Automatic generation of ultra-pure hydroxide eluent for carbohydrate analysis of environmental samples

High-performance anion-exchange chromatography coupled with pulsed amperometric detection under alkaline conditions has significantly improved our understanding of the function of carbohydrates in cycling of terrestrial carbon. The use of NaOH as an eluent has many advantages for anion analysis, but NaOH solutions are also an excellent trap for atmospheric CO2 resulting in eluent carbonate ion contamination. To minimize the carbonate contamination, eluent generation technology has been marketed that produces ultra-pure hydroxide (OH) eluents on demand. Application of this technology significantly reduced the amounts of OH present in the waste stream and provided faster chromatographic runs with greater sensitivity and precision for determining the monosaccharide composition from environmental samples. Minimizing the salt concentration to < or = 50 mm from acid hydrolysis extractions injected (25 microl injection volume) on column improved reproducibility of analyte retention times and separations. Eluent generation technology eliminated inconsistent OH eluent preparation that is often due to carbonate contamination. The contamination increases retention time variability for monosaccharides released by hydrolysis of biological samples.     

Improved determination of taurine by high-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD)

The advantages of the high selectivity of high-performance anion-exchange chromatography (HPAEC) and the sensitive response of taurine at a gold electrode with integrated pulsed amperometric detection (IPAD) have been combined, in order to establish a new analytical method for its determination in real matrices. Potential-time settings of the potential waveform were optimized in order to get the highest amperometric response. The separation of taurine in milk samples was achieved using an alkaline eluent (100 mM NaOH) containing 1 mM Ba(OAc)₂ and a column temperature of 15 °C. The inherent merits of using a barium-modified eluent, in terms of taurine separation and detection, are demonstrated. The enhancement in sensitivity under these experimental conditions makes it suitable for taurine determination in milk. Indeed, this method allows high recovery of taurine and satisfies the necessary requirements with respect to accuracy, repeatability and sensitivity with a detection limit of 50 nmol/L, which corresponds to 2.5 pmol. The taurine content in milk samples of some common mammals was evaluated, including human milk. In goats milk, the taurine content ranged from 46 to 91 mg/L, whereas human and buffalo milk samples exhibited an average content of 18 mg/L and 23 mg/L, respectively.     

Retention of alkali,alkaline earth and transition metals on an itaconic acid cation-exchange column. Eluent pH,ionic strength and temperature effects upon selectivity

The unusual selectivity of a methylene succinic (itaconic) acid modified polymeric column was investigated for the separation of alkali, alkaline earth, transition and heavy metals employing non-chelating inorganic eluents. The retention of selected metal ions on the column was investigated with simple HNO3 eluents and eluents prepared from KNO3 and KCl salts of varying pH (adjusted using HNO3). From these studies both the effect of eluent ionic strength and pH upon retention was evaluated for the itaconic acid stationary phase. The results obtained showed that despite slow exchange kinetics causing poor efficiencies, acceptable baseline separations of selected alkaline earth and transitions could be obtained under optimum conditions (the baseline separation of Mg(II), Ca(II), Mn(II), Cd(II), Zn(II) and Co(II) was possible using a 15 mM KNO3-5 mM KCl eluent at pH 3.50 in under 25 min). The use of an simple ionic strength step gradient was shown that facilitated the addition of Pb(II) to the above group of metal ions. An investigation into the effect of temperature upon peak efficiency and retention showed increased column temperature could be used to improve the resolution of closely eluting metal ions such as Ca(II) and Sr(II) and Ca(II) and Mn(II).     

Retention behavior of alkali, alkaline earth and transition metal cations in ion chromatography with an unmodified silica gel column

An unmodified silica gel (Develosil 30-5) column (150×4.6 mm I.D.) has been applied to the ion chromatographic separation of alkali, alkaline earth and transition metal cations. The retention behavior of the above cations on the bare substrate was investigated using a number of weak inorganic and organic acid eluents. During this investigation, several separations were achieved and the most suitable eluent conditions were identified. It was concluded that: (a) 1.5 mM HNO₃-0.5mM pyridine-2,6-dicar☐ylic acid eluent was the most effective for the simultaneous separation of common alkali and alkaline earth metal cations, (b) 1.5 mM oxalic acid eluent resulted in the best separation of alkali, alkaline earth, and transition metal cations, (c) 0.5 mM CuSO₄ eluent could be used for the separation of alkali metal cations alone and (d) 0.5 mM ethylenediamine-oxalic acid eluent at pH 5.5 resulted in themost efficient separation of both alkaline earth and transition metal cations.     

