Use of principal component analysis for investigation of factors affecting retention behaviour of ceramides on porous graphitized carbon column

Summary The potentiality of a porous graphitized carbon stationary phase was assessed for ceramide analysis in nonaqueous reversed phase chromatography with an evaporative light scattering detection. The influence of the nature of the mobile phase was first performed under isoeluotropic isocratic conditions. Eight mobile phase compositions in the presence or absence of triethylamine/formic acid were tested with a set of structurally relevant ceramides. Values obtained under these conditions for the peak heights, retention factors, plate numbers, asymmetry factors, relative retentions and Time-Corrected Calibrated Normalised Resolution Product were analysed by Principal Component Analysis. Principal Component Analysis was effective in attributing relationships between the chromatographic behaviour and analytical conditions. The relationship between the increase of the detector response and the presence of triethylamine/formic acid has been confirmed, and the main change of selectivity has been attributed to interactions provided by the weak solvent of the mobile phase. These conclusions have allowed the development of gradient conditions for the analysis of complex ceramide mixtures on porous graphitized carbon column.     

Using principal component analysis for the study of the retention behaviour of phenol derivatives under reversed-phase conditions

Summary The retention time of 11 ring-substituted phenol derivatives was measured on six different reversed-phase HPLC columns and the log k, theoretical plate number (N) and asymmetry factor (F) values were calculated for each solutes on each column. The similarities and dissimilarities among the columns and solutes were elucidated by principal component analysis followed with nonlinear mapping technique and cluster analysis. Calculations indicated that the retention characteristics of porous graphitized carbon stationary phase considerably deviate from those of octadecyl- and hexyl-coated silica, octadecyl-coated polystyrene-divinylbenzene polymer and polybutadiene-coated alumina. The differences among these columns were markedly smaller. The retention behaviour of aminophenols differed from those of other phenol derivatives proving the importance of molecular polarity in the retention. It was established that the mode of calculation slightly modifies the similarity and dissimilarity among the columns and solutes, therefore, the use of more than one calculation method is proposed.     

Retention of some ring-substituted aniline derivatives by porous graphitized carbon. Dependence on physico-chemical parameters

Summary The retention characteristics of 22 aniline derivatives were determined on a porous, graphitized-carbon column in unbuffered acetonitrile-water and methanol-water mixtures. Each aniline derivative gave symmetrical peaks in each eluent without buffers. Good linear correlations were found between the log k and the concentration of the organic component in the eluent. The slope and intercept values differed according to the type of organic modifier and the charcter, number and position of substituents, indicating the different selectivities of methanol and acetonitrile and the good separating power of the column. Multivariate mathematical-statistical calculations proved that the retention of ring-substituted aniline derivatives is mainly governed by electronic parameters and the hydrogen acceptor capacity of substituents.     

The calculation of retention and selectivity in reversed-phase liquid chromatography II. Methanol-water eluents

Summary To calculate retention in reversed-phase, high-performance liquid chromatography a method based on the molecular structure of the analyte and the characteristics of sorbents and mobile phases has been employed. Characteristics of different ODS-columns in water-methanol eluents have been determined.     

Molecular structure and retention behaviour of some polycyclic aromatic and perhydroaromatic hydrocarbons on graphitized carbon black

Summary On efficient glass capillary columns packed with graphitized thermal carbon black (GTCB) the stereoisomers of aromatic and saturated tricyclic hydrocarbons are completely separated and identified. The Henry's adsorption constants of the individual isomers were determined from mixtures at different temperatures. Five isomers of perhydroanthracene, five isomers of perhydrofluorene, two isomers of perhydrophenalene, four isomers of perhydroacenaphthene, as well as phenalene, dihydrophenalene, acenaphthene and acenaphthylene were investigated. The increase of the hydrogenization degree of unsaturated tricyclic hydrocarbons reduces the retention. Among the saturated tricyclic isomers the retention becomes shorter with the larger bending of the molecules, i.e. from the molecule having more equatorial bonds to the molecules having more axial connections. The experimentally determined Henry's constants were qualitatively compared with the structure of the separated molecules.     

Chromatographic behaviour of flavonoid compounds in reversed-phase HPLC systems

Summary Reversed-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.     

