Isocratic Separation of Fatty Acid Derivatives by Reversed Phase Liquid Chromatography. Influence of the Solvent on Selectivity and Rules for Elution Order

Abstract

The p-bromophenacyl esters of 16 fatty acids (C₁₂-C₂₂) have been separated by isocratic chromatography on a Radial Pak A cartridge (Reverse phase C₁₈ material). The separation factors α were measured using two solvent mixtures of comparable strength and the superiority of methanol-water to acetonitrile-water becomes evident.

Five precise rules are established, which indicates the retention of every fatty acid. They explain the chromatographic process i.e. elution order, resolution and selectivity.     

Reversed Phase Thin Layer Chromatography of Amino Acids

Abstract

The use of reversed phase layers for the thin layer chromatography of amino acids is described. Only when a modifier was added to the mobile phase was clear separation of the amino acids achieved. Ion paring with trifluoroacetic acid overcame problems with streaking and poor separation on C₂ or C₁₈ reversed phase layers. All amino acids could not be separated with a single mobile phase. Thus, three different combination of acetonitrile-0.4% trifluoroacetic acid were used to separate eighteen amino acids with derivatization. No derivative was required.     

Simple Free Amino Acid Separation by Reversed-Phase Ion-Pair Liquid Chromatography Using Column Switching Technique

Abstract

Reversed-phase ion-pair liquid chromatography with copper (II) ions (RP-IPC-Cu) was applied to develop a high speed separation of free amino acids. Dynamic gradient elution in RP-IPC-Cu could not achieve this purpose due to base line shift, therefore a column switching technique was used with a combination of different alkyl-bonded silica gel columns under isocratic elution. The flexibility of eluent components makes it easy to realize a rapid separation of a mixture of targeted amino acids.     

High Resolution Separation of Urinary Organic Acids by High Performance Liquid Chromatography

Abstract

Reverse phase, anion exchange, and two-dimensional HPLC techniques were studied in order to increase resolution of organic urinary acids for eventual quantitative measurements. Reverse phase HPLC with a phosphate buffer/acetonitrile gradient yielded a separation of over 85 components in forty minutes and a peak area reproducibility of better than 5%. Connecting two reverse phase columns together resulted in the separation of 110 components. Anion exchange chromatography was determined to be of little use in resolving urinary acids in a resonable time except as the first stage in two-dimensional chromatography where fractions from the anion exchange column were injected into a reverse phase column. Over 139 components were separated by this two-dimensional method.     

Simultaneous Determination of Maprotiline,Desipramine, and Moclobemide by Reversed-Phase High-Performance Liquid Chromatography and Statistical Optimization

Abstract

A method for separation of three antidepressants, maprotiline, desipramine, and moclobemide, by reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and validated. To find optimal conditions and estimate the impact of individual parameters on the separation, a complete set of 2³ interdependent relationships of the mobile phase composition, temperature, and the volume flow rate were examined. Full separation of the investigated components from a laboratory mixture was achieved on a Supelcosil LC-18 (120 mm × 4.6 mm, 5 µm) column, using two solvent systems, 3% ammonium ion in water/ethanol and acetonitrile, and alternating isocratic gradient–isocratic elution modes. Relevance of the proposed method for therapeutic drug monitoring is anticipated.     

Analytical Separations of Schiff Base Chelates by Reverse Phase Hplc on a 10 Micron C-18 Bonded Silica Subtrate

Abstract

Reverse phase partition chromatography on a 10 micron silica-bonded octadecyl hydrocarbon substrate is applied to the separation of neutral tetradentate chelates of copper (II), nickel (II) and palladium (II) with a range of non-fluorinated and fluorinated beta-ketoamine and salicylaldimine ligands. Variation of retention and resolution with respect to changes in ligand structure and metal chelated is investigated using acetonitrile, methanol and water solvent systems. Detection limits (254 nm) and linearity ranges of response are indicated.     

Ruthenium (II) 1,10-Phenanthroline Salts as Mobile Phase Additives for the Separation and Indirect Detection of Free Amino Acids

Abstract

Ruthenium (II) 1,10-phenanthroline, Ru(phen)²⁺ ₃, salts are used as ion interaction reagents in a basic mobile phase for the retention, resolution, and indirect photometric detection (IPD) of free amino acids on a polystyrene divinylbenzene (Hamilton PRP-1) column. Mobile phase Ru(phen)²⁺ ₃ concentration and pH and type and concentration of organic modifier and counteranion affect retention and IPD. Underivatized amino acid elution order is influenced by side chain structure typical of ion exchange processes. Detection limits for the separation and detection of free amino acids using an isocratic elution condition are about 0.1 nmole for lower retained amino acids and 0.25 nmole for higher retained amino acids for a 3:1 signal:noise ratio. Gradient elution is possible but at higher detection limits.     

