Column chromatography general

The nitration of the 2-anilino-4-phenylselenazole (103) is much more complicated. Even careful nitration using the nitrate-sulfuric acid method leads to the formation of a mixture of variously nitrated compounds in an almost violent reaction. By the use of column chromatography as well as thin-layer chromatography a separation could be made, and the compounds could be partly identified by an independent synthesis. Scheme 33 shows a general view of the substances prepared. Ring fission was not obser ed under mild conditions. 

Clinical Analysis Clinical, pharmaceutical, and forensic labs make frequent use of gas chromatography for the analysis of drugs. Because the sample s matrix is often incompatible with the GC column, analytes generally must be isolated by extraction. Figure 12.25b shows how gas chromatography can be used in monitoring blood alcohol levels. 

Column Si. Size-exclusion chromatography columns are generally the largest column on a process scale. Separation is based strictly on diffusion rates of the molecules inside the gel particles. No proteins or other solutes are adsorbed or otherwise retained owing to adsorption, thus, significant dilution of the sample of volume can occur, particularly for small sample volumes. The volumetric capacity of this type of chromatography is determined by the concentration of the proteins for a given volume of the feed placed on the column. 

Preparation of soil—sediment of water samples for herbicide analysis generally has consisted of solvent extraction of the sample, followed by cleanup of the extract through Uquid—Uquid or column chromatography, and finally, concentration through evaporation (285). This complex but necessary series of procedures is time-consuming and is responsible for the high cost of herbicide analyses. The advent of soUd-phase extraction techniques in which the sample is simultaneously cleaned up and concentrated has condensed these steps and thus gready simplified sample preparation (286). 

Three general methods exist for the resolution of enantiomers by Hquid chromatography (qv) (47,48). Conversion of the enantiomers to diastereomers and subsequent column chromatography on an achiral stationary phase with an achiral eluant represents a classical method of resolution (49). Diastereomeric derivatization is problematic in that conversion back to the desired enantiomers can result in partial racemization. For example, (lR,23, 5R)-menthol (R)-mandelate (31) is readily separated from its diastereomer but ester hydrolysis under numerous reaction conditions produces (R)-(-)-mandehc acid (32) which is contaminated with (3)-(+)-mandehc acid (33). 

High Pressure Liquid Chromatography. This modem version of the classical column chromatography technique is also used successfully for separation and quantitative analysis of dyes. It is generally faster than thin-layer or paper chromatography however, it requires considerably more expensive equipment. Visible and uv photometers or spectrophotometers are used to quantify the amounts of substances present. 

The application of pressure to the liquid phase in liquid chromatography generally increases the separation (see HPLC). Also in PIC improved efficiency of the column is observed if pressure is applied to the mobile phase (Wittmer, Nuessle and Haney Anal Chem 47 1422 1975). 

E. E. Johnson, R. Gloor and R. E. Majors, Coupled column chromatography employing exclusion and a reversed phase. A potential general approach to sequential analysis , J. Chromatogr. 149 571-585 (1978). 

Figure 12.12 Coupled SEC-RPLC separation of Plioflex rubber stock (a) SEC (b) RPLC ti ace of fraction 1, Wingstay 100 (Eive-peak pattern is representative of diarylphenylenedi-amine isomers) (c) RPLC ti ace of fraction 2, mixed disulfide and MBTS (2,2 -thiobis (ben-zothiazole)). Obtained under the same conditions as given for Eigure 12.11. Reprinted from Journal of Chromatography, 149, E. L. Johnson et al, Coupled column chromatography employing exclusion and a reversed phase. A potential general approach to sequential analysis , pp. 571-585, copyright 1978, with permission from Elsevier Science.

The alkanephosphonic acid dichlorides obtained by these methods are converted with amines, with all reactions carried out in solvents such as acetone, benzene, or diethyl ether at 10°C with triethylamine as HC1 captor. The conversion runs quantitatively followed by a purification with the help of column chromatography with chloroform/methanol in a ratio of 9 1 as mobile phase. The alkanephosphonic acid bisdiethanolamides could be obtained as pure substances with alkane residues of C8, C10, C12, and Ci4. The N-(2-hydroxyethane) alkanephosphonic acid 0,0-diethanolamide esters were also prepared in high purity. The obtained surfactants are generally stable up to 100°C. Only the alkanephosphonic acid bismonomethylamides are decomposed beneath this temperature forming cyclic compounds. 

Column chromatography is the most generally used method, and the use of several different solvents, or graded-concentration mixed solvents affords a considerable selectivity in many cases. The difficulty always remains, however, that an unidentified and unexpected species may accompany one of the carriers and give false results. This can be particularly misleading in cases where the various eluted fractions are not specifically identified, but are classified only by the polarity of the solvent. 

Diphenyl ether herbicides are generally extracted from 10 to 50 g of air-dried soil with an organic solvent such as acetone, methanol and benzene by sonication, mechanical shaking or Soxhlet extraction. If necessary, the extracts are then cleaned by column chromatography or SPE. The extract is evaporated completely to dryness and the residue is dissolved in an appropriate volume of the solvent for GC analysis. The reduced amine metabolites are extracted under alkaline conditions. 

There is a reasonably good stoichiometric control observed in these aminolysis reactions. Therefore, in principle, the extent of chlorine replacement can be regulated by the ratio of the reactants. If mixtures of products are formed, these are generally amenable to separation by column chromatography. Separation of individual stereoisomers is often accomplished by procedures such as fractional crystallization. 

A general experimental procedure57 for a diarylmethane leuco compound via a benzotriazole To a stirred solution of the corresponding (benzotriazol-l-yl-methyl)aniline (5 mmol) in methanol (30 ml) under reflux was added a solution of the appropriate aromatic compound (5 mmol) and concentrated hydrochloric acid (1 ml) in water (30 ml). The resulting mixture was heated under reflux followed by the addition of aqueous KOH (1 M, 50ml). The product was isolated by filtration or by extraction with ether, and further purified by recrystallization or by column chromatography. 

Once the protein is recovered from its producer source and concentrated it must be purified to homogeneity. In other words, all contaminant proteins and other potential contaminants of potential medical significance (discussed in Chapter 7) must be removed. Purification is generally achieved by column chromatography. 

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