Retention prediction of polycyclic aromatic hydrocarbons in reversed phase liquid chromatography with various mobile phase compositions and column temperatures

Summary The relationship between the logarithmic capacity factor measured in reversed-phase liquid chromatography and the operating conditions including the mobile phase composition and the column temperature is investigated. The strategy described herein can offer the possibility to predict the retention of polycyclic aromatic hydrocarbons without any experiments and standard materials, by utilizing equations describing the relationships between retention, temperature, mobile phase composition and physicochemical properties of the solutes previously stored in the program of the microcomputer-assisted retention prediction system.This concept is one of the most promising techniques for the optimization of the separation conditions in reversed-phase liquid chromatography.     

Design of mobile phase composition for liquid chromatography with an internal pH gradient

Summary A method is suggested for calculating the mobile phase composition for ion exchange chromatography with an internal pH gradient. An internal pH gradient of the desired steepness can be achieved providing the titration curve of the sorbent is known. The procedure has been verified by use in the design of a mobile phase composed from few anionic buffers for generation of internal pH gradient on a microcolumn packed with a cation exchanger with carboxylic functional groups.     

The role of the mobile phase in the resolution of racemic esters of aminoacids by normal phase high-performance liquid chromatography

Summary The work in this paper demonstrates the effect of the mobile phase composition on interactions involving racemic esters of aminoacids and a chiral stationary phase derived from N-formylisoleucine. Aprotic solvents have been employed to test the resolution of these racemates. An increase in the alcohol concentration of the mobile phase results in the deterioration of selectivity. These solvents seem to favour high separation factors. The solvent 1,1,2-trichlorotri fluoroethane is a suitable mobile phase modifier for normal phase liquid chromatographic resolution of enantiomers of aminoacid esters.     

Mobile phase effects in reversed-phase chromatography VI. Thermodynamic models for retention and its dependence on mobile phase composition and temperature

Summary The physico-chemical framework is examined by comparing the predictions of three models for the combined effects of the composition of the hydroorganic mobile phase and the column temperature on the retention ofn-alkylbenzenes on hydrocarbonaceous bonded stationary phases. The well-mixed model leads to expressions for the dependence of retention on three factors which are equivalent to those derived previously from linear extrathermodynamic relationships. The diachoric model stems from the assumption that the mobile phase is microscopically heterogeneous and the displacement model is identical to the retention model most widely used in chromatography with polar sorbents and less polar solvents. Over limited ranges of mobile phase composition and temperature, each model does describe retention behavior. However, only the wellmixed model describes retention well over the entire range of mobile phase composition and temperature studied here. The success of the well-mixed model, and its limits, give insight into the role of the organic solvent in determining the magnitude of chromatographic retention on non-polar stationary phases with hydro-organic eluents.Dedicated to Professor S. R. Lipsky on the occasion of his 60th birthday.     

Convenient estimation of the mobile phase volume for water-rich eluents in reversed-phase liquid chromatography

Summary Oxalodihydroxamic acid is proposed as a UV-detectable substitute for D₂O for the convenient estimation of the mobile phase volume (V_m) of water-rich hydro-organic eluents in reversed-phase liquid chromatography. The retention volume of oxalodihydroxamic acid deviates less than 6% from that of D₂O in 0 to 50% methanol-and acetonitrile-water eluents on C₁, C₈, and C₁₈ reversed-phase supports; it is independent of the amount injected from 10ng to 20μg when monitored at 254nm, although the sensitivity is about four-fold greater at 219 nm. The pH should be maintained between about 2 and 4. The deviation from the D₂O retention volume on supports of typical porosity is attributed, at least in part, to a size-exclusion effect. Simple hydroxamic acids can be used as a homologous series for the estimation of V_m over most of the methanol- and acetonitrile-water concentration ranges by the linearization of retentions of homologous series method. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Methylammonium formate as a mobile phase modifier for totally aqueous reversed-phase liquid chromatography

