Multidimensional Chromatographic Techniques Research Articles

Native Separation-Mass Spectrometry in Biopharmaceutical Analysis

Analytical methods that allow separation and identification of therapeutic proteins under native conditions play a crucial role in studying their higher-order structures and structure–function relationships. Recently, hyphenated techniques that combine native-mode separation with native mass spectrometry (nMS) have emerged as highly valuable tools for the targeted assessment of these quality attributes. This article outlines current native separation strategies coupled with nMS designed to characterize biopharmaceuticals close to their natural state. The methods provide worthwhile insights into aspects like aggregation, charge variants, conjugate stoichiometry, affinity, and conformation. As multidimensional chromatographic techniques and ion-mobility spectrometry become more accessible in laboratories, further advances in the development of native hyphenated techniques capable of simultaneously providing compositional, structural, and functional information on biopharmaceuticals can be expected.

Two-dimensional separation techniques using supercritical fluid chromatography.

High-resolution separation systems are essential for the analysis of complex mixtures in a wide variety of application areas. To increase resolution, multidimensional chromatographic techniques have been one key solution. Supercritical fluid chromatography provides a unique opportunity in these multidimensional separations based on its potential for high solvent compatibility, rapid duty cycles, and orthogonality to other separation modes. This review focuses on two-dimensional chromatography methods from the past decade that use supercritical fluid chromatography because of these advantages. Valving schemes and modulation strategies used to interface supercritical fluid chromatography with other liquid chromatography and gas chromatography techniques are described. Particular applications of multidimensional separations using supercritical fluid chromatography for the analysis of oils and chiral separations of pharmaceutical compounds are highlighted. Limitations of and a potential trajectory for supercritical fluid chromatography in this field are also discussed.

Chemometrics in comprehensive two-dimensional liquid chromatography: A study of the data structure and its multilinear behavior

Comprehensive multidimensional chromatographic techniques, such as GC×GC coupled to FID or MS and LC×LC coupled to UV or MS, have gained popularity in recent years. From the analytical perspective, these techniques allow obtaining higher peak capacities and resolution power, as well as adding selectivity from the second orthogonal dimension. From the chemometric point of view, these multidimensional techniques generate highly complex datasets, which present several challenges for their analysis. On the one hand, the selection of the appropriate chemometric data analysis tool requires the understanding of the underlying data structure and its multilinear behavior. On the other hand, peak resolution in complex samples is still a challenge, because of their possible overlapping in one or two chromatographic dimensions despite the increased resolution power.In this work, a comprehensive two-dimensional liquid chromatography method hyphenated simultaneously to PDA and MS detectors was employed for the analysis of a mixture of 31 pharmaceutical compounds. Chemometric evaluation of the obtained two-dimensional chromatograms focuses on two different goals. First, the assessment of the multilinear behavior of the high-dimensional data for each of the two detection modes (LC×LC-UV and LC×LC-MS) and, also, for the multiset data obtained by fusion of the data coming from both detectors. In addition, the chemometric resolution of peaks of overlapping compounds was evaluated using the multivariate curve resolution alternating least squares (MCR-ALS) method. Finally, the advantages of data fusion from UV and MS detectors were discussed, such as the increased ability for compound identification.

Phenolic profiling of rooibos using off-line comprehensive normal phase countercurrent chromatography × reversed phase liquid chromatography

Limited performance of one dimensional chromatographic methods provides the incentive for the development of multidimensional chromatographic techniques for the analysis of complex mixtures. Rooibos (Aspalathus linearis) represents such a complex sample, containing a range of phenolic compounds that cannot be separated and identified using a single chromatographic technique within a reasonable run time. By implementing NP high performance countercurrent chromatography (NP-HPCCC) in the first dimension (1D) and RP ultra-high pressure LC (RP-UHPLC) in the second dimension (2D), a highly orthogonal (∼80%) off-line comprehensive two-dimensional separation of rooibos phenolic compounds was achieved in a total analysis time of 17h. The use of a gradient for the 1D HPCCC separation ensured a good spread of relatively polar flavonoid di-C-glycosides and less polar mono- and di-O-glycosides, while the highly efficient UHPLC method was able to separate compounds eluting in the same 1D fraction. Analysis of green (“unfermented”) and traditional "fermented" rooibos samples enabled tentative identification of 39 phenolic compounds based on UV–vis and MS characteristics, of which 18 have not previously been reported in rooibos. Scolymoside (a flavone), hesperidin (a flavanone) and phloretin-3ʹ,5ʹ-di-C-β-d-glucopyranoside (a dihydrochalcone) were identified for the first time in rooibos by comparison with authentic reference standards.

