Chromatography Purifying Organic Compounds

High-pressure liquid chromatography (HPLC) is used to separate and purify the products of laboratory reactions. 

Even before a new organic substance has its structure determined, it must be purified by separating it from solvents and all contaminants. Purification was an enormously time-consuming, hit-g or-miss proposition in the 19th I and early 20th centuries, but pow-I erful instruments developed in I the past few decades now simplify I the problem. 

A variety of chromatographic techniques are now in common use, all of which work on a similar principle. The mixture to be separated is dissolved in a solvent, called the mobile phase, and passed over an adsorbent material, called the stationary phase. Because different compounds adsorb to the stationary phase to different extents, they migrate along the phase at different rates and are separated as they emerge (elute) from the end of the chromatography column. 

The time at which a compound is eluted is strongly influenced by its polarity. Molecules with polar functional groups are generally adsorbed more strongly and therefore migrate through the stationary phase more slowly than 

Ergocalciferol (vitamin D2) Cholecalciferol (vitamin D3) a-Tocopherol (vitamin E) a-Tocopherol acetate Phylloquinone (vitamin Ki) 

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In addition to those described, there are many other methods for purifying organic compounds. Examples which might be irientioned are sublimation, chromatography, and conversion into more crystal-lizable derivatives, such as amines into their acetyl, benzoyl, or other acyl derivatives, or acids into their chlorides, amides, esters, etc. These are the methods which are generally used in research laboratories. Although they are used also in industrial laboratories, and the technical chemist should be familiar with them, it is beyond the scope of this book to treat them in detail. 

The types of chromatography used to analyze and purify organic compounds are shown in Table 5.1. 

For pedagogical reasons, we deal only with pure organic compounds. Pure in this context is a relative term, and all we can say is the purer, the better. A good criterion of purity for a sufficiently volatile compound (no nonvolatile impurities present) is gas chromatographic homogeneity on both polar and nonpolar substrates in capillary columns. Various forms of liquid-phase chromatography (adsorption and liquid-liquid columns, thin layer) are applicable to less volatile compounds. The spectra presented in this book were obtained on purified samples. 

As stated above, the utility of silica based stationary phases does not limit its use to organic mobile phases. For many years it has been commonplace in flash chromatography to use aqueous solvents to elute analytes from silica based media. Isocratic elution with mixtures of butanol, acetic acid and water is standard protocol for the separation of amino acids and a carefully prepared combination of methanol, chloroform and water is useful for general organic compounds. Peptides are also readily purified by gradient elution on normal phase silica, moving from acetonitrile to aqueous mobile phase 3,2l This technique is particularly useful for extremely hydrophilic peptides that are not strongly retained on reversed phase media. 

The soluble fraction of the organic matter in carbonaceous chondrites can be accessed by extracting powdered meteorite samples with solvents of varying polarity. These extracts contain complex mixtures of discrete organic compounds that can then be purified further, for example by charcoal filtration, ion-exchange chromatography or sublimation, before analysis. 

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