phase separation
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The effects of mobile phase composition and of temperature on the retention behavior of phenolic acids were studied on 4 hydrosilated (type C silica) based columns in buffered aqueous acetonitrile, both in the aqueous normal phase (HILIC) and in the reversed-phase mobile phase range. The UDC cholesterol and the C₁₈ bidentate columns show significant reversed phase and normal-phase retention mechanisms, whereas very weak retention in the reversed-phase mode was observed on the silica hydride and the Diamond hydride columns. The concentration effects of the aqueous acetate buffer over the full mobile phase (HILIC and RP) composition range can be described by a simple four-parameter equation. At increasing temperature, the retention times and peak widths decrease both in the aqueous normal phase and in the reversed phase mobile phase range. Linear van't Hoff log k versus 1/T plots were observed, indicating a single retention mechanism predominating in the highly organic (HILIC), like in highly aqueous (RP) mobile phase ranges. Besides the type of the stationary phase, the separation selectivity of phenolic acids strongly depends on temperature and on the mobile phase composition. From among the 4 hydrosilated columns compared in this work, the UDC cholesterol column has high temperature stability (up to 100 °C) and is most suitable for selective and efficient separations of phenolic acids both in the HILIC and in the RP modes.
- MeSH
- acetonitrily chemie MeSH
- cholesterol chemie MeSH
- chromatografie kapalinová přístrojové vybavení metody MeSH
- chromatografie s reverzní fází MeSH
- hydrofobní a hydrofilní interakce MeSH
- hydroxybenzoáty izolace a purifikace MeSH
- oxid křemičitý chemie MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
V práci sa prezentujú výsledky získané pri použití dvoch chirálnych stacionárnych fáz (CSP),založených na báze makromolekulového antibiotika – teikoplanín s obsahom sacharidových častí(CHIROBIOTIC T) a teikoplanín bez sacharidových častí (CHIROBIOTIC TAG). Študovali saracemické zmesi 1-metyl-2-piperidínoetylesterov 2-, 3- a 4-alkoxyfenylkarbámových kyselín. Skúmalisa interakcie medzi separovanými látkami a CSP, vplyv separácie študovaných enantiomérovna hodnotu rozlišovacieho faktora (Rij) pri dodržaní rovnakých chromatografických podmienokmetódou vysokoúčinnej kvapalinovej chromatografie (HPLC). Na základe dosiahnutých výsledkovmožno konštatovať, že pre látky typu 1-metyl-2-piperidínoetylesterov 2-, 3- a 4-alkoxyfenylkarbámovýchkyselín je výhodnejšia CSP-CHIROBIOTIC TAG, pretože neobsahuje sacharidovú časť.Týmsa znižuje pravdepodobnosť nepolárnych interakcií, ktoré majú negatívny vplyv na hodnotu Rij.
The paper presents the results obtained with the use of two chiral stationary phases (CSP) basedon a macromolecular antibiotic agent – teikoplanin containing saccharide parts (CHIROBIOTIC T)and teikoplanin without saccharide parts (CHIROBIOTIC TAG). Racemic mixtures of 1-methyl-2--piperidinoethyl esters of 2-, 3-, and 4-alkoxyphenylcarbamic acids were examined. The investigationincluded interactions between separated substances and CSP, and the effect of separation of theenantiomers under study on the value of the differentiation factor (Rij) under identical chromatographicconditions with the use of the method of high-performance liquid chromatography (HPLC).On the basis of the obtained results, it is possible to report that CSP-CHIROBIOTIC TAG is moreadvantageous for the substances of the type of 1-methyl-2-piperidinoethyl esters of 2-, 3-, and4-alkoxyphenylcarbamic acids, because it does not contain a saccharide part, which decreases thepossibility of non-polar interactions which exert a negative effect on the Rij value.
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.
Na separáciu konformérov diazepamu sa použila chirálna stacionárna fáza na báze ß-cyklodextrínu. Ako mobilné fázy sa použili zmesi organického rozpúšťadla a tlmivého roztoku so zložením acetonitril/octanový tlmivý roztok (200 mmol/l) s rôznym pH (3,3; 5,5; 6,5). Následne sa skúmal vplyv prídavku chirálneho selektora (ß-cyklodextrínu) do mobilnej fázy na separáciu konformérov diazepamu. Separácia konformérov diazepamu bola komplikovaná interkonverziou diazepamu, ktorá je dôsledkom jeho konfiguračnej nestability. Detailne sa študoval vplyv prietoku, teploty, pH a iónovej sily mobilnej fázy na interkonverziu a elučné charakteristiky (retenčný faktor a selektivitný koeficient). HPLC separácia bola doplnená štandardným 1H a COSY NMR experimentom, ktorým sa overila štruktúra diazepamu v kyslom prostredí mobilnej fázy.
