Normal-phase chromatography retention

Kovat s retention index (p. 575) liquid-solid adsorption chromatography (p. 590) longitudinal diffusion (p. 560) loop injector (p. 584) mass spectrum (p. 571) mass transfer (p. 561) micellar electrokinetic capillary chromatography (p. 606) micelle (p. 606) mobile phase (p. 546) normal-phase chromatography (p. 580) on-column injection (p. 568) open tubular column (p. 564) packed column (p. 564) peak capacity (p. 554)... 

The mechanism of reversed phase chromatography can be understood by contrast with normal phase chromatography. Normal phase liquid chromatography (NPLC) is usually performed on a polar silica stationary phase with a nonpolar mobile phase, while reversed phase chromatography is performed on a nonpolar stationary phase with a polar mobile phase. In RPLC, solute retention is mainly due to hydrophobic interactions between the solutes and the nonpolar hydrocarbon stationary surface. The nonpolar... 

A less obvious example of normal-phase chromatography is the separation of saccharides and oligosaccharides in foods910 and in biological mixtures,1112 using a mobile phase consisting of acetonitrile/water or acetonitrile/dilute phosphate buffer. Although the separation mode has occasionally been misidentified as reversed phase, it is normal phase by virtue of the fact that increased aqueous levels of the mobile phase reduce carbohydrate retention, and elution order follows carbohydrate polarity.1... 

In normal-phase chromatography, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample. Therefore, the stationary phase is usually silica and typical mobile phases for normal phase chromatography are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these. In reverse phase the packing is nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as Ci8, Q, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water. In the strictest interpretation, normal and reverse phase are terms which only relate to the polarity of the column and mobile phase with respect to the sample as shown in Table 3-3 and drawn schematically in Figure 3-14. 

Bonded phases may be used in both normal and reverse phase chromatography. When normal phase chromatography is done on bonded phase packings, the packing is more polar than the mobile phase. Polar bonded phases such as the cyanopropyl and aminopropyl functionalities are popular for this use. These bonded phases are less subject to changing retention times of compounds because water is adsorbed from the mobile phase onto the stationary phase, a frequent concern when using bare silica packings for normal-phase separations. 

Since the discovery of IPC, this separation strategy has been devoted to increasing the mediocre retention of ionized samples on reversed phase stationary phases via ion-pair formation with a suitable IPR to increase the analyte hydrophobicity and, in turn, its retention. It is therefore clear that most IPC separations are performed under reversed phase conditions. Even if normal phase chromatography exploits polar... 

Detailed discussion of normal-phase chromatography process, mechanism, and retention theories, as well as types and properties of used stationary phases, is given in Chapter 5. 

Unlike the more popular reversed-phase chromatographic mode, normal-phase chromatography employs polar stationary phases, and retention is modulated mainly with nonpolar eluents. The stationary phase is either (a) an inorganic adsorbent like silica or alumina or (b) a polar bonded phase containing cyano, diol, or amino functional groups on a silica support. Tlie mobile phase is usually a nonaqueous mixture of organic solvents. As the polarity of the mobile phase decreases, retention in normal-phase chromatography... 

Figure 5-1. Hypothetical representation of the adsorption mechanism of retention in normal-phase chromatography. S denotes sample molecule, E denotes molecule of strong polar solvent, and X and Y are polar functional groups of the stationary phase. Prior to retention, the surface of stationary phase is covered with a monolayer of solvent molecules E. Retention in normal-phase chromatography is driven by the adsorption of S molecules upon the displacement of E molecules. The solvent molecules that cover the surface of the adsorbent may or may not interact with the adsorption sites, depending on the properties of the solvent. (Reprinted from reference 1, with permission.)...

