28 research outputs found
Thermoresponsive poly(N-vinylcaprolactam) as stationary phase for aqueous and green liquid chromatography
Poly(N-vinylcaprolactam) (PVCL) connected to aminopropyl silica is a new stationary phase for temperature responsive liquid chromatography (TR-LC). PVCL shows a transition from hydrophilic to hydrophobic interaction between 30 and 40 degrees C. The synthesis is described in detail. The temperature responsive characteristic of the phase is illustrated with a mixture of steroids using pure water as mobile phase. An increase in retention is observed when raising the temperature. H-u plots at different temperatures were constructed. Below the lower critical solution temperature (LCST), no optimal velocity could be measured because of substantial resistance to mass transfer. Above the LCST, u (opt) was ca. 0.3 mm s(-1) with reduced plate heights from 4 at 45 degrees C to 3 at 65 degrees C. The temperature responsive nature of the polymer is lost in green chromatography with ethanol as modifier in concentrations above 5%
Investigation into reversed phase chromatography peptide separation systems part II : an evaluation of the robustness of a protocol for column characterisation
The robustness of the Peptide Reversed Phase Chromatography (RPC) Column Characterisation Protocol was evaluated using reduced factorial design, to ascertain the degree of control required for parameters including temperature, flow rate, dwell volume, a systematic shift in the gradient, amount of formic acid in the aqueous and organic, pH of the ammonium formate and amount of acetonitrile (%MeCN) in the strong solvent, where a loss of MeCN resulted in an unacceptable variation. Mitigations have been introduced to ensure the integrity of the data to allow RPC columns to be characterised using peptides as probes, with the definitive protocol described. In addition, the instrument and column batch to batch variability were assessed with good reproducibility
Investigation into reversed phase chromatography peptide separation systems part I : development of a protocol for column characterisation
A protocol was defined which utilised peptides as probes for the characterisation of reversed phase chromatography peptide separation systems. These peptide probes successfully distinguished between differing stationary phases through the probe's hydrophobic, electrostatic, hydrogen bonding and aromatic interactions with the stationary phase, in addition, to more subtle interactions such as the phase's ability to separate racemic or isomeric probes. The dominating forces responsible for the chromatographic selectivity of peptides appear to be hydrophobic as well as electrostatic and polar in nature. This highlights the need for other types of stationary phase ligands with possibly mixed mode functionalities / electrostatic / polar interactions for peptide separations rather than the hydrophobic ligands which dominate small molecule separations. Selectivity differences are observed between phases, but it appears that it is the accessibility differences between these phases which play a crucial role in peptide separations i.e. accessibility to silanols, the hydrophobic acetonitrile / ligand layer or a thin adsorbed water layer on the silica surface
Column classification/characterisation of strong cation exchange phases for the liquid chromatographic analysis of small molecular weight bases
A simple, rapid and robust protocol for the characterisation of strong cation exchange columns for the analysis of small molecular weight bases is described. A range of ten different phases were characterised, and the resultant selectivity and retention factors analysed using Principal Component Analysis. The score plots for the first and second principal components described 83% of the variability within the dataset. Score plots highlighted the large chromatographic differences observed between the phases, the validity of which was established using a larger range of bases. All the strong cation exchange materials demonstrated a synergistic mixed mode (i.e. ion exchange and hydrophobic) retention mechanism. Principal Component Analysis also highlighted the potential difficulty in locating suitable strong cation exchange “back-up” columns for the analysis of small molecular weight bases in that the characterised columns all displayed very different selectivities. The robustness of the protocol was confirmed by a factorial design experiment
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The Use of Complimentary Stationary Phases and 2-Dimensional HPLC for the Separation of the Synthesis and Degradants of Tipredane (INN)
Investigation into reversed phase chromatography peptide separation systems part III : establishing a column characterisation database