An overview of multidimensional liquid phase separations in food analysis.

Food safety is a priority public health concern that demands analytical methods capable to detect low concentration level of contaminants (e.g. pesticides and antibiotics) in different food matrices. Due to the high complexity of these matrices, a sample preparation step is in most cases mandatory to achieve satisfactory results being usually tedious, lengthy, and prone to the introduction of errors. For this reason, many research groups have focused efforts on the development of online systems capable to do the cleanup, concentration, and separation steps at once through multidimensional separation techniques (MDS). Among several possible setups, the most popular are the multidimensional chromatographic techniques (MDC) that consist in combining more than one mobile and/or stationary phase to provide a satisfactory separation. In the present review, we selected a variety of multidimensional separation systems used for food contaminant analysis in order to discuss the instrumentation aspects, the concept of orthogonality, column approaches used in these systems, and new materials that can be used in these columns. Selected classes of contaminants present in food matrices are introduced and discussed as example of the potential applications of multidimensional liquid phase separation techniques in food safety.

Stereodifferentiation of oak lactone by using multidimensional chromatographic techniques

This work reports for the first time the use of on-line coupling of reversed phase liquid chromatography with gas chromatography for the stereodifferentiation of oak lactone, which is considered to be one of the main constituents of the aroma of different products.

Application of Chromatographic Techniques in the Detection and Identification of Constituents Formed during Food Frying: A Review

AbstractFrying is one of the most popular food processing methods. However, many physicochemical reactions occur during frying, forming complex products in both the frying oil and the fried food. Chromatographic techniques have been successfully applied to characterize the products formed during food‐frying or during a simulated frying process. In this review, the specific products analyzed by gas chromatography (GC) and high‐performance liquid chromatography (HPLC) are elucidated in detail. The most studied components are polar compounds (such as volatile compounds, hydrolysis products, oxidized triacylglycerol [TAG] decomposition products, oxidized TAG monomers, sterol oxides, oxygenated TAG polymers, and acrylamide) and nonpolar compounds (such as cyclic fatty acid monomers and trans isomers), which are classified based on their polarity. However, the definite structures of TAG polymers and sterol oxides, and polymers analyzed by GC or HPLC‐based methods are only investigated in modeled reactions. Furthermore, some of the sample pretreatments and chromatographic methods are only used to analyze the known products. A number of earlier trace amounts of undetected products need to be investigated by more effective detection techniques. The development of multidimensional chromatographic techniques and elaborate mass spectrometry detectors makes composition analysis possible for the food‐frying process, which may effectively promote the development of quality monitoring and nutrition evaluation for the fried foods.

Some aspects of multidimensional chromatographic techniques coupled with mass spectrometry applied for the separation of multicomponent mixtures of pesticides

Summary For very complex multicomponent mixtures, it is preferable to use different variants of multidimensional (planar) chromatography. In multidimensional separations, the modes of planar chromatography commonly applied are comprehensive 2D planar chromatography on mono and bilayers, coupled-layer chromatography, combination of multidimensional planar chromatographic (MDPC) techniques, and also hyphenated methods. Different multidimensional (planar) chromatography techniques and hyphenated methods may be alternative modes for separation and identification of pesticides in environmental analysis, especially after coupling with modern detection techniques. Proper identification and quantita tive analysis of the analytes are possible through the use of sensitive detection techniques such as high-resolution mass spectrometry (HRMS).