NPC is ideally suited for the analysis of compounds prone to hydrolysis because it employs nonaqueous solvents for the modulation of retention. An example of the use of NPC in the analysis of a hydrolysable analyte was demonstrated by Chevalier et al. [28] for quality control of the production of benorylate, an ester of aspirin. A major issue in benorylate production is the potential formation of impurities suspected of causing allergic side effects therefore monitoring of this step is critical to quality control. The presence of acetylsalicylic anhydride prohibited the use of RPLC since it can be easily hydrolyzed in the water-containing mobile phase. However, an analytical method based on the use of normal-phase chromatography with alkylnitrile-bonded silica as the stationary phase provided an ideal solution to the analysis. Optimal selectivity was achieved with a ternary solvent system hexane-dichloromethane-methanol, containing 0.2 v/v% of acetic acid to prevent the ionization of acidic function and to deactivate the residual silanols. The method was validated and determined to be reproducible based on precision, selectivity, and repeatability. 

In normal-phase chromatography, polar stationary phases are employed and solutes become less retained as the polarity of the mobile-phase system increases. Retention in normal-phase chromatography is predominately based upon an adsorption mechanism. Planar surface interactions determine successful use of NPC in separation of isomers. The nonaqueous mobile-phase system used in NPC has found numerous applications for extremely hydrophobic molecules, analytes prone to hydrolysis, carbohydrates, and sat-urated/unsaturated compounds. In the future, with the advent of new stationary phases being developed, one should expect to see increasingly more interesting applications in the pharmaceutical industry. 

Solutes that are labile (i.e., reacts with protic solvents) or exhibit poor solubility in aqueous media are prime candidates for normal-phase chromatography. Normal phase is well-suited for the separation of isomers and diastereomers, as well as for separating compounds with saturated and unsaturated side chains. Generally, the greater is the amount of unsaturation the greater the retention due to increased polarizabihty of double bond. 

Regardless of the exact retention mechanism — adsorption, liquid-liquid partition or their combination — the stationary phase in normal-phase chromatography is more... 

Ternary and more complex mobile phases in normal-phase chromatography contain two or more different polar solvents in a non-polar one [361. To describe the retention in ternary and more complex solvent mixtures, it is possible to use the Snyder model of... 

Some very hydrophobic samples, e.g., lipids, are strongly retained and not eluted in an acceptable time even with pure methanol or acetonitrile as the mobile phase. Such samples are usually adequately resolved by normal-phase chromatography, but they can be often equally well or even better separated by non-aqueous reversed-phase (NARP) chromatography in mixed mobile phases containing a more polar (e.g.. acetonitrile or methanol) and a less polar (e.g., tetrahydrofuran. dichloromethane. methyl-r-butyI ether) organic solvent. Ternary non-aqueous mobile phases may contain even hexane or heptane. The retention decreases with increasing concentration of the less-polar... 

In contemporary HPLC. gradient elution is by far most frequently practised in reversed-phase systems for a plethora of sample types. Special precautions required in gradient-elution normal-phase chromatography discussed in Section 1.5.5 are usually not necessary. In RPC systems where the retention Eq. (1.18) applies. Eqs. (1.34) and... 

The retention factor, Eq. (7.2), for each species / is calculated knowing the dead time, t(), and the retention time of species i at infinite dilution, /r,./- There are known methods in the literature for calculating the dead time or retention time for a non-retained peak in normal-phase, reversed-phase and ion-exchange chromatography [67]. For example, in normal-phase chromatography, pentane in 95 5 hexane-acetone is unretained. In reversed-phase chromatography, a common measure of void volume is from the refractive index response obtained when the sample solvent composition is different from the mobile-phase composition. 

Both the solvent-interaction model (Scott and Kucera, 1979) and the solvent-competition model (Snyder, 1968, 1983) have been used to describe the effects of mobile-phase composition on retention in normal-phase liquid chromatography. The solvent interaction model on the one hand provides a convenient mathematical model for describing the relationship between retention and mobile phase composition. The solvent competition model on the other hand provides a more complete, quantitative description of the relative strengths of adsorbents and organic solvents used in normal-phase chromatography. 

---