Retention selectivity

Fig. 2-14. The effect of organic modifier on retention, selectivity and resolution of methionine on teicoplanin CSP (250 x 4.6 mm). The flow rate was 1.0 mL min at ambient temperature (23 °C).

Fundamental concepts of retention, selectivity, efficiency, and peak resolution... 

First, we will explore the three fundamental factors in HPLC retention, selectivity, and efficiency. These three factors ultimately control the separation (resolution) of the analyte(s). We will then discuss the van Deemter equation and demonstrate how the particle diameter of the packing material and flow rate affect column efficiencies. 

Scrutiny of the resolution equation indicates that is controlled by three relatively independent terms retention, selectivity, and efficiency (Figure 10). To maximize R, k should be relatively large. However, a value of k over 10 will approach a point of diminishing returns as the retention term of k /(l + k ) approaches unity. No separation is possible if k = 0, since R must equal zero if k is zero in the resolution equation. 

FIGURE 10 The resolution equation, which is governed by three factors retention, selectivity, and efficiency. 

This chapter provides an overview of essential concepts in HPLC including retention, selectivity, efficiency, and resolution as well as their relationships with key column and mobile phase parameters such as particle size, column length and diameter, mobile phase strength, pH, and flow rate. The significance of several concepts important in pharmaceutical analysis such as peak capacity, gradient time, void volume, and limit of quantitation are discussed. 

The application of HPLC in routine environments, like pharmaceutical, food, or environmental analysis and particularly quality assurance, makes not only great demands on the robnstness of HPLC hardware, comprising pumps, column thermostats, and detection units, bnt in addition to the column reproducibility. Column reproducibility can be investigated at different levels of complexity Run-to-run reproducibility compares consecutive chromatographic runs, whereas long-term stability describes the column variance over several hundreds of injections. Column-to-column (batch-to-batch) reproducibility finally explores the match of independently fabricated chromatographic columns. Column characteristics that are routinely consulted for the determination of the robustness are retention, selectivity, column efficiency, and peak symmetry. 

The usefulness of protein-type CSPs has already been shown in particular Chiral-AOP and Ultron ES-OVM (see Table 2) have a very broad range of enantioselectivity. It is not, however, possible to systematically predict the resolution on such CSPs, but the overall retention, selectivity and efficiency can be modified to a certain extent by altering several key variables ... 

Multiparameter Control of Retention and Resolution. Although the key SFC variables—density (pressure), temperature, composition, and their respective gradients—can be utilized individually to vary retention, selectivity, and hence resolution, they can be employed collectively to exert even greater control. The simultaneous use of two or three variables to vary retention and selectivity instead of one is, however, an obviously more complex situation. Under ideal circumstances (in the absence of interaction), the relationships expressed in equations 2-4 might be written as... 

Although the first effect is a positive one, it is usually overshadowed by the second and third (negative) effects which are generally much larger in magnitude. For this reason it is advisable to limit the variables to only the ones believed to be important for the panicular separation of interest. To reiterate, when rapid global optimization and/or robustness of the optimum separation is desirable, the key is to consider only the most important variables. If certain variables can logically be excluded based on the retention/selectivity characteristics of the components of a particular sample, then by all means they should be. 

Temperature and pressure are rarely optimized in HPLC, but these parameters are very important in SFC, hence can alter retention, selectivity, and resolution. Toribio et al. [149] presented the chiral separation of ketoconazole and its precursors on Chiralpak AD and Chiralcel OD CSPs. The authors also reported that alcohol modifiers provided better separation than acetonitrile. Further, Wilson [143] studied the effects of composition, pressure, temperature, and flow rate of the mobile phase on the chiral resolution of ibuprofen on a Chiralpak AD CSP. It was observed that temperature affords the greatest change in resolution, followed by pressure and composition. An increase in methanol concentration, pressure, and temperature has resulted in poor chiral resolution. At first chiral resolution increased with an increase of flow rate (up to 1.5 mL/min) but then started to decrease. Contrary to this, Biermann et al. [135] described the... 

The presence of triethylamine in the aqueous portion of the mobile phase was shown to be important for the separation of enantiomers of /-Boc-amino acids, whereasa native amino acids were almost not affected by the addition of TEAA [34]. Lee and Beesley [48] observed that a change in the ionic interactions occurred when changing the acid/base ratio in reversed and polar organic mobile phases or changing the buffer pH in the reversed-phase mode. Consequently, the retention, selectivity, and peak shape is affected. 

The extent of separation can be quantified in terms of the resolution obtained between two consecutive chromatographic peaks. This resolution can be expressed in terms of three elemental characteristics of chromatographic separation retention, selectivity and efficiency. The influence of each of these three factors on resolution will be discussed. 

Carr and others have described the preparation and properties of polybutadiene (PBD) and polyethyleneimine (PEI), as well as aromatic polymer-coated and carbon-clad zirconia-based RP phases. The preparation of PBD-coated zirconia and the chromatographic evaluation of these phases have been described extensively by Carr, McNeff, and others [39 1]. From these studies, the authors conclude that at least for neutral analytes PBD zirconia-coated phases behave quite similar with respect to retention and efficiency compared to silica-based RP phases [42]. For polar and ionic analytes, however, substantial differences with respect to retention, selectivity, and efficiency have been reported [43]. 

N. Tanaka, T. Ebata, K. Hashizume, K. Hosoya, and M. Araki, Polymer-based parking materials with backbones for reversed-phase liquid chromatography. Performance and retention selectivity, /. Chromatogr. 475 (1989), 195-208. 

The basis for the analyte retention in reversed-phase chromatography is the competitive interactions of the analyte and eluent components with the adsorbent surface. The stronger the interactions of the analyte with the surface, the longer its retention. Selectivity or the ability of chromatographic system to discriminate between different analytes is also dependent on differences in the surface interactions of the analytes. 

Effect of pH on the Retention/Selectivity of the Isomers. The first step in method development is to understand the effect of pH on the separation characteristics of the method. The pKa values of the ortho and the para isomers was estimated by ACD (Advanced Chemistry Development software) to be 9.0 and 9.5, respectively. Obviously the best pH to carry out the separation would be at pH that is less than 2 units lower than the analyte that has the lowest pKa. This would be at pH values less than 7.0. However, to illustrate the effect of pH on the separation selectivity of the isomers, a controlled pH study at isocratic conditions was conducted. 

---