Cain Caitlin N, Weatherbee Shelby L, Forzano Anna V, Rutan Sarah C, Collinson Maryanne M
Department of Chemistry, Virginia Commonwealth University, 1001 W. Main St., Richmond, VA 23284-2006, USA.
Department of Chemistry, Virginia Commonwealth University, 1001 W. Main St., Richmond, VA 23284-2006, USA.
J Chromatogr A. 2020 Jul 19;1623:461177. doi: 10.1016/j.chroma.2020.461177. Epub 2020 Apr 30.
Continuous stationary phase gradients for liquid chromatography (LC) have been recently shown to be a promising method of altering selectivity. In this work, we present the first multicomponent continuous stationary phase gradient for separations involving both reversed-phase (RP) and strong cation exchange (SCX) mechanisms. These columns are fabricated using a two-step methodology based on controlled rate infusion (CRI). First, destructive CRI creates a gradient of excess silanol groups along a uniform C column. Next, these columns are infused with 3-mercaptopropyltrimethoxysilane (MPTMOS), which bonds to the excess silanol groups. The terminal thiols of the MPTMOS ligands are oxidized with HO to create the sulfonate functionality (SO) needed for SCX separations. The success of the modification procedure is characterized by thermogravimetric analysis and X-ray photoelectron spectroscopy. The stability of the C-SO gradients were found to have less than 5 % retention loss and the column-to-column reproducibility had a relative standard deviation under 9 %. The peak asymmetry factors for seven biogenic amines were found to be between 1.03 ± 0.04 to 1.30 ± 0.02, which illustrates minimal peak tailing due to poor packing and residual silanol groups. Characterization of the gradient columns using an isocratic mobile phase showed a unique elution order compared to a uniform C and SO columns. At lower counterion concentrations, more than 48 % of the overall retention on the gradient stationary phase is due to a SCX mechanism. Meanwhile, the RP mechanism was shown to predominate at higher counterion concentrations on the gradient columns (SCX retention contribution less than 40 %). Coupling the stationary phase gradient to a salt gradient in the mobile phase showed that the gradient phase provides a unique, intermediate selectivity to the uniform C and SO columns. Under an ACN mobile phase gradient, a significant increase (p < 0.003) in the retention times of three biogenic amines (15 - 16 %) was observed when the multicomponent gradient was oriented to have a high SO ligand density near the detector. This work serves as a proof-of-concept design for a multicomponent stationary phase gradient to continue fundamental studies into retention and encourage novel applications.
液相色谱（LC）的连续固定相梯度最近已被证明是一种改变选择性的有前景的方法。在这项工作中，我们展示了首个用于涉及反相（RP）和强阳离子交换（SCX）机制分离的多组分连续固定相梯度。这些色谱柱采用基于控制速率注入（CRI）的两步法制造。首先，破坏性CRI沿着均匀的C柱创建过量硅醇基团的梯度。接下来，向这些色谱柱注入3 - 巯基丙基三甲氧基硅烷（MPTMOS），它与过量的硅醇基团结合。MPTMOS配体的末端硫醇用HO氧化以产生SCX分离所需的磺酸根官能团（SO）。改性过程的成功通过热重分析和X射线光电子能谱进行表征。发现C - SO梯度的稳定性具有小于5%的保留损失，并且柱间重现性具有低于9%的相对标准偏差。发现七种生物胺的峰不对称因子在1.03±0.04至1.30±0.02之间，这表明由于装填不良和残留硅醇基团导致的峰拖尾最小。使用等度流动相表征梯度柱显示，与均匀的C柱和SO柱相比，具有独特的洗脱顺序。在较低的抗衡离子浓度下，梯度固定相上超过48%的总保留归因于SCX机制。同时，在梯度柱上较高抗衡离子浓度时，RP机制占主导（SCX保留贡献小于40%）。将固定相梯度与流动相中的盐梯度相结合表明，梯度相为均匀的C柱和SO柱提供了独特的中间选择性。在乙腈流动相梯度下，当多组分梯度定向为在检测器附近具有高SO配体密度时，观察到三种生物胺的保留时间显著增加（p < 0.003）（15 - 16%）。这项工作作为多组分固定相梯度的概念验证设计，以继续进行关于保留的基础研究并鼓励新的应用。
