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

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 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.     

Use of shift gradient in the second dimension to improve the separation space in comprehensive two-dimensional liquid chromatography

Comprehensive two-dimensional liquid chromatography (LC?×?LC) has received much attention because it offers much higher peak capacities than separation in a single dimension. The advantageous peak capacity makes it attractive for the separation of complex samples. Various gradient methods have been used in LC?×?LC systems. The use of continuous shift gradient is advantageous because it combines the peak compression effect of full gradient mode and the tailed gradient program in parallel gradient mode. Here, a comparison of LC?×?LC analysis of Chinese herbal medicine with full gradient mode and shift gradient mode in the second dimension was performed. A correlation between the first and second dimensions was found in full gradient mode, and this was significantly reduced with shift gradient mode. The orthogonality increased by 43.7 %. The effective peak distribution area increased significantly, which produced better separation.     

Comprehensive two-dimensional liquid chromatography with parallel gradients for separation of phenolic and flavone antioxidants

Various combinations of PEG-silica, phenyl-silica and C18 columns in a single-column or serial (tandem) arrangement in the first dimension and a monolithic Chromolith column in the second dimension were tested for comprehensive two-dimensional (2D) LCxLC separation of phenolic and flavone natural antioxidants. The combinations of different stationary phase chemistries provided low selectivity correlations between the first-dimension and the second-dimension separation systems. Improvement in system orthogonality, bandwidths suppression, more regular band distribution over the whole 2D retention plane and increased peak capacity in different 2D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2D separation time range. Instead of two sampling loops, two alternating trapping XTerra columns were used for sample fraction transfer from the first-dimension column to the second dimension. Stronger retention on the XTerra columns in comparison to PEG-silica or phenyl-silica columns in the first dimension allowed using focusing of sample fractions in narrow zones on the top of a trapping column and back-flushing into the second dimension in a very low volume of the mobile phase. This fraction transfer modulation provided significant bandwidth suppression in the second dimension. 2D systems with optimized stationary phase selectivity, parallel gradients and fraction transfer modulation using two trapping columns were applied for the analysis of natural antioxidants in beer and wine samples.     

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.     

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.     

Comprehensive two-dimensional gas chromatography for enhanced analysis of naphthas: new column combination involving permethylated cyclodextrin in the second dimension

A new column association using comprehensive two-dimensional gas chromatography for the detailed molecular analysis of hydrocarbon mixtures is reported in this paper. In order to compare the impact of two different secondary columns, a novel column combination relying on a GC x 2GC system was used. This system is based on a non-polar first column (PONA) combined with both a permethylated beta-cyclodextrin (beta-Dex 120) stationary phase and a polysilphenylensiloxane (BPX 50) in the second dimension. Compared to BPX 50 stationary phase, the implementation of beta-cyclodextrin columns as the second dimension was found to improve the resolution between paraffins and naphthenes in the naphtha range but not in the middle distillate range. Attempts to improve the results and to understand the interaction mechanism remained unsuccessful. Therefore, the benefits of the beta-Dex 120-column are only demonstrated on heavy naphtha cut for the quantitation of hydrocarbons.     

Comprehensive two-dimensional liquid chromatography to quantify polyphenols in red wines

A comprehensive two-dimensional liquid chromatography method has been applied for the quantification of polyphenols in red wines and compared to the most commonly employed conventional LC approach. Such methodology comprised the use of a microbore conventional HPLC column packed with totally porous particles in the first dimension and a partially porous column of conventional diameter in the second dimension. Even though a good number of applications in comprehensive LC have been reported, quantification experiments have been rarely described. To this regard, the advantages of comprehensive LC together with the employment of dedicated software capable of detecting and quantifying each peak from the 2D plot, have been taken into account for quantifying the most representative polyphenols in three different commercial Sicilian red wine samples. The optimized method has been validated in terms of linearity, sensitivity, detection and quantification limits. LODs as low as 0.02 ppm were obtained using the one-dimensional HPLC-DAD method, whereas values lower than 0.10 ppm were obtained by comprehensive LC. However, comprehensive LC allowed the quantification of a higher number of compounds with RSD lower than 10% thanks to its improved resolving power. The separation capabilities of comprehensive LC allowed the analysis of complex natural samples without any pre-treatment to effectively reduce the interferences coming from the matrix.     

Employing ultra high pressure liquid chromatography as the second dimension in a comprehensive two-dimensional system for analysis of Stevia rebaudiana extracts

Stevia rebaudiana extracts and plant materials are increasingly used as natural sweeteners. Polyphenolic and stevioside compounds contained in S. rebaudiana extracts were separated by comprehensive LC. A polyamine column operated in normal phase mode was used for the first dimension separation (D1), and a UHPLC C18 column operated in reversed phase mode was used for the second dimension separation (D2). The sub-2 μm column (2.1 mm × 30 mm, maintained at 70°C) and the UHPLC pump employed for D2 elution allowed a separation/cycle time of 20 s, with a backpressure oscillating between 805 and 922 bar at 3.4 mL/min. The reduced D2 cycle time allowed 3-12 D2 samplings for each peak eluted by D1. Polyphenolic and stevioside compounds were identified by combining the information coming from the position of the compounds in the 2D plot and UV spectra with that of reference materials.     

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.     

