Correlation between hydrophobicity of amino acids and retention data in reversed-phase liquid chromatography with micellar eluents

Summary Hydrophobic character is usually expressed in terms of the partition coefficient in 1-octanol-water (log P_O/W). However, measurement of this coefficient is often problematic. Retention in micellar liquid chromatography is mainly due to hydrophobic interactions and can also be used as an index of hydrophobicity. A hydrophobicity scale was established with retention data foro-phthalaldehyde (OPA)-N-acetyl-L-cysteine (NAC) amino acid derivatives, using the glycine derivative as reference. Since the OPA-NAC derivatives only differ in the nature of R₁ in the amino acid (R₁CH(COOH)NH₂), in the absence of electrostatic interactions the hydrophobic character of the substituent was responsible for retention. Linear relationships were obtained between log of the ratiok′ of amino acid derivatives:k′ of the glycine derivative for a given mobile phase, and logP _O/W for the R₁ substituent. Good correlations were also found for phenylthiohydantoin amino acid derivatives.     

Application of log D for the prediction of hydrophobicity in the advanced Marfey's method

In the advanced Marfey's method, the resolution between the diastereomers derivatized with 1-fluoro-2,4-dinitrophenyl-5-l-leucinamide (l-FDLA) and 1-fluoro-2,4-dinitrophenyl-5-d-leucinamide (d-FDLA) is reflected by the difference of hydrophobicity of the two functional groups at the asymmetric carbon. However, no effective method has been developed for the estimation of hydrophobicity so far. For this purpose, we introduced log D from the ACD Labs LogD and applied it to relatively simple primary amines, amino acids and secondary alcohols in the present study. It was found that the difference of the retention times (Δt_R) correlated with that of log D (Δlog D) for both diastereomers based on the obtained experimental results. Based on these results, the following procedure was proposed for the non-empirical determination of the absolute configuration of primary amines including amino acids and secondary alcohols: (1) estimate the hydrophobicity by the calculation of log D for the two substituent groups at the asymmetric carbon, (2) locate the trans-type arrangement of the two more hydrophobic substituents in the l-DLA derivative and judge the asymmetric carbon to be R or S in the trans-type that is eluted first, (3) derivatize the desired compound with l- or d-FDLA and analyze by LC/MS, and (4) compare the elution order with the prospective one and determine the absolute configuration at the asymmetric carbon. Furthermore, log D could also be used to predict the retention times of unavailable amino acids and small peptides, indicating that the combination of the advanced Marfey's method with log D would provide more reliable structural information on a mixture composed of amino acids and small peptides. The developed method is being applied to more complicated compounds.     

Determination of lipophilicity for antitumor acridinone derivatives supported by gradient high-performance liquid chromatography method

The lipophilicity values of selected acridinone (imidazoacridinone and triazoloacridinone) derivatives were measured by gradient reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 stationary phase with a water/acetonitrile mixture as a mobile phase. The retention times obtained served as input data and appropriate log k_w values (i.e., the retention factor log k_w extrapolated to 0% organic modifier) as an alternative to log P were calculated using the DryLab program. The relationships between the lipophilicity (log k_w) and the chemical structure of the studied compounds, as well as correlation between experimentally determined lipophilicities (log k_w) and log P data calculated using some commonly available software, are discussed.     

Reversed-Phase Systems for the Separation of Coumarins and Furocoumarins by Thin-Layer and High-Performance Liquid Chromatography

Abstract

The retention behaviour of eleven derivatives of counarin was investigated by reversed-phase (RP) thin-layer and high performance liquid chromatography. The linear relationships between log k'or R_M values and the content of organic modifier in the aqueous mobile phase obtained for wide composition ranges indicate that the plots can be used to determine R_Mw and log k_w values by extrapolation to pure water. The effects of individual substituants on the retention and the correlation between TLC and HPLC data was analysed.     