Although alkylammonium ionic liquids (ILs) such as ethylammonium nitrate and ethylammonium formate have been used as mobile phase "solvents" for liquid chromatography (LC), we have shown that the IL methylammonium formate (MAF), in part because of its lower viscosity as compared to other ILs, can be an effective replacement for methanol (MeOH) in reversed-phase LC. Plots of log retention factor versus the fraction of MeOH and MAF in the mobile phase indicate quite comparable solvent strength slope values of 2.50 and 2.05, respectively. Using a polar endcapped C18 column, furazolidone and nitrofurantoin using 20% MAF-80% water could be separated in 22 min but no baseline separation is possible using MeOH as the modifier, even down to 10%. Suppression of silanol peak broadening effects by MAF is important, permitting a baseline separation of pyridoxine, thiamine, and nicotinamide using 5% MAF-95% water at 0.7 mL/min. Using 5% MeOH-95% water, severe peak broadening for thiamine is evident. The compatibility of MAF as a mobile phase modifer at the 5% level for LC with mass spectrometry detection of water-soluble vitamins is also shown.     

Optimization of the mobile phase composition in gradient elution reversed-phase HPLC by stochastic prediction

Summary Quantitative analysis of more than ten compounds in a sample generally requires complex mobile phases to optimize the separation of the analytes by gradient elution reversed-phase HPLC. For this purpose, CHEOPS, a software package has been developed from the fully stochastic Computer Chromatogram Simulation Method. Calculation principles and optimization criteria are described. Experimental validation is presented with amino acids and steroids.     

Optimization by isochronal analysis I. Changes in mobile phase composition and velocity

Summary The theory of isochronal analysis (time normalization) has been extended to reversed-phase liquid chromatography. Of the possible manipulation of experimental parameters, the present paper develops a framework for optimizing the resolution by the simultaneous change of mobile phase velocity and composition. For that purpose the resolution equation was written in terms of these two parameters and under the constraint of constant analysis time. This treatment allows plotting the resolution versus the mobile phase composition and velocity. The surfaces thus obtained show the changes that the analyst must perform in order to optimize the resolution. The treatment deals with three possible α dependencies on the amount of organic modifier: constant, increasing and decreasing. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

A new method for the determination of mobile phase volume in normal and reversed-phase liquid chromatography

Summary A new method for the determination of the mobile phase volume (V _m) in liquid chromatography is presented based on the model regarding the retention of ionic solutes in the presence of eluent electrolytes. TheV _m value can be determined by measuring the retention volumes of two ions that have the same charge in two eluent electrolyte systems. Compared with the methods using isotopically labelled eluent components or inorganic salts asV _m markers, the method presented is proved to give more reasonableV _m values for both normal and reversed-phase liquid chromatography. As well as in binary mixed solvent systems, theV _m values in single solvent systems can be determined by this method.     

Effect of mobile phase composition, pH and buffer type on the retention of ionizable compounds in reversed-phase liquid chromatography: application to method development

Optimizing separation of ionizable compounds in order to find robust conditions has become an important part of method development in liquid chromatography. This work is an attempt to explain the observed variations of retention of acid and basic compounds with the organic modifier content in the mobile phase, according to various factors: the type of modifier, the type of buffer, the temperature and of course the type of solute. This is done by considering the variation of the so-called chromatographic pKa which refers to the pH measured in the aqueous medium and is determined from retention data. A procedure is described that accurately relates, from nine experiments, retention to solvent composition and pH. The limits of such a procedure are evaluated and two examples of optimized separations of basic compounds are given.     

A data base for polymer chromatography: dependence of interaction parameters on mobile phase composition

In chromatography of polymers, retention is determined by the characteristic volumes of the column (pore volume and interstitial volume), the pore diameter, and the interaction parameter. While the influence of the pore diameter is predominant in size exclusion chromatography, the key parameters in liquid adsorption chromatography are the interaction parameter and the pore surface of the column. It is shown, that the retention behaviour of polymers in liquid adsorption chromatography (LAC) can be predicted very well using the accessible volume and pore surface of the column, which can be determined very easily, and the interaction parameters from a data base.     

