Literature
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A previously reported model describes retention and band width as functions of experimental conditions, for the reversed-phase gradient elution separation of peptides and proteins. The model begins by relating separation in gradient elution to corresponding separations by isocratic elution (same high-performance liquid chromatographic system). The use of certain semi-empirical relationships then allows band width to be predicted for samples for which isocratic data are unavailable. This in turn allows the prediction of band width, peak capacity and relative peak height (or peak volume) as a function of experimental conditions such as column dimensions, gradient conditions, mobile phase flow-rate, sample molecular weight, etc. The next paper [J. Chromatogr., 327 (1985) 93] demonstrates the practical value of this approach for optimizing the separation of peptide and protein mixtures. In the present paper it is first shown that isocratic data for desamido-insulin (6000 daltons) allow the accurate prediction of band widths as a function of experimental conditions in related gradient separations. It is further shown that the present model accurately predicts how band widths vary with experimental conditions for peptides and proteins having molecular weights in the range 600–80 000 daltons.
An HPLC-method for the measurement of blood Cyclosporin A levels (CyA) of renal allograft transplanted patients within 9min is described. After a simple protein precipitation of the blood the supernatant is transferred to an HPLC-system. The short time of analysis is obtained by a step gradient elution technique and a precolumn separation of the fractions of interest followed by a backflush regeneration step of the precolumn. The analysis of the fraction of interest takes place on a column with high resolution power as long as the precolumn is regenerated. CyA is monitored by UV-absorption at 206nm. Detection of 20 to 2000ng/ml CyA allows the use of the method for patient monitoring and for research purposes.
The presently accepted theory for gradient separations of small molecules has been used to develop a predictive model for peptides and proteins as samples, using reversed-phase high-performance liquid chromatography. Given the experimental conditions (gradient time, flow-rate, temperature, etc.), the molecular weight of the sample, and certain column characteristics (Knox parameters, column dimensions, particle diameter, etc.), it is possible to calculate the overall results of a given separation by gradient elution: peak capacity or average resolution, peak volume or relative peak height, etc. This information can in turn facilitate the optimized separation of any sample. The present model assumes that isocratic and gradient retention are interrelated for peptide molecules, in the same fashion as for small molecules. This assumption has been verified for various peptides and proteins and further used to gain new insight into the control of retention and band-spacing in gradient elution.
Equipment for high-performance liquid chromatographic (HPLC) gradient elution generally distorts the gradient selected by the operator, which in turn affects the retention of solutes separated by gradient elution. A theoretical analysis describes these gradient distortions as a function of equipment design and operating conditions. Comparisons of theory with experimental data show generally good agreement. As a result, it is now possible to select gradient conditions for minimal gradient distortion, or to correct for the effect of gradient distortion on solute retention. This will be shown in later papers to allow the use of gradient elution in new ways for more efficient method development and optimization of separation by HPLC.
Under “ideal” conditions it is possible to model retention in gradient elution so as to be able to calculate retention times, tg, as a function of isocratic retention in corresponding liquid chromatographic systems. In this paper we consider various “non-ideal” processes that lead to errors in calculated values of tg. The more important of these are solvent demixing due to uptake of one mobile phase component by the column packing, non-linear plots of log k′ vs. gradient time or mobile phase composition and changes in column dead-time, t0, due to changes in mobile phase composition and flow-rate. Expressions are derived to correct for these various “non-ideal” effects, including equipment limitations discussed in the preceding paper. Calculated values of tg for reversed-phase gradient elution systems then agree with experimental values to within ± 1 % (1 standard deviation) of the total gradient time, tG. These results should prove useful in (a) improving the precision of retention in gradient elution (which should be comparable to that in isocratic elution), (b) using gradient elution for more efficient method development in isocratic procedures and (c) better understanding gradient separations of macromolecules such as proteins.
