柱后衍生用反应流高效液相色谱柱
Summary
A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.
Abstract
A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.
Introduction
再加上柱后衍生（PCD）的高效液相色谱（HPLC）是一个强大的工具，是在分析实验室解决的一些问题是有用的。它可用于检测与该套件可用1,2-探测器否则不可检测的化合物，增加目标分析物，这允许检测和定量3-5的下限或选择性的信号衍生的靶分析物，以避免基质效应6。常用的PCD反应包括胺类，如氨基酸的反应中，用邻- phthaladehyde 7-9，茚三酮9,10-或荧光胺11,12，活性氧的衍生（ROS）与2,2-二苯基1- picrylhydrazil自由基（DPPH•）13,14或2,2'-连氮基-双（3-乙基苯并噻唑-6-磺酸（ABTS）15,16，以及使用碘化物叠氮化物试剂的衍生化磺丙ontaining化合物17,18。
有，但是，众多的缺点使用PCD反应的高效液相色谱系统6。在这些主要是添加衍生试剂（S）和所述检测器，其允许时间用于混合和发生8反应的点之间的使用反应线圈。这些反应环路通常具有500微升或更多，相对于HPLC系统19的其余部分的容积是显著的卷。使用这些高容量的反应循环中增加的峰加宽的结果相比，现在将未经反应环的存在下观察到的。这将导致在具有定量和检测的更高的限制更短，更宽的峰和负效应色谱分辨率。 图1和2突出，从加入各种柱后反应循环体积导致峰形状的恶化。这种分析用94％甲醇的流动相组合物和6％Milli-Q水进行。流动相的流速为1毫升/分钟，注射体积为20μl，并在分析波长为265纳米。从20微升至1000微升变化死体积的线圈插入柱并以模拟在PCD方法反应循环死体积的效应的检测器之间。这些环被从0.5mm内径的不锈钢管来制备。该实验由一个控制器（SCL-10AVP）的HPLC系统上进行，一个低压梯度阀（FCL-10ALVP），一个泵（LC-20AD）中，喷油器（SIL-10ADVP）和PDA检测器（ SPD-M10ADVP）。流动相通过脱气引入HPLC系统之前泵送。使用250毫米×4.6mm内径5微米柱进行分离。实验条件选择为典型的最近已经在文献中公开的PCD反应。
该最简单，最常见的柱后反应器设置被称为非分段的管状反应器，它实际上是一个长，细管，通过它可以使液体流出，反应可以发生。在这个系统中的峰变宽取决于不仅添加到系统中的死体积，而且还作为强调由饭岛等人 8的管的内径上。此外，线圈的几何形状起着观察品牌变宽的一部分。斯图尔特20指出，反应器的卷绕改变二次流剖面，导致更好的混合，这意味着死体积可以被最小化。据指出，峰宽用针织线圈21一 个开放的管状时不显著。当峰加宽过大时，其它类型的反应器也可考虑20,22。这些可能包括床反应器或分段流反应器。这些反应器是反应慢了，否则会特别requir有用Ë大的反应循环。作为非分段的管状反应器是将PCD应用中最常用的类型的反应器，本文涉及这种类型的反应器装置的其余部分。
反应流程（RF）列的设计采用了多端口端部装配件，允许流动相，或者通过设在位于外柱的径向中心区域或三个端口的单个端口退出（或输入）该列列的壁区域（ 见图3）。这两个流使用含有由不可渗透的环是依次由延伸出到塔壁的外多孔玻璃料包围一个中央多孔玻璃料一个端部接头隔开。由于中央不透水环横流不是两个多孔区之间可能的。
在反应流层析，所述衍生化试剂（多个）被泵对流动相的流动方向成一个或总重量澳反应流柱的外部端口。柱洗脱剂通过自由外端口与外玻璃料的衍生试剂（多个）混合，并传递给检测器。反应流程可用于任一单一试剂衍生（1端口的衍生试剂，1端口到柱洗脱液传递给检测器和1个端口的阻塞状态）或双试剂系统（2个端口的衍生试剂和1端口通过柱洗脱剂的检测器）。从中央流的流动可以被用来检测未衍生柱洗脱液，有效地多路复用检测23，或传递给浪费。
一个主要的调整技术是可用时运行的RF-PCD色谱是中枢和外周流的比率。每个衍生的最佳比例取决于许多因素，如中央流是否将被检测或传递给浪费。因此，一旦最佳的比率已经确定，应当确保该正确流量比前正在执行每个运行来实现的。
业已发现，使用玻璃料的给列洗脱液流和射频PCD导致更有效的混合与通常采用零死体积T型件或低死体积的传统混合技术相比，衍生试剂混合W-片的两个流混合。这允许使用相对较小的反应的循环，或者在反应循环的甚至完全取消。反应回路尺寸的结果更清晰峰的减少相比传统的柱后衍生的方法。这意味着，尽管并非所有的柱洗脱液的衍生化，信噪比更大的信号被观察并且可以实现检测和定量的，因此下限。
