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Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield an

Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield an

Pavel Jandera was a world-leading analytical chemist who devoted his entire professional life to research in the field of high-performance liquid chromatography. During his scientific career, he worked at the Department of Analytical Chemistry at the University of Pardubice, Czech Republic. His greatest contribution to the field of liquid chromatography was the introduction of a comprehensive theory of liquid chromatography with programmed elution conditions. He was also involved in the research of gradient elution techniques in preparative chromatography, modeling of retention and selectivity in various phase systems, preparation of organic monolithic microcolumns, and, last but not least, in the development of theory and practical applications of two-dimensional liquid chromatography, mainly in the comprehensive form. In this review article, we have tried to capture the highlights of his scientific career and provide the readers with a detailed overview of Pavel Jandera's contribution to the evolution of separation sciences., Pavel Jandera byl předním světovým analytickým chemikem, který celý svůj profesní život zasvětil výzkumu v oblasti vysokoúčinné kapalinové chromatografie. Během své vědecké kariéry působil na katedře analytické chemie Univerzity Pardubice. Jeho největším přínosem pro obor kapalinové chromatografie bylo zavedení ucelené teorie kapalinové chromatografie s programovanými elučními podmínkami. Dále se podílel na výzkumu gradientových elučních technik v preparativní chromatografii, modelování retence a selektivity v různých fázových systémech, přípravě organických monolitických mikrokolon a v neposlední řadě na rozvoji teorie a praktických aplikací dvourozměrné kapalinové chromatografie, především v komplexní podobě. V tomto přehledovém článku jsme se pokusili zachytit nejdůležitější momenty jeho vědecké kariéry a poskytnout čtenářům podrobný přehled o přínosu Pavla Jandery k rozvoji separačních věd.

Tato diplomová práce se zabývá studiem separace alkaloidů chinovníku společně s kofeinem a jeho dvěma hlavními metabolity pomocí kapalinové chromatografie v systému s obrácenými fázemi. V teoretické části je nejprve charakterizována metoda kapalinové chromatografie, což zahrnuje instrumentaci kapalinového chromatografu, fázové systémy a popis procesu optimalizace chromatografických separací. V druhé polovině teoretické části jsou popsány jednotlivé skupiny alkaloidů, jejich vlastnosti a využití. Cíle experimentální práce spočívaly v optimalizaci izokratické separace na pěti chromatografických kolonách a výběru dvou nejvhodnějších kolon pro dané separace, které byly následně využity pro tvorbu metody za podmínek gradientové eluce., This diploma thesis deals with the study of the separation of cinchona alkaloids together with caffeine and its two main metabolites using liquid chromatography in a reversed-phase system. In the theoretical part, at first, the method of liquid chromatography is characterized, which includes the instrumentation of the liquid chromatograph, phase systems and a description of the optimization process of chromatographic separations. In the second half of the theoretical part, individual groups of alkaloids are described including their properties and uses. The goals of the experimental work consisted of the optimization of isocratic separation on five chromatographic columns and the selection of the two most suitable columns for the given separations, which were subsequently used to create a method under gradient elution conditions., Fakulta chemicko-technologická, Studentka přednesla výsledky své diplomové práce, poté byla seznámena s posudky vedoucího a oponenta diplomové práce. Studentka zodpověděla dotazy oponenta a členů komise., Dokončená práce s úspěšnou obhajobou

Acetaminophen (paracetamol, APAP) is one of the most widely used drugs worldwide. Unfortunately, its overdose, which is caused by predominant oxidation of APAP, can lead to acute liver injury. In liver, oxidized APAP is conjugated with glutathione, leading to APAP-glutathione conjugate, which is metabolized to APAP-cysteine and APAP-N-acetylcysteine conjugates. Thus, all of those compounds could be used to monitor APAP metabolism in the overdosed patients. To date, only a limited number of rapid and accurate methods have been reported for the assessment of APAP oxidation metabolites using simple instrumentation, and thus this work was aimed at developing a fast and convenient gradient HPLC-UV/MS method. For this purpose, APAP conjugates with glutathione, cysteine, and N-acetylcysteine were synthesized, purified by preparative liquid chromatography, and characterized by NMR and high-resolution mass spectrometry. The gradient elution conditions were optimized using the window diagram approach and the effects of mobile phase composition and additives on separation and detection sensitivity were evaluated using two, i.e., linear and non-linear isocratic retention models. Quantitative parameters of the developed method were evaluated and the effectiveness, sensitivity, and specificity of the method were demonstrated on the analysis of human kidney HK-2 cell lysates, confirming the suitability of the method for routine use in studies on APAP toxicity., Paracetamol (APAP) je jedním z celosvětově nejpoužívanějších léků. Bohužel jeho předávkování, které je způsobeno převažující oxidací APAP, může vést k akutnímu poškození jater. V játrech je oxidovaný APAP konjugován s glutathionem, což vede ke konjugátu APAP-glutathion, který je metabolizován na konjugáty APAP-cystein a APAP-N-acetylcystein. Všechny tyto sloučeniny by tedy mohly být použity ke sledování metabolismu APAP u předávkovaných pacientů. Doposud byl popsán pouze omezený počet rychlých a přesných metod pro hodnocení oxidačních metabolitů APAP s využitím jednoduchého přístrojového vybavení, a proto byla tato práce zaměřena na vývoj rychlé a pohodlné gradientové HPLC-UV/MS metody. Za tímto účelem byly syntetizovány konjugáty APAP s glutathionem, cysteinem a N-acetylcysteinem, přečištěny preparativní kapalinovou chromatografií a charakterizovány pomocí NMR a hmotnostní spektrometrie s vysokým rozlišením. Podmínky gradientové eluce byly optimalizovány pomocí metody okenního diagramu a vliv složení mobilní fáze a aditiv na separaci a citlivost detekce byl vyhodnocen pomocí dvou, tj. lineárního a nelineárního isokratického retenčního modelu. Kvantitativní parametry vyvinuté metody byly vyhodnoceny a účinnost, citlivost a specifičnost metody byly prokázány na analýze buněčných lyzátů lidských ledvin HK-2, což potvrdilo vhodnost metody pro rutinní použití ve studiích toxicity APAP.

Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield an

Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield an

Ion-pair chromatography is the de facto standard for separating oligonucleotides and related impurities, particularly for analysis but also often for small-scale purification. Currently, there is limited understanding of the quantitative modeling of both analytical and overloaded elution profiles obtained during gradient elution in ion-pair chromatography. Here we will investigate a recently introduced gradient mode, the so-called ion-pairing reagent gradient mode, for both analytical and overloaded separations of oligonucleotides. The first part of the study demonstrates how the electrostatic theory of ion-pair chromatography can be applied for modeling gradient elution of oligonucleotides. When the ion-pair gradient mode is used in a region where the electrostatic surface potential can be linearized, a closed-form expression of retention time can be derived. A unified retention model was then derived, applicable for both ion-pair reagent gradient mode as well as co-solvent gradient mode. The model was verified for two different experimental systems and homo- and heteromeric oligonucleotides of different lengths. Quantitative modeling of overloaded chromatography using the ion-pairing reagent gradient mode was also investigated. Firstly, a unified adsorption isotherm model was developed for both gradient modes. Then, adsorption isotherms parameter of a model oligonucleotide and two major synthetic impurities were estimated using the inverse method. Secondly, the parameters of the adsorption isotherm were then used to investigate how the productivity of oligonucleotide varies with injection volume, gradient slope, and initial retention factor. Here, the productivity increased when using a shallow gradient slope combined with a low initial retention factor. Finally, experiments were conducted to confirming some of the model predictions. Comparison with the conventional co-solvent gradient mode showed that the ion-pairing reagent gradient leads to both higher yield an

Structural elucidation of compounds detected with liquid chromatography coupled to high resolution mass spectrometry is a challenging and time-consuming step in the workflow of non-targeted analysis and often requires manual validation of the results. Retention time, alongside exact mass, isotope pattern, fragmentation spectra, and collision cross-section, is valuable information for ruling out unlikely structures and increasing the confidence in others. Different approaches to predict retention times have been used previously for reversed phase chromatography and hydrophilic interaction liquid chromatography (HILIC), but application is limited to a small set of mobile phases and gradient profiles. Here, we expand the toolbox available for retention time predictions by developing a random forest regression model for predicting retention times for four column types and twenty mobile phase systems. MultiConditionRT was built using a dataset containing 78 compounds analyzed with C18 reversed phase, mixed mode, HILIC, and biphenyl columns. In addition, different eluent compositions were used: both methanol and acetonitrile were combined with different aqueous phases with pH from 2.1 to 10.0 (formic acid, acetic acid, trifluoroacetic acid, formate, acetate, bicarbonate, and ammonia). The root mean square error (RMSE) of the test set predictions was 1.55 min for C18 reversed phase, 1.79 min for mixed-mode, 1.93 min for HILIC, and 1.56 min for biphenyl column. Additionally, MultiConditionRT can be applied to different gradient profiles with a general additive model-based calibration approach. The approach of MultiConditionRT was validated externally and internally with 356 and 151 compounds respectively, yielding an RMSE of 2.68 and 2.32 min. 324 and 84 of these compounds were not in the dataset used in the model development.

This paper presents a method for prediction of the gradient retention times of six samples of benzodiazepines - phenazepam, pyrazolam, flubromazepam, meclonazepam, diclazepam, and diazepam. Isocratic and gradient separation of the compounds of interest was achieved on a Luna Omega C18 column followed by RP-LC/MS detection. The results have shown good agreement between the predicted and experimental retention data; however, higher differences were obtained for fast gradients in the ultrahigh performance separation mode., Tento článek představuje metodu předpovědi gradientových retenčních časů šesti vzorků benzodiazepinů - fenazepamu, pyrazolamu, flubromazepamu, meklonazepamu, diklazepamu a diazepamu. Izokratické a gradientové separace sledovaných sloučenin bylo dosaženo na koloně Luna Omega C18 s následnou RP-LC/MS detekcí. Výsledky ukázaly dobrou shodu mezi předpovězenými a experimentálními retenčními údaji; vyšší rozdíly však byly získány pro rychlé gradienty v režimu ultravysokoúčinné separace.

This paper presents a method for prediction of the gradient retention times of six samples of benzodiazepines – phenazepam, pyrazolam, flubromazepam, meclonazepam, diclazepam, and diazepam. Isocratic and gradient separation of the compounds of interest was achieved on a Luna Omega C18 column followed by RP-LC/MS detection. The results have shown good agreement between the predicted and experimental retention data; however, higher differences were obtained for fast gradients in the ultrahigh performance separation mode.

A method for the analysis of weak anionic surfactants based on N-acyl amino acids was developed. The surfactants were derivatized using 2,4′-dibromoacetophenone yielding 4′-bromophenacyl esters suitable for spectrophotometric detection. Surfactants containing glycine, threonine and glutamic acid were analyzed after derivatization using reversed-phase liquid chromatography with UV/Vis and MS detection. The gradient profile was optimized using isocratic retention data of N-acyl-linked fatty acid homologues. The relative content of the homologues of N-acyl-linked fatty acids was expressed using the determined method. The intraday repeatability and stability of the prepared derivatives was tested. The relative content of fatty acids in the surfactants was correlated with the most common sources of fatty acids, showing high Pearson’s correlation coefficients with the typical fatty acids profile of a coconut oil., Byla vyvinuta metoda pro analýzu slabých aniontových tenzidů na bázi N-acylaminokyselin. Tenzidy byly derivatizovány pomocí 2,4'-dibromacetofenonu za vzniku korespondujících 4'-bromfenacylesterů vhodných pro spektrofotometrickou detekci. Pomocí kapalinové chromatografie s UV a MS detekcí byly po derivatizaci analyzovány tenzidy obsahující glycin, threonin a glutamovou kyselinu. Profil gradientu byl optimalizován pomocí isokratických retenčních dat homologů mastných kyselin a s použitím vyvinuté metody byl charakterizován relativní obsah homologů mastných kyselin. Byla testována opakovatelnost a stabilita přípravy derivátů. Relativní obsah mastných kyselin byl porovnán s nejběžnějšími zdroji mastných kyselin prokazující nejvyšší hodnotu Pearsonova korelačního koeficientu s typickým profilem kokosového oleje.

A combined experimental and theoretical study was performed to understand how the pore size of packing materials with pores 60-300 angstrom in size affects the separation of 5-50-mer oligonucleotides. For this purpose, we developed a model in which the solutes were described as thin rods to estimate the accessible surface area of the solute as a function of the pore size and solute size. First, an analytical investigation was conducted in which we found that the selectivity increased by a factor of 2.5 when separating 5- and 15-mer oligonucleotides using packing with 300 angstrom rather than 100 angstrom pores. We complemented the analytical investigation by theoretically demonstrating how the selectivity is dependent on the column's accessible surface area as a function of solute size. In the preparative investigation, we determined adsorption isotherms for oligonucleotides using the inverse method for separations of a 9- and a 10-mer. We found that preparative columns with a 60 angstrom-pore-size packing material provided a 10% increase in productivity as compared with a 300 A packing material, although the surface area of the 60 angstrom packing is as much as five time larger.

A combined experimental and theoretical study was performed to understand how the pore size of packing materials with pores 60-300 angstrom in size affects the separation of 5-50-mer oligonucleotides. For this purpose, we developed a model in which the solutes were described as thin rods to estimate the accessible surface area of the solute as a function of the pore size and solute size. First, an analytical investigation was conducted in which we found that the selectivity increased by a factor of 2.5 when separating 5- and 15-mer oligonucleotides using packing with 300 angstrom rather than 100 angstrom pores. We complemented the analytical investigation by theoretically demonstrating how the selectivity is dependent on the column's accessible surface area as a function of solute size. In the preparative investigation, we determined adsorption isotherms for oligonucleotides using the inverse method for separations of a 9- and a 10-mer. We found that preparative columns with a 60 angstrom-pore-size packing material provided a 10% increase in productivity as compared with a 300 A packing material, although the surface area of the 60 angstrom packing is as much as five time larger.

