Normal-Phase Chromatography NPC

Figure 1.3. Schematic diagrams depicting separation modes of (a) normal-phase chromatography (NPC) and (b) reversed-phase chromatography (RPC).

In normal-phase chromatography (NPC), the stationary phase is polar. A nonpolar mobile phase is used, such as n-hexane, methylene chloride, or chloroform. The stationary phase is a bonded siloxane with a polar functional group (polarity order cyano < diol < amino < dimethylamino). These phases retain polar compounds in preference to nonpolar compounds. 

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]. 

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. 

RPC = reversed-phase chromatography, IEC = ion-exchange chromatography, NPC = normal-phase chromatography, SEC = size-exclusion chromatography, NARP = nonaqueous reversed-phase, PAD = pulsed amperometric detector, ELSD = evaporative light scattering detector. Pre-column or post-column derivatization required. 

A wide range of organic compounds can dissolve in the mixed water-organic solvent phases and be separated, so RPC is by far the most popular form of HPLC. For those that can be separated either by NPC or RPC, the elution order is generally reversed, although not always exactly. Of course, if a sample is very nonpolar and insoluble in water mixtures, normal-phase chromatography is used. Otherwise, reversed-phase chromatography is used. 

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