Liquid chromatography mixture separation

Link, J.C. (2004). Development and application of gradient ultrahigh pressure liquid chromatography for separations of complex biological mixtures Dissertation, University of North Carolina, Chapel Hill. Available from UMI ProQuest Digital Dissertations, Ann Arbor, MI, AAT 3156171. 

Alternatively, one may monitor on-line and fractionate the eluate accordingly. This is generally used at the later stages of separation for separations of less complex mixtures, typically on high-performance liquid chromatography (HPLC) separations monitored by UV, where one can identify and isolate material corresponding to individual peaks. 

Using the purified (S)-3-hydroxycarboxylate oxidoreductase (107) and two forms of NADPH regeneration led to the chiral 3-hydroxy carboxylates shown in Table 28 (83). All products of the bioreduction procedures showed ee values greater than 98 %. On the basis of the optical rotation value reported (108) the synthesized 3-hydroxybutyrate was the (5)-enantiomer. It was assmned that the configuration of the other prepared 3-hydroxycarboxylates was the same. As checked with racemic mixtures of the derivatives of the four 3-hydroxy acids mentioned in Table 28, the applied chiral gas/liquid chromatography columns separated the enantiomers. For details see (83). 

Separation and Purification. Once the proteins have been isolated, one can then begin to separate and purify them. A common method is to use affinity or liquid chromatography to separate out specific proteins or a family of related proteins through the binding of proteins to specific substrates, which will elute out specific proteins from a mixture. Certain technologies have made it possible to extract very small quantities of proteins from a mixture. 

A few reports are available on chiral separations of pollutants using this modality of liquid chromatography. The separated chiral pollutants are 2-(2-chlorophenoxy)propionic acid and 2-(4-chlorophenoxy)propionic acid on n-alkyl-)8-D-glucopyranoside [17], ibuprofens on vancomycin [18] and PCBs on y-cyclodextrin [19]. Marina etal. [20] reported chiral separations of polychlorinated biphenyls (PCBs) 45, 84, 88, 91, 95, 132, 136, 139, 149, 171, 183 and 196 by MEKC using cyclodextrin chiral selectors. Mixtures of and y-cyclodextrins were used as chiral modifiers in a 2-(yV-cyclohexylamino)ethanesulfonic acid (CHES) buffer containing urea and sodium dodecyl sulfate (SDS) micelles. A mixture of PCBs 45, 88, 91, 95, 136, 139, 149 and 196 was separated into all 16 enantiomers in an... 

FIGURE 7.13 High-performance liquid chromatography (HPLC) separation of a mixture of standards of possible pigments present in an extract of algal origin. Peaks 1, chlorophyUide a 2, chlorophyll c 3, pheophorbide a 4, fiicoxanthin 5, diadinoxanthin 6, lutein 7, chlorophyll h 8, chlorophyll a 9, asteroidenone 10, 3-carotene 11, pheophytin a. 

FIGURE 7.18 High-performance liquid chromatography (HPLC) separation of a mixture of standards of pheophytins and their oxidation products. Peaks 1,15-glyoxilic acid-pheophytin b 2, 15-gJyoxilic acid-pheophytin a 3, 15-OH-lactone-pheophytin b 4, 15-OH-lactone-pheophytin a 5, pheophytin b 5, pheophytin b C-13 epimer 6, pheophytin a, 6, pheophytin a C-13 epimer 7, phytol-purpurin 18 b ester 8, phytol-purpurin 18 a ester. 

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). 

Liquid chromatography is a separation technique based on the selective adsorption on a solid, siiica or alumina for example, or a mixture of the two, of the different components of a liquid mixture. 

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. 

For mixture.s the picture is different. Unless the mixture is to be examined by MS/MS methods, usually it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography. In the latter, small quantities of emerging mixture components dissolved in elution solvent would be laborious to deal with if each component had to be first isolated by evaporation of solvent before its introduction into the mass spectrometer. In such circumstances, the direct introduction, removal of solvent, and ionization provided by electrospray is a boon and puts LC/MS on a level with GC/MS for mixture analysis. Further, GC is normally concerned with volatile, relatively low-molecular-weight compounds and is of little or no use for the many polar, water soluble, high-molecular-mass substances such as the peptides, proteins, carbohydrates, nucleotides, and similar substances found in biological systems. LC/MS with an electrospray interface is frequently used in biochemical research and medical analysis. 

Although simple solutions can be examined by these techniques, for a single substance dissolved in a solvent, straightforward evaporation of the solvent outside the mass spectrometer with separate insertion of the sample is usually sufficient. For mixtures, the picture is quite different. Unless the mixture is to be examined by MS/MS methods, it will be necessary to separate it into its individual components. This separation is most often done by gas or liquid chromatography (GC or LC). 

LC, or sometimes HPLC (high-pressure liquid chromatography), is a means of separating components of mixtures by passing them in a solvent through a chromatographic column so that they emerge sequentially. 

Gas-liquid chromatography has been widely used for the identification of reaction mixtures and for the separation of heterocycles. Some typical conditions are shown in Table 34. 

Ion-exchange chromatography involves an electrostatic process which depends on the relative affinities of various types of ions for an immobilised assembly of ions of opposite charge. The stationary phase is an aqueous buffer with a fixed pH or an aqueous mixture of buffers in which the pH is continuously increased or decreased as the separation may require. This form of liquid chromatography can also be performed at high inlet pressures of liquid with increased column performances. 

The separation of mixtures involving N-methyl-JLtetrahydropyridines into their pure components by means of gas-liquid chromatography was discussed in a report by Holik et al. (87). They found that, using tris(/3-cyanoethoxymethyl)-y-picoline as the stationary phase, the primary factors involved in the specific retention volumes of these enamines is the electronic effect of a methyl substituent and the nitrogen atom on the carbon-carbon double bond. It was observed that 1,3-dimethyl-Zl -tetrahydropyridine (141) has a smaller specific retention volume and, hence, is eluted before... 

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