Ion chromatographic separation of alkylsulphonic acids with conductivity detection

Summary An ion chromatographic (IC) method has been developed for the separation of alkylsulphonic acid. Two different stationary phases, silica-based and polymer-based ion-exchange resins, were studied using pure ion exchange and/or hydrophobic interaction mechanisms. Correlations between analyte hydrophobicity and eluent polarity were made in order to investigate the possibility of changing the dominant separation mechanism by varying the eluent composition. The alkyl chain lengths of the sulphonic acids analysed ranged from C₁ to C₉. Detection limits in the submicromolar range were obtained by suppressed conductivity detection.     

Analysis of alkyl sulfonates and carboxylates using high performance ion chromatography

Summary A simple and general method for the determination of both C₁−C₁₀ alkyl sulfonates and alkyl carboxylates using the same basic methodology, based on High Performance ion Chromatography, using relatively simple eluent systems allowing fast analysis with both high sensitivity and accuracy is presented.     

Peak parking technique for the simultaneous determination of anions and cations

An ion chromatography method is described for the simultaneous determination of anions (Cl^–, NO₃^–, and SO₄^2–) and cations (Na⁺, NH₄⁺, K⁺, Mg²⁺, and Ca²⁺) using a single pump, a single eluent and a single detector. An anion-exchange column modified with chondroitin sulfate C facilitated the elution of the above three anions using 5 mM tartaric acid as the eluent in isocratic mode, whereas the same eluent facilitated the separation of the above five cations on a commercially-available cation-exchange column. The separation columns were connected in series via two six-port switching valves, so the required cation-exchange or anion-exchange separation could be carried out by selecting the appropriate positions for the switching valves. The separations were completed in 30 min.     

Retention of some glucosinolates in anion exchange chromatography. Part I: Qualitative study on a silica-trialkylammonium exchanger

Summary The retention and separation of glucosinolates, as organic anions, were studied on a silica-based strong anion exchanger under isocratic elution conditions. All glucosinolates carry the same functional ionic group (-OSO ₃ ^– ), however they do not have the same retention in anion exchange chromatography. The plots of capacity factors of organic anions versus the reciprocal of eluent ion concentration show good linearity. From the slope and y-intercept data the major retention mechanisms are interpreted as ion exchange and reversed-phase interactions. The effects of nature and concentration of the eluent ion and the influence of organic modifier addition to the aqueous buffered mobile phase are also investigated. Direct and indirect UV detection were used.Our results open the way for the development of new systems for intact glucosinolate analysis which are easier to use than the present ion-pairing chromatographic method.     

Histidine as a dipolar eluent in ion chromatography of aliphatic amines

Summary Acidified L-histidine was found to be a suitable eluent in membrane-suppressed cation chromatography for the separation of aliphatic mono-amines (methyl, ethyl, trimethyl, triethyl, tripropylamines). Based on the protonation equilibrium of amines with the histidine eluent, a simplified separation method was developed, in which the eluent can act as a source of diprotic cations and analytes as monoprotic amines. On detection, the suppressor converts the eluent by deprotonation to its dipolar form with minimum conductivity at the pH of the isoelectric point of histidine, (pl 7.56). Efficient separations were observed under isocratic development with an eluent concentration of C_HIS≤2 mM and a pH below 2.0. The effect of eluent concentration and number of carbon atoms in the aliphatic chain on the retention and the resolution was determined. Selectivity data of the separation system are also presented. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Study of pH dependence using molecular modeling in reversed-phase liquid chromatography of 1-phenyl-3-methyl-5-pyrazolone derivatives of aldose

A series of 1-phenyl-3-methyl-5-pyrazolone (PMP) aldose was eluted on a C₁₈ column with an acetonitrile-phosphate buffer over the range of pH 2-10 in reversed phase liquid chromatography. The relationship between the retention factor, k, and the eluent pH was expressed by an equilibrium scheme representing the probable retention process. The experimental data were fitted to the correlation curve drawn according to the equation containing the formation of the hydrogen bond with a hydroxonium ion in the eluent and the ionization of the oxygen atoms in the pyrazolone rings of the solute.The conformational changes of PMP-aldose according with the eluent pH were corroborated by computer-aided molecular modeling using quantum chemical calculations. The retention behavior was elucidated as a function of the geometrical feature of the PMP-aldose.     

