Development of an ion chromatographic gradient retention model from isocratic elution experiments

When facing separation problems in ion chromatography, chromatographers often lack guidelines to decide a priori if isocratic elution will give enough separation in a reasonable analysis time or a gradient elution will be required. This situation may be solved by the prediction of retention in gradient elution mode by using isocratic experimental data. This work describes the development of an ion chromatographic gradient elution retention model for fluoride, chloride, nitrite, bromide, nitrate, sulfate and phosphate by using isocratic experimental data. The isocratic elution retention model was developed by applying a polynomial relation between the logarithm of the retention factor and logarithm of the concentration of competing ions; the gradient elution retention model was based on the stepwise numerical integration of the corresponding differential equation. It was shown that the developed gradient elution retention model was not significantly affected by transferring data form isocratic experiment. The root mean squared prediction error for gradient elution retention model was between 0.0863 for fluoride and 0.7027 for bromide proving a very good predictive ability of developed gradient elution retention model.     

Relationship between isocratic and gradient retention times in the high-performance ion-exchange chromatography of proteins. Theory and experiment

Using isocratic retention parameters, the gradient elution retention time for several proteins has been calculated. The gradient retention time calculation is based on fitting the isocratic retention data to an equation of the form: log k' = m log (1/[Ca2+]) + log K and on applying well-established principles of gradient elution. A good correlation between the observed and calculated retention times for several test proteins was obtained at various total gradient times and column flow-rates. Conversely, isocratic retention parameters characterizing protein retention can be calculated from gradient elution retention data. However, even with retention data of high quality, small errors are amplified by the log-log nature of the ion-exchange isocratic retention model employed. Based on the close correlation between predicted and observed gradient retention times, no evidence for protein denaturation resulting from immobilization of the protein at high initial k' values at or near the column inlet was observed.     

Rapid and Complete Separation of Inorganic Polyphosphates from Monomer to 35-Mer by Hplc

Abstract

The on-line coupling system of high-performance liquid chromatography (HPLC) and flow injection analysis (FIA) was developed for the rapid separation and sensitive detection of inorganic polyphosphates. Effects of gradient elution conditions on the chromatographic behavior were studied. The column temperature as well as a chloride concentration gradient were found to be very effective for the improvement of resolution(1). To minimize analysis time and to maximize resolution, the gradient elution conditions were optimized by use of a computer-assisted retention prediction system(2). More than 35 kinds of inorganic polyphosphates could be separated completely within 200 min under the optimum conditions as shown in Fig. 1.     

人参皂甙的反相高效液相色谱多台阶梯度优化方法

建立了一种反相高效液相色谱多台阶梯度分离人参皂甙的方法.该方法以乙腈-水溶液为流动相,通过一系列等度实验,获得了8种人参皂甙Rg1,Re,Rf,Rg2,Rb1,Rc,Rb2和Rd的色谱保留参数,发现两参数保留方程不适合用于人参皂甙这种天然产物的分离条件的优化,而三参数保留方程的高精度才可满足预测的要求.在三参数保留方程的基础上,通过计算确定了8种人参皂甙(包括3台阶梯度)的液相色谱分离条件.通过实验对此优化条件进行了验证,实验结果显示了较好的预测精度和分离度.将本方法用于分离人参皂甙,分析时间短且分离度高,显示了等度台阶梯度优化方法对确定色谱分离条件的优越性.     

Use of temperature programming to improve resolution of inorganic anions, haloacetic acids and oxyhalides in drinking water by suppressed ion chromatography

Temperature programming was used to improve selectivity in the suppressed ion chromatographic (IC) separation of inorganic anions, haloacetic acids and oxyhalides in drinking water samples when using NaOH gradient elution. The programme exploited varying responses of these anions to changes in temperature. Heats of adsorption (deltaH, kJ/mol) for 17 anionic species were calculated from van't Hoff plots. For haloacetic acids, both the degree of substitution and log P (log of n-octanol-waterpartition coefficient) values correlated well with the magnitude of the temperature effect, with monochloro- and monobromoacetic acids showing the largest effect (deltaH= -10.4 to -10.7 kJ/mol), dichloro- and dibromoacetic acids showing a reduced effect (deltaH= -6.8 to -8.4 kJ/mol) and trichloro-, bromodichloro- and chlorodibromoacetic acids showing the least effect (deltaH= -4.7 to -2.4 kJ/mol). The effect of temperature on oxyhalides ranged from deltaH= 8.4 kJ/mol for perchlorate to deltaH= -9.1 kJ/mol for iodate. The effectiveness of two commercial column ovens was investigated for the application of temperature gradients during chromatographic runs, with the best system applied to improve the resolution of closely retained species at the start, middle and end of the separation obtained using a previously optimised hydroxide gradient, in a real drinking water sample matrix. Retention time reproducibility of the final method ranged from 0.62 to 3.18% RSD (n = 30) showing temperature programming is indeed a practically important parameter to manipulate resolution.     

