The effects of pH, ionic strength and buffer concentration of mobile phase onR F of acidic compounds in ion-pair TLC

Summary The effects of some factors important in ion-pair, high-performance liquid chromatography were studied in ion-pair, thin-layer chromatography. Tetramethyl and cetyltrimethylammonium salts were used as ion-pairing reagents. As stationary phases, silica gel and chemically bonded reversed-layers (C₁₈) were used. Layers were impregnated with ion-pairing reagent prior to chromatography. In some cases the stationary phase was treated with buffer at different concentrations. The mobile phase contained methanol and water, in one set of experiments buffer, salt for adjustment of ionic strength and ion-pairing reagent were added. The migration behaviour of different benzoic acids was studied. Several problems of ion-pair thin-layer chromatography are discussed. Passed away on 13^th of April, 1998 Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Identification of organic colourants in cosmetics by HPLC-diode array detection

Summary A method for the analysis of organic cosmetic colourants was developed. The colourants extracted from the cosmetic products were analysed by HPLC followed by diode-array detection across the wavelength range 275 nm–760 nm. The chromatographic separation was performed on a column with a polymeric packing using gradient elution with a mobile phase composed of citrate buffer, the ion-pairing reagent tetrabutylammonium hydroxide, acetonitrile and tetrahydrofuran. The colourants were identified on the basis of their retention times as well as their UV-visible spectra. A spectral library consisting of retention times and UV-visible spectra of 130 organic cosmetic colourants has been built for the purpose of identifying of colouring matter in cosmetic products. Solid-phase extraction methods using C-18 silica and aminobonded silica have been developed for the extraction of colourants from various types of cosmetic products. The method for the identification of colourants in nail varnishes and lipsticks has been optimized for routine analysis. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

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.     

Retention modeling under organic modifier gradient conditions in ion-pair reversed-phase chromatography. Application to the separation of a set of underivatized amino acids

The combined effect of the ion-pairing reagent concentration, C _ipr, and organic modifier content, φ, on the retention under φ-gradient conditions at different constant C _ipr was treated in this study by using two approaches. In the first approach, the prediction of the retention time of a sample solute is based on a direct fitting procedure of a proper retention model to 3-D φ-gradient retention data obtained under the same φ-linear variation but with different slope and time duration of the initial isocratic part and in the presence of various constant C _ipr values in the eluent. The second approach is based on a retention model describing the combined effect of C _ipr and φ on the retention of solutes in isocratic mode and consequently analyzes isocratic data obtained in mobile phases containing different C _ipr values. The effectiveness of the above approaches was tested in the retention prediction of a mixture of 16 underivatized amino acids using mobile phases containing acetonitrile as organic modifier and sodium dodecyl sulfate as ion-pairing reagent. From these approaches, only the first one gives satisfactory predictions and can be successfully used in optimization of ion-pair chromatographic separations under gradient conditions. The failure of the second approach to predict the retention of solutes in the gradient elution mode in the presence of different C _ipr values was attributed to slow changes in the distribution equilibrium of ion-pairing reagents caused by φ-variation.     

Ion-Pair Chromatography of Metal Complexes of Unithiol in the Presence of Quaternary Phosphonium Salts

The chromatographic behavior of heavy-metal (Pb, Cd, Cu, Hg, and Ni) complexes of unithiol was studied by ion-pair reversed-phase chromatography using new ion-pair reagents: tetrabutylphosphonium and tributylhexadecylphosphonium bromides. The dependence of the retention of metal unithiolates on the nature and concentration of ion-pair reagents, concentrations of an organic solvent and inorganic salts in the mobile phase, and the pH of the mobile phase was studied. It was found that the retention of complexes increases upon increasing the hydrophobicity and concentration of the ion-pair reagent and upon decreasing the concentrations of acetonitrile and inorganic salts (NaClO₄ and NaNO₃). Optimal conditions of the separation of Pb, Cd, Cu, Hg, and Ni unithiolates were selected, and the possibility of their separate determination in a mixture was demonstrated. Concentrations of metals in process water were determined.     