Separation of urea and other polar compounds by HPLC

Summary Urea, glycolic and aminoxyacetic acid amides are the polar metabolites of 2-acetyl-3-phenyl-tetrahydro-1,2,4-oxadiazin-5-one (RGH 4615). They cannot be separated on octadecyl-, cyanosilyl- or amino-phase columns, but silica, used with C₃-C₄ alcohol and water mixtures as the eluent permits their separation. Besides refractive index detection and on-line radioactivity measurement the metabolic formation of¹⁴C-urea was proved by enzymatic cleavage into¹⁴CO₂.     

Portable,lightweight, low power,ion chromatographic system with open tubular capillary columns

Basic operation principles of a lightweight, low power, low cost, portable ion chromatograph utilizing open tubular ion chromatography in capillary columns coated with multi-layer polymeric stationary phases are demonstrated. A minimalistic configuration of a portable IC instrument was developed that does not require any chromatographic eluent delivery system, nor sample injection device as it uses gravity-based eluent flow and hydrodynamic sample injection adopted from capillary electrophoresis. As a detection device, an inexpensive commercially available capacitance sensor is used that has been shown to be a suitable substitute for contactless conductivity detection in capillary separation systems. The built-in temperature sensor allows for baseline drift correction typically encountered in conductivity/capacitance measurements without thermostating device. The whole instrument does not require any power supply for its operation, except the detection and data acquisition part that is provided by a USB port of a Netbook computer. It is extremely lightweight, its total weight including the Netbook computer is less than 2.5 kg and it can be continuously operated for more than 8 h. Several parameters of the instrument, such as detection cell design, eluent delivery systems and data treatment were optimized as well as the composition of eluent for non-suppressed ion chromatographic analysis of common inorganic cations (Na⁺, NH₄⁺, K⁺, Cs⁺, Ca²⁺, Mg²⁺, transition metals). Low conductivity eluents based on weakly complexing organic acids such as tartaric, oxalic or pyridine-2,6-dicarboxylic acids were used with contactless capacitance detection for simultaneous separation of mono- and divalent cations. Separation of Na⁺ and NH₄⁺ cations was optimized by addition of 18-crown-6 to the eluent. The best separation of 6 metal cations commonly present in various environmental samples was accomplished in less than 30 min using a 1.75 mM pyridine-2,6-dicarboxylic acid and 3 mM 18-crown-6 eluent with excellent repeatability (below 2%) and detection limits in the low micromolar range. The analysis of field samples is demonstrated; the concentrations of common inorganic cations in river water, mineral water and snow samples were determined.     

Application of Artificial Neural Network and Multiple Linear Regression Retention Models for Optimization of Separation in Ion Chromatography by Using Several Criteria Functions

This work focuses on problems regarding empirical retention modelling and optimization of separation in ion chromatography. Influences of eluent flow rate and concentration of eluent competing ion (OH^–) on separation of seven inorganic anions (fluoride, chloride, nitrite, sulphate, bromide, nitrate, and phosphate) were investigated. Artificial neural networks and multiple linear regression retention models in combination with several criteria functions were used and compared in global optimization process. It can be seen that general recommendations for optimization of separation in ion chromatography is application of chromatography exponential function criterion in combination with artificial neural networks retention model.     

