Normal phase chromatography compounds

In contrast to reversed-phase, the stationary phase in normal-phase chromatography is polar, usually silica or alumina, and uses nonpolar solvents, e.g., hexane and ethylacetate, that are not compatible with the API processes nsed in LC-MS. In normal-phase chromatography compounds elute progressively from the least to the most polar. The technique is not applicable to the highly polar compounds encountered... 

When bnffer is not present, i.e. when normal-phase chromatography is being nsed, thermospray ionization is not possible and a filament or discharge electrode is nsed to generate a plasma in which Cl-type processes can occnr. In addition to allowing ionization nnder these conditions, it is found that the ionization of compounds may be enhanced under conditions in which true thermospray can operate. 

Cyano Intermediate Hydrophobic, dipole-dipole, and pi-pi Resolves polar organic compounds by reversed-phase or normal-phase chromatography... 

With all these tools, you can usually find a way to separate the components of a mixture if it does not contain too many compounds. If reversed-phase chromatography fails, normal-phase chromatography or one of the methods in Chapter 26 could be appropriate. Method development is part science, part art, and part luck. 

Adsorption Chromatography—Separation mode resulting from compounds that have different adhesion rates for the packing surface. (See Normal-Phase Chromatography.)... 

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. 

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. 

The term normal phase refers to a chromatographic system that utilizes a polar stationary phase and a nonpolar mobile phase. Normal-phase chromatography usually succeeds in the separation of compounds that differ in the... 

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. 

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. 

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