Mobile-phase composition, optimal determination

Morita et al. [69] optimized the mobile phase composition using the PRISMA model for rapid and economic determination of synthetic red pigments in cosmetics and medicines. The PRISMA model has been effective in combination with a super modihed simplex method for fadhtating optimization of the mobile phase in high performance thin layer chromatography (HPTLC). 

When developing separation methods for analytes with known pKa values, determination of the starting mobile-phase pH is highly advisable. This estimation may help to avoid strange analyte retention behavior during further method optimization and variation of the mobile-phase composition. Below we include several examples where the methodology of the combined pH and pKa shift evaluation is outhned. 

In [84] a variable size simplex is u.sed to optimize the mobile phase composition in RP chromatography. The fractions of methanol and water in a methanol/water/acetonitrile mobile phase were optimized. The feasible experimental area was determined by carrying out a gradient elution separation and then calculating the boundaries of the variable space in which the simplexes should move to find an optimized isocratic separation. 

While all detectors place some limitations on the mobile phase composition, in electrochemical detection, it is essential to recognize that a complex surface reaction is involved, which depends on both the physical and chemical properties of the medium. To optimize an LCEC determination, it is necessary to consider both chromatographic and electrochemical requirements simultaneously. Fortunately, most commonly applied chromatographic techniques fall into the category of reverse phase separations, the mobile phase requirements of which are consistent with the requirements for electrochemistry. The primary requirement for electrochemical detection is that the... 

Step 4 - determination of optimal mobile phase composition... 

For the stationary and the mobile phase, the presence of ionizable or polar groups and the relative hydrophobicity are determining factors to modulate the specific interactions with the template. Thus, to obtain well optimized separations, it is necessary to take into the account not only the mobile phase composition, but also the structure and the physico-chemical properties of the template and of the stationary phase. 

The optimization of mobile phase composition should take into account not only the retention of the compound to be analyzed, but also the retention of the matrix. The determination of anticancer 6-thiopurine drugs and Iheir metabolites in untreated serum is a usefiil example (Fig. 11.6) [ 19]. With a mobile phase of 0.04 M SDS in 0.01 M phosphate buffer at pH 2.2, the blank serum produced a background response that had completely eluted after 6 min, with the exception of a peak at 8 min. The peaks of 6-thioguanidine riboside and 6-thioguanine were well resolved, but unfortunately, the three earlier-eluted compounds (6-mercaptopurine riboside, 6-thioxanthine and 6-mercaptopurine) were overlapped by the matrix peaks. A lower pH (2.0) permitted complete separation of 6-mercaptopurine from the serum background signal. Under these conditions, 6-thioguanine eluted too late (at around 40 min). 

Mobile phase parameters should be properly chosen to improve the selectivity of the separation between a drug and the endogeneous components in the physiological fluids. Optimal control of mobile phase composition, including pH and type and concentration of surfactant and modifier, permits the development of MLC procedures for the determination of virtually any drug in these samples. 

In most cases, solute separations can be achieved in the isocratic mode by optimizing the mobile phase composition. Nevertheless, for some complex cases of solutes possessing very different polarities, separations can require a gradient elution mode in such cases, mobile phase compositions and gradient slopes have to be determined. 

The optimization process as described in Chapter 2 is a critical one for the analyst. Successful method optimization means that targeted, defined criteria (e.g., resolution, elution time, sensitivity) have been met through the use of an experimentally determined set of variables (flow rate, mobile phase composition, system temperature, detector settings, column type, etc.). 

A separation involving a mobile phase of constant composition (irrespective of the number of components it contains) is termed isocratic elution, while that in which the composition of the mobile phase is changed is termed gradient elution. In the latter, a mobile phase is chosen which provides adequate separation of the early eluting analytes and a solvent which is known to elute the longer-retained compounds is added over a period of time. The rate at which the composition is changed may be determined by trial and error , or more formal optimization techniques may be used [5-7]. 

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