Reverse-Polarization High-Performance Layer Electrochromatography-A New Approach to Anion Separation.

High-performance layer electrochromatography (HPLEC) combines the advantages of overpressured-layer chromatography (OPLC) and pressurized planar electrochromatography (PPEC) while overcoming some of their limitations. HPLEC equipment can work in various HPLEC, OPLC, and PPEC modes. The equipment enables HPLEC analysis also with an electroosmotic effect directed against the hydrodynamic flow of the mobile phase. The change in the electric field direction in the separation system does not result in a change in either the direction of the mobile phase flow or the direction of solute migration. The hydrodynamic flow generated by the pump dominates the electroosmotic effect and enables separation against the direction of the latter. Reversed-polarization HPLEC may be advantageous for the analysis of anionic compounds, as it facilitates faster and more selective separation than OPLC performed in similar conditions. This separation mode provides a new possibility to develop and optimize separation methods by performing separation against the electroosmotic effect and without need of any modification of the adsorbent surface. A drawback of this separation mode is the increase in the backpressure at the mobile phase inlet and the limitation of the mobile phase flow rate. Currently, contrary to the single-channel mode, multi-channel reverse-polarity HPLEC still requires some technical and methodological improvements.

Comparison of Overpressured-Layer Chromatography and High-Performance/High-Pressure Layer Electrochromatography Using the New Prototype Equipment in Various Operational Modes.

In our previous paper we have presented the new prototype equipment and introduced a new analytical technique-high-performance/high-pressure layer electrochromatography (HPLEC), a combination of overpressured-layer chromatography (OPLC) and pressurized planar electrochromatography (PPEC). In this paper, the work of the equipment in various operational modes is investigated. Some difficulties and challenges related to various aspects of separation are discussed. The OPLC and HPLEC techniques are compared in terms of selectivity and performance. The results show that our equipment can be successfully used for singe- and multichannel OPLC and HPLEC separations in various sample application and detection modes. It includes the high-throughput, multichannel, and fully automated online separation of multiple samples simultaneously. The equipment allows for the independent optimization of various operational parameters. HPLEC combines the advantages of column/capillary and planar separation techniques while overcoming their limitations. It also combines the advantages and overcomes the drawbacks of OPLC and PPEC. It provides hydrodynamic flow of the mobile phase, irrespective of the voltage used and/or the mobile phase composition. Thus, any optimization of the composition and the voltage can be used independently. Both can be used to obtain the required selectivity of separation. The voltage can be used to facilitate the mobile phase flow and accelerate the analysis.

High Performance (High Pressure) Layer Electrochromatography Separation Technique: Equipment and Preliminary Results.

We present a new prototype device and propose a new analytical technique: high performance (high pressure) layer electrochromatography (HPLEC). The equipment provides a combination of overpressured layer chromatography (OPLC) and pressurized planar electrochromatography (PPEC), yet it still enables researchers to perform each of these analyses separately. In comparison to PPEC, HPLEC provides hydrodynamic flow of the mobile phase, irrespective of the voltage used and the mobile phase composition. The advantages of HPLEC over OPLC include the possibility of the use of the electrophoretic effect to influence the selectivity of separation and the use of the electroosmotic effect to facilitate the mobile phase flow in order to decrease backpressure and increase the flow velocity. Many operational parameters can be freely adjusted and optimized independently. The equipment is fully automated and can work in various separation/operational modes, including combinations of online/offline sample application and detection. We present preliminary results of simultaneous, fully online, multichannel HPLEC separation of analgesic drugs (including acetaminophen, ibuprofen, and tramadol) as an example of increasing analysis throughput.

Solvent Front Position Extraction and some conventional sample preparation techniques for the determination of coccidiostats in poultry feed by LC-MS/MS.

Solvent Front Position Extraction is a novel technique developed for effective sample preparation of biological samples containing coccidiostats prior to LC-MS/MS. In this study the technique was used for isolation and determination of seven coccidiostats, from both main groups being: ionophores and chemical coccidiostats. Its effectiveness was evaluated by comparing with other sample preparation procedures, used in European routine laboratories. Results obtained by Solvent Front Position Extraction were very satisfactory (linearity R2 ≥ 0.971, recovery 90.1-111.1%, RSDr: 8.7-16.6%, RSDR: 9.0-17.7%) and fulfilled requirements described in Commission Regulation (EU) 2021/808 of 22 March 2021, which showed great potential of the technique in sample preparation of coccidiostats in poultry feed.

Preparation of Antihypertensive Drugs in Biological Matrix with Solvent Front Position Extraction for LC-MS/MS Analysis.

