Liquid Chromatographic Behavior of Nitrogen Compounds

Abstract

Chromatographic behavior of nitrogen compounds differed from others. The column efficiency was poor for the compounds and sometimes solutes were not eluted out from a column. Therefore, the elution volume of alkylamines, anilines, pyridines, pyrazines, quinolines and aminopolyaromatic hydrocarbons was measured on a methacrylate gel and octadecyl bonded silica gels in pH controlled acetonitrile/water mixtures. The solvent effect on the dissociation constant differs from that obtained for aromatic acids. The values in acetonitrile/water mixtures are smaller than those obtained in 100% water. The linear relation between log P and log k′ values is obtained in eluents of pH 7 where the retention of these compounds is maximized. Some hydrophobic fragmental constants are proposed from this result. Prediction of retention time of these compounds from their log P values can be done in the individual groups on octadecyl bonded silica gels in pH controlled acetonitrile/water mixtures.     

An approach to the prediction of retention times in liquid chromatography

The utility of Rekker's hydrophobic fragmental constant has been examined for optimization of reversed-phase mode liquid chromatographic separations. The chromatographic behavior of about 60 non-ionic compounds was measured in different acetonitrile/water mixtures and the logarithm of their capacity factors (log k) was correlated with their calculated hydrophobicities (log P). Linear relations were found in each case between log k and log P. The slope of the various lines was related to the percentage concentration of acetonitrile in the mobile phase. It was shown that, by using nine stand ard compounds and measuring their capacity factors in five eluents with different acetonitrile concentrations, the retention time could be predicted for 60 compounds. Calculation of the concentration of the organic modifier was also possible in a system of well coated octadecyl bonded packings with acetonitrile/water mixtures as eluent. Prediction of the capacity factor was accomplished to within 5% error.     

The effect of stationary phase on lipophilicity determination of beta-blockers using reverse-phase chromatographic systems

Evaluation of lipophilicity parameters for basic compounds using different chromatographic stationary phases is presented. An HPLC method for determination of lipophilic molecule-stationary phase interactions was based on gradient analysis. Differences in correlation between the lipophilicity of compounds and experimental chromatographic results obtained in pseudo-membrane systems showed a strong influence of stationary phase structure and physico-chemical properties. beta-Blocker drugs with varying lipophilicity and bio-activity were chosen as test compounds. The stationary phases used for the study were monolithic rod-structure C18 and silica gel octadecyl phase SG-C18 as reference material. The second group was silica gel-based polar-embedded alkylamide and cholesterolic phases. The mobile phase was composed of acetonitrile or methanol with ammonium acetate, and a linear gradient of methanol and acetonitrile in mobile phase was performed. A linear correlation of plots of log k(g) = f(log P) was observed, especially for polar-embedded phases, and this allowed log P(HPLC) to be calculated. The behavior of stationary phases in methanol and acetonitrile buffer showed differences between obtained log P(HPLC) values.     

Study of qualitative analysis by liquid chromatography using porous polymer gel

Summary The possibility of qualitative analysis by liquid chromatography is discussed in systems of polystyrene gel or chemically bonded octadecyl packing with alcohols as standard compounds. Rekker's hydrophobic fragmental constants are used for identification purposes. Capacity ratios for alcohols can be calculated from the hydrophobicities in spite of changes in solvent mixture ratios if the capacity ratio for an internal standard compound is measured.     

Correlation of HPLC Retention Data of Triazines with the Inhibition of DHFR from L1210 Cells: Development of New Chromatographic Adsorption Parameter in QSAR

Abstract

The use of chromatographic retention measurements for quantitative structure—activity relationships (QSAR) was investigated by the development of an adjusted logarithmic retention factor. Retention data of 15 triazine derivatives, which inhibit dihydrofolate reductase were measured by HPLC on octyl-silica and silica gel columns. From these data two parameters were calculated. First, the intercept of the plot of the logarithmic retention factor versus acetonitrile concentration was calculated from the reversed-phase retention measurements (log k'o) and, second, the difference between the logarithmic retention factor at high ammonium salt concentration and low ammonium salt concentration in the mobile phase was calculated from retention measurements on the silica gel column. The first parameter represents hydrophobic properties and the second parameter characterizes adsorption properties of the triazine derivatives. The results show a correlation between the two above mentioned parameters and the inhibition of L1210/71 cell growth.     

