Shulamit Levin

Continuous Separation of Proteins in Electrical Split-Flow Thin (SPLITT) Cell with Equilibrium Operation

Abstract The continuous separation of charged species in an electrical split-flow thin (SPLITT) cell is described. By operating the electrical SPLITT system in an equilibrium mode at a solution pH lying between the isoelectric points of two proteins, a mechanism is available for the rapid and complete separation of the proteins. The theoretical conditions necessary for such a separation are established. The apparatus constructed to test this concept is described. Preliminary experimental results are reported for several proteins, and the complete resolution of a binary mixture of ferritin and cytochrome C is demonstrated.

Resolution of chiral cannabinoids on amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase: Effects of structural features and mobile phase additives

The separation of six pairs of chiral cannabinoids was achieved using a dimethylphenylcarbamate derivative of amylose, immobilized on silica gel (ChiralPak AD, Daicel), using 2-propanol and ethanol as the modifiers of n-hexane in the mobile phase. Good separation was achieved for most of the solutes in both solvent systems under various conditions. The chromatographic parameters of various cannabinoids in the two solvent systems were determined. The pairs differ from each other in small structural features such as the degree of saturation, position of a double bond and closure of a pyran ring. Therefore, a comparative study could give some clues regarding the mechanism of discrimination between the enantiomeric pairs on the chiral stationary phase. Preliminary measurements of limit of determination showed that it was possible to assess 99.9% enantiomeric purity of the cannabinoids, owing to the high efficiency of the separation. Enantiomers of two monoterpenes, used as intermediates or as starting materials in the chiral synthesis of cannabinoids, were also separated, hence the described procedure is capable of assessing whether the chiral centres in the molecules were sustained throughout the synthetic procedures.

Separation of amino acids on reversed-phase columns as their copper(II) complexes☆

Abstract We report the separation of amino acids on reversed-phase columns using aqueous mobile phases containing copper ions. Cu(II) forms complexes with the amino acids and therefore influences their retention times. More importantly the Cu—amino acid complex absorbs UV radiation with λ max around 230 nm. Thus the solutes can be detected at relatively long wavelengths. The linearity of the detector signal and the detection limits were studied. Ssamples as small as 10 ng per 10 μI can be detected and the useful range of detection is oer four orders of magnitude. The effects of the pH of the mobile phase were also studied: retention increases with pH, at least over the range investigated.

Comparison of the enantioseparation of racemic uridine analogs on Whelk-O 1 and ChiralPak-AD columns

The commercially available, brush-type (S,S)-Whelk-O 1 chiral stationary phase (CSP) has been used to separate 10 racemates of structurally related uridine analogs, potentially anti-viral agents, under various mobile phase compositions, using various temperatures. The enantioseparation was evaluated by comparing the Whelk-O 1 column performance with that of ChiralPak-AD column, reported previously. The comparison involved the role of some distinctive structural features of the racemates, type and composition of the solvent modifiers, as well as effect of temperature on the chiral discrimination. Despite the fact that both columns separate almost all the uridine analogs, significant differences were observed in their chiral recognition, as revealed from their retention, selectivity, resolution and elution order. The chiral recognition processes, responsible for enantioseparation on the Whelk-O 1 column, were relatively more systematic and easier to manipulate than on ChiralPak-AD column. Enantioseparation on the latter are of more complex nature and frequently gave results that were contradictory to the expectations. On the other hand, the performance in the ChiralPak-AD column was superior to that of the Whelk-O 1 column. Limitations in column handling and maintenance (pressure and temperatures) as well as limited solvent choice lead to the preference of the Whelk-O 1 column, in spite of its lower (but adequate) performance.

Role of hydroxyl groups in chiral recognition of cannabinoids by carbamated amylose

Abstract The enantioselective retention of four pairs of enantiomeric cannabinoids that have hydroxyl groups was compared with that for the corresponding acetylated compounds, using amylose tris(3,5-dimethylphenylcarbamate) in the stationary phase. According to this study the hydroxyl groups were essential to the chiral discrimination by the amylose stationary phase, since blocking them by acetylation was detrimental to the enantioselective separation. Three of the four enantiomeric pairs had a relatively rigid tricyclic backbone, whereas the fourth, the cannabidiol, was a flexible compound. In contrast to the other three enantiomeric pairs, the resolution of the acetylated cannabidiol was not completely lost as a result of the acetylation, but it was decreased and the elution order was reversed. Conformational analysis of the acetylated and non-acetylated enantiomeric pairs was systematically performed, using molecular mechanics, in order to examine the effect of acetylation on the conformation of the molecules. The results indicated that acetylation did not change the conformations substantially and therefore the loss of resolution was attributed to the blockage of the hydroxyl groups. The molecular mechanics approach was validated by comparing the energy-minimized structure of cannabidiol with its X-ray crystallographic structure taken from the literature.