Simple and Rapid Determination of Denaturants in Alcohol Formulations by Hydrophilic Interaction Chromatography

A hydrophilic interaction chromatography (HILIC) technique has been developed and validated for determination of common denaturants (denatonium benzoate, crystal violet and methylene blue) in denaturated alcohol formulations. Among the three different polar stationary phases (i.e., aminopropyl, cyanoethyl and silica) studied the cyanoethyl phase provided much stronger retention for the organic cations. It was shown that high efficiencies were reached only with anionic ion-pairing reagent that reduces the interactions with the silanol groups. The anion ion-pairing strength under HILIC conditions was: acetate < formate < trifluoroacetate < perchlorate. This study also investigated the effect of various experimental factors on the retention of the cyanoethyl stationary phase, such as acetonitrile content, pH and ion-pairing anion concentration in the mobile phase. The separation of three denaturants was achieved in about 8 min with a mobile phase containing 60% (v/v) acetonitrile and 10 mmol L⁻¹ HClO4. The proposed method was validated and applied to the determination of danaturating agents in various Lithuanian denaturated alcohol formulations.     

Determination of Cyanuric Acid in Milk Powder and Swimming Pool Water by Ion‐pair Reversed‐phase Liquid Chromatography

An ion‐pair reversed‐phase high‐performance liquid chromatographic method, using tetrabutylammonium bromide (TBAB) as ion‐pair reagent, has been developed for the analysis of cyanuric acid (CA) in milk powder and swimming pool water. It was found that the effect of the concentrations of ion‐pair reagent on the retention of cyanuric acid was different for standard solution and different real samples. The separation was carried out on a reversed‐phase C₁₈ column with 85:15 (V/V) water‐acetonitrile (ACN) containing different concentration of TBAB as mobile phase for different samples. The linear range of the calibration curve for CA was 0.1–100 mg·L^?1. The detection limits calculated at S/N=3 was 0.11 mg·L^?1 for the analysis of milk powder and 0.31 mg·L^?1 for the analysis of swimming pool water, respectively. The method was successfully applied to the analysis of CA in milk powder and swimming pool water.     

Positive mode electrospray ionization mass spectrometry of bisphosphonates using dicationic and tricationic ion-pairing agents

A general method for detecting bisphosphonate drugs by ESI-MS and LC-ESI-MS as positive ions has been developed. Bisphosphonates can have multiple negative charges in solution. Tricationic ion-pairing reagents were paired with bisphosphonates to form a positively charged complex. It was clear that this facile pairing method worked. However, an appreciable presence of −1 bisphosphonate species were observed in positive mode ESI-MS (i.e. as the +2 complex with tricationic reagents). This led to an extended investigation on the use of dicationic pairing agents. The use of dicationic reagents improved the detection sensitivity for all of the bisphosphonates. Tandem mass spectrometry also improved the limits of detection for most of the bisphosphonates using both the tricationic and dicationic pairing reagents. A tricationic reagent also was used as an ion-pairing reagent in chromatography experiments. Thus the addition of a single reagent produced benefits in that it increased chromatographic retention and enhanced the ESI-MS detection of bisphosphonates.     

反相离子对色谱-电感耦合等离子体质谱联用技术测定水中痕量Cr(Ⅲ)与Cr(Ⅵ)

建立了反相离子对色谱(RPIPC)与电感耦合等离子体质谱(ICP-MS)联用技术快速分离测定水中痕量Cr(Ⅲ)和Cr(Ⅵ)的方法.通过考察流动相的pH值、离子对试剂及甲醇的浓度和EDTA的添加等对不同形态铬的保留时间及分离度的影响,确定当流动相组成为2.0 mmol/L TBA,5%(V/V)甲醇,pH=5.5时,Cr(Ⅲ)与Cr(Ⅵ)可达最佳分离.ICP-MS测定时选用碰撞池技术以消除40Ar12C+与35Cl16OH+对52Cr+的谱学干扰;进样100 μL时,Cr(Ⅲ)与Cr(Ⅵ)的检出限分别为0.15 μg/L和0.16 μg/L.加标回收率在93.6%～106.2%之间; RSD<4%(n=3).以本方法分析了某市自来水、雨水及某品牌纯净水中Cr(Ⅲ)与Cr(Ⅵ)的含量,结果令人满意.     

