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PubMed2005

Orthogonal method development using hydrophilic interaction chromatography and reversed-phase high-performance liquid chromatography for the determination of pharmaceuticals and impurities.

A hydrophilic interaction chromatography (HILIC) method has been developed and validated as a secondary or orthogonal method complementary to a reversed-phase HPLC (RP-HPLC) method for quantitation of a polar active pharmaceutical ingredient and its three degradation products. The HILIC method uses a diol column and a mobile phase consisting of acetonitrile/water and ammonium chloride. The compounds of interest show significant differences in retention behaviors with the two very different chromatographic systems, which are desired in developing orthogonal methods. The HILIC method is validated and has met all validation acceptance criteria for the support of drug development activities.

Acetonitriles↗

PubMed2005

Multidimensional gas chromatography for the detailed PIONA analysis of heavy naphtha: hyphenation of an olefin trap to comprehensive two-dimensional gas chromatography.

A multidimensional method providing the composition of a heavy naphtha in paraffins, isoparaffins, olefins, naphthenes, and aromatics (PIONA) in the C8-C14 range is presented. The analytical set-up consists in a silver modified silica olefin trap on-line coupled to comprehensive two-dimensional gas chromatography (GC x GC). In this configuration, hydrocarbons are separated, in gaseous state, in two fractions, saturate and unsaturate, each fraction being subsequently analysed by GC or by GC x GC. The resolution between saturates and olefins was found to be improved compared to a single GC x GC run. The characterisation of the olefin trap highlights the benefits and the limits related to the use of that stationary phase as a double bond selective fractionation medium.

Alkanes↗

PubMed2005

Critical contribution of nonlinear chromatography to the understanding of retention mechanism in reversed-phase liquid chromatography.

The retention of most compounds in RPLC proceeds through a combination of several independent mechanisms. We review a series of recent studies made on the behavior of several commercial C18-bonded stationary phases and of the complex, mixed retention mechanisms that were observed in RPLC. These studies are essentially based on the acquisition of adsorption isotherm data, on the modeling, and on the interpretation of these data. Because linear chromatography deals only with the initial slope of the global, overall, or apparent isotherm, it is unable fully to describe the complete adsorption mechanism. It cannot even afford clues as to the existence of several overlaid retention mechanisms. More specifically, it cannot account for the consequences of the surface heterogeneity of the packing material. The acquisition of equilibrium data in a wide concentration range is required for this purpose. Frontal analysis (FA) of selected probes gives data that can be modeled into equilibrium isotherms of these probes and that can also be used to calculate their adsorption or affinity energy distribution (AED). The combination of these data, the detailed study of the best constants of the isotherm model, the determination of the influence of experimental parameters (e.g., buffer pH and pI, temperature) on the isotherm constants provide important clues regarding the heterogeneity of the adsorbent surface and the main properties of the adsorption mechanisms. The comparison of similar data obtained for the adsorption of neutral and ionizable compounds, treated with the same approach, and the investigation of the influence on the thermodynamics of phase equilibrium of the experimental conditions (temperature, average pressure, mobile phase composition, nature of the organic modifier, and, for ionizable compounds, of the ionic strength, the nature, the concentration of the buffer, and its pH) brings further information. This review provides original conclusions regarding retention mechanisms in RPLC.

Adsorption↗

PubMed2006

Optimization of elution salt concentration in stepwise elution of protein chromatography using linear gradient elution data. Reducing residual protein A by cation-exchange chromatography in monoclonal antibody purification.

Our simple method for optimization of the elution salt concentration in stepwise elution was applied to the actual protein separation system, which involves several difficulties such as detection of the target. As a model separation system, reducing residual protein A by cation-exchange chromatography in human monoclonal antibody (hMab) purification was chosen. We carried out linear gradient elution experiments and obtained the data for the peak salt concentration of hMab and residual protein A, respectively. An enzyme-linked immunosorbent assay was applied to the measurement of the residual protein A. From these data, we calculated the distribution coefficient of the hMab and the residual protein A as a function of salt concentration. The optimal salt concentration of stepwise elution to reduce the residual protein A from the hMab was determined based on the relationship between the distribution coefficient and the salt concentration. Using the optimized condition, we successfully performed the separation, resulting in high recovery of hMab and the elimination of residual protein A.

Antibodies, Monoclonal↗

PubMed2006

Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase liquid chromatography.