反应流层析已经开发克服与PCD反应的适应性困难s至因造成的，需要采用大体积反应循环大柱后死体积现代HPLC柱和系统，特别是在致频带加宽效率的损失。相比于常规的PCD中的RF-PCD的更有效的混合过程意味着较小的反应环体积可以采用导致的增加观察到的分离效率。此外射频-PCD色谱同时显示增加的信号并进行比较，以产生相对于常规的PCD方法检测和定量的下限常规的PCD技术减少噪音。相比于常规的PCD方法的RF-PCD的另一个优点是，以监视来自RF列的中央端口以及从所述塔的外围区域洗脱衍生的流洗脱未衍生流的能力。 RF-PCD是一个相对较新的，但有前途的技术，该技术相对于传统的PCD方法显示许多优点。
<p class="jove_content">射频柱的连接中几乎相同的方式与主要差别是端部装配件的一个射频列的数目的常规HPLC柱实现。用于连接标准HPLC柱的HPLC系统接头能够被用于一个射频柱连接到HPLC系统。Protocol
Representative Results
Discussion
RF-PCD允许与HPLC流出柱后衍生化试剂在不使用反应线圈的有效混合，减少频带加宽的影响，并提高分离性能。的RF-PCD方法也示于相对于检测方法的信号响应的改善。 Camenzuli 等 28是第一个报道的意式咖啡样品中DPPH•使用反应流列用于检测ROS的。他们的研究中所涉及的分析和射频条件的优化，以达到最大的性能，测试了一系列的DPPH•浓度与各种DPPH•试剂流速?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This work was supported by UWS and ThermoFisher Scientific. One of the authors (DK) acknowledges the receipt of an Australian Postgraduate Award.
Materials
| HPLC instrument | Agilent | 1290 Series HPLC | |
| Additional Pump(s) for derivatisation system | Shimadzu | LC-20A | |
| RF colum | Non-commercial | ||
| PEEK tubing | Sigma Aldrich | Z227307 | |
| Column stoppers | Provided with column | ||
| PEEK tube cutter | Sigma Aldrich | Z290882 | |
| Analytical Scale Balance | 4-point analytical balance | ||
| Stop watch | Non-Scientific equiptment | ||
| Eluent collection vials | Any Small vial with a flat bottom will do e.g. HPLC vials | ||
| HPLC Vials | Will depend on instrument used | ||
| Vessels for mobile phase and derivatisation solution(s) | Sigma Aldrich | Z232211 | |
| General Laboratory glassware | Volumetric Flasks, pippettes, etc. Quantity and volumes will depend on sample preparation method | ||
| Methanol | Sigma Aldrich | 34860 | |
| DPPH | Sigma Aldrich | D9132 | |
| Ammonium Acetate | Sigma Aldrich | 17836 | |
| Ammonia | Sigma Aldrich | 320145 | Corrosive |
| Acetonitrile | Sigma Aldrich | 34998 | |
| Fluorescamine | Sigma Aldrich | F9015 | |
| 4-aminoantipyrene | Acros Organics BVBA | AC103151000 | |
| Potassium ferricyanide | AnalaR | B10204-30 |
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