Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale
Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale
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DOI：
10.1021/acs.analchem.9b03408
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发表时间：
2019-11-05
影响因子：
7.4
通讯作者：
Regalado, Erik L.
中科院分区：
文献类型：
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作者：
Liu, Jinchu;Makarov, Alexey A.;Regalado, Erik L.
使用常规分离方法对复杂的高极性分析物混合物进行色谱分离、分析和表征通常非常具有挑战性。亲水性化合物的分析和纯化一直由液相色谱（LC）和离子交换色谱（IC）主导，亚/超临界流体色谱（SFC）正朝着这些超越传统手性分离的新应用方向发展。然而，超临界二氧化碳（CO2）的低极性限制了SFC在生物分析空间中用于分离和纯化的使用，特别是在制备规模。即使在醇改性剂/CO2基洗脱液中使用极性添加剂，高极性物质的反应混合物也能被强烈保留。在这里，我们克服了这些问题，通过引入超液效应在SFC分离使用非传统的移动的相混合物组成的氢氧化铵结合高浓度的水在醇改性剂和二氧化碳。在此阐明了分离机制的基础上进行的SFC条件下，表明原位形成的碳酸氢盐counterparts（HCO 3-）的环肽的广泛的IC-CD（IC耦合到电导检测）分析。与其他盐相比，HCO 3-被发现发挥关键作用，作为一种离液剂，破坏不需要的氢键相互作用，这是由尺寸排阻色谱法结合差分氢-氘交换-质谱实验（SEC-HDX-MS）证明。此外，将在富含水的MeOH改性剂中使用NH 4 OH与其他常用的碱性添加剂（二乙胺、三乙胺和异丁胺）进行了比较，在峰形、保留、选择性和电离方面显示出无与伦比的色谱和MS检测性能，以及完全不同的选择性和保留行为。此外，相对于富水甲醇改进剂中的甲酸铵和乙酸铵，水添加剂中的氢氧化铵表现出更好的色谱性能，提高了灵敏度。NH 4 OH和H2O水平的进一步优化与MeOH/CO2一起用于提供通用改性剂（0.2%NH4OH，5%H2O在MeOH中），其使得SFC能够广泛过渡到先前被认为超出其范围的域。这种方法被广泛应用于分离，分析和纯化的多组分反应混合物的密切相关的极性药物使用现成的SFC仪器。本文所述的实施例涵盖了生物分析和制药应用的广泛范围，包括有机卤化物质、核碱基、核苷、核苷酸、磺酰胺和环肽以及其他高极性物质的分析和制备色谱。
Chromatographic separation, analysis and characterization of complex highly polar analyte mixtures can often be very challenging using conventional separation approaches. Analysis and purification of hydrophilic compounds have been dominated by liquid chromatography (LC) and ion-exchange chromatography (IC), with sub/supercritical fluid chromatography (SFC) moving toward these new applications beyond traditional chiral separations. However, the low polarity of supercritical carbon dioxide (CO2) has limited the use of SFC for separation and purification in the bioanalytical space, especially at the preparative scale. Reaction mixtures of highly polar species are strongly retained even using polar additives in alcohol modifier/CO2 based eluents. Herein, we overcome these problems by introducing chaotropic effects in SFC separations using a nontraditional mobile phase mixture consisting of ammonium hydroxide combined with high water concentration in the alcohol modifier and carbon dioxide. The separation mechanism was here elucidated based on extensive IC-CD (IC couple to conductivity detection) analysis of cyclic peptides subjected to the SFC conditions, indicating the in situ formation of a bicarbonate counterion (HCO3-). In contrast to other salts, HCO3- was found to play a crucial role acting as a chaotropic agent that disrupts undesired H-bonding interactions, which was demonstrated by size-exclusion chromatography coupled with differential hydrogen-deuterium exchange-mass spectrometry experiments (SEC-HDX-MS). In addition, the use of NH4OH in water-rich MeOH modifiers was compared to other commonly used basic additives (diethylamine, triethylamine, and isobutylamine) showing unmatched chromatographic and MS detection performance in terms of peak shape, retention, selectivity, and ionization as well as a completely different selectivity and retention behavior. Moreover, relative to ammonium formate and ammonium acetate in water-rich methanol modifier, the ammonium hydroxide in water additive showed better chromatographic performance with enhanced sensitivity. Further optimization of NH4OH and H2O levels in conjunction with MeOH/CO2 served to furnish a generic modifier (0.2% NH4OH, 5% H2O in MeOH) that enables the widespread transition of SFC to domains that were previously considered out of its scope. This approach is extensively applied to the separation, analysis, and purification of multicomponent reaction mixtures of closely related polar pharmaceuticals using readily available SFC instrumentation. The examples described here cover a broad spectrum of bioanalytical and pharmaceutical applications including analytical and preparative chromatography of organohalogenated species, nucleobases, nucleosides, nucleotides, sulfonamides, and cyclic peptides among other highly polar species.