Separation of unmodified α-amino acid enantiomers by reverse phase HPLC

Summary A novel chiral phase system is presented for the resolution of unmodified α-amino acid racemates which is composed of a reverse phase packing coated with N-alkyl-L-hydroxyproline (where alkyl is n-C₇H₁₅-, n-C₁₀H₂₁- and n-C₁₆H₃₃-) and a hydro-organic eluent containing copper(II)acetate. The factors controlling retention and enantioselectivity such as concentration of Cu(II)ion and pH of the eluent, addition of NH₄Ac to the eluent, type and content of organic solvent of hydro-organic eluent and column temperature were examined. The extremely high enantioselectivity observed (α up to 16) is assumed to be caused by a three site sorbate-sorbent interaction involving bidentate coordination of two amino acids to a Cu(II)ion and hydrophobic attractions between hydrocarbon side chains of amino acids and the n-octadecyl groups of the support. The efficiency and selectivity of the system permits resolution of up to 7 racemic amino acids into enantiomers within 35 minutes on a conventional HPLC apparatus. 13th publication in the series dealing with the ligand-exchange chromatography. Previous publication: ref. [1].     

Fast ion chromatography using short anion exchange columns

A fast ion chromatographic system is described which uses shorter column lengths and compares various eluent profiles in order to maximise the performance without sacrificing the chromatographic resolution. Both isocratic and gradient elution profiles were considered to find the most efficient mode of separation. The separation and determination of seven target anions (chloride, chlorate, nitrate, chromate, sulfate, thiocyanate and perchlorate) was achieved using a short (4 mm ID, 50 mm long) column packed with Dionex AS20 high-capacity anion exchange material. A hydroxide eluent was used at an initial concentration of 25 mM (at a flow-rate of 1.0 mL/min) and two performance maxima were found. The maximum efficiency occurred at a normalised gradient ramp rate of 5 mM/t₀, resulting in a peak capacity of 16, while the fastest separation (<3 min) occurred at a normalised ramp rate of 30 mM/t₀. The retention time, peak width and resolution using the different eluent profiles on varying column lengths is also compared. Further investigations in this study determined that the highest peak capacity separation under gradient conditions could be approximated using an isocratic separation. The advantage of using this novel approach to approximate the maximum efficiency separation removes the need for column re-equilibration that is required for gradient elution resulting in faster analyses and enhanced sample throughput, with benefits in particular for multidimensional chromatography.     

Anion chromatography in open tubular capillary columns with indirect fluorometric detection

Summary A C₈ reversed-phase open tubular capillary column is dynamically modified with cetylpyridinium chloride to provide anion-exchange sites. Sodium salicylate is used as the eluent at the micromolar concentration range. The low capacity of the dynamically modified column allows the elution of common anions with k′ values similar to those in conventional ion chromatography. The reduced salicylate concentration allows good detectability in the indirect fluorescence mode, even though the baseline noise increased because the fluorescence intensity is lower. The noise level is equivalent to one picogram of non-fluorescing anions.     

Use of Database of Plots of Pesticide Retention (R F ) Against Mobile-Phase Compositions for Fractionation of a Mixture of Pesticides by Micropreparative Thin-Layer Chromatography

Relationships between R _F values and mobile-phase composition have been determined for urea herbicides and fungicides in normal-phase systems (NP) of the type silica-nonpolar or weakly polar diluent (heptane, toluene, diisopropyl ether) – polar modifier (ethyl acetate, tetrahydrofuran, dioxane, ethyl-methyl ketone and 2-propanol). These relationships constitute a retention database which has enabled to choose optimum systems for preliminary fractionation of a multicomponent mixture of pesticides by zonal micropreparative TLC. The mixture was applied from the edge of the layer in the frontal + elution mode which increased the separation efficiency because or displacement effects. The separated simpler fractions were applied to a silica plate and rechromatographed. The plate was videoscanned, furnishing a real picture of the plate showing preliminary separation of the simpler pesticide fractions. Complete separation of the fractions was carried out by two-dimensional thin-layer chromatography on plates with chemically bonded-cyanopropyl silica stationary phase using non-aqueous eluent in the first direction and aqueous reversed-phase eluent in the second direction.     

