Normal-phase mode

Also for analysis of some pharmaceutical substances a normal-phase mode of HPLC and a lot of organic solvents are needed, especially if it is used in routine analysis. 

The column was operated in the normal phase mode using mixtures of n-hexane and ethanol as the mobile phase. Equation (13) is validated by the curves relating the corrected retention volume to the reciprocal of the volume fraction of ethanol in Figure 19. It is seen that an excellent linear relationship is obtained between the corrected retention volume and the reciprocal of the volume fraction of ethanol. 

The sulfonate content was determined either by the well-known technique of two-phase titration with hyamine or by liquid chromatography (HPCL). Nonionic surfactants were analyzed by HPLC (16) in the reverse or normal phase mode depending on whether the aim was to determine their content in effluents or to compare their ethylene oxide distribution. 

In achiral-chiral LC-LC, the mobile phases used with the achiral and chiral columns must be miscible with one another. Since the enantiomeric separation is usually the most difficult to optimize, it is usually the separation that dictates the mode of operation of the total analysis. Thus, it makes sense that a chiral column that operates in the normal phase mode would require an achiral column that also works in the normal phase mode. Polar organic mode chiral separations are universal in that they can be paired with an achiral column that operates in either the reverse phase or normal phase mode. The choice of the achiral column is always determined after selecting the chiral column and the mode of operation. As with traditional liquid chromatography, different achiral columns will give different selectivity. 

Additionally, the inj ected matrix must also be miscible with the solvents used in the separations. For normal phase mode separations, all water must be removed from the injected matrix. Since many of the complex matrixes, such as plasma, urine, and other biological fluids contain a large amount of water, this requires more time consuming sample preparation. However, water can be injected into a polar organic or reverse phase mode separation. Even within the same mode, mobile phases that are very different can cause large disturbances in the baseline. Oda et al., (1991) solved this problem by inserting a dilution tube followed by a trap column in order to dilute the mobile phase used on the achiral column. Following the dilution tube, a trap column was used to reconcentrate the analyte of interest before the enantiomeric separation. 

In another study, the authors reported a comparative study of the enantiomeric resolution of miconazole and the other two chiral drugs by high performance liquid chromatography on various cellulose chiral columns in the normal phase mode [79], The chiral resolution of the three drugs on the columns containing different cellulose derivatives namely Chiralcel OD, OJ, OB, OK, OC, and OE in normal phase mode was described. The mobile phase used was hexane-isopropanol-diethylamine (425 74 1). The flow rates of the mobile phase used were 0.5, 1, and 1.5 mL/min. The values of the separation factor (a) of the resolved enantiomers of econazole, miconazole, and sulconazole on chiral phases were ranged from 1.07 to 2.5 while the values of resolution factors (Rs) varied from 0.17 to 3.9. The chiral recognition mechanisms between the analytes and the chiral selectors are discussed. 

There are two commonly used ways to elute a given compound in HPLC the normal-phase mode (t)s><5m) and the reversed-phase mode (<5m><5s). Reversed-phase systems offer superior general selectivity. Solutes are eluted in ascending order of polarity in normal-phase systems and in descending order of polarity in reversed-phase systems. 

The elution volumes of polystyrene and benzene in the size-exclusion mode were 0.98 and 1.78 ml, respectively (Figure 1.4A). This means that separations by molecular size can be achieved between 0.98 and 1.78 ml in this system. In the normal phase mode the elution volumes of octylbenzene and benzene were 1.98 and 2.08 ml, respectively, in n-hexane solution (Figure 1.4B). This type of chromatography is called adsorption or non-aqueous reversed-phase liquid chromatography. These are adsorption liquid chromatography and non-aqueous reversed-phase liquid chromatography. The elution order of the alkylbenzenes in the reversed-phase mode using acetonitrile was reversed... 

Aboul-Enein, H.Y. and Ali, I., A comparative study of the enantiomeric resolution of several tetralone derivatives on macrocyclic antibiotic chiral stationary phases using HPLC under normal phase mode, Pharmazie, 334, 258, 2001. 

Pirkle-type CSPs do not involve ionic interactions and therefore are almost exclusively operated in the normal-phase mode. The use of subcritical carbon dioxide based mobile phase, i.e., subcritical fluid... 

Cellulose and amylose derivative CSPs are mostly used, in the normal-phase mode, with n-hexane-based mobile phases containing some alcohol as modifier. Chromatographic performances, retention and selectivity, are reported to be affected by the composition of the mobile phase... 

Since acrylamide-based monoliths generally represent polar support materials, they are predominately also used for separation in normal-phase mode. For that purpose, monolithic polymers were prepared by polymerization of mixtures containing piperazine diacrylamide as cross-linking agent and methacrylamide, A-isopropylacrylamide or 2-hydroxyethyl methacrylate, and vinylsulfo-nic acid as monomers. 

Chiral Separation in Biological Matrices Using the Normal-Phase Mode... 

Only a limited number of biomedical applications have been published in the normal-phase mode, as can be seen in Table 17.4. However, the sensitivity of the methods seems to be comparable to the reversed-phase and polar-organic mode applications, although a detailed comparison is not feasible since the LOQ data are missing for the few substances that have been analyzed in both modes. The majority of the methods are based on MS detection, and APCl seems to be the predominant ionization mode for the applications in normal-phase mode. 

In any of its different modes, LC behaves as a dynamic adsorption process. Analyte molecules, while moving through the porous packing bead, tend to interact with the surface adsorption sites. Depending on the LC mode, different types of adsorption forces may be included in the retention process. Hydrophobic (nonspecific) interactions are the main ones in reversed-phase separations. Polar interactions including dipole-dipole, and dipole-induced dipole forces dominate in the normal-phase mode, whereas ionic interactions are responsible for the retention... 

A mixture of acetonitrile and water is capable of separating sugars on amino phase. Problems with the determination of reducing sugars can occur, since the reaction of the keto group with the amino moiety is well known. In a similar way, an amino phase in normal-phase mode should not be used with acetone. NH2 packing can be used as a weak anion exchanger. The diol... 

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