Ion-pair Chromatographic System Research Articles

Regeneration of Tetrabutylammonium Ion-Pairing Reagent Distribution in a Gradient Elution of Reversed Phase Ion-Pair Chromatography

The regeneration of ion-pairing reagent distribution on liquid chromatography columns after gradient elution has been well recognized as the cause for long column equilibration time, a major drawback associated with gradient elution reverse phase ion-pair chromatography. To date, the majority of studies have focused on optimizing the separation conditions to shorten the equilibration time. There is limited understanding of the ion-pairing reagent distribution process between the mobile phase and stationary phase in the course of gradient elution, and subsequent column re-equilibration. The focus of this work is to gain a better understanding of this process. An ion-pair chromatographic system, equipped with a YMC ODS C(18) column and a mobile phase containing tetrabutylammonium (TBA) hydroxide as the ion-pairing reagent, was used in the study. The TBA distribution profile was established by measuring its concentration in the eluent fractions collected during the gradient cycle using different column equilibration times with an ion chromatographic method. Furthermore, the analyte retention time was evaluated as the function of the column equilibration time and TBA concentration in the mobile phase. The column equilibration and its impact on the method robustness will also be discussed.

UPLC-ICP-MS—a fast technique for speciation analysis

Ultra performance liquid chromatography is a new development of the HPLC separation technique that allows separations on column materials at high pressures up to 108 Pa using particle diameters of 1.7 μm. This increases the efficiency, the resolution and the speed of the separation. Four aqueous selenium standards were separated within 1.2 min on a 1.00 id × 50 mm reversed phase column in an ion-pair chromatographic system using a flow rate of 200 μL min−1. Hence, analysis times could be reduced to 1/10 compared with ordinary HPLC for aqueous standards. The precision and detection limits were comparable to values obtained by HPLC. Detection limits were better than 0.4 μg Se L−1. A urine sample was analysed on a 1.0 id × 100 mm column within 5 min using a flow rate of 100 μL min−1. The improved separation efficiency, owing to the use of 1.7 μm column particles, allowed the use of short columns. Hence, analysis times could be halved without loss of separation efficiency in this biological sample.

Selenium speciation in human urine samples by LC- and CE-ICP-MS—separation and identification of selenosugars

Human urine samples were analysed by a reversed-phase chromatographic system and an ion-pair chromatographic system. The chromatographic system was connected to the ICP-MS either by a microconcentric nebulizer (MCN) in combination with a cyclonic spraychamber or by a modified direct injection nebulizer (MDIN). The sensitivity of the latter was better than the sensitivity of the MCN, which on the other hand was more robust for the analysis of samples with high concentrations of dissolved solids. Urine sample composition did not seem to change when urine was exposed to evaporation under nitrogen at ambient temperature and methanol extraction. A pre-concentration factor of 10 was achieved with this procedure. On occasions when a pre-concentration factor of 100 was obtained by lyophilsation and methanol extraction, at least 10 selenium compounds were separated in the urine sample. Urine samples were collected from two healthy volunteers who had been supplied with 1000 µg and 2000 µg of selenium, respectively, in the form of selenized yeast. When samples were spiked with 8 different standards, only two standards co-eluted with compounds in urine in both chromatographic systems: the major urinary metabolite Se-methyl-N-acetylgalactosamine and Se-methyl-N-acetylglucosamine. The presence of Se-methyl-N-acetylglucosamine in urine was verified by co-migration with the standard in capillary electrophoresis after fractionation by preparative reversed-phase chromatography. Se-methyl-N-acetylglucosamine is only a minor metabolite as its concentration was less than 2% of the concentration of Se-methyl-N-acetylgalactosamine. The presence of this metabolite in urine has, to our knowledge, not been suggested before. Trimethylselenonium, selenomethionine, Se-methylselenocysteine, Se-methylselenomethionine and selenocystamine were not detected in these samples.

