Liquid chromatography fast separations

The experiments within the frame of the REHE project were performed in the aqueous phase in a discontinuous, batch-wise manner. It was necessary, in order to get a statistically significant result, to repeat the same experiment several hundred or even several thousand times with a cycle time of typically 45 s. These studies were performed with the Automatic Rapid Chemistry Apparatus (ARCA) II (SchSdel et al, 1989), a computer-controlled apparatus for fast, repetitive high-performance liquid chromatography (HPLC) separations. A schematic of the ARCA II components is shown in Figure 6.2. 

Automated separations with ARCA II were performed with element 104 (Gunther et al. 1998 Strub et al. 2000), element 105 (Kratz et al. 1989 Gober et al. 1992 Kratz et al. 1992 Schadel et al. 1992 Zimmermann et al. 1993 Paulus et al. 1999a, b), and element 106 (Schadel et al. 1997a, b, 1998). ARCA II is a computer-controlled apparatus for fast, repetitive High Performance Liquid Chromatography (HPLC) separations (Schadel et al. 1989). A schematic representation of ARCA 11 is shown in O Fig. 20.6. 

Horvath, C. G., Preiss, B. A., and Lipsky, S. R., Fast liquid chromatography an investigation of operating parameters and the separation of nucleotides on pellicular ion exchangers, Anal Chem., 39, 1422, 1967. 

Others have examined the necessary parameters that should be optimized to make the two-dimensional separation operate within the context of the columns that are chosen for the unique separation applications that are being developed. This is true for most of the applications shown in this book. However, one of the common themes here is that it is often necessary to slow down the first-dimension separation system in a 2DLC system. If one does not slow down the first dimension, another approach is to speed up the second dimension so that the whole analysis is not gated by the time of the second dimension. Recently, this has been the motivation behind the very fast second-dimension systems, such as Carr and coworker s fast gradient reversed-phase liquid chromatography (RPLC) second dimension systems, which operate at elevated temperatures (Stoll et al., 2006, 2007). Having a fast second dimension makes CE an attractive technique, especially with fast gating methods, which are discussed in Chapter 5. However, these are specialized for specific applications and may require method development techniques specific to CE. 

Nimura, N., Itoh, H., Kinoshita, T., Nagae, N., Nomura, M. (1991). Fast protein separation by reversed-phase high-performance liquid-chromatography on octadecylsilyl-bonded non-porous silica-gel — effect of particle-size of column packing on column efficiency. J. Chromatogr. 585(2), 207-211. 

Asperger A. et al., 2002. Trace determination of priority pesticide in water by means of high-speed online solid-phase extraction-liquid chromatography-tandem mass spectrometry using turbulent-flow chromatography columns for enrichment and a short monolithic column for fast liquid chromatographic separation. J Chromatogr A 960 109. 

Development of fast, accurate, and reproducible high-performance liquid chromatography (HPLC) methods has offset the use of traditional open-column and TLC methods in modern chlorophyll separation and analysis. A number of normal and reversed-phase methods have been developed for analysis of chlorophyll derivatives in food samples (unit F4.4), with octadecyl-bonded stationary phase (C]8) techniques predominating in the literature (Schwartz and Lorenzo, 1990). Inclusion of buffer salts such as ammonium acetate in the mobile phase is often useful, as this provides a proton equilibrium suitable for ionizable chlorophyllides and pheophorbides (Almela et al., 2000). 

In this unit, methods for reversed-phase high-performance liquid chromatography (HPLC) are described for the analysis of polyphenolics. HPLC analysis can be employed in an easy and fast manner to obtain an accurate elucidation and quantification of individual polyphenolic compounds found in plant-based materials. The separation of each polyphenolic is based on the polarity differences among polyphenolics with structural similarities and uses various combinations of mobile and stationary phases. 

---