Group separation of non-sulphated and 3-sulphated bile acids by disposable anion-exchange cartridges

Summary A rapid and simple method has been developed for the group fractionation of the major unsulphated and mono-sulphated bile acids in human body fluids. After extraction with Bond Elut C₁₈ cartridges, the bile acids are separated into the unconjugated, glycine-, taurine- and sulphate-conjugated forms on pre-packed Bond Elut SAX columns by increasing the ionic strength of the methanol-acetate buffer eluent. The procedure was found to be accurate and reproducible and to give complete resolution between the different groups. The levels of 3-sulphate bile acids in human serum and urine from patients with liver disease were determined by high-performance liquid chromatography, after group separation and solvolysis of the sulphate fraction.     

Separation of Amines as their 6-Methyl-2-phenyl-4-quinolinecarboxylic Acid N-Hydroxysuccinimide Ester Derivatives by High Performance Liquid Chromatography

6-Methyl-2-phenyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester (MPQC-OSu) has been developed for precolumn derivatization of aliphatic amines in HPLC. The great difference between the fluorescence quantum yield of MPQC-OSu derivative and that of MPQC-OSu hydrolysate, 6-methyl-2-phenyl-4-quinolinecarboxylic acid (MPQC) makes this reagent suitable for amine-labeling, followed by chromatographic separation. In pH8.5 borate buffer, MPQC-OSu reacted with amines at 60°C for 12 min to form highly fluorescent carboxamides and excess reagent hydrolysed to MPQC. The chromatographic behavior of amine derivatives with MPQC-OSu has been investigated using HPLC on C₁₈ and C₈ columns, respectively, with fluorescence detection at 340–402nm. A baseline separation of isopropanolamine, methylamine, ethylamine, n-propylamine, n-butylamine, n-amylamine and n-hexylamine was obtained in 25min using isocratic elution on a C₁₈ column using methanol-water (70:30, v/v) as eluent. Detection limits were in the range 0.13–1 nM.Acknowledgements This research was supported by the National Natural Science Foundation of China, the Foundation of Education Ministry of China and Chengguang Project of Wuhan, China.     

Prediction of the effects of methanol and competing ion concentration on retention in the ion chromatographic separation of anionic and cationic pharmaceutically related compounds

The mixed-mode separation of a selection of anionic and cationic pharmaceutically related compounds is studied using ion-exchange columns and eluents consisting of ionic salts (potassium hydroxide or methanesulfonic acid) and an organic modifier (methanol). All separations were performed using commercially available ion-exchange columns and an ion chromatography instrument modified to allow introduction of methanol into the eluent without introducing compatibility problems with the eluent generation system. Isocratic retention prediction was undertaken over the two-dimensional space defined by the concentration of the competing ion and the percentage of organic modifier in the eluent. Various empirical models describing the observed relationships between analyte retention and both the competing ion concentration and the percentage of methanol were evaluated, with the resultant model being capable of describing the separation, including peak width, over the entire experimental space based on six initial experiments. Average errors in retention time and peak width were less than 6% and 27%, respectively, for runs taken from both inside and outside of the experimental space. Separations performed under methanol gradient conditions (while holding the competing ion concentration constant) were also modelled. The observed effect on retention of varying the methanol composition differed between analytes with several analytes exhibiting increased retention with increased percentage methanol in the eluent. An empirical model was derived based on integration of the observed t_R vs. %methanol plot for each analyte. A combination of the isocratic and gradient models allowed for the prediction of retention time using multi-step methanol gradient profiles with average errors in predicted retention times being less than 4% over 30 different 2- and 3-step gradient profiles for anions and less than 6% over 14 different 2- and 3-step gradient profiles for cations. A modified peak compression model was used to estimate peak widths under these conditions. This provided adequate width prediction with the average error between observed and predicted peak widths being less than 15% for 40 1-, 2- and 3-step gradients for anions and less than 13% over 14 1-, 2- and 3-step gradients for cations.     

