comprehensive two-dimensional chromatography
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Two-dimensional liquid chromatography largely increases the number of separated compounds in a single run, theoretically up to the product of the peaks separated in each dimension on the columns with different selectivities. On-line coupling of a reversed-phase column with an aqueous normal-phase (hydrophilic interaction liquid chromatography) column yields orthogonal systems with high peak capacities. Fast on-line two-dimensional liquid chromatography needs a capillary or micro-bore column providing low-volume effluent fractions transferred to a short efficient second-dimension column for separation at a high mobile phase flow rate. We prepared polymethacrylate zwitterionic monolithic micro-columns in fused silica capillaries with structurally different dimethacrylate cross-linkers. The columns provide dual retention mechanism (hydrophilic interaction and reversed-phase). Setting the mobile phase composition allows adjusting the separation selectivity for various polar substance classes. Coupling on-line an organic polymer monolithic capillary column in the first dimension with a short silica-based monolithic column in the second dimension provides two-dimensional liquid chromatography systems with high peak capacities. The silica monolithic C18 columns provide higher separation efficiency than the particle-packed columns at the flow rates as high as 5 mL/min used in the second dimension. Decreasing the diameter of the silica monolithic columns allows using a higher flow rate at the maximum operation pressure and lower fraction volumes transferred from the first, hydrophilic interaction dimension, into the second, reversed-phase mode, avoiding the mobile phase compatibility issues, improving the resolution, increasing the peak capacity, and the peak production rate.
- Klíčová slova
- comprehensive two-dimensional chromatography, monolithic columns, polymethacrylate monoliths, silica-based monolithic columns,
- Publikační typ
- časopisecké články MeSH
Recent advances in applications of gradient elution in two-dimensional liquid chromatography are reviewed. Benefits of simultaneous programmed elution in two-dimensional liquid chromatography are shown, with emphasis on fast gradient elution in second dimension using various gradient types and profiles. The theory of gradient elution is shortly overviewed with attention to its impacts on two-dimensional separations. Various two-dimensional gradient systems are compared in terms of peak capacity and peak production rate: off-line, stop-and-go and real-time comprehensive approaches. Selection of stationary phases in combination with gradient elution and the effects of gradient elution on improved regularity of two-dimensional space coverage, orthogonality and peak capacity are discussed. Not only two-dimensional reversed-phase systems, but also other HPLC modes such as HILIC, ion-exchange or normal-phase chromatography with organic mobile phases are included. Fraction modulation in gradient elution setups, mobile phase compatibility and solvent effects in the fraction transfer between the first and the second dimension are addressed. Design and optimization of two-dimensional gradient separation systems for particular ordered and non-ordered samples are dealt with in detail. The discussed principles are illustrated by practical examples.
- MeSH
- chromatografie kapalinová metody MeSH
- organické látky chemie MeSH
- rozpouštědla chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- organické látky MeSH
- rozpouštědla MeSH
Two-dimensional liquid-phase separations have gained increasing attention for their ability to separate complex sample mixtures. Among the experimental setups used, an on-line approach is preferred to reduce the probability of sample contamination, for easier automation and high-sample throughput. The interfacing of the separation techniques in the on-line mode brings additional demands on proper optimization of the two-dimensional system. In this review, the possibilities of the on-line coupling of liquid chromatography and liquid chromatography with capillary electrophoresis in two-dimensional systems are discussed. Special attention is paid to the fraction transfer process, which includes an overview of interfaces and experimental setups applied, the compatibility issues of separation systems, and instrumental parameters. The benefits and drawbacks of using electromigration separations in combination with liquid chromatography are presented as well.
- Klíčová slova
- capillary electrophoresis, comprehensive liquid chromatography, fraction transfer, liquid chromatography, two-dimensional separations,
- MeSH
- chemické techniky analytické normy trendy MeSH
- chromatografie kapalinová * MeSH
- elektroforéza kapilární * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The separation in liquid chromatography is defined either by the space domain where it proceeds until the least retained analyte reaches the outlet of the column or by the time when individual analytes elute out of the column. These two approaches lead to the four possible combinations of two-dimensional liquid chromatography with online space x time coupling being the least experimentally feasible. Here, we show the development of a novel two-dimensional liquid chromatography method combining separation defined by space and the conventional elution-based separation. First-dimension column consisted of four capillary segments coupled serially via two-position six-port valves allowing an online and comprehensive transfer of analytes from the first to the second dimension. After initial experiments using homemade monolithic capillary columns, we tested commercially available columns in both dimensions. We ended with the combination of packed capillary columns in the first dimension and monolithic capillary column in the second dimension. We used a reversed-phase retention mechanism in the first spatial dimension, while HILIC was in the second, time-based dimension. We also developed a theoretical model to describe the proposed two-dimensional separation that was further confirmed by utilizing both an isocratic and gradient elution in the second dimension. Finally, we applied our experimental setup to separate neurotransmitters contained in human urine.
