Liquid chromatography isomer separation

Resolution of a-substituted aldehydes. The SASP hydrazones of a-substituted aldehydes can be resolved by high-performance liquid chromatography. The separability factors are sufficient for analytical and preparative purposes. The (S,S)-isomer elutes consistently before the (S,R)-isomer. Both isomers can be cleaved to the enantiomerically pure aldehydes by ozonolysis or acid hydrolysis, with resolution yields of 35-70%. 

FIGURE 7.12 High-performance liquid chromatography (HPLC) separation of pigments present in a green tissue. Peaks 1, neoxanthin 1, neoxanthin isomer 2, violaxanthin 2, violaxanthin isomer 3, luteoxanthin 4, anteraxanthin 5, lutein 5 and 5", lutein isomers 6, chlorophyll b 6, chlorophyll b C-132 epimer 7, chlorophyll a 7, chlorophyll a C-13 epimer 8, (i-carotene 8, cw-P-carotene isomer. 

FIGURE 7.15 High-performance liquid chromatography (HPLC) separation of pigments present in canned peas. Peaks 1, pheophorbide b 1, pheophorbide b C-13 epimer 2, pheophorbide a 2, pheophorbide a C-13 epimer 3, neochrome 3, neochrome isomer 4, piropheophorbide b 5, piropheophorbide a 6, auroxanthin 6, auroxanthin isomer 7, muta-toxanthin 8, lutein 8 and 8", lutein isomers 9, pheophytin b 9, pheophytin b C-13 epimer 10, (3-carotene 11, pheophytin a 11, pheophytin a C-13 epimer 12, pyropheoph3din b 13, pyropheophytin a. 

FIGURE 7.19 High-performance liquid chromatography (HPLC) separation of pheophytins a and b from their respective Cu and Zn complexes. Peaks 1, Zn-pheophytin b 2, Zn-pheophytin a 3, Cu-pheophytin b 3 Cu-pheophytin b isomer 4, pheophytin b 4, pheophy-tin b C-13 epimer 5, pheophytin a 5, pheophytin a C-13 epimer 6, Cu-pheophytin a 6, Cu-pheophytin a isomer. 

Methano-indene-fullerene, C6o(CH2)(Ind) (26), was synthesized in 47% yield by the Diels-Alder reaction of indene with 24. The product 26 is a mixture of regioisomers two isomers were successfully isolated by high-performance liquid chromatography (HPLC) separation and one (Cg-isomer) of two was crystallographically characterized (Figure 3.4). 

For most samples liquid-solid chromatography does not offer any special advantages over liquid-liquid chromatography (LLC). One exception is for the analysis of isomers, where LLC excels. Figure 12.32 shows a typical LSC separation of two amphetamines on a silica column using an 80 20 mixture of methylene chloride and methanol containing 1% NH4OH as a mobile phase. Nonpolar stationary phases, such as charcoal-based absorbents, also may be used. 

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. 

Chen, X. (1991). An electrochemical, EPR study of carotenoids and high performance liquid chromatography separation of cis-trans isomers of canthaxantin. MS thesis, The University of Alabama, Tuscaloosa, AL. 

One application in liquid chromatography which does alter the separation process is the use of a specific series of derivatives to enable the separation of chiral (optical isomers) forms of alcohols, amines and amino acids using reverse-phase separation. FLEC is available in the two chiral forms (+)-l-(9-fluorenyl) ethyl chloroformate and (—)-l-(9-fluorenyl) ethyl chlorofor-mate (Figure 3.12). Reaction of two stereoisomers of a test compound (e.g. T+ and T—) with a single isomer of the derivatizing reagent (e.g. R+) will result in the formation of two types of product, T+R+ and T—R+. It is possible to separate these two compounds by reverse-phase chromatography. 

Normal-phase liquid chromatography is thus a steric-selective separation method. The molecular properties of steric isomers are not easily obtained and the molecular properties of optical isomers estimated by computational chemical calculation are the same. Therefore, the development of prediction methods for retention times in normal-phase liquid chromatography is difficult compared with reversed-phase liquid chromatography, where the hydrophobicity of the molecule is the predominant determinant of retention differences. When the molecular structure is known, the separation conditions in normal-phase LC can be estimated from Table 1.1, and from the solvent selectivity. A small-scale thin-layer liquid chromatographic separation is often a good tool to find a suitable eluent. When a silica gel column is used, the formation of a monolayer of water on the surface of the silica gel is an important technique. A water-saturated very non-polar solvent should be used as the base solvent, such as water-saturated w-hexane or isooctane. 

Example 2 Chromatography of nitroaniline isomers. The elution order of the nitroaniline isomers was ortho, meta, and para in normal-phase liquid chromatography using H-butanol-w-hexane mixtures as the eluent, when the stationary phase material was either silica gel, alumina, an ion-exchanger, polystyrene gel, or octadecyl-bonded silica gel. The results indicate that the separation of these compounds can be performed on a range of different types of stationary phase materials if the correct eluent is selected. The best separation will be achieved by the right combination of stationary phase material and eluent.68... 

Reversed-phase liquid chromatography shape-recognition processes are distinctly limited to describe the enhanced separation of geometric isomers or structurally related compounds that result primarily from the differences between molecular shapes rather than from additional interactions within the stationary-phase and/or silica support. For example, residual silanol activity of the base silica on nonend-capped polymeric Cis phases was found to enhance the separation of the polar carotenoids lutein and zeaxanthin [29]. In contrast, the separations of both the nonpolar carotenoid probes (a- and P-carotene and lycopene) and the SRM 869 column test mixture on endcapped and nonendcapped polymeric Cig phases exhibited no appreciable difference in retention. The nonpolar probes are subject to shape-selective interactions with the alkyl component of the stationary-phase (irrespective of endcapping), whereas the polar carotenoids containing hydroxyl moieties are subject to an additional level of retentive interactions via H-bonding with the surface silanols. Therefore, a direct comparison between the retention behavior of nonpolar and polar carotenoid solutes of similar shape and size that vary by the addition of polar substituents (e.g., dl-trans P-carotene vs. dll-trans P-cryptoxanthin) may not always be appropriate in the context of shape selectivity. 

Bell, C.M., Sander, L.C., Fetzer, J.C., and Wise, S.A., Synthesis and characterization of extended length aUcyl stationary phases for liquid chromatography with application to the separation of carotenoid isomers, J. Chromatogr. A, 753, 37, 1996. 

Price, W.P., Jr., and Deming, S.N. (1979), Optimized Separation of Scopoletin and Umbelliferone and cis-trans Isomers of Ferulic and p-Coumaric Acids by Reverse-Phase High-Performance Liquid Chromatography, Anal. Chim. Acta, 108, 227-231. 

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