Theoretical background of short chromatographic layers. Optimization of gradient elution in short columns

Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions.     

Quasi-linear gradients for capillary liquid chromatography with mass and tandem mass spectrometry

Gradient elution, capillary liquid chromatography mass spectrometry was performed with linear, static gradients constructed by laminar flowing ten, 1.5 microL volume steps of decreasing organic concentration into tubing of small internal diameter. Sample loading, gradient formation, and sample elution were accomplished entirely by means of a commercially available micro-autosampler and single-syringe drive pump. The procedure was simple, fast, stable, and reproducible. Essentially linear gradients were produced without the use of additional valves, mixers, pumps or software. It took less than 10 minutes to form a gradient and less than 30 minutes to construct the set of individual buffer vials. The gradients were shown to be stable to storage. One hour after forming, peak retention times were reproduced to +/-0.5%. Long-term retention time reproducibility was found to vary by +/-2%. Chromatographic resolution was comparable or superior to that obtained by gradient elution with conventional dynamic mixing and split flow. The procedure was adapted with a 'peak parking' method which extended the time for generating peptide fragmentation data up to 10 minutes per peptide with the triple quadruple mass spectrometer. Using this technique, collision data were collected at the 25 femtomole level on nine of ten tryptic peptides in a single run.     

Effect of subambient temperature on RP-HPLC of β-carotene isomers

Summary (E)-β-carotene and three mono-(Z)-isomers (9(Z)-, 13(Z)-, 15(Z)-) were eluted isocratically on a VYDAC 201TP54 column, using column temperatures between +30°C and −7°C. In this temperature range, their elution order changed dramatically with decreasing temperature. Nearly baseline separation of the four isomers within one hour was achieved at −7°C. For laboratory routine, temperatures of 9±2°C can be recommended. Thus, a good resolution of β-carotene- and lycopene-isomers (e.g. in tomato products) within 30 minutes is possible.     

High-speed analysis of dansyl derivatives of polyamines

Summary Analytical conditions for the high-speed, reversed-phase, liquid chromatographic of a diamine (putrescine) and polyamines (spermine and spermidine) were determined. Various elution modes were employed using the same mobile phase constituents: 20mM sodium heptane sulfonate and 20mM acetic acid as solvent A and, pure acetonitrile as solvent B. Samples were derivatized with dansyl chloride before injection.Under isocratic conditions, the separation of the three polyamines was achieved in 7 min. The use of a linear elution gradient led to the same analytical time but with a better resolution of the putrescine peak from non-polyamine frontal peaks. For these measurements, the sample size was 5l. This volume was increased to 20l and the use of a steep gradient combined with the peak-compression technique allowed a fast analysis in 2–3 minutes, which may be compared with a 30 min run time necessary when a conventional column is used.     

Prediction of peptide retention times in normal-phase liquid chromatography with only a single gradient run.

Previous studies of peptide separation by normal-phase liquid chromatography have shown a linear relationship between the logarithm of the capacity factor and the logarithm of the volume fraction of modifier in the mobile phase. This permitted the use of a model to predict isocratic and gradient retention times based on data obtained by two initial gradient runs. In the present study, chromatographic behavior of 25 peptides in normal-phase liquid chromatography with isocratic elution have been studied and a linear relationship between the slope (S) and intercept [log k(0)] was obtained. This relationship was combined with the algorithm of prediction reported in the previous paper. The prediction of peptide retention times with only a single experimental gradient retention data was investigated.     

Axial temperature gradient and mobile phase gradient in microcolumn high-performance liquid chromatography

The effect of axial temperature gradient (ATG) along a microcolumn on the separation performance at both isocratic and gradient elution mode was investigated. A thermostat system was designed to form an ATG along the packed column. Polycyclic aromatic hydrocarbons (PAHs) were separated on a 0.53 mm  × 150 mm i.d. 5 μm C₁₈ microcolumn, with water and acetonitrile as mobile phase. The separation results obtained at mobile phase gradient (MPG) and ATG in microcolumn HPLC were compared with the results performed at ambient conditions. Extrapolated curves of peak width at half height (wh)versus lnk showed that wh is narrower at the same retention time when ATG was applied in addition to MPG. The column efficiency was enhanced 20-30% and the resolution was slightly reduced because of reduction of selectivity at elevated temperature at ATG condition. The RSD of retention time in ATG mode was less than 2.5%.     

