Inverz módszerek kidolgozása a molekuláris kölcsönhatások vizsgálatára folyadékkromatográfiában = Developing inverse methods to study the molecular interactions in liquid chromatography

Elsősorban a fordított fázisú folyadékkromatográfia területén tanulmányoztuk különböző vegyületek megkötődésének retenciós mechanizmusát, illetve kinetikáját, de vizsgálatainkat kiterjesztettük az ioncsere-kromatográfia, a királis elválasztások és a méretszerinti kromatográfia területére is. Numerikus eljárást dolgoztunk ki, amellyel a csúcsok alakjából a retenciós tulajdonságok meghatározhatók. A kromatográfia sztochasztikus modelljének alkalmazhatóságát bizonyítottuk ezeken a területeken. Összehasonlítottuk a mikroszkopikus és makroszkopikus modellek alkalmazhatóságát. Egyensúlyi izotermák, illetve oldószerelegyek többletizotermáinak mérésével jellemeztük a metanol és acetonitril adszorpcióját nagyszámú állófázison, és így a molekuláris kölcsönhatásokat fordított fázisú folyadékkromatográfiában. | We have studied the retention mechanism and kinetics of a number of solutes in reversed phase chromatography as well as in chiral, ion exchange, and size exclusion chromatography. We developed a numerical method to estimate the retention properties on the basis of peak shape analysis. We have proved that the stochastic model of chromatography can successfully be applied for this purpose. We have compared the results obtained with microscopic and macroscopic modeling. We have measured the adsorption isotherms of a number of solutes to characterize various HPLC stationary phases. Furthermore we used the measurement of excess isotherms of methanol and acetonitrile to characterize molecular interactions in reversed phase liquid chromatography

Multilayer adsorption on fractal surfaces

AbstractMultilayer adsorption is often observed in liquid chromatography. The most frequently employed model for multilayer adsorption is the BET isotherm equation. In this study we introduce an interpretation of multilayer adsorption measured on liquid chromatographic stationary phases based on the fractal theory. The fractal BET isotherm model was successfully used to determine the apparent fractal dimension of the adsorbent surface. The nonlinear fitting of the fractal BET equation gives us the estimation of the adsorption equilibrium constants and the monolayer saturation capacity of the adsorbent as well. In our experiments, aniline and proline were used as test molecules on reversed phase and normal phase columns, respectively. Our results suggest an apparent fractal dimension 2.88–2.99 in the case of reversed phase adsorbents, in the contrast with a bare silica column with a fractal dimension of 2.54

The correctness of van 't Hoff plots in chiral and achiral chromatography

Abstract van 't Hoff plots (logarithm of the retention factor, ln k, vs. the reciprocal of absolute temperature, 1/T) are commonly used in chromatographic studies to characterize the retention mechanisms based on the determined enthalpy (ΔH∘) and entropy (ΔS∘) change of analyte adsorption. In reversed phase liquid chromatography, the thermodynamic parameters could help to understand the retention mechanism. In chiral chromatography, however, the conclusions drawn based on van 't Hoff plots can be deceptive because several different types of adsorption sites are present on the surface of stationary phase. The influence of heterogeneity, however, cannot be studied experimentally. In this study, we employed two reversed phase columns with different retention mechanisms to show that by serially coupling the columns, the obtained thermodynamic parameters are not related to the results obtained on the respective individual columns. Furthermore, our results show that the experimental conditions – such as flow-rate or choice of instrument – will strongly influence the calculated enthalpy and entropy values

Competitive adsorption of sample solvent and analyte in supercritical fluid chromatography

