Reversed-phase chromatography columns

A mixture of rhodium II) acetate (228 mg, 0.516 mmol), the imidazolidinone (1.70 g, 6.15 mmol), and dry chlorobenzene (20 mL) is heated under reflux for 18 h in a flask fitted with a Soxhlet extraction apparatus into which a thimble is placed containing an oven-dried mixture of sodium carbonate and sand (2 1, 5 g). The progress of the ligand-exchange reaction can be monitored by HPLC (p-Bondapak-CN column, methanol). The resulting blue solution is concentrated under reduced pressure, and the residue is purified by column chromatography (reversed phase silica, Bakerbond Cyano 40 mm prep. LC packing, methanol). 

Column chromatography reversed phase TLC and separation on AgNOg-impregnated layers)... 

Emenhiser, C. et al.. Separation of geometrical carotenoid isomers in biological extracts using a polymeric Cjq column in reversed-phase liquid chromatography, J. Agric. Food Chem., 44, 3887, 1996. 

Haliclonacyclamine E (13) and arenosclerins A (14), B (15), and C (16) have been isolated from the marine sponge Arenosclera brasiliensis, endemic in Brazil. Crude extracts of this sponge displayed potent cytotoxic and antibiotic activities, and were subjected to fractionation by sihca-gel flash chromatography, medium pressure chromatography on a SiOH cyanopropyl-bonded column, and reversed-phase Cis column chromatography to give compounds 13-16 [18]. The structure elucidation was based on spectroscopic analysis, including HRFABMS, COSY, HSQC, HSQC-TOCSY, and HMBC NMR... 

Crego, A. L., Diez-Masa, J. C., and Dabrio, M. V., Preparation of open tubular columns for reversed-phase liquid chromatography, Anal. Chem., 65, 1615, 1993. 

A comprehensive 2D HPLC can be carried out with two very similar columns in reversed-phase liquid chromatography (Ikegami et al., 2005). A mixture of water and tetrahydrofuran was used as a mobile phase in the lst-D separation, and a mixture of water and methanol (CH3OH) in the 2nd-D separation with a common Ci8 stationary phase. 

Minakuchi, H., Nakanishi, K., Soga, N., Ishizuka, N., Tanaka, N. (1997). Effect of skeleton size on the performance of octadecylsilylated continuous porous silica columns in reversed-phase liquid chromatography. J. Chromatogr. A 762, 135-146. 

A novel development for HPLC is something called bonded reversed-phase columns, where the stationary phase is a nonpolar hydrocarbon, chemically bonded to a solid support. You can use these with aqueous eluents, usually alcohol-water mixtures. So you have a polar eluent and a nonpolar stationary phase, something that does not usually occur for ordinary wet-column chromatography. One advantage is that you don t need to use anhydrous eluents (very small amounts of water can change the character of normal phase columns) with reversed-phase columns. 

Fig. 1.43 Strategies for protein identification. (A) 2D gel electrophoresis approach. (B) 2D liquid chromatography approach. lEF Isoelectric focusing, sex strong cation exchange column, RP reverse phase column, SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis.

In liquid chromatography, reversed-phase materials such as Cig and Cg are the most commonly used sorbents (429, 430, 434, 438, 446, 447, 453, 454). Examples of baseline separations with reversed-phase columns of several groups of anabolics including stilbenes, resorcyclic acid lactones, and other, frequently used anabolics have been reported (463-466). In addition to reversed-phase separations normal-phase separations of anabolics using either Hypersil (467) and Brownlee (456) silica or diol-modified silica have been reported. Although not all analytes were completely separated, the latter column could be efficiently used to differentiate between estrogenic and androgenic compounds within a mixture of 15 anabolics and their metabolites (468). 

T. Okuda, Y. Nakagawa and M. Motohashi, Complete two-dimensional separation for analysis of acidic compounds in plasma using column-switching reversed-phase liquid chromatography ,/. Chromatogr. B726 225-236 (1999). 

Qin F, Zhao Y, Sawyer MB, Li X (2008) Column-switching reversed phase-hydrophilic interaction liquid chromatography/tandem mass spectrometry method for determination of free estrogens and their conjugates in river water. Anal Chim Acta 627 91-98... 

Buszewski, B., Bocian, S., Felinger, A. Excess isotherms as a new way for characterization of the columns for reversed-phase liquid chromatography. J. Chromatogr. A 1191, 72-77 (2008)... 

Extraction chromatography (reversed phase partition chromatography) has been used in analytical and biochemistry to effect chemical separations. It is a method which combines the simplicity of ion exchange and the selectivity of solvent extraction. Ion exchange theory may be used to calculate the number of theoretical plates in the column and the enrichment coefficient. Extraction chromatography as a separation method has been recently reviewed by Cerrai (J) and Katykhin (7). 

S.6.4.2 Reversed-phase ion-pair liquid chromatography. Reversed-phase ion-pair chromatography is an alternative approach for controlling the retention of ionic compounds. This approach is particularly useful for the separation of amines on silica-based columns and it has had a profound effect on the analysis of this class of compounds. In particular, the combination of reversed-phase ion-pair liquid chromatography and electrochemical detection revolutionized the analysis of neurotransmitters in the brain (Tomlinson et al., 1978 and refs, therein). 

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. 

Figure 3 Schematic drawing showing the immunoaffinity chromatography/reverse-phase chromatography/atmospheric pressure ionization mass spectrometry experiment. System components are represented as follows P, HPLC pump IAC, immunoaffinity column HPLC, analytical column MS, mass spectrometer with atmospheric pressure ionization interface DS, data system and W, waste. Switching valves are represented as .

The selection of mobile phases to be used in conjunction with these columns is dependent on the mode of chromatography (reversed phase versus normal phase) and on the physicochemical properties of the API (and related substances). However, in all instances, it is important to ensure that the API and all related substances can be monitored and are separated. Accordingly, it is of paramount importance that the initial chromatographic conditions elute all the individual components from the column and that no components are eluted at the solvent front (i.e., all compounds of interest are retained). In practice, this dictates the use of broad (5-90% strong solvent in 60 min) gradients. 

The separation of acidic or basic products may cause difficulties in adsorption chromatography. Reversed-phase or ion-exchange chromatography would be more suitable but solubility problems may arise. One answer is to use buffered silica, as shown in Figure 9.9. The silica is treated either as bulk material or in the column with a buffer solution of a suitable pH (acidic buffer for acidic samples, basic buffer for... 

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