High Performance Liquid Chromatography Separations of Nitrosamines. I. Cyclic Nitrosamines

Abstract

The separation of cyclic nitros amines by HPLC was achieved using a cyclobond I column and a mobile phase of methanol/water. The effect of in-the-ring and on-the-ring substitutions on retention times were studied. The results show that the longer the chain of the substituent, the longer the retention time, which is consistent with the inclusion mechanism of separation. Also, the larger the ring, the longer the retention time. The results also show that the in-the-ring substitution has a dramatic effect on retention.     

Application of HPLC and Dual Wavelength Detection to the Analysis of Phenolic Compounds in Apples

Abstract

High-performance liquid chromatography, coupled with a programmable UV/visible detector, was applied to the identification and quantification of the phenolic compounds of apple tissue. The identity and purity of unknown components eluted from the HPLC column were established by comparing retention times and absorbance ratios with those of authentic compounds. In Golden Delicious apples, components with retention times agreeing with those of authentic catechin, epicatechin, chlorogenic acid, protocatechuic acid, p-hydroxybenzoic acid, p-coumaric acid, ferulic acid, and sinapic acid were found. However, comparison of absorbance ratios with those of authentic compounds suggested that many of the components that eluted in this system as single, symmetrical peaks were mixtures rather than individual compounds. This method helped to eliminate inaccurate identifications or faulty assumptions about homogeneity of HPLC “peaks”, errors that can occur when traditional HPLC methods that rely solely on detection at a single wavelength are employed. The method should have general applicability to plant extracts.     

Modeling of chromatographic retention of the selected antiarrhythmics and structurally related compounds in the hydrophilic interactions under the TLC and HPLC conditions

Abstract

High-performance liquid chromatography (HPLC) plays an important role in testing the pharmaceutically active compounds. In despite of the advantages of HPLC, thin-layer chromatography (TLC) retains its applicability to the different experimental tasks. The experimental conditions which allow hydrophilic interactions in the chromatographic system were tested in the HPLC and TLC systems for ivabradine, its related compounds, diltiazem and verapamil. Under the TLC conditions, retention behavior of the investigated compounds was tested on silica gel modified with cyanopropyl ligands as stationary phase and acetonitrile?+?methanol containing 25% v/v formic acid. Under the HPLC conditions, we used silica gel modified with cyanopropyl ligands as a column packing and the acetonitrile + 0.25% aqueous solution of formic acid as mobile phase. Retention behavior of the investigated analytes depending on the changing volume fractions of the mobile phase modifier was characterized both for TLC and HPLC data sets by the Soczewiński–Wachtmeister equation. Linear relationships were established between the retention coefficients characterizing the retention mechanism (R_M⁰/m, logk₀/m) and molecular properties of the investigated compounds. The Quantitative Structure Retention Relationship (QSRR) modeling was performed with the use of the stepwise multiple linear regression, in order to select molecular properties which influence retention.     

On the Determination of the Hold-Up Time in Reversed Phase Liquid Chromatography

Abstract

The determination of the hold-up time in reversed phase liquid chromatography has been studied extensively for the mobile phase system methanol-water. Hold-up times obtained by static methods, linearization of homologous series and so-called “unretained compounds” are discussed and mutually compared. Several n-alkyldimethylsilyl bonded phases have been used for this investigation.

A rough estimate of the hold-up time can be obtained by using components of the mobile phase or highly concentrated salt solutions, but only for mobile phase compositions around 60% (v/v) methanol. Hold-up times accurate to 1% can be obtained over the complete range of mobile phase compositions from the linearization of net retention times of homologous series.     

Molecular Modeling of Cyclodextrin-Guest Molecule Interactions

Abstract

Inclusion complexes formed with substituted aromatic compounds and ß-cyclodextrin were studied using molecular modeling techniques. Energies of the MM2 optimized structures were correlated with HPLC retention times. A direct correlation between the inclusion complex stability and the HPLC retention times for the bonded ß-cyclodextrin was found within a series of disubstituted aromatic compounds. Evidence for a secondary mechanism of separation was found.     

