Reverse phase chromatography octadecyl

Fio. 36. Vloi t Hoff plou of the retention bctors of aromatic acids in reversed-phase chromatography using octadecyl silica as the stationaiy phase and neat aqueous 30 taM NaHiPO buffer (pH 2.0) (open symbols), or the same buffer containing 696 (v/v) of aceloni ti (closed symbols) as the eluent. Column S imSpherisorbODS, 230 x 4.6 mm. Eluites 3.4xlihydroxymandelic acid (O. ) 4 hydroxymandelic acid ( , ) 4-hydroxyphenylacetic acid (7. ) 3,4-dihydroxyphenylacetic acid (A, A). Reprinted with permission from Me-lander tt at. U77). 

At present, liquid chromatography has become the most widely used technique for determining anticoccidial drugs in edible animal products (Table 29.11). The separation of these drugs is generally done on nonpolar reversed-phase columns (octadecyl, octyl and phenyl), the preferred type being the octadecyl bonded silica. However, polar (389, 390, 392, 398, 400) columns have also been used for the determination of the polyether antibiotics in edible animal products. 

If solutes dissolve only in nonpolar or weakly polar solvents, the decision tree suggests that we try reversed-phase chromatography. Our choices include bonded phases containing octadecyl (CJg), octyl, butyl, ethyl, methyl, phenyl, and cyano groups. 

Several new phases applied to maltosaccharide analysis that seem to be promising have been described in the literature, for instance, a silica-phase covered with polymeric polyamine resin, and an entirely polymeric resin containing an amide function (35). In any case, partition chromatography is restricted mainly to the utilization of polar-bonded phases, as already described. Nevertheless, there have been a certain number of applications of reversed-phase chromatography that permit relatively simple separations to be achieved. Octadecyl-bonded silica phases are the most widely used, although few applications involve carbohydrate analysis. Their interest lies rather in the analysis of derivated sugars, where the selectivity increases (36). 

Silica can be drastically altered by reaction with organochlorosilanes or organoalkoxysilanes giving Si—O—Si—R linkages with the surface. The attachment of hydrocarbon chains to silica produces a non-polar surface suitable for reversed-phase chromatography where mixtures of water and organic solvents are used as eluents. The most popular material is octadecyl-silica (ODS-silica) which contains C g chains, but materials with Cj, C, Cg, and C22 chains are also available. 

Olieman et al40 found that reversed-phase ion-pair chromatography was the most suitable technique to analyze a series of morphinan derivatives. Normal reversed-phase chromatography on octadecyl columns, as well as straight-phase systems gave too much tailing -particularly for alkaloids with a high retention time (Table 7.4). 

Reversed-phase chromatography is now widely used for the fractionation of biological molecules. The technique is based on the use of a non-polar stationary phase and a polar mobile phase. The stationary phase is usually made of a hydrocarbonaceous layer, either n-octyl. Cfl, or n-octadecyl, Cib, ligands, chemically bonded to the surface of a silica matrix via siloxane bonds. Separation Is achieved by exploiting the difference in the hydrophobic properties of the molecules. 

Fig. 2.4. Surface groups used for stationary phases in reversed phase chromatography range from ethyl silane (C2) to n-octadecyl silane (Cig).

Reverse-phase chromatography - in which the amino acids react with the reagent to form fluorescent or ultraviolet-absorbing derivatives, which are then separated using a more polar mobile phase (e.g. acetate buffer with a gradient of acetonitrile) and a less polar stationary phase (e.g. octadecyl-bonded silica). The availability of amino adds to the animal can be estimated by chemical methods. For example, for lysine there are colorimetric methods that depend on the formation of compounds between lysine and dyes (see Chapter 13). 

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

Dibenzyl-14-crown-4 (lithium ionophore VI 6,6-dibenzyl-l,4,8,ll-tetra-oxa-cyclo-tetradecane) [106868-21-7] M 384.5, m 102-103°. Dissolve in CHCI3, wash with saturated aqueous NaCl, dry with MgSOa, evaporate and purify by chromatography on silica gel and gradient elution with C6Hg-MeOH followed by preparative reverse phase HPLC on an octadecyl silanised silica (ODS) column and eluting with MeOH. It can be crystd from MeOH (v Br 120 cm , C-O-C). [7 Chem Soc Perkin Trans 1 1945 1986.]... 

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