Separation and Quantification of 1,4-benzodiazepines: HPLC versus CZE

The goal of the present study was to separate a set of benzodiazepines, namely bromazepam, oxazepam, nitrazepam, chlordiazepoxide, flunitrazepam, lormetazepam and diazepam by analytical scale HPLC and CZE. The both methods for separation of these seven compounds from the 1,4-benzodiazepine group were optimized and compared. LODs and LOQs were determined under the optimized conditions in the both methods. The corresponding LOD and LOQ values are approximately three orders of magnitude lower in HPLC than in CZE. As expected, elution order was found to be different for the both techniques. As a result of a critical collation of all the parameters considered, RP-HPLC was found to be more suitable for determination of the set of benzodiazepines. A real sample analysis was performed under optimized conditions to demonstrate applicability of the proposed analytical methods. (doi: 10.5562/cca1738

Performance comparison of three trypsin columns used in liquid chromatography

Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300 Å pores, packed in 2.1 × 30 mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using Nα-Benzoyl-L-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37 °C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented

Study of interactions participating in the separation mechanism of chromatographic separation systems

Charles University in Prague Faculty of Science Department of Physical and Macromolecular Chemistry Study of interactions participating in the separation mechanism of chromatographic separation systems The PhD Thesis Report Květa Kalíková Prague 2009 The PhD thesis was carried out within the PhD study during years 2005-2009 at the Department Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague. Supervisor: Doc. RNDr. Eva Tesařová, CSc. 2 Content 1. Introduction 4 2. Aims of the thesis 4 3. Results and discussion 5 3.1 Characterization of separation system using LFER model (publication I) 5 3.2 Occurrence and behavior of system peaks in RP HPLC with solely aqueous mobile phases (publication II) 6 3.3 Examples of optimized separation systems and their application 8 3.3.1 Achiral separation systems 8 3.3.1.1 HPLC separation of estrogens (publication III) 8 3.3.1.2 HPLC method for separation of 1,4-benzodiazepines (publication in progress) 9 3.3.2 Chiral separation systems (publication IV) 10 3.3.2.1 Optimization and validation of HPLC method for enantioseparation of (±)-cloprostenol (publication V) 11 4. Conclusions 12 5. References 13 6. List of publications 14 7. List of presentations 15 8. Curriculum Vitae 17 3 1. Introduction High performance liquid chromatography (HPLC)..

Study of interactions participating in the separation mechanism of chromatographic separation systems

The Thesis combines a theoretical part focused on better understanding of separation processes and interaction mechanisms with experimental applications for specific analytical purposes. HPLC is one of the preferently used separation techniques. As various interaction types can participate in the retention mechanism of analytes, the choice of the optimal separation system is often ambiguous. The selection of suitable separation conditions is very often made empirically. Such approach is rather time consuming. Therefore, it could be advantageous to utilize some physicochemical characteristics of the separation system. Linear free energy relationship (LFER) is one of the comprehensive semiempirical methods that allows characterization and comparison of stationary phases/separation systems and yield to better understanding of the intermolecular interactions, which play role in the separation processes. The optimization of separation processes can be disturbed or complicated by phenomena called system peaks. In the case of detectable system zone the resultant chromatogram includes more peaks than is the number of analytes in the sample. A serious problem that can arise if an analyte interacts with the system zones is resonance. This phenomenon can disturb completely the separation results and make...Předkládaná práce kombinuje teoretické přístupy směřující k objasnění separačních systémů a interakčních mechanizmů s aplikacemi pro konkrétní analytické účely. HPLC se řadí mezi nejčastěji používané separační metody. Volba vhodného separačního systému se často provádí empiricky, na základě zkušeností experimentátora, podle informací získaných z literatury, s využitím některých optimalizačních programů nebo čistě postupem pokus-omyl. Pro výběr optimálních separačních podmínek, tj. výběr vhodné stacionární fáze a mobilní fáze je však nutné hlubší poznání interakčních a retenčních mechanizmů. Jedním z možných semiempirických postupů charakterizace separačních systémů je metoda lineárních vztahů volných energií (LFER - Linear Free Energy Relationship). Proces optimalizace separace může velmi zkomplikovat výskyt systémových píků. Existují případy, kdy je daná systémová zóna detekovatelná a ve výsledném chromatogramu se objeví více píků než odpovídá počtu analyzovaných látek anebo systémová zóna "interaguje" se zónou analytu a nastává tzv. rezonance. Porozumění jevům probíhajícím v chromatografických separačních systémech má zásadní vliv jak na optimalizaci separačních podmínek tak na správnou interpretaci analytických dat, a tudíž výsledky analýzy. Model LFER byl použit k porovnání tří chirálních...Department of Physical and Macromolecular ChemistryKatedra fyzikální a makromol. chemieFaculty of SciencePřírodovědecká fakult

Study of interactions participating in the separation mechanism of chromatographic separation systems