Reversed-phase ion-pair chromatographic analysis of tetracycline antibiotics: Application to discolored teeth

A high-performance liquid chromatographic method with diode array detection was developed to simultaneously separate tetracycline antibiotics and applied to the analysis of discolored teeth. By a reversed-phase ion-pair chromatographic system using pentanesulfonate as a counter ion, minocycline, oxytetracycline, tetracycline and demeclocycline were eluted in this order, and they showed base-line separation within 9 min. When using oxytetracycline as an internal standard, the quantitative ranges were between 2.5 ng/ml and 7.5 μg/ml. Powdered dentine (10 mg) and enamel (40 mg) prepared from discolored primary teeth were sonicated in 0.25 ml of 10 m M HCl containing oxytetracycline (0.75 μg/ml) and 50 m M EDTA-2Na, thereafter the supernatants were chromatographed. Eluates from both discolored tooth samples were identified as minocycline based on diode array spectra of their peaks, while minocycline was not detected in any samples from nondiscolored normal teeth, indicating that discoloration of the tested teeth was due to minocycline incorporated into dentine and enamel. Replicate quantitative analyses of the identical tooth substances showed that intra- and inter-assay C.V.s were 2.63 and 4.95% for dentine, and 5.42 and 10.88% for enamel. Application of the developed method to nine discolored teeth revealed that the incorporated minocycline ranged from 20.13 to 84.62 ng/mg of dentine and 0.89 to 7.87 ng/mg of enamel.

The impact of column temperature in the high performance liquid chromatographic analysis of topotecan in rat and dog plasma

A sensitive high performance liquid chromatographic (HPLC) assay has been developed and validated for the quantitation of the novel anticancer agent topotecan and topotecan as its lactone plus carboxylate forms in rat and dog plasma. Linear responses in analyte standard peak areas were observed over the concentration ranges 0.10–10 ng ml −1 using 100 μl of rat plasma and 0.2–100 ng ml −1 using 100 μl of dog plasma. Due to the instability of the drug in the biological matrix it was necessary to obtain the plasma fraction within 5 min after blood sampling by centrifugation, immediately followed by protein precipitation with cold methanol (−30°C). For the determination of total drug levels (lactone plus lactone ring-opened form), plasma samples were deproteinated with methanol and subsequently acidified with 2% (v/v) perchloric acid. The samples were analysed by HPLC using a Zorbax SB-C18 Stable Bond column and methanol-0.1 M hexane-1-sulfonic acid in methanol-0.01 M N, N, N′, N′-tetramethylethylenediamine in distilled water pH 6.0 (25:10:65, v/v/v) as the mobile phase. The detection was performed fluorimetrically. The analytical column was thermostated at 19–21°C to obtain baseline resolution between an interfering endogenous compound in rat and dog plasma and topotecan. This endogenous peak was absent in human plasma. Variation of chromatography temperature appeared to be a very useful tool in the bioanalysis of topotecan. It allowed optimization of the separation between the endogenous compound and the analyte; different mechanisms of solute interactions are apparently involved in this reversed-phase ion-pair chromatographic system.

Reversed-phase chiral ion-pair chromatography at a column temperature below 0°C using three generations of Hypercarb as solid-phase

Enantiomeric separations using chiral ion-pair chromatography have been investigated with porous graphitic carbon, Hypercarb®, as the solid-phase. The enantiomers of several aminoalcohols were succesfully separated as diastereomeric ion pairs with N-benzyloxycarbonyl-glycyl- l-proline ( l-ZGP) or N-benzyloxycarbonyl-lycygglycyl- l-proline ( l-ZGGP) dissolved in polar solvents e.g., methanol as the mobile phase. The influence of the solute structure as well as the counter ion structure on the chiral recognition were examined. The position of substituents in the aromatic ring, type of alkyl group attached to the nitrogen and the number of methylene groups between the asymmetrical carbon atom and the nitrogen atom were studied. The present study shows that a column temperature below 0°C improved the enantioselective resolution. A stable and robust chromatographic system with a short equilibration time is presented. The chiral ion-pair chromatographic system could be used for the determination of enantiomeric impurities of less than 0.1% and is possible even if the enantiomer in low concentration is eluted after the main enantiomer.

