Ion-interaction Chromatography Research Articles

Separation and determination of trace level terbium in gadolinium matrix by ion interaction chromatography

ABSTRACT A gradient elution-based ion interaction chromatography (IIC) method was developed for the determination of trace terbium (Tb) in a gadolinium (Gd) matrix. A reverse-phase column dynamically coated with camphor sulfonic acid (CSA) and α-hydroxybutyric acid (α-HIBA) as the eluent was utilized. Through a comprehensive study, an optimized gradient profile was established, which improved the separation between Tb and Gd. The limit of detection (LOD) for Tb was 2 ng/mL, and the standard deviation was 2% (n = 3) for 0.1 µg/mL Tb in the presence of 1084 µg/mL Gd. With the developed method, trace Tb determination without prior matrix separation was possible.

Regulatory Perspective Reverse Engineering Analysis of the Mast Cell Stabilizer and the Histamine Receptor Antagonist (Olopatadine HCl): Instrumental and Classical Methods for Multiple Formulations.

This study evaluates the unknown qualitative (Q1) and quantitative (Q2) formulas for nasal spray and ophthalmic solution formulations of olopatadine HCl by classical and instrumental techniques to match the generic formula with reference-listed drugs to avoid clinical study. Reverse engineering of olopatadine HCl nasal spray 0.6% and ophthalmic solution 0.1, 0.2% formulations was accurately quantified using a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. Both formulations possess similar components, namely ethylenediaminetetraacetic acid (EDTA), benzalkonium chloride (BKC), sodium chloride (NaCl), and dibasic sodium phosphate (DSP). These components were qualitatively and quantitatively determined using the HPLC, osmometry, and titration techniques. With derivatization techniques, EDTA, BKC, and DSP were determined by ion-interaction chromatography. NaCl in the formulation was quantified by measuring the osmolality and using the subtraction method. A titration method was also used. All the employed methods were linear, accurate, precise, and specific. The correlation coefficient was >0.999 for all components in all the methods. The recovery results ranged from 99.1 to 99.7% for EDTA, 99.1-99.4% for BKC, 99.8-100.8% for DSP, and 99.7-100.1% for NaCl. The obtained % relative standard deviation for precision was 0.9% for EDTA, 0.6% for BKC, 0.9% for DSP, and 1.34% for NaCl. The specificity of the methods in the presence of other components, diluent, and the mobile phase was confirmed, and the analytes were specific.

Separation of Rare Earth Elements (REE) by Ion Interaction Chromatography (IIC) Using Diglycolic Acid (ODA) as a Complexing Agent

The possibility of rare earth elements (REE) separation by ion interaction chromatography (IIC) employing their complexes with diglycolic acid (ODA) in anion exchange mode has been studied theoretically and experimentally. Calculations, assuming that only trivalent complex is significantly uptaken by the stationary phase, indicated that at at pH 4–6, the retention in the lanthanide series should increase from La to the Tb–Dy–Ho region with yttrium showing apparent atomic number (App.At.No.) of 67½ and then decrease with further increase of atomic number. Chromatographic experiments in the system: Column: Eternity C18—mobile phase 5 mM ODA/8.6 mM TBAOH/0.6 mM HNO3; pH 4.60 confirmed theoretically predictions. It was found that scandium at pH ≥ 4.0 elutes in front of the whole REE group but at low pH enters the region of light lanthanides. The non-monotonical change of affinity of the REE with the increase of atomic number results in quite unusual order of elution of REE namely: Sc < La < Ce < Lu < Pr < Yb < Nd < Tm < Sm < Eu < Er≈Y < Gd < Ho < Tb≈Dy.

Fluoroalkylamines: Novel, Highly Volatile, Fast-Equilibrating, and Electrospray Ionization-Mass Spectrometry Signal-Enhancing Cationic Ion-Interaction Reagents.