A new measure of orthogonality for multi-dimensional chromatography

Multi-dimensional chromatographic techniques, such as (comprehensive) two-dimensional liquid chromatography and (comprehensive) two-dimensional gas chromatography, are increasingly popular for the analysis of complex samples, such as protein digests or mineral oils. The reason behind the popularity of these techniques is the superior performance, in terms of peak-production rate (peak capacity per unit time), that multi-dimensional separations offer compared to their one-dimensional counterparts. However, to fully utilize the potential of multi-dimensional chromatography it is essential that the separation mechanisms used in each dimension be independent of each other. In other words, the two separation mechanisms need to be orthogonal. A number of algorithms have been proposed in the literature for measuring chromatographic orthogonality. However, these methods have their limitations, such as reliance on the division of the separation space into bins, need for specialist software or requirement of advanced programming skills. In addition, some of the existing methods for measuring orthogonality include regions of the separation space that do not feature peaks. In this paper we introduce a number of equations which provides information on the spread of the peaks within the separation space in addition to measuring orthogonality, without the need for complex computations or division of the separation space into bins.

2D-LC/SEC-(MALDI-TOF)-MS Characterization of Symmetric and Nonsymmetric Biocompatible PEOm–PIB–PEOn Block Copolymers

Complex copolymer mixtures can be directly analyzed via multidimensional chromatographic techniques after successful synthesis. High-performance liquid chromatography coupled to size exclusion chromatography (LC/SEC) revealed detailed information on the chemical composition, polymeric structure, and molar mass distribution of copolymer mixtures, in particular of symmetric and nonsymmetric α-TEO-ω-PEO telechelic PIB copolymers. A series of azide/alkyne “click” reactions after living polymerization reactions were used to prepare the either symmetric or nonsymmetric PIB–PEO-based triblock copolymers of the general structure (PEOn–PIB–PEOm BCPs (with n = 3; m = 3, 12, or 17)). In order to demonstrate the efficiency of the “click” reaction and thus the purity of the final triblock copolymers, the critical conditions of the PIB-homopolymers (Mn = 3–30 kg mol–1) in the isocratic elution mode (LCCC) were investigated. Thus, it was possible to separate the final polymers from their intermediates using a reversed-p...

Determination of trans fat in edible oils: current official methods and overview of recent developments

The adverse effects of dietary trans fat on biomarkers of chronic disease are well documented. Regulatory authorities in many countries have enacted legislation aimed at reducing trans fat content of their food supplies, either by requiring trans fat labeling on pre-packaged foods or by limiting the amount of trans fat in oils used for food production. Increased use by the food industry of oils with a low trans fat content necessitates reevaluation of official methods used by the food industry and regulatory agencies for the determination of total trans fat. Attenuated total reflection-Fourier-transform infrared (ATR-FTIR) spectroscopy and gas chromatography with flame ionization detection (GC-FID) are two techniques used in official methods approved by method-endorsing organizations, for example AOAC International and the American Oil Chemists' Society. Here, we review current official ATR-FTIR and GC-FID methods for determination of trans fat, with a focus on factors affecting quantification of low levels of trans fat. We include new data on method performance that have only recently become available, and provide an overview of notable recent developments in lipid analysis (e.g. IR spectroscopy procedures, ionic-liquid GC columns, and multidimensional chromatographic techniques) that have the potential to substantially improve the accuracy, sensitivity, and/or speed of trans fat determination.

Multidimensional Characterization of α,ω-Telechelic Poly(ε-caprolactone)s via Online Coupling of 2D Chromatographic Methods (LC/SEC) and ESI-TOF/MALDI-TOF-MS