A combined experimental and theoretical study was performed to understand how the pore size of packing materials with pores 60-300 angstrom in size affects the separation of 5-50-mer oligonucleotides. For this purpose, we developed a model in which the solutes were described as thin rods to estimate the accessible surface area of the solute as a function of the pore size and solute size. First, an analytical investigation was conducted in which we found that the selectivity increased by a factor of 2.5 when separating 5- and 15-mer oligonucleotides using packing with 300 angstrom rather than 100 angstrom pores. We complemented the analytical investigation by theoretically demonstrating how the selectivity is dependent on the column's accessible surface area as a function of solute size. In the preparative investigation, we determined adsorption isotherms for oligonucleotides using the inverse method for separations of a 9- and a 10-mer. We found that preparative columns with a 60 angstrom-pore-size packing material provided a 10% increase in productivity as compared with a 300 A packing material, although the surface area of the 60 angstrom packing is as much as five time larger.

A combined experimental and theoretical study was performed to understand how the pore size of packing materials with pores 60-300 angstrom in size affects the separation of 5-50-mer oligonucleotides. For this purpose, we developed a model in which the solutes were described as thin rods to estimate the accessible surface area of the solute as a function of the pore size and solute size. First, an analytical investigation was conducted in which we found that the selectivity increased by a factor of 2.5 when separating 5- and 15-mer oligonucleotides using packing with 300 angstrom rather than 100 angstrom pores. We complemented the analytical investigation by theoretically demonstrating how the selectivity is dependent on the column's accessible surface area as a function of solute size. In the preparative investigation, we determined adsorption isotherms for oligonucleotides using the inverse method for separations of a 9- and a 10-mer. We found that preparative columns with a 60 angstrom-pore-size packing material provided a 10% increase in productivity as compared with a 300 A packing material, although the surface area of the 60 angstrom packing is as much as five time larger.

A combined experimental and theoretical study was performed to understand how the pore size of packing materials with pores 60-300 angstrom in size affects the separation of 5-50-mer oligonucleotides. For this purpose, we developed a model in which the solutes were described as thin rods to estimate the accessible surface area of the solute as a function of the pore size and solute size. First, an analytical investigation was conducted in which we found that the selectivity increased by a factor of 2.5 when separating 5- and 15-mer oligonucleotides using packing with 300 angstrom rather than 100 angstrom pores. We complemented the analytical investigation by theoretically demonstrating how the selectivity is dependent on the column's accessible surface area as a function of solute size. In the preparative investigation, we determined adsorption isotherms for oligonucleotides using the inverse method for separations of a 9- and a 10-mer. We found that preparative columns with a 60 angstrom-pore-size packing material provided a 10% increase in productivity as compared with a 300 A packing material, although the surface area of the 60 angstrom packing is as much as five time larger.

This article surveys the research activities and contributions of the Department of Analytical Chemistry (first at the Institute of Chemical Technology, later at the Faculty of Chemical Technology of the University of Pardubice), to the field of High Performance Liquid Chromatography in the half a century period between 1969 and 2018. The research topics investigated include various theoretical and practical analytical or preparative techniques, in agreement with the general development trends in the field. The attention is focused on the mechanism and predictive modelling of the retention and separation selectivity in the reversed-phase, organic and aqueous normal phase, ion-pairing, salting-out, micellar and mixed-mode liquid chromatography modes. The effects of the stationary phase, of the mobile phases, and of the sample structure on the retention were investigated in detail. The principal point of the research was the development and verification of the new theory of gradient elution. An important part of the recent research activities has been oriented on the theory and practical applications of the new two-dimensional liquid chromatography technique. During the past half a century, new applications of liquid chromatography were developed in the environmental analysis, in the analysis of drugs and their metabolite products, industrial dyes, intermediates, surfactants, of the phenolic antioxidants and plant oils in food and beverages; some attention being also paid to the separation of chiral compounds.

Příspěvek shrnuje a diskutuje tři nejčastěji používané přístupy k dosažení co nejvyššího rozlišení v kapalinové chromatografii a to příprava vhodné stacionární fáze (sub-2um stacionární fáze, povrchově porézní částice, monolitické kolony), použití gradientové eluce a koncept vícerozměrných separačních technik., The contribution reviews and discuss three most common approaches used for achievement of the highest resolution in liquid chromatography. These approaches include new stationary phases (sub-2 um particles, superficially porous particles, monolithic columns), application of gradient elution and concept of multidimensional separation techniques.

Correct adjustment of the mobile phase is equally important as the selection of the appropriate column for the separation of polar compounds in LC. Both solvophobic and selective polar interactions control the retention in the Reversed Phase and Hydrophilic Interaction modes. The retention models describing the effects of the volume fraction of the strong eluent component in binary mobile phases on the sample retention factors apply in a limited mobile phase composition range. We introduced a three-parameter retention model, which provides improved prediction of retention over a broad mobile phase range, under isocratic and gradient elution conditions. The model does not imply any assumptions concerning either adsorption or partition distribution mechanism, but allows estimating retention in pure strong and in pure weak mobile phase components. The experimental retention data for phenolic acids and flavones on several core-shell columns with different types of stationary phases agree with the theory. Many polar columns with important structural hydrophobic moieties show dual retention mechanism, (Reversed Phase in water rich mobile phases and Hydrophilic Interaction at high acetonitrile concentrations). It is possible to select the mobile phase compositions in each of the two modes for separations of samples containing compounds largely differing in polarity. The three-parameter model describes the retention in each mode, with separately determined best-fit parameters. We applied the two-mode model to the retention data of sulfonamides and benzoic acid related compounds on a new polymethacrylate zwitterionic monolithic micro-column., Pro separaci polárních látek pomocí kapalinové chromatografie je správné složení mobilní fáze stejně důležité jako výběr vhodné kolony. V chromatografii na obrácených fázích a v HILIC chromatografii je retence řízena solvofobními a selektivě polárními interakcemi. Retenční modely popisují vliv obsahu silnější eluční složky v mobilní fázi na retenčních faktory jednotlivých látek. V této publikaci uvádíme tří parametrový retenční model, který poskytuje lepší předpověď retence v širokém rozmezí složení mobilní fáze za izokratických i gradientových podmínek. Mnoho polárních chromatografických kolon s hydrofobickými vlastnostmi vykazuje duální retenční mechanismus (reversní mechanismus ve vodných mobilních fázích a HILIC mechanismus v mobilních fázích obsahující vysoké koncentrace acetonitrilu).