Supercritical fluid chromatography (SFC) has gained increased attention in the fields of theoretical research, purification processes and separation sciences over the last few years due to the latest technological advancements. The method is well-known for being capable of faster separations and reduced solvent consumption compared to liquid chromatography (LC) while also achieving high efficiency. Due to the nature of carbon-dioxide serving as mobile phase, eluent strength is well-tunable by adjusting temperature, pressure and the amount of organic modifier. However, SFC is considered a complementary technique besides liquid and gas chromatography since there is always a trade-off in terms of the advantages [1]. Recently, Gritti experienced an anomaly in the retention behaviour of alkylbenzenes using a heavily modified Waters ACQUITY UPC2 System [2]. These small molecules are ideal for studying the processes taking place in the chromatographic column. Therefore, the aim of this work is to study the retention of several n-alkylbenzenes on an alkylamide stationary phase using a standard UPC2 System and assessing the effect of different sample solvents on chromatographic efficiency. The results show that even a small amount of methanol overloads the column and a competitive adsorption takes place between the analytes and the sample solvent. This is indicated by the changes in column efficiency, retention factors and peak widths. The selected experimental conditions guarantee that the density of the mobile phase does not change significantly along the column, so the phenomenon can be modeled as in LC. The concentration of the analytes is negligible compared to the amount of methanol – but their adsorption is influenced by the solvent – while the adsorption of methanol remains unaffected by the alkylbenzenes. The competition was described by determining the singlecomponent adsorption isotherms for both the analytes and the solvent then competitive isotherms were calculated. The solvent effect was modeled by a numerical method created inhouse where the differential mass balance equation was integrated using the Rouchon algorithm. The experimental observations were confirmed by in silico experiments and further cases involving hypothetical analytes were studied as well

Szénkalitka (fullerén) és hosszú szénláncú, (C30) fordított fázisú szorbensek és szorpciós viszonyok a folyadékkromatográfiában = Carbon-cage (fullerene) and long chain reversed phase adsorbents (C30) and investigation of the sorption equilibrium in liquid chromatography

A kutatás negyedik évében a fullerén kutatások területén két területen értünk el jelentős előrelépést. Fullerének, mint kromatográfiás állófázisok vizsgálatában kimutattuk, hogy szilikagél alapú, fullerén C60 borított szorbensek kiválóan alkalmazhatók hidrofil biomolekulák (foszfopeptidek, glikált peptidek, flavonoidok) koncentrálására, sómentesítésére, frakcionálására. Vizsgáltuk ezen újszerű szorbensek terhelhetőségét, alkalmasságát fehérjeszerkezet meghatározásában. Megállapítottuk, hogy a szokásos fordított fázisú szorbensekhez hasonlítva hidrofil biomolekulák prekoncentrálására sokkal hatásosbban alkalmazhatóak. Fullerének, mint a MALDI-TOF tömegspekrometriában alkalmazható mátrixvegyületek vizsgálatánál bizonyítottuk, hogy a MALDI Tof eljárással csak nehezen analizálható szteroidhormonok (ösztrogének, androgének) ionizálására kitűnően felhasználhatók. C30-as tölteteinket további alkalmazásokban vörösboranalitikában alkalmaztuk. Nagyszámú borminta analízisével kimutattuk, hogy a vörösborok polifenoltartalma erősebben függ a borkészítés technológiájától (a borásztól), mint földrajzi, vagy időjárási körülményektől. Kísérletesen, a C30 állófázisok hatékony elválasztási tulajdonságát kihasználva, elsőként bizonyítottuk, hogy a transz rezveratrol (mely a vörösborok fő kardioprotektív hatóanyaga) oxidádációs terméke közeli UV fény hatására az eddig ismert cisz-rezveratrol mellett egy a szénláncban hármas kötéssel rendelkező polifenol (3,4',5-trihidroxidifenilacetilén). | In the fourth year of our investigations we achieved new results in two fields of fullerene research. It has been demonstrated, that C60 fullerene coated silica stationary phases are excellent for the preconcentration, desalting, fractionating of hydrophilic biomolecules (flavonoids, phosphopeptides, glycopeptides). The loadability of these new phases has been studied. The new phases were found to be excellent for sequence coverage studies of proteins and tryptic digest of proteins. Fullerenes have been used for matrix materials in MALDI TOF mass spectrometry. The ionization of estrogens and androgens were more affective by using C60 or C70 fullerenes as matrices, than any other commonly used matrix material. Our new C30 packings have been extensively used for red wine analysis. It has been found that the most important factor in wine making for high polyphenol contentof wine is the oenologist, rather than geographic or meteorological factors. Exploiting the high separation power of the new C30 phase a new oxidation product of trans-resveratrol has been found. By irradiation of trans resveratrol not only the well known cis-resveratrol has been formed but we detected and described a new tripe-bond polyphenol: 3,4',5-trihydroxy-diphenylacetylene