The focus of the current study is the development of chromatographic methods to understand the end-group modification of poly(ε-caprolactones) (pCLs) prepared via azide/alkyne “click” chemistry. In order to prove the efficiency of the “click” reaction with increasing molar mass of the pCLs, a detailed characterization method via several multidimensional chromatographic techniques (2D LC/SEC, coupled to mass spectrometry) was developed. In particular, we have investigated the separation of low molar mass pCLs (Mn ≤ 35 000 g·mol–1) using a gradient elution chromatographic method (eluent: ACN/THF) monitoring the end-group functionalization of the separated polymers online via ESI-TOF and/or MALDI-TOF mass spectrometry, the latter coupled via a transfer module sprayer (TM sprayer). Because of the poor solubility of high molar mass pCLs (Mn above 35 000 g·mol–1), a second chromatographic method was employed, enabling the separation of end-group-functionalized pCLs up to a molar mass of 115 400 g·mol–1. Based on the critical solution conditions of the polymers, different solvent compositions and chromatographic columns were investigated in an isocratic mode to determine the critical conditions of α,ω-dihydroxy-telechelic pCLs. Finally, the critical conditions were optimized on a silica gel modified aminopropyl column (NUC-NH2) using a solvent mixture ratio of DCM/THF of 92/8 at 35 °C. A combination of the developed LCCC methods with SEC revealed the chemical composition as well as the molar mass of all of the modified pCLs, from the low molar mass polymers up to Mn = 115 400 g·mol–1.

In-Depth LCCC-(GELC)-SEC Characterization of ABA Block Copolymers Generated by a Mechanistic Switch from RAFT to ROP

A recently introduced procedure involving a mechanistic switch from reversible addition–fragmentation chain transfer (RAFT) polymerization to ring-opening polymerization (ROP) to form diblock copolymers is applied to synthesize ABA (star) block copolymers. The synthetic steps include the polymerization of styrene with R-group designed RAFT agents, the transformation of the thiocarbonyl thio end groups into OH functionalities, and their subsequent chain extension by ROP. The obtained linear ABA poly(ε-caprolactone)-block-poly(styrene)-block-poly(ε-caprolactone) (pCL-b-pS-b-pCL) (12 500 g mol–1 ≤ Mn ≤ 33 000 g mol–1) and the star-shaped poly(styrene)-block-poly(ε-caprolactone) (Mn = 36 000 g mol–1) copolymers were analyzed by size exclusion chromatography (SEC), nuclear magnetic resonance (NMR), infrared (IR) spectroscopy, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The focus of the current study is on the detailed characterization of the ABA (star) block polymers via multidimensional chromatographic techniques specifically high performance liquid chromatography coupled to size exclusion chromatography (HPLC-SEC). In particular, we demonstrate the first time separation of poly(ε-caprolactone) (pCL) homopolymer and additionally poly(styrene) (pS) from the ABA poly(ε-caprolactone)-b-poly(styrene)-b-poly(ε-caprolactone) and star-shaped poly(styrene)-b-poly(ε-caprolactone) block copolymer utilizing critical conditions (CC) for pCL with concomitant gradient elution liquid chromatography (GELC).

ガスクロマトグラフと加速器質量分析計の組み合わせ（ＧＣ‐ＡＭＳ）による個々の化合物の放射性炭素年代測定

Development of key techniques for compound-specific radiocarbon analysis by the combination of multi-dimensional chromatographic techniques, especially preparative capillary gas chromatograph, with accelerator mass spectrometry were briefly reviewed together with some recent application to environmental researches.

Advanced and innovative chromatographic techniques for the study of citrus essential oils

AbstractA brief description is given of advanced chromatographic techniques currently employed in citrus essential oil analysis (the interactive use of linear retention indices and MS spectra probability matching in GC–MS; on‐line ‘heart cutting’ multidimensional chromatographic techniques, comprehensive GC and LC; fast GC and GC–MS; fast HPLC; HPLC–MS). A series of applications are reported, demonstrating both the valid and the potential of these methods. Copyright © 2005 John Wiley & Sons, Ltd.

Different multidimensional chromatographic approaches applied to the study of wine malolactic fermentation

Different multidimensional chromatographic techniques were used to study wine aroma pattern changes during malolactic fermentation (MLF). Ethyl lactate enantiomeric ratios were determined using on-line multidimensional gas chromatography. The values found agree with a spontaneous MLF. Off-line multidimensional HPLC/GC was used to deconvolute and enrich the sample and ease enantioselective chromatography. Chiral compound enantiomeric ratio changes during MLF were monitored. Evaluation of enantiomeric ratio changes during MLF has never been studied. ( R, R), ( S, S) and meso-butane-2,3-diol and pentane-2,4-diol (reported in wines for the first time) were submitted to untrained sensory panel tests. All stereoisomers revealed different sensory notes; pentane-2,4-diol showed an aromatic impact.

