High performance liquid chromatography fast separation

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. 

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. 

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. 

High-performance liquid chromatography (HPLC) is a fast technique that, with high precision and specificity, separates mixtures into individual ingredients. Used as a routine procedure, it has several advantages It can be completely automated sample cleanup and preparation are simple and the reproducibility of the packing material means that the analytical conditions remain the same for a new column. 

High-performance liquid chromatography (HPLC) and fast protein liquid chromatography (FPLC) rely on the same separation principles as the traditional chromatography columns, but tend to be much faster because of high flow rates that are possible due to the uniform bead size and the mechanical strength of the beads. See also Chapter 4, section 1.2.2. 

Some methods for fast chromatographic separation and detection of PolyPs in food, biological samples or water have been proposed (Halliwell et al., 1996 Baluyot and Hartford, 1996 Svoboda and Schmidt, 1997 Bewsler et al., 2001). A single-column chromatographic system with indirect UV detection was elaborated, and the dependencies of PolyP retention on the concentrations of pyromellitic acid and ethylenediaminetetraacetic acid (EDTA) in the mobile phase and on the pH of the eluent were determined (Svoboda and Schmidt, 1997). A high performance liquid chromatography (HPLC) method has also been used for the separation of PolyPs (Lorenz and Schroder, 1999). 

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