- Klíčová slova
- Modulation, Polymer monoliths, Retention modeling, Spatial separation, Two-dimensional liquid chromatography,
- MeSH
- chromatografie kapalinová metody MeSH
- chromatografie s reverzní fází * metody MeSH
- lidé MeSH
- ověření koncepční studie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Three different comprehensive 2-D HPLC systems for the separation of phenolic antioxidants have been developed on the basis of different selectivities of a PEG-silica column in the first dimension and a packed or monolithic C18 or a ZR-CARBON column, respectively, in the second dimension. Two-dimensional comprehensive liquid chromatography using a serially connected short PEG-silica column and a conventional C18-silica or a ZR-CARBON column in the second dimension was tested to improve the resolution of the earlier eluting compounds in the first dimension. Various types of interface were used to connect the columns in the first and in the second dimension: i) two injection sampling loops of 100 microL in conventional arrangement; ii) a 10-port 2-position valve equipped with two trapping X-Terra columns instead of loops; and iii) two analytical D2 columns in parallel. The mobile phase in the first dimension has a lower elution strength than in the second dimension, allowing band compression of the solutes transferred from the first to the second dimension. This effect was enhanced using trapping columns instead of sampling loops as the interface between the two dimensions, thus allowing a decrease in the time of analysis. These systems were used for the analysis of beer samples. The relative location of the components in the 2-D retention plane varied in relation to their chemical structure in each instrumental set-up and allowed positive peak identification.
- MeSH
- 2D gelová elektroforéza MeSH
- analýza potravin metody MeSH
- antioxidancia analýza chemie izolace a purifikace MeSH
- chemické techniky analytické přístrojové vybavení metody MeSH
- chromatografie kapalinová metody MeSH
- chromatografie metody MeSH
- fenol analýza chemie izolace a purifikace MeSH
- kinetika MeSH
- oxid křemičitý chemie MeSH
- pivo MeSH
- polyethylenglykoly chemie MeSH
- regresní analýza MeSH
- uhlík chemie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zirkonium chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antioxidancia MeSH
- fenol MeSH
- oxid křemičitý MeSH
- polyethylenglykoly MeSH
- uhlík MeSH
- zirconium oxide MeSH Prohlížeč
- zirkonium MeSH
In-line coupled comprehensive HILIC×RP systems should offer larger selectivity differences and better two-dimensional orthogonality than coupled RP×RP systems. However, this may not apply for all systems. The HILIC selectivity depends on the mix of selective polar and non-polar interactions with the functional groups, but also with the matrix of polar columns and depends on the sample type. We synthesized a new polar monolithic sulfobetaine polymethacrylate capillary column with excellent efficiency for low-molecular compounds. When used in the first, HILIC dimension coupled to core-shell or monolithic RP columns in the second dimension, this column provides much improved orthogonality for two-dimensional separations of phenolic and flavonoid compounds, in comparison to silica-bonded Diol, Polyethylene glycol or Zwitterionic columns. We investigated the performance of 11 short 5 cm and 3 cm columns for fast (1-2 min) gradient second-dimension separations. Band broadening or distortion may occur in directly coupled comprehensive HILIC×RP systems, due to strong solvent-strength differences between the mobile phases used in the first and in the second dimension. To suppress this effect, low fraction volumes were collected from a 0.5mm I.D. capillary monolithic sulfobetaine column at the flow-rate of a few microliters per min, coupled in-line with various core-shell columns operated at the maximum flow-rate. This setup with simultaneous gradient elution in the HILIC and in the RP dimension provided successful separation of natural antioxidants.