A new class of cationic ion-interaction reagents for reversed-phase chromatography is introduced in the present work. Compounds belonging to a homologous series of linear fluoroalkyl chains including trifluoroethylamine (TFEAm), pentafluoropropylamine (PFPAm), heptafluorobutylamine (HFBAm), and nonafluoropentylamine (NFPAm) were tested and compared with ammonia and triethylamine (TEA) for the separation of selected organic acids of general interest such as the herbicides glyphosate, ethephon, and fosamine and arsenic metabolites methylarsonic acid and dimethylarsinic acid as well as other compounds. Depending on the carbon and fluorine atom number, the fluoroalkylamines were shown to be effective cationic ion-interaction reagents, significantly enhancing the retention of organic acids on a C18 reversed-phase column. Contrary to the general behavior of ion-interaction reagents (a broader term than ion-pairing reagent), significant (up to 5-fold) and consistent enhancement in the electrospray ionization mass spectrometry signal (ESI-MS) was observed relative to ammonia and triethylamine. Overall, among the tested series HFBAm was found to offer the best overall properties among the tested series as it provided a good compromise between column equilibration time (ca. 25 column volumes) and retention behavior (up to a 10-fold increase in the retention factor of acids relative to ammonia) while providing the same general advantages found for the fluoroalkylamines such as fast washout times from the ESIMS system (ca. 30 min) and a 3–5-fold signal enhancement. The fluoroalkylamines are a new class of cationic ion-interaction reagents with clear advantages over the currently employed alkylamines and may revive the general interest in ion-interaction chromatography.

Selective separation of yttrium from rare earth elements by ion interaction chromatography. Analytical and larger scale applications

ABSTRACTNew ion interaction chromatographic (IIC) method employing Kromasil 100 C18 column and tetra-n-butylammonium hydroxide (TBAOH) as an ion interaction reagent (IIR) is able to separate yttrium from Rare Earth Elements (REE) as anionic complexes with nitrilotriacetic acid (NTA). New method for the chromatographic determination of Y in REE mixture was devised and validated by the analysis of the certified reference material (CRM). Potential possibilities of the new chromatographic system for larger scale applications including macro-micro events were demonstrated.

Ion interaction chromatography of anions in alkaline solutions: effect of temperature and the kind of stationary phase

ABSTRACTEffect of temperature (5°–65°C) on the separation of 11 inorganic anions by ion interaction chromatography (IIC) was studied employing RP C18 and C PhenylHexyl columns and aqueous mobile phase: 2.8 mM NaHCO3 + 0.7 mM TBAOH (tetra-n-butylammonium hydroxide). The apparent enthalpy changes, ΔH for hydrophobic ions like I−, SCN−, and ClO4 − largely exceeded 3 kcal/mole suggesting that added to ion exchange they are retained by hydrophobic adsorption. Unlike conventional strongly basic anion exchangers, our system can be used at elevated temperatures with alkaline eluents without irreversible damaging the column.

Determination of rare earth elements at trace levels in uranium process stream samples by ion-interaction chromatography

Determination of rare-earth elements like Ce, Sm, Eu, Gd, Dy, Er at trace levels in uranium process stream samples has been investigated by ion-interaction chromatography using C18 column, cation exchange based pre-concentration column (PCC). A mixture of α-hydroxy-iso-butyric acid and octane sulfonic acid is used as eluent after separation of uranium by solvent extraction. The trapping of analytes by PCC is found to be maximum in the pH range of 2.9–3.7 of sample solution. The spiking standard recovery for each analyte was found to be more than 90% and the method can be employed regularly.

Identification and determination of selenohomolanthionine – The major selenium compound in Torula yeast

Torula yeast (Candida utilis) was found to metabolize selenium in a totally different way to Brewer’s yeast (S. cerevisiae) leading to the biosynthesis of selenohomolanthionine (SeHLan), a major selenium compound accounting for 60–80% of the total selenium. The identity of SeHLan was confirmed by retention time matching in hydrophilic ion interaction chromatography (HILIC) with inductively coupled plasma mass spectrometric detection (ICP MS) using a custom synthesized standard molecule and by HILIC – Orbitrap MS and MS-MS fragmentation. Selenohomolanthionine escapes the current assays for the organic character of Se-rich yeast based on the protein-bound selenomethionine determination. A HILIC – ICP MS method was developed for the quantitative determination of selenohomolanthionine in yeast supplements with a detection limit of 146ng/g.

