Comprehensive characterization of Stevia Rebaudiana using two-dimensional reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography

Two-dimensional reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography (2D-RPLC/HILIC) system was successfully applied for comprehensive characterization of steviol glycosides from Stevia rebaudiana. The experiments were performed in offline mode using an XCharge C18 column in first dimension and an XAmide column in second dimension. In first dimension, preliminary separation of Stevia aqueous extract was accomplished and 30 fractions were collected. Then fractions 1-20 were selected for further purification and 13 compounds with high purity were obtained in second dimension. Comprehensive characterization of these compounds was completed by determination of their retention time, accurate molecular weight, diagnostic fragmentation ions, and nuclear magnetic resonance spectroscopy. As a result, all nine known steviol glycosides, as well as other four steviol glycosides were fully purified. The result demonstrated that this procedure is an effective approach for the preparative separation and comprehensive characterization of steviol glycosides in Stevia. This 2D-RPLC/HILIC method will be a promising tool for the purification of low-abundance compounds from natural products.     

Gradient separations of complex mixtures by micro high-performance liquid chromatography

High-performance micro packed fused-silica and open-tubular glass capillary columns were prepared and applied to separations of complex mixtures. Solvent-gradient elution proved quite useful for the separation of solutes with wide polarity. Instruments and some applications are described.     

Comprehensive coupled reversed-phase reversed-phase separations of a complex isomeric mixture

Using predictions based on results obtained from Information Theory and Factor Analysis for the two-dimensional separation of a complex isomeric mixture, a practical experimental comprehensive coupled reversed phase-reversed phase chromatographic system was developed. In total four reversed phase-reversed phase systems were studied, each of which theory predicted would be able to resolve essentially equal numbers of components. However, in practice only one of these coupled systems realised the theoretical potential. This system employed as the first dimension, a C18 stationary phase with methanol as the mobile phase and as the second dimension, carbon clad zirconia as the stationary phase and acetonitrile as the mobile phase. In this system, 27 of the 32 isomers of a mixture of oligostyrenes were resolved. Failure of the remaining coupled systems to achieve the theoretical potential was attributed to high solute crowding, low efficiency of separation space utilisation and long analysis times in the second dimension.     

"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.     

Optimization of two-dimensional gradient liquid chromatography separations

An almost orthogonal comprehensive two-dimensional liquid chromatography was developed for the separation of phenolic and flavone natural antioxidants by using combinations of a polyethylene glycol silica micro-column in the first dimension and a porous-shell fused-core C18 column in the second dimension, both in the reversed-phase mode. System orthogonality was improved using parallel gradients of acetonitrile in buffered mobile phase. A new approach was proposed to optimize matching segmented gradient profiles in the two dimensions. An algorithm was developed for automatic corrections of the shifts in retention in the second dimension induced by the parallel two-dimensional gradient operation technique. Using the porous-shell C18 column in the second dimension at elevated temperature (60 degrees C) and high pressure (480 bar) with optimized segmented profiles of the parallel gradients in the two dimensions, the overall separation time for comprehensive LC x LC was reduced to 30 min.     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

Comprehensive two-dimensional liquid chromatography

Having nearly exhausted the possibilities for generating peak capacity through improvements in column technology, chromatographers are increasingly looking to alternative ways of maximising chromatographic separation. In recent years there has been increasing activity in the field of comprehensive multidimensional separations to meet analysis demands. Comprehensive two-dimensional liquid chromatography (LC×LC) approaches offer high peak capacity which leads to significantly improved analytical performance over single-column liquid chromatography. There are several closely related avenues available for achieving an LC×LC separation and this review pays special attention to the different valve-based interfaces that have been used to comprehensively couple the first and second dimension columns in LC×LC systems. A brief discussion of column choices for selected applications and the conditions employed is also presented.     