The role of ion pairing in the chromatographic study of propranolol analogues

Summary The partition coefficients, logP _app ^RPLC, for a series of propranolol analogues have been determined by reversed phase high performance liquid chromatography. The hydrochloride salts and free base logP _app ^RPLC show that charged and uncharged species can partition into the reversed phase. An extrathermodynamic relationship was found for logP _app ^RPLC measured in two different column stationary phases, C8 and PMOS, suggesting that the same set of compounds experience a similar trend in these elution systems. The importance of using phosphate and/or MOPS buffer is due to the fact that the latter can avoid ion-pairing partitioning, but the former does play the same role for PMOS column when the free base is considered.     

Quantitative structure activity relationships of 5-nitrofuran derivatives

Summary Quantitative structure-activity relationships were performed on a set of 5-nitrofuran derivatives. The correlation parameters used were hydrophobicity and connectivity. The former was measured by means of the capacity factor (log k _o ) obtained by HPLC and the latter through the molecular valence connectivity index^{1 v} obtained by computational methods. The biological activity data considered were growth inhibition percentages ofT. cruzi (GI % (48 h)) and half-maximal inhibitory concentration of Glutathione Reductase (I_{0.5 (max)}). Good adjustment, r=0.98, was obtained if a logarithmic model is used for I_{0.5 (max)} correlations. When GI% is considered, correlations were poorer and the consideration of both structural parameters was necessary for their improvement, r=0.94.     

Separation of carotenoids by high-performance liquid chromatography with polymeric and monomeric octadecylsilica stationary phases

Summary In addition to their value in the nutritional context, the carotenoids have other important functions, including some epidemiological significance in disease prevention. With increasing interest in the carotenoids methods for their characterization and quantification in various matrices are essential particularly with regard to epidemiologic studies dealing with diet and health. Since high-performance liquid chromatography (LC) is the most promising analytical method for this purpose, having high reliability, high selectivity and quantification ability, this work presents the evaluation of reversed-phase LC methods using polymeric and monomeric octadecylsilica (ODS) stationary phases for the highly selective separation of carotenoids. It has been found that the polymeric ODS has a better selectivity for carotenoids, taking account of their molecular shape and size recognition, than the monomeric ODS phase and that the former is more suitable than the latter for separations of the carotenoids.     

High performance liquid chromatographic separation of polar and non-polar chlorophyll pigments from algae using a wide pore polymeric octadecylsilica column

A method employing a wide pore polymeric reversed phase column has been developed for the separation of most of the chlorophylls and related compounds previously described as occurring in marine microalgae. The high selectivity toward molecular shape of this kind of stationary phase has enabled compounds of very similar structure, such as chlorophylls c₁, c₂ and Mg-divinylpheoporphyrin a₅ monomethyl ester, and chlorophyll a and the phytol-substituted chlorophyll c-like pigments, which commonly coelute on monomeric bonded phases, to be resolved in a single run. Some of these pigments, formerly thought to be a single compound, have, in fact, been demonstrated to be groups of two or more. The method has been successfully applied to both algal cultures and natural sea water samples. When visible light absorbance detection was used, the method proved suitable for separation of various carotenoids.     

Thermodynamic parameters of adsorption of adamantane and its derivatives on graphitized thermal carbon black

The adsorption equilibrium constants for adamantane, 1-fluoro-, 1-chloro-, 1,3-difluoro-, 1,3-dichloro-, 1,3-dibromo-, and 1-hydroxyadamantane, and methyl 1-adamantyl ketone were determined by gas chromatography. The results were compared with molecular statistical calculations based on the known atomic-atomic potentials of the interaction of atoms of the sorbate molecule with the C atom of graphitized thermal carbon black (GTCB). The experimental adsorption heats exceed the calculated values by 3-10 kJ mol^-1. The reasons for this divergence are discussed. The changes in the adsorption entropy show that the molecules of the studied compounds form a layer of the ideal dimeric gas on the GTCB surface upon adsorption.     

Effects of stationary phase structure on retention and selectivity in reversed-phase liquid chromatography of flavonoid compounds with methanol as organic modifier

Summary Retention of an extended set of flavonoid compounds by octyl, octadecyl, phenyl and cyanopropyl-bonded, reversed-phase columns, with methanol, as the organic mobile phase modifier and acetic acid as the acid modifier is reported. Solvent strengths and useful ranges for both isocratic and gradient elution are determined. Relative retention is found to be independent of methanol volume fraction in the mobile phase for all the columns examined. Correlations between retention on different columns, specific selectivity effects and their dependence on molecular structure are analyzed. Practical applications for separation and identification are discussed.     

