Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency. The investigation of trypsin reactor activity was performed by on-line digestion of N-α-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA), followed by separation of the digests on the mixed-mode column. Two trypsin column reactors with the different trypsin coverage on the bridged ethylene hybrid particles were evaluated. To ensure optimal trypsin activity, the separation temperature was set at 37.0 °C and the pH of the mobile phase buffer was maintained at 8.5. The on-line digestion itself ongoing during the initial state of gradient was carried out at a low flow rate using a mobile phase that was free of organic modifiers. Proteins such as cytochrome C, enolase, and myoglobin were successfully digested on-line without prior reduction or alkylation, and the resulting peptides were separated using a mixed-mode column. Additionally, proteins that contain multiple cysteines, such as α-lactalbumin, albumin, β-lactoglobulin A, and conalbumin, were also successfully digested on-line (after reduction and alkylation). Moreover, trypsin immobilized enzymatic reactors were utilized for over 300 injections without any noticeable loss of digestion activity.

• Klíčová slova
• Immobilized enzymatic reactor, Mass spectrometry, Mixed-mode column, On-line protein digestion, Trypsin digestion,
• MeSH
• alkylace MeSH
• enzymy imobilizované MeSH
• laktalbumin * MeSH
• proteolýza MeSH
• proteomika * MeSH
• trypsin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• enzymy imobilizované MeSH
• laktalbumin * MeSH
• trypsin MeSH

In this work, two mixed-mode columns from a different manufacturers and one marketed as a reversed-phase column were characterized and compared in the terms of their interaction abilities, retentivity, peak symmetry, and applicability for peptide separation. All the tested columns contain octadecyl ligand and positively charged modifier, i.e. pyridyl group for the reversed-phase column XSelect CSH C18, quaternary alkylamine for mixed-mode column Atlantis PREMIER BEH C18 AX, and permanently charged moiety (details not available from the manufacturer) for mixed-mode column Luna Omega PS C18. For detailed characterization and comparison of their interaction potential, several approaches were used. First, a simple Walters test was performed to estimate hydrophobic and silanophilic interactions of the tested columns. The highest values of both parameters were observed for column Atlantis PREMIER BEH C18 AX. To investigate the effect of pH and buffer concentration on retention, mobile phases composed of acetonitrile and buffer (ammonium formate, pH 3.0; ammonium acetate pH 4.7 and pH 6.9) in various concentrations (5mM; 10mM; 15mM and 20mM) were used. The analysis of permanently charged compounds was used to describe the electrostatic interaction abilities of the stationary phases. The most significant contribution of electrostatic interactions to the retention was observed for Atlantis PREMIER BEH C18 AX column in the mobile phase with buffer of pH 3.0. A set of ten dipeptides, three pentapeptides and one octapeptide was used to investigate the effects of pH and buffer concentration on retention and peak symmetry. Each of the tested columns provides the optimal peak shape under different buffer pH and concentration. The gradient separation of the 14 tested peptides was used to verify the application potential of the tested columns for peptide separation. The best separation was achieved within 4 minutes on column Atlantis PREMIER BEH C18 AX.

• Klíčová slova
• Mixed-mode chromatography, Mixed-mode columns, Peptide separation, RP/anion-exchange mechanism, Reversed-phase column,
• MeSH
• chromatografie s reverzní fází přístrojové vybavení   MeSH
• hydrofobní a hydrofilní interakce MeSH
• koncentrace vodíkových iontů MeSH
• peptidy izolace a purifikace   MeSH
• pufry MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• peptidy MeSH
• pufry MeSH

