Liquid chromatography ultra-performance

Lhve D. Neue, Eric S. Crumbach, Marianna Kele, Jeffrey R. Mazzeo, and Dirk Sievers 

There is a continued need to improve the performance of HPLC separations. On the one hand, people want faster analyses due to increased pressure to get rapid results. Such a situation is often encountered in the analysis of animal or human plasma or urine samples in the pharmaceutical industry. On the other hand, a higher separation performance is often needed. In some cases, the higher separation power simplifies method development. In other cases, such as complex peptide maps, it is desirable to obtain meaningful results in a reasonable time frame and analysis times of the order of 24 h, which could result in optimal separations, are not tolerable. 

In Chapter 1.2 of this book, we discussed the issue of fast gradient separations and showed that one can accomplish high-quality rapid separations with short columns packed with small particles. How fast one can go at a stiU reasonable performance is limited by the available pressure. A similar situation arises if one wants to accomplish a truly high-powered separation within a reasonable time frame. Once again, the available pressure is the limiting factor. Thus, itis absolutely clear that better separations can be accomplished if more pressure can be applied to drive the LC separation. 

The principles of how to operate an HPLC column under optimal isocratic conditions were outlined a long time ago by Guiochon and coworkers [4]. The maximum column performance at the lowest pressure is always achieved around the minimum of the van Deemter curve. If we reduce the particle size, the back-pressure increases - at equal velocity - inversely proportionally to the square of the particle diameter. At the same time, the velocity at the minimum of the van Deemter curve increases with decreasing particle diameter. Thus, the pressure at the optimum rises with the third power of the reduction in particle diameter dp. 

Note that the pressure at this optimum is, to a first approximation, independent of the viscosity of the mobile phase or of the temperature. The performance, on the other hand, increases by two separate factors. On the one hand, resolution increases inversely proportionally to the particle diameter, and on the other hand, the analysis time decreases with the reduction in particle size. Both of these features are highly desirable higher performance in a shorter time. This is the fundamental driving force behind UPLC. 

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Mass spectrometry has become a very important technique in the identification and quantification ofphenolics in fruit and vegetables. Different factors, such as sensitivity and specificity, have been cited to explain the acceptance of this method by the scientific community. Additionally, this technique might easily combine with different separation techniques such as CE, gas chromatography (GC), and liquid chromatography (LC), including HPLC and UPLC (ultra performance liquid chromatography). 

Today s personalized medicine requires analysis of a large number of biological samples in a short period on the day they are collected from patients so that a proper informed dose adjustment can be made before subsequent dosing. The high-throughput analytical procedures developed to meet this demand are reviewed in subsequent sections covering immunoassays, HPLC alone and combined with tandem mass spectrometry detection (HPLC-MS/MS), and ultra-performance liquid chromatography with MS/MS detection (UPLC-MS/MS). 

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