Solvent front position extraction procedure was used to prepare biological samples containing selected antihypertensive drugs (ramipril, lercanidipine, indapamide, valsartan, hydrochlorothiazide, perindopril, and nebivolol). Substances separated from the biological matrix components (bovine serum albumin) were quantified by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Sample preparation process was performed with the use of a prototype horizontal chamber with a moving pipette driven by a 3D printer mechanism enabling a controlled eluent flow velocity. Application of this device was advantageous for simultaneous preparation of several samples for further quantitative analysis, with a synchronized reduction of manual operations and solvent consumption. Quantitative results obtained for the majority of the investigated antihypertensive drugs in a complex biological matrix were satisfactory. The values of the %RSD were around 5% for six of the seven substances (with the exception of indapamide). The method exhibits a suitable accuracy (the relative error percentage was below 10% for most drugs). The values of LOD and LOQ were in the range of 1.19 µg/L-8.53 µg/L and 3.61 µg/L-25.8 µg/L, respectively.

Optimization of Adsorbent Layer Type and Developing Solvent in Coccidiostats Sample Preparation with Procedure of Solvent Front Position Extraction.

Coccidiostats are drugs used against coccidiosis, a common disease among breeding animals. Their widespread application leads to the appearance of their residues in food, which is potentially harmful for human health and life. The European Union has established limits of concentrations of these drugs in premixtures and food. Nowadays, there are many methods for monitoring coccidiostats' presence in market products, but their frequent weakness is sample preparation. Solvent Front Position Extraction is a planar chromatography-based sample preparation method that allows for effective assay of samples with coccidiostats when coupled with LC-MS/MS. The purpose of this research was to find common conditions for the effective isolation of eight coccidiostats from biological sample components with both lower and higher retention than the substances of interest. The acquired results were used for effective isolation of monensin and salinomycin from the premixture samples and allowed for their quantitative determination. The application of a semi-automatic device for the development of chromatograms positively impacted the results, confirming the effectiveness of the method for determining coccidiostats in biological samples.

Reversed-phase stepwise gradient thin-layer chromatography of test dye mixtures with controlled developing solvent velocity.

The new approach to the development of planar chromatogram with controlled developing solvent velocity was implemented for gradient reversed-phase chromatography of a test mixture of dyes. The developing solvent components were directly delivered onto the surface of the C18 type of silica adsorbent layer with controlled velocity by moving pipette combined with two syringe pumps. The eluent components were mixed onto the adsorbent layer, so in this way, there was no dwell volume of the developing solvent during gradient chromatogram development. The delivery of the developing solvent solution to the adsorbent layer was adjusted to its absorption rate by the adsorbent layer. Under such conditions, there was no excess of the eluent solution on the surface of the adsorbent layer, so the high performance of the chromatographic system could be obtained. In the paper, the results of the repeatability and reproducibility test of migration distances of selected separated solute zones are presented.

Solvent Front Position Extraction with Semi-Automatic Device as a Powerful Sample Preparation Procedure Prior to Quantitative Instrumental Analysis.

The new prototype device is applied to the Solvent Front Position Extraction (SFPE) sample preparation procedure. The mobile phase is deposited onto the chromatographic plate adsorbent layer by the pipette, which is moved, according to programmed movement path, by a 3D printer mechanism. The application of the prototype device to SFPE procedure leads to the increased repeatability of the results and significant reduction of the analysis time in comparison to the classical procedure of chromatogram development. Additionally, the new equipment allows use procedures that are not possible to run using the classic chromatogram development. In this paper, the results of manual and semi-automatic sample preparation with SFPE are compared and the possible application of this prototype device is discussed.

Two-dimensional high-performance thin-layer chromatography of tryptic bovine albumin digest using normal- and reverse-phase systems with silanized silica stationary phase.

Among many advantages of planar techniques, two-dimensional (2D) separation seems to be the most important for analysis of complex samples. Here we present quick, simple and efficient two-dimensional high-performance thin-layer chromatography (2D HPTLC) of bovine albumin digest using commercial HPTLC RP-18W plates (silica based stationary phase with chemically bonded octadecyl ligands of coverage density 0.5µmol/m(2) from Merck, Darmstadt). We show, that at low or high concentration of water in the mobile phase comprised methanol and some additives the chromatographic systems with the plates mentioned demonstrate normal- or reversed-phase liquid chromatography properties, respectively, for separation of peptides obtained. These two systems show quite different separation selectivity and their combination into 2D HPTLC process provides excellent separation of peptides of the bovine albumin digest.