Influence of Stationary Phase

The influence of the stationary phase on the sensitivity in terms of the selectivity, retention time, peak shape, and particle size, is very variable. 

Assuming that only the particle size of the silica gel is changed and thus all other factors are constant, sensitivity changes according to the following formula  

Example In place of a column with 5 im packing, one is used with 3 im packing  

All three factors cooperate. By changing from a classical column (250 x 4 mm, 5 pm) to a short, modern column (50 x 2 mm, 3 pm), the gain in sensitivity is 

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Sander, L.C. and Wise, S.A., The influence of stationary-phase chemistry on shape recognition in liquid chromatography. Anal. Chem., 67, 3284, 1995. 

Limsavam, L. Dorsey, J.G. Influence of stationary phase solvation on shape selectivity and retention in reversed-phase hquid chromatography. J. Chromatogr. A, 2006,1102,143-153. 

Buszewski,B., Kowalska,S. Stepnowski,P. (2006). Influence of stationary phase properties on th separation of ionic liquid cations by RP-HPLC, J.Sep.Sci. Vol. 29 (No.8) 1116-1125. 

B. Waiczak, L. Morin-Allory, M. Chrdtien, M. Lafosse and M. Dreux, Factor analysis and experiment design in high-performance liquid chromatography. III. Influence of mobile phase modifications on the selectivity of chalcones on a diol stationary phase. Chemom. Intell. Lab. Syst., I (1986) 79-90. 

The physical and chemical aspects of liquid chromatography, in addition to mechanical aspects, are briefly described in this chapter. Theoretical approaches are explained in detail in later chapters. The effect of stationary phase materials on the chemical selectivity is described in Chapter 3, and the influence of the eluent components is covered in Chapter 4. The plate number theory is discussed in Chapter 5. Quantitative optimization is explained in Chapter 6. 

Eddy diffusion is a result of the presence of particles of stationary phase material in a column, and depends on the stationary phase conditions, shape of the column, and the structure of the stationary phase material. The influence of the stationary phase material can be divided into the particle size (dp), the shape of the particles, and the porosity of the particles. The standard deviation for peak broadening due to the particles is described by [Pg.102]

Dittmann, M. M., and Rozing, G. P. (1997). Capillary electrochromatography investigation of the influence of mobile phase and stationary phase properties on electroosmotic velocity, retention, and selectivity. J. Microcolumn Sep. 9, 399-408. 

Walczak, B., Chretien, J.R., Dreux, M., Morin-Allory, L., and Lafosse, M. (1987), Factor Analysis and Experiment Design in High-performance Liquid Chromatography. IV. Influence of Mobile Phase Modifications of the Selectivity of Chalcones on an ODS Stationary Phase, Chemom. Intel. Lab. Sys., 1, 177-189. 

An analyte migrates up or across a layer of stationary phase (most commonly silica gel), under the influence of a mobile phase (usually a mixture of organic solvents), which moves through the stationary phase by capillary action. The distance moved by the analyte is determined by its relative affinity for the stationary vs the mobile phase. 

The choice of stationary phase will be influenced by the polar character of the components of the mixture. In general, mixtures with components of high polarity separate better on chromatography when the more polar stationary phases are used. The chromatographic separation of a mixture is judged to be successful... 

As noted, and as detailed in Table 2, a large variety of stationary-phase and mobile-phase factors influence the selectivity, recovery, and stability of proteins and other biomacromolecules in the adsorptive modes of HPLC. Batch adsorption pilot experiments provide an expedient approach to ascertain the effect of many parameters, such as the pH, nature, and concentration of organic solvent or ionic additives in the mobile phase, the temperature- or the static-binding capacity with a defined sorbent. Similarly, the influence of... 

Fig. 9.34. Influence of chromatographic conditions on retention and/or enantioseicctivity using CSP I (see Fig. 9.32) a.s the stationary phase (conditions T. 2.5°C flou rate. I ml/min UV detection), (a) Influence of mobile phase pH on k , and u of B/-lcucine (mohilc phase McOH-ammonium acetate (80 20) pHa adjusted with AcOH). (b) Influence of buffer concentration on retention (In/ , ) and enantioseicctivity (Inor) of B/-leucine (mobile phase MeOH-ammonium acetate (80 20) pH., = 6.0). (c) Influence of mobile phase solvent type on enantioseicctivity (Ina) of 3-phenyl lactic acid at different buffer cxincentrations (mobile phases water, methanol, acetonitrile, respectively, and acetic acid (mM sec plot -triethylaniine = 4 I). (d) Influence of temperature on Ina of DNS-valine (mobile phase MeOH-O.I M ammonium aeetate (80 20) pHa = 6.0) (reprinted with permission from Ref. 1388 ),...

In (24-35) the term V /4 is influenced by many factors [Equation (24-14)], but mainly reflects the characteristics of a particular column and its mode of operation. This plate-munber factor is described in the preceding sections (24-2 and 24-3). The factors of relative retention and partition ratio in (24-35) depend on the solutes, the nature of the mobile and stationary phases, and the temperature. The factor A /(l -I- k) also strongly depends on the dead-space volume in a column. Figure 24-8 illustrates how it varies according to the dead-space volume per gram of stationary phase. When the specific retention volume of a solute is relatively low and a column... 

Increasing a. If a is close to 1.0, the greatest increase in Rs can be obtained by changing those parameters which influence a (i.e., the mobile-phase composition, the choice of stationary phase, the temperature, or, less frequently, the pressure). Increasing a from 1.1 to 1.2 increases Rshy more than 80%. However, as a increases, the amount of increase in decreases, so that increasing a from 2.1 to 2.2 increases Rg by only about 4%. 

The maximal influence on RI values is the nature of stationary phase in the chromatographic column. Use of these parameters as the constants of chemical compounds (similar to other known physicochemical constants like boiling points, T, refractive index, density, df, etc.) requires the choice of standard phases for their determination. In accordance with the criteria of the most often used application in practice, two types of phases may be classified as standards ... 

Tesarova and Bosakova [58] proposed an HPLC method for the enantio-selective separation of some phenothiazine and benzodiazepine derivatives on six different chiral stationary phases (CSPs). These selected CSPs, with respect to the structure of the separated compounds, were either based on b-CD chiral selectors (underivatized (J>-CD and hydroxypropyl ether (3-CD) or on macrocyclic antibiotics (vancomycin, teicoplanin, teicoplanin aglycon and ristocetin A). Measurements were carried out in a reversed-phase separation mode. The influence of mobile phase composition on retention and enantio-selective separation was studied. Enantioselective separation of phenothiazine derivatives, including levopromazine (LPZ), promethazine and thioridazine, was relatively difficult to achieve, but it was at least partly successful with both types of CSPs used in this work (CD-based and glycopeptide-based CSP), except for levomepromazine for which only the [CCD-based CSP was suitable. 

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