Influence of mobile phase composition on the adsorption of hen egg-white proteins to anion-exchange cellulose

Summary Factors influencing the capacity of Whatman QA52 anion-exchange cellulose for hen egg-white proteins have been investigated. While simply increasing the concentration of protein in the feedstock increases the loading there is a concomitant reduction in binding efficiency in a single pass through the column. Substitution of buffer by water in the mobile phase during sample preparation, thereby reducing the ionic strength of the feedstock, had the effect of significantly increasing protein capacity whilst improving binding efficiency. During column loading large changes in pH of the mobile phase were observed although this had no apparent effect on the chromatography for this system.     

Retention characteristics of porous graphitic carbon in subcritical fluid chromatography with carbon dioxide-methanol mobile phases

Numerous relationships usually used in high-performance liquid chromatography (HPLC) for describing the retention on porous graphitic carbon (PGC) have been applied in subcritical fluid chromatography, with CO2-methanol mobile phases. As reported in HPLC, octanol-water partition coefficient failed to fit the retention, whereas satisfactory results were obtained with the sum of partial negative charges. A better fit was reached by using the solvation parameter model, allowing a better understanding of the interactions developed between the solute, the stationary and the mobile phases. Results show that the dominant contribution to retention was given by the polarizability (E) and the volume (V), while the hydrogen-bond basicity (B) was not selected in the retention model, whatever the methanol content. The increase in methanol percentage favours the retention decrease, mainly through the volume for hydrophobic compounds, and through the hydrogen-bond acidity for polar compounds.     

Optimization of a new mobile phase to know the complex and real polyphenolic composition: towards a total phenolic index using high-performance liquid chromatography

An HPLC method is reported for the separation and quantification of five major polyphenolic groups found in fruits and related products: single ring phenolic acids (hydroxybenzoic acid and hydroxycinnamic acid derivatives), flavan-3-ols, flavonols, anthocyanins, and dihydrochalcones. A binary mobile phase consisting of 6% acetic acid in 2 mM sodium acetate aqueous solution (v/v, final pH 2.55) (solvent A) and acetonitrile (solvent B) was used. The use of sodium acetate was new and key to the near baseline separation of 25 phenolics commonly found in fruits. A photodiode array detector was used and data were collected at four wavelengths (280, 320, 360, and 520 nm). This method was sensitive and gave good separation of polyphenolics in apple, cherry, strawberry, blackberry, grape, apple juice, and a processing by-product. The improved separation has led to better understanding of the polyphenolic profiles of these fruits. Individual as well as total phenolic content was obtained, and the latter was close to and correlated well with that obtained by the Folin-Ciocalteu method (FC). The HPLC data can be used as a total phenolic index (TPI) for quantification of fruit phenolics, which is advantageous over the FC because it has more information on individual compounds.     

Theoretical and experimental comparison of mobile phase consumption between ultra-high-performance liquid chromatography and high performance liquid chromatography

Ultra performance liquid chromatography (UPLC) using small particles and very high pressure has demonstrated higher resolution and speed compared with conventional HPLC. An additional benefit of UPLC is the significantly reduced consumption of mobile phase. This report discusses how column length, particle size, inner column diameter, extra column void volume, and capacity factor contribute to the reduction of mobile phase consumption in UPLC compared with HPLC. In addition, theoretical and experimental comparison of mobile phase consumption was made between isocratic HPLC and UPLC as well as between gradient HPLC and UPLC. Both theoretical and experimental results indicate that UPLC typically saves at least 80% of mobile phase in isocratic and gradient conditions when compared with HPLC.     

