Capillary Electrochromatography as a Method Development Tool for the Liquid Chromatographic Separation of DUP 654 and Related Substances

Capillary Electrochromatography (CEC) offers a rapid, economical, and efficient means for resolving nonionic compounds in the reversed phase mode on octadecylsilane (ODS) columns. A CEC optimization on a Hypersil ODS capillary column was employed to identify a suitable mobile phase for the pressure-driven (reversed phase ODS) separation of the anti-inflammatory 2-phenylmethyl-1-naphthol (DUP 654), and its related substances. The proportions of mobile phase modifiers methanol, acetonitrile, and water as well as pH were employed as variables in a stacked mixture design. Comparable response surface profiles were obtained for the CEC separations at pH 4 and pH 8. However, subtle differences were evident in the quality of separations obtained in the liquid chromatographic (LC) mode when using a specially-prepared column packed with exactly the same stationary phase as used in the CEC experiments. A mapping of the response surface for separations on a commercially available Hypersil ODS LC column revealed obvious differences. The differences indicate that the transfer of ODS based separation methods between CEC and LC involves more than simply transferring the conditions from one mode to the other.     

Microbore polypropylene capillary channeled polymer (C-CP) fiber columns for rapid reversed-phase HPLC of proteins

The performance of microbore columns with polypropylene (PP) capillary-channeled polymer (C-CP) fibers as the support/stationary phase for separation of macromolecules has been investigated. Polypropylene C-CP fibers (40 μm diameter) were packed in fluorinated ethylene propylene (FEP) tubing of inner diameter 0.8 mm and lengths of 40, 60, 80, and 110 cm. The performance of PP fiber packed microbore columns (peak width, peak capacity, and resolution) was evaluated for separation of a three-protein mixture of ribonuclease A, cytochrome c, and transferrin under reversed-phase gradient conditions. The low backpressure characteristics of C-CP fiber columns enable operation at high linear velocities (up to 75 mm s(-1) at 1.5 mL min(-1)). In contrast with the performance of other phases, such velocities enable enhanced resolution of the three-protein mixture, because peak widths decrease with velocity. Increased column length resulted in increased resolution, because the peak widths remained essentially constant, although retention times increased. In addition, it was found that the peak capacity increased with column length and linear velocity. Radial compression of the microbore tubing enhanced the homogeneity of the packing and, thereby, separation efficiency and resolution. Radial compression of columns resulted in a decrease in the interstitial fraction (~5%), but increased resolution of ~14% between ribonuclease A and cytochrome c. Even so, a linear velocity of 75 mm s(-1) required a backpressure of 9.5 MPa only. It is clear that the fluid and solute-transport properties of the C-CP fiber microbore columns afford far better performance than is obtainable by use of standard format columns. The ability to achieve high separation efficiencies, rapidly and with low volume flow rates, holds promise for high-capacity protein separations in proteomics applications.     

Fast supercritical fluid chromatography of polymers using packed capillary columns

Summary Fast separations of perfluorinated polyethers and polymethylsiloxanes that are composed of 50–80 oligomers were demonstrated in packed capillary column supercritical fluid chromatography (SFC) using a carbon dioxide mobile phase. Separations were accomplished within 10 min using a 13 cm×250 μm i.d. column packed with 2 μm porous octadecyl bonded silica (ODS) particles. Effects of particle diameter of the packing material and pressure programming on separation were investigated, and packed column SFC was compared with open tubular column SFC. Results show that as the particle diameter was decreased from 5 to 3 to 2 μm and the column length was reduced from 85 to 43 to 13 cm, the separation time could be reduced from 70 to 20 to 10 min while still maintaining similar separation (resolution). Short columns packed with small porous particles are very suitable for fast SFC separations of polymers.     

High Performance Liquid Chromatography Separations Using Short Columns Packed with Spherical and Irregular Shaped ODS Particles

Abstract

In a comparative study of the separation of a mixture of polycyclic aromatic hydrocarbrons on 5 cm and 10 cm columns packed with 3μ and 5μ ODS spherical particles, the separations achieved on the 10 cm column were not significantly better than those on the 5 cm column. Although, columns packed with 3μ ODS spherical particles gave slightly better resolution than those packed with 5μ ODS spherical particles having the same physical properties, differences were observed when columns packed with ODS spherical particles were compared with columns packed with ODS irregular shaped particles of the same size.