The Peptide RPC Column Characterisation Protocol was applied to 38 stationary phases, varying in ligand chemistry, base silica, end capping and pore size, which are suitable for the analysis of peptides. The protocol at low and intermediate pH is based on measuring retention time differences between peptides of different functionality to calculate selectivity delta values. The characterisation was designed to explore increases / decreases in positive or negative charge (deamidation), steric effect (i.e. racemisation / switch in amino acid order), oxidation and addition / removal of aromatic moieties. The necessity of developing a characterisation protocol specifically for peptide analysis was highlighted by the fact that the small molecule databases (Snyder's Hydrophobic Subtraction Model and the extended Tanaka protocol) failed to correlate with the Peptide RPC Column Characterisation Protocol. Principal Component Analysis was used to demonstrate that the protocol could be used to identify columns with similar or dissimilar chromatographic selectivity for the purpose of selectivity back-up or method development columns respectively. This was validated using peptide fragments derived from the tryptic digest of bovine insulin and carbonic anhydrase. It was also demonstrated that the presence of positively charged functional groups on the stationary phase was advantageous as it yielded very different chromatographic selectivity and improved peak shape
Overloading study of bases using polymeric RP-HPLC columns as an aid to rationalization of overloading on silica-ODS phases
The separation of ionized bases by reversed-phase liquid chromatography with alkyl silica columns often leads to severely tailed bands that are highly detrimental. Band shape and its dependence on sample mass are notably different when mobile-phase pH is changed, and this behavior has not been previously explained. Ionized silanols present in the stationary phase have been credited with a role in determining peak shape. In the present study, separations on two different polymer columns were compared with those previously obtained on alkyl silica phases. Because silanols are absent from polymer columns, this comparison enabled us to assess the role of silanols in separations on alkyl silica phases and to offer an explanation of why band shape changes with sample size and mobile-phase pH for both polymer and silica-based phases
A study of retention and overloading of basic compounds with mixed-mode reversed-phase/cation-exchange columns in high performance liquid chromatography
The retention and overload of bases were studied on two new mixed-mode, silica based phases possessing ionic carboxylate functionalities of different acidity embedded within a hydrophobic ligand (SiELC Primesep™). At low pH, good peak shapes were obtained for small solute mass, suggesting that the mere presence of a mixed-mode hydrophobic/ionic retention mechanism is not responsible for the poor peak shape that can occur on conventional reversed-phases with ionised silanols. Somewhat inferior, but still acceptable peak shape for bases was obtained on a column containing a mixture of discrete ion exchange and reversed-phase particles (Hypersil Duet). In both types of column, the ionic sites favourably increased the capacity for ionised bases, reducing considerably the deterioration of peak shape with load observed with conventional RP columns. The combined ionic and reversed-phase interaction can give strong retention of bases under certain conditions, necessitating careful choice of stationary and mobile phase. © 2006 Elsevier B.V. All rights reserved
Analysis of basic compounds by reversed-phase high-performance liquid chromatography using hybrid inorganic/organic phases at high pH
The retention and mass overload of five bases and a quaternary ammonium compound were studied on a bridged ethyl hybrid inorganic/organic phase (XBridge C18) over a pH range 2.7-12.0 using acetonitrile-phosphate and carbonate buffers. Some comparisons were drawn with results on a methyl hybrid (XTerra) phase. At low pH, rapid mass overload was observed with severe decreases in efficiency as sample mass was increased over the range 0.04-5 ÎĽg of solute. At intermediate pH (wspH 7 .0), generally good peak shapes for small sample mass were still obtained on the ethyl hybrid, but with somewhat increased tailing of bases compared with wspH 2.7. At higher pH (wspH 10), good peak shape and improved loadability were obtained for moderately strong bases, due to their occurrence mostly as neutral species. However, stronger bases gave poor efficiency, attributed to interaction of the charged basic solute with increasingly ionised column silanol groups. Results were broadly similar on the methyl hybrid at wspH 10. At wspH 11, unusual profiles of increasing followed by decreasing efficiency were obtained on the ethyl hybrid for some bases as sample mass was increased; improved results were obtained at wspH 12. While the column loadability increased substantially at the highest pH studied, tailing for small sample mass was still more severe than at low pH, even though all compounds wer