Joachim Kinkel

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography II. Performance of non-porous monodisperse 1.5-μm Silica beads in the separation of proteins by reversed-phase gradient elution high-performance liquid chromatography

Columns of dimensions 36 × 8 mm I.D., packed with n-octyl-bonded nonporous monodisperse silica particles of diameter 1.5 μm ± 2%, were tested for their performance in the reversed-phase gradient elution of proteins, employing methanol, acetonitrile, 2-propanol and 1-propanol as organic solvents and 0.01–0.02 M trifluoroacetic acid or perchloric acid as eluents. The retention of proteins over a wide range of volume fractions of the organic solvent modifier follows the same dependences as are observed on porous reversed-phase packings, with one noticeable exception: the slope of the linear dependence of the logarithmic capacity factors on the composition of the binary eluent yields values between 4 and 7 for the proteins studied, whereas S is usually a function of the molecular weight and approaches values of up to 100. This peculiarity is assumed to be associated with the retention of proteins on a surface with easily accessible n-alkyl ligands and free from any pore diffusion effects. The 1.5-μm particles provide an excellent column performance, enhancing both the resolution and the detection sensitivity. Gradient times of 3 min and flow-rates of 1.5 ml/min allow the rapid separation of proteins. The sample capacity was found to be ca. 1 mg of protein per column volume, without a loss of resolution.

Role of the functional group in n-octydimethylsilanes in the synthesis of C8 reversed-phase silica packings for high-performance liquid chromatography

Abstract C8 reversed-phase packings have been sysnthesised by reaction of a 25-nm pore-size high-performance liquid chromatographic silica (10 μm, as(BET) = 297 m2 g-1) with 2,4-lutidine as base and dichloromethane and N,N-dimethylflormamide as solvents, or without solvents and with the following silanes: n-octyldimenthylchlorosilane (C8-Cl), n-octyldimethylhydroxysilane (C8-OH), n-octyldimethylmethoxysilane (C8-OCH3), n-octyldimethylethoxysilane (C8-OC2H5), n-octyldimethyl(dimethylamino) silane [C8-N(CH3)2], n-octyldimethyl(trifluoroacetoxy)silane (C8- OCOCF3), and bis-(n-octyldimethylsiloxane) (C8-O-C8). C8-Cl, C8-OH and C8-OCH3 each form a reactive intermediate with 2,4-lutidine, favouring the reaction with the hydroxyl groups of the silica. In the C8-Cl reaction with silica, an amount of water larger than half of the stoichiometric amount of C8-Cl prresent in the starting reaction leads to a drastic decline in the ligand density of bonded C8 groups. This is associated with the formation of C8-O-C8, the latter exhibiting an extremely low reactivity. The highest reactivity of the silanes studied was observed for C8-N(CH3)2, since this already contains a reactive Si bond and furthermore eliminates the volatile dimethylamine, which is a co-product of silanization. The ligand density of the bonded C8 groups in the reaction of C8-N(CH3)2 with silica was affected scarcely at all by the thermal pretreatment temperature (up to 800 K) of the silica, and the hydroxyl group concentration of the silica showed an approximately linear decrease at temperatures of 500–900 K. Under comparable conditions, the reactives of the silanes followed the sequence C8-N(CH3)2 > C8-OCOCF3 > C8-Cl ⋍ C8-OH ≈ C8-OCH3 ≈ C8-OC2H5 ⋍ C8-O-C8, with a maximum lignad density of 4.10 μmol m-2 for C8-N(CH3)2. In accordance with their reaction mechanisms, distinct patterns in the course of the ligand densities achieved by the silanes as a function of the reaction temperature were obtained.

Preparative chromatographic resolution of racemates on chiral stationary phases on laboratory and production scales by closed-loop recycling chromatography

Abstract The time-saving, economical and practical advantages that are obtained when closed-loop recycling chromatography under overload conditions and peak shaving is applied for the preparative chromatographic separations of enantiomers on chiral stationary phases are described. The results of this study indicate that this kind of operation presents an alternative means for the preparation of pure enantiomeric compounds on a large-scale starting from racemic mixtures to the stereoselective synthetic and enzymatic routes currently available. The straightforward scaling up of the procedure from an analytical scale to gram amounts per day in the laboratory or even multi-kilogram separations by automated industrial production chromatography was demonstrated using mixtures of synthetically prepared racemates mainly of pharmaceutical interest.