Guanidine improves DEAE anion exchange-based analytical separation of plasmid DNA

Elution of strong and weak anion exchangers with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA. Gradient elution of a weak anion exchanger (diethylaminoethyl) in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open-circular (oc) and supercoiled (sc) isomers. It also improves resolution among sc, linear, and multimeric/aggregated forms. Sharper elution peaks with less tailing increase sensitivity about 30%. However, elution with an exclusively Gdn gradient to 900 mM causes more than 10% loss of plasmid. Elution with a sodium chloride gradient while maintaining Gdn at a level concentration of 300 mM achieves close to 100% recovery of sc plasmid while maintaining the separation improvements achieved by exclusively Gdn elution. Corresponding improvements in separation performance are not observed on a strong (quaternary amine) anion exchanger. Other chaotropic salts do not produce a favorable result on either exchanger, nor does the inclusion of surfactants or EDTA. Selectivity of the diethylaminoethyl-Gdn method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than CE, and resolution approaching CE.     

Prediction of peptide retention times in reversed-phases high-performance liquid chromatography during linear gradient elution

The retention behavior of 100 peptides was studied during high-performance liquid chromatography on a C₁₈ column using aqueous trifluoroacetic acid as the mobile phase and acetonitrile as the mobile phase modifier in a linear gradient elution system. Retention times of the peptides were linearly related to the logarithm of the sum of Rekker's constants (R.F. Rekker, The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977, p. 301) for the constituent amino acid. Assuming this relationship, the best fit constants for this system were computed by non-linear multiple regression analysis. Using the new constants, it is possible to predict retention times for a wide variety of peptides at any slope of linear gradient, if the amino acid composition is known. It also enables accurate prediction of the retention time of peptides, whose amino acid composition in not known, after an analytical run with an alternate gradient.     

Comparison between the isocratic and gradient retention behaviour of polypeptides in reversed-phase liquid chromatographic environments.

The isocratic and gradient elution behaviour of beta-endorphin and glucagon, two polypeptides known to exist in amphipathic alpha-helical conformations in lipophilic environments, have been examined under reversed-phase high-performance liquid chromatographic (RP-HPLC) conditions with low pH, aquo-acetonitrile mobile phases. The effects of changes in the volume fraction, psi, of the organic solvent modifier and temperature, T, on the magnitudes of the S and log k(o) values of these two polypeptides, obtained from the plots of logarithmic capacity factor (log k') vs. psi using isocratic elution conditions have been determined. These data have then been compared to the corresponding S and log k(o) values, obtained from the plots of logarithmic median capacity factor (log k) versus the median volume fraction of the organic solvent modifier (psi) derived from the linear gradient elution data, using the same n-butyl silica sorbent and related aquo-acetonitrile mobile phase conditions. As apparent from these studies, substantial differences occur in the temperature-dependent trends and magnitudes of the corresponding S and S values, or the log k(o) and log k(o) values, when these parameters are derived from experimental data acquired by these two different elution methods. Moreover, when gradient elution data for beta-endorphin and glucagon are utilised, the extrapolated values of the intercept and slope of the plots of log k vs. 1/T (corresponding to an apparent change in the median enthalpy of association, deltaH(o)assoc, or an apparent change in the median entropy of association, deltaS(o)assoc) substantially deviated from the values obtained for the thermodynamic parameters, deltaH(o)assoc and deltaS(o)assoc, derived from the log k' vs. 1/T plots using the corresponding isocratic data. These findings thus have important implications for biophysical and thermodynamic investigations when gradient elution data are employed to assess the molecular basis of the interaction of polypeptides with non-polar ligates.     

Application of Multilinear Gradient Elution for Optimization of Separation of Chlorophenols Using Derringer’s Desirability Function

Multilinear gradient elution was applied for the simultaneous optimization of resolution and analysis times of nine chlorophenols separated by HPLC. The first relationship of ln k versus φ was determined using the isocratic retention time for each analyte. For prediction of gradient retention times of analytes, the fundamental equation of gradient elution was numerically solved. Then a grid search program was used to simulate chromatograms under each new condition. Two different chromatographic goals, analysis time and minimum distance between adjacent peaks, were evaluated simultaneously using Derringer’s desirability function for each chromatogram. The sigmoid function was used to transform the optimization criteria to desirability values. Under optimal conditions, a good agreement was observed between predicted and experimental values of the chromatographic response function when analysis time was less than 40 min.     

