"Orthogonal" separations for reversed-phase liquid chromatography

A general procedure is proposed for the rapid development of a reversed-phase liquid chromatographic (RP-LC) separation that is "orthogonal" to a pre-existing ("primary") method for the RP-LC separation of a given sample. The procedure involves a change of the mobile-phase organic solvent (B-solvent), the replacement of the primary column by one of very different selectivity, and (only if necessary) a change in mobile phase pH or the use of a third column. Following the selection of the "orthogonal" B-solvent, column and mobile phase pH, further optimization of peak spacing and resolution can be achieved by varying separation temperature and either isocratic %B or gradient time. The relative "orthogonality" of the primary and "orthogonal" RP-LC methods is then evaluated from plots of retention for one method versus the other. The present procedure was used to develop "orthogonal" methods for nine routine RP-LC methods from six pharmaceutical analysis laboratories. The relative success of this approach can be judged from the results reported here.     

Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions

The application of a silica hydride modified stationary phase with low organic loading has been investigated as a new type of chromatographic material suitable for the separation and analysis of peptides with electrospray ionization mass spectrometric detection. Retention maps were established to delineate the chromatographic characteristics of a series of peptides with physical properties ranging from strongly hydrophobic to very hydrophilic and encompassing a broad range of pI values (pI 5.5-9.4). The effects of low concentrations of two additives (formic acid and acetic acid) in the mobile phase were also investigated with respect to their contribution to separation selectivity and retention under comparable conditions. Significantly, strong retention of both the hydrophobic and the hydrophilic peptides was observed when high-organic low-aqueous mobile phases were employed, thus providing a new avenue to achieve high resolution peptide separations. For example, simultaneous separation of hydrophobic and hydrophilic peptides was achieved under aqueous normal phase (ANP) chromatographic conditions with linear gradient elution procedures in a single run, whilst further gradient optimization enabled improved peak efficiencies of the more strongly retained hydrophobic and hydrophilic peptides.     

Separation of potentially therapeutic peptide hormones by liquid chromatography. Optimisation of the composition and pH of the mobile phase

The aim of this work is to optimise the proportion of the organic modifier and the pH of the mobile phase, in order to separate a series of peptide hormones with therapeutic interest in the molecular mass range from 500 to 6000. The composition of the mobile phase was optimised by establishing relationships between retention parameters and either the scale of solvent polarity, or the Kamlet–Taft multiparameter solvent scale of the eluent, using linear solvation energy relationships. Likewise, linear correlations between the chromatographic retention and Reichardt’s E^N_T parameter were obtained. These relationships allowed an important reduction of the experimental retention data needed for developing a given separation. In addition, a model describing the effect of the correctly measured pH of the mobile phase on retention in LC was established and tested for the series of selected peptides using an octadecylsilica column. The proposed equations permit the prediction of the optimum pH and also permit the determination of the acidity constants of the peptides in the hydro-organic mixtures using a minimum number of measurements.     

The Behaviour of Reduced, Alkylated and Native Proteins in a pH-Gradient LC System

Two dimensional (2D) liquid chromatography (LC) separations of proteins can be obtained faster and more automated than traditional 2D gel electrophoresis. Previously we have described a 2D LC method for separation of native proteins with separation according to pI by pH-gradient strong anion exchange (SAX) chromatography in the first dimension, and according to hydrophobicity by reversed phase chromatography in the second dimension. Since there are few literature reports on the combination of reduced/alkylated proteins and modern LC, a basic study of the chromatographic properties of a few reduced /alkylated proteins was undertaken with a pH-gradient SAX chromatographic system. Proteins where the disulfide groups were reduced, but not alkylated, were also included. The conditions that separated native proteins according to pI could not be used for neither reduced nor reduced/alkylated proteins. High concentrations of urea (4–8 M) were needed in the mobile phase in order to obtain good peak shapes. Addition of urea had an undesired impact on both the retention of the proteins and the pH gradient profile, with the effect that little correlation between reported pI values and elution pH was found. The conclusion was that proteins should be separated in the native state if good pI–pH correlations are important, and in the alkylated state with urea if other considerations are more important.     

