反向高效液相色谱法检测污泥中含硫氨基酸

Determination of sulfur amino acids in sludge by RP-HPLC

含硫氨基酸是污水厂污泥厌氧消化过程恶臭气体H2S和VOSCS的主要前体物质,以污水厂浓缩污泥为研究对象,针对污泥成分复杂,其含硫蛋白难水解、难提纯和易氧化而导致的尚无法定量检测分析的难点,通过优化预处理条件、色谱条件参数等,建立了一种污泥中含硫氨基酸反相高效液相检测的新方法.主要步骤为:50mg冷干污泥经1.5mL过甲酸氧化30min,将其中的半胱氨酸和蛋氨酸氧化为磺基丙氨酸和蛋氨酸砜,于水解管进行酸水解,3k D Millipore超滤离心,采用邻苯二甲醛进行柱前衍生,经氨基柱分离,通过FLD检测器检测.测定结果表明,磺基丙氨酸和蛋氨酸砜的定量限分别为0.24和1.21μmol/L,并且在5~500μmol/L范围内均具有良好线性关系(R^2≥0.9997),加标回收率均在90%以上.采用该方法测定污泥中的含硫氨基酸结果表明:浓缩污泥半胱氨酸和蛋氨酸含量分别为3.86和6.20mg/(g DS),160℃热水解后,二者含量分别降低了48.0%和51.9%,且厌氧消化后两种条件下含硫氨基酸的含量存在明显差异,为探究厌氧消化体系下污泥中硫的代谢途径和转化机制提供了方法支撑.

Sulfur-containing amino acids are the main precursor of H2S and VOSCS in sludge anaerobic digestion process.As the sulfur-containing protein in sewage sludge is difficult to be hydrolyzed,difficult to be extracted and easy to be oxidized,thus it is difficult to be quantified.Based on the optimal parameters of oxidation conditions,chromatographic conditions and etc.,a quantitative analysis method of sulfur-containing amino acids in sewage sludge was established by reverse-phase high performance liquid chromatography coupled with pre-column derivatization.The main procedures are as following:Firstly,the cysteine and methionine in sludge(50mg)were respectively oxidized to cysteic acid and methionine sulfone by performic acid(1.5mL,30min).Then,after ultrafiltration and derivatization by OPA,they were sequentially separated by NH2column and finally detected by FLD detector.A good linear relationship(corresponding linear correlation coefficient(R2)greater than0.9997)was obtained with the high recoveries of cysteine and methionine(above90%).In addition,the quantitation limits of cysteine and methionine were0.24and0.12μmol/L respectively.The sulfur-containing amino acids in different sludge samples were quantified by using this method.In raw sludge,the cysteine and methionine contents were3.86and6.20mg/(g DS)respectively,and decreased by48.0%and51.9% respectively after pyrohydrolysis treatment at160℃.Moreover,a significant difference of sulfur-containing amino acids content between pyrohydrolysis treatment and non-pyrohydrolysis treatment can be found,which suggest that the generation and utilization mechanisms of sulfur-containing amino acids were different in different anaerobic digesters.However,the established method of this study can provide an effective support to study the metabolic pathways and transfer mechanism in digestion systems.