Liquid chromatographic studies of the effect of phosphate on the binding properties of silica-immobilized bovine serum albumin

High-performance liquid chromatography has been used to examine how phosphate ions affect the binding properties of bovine serum albumin (BSA) immobilized to porous silica. In doing this, the time dependence of the protein to reach conformational equilibrium is measured as a function of the concentration of phosphate in the eluent using the D- and L-isomers of tryptophan and kynurenine as solutes. The overall binding and chiral selectivity (alphaD,L) of the protein toward these solutes appear to be related to two types of effects: one being those that are site-selective and only influence the retention of the L-isomers and the other being those that are nonselective and influence the retention of both enantiomers. An interesting feature of the concentration-dependent data is a maximum in alphaD,L at intermediate phosphate concentrations (i.e., 10 to 50mM phosphate) indicative of both cooperative and antagonistic binding effects. Phosphate eluents within this concentration range provide selectivity advantages, and those at higher concentrations decrease the time required for the protein or column to reach equilibrium. A final set of studies has also been carried out using four alternate buffer systems (i.e., borate, carbonate, acetate, and arsenate eluents). Although the borate eluents affect the BSA's binding properties and alphaD,L similar to the phosphate eluents, the other buffers result in poor separations. Observations from this study are useful in helping to optimize separations carried out on immobilized BSA as well as addressing biological and mechanistic questions related to how anions influence the native binding properties of serum albumins.     

A versatile method for stable carbon isotope analysis of carbohydrates by high-performance liquid chromatography/isotope ratio mass spectrometry

We have developed a method to analyze stable carbon isotope ((13)C/(12)C) ratios in a variety of carbohydrates using high-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS). The chromatography is based on strong anion-exchange columns with low strength NaOH eluents. An eluent concentration of 1 mM resulted in low background signals and good separation of most of the typical plant neutral carbohydrates. We also show that more strongly bound carbohydrates such as acidic carbohydrates can be separated by inclusion of NO(3) (-) as an inorganic pusher ion in the eluent. Analyses of neutral carbohydrate concentrations and their stable carbon isotope ratios are shown for plant materials and marine sediment samples both at natural abundance and for (13)C-enriched samples. The main advantage of HPLC/IRMS analysis over traditional gas chromatography based methods is that no derivatization is needed resulting in simple sample treatment and improved accuracy and reproducibility.     

High sensitivity separation and detection of heparan sulfate disaccharides

Eight Delta-disaccharide standards from heparan sulfate/heparin were derivatized with the fluorophore 4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, hydrazide (BODIPY) via formation of a Schiff's base and separated using HPAEC on a Propac PA1 column with a linear salt gradient and isocratic 150 mM NaOH. Detection was with an in-line fluorescence detector. The standard deviation (sigma(n-1)) in retention times were 0.7-2% over nine runs. The limit of detection, was 100 fmol (100 x 10(-15)mol) of BODIPY labeled Delta-disaccharides, representing considerably improved detection compared to other fluorophore labeled derivatives and, unlike these, required no further purification steps. Separation and improved detection of BODIPY-Delta-disaccharide conjugates will assist the structural analysis of HS and the development of improved sequencing methodologies.     

Double gradient ion chromatography using short monolithic columns modified with a long chained zwitterionic carboxybetaine surfactant

The rapid separation of inorganic anions on short monolithic columns permanently coated with a long chained zwitterionic carboxybetaine-type surfactant is shown. The surfactant, N-dodecyl-N,N-(dimethylammonio)undecanoate (DDMAU), was used to coat 2.5, 5.0 and 10 cm long reversed-phase silica monoliths, resulting in a permanent zwitterionic exchange surface when used with aqueous based eluents. The unique structure of the surfactant results in a charge double layer structure on the surface of the stationary phase, with strong internal anionic and weak external cationic exchange groups. The dissociation of the weak external carboxylic acid group acts to shield the inner anionic exchange site, resulting in substantial effective capacity changes with eluent pH. Utilising this effect with the application of an eluent pH gradient, simultaneously combined with eluent flow-rate gradients, very rapid simultaneous separations of both weakly retained anions and strongly retained polarisable anions was possible, with up to 10-fold decreases in overall run times. Coating stability and retention times under isocratic and isofluentic eluent conditions were shown to be reproducible over >450 repeat injections, with peak efficiency values averaging 29,000 N/m for the 2.5 cm column and 42,000 N/m for the 10 cm monolithic column, again under isocratic elution conditions.     