The results show that separations on a 5μ or 3μ packed columns takes place at the first few centimeters.     

Rapid, high-resolution high-performance liquid chromatographic analysis of antibiotics

A HPLC column devised for high separation speed combined with highly practical operating features has been found useful for separating antibiotics. Important characteristics involve compromises in packing particle size, column configuration and support-stationary phase combinations. We determined that these columns are useful for rapid, high-resolution separations with unmodified state-of-the-art HPLC equipment without the extra-column band-broadening effects typical of so-called “fast” HPLC columns. The proposed columns feature efficient sterically-protected monofunctional silane stationary phases that provide good separation reproducibility and high column stability. The combination of these unique bonded silanes and a highly purified, less-acidic silica support give superior peak shapes for antibiotic compounds. The proposed column configuration can halve separation times and double peak heights without loss in resolution, compared to widely used analytical columns. Increased mobile phase flow-rates permit even faster separations of antibiotics with only modest loss in resolution and peak heights for trace analyses in biological systems.     

Hypercrosslinking: new approach to porous polymer monolithic capillary columns with large surface area for the highly efficient separation of small molecules

Monolithic polymers with an unprecedented surface area of over 600 m(2)/g have been prepared from a poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) precursor monolith that was swollen in 1,2-dichloroethane and hypercrosslinked via Friedel-Crafts reaction catalyzed by ferric chloride. Both the composition of the reaction mixture used for the preparation of the precursor monolith and the conditions of the hypercrosslinking reaction have been varied using mathematical design of experiments and the optimized system validated. Hypercrosslinked monolithic capillary columns contain an array of small pores that make the column ideally suited for the high efficiency isocratic separations of small molecules such as uracil and alkylbenzenes with column efficiencies reproducibly exceeding 80,000 plates/m for retained compounds. The separation process could be accelerated while also improving peak shape through the use of higher temperatures and a ternary mobile phase consisting of acetonitrile, tetrahydrofuran, and water. As a result, seven compounds were well separated in less than 2 min. These columns also facilitate separations of peptide mixtures such as a tryptic digest of cytochrome c using a gradient elution mode which affords a sequence coverage of 93%. A 65 cm long hypercrosslinked capillary column used in size exclusion mode with tetrahydrofuran as the mobile phase afforded almost baseline separation of toluene and five polystyrene standards.     

A graphical method for understanding the kinetics of peak capacity production in gradient elution liquid chromatography

A novel graphical method for assessing the compromise between conditional peak capacity and separation speed for packed bed columns under gradient conditions has been developed and applied to the separation of peptides. This approach is analogous to and complements the conventional "Poppe plot" used to study plate count in isocratic separations. The use of the new plot can assist the design of appropriate column formats (e.g. particle size and column length) for both dimensions in gradient elution two-dimensional liquid chromatography (2DLC). Particularly for the second dimension of 2DLC, we find that smaller particles provide faster separations even though fast separations based on particles smaller than 2 microm are practically limited by the required miniscule column length. We also find that high temperatures strongly enhance the kinetics of peak capacity production whereas higher pressures help achieve larger absolute peak capacities albeit at the cost of longer analysis time.     

High Performance Liquid Chromatography Separations Using Columns Packed with Spherical ODS Particles. III. Effect of Column Dimensions on the Resolution of a Complex Mixture

Abstract

Separations on short columns, (3 and 5 cm, packed with 3 μ ODS spherical materials) and somewhat larger ones (10 cm and 20 cm columns having 2.1 mm and 4.6 mm diameters packed with 5 μ ODS spherical materials) were compared using Aroclor 1254. With simple mixtures, the results showed that short columns can give separations comparable with those on longer columns when the percentage of the organic modifier in the mobile phase is adjusted. This was not so with more complex mixture. The results also showed that columns which have a comparable volume do not produce comparable separation. The longer column, 200 mm × 2.1 mm gave better resolution than the shorter 50 mm × 4 mm column. Also a shorter column, (100 mm × 4.6 mm), which had double the volume of a longer column. (200 mm × 2.1 mm), gave better resolution of the Aroclor 1254 test solution.     