- MeSH
- antioxidancia analýza MeSH
- chromatografie s reverzní fází přístrojové vybavení metody MeSH
- fenoly analýza MeSH
- flavonoidy analýza MeSH
- hydrofobní a hydrofilní interakce MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antioxidancia MeSH
- fenoly MeSH
- flavonoidy MeSH
A new continuous comprehensive two-dimensional liquid chromatography-electrospray ionization mass spectrometry method has been developed for the lipidomic characterization of complex biological samples. The reversed-phase ultra-high-performance liquid chromatography with a C18 column (150 mm × 1 mm, 1.7 μm) used in the first dimension makes the separation of numerous lipid species differing in their hydrophobic part of the molecule, mainly fatty acyl chain lengths and the number and positions of double bonds, possible. Coeluted lipid species in the first dimension are resolved by the fast hydrophilic interaction liquid chromatography separation (50 mm × 3 mm, 2.7 μm, core-shell particles) of lipid classes according to their different polarities in the second dimension. Retention times in both dimensions, accurate m/z values, and tandem mass spectra provide high confidence in the identification of lipid species. The retention behavior of individual lipids in reversed-phase mode follows the equivalent carbon number pattern, which provides an additional tool for unambiguous identification. This analytical method is applied for the lipidomic characterization of total lipid extracts of human plasma and porcine brain samples, which resulted in the identification of 143 lipid species from four lipid categories and ten lipid classes.
- MeSH
- chromatografie s reverzní fází metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- hydrofobní a hydrofilní interakce MeSH
- lidé MeSH
- lipidy analýza krev izolace a purifikace MeSH
- mozek - chemie * MeSH
- prasata MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- lipidy MeSH
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.
- MeSH
- antioxidancia izolace a purifikace MeSH
- chromatografie kapalinová metody MeSH
- fenoly izolace a purifikace MeSH
- flavony izolace a purifikace MeSH
- senzitivita a specificita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antioxidancia MeSH
- fenoly MeSH
- flavony MeSH
Gradient elution provides significant improvement in peak capacity with respect to isocratic conditions. In the second dimension, gradients are limited to a short-time period available for separation. Various types of second-dimension gradients in comprehensive LC x LC are compared: (i) "full in fraction", (ii) "segment in fraction" and (iii) "continuously shifting" gradients, applied in orthogonal LC x LC separations of phenolic acids and flavones on a polyethylene glycol column in the first dimension and two types of porous shell fused-core C18 columns in the second dimension (Ascentis Express and Kinetex). The porous shell columns provide narrow bandwidths and fast second-dimension separations at moderate operating pressure that allows important savings of the overall separation time in comprehensive LC x LC separations. The effects of the gradient type on the bandwidths, theoretical peak capacity, separation time and column pressure in the second dimension were investigated. The type of gradient program controls the range of lipophilicity of sample compounds that can be separated in the second-dimension reversed-phase time period. This range can be calibrated using alkylbenzene standards, to design the separation conditions for complete sample separation, avoiding harmful wrap around of non-eluted compounds to the subsequent second-dimension fractions.
- MeSH
- chromatografie kapalinová metody MeSH
- flavony izolace a purifikace MeSH
- hydroxybenzoáty izolace a purifikace MeSH
- kalibrace MeSH
- poréznost MeSH
- povrchové vlastnosti MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- flavony MeSH
- hydroxybenzoáty MeSH
- phenolic acid MeSH Prohlížeč
Head-space solid phase microextration (SPME), followed by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOFMS), has been implemented for the analysis of honey volatiles, with emphasis on the optimal selection of SPME fibre and the first- and second-dimension GC capillaries. From seven SPME fibres investigated, a divinylbenzene/Carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 microm fibre provided the best sorption capacity and the broadest range of volatiles extracted from the headspace of a mixed honey sample. A combination of DB-5ms x SUPELCOWAX 10 columns enabled the best resolution of sample components compared to the other two tested column configurations. Employing this powerful analytical strategy led to the identification of 164 volatile compounds present in a honey mixture during a 19-min GC run. Combination of this simple and inexpensive SPME-based sampling/concentration technique with the advanced separation/identification approach represented by GCxGC-TOFMS allows a rapid and comprehensive examination of the honey volatiles profile. In this way, the laboratory sample throughput can be increased significantly and, at the same time, the risk of erroneous identification, which cannot be avoided in one-dimensional GC separation, is minimised.