The Purification of Amino Coumarin Dyes by Reverse Phase High Performance Liquid Chromatography

Abstract

A reliable method for the separation of amino coumarin dyes from associated impurities has been developed using reverse phase high performance liquid chromatography using an ODS-18 column and mixtures of methanol and water. Further, the relation between the retention times and structure of amino coumarines has also been studied.     

High-Performance Liquid Chromatography of 2′-5′Oligoadenylates and Related Oligonucleotides

Abstract

HPLC has been used for the analysis and separation of the components of p_x (A2′p)_n A (x = 1 to 3, n = 1 to ≥4). Weak anion exchange columns give excellent resolution, but their instability in phosphate buffers makes them impractical for routine use. Reverse phase chromatography using C18 columns provides a satisfactory alternative method. For preliminary analysis of crude material, ammonium phosphate pH7.0 with a linear 1:1 methanol/H₂O gradient gives a good basic separation of the individual oligomers. Resolution of the 5′ mono-, di-and triphosphorylated oligomers or of the nonphosphorylated components can be obtained using ammonium phosphate pH6.0 and potassium phosphate pH6.5 buffers respectively. The C18 columns are very stable and any one column will give retention times reproducible within 0.2%.     

Control of Retention Times in the Ion-Paired liquid chromatographic analysis of Bilirubin

Abstract

Fluctuations in bilirubin levels are biologically significant and the development of simple, reliable, and flexible chromatographic analyses are required for many research fields. Bilirubin's short retention time using reverse phase HPLC combined with the presence of acidic groups on bilirubin suggests ion-paired chromatography would produce the desired analytical procedures. Chromatography using dibutylamine as the ion-pair reagent proved to be successful and the capacity factor of bilirubin was reproducibly controlled by the concentration of dibutylamine, pH, and per cent of methanol in the mobile phase. The results, analyzed by a multilinear regression analysis, produced a mathematical model which calculated the capacity factor based on the dibutylamine concentration, methanol concentration and the hydrogen ion concentration. The analytical system was flexible, reproducible and easy to use. Manipulation of these factors should allow the facile separation of bilirubin from other similar compounds.     

Electrochemically Controlled Liquid Chromatography on Conducting Polymer Stationary Phases

Abstract

Electrochemically controlled high performance liquid chromatography (EC/HPLC) using a conducting polymer stationary phase, has been investigated in the present paper. Polypyrrole coated reticulated vitreous carbon (RVC) particles have been employed as the stationary phase. Chromatographic characterization has been carried out using various molecular probes. The results indicate that chromatographic retention can be altered by the imposition of small electrical potentials on the stationary phase and that such effects can be used to improve the separation of certain compounds.     

Retention characteristics of porous graphitic carbon in subcritical fluid chromatography with carbon dioxide-methanol mobile phases

Numerous relationships usually used in high-performance liquid chromatography (HPLC) for describing the retention on porous graphitic carbon (PGC) have been applied in subcritical fluid chromatography, with CO2-methanol mobile phases. As reported in HPLC, octanol-water partition coefficient failed to fit the retention, whereas satisfactory results were obtained with the sum of partial negative charges. A better fit was reached by using the solvation parameter model, allowing a better understanding of the interactions developed between the solute, the stationary and the mobile phases. Results show that the dominant contribution to retention was given by the polarizability (E) and the volume (V), while the hydrogen-bond basicity (B) was not selected in the retention model, whatever the methanol content. The increase in methanol percentage favours the retention decrease, mainly through the volume for hydrophobic compounds, and through the hydrogen-bond acidity for polar compounds.     

Reversed Phase Liquid Chromatographic Separation of Lysergic Acid Diethylamide (LSD) and Lysergic Acid Methylpropylamide (LAMPA)

Abstract

A fast and convenient procedure is described for the HPLC separation of LSD and LAMPA. These compounds are separated by a reversed phase (C18) procedure using a binary solvent system of methanol and pH 3 phosphate buffer. Under these conditions all compounds are eluted in a retention volume of 27 mL or less. This procedure allows a forensic sample to be identified as LSD and to eliminate LAMPA as a possibility.     