Chiral ß-cyclodextrin was used to separate diazepam conformers. Several mobile phases of the composition acetonitrile/acetate buffer 200 mmol/l (pH=3.3, 5.5, 6.5) were employed for this purpose. As follows, the influence of addition of chiral ß-cyclodextrin to the mobile phase on diazepam separation was studied. The interconversion was a concurrence process of separation, resulting from stereolability of the diazepam molecule. The influences of temperature, flow rate, pH, and ionic strength of the mobile phase on interconversion and chromatographic parameters (retention factor and selectivity coefficient) were studied. Complementary off-line NMR measurements were carried out with the goal to confirm the structure of diazepam in the presence of an acid mobile phase.
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.
- 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
We synthesized 8 polymethacrylate monolithic capillary columns using laurylmethacrylate functional monomer and various cross-linking monomers differing in the polarity and size. The efficiency of monolithic columns for low-molecular compounds significantly improved with increasing number of repeat non-polar methylene groups in the cross-linker molecules, correlating with greater proportion of small pores with size less than 50 nm. The best efficiency with HETP=25 μm for alkylbenzenes was achieved for columns prepared using hexamethylene dimethacrylate (HEDMA). Columns prepared with polar (poly)oxyethylene dimethacrylate cross-linkers show also improved efficiency with increasing chain length and generally better performance in comparison to the (poly)methylene dimethacrylate cross-linkers of comparable size, however with less apparent effects of the chain lengths on the pore distribution. The monolithic columns prepared with tetraoxyethylene dimethacrylate (TeEDMA) showed the best efficiency of all the columns tested, corresponding to HETP=15 μm (approx. 70,000 theoretical plates/m), show excellent column-to-column reproducibility with standard deviations of 2.5% in retention times, good permeability and low mass transfer resistance, so that is suitable for fast separation of low-molecular compounds in 2 min or less. By modification of the fused-silica capillary inner walls pre-treatment procedure, very good long-term stability was achieved even in 0.5 mm i.d. capillary format. The TeEDMA column can be also used for size-exclusion chromatography of lower non-polar synthetic polymers, whereas it is less suitable for separations of proteins than the HEDMA column.
- MeSH
- benzenové deriváty izolace a purifikace MeSH
- chromatografie s reverzní fází přístrojové vybavení MeSH
- kyseliny polymethakrylové chemie MeSH
- polymerizace MeSH
- poréznost MeSH
- proteiny izolace a purifikace MeSH
- reagencia zkříženě vázaná chemie MeSH
- reprodukovatelnost výsledků MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We studied sequence-dependent retention properties of synthetic 5'-terminal phosphate absent trinucleotides containing adenine, guanine and thymine through reversed-phase liquid chromatography (RPLC) and QSRR modelling. We investigated the influence of separation conditions, namely mobile phase composition (ion interaction agent content, pH and organic constituent content), on sequence-dependent separation by means of ion-interaction RPLC (II-RPLC) using two types of models: experimental design-artificial neural networks (ED-ANN), and linear regression based on molecular dynamics data. The aim was to determine those properties of the above-mentioned analytes responsible for the retention dependence of the sequence. Our results show that there is a deterministic relation between sequence and II-RPLC retention properties of the studied trinucleotides. Further, we can conclude that the higher the content of ion-interaction agent in the mobile phase, the more prominent these properties are. We also show that if we approximate the polar component of solvation energy in QSRR by the electrostatic work in transferring molecules from vacuum to water, and the non-polar component by the solvent accessible surface area, these parameters best describe the retention properties of trinucleotides. There are some exceptions to this finding, namely sequences 5'-NAN-3', 5'-ANN-3', 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' (N stands for generic nucleotide). Their role is still unknown, but since linear regression including these specific constellations showed a higher observable variance coverage than the model with only the basic descriptors, we may assume that solvent-analyte interactions are responsible for the exceptional behaviour of 5'-NAN-3' & 5'-ANN-3' trinucleotides and some intramolecular interactions of neighbouring nucleobases for 5'-TGN-3', 5'-NTA-3'and 5'-NGA-3' trinucleotides.