Applicability of linear and nonlinear retention‐time models for reversed‐phase liquid chromatography separations of small molecules,peptides, and intact proteins

The applicability and predictive properties of the linear solvent strength model and two nonlinear retention‐time models, i.e., the quadratic model and the Neue model, were assessed for the separation of small molecules (phenol derivatives), peptides, and intact proteins. Retention‐time measurements were conducted in isocratic mode and gradient mode applying different gradient times and elution‐strength combinations. The quadratic model provided the most accurate retention‐factor predictions for small molecules (average absolute prediction error of 1.5%) and peptides separations (with a prediction error of 2.3%). An advantage of the Neue model is that it can provide accurate predictions based on only three gradient scouting runs, making tedious isocratic retention‐time measurements obsolete. For peptides, the use of gradient scouting runs in combination with the Neue model resulted in better prediction errors (<2.2%) compared to the use of isocratic runs. The applicability of the quadratic model is limited due to a complex combination of error and exponential functions. For protein separations, only a small elution window could be applied, which is due to the strong effect of the content of organic modifier on retention. Hence, the linear retention‐time behavior of intact proteins is well described by the linear solvent strength model. Prediction errors using gradient scouting runs were significantly lower (2.2%) than when using isocratic scouting runs (3.2%).     

Sub-ambient temperature effects on the separation of monosaccharides by high-performance anion-exchange chromatography with pulse amperometric detection: Application to marine chemistry

The effects of column temperature in the range 10–45°C using high-performance anion-exchange chromatography (HPAEC) and pulse amperometric detection are described for the determination of monosaccharides. The influence of temperature was tested with an isocratic elution of NaOH at concentrations varying from 2.5 to 20 mM and with a post-column addition of 1 M NaOH. The results showed that small changes of temperature greatly affect retention times and resolution (R_s) of monosaccharides and particularly those of the both pairs xylose–mannose and rhamnose–arabinose which cannot be simultaneously detected at usual room temperature (25°C). Our results suggest that a subambient temperature of 17°C and an eluent concentration of 19 mM are the more appropriate conditions for an acceptable separation (R_s rha/ara=1.02, R_s man/xyl=0.70) in a short analytical run time (35 min). The results showed that within the range of temperatures studied, enthalpy and entropy are invariant of temperature indicating that changes in the retention processes are mainly due to temperature than other associated changes in the system. This study demonstrated the importance of controlling temperature during HPAEC of monosaccharides, both to accomplish highly reproducible retention times and to achieve optimal separation of sugars. This method gave acceptable results for detection of marine sugars.     

Separation and determination of carbohydrates in drinks by ion chromatography with a self-regenerating suppressor and an evaporative light-scattering detector

Analysis of glucose and other carbohydrates are often performed by use of normal phase HPLC methods with acetonitrile as major eluent coupled with evaporative light-scattering detector (ELSD) or by use of anion-exchange ion chromatography (IC) methods with NaOH as eluent coupled with pulsed amperimetric electrochemical detector. In this work, a novel method for the determination of carbohydrates by IC in conjunction with a self-regenerating suppressor and an ELSD detector was investigated. Three carbohydrates (glucose, fructose, and sucrose) were separated using a KOH eluent generator to avoid the effect of carbon dioxide absorption in the alkaline eluent. Due to the use of the suppressor, non-volatile components were removed and a low salt background (K+ approximately 0.070 microg/mL) can be obtained so the suppressed eluent could directly go into an ELSD detector without obvious interference of inorganic salts. After examining the changes in retention and resolution, an optimized method was established (for IC: using 32 mM KOH as the eluent at a flow rate of 1 mL/min; for ELSD: operated at 95 degrees C, 4.0 bar nitrogen with a gas flow rate of 2.0 L/min) and the linearity, reproducibility, and the limit of detection (LOD) for the three carbohydrates were further evaluated. Regression equations revealed acceptable linearity (correlation coefficients=0.994-0.998) across the working-standard range (100-1000 microg/mL for glucose and sucrose, 150-1000 microg/mL for fructose) and LODs of glucose, fructose, and sucrose were 93, 126, and 90 microg/mL, respectively. This method has successfully been applied to the determination of the three carbohydrates in carbonated cola drinks and fruit juices. The recoveries were between 95 and 113% (n=3) for different carbohydrates.     