Effects of first dimension eluent composition in two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC×LC) has received a great deal of attention during the past few years because of its extraordinary resolving power. The biggest advantage of this technique is that very high peak capacities can be generated in a relatively short time. Numerous approaches to maximize the peak capacity in LC×LC have been employed. In this work we investigate the impact of the first dimension mobile phase on selectivity. LC×LC has several potential advantages over one-dimensional LC (1DLC) in that unconventional solvents, at least in reversed-phase LC, can be used. For example, solvents which strongly adsorb in the UV in the first dimension are not problematic in LC×LC. This so because the UV detector is placed after the second dimensional column, as pulses of the first dimension eluent arrive at the second dimensional column, they elute well before the solutes of interest and therefore do not interfere at all with detection of solute peaks. So far, the most widely used solvents in reversed-phase 1DLC are methanol and acetonitrile. However, the "UV advantage" of 2DLC allows us to employ UV active solvents, such as acetone. We compare their differential selectivities to that of acetonitrile for the separation of 23 indole acetic acids of interest in plant biology. We also apply them to the separation of a maize seed extract, a very complex sample. In both sample sets, mobile phase composition can be an important parameter to increase the orthogonality of the two dimensions and thus, to increase the effective peak capacity of LC×LC.     

Predicting retention in reverse‐phase liquid chromatography at different mobile phase compositions and temperatures by using the solvation parameter model

The prediction capability of the solvation parameter model in reverse‐phase liquid chromatography at different methanol‐water mobile phase compositions and temperatures was investigated. By using a carefully selected set of solutes, the training set, linear relationships were established through regression equations between the logarithm of the solute retention factor, logk, and different solute parameters. The coefficients obtained in the regressions were used to create a general retention model able to predict retention in an octadecylsilica stationary phase at any temperature and methanol‐water composition. The validity of the model was evaluated by using a different set (the test set) of 30 solutes of very diverse chemical nature. Predictions of logk values were obtained at two different combinations of temperature and mobile phase composition by using two different procedures: (i) by calculating the coefficients through a mathematical linear relationship in which the mobile phase composition and temperature are involved; (ii) by using a general equation, obtained by considering the previous results, in which only the experimental values of temperature and mobile phase composition are required. Predicted logk values were critically compared with the experimental values. Excellent results were obtained considering the diversity of the test set.     

Use of mobile phase 18-crown-6 to improve peak resolution between mono- and divalent metal and amine cations in ion chromatography

It is difficult to quantify NH4+ by ion chromatography in the presence of high concentrations of Na+ due to peak overlap. The Dionex IonPac CS15 column, which contains phosphonate, carboxylate, and 18-crown-6 functional groups, was originally developed to overcome this problem. We have found that the addition of 18-crown-6 to the eluent promotes improved peak resolution between Na+ and NH4+ even at concentrations as high as 60,000 to 1 using this column. Its use also improves the separation of alkali and alkaline earth metal and amine cations. Mobile phase 18-crown-6 increased the retention times of CH3NH3+, NH4+, and K+, and decreased the retention time of Sr2+. The retention times of Li+, Na+, Mg2+, Ca2+, (CH3)2NH2+, and (CH3)3NH+ were not affected. This method makes possible the direct analysis of ammonia from nitrogenase, the enzyme responsible for biological nitrogen fixation. The resolution of the NH4+ peak from the Na+ and Mg2+ peaks improved from zero resolution to values of 6.19 and 5.65, respectively. This technique considerably reduces the analysis time of NH4+ in the presence of high concentrations of Mg2+ and Na+ over traditional indophenol measurements.     

Retention prediction and computer-assisted optimization for the separation of PTH-amino acids in isocratic reversed-phase liquid chromatography

Summary Retention prediction of phenythiohydantoin amino acid derivatives in isocratic reversed-phase liquid chromatography was investigated. The predicted retention data of all derivatives were evaluated by comparing them with actually measured retention data. Excellent agreements between these data were found. The optimized conditions to separate overlapping components can also be predicted using the developed computer-assisted optimization system with the concept of retention prediction.