Glycoproteomics is a challenging branch of proteomics because of the micro- and macro-heterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous alternative to reversed-phase chromatography for intact glycopeptide separation prior to their identification by mass spectrometry. Nowadays, several HILIC columns differing in used chemistries are commercially available. However, there is a lack of comparative studies assessing their performance, and thus providing guidance for the selection of an adequate stationary phase for different glycoproteomics applications. Here, we compare three HILIC columns recently developed by Advanced Chromatography Technologies (ACE)- with unfunctionalized (HILIC-A), polyhydroxy functionalized (HILIC-N), and aminopropyl functionalized (HILIC-B) silica- with a C18 reversed-phase column in the separation of human immunoglobulin G glycopeptides. HILIC-A and HILIC-B exhibit mixed-mode separation combining hydrophilic and ion-exchange interactions for analyte retention. Expectably, reversed-phase mode successfully separated clusters of immunoglobulin G1 and immunoglobulin G2 glycopeptides, which differ in amino acid sequence, but was not able to adequately separate different glycoforms of the same peptide. All ACE HILIC columns showed higher separation power for different glycoforms, and we show that each column separates a different group of glycopeptides more effectively than the others. Moreover, HILIC-A and HILIC-N columns separated the isobaric A2G1F1 glycopeptides of immunoglobulin G, and thus showed the potential for the elucidation of the structure of isomeric glycoforms. Furthermore, the possible retention mechanism for the HILIC columns is discussed on the basis of the determined chromatographic parameters.

• Klíčová slova
• Glycopeptides, Glycoproteomics, Hydrophilic interaction liquid chromatography, Immunoglobulin G, Mixed-mode chromatography,
• MeSH
• chromatografie iontoměničová metody   MeSH
• chromatografie s reverzní fází metody   MeSH
• glykopeptidy izolace a purifikace   MeSH
• hydrofobní a hydrofilní interakce MeSH
• imunoglobulin G izolace a purifikace   MeSH
• isomerie MeSH
• lidé MeSH
• proteomika MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• glykopeptidy MeSH
• imunoglobulin G MeSH

A set of new mixed-mode ion-exchange stationary phases is presented. The backbone of organic selectors is formed by a linear hydrocarbon chain, which is divided into two parts of various lengths by a heteroatom (oxygen or nitrogen). In all studied cases, there is a sulfonic acid moiety as the terminal group. Therefore, selectors bearing oxygen gave rise to strong cation ion-exchange stationary phases, while selectors with an embedded nitrogen atom (inducing a weak anion exchange capacity) were used to create zwitterion ion-exchange stationary phases. The new mixed-mode stationary phases were chromatographically evaluated in high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) using isocratic elution conditions to disclose their chromatographic potential. In HPLC mode, aqueous-rich reversed phase chromatography, acetonitrile-rich hydrophilic interaction liquid chromatography and methanolic ion-exchange chromatography mobile phases were employed. In these chromatographic modes, retention factors and selectivity values for a test set of basic and zwitterionic analytes were determined. The results were compared and principal component analysis for each chromatographic mode was performed. For all chromatographic modes, the component 1 in the principal component analysis reflected the elution order. The application of different mobile phases on a particular column resulted not only in variation in retention, but also in modified selectivity, and different elution order of the analytes. The orthogonality of the elution order depending on the employed mobile phase conditions was especially reflected for structurally closely related analytes, such as melatonin and N-acetyl-serotonin, tryptamine and serotonin or noradrenalin and octopamine. However, ion-exchange interactions remain the main driving force for retention. From all investigated stationary phases, the SCX 2 (C5-linker and C4-spacer) seems to be the best choice for the separation of basic analytes using different mobile phase conditions.

