The Use of an α-Cyclodextrin Mobile Phase in the Thin-Layer Chromatographic Separation of Ortho,Meta, and Para Substituted Phenols

Abstract

An aqueous solution of α-cyclodextrin (cyclohexaamylose) is demonstrated to be a very effective mobile phase in thin-layer chromatographic separations. The chromatographic behaviors of twenty-six substituted phenolic and naphtholic compounds using polyamide thin-layer stationary sheets are described. The R_f values were found to be dependent upon both the structural features of the phenolic compounds and the concentration of α-cyclodextrin in the mobile phase. A possible mechanism that accounts for the observed chromatographic behavior is presented. The advantages and disadvantages of the aqueous α-cyclodextrin mobile phase over the traditional pure or mixed organic solvent systems typically employed are discussed.     

Isocratic HPLC Separation of Scopoletin and Cis/Trans Isomers of Ferulic Acid as Well as Isoscopoletin and Cis/Trans Isomers of Isoferulic Acid

Abstract

Cis/trans isomers of ferulic and isoferulic acids and their corresponding coumarins, scopoletin and isoscopoletin, were separated by isocratic High Performance Liquid Chromatography using RP-8 (5 μm) as a stationary phase and aqueous methanol or aqueous acetonitrile as a mobile phase. The UV spectrum of cis-isoferulic acid was obtained by a photodiode array detector.     

HPLC of Steroids in Non-aqueous Mobile Phase at Subambient Temperature

Abstract

Increased resolution of steroid mixtures was found in high performance liquid chromatography of steroids at subambient temperatures. With aqueous mobile phase using a reversed phase column it was not possible to decrease temperatures below ?10°C due to increased viscosity. This report describes further increase in resolution at ?50°C using a non-aqueous mobile phase for the separation of steroids. Retention times were shorter several fold while resolution was improved.     

Reversed-Phase High Performance Liquid Chromatography of Carbon Dioxide

Abstract

An investigation of the qualitative aspects of the liquid chromatography of carbon dioxide is described. It is demonstrated that although injected as a high pressure liquid, carbon dioxide dissolves in aqueous methanol mobile phases and elutes as a solution. The detector response is discussed in terms of the possible chemical interactions between the carbon dioxide molecule and the mobile phase; the effect of eluent pH upon the response is described. The variation of relative retention with mobile phase composition is detailed and the results discussed in terms of Horvath's solvophobic theory.     

Enhanced Fluorescence And Room Temperature Liquid Phosprorescence Detection In Pseudophase Liquid Chromatography (Plc)

Abstract

The effect of a micellar mobile phase on the fluorescence detection of a variety of polynucleararomatic hydrocarbons (PAH's) separated via HPLC is examined. It was found that the “fluorescence peaks” of the separated PAH's were enhanced up to ten times by the sodium dodecylsulfate micellar mobile phase. Furthermore, one can use room temperature liquid phosphorescence detection when the mobile phase consists of a deoxygenated aqueous solution of thallium and sodium dodecylsulfate micelles. The phosphorescence intensity was relatively weak and difficult to obtain compared to the fluorescence.     

Thin Layer Chromatographic Separation of Ortho,Meta, and Para Substituted Benzoic Acids and Phenols with Aqueous Solutions of α-Cyclodextrin

Abstract

The use of aqueous solutions of α-cyclodextrin as the mobile phase in thin layer chromatography (TLC) is described. A series of eighteen substituted benzoic acid compounds were chromatographed on polyamide thin layer sheets. The R_f values were dependent on the concentration of α-cyclodextrin in the mobile phase as well as the structure and size of the individual molecules. Possible advantages of this technique over those which use pure or mixed solvent systems as the mobile phase are discussed.     

Adsorption of an Anionic Surfactant on an Octadecyl Bonded Phase from a Pure Aqueous Mobile Phase

Abstract

Difficulties encountered when using pure aqueous mobile phases in ion pair reversed phase liquid chromatography are described.

The results presented in this paper show the influence of the structure and wettability of the stationary phase surface on the adsorption of an anionic surfactant (sodium octyl sulfate).     

Purification of Solid-Phase Synthesized Peptides on the Coil Planet Centrifuge

Abstract

The analytical flow-through coil planet centrifuge, an instrument for countercurrent chromatography, performs the preparative purification of synthetic peptides. Various two-phase solvent systems have been tried with either phase mobile to purify many synthesized peptides. A series of N-terminal fragment peptides of cholecystokinin octapeptide (CCK 26–33) were synthesized by solid-phase techniques and purified on the coil planet centrifuge. The peptides were sulfated and chromatographed again. For hydrophobic peptides, purification is effected in solvent systems with a mobile aqueous phase. The n-butanol, acetic acid and water system (4:1:5 by volume) with the lower phase mobile was utilized. For sulfated peptides, the neutral system, 0.2 M ammonium acetate and n-butanol was generally applied.     

