Stationary-Phase Considerations

Three of the most common nonpolar organic substituents employed in stationary phases for RP-HPLC with aqueous/polar mobile phases are as follows  

The larger the substituent group, the more strongly and longer will it retain the nonpolar analyte, so retention will vary inversely to the order listed here. 

Three examples of common polar organic substituents (incorporating functional groups to confer the desired polarity) for NP-HPLC with less-polar mobile phases are the following  

it is the functional groups that dominate retention. The amino and diol phases will retain compounds with pronounced hydrogen-bonding potential more strongly, while the cyano phase interacts most strongly with analytes whose polar interactions result from strong dipole moments. Complex structures may interact in a combination of these mechanisms, so relative retention is less easy to predict than with the RP columns. 

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Stationary phase considerations. The retention mechanism(s) by which bonded phases retain solutes is complex and not fully understood [71-73]. It may be that it is comparable in behaviour and function to conventional partitioning in liquid-liquid phase systems or there may be competition between eluant and solute molecules for position on the stationary phase. It is likely that there are several mechanisms operating however it is adequate to regard the stationary phase as a conventional, physically retained liquid with which analytes interact by the conventional sorption processes. 

The product p (1-p) reaches a maximum atp = 0.5. p = 0.5 characterizes substances which stay equal periods of time in the mobile and the stationary phases. Considerably larger p values signify extremely short retention times and are not advisable for gas-chromatographic separations. Most separations on packed columns are conducted with retention times tf s equal to... 

Cole, LA. Dorsey, J.G. Temperature dependence of retention in reversed-phase liquid chromatography. 1. Stationary-phase considerations. Anal. Chem. 1992,64, 1317-1323. 

Concentrations of moderator at or above that which causes the surface of a stationary phase to be completely covered can only govern the interactions that take place in the mobile phase. It follows that retention can be modified by using different mixtures of solvents as the mobile phase, or in GC by using mixed stationary phases. The theory behind solute retention by mixed stationary phases was first examined by Purnell and, at the time, his discoveries were met with considerable criticism and disbelief. Purnell et al. [5], Laub and Purnell [6] and Laub [7], examined the effect of mixed phases on solute retention and concluded that, for a wide range of binary mixtures, the corrected retention volume of a solute was linearly related to the volume fraction of either one of the two phases. This was quite an unexpected relationship, as at that time it was tentatively (although not rationally) assumed that the retention volume would be some form of the exponent of the stationary phase composition. It was also found that certain mixtures did not obey this rule and these will be discussed later. In terms of an expression for solute retention, the results of Purnell and his co-workers can be given as follows,... 

It is seen from equation (22) that there will, indeed, be a temperature at which the separation ratio of the two solutes will be independent of the solvent composition. The temperature is determined by the relative values of the standard free enthalpies of the two solutes between each solvent and the stationary phase, together with their standard free entropies. If the separation ratio is very large, there will be a considerable difference between the respective standard enthalpies and entropies of the two solutes. As a consequence, the temperature at which the separation ratio becomes independent of solvent composition may well be outside the practical chromatography range. However, if the solutes are similar in nature and are eluted with relatively small separation ratios (for example in the separation of enantiomers) then the standard enthalpies and entropies will be comparable, and the temperature/solvent-composition independence is likely be in a range that can be experimentally observed. 

For the GPC separation mechanism to strictly apply, there must be no adsorption of the polymer onto the stationary phase. Such adsorption would delay elution of the polymer, thereby resulting in the calculation of too low a molecular weight for the polymer. The considerable variety of undesirable interactions between polymers and column stationary phases has been well reviewed for GPC by Barth (1) and this useful reference is recommended to the reader. Thus, the primary requirement for ideal GPC is that the solvent-polymer interaction be strongly thermodynamically favored over the polymerstationary phase interaction. 

However, in LC solutes are partitioned according to a more complicated balance among various attractive forces solutes interact with both mobile-phase molecules and stationary-phase molecules (or stationary-phase pendant groups), the stationary-phase interacts with mobile-phase molecules, parts of the stationary phase may interact with each other, and mobile-phase molecules interact with each other. Cavity formation in the mobile phase, overcoming the attractive forces of the mobile-phase molecules for each other, is an important consideration in LC but not in GC. Therefore, even though LC and GC share a considerable amount of basic theory, the mechanisms are very different on a molecular level. This translates into conditions that are very different on a practical level so different, in fact, that separate instruments are required in modern practice. 

At the current time, there is considerable interest in the preparative applications of liquid chromatography. In order to enhance the chromatographic process, attention is now focused on the choice of the operating mode [22]. SMB offers an alternative to classical processes (batch elution chromatography) in order to minimize solvent consumption and to maximize productivity where expensive stationary phases are used. 

The selectivity (separation capability) of an HPLC system is dependent upon the combination of mobile and stationary phases. Since ions are being generated directly from the mobile phase by electrospray, its composition, including the identity and concentration of any buffer used, and its flow rate are important considerations. 

Optimized HPLC separation allows most betaxanthins to be separated on a Cl8 reversed phase stationary phase according to their respective polarities. - Considerable progress was achieved by the introduction of a highly polar silica-based column, which allowed major improvement of peak resolution, especially at early... 

Solvent selectivity is seen as the factor that distinguishes individual solvents that have solvent strengths suitable for separation. In reality, separations result from the competition between the mobile and stationary phases for solutes based on the differences of all intermolecular interactions with the solute in both phases. Solvents can be organized on selectivity scales that are useful for initial solvent selection, but in a chromatographic separation the properties of the stationary phase must be taken into consideration. Methods that attempt to model chromatographic separation need to consider simultaneously mobile and stationary phase properties [38]. 

The HcReynolds abroach, which was based on earlier theoretical considerations proposed by Rohrschneider, is formulated on the assumption that intermolecular forces are additive and their Individual contributions to retention can be evaluated from differences between the retention index values for a series of test solutes measured on the liquid phase to be characterized and squalane at a fixed temperature of 120 C. The test solutes. Table 2.12, were selected to express dominant Intermolecular interactions. HcReynolds suggested that ten solutes were needed for this purpose. This included the original five test solutes proposed by Rohrschneider or higher molecular weight homologs of those test solutes to improve the accuracy of the retention index measurements. The number of test solutes required to adequately characterize the solvent properties of a stationary phase has remained controversial but in conventional practice the first five solutes in Table 2.12, identified by symbols x through s have been the most widely used [6). It was further assumed that for each type of intermolecular interaction, the interaction energy is proportional to a value a, b, c, d, or e, etc., characteristic of each test solute and proportional to its susceptibility for a particular interaction, and to a value x, X, Z, U, s, etc., characteristic of the capacity of the liquid phase... 

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