Petr Česla

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.

Optimization of comprehensive two-dimensional gradient chromatography coupling in-line hydrophilic interaction and reversed phase liquid chromatography

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.

Multidimensional LC×LC analysis of phenolic and flavone natural antioxidants with UV‐electrochemical coulometric and MS detection

A comprehensive 2-D LC x LC system was developed for the separation of phenolic and flavone antioxidants, using a PEG-silica column in the first dimension and a C(18) column with porous-shell particles or a monolithic column in the second dimension. Combination of PEG and C18 or C8 stationary phase chemistries provide low selectivity correlations between the first dimension and the second dimension separation systems. This was evidenced by large differences in structural contributions to the retention by -COOH, -OH and other substituents on the basic phenol or flavone structure. Superficially porous columns with fused core particles or monolithic columns improve the resolution and speed of second dimension separation in comparison to a fully porous particle C(18) column. Increased peak capacity and high orthogonality in different 2-D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2-D separation time range. Multi-dimensional set-up combining the LC x LC separation on-line with UV and multi-channel coulometric detection and off-line with MS/MS technique allowed positive peak identification. The Coularray software compensates for the effects of the baseline drift during the gradient elution and is compatible with parallel gradient comprehensive LC x LC technique. Furthermore, it provides significant improvement in the sensitivity and selectivity of detection in comparison to both UV and MS detection. The utility of these systems has been demonstrated in the analysis of beer samples.

Orthogonality of silver‐ion and non‐aqueous reversed‐phase HPLC/MS in the analysis of complex natural mixtures of triacylglycerols

The goal of this work is the study of possibilities of two basic separation modes used in the analysis of complex triacylglycerol (TG) samples of plant oils and animal fats, i.e. non-aqueous reversed-phase (NARP) and silver-ion HPLC coupled with atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The orthogonality of both separation modes is tested for complex TG mixtures containing fatty acids (FAs) with different acyl chain lengths, different number, positions and geometry of double bonds (DBs) and different regioisomeric positions of FAs on the glycerol skeleton. The retention in NARP mode is governed by the equivalent carbon number, while the retention in silver-ion chromatography increases with the increasing number of DBs with a clear differentiation between cis- and trans-FAs. Moreover, silver-ion mode enables at least the partial resolution of regioisomeric TG mixtures including cis-/trans-regioisomers, as illustrated on two examples of randomization mixtures. Off-line 2D coupling of both complementary modes (NARP in the first dimension and silver-ion in the second dimension) yields the superior chromatographic selectivity resulting in the highest number of identified TGs ever reported for studied samples. Off-line 2D chromatograms are processed with the home-made software providing various ways of data visualization.

Capillary electrophoretic chiral separation of Cinchona alkaloids using a cyclodextrin selector

A new capillary electrophoretic method for the chiral separation of four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) was developed using heptakis-(2,6-di-O-methyl)-beta-cyclodextrin as the chiral selector. The inner walls of the separation capillary were modified with a thin polyacrylamide layer, which substantially reduced the electroosmotic flow and improved the chiral resolution and the reproducibility of the migration time of the analytes. Various operation parameters were optimised, including the pH, the capillary temperature, the concentration of the background electrolyte, and the concentration of the chiral selector. Baseline separation of the two diastereomer pairs was achieved in 12 minutes in ammonium acetate background electrolyte pH 5.0 with addition of cyclodextrin in a concentration of 3 mM or higher.

Separation of phenolic acids and flavone natural antioxidants by two-dimensional method combining liquid chromatography and micellar electrokinetic capillary chromatography.

A 2‐D method was developed for separation of phenolic acids and flavone compounds combining LC with MEKC. The effect of substituted neutral and anionic CD additives to the background electrolyte on the quality of MEKC separation was investigated. The best selectivity of the MEKC separation was achieved in 25 mmol/L borate background buffer at pH 9.05 with the addition of 10 g/L SDS and 1.85 g/L heptakis (6‐O‐sulfo)‐β‐CD. These conditions were used in the second dimension of 2‐D combination of LC and MEKC separation in combination with a PEG column in the first dimension, providing the best orthogonality (the lowest degree of correlation between the selectivity of separation) in the two dimensions. A CE autosampler was employed as the interface between LC and MEKC steps based on automated fraction collection before the re‐analysis of the collected LC fractions in the second, MEKC dimension. The 2‐D method under optimized conditions was applied for the separation of natural antioxidants in the samples of green tea.