Temperature optimization for the separation of PAHs on micropacked LC ODS columns

The effect of column temperature on the separation of the sixteen polycyclic aromatic hydrocarbons (PAHs) of mixture SRM 1647a of the US Environmental Protection Agency has been studied on different micropacked ODS columns. Isothermal temperature optimization was successfully used for complete separation of the PAHs on a polymeric ODS stationary phase, whereas temperature programmed conditions were selected for separation on a monomeric ODS stationary phase.     

Methodology for porting retention prediction data from old to new columns and from conventional-scale to miniaturised ion chromatography systems

Several procedures are available for simulating and optimising separations in ion chromatography (IC), based on the application of retention models to an extensive database of analyte retention times on a wide range of columns. These procedures are subject to errors arising from batch-to-batch variability in the synthesis of stationary phases, or when using a column having a different diameter to that used when the database was acquired originally. Approaches are described in which the retention database can be recalibrated to accommodate changes in the stationary phase (ion-exchange selectivity coefficient and ion-exchange capacity) or in the column diameter which lead to changes in phase ratio. The entire database can be recalibrated for all analytes on a particular column by performing three isocratic separations with two analyte ions. The retention data so obtained are then used to derive a "porting" equation which is employed to generate the required simulated separation. Accurate prediction of retention times is demonstrated for both anions and cations on 2mm and 0.4mm diameter columns under elution conditions which consist of up to five sequential isocratic or linear gradient elution steps. The proposed approach gives average errors in retention time prediction of less than 3% and the correlation coefficient was 0.9849 between predicted and observed retention times for 344 data points comprising 33 anionic or cationic analytes, 5 column internal diameters and 8 complex elution profiles.     

Hydrophilic interaction chromatography coupled to electrospray mass spectrometry for the separation of peptides and protein digests

In this study, the analysis of a peptide set, chosen for their differences in hydrophilicity, and the tryptic digests of bovine cytochrome c and β-lactoglobulin by hydrophilic interaction chromatography–electrospray ionisation mass spectrometry (HILIC–ESI-MS) is demonstrated. Two different types of HILIC phases, i.e., an amide- and an amino-modified silica-based phase, packed into narrow bore or capillary columns, were investigated with separations conducted under either low pH or neutral pH conditions. The separation performance of the two HILIC columns with respect to peak efficiency and selectivity have been documented under these different mobile phase conditions, and the results compared with the performance of a conventional capillary reversed-phase C18 column of similar dimensions. It was found that very good separation of the peptide set could be achieved by using the amide-modified silica column over a broad pH range. Moreover, with the protein digest samples, excellent separation of the tryptic digests was obtained with the amide-modified HILIC column under neutral pH conditions. Compared to the conventional reversed-phase C18 separations, the use of these HILIC columns not only provided complementary separation selectivity, but also offered the capability to identify unique peptides using tandem HILIC–mass spectrometric techniques. These studies therefore highlight the potential of HILIC procedures for future proteomic applications.     

Development of capillary column packed with thiol-modified gold-coated polystyrene particles and its selectivity for aromatic compounds

Three types of thiol compounds (n-octadecanethiol, thiophenol, and 2-phenylethanethiol) were used to modify the gold-coated polystyrene particles (dp. 5microm) to prepare a stationary phase for capillary liquid chromatography through the formation of self-assembled monolayer. The column with n-octadecanethiol-modified gold-coated polystyrene particles (C18-Au) demonstrated the higher affinity to phenanthrene and anthracene than small aromatics compared to the ODS column. In addition, the shape selectivity between phenanthrene and anthracene in the C18-Au column was much higher than that in the ODS column (separation factors: 1.82 and 1.14, respectively). The relationship between the retention factor and acetonitrile content in the mobile phase revealed that the retention behaviors in the C18-Au column was more sensitive on the acetonitrile content than those in the ODS column. Relatively higher affinity for phenanthrene and anthracene was commonly observed in all the three thiol-modified Au columns than that for the conventional ODS column, whereas separations of benzene and nitro- and chlorobenzenes were quite different among the three thiol-modified Au and ODS columns.     

