Comprehensive two-dimensional chromatography in food analysis

Comprehensive two-dimensional (2D) chromatographic techniques can be considered innovative methods, only quite recently developed. Since their introduction to the chromatographic community, these techniques have been used in several fields and have gained an excellent reputation as valuable and powerful analytical tools. The revolutionary aspect of comprehensive multidimensional (MD) techniques, in respect to classical MD chromatography, is that the entire sample is subjected to the 2D advantage. The resulting unprecedented separating capacity makes these approaches prime choices when analysts are challenged with highly complex mixtures. Furthermore, in the case of automated systems, instrumental analysis times are roughly the same as in monodimensional applications. The present review reports various comprehensive chromatographic applications on different food matrices. The GC x GC section highlights two fundamental aspects for component separation/identification: the exceptional peak capacity and the formation of group types on the 2D space plane. The LC x LC section reports the employment in food analysis of a recently developed multidimensional normal-phase (NP)-reversed-phase (RP) high performance liquid chromatography (HPLC) system. Also reported are comprehensive LC x GC and packed column supercritical fluid chromatography (pSFC x pSFC) applications in this field.     

Comprehensive two-dimensional liquid chromatography combined with mass spectrometric detection in the analyses of triacylglycerols in natural lipidic matrixes

Triacylglycerols (TAGs), the major components in fats and oils, are a good example of natural complex mixtures. The best technique for the separation of such samples is certainly high performance liquid chromatography (HPLC). Monodimensional HPLC separations are often not sufficient to resolve all the components of interest. The present investigation reports the employment of a comprehensive LC system, based on the different separation mechanisms of silver ion (Ag) and non-aqueous reversed phase (RP) HPLC, used respectively in the first and second dimension, and applied to the analysis of plant-derived natural lipidic matrixes. The results obtained show that the approach enables both the separation of a high number of components and the attainment of structural information due to the formation of group-type patterns on the bidimensional (2D) plane. The employment of atmospheric pressure chemical ionisation (APCI) mass spectrometry as detection system was of substantial support for reliable TAG assignment, thus increasing the identification power of this comprehensive chromatographic approach.     

Comprehensive multidimensional liquid chromatography: theory and applications

Comprehensive two-dimensional (2D) liquid chromatographic (LC x LC) techniques can be considered innovative methods only recently developed and adopted in many configurations. The revolutionary aspect of comprehensive two-dimensional techniques, with respect to classical multidimensional (MD) chromatography, is that the entire sample is subjected to the 2D advantage. The major benefit is that the separation capacities of each dimension are multiplied, offering a high peak capacity to resolve samples of great complexity. The first part of the present review briefly describes the theoretical and practical aspects related to the development of a multidimensional comprehensive liquid chromatographic method. Applicational experiences in comprehensive liquid chromatography are then described, divided into four groups, according to the HPLC modes used in the two dimensions and to the nature of the samples analyzed.     

Infrared methods for high throughput screening of metabolites: food and medical applications

Chemical and physiological properties are related to individual or bioactive compounds such as essential oils, terpenoids, flavonoids, volatile compounds and other chemicals which are present in natural products in low concentrations (e.g. ppm or ppb). For many years, classical separation, chromatographic and spectrometric techniques such as high performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography (LC) and mass spectrometry (MS) have been used for the elucidation of isolated compounds from different matrices. Hence, the use of standard separation, chromatographic and spectrometric methods was found useful in chemical and both plant and animal physiology studies, for fingerprinting and comparing natural and synthetic samples, as well as to identify single active compounds. It has been generally accepted that a single analytical technique will not provide sufficient visualization of the metabolome, hence holistic techniques are needed for comprehensive analysis. In the last 40 years near infrared (NIR) spectroscopy became one of the most attractive and used methods of analyzing agricultural related products and plant materials which provide simultaneous, rapid and non-destructive quantitation of major. This technique has been reported to determine other minor compounds in plant materials such as volatile compounds and elements. The aim of this short review is to describe some recent applications of NIR spectroscopy combined with multivariate data analysis for high throughput screening of metabolites with an emphasis on food and medical applications.     