Effect of Temperature and Mobile Phase Composition on RP-HPLC Separation of Cephalosporins

Abstract

The effects of mobile phase composition and column temperature on resolution in reversed-phase high performance liquid chromatography (RP-HPLC) were used to separate the following cephalosporins: cefonicid, cefaclor, cephazolin, cefodizime, cephaloridine, cephamandole and cephalotin, from a single sample. The capacity factor (k') was described as a function of temperature and mobile phase composition. Semi-empirically estimated values of k' were determined using a small number of experimental data for different temperatures between 20°C and 60°C, and mobile phase compositions (acetate buffer/isopropanol). The capacity factor of each cephalosporin was observed to decrease with increasing temperature and the volume fraction (V) of isopropanol in the mobile phase; linear relationships were obtained for plots of In k' versus 1/T and log k' versus log V. The method developed, while simple, reveals the optimal isocratic elution conditions for column temperature and eluent concentration for the complete separation and rapid analysis of the cephalosporins studied.     

An Efficient Strategy for Optimization of Isocratic Mobile Phase Conditions for HPLC Separation of a Complex Mixture

Abstract

Chromatographic resolution of a complex mixture is often a trial and error process. An efficient strategy for the optimization of previously reported isocratic mobile phase conditions utilizes a factorial design and multivariate regression equations to model the dependence of elution time of each component on chromatographic parameters. Several parameters are varied simultaneously, with extreme and central values to allow interpolation in the modeling. Run order is scrambled to minimize time dependent errors. Reported here is the optimization of the separation of monoamine neurotransmitters and metabolites using isocratic reverse phase HPLC chromatography with serial oxidation and reduction electrochemical detection. The predicted retention times closely follow the experimental retention times for a set of conditions not included in the calculation of the model. The results enable the rational adjustment of parameters to meet subsequent chromatographic needs.     

Automated Analysis of O-Phthalal-Dehyde Derivatives of Amino Acids in Physiological Fluids by Reverse Phase High Performance Liquid Chromatography

Abstract

An automated method is described for the determination of free amino acids in biological fluids using precolumn deriva-tization with o-phthalaldehyde and reverse phase high performance liquid chromatography. Chromatographic separation of amino acids is accomplished in 24 minutes (cycle time 44 minutes). As little as 1.5 pmol of most commonly occurring amino acids can be accurately quantified. Accuracy and reproducibility are optimized by automating the derivatization-injection sequence and by correcting for variations in the fluorescence response of each amino acid in each run. A total of 31 analyses can be completed in 24 hours on a single column (7 standards and 24 unknowns). The method can be used in the general determination of free amino acids in biological fluids, or can be further accelerated and used for the quantitation of specific amino acids simply by altering the elution conditions.     

The Quantitative Separation of Alkyl and Phenethyl Halides by HPLC

Abstract

A method is described for the quantitative separation of alkyl and phenethyl halides in mixtures containing benzene or toluene. This method involves the isocratic chromatography separation of the mixture, using a ODS column and the detection of the compounds by means of a RI detector.     

Separation of Chlorinated Phenols by Isocratic High-Performance Liquid Chromatography on Reverse Phase Column

Abstract

A method is described for isocratic highperformance liquid chromatographic separation of chlorinated phenols using methanol : phosphate buffer - pH 7.20 (50:50) solvent system. 13 out of 15 congeners of chlorophenols and phenol have been separated in 32 minutes at a flow rate of 0.8 ml per minute. The system is found to be useful for the separation of chlorophenols extracted from mouse liver fed with hexachlorocyclohexane.     

Rapid Separation and Determination of Thyromimetic Iodoamino Acids by Gradient Elution Reverse Phase Liquid Chromatography with Electrochemical Detection

Abstract

The usefulness and capabilities of gradient elution techniques with electrochemical detection are shown with a rapid separation of D,L-tyrosine (Tyr), 3-iodo-L-tyrosine (MIT), 3,5-diiodo-L-tyrosine (DIT), D,L-thyronine (T₀), 3,5-diiodo-D,L-thyronine (T₂), 3,3′,5-triiodo-L-thyronine (T₃), and L-thyroxine (T₄) by reverse phase liquid chromatography. A rapid (five minute) isocratic separation of T₀, T₂, T₃, and T₄ is also reported. Limits of detection are in the sub-nanogram range with a linear dynamic range to 500 ng for T₂ and T₃, and 1000 ng for T₄. Analysis of levothyroxine sodium tablets and injectable intravenous samples is described.     

Reverse Phase HPLC Determination OF 5,6-Dihydro-5-azacytidine in Biological Fluids

Abstract

A sensitive and specific reverse phase HPLC method which allows measurement of the new antitumor agent 5,6-dihydro-5-azacytidine (DHAC) in biological fluids at concentrations as low as 50 ng/ml (2 × 10^?7 M) has been developed. After addition of 5′-chloro-5′-deoxy-5,6-dihydro-5-azacytidine as an internal standard, sequential ultrafiltration, boronate gel affinity chromatography and cation exchange chromatography are employed to isolate DHAC from plasma or urine. DHAC is then reacted with N,N-dimethylformamide diethylacetal to form a dimethylaminomethylene derivative with enhanced UV detectability (λmax = 264 nm, log ε = 4.3) and better retention on a reverse phase column. Isocratic separation is then accomplished on a fully loaded and end-capped ODS column with 0.050 M formic acid in 20% acetonitrile/water. This assay has been used to determine the plasma pharmacokinetics of DHAC in rats given a single i.v. bolus dose of 50 mg/kg. Analysis of the drug in human plasma indicates that this method is suitable for determining DHAC disposition and pharmacokinetics in human subjects.     