A Comparative Study of Pentafluorophenyl and Octadecylsilane Columns in High‐throughput Profiling of Biological Fluids

In high‐throughput metabolomic profiling, chromatographic separation is crucial because a well‐performed chromatographic separation may reduce signal suppression from complex biological matrices and improve the discoverability of low‐abundance metabolites. We compared the performance of pentafluorophenyl (PFP )‐ and octadecylsilane (ODS )‐based columns in profiling biological fluids. Peak resolutions and consistencies were acquired using several reversed‐phase columns and were evaluated. Total and extracted ion chromatograms demonstrated that the PFP column achieved better analyte separations than the ODS column. Low relative standard deviations on peak areas and retention times (<10.2 and <0.9%, respectively) acquired using the PFP column evidenced the high reproducibility and consistency of this column. In our study, a PFP column was used for profiling metabolomes extracted from urine and serum samples. Metabolomic study revealed a metabolome difference in normal and overweight participants. In total, 26 lipid species were significantly perturbed and further identified. Choline‐containing lipids were the most abundant perturbed lipidome in overweight participants, followed by sphingolipids and various phospholipids. We recommend the use of PFP columns in high‐throughput metabolomic analysis to promote the development of basic biological and clinical research in the future.     

Selectivity comparisons of monolithic silica capillary columns modified with poly(octadecyl methacrylate) and octadecyl moieties for halogenated compounds in reversed-phase liquid chromatography

Stationary phase selectivities for halogenated compounds in reversed-phase HPLC were compared using C18 monolithic silica capillary columns modified with poly(octadecyl methacrylate) (ODM) and octadecyl moieties (ODS). The preferential retention of halogenated benzenes on ODM was observed in methanol/water and acetonitrile/water mobile phases. In selectivity comparison of selected analytes on ODM and ODS, greater selectivities for halogenated compounds were obtained with respect to alkylbenzenes on an ODM column, while similar selectivities were observed with a homologous series of alkylbenzenes on ODM and ODS columns. These data can be explained by greater dispersive interactions by more densely packed octadecyl groups on the ODM polymer coated column together with the contribution of carbonyl groups in ODM side chains. For the positional isomeric separation of dihalogenated benzenes (ortho-, meta-, para-), the ODM column also provided better separation of these isomers for the adjacently eluted isomers that cannot be completely separated on an ODS column in the same mobile phase. These results imply that the ODM column can be used as a better alternative to the ODS column for the separation of other halogenated compounds.     

Selectivity enhancement for the separation of tocopherols and steroids by integration of highly ordered weak interaction sites along the polymer main chain

A novel alternating copolymer-based organic phase was synthesized using a new N-substituted maleimide monomer for the development of alternating copolymer-grafted silica for high-performance liquid chromatographic applications. This new monomer (DGMI) was copolymerized with octadecyl acrylate (ODA) from 3-mercaptopropyltrimethoxysilane-grafted silica to produce Sil-poly(ODA-alt-DGMI). The organic phase was characterized by the elemental analysis and the diffuse reflectance infrared Fourier transform spectroscopy. Tocopherol isomers and steroids were used as analytes for the evaluation of the chromatographic selectivity profiles of this novel stationary phase. The selectivity of this column was then compared with a polymeric ODS column and previously developed another alternating copolymer-grafted silica (without the glutamide-derived moiety) column, Sil-poly(ODA-alt-N-octadecylmaleimide). The complete baseline separation of tocopherol isomers in an isocratic mode has been achieved within 25 min with the Sil-poly(ODA-alt-DGMI). The separation of eight kinds of estrogenic steroids and corticoids has also been achieved in an isocratic mode with this column. Significant differences in separation selectivity between Sil-poly(ODA-alt-DGMI) and polymeric ODS columns were observed towards the steroids, and compared with the reference columns, a better separation profile for these analytes was obtained with the Sil-poly(ODA-alt-DGMI). The results of this investigation indicated that the enhancement of selectivity of Sil-poly(ODA-alt-DGMI) towards the test analytes arose from the multiple interaction mechanism such as hydrophobic effect, carbonyl-π and hydrogen-bonding interactions, and such integrated interactions originated from the addition of two amide groups in the N-substituted maleimide monomer.     