An ion-pairing liquid chromatography/tandem mass spectrometric method for the determination of cytarabine in mouse plasma

An ion-pairing high-performance liquid chromatographic (IP-HPLC) system interfaced with an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) with minimal sample preparation was developed for the determination of cytarabine (ara-C), a very hydrophilic anticancer drug, in mouse plasma. A conventional reversed-phase chromatographic column in combination with two ion-pairing reagents was adapted for retention and separation of ara-C from the endogenous interferences in mouse plasma. The effects of the experimental conditions such as the fraction of ion-pairing reagents and organic solvents in the mobile phase on the chromatographic performance and the ionization efficiency of ara-C were investigated. The potential of ionization suppression resulting from the endogenous biological materials on the IP-HPLC/MS/MS method was evaluated using the post-column infusion technique. Furthermore, the feasibility of the proposed IP-HPLC/MS/MS procedure for analysis of ara-C in the mouse plasma was demonstrated by comparison with those obtained by the porous graphite carbon column (PGC) HPLC/MS/MS method.     

The Use Of Soft Acid Metal Salts Of Carboxylic Acids For Retention Of Hydrophilic Peptides In Reversed-Phase High Performance Liquid Chromatography

Abstract

The effect of various soft acid cation acetate salts on the retention of the peptides Phe-(Gly)_n, where n=1–4, and on phenylalanine, indicates a correlation between the softness character of the cation of the acetate salt used in the mobile phase and retention in reversed-phase high performance liquid chromatography. Inorganic soft acid cation salts did not exhibit the same increased retention properties. By using silver(I) valerate or silver(I) octanoate as ion-pairing reagents in the mobile phase for RP-HPLC, static retention of peptides was achieved.     

Insights into the mechanism of separation of heparin and heparan sulfate disaccharides by reverse-phase ion-pair chromatography

Reverse-phase ion-pair high performance liquid chromatography (RPIP-HPLC) and ultra-performance liquid chromatography (RPIP-UPLC) are increasingly popular chromatographic techniques for the separation of organic compounds. However, the fine details of the RPIP separation mechanism are still being debated. Many factors including type and concentration of the ion-pairing reagent, mobile phase pH, organic modifier, ionic strength, and stationary phase all play a role in the overall efficiency and optimization of ion-pairing separations. This study investigates the role that competition between ion-pairing reagents with different steric bulk and hydrophobicity plays in the separation of structural isomers of heparin and heparan sulfate (HS) disaccharides. In addition to providing insights into the mechanism by which RPIP-HPLC can resolve closely related disaccharides, the use of competition between ion-pairing agents could lead to new methods for the separation of larger heparin and HS oligosaccharides. This approach should also be applicable to the analysis of other compound classes, and could lead to a general approach for the chromatographic resolution of mixtures of charged analytes having similar structures.     

Determination of Nimesulide by Ion Pair High-Performance Liquid Chromatography Using Tetrabutylammonium as the Counterion

A new method for nimesulide was developed using ion-pair reversed phase liquid chromatography and tetrabutylammonium hydrogen sulfate as the ion-pairing reagent. The influence of the ion pair forming reagent concentration, pH, and mobile phase composition on the retention time of nimesulide were studied. The optimum experimental conditions included a C18 column, a mobile phase of a 50/50 (v/v) mixture of acetonitrile and 15 mM phosphate buffer (pH 8.00) containing 6 mM tetrabutylammonium hydrogen sulfate, 25°C, isocratic elution, a flow rate of 1 mL/min, a run time of 10 minutes, and photodiode array detection at 404 nm. From the analysis of the results, the mechanism for the separation of nimesulide was also established. The retention time for nimesulide was 4.76 ± 0.05 min. The method was linear between concentrations of 9 µg/mL to 64 µg/mL, with limits of detection and quantification of 1.111 µg/mL and 3.390 µg/mL, respectively. The method is simple, rapid, accurate, and precise, and successfully applied for the determination of nimesulide in pharmaceutical products.     