Ultrasonically assisted solid‐phase extraction and GC analysis of filbertone in hazelnut oil

AbstractUltrasound was used to assist solid‐phase extraction (SPE) of filbertone (E‐5‐methylhept‐2‐en‐4‐one) from hazelnut oil. Interferences from TG were reduced for effective separation and detection during chromatographic analysis. The enantiomeric distribution of filbertone was determined. Different sorbent materials, sample volumes, and eluents were tested, and the effect on filbertone recovery by ultrasound during the elution step was evaluated. Experimentation performed on a 2‐mL volume of oil diluted with a 2‐mL volume of n‐hexane, using a silica modified with either a cyano or phenyl group during the extraction, and ultrasound‐assisted elution with a 1‐mL volume of chloroform allowed filbertone recoveries of up to 94.4% (relative SD 6.5%) vs. 11% obtained when ultrasound was not applied. This improvement is most likely due to a mechanism of cavitation. Ultrasonically assisted SPE is proposed as an accesible and simple alternative to multidimensional chromatographic techniques to accomplish the reliable determination of the enantiomeric excess of chiral compounds in complex matrices, such as hazelnut oil, since experimental conditions that can bring about racemization are not required.

Formation and Lifetime of Polycyclic Aromatic Compounds During a National-Scale Transient Pollution Episode

Concentrations of selected particle-borne parent, alkylated, oxygenated, and nitrated polycyclic aromatic compounds (PACs) have been monitored before, during, and after a large national-scale wood-burning pollution episode (5 November 1994 and 4-5 November 1995). Toluene-modified supercritical CO 2 was used for the extraction of PACs from the collected particulate matter. Analysis was performed using an on-line multidimensional chromatographic technique using coupled liquid and gas chromatography (LC-GC) with loop interface. Comparison of average urban and wood smoke particulate PAC profiles identified a marked change in the ambient PAC profile for the pollution episode period, caused by the change in emission source from predominantly vehicular to wood smoke. A chemical mass balance model has been used to calculate the relative contributions from general urban and wood smoke PAC sources and from them, an estimation of the atmospheric life-time of the PAC emissions has been made. When monitored with 24 hr resolution in 1994, the concentrations of quinone-PAC oxidation products were found to peak 1 day after the maximum concentrations in the parent PAH were recorded, consistent with photochemically driven reactions with singlet state molecular oxygen. When concentrations were monitored on a more frequent basis in 1995, the concentrations of oxygenated species were seen to begin increasing in concentration before sunrise but still peaking after the maximum concentrations of parent PAH were recorded. The nighttime rise in product PAC suggests dark reactions with ozone or the NO 3 radical also may be occurring.

Multi-dimensional chromatography using different developing methods

A new multi-dimensional chromatographic technique is described, μg to mg quantities from the gas chromatogram are placed on the start-line of a thin layer of adsorbent or of a sheet of chromatographic paper, which is moved past the orifice of the gas chromatograph and then developed in the usual manner. Thus, separation of the materials is obtained by exploiting the two most extreme possibilities given by the existing chromatographic methods, viz. gas—liquid chromatography, which separates according to the relative volatility of compounds (in the direction of the time-axis along the start), and thin-layer chromatography or paper chromatography, which separate according to the type of the functional group (in the vertical direction). After the extraction of the separated materials from the thin-layer or paper, GLC, on a suitably selected stationary phase, may be repeated if necessary. The theor for separation and experimental details are given. Further possibilities are obtainable by programming the driving velocity of the plate (paper) which acts in the same manner as temperature programming of a GC column. The method described was used successfully in the research on the composition of coal tar, but can be applied to other complex mixtures of compounds of different chemical character.