Hydrophilic interaction liquid chromatography on polar columns in aqueous-organic mobile phases has become increasingly popular for the separation of many biologically important compounds in chemical, environmental, food, toxicological, and other samples. In spite of many new applications appearing in literature, the retention-mechanism is still controversial. This review addresses recent progress in understanding of the retention models in hydrophilic interaction liquid chromatography. The main attention is focused on the role of water, both adsorbed by the column and contained in the bulk mobile phase. Further, the theoretical retention models in the isocratic and gradient elution modes are discussed. The dual hydrophilic interaction liquid chromatography reversed-phase retention mechanism on polar columns is treated in detail, especially with respect to the practical use in one- and two-dimensional liquid chromatography separations., Kapalinová chromatografie založená na hydrofilních interakcí probíhající na polárních chromatografických kolonách se stále více používá na separaci biologicky důležitých látek, které se nacházejí v různých environmentálních, potravinářských, toxikologických a dalších vzorcích. Navzdory stále nových publikací týkající se HILIC chromatografie, je mechanismus retence stále nejasný. Tato publikace se zabývá nedávnými pokroky v chápání retenčních modelů používaných v chromatografii hydrofilních interakcí. Hlavní pozornost je věnována roli vody, jednak adsorbované na stacionární fázi, tak množství vody přítomné v mobilní fázi.

The antibody immunoglobulin G (IgG) can be tryptically digested into smaller peptides. This study attempted to develop a separation method for those peptides using RP-HPLC with a C18 column at room temperature. Optimizing separation of trypsin cleaved cytochrome C was used as a guideline before analyzing IgG. The optimized analysis of Cytochrome C was performed at wavelength 280nm (UV) and methanol was used as an organic solvent in mobile phase (B). A fast gradient to 100% mobile phase B with low flow rate gave favorable results for cytochrome C. A slow gradient to 100% mobile phase B was suited for IgG separation. The optimized gradient elution of cytochrome C and IgG was performed at 0.3 and 0.8 ml/min, respectively., Antikroppen Immunoglobulin G (IgG) kan klyvas till peptider med enzymet trypsin. Under denna studie ska utvecklades en separationsmetod för dessa peptider med RP-HPLC. Separationen utfördes med en C18 kolonn i rumstemperatur. Först optimerades en separation av trypsin-klyvt cytokrom C vars optimerade parametrar sedan användes som grund för IgG-separation. Optimeringen utfördes vid våglängden 280 nm(UV) och metanol användes som organiskt lösningsmedel i mobil fas (B). En snabb gradient upp till 100% B med låg flödeshastighet gav mest gynnsamt resultat för cytokrom C. Separationen av IgG gynnades av ett högt flöde och en långsam gradient till 100% B. Den optimerade gradientelueringen för cytokrom C och IgG gjordes vid flödet 0.3 respektive 0.8 ml/min.

In this paper, an efficient way for robustness testing of gradient elution liquid chromatographic methods is proposed and tested on model mixtures comprising cilazapril, hydrochlorothiazide, and their degradation products, solutes that differ not only in polarities, but also in solubility and absorption characteristics. In general, the robustness could be tested with respect to various responses: resolution, retention factor, selectivity factor, change of detector response, etc. In chromatographic methods, the separation of the adjacent peaks is mandatory, and, consequently, the resolution is usually used as response. In isocratic elution methods, the resolution threshold depends on many factors, such as sizes of adjacent peaks, peak shapes, and asymmetry factor. At the same time, the situation is even more complex in gradient elution methods, because separation depends on a larger number of parameters, such as gradient profile, column geometry, mobile phase flow rate, column equilibration between gradient runs, etc. To ensure baseline separation, the authors propose application of separation criterion (s) as response and indirect modeling in the robustness evaluation. Examined response in this approach is represented by the difference between the retention time of the beginning of the peak and the retention time of the end of the previously eluting peak of the critical pair. Moreover, the proposed methodology included reusing experiments from the optimization phase to define a robust chromatographic region without increasing the number of experiments. It was shown that method robustness can be easily and efficiently evaluated by this methodology.

The antibody immunoglobulin G (IgG) can be tryptically digested into smaller peptides. This study attempted to develop a separation method for those peptides using RP-HPLC with a C18 column at room temperature. Optimizing separation of trypsin cleaved cytochrome C was used as a guideline before analyzing IgG. The optimized analysis of Cytochrome C was performed at wavelength 280nm (UV) and methanol was used as an organic solvent in mobile phase (B). A fast gradient to 100% mobile phase B with low flow rate gave favorable results for cytochrome C. A slow gradient to 100% mobile phase B was suited for IgG separation. The optimized gradient elution of cytochrome C and IgG was performed at 0.3 and 0.8 ml/min, respectively., Antikroppen Immunoglobulin G (IgG) kan klyvas till peptider med enzymet trypsin. Under denna studie ska utvecklades en separationsmetod för dessa peptider med RP-HPLC. Separationen utfördes med en C18 kolonn i rumstemperatur. Först optimerades en separation av trypsin-klyvt cytokrom C vars optimerade parametrar sedan användes som grund för IgG-separation. Optimeringen utfördes vid våglängden 280 nm(UV) och metanol användes som organiskt lösningsmedel i mobil fas (B). En snabb gradient upp till 100% B med låg flödeshastighet gav mest gynnsamt resultat för cytokrom C. Separationen av IgG gynnades av ett högt flöde och en långsam gradient till 100% B. Den optimerade gradientelueringen för cytokrom C och IgG gjordes vid flödet 0.3 respektive 0.8 ml/min.

In electrostatic repulsive interaction chromatography, using a charged surface hybrid sorbent carrying positive charges can improve the peak shape of peptides in reversed-phase liquid chromatography (RP-LC), especially in overloaded conditions, compared with standard C-18 sorbents. However, the positive surface charges can interact with anionic additives commonly used in peptide separations, e.g., trifluoroacetic acid (TFA), complicating adsorption isotherm estimation. We investigated how the competition for available adsorption sites between TFA and two peptides influenced the adsorption isotherm in gradient elution. A model accounting for the competition with TFA was compared with a model neglecting TFA adsorption. We found that the two models predicted elution profiles with the same accuracy. We also found that the adsorption isotherms were extremely similar in shape, leading to the conclusion that neglecting the competition with TFA is a valid approximation enabling faster and more robust adsorption isotherm estimation for the studied type of sorbent.

In electrostatic repulsive interaction chromatography, using a charged surface hybrid sorbent carrying positive charges can improve the peak shape of peptides in reversed-phase liquid chromatography (RP-LC), especially in overloaded conditions, compared with standard C-18 sorbents. However, the positive surface charges can interact with anionic additives commonly used in peptide separations, e.g., trifluoroacetic acid (TFA), complicating adsorption isotherm estimation. We investigated how the competition for available adsorption sites between TFA and two peptides influenced the adsorption isotherm in gradient elution. A model accounting for the competition with TFA was compared with a model neglecting TFA adsorption. We found that the two models predicted elution profiles with the same accuracy. We also found that the adsorption isotherms were extremely similar in shape, leading to the conclusion that neglecting the competition with TFA is a valid approximation enabling faster and more robust adsorption isotherm estimation for the studied type of sorbent.