Reduced plate height (h) vs. reduced velocity (v) plots have been measured over a wide range of v for 36 high-performance liquid chromatographic systems. Column type was varied over wide limits and solute capacity factor (k′) values were changed over the range 0.6–22. Resulting data can be accurately described by the Knox equation h = Av1/3 + B/v + Cv, where A is roughly constant (A = 0.5–0.8) for all columns studied, but values of B and C are strongly dependent on column type and solute k′ values. These observations can be rationalized by a quantitative model that recognizes two effects: (a) surface diffusion of solute molecules in the stationary phase (along the pore wall) and (b) restricted diffusion of small solute molecules within particles having narrow pores and long alkyl chains bonded to their surface.
Analysis of proteins in solution by high-performance liquid chromatography is presented with respect to structural changes in solution, adsorption processes and differentation concerning specific activities. Trypsin and cellulases were taken as examples.
High-Performance Liquid Chromatography: Advances and Perspectives, Volume 1 deals with the fundamental aspects of high-performance liquid chromatography, a technique used in chemical analysis. The publication provides accounts, presented by experts in the field, of a variety of topics in high-performance liquid chromatography. Each chapter covers interesting subjects such as the evolution of liquid chromatography; the use of bonded phases in high-performance chromatography; effects of ionization and complex formation on retention and selectivity in reversed-phase chromatography; and gradient elution. Chromatographers, chemists, and researchers in the field of chemical analysis will find this book a valuable reference material.
The capacity factors of 32 compounds were measured on octadecyl-silica columns by using neat aqueous phosphate buffer, pH 2.1, as the eluent. The tabulated results allow the estimation of the effect of various substituents on the retention of catecholamines and related compounds under similar chromatographic conditions.
Microparticulate non-polar stationary phases, such as octadecyl-silica offer a rapid and efficient means for the separation of peptides and amino acids by high-performance liquid chromatography. Retention is attributed to hydrophobic interaction between the solutes and the hydrocarbonaceous functions covalently bound to the stationary phase surface. Consequently the species are eluted in the order of increasing hydrophobicity. Various peptide mixtures were analyzed by using gradient elution with increasing acetonitrile concentration in the eluent and monitoring the column effluent at 200 or 210 nm with an UV detector. The separation of angiotensins and enzymic digest of polypeptides illustrates the speed of the method which can be used to assay the purity of peptide hormones such as α-melanotropin and gramicidin or to analyze the composition of reaction mixtures involving peptides. The efficiency of the method is superior to that obtained on the conventionally used ion-exchanger columns, except for hydrophilic amino acids and peptides that are poorly retarded. Nevertheless, with a suitable ionic surfactant in the mobile phase, non-polar stationary phases can be used for the separation of these species as well.
Peptides : proceedings of the 5th American Peptide Symposium -conference_proceeding
The effect of solute ionization on the retention of weak acids, bases, and ampholytes on octadecylsilica was investigated both theoretically and experimentally. The retention was attributed to a reversible association of the solutes with the hydrocarbonaceous ligand of the stationary phase. A phenomenological treatment of the corresponding equilibria was developed for various types of ionogenic substances. The energetics of the association process was analyzed in a rigorous fashion in the light of the solvophobic theory and a semi-empirical extension of the Debye-Hiickel theory to high ionic strength. The predicted effect of solute ionization on the capacity factors was substantiated by experimental data. The observed dependence of the capacity factors on the ionic strength of the eluent and the hydrophobic surface of the solute molecules showed good agreement with the theory. The advantages of the technique in the separation of biological substances are illustrated.
Over a hundred acidic urinary constituents were separated within 30 min by using 5-μm octadecyl-silica columns and gradient elution with increasing acetonitrile concentration in dilute aqueous phosphoric acid solution at 70°. The column effluent was monitored with a UV detector at 280 nm or with a fluorescence detector at 260 nm excitation and 340 nm emission wavelengths. The high sensitivity and speed of analysis, the excellent reproducibility and adequate resolution obtained suggest that this technique may be useful to obtain metabolic profiles in routine clinical work.