Determination of Phthalic Anhydride In Workplace Air Using Reverse Phase High Performance Liquid Chromatography

Abstract

A reverse phase high performance liquid chromatographic procedure is described for the analysis of phthalic anhydride in workplace air. A glass fibre filter was used to collect the airborne phthalic anhydride and the analyte was then desorbed and hydrolyzed with dilute aqueous sodium hydroxide. Subsequent acidification of this solution with the mobile phase enabled the phthalic anhydride to be determined as phthalic acid using UV detection.     

Use of Aqueous Micellar Mobile Phases in Reverse Phase TLC

Abstract

Aqueous micellar solutions can be used in reverse phase TLC providing the ionic strength of the solution is sufficiently high to prevent the destruction of the stationary phase. Stability curves have been determined for sodium dodecyl sulfate and cetyltrimethylammonium chloride containing aqueous mobile phases. These “pseudophase” solutions allow the unique separation of hydrophobic from hydrophilic compounds. Indeed one can estimate the relative hydrophobicity of a compound by observing its chromatographic behavior in this system.     

A Rapid Stability-Indicating HPLC Assay for the Arabinosylcytosine Prodrug,Cyclocytidine

Abstract

Hydrolysis of cyclocytidine in aqueous solutions produced arabinosylcytosine which, in some cases, further reacted to form arabinosyluracil. No other degradation products were detected. A rapid isocratic reverse-phase HPLC assay for all three components in mixtures arising from cyclocytidine hydrolysis was developed. The analysis employs a 4.6 cm column together with a low methanol mobile phase containing 1-heptane sulfonic acid at pH 2.9. The ion-paring of cyclo-C, a cation, was independent of pH. However, ion-paring of arabinosylcytosine was controlled by adjusting the pH to 2.9 which is below its pKa of 4.2. The retention time of neutral arabinosyluracil (pKa = 9.2) was not affected by either the pH or the ion-pairing agent. Its separation was achieved by using a primarily aqueous mobile phase with the minimum methanol required for the other components. The time courses for cyclocytidine and its hydrolysis products were successfully defined under a variety of aqueous conditions.     

High Performance Liquid Chromatographic Assay for Basic Amine Drugs in Plasma and Urine Using a Silica Gel Column and an Aqueous Mobile Phase. II. Chlorpheniramine

Abstract

A high performance liquid chromatographic [HPLC] method that involves the use of a silica gel column and an aqueous mobile phase for quantitation of chlorpheniramine in plasma and urine is presented. Alkalinized samples are cleaned by extraction with pentane [containing 1% CH₃CN], and the extraction is followed by evaporating the solvent and reconstituting the residue in a small amount of mobile phase. An aliquot of this solution is analyzed by an HPLC system with an Ultrasphere Si Column, an aqueous mobile phase at pH 7 containing 60% CH₃CN and 7.5 mM [NH₄]₂HPO₄, and UV detection at 200 nm. Although the average recovery of extraction is 58% ± SD 10%, the detection limit for the method is 0.7 ng/ml in plasma and 100 ng/ml in urine [s/n = 3] for 0.5 ml samples. The coefficients of variation [CV] on the results of samples run to measure interday and intraday precision and the bias on control samples were all 10% or less. We have used the method in a bioavailability study of a controlled release formulation involving over 1000 samples.     

Size Exclusion Chromatography of Nonionic and Anionic Copolymers of Vinylpyrrolidone

Abstract

Size exclusion chromatography (SEC) of poly(vinylpyrrolidoneco-vinylacetate), PVPVA, and poly(vinylpyrrolidone-co-dimethylamincethylmethacrylate-co-vinylcaprolactan),PVPDMMAEMAVC, is evaluated in terms of resolution between polymer and solvent peaks using aqueous and nonaqueous mobile phases. A 1:1 (v/v) water/methanol, 0.1M LiNO₂ mobile phase is used with four Waters Ultrahydrogel columns with pore sizes of 120Å, 500Å, 1000Å, and 2000Å. DMF, 0.1M LiNO₃ mobile phase is used with three different two column sets. Two of the column sets are comprised of a mixed bed packing of poly(styrene-co-divinyl benzene), obtained from Shodex (KD80M), followed by either an Ultrahydrogel 120Å or a PLge1 100Å. The third two column set is a PLge1 10⁴Å followed by a PLgel 500Å. Any of the second columns in these sets improve separation between the trailing end of the polymer peak and the leading end of the solvent peak but the column set using an Ultrahydrogel 120Å yields a separation of these peaks whose valley is closer to the baseline. The aqueous mobile phase with the four column set yields a separation between the trailing end of the polymer peak and the leading end of the solvent peak whose valley is closest to the baseline. Recovery of PVPVA and PVPDMAEMAVC from the columns evaluated is 100%. Vinylpyrrolidone compositions of PVPVA ranging from 30 to 70 mole % were studied using both mobile phases. SEC of     