Radioprotective mechanisms of Rutin (RUT) and Quercetin (QRT) against gamma radiation was studied by investigating recovery of endogenous spleen colony-forming units, haematological alterations in peripheral blood in Swiss albino mice. Radiation induced haematological alterations in peripheral blood was studied; mice were treated with RUT (10mg/kg.b.wt.) and QRT (20mg/kg.b.wt.) once daily for five consecutive days and exposed to 7.5 Gy of gamma radiation after the last administration. RUT and QRT treatment before exposure to 7.5 Gy of gamma radiation resulted in a significant (p 0.01) increase in cellular components of blood at various post-treatment time points when compared with the respective irradiated groups. Irradiation of mice reduced the spleen colony forming units in the spleen and the number of spleen nodules was inversely related with the increase in radiation dose. The potent antioxidant nature of RUT and QRT mitigate the oxidative stress induced by gamma radiation and thus protect the mice from hemopoietic damage.

Radioprotective mechanisms of Rutin (RUT) and Quercetin (QRT) against gamma radiation was studied by investigating recovery of endogenous spleen colony-forming units, haematological alterations in peripheral blood in Swiss albino mice. Radiation induced haematological alterations in peripheral blood was studied; mice were treated with RUT (10mg/kg.b.wt.) and QRT (20mg/kg.b.wt.) once daily for five consecutive days and exposed to 7.5 Gy of gamma radiation after the last administration. RUT and QRT treatment before exposure to 7.5 Gy of gamma radiation resulted in a significant (p 0.01) increase in cellular components of blood at various post-treatment time points when compared with the respective irradiated groups. Irradiation of mice reduced the spleen colony forming units in the spleen and the number of spleen nodules was inversely related with the increase in radiation dose. The potent antioxidant nature of RUT and QRT mitigate the oxidative stress induced by gamma radiation and thus protect the mice from hemopoietic damage.

In electrostatic repulsive interaction chromatography, using a charged surface hybrid sorbent carrying positive charges can improve the peak shape of peptides in reversed-phase liquid chromatography (RP-LC), especially in overloaded conditions, compared with standard C-18 sorbents. However, the positive surface charges can interact with anionic additives commonly used in peptide separations, e.g., trifluoroacetic acid (TFA), complicating adsorption isotherm estimation. We investigated how the competition for available adsorption sites between TFA and two peptides influenced the adsorption isotherm in gradient elution. A model accounting for the competition with TFA was compared with a model neglecting TFA adsorption. We found that the two models predicted elution profiles with the same accuracy. We also found that the adsorption isotherms were extremely similar in shape, leading to the conclusion that neglecting the competition with TFA is a valid approximation enabling faster and more robust adsorption isotherm estimation for the studied type of sorbent.

In electrostatic repulsive interaction chromatography, using a charged surface hybrid sorbent carrying positive charges can improve the peak shape of peptides in reversed-phase liquid chromatography (RP-LC), especially in overloaded conditions, compared with standard C-18 sorbents. However, the positive surface charges can interact with anionic additives commonly used in peptide separations, e.g., trifluoroacetic acid (TFA), complicating adsorption isotherm estimation. We investigated how the competition for available adsorption sites between TFA and two peptides influenced the adsorption isotherm in gradient elution. A model accounting for the competition with TFA was compared with a model neglecting TFA adsorption. We found that the two models predicted elution profiles with the same accuracy. We also found that the adsorption isotherms were extremely similar in shape, leading to the conclusion that neglecting the competition with TFA is a valid approximation enabling faster and more robust adsorption isotherm estimation for the studied type of sorbent.

In electrostatic repulsive interaction chromatography, using a charged surface hybrid sorbent carrying positive charges can improve the peak shape of peptides in reversed-phase liquid chromatography (RP-LC), especially in overloaded conditions, compared with standard C-18 sorbents. However, the positive surface charges can interact with anionic additives commonly used in peptide separations, e.g., trifluoroacetic acid (TFA), complicating adsorption isotherm estimation. We investigated how the competition for available adsorption sites between TFA and two peptides influenced the adsorption isotherm in gradient elution. A model accounting for the competition with TFA was compared with a model neglecting TFA adsorption. We found that the two models predicted elution profiles with the same accuracy. We also found that the adsorption isotherms were extremely similar in shape, leading to the conclusion that neglecting the competition with TFA is a valid approximation enabling faster and more robust adsorption isotherm estimation for the studied type of sorbent.

Preparative chromatography is the best generic method today for the purification of small drugs and valuable chemical components at <10-kg level. Recent progress in computer technology and the development of new nonchiral/chiral stationary phases, as well as numerous improvements in reliability and economic performance, have considerably increased the interest in modeling in academic and industrial communities. This chapter serves as an introduction to modeling of preparative liquid chromatography where the aim is to improve process purification of valuable chemical components, such as drugs and chiral components. We go through the most important column and adsorption models and methods for determination of the essential thermodynamic adsorption data for both column characterization and process improvement. We also cover important operational modes, such as separation in gradient mode and cases where additives are present, and operational conditions sometimes neglected in the modeling process, such as the impact of injection profiles.

In this work, we have investigated the predictive properties of mixed-mode retention model and oligomeric mixed-mode model, taking into account the contribution of monomeric units to the retention, in hydrophilic interaction liquid chromatography. The gradient retention times of native maltooligosaccharides and their fluorescent derivatives were predicted in the oligomeric series with number of monomeric glucose units in the range from two to seven. The maltooligosaccharides were separated on a packed column with carbamoyl-bonded silica stationary phase and 15 gradient profiles with different initial and final mobile phase composition were used with the gradient times 5; 7.5 and 10 min. The predicted gradient retention times were compared for calculations based on isocratic retention data and gradient retention data, which provided better accuracy of the results. By comparing two different mobile phase additives, the more accurate retention times were predicted in mobile phases containing ammonium acetate. The acidic derivatives, prepared by reaction of an oligosaccharide with 2-aminobenzoic acid or 8-aminonaphthalene-1,3,6-trisulfonic acid, provided more accurate predictions of the retention data in comparison to native oligosaccharides or their neutral derivatives. The oligomeric mixed-mode model allowed prediction of gradient retention times using only one gradient profile, which significantly speeded-up the method development., V této práci jsme prozkoumali prediktivní vlastnosti směsného retenčního modelu a oligomerního modelu uvažujícího příspěvky monomerních glukozových jednotek k retenci v chromatografii hydrofilních interakcí. Byly predikovány gradientové retenční časy nativních maltooligosacharidů a jejich fluorescenčních derivátů v oligomerních sériích s počtem glukozových jednotek od dvou do sedmi. Maltooligosacharidy byly separovány na kolonách plněných silikagelovými částicemi s vázaným karbamoylem s použitím 15 gradientových profilů s rozdílnou počáteční a koncovou koncentrací mobilní fáze a gradientovými časy 5; 7,5 a 10 min. Predikované gradientové retenční časy byly porovnány pro výpočet na základě izokratických vstupních dat a gradientových dat, které poskytly přesnější výsledky predikce. Přesnějších retenčních časů bylo dosaženo v mobilních fázích s přídavkem octanu amonného. Kyselé deriváty připravené reakcí oligosacharidu s 2-aminobenzoovou kyselinou nebo 8-aminonaftalen-1,3,6-trisulfonovou kyselinou poskytly přesnější predikovaná data v porovnání s nederivatizovanými oligosacharidy nebo jejich neutrálními deriváty. Směsný oligomerní retenční model umožnil předpověď retenčních časů s použitím pouze jednoho gradientového profilu což výrazně urychlilo vývoj metody.