Automated dual two-dimensional liquid chromatography approach for fast acquisition of three-dimensional data using combinations of zwitterionic polymethacrylate and silica-based monolithic columns

A monolithic sulfobetaine polymethacrylate micro-column BIGDMA-MEDSA designed in our laboratory, shows dual retention mechanism: In acetonitrile-rich mobile phase, hydrophilic interactions control the retention (HILIC system), whereas in more aqueous mobile phases the column shows essentially reversed-phase behavior with major role of hydrophobic interactions. The zwitterionic polymethacrylate micro-column can be used in the first dimension of two-dimensional LC in alternating reversed-phase (RP) and HILIC modes, coupled with an alkyl-bonded core-shell or silica-based monolithic column in the second dimension, for HILIC×RP and RP×RP comprehensive two-dimensional separations. During the HILIC×RP period, a gradient of decreasing acetonitrile gradient is used for separation in the first dimension, so that at the end of the gradient the polymeric monolithic micro-column is equilibrated with a highly aqueous mobile phase and is ready for repeated sample injection, this time for separation under reversed-phase gradient conditions with increasing concentration of acetonitrile in the first dimension. The fast repeating reversed-phase gradients on a short silica-monolithic or core-shell column in the second dimension can be optimized independently of the actual running first-dimension gradient program. As the alternating HILIC and RP separations on the first-dimension zwitterionic methacrylate column are based on complementary retention mechanisms, the instrumental setup essentially represents two coupled two-dimensional systems. It is first time that such an automated dual LCxLC approach is reported. The novel system allows obtaining three-dimensional data in a relatively short time and can be applied not only to multidimensional gradient separations of flavones and related polyphenolic compounds.

Improvement of the sensitivity of 2D LC-MEKC separation of phenolic acids and flavonoids natural antioxidants using the on-line preconcentration step.

A 2D method was developed for separation of phenolic acids and flavonoids natural antioxidants combining LC with MEKC. The in‐capillary preconcentration step was applied for the improvement of the sensitivity of 2D method before the second dimension MEKC analysis. The influence of first dimension LC mobile phase composition on migration times in the second MEKC dimension was evaluated. When gradient elution is applied in the first dimension of 2D LC‐MEKC system, increasing concentration of organic solvent in the mobile phase and in fractions transferred from LC influences the electroosmotic flow, partitioning equilibria of samples in micelles and properties of the micelles, which results in shifts of migration times during the consecutive runs in the second MEKC separation dimension. The shifts of migration times caused by the influence of increasing concentration of ACN on MEKC separation in second dimension of 2D LC‐MEKC system were compensated by aligning the time axis using electroosmotic flow and micellar marker migration times. The optimized LC‐MEKC method was applied on the separation of natural antioxidants in the plant extracts samples.

Fraction transfer process in On‐line comprehensive Two‐dimensional liquid‐phase separations

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.

Comparison of isocratic retention models for hydrophilic interaction liquid chromatographic separation of native and fluorescently labeled oligosaccharides.

In this work, we have investigated retention of maltooligosaccharides and their fluorescent derivatives in hydrophilic interaction liquid chromatography using four different stationary phases. The non-derivatized maltooligosaccharides (maltose to maltoheptaose) and their derivatives with 2-aminobenzoic acid, 2-aminobenzamide, 2-aminopyridine and 8-aminonaphthalene-1,3,6-trisulfonic acid were analyzed on silica gel, aminopropyl silica, amide (carbamoyl-bonded silica) and ZIC-HILIC zwitterionic sulfobetain bonded phase. The partitioning of the analytes between the bulk mobile phase and adsorbed water-rich layer, polar and ionic interactions of analytes with stationary phase have been evaluated and compared. The effects of the mobile phase additives (0.1% (v/v) of acetic acid and ammonium acetate in concentration range 5-30 mmol L(-1)) on retention were described. The suitability of different models for prediction of retention was tested including linear solvent strength model, quadratic model, mixed-mode model, and empirical Neue-Kuss model. The mixed-mode model was extended to the parameter describing the contribution of monomeric glucose unit to the retention of non-derivatized and derivatized maltooligosaccharides, which was used for evaluation of contribution of both, oligosaccharide backbone and end-group to retention.