Automated Programmable Generation of Broad pH Range Volatile Ionic Eluents for Liquid Chromatography
Automated Programmable Generation of Broad pH Range Volatile Ionic Eluents for Liquid Chromatography
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自动可编程生成用于液相色谱的宽 pH 范围挥发性离子洗脱液
DOI：
10.1021/acs.analchem.0c05089
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发表时间：
2021
影响因子：
7.4
通讯作者：
Dasgupta, Purnendu K.
中科院分区：
文献类型：
--
作者：
Shelor, Charles Phillip;Yoshikawa, Kenji;Dasgupta, Purnendu K.
在液相色谱（包括质谱）中的许多通用检测器在进一步处理和检测分析物之前必须首先使色谱洗脱液完全挥发。基本要求是洗脱液不含非挥发性溶解组分。然而，如果生物分子的生物活性必须在分离过程中存活，则生物分子的分离必须在具有相容pH和离子强度的主要水性介质中进行。氨与乙酸和甲酸的组合通常用作用于此目的的洗脱剂，但通常可耐受的最大浓度相对较低。此外，缓冲仅在有限的pH范围内是良好的。我们描述了一个系统，其中洗脱液是在一个自动的压力程序化的方式从高纯度的气态NH3和CO2通过气体渗透膜装置产生。这可以通过在移动的相中预先存在甲酸/乙酸来辅助，以延长可达到的低pH限度。我们概述了基本的pH值，离子强度和缓冲强度的考虑，并证明了这种洗脱液在分离氨基酸，蛋白质和单克隆抗体的应用。我们还证明了使用溶解的CO2作为离子配对剂在手性胺的分离。
Many of the universal detectors in liquid chromatography, including mass spectrometry, must completely volatilize the chromatographic eluent first before further processing and detection of the analytes. A basic requirement is that the eluent does not contain a nonvolatile dissolved component. However, separation of biomolecules must be conducted in mostly aqueous media of compatible pH and ionic strength if their biological activity must survive the separation process. Combinations of ammonia with acetic and formic acids are commonly used as eluent for this purpose but generally maximum concentrations that can be tolerated are relatively low. Further, buffering is good only over a limited pH range. We describe a system where the eluent is generated in an automated pressure-programmed manner from high-purity gaseous NH3and CO2through gas-permeable membrane devices. This can be aided by the prior presence of formic/acetic acids in the mobile phase to extend the attainable low pH limit. We outline the fundamental pH, ionic strength, and buffer intensity considerations and demonstrate the application of such eluents in the separation of amino acids, proteins, and monoclonal antibodies. We also demonstrate the use of dissolved CO2as an ion-pairing agent in the separation of chiral amines.