Normal-phase column chromatography

Flash column chromatography, normal phase, analgesics. 

For flavones in citrus peel oils, separations were accomplished with isocratic mobile phases of 38% and 40% acetonitrile in H20 (1). The extracts of peel and cold-pressed peel oils were diluted in ethanol and analyzed by reversed-phase on various C18 columns with good results. For the dilute citrus oils, gradient elution was preferred, to prevent the accumulation of terpenes on the column. With normal-phase chromatography, the elution order is reversed terpenes elute with the solvent front and are not a problem. 

A. W. Salotto, E. L. Weiser, K. P. Caffey, R. L. Carty, S. C. Racine, and R. L. Snyder, Relative Retention and column selectivity for the common polar bonded-phase columns The diol-silica column in normal-phase high-performance liquid chromatography,/. Chromatogr. 498 (1990), 55-65. 

The mode of separation in the HPLC depends on the selection of the stationary and mobile phases. In HPLC of lipids, normal- and reversed-phase modes are primarily used, with the reverse phase being more common than the normal phase. Separation in the re-versed-phase mode is mainly by partition chromatography, whereas separation in the normal phase mode is primarily by adsorption chromatography. Normal-phase HPLC is used for the separation of the lipids into classes of Upids [1,F]. Reversed-phase HPLC (RP-HPLC), on the other hand, is mainly used to separate each lipid class into individual species [2,B1]. For example, several triglycerides were separated from each other via nonaqueous reversed-phase HPLC, involving an octadecyl (ODS) column and a nonpolar (non-aqueous) mobile phase. RP-HPLC alone can be used to separate the fat molecules into classes and species [2,B1]. 

Typical thin-layer separations are performed on a glass plate that is coated with a thin and adherent layer of finely divided particles this layer constitutes the stationary phase. The particles are similar to those described in the discussion of adsorption, normal- and reversed-phase partition, ion-exchange, and size-exclusion column chromatography. Mobile phases are also similar to those employed in high-performance liquid chromatography. 

The principle of adsorption chromatography (normal-phase chromatography) is known from classical column and thin-layer chromatography. A relatively polar material with a high specific surface area is used as the stationary phase, silica being the most popular, but alumina and magnesium oxide are also often used. The mobile phase is relatively nonpolar (heptane to tetrahydrofuran). The different extents to which the various types of molecules in the mixture are adsorbed on the stationary phase provide the separation effect. A nonpolar solvent such as hexane elutes more slowly than a medium-polar solvent such as ether. 

Poor reproducibility in adsorption chromatography (normal-phase chromatography) is, aside from the limited solubility of polar compounds in organic solvents, the main problem of these systems. Almost always, the problem is with the (uncontrolled) water content in the total system, i.e. the HPLC instrument, the sample, the mobile phase and the column. 

RP systems dominate analytical applications because of their robustness and reduced equilibration time. This fast column equilibration makes RP phases applicable for gradient operation. For preparative chromatography, normal phase systems are preferred because of some disadvantages of the RP systems. Although water is a very cheap eluent it is not an ideal mobile phase for chromatography because it evaporates at higher temperatures, the enthalpy for evaporation is quite... 

In order for the separation to take place, a more polar solvent (than the original sample matrix) will be used to effect the desorption of the analyte molecules from the packing material. Examples of packing material include silica bonded with cyano, amine, and diol groups, as with the stationary phase of columns in normal phase chromatography (see Chapter 4 for further explanation). 

See also Extraction Solvent Extraction Principles. Infrared Spectroscopy Overview. Ion Exchange Overview Principles. Liquid Chromatography Overview Column Technology Normal Phase Reversed Phase. Nuclear Magnetic Resonance Spectroscopy-Applicable Elements Phosphorus-31. Thin-Layer Chromatography Overview. 

Figure 2 Effects of the concentration of 2-propanol,

Figure 4 Breakthrough curve of 2-propanol in heptane on a Separon SGX silica gel column in normal-phase gradient-elution HPLC calculated using the experimental isotherm data. Gradient 0-50% 2-propanol in 30min, 1 ml min" . V- milliliters of eluate from the start of gradient elution,

Liquid chromatography Normal-phase systems have the advantage of being directly compatible with extracts in hexane. Silica, alumina, and lime (calcium hydroxide) are all particularly suited to the resolution of carotenoid geometrical isomers cis-trans) and diastereoisomers, but not positional isomers oc/fi-carotene). However, silica may cause on-column artifacts, reproducible retention on alumina is strongly dependent on a rigorous control of the water content of the eluent, and lime columns are not commercially available. 