Comparison of Octadecyl-Bonded Alumina and Other Stationary Phases for Lipophilicity Estimation by High Performance Liquid Chromatography

Abstract

A recently developed octadecyl-bonded alumina stationary phase (ODA) was evaluated for determining the lipophilicities of organic compounds by high performance liquid chromatography. Using a column packed with this material and mobile phases consisting of methanol and aqueous buffers, the correlation between the octanol-water partition coefficients (log P's) of compounds of various chemical classes and the logarithms of their chromatographic capacity factors (log k's) was found to be superior to that obtained using columns packed with octadecylsilica, poly butadiene-coated alumina or octadecyl-derivanzed polystyrene-divinylbenzene copolymer. In contrast to results obtained with other columns, phenols and other hydrogen-bonding compounds did not need to be treated as a separate data set on the ODA column to obtain good correlations between log k's and log P's. The resistance of ODA to degradation by alkaline solvents allowed the use of a basic mobile phase (pH > 10) for suppressing ionization and determining the lipophilicities of organic bases which could not be evaluated within the stable pH range of ODS (pH 2–8). The log P's of five basic pharmaceutical compounds determined in this manner were found to be significantly higher than previously reported values. Evidence is presented which indicates that the previously reported log P values of these compounds are inaccurate, due to improper correction for ionization.     

Study of the Lipophilic Character of Xanthine and Adenosine Derivatives: II. Relationships Between Log K', RM and Log P Values

Abstract

The log k' values of a series of xanthine and adenosine derivatives were measured by means of a reversed-phase HPLC. The HPLC data were shown to be well correlated with previously reported R_M and R_MC18 values. The equations describing the relationships log k'/R_M and log k'/R_MC18 allowed the calculation of the log k' values of some compounds, which were not tested in the HPLC system. Since the relationship log k'/log P is very close to the previously described relationships R_M/log P and R_MC18/log P one can conclude that reversed-phase TLC and HPLC are very similar in describing the lipophilicity of the compounds.     

Comparison of phenyl- and octadecyl-modified silica gel as stationary phase for the prediction of n-octanol-water partition coefficients by high-performance liquid chromatography

The correlation between the retention of 29 aromatic compounds on phenyl and octadecyl silica gel with their n-octanol-water partition coefficients has been studied. The work also covered the influence of mobile phase buffers and the effect on the correlation of using k′ values extrapolated to 100% water. The results show that the retention on phenyl silica correlates significantly better than that on octadecyl silica. The error in the prediction of log P, ranging from 1 to 8, from retention data on phenyl silica at a 95% confidence limit was found to be ±0.04 log units at log P = 4, and ±0.07 log units at log P = 7 when extrapolated k′ values were used. When k′ values measured at one fixed mobile phase composition were used, the error was found to be ±0.10 and ±0.16 log units at log P = 4 and 7, respectively.     

