P. Reschiglian

Evaluation of the number of components in multi-component liquid chromatograms of plant extracts

Optimization of the chromatographic separation of flavonoid compounds in camomile extracts by the simplex method and the Monte Carlo method is described. Evaluation fo the number of peaks at unit resolution (Rs = 1) is used as the criterion of separation quality. The Davis-Giddings theory is applied in calculating the number of components and the results are validated by numerical simulations. Peak-purity checks for three identified compounds in commercial sample extracts are reported. Capacity factor patterns for 18 flavonoid and phenolic compounds over an extended range of methanol/aqueous buffer (pH 2.8) mobile phases are described.

Solvent selectivity effects in reversed-phase high-performance liquid chromatography of flavonoid compounds

Abstract The selectivity properties of methanol, acetonitrile and tetrahydrofuran as organic modifiers in the reversed-phase high-performance liquid chromatographic separation of flavonoid compounds was studied. Conditions for achieving separations between compound classes are described, in particular the possible separation of glycosides from aglycones in acetonitrile and tetrahydrofuran. Eleven retention contributions as Δlog k ′ are reported, and their dependence on mobile-phase composition is described. Solvent strength values and useful gradient elution conditions are given.

Chromatographic behaviour of flavonoid compounds in reversed-phase HPLC systems

SummaryReversed-phase HPLC retention behaviour of different flavonoid compounds in different columns and with a range of methanol-buffer eluents was examined. Repeatibility and reproducibility of logk′ and Δlogk′ values were analyzed. Sharp group contributions, expressed as Δlogk′, were observed. These did not depend significantly on acid modifier type, column packing characteristics (C8 and C18) and dimensions (5,7 and 10μm) or the percentage of methanol in the mobile phase. A list of twelve group contributions is presented which is in agreement with literature data.

Comparison of the sequential simplex method and linear solvent strength theory in hplc gradient elution optimization of multicomponent flavonoid mixtures

SummaryUsing a camomile flavonoid extract as the sample and four different reversed-phase partition systems, the ability of the Simplex procedure to produce optimum gradient separation of unknown multicomponent mixtures was checked against the linear solvent strength (LSS) gradient elution theory. On the same partition systems the mean solvent strenghts were measured by experimentally determined logk′ vs. mobile phase composition plots. These mean solvent strengths are compared to those inferred from the optimum gradients and the usefulness of LSS theory in multicomponent mixture gradient elution optimization is discussed.

Effects of different organic modifiers in optimization of reversed-phase high-performance liquid chromatographic gradient elution of a mixture of natural secoiridoid compounds

Abstract Optimization of the linear gradient elution of a crude extract of Swertia herb (Gentianaceae) was performed by means of sequential methods (Fibonacci and simplex) using a reversed-phase partition system and three different organic modifiers. The optimum gradient elution separations obtained with methanol, acetonitrile and tetrahydrofuran as different organic modifiers are discussed and compared by using the linear solvent strength theory. The Poisson character of the optimized separations was checked and the Davis-Giddings statistical overlap theory was applied in order to calculate the number of components. The value obtained is independent of both the organic modifier and column type. The quality of the best separation obtained was close to the absolute optimum expected from the Davis-Giddings theory. The usefulness of dealing with two or more optimized gradient separations is discussed. Solvent strengths are reported for selected compounds (amarogentin, amaroswerin and gentiopicroside).

Comparison of reversed-phase thin-layer and high-performance liquid chromatography of flavonoid compounds

Abstract The reversed-phase high-performance liquid (HPLC) and thin-layer chromatographic (TLC) behaviour of flavonoids with methanol, tetrahydrofuran and acetonitrile as organic modifiers was compared. Twenty-six different correlation cases, together with their statistical parameters, are presented and discussed. Both TLC and HPTLC plates were considered. The dependence of TLC incremental Rm, values from different group substitutions on solvent type, solvent composition and type of plate is considered. A method for establishing HPLC gradient elution conditions by using TLC data is discussed.

Retention behaviour of selected flavonoid compounds in NP-HPLC

SummaryHPLC retention data for a number of flavonoid compounds have been evaluated on normal phase silica and cyano colums according to the Soczewinski-Snyder (S.-S.) model. On the silica phase most of the flavones examined exhibited asymetrical peak shapes indicating strong and mixed retention mechanisms; on the cyano phase the intercept of the S.-S. relationship was independent of any hydroxy substitution pattern or of molecular planarity. A similar behaviour, but with different intercept values, was observed with coumarin and quinone compounds.

Stochastic Theory of Chromatography: The Characteristic Function Method and the Approximation of Chromatographic Peak Shape

The Stochastic Theory of Chromatography is reviewed in its fundamental aspects and achievements. Basic elements of the probability theory for linear-finite-time chromatographic model description are presented and the mathematical method of “Characteristic Function” (CF) for solving probabilistic models is described. Solutions are found in terms of CF for general chromatographic models of increasing complexity. These models are the constant mobile phase velocity model with general mechanisms of stationary phase entry and desorption process and non-constant mobile phase processes with Poisson mechanism of the stationary phase entry process. Among the solved models those belonging to the class of stochastic processes with stationary and independent increments are identified for which general description of the peak profile by the Edgeworth-Cramer series holds true. The convergence towards the Gaussian Law through the Berry-Esseen Theorem is discussed and the basic conditions for peak parameter determination by non-linear least squares fitting are discussed.

Correlations between high-performance liquid chromatographic retention, X-ray structural and 13C NMR spectroscopic data of flavonoid compounds

Abstract The retention behaviour of selected flavonoid compounds was analysed and correlated with structural properties, as elucidated by X-ray crystallographic and 13 C NMR spectroscopic studies. A detailed analysis of group retention contribution (Δlog k′ ) makes it possible to establish how the introduction of an OH group in the molecule affects the overall solute polarity and, therefore, its retention, in relation to the position of the OH group and the molecular environment. X-ray structural information and 13 C NMR chemical shifts show the formation of strong intramolecular hydrogen bonding (HB) in 5-hydroxy-flavones and a weaker intramolecular HB in 3-hydroxy-substituted compounds. Δlog k′ values may be explained in terms of these intramolecular HB properties; moreover a quantitative relationship Δlog k′ values and chemical shifts was found.