Venkatramani Cadapakam J, Zelechonok Yury
Pharmacia Corporation, Global Chemical Process R & D, 4901 Searle Parkway, Skokie, Illinois 60077, USA.
Anal Chem. 2003 Jul 15;75(14):3484-94. doi: 10.1021/ac030075w.
A simple approach to two-dimensional liquid chromatography has been developed by coupling columns of different selectivity using a 12-port, dual-position valve and a standard HPLC system. The valve at the junction of the two columns enables continuous, periodic sampling (injection) of the primary column eluent onto the secondary column. The separation in the primary dimension is comparable to conventional HPLC, whereas the secondary column separation is fast, lasting several seconds. The high-speed separation in the secondary dimension enables the primary column eluent to be sampled with fidelity onto the secondary column throughout the chromatographic run. One might expect a coupled column liquid chromatography system operating in reverse-phase mode to be strongly correlated and, hence, inefficient. However, by applying a solvent gradient in the primary dimension and by progressively incrementing the solvent strength in the secondary dimension (tuning), the inefficiency or cross correlation between the two dimensions is minimized. In a tuned two-dimensional system, the influence of primary column retention (usually hydrophobicity) is minimal on secondary column retention. This enables subtle differences in component interaction with the two stationary phases to dominate the secondary column retention. The peaks are randomly dispersed over a retention plane rather than along a diagonal, resulting in an orthogonal separation. The peak capacity is multiplicative, and each component has a unique pair of retention times, enabling positive identification. In addition, the location of the component provides two independent measures of molecular properties. The 2D-LC system was evaluated by analyzing a test mixture made of some aromatic amines and non-amines on different secondary columns (ODS-AQ/ODS monolith, ODS/amino, ODS/cyano). The relative location of sample components in the two-dimensional plane varied significantly with change in secondary column. Among the secondary columns, the amino and cyano columns offered the most complementary separation, with the retention order of several components reversed in the secondary dimension. The theoretical peak capacity of the 2D-LC system was around 450 for a separation lasting 30 min. A 2D-LC system involving amino and cyano columns resulted in a high-speed separation of the test mixture, with most of the chemical components resolved within a few minutes.
通过使用一个12端口、双位置阀和一个标准高效液相色谱（HPLC）系统连接不同选择性的色谱柱，开发出了一种二维液相色谱的简单方法。两根色谱柱连接处的阀门能够将主色谱柱洗脱液连续、周期性地进样到次色谱柱上。主维度的分离与传统HPLC相当，而次色谱柱的分离速度很快，持续几秒钟。次维度的高速分离使得在整个色谱运行过程中，主色谱柱洗脱液能够准确无误地进样到次色谱柱上。人们可能会认为，以反相模式运行的联用色谱柱液相色谱系统会有很强的相关性，因此效率低下。然而，通过在主维度上应用溶剂梯度，并在次维度上逐步增加溶剂强度（调整），两个维度之间的低效率或交叉相关性被最小化。在一个经过调整的二维系统中，主色谱柱保留（通常是疏水性）对次色谱柱保留的影响最小。这使得组分与两种固定相之间相互作用的细微差异主导了次色谱柱的保留。峰随机分散在保留平面上，而不是沿着对角线分布，从而实现正交分离。峰容量是相乘的，每个组分都有一对独特的保留时间，能够进行可靠的鉴定。此外，组分的位置提供了分子性质的两个独立测量值。通过在不同的次色谱柱（ODS - AQ/ODS整体柱、ODS/氨基柱、ODS/氰基柱）上分析由一些芳香胺和非胺类组成的测试混合物，对二维液相色谱系统进行了评估。样品组分在二维平面中的相对位置随次色谱柱的变化而显著不同。在这些次色谱柱中，氨基柱和氰基柱提供了最互补的分离，几种组分在次维度上的保留顺序发生了反转。对于持续30分钟的分离，二维液相色谱系统的理论峰容量约为450。涉及氨基柱和氰基柱的二维液相色谱系统实现了测试混合物的高速分离，大多数化学成分在几分钟内就得到了分离。