Optimization of a high-performance liquid chromatography method for the analysis of complex polyphenol mixtures and application for sainfoin extracts (Onobrychis viciifolia)

A pentafluorophenylpropyl (PFP) stationary phase was tested for the simultaneous determination of several classes of phenolic compounds. The chromatographic results were compared with those obtained by using a bifunctional phase constituted of octadecyl and phenylpropyl bonded silica and three conventional C18 columns. The elution gradient was optimized with 5% formic acid and sodium acetate in combination with acetic acid as additives and methanol as solvents. For these evaluations, a complex phenolic extract of Onobrychis viciifolia (sainfoin) and test mixtures containing 54 standard substances including 2 simple phenolic compounds, 1 amino acid, 4 hydroxybenzoic acids (HBA), 6 hydroxycinnamic acids (HCA), 3 flavan-3-ols, 9 anthocyanins, 2 dihydroflavonols, 1 chalcone, 4 flavones, 1 isoflavone and 21 flavonols have been assayed. The perfluorinated column showed good resolution for the studied phenolic compounds which have the following elution order: HBA, HCA, flavan-3-ols, anthocyanins, dihydroflavonols, flavones, flavonols and isoflavones. Compared with other columns, it provides longer elution ranges for HBA, HCA and flavan-3-ols and increased retention times for all compound classes except anthocyanins which were similarly retained on a C18 column. Its selectivity is different from C18 and bifunctional phases. A high-performance liquid chromatography (HPLC) method with diode array detection (DAD) and post-column derivatization with p-dimethyl-aminocinnamic aldehyde (DMACA) has been validated for the analysis of individual phenolic compounds from a sainfoin plant extract (O. viciifolia).     

Pharmaceutical Analysis for Environmental Samples: Individual and Simultaneous Determination of Ciprofloxacin,Ofloxacin and Norfloxacin Using an HPLC with Fluorescence and UV Detection with a Wetland Soil Matrix

Abstract

Two HPLC methods were developed for individual and simultaneous determination of ciprofloxacin, norfloxacin and ofloxacin for use in laboratory experiments producing large numbers of samples (100 s to 1000 s). Individual compound detection produced retention times between 1.5 and 2 min and simultaneous detection between 6.5 to 8 min. The methods are compatible with complex geomatrices, e.g. a wetland soil. These methods provide 1) detection limits in the low parts per-billion range; 2) decrease in retention times of 5–10 times for single compounds, and up to 2 times for simultaneous detection over published methods; and 3) require no solid phase extraction.     

Reversed-Phase High Performance Liquid Chromatography (HPLC) of Nucleosides in Cell Extracts with Special Reference to Deoxythymidine

Abstract

A reversed-phase high performance liquid chromatography (HPLC) system was designed to assay the nucleoside concentrations, especially deoxythymidine, of tissue cultured cells. Concentration and purification of the acid extracted cell samples were achieved by utilizing a Sep-Pak C₁₈ cartridge. After adsorption of the sample, the cartridge was washed with 0.5 ml of water followed by 2 × 0.5 ml of 2.5% methanol. The compounds of interest were subsequently eluted in 2 × 0.5 ml of methanol. The cartridge procedure was found to be fast, inexpensive and showed good recoveries for most tested nucleosides. The nucleosides uridine, deoxythymidine and adenosine could be detected in green monkey kidney cells. Ribonucleotides and deoxyribonucleotides could be separated from each other with the HPLC system used.     

Sample Solvent as Analyte in High Performance Liquid Chromatography

Abstract

Solvents vary in their behavior in high performance liquid chromatography (HPLC). Water and methanol, among others, are widely used in the mobile phase as well as solvents for the solute. Few reports indicate that the solvent used for the solute can behave as an analyte. Normally, it is generally accepted that the solute solvent, a non-constituent of the mobile phase will be the first eluent. However, a solvent which is a component of the sample can show up as an unexpected peak with its own identity. This solvent may show a similar retention time as some of the unknown components of the sample. This indicates that in some cases the quantitative results may be the sum of the absorptivity of the solute and solvent used for the sample. It is assumed that some solvents show no absorption in the ultraviolet region at which the analysis is being conducted. Depending on the mobile phase composition some solvents can be detected at the wavelengts or wavelengths used for analysis. Water, ethylacetate, and methanol showed absorption at 210 nm when present in the sample being analyzed with a mobile phase of acetonitrile-methanol using a C₁₈ column. These solvents overlapped or showed retention times the same as estriol and testosterone.     

