Chromatographic performance

Methods involving chromatographic separations prior to detection require optimization of the chromatographic conditions for best performance. A Gaussian peak shape (no tailing or fronting), resolution (baseline separation from nearby peaks) and reproducible retention time are necessary for identification of the analytes of interest (Goldberger et al., 1997). 

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Zorbax PSM particles are made from small (80-2000 A), extremely uniform colloidal silica sol beads. In a patented polymerization process, these beads are agglutinated to form spherical particles. The size of the Zorbax PSM particles is controlled by the polymerization process, and the pore size is determined by the size of the silica sol beads. After polymerization, the silica is heated to remove the organic polymer and sinter the particles. The result is a spherical, porous, mechanically stable, pure silica particle that provides excellent chromatographic performance (Pig. 3.1). 

PSS columns for fluorinated eluents PSS PFG columns were developed by PSS because users worldwide were unsatisfied with the stability of conventional organic SEC columns when running solvents such as hexafluroisopropa-nol (HFIP). Because polymeric gels tend to be unstable in fluorinated media, PSS modified silica to achieve better stability while maintaining perfect chromatographic performance. 

The interface should cause no reduction in chromatographic performance. This is particularly important for the analysis of complex multi-component mixtures (although the specificity of the mass spectrometer may, in certain circumstances, compensate for some loss of performance - see Chapter 3). 

In contrast to GC, in which, particularly at high temperatures, the stationary phase may give rise to a continuous background at the detector, this is not normally observed in HPLC unless the pH of the mobile phase is such that degradation of the stationary phase occurs. Under these circumstances, both an increased background and a reduction in chromatographic performance may be observed. 

This can potentially be overcome by the use of microbore HPLC columns with flow rates which are directly compatible with mass spectrometer operation, although the necessary decrease in injection volume results in little overall gain in the concentration of sample reaching the mass spectrometer. In addition, at the time that the DLI was available, the use of microbore HPLC, which introduces another set of potential problems related to chromatographic performance, was probably as widespread as the use of LC-MS It has been assessed [2] that in around 25% of the reported applications of DLI, microbore HPLC has been utilized. 

The use of microbore columns is not yet routine as much more rigorous control of parameters, such as flow rate and instrument dead volume, is required to ensure that degradation of chromatographic performance does not occur. It is therefore experimentally more difficult. 

Quantitative methodology employing mass spectrometry usually involves selected-ion monitoring (see Section 3.5.2.1) or selected-decomposition monitoring (see Section 3.4.2.4) in which a small number of ions or decompositions of ions specific to the compound(s) of interest are monitored. It is the role of the analyst to choose these ions/decompositions, in association with chromatographic performance, to provide sensitivity and selectivity such that when incorporated into a method the required analyses may be carried out with adequate precision and accuracy. 

These chromatographic performance tests are carried out in order to ensure that all impurities to be controlled are well separated from the substance to be examined (HPLC, GC andTLC). Forthis reason, preferably such reference substances are chosen which elute dose to the main compoxmd (HPLC, GC) or which have a similar Rf-value (TLC) but can stiU be separated. These may be structurally related compoimds which shall be separated with a minimum requirement for the resolution using the chromatographic system described, e.g. such as in the monograph for desmopressin (Monograph 07121999), Figure 5.3. 

Leinweber, F.C., Tallarek, U. (2003). Chromatographic performance of monolithic and particulate stationary phases hydrodynamics and adsorption capacity. J. Chromatogr. A 1006, 207-228. 

The study concluded that Once wash steps are optimized, samples prepared by solid phase extraction are cleaner than those prepared by protein precipitation. Samples prepared by extraction with a Multi-SPE plate resulted in lower LOQs than samples prepared by solvent precipitation. Drug recoveries were acceptable (>80%) for both the SPE and the solvent precipitation methods. Well-to-well reproducibility of samples was slightly better with extraction with a Multi-SPE plate. Evaporation and reconstitution, while more time-consuming, yield better chromatographic performance, allow analysis of lower concentration samples, and require optimization for good analyte recovery. 

Figure 7.1 Total ion current (TIC) chromatogram obtained by GC-MS analysis of a resin (Pinus sylvestris). The diterpenoid resin acids were methylated (using diazomethane) to improve chromatographic performance. Peak identities 1, Methyl pimarate 2, Methyl sandaracopimarate 3, Methyl isopimarate 4, Methyl palustrate 5, Methyl dehydroabietate 6, Methyl abietate 7, Methyl neoabietate. For GC-MS operating conditions, see Heron and Pollard (1988).

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