Reversed-phase HPLC selected applications

The understanding of retention and selectivity behaviour in reversed-phase HPLC in order to control and predict chromatographic properties ai e interesting for both academic scientists and manufacturers. A number of retention and selectivity models are the subject of ongoing debate. The theoretical understanding of retention and selectivity, however, still lags behind the practical application of RP HPLC. In fact, many users of RP HPLC techniques very often select stationary phases and other experimental conditions by experience and intuition rather than by objective criteria. 

An on-line concentration, isolation, and liquid chromatographic separation method for the analysis of trace oiganics in natural waters has been described (63). Concentration and isolation are accomplished with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is applicable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for determination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

McCalley, D.V. Selection of suitable stationary phases and optimum conditions for their application in the separation of basic compounds by reversed-phase HPLC. J. Sep. ScL 2003, 26, 187-200. 

In addition to the examples discussed above, these various reversed-phase HPLC mapping procedures have subsequently found numerous other advocates. Selected recent achievements include application to human hemoglobin variants 9a, 182, 185), the a- and j8-chains of rat hemoglobin 186), polypeptide hormones 9a, 99, 163), porcine C5a anaphylatoxin 187), Aplysia neuroactive polypeptide 188), ACTH/jS-lipotropin precursors 97, 99, 189, 190), oncoproteins 157), chick liver dihydrofolate reductase 191), limulin 192), ATP-citratelyase 193), spore-specific proteins 194), cAMP-dependent protein kinases (/95, 1%), interferons 197), complement components 9a, 198), aj-macroglobulin 198a), lectins 199), phycobiliproteins 200), bovine mitochondrial-F, ATPase 201), collagens, tubulins, and other structural proteins 9a, 202). 

It appears advantageous to use for molecular imprinting an in-situ method that has been developed previously for reversed-phase HPLC [127]. In this case, the polymer is directly prepared inside the colurrm. It has been shown that this method is applicable for molecular imprinting [54, 79, 127, 128] but the selectivity for separation in these columns is somewhat reduced. 

Most of the applications are performed under typical reversed-phase HPLC conditions. Nondeuterated solvents such as MeOH or MeCN are used. Great care in selecting the appropriate solvent grades is important since most solvents contain small impurities such as stabilizing chemicals, which will not be detected in UV but will cause interferences in the LC/ NMR spectra. Water is usually replaced by D2O, which is a relatively cheap deuterated solvent, and gives better quality spectra. 

Thus, a complex mixture is first separated with a microbore system, thereby providing a low flow rate of about 60 llLVmin. This low flow rate enables a connection without a splitter by transferring the selected HPLC fractions directly to the TLC plate with the aid of a special application device (CAMAG Linomat C or ATS III). The coupling of a reversed phase HPLC system with a normal phase TLC system is leading to an enormously efficient method. 

Not only in HPLC, but also in modem thin-layer chromatography, the application of reversed-phase stationary phases becomes increasingly important. The advantage of the hydrophobic layers in comparison with the polar, surface-active stationary phases is the additional selectivity and a reduced hkehhood of decomposition of sensitive substances. 

The PRISMA model was developed by Nyiredy for solvent optimization in TLC and HPLC [142,168-171]. The PRISMA model consists of three parts the selection of the chromatographic system, optimization of the selected mobile phases, and the selection of the development method. Since silica is the most widely used stationary phase in TLC, the optimization procedure always starts with this phase, although the method is equally applicable to all chemically bonded phases in the normal or reversed-phase mode. For the selection of suitable solvents the first experiments are carried out on TLC plates in unsaturated... 

Most HPLC applications are performed with non-polar columns, thus in the reversed-phase mode (RPLC), since it allows simple and versatile conditions. Another advantage is that in general the applied mobile phase is an aqueous buffer. Moreover in RPLC chemical equilibria such as ion suppression, ion-pair formation, metal complexation, and micelle formation can easily be exploited to optimize separation selectivity. This explains the large number of commercially available non-polar HPLC columns. " ... 

---