Normal-phase chromatography bonded phases

NPC = normal phase chromatography (bonded phase separation) RPC = reverse phase chromatography (bonded phase separation) lEC = ion exchange chromatography (bonded phase ion exchange) SEC = size exclusion chromatography... 

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. 

Normal Phase Chromatography. Normal phase mode involves the polar interaction of sample molecules with a polar stationary phase, commonly silica-based packings with an aminopropyl bonded phase. Alternatively, pure silica gel modified in situ by equilibration (and simultaneous regeneration) with a mobile phase containing a polyfunctional amine that is adsorbed could be used (47). 

The most common technique used for agrochemicals is reversed-phase SPE. Here, the bonded stationary phase is silica gel derivatized with a long-chain hydrocarbon (e.g. C4-C18) or styrene-divinylbenzene copolymer. This technique operates in the reverse of normal-phase chromatography since the mobile phase is polar in nature (e.g., water or aqueous buffers serve as one of the solvents), while the stationary phase has nonpolar properties. 

Liquid-solid chromatography (LSC), sometimes referred to as normal phase or straight phase chromatography, is characterized by the use of an inorganic adsorbent or chemically bonded stationary phase with polar functional groups and a nonaqueous mobile phase... 

The TLC process is an off-line process. A number of samples are chromatographed simultaneously, side-by-side. HPTLC is fast (5 min), allows simultaneous separation and can be carried out with the same carrier materials as HPLC. Silica gel and chemically bonded silica gel sorbents are used predominantly in HPTLC other stationary phases are cellulose-based [393]. Separation mechanisms are either NPC (normal-phase chromatography), RPC (reversed-phase chromatography) or IEC (ion-exchange chromatography). RPC on hydrophobic layers is not as widely used in TLC as it is in column chromatography. The resolution capabilities of TLC using silica gel absorbent as compared to C S reversed-phase absorbent have been compared for 18 commercially available plasticisers, and 52 amine and 36 phenolic AOs [394]. 

Hydrophilic interaction chromatography (HB IC) or aqueous normal-phase chromatography (ANP) refers to the use of polar stationary phases (e.g., bare silica, silica, or polymeric phases with bonded zwitterionic ligands, diol phases) in combination with a mobile phase rich in organic solvent but containing a significant amount of water (typically at least 3%). Bell [97] summarized the advantages of this technique as follows ... 

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. 

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... 

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. 

Normal-Phase Chromatography Example. Vitamin E, an antioxidant, is a complex made up of tocopherols and tocotrienols (Figure 8-62), which are sometimes used to stabilize formulations. Tocopherols are a series of related benzopyranols with a C16 saturated side chain. Tocotrienols contain three double bonds on the C16 side chain [32]. 

There are a number of modes or mechanisms into which chromatography is divided. These include adsorption, normal-phase partition, reversed-phase partition, and ion exchange. Often, the term partition is deleted from the discussions of the differences and similarities of these modes. The word partition initially arose when supports had to be coated with a liquid phase (and the mobile phases saturated with them) to accomplish separations with these two modes. Today, bonded-phase versions of these liquid phases are available, making them easier to use with greater reproducibility. Perhaps it has been the use of these bonded supports that have enabled the name of the mode to be simplified. 

Hydrophilic interaction chromatography (HILIC) is a variation of normal-phase chromatography in which solutes are retained on a polar bonded phase under high concentrations (80-90%) of organic solvent and released during a gradient to a more aqueous solvent. The organic mobile phase usually causes at least partial denaturation of proteins. 

There are many applications of normal-phase chromatography to separate all eight vitamers using silica or bonded silica phases such as diol or amino (Table 1.5 Figure 1.4). The separation of 3- and y-tocopherols and tocotrienols is the most difficult task, but it can be achieved with normal-phase HPLC. In addition to its... 

The extraction of aromatic hydrocarbons (Fig. 5.5) from crude oil uses two sorbents in series, first a cyanopropyl column attached to a second silica column. In this procedure, aromatic hydrocarbons are sorbed on both the cyanopropyl sorbent and on the silica sorbent. The heteroatom hydrocarbons (containing nitrogen, oxygen, and sulfur) are trapped on the cyanopropyl sorbent and eluted as a separate fraction from the silica column. Because the major interaction of aromatic heterocyclic hydrocarbons is through hydrogen bonding to the surface of the sorbent, the cyanopropyl sorbent is easier to elute than a silica sorbent alone. For this reason, the cyanopropyl column is used before the silica column. The separation of hydrocarbons from crude oil is an example of normal-phase chromatography that has been performed for many years on silica gel prior to the introduction of SPE. 

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