Comprehensive two-dimensional liquid chromatography separations of pharmaceutical samples using dual Fused-Core columns in the 2nd dimension

This paper focuses on the application of RPLC × RPLC to pharmaceutical analysis and addresses the specific problem of separating co-eluting impurities/degradation products that maybe “hidden” within the peak envelope of the active pharmaceutical ingredient (API) and thus may escape detection by conventional methods. A comprehensive two-dimensional liquid chromatograph (LC × LC) was constructed from commercially available HPLC equipment. This system utilizes two independently configurable 2nd dimension binary pumping systems to deliver independent flow rates, gradient profiles and mobile phase compositions to dual Fused-Core secondary columns. Very fast gradient separations (30 s total cycle time) were achieved at ambient temperature without excessive backpressure and without compromising optimal 1st dimension sampling rates. The operation of the interface is demonstrated for the analysis of a 1 mg/ml standard mixture containing 0.05% of a minor component. The practicality of using RPLC × RPLC for the analysis of actual co-eluting pharmaceutical degradation products, by exploiting pH-induced changes in selectivity, is also demonstrated using a three component mixture. This mixture (an API, an oxidation product of the API at 1.0%, w/w, and a photo degradant of the API at 0.5%, w/w) was used to assess the stability indicating nature of an established LC method for analysis of the API.     

Comprehensive two-dimensional liquid chromatography of polymers

The need for and the emergence of comprehensive two-dimensional liquid chromatographic separations of synthetic polymers are reviewed in this paper. LC×SEC is shown to be a particularly valuable two-dimensional technique in this domain. An improved (symmetrical) configuration based on a single 10-way switching valve is described. The use of LC×SEC to understand and optimize one-dimensional separations is illustrated, as well as the potential of the technique for the separation and characterization of functional polymers and copolymers.     

On-line analysis of complex hydrocarbon mixtures using comprehensive two-dimensional gas chromatography

This paper discusses the first setup for on-line qualitative and quantitative comprehensive two-dimensional gas chromatography (GC × GC) of complex hydrocarbon mixtures. A built-in 4-port 2-way valve allows switching between flame ionization detection (FID) and time-of-flight mass spectrometry (TOF-MS) between runs, without the need to cool down and vent the MS. Proper selection of GC carrier gas flow rates enables maximal agreement between the obtained chromatograms in both configurations. For on-line analysis of reactor effluents, a dedicated sampling system allows automatic sampling of the hot reactor effluent gases and immediate injection of the sample on the GC × GC. To determine a complete effluent composition in a single run of the GC × GC, a subzero oven starting temperature was employed. Modulation is started when the oven temperature reaches 40 °C, thus dividing the chromatogram in a conventional 1D and a comprehensive 2D part. This work illustrates the mature and robust character of GC × GC, extending its capabilities from mere laboratory use to on-line routine analysis for industrial processes in the (petro-)chemical industry.     

Optimisation of multilinear gradient elutions in reversed-phase liquid chromatography using ternary solvent mixtures

The multilinear gradient elution theory for binary mobile phases in reversed-phase liquid chromatography presented in [P. Nikitas, A. Pappa-Louisi, A. Papageorgiou, J. Chromatogr. A 1157 (2007) 178] is extended to ternary gradients. For the evaluation of this theory and the performance of the various fitting and optimisation algorithms we used 13 o-phthalaldehyde (OPA) derivatives of amino acids with mobile phases modified by acetonitrile and methanol. It is shown that the theory can lead to high quality predictions of the retention times under gradients elutions and optimisation of ternary gradients provided that we use a six-parameter expression for the logarithm of the retention factor, lnk, and the adjustable parameters of this expression are determined from ternary isocratic data.     

Retention equilibrium and mass transfer characteristics in reversed-phase liquid chromatography using methanol-water mixtures

Pulse response experiments (i.e., elution chromatography) were made in reversed-phase liquid chromatography (RPLC) using a C18 silica gel column and methanol-water mixtures of different compositions (phi). The moment analysis of the elution peak profiles measured in the RPLC system provided some items of information about four parameters characterizing the retention equilibrium and the mass transfer kinetics in the column, i.e., adsorption equilibrium constant, isosteric heat of adsorption, surface diffusion coefficient and activation energy of surface diffusion. Characteristics of the chromatographic behavior were studied by analyzing the dependence of the four parameters on phi and the correlation between them. It was found that surface diffusion was one of the important processes of molecular migration having a significant contribution to the mass transfer kinetics in the column. Both the adsorption equilibrium constant and the surface diffusion coefficient varied depending on phi. The direction of their changes was approximately opposite, suggesting that the mass transfer in the manner of surface diffusion was restricted owing to the retention of the sample molecules on the stationary phase.     

