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. 2018 Oct 5;1570:82-90. doi: 10.1016/j.chroma.2018.07.073. Epub 2018 Jul 27.
The use of stationary phase gradients for liquid chromatography (LC) is a promising new strategy to allow for specific control over the selectivity of a separation by having a gradual change in the ligand density along the length of the column. Unfortunately, there have been very few, if any, methods to prepare continuous stationary phase gradients on traditional packed LC columns. In this work, destructive methodologies are used to create stationary phase gradients on commercial C columns by infusing trifluoroacetic acid (TFA) onto the column through controlled rate of infusion (CRI). The introduction of TFA via CRI while the column is heated at 80 °C promotes acid hydrolysis of the alkylsilane ligand in a gradient fashion. Characterization with scanning electron microscopy and Barrett-Joyner-Halenda pore size distributions of the stationary phase after fabrication of the destructive gradient establishes that the chromatographic support was not damaged during the procedure. The shape of the gradient was examined using thermogravimetric analysis (TGA) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. TGA and ATR-FTIR showed an increase in the percent carbon loss along the length of the column, indicating that there was an increase in the C ligand from the front to the end of the column. Two selectivity tests demonstrated a decrease in the hydrophobicity and increase in the silanol activity of the stationary phase gradient from the uniform C counterpart. Additionally, the fabrication of the destructive stationary phase gradient resulted in two different surface functionalities allowing hydrophobic and hydrophilic interactions with analyte species depending on the mobile phase composition. Plots of the log of retention factor versus percent acetonitrile illustrated that these stationary phase gradients have two separation mechanisms: reversed-phase (RP) and hydrophilic interaction. Coupling the stationary phase gradient with a mobile phase gradient shows differences in the peak widths and the resolution of phenolic compounds, indicating that the orientation of the stationary phase gradient has the potential to enhance the resolution of a separation. With this methodology, stationary phase gradients can be fabricated on previously used RP columns, allowing for these columns to be repurposed.
液相色谱（LC）中使用固定相梯度是一种很有前途的新策略，可以通过沿柱长逐渐改变配体密度来对分离的选择性进行特定控制。不幸的是，在传统填充 LC 柱上制备连续固定相梯度的方法很少，如果有的话。在这项工作中，通过受控速率输注（CRI）将三氟乙酸（TFA）注入柱中，使用破坏性方法在商业 C 柱上创建固定相梯度。在 80°C 加热的同时通过 CRI 引入 TFA 会以梯度方式促进烷基硅烷配体的酸水解。在制造破坏性梯度后，通过扫描电子显微镜和 Barrett-Joyner-Halenda 孔径分布对固定相进行的特性研究表明，在该过程中色谱支撑体未损坏。通过热重分析（TGA）和衰减全反射-傅里叶变换红外（ATR-FTIR）光谱检查梯度的形状。TGA 和 ATR-FTIR 显示柱长上的碳损失百分比增加，表明柱前端到柱末端的 C 配体增加。两种选择性测试表明，固定相梯度的疏水性降低，硅醇活性增加，与均匀的 C 相比。此外，制造破坏性固定相梯度会导致两种不同的表面官能团，根据流动相组成，允许与分析物物种发生疏水和亲水相互作用。保留因子的对数与乙腈百分比的关系图表明，这些固定相梯度具有两种分离机制：反相（RP）和亲水相互作用。将固定相梯度与流动相梯度耦合表明酚类化合物的峰宽和分辨率存在差异，表明固定相梯度的方向有可能提高分离的分辨率。使用这种方法，可以在以前使用的 RP 柱上制造固定相梯度，从而可以重复使用这些柱。
