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甲、乙、丙、丁酸在土壤圈广泛存在，通常含量较低，测定时易受基质中杂质干扰，难以准确定性定量。采用偶联酰胺化法将具有紫外高灵敏响应的吲哚基团引入其结构中，生成酰胺类衍生物，即可在液相色谱-二极管阵列/紫外检测器（HPLC-DAD/UV）下间接高灵敏度检测，达到准确测定的目的。衍生化操作步骤如下：先将适量样品和2-（N-吗啉代）乙磺酸（MES）水溶液加入反应小瓶中，使反应体系pH保持约5.5，再加入适量的1-乙基-（3-二甲基氨基丙基）碳二亚胺（EDC）水溶液，交联反应20 min后再加入适量的N-羟基丁二酰亚胺（NHS）水溶液，之后经偶联反应适当时间后再加入色胺的乙腈水溶液，酰胺化反应过夜，第二天用超纯水定容终止反应，待测。实验表明：在衍生化过程中，当保持MES：EDC：NHS：色胺：羧基≈10︰7︰3︰7︰1（摩尔比）的比例，加入NHS后偶联反应45 min时效果较佳。该方法中四种有机酸的检测限范围为0.008~0.046 mg·L–1，标准工作曲线的线性决定系数R2为0.991~0.998，平均回收率为85.9%~123%，方法精密度为1.69%~8.41%，可以满足有机分析要求。与液相色谱法直接测定小分子单羧酸相比，发现直接法测试结果与衍生-液相色谱法测试结果基本一致，但衍生法可以修正直接法中杂质干扰带来的阳性误差，同时提升低浓度单羧酸样品中有机酸的检出率，定性与定量结果更可靠。
Objective Formic, acetic, propionic and butyric acid exist widely in the natural ecological environment, but their contents are usually low. Thus, their accurate qualitative and quantitative determination is often hindered by impurities in the sample matrix.Method Using the coupling amidation method, low molecular monocarboxylic acids (LMMAs) can form new derivatives with high sensitivity to ultraviolet (UV) light. This was achieved by incorporating the indole group into LMMAs' structure, which made it possible for the low-level LMMAs to be detected by high-performance liquid chromatography with diode array detector (HPLC-DAD) under a specific wavelength UV light with high response. As a result, we observed the accurate determination of LMMAs. Through experimentations, we propose the following steps: first, a suitable amount of sample and MES (2-Morpholinoethanesulfonic Acid) aqueous solution were added into a small bottle with the reaction system adjusted to about pH≈5.5. This was followed by the addition of EDC (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) aqueous solution. After 20 minutes of cross-linking reaction, a suitable amount of NHS (N-Hydroxy succinimide) aqueous solution was added to promote coupling reactions. After an appropriate period, a tryptamine aqueous solution (containing acetonitrile) was added into the reaction system for amidation overnight. This was followed by the addition of ultrapure water to a certain volume and prepared for determination.Result The experimental results showed that for an optimized observation, the mole ratio of MES: EDC: NHS: tryptamine: carboxylic acid should be maintained at about 10 : 7 : 3 : 7 : 1 around and after 45 minutes of coupled reaction and the addition of tryptamine aqueous solution for amidation. The detection limit for the four organic acids in this method was from 0.008 to 0.046 mg·L–1, and the linear determinate coefficient R2 of the standard working curve was around 0.991~0.998. The average recovery rate range was 85.9%~123%, and the precision of the method ranged between 1.69%~8.41%, which meets the requirements of organic analysis.Conclusion The actual sample test results showed that the data of the derivation method and the direct method were the same. However, compared with the HPLC direct detection method, the derivation-HPLC method could correct the positive error caused by the impurities in complex samples, and improve the detection rates of low concentration monocarboxylic acid samples. So the qualitative and quantitative results of the derivation-HPLC method were more reliable.
陈虹,唐昊冶,钟明,刘晓东,王如海,俞元春.偶联酰胺化衍生-液相色谱法高灵敏度检测土壤圈样品中小分子单羧酸[J].土壤学报,2022,59(6):1594-1605. DOI:10.11766/trxb202103080130 CHEN Hong, TANG Haoye, ZHONG Ming, LIU Xiaodong, WANG Ruhai, YU Yuanchun. A Coupled Amidation - Liquid Chromatography Technique for Detecting Low Molecular Monocarboxylic Acid in Pedosphere Samples with High Sensitivity[J]. Acta Pedologica Sinica,2022,59(6):1594-1605.复制