Protein and alkaloid patterns of the floral nectar in some solanaceous species

The family Solanaceae includes several melliferous plants, which tend to produce copious amounts of nectar. Floral nectar is a chemically complex aqueous solution, dominated by sugars, but minor components such as amino acids, proteins, flavonoids and alkaloids are present as well. This study aimed at analysing the protein and alkaloid profile of the nectar in seven solanaceous species. Proteins were examined with SDS-PAGE and alkaloids were analyzed with HPLC. The investigation of protein profile revealed significant differences in nectar-protein patterns not only between different plant genera, but also between the three Nicotiana species investigated. SDS-PAGE suggested the presence of several Nectarin proteins with antimicrobial activity in Nicotiana species. The nectar of all tobacco species contained the alkaloid nicotine, N. tabacum having the highest nicotine content. The nectar of Brugmansia suaveolens, Datura stramonium, Hyoscyamus niger and Lycium barbarum contained scopolamine, the highest content of which was measured in B. suaveolens. The alkaloid concentrations in the nectars of most solanaceous species investigated can cause deterrence in honeybees, and the nectar of N. rustica and N. tabacum can be considered toxic for honeybees

Overloading study of zwitterionic chiral stationary phases based on cinchona alkaloids

Nowadays, chirality has great importance in the life sciences as well as in the pharmaceutical industry. Usually the enantiomers have diverse activity in the living systems: one of the enantiomers possesses positive effect, while the other may be inactive or toxic. Thus, the separation of the enantiomers is very important. On the Cinchona alkaloid-based ZWIX(+) and ZWIX(-) zwitterionic chiral stationary phases, simultaneous anion- and cation-exchange takes place. These stationary phases are good choice for the separation of the enantiomers of chiral acids, amines, amino acids and peptides. Nonlinear chromatography was used for the investigation of the retention mechanisms. The stationary phases were overloaded by large volume injection of DL-tert-Leucine. BiLangmuir isotherm was used for modeling the adsorption because of the heterogeneous surface of the zwitterionic stationary phases. The isotherm parameters were determined by the inverse method. In the optimization of the enantioseparation, the acid and base additives play an important role, they ensure the ionization of the selector and the selectand. Therefore, the effect of the nature and the ionic strength of the additives on the overloaded chromatographic bands were investigated

Investigation of interactions on chiral zwitterionic stationary phases in enantiomer separation using HPLC and SFC methods

Most biologically and pharmacologically important molecules (amino acids, carbohydrates, pharmaceuticals, etc.) are chiral compounds that may cause various biological effects; therefore increased attention is placed on their separation, qualitative and quantitative analysis. As a result of this knowledge, in this work the theoretical background of chiral chromatographic separation was investigated by high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). For our measurements, a new generation chiral zwitterionic stationary phase (Zwix (-) column) was used to separate the enantiomers one of the antimalarial drugs, mefloquine. According to the stochastic model of chromatography [1,2], the peak formed during separation is a combination of the random walk of a molecule through the column and the slow adsorption-desorption processes. Using this theory, it is possible to determine the residence time of the molecules in the stationary phase and the average number of adsorption steps from which the retention factor can be calculated. For chromatographic measurements, van 't Hoff plot [3] is used to explain the retention mechanism. In our case, the values of the retention factors were determined in two ways, on the one hand by classical calculations, on the other hand with stochastic model, which assumes multiple binding. Finally, the thermodynamic parameters were compared for the two chromatographic methods, high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC)