Stationary phase selections, HPLC development

HPLC has been one ofthe most widely used analytical methods for determining PAHs in complex environmental samples. The development of a chemically nonpolar stationary phase for HPLC has provided a unique selectivity for separation of PAH isomers that are often difficult to separate by GC columns. For example, chrysene, benz[a]anthracene, and triphenylene are baseline resolved with a C-18 reverse phase column packing. A detection limit of subpicogram to picogram levels of PAHs per sample has been achieved by HPLC with fluorescence detector (For and Staley 1976 Furuta and Otsuki 1983 Futoma et al. 1981 Golden and Sawicki 1978 Lawrence and Weber 1984 Marcomini et al. 1987 Miguel and De Andrade 1989 Nielsen 1979 Risner 1988 Tomkins et al. 

The use of peptide-derived lipids (Glu-1, Phe-1, and Phe-2) as organic stationary phases also has been described in this entry. These lipids not only have high potential ability as a self-assembling system but also are attractive as a carbonyl Tr-electron source. As a result, double-alkylated L-glutamide-derived stationary phase has been developed and extremely high selectivity is detected in HPLC as predicted. Polymeric types of peptide-derived hpids were also considered and developed, but their selectivity could not exceed that of the monomeric type hpids. This is probably due to the fact that radical polymerization of peptide-derived monomers disturb the stereoregularity of the resultant polymer main chain, and thus, sufficient molecular ordering is not obtained to increase the selectivity (multiple TT-TT interaction). Finally, it is concluded that subsidiary weak interactions, such as tt-tt interaction, can... 

When one develops new reversed-phase (RP)-HPLC methods, one usually uses the selectivity of the mobile phase as the primary method development tool. The chromatographic separation can be influenced by the choice of the organic solvent (mainly methanol and acetonitrile), or by variation of pH or buffer type. Schemes for method development using these parameters have been described in the literature [1,2]. Most important are the selectivity changes caused by pH changes, which are well-understood and easily predictable (3). It is well known that the stationary phase influences the selectivity as well, but this effect is often not very well understood. The primary reason for this is the fact that reliable methods for the description of the stationary phase selectivity have only become available fairly recently. In the last few years, several papers have been published that deal with the subject of selectivity in a fimdamental way [4—9] or represent a data collection based on older methods [10-15]. In this chapter, we describe in detail the method used in our laboratory. We then look at our selectivity charts and discuss our results. It needs to be pointed out in advance that selectivity charts only accurately represent the properties of a stationary phase under the conditions of the measurement. If we depart from the mobile phase composition of the test, the relationships between different columns will change, since selectivity arises from a combined effect of the mobile phase and the stationary phase. 

Currently employed HPLC methods for pantothenic acid and/or pantothenates have been applied solely to pharmaceuticals and simple matrices such as fortified infant formulas, whereas assays of coenzyme A and its acyl analogs have also been successfully performed on animal tissues. In the last few years, chiral stationary phases have been developed for optical resolution of pantothenic acid and related compounds by HPLC, and also HPLC-MS has become a promising technique. However, the newly developed HPLC procedures still require increased sensitivity and selectivity to make them applicable for the analysis of the total vitamin content in complex matrices such as foods and feeds. 

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

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... 

Packed column SFC and CE are both able to make inroads into the application area served by HPLC, but from opposite extremes of polarity and with little overlap. CE is likely to be more efficient and faster, but mostly applicable to very polar molecules and ions. SFC qualifies as a more reproducible, trace technique, with greater selectivity and multiple detection options. HPLC and CE have been compared [365], Owing to their orthogonality, CZE and SFC are worth developing, not in competition or as an alternative to HPLC, but as an additional method in order to augment the information obtained from the analysis. With the broad scope of possible eluents and stationary phases, HPLC has fewer constraints than SFC and CZE. The parameters influencing selectivity may be used as a guide to optimisation (Table 4.44). 

Since selectivity in HPLC involves both the stationary and mobile phases [5-9,58-60], it is important to note that the solvent strength of the mobile phase, as compared to the stationary phase, (composed of mobile-phase components reversibly retained by the bonded phase and silica support) determines the elution order or k of the retained components. Unfortunately, the columns with the same stationary phase can exhibit significant variabilities from one manufacturer to another and even from the same manufacturer [5-8]. Based on discussions heard at various scientific meetings, this situation has not changed much. Variabilities can occur in the packing process even where all other conditions are supposedly constant. These factors have to be considered prior to developing an understanding as to how separations occur in HPLC. 

Non-silica-based RP-HPLC stationary phases have also been developed and their separation capacity has been compared with those of silica-based ones. The porous structure of crosslinked polymer gels may be responsible for the markedly different selectivity and retention characteristics. Up till now, the mode of separation on polymer stationary phases is not entirely understood at the molecular level. It has been established that the size-exclusion effect may influence the retention of analyses on polymer gels. 

A typical example of HPLC method development and validation was provided by Boneschans et al. [9]. They developed an HPLC method for piroxicam benzoate and its major hydrolytic degradation products, piroxicam and benzoic acid. The authors utilised a robust stationary phase (Phenomenex Luna, Cig), with an optimised mobile phase comprising of acetonitrile/water/acetic acid (45/7/8 v/v), and a flow rate of 1.5 ml/min. The operating pH of the mobile phase (pH 2.45) was selected on the basis that it is ca. 2 pH units from the pKa of the drug, and hence reasonably insensitive to changes in mobile-phase preparation. The injection volume was 20 pi with a detection wavelength of 254 nm. They utihsed... 

The application of biosensors for process monitoring has been dogged by problems with fouling, selectivity, degrading bioactivity and long-term stability. Recent developments in molecularly imprinted polymers (MIPs) show some promise as synthetic receptors and have been used for this purpose for assays and as HPLC stationary phases. Recent work has shown that MIPs show increased robustness, storage endurance and lower cost compared with biosensors, which... 

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