Effect of anionic ion-pairing reagent concentration (1-60 mM) on reversed-phase liquid chromatography elution behaviour of peptides

The homologous series of volatile perfluorinated acids-trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--continue to be excellent anionic ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) after more than two decades since their introduction to this field. It was felt that a thorough, step-by-step re-examination of the effects of anionic ion-pairing reagents over a wide concentration range on RP-HPLC peptide elution behaviour is now due, particularly considering the continuing dominance of such reagents for peptide applications. Thus, RP-HPLC was applied over a range of 1-60 mM phosphoric acid, TFA, PFPA and HFBA to two mixtures of 18-residue synthetic peptides containing either the same net positive charge (+4) or varying positive charge (+1, +2, +3, +4). Peptides with the same charge are resolved very similarly independent of the ion-pairing reagent used, although the overall retention times of the peptides increase with increasing hydrophobicity of the anion: phosphate < TFA- < PFPA- < HFBA-. Peptides of differing charge move at differing rates relative to each other depending on concentration of ion-pairing reagents. All four ion-pairing reagents increased peptide retention time with increasing concentration, albeit to different extents, again based on hydrophobicity of the anion, i.e., the more hydrophobic the anion, the greater the increase in peptide retention time at the same reagent concentration. Interestingly, phosphoric acid produced the best separation of the four-peptide mixture (+1 to +4 net charge). In addition, concentrations above 10 mM HFBA produced a reversal of the elution order of the four peptides (+1 < + 2 < + 3 < + 4) compared to the elution order produced by the other three reagents over the entire concentration range (+4 < + 3 < + 2 < + 1).     

LC and SPE Determination of Chlorophacinone in Corn Grains, Soil and Rat Plasma

A liquid chromatographic method was used for the analysis of chlorophacinone in corn, soil and rat plasma. Sample preparation differed from corn, soil and serum. Simple liquid extraction procedures were applied to soil and corn; solid phase extraction using Oasis HLB cartridges and easy protocol was used for plasma. Liquid chromatography was on X-terra C18 column. The mobile phase used did not contain ion-pairing reagent, it was constituted with methanol/ammonium bicarbonate (0.01 M, pH = 7), 70/30, v/v at 1 mL min⁻¹. UV detection was by using photodiode detector operating at 282 nm. Complete validation of the proposed procedure for corn, soil and serum was made.     

Contribution of the lipophilic buffer to chiral recognition under reversed-phase conditions

Summary A lipophilic buffered aqueous mobile phase, without organic modifier, was used for the high performance liquid chromatographic enantioseparation of D,L-lactic acid on a Merck ChiraSpher (250 mm×4 mm i.d.) column in which the chiral selector is poly(N-acryloyl-S-phenylalanine ethyl ester) bonded to a spherical silica particle. The lipophilicity of the buffer was achieved by addition of triethylammonium phosphate, the ‘ethyl’ apolar chains of which dynamically modified the ChiraSpher stationary phase and increased its hydrophobic character. The ion-paired (cethyltrimethylammonium bromide) analyte enantioseparation was realized by hydrogen-bonding and dipole-dipole complexation on the ChiraSpher stationary phase, superimposed on simultaneous reversed-phase partitioning. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Reversed phase ion-pair high performance liquid chromatographic gradient separation of related impurities in 2,4-disulfonic acid benzaldehyde di-sodium salt

A reversed phase ion-pair gradient liquid chromatographic method has been developed and validated for purity determination of the hydrophilic compound 2,4-disulfonic acid benzaldehyde di-sodium salt (2,4-DSAD) containing both hydrophilic and more lipophilic related impurities. Mixtures of acetonitrile-phosphate buffer containing tetrahexylammonium hydrogen sulfate as the ion-pairing reagent were used as the mobile phase. A linear gradient, which generated simultaneous change in the concentration of organic modifier, buffer concentration and the concentration of ion-pairing reagent, was applied. The method allows detection of impurities at low levels (0.01% w/w). Excellent repeatability for both retention time (RSD< or =0.3%, n = 6) and detector response (RSD = 0.03%, n = 6 for the main peak and RSD = 6%, n = 6, for an impurity at 0.01 area% level) was obtained. The method was shown to be robust for routine analysis and has been successfully transferred to the quality control laboratory.     