• Klíčová slova
• Biogenic amines, Ion-exchange stationary phases, Liquid chromatography, Mixed-mode stationary phases, Supercritical fluid chromatography,
• MeSH
• acetonitrily chemie   MeSH
• anionty chemie   MeSH
• biogenní aminy izolace a purifikace   MeSH
• chromatografie metody   MeSH
• farmaceutická chemie metody   MeSH
• hydrofobní a hydrofilní interakce MeSH
• iontová výměna MeSH
• kationty chemie   MeSH
• léčivé přípravky chemie   izolace a purifikace   MeSH
• methanol chemie   MeSH
• voda chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetonitrile MeSH Prohlížeč
• acetonitrily MeSH
• anionty MeSH
• biogenní aminy MeSH
• kationty MeSH
• léčivé přípravky MeSH
• methanol MeSH
• voda MeSH

Polar columns used in the HILIC (Hydrophilic Interaction Liquid Chromatography) systems take up water from the mixed aqueous-organic mobile phases in excess of the water concentration in the bulk mobile phase. The adsorbed water forms a diffuse layer, which becomes a part of the HILIC stationary phase and plays dominant role in the retention of polar compounds. It is difficult to fix the exact boundary between the diffuse stationary and the bulk mobile phase, hence determining the column hold-up volume is subject to errors. Adopting a convention that presumes that the volume of the adsorbed water can be understood as the column stationary phase volume enables unambiguous determination of the volumes of the stationary and of the mobile phases in the column, which is necessary for obtaining thermodynamically correct chromatographic data in HILIC systems. The volume of the aqueous stationary phase, Vex, can be determined experimentally by frontal analysis combined with Karl Fischer titration method, yielding isotherms of water adsorbed on polar columns, which allow direct prediction of the effects of the composition of aqueous-organic mobile phase on the retention in HILIC systems, and more accurate determination of phase volumes in columns and consistent retention data for any mobile phase composition. The n phase volume ratios of 18 columns calculated according to the new phase convention strongly depend on the type of the polar column. Zwitterionic and TSK gel amide and amine columns show especially strong water adsorption.

• Klíčová slova
• hydrophilic interaction, liquid chromatography, polar columns, stationary and mobile phase,
• Publikační typ
• časopisecké články MeSH

A method for separation of cytokinins from auxin and abscisic acid, which allows further separation of cytokinin ribotides from cytokinin bases, ribosides and glucosides and their purification on a single Oasis MCX column was developed. Due to the mixed reversed-phase and cation-exchange mode of the Oasis MCX sorbent the cationic cytokinin bases, ribosides and glucosides as well as the anionic auxin, abscisic acid and cytokinin ribotides are retained and can be sequentially eluted by solvents containing different concentrations of methanol and ammonium hydroxide. Characteristics of the method are high recoveries of analyzed phytohormones and their sufficient purity for quantification by HPLC-ELISA (RIA) or HPLC-MS.

• MeSH
• cytokininy izolace a purifikace   MeSH
• ELISA metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• kyselina abscisová izolace a purifikace   MeSH
• kyseliny indoloctové izolace a purifikace   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• kyselina abscisová MeSH
• kyseliny indoloctové MeSH

Adopting a stationary phase convention circumvents problematic definition of the boundary between the stationary and the mobile phase in the liquid chromatography, resulting in thermodynamically consistent and reproducible chromatographic data. Three stationary phase definition conventions provide different retention data, but equal selectivity: (i) the complete solid phase moiety; (ii) the solid porous part carrying the active interaction centers; (iii) the volume of the inner column pores. The selective uptake of water from the bulk aqueous-organic mobile phase significantly affects the volume and the properties of polar stationary phases. Some polar stationary phases provide dual-mode retention mechanism in aqueous-organic mobile phases, reversed-phase in the water-rich range, and normal-phase at high concentrations of the organic solvent in water. The linear solvation energy relationship model characterizes the structural contributions of the non-selective and selective polar interactions both in the water-rich and organic solvent-rich mobile phases. The inner-pore convention provides a single hold-up volume value for the retention prediction on the dual-mode columns over the full mobile phase range. Using the dual-mode monolithic polymethacrylate zwitterionic micro-columns alternatively in each mode in the first dimension of two-dimensional liquid chromatography, in combination with a short reversed-phase column in the second dimension, provides enhanced sample information.