nships QSPR) predstavlja matematičko modelovanje kojim se predviđaju fizičke, hemijske ili biološke osobine grupe analita na osnovu njihovih molekulskih deskriptora. Molekulski deskriptor je broj koji može biti rezultat logičke i matematičke procedure kojom se transformiše hemijska informacija koju nosi simbolički prikaz molekula u upotrebljiv broj (teorijski deskriptor), ili rezultat standardizovanog eksperimenta (eksperimentalni deskriptor). QSRR su matematički dobijene veze između hromatografskih odgovora određenih za seriju analita u datom hromatografskom sistemu i molekulskih deskriptora. Mogu se primeniti u cilju predviđanja retencije, kao i identifikovanja deskriptora koji najbolje opisuju retenciono ponašanje. Ukoliko se kao ispitivani faktori uključe i hromatografski parametri, QSRR metodologija se može primeniti za optimizaciju hromatografske metode, kao i sticanje uvida u molekulske mehanizme razdvajanja u ispitivanom hromatografskom sistemu između analita i stacionarne faze. QSPR u kojima je odgovor koji se prati intenzitet signala u masenom spektrometru je relativno nov koncept osmišljen u cilju predviđanja odgovora, optimizacije uslova u cilju povećanja osetljivosti metode, identifikacije deskriptora koji najbolje opisuju proces jonizacije u masenom spektrometru, kao i sticanje uvida u mehanizme formiranja jona pri ispitivanim hromatografskim uslovima. Hemometrija je matematičko-statistička disciplina koja omogućava dizajn i selekciju optimalnih procedura merenja i eksperimenata, i dobijanje što više relevantnih informacija iz hemijskih podataka..., Quantitative Structure – Property Relationships (QSPR) is a mathematical modeling which enables prediction of physical, chemical or biological properties of the groups of analytes based on their molecular descriptors. Molecular descriptor is a number that can be the result of logical and mathematical procedure that transforms chemical information carried by the symbolic representation of molecules in a usable number (theoretical descriptor), or the result of a standardized experiment (experimental descriptor). QSRR are mathematically derived relationships between chromatographic response determined for a representative series of analytes in given chromatographic system and the molecular descriptors. They can be used to predict retention, as well as to identify descriptors that best describe the retention behavior. If chromatographic parameters are also included as inputs, QSRR methodology can be applied to optimize chromatographic methods, as well as gain insight into the molecular mechanisms of separation in the investigated chromatographic system between the analytes and the stationary phase. QSPR in which mass spectrometric signal intensity is the observed response is a relatively new concept designed to predict response, optimize the conditions in order to increase the sensitivity of the method, identify the descriptors that best describe the process of ionization in a mass spectrometer, as well as gain insight into the mechanisms of formation of ions under the tested chromatographic conditions. Chemometrics is a mathematical – statistical discipline that enables design and selection of optimal measurement procedure and experiments, and obtaining as much relevant information from chemical data...

Health benefits of wine, especially with red wine, have been linked to the presence of a wide range of phenolic antioxidants. Thus, the aim of this study was to develop a simple, high performance thin layer chromatographic (HPTLC) method combined with high resolution digital plate images to visually compare multiple wine samples simultaneously on a single chromatographic plate and to quantify levels of gallic acid, caffeic acid, resveratrol and rutin, as representatives of the four different classes of phenolics found in wines. We also wanted to investigate the contribution of the investigated phenolic compounds to the total polyphenolic content (TPC) and total antioxidant capacity (TAC) of the wine samples. The average concentrations of caffeic acid, gallic acid, resveratrol, and rutin in the red wines were 2.15, 30.17, 0.59 and 2.47 mg/L respectively with their concentration below limit of quantification in the white wine samples. The highest concentration of resveratrol and rutin is found in the Cabernet and Shiraz wine samples. The amounts of gallic acid are correlated with TPC (r = 0.58). Italian wines have the highest correlation between TPC and TAC (r = 0.99) although they do not contain detectable amounts of resveratrol, they contain significant amount of rutin. Therefore, antioxidant properties might be associated with the presence of flavanols in these wines.

Tato prace se zabyva predikci reten.nich dat v rychlych gradientech pomoci izokratickych reten.nich model. na povrchov. poreznich kolonach s velikosti častic 5 mikrom. K analyzam byly použity standardy alkylbenzenů. a fenolickych latek, ktere byly separovany na povrchově poreznich kolonach s chemicky vazanymi stacionarnimi fazemi C18, fenyl-hexyl a bifenyl. Vybrane povrchově porezni kolony byly použity ve druhe dimenzi celostni dvoudimenzionalni kapalinove chromatografie k separaci fenolickych latek, zatimco k separaci v prvni dimenzi byla použita kapilarni monoliticka zwitteriontova kolona., This thesis deals with possibilities of prediction of gradient elution data using three different isocratic retention models on 5micro m core-shell columns. Alkylbenzenes, phenolic acid a flavone standards were separated in fast gradient times between 1-10 min with different gradient range on three columns with different stationary phases (octadecyl, phenyl-hexyl and biphenyl). Furthermore, core-shell columns were employed in the second dimension of comprehensive two-dimensional liquid chromatography coupled with monolithic zwitterionic microcolumn in the first dimension., Katedra analytické chemie, Student(ka), přistoupil(a) k obhajobě diplomové práce, zodpověděl(a) dotazy oponenta a obhájil(a) diplomovou práci.

This is the first investigation in a series that aims to enhance the scientific knowledge needed for reliable analytical method transfer between HPLC and UHPLC using the quality by design (QbD) framework. Here, we investigated the differences and similarities from a thermodynamic point of view between RP-LC separations conducted with 3.5 μm (HPLC) and 1.7 μm (UHPLC) C18 particles. Three different model solutes and one pharmaceutical compound were used: the uncharged cycloheptanone, the cationic benzyltriethylammonium chloride, the anionic sodium 2-naphatlene sulfonate and the pharmaceutical compound omeprazole, which was anionic at the studied pH. Adsorption data were determined for the four solutes at varying fractions of organic modifier and in gradient elution in both the HPLC and UHPLC system, respectively. From the adsorption data, the adsorption energy distribution of each compound was calculated and the adsorption isotherm model was estimated. We found that the adsorption energy distribution was similar, with only minor differences in degree of homogeneity, for HPLC and UHPLC stationary phases. The adsorption isotherm model did not change between HPLC and UHPLC, but the parameter values changed considerably especially for the ionic compounds. The dependence of the organic modifier followed the same trend in HPLC as in UHPLC. These results indicates that the adsorption mechanism of a solute is the same on HPLC and UHPLC stationary phases which simplifies design of a single analytical method applicable to both HPLC and UHPLC conditions within the QbD framework.