Reversed-phase ion-pair chromatographic separation of selenite and selenate with selenium-specific detection

Tetraalkylammonium salts [R₃RN]⁺X^– (R=R, XCH₃, Cl; C₂H₅, Br; C₃H₇, Br; C₄H₉, H₂PO₄; C₅H₁₁ Br; R, R, XCH₃, C₁₆H₃₃, Br) were investigated as ion-pairing reagents for the reversed-phase ion-pair chromatographic separation of selenite and selenate. Other chromatographic parameters such as composition and pH of the mobile phase and concentration of the ion-pairing reagent were also investigated. The compatibility of the proposed chromatographic procedures with various selenium-specific detectors is discussed. The absolute detection limits were found to be 31 ng Se for selenite and 51 ng Se for selenate (100 l injection) with a solution of 5 mM tetrabutylammonium dihydrogen phosphate in 5050 (v/v) methanol/water as mobile phase at a flow rate of 1 ml/min when a flame atomic absorption spectrometer was used as detector. The HPLC/FAAS system was employed for the determination of selenite in solutions serving as selenium supplement for animals.     

Effect of chromatographic conditions on the separation and system efficiency for HPLC of selected alkaloids on different stationary phases

Retention parameters of alkaloid standards were determined on different stationary phases, i.e., octadecyl silica, base-deactivated octadecyl silica, cyanopropyl silica, preconditioned cyanopropyl silica, and pentafluorophenyl, using different aqueous eluant systems: acetonitrile-water mixtures; buffered aqueous mobile phases at pH 3 or 7.8; and aqueous eluants containing ion-pairing reagents (octane-1-sulfonic acid sodium salt and pentane-1-sulfonic acid sodium salt) or silanol blockers (tetrabutyl ammonium chloride and diethylamine). Improved peak symmetry and separation selectivity for basic solutes was observed when basic buffer, ion-pairing reagents, and, especially, silanol blockers as mobile phase additives were applied. The best separation selectivity and most symmetric peaks for the investigated alkaloids were obtained in systems containing diethylamine in the mobile phase. The influence of acetonitrile concentration and kind and concentration of ion-pairing reagents or silanol blockers on retention, peak symmetry, and system efficiency was also examined. The most efficient and selective systems were used for separation of the investigated alkaloids and analysis of Fumaria officinalis and Glaucium flavum plant extracts.     

Fluorometric Determination of Tryptophan and Its Brain Indoleamine Metabolites by Ion-Pair HPLC

Abstract

The reverse phase HPLC separation of tryptophan, serotonin, 5-hydroxyindoleacetic acid, tryptamine and indoleacetic acid using two solvent systems (one of them containing an ion-pairing reagent) is reported. The two low concentration eluents employed are either formic acid/water or 0.005 mol/L solution of 1-pentane sulfonn ic acid. In both cases chromatographic separation was achieved through a linear gradient elution using methanol/water (7/3) as the high concentration eluent. We also describe the variation of retention volumes of these compounds as a function of the pH of the mobile phase. pH values ranged from 1.5 to 4.0 and were obtained by adding either formic acid or NaOH respectively to the low concentration eluent. Tryptophan and its metabolites were detected fluorometrically. All compounds show a linear response in the pg to μg range. The chromatographic separation achieved allows a concurrent measurement of tryptophan and its main indoleamine metabolites which coupled to the high sensitivity of fluorometric detection permits a direct estimation of these metabolic pathways in brain tissue.