- MeSH
- adenin analogy a deriváty izolace a purifikace MeSH
- chromatografie s reverzní fází MeSH
- guanin analogy a deriváty izolace a purifikace MeSH
- kvantitativní vztahy mezi strukturou a aktivitou MeSH
- neuronové sítě MeSH
- oligonukleotidy izolace a purifikace MeSH
- rozpouštědla MeSH
- simulace molekulární dynamiky MeSH
- statická elektřina MeSH
- thymin analogy a deriváty izolace a purifikace MeSH
- voda MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
Thermally induced phase separation (TIPS) based methods are widely used for the fabrication of porous scaffolds for tissue engineering and related applications. However, formation of a less-/non-porous layer at the scaffold's outer surface at the air-liquid interface, often known as the skin-effect, restricts the cell infiltration inside the scaffold and therefore limits its efficacy. To this end, we demonstrate a TIPS-based process involving the exposure of the just quenched poly(lactide-co-caprolactone):dioxane phases to the pure dioxane for a short time while still being under the quenching strength, herein after termed as the second quenching (2Q). Scanning electron microscopy, mercury intrusion porosimetry and contact angle analysis revealed a direct correlation between the time of 2Q and the gradual disappearance of the skin, followed by the widening of the outer pores and the formation of the fibrous filaments over the surface, with no effect on the internal pore architecture and the overall porosity of scaffolds. The experiments at various quenching temperatures and polymer concentrations revealed the versatility of 2Q in removing the skin. In addition, the in vitro cell culture studies with the human primary fibroblasts showed that the scaffolds prepared by the TIPS based 2Q process, with the optimal exposure time, resulted in a higher cell seeding and viability in contrast to the scaffolds prepared by the regular TIPS. Thus, TIPS including the 2Q step is a facile, versatile and innovative approach to fabricate the polymer scaffolds with a skin-free and fully open porous surface morphology for achieving a better cell response in tissue engineering and related applications.
- MeSH
- analýza selhání vybavení MeSH
- biokompatibilní materiály chemická syntéza MeSH
- chemická frakcionace metody MeSH
- design vybavení MeSH
- polyestery chemie MeSH
- polymery chemie MeSH
- poréznost MeSH
- povrchové vlastnosti MeSH
- testování materiálů MeSH
- tkáňové inženýrství přístrojové vybavení metody MeSH
- tkáňové podpůrné struktury * MeSH
- vytápění metody MeSH
- změna skupenství MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Improvements in stationary phase stability have been and remain a great task for research of new stationary phases. Metal oxide-based stationary phases appear as one of perspective alternatives to classical silica based stationary phases regarding to their similar effectiveness, different selectivity, different retention mechanism and mainly better chemical and thermal stability. In this study, the retention behaviour of ondansetron and its five pharmacopoeial impurities on TiO(2)-based reversed phase was investigated. The influence of buffer type, pH and concentration on retention was studied. Different types and amount of organic solvent in mobile phase were tested. The effect of temperature and flow rate on separation was investigated. The separation conditions were optimized and developed method validated. The retention parameters - retention time (t(R)), retention factor (k'), theoretical plate number (N), resolution between peaks due to nearby peaks (R(s)) and symmetry factor (A(s)) have been compared to parameters achieved on polybutadiene-coated zirconia column. The thermodynamic parameters of retention of analysed compounds - enthalpy, entropy and Gibbs free energy - were calculated and compared to those achieved on polybutadiene-coated zirconia column. This work proves similarity of retention behaviour of ondansetron and its five related compounds on zirconia-based and titania-based stationary phases and potential utilisation of polyethylene covered TiO(2)-based reversed stationary phase as an alternative to polybutadiene-coated ZrO(2) stationary phase in pharmaceutical analysis of ondansetron.
Nuclear phosphoinositides are recognized as regulators of many nuclear processes including chromatin remodeling, splicing, transcription, DNA repair and epigenetics. These processes are spatially organized in different nuclear compartments. Phase separation is involved in the formation of various nuclear compartments and molecular condensates separated from surrounding environment. The surface of such structures spatiotemporally coordinates formation of protein complexes. PI(4,5)P2 (PIP2) integration into phase-separated structures might provide an additional step in their spatial diversification by attracting certain proteins with affinity to PIP2. Our laboratory has recently identified novel membrane-free PIP2-containing structures, so called Nuclear Lipid Islets (NLIs). We provide an evidence that these structures are evolutionary conserved in different organisms. We hypothesize that NLIs serve as a scaffolding platform which facilitates the formation of transcription factories, thus participating in the formation of nuclear architecture competent for transcription. In this review we speculate on a possible role of NLIs in the integration of various processes linked to RNAPII transcription, chromatin remodeling, actin-myosin interaction, alternative splicing and lamin structures.