Evaluating the utility of ion mobility separation in combination with high-pressure liquid chromatography/mass spectrometry to facilitate detection of trace impurities in formulated drug products

Many formulated products contain complex polymeric excipients such as polyethylene glycols (PEGs). Such excipients can be readily ionized by electrospray and may be present at very high concentrations, thus making it very difficult to identify trace level impurities such as degradants in samples, even if hyphenated techniques such as liquid chromatography/mass spectrometry (LC/MS) are used. Ion mobility (IM) spectrometry is a very rapid gas-phase separation technique and offers additional separation capability within the LC timeframe. This work investigates the use of an IM separator in combination with high-pressure liquid chromatography (HPLC) and MS, to improve the separation of drug-related materials from excipients, thus aiding the identification of trace-level impurities in an anti-HIV medication, Combivir.     

Separation of triacylglycerols in a complex lipidic matrix by using comprehensive two-dimensional liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometric detection

The present investigation describes the employment of a comprehensive 2-D HPLC system, based on the combination of a silver ion and an RP column, for the characterization of the triacylglycerol (TAG) fraction of a very complex lipidic sample: donkey milk fat. The TAGs were grouped on the resulting bidimensional contour plot according to their double bond numbers (aligned along vertical bands) and according to their partition numbers (aligned along horizontal bands). Peak assignment was supported by using atmospheric pressure chemical ionization mass spectrometric (APCI-MS) detection. The combination of the enhanced resolving power of comprehensive multidimensional LC, the formation of ordered 2-D patterns, and APCI-MS detection proved to be an effective tool for the characterization of the complex matrix, enabling the separation and identification of nearly 60 TAGs.     

Efficient method development strategy for challenging separation of pharmaceutical molecules using advanced chromatographic technologies

In this paper, we describe a strategy that can be used to efficiently develop a high-performance liquid chromatography (HPLC) separation of challenging pharmaceutical molecules. This strategy involves use of advanced chromatographic technologies, such as a computer-assisted chromatographic method development tool (ChromSword) and an automated column switching system (LC Spiderling). This process significantly enhances the probability of achieving adequate separations and can be a large time saver for bench analytical scientists. In our study, the ChromSword was used for mobile phase screening and separation optimization, and the LC Spiderling was used to identify the most appropriate HPLC columns. For proof of concept, the analytes employed in this study are the structural epimers betamethylepoxide and alphamethylepoxide (also known as 16-beta methyl epoxide and 16-alpha methyl epoxide). Both of these compounds are used in the synthesis of various active pharmaceutical ingredients that are part of the steroid pharmaceutical products. While these molecules are relatively large in size and contain various polar functional groups and non-polar cyclic carbon chains, their structures differ only in the orientation of one methyl group. To our knowledge, there is no reported HPLC separation of these two molecules. A simple gradient method was quickly developed on a 5 cm YMC Hydrosphere C(18) column that separated betamethylepoxide and alphamethylepoxide in 10 min with a resolution factor of 3.0. This high resolution provided a true baseline separation even when the concentration ratio between these two epimers was 10,000:1. Although outside of the scope of this paper, stability-indicating assay and impurity profile methods for betamethylepoxide and for alphamethylepoxide have also been developed by our group based on a similar method development strategy.     

Sequential simplex optimization and multichannel detection in HPLC: Application to method development

Summary The sequential simplex procedure for the optimisation of mobile phase composition in high-performance liquid chromatography (HPLC) is combined with multichannel detection. The operating software already available in a commercial LC system is exploited and extended to incorporate a number of new routines developed to facilitate HPLC method development. An efficient stop criterion for automated simplex procedures is proposed, based on continuous comparison of the chromatographic response function attained with that predicted. The theoretical basis is developed for a new peak homogeneity test, based on the wavelength sensitivity of the chromatographic peak maximum. An algorithm for assigning peak elution order, based on peak areas at multiple wavelengths, is developed for cases where multiple optima are recorded. The simplicity and practical utility of these techniques are demonstrated in the context of an HPLC separation method developed for a model system of six solutes.     