Simultaneous micellar liquid chromatographic analysis of seven water-soluble vitamins: optimization using super-modified simplex

A super-modified simplex (SMS) method has been used to optimize the mobile phase used for separation of seven water-soluble vitamins in multivitamin tablets by gradient micellar liquid chromatography (MLC) with ultraviolet (UV) detection at 254, 295, and 361 nm. Effect of column temperature and addition of organic modifier to the mobile phase on separation efficiency were investigated: the appropriate conditions used were a temperature of 35 degrees C and 1-butanol modifier. The sodium dodecyl sulfate (SDS) concentration, pH, and 1-butanol% in the mobile phase were chosen for simultaneous optimization using the SMS method. The optimum mobile phase was found to be 16 mmol L(-1) (mM) SDS, 0.02 M phosphate buffer, pH 3.6, and a gradient of 3.5-10% (v/v) butanol. The total analysis time for vitamins was 75 min. The analytical parameters including linearity ( r>0.9970), limit of detection (0.12-50 micro g mL(-1)), precision of method (relative standard deviation (RSD) <8.90%), and accuracy obtained by the recovery assay (88-103%) support the usefulness of the proposed method for the determination of the water-soluble vitamins.     

pH gradient high-performance liquid chromatography: theory and applications

pH gradient high-performance liquid chromatography (HPLC) is a method of reversed-phase high-performance liquid chromatography suitable for ionogenic substances. It consists in programmed increase during the chromatographic process of the eluting strength of eluent with respect to the analytes separated. On the analogy of the conventional organic modifier gradient reversed-phase HPLC, in the pH gradient approach the eluting strength of the mobile phase increases due to its changing pH: increasing in case of acids or decreasing in case of bases. At the same time the content of organic modifier remains constant. A theory of the pH gradient HPLC has been elaborated. The resulting mathematical model is easily manageable. Its ability to predict changes in retention and separation of analytes following the changes in chromatographic conditions is demonstrated. The pH gradient method is uniquely suitable to determine pKa values of analytes. An equation is presented allowing to calculate pKa values basing on appropriate retention data. The effects on pKa are discussed of the concentration of methanol in the mobile phase. The RP HPLC-derived pKa data correlate to the reference pKa values (w(w)pKa) but are not identical. That may be explained by the effects on the chromatographically determined pKa of the specific interactions of analytes with stationary phases. The proposed pH gradient RP HPLC procedure offers a fast and convenient means to get comparable acidity parameters for larger series of compounds, like drug candidates, also when the analytes are available only in minute amounts and/or as complex mixtures.     

Low‐molecular‐weight color pI markers to monitor on‐line the peptide focusing process in OFFGEL fractionation

High‐throughput mass spectrometry‐based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low‐molecular‐weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24‐wells device encompassing the pH range 3–10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI‐TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on‐line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom‐up proteomic approach with or without iTRAQ labeling.     

Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions

Two-dimensional high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than single-dimensional LC. The most popular 2D-HPLC approach used today for proteomic research combines strong cation exchange and reversed-phase HPLC. We have evaluated an alternative mode for 2D-HPLC of peptides, employing reversed-phase columns in both separation dimensions. The orthogonality of 2D separation was investigated for selected types of RP stationary phases, ion-pairing agents and mobile phase pH. The pH appears to have the most significant impact on the RP-LC separation selectivity; the greatest orthogonality was achieved for the system with C18 columns using pH 10 in the first and pH 2.6 in the second LC dimension. Separation was performed in off-line mode with partial fraction evaporation. The achievable peak capacity in RP-RP-HPLC and overall performance compares favorably to SCX-RP-HPLC and holds promise for proteomic analysis.     