Reversed phase,high-performance liquid chromatography: Effect of the structure of the chemically bonded hydrocarbon ligand on retention and selectivity

Summary Chemically bonded stationary phases (CBSP) were prepared by reaction of di- and trialkylchloro- and methoxy-silanes, R¹R²SiCl₂ and R¹R²Si(OMe)₂ with varying chain length of the ligands R¹ and R² with Lichrosorb Si 100, 10 μm, Merck. The influence of the structure of the CBSPs on the retention and selectivity in reversed phase chromatography (RPC) was investigated and compared with results obtained with monoalkyl-CPSPs. Plots of log k′ versus the percentage carbon yield straight lines. The slopes and intercepts are dependent on the structure of the ligands and the structure of the solute. An attempt was made to explain how structure of the CBSP influences the selectivity in RPC.     

Evaluation of N-Substituted 2,4-Dihydroxyphenylthioamide Fungicide Lipophilicity Using the Chromatographic Techniques HPLC and HPTLC

2,4-Dihydroxyphenylthioamide derivatives modified on the N-aryl ring have substantial fungicidal activity. To determine their quantitative structure–activity relationships their lipophilicity was determined by use of the chromatographic methods column liquid chromatography and thin-layer chromatography. Methanol–water systems were used as mobile phases and the linear dependences of retention (R_M and log k) on volume fraction of organic modifier, φ, were determined. This enabled precise determination of lipophilicity (R_Mw and log k_w) by extrapolation. Correlations were found between quantities characterizing the lipophilicity of the compounds. Deviations enabled discovery of compound structural features which increase or reduce lipophilicity. When these data were correlated with biological activity against the phytopathogenic fungi Alternaria alternata and Botrytis cinerea parabolic dependences were obtained.     

Application of perfluorinated acids as ion-pairing reagents for reversed-phase chromatography and retention-hydrophobicity relationships studies of selected β-blockers

The addition of the homologous series of perfluorinated acids-trifluoroacetic acid (TFAA), pentafluoropropionic acid (PFPA), heptafluorobutyric acid (HFBA) to mobile phases for reversed-phase high-performance liquid chromatography (RP-HPLC) of β-blockers was tested. Acidic modifiers were responsible for acidification of mobile phase (pH 3) ensuring the protonation of the β-blockers and further ion pairs creation. The effect of the type and concentration of mobile phase additives on retention parameters, the efficiency of the peaks, their symmetry and separation selectivity of the β-blockers mixture were all studied. It appeared that at increasing acid concentration, the retention factor, for all compounds investigated, increased to varying degrees. It should be stressed that the presence of acids more significantly affected the retention of the most hydrophobic β-blockers. Differences in hydrophobicity of drugs can be maximized through variation of the hydrophobicity of additives. Thus, the relative increase in the retention depends on either concentration and hydrophobicity of the anionic mobile phase additive or hydrophobicity of analytes. According to QSRR (quantitative structure retention relationship) methodology, chromatographic lipophilicity parameters: isocratic log k and log k_w values (extrapolated retention to pure water) were correlated with the molecular (log P_o/w) and apparent (log P_app) octanol–water partition coefficients obtained experimentally by countercurrent chromatography (CCC) or predicted by Pallas software. The obtained, satisfactory retention-hydrophobicity correlations indicate that, in the case of the basic drugs examined in RP-HPLC systems modified with perfluorinated acids, the retention is mainly governed by their hydrophobicity.     

Pyridoxal-5′-phosphate-dependent catalytic antibodies

Cofactors—i.e., metal ions and coenzymes—extend the catalytic scope of enzymes and might have been among the first biological catalysts. They may be expected to efficiently extend the catalytic potential of antibodies. Monoclonal antibodies (MAbs) against N^α-phosphopyridoxyl-l-lysine were screened for 1) binding of 5′-phosphopyridoxyl amino acids, 2) binding of the planar Schiff base of pyridoxal-5′-phosphate (PLP) and amino acids, the first intermediate of all PLP-dependent reactions, and 3), catalysis of the PLP-dependent α, β-elimination reaction with β-chloro-D/L-alanine. Antibody 15A9 fulfilled all criteria and was also found to catalyze the cofactor-dependent transamination reaction of hydrophobic D-amino acids and oxo acids (k′ _cat=0.42 min⁻¹ with D-alanine at 25°C). No other reactions with either D- or L-amino acids were detected. PLP markedly contributes to catalytic effecacy—it is a 10⁴ times more efficient acceptor of the amino group than pyruvate. The antibody ensures reaction specificity, stereospecificity, and substrate specificity, and further accelerates the transamination reaction (k′ _cat(Ab)/k′ _cat(PLP)=5×10³). The successive screening steps simulate the selection criteria that might have been operative in the evolution of protein-assisted psyridoxal catalysis.     

