Hplc

Analytes Prone to Hydrolysis 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 alkylnitrilebonded 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. Another application that demonstrates the advantages of using NPC for the separation of analytes prone to hydrolysis is the reaction monitoring for the formation of 9,10-anthraquinone [29]. Anthraquinone is an important intermediate in the manufacturing of various dye products but also is used as a catalyst in the isomerization of vegetable oils. It is produced in large amount by Friedel–Crafts reaction of phthalic anhydride with benzene in the presence of AlCl3 catalyst.

The development of a normal-phase HPLC method was warranted due to the presence of phthalic anhydride, which is unstable in water. Analysis in organoaqueous solvent systems that are used in RPLC would lead to an on-column reaction forming the respective carboxylic acid degradation product. Figure 5-5 shows the chromatogram obtained for the separation of 9,10-anthraquinone from the reactants and impurities on a silica column. The method was successfully applied to monitor the reaction conversion and also to determine the stability of 9,10-anthraquinone at the specified storage conditions. In addition, sometimes a normal-phase HPLC method at subambient temperature must be applied for analytes that are extremely prone to hydrolysis. In the synthesis of leukotriene D4 antagonist, accurate quantitation of mesylate intermediate is essential for process optimization. Owing to its inherent

instability, analysis of mesylate intermediate must be carried out under normal-phase conditions with nonprotic solvents; however, significant cyclization of mesylation was still observed in such condition at room temperature. The authors concluded that the on-column reaction of the mesylate was silicacatalyzed cyclization. By conducting the normal-phase HPLC analysis at −30°C, it was demonstrated that on-column cyclization was adequately inhibited [30].

Extremely Hydrophobic Compounds

NPC has been used in the analysis hydrophobic compounds such as polyaromatic hydrocarbons [31–33]. An interesting example of an application of NPC involving extremely hydrophobic compounds was recently offered by Liu and NORMAL-PHASE

Typical chromatogram of a reaction mixture collected during the course of reaction of phthalic anhydride with benzene in the presence of AlCl3, as catalyst. Peaks: 1, benzene; 2, anthraquinone; 3, phthalic anhydride; 4, maleic anhydride; 5, unknown. Chromatographic conditions: Column: Spherisob silica, 250 4.6mm, 10 m; mobile phase, n-heptane–ethanol–chloroform–acetic acid (89 : 5 : 5 : 1, v/v/v/v); flow rate, 1mL/ min; detection, UV at 254 nm; temperature, 27°C. (Reprinted from reference 29, with permission.)

Warmuth [34] when they adopted NPC for the analysis of supermolecules, such as hemicarcerplexes. Hemicarcerplexes are complexes formed with hemicarcerand host and guest molecules. As shown in Figure 5-6, hemicarcerands possess a very hydrophobic structure with molecular weight over 2000 and is insoluble in protic solvents. A normal-phase HPLC method was developed using a silica column with dichloromethane and diethylether as the mobilephase system.The authors demonstrate that the chromatographic retention of hemicarcerplexes is mainly dominated by its size. Furthermore, a linear relationship between the logarithmic retention factor and the size of the hemicarcerplexes was observed for linear guest molecules independent of their polarity.

Separation of Isomers

With more and more complex molecules being investigated as drug candidates, isomer separation has become increasingly challenging. Despite being a workhorse analytical tool, reversed-phase chromatography is limited in its ability to distinguish between isomers [35–39]. On the other hand, NPC has established itself as the technique of choice for the separation of positional isomers as well as stereoisomers due to the specific nature of interactions.The separation of positional isomers of alkyl-substituted polyaromatic hydrocarbons (PAHs) in the petroleum industry is an example where NPC has been employed successfully. Alkyl substitution significantly increases PAH retention in RPC so that alkyl-substituted PAHs with low ring number have retention times close to some of the nonsubstituted higher-ring-numbered PAHs, resulting in co-elution.Wise et al. [40] reported that aminopropyl-bonded silica

A schematic of hemicarcerplex. (Reprinted from reference 34, with permission.) phase yields a separation sequence of PAHs solely based on the number of

conjugated rings independent of the type of alkyl substitution. Separations involving cis/trans isomers can also be accomplished by employing NPC. An example of this application is the separation of tricyclic antidepressant doxepin, which is marketed as a mixture of geometric isomers in a cis/trans ratio of 15 : 85 [41].When a spherisorb silica column is used with a hexane–methanol–nonylamine mobile-phase system, the cis isomer of doxepin elutes first. The structures of the two isomers and the chromatographic separation are shown in Figure 5-7. NPC has also been successfully employed in the separation of cis/trans isomers of steroids. Four diastereomers NORMAL-PHASE