A new approach to high speed, comprehensive online dual gradient elution 2DLC (LCxLC) based on the use of ultra-fast, high temperature gradient elution reversed phase chromatography is described. Entirely conventional gradient elution instrumentation and columns are assembled in a system which develops a total peak capacity of about 900 in 25 min; this is equivalent to roughly one peak/2 s. Each second dimension gradient is done in a cycle time of 21 s and the peak retention times measured for a set of twenty six indole-3-acetic acid (IAA) derivatives are reproducible to 0.2 s. Each peak eluting from the first dimension column is sampled at least twice across its width, as the corresponding peaks on the second dimension column appear in two or three consecutive second dimension chromatograms, clearly indicating that there is little loss in the resolution gained in the first dimension separation. Application to the separation of the low molecular weight components of wild-type and mutant maize seedlings indicates the presence of about 100 peaks on a timescale of 25 min. Compelling illustrations of the analytical potential of fast, high temperature 2DLC are evident in the clear presence of nine distinct peaks in a single second dimension chromatogram from a single quite narrow first dimension peak, and the great power of 2DLC to solve the "analytic dynamic range" problem inherent in the measurement of small peaks that are neighbors to a gigantic peak.

Chromatography, High Pressure Liquid↗

PubMed2006

On-fibre solid-phase microextraction coupled to conventional liquid chromatography versus in-tube solid-phase microextraction coupled to capillary liquid chromatography for the screening analysis of triazines in water samples.

This paper compares the advantages and disadvantages of two different configurations for the extraction of triazines from water samples: (1) on-fibre solid-phase microextraction (SPME) coupled to conventional liquid chromatography (LC); and (2) in-tube SPME coupled to capillary LC. In-tube SPME has been effected either with a packed column or with an open capillary column. A critical evaluation of the main parameters affecting the performance of each method has been carried out in order to select the most suitable approach according to the requirements of the analysis. In the on-fibre SPME configuration the fibre coating was polydimethylsiloxane (PDMS)-divinylbenzene (DVB). The limits of detection (LODs) obtained with this approach under the optimized extraction and desorption conditions were between 25 and 125 microg/L. The in-tube SPME approach with a C18 packed column (35 mm x 0.5 mm I.D., 5 microm particle size) connected to a switching micro-valve provided the best sensitivity; under such configuration the LODs were between 0.025 and 0.5 microg/L. The in-tube SPME approach with an open capillary column coated with PDMS (30 cm x 0.25 mm I.D., 0.25 microm of thickness coating) connected to the injection valve provided LODs between 0.1 and 0.5 microg/L. In all configurations UV detection at 230 nm was used. Atrazine, simazine, propazine, ametryn, prometryn and terbutryn were selected as model compounds.

Chromatography, Liquid↗

PubMed2006

Application of polymer based stationary phases in high performance liquid chromatography and capillary high performance liquid chromatography hyphenated to microcoil 1H nuclear magnetic resonance spectroscopy.

The increased demand for chromatographic materials that are able to achieve a fast separation of large quantities of structure analogues is a great challenge. It is known that polymer based chromatographic materials have a higher loadability, compared to silica based sorbents. Unfortunately these polymer materials cannot be used under high pressure which is necessary in order to obtain high flow rates, and hence long times are needed to perform a separation. However, by immobilizing a polymer on a mechanically stable porous silica core, this problem can be circumvented and higher flows become feasible on these materials. Especially for capillary liquid chromatography hyphenated with nuclear magnetic resonance a high loadability is of great importance in order to obtain sharp, resolved, and concentrated peaks thus resulting in a good signal to noise ratio in the NMR experiment. Therefore, a highly shape selective chromatographic sorbent was developed by covalently immobilizing a poly(ethylene-co-acrylic) acid copolymer (-CH(2)CH(2)-)(x)[CH(2)CH(CO(2)H)-](y) (x=119, y=2.4) with a mass fraction of acrylic acid of 5% as stationary phase on silica via a spacer molecule (3-glycidoxypropyltrimethoxysilane). First, the loadability of this sorbent compared to C(30) is demonstrated by the HPLC separation of two xanthophyll isomers. Subsequently, it has been successfully employed in the hyphenation of capillary HPLC with microcoil (1)H NMR spectroscopy by separating and identifying a highly concentrated solution of the tocopherol homologues.