Testing the capability of a polynomial-modified gaussian model in the description and simulation of chromatographic peaks of amlodipine and its impurity in ion-interaction chromatography.

In this paper, the capability of a polynomial-modified Gaussian model to relate the peak shape of basic analytes, amlodipine, and its impurity A, with the change of chromatographic conditions was tested. For the accurate simulation of real chromatographic peaks the authors proposed the three-step procedure based on indirect modeling of peak width at 10% of peak height (W0.1), individual values of left-half width (A) and right-half width (B), number of theoretical plates (N), and tailing factor (Tf). The values of retention factors corresponding to the peak beginning (k(B)), peak apex (k(A)), peak ending (k(E)), and peak heights (H0) of the analytes were directly modeled. Then, the investigated experimental domain was divided to acquire a grid of appropriate density, which allowed the subsequent calculation of W0.1, A, B, N, and Tf. On the basis of the predicted results for Tf and N, as well as the defined criteria for the simulation the following conditions were selected: 33% acetonitrile/67% aqueous phase (55 mM perchloric acid, pH 2.2) at 40°C column temperature. Perfect agreement between predicted and experimental values was obtained confirming the ability of polynomial modified Gaussian model and three-step procedure to successfully simulate the real chromatograms in ion-interaction chromatography.

Surface-Enhanced Raman Spectroscopy Detection of Ionic Solutes by Surfactant-Mediated Adsorption to a Hydrophobic Surface

Chemical modification of the surfaces of surface-enhanced Raman spectroscopy (SERS)-active metals with thin molecular layers expands the variety of molecular species that can be attracted to the SERS surface from solution. This approach can provide selective detection of new classes of molecules that would not otherwise be detectable through direct interaction with a SERS-active metal. For example, polycyclic aromatic compounds can be attracted from aqueous solution to gold or silver SERS substrates that are modified with alkylsilanes or alkanethiols. While n-alkane monolayers attract hydrophobic solutes to a SERS-active surface, they are not well suited to adsorbing more water-soluble, ionized species from solution. In this work, we address SERS detection of ionic solutes by applying the principles of ion-interaction chromatography, where a charged surfactant is added to solution and adsorbs to an n-alkane-modified, SERS-active surface. The adsorbed charged surfactant serves to attract an ionic solute of opposite charge to the surface, where it can be detected. This concept was tested with a model anionic solute, 3-nitrobenzenesulfonate (NBS(-)), with a cationic surfactant, cetylpyridinium (CP(+)), that adsorbs to a 1-dodecanethiol (C12)-modified silver surface. The interfacial populations of both the surfactant and anionic solute can be determined simultaneously from SERS spectra. The adsorption equilibrium of CP(+) to the C12 surface was fit to a Langmuir model, and the effect of supporting electrolyte on its adsorption equilibria was also investigated by SERS. The retention of NBS(-) at the C12 surface depends on the concentration of CP(+). The binding of NBS(-) to adsorbed CP(+) is described by an ion-interaction model that includes competition for the NBS(-) population due to association with surfactant ions in solution. While the strength of this binding interaction is not as great as the hydrophobic interactions that drive aromatic hydrocarbons to hydrophobic SERS surfaces, SERS detection of analyte ions by this approach could be accomplished at concentrations two orders of magnitude lower compared with Raman detection in free solution.