Variables Influencing the Direct Determination of Haloacetic Acids in Water by Reversed-Phase Ion-Pair Chromatography with Indirect UV Detection

Abstract A reversed-phase ion-pair chromatographic system for the direct determination of haloacetic acids in water has been optimized utilizing indirect photometric detection. Acetic, chloroacetic, bromoacetic, iodoacetic, dichloroacetic and dibromoacetic acids were used to characterize the chromatographic system. The effect of temperature on retention time shows a non linear van't Hoff behavior indicating a change in the mechanism of retention at about 30°C. Above 30°C, retention times decreased proportionally to increases in the temperature of the column. Separations are achieved in the pH range of 3.5 to 6.3 with an optimum at ca. pH 5.4. Increases in the concentrations of KH2PO4, l-hexanesulfonate (competing ion) and acetonitrile result in proportional decreases in capacity factors with some selectivity variations depending on the analyte. Increases in the concentration of benzyltributylammonium ion (ion interaction reagent) resulted in increases in capacity factors with a usable range from 8 to 12 mM. Of the reversed phases studied, Spherisorb C-18 yielded the optimal results for the acids studied. Linear calibration curves for these acids were obtained utilizing indirect UV detection with detection limits as low as 2 parts per million.

Chromatographic Behavior of the Anthelmintic Fenbendazole and Its Major Metabolite Oxfendazole in Various Ion-Pair Liquid Chromatographic Systems

Abstract The chromatographic behavior of fenbendazole (FBZ) and oxfendazole (OFZ) in various reversed-phase liquid chromatographic (LC) systems has been investigated. The addition of negative and/or positive charged ion-pair reagents in the mobile phase has been examined, whereas the influence of mobile phase pH, mobile phase composition, and column temperature on retention and peak height has been evaluated. The observed behavior of the analytes during the various chromatographic processes has been discussed.

Comparison between Experimental and Theoretical Profiles of High Concentration Elution Bands and Large System Peaks in Nonlinear Chromatography

A detailed experimental investigation of the profiles of system peaks and single solute bands at high concentrations was conducted under experimental conditions leading to severe interference between the primary additive system peak and the additive system peak associated with the solute band. These conditions trigger the formation of extremely unusual band shapes. Both additive and solute individual profiles have been recorded separately, using a diode-array detector operating at different wavelengths selected for the selectivity of the response to the two different compounds. The profiles recorded are in very good agreement with the profiles calculated from the isotherms of the two compounds, using the equilibrium-dispersive model of chromatography. This result confirms that major distortions of band profiles occur whenever the additive is more strongly adsorbed than the solute by the stationary phase, but its concentration in the mobile phase is high enough for the additive primary system peak to be eluted before the solute peak, with a separation factor close to unity. Although the experiments were carried out using a reversed-phase ion-pair chromatographic system, the same phenomenon has been shown to occur in normal-phase adsorption chromatography as well. 36 refs., 5 figs.

Electrostatic retention model of reversed-phase ion-pair chromatography

The theoretical foundation of the electrostatic theory of ion-pair chromatography derives from colloid and surface chemistry. In the first part of this paper, the basic concepts of the theory are discussed with emphasis on the physical principles. The theory can predict retention changes of a charged solute when varying experimental parameters in ion-pair chromatographic systems. However, because of the interplay between the different parameters, such a prediction is only feasible when using iterative numerical procedures. Therefore, a simplified theory is developed in the second part where a relationship is derived which separates the contributions of various parameters, such as type and concentration of ion-pairing reagent, ionic strength, concentration of organic modifier and eluent pH. At high surface concentrations of the ion-pairing reagent, competition between the solute and ion-pairing reagent for the limited area of the stationary phase available may occur. It is shown in the third part of the paper that this results in a maximum in the relationship between capacity factor and concentration of ion-pairing reagent in the eluent. In the final section, an extended version of the electrostatic theory is developed. It accounts for the effect of accumulation of solute ions in the electrical double layer on the capacity factor. The extended form of the electrostatic theory provides the most complete treatment of the retention of charged solutes. However, this is achieved at the cost of developing a complex mathematical formulation.