Gas chromatographic analysis of C1–C4 nitroparaffins. Separation and identification

Summary The separation of C₁–C₄ nitroparaffins on SE-30, PEGA and Porapak R stationary phases was investigated. The nitroparaffin mixture was obtained from a real technological process. The best separation was achieved on Porapak R at 190°C. The fact that each peak obtained from this column corresponded to a single compound was checked by utilizing the linear relationship between the peak widths and the adjusted retention times (t _R ). Peak identification was carried out using the chromatographic data only, with help of plots of log t _R vs. carbon number and log t _R vs. boiling point; some of the results were also confirmed with comparing the retention times with those of pure substances and IR investigation of the collected chromatographic fraction.     

Rm Values, retention times and π values of a series of potentially mutagenic nitroimidazo[2,1-b]thiazoles

The R_m values and retention times of a series of nitroimidazo[2,1-b]thiazoles were obtained by means of thin-layer and high-performance liquid chromatography, respectively. Extrapolated R_m and log k′ values were correlated in a statistically significant way with π values calculated according to the literature.     

Determination of Retention Factors of s-Triazines Homologous Series in Water Using a Numerical Method Basing on Ościk's Equation

The retention factors in pure water for a homologous series of s-triazines were calculated by a numerical method basing on Ościk's equation and were correlated with log k _w values obtained by linear and parabolic extrapolation. Chromatographic data (log k _w ) were compared with the software-calculated partition coefficients in the n-octanol/water system (Alog P, IAlog P, clog P, log P _Kowin , xlog P, log P _ACD and log P _Chem.Off.) as alternative hydrophobicity indices. The effect of organic modifier (methanol and acetonitrile) and its concentration in the mobile phase used for log k _w evaluation were investigated. Very good linear correlations were found between log k _w values calculated by the numerical method and log P _ACD , log P _Chem.Off . and clog P values, independent of organic modifier type.

     

Driving force prediction for inclusion complexation of α-cyclodextrin with benzene derivatives by a wavelet neural network

A wavelet neural network (WNN) has been constructed and used to predict the binding constant for the inclusion of α-cyclodextrin with benzene derivatives from the substituent molar refraction (R_m) and hydrophobic constant (π). The ln K_a values predicted by WNN from R_m and π are close to the experimental data. The results indicate that the inclusion complexation of α-cyclodextrin with mono- and 1,4-disubstituted benzene is dominantly driven by the van der Waals force and hydrophobic interactions.     

Studies on the retention behaviour of some thiazolylazo derivatives in reversed phase liquid chromatography

The retention behaviour of thiazolylazo derivatives, 4-(2-thiazolylazo) resorcinol (TAR), 4-(2-thiazolylazo)-orcinol (TAO), 2-(2-thiazolylazo)-4-methylphenol (TAC) and 1-(2-thiazolylazo)-2-naphtol (TAN) was studied by reversed phase liquid chromatography. The optimum conditions for the separation of four thiazolylazo derivatives were examined with respect to column, flow rate, mobile phase composition and pH of mobile phase. These derivatives were separated simultaneously on Symmetry C₈ column using composition of acetonitrile/water (60/40, v/v) as mobile phase. The capacity factor (k′) has been decreased at higher pH than pK_a of solute which may due to the increasing concentration of the ionized species as increase the pH of mobile phase. The dependence of log k′ on the volume faction of water in the binary mobile phase and k′ on the liquid–liquid extraction distribution ratio (D_c) in acetonitrile–water (60/40, v/v)/n-octane extraction system for thiazolylazo derivatives were obtained good linear relationship. The results showed that the retention behaviour of these derivatives was mainly affected by the hydrophobic interaction between thiazolylazo as solute and mobile phase.     

