Shock layer thickness and optimum linear velocity in displacement chromatography

The results of a study of the optimization of the experimental conditions for maximum production rate in overloaded elution and displacement chromatography are discussed. This study is based on the use of the equilibrium-dispersive model of chromatography and the competitive Langmuir isotherms to calculate individual band profiles in the elution and displacement modes, and of a simplex algorithm to optimize the production rate. The operating parameters (sample size, mobile phase velocity, and the displacer concentration in the displacement model) and the column design parameters (column length and average particle diameter) are optimized simultaneously. Binary mixtures having relative concentrations 3:1 and 1:3, and separation factors of 1.2 to 1.8 are investigated. One of our major results is that, in both modes of chromatography, the maximum production rate is achieved at very low values of the retention factors, k', much lower than those used in current practice. In all cases, unless k' exceeds greatly that optimum value, the production rate is higher in overloaded elution than in displacement chromatography. This is particularly true for the extraction of a minor component, which is eluted second.

Development of mass transfer model for chromatographic separation of free lutein and fatty acids in de-esterified marigold lutein

Displacement chromatography is a predominantly nonlinear mode of chromatography, which has certain advantages over the elution mode for preparative bioseparations. Whereas continuous production (and separation) processes have their theoretical benefits in this context, protein displacement chromatography has up to now only been performed in the batch mode. In this contribution, we demonstrate that the principle of continuous annular chromatography can be adapted to displacement chromatography. Separations of up to three standard proteins (two whey proteins, soybean trypsin inhibitor) were developed and optimized using a small (4 x 250 mm) batch column. These separations were subsequently transferred directly to the continuous system (500-mL column). Separations of similar quality in terms of final product purity and recovery yield were obtained using the continuous system.

Shock layer temperature profiles are obtained through analysis of radiation from shock layers produced by a blunt body inserted in arc jet flow. Spectral measurements have been made in a nitrogen flow of 54.4 gm/s at an enthalpy of 8.72 MJ/kg. Vibrational temperatures for N2+ are obtained by matching spectral regions from arc jet spectra with spectra generated using the NEQAIR code. Temperature profiles obtained from the radiation layers show a vibrational temperature higher than the rotational temperature near the front of the shock and both temperatures decrease as the flow approaches the body. The spectral measurements are made and analysis completed for four distances, from the surface of the blunt body. The corresponding shock layer thickness is approximately 3.6 cm. Although the shock layer appears to be in thermal nonequilibrium, the measured rotational temperature approaches the single temperature results of viscous shock layer calculations at this test condition.

Cooperation of solid granule and solvent as porogenic agents Novel porogenic mode of biporous media for protein chromatography

Capillary electrochromatography with macroporous particles

Segmented flow and curtain flow chromatography: Overcoming the wall effect and heterogeneous bed structures

Gradient elution separation and peak capacity of columns packed with porous shell particles

Several HPLC columns that have been recommended for the separation of fullerenes were compared. Each column (i.e., stationary phase) had a different optimum mobile phase. In general, mobile phases consisted of binary mixtures of a “good” fullerene solvent and a “poorer” fullerene solvent. Reversed phase stationary phases with alkyl (aliphatic) substituents produced superior analytical separations for all fullerenes. However, stationary phases with aromatic substituents were better for preparative separations. Increasing the proportion of the “poor” fullerene solvent in the mobile phase generally increased resolution and retention but decreased the mass load. Injecting too high a concentration of fullerenes in any column caused peak splitting to occur. The fullerene retention order was the same on all columns and with all mobile phases. The fullerene separation ability of all columns tended to deteriorate with time. Overloading the column or doing preparative separations greatly accelerated the deterioration process. It is believed that irreversible adsorption of fullerenes or fullerene by-products is responsible for the degradation of column performance.

We developed a micellar liquid chromatographic (MLC) procedure for the determination of three extensively monitored antiepileptics in serum samples: carbamazepine, phenobarbital, and phenytoin. We determined the composition of the mobile phase after modeling the elution behavior of the antiepileptics in hybrid micellar mobile phases of sodium dodecyl sulfate (SDS) with different organic modifiers (propanol, butanol, or pentanol) in an experimental design that used five mobile phases, a C(18) column, and ultraviolet detection. In the micellar chromatographic system, the serum samples can be injected directly. The optimum mobile phase was 70 mL/L butanol in 0.05 mol/L SDS, pH 7, in which the three antiepileptics were resolved in <10 min. Intra- and interday precision was evaluated at four different drug concentrations within the therapeutic range (n =10); CVs were <2.1%. The method was applied to the analysis of 120 serum samples, and results were similar to those obtained by the TDx method. The MLC method allows chromatographic determination of three antiepileptics, using an interpretative strategy of optimization, without pretreatment of the serum samples and with direct injection in a hybrid micellar mobile phase of SDS-butanol. The method provides complete resolution and quantification of mixtures of two and three antiepileptics.

Reversed-phase high-performance liquid chromatography of mouse epidermal growth factor and its congeners: mobile phase optimization with ion-pairing additives

Structural radial heterogeneity of a silica-based wide-bore monolithic column

A quantitative liquid chromatography/ion trap mass spectrometry method for the simultaneous determination of paclitaxel, 6alpha-hydroxypaclitaxel and p-3'-hydroxypaclitaxel in human plasma has been developed and validated. 6alpha-,p-3'-Dihydroxypaclitaxel was also quantified using paclitaxel as a reference and docetaxel as an internal standard. The substances were extracted from 0.500 mL plasma using solid-phase extraction. The elution was performed with acetonitrile and the samples were reconstituted in the mobile phase. Isocratic high-performance liquid chromatography analysis was performed by injecting 50 microL of reconstituted material onto a 100 x 3.00 mm C12 column with a methanol:1% trifluoroacetic acid/ammonium trifluoroacetate in H(2)O 70:30 mobile phase at 350 microL/min. The [M+H](+) ions generated in the sonic spray ionization interface were isolated and fragmented using two serial mass spectrometric methods: one for paclitaxel (transition 854 --> 569 & 551) and the dihydroxymetabolite (transition 886 --> 585 & 567) and one for the hydroxy metabolites (transition 870 --> 585 & 567; transition 870 --> 569 & 551) and docetaxel ([M+Na](+), transition 830 --> 550). Calibration curves were created ranging between 0.5 and 7500 ng/mL for paclitaxel, 0.5 and 750 ng/mL for 6alpha-hydroxypaclitaxel, and 0.5 and 400 ng/mL for p-3'-hydroxypaclitaxel. Adduct ion formation was noted and investigated during method development and controlled by mobile phase optimization. In conclusion, a sensitive method for simultaneous quantification of paclitaxel and its metabolites suitable for analysis in clinical studies was obtained.

Peak tailing and mass transfer kinetics in linear chromatography: Dependence on the column length and the linear velocity of the mobile phase

Determination of the kinetic and equilibrium parameters of the n-boc-rolipram enantiomers on a chiral stationary phase by high performance liquid chromatography