Quantitative Confocal Raman Microscopy Study of Ion-Interaction Retention at Reversed-Phase Chromatographic Interfaces

Ion-interaction chromatography utilizes the addition of amphiphilic (surfactant) molecules to the mobile-phase solution in order to bring about the retention and separation of hydrophilic ionic analytes on reversed-phase liquid chromatographic stationary phases. The mechanism by which retention and separation in ion-interaction chromatography is accomplished remains unclear, although two primary mechanisms of this phenomenon have been proposed: preadsorption of the hydrophobic counterions to the hydrophobic stationary phase followed by interaction with the ionic analyte or adsorption of ion-paired species to the hydrophobic surface. There is evidence that both mechanisms contribute to ionic retention and separations. In this work, ion interactions at a C(18) stationary-phase interface are investigated by confocal Raman microscopy of individual stationary-phase particles, which is capable of quantifying surface concentrations of surfactants and model solutes. Raman spectra taken within the interior of the particle indicate that preadsorption of the hydrophobic surfactant cetylpyridinium to the stationary-phase interface is sensitive to ionic strength and concentration of organic modifier in the mobile phase. It was also determined that the presence of low concentrations of a model, ionic solute, nitrobenzenesulfonate, leads to greater adsorption of cetylpyridinium ion at the hydrophobic interface. As the concentration of nitrobenzenesulfonate is increased, the 1:1 increase in the cetylpyridinium ion interfacial concentration provides evidence of ion pairs forming at the stationary-phase surface.

Bioactivity and pharmacokinetics of two human serum albumin-thymosin alpha1-fusion proteins, rHSA-Talpha1 and rHSA-L-Talpha1, expressed in recombinant Pichia pastoris.

Thymosin-alpha1 (Talpha1) is indicated for the treatment of certain viral infections, including hepatitis B and C, and cancers, such as melanoma. In this paper, the fusion genes encoding human serum albumin (HSA) and Talpha1 with (rHSA-L-Talpha1) and without a linker peptide (rHSA-Talpha1) were constructed and overexpressed in P. pastoris. Through the process of ion interaction chromatography (Q-Sepharose F.F), hydrophobic interaction chromatography (Phenyl Sepharose HP) and affinity chromatography (Blue Sepharose F.F), the purity of fusion proteins was greater than 97%. In contrast to the reactivity of normal spleen cells to Con A, the data of in vitro murine spleen lymphocytes proliferation experiment suggested that spleen cells achieved a higher degree of T cell maturation after rHSA-L-Talpha1, rHSA-Talpha1 and Talpha1 treatments, respectively. Moreover, rHSA-L-Talpha1, rHSA-Talpha1 and Talpha1 can also antagonize dexamethasone-induced apoptosis of thymocyte sub-populations. In hydrocortisone-induced immunosuppression mice (in vivo experiments), after subcutaneous injections with two fusion proteins and Talpha1 for seven consecutive days, the net increment of body weight, the spleen index and the thymus index were significantly improved. Simultaneously, the increase in SOD level and the decrease in MDA level in plasma were observed. The pharmacokinetic data of rHSA-L-Talpha1 and rHSA-Talpha1 administered in rats showed an improved pharmacokinetic profile with a conspicuous prolonged half life. The analysis of bioactivity and pharmacokinetics suggested that fusion proteins rHSA-L-Talpha1 and rHSA-Talpha1 were new drug candidates.

Rapid Analysis of Nitrate and Nitrite by Ion-Interaction Chromatography on a Monolithic Column

Ion-interaction chromatography with direct conductivity detection has been used for analysis of nitrate and nitrite. Chromatographic separation was performed on a monolithic silica-based C18 column dynamically modified with tetrabutylammonium (TBA+). Using the optimized mobile phase, containing 2.0 mmol L−1 TBA+ and 0.8 mmol L−1 citrate (pH 6.0), delivered at a flow rate of 6.0 mL min−1, separation of five anions (chloride, nitrite, bromide, nitrate, and sulfate) was achieved in only 40 s at a column temperature of 30 °C. The detection limits for nitrate and nitrite were 0.74 and 0.92 mg L−1, respectively. The relative standard deviation (RSD, n = 5) of the retention times of nitrate and nitrite was 0.1% and RSD of chromatographic peak areas were 0.4 and 0.2%, respectively. The method was successfully used for analysis of the anions in groundwater. Recovery of nitrate and nitrite was 99.1 and 105%, respectively.