Effect of temperature on partial molar volumes and viscosities of aqueous solutions of α-dl-Aminobutyric acid,dl-Norvaline and dl-Norleucine

In this work, we present experimental results for partial molar volumes and viscosities of aqueous solutions of α-dl-aminobutyric acid, dl-norvaline and dl-norleucine at 288.15, 293.15, 298.15 and 303.15?K. The thermodynamic behavior of aqueous amino acid solutions is compared with that reported for glycine and α-alanine in water and is discussed in terms of group additivity and electrostriction.

The temperature dependence of the infinite dilution partial molar volumes and the B viscosity coefficients are interpreted in terms of amino acid hydration. According to the usual hydrophobicity criteria, the amino acids considered do not have a hydrophobic character and their behavior is dominated by the polar groups.     

Surfactant effect on the lower critical solution temperature of poly(organophosphazenes) with methoxy-poly(ethylene glycol) and amino acid esters as side groups

 The surfactant effect on the lower critical solution temperature (LCST) of thermosensitive poly(organophosphazenes) with methoxy-poly(ethylene glycol) and amino acid esters as side groups was examined in terms of molecular interactions between the polyphosphazenes and surfactants including various anionic, cationic, and nonionic surfactants in aqueous solution. Most of the anionic and cationic surfactants increased the LCST of the polymers: the LCST increased more sharply with increasing length and hydrophobicity of the hydrophobic part of the surfactant molecule. The ΔLCSTs (T _0.03M− T _0M), the change in the LCST by addition of 0 and 0.03 M sodium dodecyl sulfate (SDS), were found to be 7.0 and 14.5 °C for the polymers bearing ethyl esters of glycine and aspartic acid, respectively. The LCST increase of poly(organophosphazene) having a more hydrophobic aspartic acid ethyl ester was 2 times larger compared with that of the polymer having glycine ethyl ester as a side group. The binding behavior of SDS to the polymer bearing glycine ethyl ester as a hydrophobic group was explained from the results of titration of the polymer solutions containing SDS with tetrapropylammonium bromide. Graphic models for the molecular interactions of polymer/surfactant and polymer/surfactant/salt in aqueous solutions were proposed. Received: 17 February 2000/Accepted: 25 April 2000     

Synthesis of Some Novel Benzylidenehydantoins: Amino Acids Derivatives

5-Benzylidenehydantoin reacts with chlorosulfonic acid to give the corresponding p-sulfonyl chloride. Condensation of the latter with amino acids leads to sulfonylamino acid derivatives, which on coupling with glycine methyl ester hydrochloride in THF-Et₃N using the dicyclohexylcarbodiimide method furnish the desired dipeptide methyl esters. The spectral data of the compounds are briefly discussed.     

Optimized Method for the Determination of Flavonoid Glycosides and Aglycones

The relationship R_M=f (log % S; S- modifier) of several flavonoids and their sugar derivatives was examined by using chromatographic systems chosen by preliminary experiments. The dependence for flavonoid aglycones was flat in the whole mobile phase concentration. Two types of the R_M=f (log % S) plots, related to the size of sugar molecule, were found. For glycosides with smaller sugar moiety (arabinose or glucose) the plot was almost parallel, while plots of derivatives with larger sugar moiety (rutose) were convergent. After plotting the relationship R_M=f (log % S), even if standards are not available, it is possible to qualify the examined compound as containing single or complex sugar moiety. For all mentioned above compounds the R_M=f (log %S) values were given. Silica gel Si60 was used as polar adsorbent. Mobile phase composition was determined in preliminary experiments; it was composed of two solvents: methanol in ethyl acetate in increasing concentration of methanol: 5 – 20%.