Bonded stationary phases acid-base properties

The IUPAC Compendium of Chemical Technology defines Normal Phase as an elution procedure in which the stationary phase is more polar than the mobile phase . In practice, the most widely used stationary phases for preparative HPLC are based on silica and the polarity of the underlying silyl ether and silanol provides the required hydrophilic surface. Amino and cyano bonded silica are also commonly used in normal phase mode though the latter also has some reversed phase properties. The predominant mechanism of interaction is hydrogen bonding. However, the silanol is mildly acidic so the silica surface will also have mild cation exchange properties. 

Polystyrene/divinylbenzene-based ion-exclusion columns are also offered by Hamilton Co. (Reno, NV, USA) under the trade name PRP-X300. This is a 10-pm material with an exchange capacity of 0.2 mequiv/g [4], It is obtained by sulfonation of PRP-1, a macroporous PS/DVB polymer with reversed-phase properties. Fig. 4-2 shows the separation of various organic acids on this stationary phase. Dilute sulfuric acid was used as the eluent. The much higher retention of succinic acid compared to acetic acid reveals that the retention of organic acids is chararcterized, apart from reversed-phase effects, by the formation of hydrogen bonds. 

A historical survey of the more successful stationary phases will demonstrate that a blend of chemical properties should ofl er the best potential for increasing nucleic acid selectivity. OEAE cellulose, a weak anion-exchange support, has for some time been known to Mparate single-stranded oligonucleotides up to 10 bases (55-55). Alternately, the derivatization of agarose by the covalent bonding of spermine, an aliphatic tetraamine, has been used to separate rRNAs (5(5). In each case it is likely that both the anion exchange and hydrophilic character of each support influence selectivity. 

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