• Klíčová slova
• dual retention mechanism, hydrophilic interaction liquid chromatography, mixed-mode columns, phase volume convention, two-dimensional separation,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

A recently presented new type of "multilayered" organic-inorganic hybrid silica particle packed column YMC-Triart C18 (50 mm × 4.6 mm, 5 μm) was used for the development of a sequential injection chromatography method for determination of five azo dyes (Sudan I, Sudan II, Sudan III, Sudan orange G, and para red) in selected food seasonings. The use of a novel sorbent brings attractive features, reduced backpressure, and broader chemical stability together with high separation performance, which are discussed and compared with that of three types of columns typically used in medium-pressure flow chromatography techniques (classic monolithic, narrow monolithic, and core-shell particle columns). The separation was performed in gradient elution mode created by the zone mixing of two mobile phases (acetonitrile/water 90:10, 1.5 mL + acetonitrile/water 100:0, 2.3 mL) at a flow rate of 0.60 mL/min and time of analysis <9.5 min. The spectrophotometric detection wavelengths were set to 400, 480, and 500 nm. The high performance of the developed method with multilayered particle column was well documented and the results indicate a broad capability of sequential injection chromatography.

• Klíčová slova
• azo dyes, columns comparison, multilayered particle column, sequential injection chromatography,
• Publikační typ
• časopisecké články MeSH

In this study, the selectivity, retention properties, peak shape and loading capacity for bases were practically evaluated using two UHPLC mixed-mode hybrid CSH stationary phases modified by C18 or Phenyl group. The data were compared with the data obtained on other UHPLC hybrid stationary phases (BEH C18, BEH C8, BEH Phenyl and BEH Shield RP18) at both basic and acidic conditions using conventional HPLC buffers (50mM ammonium formate/acetate) as well as low ionic-strength additives such as, e.g. 0.1-0.01% formic/acetic acid and 1mM solution of ammonium formate/acetate, which are widely used in LC-MS applications. Ten pharmaceutically important compounds encompassing acids, bases and neutral were included into the study. Due to properties of CSH sorbent (which possess positively charged surface besides RP group), much improved peak shapes and weaker retention was obtained for bases even at very low concentration of acidic additives. Such conditions are ideally suited for LC-MS analysis of bases, where typical RP chromatographic separation (retention and good selectivity at basic pH) and LS-MS conditions (efficient ionization at acidic pH) are not in agreement. On the other hand, acids were more strongly retained and for some compounds the peak shape was influenced negatively due to ion-exchange mechanism. Further, the behavior of acidic, basic and neutral solutes is discussed using various additives at both basic and acidic pH for all above stated columns. The robustness of retention times after pH change from basic to acidic was also evaluated. The new CSH stationary phases represent an interesting selectivity tool preferably for separation of basic compounds.

• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We investigated the retention of phenolic acid and flavone antioxidants on five polar columns in buffered aqueous ACN mobile phases. All columns show mixed retention mechanism: RP in highly aqueous mobile phases and normal phase (hydrophilic interaction LC, HILIC) in mobile phases with high concentration of ACN. The Silica Hydride and the ZIC HILIC sulfobetaine zwitterionic columns show rather limited retention in the RP mode. The Luna HILIC column shows higher retention in both the HILIC and the RP modes in comparison to the PEG and DIOL columns. We characterized the selectivity of various HILIC systems using linear solvation energy relationship model with molecular structure descriptors characterizing selective molecular size, dipole-dipole and proton-donor/proton-acceptor interactions and we investigated the effects of the mobile phase composition on the linear solvation energy relationship characteristics of the separation phase systems to select suitable conditions for orthogonal HILIC separations in combination with RP systems. Dual retention mechanism offers possibilities for using complementary selectivity in the HILIC and the RP modes for sequential 2-D separations of natural antioxidants on a single Luna HILIC column. Column equilibration time of 15 min between alternating RP and HILIC gradient runs is sufficient for reproducible results.

• Publikační typ
• časopisecké články MeSH