De Vos Jelle, Desmet Gert, Eeltink Sebastiaan
Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium.
Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050 Brussels, Belgium.
J Chromatogr A. 2016 Jul 15;1455:86-92. doi: 10.1016/j.chroma.2016.05.046. Epub 2016 May 25.
We developed earlier the post-column refocusing strategy for isocratic separations, which employs trapping target analytes after an analytical separation and additionally focusing them using a strong remobilization solvent prior to detection, and have now extended it to high-speed gradient LC. A gradient separation of antibiotics and its metabolites, applying a linear aqueous acetonitrile gradient from 2 to 65% (v/v) ACN containing 0.1% FA in 10min, performed on an analytical column was selected as an application. Eluted heart-cut fractions were directed from the analytical silica C18 column to a trap column packed with Hypercarb particles. The remobilization of the target analytes was performed in back-flush mode using solvent mixtures tuned to maximize the solvent strength by mixing isopropanol into the remobilization solvent containing acetonitrile. Additionally, a viscosity-calibration experiment showed that the viscosity difference between trapping and remobilization solvents should be smaller than 0.15mPa·s to prevent viscous fingering. To keep the viscosity difference below this limit, during the gradient separation performed on the analytical column, the composition of the remobilization solvent was changed in time. An empirical equation is provided that allows for the selection of the optimal remobilization-solvent composition. To maximize the signal enhancement, the loading time of target analytes on the trap column should be optimized. Peak dispersion was further minimized by applying a flow rate that corresponded to the optimal van-Deemter flow rate of the trap column (20μL/min). Finally, decreasing the diameter of the trap column from 1mm to 0.3mm led to a significant enhancement of the detection sensitivity with more than one order of magnitude. Using an optimized trap configuration and elution/remobilization conditions, a signal enhancement of a factor of 14 was achieved for sulfaguanidine (early-eluting compound in the gradient separation) and 7.3 for furazolidone (late-eluting compound).
我们之前开发了用于等度分离的柱后重新聚焦策略，该策略是在分析分离后捕获目标分析物，并在检测前使用强流动溶剂对其进行额外聚焦，现在我们已将其扩展到高速梯度液相色谱。选择了在分析柱上进行的抗生素及其代谢物的梯度分离作为应用实例，该分离采用线性乙腈水溶液梯度，在10分钟内从2%（v/v）乙腈线性变化至65%（v/v）乙腈，并含有0.1%甲酸。从分析型硅胶C18柱洗脱的中心切割馏分被导向填充有Hypercarb颗粒的捕集柱。目标分析物的重新聚焦采用反冲模式，通过将异丙醇混入含乙腈的流动溶剂中来调整溶剂混合物，以最大化溶剂强度。此外，粘度校准实验表明，捕获溶剂和流动溶剂之间的粘度差应小于0.15mPa·s，以防止粘性指进。为了将粘度差保持在此限值以下，在分析柱上进行梯度分离期间，流动溶剂的组成会随时间变化。提供了一个经验方程，用于选择最佳的流动溶剂组成。为了最大化信号增强，应优化目标分析物在捕集柱上的加载时间。通过应用与捕集柱的最佳范德姆特流速（20μL/min）相对应的流速，进一步最小化了峰展宽。最后，将捕集柱的直径从1mm减小到0.3mm，检测灵敏度显著提高，提高了一个多数量级。使用优化的捕集配置和洗脱/重新聚焦条件，对于磺胺胍（梯度分离中早洗脱的化合物）实现了14倍的信号增强，对于呋喃唑酮（晚洗脱的化合物）实现了7.3倍。