Liquid Chromatographic Analysis of Some N-Alkyl-3,4-methylenedioxyamphetamines

Abstract

The liquid chromatographic separation of a series of low molecular weight N-alkyl derivatives of 3,4-methylenedioxyamphetamine (MDA) is reported. The N-methyl-, N-ethyl- and N,N-dimethyl-derivatives of MDA have appeared as street drugs in recent years. These compounds are becoming significant forensic problems due to their unique pharmacological properties and relative ease of chemical synthesis. These amines were synthesized via reductive amination of the corresponding ketone with alkylamines. The UV absorption spectra for these compounds produced almost equally intense absorbance at 234 and 285 nm. The compounds were separated by reversed-phase (C₁₈) HPLC procedures using a mobile phase of aqueous methanol at low pH.     

Large Volume Injection of Biological Samples Dissolved in a Non-Eluting Solvent: A Way to Increase Sensitivity and a Means of Automating Drug Determination Using Hplc

Abstract

The injected volume of a sample dissolved in the mobile phase of an HPLC system must be maintained as small as possible so as to minimize the loss in efficiency. Generally this requirement limits the sensitivity of HPLC methods devoted to trace quantity determinations of drugs in biological fluids. In order to avoid this limitation and to increase the effective sensitivity of HPLC methods for determination of drugs such as antrafenine, nifuroxazide and cipropride, the samples were dissolved in a non-eluting solvent and a large volume (> 100 μl) was injected on to the chromatographic column.

The above-mentioned compounds and their internal standards were dissolved in a series of eluting and non-eluting solvents and increasing volumes (5 to 1000 μl) were injected. Peaks corresponding to injections made in an eluting solvent showed retention times independent of the injection volume but their variances increased with the volume injected. In contrast, peaks corresponding to injections made in a non-eluting solvent, similar to the mobile phase, had a variance independent of the injection volume but their retention times increased linearly with the injection volume. The repeated injection of such non-eluting solvents had no influence on chromatographic behaviour. Peaks corresponding to compounds injected in a non-eluting solvent made with components different from those of the mobile phase had a variance independent of the injection volume but their retention times varied both with the injection volume and with the interval between injection.

The application of non-eluting solvents has been defined theoretically and it has been demonstrated that solutions composed of 25% of the mobile phase diluted with the least eluting of its components act as non-eluting solvents and can be injected in large volume without loss in efficiency. This feature could be used to inject all the samples volume or only part of it, manually or automatically, since any automatic injector can be used with large volumes.

Thus, using the relatively simple procedure of making injections with a non-eluting solvent it is possible to increase both sensitivity and the rate of sample analysis.     

The Influence of Reversed-Phase n-Alkyl Chain Length on Protein Retention,Resolution and Recovery: Implications for Preparative HPLC

Abstract

High-performance liquid chromatography (HPLC) of proteins on reversed-phase columns of varying n-alkyl chain length (C₂ to C₂₂ was studied using a trifluoroacetic acid/2-propanol mobile phase system. Protein resolution was influenced by chain length but retention times for proteins, unlike those of small molecules, were relatively constant, independent of chain length or carbon loading. Loading capacities were found to be affected by chain length, and aspects of protein interaction with stationary phase are discussed.     

Optimization of HPLC Analysis of Biogenic Amines with Reference to Molecular Structure

Abstract

The selectivity and molecular connectivity indices (x) up to the sixth order were calculated and compared with measured reversed-phase high pressure liquid chromatographic (HPLC) retention data for biogenic amines common in foodstuffs and animal feed. The separation of seventeen dansylated amines including primary, secondary and polyamines, was investigated using an isoselective multisolvent gradient (IMGE), reversed-phase HPLC method. The mobile phase was optimized with the “PRISMA” model testing thirteen selectivity points. The compounds were divided into two groups according to their retention; the low-order valence level indices best described the retention. As the high correlations between the calculated and observed retention indicate, retention could be predicted in different selectivity points with a high degree of accuracy by the molecular connectivity indices.