitrogen, the buffer solution in chromatographic conditions, the mobile phase pH? The standard curve of tryptophan was established by studying the organic elution solvent, the composition of mobile phase, the flow rate of mobile phase, the temperature of chromatographic column and the detection wavelength. The correlation coefficient was 0.9998, the detection limit was 0. Llmg / L, and the recovery rate was 93. 5%. The results showed that the method was simple, without interference, had better accuracy and sensitivity, and could be used for quantitative analysis of amino acids in feed.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816.17

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