Stoll Dwight R, O'Neill Kelly, Harmes David C
Gustavus Adolphus College, 800 West College Avenue, St. Peter, MN 56082, United States.
Gustavus Adolphus College, 800 West College Avenue, St. Peter, MN 56082, United States.
J Chromatogr A. 2015 Feb 27;1383:25-34. doi: 10.1016/j.chroma.2014.12.054. Epub 2014 Dec 27.
Two persistent impediments to wider adoption of two-dimensional liquid chromatography (2D-LC) are the perceptions that 2D methods are generally less sensitive than 1D ones, and that coupling of certain separation modes in a 2D system is difficult because of the negative impact of the effluent of the first separation on the second separation. In this work we address these problems in the specific case where reversed-phase separations are used in both dimensions of a 2D-LC system, but the pH is varied such that the ionization state of carboxylic acid analytes is different (i.e., neutral or negatively charged, in eluents buffered at pH 2 or 7) in the two columns. We first demonstrate that the effect of first dimension ((1)D) effluent on the performance of second dimension ((2)D) separation of ionogenic solutes is much more serious than it is for neutral compounds where the pH of the eluent does not play a role in retention. We have systematically varied the properties of the sample solution injected into the (2)D column (i.e., the (1)D effluent), as well as the (2)D eluent, with the goal of establishing guidelines for conditions that yield acceptable (2)D performance. We find that the organic solvent content of the (1)D effluent and (2)D eluent is not as important as the buffer concentrations in these two solutions, and that the greater the ratio of buffer concentration in the (1)D effluent relative to the (2)D eluent, the smaller the volume one can inject into the (2)D column before dramatic peak splitting occurs. We have then used the information from these simple experiments to guide both 1D experiments that mimic the (2)D separation, and actual 2D separations, to demonstrate that online adjustment of the properties of the (1)D effluent by dilution with a buffered solvent prior to injection into the (2)D column is a very effective solution to the pH mismatch problem. We find that when the buffer capacity of the diluent is high enough to effectively titrate the (1)D effluent such that its pH approaches that of the (2)D eluent, excellent (2)D peak shape is obtained for the carboxylic acid analytes, even when the volume of injected sample solution exceeds the (2)D column volume.
二维液相色谱法（2D-LC）更广泛应用面临两个持续存在的障碍，一是人们认为二维方法通常不如一维方法灵敏，二是二维系统中某些分离模式的联用存在困难，因为第一次分离的流出物会对第二次分离产生负面影响。在这项工作中，我们针对二维液相色谱系统的两个维度均采用反相分离，但改变pH值以使羧酸分析物在两根色谱柱中的电离状态不同（即在pH 2或7缓冲的洗脱液中呈中性或带负电荷）这种具体情况来解决这些问题。我们首先证明，一维（(1)D）流出物对离子型溶质二维（(2)D）分离性能的影响比对中性化合物严重得多，在中性化合物中洗脱液的pH值对保留没有作用。我们系统地改变注入二维色谱柱（即一维流出物）的样品溶液的性质以及二维洗脱液的性质，目的是为获得可接受的二维性能的条件制定指导原则。我们发现，一维流出物和二维洗脱液中的有机溶剂含量不如这两种溶液中的缓冲液浓度重要，并且一维流出物中缓冲液浓度与二维洗脱液中缓冲液浓度的比值越大，在二维色谱柱出现明显峰分裂之前能够注入的体积就越小。然后，我们利用这些简单实验获得的信息来指导模拟二维分离的一维实验以及实际的二维分离，以证明在注入二维色谱柱之前用缓冲溶剂稀释对一维流出物的性质进行在线调节是解决pH不匹配问题的一种非常有效的方法。我们发现，当稀释剂的缓冲容量足够高，能够有效滴定一维流出物使其pH值接近二维洗脱液的pH值时，即使注入的样品溶液体积超过二维色谱柱的体积，羧酸分析物也能获得出色的二维峰形。