Synthesis of porous divinylbiphenyl copolymers and their sorptive properties towards phenol and its derivatives

Seven porous divinylbiphenyl polymers having the same nominal crosslinking degree (51.8 wt.%) have been synthesized using suspension polymerization method in the presence of the following inert diluents or their mixtures: toluene, heptane, dodecane, isooctane. The use of various inert diluents was aimed at changing the extent of polymeric network-diluent interactions. The obtained polymers have specific surface area in the range 50-300 m²/g depending on the type and amount of inert diluents used during polymerization. Their sorptive properties have been studied using dilute (0.5 mmol/l) aqueous solutions of phenol and its derivatives (2-chlorophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,6-dimethylphenol, 4-hydroxyphenol). It has been found that sorption, at low equilibrium concentration, follows the order: 2,4,5-trichlorophenol > 2,4-dichlorophenol > 2-chlorophenol > 2,6-dimethylphenol > phenol > 4-hydroxyphenol. Full characteristic of the porous structure of polymers has been obtained by nitrogen adsorption at 77K.     

Structure-retention studies on the inclusion complex formation of some polycyclic aromatic hydrocarbons with β-cyclodextrin

Summary The retention behaviour of a group of polycyclic aromatic hydrocarbons having nearly equal ionization potentials but different molecular polarizability values was investigated using reversed-phase HPLC. Methanol-water binary systems containing -cyclodextrin were applied as the mobile phase. The relationships between the capacity factors, molecular polarizabilities and the shape parameter of solute molecules are discussed.     

Retention studies of sulphated glycosaminoglycan disaccharides on porous graphitic carbon capillary columns

Summary The retention behaviour of highly polar and charged disaccharide isomers has been studied on porous graphitic carbon columns and experimental parameters were varied over wide a range, including concentration and type of mobile phase constituents as well as temperature. The hydrophilic and anionic sugar analytes were highly retained on this stationary phase compared to the octadecyl-derivatized silica packings more commonly used. E.g., an increase in retention with polarity of a solute and with temperature was observed. By isotherm measurements and nonlinear fitting of Langmuirian expressions to the experimental data the graphite surface appeared homogeneous with only one kind of active adsorption site for these kinds of compound which was furthermore supported by the linear Van't Hoff plots obtained by varying the temperature. The gain in free energy was found to be entropically driven after determination of the ΔH° and ΔS° values. However, enthalpy-entropy compensation behavior was not met.     

Temperature effect on HPLC retention of PCBs on porous graphitic carbon

Summary The effect of column temperature on solute retention was investigated for 10 PCBs with different degrees of planarity (degree of ortho position substitution). The variation of retention values (lnk) with temperature was measured using two solvents, n-hexane and dichloromethane, as mobile phase: retention decreases as temperature increases. The relationship between lnk values and temperature (1/T) exhibits a good linearity. The ΔH ⁰ values calculated are in the −2 to −5 kcal mole^{− 1} range: they are very similar for compounds with the same degree of ortho-substitution and are significantly higher for non-ortho substituted congeners. High temperature significantly improves separation efficiency (plate number significantly increases): this effect is the dominant factor controlling chromatographic resolution, which improves with increasing temperature. Operating at 40 °C, an efficient separation of planar PCBs was obtained with low solvent consumption.     

Calculation of amino acid hydrophilicity indices for retention of peptides on amide, diol and silica columns in normal-phase liquid chromatography

Summary A set of hydrophilicity parameters in a normal-phase liquid chromatography of peptides is presented in order to clarify the contribution of individual amino acid residues to peptide retention and to predict retention times. The retention of 100 peptides was studied using normal-phase liquid chromatography on amide, diol and silica columns. An acetonitrile-water mixed solution containing 0.2% trifluoroacetic acid +0.2% triethylamine was used as the mobile phase in a linear gradient elution system. The contribution of each residue upon retention was calculated by linear multiple regression analysis. This paper described the contribution values as “hydrophilicity retention coefficients”. Using these hydrophilicity retention coefficients, retention times could be predicted for peptides of known amino acid content and sequence. A set of hydrophilicity retention coefficients on each column was successfully explained by contributions to the degree of retention.