Interconversion of gradient and isocratic retention data in reversed-phase liquid chromatography: Effect of the uptake of eluent modifier on the retention of analytes

The experimental technique of mass spectrometric tracer pulse chromatography was used to study the effect of the sorption of eluent components by a C₁₈-bonded silica RPLC packing on the retention of a series of test analytes during isocratic and gradient elution experiments. The analytes of interest were a substituted phenol, a substituted nitroaniline, an anti-malaria drug, tetrahydrofuran, and methanol. The eluent used was a mixture of acetonitrile and water. The solutes and isotopically labeled eluent components were injected at fixed time intervals during each gradient run. The mass specific detector allowed the assignment of individual analyte peaks even when there was overlap in the chromatograms from successive injections. Thus, the retention time of each analyte could be determined as a function of gradient slope and initial eluent composition at the time of each injection. Experimental gradient retention time data were then compared with the calculated results from two theoretical models. The first model assumed the velocity of the mobile phase and eluent were equal. The second and most realistic model assumed the velocity of the eluent was less than the velocity of the mobile phase due to the uptake of eluent by the stationary phase. Gradient retention times predicted by the two models were reasonably accurate with the sorption model giving slightly more accurate values. Inverse calculations, i.e., calculation of isocratic retention factors from gradient elution data were also carried out with very similar results. That is, the model allowing for the uptake of eluent was slightly more accurate than the model assuming no eluent-stationary phase interaction.     

Characterization and separation of Al13 species using gel-filtration chromatography

A polyaluminum chloride (PAC) sample was prepared using a slow alkaline titration method. The Bio-Gel P-100 gel column chromatographic technique was used to separate and characterize the various forms of aluminum present in the prepared PAC solution. The effluents from a gel column were monitored using online chemical method: Al-Ferron timed complexation spectrophotometry and by ²⁷Al nuclear magnetic resonance (NMR) spectroscopy. Effects of different experimental conditions such as eluent flow rate, ionic strength and pH on separation of Al₁₃ were investigated. Experimental results indicated that molecule size exclusion was not the only parameter affecting the column chromatographic separation efficiency of Al₁₃ but molecule charge as well. Reducing the eluent flow rate, increasing the ionic strength and suitable pH resulted in increase in the separation efficiency. Experimental results clearly indicated that by varying the experimental conditions, it is possible to produce pure Al₁₃ species using a gel column chromatographic technique.     

Study of two-dimensional liquid chromatography with high temperature NPLC and room temperature RPLC

To overcome the peak band broadening and to increase the peak capacity and separation efficiency of a two-dimensional liquid chromatographic system, a high-temperature normal phase liquid chromatography (HTNPLC) was used as the first dimension (1^st-D), and a RPLC was used as the second dimension (2^nd-D). The sample was first separated on the 1^st-D CN column and the primary eluent stored in the sampling-loop system alternatively (in HTNPLC×RPLC mode) or selectively (in HTNPLC/RPLC mode) and was then transferred to 2^nd-D C₁₈ column for further separation. The resolution and separation efficiency of the systems were greatly improved. The systems were evaluated by analyzing several polycyclic aromatic hydrocarbons and Glycyrrhiza uralensis extract. __________ Translated from Journal of Instrumental Analysis, 2008, 27(1) (in Chinese)     

Mixed-mode liquid chromatography of aliphatic anionic surfactants with a naphthalenetrisulfonate mobile phase

Summary Separation of three classes of anionic surfactants (alkyl sulfates, alkanesulfonates, and alkyl phosphates) are achieved on a mixed-mode reversed phase (RP) phenyl/-anion exchange column using a naphthalenetrisulfonate (NTS)-acetonitrile (ACN) mobile phase via indirect photometric, indirect fluorometric, direct or indirect conductivity detection. Mixtures of C₅–C₁₈ sulfates, C₆–C₁₈ sulfonates, and C₁–C₄ phosphates (mono- and di-ester) can be separated in less than 20, 24, and 20 min respectively. Although hydrophobic effects are more pronounced in mixed-mode chromatography, equivalent-per-equivalent exchange of analysis and eluent ion is still required for sensitive indirect photometric, fluorometric, or conductivity detection. The detection limits of alkyl sulfates and alkanesulfonates are in the range of 3–15 pmoles which are at least an order of magnitude better than suppressed conductivity detection. The determination of surfactants in a variety of real samples is also presented.