Column reversed-phase

Reversed-phase columns are used to separate polar substances. Although in LC the stationary phase is a solid, it is necessary to bear in mind that there may be a thin film of liquid (e.g water) held on its surface, and this film will modify the behavior of sample components equilibrating between the mobile and stationary phases. A textbook on LC should be consulted for deeper discussion on such aspects. 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

The separation of xanthates by ion-interaction reversed-phase column chromatography is described for the determination of eight different xanthates in reagents commonly used in flotation plants. The separated species were detected spectroscopically at a wavelength of 305 nm (96). 

The stationary phase constitutes about 12% of the column volume, which is equivalent to only about 17% of the mobile phase content of the column. The values given in Table 2 are probably representative of most reverse phase columns but will differ significantly with extremes of pore size and pore volume. 

The dispersions of 12 solutes were then measured in a packed reverse phase column using the same mobile phase as that used in the determination of their diffusivities acetonitrile/water). The column dispersion (H) was then plotted against l/Dm... 

Inorganic packings (silica, alumina, etc.) are very stable (yet brittle) and show very high pore volumes (i.e, efficiency). However, their chemical stability is very limited and the surface is very active (this is also true for reversed-phase columns), allowing their use in special applications only. 

The mixture of free amino acids is reacted with OPA (Fig. 7-8) and a thiol compound. When an achiral thiol compound is used, a racemic isoindole derivative results. These derivatives from different amino acids can be used to enhance the sensitivity of fluorescence detection. Figure 7-9 shows the separation of 15 amino acids after derivatization with OPA and mercaptothiol the racemic amino acids may be separated on a reversed-phase column. If the thiol compound is unichiral, the amino acid enantiomers may be separated as the resultant diastereomeric isoindole compound in the same system. Figure 7-10 shows the separation of the same set of amino acids after derivatization with the unichiral thiol compound Wisobutyryl-L-cysteine (IBLC). 

The alternative technique for analyzing 1,4-dioxane is HPLC. Scalia proposed a method by solid-phase extraction using octadecyl-bonded silica cartridges and analyzed directly on a reverse phase column with UV detection at 200 nm and acetonitrile-water as eluent [328-330]. 

Many racemic mixtures can be separated by ordinary reverse phase columns by adding a suitable chiral reagent to the mobile phase. If the material is adsorbed strongly on the stationary phase then selectivity will reside in the stationary phase, if the reagent is predominantly in the mobile phase then the chiral selectivity will remain in the mobile phase. Examples of some suitable additives are camphor sulphonic acid (10) and quinine (11). Chiral selectivity can also be achieved by bonding chirally selective compounds to silica in much the same way as a reverse phase. A example of this type of chiral stationary phase is afforded by the cyclodextrins. 

The pore structure of most cross-linked polystyrene resins are the so called macro-reticular type which can be produced with almost any desired pore size, ranging from 20A to 5,000A. They exhibit strong dispersive type interaction with solvents and solutes with some polarizability arising from the aromatic nuclei in the polymer. Consequently the untreated resin is finding use as an alternative to the C8 and Cl8 reverse phase columns based on silica. Their use for the separation of peptide and proteins at both high and low pH is well established. 

Polymer C18 Column ODS-A 120A Silica Based Reverse Phase Column... 

It is seen that the polymer resin does not have the same retentive capacity as the conventional reverse phase column and thus, will not exhibit the same resolution or the equivalent loading capacity. Nevertheless, the polymer column will function over a wide range of pH whereas the silica based columns will be restricted to operating within a pH of 4.0 to 8.0 at the most. 

The overall sensitivity of the sampling and concentrating system was examined using acetophenone as the solute. Purified water (water that had been passed through a reversed-phase column) was used to make... 

As reverse phase column ( LC-8) was used (octyldimethyl chains), the interactions with the stationary phase that controlled retention, and thus, the separation, were predominantly dispersive. It should be remembered, however, that although dispersive interactions dominate in the stationary phase, the separation is controlled by adjusting the competing dispersive interactions together with the polar interactions that take place in the mobile phase by the solvent composition. 

Finally an example is included of the use of a Cl 8 reverse phase column. The packing is also a silica based but is contained in a short column 3.3 cm long, 4.6 mm in diameter and packed with particles 3 Jim in diameter. The example of its use is in the separation of mixture of growth regulators which is shown in figure 9. 

However, complete hydrolysis of carotenoid esters sometimes is not achieved in 1 to 3 hr. The saponification degree can be verified easily by the presence of carotenol ester peaks eluting later than the peaks of P-carotene on reversed phase columns. Retinol palmitate, added as an internal standard to orange juice, also serves to indicate whether saponification is complete, since it is converted to retinol which elutes at lower retention time. The mixture is subsequently washed with water until free of alkali in a separatory funnel. Other more polar solvents such as CH2CI2 or EtOAc, and diethyl ether alone or mixtured with petroleum ether can be used to increase the recovery of polar xanthophylls from the water phase. 

HPLC Systems Employing Reversed Phase Columns for Separation of Carotenoids... 

In recent years, the methods for carotenoid determination without saponification have increased. Independently of the mobile phase and food composition, there are similar patterns of chromatographic separation on reversed phase columns. A chromatograph can be divided roughly into four zones the first zone corresponds to free xanthophyUs, the second zone to monoesterified pigments, the third zone contains carotenes, and finally the fourth zone corresponds to diesterified carotenoids. - ... 

Independently of the reversed phase column, the addition of TEA to the mobile phase increases carotenoid recovery from the column." Increased recoveries of 18% lutein, 33% zeaxanthin, 33% P-cryptoxanthin, 53% lycopene, 30% a-carotene, and 42% P-carotene in a Vydac column were observed after the addition of 0.1% TEA to the mobile phase. Recovery on a C30 column was also enhanced by the addition of 0.1% TEA to the mobile phase, with the peak area of lutein increasing by 26%, that of zeaxanthin by 42%, that of P-cryptoxanthin by 55%, that of lycopene by 21%, and those of a-carotene and P-carotene by 47 and 64%, respectively. ... 

The most popnlar system is a reversed phase column (Cl8), on a silica base column. However, the use of C18 on a polymer-based column has been reported to provide better resolution, especially for the separation of complex anthocyanin mixtures containing acylated pigments. - Polymer-based columns also show better stability at low pH operating conditions. 

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. 

Dias, N. C., Nawas, M. I., Poole, C. F. Evaluation of a reversed-phase column (Supelcosil LC-ABZ) under isocratic and gradient elution conditions for estimating octanol-water partition coeffidents. 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Figure 4.6 Separation of SRM 1647 and SRM 869 polycyclic aromatic hydrocarbon test mixtures on a series of polymeric octadecylsiloxane reversed-phase columns differing in pore diameter. (Reproduced with permission from ref. 36. Copyright Elsevier Scientific Publishing Co.)...

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