Coupling liquid chromatography to Orbitrap mass spectrometry

The Orbitrap mass analyzer has become a mainstream mass spectrometry technique. In addition to providing a brief introduction to the Orbitrap technology and its continuing development, this article reviews the most recent publications quoting the use of the Orbitrap detection for a variety of chromatographic separation techniques. Its coupling to reversed-phase liquid chromatography (LC) represents undoubtedly the most ubiquitous approach to both small molecule and proteomic analyses. Multi-dimensional LC separations have an important role to play in the proteomics applications while an ultra-high-pressure LC is more frequently encountered in the area of metabolomics and metabolite analysis. Recently, special chromatographic techniques such as hydrophilic interaction chromatography and its variations have also been also cited with the Orbitrap detection.     

Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages

In this study, 1-D and 2-D liquid chromatographic systems, namely, conventional HPLC, UPLC, HPLC x HPLC and HPLC x UPLC systems were developed and evaluated for the separation of phenolic acids in wine and juices. In the LC x LC studies, the first dimension separation was based on RPLC and the second dimension was performed with ion-pair chromatography. Three different columns, namely two short columns packed with either 2.5 or 1.7 microm particles and a monolithic column, were tested for the fast second dimension separation. The best results were obtained when the monolithic column was applied for the second dimension separation. The peak capacities for comprehensive 2-D systems varied from 330 to 616.     

Comprehensive chromatographic separations in proteomics

In such a complicated field as proteomic analysis, scientists are more and more challenged in implementing separation systems capable to provide enhanced separation power, as well as sensitivity of detection for adequate identification and, to a lesser extent, quantification of the separated compounds. To address such issues, several combinations of different separation modes have been investigated in comprehensive liquid chromatographic platforms, in which the entire sample eluted from the first dimension is subjected to a secondary chromatographic separation. The different applications exploited for comprehensive LC analysis of intact or digested proteins are the focus of this review, in which advantages and disadvantages of the different columns combinations, interfaces, and operating modes are pointed out. The combination with mass spectrometry as part of the total system is stressed, and illustrated in more detail. Theoretical concerns and practical requirements will be briefly discussed, as well.     

New Developments in Multidimensional Chromatography of Complex Polymers

Summary: Complex polymers are distributed in more than one direction of molecular heterogeneity. In addition to the molar mass distribution, they are frequently distributed with respect to chemical composition, functionality, and molecular heterogeneity. One approach for the analysis of the heterogeneity of complex polymers is their chromatographic separation by combining different separation mechanisms. A typical experimental protocol includes the separation of the sample according to composition to yield fractions that are chemically homogeneous. These fractions are transferred to a size‐selective separation method and analyzed with respect to molar mass. As a result of this two‐dimensional (2D) separation, information on both types of molecular heterogeneity is obtained. So far, 2D chromatography has been applied mostly to polymers that are soluble in organic solvents. There are several problems related to the use of aqueous mobile phases in polymer chromatography. These problems relate to the very polar or ionic character of the polymers and the experimental conditions, including the use of salt‐containing eluents. The present paper addresses the different parameters that influence the chromatographic experiments. For a model polymer system resulting from the grafting of methacrylic acid (MAA) onto poly(ethylene glycol) (PEG), i.e., PEG‐g‐PMAA, it will be shown that different chromatographic techniques including SEC, LC‐CC, and 2D chromatography, as well as coupled LC‐CC/FT‐IR can be used to analyze the molecular complexity of the copolymers.

LC‐CC/FT‐IR spectra of a PEG‐g‐PMAA sample as function of the elution volume.     