Acrylic Resins↗

PubMed2000

Simultaneous separation and enantioseparation of thalidomide and its hydroxylated metabolites using high-performance liquid chromatography in common-size columns, capillary liquid chromatography and nonaqueous capillary electrochromatography.

The separation of thalidomide (TD) and its hydroxylated metabolites including their simultaneous enantioseparation was studied using three different polysaccharide-type chiral stationary phases (CSPs) in combination with polar organic mobile phases. Three different techniques, high-performance liquid chromatography in common-size columns, capillary LC and nonaqueous capillary electrochromatography were compared in terms of separation. As this study illustrates, polar organic mobile phases represent a valuable extension for less polar and polar aqueous-organic mobile phases in combination with polysaccharide CSPs. Chiralpak AD consisting of 25% of amylose-tris(3,5-dimethylphenylcarbamate) coated on wide-pore aminopropylsilanized silica gel exhibited higher resolving ability compared to the similar cellulose derivative (Chiralcel OD) as well as to cellulose-tris(4-methylbenzoate) (Chiralcel OJ) CSPs for this particular set of chiral analytes. Baseline separation and simultaneous enantioseparation of all three compounds could be achieved under optimized separation conditions.

Chromatography, High Pressure Liquid↗

PubMed2000

Hold-up time in gas chromatography. V. Dependence of the retention of n-alkanes on the chromatographic variables in isothermal gas chromatography.

The chromatographic behaviour of n-alkanes and other homologous series in isothermal gas chromatography has been shown to depart from the "linear" representation of the logarithms of the adjusted retention times vs. carbon number. One of the expressions proposed to describe this behaviour is tR(z)=A+exp(B+CzD). In this paper, a regression analysis shows that three of the parameters of the equation depend on different chromatographic variables such as hold-up time, average linear gas velocity, volume and polarity of the stationary phase and temperature of the column. The fourth parameter (D), responsible for the departure from the "linearity", does not depend on any chromatographic variable, and represents a gradual decrease of the contribution of the methylene groups to the general properties of n-alkanes, with no relation to the chromatographic phenomenon.

Alkanes↗

PubMed2000

Analysis of anthocyanins in foods by liquid chromatography, liquid chromatography-mass spectrometry and capillary electrophoresis.

This article reviews recent developments in the methodology for the measurement of anthocyanins that offer several advantages over classical methods of analysis. The use of UV-diode array and mass spectrometric (MS) detectors, with improved methods of liquid chromatography analysis has facilitated identification of these analytes. The use of capillary electrophoresis (CE) analysis of the anthocyanins under acid conditions has significantly increased peak resolution and improved the detection limits by several orders of magnitude. CE offers the advantage of economies of very small sample size, very small solvent consumption, and short analysis times along with the future possibility of being combined with MS detection.

Anthocyanins↗

PubMed2000

Determination of chlorophacinone and diphacinone in commercial rodenticides by liquid chromatography-UV detection and liquid chromatography-electrospray ionization mass spectrometry.

This work describes a simple method for the determination and mass-spectrometric confirmation of the indanediones in commercial rodenticides. The sample is sonicated in methanol containing 2% formic acid and analyzed by liquid chromatography-UV detection. Once retention time and UV-Vis spectrum provide tentative identification, mass-spectrometric confirmation is obtained by analyzing a second aliquot by LC and electrospray ionization mass spectrometry (LC-ESI-MS). The extensive fragmentation of the indanedione molecule under MS/MS conditions provides sufficient structural information for positive identification on analyte levels as low as 20 ng on column.

Chromatography, Liquid↗

PubMed2000

Characterization of odorant compounds of mussels (Mytilus edulis) according to their origin using gas chromatography-olfactometry and gas chromatography-mass spectrometry.

Gas chromatography-olfactometry consists of sniffing the effluent of a gas chromatograph and leads to the direct determination of potent odorants in food. GC-olfactometry and GC-MS were applied in order to identify volatile compounds, and to characterize potent odorants of cooked wild mussels and bouchot mussels. Eighty-five volatiles were identified by GC-MS, among those the majority were identified for the first time in mussels. Using GC-olfactometry, the main contributors of cooked mussels aroma were characterized. Of the 85 volatiles identified in the flavor, only 33 were odor-active and contribute to the overall aroma of mussels. Dimethyl disulfide (sulfury odor) was the odorant the most differently perceived between the two extracts and seems to be characteristic of wild mussels. Combined GC-MS and GC-olfactometry made it possible to point out odorants which actually contribute to the aroma of cooked mussels and those which showed typical dependence on the origin of mussels.