Ion Chromatographic Analysis of Sulfate and Nitrate in Ambient Aerosols

Abstract

Ion exchange has been known to provide excellent separation of ions since 1850, and ion exchange chromatography has been in use since 1940. However, ion exchange chromatography has not been widely used for the automated analysis of eluted ions because of the background produced by the electrolyte used for elution. H. Small, T. S. Stevens, and W. C. Bauman, (Anal. Chem., 47, 1801 (1975)) recently developed a technique whereby the background is reduced to a minimum with eluant suppression. Eluant suppression allows the use of ion exchange chromatography with conductimetric detection as a sensitive and selective means for the analysis of practically all ionic species. This communication describes the first successful application of ion chromatography (IC) to the analysis of total water soluble sulfate and nitrate in ambient aerosols. Analytical conditions and data on sensitivity, selectivity, accuracy and repeatability are described.

The application of this technique to the analysis of atniospheric anions and cations and their precursors is to be investigated in future studies.     

Rapid Separation of Testosterone and its Microsomal Metabolites by Reverse-Phase High Performance Liquid Chromatography

Abstract

High performance liquid chromatography has been used to separate testosterone from its oxidative metabolites (7α-, 16α -and 6 β-hydroxytestosterone and androstenedione) following in vitro incubation with rat liver microsomes. The separation was accomplished in less than 18 minutes on a radially compressed C₁₈ reverse-phase column using isocratic elution with tetra-hydrofuran: water (24/76, v/v).     

Reversed-Phase,Ion-Pair Separation of L-Methionine and L-Methionine Dipeptides Complexed with Palladium (II)

Abstract

A high performance liquid chromatographic (HPLC) method is described for separation of L-methionine and L- methionine dipeptides complexed with palladium (II). Under isocratic conditions, at room temperatures, with the appropriate selection of counter-ion (cetyltrimethylammonium or trioctylmethylammonium), it was possible by ion-pairing reversed phase chromatography to resolve the palladium (II) complexes studied. Stainless steel and polythylene columns were used. The chroma-tograms from both the two different materials made columns indicate about the same ratio of capacity factor of the palladium (II) complexes.     

Separation of Methylated Purines by High Pressure Liquid Chromatography

Abstract

This paper reports a method for the separation and measurement of methylated purines of interest to carcinogenesis studies by high-pressure liquid chromatography (HPLC) following their column chromatographic isolation from collected urine samples. HPLC was evaluated on three different cation-exchange columns, with optimum conditions obtained on a Partisil 10-SCX column employing isocratic elution with 0.25M (NH₄)H₂PO₄ at pH 4.0. This column was also found to be useful for the separation of mono-methylguanine isomers. Application is shown to the analysis of rat urine following animal treatment with methyl methanesulfonate.     

Application of Thin-Layer Chromatography to High-Performance Liquid Chromatographic Separation of Steroidal Hormones and Cephalosporin Antibiotics

Abstract

High-performance liquid chromatographic (HPLC) separation of steroidal hormones and cephalosporin antibiotics was investigated by adsorption chromatography and reversed-phase chromatography, respectively.

Prior to the HPLC separation of these pharmaceuticals, silica gel thin-layer adsorption chromatography of steroidal hormones and reversed-phase thin-layer partition chromatography of cephalosporin antibiotics with chemically bonded dimethylsilyl silica gel were performed in order to obtain suitable HPLC separation systems.

In the separation of steroidal hormones, the same binary mobile phase ratios of TLC did not give satisfactory results in HPLC. For the sharp separation in HPLC, solvent strength in the binary solvent mixture used for TLC had to be decreased.

The difference in solvent strength for efficient separation between TLC and HPLC might be attributed to the fact that in HPLC the solvent elution power acts in an isocratic manner while in TLC it acts in a gradient manner.

On the other hand, a correlation of mobility between TLC and HPLC separation for cephalosporin antibiotics was obtained, and the possibility of direct transfer of chromatographic systems from TLC to HPLC for separation of these antibiotics was confirmed.     

Buffer Choice for Tuning the Selectivity in Reverse Phase Chromatography

Abstract

Retention behaviour of ionogenic species in high-performance liquid chromatography on reversed phase materials was studied, specifically dependence of buffer quality applied to mobile phases. The buffers' effect on retention of organic acids, amino acids and dipeptides is quantified by modelling capacity factors as a function of pH-values. At constant ionic strength, increasing capacity factors were observed going from phosphate to less polar citrate buffer, modification of accessible silanol groups of the stationary phase being responsible for this effect. Application of citrate buffer for separation of a seven-component mixture is demonstrated on the basis of a computerized search for optimum chromatographic performance. The evaluated factor levels (pH, methanol content and ionic strength) differ from those found using phosphate buffer-containing mobile phases.