(a) Structures of cis and trans isomers of Doxepin and (b) normal-phase chromatographic separation of isomers of doxepin. I, cis-doxepin; II, transdoxepin; III, nortriptyline; IV, cis-N-desmethyldoxepin; V, trans-N-desmethyldoxepin. Chromatographic conditions: Column: Spherisob silica, 150 4.5mm, 3 m. Hexane: methanol :

nonylamine, 95 : 5 : 0.3 (v/v/v); flow rate, 1.0 mL/min; detection, 254 nm; temperature, 23°C. (Reprinted from reference 41, with permission.) consisting of two pairs of cis/trans isomers (see Figure 5-8 for structures) were separated using a silica column and hexane-dichloromethane-2-propanol mobile-phase system as shown in Figure 5-9 [42]. Other interesting examples of positional isomer separation involving NPC are (a) the separation of dihydrodipyridopyridopyrazines, a new family of antitumor agents, on a silica Nucleosil 50 A-10 m column [43] and (b) the separation of celecoxib isomers by Chiralpak AD column [44]. NPC was also employed successfully for the resolution of (a) four configurational isomers of a steroidal calyx pyrrole [45], (b) regio- and stereoisomers of eicosanoids [46], (c) retinal and retinol isomers [47], and (d) several E/Z isomers pairs of vitamin A [48]. In certain cases, such as the separation of PAHs obtained from a coal liquefaction process, using reversed-phase HPLC is complicated as sample preparation is elaborate.This is due in large part to the fact that most complex fuel-related materials contain compounds that are not usually soluble in acetonitrile, the solvent of choice in reversed-phase HPLC. Here, NPC, which employs a variety of solvents, offers an alternative to the analysis of such samples. Separation of five well-studied coal liquefaction process stream samples was achieved and 19 isomers were resolved when NPC was used [33]. The method employed a tetrachlorophthalimidopropyl-modified silica column (TCPP) with a charge-transfer mechanism. One of the most challenging tasks in isolating secondary metabolites from fermentation broths is the removal of numerous structural analogs of the desired product formed by the host organism. Pneumocandin B0 is a potent antifungal agent produced as a recently discovered secondary metabolite by the fermentation of Zalerion arboricola [49]. Pneumocandin B0 is the product of interest, with a molecular weight of 1069Da.Pneumocandin C0, which differs from B0 only by a single carbon shift of hydroxyl group, is a key impurity coproduced by the fermentation. This impurity is proved to be intractable by reversed-phase chromatography or crystallization.The isomer was successfully

Structures of cis and trans isomers of steroids. (Reprinted from reference 42, with permission.) separated in NPC mode by employing LiChrospher Silica stationary phase and an ethyl acetate/methanol/water mixture (86/7/7) mobile-phase system.

Carbohydrates NPC has also found some applications in the field of carbohydrate analysis. Typical stationary phases used for this application are alkyl amine-, diol-, or polyol-bonded silicas [50–53]. Alkyl amino-bonded silicas are commonly used for the separation of saccharides and oligosaccharides in various matrixs, such as food or biological fluids. Although water is used as part of mobile phase, the retention behavior of carbohydrate follows the NPC retention behavior. NORMAL-PHASE HPLC

Chromatograms of isomers 3–6. Chromatographic conditions: Column: APEX silica, 250 4.6mm, 5 m (Jones chromatography); mobile phase, hexanedichloromethane-2-propanol. (a) 82 : 10 : 8 (v/v/v), (b) 84 : 10 : 6 (v/v/v); flow rate, 1 mL/min; detection, 280 nm; temperature, ambient. (Reprinted from reference 42, with permission.) Carbohydrates are eluted in the order of increasing polarity, and retention

decreases when water content increases.With an aminopropyl silica column, Koizumi et al. [54] showed the resolution of d-glycooligosaccharides up to a degree of polymerization of 30–35 under isocratic conditions, with a binary mixture of acetonitrile/water. Similarly, with the use of a polyamine polymer resin-bonded silica or an amino-bonded silica, separation of maltooligosaccharides up to a degree of polymerization of 28 was achieved [55].

Separation of Saturated/Unsaturated Compounds

The surface of the silica may be dynamically coated with transition metals, and the selectivities observed can be attributed to the complexes between the metal ions and the analyte species [56]. The use of silver-impregnated silica (adsorption of salts of transition metals on the silica surface) has been used for the analysis of saturated and unsaturated fatty acid methyl esters (FAME) and triacylglycerols (TAG) [57]. The retention of the unsaturated FAME and TAG can be attributed to the stability of the complex that is formed between the electrons of the carbon–carbon double bonds and the silver ions. The predominant interaction for saturated analytes is with the polar silanol groups. The secondary interactions are those of the silver ions with the unpaired electrons of the carbonyl oxygens of the analytes. The amount of silver adsorbed onto the silica and the pH (employment of acidic or basic modifiers) have been determined to have an effect on the retention and resolution of certain acidic and basic compounds and fatty acids [58].

CONCLUSIONS

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 saturated/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.