The Thesis combines a theoretical part focused on better understanding of separation processes and interaction mechanisms with experimental applications for specific analytical purposes. HPLC is one of the preferently used separation techniques. As various interaction types can participate in the retention mechanism of analytes, the choice of the optimal separation system is often ambiguous. The selection of suitable separation conditions is very often made empirically. Such approach is rather time consuming. Therefore, it could be advantageous to utilize some physicochemical characteristics of the separation system. Linear free energy relationship (LFER) is one of the comprehensive semiempirical methods that allows characterization and comparison of stationary phases/separation systems and yield to better understanding of the intermolecular interactions, which play role in the separation processes. The optimization of separation processes can be disturbed or complicated by phenomena called system peaks. In the case of detectable system zone the resultant chromatogram includes more peaks than is the number of analytes in the sample. A serious problem that can arise if an analyte interacts with the system zones is resonance. This phenomenon can disturb completely the separation results and make..

Enantiomeric Ratio of Amino Acids as a Tool for Determination of Aging and Disease Diagnostics by Chromatographic Measurement

Occurrence of d-amino acids in living organisms is a useful indicator of various changes, diseases, or disorders. Determination of amino acid enantiomers, namely the enantiomeric ratio of amino acids or excess of certain d-amino acids, represents a useful tool in the studies of aging processes or biomarkers in disease/disorder diagnosis in humans. The amount of d-amino acids is usually very low. Therefore, suitable sample pretreatment, often derivatization, and highly selective and sensitive separation methods are essential for d-amino acid analysis in this field. Chromatographic techniques offer appropriate choices for solving these tasks. This review covers the advances in methodology and development of improved instrumental chromatographic methods focused on d,l-amino acid separation and determination. New findings in the area of possible d-amino acid biomarkers are also included

Study of interactions participating in the separation mechanism of chromatographic separation systems

The Thesis combines a theoretical part focused on better understanding of separation processes and interaction mechanisms with experimental applications for specific analytical purposes. HPLC is one of the preferently used separation techniques. As various interaction types can participate in the retention mechanism of analytes, the choice of the optimal separation system is often ambiguous. The selection of suitable separation conditions is very often made empirically. Such approach is rather time consuming. Therefore, it could be advantageous to utilize some physicochemical characteristics of the separation system. Linear free energy relationship (LFER) is one of the comprehensive semiempirical methods that allows characterization and comparison of stationary phases/separation systems and yield to better understanding of the intermolecular interactions, which play role in the separation processes. The optimization of separation processes can be disturbed or complicated by phenomena called system peaks. In the case of detectable system zone the resultant chromatogram includes more peaks than is the number of analytes in the sample. A serious problem that can arise if an analyte interacts with the system zones is resonance. This phenomenon can disturb completely the separation results and make..

Enantiomeric Ratio of Amino Acids as a Tool for Determination of Aging and Disease Diagnostics by Chromatographic Measurement

Occurrence of d-amino acids in living organisms is a useful indicator of various changes, diseases, or disorders. Determination of amino acid enantiomers, namely the enantiomeric ratio of amino acids or excess of certain d-amino acids, represents a useful tool in the studies of aging processes or biomarkers in disease/disorder diagnosis in humans. The amount of d-amino acids is usually very low. Therefore, suitable sample pretreatment, often derivatization, and highly selective and sensitive separation methods are essential for d-amino acid analysis in this field. Chromatographic techniques offer appropriate choices for solving these tasks. This review covers the advances in methodology and development of improved instrumental chromatographic methods focused on d,l-amino acid separation and determination. New findings in the area of possible d-amino acid biomarkers are also included

The Enantioselective Potential of NicoShell and TeicoShell Columns for Basic Pharmaceuticals and Forensic Drugs in Sub/Supercritical Fluid Chromatography

The enantioselective potential of two macrocyclic glycopeptide-based chiral stationary phases for analysis of 28 structurally diverse biologically active compounds such as derivatives of pyrovalerone, ketamine, cathinone, and other representatives of psychostimulants and antidepressants was evaluated in sub/supercritical fluid chromatography. The chiral selectors immobilized on 2.7 μm superficially porous particles were teicoplanin (TeicoShell column) and modified macrocyclic glycopeptide (NicoShell column). The influence of the organic modifier and different mobile phase additives on the retention and enantioresolution were investigated. The obtained results confirmed that the mobile phase additives, especially water as a single additive or in combination with basic and acidic additives, improve peak shape and enhance enantioresolution. In addition, the effect of temperature was evaluated to optimize the enantioseparation process. Both columns exhibited comparable enantioselectivity, approximately 90% of the compounds tested were enantioseparated, and 30% out of them were baseline enantioresolved under the tested conditions. The complementary enantioselectivity of the macrocyclic glycopeptide-based chiral stationary phases was emphasized. This work can be useful for the method development for the enantioseparation of basic biologically active compounds of interest