Theoretical opportunities and actual limitations of pH gradient HPLC

In a series of reports published recently by our laboratory comprehensive theory and experimental conditions were established for reversed-phase high-performance liquid chromatography (RP HPLC) employing the programmed pH gradient of mobile phase. A procedure was developed providing, rapidly and conveniently, the acidity (pK_a) of weak acids and bases and their lipophilicity (hydrophobicity) log k_w. The basis of the double-gradient RP HPLC, employing simultaneous gradients of organic modifier content and mobile phase pH, was also elaborated. The fundamentals of the approach are presented briefly and systematically and its advantages and limitations are discussed. It is demonstrated that the newly introduced pH gradient method increases the analytical versatility of RP HPLC and our understanding of its physicochemical basis.     

Modelling retention in liquid chromatography as a function of solvent composition and pH of the mobile phase

The aim of this work was to develop a model that accurately describes retention in liquid chromatography (LC) as a function of pH and solvent composition throughout a large parameter space. The variation of retention as a function of the solvent composition, keeping other factors constants, has been extensively studied. The linear relationship established between retention factors of solutes and the polarity parameter of the mobile phase, E(N)T, has proved to predict accurately retention in LC as a function of the organic solvent content. Moreover, correlation between retention and the mobile phase pH, measured in the hydroorganic mixture, can be established allowing prediction of the chromatographic behavior as a function of the eluent pH. The combination of these relationships could be useful for modelling retention in LC as a function of solvent composition and pH. For that purpose, the retention behavior on an octadecyl silica column of a group of diuretic compounds covering a wide range of physico-chemical properties were studied using acetonitrile as organic modifier. The suggested model accurately describes retention of ionizable solutes as concomitant effects of variables included and is applicable to all solutes studied. We also aimed to establish an experimental design that allows to reproduce to a good approximation the real retention surface from a limited number of experiments, that is from a limited number of chromatograms. Ultimately, our intention is to use the model and experimental design for the simultaneous interpretive optimization of pH and proportion of organic solvent of the mobile phase to be used in the proposed separation.     

Systematic approach to treatment of enantiomeric separations in capillary electrophoresis and liquid chromatography II. A study of the enantiomeric separation of fluoxetine and norfluoxetine

A systematic approach to enantiomeric separations in capillary electrophoresis (CE) and liquid chromatography (LC) with chiral mobile phase additives (MPA) or a chiral stationary phase (CSP) is used in the study of fluoxetine and norfluoxetine with cyclodextrins as chiral selectors. Binding constants and selectivities are determined under the same experimental conditions (mobile phase, buffer composition). Good agreement is found between results from the three techniques. The role of the buffer salt is investigated by comparison of binding constants obtained with triethylammonium and sodium acetate buffers.

Investigation of the effects of derivatisation of the selector in CE and LC with MPA demonstrates the appropriate choice of cyclodextrin type for use in LC. By studying the influence of organic modifier content on separation parameters, CE can predict a useful solvent working range for a CSP.     

Development and validation of a pre-column reversed phase liquid chromatographic method with fluorescence detection for the determination of primary phenethylamines in dietary supplements and phytoextracts

A sensitive and selective reversed-phase liquid chromatographic (RP-LC) method was developed and validated to determine octopamine, tyramine and Tyrosine (Tyr) in complex matrices as formulations and phytoextracts (Citrus aurantium), after pre-column derivatization with o-phthaldialdehyde (OPA) reagent. The chromatographic separations were performed at room temperature on a Phenomenex Luna C18 column using methanol and sodium acetate buffer (pH 5.5) by varying composition gradient elution as mobile phase and detected flurometrically at λ(em)=455 nm with λ(ex)=340 nm. The results obtained by the proposed method were compared with those achieved by a validated direct RP-LC method with fluorescence detection at λ(em)=310 nm with λ(ex)=275 nm, as reference method, using a Phenomenex Gemini C18 column under isocratic elution conditions with acetonitrile and sodium 1-heptanesulphonate (pH 3), as mobile phase. The higher sensitivity of the derivatization method (detection limit about 0.06 pmol) allowed the sure determination of octopamine present in traces in the examined samples. The repeatability of method (RSD) was ≤1.90% and there was no significant difference between repeatability and intermediate precision data. Recovery studies showed good results 99.5-101.3% with RSD ranging from 0.8 to 1.2%. All analyses were performed by mild conditions in absence of preliminary difficult extraction methodologies or laborious step of sample pre-treatment.     

High-throughput evaluation of lipophilicity and acidity by new gradient HPLC methods

There is a need for fast testing of drug candidates for properties of pharmacokinetics and pharmacodynamics importance, in particular lipophilicity and acidity. These two parameters can conveniently be estimated by gradient reversed-phase HPLC. Appropriate conventional organic solvent gradient and the new pH gradient HPLC procedures are presented. The chromatographic parameter of lipophilicity, log kw, can be determined from two organic solvent gradient runs instead of 6-8 runs necessary in the standard isocratic (polycratic) approach. The newly introduced pH gradient reversed-phase HPLC consists in a programmed increase during the chromatographic run of the eluting power of the mobile phase with regards to ionizable analytes. The eluting strength of the mobile phase increases due to its increasing (in case of acidic analytes) or decreasing (basic analytes) pH, whereas the content of organic modifier remains constant. It has been theoretically and experimentally demonstrated that the pKa and log kw values can be evaluated based on retention data from a pH gradient run, combined with appropriate data from two organic solvent gradient runs. The gradient HPLC-derived log kw parameters correlate well with analogous parameters determined isocratically as well as with reference lipophilicity parameter log P (logarithm of n-octanol/water partition coefficient). Also, the HPLC-derived pKa parameters correlate to the literature pKa values (w(w)pKa), conventionally determined by titrations in water. The approach described allows rapid and high-throughput assessment of log kw and pKa for large series of drugs candidates, also when the analytes are available in a form of mixture, e.g. produced by combinatorial synthesis.     

Affinity partitioning for membrane purification exploiting the biotin-NeutrAvidin interaction. Model study of mixed liposomes and membranes

The linear-solvent strength (LSS) model of gradient elution has been applied to estimate parameters of lipophilicity and acidity of a series of drugs and model chemicals. Apparent pKa values and log kw values for individual analytes were determined in 2-3 gradient runs. The first experiment (or first two experiments) uses a wide-range organic modifier gradient with pH chosen for suppressed ionization of the analyte. The result of this experiment allows an estimate of contents of organic modifier of the mobile phase (%B) providing the required retention coefficient, k, for the non-ionized analyte. The following experiment is carried out with the latter %B and a pH-gradient of the aqueous component of the eluent that is sufficient to overlap the possible pKa-value of the analyte. The initial pH of the buffer used to make the mobile phase is selected to insure that the analyte is in non-ionized form. The resulting retention time allows an estimate of PKa in a solvent of the selected %B. At the same time, estimates of log kw can also be obtained. The log kw parameter obtained from gradient HPLC by the approach proposed correlated well with the corresponding value obtained by standard procedure of extrapolation of retention data determined in a series of isocratic measurements. Correlation between log kw and the reference parameter of lipophilicity, log P, was very good for a series of test analytes and satisfactory for a structurally diverse series of drugs. The approach supported with specific detection procedures can be recommended for fast screening of lipophilicity of individual components of complex mixtures like those produced by combinatorial chemistry. The values of pKa obtained in a study were found to correlate with the literature pKa data determined in water for a set of aniline derivatives studied. In case of a series of drugs the correlation was less than moderate if the general procedure of pKa determination was applied.     

Retention time and peak width in the combined pH/organic modifier gradient high performance liquid chromatography

The purpose of this work was to test the applicability of the current theory to predict the peak retention time and the peak width in the combined pH/organic modifier gradient reversed phase high performance liquid chromatography (RP HPLC). A series of 38 isocratic measurements have been conducted for a wide range of pH and methanol contents for ketoprofen (weak acid) and papaverine (weak base). It served to find the model describing dependence of retention factor and the height equivalent of a theoretical plate (HETP) on pH and organic modifier content. The information gathered in the isocratic mode was used to simulate retention times and peak widths for 30 various methanol gradients, 25 pH gradients, and 3 combined pH/methanol gradients. The simulations were compared with the experimental data. We also proposed a simplified version of this model that was parameterized based on 12 initial organic modifier gradients carried out for different pHs and for the 20 min and 60 min gradient development times. The full and the simplified model described the experimental data very well. In conclusion, the proposed modeling approach allowed predicting analyte retention times and peak width for various pH and organic modifier changes. Its simplified version required only 12 initial experiments and seems to be very promising in the optimization RP HPLC separations for complex samples and for conditions providing peak compression.     

Optimization of the LC Separation of 15 Phenolic Compounds with Plackett–Burman Designs

In this study Plackett–Burman designs have been used to find the optimum conditions for separation of 15 phenolic compounds by LC. The responses used were the minimum resolution between two peaks and the analysis time. The adopted factors and experimental field were organic modifier (MeOH or ACN), percentage of acetic acid in the aqueous phase, flow rate, gradient with ten steps of percentage increase of the organic component of the mobile phase, and ten factors representing the duration of the steps. Use of Plackett–Burman designs achieved separation of the phenolic compounds within 45 min.     

Multiple Step Gradient Analysis in Stationary Phase Optimised Selectivity LC for the Analysis of Complex Mixtures

Stationary phase optimised selectivity liquid chromatography (SOSLC) is an approach to tune a given LC separation by combining different stationary phases in a multi- segment column set-up. The presently available SOSLC optimisation procedure and algorithm are, however, only applicable to isocratic conditions. This is a severe limitation for the analysis of mixtures composed of components covering a broad hydrophobicity range. A strategy is described to circumvent this limitation. The components of a mixture are divided into different groups according to hydrophobicity as elucidated by a gradient analysis on a C₁₈ reversed-phase column. Each group separation is then individually optimised with a specific isocratic mobile phase composition using the original SOSLC strategy. The mobile phase composition thereby only differs in the percentage of organic modifier between the various groups. Finally, a combination of stationary phases that guarantees sufficient selectivity for all the groups is selected and the separation is performed by a multiple step gradient, whereby each level consists of the mobile phase composition applied for the SOSLC optimisation of the individual groups. The multi step gradient approach is demonstrated through the analysis of a mixture of 27 steroids covering a wide range of hydrophobicity.     

Influence of the mobile phase on salmon calcitonin analysis by reversed-phase liquid chromatography

The retention properties of calcitonins on a reversed-phase column are examined using salmon calcitonin as the model compound. The effect of the concentration of organic modifier, buffer strength, pH of the mobile phase, and ion-pair reagent are studied. In the absence of an ionic modifier in the eluent the calcitonin peak shapes are not symmetrical. The addition of 0.1% trifluoroacetic acid (TFA), however, results in good peak characteristics without the need to add nonvolatile salts. The retention of the calcitonins was found to be very sensitive to the concentration of the organic modifier (acetonitrile) present in the mobile phase. A change of pH between 2 and 5 has only a slight effect of the k' of salmon calcitonin, but the k' increases significantly at higher pH values. The addition of a phosphate buffer to the mobile phase and an increase in the buffer concentration (0-0.2 M) causes a decrease in the retention of salmon calcitonin. Evidence shows that reproducible, quantitatively measurable data can be obtained using reversed-phase chromatography if the ion-pairing reagent and organic modifier concentrations are carefully controlled. The system also shows a good selectivity for the calcitonin series. Therefore, both highly selective methods (qualitative) as well as quantitative methods for analytical, pharmaceutical, and manufacturing use can be developed by adjusting the high-performance liquid chromatography (HPLC) conditions as discussed.     

Column selectivity in reversed-phase liquid chromatography II. Effect of a change in conditions

The isocratic retention of 67 widely-different solutes in reversed-phase liquid chromatography (RP-LC) has been investigated as a function of temperature and mobile phase composition (% B) for three different C18 columns. Similar studies were also carried out in a gradient mode, where temperature, gradient time and solvent type were varied. These results show that changes in retention with these conditions are similar for each of these three columns. This suggests that relative column selectivity as defined by experiments for one set of experimental conditions will be approximately applicable for other conditions, with the exception of changes in mobile phase pH-which can affect values of the column parameter C (a measure of silanol ionization). Column selectivity as a function of pH was explored for several columns.