Measurements of size distribution and density of a pharmaceutical fat emulsion, using field-programmed sedimentation field-flow fractionation (SdFFF)

AbstractPurpose. The main goal was to establish that sedimentation field-flow fractionation (SdFFF), operated with power based field programming, is effective in the characterization of a commercial emulsion, Medialipide®. This emulsion is used clinically for total parenteral nutrition and it is consisted of a mixture of long-chain triglycerides (LCT, soybean oil) with medium-chain triglycerides (MCT) emulsified by phospholipids. Methods. Different field programming methods were used in the analysis to establish the limits of applicability of the technique. Results. Identical size distribution profiles were obtained under various conditions of the analysis. The density of the droplets was determined by collecting fractions from the SdFFF eluting bands, and analyzing them by photon correlation spectroscopy. The value of density of the oil droplets was changed in the SdFFF data, until best agreement with the PCS values was achieved. The value of density corresponding to the best agreement was considered as the oil density, and it was closed to the weighted average value between soybean and MCT oils. Conclusions. Field programming extends the capabilities of sedimentation field-flow fractionation in handling and characterizing complex and delicate samples as Medialipide®.

Adsorption isotherms of phenylalanine in a chromatographic column measured simultaneously by system peaks analysis and frontal analysis

Abstract Adsorption isotherms of phenylalanine dissolved in acetate buffer of three concentrations, 0.001, 0.01 and 0.1 M , were measured using frontal analysis and system peaks analysis simultaneously. The adsorption isotherms measured by the two methods were identical. System peaks were induced by injecting a small vacancy, pure water, just after the plateau was reached in each step of the frontal analysis. The capacity factor of the system peak corresponding to phenylalanine was used in the calculation of the adsorption isotherm. The use of system peaks for the measurement of adsorption isotherms is promising, especially for multi-component systems where mixed isotherms are needed for rational formulation of a preparative separation.

Structural features affecting chiral discrimination of terpene derivatives, on a carbamated amylose stationary phase

The chiral discrimination of enantiomeric derivatives of alpha-pinene was studied using amylose tris(3,5-dimethylphenylcarbamate) as a chromatographic stationary phase. The effect of structural features of these enantiomeric pairs on their chromatographic resolution was systematically studied to understand further the correlation between these features and chiral discrimination by the carbamated amylose. Structural analysis by molecular mechanics indicated that the conformation of the alpha-pinene skeleton was preserved with substitution in all its derivatives. However, in spite of the rigidity of the molecular backbone of these molecules, their resolution capabilities were different. Apparently, the site of the hydrogen-bonding substituents affected chiral discrimination by the stationary phase rather than conformational changes. Separation was achieved in spite of the fact that none of the members in the series had an aromatic moiety, and therefore pi-pi interactions with the stationary phase were insignificant. Hence it was concluded that the most important interaction of the terpene derivatives with the carbamated amylose was hydrogen bonding.

Use of system peaks for the determination of the distribution of resorcinol, catechol and phenol in liquid chromatography

Abstract The single-component adsorption isotherms of resorcinol, catechol and phenol between aqueous solutions and LiChrosorb RP-18 were determined using the system peaks of these components. The single-component isotherms obtained agree well with those derived by frontal analysis. For multi-component solutions at low concentrations, the system peaks obtained are the combination of those observed for the different single-component systems (linear range). When the concentration increases, the retention times and the areas of the different system peaks depend on the nature and concentration of all the system components (non-linear range). The experimental results agree well with the results predicted using the Langmuir competitive isotherms derived from the single-component isotherms. The dependence of the system peak areas on these concentrations is especially complex. The experimental results agree well, however, with those of calculations based on the use of the equilibrium-dispersive model.

Factors controlling the separation of amino acids in isocratic reversed-phase liquid chromatography

Abstract Amino acids can be separated with a simple isocratic liquid chromatographic system in which no pre- or post-column derivatization of the amino acids is needed. Copper ions and alkylsulphonate additives are used to effect both the selectivity and the retention of the solutes. The effect of several operating conditions on the performance of the system was examined, especially the nature and concentration of the alkylsulphonate, the concentration of copper ions, the ionic strength of the buffer and temperature. Guidelines are given for optimization of the separation. the deleterious effects of system peaks and means of overcoming them are discussed.