Normal-phase chromatography selectivity

The mobile phases used in normal-phase chromatography are based on nonpolar hydrocarbons, such as hexane, heptane, or octane, to which is added a small amount of a more polar solvent, such as 2-propanol.5 Solvent selectivity is controlled by the nature of the added solvent. Additives with large dipole moments, such as methylene chloride and 1,2-dichlor-oethane, interact preferentially with solutes that have large dipole moments, such as nitro- compounds, nitriles, amines, and sulfoxides. Good proton donors such as chloroform, m-cresol, and water interact preferentially with basic solutes such as amines and sulfoxides, whereas good proton acceptors such as alcohols, ethers, and amines tend to interact best with hydroxylated molecules such as acids and phenols. A variety of solvents used as mobile phases in normal-phase chromatography are listed in Table 2.2, some of which may need to be stabilized by addition of an antioxidant, such as 3-5% ethanol, because of the propensity for peroxide formation. 

This equation shows that we should ideally select a stationary phase with a polarity that is very different from that of the solute. Indeed, the recommendation to use normal phase chromatography (high 5,) for non-polar solutes (low 5() and reversed phase chromatography (low 8) for the separation of polar solutes (high 8 is not new. However, this rule of thumb is much too simple. A complication is caused by the availability of appropriate mobile phases. For instance, to satisfy eqn.(3.30) for the elution of non-polar solutes (5, 7) from a silica column (5, 16), a mobile phase with 8mx -2 would be required. 

An improvement in the HPLC method should be realized by collection of the cannabinoid elution region of the reverse phase separation followed by normal phase chromatography of the fraction. This should allow use of the excellent selectivity of the normal phase... 

In order to accomplish the desired separation, the selection of appropriate stationary phase and eluent system is imperative. The most commonly used stationary phases in normal-phase chromatography are either (a) inorganic adsorbents such as silica and alumina or (b) moderately polar chemically bonded phases having functional groups such as aminopropyl, cyanopropyl, nitrophenyl, and diol that are chemically bonded on the silica gel support [16]. Other phases that are designed for particular types of analytes have also... 

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. 

The first step in method development is selecting an adequate HPLC mode for the particular sample. This choice depends on the character of the sample compounds, which can be either neutral (hydrophilic or lipophilic) or ionic, low-molecular (up to 2000 Da) or macromolecular (biopolymers or synthetic polymers). Many neutral compounds can be separated either by reversed-phase or by normal-phase chromatography, but a reversed-phase system without ionic additives to the aqueous-organic mobile phase is usually the best first choice. Strongly lipophilic samples often can be separated either by non-aqueous reversed-pha.se chromatography or by normal-phase chromatography. Positional isomers are usually better separated by normal-phase than by reversed-phase chromatography and the separation of optical isomers (enantiomers) requires either special chiral columns or addition of a chiral selector to the mobile phase. 

The solvent-strength parameters for the common solvents used in normal phase chromatography on carbon are quite different from those of silica or alumina. Thus carbon offers quite different selectivities than alumina and silica for normal-phase chromatography. However, the lack of a reproducible commercial source for carbon was for many years a significant limitation to its widespread application. In addition the sensitivity of carbon to changes in solvent strength is much less than that of silica or alumina. 

The main use of unmodified silica in HPLC is as an adsorbent in normal phase chromatography where the mobile phase is less polar than the stationary phase, and in this mode a non-aqueous mobile phase is commonly selected. Due to the high polarity of water and its strong affinity for free silanols on the silica surface, the presence of even very... 

Retention in normal-phase chromatography increases as the polarity of the mobile phase decreases. The selectivity of the analytes may arise from the differences in solvent strengths (eq), acidity, basicity, and dipolar nature of the mobile phase. Furthermore, solvent localization of the mobile phase plays a major role in the retention of the analytes [15,16]. These solvent strengths have been shown to be different when used with varied stationary-phase packings such as alumina, diol, and silica [3,17]. 

There are also stationary phases that effectively partition solutes in either reversed-phase or normal-phase mode. These stationary phases are typically silica particles derivatized with cyano, diol, or amino functional groups. Particles with a cyano-functionality separate based on polarity utilizing nitrile interactions between the stationary phase and the solute. The amino group of typical amino stationary phases interacts primarily with anionic and organic acid portions of the solute. Diols utilize hydroxyl interactions similar to underivatized silica but offer a slightly different selectivity. These and other bonded-silica phases offer alternatives to underivatized silica, but they are used much less frequently. The mobile phases employed with these stationary phases are the same as used in standard reversed-phase or normal-phase chromatography. 

Selection of a mobile phase for LC is more difficult than for GC. In LC, solute interactions with the mobile phase affect solute retention dramatically. The goal is to optimize the interactions between the solutes and the mobile phase and the solutes and the stationary phase to accomplish separation. If the solutes are to be separated based on differences in polarity, then a common approach is to use a polar stationary phase with a less polar mobile phase this approach is termed normal-phase chromatography. If the solutes are to be separated based on differences in polarizabilities, then a common approach is to use a nonpolar stationary phase and a polar mobile phase this approach is termed reverse-phase chromatography. 

Fig. 19 Selectivity triangles for preferred solvents in reversed-phase and normal-phase chromatography ., reversed phase --------, normal phase. (From Ref. 97. Re-...

Normal-phase, bonded-phase columns are likely underutilized for separations where they should be the method of choice. This is due both to the ease of use of reversed-phase, bonded-phase columns, discussed next, and also to the many problems inherent in the use of bare silica and alumina. Very straightforward method development in normal-phase chromatography can be performed by combining the solvent and stationary-phase selectivity triangles. The three columns, each used with the three recommended modifiers, should provide the maximum difference in selectivity available. These nine experiments, used in conjunction with chemometric optimization schemes, should then provide a ratio-... 

---