Histidine as a dipolar eluent component in cation chromatography: II. Prediction of retention data for alkaline and alkaline-earth ions

The utility of cation chromatography has been developed by the application of -histidine as a multiprotic and dipolar (zwitterionic) eluent component. The method simplifies the cation analysis. The chromatographic characteristics of this system were studied in detail with a view to determining the selectivity and the mechanism by which the cations (Na⁺, K⁺, Mg²⁺, Ca²⁺) are retained. Complete separations were observed in the isocratic run over the eluent concentration range 3.0–6.0 mM at pH below 2.0. Sensitive detection was achieved using suppressed conductivity at the pH of isoelectric point of the histidine. Retention equations are derived for mono- and divalent cations eluted from ion-exchange separation column with multiple ionic eluents. The theory is based on the extension of ion-exchange equilibrium by protonation equilibria. The selectivity data for analyte and eluent species are determined using the model from the experimental retention data by computer-assisted iterative calculations. The model was utilized to predict retention data. The results in three-dimensional retention surfaces together with species distribution graphs are presented.     

Ion-exclusion/cation-exchange chromatographic determination of common inorganic ions in human saliva by using an eluent containing zwitterionic surfactant

Ion-exclusion/cation-exchange chromatography with an eluent containing the bile salt-type zwitterionic surfactant CHAPS was performed in order to evaluate variations in anion (SO(4)(2-), NO(3)(-), and SCN(-)) and cation (Na(+), K(+), NH(4)(+), Mg(2+), and Ca(2+)) concentrations in human saliva. CHAPS prevents the adsorption of proteins to the stationary phase, i.e., weakly acidic cation-exchange resin, since it aggregates proteins without denaturing them. Addition of 1mM CHAPS to the eluent comprising 6mM tartaric acid and 7 mM 18-crown-6 yielded reproducible separations of anions and cations in protein-containing saliva. The resolutions of anions and cations were not significantly affected by the addition of CHAPS to the eluent. The concentrations of Na(+) and K(+) varied before and after meals; or that of SCN(-), upon smoking. The relative standard deviations of peak areas ranged from 0.3 to 5.1% in 1 day (n=20) and from 1.4 to 5.8% over 6 days (n=6).     

Determination of mono- and disaccharides in milk and milk products by high-performance anion-exchange chromatography with pulsed amperometric detection

A simple and sensitive liquid chromatographic method for the separation and quantification of mono- and disaccharides in raw- and processed-milk is described. Samples of cows’, buffalos’, sheeps’ and goats’ milk were analyzed upon clarification and appropriate dilution for the quantification of lactose, galactose, glucose and N-acetylglucosamine (GlcNAc). The separation was accomplished by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), using a gold working electrode and dilute alkaline eluents modified by a millimolar concentration of barium acetate. The eluent composition employed was designed to provide optimum separation with respect to the selected sample, without interference from the matrix components. The analytical method was successfully employed for the determination of mono- and disaccharides naturally occurring in dairy milk, mozzarella cheese and whey samples, with high sensitivity and accuracy.     

Determination of trace alkaline earth metals in brines using chelation ion chromatography with an iminodiacetic acid bonded silica column

The chromatographic behaviour of alkaline earth metals on iminodiacetic acid bonded silica was studied. It was found that the ionic strength of the eluent greatly affected both retention time and selectivity by controlling the extent to which either simple ion exchange or surface complexation was responsible for retention. With a 0.1 M KNO3 eluent, the retention order was Mg(II), Sr(II), Ca(II) and Ba(II), indicating a strong contribution to retention from ion exchange. However, when using a 1.5 M KNO3 eluent, Ba(II) was found to elute first, indicating complexation to be more dominant under these conditions (pH 4.2). The effect of the ionic strength of the sample was also studied and it was found that by matching the eluent cation with that of the sample matrix, efficient separations of alkaline earth metals in 1.0 M NaCl and KCl brines could be obtained without matrix system peaks. Using post-column reaction with o-cresolphthalein complexone, trace levels of Ca(II) and Mg(II) were determined in medicinal NaCl saline solution and laboratory-grade KCl.     