Experimental Van Deemter plots of shear-driven liquid chromatographic separations in disposable microchannels

We present a new stationary phase coating method, yielding a monolayer of densely arrayed porous HPLC beads (d(p)=4 microm) for use in a disposable shear-driven flow LC system. The system is inherently suited for whole-column detection through the small voids between the individual particles of the layer. The chromatographic performance of the system has been characterized by performing a series of coumarin dye separation experiments (reversed-phase mode) and by measuring the theoretical plate height as a function of the mobile phase velocity. The resulting Van Deemter curve, yielding a value of about 90,000 plates/m near the u=u(opt) velocity, shows good agreement with the theoretical expectations, and hence constitutes the first full validation of the theory of shear-driven chromatography.     

Liquid chromatographic separation of terpenoid pigments in foods and food products.

The newest achievements in the use of various liquid chromatographic techniques such as adsorption and reversed-phase thin-layer chromatography and HPLC employed for the separation and quantitative determination of terpenoid-based color substances in foods and food products are reviewed. The techniques applied for the analysis of individual pigments and pigments classes are surveyed and critically evaluated. Future trends in the separation and identification of pigments in foods and food products are delineated.     

Description and validation of a staggered parallel high performance liquid chromatography system for good laboratory practice level quantitative analysis by liquid chromatography/tandem mass spectrometry.

The Aria LX4 staggered parallel high performance liquid chromatography (HPLC) system is evaluated for application to good laboratory practice (GLP) level quantitative analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This system consists of four fully independent binary HPLC pumps, a modified autosampler, and a series of switching and selector valves all controlled by a single computer program. The system improves sample throughput without sacrificing chromatographic separation or data quality. Validation results for four different compounds, each analyzed on a separate channel of the Aria system, show precision and accuracy equivalent to that required of a single-channel system. The results show that sample throughput can be increased nearly four-fold without requiring significant changes in current analytical procedures. The flexibility and ease of use of the Aria system suggest that it should be possible to quickly implement it in any analytical LC/MS/MS environment.     

Column properties and flow profiles of a flat, wide column for high-pressure liquid chromatography

The design and the construction of a pressurized, flat, wide column for high-performance liquid chromatography (HPLC) are described. This apparatus, which is derived from instruments that implement over-pressured thin layer chromatography, can carry out only uni-dimensional chromatographic separations. However, it is intended to be the first step in the development of more powerful instruments that will be able to carry out two-dimensional chromatographic separations, in which case, the first separation would be a space-based separation, LC(x), taking place along one side of the bed and the second separation would be a time-based separation, LC(t), as in classical HPLC but proceeding along the flat column, not along a tube. The apparatus described consists of a pressurization chamber made of a Plexiglas block and a column chamber made of stainless steel. These two chambers are separated by a thin Mylar membrane. The column chamber is a cavity which is filled with a thick layer (ca. 1mm) of the stationary phase. Suitable solvent inlet and outlet ports are located on two opposite sides of the sorbent layer. The design allows the preparation of a homogenous sorbent layer suitable to be used as a chromatographic column, the achievement of effective seals of the stationary phase layer against the chamber edges, and the homogenous flow of the mobile phase along the chamber. The entire width of the sorbent layer area can be used to develop separations or elute samples. The reproducible performance of the apparatus is demonstrated by the chromatographic separations of different dyes. This instrument is essentially designed for testing detector arrays to be used in a two-dimensional LC(x) x LC(t) instrument. The further development of two-dimension separation chromatographs based on the apparatus described is sketched.     