Explanation of the selectivity differences between reversed-phase ion-pair chromatographic systems containing trifluoroacetate or heptafluorobutyrate as pairing ion

The well documented selectivity differences found between reversed-phase ion-pair chromatographic systems containing trifluoroacetate or heptafluorobutyrate as pairing ion were explained after determining sorption isotherms for trifluoroacetate and heptafluorobutyrate on Nucleosil 100-5 C 18 from a solution similar to the eluents used for the separation of transmitteramines and peptides. Based on the isotherms and retention data obtained with reversed-phase, ion-exchange and reversed-phase ion-pair chromatographic systems, it is proposed that the selectivity differences between the systems studied are caused by the fact that trifluoroacetate and heptafluorobutyrate are not interchangeable in terms of their surface concentrations at the practical eluent concentrations of the pairing ions concerned.

Analysis of steroids : XLI. Ion-pair high-performance liquid chromatographic separation of quaternary ammonium steroids on silica

A normal-phase ion-pair chromatographic system has been developed for the high-performance liquid chromatographic investigation of pipecuronium bromide (2β,16β-bis(N′-dimethyl-1-piperazinyl)-3α, 17β-diacetoxy-tα-androstane dibromide) and related quaternary ammonium steroids. The use of silica as the stationary phase and a 96:4 mixture of acetonitrile and water containing 0.1 mol/dm 3 sodium perchlorate as the eluent with detection at 213 nm enable the potential impurities as well as the hydrolic and oxidative degradation products of pipecuronium bromide to be separated and detected down to the 0.01% level. The above system is also applicable to the high-performance liquid chromatographic investigation of other quanternary ammonium steroids (pancuronium bromide, vecuronium bromide).

High-performance reversed-phase ion-pair chromatographic study of myo-inositol phosphates: Separation of myo-inositol phosphates, some common nucleotides and sugar phosphates

A detailed study of all the major chromatographic variables affecting the retention behaviour and separation of myo-inositol phosphates in reversed-phase ion-pair chromatographic systems was carried out. The parameters studied included the eluent concentration of the pairing ion, the eluent concentration of the organic modifier and the buffer salt, the pH of the eluent, the minimum column plate count necessary for the separation of the inositol trisphosphate isomers and isocratic and gradient modes of separation. The retention behaviour of some common nucleotides and sugar phosphates was also investigated as these phosphates present chromatographic interference problems in biochemical studies based on the cellular incorporation of [ 32P]P i. The separation methods developed appear to be superior to established anion-exchange separation techniques in terms of separation speed and “mildness” of the chromatographic conditions.

Separation and determination of metals as complexes of 1-nitroso-2-naphthol-6-suiphonic and 2-nitroso-1-naphthol-6-sulphonic acids. II. Liquid chromatography on c18 bonded and copolymer stationary phases

An ion-pair Chromatographic system for the separation of copper(II), palladium(II), iron(III) and cobalt(III) as ion-associates of their l-nitroso-2-naphthol-6-sulphonate and 2-nitroso-l-naphthol-6-sulphonate anionic complexes with organic ammonium compounds and inorganic cations has been studied. Isocratic and gradient elution methods were used, and the effects of column material, organic and aqueous modifiers, and pH of the eluent on the retention were examined. The elution time for the metal complex anions depends on the eluent, the proportion of organic solvent in the mobile phase, the pH of the eluent and the extraction coefficient of the compounds. The compounds were identified photometrically with a diode-array detector at wavelengths of 229, 254, 260, 298, 320 and 400 nm. The detection limits for the metals are at the ng/ml level.