Tato diplomová práce se zabývá studií kolon s povrchově pórovitými částicemi s různými stacionárními fázemi v jednorozměrné vysokoúčinné kapalinové chromatografii a dvourozměrné kapalinové chromatografii. Jejím cílem je nalézt povrchově pórovitou kolonu vhodnou pro použití ve druhé dimenzi úplné dvourozměrné kapalinové chromatografie k analýze přírodních antioxioxidantů., Subject of the studies performed within this thesis was investigation of columns with surface-porous particles with different stationary phases in one-dimensional high performance liquid chromatography and two-dimensional liquid chromatography. The purpose of the above mentioned acitvities within this thesis was to find surface-porous column most suitable for usage in the second dimension of comprehensive liquid chromatography for natural antioxidants analysis., Katedra analytické chemie, Student přednesl obhajobu své diplomové práce a zodpověděl dotazy oponenta a členů komise.

Many recent efforts in the field of microfluidics have been focused on reducing the size and the complexity of devices and on simplifying the methods of analysis performed with them. Gradient elution moving boundary electrophoresis (GEMBE) is a recently described counterflow electrophoresis method that was developed to simplify the analysis of ions in complex matrices. In this thesis, the improvement of the limit of detection of GEMBE and reduction of the GEMBE channel length is investigated. Integration of simple and robust device components required for the successful adaptation of many analytical methods to multiplexed and field-portable devices often has negative effects on detection sensitivity, such as in the optical detection components in a capillary electrophoresis (CE) system. One of the simplest methods to improve sensitivity in the CE field is known as sample stacking. This method involves preparing the sample in a buffer with a different concentration (and conductivity) than that of the run buffer, such that when an electric field is applied the analyte concentration is increased at the boundary between the two different buffer concentrations. A method in which the sample is prepared in a buffer at a lower concentration than the run buffer has been implemented. This method achieves a significantly greater signal enhancement than expected for sample stacking. The concentration enhancement ability of this method is demonstrated utilizing GEMBE with channel current detection. Current GEMBE device construction methods impose limitations on the minimum length of the separation channel. One technique well suited for minimizing the size of the GEMBE separation channel is multiphoton absorption polymerization (MAP). Because MAP is a non-linear optical fabrication method, polymerization is limited to a small region near the focal point of a laser beam. As a result, three-dimensional structures with small feature sizes can be easily created. The 3D capabilities o

The simultaneous pharmaceutical analysis of multi-component drugs represents a challenge due to a large total number of analytes present in the sample. These analytes are not only the active pharmaceutical ingredients, but also the impurities that might follow the active substances. The aim of this study was to develop an efficient reversed-phase LC method for the simultaneous analysis of antiparkinsonian drugs levodopa, carbidopa and entacapone along with their six related impurities. For the achievement of desirable separation, different acids with anions possessing different properties according to Hofmeister classification (ortho-phosphoric, trifluoroacetic and perchloric acid) were tested. Finally, in order to draw the unbiased conclusions when optimizing the analytical method, for the final tuning of the gradient program, Box-Behnken experimental design and Derringer's desirability function were used. The experiments were performed on Zorbax Extend C18, 150 mm x 4.6 mm, 5 mu m particle size column with the UV detection at 280 nm and mobile phase flow rate of 1 mL/min. The optimal mobile phase consisted of methanol and 20 mM trifluoroacetic acid (pH 2.0 adjusted with NaOH), while their ratio is changed according to previously defined gradient program. The method was tested for selectivity, sensitivity, linearity, accuracy and precision, and proved to be suitable for routine qualitative and quantitative analysis of levodopa, carbidopa, entacapone and their impurities in their mixture.

Experimental competitive adsorption isotherms were successfully determined directly from overloaded elution profiles in gradient elution mode using an extended inverse method. This approach differs from the existing methods in one important aspect – no isocratic experiments are necessary which makes it possible to study adsorption of substances whose retention factors vary strongly with the mobile-phase composition. The approach was verified with simulated binary data and with experimental data from gradient separations of a cyclohexanone/cycloheptanone mixture. For the synthetic data, the original adsorption isotherm parameters were found using a two-step estimation procedure. In the first step analytical peaks were used to estimate the “analytical” part of the Langmuir equation and in the second step the association equilibrium parameters were estimated from two simulated overloaded elution profiles. For the experimental data, a three-step approach was used. The two first steps were used to reduce the calculation time so that parameter estimation could be performed on an ordinary computer. In the first step, analytical peaks were used to estimate the “analytical” part of the bi-Langmuir equation. In the second step, initial guesses for all other parameters were determined separately for each solute using the faster Rouchon algorithm. In the final and third step, the more accurate orthogonal collocation on finite elements algorithm, was used to fine-tune the isotherm parameters. The model could accurately predict the shape of overloaded elution profiles. The shape of the adsorption isotherms agreed well with those determined with the standard isocratic method, although the numerical values were not the same. The extended inverse method is well suited for process optimization where few experiments and accurate predictions are important.

The inverse method is a numeric method for fast estimation of adsorption isotherm parameters directly from overloaded elution profiles. However, it has previously only been used for isocratic experiments. Here we will extend the inverse method so it can be used for gradient elution too. This extended inverse method will make it possible to study the adsorption of substances whose retention factor vary strongly with the mobile-phase composition, like peptides and proteins, where the classic methods will fail. Our extended inverse method was verified using both simulations and real experiments. For simulated overloaded elution profiles we were able to determine almost exact Langmuir adsorption isotherm parameters with the new approach. From real experimental data, bi-Langmuir adsorption parameters were estimated using both the perturbation peak method and the extended inverse method. The shape of the acquired adsorption isotherms did match over the considered concentration range; however, the adsorption isotherm parameters found with the two methods were not the same. This is probably due to the fact that adsorption isotherm estimated with the inverse method is only a good approximation up to the highest eluted concentration in the used chromatograms. But this is not a serious drawback from a process point of view where the main objective is to make accurate predictions of elution profiles. The bi-Langmuir adsorption isotherm obtained with both methods could accurately predict the shape of overloaded elution profiles.