Serial coupled columns reversed-phase separations in high-performance liquid chromatography. Tool for analysis of complex real samples

High-performance liquid chromatography is considered as the selected analytical tool for a huge number of applications, including the carotenoid analysis. However, due to the great complexity of some natural samples containing this kind of compounds, conventional LC could not have enough separation power. In this work, serial connection of several columns is proposed as an alternative to LC and also to comprehensive two-dimensional LC for the analysis of carotenoids from complex real matrices. Different parameters regarding these separation procedures are studied and discussed, such as the stationary phase used or the application of high separation temperatures. The applicability of connecting two C30 columns to significantly increase the separation power, resolution and peak capacity for the analysis of carotenoids has been demonstrated for the first time. In fact, a peak capacity of 79 was obtained when using two C30 serial coupled columns, compared to 61 achieved using a single column. Similar improvements were also observed for the other serial couplings studied. The present methodology could be applied to the analysis of carotenoids in a great variety of samples. To the best of our knowledge, this is the first development carried out to study natural products such as carotenoids using columns coupled in series.     

Phase system selectivity and two-dimensional separations in liquid column chromatography

Correlations between the separation selectivity in aqueous and non-aqueous reversed-phase systems and in normal-phase LC systems were investigated for samples containing different numbers of two repeat structural elements. Such samples are best separated in "orthogonal" two-dimensional chromatographic systems, showing selectivity for one type of the repeat structural element only in the first dimension and for the other structural element only in the second dimension. The number of resolved compounds improves as the degree of orthogonality of the separation systems increases with decreasing correlation between the selectivities for the sample structural distribution in the two dimensions. Orthogonal systems with non-correlated selectivities for each repeat structural element provide the highest number of separated peaks and regular arrangement of the peaks over the two-dimensional retention space according to the individual structural element distribution and the best use of the available peak capacity. Fully orthogonal systems are difficult to find in practice. Partially orthogonal system with correlated selectivities for one structural type distribution, but with one system non-distinguishing the distribution for the other structural element are still useful for the two-dimensional separations. The correlations between the selectivities for repeat regular structural increments were employed to evaluate the suitability of phase systems for two-dimensional HPLC separations. The selectivity correlation in various reversed-phase and normal-phase systems was evaluated for two sample types: (1) Various RP columns show significantly inversely correlated selectivities for acyl lengths and numbers of double bonds distribution, but the differences in the double bond selectivity can be used for practical separations of triacylglycerols with the same equivalent carbon numbers. (2) Synthetic EO-PO block (co)oligomers with two-dimensional distribution of oxyethylene and oxypropylene monomer units were separated according to the two distribution types using on-line two-dimensional reversed-phase-normal-phase LC with a C18 column in the first dimension and an aminopropyl silica column in the second dimension.     

Optimizing separations in reversed-phase liquid chromatography by varying pH and solvent composition

Summary An interpretive optimization procedure in which pH can be one of the variables is presented with the emphasis on optimizing separations. When varying the pH in reversed-phase liquid chromatography the retention of ionogenic solutes will change. Thus, the selectivity between ionogenic and neutral solutes or between ionogenic solutes mutually can be optimized. However, pH also greatly affects the efficiency (plate count) and peak shape (asymmetry). Optimum selectivity (i.e. large differences in retention times) may be observed under conditions where peaks are broad and asymmetrical. Thus, it is essential to simultaneously consider retention, peak width and peak shape and their effects on separation (effective resolution) in pH-optimization studies. A procedure in which this is done is presented and applied to optimizing the separation of a synthetic mixture of selected pharmaceuticals. After initial experiments to establish the parameter space (boundaries for pH and binary methanol — water composition), twelve experiments are performed according to a 3×4 experimental design. At each loaction the retention, peak height, peak area and peak symmetry are recorded for each solute. These data are then used to build models for each of the four characteristics and for each solute. From this set of models the response surface, describing the quality of separation as a function of pH and composition, can be calculated. A variety of optimization criteria (quantifying quality of separation) can be used. The optimum corresponds to the highest point on the response surface.     