In our laboratory we make our own columns for normal-phase chromatography (diol-modified silica). We have noticed that by packing the columns under a high pressure, that is, 950-1000 bar, instead of at the recommended 500-600 bar, we obtained far better separation of the phospholipid classes. Accordingly, this way of packing columns may be a way to achieve better separation of intact polar lipids, if choice of column packing, solvent mixture gradient and sample clean-up have already been optimized. 

Figure 27. Separation of P-carotin isomers on an alumina column in normal phase chromatography [78]...

Clearly, the capabilities of modem chromatographic techniques have been vastly improved. Packed column GC has been replaced by capillary column GC. Similarly, the large columns of normal-phase liquid chromatography (LC) are replaced by microcolumn reverse phase LC columns. Capillary electrophoresis (CE) is an entirely new means of separating small amounts of more complex, and charged sample molecules, and has evolved into several distinct forms with unique capabilities. Mass spectrometry coupled with different forms of chromatography is now applied to the analysis of many mixtures, of higher complexity, and more disparate sample types. 

Kovat s retention index (p. 575) liquid-solid adsorption chromatography (p. 590) longitudinal diffusion (p. 560) loop injector (p. 584) mass spectrum (p. 571) mass transfer (p. 561) micellar electrokinetic capillary chromatography (p. 606) micelle (p. 606) mobile phase (p. 546) normal-phase chromatography (p. 580) on-column injection (p. 568) open tubular column (p. 564) packed column (p. 564) peak capacity (p. 554)... 

E. G. McEauglilin and J. D. Henion, Determination of dexamethasone in bovine tissues by coupled-column normal-phase liigh-performance liquid cliromatography and capil-laiy gas chromatography-mass specti ometiy , 7. Chromatogr. 529 1-19 (1990). 

One example of normal-phase liquid chromatography coupled to gas chromatography is the determination of alkylated, oxygenated and nitrated polycyclic aromatic compounds (PACs) in urban air particulate extracts (97). Since such extracts are very complex, LC-GC is the best possible separation technique. A quartz microfibre filter retains the particulate material and supercritical fluid extraction (SPE) with CO2 and a toluene modifier extracts the organic components from the dust particles. The final extract is then dissolved in -hexane and analysed by NPLC. The transfer at 100 p.1 min of different fractions to the GC system by an on-column interface enabled many PACs to be detected by an ion-trap detector. A flame ionization detector (PID) and a 350 p.1 loop interface was used to quantify the identified compounds. The experimental conditions employed are shown in Table 13.2. 

The same concepts apply to column chromatography, where the stationary phase is normally small particles of silica, Si02, or alumina, A1,0 . These substances are not very reactive and have specially prepared surfaces to increase their adsorption ability. The column is saturated with solvent, and a small volume of solution containing the solutes is poured onto the top. As soon as it has soaked in, more solvent is added. The solutes travel slowly down the column and are eluted (removed as fractions) at the bottom (Fig. 2). If the mobile phase is less polar than the stationary phase, the less polar solutes will be eluted first and the more polar ones last. 

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

Compared with liquid column chromatography, in PLC there is a certain limitation with respect to the composition of the mobile phase in the case of reversed-phase chromatography. In planar chromatography the flow of the mobile phase is normally induced by capillary forces. A prerequisite for this mechanism is that the surface of the stationary phase be wetted by the mobile phase. This, however, results in a Umitation in the maximum possible amount of water applicable in the mobile phase, is dependent on the hydrophobic character of the stationary RP phase. To... 

Recently, Janjic et al. published some papers [33-36] on the influence of the stationary and mobile phase composition on the solvent strength parameter e° and SP, the system parameter (SP = log xjx, where and denote the mole fractions of the modiher in the stationary and the mobile phase, respectively) in normal phase and reversed-phase column chromatography. They established a linear dependence between SP and the Snyder s solvent strength parameters e° by performing experiments with binary solvent mixtures on silica and alumina layers. 

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