Comprehensive two-dimensional liquid chromatography for the characterization of functional acrylate polymers

Comprehensive two-dimensional liquid chromatography-size-exclusion chromatography (LC x SEC) was investigated as a tool for the characterization of functional poly(methyl methacrylate) (PMMA) polymers. Ultraviolet-absorbance and evaporative light-scattering detection (ELSD) were used. A simple method to quantify ELSD data is presented. Each data point from the ELSD chromatogram can be converted into a mass concentration using experimental calibration curves. The qualitative and quantitative information obtained on two representative samples is used to demonstrate the applicability of LC x SEC for determining the mutually dependent molar-mass distributions (MMD) and functionality-type distributions (FTD) of functional polymers. The influence of the molar mass on the retention behavior in LC was investigated using LC x SEC for hydroxyl-functional PMMA polymers. The critical conditions, at which retention is--by definition--independent of molar mass, were not exactly the same for PMMA series with different end-groups. Our observations are in close agreement with theoretical curves reported in the literature. However, for practical applications of LC x SEC it is not strictly necessary to work at the exact critical solvent composition. Near-critical conditions are often sufficient to determine the mutually dependent distributions (MMD and FTD) of functional polymers.     

Comprehensive two-dimensional liquid chromatography (NPLCxRPLC) with vacuum-evaporation interface

A comprehensive two-dimensional liquid chromatographic system incorporating a vacuum-evaporation interface was developed. Normal-phase liquid chromatography with a CN microcolumn was used as the first dimension (1(st)-D), and reversed-phase liquid chromatography with a C(18) monolithic column was used as the second dimension (2(nd)-D). An electronically controlled dual-position, ten-port valve with two identical storage loops served as the interface and the analysis time in the 2(nd)-D was 1.5 min. The solvent in the loops of the interface was evaporated at 25 degrees C under vacuum conditions, leaving the analytes on the inner wall of the loops. The mobile phase of the 2(nd)-D dissolved the analytes in the loop and injected them onto the second column, allowing an on-line solvent exchange of the fractions from the 1(st)-D to the 2(nd)-D. The chromatographic resolution of analytes on the two dimensions was evaluated. Sample loss due to evaporation in the interface was investigated with standard samples having different boiling points. The usefulness of the comprehensive 2-DLC system was demonstrated in the analysis of a traditional Chinese medicine Radix salviae miltiorrhiza bage extract.     

Ultra rapid liquid chromatography as second dimension in a comprehensive two-dimensional method for the screening of pharmaceutical samples in stability and stress studies

Separation orthogonality has been explored with respect to comprehensive two-dimensional liquid chromatography (2D-LC) for different reversed-phase stationary phases. The outcome of this study points out an SB-CN x BEH-C18 combination, used in the first and the second dimension, respectively, as the most orthogonal chromatographic system for the samples assayed. The present investigation reports the employment of an ultra high pressure liquid chromatography system (UPLC) as the second chromatographic dimension, increasing the sensitivity and the speed, completing the whole chromatographic separation in a reasonable time frame. Finally, an off-line 2D-LC method with diode array detection based on the UPLC has been optimized, allowing the separation and minimizing the run time. SB-CN and BEH-C18 were employed as first and second dimension, respectively, with gradient elution applied in each dimension. Alprazolam degraded tablets were studied as a proof of concept of the utility of this type of setups for impurity profiling of complex samples.     

The development of an evaluation method for capture columns used in two-dimensional liquid chromatography

Capture columns are important interface tools for on line two-dimensional liquid chromatography (2D-LC). In this study, a systematic method was developed to evaluate and optimize the capture ability of capture columns by off-line method. First, the parameter Δt_R (Δt_R = t₂−t₁−t₀−W) was introduced to quantitatively represent the capture ability of the capture column by connecting a capture column behind the first dimensional column. Based on the value of Δt_R, an appropriate capture column was selected after the first dimensional column was fixed. Then, the capture ability of the selected column was promoted by adjusting the mobile phase of the first dimensional column. Capture ability was also optimized using complex sample analysis software system (CSASS) software. Second, the elution mode of the trapped compounds on the capture column was investigated by connecting the capture column before the second dimensional column. More specifically, in mode I, capture column was connected to the second dimension without changing the flow rate direction and the trapped compounds must pass through the capture column and be eluted into the second dimensional column. The contrary connection mode was mode II. It was found that mode I is more suitable method for 2D-LC. Finally, an off-line reversed-phase/hydrophilic interaction liquid chromatography two-dimensional liquid chromatography (RP/HILIC 2D-LC) system with a C18 capture column was developed to demonstrate the practical application of this method.     

Comprehensive two-dimensional high-performance liquid chromatography for the separation of polycyclic aromatic hydrocarbons

A comprehensive two-dimensional HPLC system for the separation of polycyclic aromatic hydrocarbons was developed using a pentabromobenzyl column as the first dimension and two short monolithic C18 columns as the second dimension. The primary column and two secondary columns were coupled by a 10-port 2-position valve. The effluent from the first dimension was repetitively injected into the second dimension every 12 s. Due to its resolution, this technique is a powerful tool for the separation of polycyclic aromatic hydrocarbons in a complex matrix such as environmental samples.     

Monolithic columns in high-performance liquid chromatography

Monolithic media have been used for various niche applications in gas or liquid chromatography for a long time. Only recently did they acquire a major importance in high-performance column liquid chromatography (HPLC). The advent of monolithic silica standard- and narrow-bore columns and of several families of polymer-based monolithic columns has considerably changed the HPLC field, particularly in the area of narrow-bore columns. The origin of the concept, the differences between their characteristics and those of traditional packed columns, their advantages and drawbacks, the methods of preparation of monoliths of different forms, and the current status of the field are reviewed. The actual and potential performance of monolithic columns are compared with those of packed columns. Monolithic columns have considerable advantages, which makes them most useful in many applications of liquid chromatography. They are extremely permeable and offer a high efficiency that decreases slowly with increasing flow velocity.