The importance of dead volume in the optimization of separations in reversed-phase liquid chromatography using temary solvents

Summary Described in this paper is a simple method for optimization of separations in reversed-phase liquid chromatography using ternary solvents. The graphical procedure is based on the linearity of the plots of log k against solvent composition. This relationship was found to depend critically both on the relative composition of the binary solvents used to prepare the ternary mixtures and the method chosen for dead volume determination.     

Comprehensive two-dimensional gas chromatography using a high-temperature phosphonium ionic liquid column

A high-temperature ionic liquid, trihexyl(tetradecyl)phosphonium bis(trifluoromethane)sulfonamide, was used as the primary column stationary phase for comprehensive two-dimensional gas chromatography (GC × GC). The ionic liquid (IL) column was coupled to a 5% diphenyl/95% dimethyl polysiloxane (HP-5) secondary column. The retention characteristics of the IL column were compared to polyethylene glycol (DB-Wax) and 50% phenyl/50% methyl polysiloxane (HP-50+). A series of homologous compounds that included hydrocarbons, oxygenated organics, and halogenated alkanes were analyzed with each column combination. This comparison showed that the ionic liquid is less polar than DB-Wax but more polar than HP-50+. The most unique feature of the IL × HP-5 column combination is that alkanes, cyclic alkanes, and alkenes eluted in a narrow band in the GC × GC chromatogram; whereas, these compounds occupied a much larger portion of the DB-Wax × HP-5 and the HP-50+ × HP-5 chromatograms. Each column combination was used to analyze diesel fuel. The IL × HP-5 chromatogram displayed narrow bands for three major compound classes in diesel fuel: saturates, monoaromatics, and diaromatics. The IL column was used at temperatures as high as 290 °C for several months without any noticeable changes in column performance.     

Automated off-line optimisation of programmed elutions in reversed-phase high-performance liquid chromatography using ternary solvent mixtures

A new system for the unattended optimisation of gradient elutions in reversed-phase high-performance liquid chromatography has been developed. The system is based on the simulation of retention times under conditions of ternary solvent mixtures and ternary gradient programmes. This model is constructed departing from a few experimental data obtained in isocratic elutions and validated against experimental gradient elutions. Once validated, this retention model can be used for the unattended search of an optimum separation. The optimisation process is driven by an evolutionary algorithm (EA), specially developed to map the ternary gradient elutions problem. The development and characteristics of the retention modelling as well as those of the EA and its particularities are discussed and some real world examples of separation presented.     

Purification of saponins from leaves of Panax notoginseng using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography

Saponins are widely distributed in the plant kingdom and have been shown to be active components of many medicinal herbs. In this study, a two-dimensional purification method based on reversed-phase liquid chromatography coupled with hydrophilic interaction liquid chromatography was successfully applied to purify saponins from leaves of Panax notoginseng. Nine saponin reference standards were used to test the separation modes and columns. The standards could not be resolved using C₁₈ columns owing to their limited polar selectivity. However, they were completely separated on a XAmide column in hydrophilic interaction liquid chromatography mode, including two pairs of standards that were coeluted on a C₁₈ column. The elution order of the standards on the two columns was sufficiently different, with a correlation coefficient between retention times on the C₁₈ and XAmide columns of 0.0126, indicating good column orthogonality. Therefore, the first-dimension preparation was performed on a C₁₈ column, followed by a XAmide column that was used to separate the fractions in the second dimension. Fifty-four fractions were prepared in the first dimension, with 25 fractions rich in saponins. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from three representative fractions. This procedure was shown to be an effective approach for the preparative isolation and purification of saponins from leaves of P. notoginseng. Moreover, this method could possibly be employed in the purification of low-content and novel active saponins from natural products.