A reversed-phase ion-interaction chromatographic method for in-vitro estimation of the percutaneous absorption of water-soluble UV filters

An analytical method based on ion-interaction chromatography with UV detection for simultaneous in-vitro estimation of the percutaneous absorption of the most used water-soluble UV filters in sunscreen cosmetics is proposed. These UV filters were phenylbenzimidazole sulfonic acid, disodium phenyl dibenzimidazole tetrasulfonate, benzophenone-4, and terephthalylidene dicamphor sulfonic acid. The methodology is based on applying the sunscreen containing the target UV filters to human epidermis in a diffusion cell. Analytes are determined in the receptor solution. To ensure skin integrity, screening of the cells was carried out by analytical determination of a marker. Analytical variables such as percentage ethanol, concentration of ion-pairing agent, pH of the mobile phase, and temperature were studied in order to achieve high resolution of the chromatographic peaks in the lowest possible time of analysis. The conditions selected consisted of a mobile phase composed of 35:65 (v/v) ethanol-ammonium acetate buffer solution (pH 4, containing 50 mmol L(-1) tetra-n-butylammonium bromide). The chromatographic determination was carried out with the analytical column at 50 degrees C. UV detection was carried out at the maximum absorption wavelength for each analyte. The limit of detection (3s(y/x)/b) ranged from 16 to 65 ng mL(-1), depending on the analyte.

Ion-interaction Chromatography of Several Metallocyanide Complexes Stabilized by Adding Cyanide in a Neutral Mobile Phase

Standard solutions (at 10(-5) M levels) of Cu(I)- and Fe(II)-cyanide complexes were stabilized for at least 5 h using 0.5 mM cyanide solution (around pH 9) as a medium. Complexes of Cu(I)- and Fe(III)-cyanide also could be stabilized without any dissociation by adding 1 mM cyanide to an acetonitrile-water (18:82, v/v) mobile phase (pH 7.0) containing 10 mM tetra-n-propylammonium salt (TPA). Under the optimal conditions, the six complexes of Cu(I)-, Ag(I)-, Ni(II)-, Fe(II)-, Fe(III)- and Au(I)-cyanides were resolved from their mixtures within about 45 min, with well-shaped chromatographic peaks.

Separation and determination of metallocyanide complexes of Fe(II), Ni(II) and Co(III) by ion-interaction chromatography with membrane suppressed conductivity detection applied to analysis of oil refinery streams (sour water)

A separation and determination method for the analysis of cyanometallic complexes of Fe(II), Ni(II) and Co(III) was developed to be applied to the analysis of petroleum refinery streams (sour water). Ion-interaction chromatography was used employing an analytical column IonPac NS1 10 μm and a chromatographic system ICS 2500 equipped with a membrane conductivity suppression ASRS ultra 4 mm, both supplied by Dionex Corporation. The mobile phase was composed of 2 mmol l −1 TBAOH, 1 mmol l −1 Na 2CO 3, 0.1 mol l −1 NaCN and ACN (77:23, v/v), flowing at 0.7 ml min −1. At the optimized conditions, detection limits estimated by the calibration curve parameters and relative standard deviation were: 0.002 mg CN l −1 and 3.1% for Fe(CN) 6 4−; 0.003 mg CN l −1 and 2.5% for Ni(CN) 4 2− and 0.003 mg CN l −1 and 2.8% for Co(CN) 6 3−. Sour water samples without any pretreatment (except membrane filtration) from a petroleum refinery in Brazil were analyzed successfully by external calibration method.

Separation of Fexofenadine, Pseudoephedrine, Potential Impurities, and Degradation Products Using Ion Interaction Chromatography

Ion interaction chromatography has been demonstrated to be a viable separation scheme for a wide variety of small molecules. This separation mechanism has been shown to be effective for a complex mixture of analytes that range from uncharged to positively and/or negatively charged. The analytes present in fexofenadine‐D tablets are either uncharged or cationic. Therefore, if a single HPLC method is to be used for this difficult separation, a dual mechanistic system must be present so that the charged and neutral components are retained and separated. An ion interaction liquid chromatographic system that contains an anionic surfactant, such as sodium dodecyl sulfate (SDS), meets both of these requirements: a reversed stationary phase for a hydrophobic analyte and a fixed anionic charge site for a positively charged analyte. The effect that each mobile phase parameter may have on this complex separation was found to be crucial. These variables were studied and include: concentration of SDS, ionic strength, pH, concentration of phosphoric acid, concentration of organic modifier, column temperature, and different gradients. The results obtained in this study and the optimized separation is discussed.