Tato diplomová práce se zabývá využitím úplné multidimenzionální kapalinové chromatografie pro analýzu přírodních antioxidantů. Cílem této práce bylo nalézt vhodnou povrchově porézní kolonu pro separaci v druhé dimenzi (v systému s obrácenými fázemi) na základě získaných dat z jednorozměrných separací fenolických kyselin a flavonů. Na těchto kolonách byl dále studován vliv objemu a složení rozpouštědla ve frakcích převáděných do druhé dimenze. Optimalizované podmínky byly použity na dvoudimenzionální separace standardů fenolických kyselin a flavonů a na vzorek čaje., The use of full multi-dimensional chromatography for the analysis of natural antioxidants was main theme of this thesis. The selection of suitable surface-porous column for separation in the second dimension (in reversed-phase system) based on data obtained from one-dimensional separation of phenolic acids and flavones was the aim of this work. The effects of volume and solvent composition of fractions transferred to second dimension on these columns were also studied. For two-dimensional separation of standards of phenolic acids, flavones and sample tea were the optimized conditions used., Katedra analytické chemie, Studentka přednesla obhajobu své diplomové práce a zodpověděla dotazy oponenta a členů komise.

Methacrylate monolithic stationary phases were produced in fused-silica chips by UV initiation. Poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA) and poly(lauryl methacrylate-co-ethylene dimethacrylate) (LMA) monoliths containing 30, 35 and 40% monomers were evaluated for the separation of peptides under gradient conditions. The peak capacity was used as an objective tool for the evaluation of the separation performance. LMA monoliths of the highest density gave the highest peak capacities (approximate to 40) in gradients of 15 min and all LMA monoliths gave higher peak capacities than the BMA monoliths with the same percentage of monomers. Increasing the gradient duration to 30 min did not increase the peak capacity significantly. However, running fast (5 min) gradients provides moderate peak capacities (approximate to 20) in a short time. Due to the system dead volume of 1 mu L and the low bed volume of the chip, early eluting peptides migrated over a significant part of the column during the dwell time under isocratic conditions. It was shown that this could explain an increased band broadening on the monolithic stationary phase materials used. The effect is stronger with BMA monoliths, which partly explains the inferior performance of this material with respect to peak capacity. The configuration of the connections on the chip appeared to be critical when fast analyses were performed at pressures above 20 bar. (C) 2011 Elsevier B.V. All rights reserved.

V této disertační práci jsou shrnuty poznatky týkající se přípravy, modifikace a charakterizace kapilárních monolitických kolon na bázi polystyrénu a možnosti predikce retenčních dat proteinů na polystyrenových kapilárních monolitických kolon a různých typech komerčně dostupných stacionárních fází při separacích v HPLC s obrácenými fázemi., The preparation, modification and characterization of polystyrene based capillary monolithic columns is stated in this dissertation as well as the possibility of protein retention data prediction using the polystyrene capillary monolithic columns and various types of commercially available stationary phases used for the separation compounds in reversed-phase HPLC., Katedra analytické chemie, Předseda seznámil přítomné se životopisnými daty dizertantky. Následně vedoucí školitel dizertační práce seznámil komisi se svým posudkem. Konstatováno souhlasné stanovisko vedoucího školícího pracoviště. Poté dizertantka seznámila přítomné s tezemi a výsledky své dizertační práce. Za nepřítomnou oponentku prof. Pacákovou přečetl oponentský posudek předseda komise. Přítomný prof. Lemr přečetl posudek osobně . Dizertantka uspokojivě zodpověděla připomínky a dotazy oponentů. Konstatováno, že nepřišly žádné připomínky zvenčí. Následovala diskuze k práci: - prof. Ventura: Proč jako vzorky použily benzen a ethylbenzen? - ing. Česla: Jaká byla použita MF v CEC? Revisibilita EOF? - doc. Fischer: Jak vzniká EOF na nepolárních částicích? Následovala neveřejná část obhajoby, kde byla konstatována odpovídající kvalita dizertační práce. Následovalo hlasování a vyhlášení výsledků.

Disertační práce se zabývá možnostmi úplné dvoudimenzionální chromatografie na analýzu přírodních fenolických látek. Výzkum byl zaměřen na volbu stacionárních fází, s cílem dosažení co největší ortogonality systému a rozdílné selektivity separace v obou dimenzích. Testovány byly RP a HILIC stacionární fáze. U HILIC kolon byl pozorován duální retenční mechanismus. Byly porovnávány různé gradienty, použitelné v úplné dvoudimenzionální chromatografii, pro dosažení co největší píkové kapacity a ortogonality 2D systému. Gradienty v první i v druhé dimenzi byly optimalizovány pomocí okénkových diagramů. Dále byl studován vliv dávkovaného objemu a složení rozpouštědla na separaci., The comprehensive two dimensional liquid chromatography for analysis of natural antioxidants was studied in this disertation. The combination of stationary phases in two dimensional system was selected to provide very different chromatographic selectivity in both dimensions and high degree of orthogonality of 2D system. The RP and HILIC stationary phases were tested. The dual HILIC-RP retention mechanism was observed on all polar columns tested. Various types of second-dimension gradients in comprehensive LCxLC are compared. The effects of the gradient type on the bandwidths, peak capacity, orthogonality and separation time were investigated.The gradients in the first and second dimension were optimized using the windowdiagram method. The effect of the volume of samples on the band broadening and peak splitting was studied., Katedra analytické chemie, Předseda komise prof. Ventura představil disertanta a seznámil komisi s jeho základními životopisnými daty. Přečteny posudky školitele a vedoucího školícího pracoviště. Následně disertant přednesl krátký výklad své disertační práce. Přítomní oponenti přečetli své oponentní posudky a disertant se vyjádřil k jejich připomínkám. Konstatováno, že nepřišly žádné připomínky zvenčí. Následovala diskuse: doc. Fischer - otázka kvantifikace a limitu detekce prof. Jandera, doc. Mikšík - transfer frakcí mezi 1. a 2. dimenzí doc. Červenka - aplikace na reálné vzorky (víno). Následovala neveřejná část diskuse, kde členové komise se kladně vyjádřili k vystoupení disertanta. Obhajoba byla zakončena tajným hlasováním., Dokončená práce s úspěšnou obhajobou

Cílem této práce bylo zjistit možnosti využití programované teploty a programovaného složení mobilní fáze pro zlepšení separace fenolických a flavonoidních látek v kapalinové chromatografii v systému s obrácenými fázemi. Měření byla prováděna jak izotermálně tak při gradientu teploty. Všechna měření byla provedena v mobilní fázi složené z metanolu a pufru. Separace flavonoidních látek byla provedena při gradientu teploty i při gradientové eluci a při spojení gradientové eluce s programovanou teplotou., A goal of this thesis was to determine the possibilities of the temperature-programmed and solvent-programmed separation of phenolic acids and flavonoids using reversed-phase high performance liquid chromatography. The measurements were carried out with constant temperature as well as with temperature gradient. All the measurements were carried out in methanol-buffer mobile phase. The separation of flavonoids was accomplished using the gradient of the temperature, the gradient of the solvent and simultaneous gradients of the temperature and the solvent., Katedra analytické chemie, Dokončená práce s úspěšnou obhajobou