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for anionic analytes and mobile phase components on polystyrene polymers using an alkaline mobile phase

Retention models for anionic analytes and mobile phase compounds in reversed-phase ion-pair chromatographic systems have been studied. The solid phase was a polystyrene-divinylbenzene copolymer. The analytes were mono- and divalent anions monitored by indirect detection technique. The mobile phase was highly alkaline and contained a detectable anion, sulfanilic acid.

Bioanalysis of fluorouracil applying liquid chromatography and valve switching

Fluorouracil has been analyzed with a reversed-phase ion-pair liquid chromatographic system applying PRP-1 as the stationary phase. A valve switching technique was applied for the separation of the analyte and some other chemotherapeutic agents. The described procedure gives a rapid screening for the analyte in urine and plasma samples without interferences from late eluting solutes. Biological samples are pretreated by means of a liquid-liquid extraction resulting in a recovery of about 52% for plasma and urine with minimum detectable amounts of about 5ng/ml. The sensitivity and the selectivity of the procedure allows the monitoring of fluorouracil in body fluids and subsequent pharmaco-kinetic studies of the fluoropyrimidine.

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for ionic compounds on μ Bondapak phenyl

Retention models for ionic compounds in reversed-phase ion-pair chromatographic systems have been developed on the basis of the capacity ratios of solutes and ionic mobile phase components using indirect detection. The binding to the adsorbent, μBondapak Phenyl, has been expressed by ion-pair distribution constants and tested in the retention models by non-linear regression.

Ion-pair high-performance liquid chromatographic procedure for the quantitative analysis of theophylline in serum samples

An ion-pair chromatographic system is described for the separation of theophylline and related xanthines from serum samples. The mobile phase consisted of 0.02 M tetrabutylammonium ion and 0.015 M Tris buffer in water-acetonitrile-methanol (93:3.5:3.5, v/v/v) at a precisely controlled pH (7.50 ± 0.02, adjusted with hydrochloric acid). The flow-rate was 1.2 ml/min through a 15 cm × 4.6 mm I.D., 5-μm reversed-phase column (Ultrasphere C 18 ion pair). Xanthines were extracted from serum (100 μl) with 1 ml of acidified chloroform-isopropanol (95:5, v/v). After reconstitution in 200 μl of mobile phase, the extracted xanthines, including theophylline, caffeine, theobromine and 1,7-dimethylxanthine, were baseline-resolved in less than 15 min. The method correlates well with a common clinical immunoassay for theophylline (EMIT Syva, r 2 = 0.999) and yields excellent recovery and precision (98–101% and better than 2% at therapeutic levels, respectively). In addition, the use of the ion-pair chromatography mode eliminates many of the interferences noted in the published literature for the common reversed-phase separations of theophylline.

Separation of Enantiomers in Ion-Pair Chromatographic Systems

Abstract Enantiomers of amines and acids have been separated in ion-pair chromatographic systems with chiral agents present in mobile or stationary phase. Chiral counter ions such as quinine or camphorsulfonate have been used in liquid solid systems with organic mobile phases, while the chiral complexing agents albumin and (+)-n-dibutyltartrate have been used in reversed phase systems. Regulation of retention and stereoselectivity as well as improvement of detection sensitivity by the composition of the system is discussed.

Determination of inorganic anions by ion-pair chromatography : Hetaeron adsorption characteristics on some alkyl-bonded silica stationary phases

A non-buffered ion-pair chromatographic system with alkyl-bonden silica was used to determine inorganic anions. Some octyl-bonded stationary phases showed unexpected behaviour such as a dependence on pre-wetting and on the flow-rate during the dynamic equilibrium. On the other hand, octadecyl-bonded stationary phases were not influence by these effets. The influence of an organic co-solvent and the efficiency with 3-μm particles in ion-pair chromatography were also studied.