New Chromatographic and Hyphenated Techniques for Hydrophilic Copolymers

Most synthetic polymers are distributed in more than one parameter of molecular heterogeneity. For hydrophobic copolymers there are different chromatographic techniques available to analyse these distributions. As a result of the increasing interest in hydrophilic polymers and copolymers new chromatographic techniques are developed for the characterization of these polymers as well. However, very frequently these polymers contain highly polar or charged functional groups making them soluble only in aqueous mobile phases. There are several problems related to the use of aqueous mobile phases in polymer chromatography. Even the SEC analysis of such copolymers is not straightforward. As for HPLC in aqueous mobile phases, there are only a few applications in the literature so far. In addition to the fact that only a very limited number of stationary phases is available for aqueous HPLC of polymers, the interactions of polyelectrolytes in such chromatographic systems are not well understood. The present paper addresses the problems related to the application of SEC and HPLC in aqueous mobile phases. For graft copolymers with a polyethylene oxide backbone, e.g. PEG-g-polymethacrylic acid and PEG-g-polyvinyl alcohol, it will be shown that methods can be developed that give accurate molar mass and chemical composition information. Two-dimensional chromatography where aqueous HPLC and SEC are coupled on-line will be shown to be the most powerful analysis tool for the analysis of such copolymers. The hyphenation of the chromatographic separation techniques with spectroscopic detection techniques provides further insight into the molecular complexity of these copolymers.     

Identification and compatibility of the major active principles in some new natural origin antiseptics

The newly established instrumentation of HPLC/DAD, FTIR, and NMR techniques have been applied for simultaneous identification and physicochemical compatibility determination of the potential major antiseptic constituents (Hypericum perforatum L. and Usnea barbata extracts) which can be present in some new origin pharmaceutical preparation. Based on the obtained results the conclusion is that a simultaneous use of the analyzed constituents in production of some new preparations with antiseptic properties is possible. The chromatographic separation of antiseptic mixture was performed on a RP-HPLC C18 column. For the NMR detection, the analytes eluted from LC column were trapped and hereafter transported into the NMR flow-cell. The NMR and FTIR techniques allowed the characterization of the major constituent of Hypericum perforatum L., mainly hypericin, as well as of Usnea barbata, mainly usnic acid. The article is published in the original.     

Instability of hexane-acetonitrile mobile phases used for the chromatographic analysis of triacylglycerides

Comprehensive 2-D LC is an emerging separation technique that has seen a rapid increase in applications in the last decade. The technique has been applied for the separation of numerous complex mixtures including triacylglycerides (TAG). Determination of TAG in food products such as rice, palm, and canola oils have been previously described and the technique of choice utilizes a silver-modified silica column with hexane-ACN as the mobile phase. Repeated retention time inconsistencies were experienced in our studies when this mobile phase was applied to the separation of natural and synthetic mixtures containing TAG. The present report summarizes a study performed to determine the relative stability of ACN, propionitrile (PCN), and butyronitrile (BCN) at concentrations ranging from 0.43 to 2.8% in hexane and heptane. The data obtained suggest that unless evaporative loss of the mobile phase is prevented, TAG retention time irreproducibility can be significant when using mobile-phase mixtures prepared with ACN or PCN. BCN should be used as the solvent modifier in cases where evaporation cannot be prevented.     

Analysis of amino acid 13C abundance from human and faunal bone collagen using liquid chromatography/isotope ratio mass spectrometry

The scope of compound-specific stable isotope analysis has recently been increased with the development of the LC IsoLink which interfaces high-performance liquid chromatography (HPLC) and isotope ratio mass spectrometry (IRMS) to provide online LC/IRMS. This enables isotopic measurement of non-volatile compounds previously not amenable to compound-specific analysis or requiring substantial modification for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), which results in reduced precision. Amino acids are an example of such compounds.We present a new chromatographic method for the HPLC separation of underivatized amino acids using an acidic, aqueous mobile phase in conjunction with a mixed-mode stationary phase that can be interfaced with the LC IsoLink for compound-specific delta13C analysis. The method utilizes a reversed-phase Primesep-A column with embedded, ionizable, functional groups providing the capability for ion-exchange and hydrophobic interactions. Baseline separation of 15 amino acids and their carbon isotope values are reported with an average standard deviation of 0.18 per thousand (n = 6). In addition delta13C values of 18 amino acids are determined from modern protein and archaeological bone collagen hydrolysates, demonstrating the potential of this method for compound-specific applications in a number of fields including metabolic, ecological and palaeodietary studies.