R.W. Stout

Separation of macromolecules by reversed-phase high-performance liquid chromatography : Pore-size and surface-area effects for polystyrene samples of varying molecular weight

The separation of polystyrenes of varying molecular weight by reversed-phase high-performance liquid chromatography has been studied, using both isocratic and gradient elution with tetrahydrofuran—water mobile phases. Bonded-phase Zorbax® and Zipax® particles (C18-silica) of different pore-diameters were used: 6, 15, 30 and 100 nm. The results confirm that the normal exclusion of high-molecular-weight random-coil polymers from small pores (e.g., 6 nm) has little effect on the reversed-phase retention process. That is, effectively all of the bonded phase within the particle is available for interaction with these sample molecules. Quantitative relationships are derived which permit the interpretation of the present data and its extrapolation for the optimization of reversed-phase separations of polystyrene mixtures.

Study of the retention mechanism for basic compounds on silica under “pseudo-reversed-phase” conditions

Abstract The performance characteristics of a set of nitogenous bases were studied on a number of silicas using aqueous organic mobile phases. The retention characteristics were complex functions of the organic solvent and buffer concentrations as well as pH. The retention mechanisms were shown to be a combination of ion-exchange and interaction with siloxane and silanol groups over the entire range of concentration of organic solvent. The differences in retention on silica were due largely to the differences in ion-exchange strength of the silanol groups and the surface concentration of the siloxane bridges. The same solutes were studied on a range of C8-bonded phases of varying surface coverage, where the same interactions were observed as seen on silica with the addition of reversed-phase interactions. The unexpected relation between capacity factor and bonded-phase coverage was explained by interactions of the several retention mechanisms involved. The retention of small proteins on pure silica was also related to its ion-exchange strength. The performance of the silicas was related to that of the bonded-phase packings prepared from them, the retention of the proteins again being related to the ion-exchange characteristics.

High-performance liquid chromatographic column efficiency as a function of particle composition and geometry and capacity factor

Reduced plate height (h) vs. reduced velocity (v) plots have been measured over a wide range of v for 36 high-performance liquid chromatographic systems. Column type was varied over wide limits and solute capacity factor (k′) values were changed over the range 0.6–22. Resulting data can be accurately described by the Knox equation h = Av1/3 + B/v + Cv, where A is roughly constant (A = 0.5–0.8) for all columns studied, but values of B and C are strongly dependent on column type and solute k′ values. These observations can be rationalized by a quantitative model that recognizes two effects: (a) surface diffusion of solute molecules in the stationary phase (along the pore wall) and (b) restricted diffusion of small solute molecules within particles having narrow pores and long alkyl chains bonded to their surface.

A new, stabilized, hydrophilic silica packing for the high-performance gel chromatography of macromolecules

Abstract A new approach for the stabilization of bonded-phase silica packings for the gel chromatography of proteins has been examined. Porous silica microspheres were stabilized by a zirconium salt treatment and then covalently bonded with a hydrophilic organo-silane. The resultant product (Zorbax® Bio Series GF-250) is a high-resolution, high-speed chromatographic packing with enhanced bonded-phase stability in aqueous buffered mobile phases. Packed columns of this material fractionate proteins roughly by molecular weight (MW) in the range of from 12 000 to 120 000 Daltons with approximately log MW-elution volume linearity. The zirconium-treated surface presents no unusual restraints in operating conditions and even permits short-term use of buffered eluents at pH 9. The useful operating lifetime of this packing has been shown to surpass some other currently available products.

Separation of proteins by gradient elution from ion-exchange columns : Optimizing experimental conditions

Abstract A model has been described previously for the gradient elution separation of large biomolecules by reversed-phase high-performance liquid chromatograph

Larger-diameter small-particle columns for fast high-performance liquid chromatographic separations with conventional equipment

Abstract High-performance liquid chromatographic columns packed with 3-μm particles can achieve identical separations as for larger-particle columns (e.g., 5-6-μm particles) in about half the total time. However these 3-μm columns also produce narrower solute bands that require very-low-dead-volume liquid chromatographic systems. This paper describes a new column design (DuPont Golden Zorbax Series®) based on wider-diameter columns (8 x 0.62 cm) plus 3-μm particles of various compositions (silica, C8, C18). These new columns provide all of the advantages of 3-μm-particle columns and can be used with present liquid chromatographic equipment, usually without any system modifications. A number of fundamental questions relating to the use of these new columns were examined experimentally and shown not to compromise their performance. A number of applications of Golden Zorbax Series columns are presented, including comparisons of separations with corresponding 6-μm columns. Relative retention was found to be generally identical on both 3- and 6-μm columns.