An HPLC Procedure for the Analysis of Furosemide in Pharmaceuticals-Analysis of Furosemide Tablets and Furosemide Injection

Abstract

A simple and specific procedure was developed for the analysis of furosemide from tablets and injections. The procedure consists of extracting furosemide into aqueous sodium hydroxide, addition of the internal standard, appropriate dilution and injection onto a u Bondapak C₁₈ reversed phase column. The mobile phase consisted of a solvent containing acetonitrile and aqueous sodium acetate and the eluate was monitored by either U.V. absorption or spectrofluorimetry. A standard linear calibration curve was obtained for direct standard solutions containing 75 ng to 500 ng on column. This procedure was successfully used to analyze furosemide tablets (individual assay) and injections.     

Separation of Biological Pyridines by High Pressure Liquid Chromatography

Abstract

The separation of the six pyridine compounds which comprise the pyridine nucleotide cycle, nicotinamide adenine dinucleotide phosphate and para-aminobenzoic acid, a compound biologically related to these pyridines, can be achieved rapidly utilizing high pressure liquid chromatography. Optimum separation is accomplished using ion-ion pairing in reverse phase chromatography with a C₁₈ stationary phase and an aqueous mobile phase of 5mM pentanesulfonic acid and 25 mM KH₂PO₄. The effect of temperature on the separation is minimal. As little as 10 ng of these compounds is detected via absorption of ultraviolet light at a wavelength of 254 nm.     

The Identification of Uridine in Cacao Extracts

Abstract

Uridine has been identified as a minor component of defatted Ecuadorian cacao liquor through reverse phase HPLC of aqueous extracts. The nucleoside was identified by comparison of its behavior in a variety of mobile phases and column types as well as its absorbance ratios at 245 and 270 nm. This work has extended the previously reported methodology to include pyrimidine nucleosides.     

Ion Chromatographic Determination of Sodium Isethionate

Abstract

Single column anion chromatography with conductivity detection has been used for the determination of sodium isethionate. A mobile phase of aqueous phthalic acid - methanol (pH 2.5) allows for the separation of isethionate from chloride and alkyl isethionate ester surfactants. Commercially available samples of sodium isethionate and alkyl esters were analyzed by the described procedure.     

A Simultaneous Analysis by High Performance Liquid Chromatography of Bamipine Combined with Tricyclic Antidepressants and/or Antipsychotics in Dosage Forms

Abstract

A reversed phase high-performance liquid chromatographic method is described for the simultaneous determination of antihistamines, tricyclic antidepressants and anti-psychotics in pharmaceutical formulations and in spiked placebos. The separation was performed on an octadecyl-silica column using acetonitrile: tetrahydrofuran: 0.015 M aqueous ammonium acetate (53:42: 5) as mobile phase. The presence of ammonium acetate both shortens the elution time and improves the symmetry of the chromatographic peaks. Measurements were made at 251 nm.     

Determination of Some Penicillin Derivativies Using High Performance Liquid Chromatography

Abstract

A rapid, precise and selective method is described for determination of penicillin derivatives. Penicillin derivatives are separated from closely related degradation products by high performance liquid chromatography at ambient temperature using 10cm column packed with 5um Nucleosil RP₁₈ and buffered aqueous acetonitrile as mobile phase. The eluate is monitored at 254nm. The procedure is suitable for determination of 8 penicillin derivatives either in raw material or phamaceutical dosage forms.     

Analysis of Allopurinol and Oxipurinol in Plasma by Reversed Phase HPLC

Abstract

A reversed phase HPLC assay is described for the quantitation of allopurinol and oxipurinol in human plasma. The strategies for the development of this method are discussed. The analysis is performed on a C-18 column using an acidic aqueous mobile phase. Sample preparation consists of protein precipitation with a mixture of trichloracetic acid and perchloric acid. The detection limit of the assay for both substances is in the region of 30ng/ml. This method has been applied to an investigation of the relative bioavailability of two commercial preparations of 300mg allopurinol tablets in eight healthy volunteers.