Packing and performance of microbore columns for adsorption and partition chromatography

Summary Microbore columns of 1 mm i.d. have turned out to be very suitable for the achievement of efficient columns.The packing procedure for stainless steel 1 mm i.d. columns from 5 to 100 cm in length was studied. Stationary phases used were: pure silica gel, octyl, octadecyl and amino bonded silicas. The main parameters (slurry composition, packing system, choice of materials) are discussed.Short columns packed with 3 or 5m particles allow high speed separations. A separation in 18 seconds is described.Very high plate numbers can be obtained with long columns. With 7–8m particles, a 1 m column can produce 50,000 plates (h=3). Columns can be joined without loss of efficiency. 270 000 theoretical plates were obtained on a 6 m adsorption column with a test mixture. In reversed-phase chromatography, bile acid sodium salts can be separated on a 1 m column. In adsorption chromatography, details are given of the separation of a polystyrene oligomer sample, as well as a light and a heavy petroleum distillate samples on a 2 m column with refractive index detection in the last-case.     

Preparative high-performance liquid chromatography with reversed phase packed glass columns

An efficient system for preparative reversed-phase separations with packed glass columns is described. The advantage of this system is the use of relatively simple and inexpensive equipment. Column performance, load capacity, effect of the feed volume and the feed concentration on peak broadening are shown. The influence of the selectivity and the capacity factors on column load have been measured. The effect of the column dimensions is demonstrated by means of practical applications. The loading capacity of a column depends on the thermodynamic proporties of the separation system used. It is therefore not expedient in preparative chromatography to correlate the loading capacity of a column by means of grams dissolved per grams of adsorbent.     

Elevated temperature-extended column length conventional liquid chromatography to increase peak capacity for the analysis of tryptic digests

High efficiency separations (200 000 plates) were obtained on conventional LC equipment by coupling 8 x 25 cm x 2.1 (or 4.6) mm id x 5 microm d(p) ODS columns (total length 2 m) and operation at 60 degrees C using a dedicated LC oven. The peak capacity in this 1-D set-up was 900 for the separation of human serum tryptic peptides analyzed after depletion of six highly abundant proteins. The chromatographic performance of an elevated temperature-extended column length conventional LC is highlighted.     

Comparison of Octadecyl-Bonded Alumina and Other Stationary Phases for Lipophilicity Estimation by High Performance Liquid Chromatography

Abstract

A recently developed octadecyl-bonded alumina stationary phase (ODA) was evaluated for determining the lipophilicities of organic compounds by high performance liquid chromatography. Using a column packed with this material and mobile phases consisting of methanol and aqueous buffers, the correlation between the octanol-water partition coefficients (log P's) of compounds of various chemical classes and the logarithms of their chromatographic capacity factors (log k's) was found to be superior to that obtained using columns packed with octadecylsilica, poly butadiene-coated alumina or octadecyl-derivanzed polystyrene-divinylbenzene copolymer. In contrast to results obtained with other columns, phenols and other hydrogen-bonding compounds did not need to be treated as a separate data set on the ODA column to obtain good correlations between log k's and log P's. The resistance of ODA to degradation by alkaline solvents allowed the use of a basic mobile phase (pH > 10) for suppressing ionization and determining the lipophilicities of organic bases which could not be evaluated within the stable pH range of ODS (pH 2–8). The log P's of five basic pharmaceutical compounds determined in this manner were found to be significantly higher than previously reported values. Evidence is presented which indicates that the previously reported log P values of these compounds are inaccurate, due to improper correction for ionization.     

High performance liquid chromatographic separations of gas oil samples and their hydrotreated products using commercial normal phases

Three commercially available high performance liquid chromatography columns are used in normal phase or quasi-normal phase mode for the separation of gas oil samples. The columns are tested with 20 analytical standards to determine their suitability for separations of petroleum samples and their ability to separate the nitrogen group-types (pyrrole and pyridine) found in petroleum. The columns studied are polymeric hypercrosslinked polystyrene (HGN), a biphenyl phase, and a Chromegabond "DNAP" column from ES Industries. The HGN column separates gas oils based on both ring structure and heteroatom, while the biphenyl phase has low retention of most compounds studied in quasi-normal phase mode. The "DNAP" column is selective for nitrogen-containing compounds, separating them from PAHs as well as oxygen and sulphur compounds. Retention data of standards on all three columns is shown, along with chromatograms of gas oil samples on the HGN and "DNAP" columns.