New ion-exchange packing based on zirconium oxide surface-stabilized, diol-bonded, silica substrates

Abstract A new family of silica-based ion-exchange packing materials has been compared in stability and chromatographic performance to similar organic based materials. The new materials are derivatives of the zirconium oxide-stabilized Zorbax® Bio Series GF-250 and GF-450. Long-term stability of the cation-exchange packings has been determined with no detectable loss of the organic surface material. Using a previously published theoretical model, the surface of these materials has been tested with various proteins. Although many results correlate with the model, certain modifications may be necessary if it is to described all observations. In a practical application of these new packings, monoclonal antibody was purified from ascites fluid and was further analyzed by sodium dodecyl sulphate—polyacrylamide gel electrophoresis. These new ion-exchange packing materials function in a manner very similar to packings based on synthetic on synthetic organic polymers.

Separation and purification of oligonucleotides using a new bonded-phase packing material

We describe a new bonded-phase packing material, based upon surface-stabilised microparticulate silica, suitable for the rapid separation and purification of oligonucleotides. Columns packed with this material were demonstrated to give rapid separations of individual oligonucleotide species of up to 44 base units with high purity; agarose gel electrophoresis showed that the products were essentially single bands, with only trace quantities of the (n-1)-mer present. Baseline resolution of the desired oligomer from (n +/- 1)-mer was achieved under preparative loading conditions, where up to 200-300 micrograms of oligonucleotide could be separated. The separation was essentially independent of structure or sequence of the oligonucleotides. The retention mechanism of the oligonucleotides was investigated, and the results used to determine the optimum column configuration and separation conditions.

Effects of solutions used for storage of size-exclusion columns on subsequent chromatography of peptides and proteins.

The effects of storage of size-exclusion column packing materials in methanolic or azide-water solutions on subsequent separations were tested. Three commercially available columns were used in these studies; the Toyo-Soda Bio-Sil TSK 125, Bio-Sil TSK 250 and the DuPont Bio-Series GF-250. Upon initial chromatography, all three columns bound up to 760 micrograms of cytochrome c tryptic peptides. Sample binding to packing material is probably a function of the positively charged basic groups on peptides or proteins interacting with silanol groups. The larger the peptide, the less the opportunity for silanol-charged group interaction, hence, less binding. Initial samples introduced to a new column occupy the binding sites. Equilibration with neat methanol removes the bound protein revealing sites which bind sample. After absorption of peptides to binding sites on the packing material, storage in neat methanol regenerates the binding sites. Storage in 10% methanol diminished the binding phenomenon, but storage in azide-water reduced binding to a range below detection at the microgram level. Our recommendation to users of size-exclusion chromatographic columns is that one satisfy the absorption capacity of a new column by injecting a sufficient quantity of a basic peptide standard or other convenient sample to reduce available binding sites before using the column for important separations. Store columns in azide-water or 10% methanol to prevent the regeneration of exposed silanol groups.

Surface treatment and porosity control of porous silica microspheres

SummaryPorous silica microspheres (PSM) have been treated with ammonium bifluoride to adjust porosity, pore size, remove surface impurities, and minimize surface acidity. The porosities of four silicas having mean pore diameters ranging from 150 to 750 Å have been altered from initial values to the point at which the mechanical strength is insufficient to allow packed columns with acceptable performance. It is shown that a linear relationship exists between a change in porosity and the relative amount of ammonium bifluoride used to treat the silica. This reagent removes silica homogeneously from all pores in a given microsphere in a controllable and predictable manner. This treatment increases the peak capacity and improves chromatographic performance. The surfaces of treated silicas were probed with thiamine in the ion-exchange chromatographic mode. The slopes and intercepts of plots in which retention is plotted against the reciprocal of buffer concentration were both significantly reduced indicating that surface acidity is minimized by this treatment.