Theory Of Gradient Elution Research Articles

High-performance liquid chromatography of amino acids, peprtides and proteins : LXIX. Evaluation of retention and bandwidth relationships of myosin-related peptides separated by gradient elution reversed-phase high-performance liquid chromatography

The gradient elution behaviour of ten peptide analogues encompassing the primary amino acid sequences adjacent to the serine-19 phosphorylation site of myosin light chain has been investigated using a 30-nm pore diameter n-butilsilica stationary phase and 0.1% trifluoroacetic acid—water—acetonitrile mobile phases. Quantitative expressions derived from linear solvent strength theory and general plate height theory have been used to assess the influence of gradient time and flow-rate on the relative retentions and bandwidths of these peptides. The retention behaviour of these peptide analogues was found to closely mirror the predictions of linear solvent strength gradient elution theory. Although the experimentally observed bandwidth changes over a conventional range of gradient conditions ( i.e. t G = 30–60 min) also correlated with bandwidth changes predicted on the basis of plate theory derived for low molecular weight organic molecules, for very steep and very shallow gradients divergencies from theory were observed. The significance of these discrepancies is discussed in relation to the influence of amino acid sequence changes rather than composition of these peptide analogues. These data allow the importance of sequential effects on the chromatographic behaviour and in particular, the resolution optimisation of these closely related peptides to be evaluated.

The theory of gradient elution under hydrostatic equilibrium in liquid chromatography. Part 3: Using solvents of different densities

AbstractGradient elution under hydrostatic equilibrium is extended to the treatment of the case where the two liquids have different densities. Analytic expressions are derived only for the cases where both mixing chamber and reservoir have constant cross‐sectional areas.As in the case where the densities of the liquids are equal, pressigned concentration gradients can be duplicated quite satisfactorily by combining a mixing chamber having a constant cross‐sectional area and a reservoir having an area which changes linearly with reservoir height. A numerical example is given to illustrate the use of the derived equations.

High-performance liquid chromatography of amino acids, peptides and proteins: LXVII. Evaluation of bandwidth relationships of peptides related to human β-endorphin, separated by gradient-elution reversed-phase high-performance liquid chromatography

The bandwidths of several polypetides related to human β-endorphin have been investigated with different n-alkylsilica stationary phases and different elution gradients of 0.1% trifluoroacetic acid—water—acetonitrile mobile phases. In particular, we have examined the influence of changes in the gradient steepness parameter, b, on peakwidth with five different octadeccylphases, chemically bonded to porous spherical silica particles of nominally 4 μm and 6 μm average particle diameter, respectively. The effect on the zone dispersion of these polypetide solutes as the average more diameter of the silica matrix was increased from 7.3 nm to 30 nm with stationary phases of similar ligand densities packed into columns of identical configuration has been further documented. The experimental data on solute bandwidths and peak capacities are comparable with the corresponding bandwidth and peak capacity values calculated from analytical equations, derived from the general plate height theory and from gradient elution theory. These comparisons clearly demonstrate that anomalous bandbroadening phenomena may occur when polypeptides are eluted with steep gradients, i.e. with gradients of large b values. Moreover, as the relative chromatographic residence times of B-endorphin peptides capable of forming a C-terminal amphiphilic secondary structures is increased, i.e. as the dwell times and median capacity factors, k ¯ for such peptides are increased, significant divergencies arise between the observed peakwidth behaviour and the behaviour predicted by analytical relationships which describe ether the dependency of peak bandwidth (as 4σ v) on the gradient steepness parameter, b, or the dependency of peak capacity on gradient time, t G , median capacity factor, k ¯ , and the Knox parameter, C, respectively. The importance of these divergences from the predicted bandwidth and peak capacity behaviour for polypeptides separated on reversed phases, and for resolution optimisation in particular, is evaluated. These investigations thus enable further assessment of the quantitative relevance of current models that describe polypeptide zone migration under gradient elution reversed-phase chromatographic conditions in which solute-dependent slow equilibria, mediated by conformational or solvation effects, mayu still occur.

High-performance liquid chromatography of amino acids, peptides and proteins LXVIII . Evaluation of retention and bandwidth relationships of peptides related to luteinising hormone-releasing hormone and growth hormone-releasing factor, separated by gradient elution reversed-phase high-performance liquid chromatography

The gradient-elution behaviour of six peptides related to luteinising hormone-releasing hormone and three peptides related to growth hormone-releasing factor has been investigated using a 30-nm pore diameter n-butylsilica stationary phase and water—acetonitrile mobile phases containing 0.1 % trifluoroacetic acid. The effect of gradient time and flow-rate on the relative retentions and bandwidths of these peptides have been assessed using quantitative expressions, derived from linear solvent strength theory and general plate height theory. Whilst the retention data for these hormonal peptides appear to be adequately described by gradient elution theory for the range of operational conditions used in this study, the experimental band broadening data are not in agreement in all cases with bandwidths predicted on the basis of plate theory derived for low-molecular-weight organic molecules. Possible causes of the “non-ideal” peak shapes observed for these peptides are discussed.

Gradient elution under hydrostatic equilibrium in liquid chromatography. Part 1

AbstractThe theory of gradient elution under hydrostatic equilibrium is developed for the case where only one reservoir and one mixing chamber are used and where both solvents have equal densities. Given the shapes of the two vessels, effluent concentration curve equations are deduced for different mixing chamber‐reservoir combinations. On the other hand, given the equation for the effluent concentration curve, the reservoir cross‐sectional area, as a function of height, can be deduced when the mixing chamber has a constant cross‐section. The varying cross‐section of the reservoir is accomplished by inserting thin discs of different areas on top of one another inside a regular vessel of constant cross‐section.Except for complicated gradients, a given gradient can be accomplished using a reservoir with a cross‐sectional area which varies linearly with height. In the case of complex gradients, a reservoir having two or more linear segments becomes necessary. A numerical method is given for the calculation of the continuously varying reservoir cross‐section for the exact duplication of a given gradient.

Theory of Gradient Elution through Ion Exchangers

Theory of Gradient Elution through Ion Exchangers