Multi-linear gradient elution optimization for separation of phenylthiohydantoin amino acids using pareto optimality method

Multi-linear gradient elution was applied for simultaneous optimization of resolution and analysis times for ten phenylthiohydantoin amino acids (PTH-AAs) in liquid chromatography. Relation of lnK upon φ for each analyte was determined using isocratic retention time data, and gradient retention time of analytes was predicted using fundamental equation of gradient elution. Then a grid search program was used to predict retention time of solutes in variable space. Two different chromatographic goals-analysis time and minimum difference between adjacent peaks- were simultaneously evaluated using Pareto optimality method. Gradient program in optimum condition was: initially 24% CH₃OH/Water for 10 min, linear ramp to 34% over 5 min, to 29% over 5 min, and to 70% over 20 min. The average of calculated relative error in the prediction of the retention time in optimal conditions was -1.67% that shows a good agreement between predicted and experimental values of the chromatographic retention time in optimal condition.     

Retention behavior of oligomeric proanthocyanidins in hydrophilic interaction chromatography

A novel method was developed for the separation of proanthocyanidins (PAs; oligomeric flavan-3-ols) by hydrophilic interaction chromatography (HILIC) using an amide-silica column eluting with an aqueous acetonitrile mobile phase. The best separation was achieved with a linear gradient elution of acetonitrile-water at ratios of 9:1 to 5:5 (v/v) for 60 min at a flow rate of 1.0 ml/min. Under these HPLC conditions, a mixture of natural oligomeric PAs (from apple) was separated according to degree of polymerization (DP) up to decamers. The DP of each separated oligomer was confirmed by LC/electrospray ionization MS. In further HILIC separation studies of 15 different flavan-3-ol and oligomeric PA (up to pentamer) standards with an isocratic elution of acetonitrile-water (84:16), a high correlation was observed between the logarithm of retention factors (log k) and the number of hydroxyl groups in their structures. The coefficient of this correlation (r2=0.9501) was larger than the coefficient (r2=0.7949) obtained from the correlation between log k and log P(o/w) values. These data reveal that two effects, i.e. hydrogen bonding between the carbamoyl terminal on the column and the hydroxyl group of solute oligomer and hydrophilicity based on the high-order structure of oligomeric PAs, corporately contribute to the separation, but the hydrogen bonding effect is predominant in our HILIC separation mode.     

An initial assessment of the use of gradient elution in microemulsion and micellar liquid chromatography

Novel microemulsion and micellar HPLC separations have been achieved using gradient elution and columns packed with reverse phase material. Initial attempts at gradient microemulsion liquid chromatography proved impossible on use of a microemulsion successfully used in capillary electrophoresis. Optimisation of the microemulsion composition allowed the generation of stable microemulsions to achieve separations in HPLC. The novel use of organic-solvent micellar chromatography in gradient elution mode was shown to give efficient separations. A range of efficient separations of pharmaceuticals and related impurities were obtained. Acidic, basic, and neutral solutes were resolved covering a wide range of water solubilities and polarities. Elution times were in the order of 4-15 minutes. Separations were briefly compared to those accomplished with a micellar HPLC system. It is proposed that gradient elution in both microemulsion and micellar HPLC can be regarded as a highly successful means of achieving resolution of complex mixtures and should be considered for routine analysis and further investigation.     

Retention prediction of a set of amino acids under gradient elution conditions in hydrophilic interaction liquid chromatography

The analysis of amino acids presents significant challenges to contemporary analytical separations. The present paper investigates the possibility of retention prediction in hydrophilic interaction chromatography (HILIC) gradient elution based on the analytical solution of the fundamental equation of the multilinear gradient elution derived for reversed‐phase systems. A simple linear dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier (φ) in a binary aqueous‐organic mobile is adopted. Utility of the developed methodology was tested on the separation of a mixture of 21 amino acids carried out with 14 different gradient elution programs (from simple linear to multilinear and curved shaped) using ternary eluents in which a mixture of methanol and water (1:1, v/v) was the strong eluting member and acetonitrile was the weak solvent. Starting from at least two gradient runs, the prediction of solute retention obtained under all the rest gradients was excellent, even when curved gradient profiles were used. Development of such methodologies can be of great interest for a wide range of applications.     

Optimisation and validation of a generic method for the LC assay of six corticosteroids and salicylic acid in dermopharmaceutical forms

Summary A generic method was developed for the liquid chromatographic (LC) assay of six corticosteraids and salicylic acid in different dermatological formulations containing methylparaben and propylparaben or sorbic acid as preservatives. An adequate separation was obtained using octadecylsilica as stationary phase and a mixture of acetonitrile (MeCN) and 25 mM phosphate buffer of pH 3.5 as mobile phase. Segmented gradient elution was applied from 30 to 45% of MeCN in 9.9 minutes, then from 45 to 96% in 19.0 minutes. UV detection was performed at 240 nm. The method was validated. Very good results were obtained with respect to linearity, selectivity, precision and accuracy.