Nonpolar steroids, separation

Nicotinic acid (vitamin B3), 1048 Nondestructive detection methods for carbohydrates, 499-501 Nonpolar extraction, 13 Nonpolar steroids, separation of, 974 Normal chamber RPC (N-RPC), 325-326 Nuclear magnetic resonance (NMR), 4,33 Nucleic acids and their derivatives, 921-969 detection general methods, 931-935 reliability, 934 sensitivity, 934... 

Two important groups of nonpolar steroids are cholesterol (plus related compounds) and Vitamins D. Several methods have been reported for the separation of Vitamins D (see ref. 1. for comprehensive review). Some are suitable for the most difficult separation problems. The members of the Vitamins D group are very sensitive to experimental conditions, and special precautions are required to obtain reproducible results. The situation is different in the case of the separation of sterols, because these compounds are more stable. However, achieving separation between structurally related sterols is a difficult problem. Several methods have been published for improving separation efficiency. For example, a procedure utilizing O-complex formation between silver (I) ions and the double bonds occurring in various locations of the sterol molecules should be mentioned (19). 

The recent development of mixed-mode stationary phases may provide new approaches for the separation of neuroactive steroids from biological matrices. These stationary phases typically embed a charged functional group within the nonpolar carbon chains typically employed in RP separations, and as such separations based on both RP and ion-exchange characteristics can be designed. Examples of this column type are the Primesep line of columns from SIELC Technologies. This separation approach maybe quite useful for the separation of steroid sulfates from complex mixtures. 

Of course, manv separalion problems can be snlvpil e< n ill well Im usiuti iiioie tliaii one separation system, t hus, for example, the separation of steroids is effected by adsorption on polar as well as on nonpolar sta-lionarv phases or by pariilinning in n tc-miiry sysieni, The choke ol the chromatographic system depends not only on the nature of the compounds to be separated, but also on the familiarity of the analyst with a particular separation system. 

In some instances, combinations of Cig and silica columns are also used for better purification of the crude extracts (431, 445). A combination of Cg, silica, and amino solid-phase extraction columns has been successfully employed to fractionate anabolic and catabolic steroid hormone residues from meat in polar and nonpolar neutral and phenolic compounds, and to purify further each fraction effectively (452). Another combination of two solid-phase extraction columns, one using a graphitized carbon black sorbent and the other Amberlite resin in the hydroxyl form, allowed neutral anabolics to be isolated and separated from acidic anabolics and their metabolites (453). A combination of basic alumina column placed in tandem with an ion-exchange column has also been applied for the purification of the crude extracts in the determination of diethylstilbestrol and zeranol (427), and estradiol and zeranol in tissues (450). 

The main functional groups on paper are hydroxyls with acids and aldehydes next in amount. These, along with the adsorbed water, make the system polar and not particularly good to separate nonpolar compounds. To obtain a nonpolar stationary phase (reversed phase), the paper is first dried thoroughly. It is then dipped in a nonpolar liquid and dabbed dry with paper towels. This produces a "reversed phase" suitable for separating water-insoluble substances like steroids, long chain fatty acids, or chlorinated insecticides. Figure... 

An aqueous sluny of acidic alumina will have a pH of approx 4.0 and is most useful for the separation of acidic compounds such as carboxylic acids. Basic alumina (pH approx 10.0) is useful for the separation of such basic compounds as alkaloids. Neutral alumina (pH approx 7.0) is most often used for the separation of relatively nonpolar compounds such as steroids. 

Lipids are chemical components of the cell that are insoluble in water but soluble in nonpolar solvents. Lipids include fatty acids, fats, oils, phospholipids, glyco-lipids, and steroids. Insolubility in water makes lipids an ideal structural component of cell membranes. Lipids make up the containers that separate the interior of the cell from its external environment. Lipids are also used for long-term energy storage and for insulation. We all store extra calories from food as lipids, some of us more than others. 

An especially pronounced selectivity of the diol-modified precoated layers exists for steroids. An example of this is the separation of some anabolic agents as shown in Fig. 7. Vicinal diol groups are fixed to the silica gel matrix by a quite nonpolar spacer. Therefore, an RP mechanism on diol... 

Sex hormones, corticosteroids, and sapogenins are the most important medium polar steroids and numerous solvent systems have been used to separate them. Based on the work of Hara and Mebe (18), the following conclusions can be drawn. Medium polar steroids can be separated using a medium polar system most of the applicable solvent systems contain one or two nonpolar solvents such as hexane, benzene or toluene and one medium polar solvent, e.g., chloroform, acetone, etc. 

Separation of steroid epimers (including so-called critical pairs and positional and structural isomers) is an important goal in steroid analysis. The separation of steroid isomers (R- and S-budenoside, syn-and anti-isomers of norgestimate, -norgestreloxime, and of -6-hydroxynorgestimate) has been reported on an amino-bonded silica using a nonpolar eluent (chloroform-diethyl ether-methyl ethyl ketone 4 3 3, v/v)). This system provides better selectivity and resolution for the steroids investigated than did the use of silica with the same mobile phase (34). 

The choice of a suitable developing system must be based on the characteristics of the mixture to be separated. Combinations of SkeUysolve B, ethylene chloride, acetone, and ethanol have given the most satisfactory results in the separation of steroid concentrates when developed in silicate adsorption columns. Speaking in general terms, the SkeUysolve B will remove the lipids and very nonpolar chromogenic material, and the ethylene chloride will remove the bulk of the less polar nonsteroidal impurities. The addition of as little as 5% acetone to the ethylene chloride developer of a column will permit the elution of the adrenal steroids in an order related to their polarity, as shown by the characterization of a neutral adrenal extract (see Section VI,2,B). 

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