Comparison of the specific productivity of different chiral stationary phases used for simulated moving-bed chromatography

Abstract The use of preparative chromatography for the separation of enantiomers is now a well established technique. With the technical setup of simulated moving bed-chromatography significant improvements can be achieved regarding throughput and eluent consumption. To find the optimum separation system, the determination of some parameters with analytical chromatographic methods is sufficient. This article gives a procedure to optimize the separation conditions for a precursor of the novel Ca-sensitizing drug (EMD 53998) on four different chiral stationary phases. This analytical procedure led to a maximum specific productivity for the desired enantiomer of 430 g enantiomer/d·kg chiral stationary phase.

Evaluation of advanced silica packings for the separation of biopolymers by high-performance liquid chromatography : IV. Mobile phase and surface-mediated effects on recovery of native proteins in gradient elution on non-porous, monodisperse 1.5-μm reversed-phase silicas

The reversed-phase chromatography of proteins by gradient elution with acidic, low-ionic-strength aqueous-organic eluents is often associated with losses of the biological activity of the protein. In this study, the enzymatic activities of catalase, horseradish peroxidase and pepsin were examined under static and dynamic column conditions on non-porous, monodisperse 1.5-microns reversed-phase silicas with various n-alkyl ligands. Catalase readily lost its enzymatic activity under the influence of the acidic aqueous-organic eluents in the absence of the reversed-phase packing, whereas peroxidase was partially deactivated as a result of combined mobile phase and stationary phase effects but regained its activity on storage after elution. The enzymatic activity of pepsin was found to be very dependent on the column residence time and on the type of bonded n-alkyl ligand employed.

Packing technology, column bed structure and chromatographic performance of 1-2-μm non-porous silicas in high-performance liquid chromatography

This work is aimed at further elucidating the aggregation behaviour of micron- and submicron-size non-porous silicas and the column performance of 1–2-μm C18 silicas in reversed-phase high-performance liquid chromatography of low-molecular weight compounds. It is demonstrated that highly ordered, dense, porous aggregates of such silica beads were obtained by gravity settling and centrifugation. The slurry techniques applied at constant flow-rate and a pressure up to 50 MPa provided less-ordered aggregates, but generated an acceptable performance of columns when 1–2-μm C18 silica beads were employed. To operate columns of 53 mm × 4.6 mm I.D., the maximum flow-rate needs to be ca. 2.5 ml/min at an inlet pressure of 50 MPa. To keep extra-column effects to a minimum, the injection volume should be about 0.6μl, the volume of the detector cell about 0.3μl and the time constant of the detector <50 ms. Such columns enable fast separations of mixtures of low-molecular-weight substances in less than 30 s.

Scale-up of protein purifications using aqueous two-phase systems: Comparing multilayer toroidal coil chromatography with centrifugal partition chromatography

Two different laboratory scale liquid-liquid extraction processes using aqueous two-phase systems (ATPS) are compared: centrifugal partition chromatography (CPC) and multilayer toroidal coil chromatography (MTCC). Both use the same phase system, 12.5% (w/w) PEG-1000:12.5% (w/w) K(2)HPO(4), the same flow rate of 10 mL/min and a similar mean acceleration field of between 220 × g and 240 × g. The main performance difference between the two processes is that there is a continuous loss of stationary phase with CPC, while for MTCC there is not - even when sample loading is increased. Comparable separation efficiency is demonstrated using a mixture of lysozyme and myoglobin. A throughput of 0.14 g/h is possible with CPC despite having to refill the system with stationary phase before each injection. A higher throughput of 0.67 g/h is demonstrated with MTCC mainly due to its ability to tolerate serial sample injections which significantly reduces its cycle time. While CPC has already demonstrated that it can be scaled to pilot scale, MTCC has still to achieve this goal.

Unusual peak distortion in ligand-exchange chromatography of enantiomers under overloaded conditions

Abstract The separation of amino acid enantiomers by means of ligand-exchange chromatography on an adsorbed chiral stationary phase was investigated under overloaded conditions. The selectivity of separation, the limit of mass loading and the peak shapes of enantiomers are strongly affected by the copper(II) concentration in the eluent. Under the optimum conditions it was possible to separate up to 2000 μg of amino acid on a 125 × 4 mm I.D. column. With increase in mass loading the peak shape of the enantiomers changed from symmetrical Gaussian into fronting and subsequently into trapezoidal. During this transformation, peak compression was observed. The complex changes in solute peak shape observed in ligand-exchange chromatography of amino acid enantiomers imply a complex form of adsorption isotherm in these systems.

Chromatographic resolution of synthetically useful chiral glycine derivatives by high-performance liquid chromatography

Abstract The preparation of one oxo-oxazolidine and two dihydro-imidazole derivatives, useful chiral building blocks for amino acid syntheses, in racemic form is described. The racemic mixtures were separated on an analytical and preparative scale using high-performance liquid chromatography on chiral stationary phases. Detailed procedures for the separations are given, the results obtained are discussed, and the enantiopure compounds are fully characterized.