Animals↗

PubMed2000

Observation of exchangeable protons by high-performance liquid chromatography-nuclear magnetic resonance spectroscopy and high-performance liquid chromatography-electrospray ionization mass spectrometry: a useful tool for the hyphenated analysis of natural products.

The first high-performance liquid chromatography-nuclear magnetic resonance (HPLC-NMR) investigation of exchangeable protons of low-molecular-mass natural products is reported. Model alkaloids or crude plant extracts were dissolved in 2H2O-1H2O-MeCN (deuterium oxide-water-acetonitrile) or 2H2O-MeCN and, after direct injection or chromatographic separation, examined in a 60-microl NMR flow probe. Exchangeable amino protons initially detected by HPLC-electrospray ionization mass spectrometry were subsequently identified and investigated by stop-flow 1H-NMR, two-dimensional (2D) total correlation spectroscopy (TOCSY), and 2D nuclear Overhauser effect spectroscopy (NOESY). These experiments extend the applicability of HPLC-NMR for the investigation and structure elucidation of natural products.

Biological Products↗

PubMed1988

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXVIII. Calculation of the average distance between protein solutes and the stationary phase during isocratic anion-exchange chromatography.

This investigation deals with protein retention behaviour in high-performance anion-exchange chromatography in terms of the average distance of approach between the protein solute and the positively charged anion-exchange stationary-phase surface. The theoretical treatment is based on a modified Debye-Hückel theory for spherical impenetrable ions, where the electrostatic potential energy has been related to the chromatographic capacity factor, k'. Results are presented for three globular proteins, eluted isocratically from a Mono-Q strong anion-exchange resin with sodium chloride as the displacer salt by a mobile phase with pH in the range 5.50-9.60. Analysis of experimental retention data indicates that topographically predefined, charged regions on the protein surface, called ionotopes, control the orientation and approach distance of the protein solute.

Amino Acids↗

PubMed1978

High-pressure liquid chromatography of amino acids, peptides and proteins. V. Separation of thyroidal iodo-amino acids by hydrophilic ion-paired reversed-phase high-performance liquid chromatography.

The separation of thyroidal iodoamino acids has been carried out by high-performance liquid chromatography in phase systems consisting of chemically bonded C18-hydrophobic supports as the stationary phase and water-organic solvent mixtures containing phosphoric acid or other ion-pairing reagents as the mobile phase. Under conditions of hydrophilic ion-pair formation, excellent resolution of the iodoamino acids is observed. This method permits the rapid separation and, hence, analysis of mixtures containing thyroxine, 3,3',5-triiodothyronine and 3,3',5'-triiodothyronine and related compounds in ca. 30 min with sensitivity, using a UV monitor at 210 nm, at the 1-10-pmole level.

Chromatography, High Pressure Liquid↗

PubMed1985

Microbore reversed-phase chromatography of proteins with conventional gradient equipment for high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography with microbore columns (50 X 1.0 mm) was used effectively for the separation and analysis of proteins down to 1 ng at flow-rates of 0.1-0.2 ml/min. With the use of standard low-pressure gradient HPLC equipment, the peak volumes were five times smaller when compared with a conventional column at equal chromatographic efficiencies and analysis time. The sensitivity of detection was further increased by a reduction in solvent peaks, resulting in a 20-fold overall increase.

Acetonitriles↗

PubMed1975

Improvements in the thin-layer chromatography of natural products. I. Thin-layer chromatography of the aflatoxins.

During the period which has elapsed since the aflatoxins were first isolated, one of the main problems has been the separation of the individual aflatoxins in pure form from aflatoxin-containing extracts. This separation has been best effected by thin-layer chromatography, and in this paper we describe how some of the difficulties may be overcome by using an appropriate combination of solvent system and silica gel preparation. For the examination of aflatoxin-containing extracts from the mycelia of Aspergillus flavus moulds, an initial freeze-drying step has been found to improve appreciably the quality of the chromatograms obtained.

Acetates↗

PubMed2001

Affinity chromatography of polyhistidine tagged enzymes. New dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions.

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta-galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein, on these IMAC supports via multipoint weak adsorptions; (b) the multimeric poly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme from the support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are now adsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins (80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface.

Adsorption↗