Simultaneous determination of Cr(III) and Cr(VI) with prechelation of Cr(III) using phthalate by ion interaction chromatography with a C-18 column

Ion interaction chromatography has been successfully used for the simultaneous determination of Cr(III) and Cr(VI) in waste water. A C-18 column which had been dynamically coated with octylamine was used for the separation of Cr(III) and Cr(VI) based on anionic interaction. Cr(III) was chelated with potassium hydrogen phthalate (KHP) before injecting into the column since the Cr(III) did not exist in an anionic form like the Cr(VI) (Cr 2O 7 2−) presented at the optimum condition. The analytes were detected at 200 nm and linear relationship between absorption with the concentration of Cr(III) or Cr(VI) was 0.1–50 mg/L. Most of the interested interferences including alkali metals, heavy metals and organic materials have no significant effect on Cr(III)–KHP complexation and Cr(VI) stability, only NH 4 + and ascorbic acid yielded the serious effect on the Cr(VI) stability. The relative standard deviations calculated from both of peak area and retention time were 0.75–2.20%. The sensitivity of the method at the level concentration of sub mg/L enabled the simultaneous determination of Cr(III) and Cr(VI) contents in waste water samples without any special sample preparation step.

The Fractional Charge Approach in Ion‐Interaction Chromatography of Zwitterions: Influence of the Stationary Phase Concentration of the Ion Interaction Reagent and pH

The fractional charge approach to Ion‐interaction chromatography (IIC) of zwitterions was used to quantitatively explain their retention behavior as a function of the Ion Interaction Reagent (H) concentration in the stationary phase and also to account for the influence of the zwitterion ionization degree, according to the mobile phase pH. The theory was validated using two Hs and two reversed phase columns. The good predictive abilities of the retention equations lend strong support to the hypotheses made by the fractional charge approach in the IIC of zwitterions. For the investigated homologous series the increase in retention upon H addition can be quantitatively related to the estimated fractional charge. The estimates of the operative charges and bonded phase coverages are very reasonable since they make sense physically. This way, they offer a cross validation of the fractional charge approach, since they compare well with those obtained from the fitting of parallel retention data, obtained as a function of the mobile phase concentration of H. This confirms that retention modelling may instruct the chromatographer through the optimization procedure in the parameter space.

Investigation of the retention/pH profile of zwitterionic fluoroquinolones in reversed-phase and ion-interaction high performance liquid chromatography

The retention/pH profiles of three fluoroquinolones, ofloxacin, norfloxacin and ciprofloxacin, was investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC) and reversed-phase ion-interaction chromatography (RP-IIC), using an octadecylsilane stationary phase and acetonitrile as organic modifier. Sodium hexanesulphonate and tetrabutylammonium hydroxide were used as sources of counter ions in ion-interaction chromatography. The retention/pH profiles under in RP-HPLC were compared to the corresponding lipophilicity/pH profiles. Despite the rather hydrophilic nature of the three fluoroquinolones positive retention factors were obtained while there was a shift of the retention maximum towards more acidic pH values. This behavior was attributed mainly to non-hydrophobic silanophilic interactions with the silanized silica gel material of the stationary phase. In ion-interaction chromatography the effect of counter ions over a broad pH range was found to be ruled rather by the ion pair formation in the mobile phase which led to a drastic decrease in retention as a consequence of the disruption of the zwitterionic structure and thereupon the deliberation of a net charge in the molecules. At pH values at which zwitterionic structure was not favored both the ion-exchange and ion pair formation mechanisms were assumed to contribute to the retention.