HPLC separation of all aldopentoses and aldohexoses on an anion-exchange stationary phase prepared from polystyrene-based copolymer and diamine: the effect of NaOH eluent concentration

To investigate the separations of all aldopentoses (ribose, arabinose, xylose and lyxose) and aldohexoses (glucose, galactose, allose, altrose, mannose, gulose, idose and talose) on the D? stationary phase prepared by the reaction of chloromethylated styrene-divinylbenzene copolymer and N,N,N',N'-tetramethyl-1,6-diaminohexane, we examined the effect of varying the concentration of the NaOH eluent on the elution orders. Separations of these aldoses were achieved using a 20 mM NaOH eluent. The elution behaviors of the aldoses were probably due to not only the individual pK(a) values, but also the chemical structures of the cyclic aldoses.     

Analysis of oligosaccharides using aminobonded silica gel and a ternary eluent with evaporative light scattering detection

Using two commercial, amino bonded, silica gel columns, binary eluents (methanol/water, acetonitrile/methanol) and ternary eluents (acetonitrile/methanol/water) are evaluated for their ability to separate oligosaccharides in comparison with classical acetonitrile/water eluents. Chromatographic data on glucose, maltose, raffinose, stachyose and maltodextrin are reported. Mobile phases consisting of acetonitrile/water or acetonitrile/methanol/water produce the best separations. Phase hydrolysis is evaluated using an evaporative light scattering detector. The reduction of the water content in the eluent stabilizes the aminocolumns and the life time of the amino columns should be improved with the ternary eluent. Moreover, a better detection limit can be obtained. Capabilties to run gradient elution without baseline drift are clearly demonstrated.     

Zwitterionic ion chromatography with carboxybetaine surfactant-coated particle packed and monolithic type columns

Both particle packed (25 cm x 0.46 cm I.D. SUPELCOSIL 5 microm C18) and monolithic type (10 cm x 0.46 cm I.D. Merck Chromolith Performance C18) reversed-phase substrates were dynamically coated with a carboxybetaine type zwitterionic surfactant ((dodecyldimethyl-amino) acetic acid) and investigated as stationary phases for use in zwitterionic ion chromatography (ZIC). Investigations into eluent concentration and pH were carried out using KCl eluents containing 0.2 mM of the carboxybetaine surfactant to stabilise the column coatings. It was found that eluent concentration decreased anion retention whilst simultaneously increasing peak efficiencies, which may be due to the dissociation of intra- and inter-molecular salts of the carboxybetaine surfactant under higher ionic strength conditions. The Effect of eluent pH was an increase in anion retention with decreased eluent pH due to the increased protonation of the weak acid terminal group of the carboxybetaine, causing both a relative increase in the positive charge of the stationary phase and less repulsion of the anions by the dissociated weak acid group. The carboxybetaine-coated monolithic phase was applied to rapid anion separations using elevated flow rates and flow rate gradients.     

On-line coupling of supercritical fluid extraction with high-performance liquid chromatography for the determination of explosives in vapour phases

An investigation into the selectivity of an iminodiacetic acid (IDA) modified silica gel column for transition and heavy metal ions using non-chelating inorganic eluents has been carried out. A number of eluent parameters were investigated to determine the exact retention mechanism taking place and to control selectivity. The parameters studied were eluent ionic strength and the nature of the inorganic salt used, eluent pH and eluent temperature. The results obtained showed how despite certain metal ions exhibiting similar stability constants with the bonded IDA groups, careful control of each of the above parameters, in particular eluent chloride ion concentration and eluent temperature, could result in large changes in selectivity. Optimal conditions for the isocratic and gradient separation of Mg(II), Ca(II), Mn(II), Cd(II), Co(II), Zn(II) and Pb(II) were determined. An isocratic method using a 0.035 M KCl, 0.065 M KNO3 (pH 2.5) eluent was successfully applied to the determination of Mn(II), Cd(II), Co(II) and Zn(II) at concentrations between 20 and 121 microg/l in a freshwater certified reference material (NIST 1640).     

Comparison of void volume, mobile phase volume and accessible volume determined from retention data for oligomers in reversed-phase liquid chromatographic systems

The experimental technique of mass spectrometric tracer pulse chromatography was used to determine the void volume, i.e., the total volume of eluent in the column, and the volume of eluent moving freely through the column, i.e., mobile phase volume, for a series of eluents with a C(18)-bonded RPLC column. The interstitial volume of the column was determined by size exclusion chromatography. In order to evaluate the utility of the accessible volumes determined from the retention volumes of homologous solutes, the accessible volume of the column was determined as a function of eluent composition and temperature with polystyrene and polyethylene glycol samples using Martin's Rule. Comparison of these four measured volumes indicated that the experimentally measured accessible volumes did not correspond to either the void volumes, mobile phase volumes or interstitial volumes.