高效液相色谱槲皮素键合硅胶固定相分离极性化合物

槲皮素是一种植物体中含量丰富、价格较便宜的黄酮类化合物,本研究以γ-[(2,3)-环氧丙氧]丙基三甲氧基硅烷(KH-560)为偶联剂,将其化学键合到硅胶上,得到一种含天然配体的槲皮素键合硅胶固定相(QUSP)。 采用红外光谱、热重分析、元素分析及固体核磁对其结构进行表征,测得硅胶表面槲皮素的键合量为0.139 mmol/g。 采用不同结构的溶质作探针,在评价固定相反相液相色谱疏水作用性能的基础上,侧重研究新固定相对极性芳香族化合物的分离能力,探讨了新固定相的色谱分离机理。 研究表明,仅采用甲醇或乙腈-水简单流动相,无需用缓冲液精确控制pH值,QUSP就能分别实现吡啶类、芳胺类、苯酚类、苯甲酸类和黄酮类等极性化合物的快速基线分离。 QUSP键合的槲皮素除含疏水性的C6-C3-C6骨架外,黄酮环还能为溶质提供氢键、偶极、π-π、电荷转移等多种作用位点,各种协同作用有利于提高色谱分离选择性,尤其对极性的可离子化的酸性和碱性化合物。     

Thermodynamics of sorption of 1,3,4-oxadiazoles and 1,2,4,5-tetrazines according to reversed phase high-performance liquid chromatography

The thermodynamics of sorption of several 1,3,4-oxadiazoles and 1,2,4,5-tetrazines on octadecyl silica gel from aqueous acetonitrile solutions of different compositions has been studied. The thermodynamic characteristics of the chromatographic systems under study were calculated. The dependences of the standard molar change in the entropy term of the dependence lnk-1/T and the enthalpy after the transfer of the sorbate from bulk solution to the surface layer on the content of the organic component in the eluent were analyzed for the compounds. It was shown that the structure of sorbate molecules affected the thermodynamic characteristics of their quasichemical sorption and solvation. Tendencies of compensation were found for sorption and solvation of the heterocyclic compounds under study.     

Thermal decomposition of immobilized compounds for the generation of gaseous standard mixtures containing ammonia and amines

The thermal decomposition of phthalamic acid derivatives chemically bonded to the surface of silica gel was examined and utilized for the generation of single-component gaseous standard mixtures of ammonia, methylamine, diethylamine and triethylamine. The conditions of the thermal decomposition (temperature, time, diluent gas flow rate) were optimized to ensure complete liberation and rapid elution of the compounds from the bed of modified silica gel. The total amounts of these four compounds that can be released from unit mass of the modified silica gel are in the range of several mg.     

Influence of Physicochemical Parameters of Some Ring-Substituted Phenol Derivatives on Their Retention on a Porous Graphitized Carbon Column

Abstract

The retention characteristics of 29 phenol derivatives were determined on a porous graphitized carbon column in unbuffered acetonitrile—water and methanol—water eluent mixtures at various organic phase concentrations. Each phenol derivative showed symmetric peaks in each eluent without buffers. Good linear correlations were found between the log k' value and the organic mobile phase concentration in the eluent. Principal component analysis indicated that methanol and acetonitrile expose different selectivities. Stepwise regression analysis proved that the retention of ring—substituted phenol derivatives is mainly governed by the sterical parameters, electron-withdrawing power and hydrogen donor capacity of substituents. According to the results of Free-Wilson analysis, the substituents with large steric parameters, strong electron-withdrawing power and hydrogen donor capacity have the highest impact on the retention. The lipophilicity of phenol derivatives did not affect significantly the retention, although the eluents were typical reversed-phase eluents.     

RP-HPLC Retention Data of New 2-Amino-2-Oxazolines. An Approach of Their Lipophilic Properties

Abstract

A RP-HPLC procedure has been developed for measuring the capacity factor k' of a series of new 5-substituted-2-amino-2-oxazolines. the chromatographic behaviour measured on a μBondapack C₁₈ column with methanol-aqueous buffer as mobile phase was related to the volume fraction of methanol φ. the log k' value extrapolated to 0% organic modifier in the eluent (log k'_w) was chosen as a measure of the solute lipophilicity. A good correlation was found between the slope S and the intercept value (log k'_w) of the log k' versus φ straight lines for all these structurally related 2-amino-2-oxazolines.

Some parameters related either to chromatographic conditions or to chemical structure were shown to influence the capacity factor. A study made at different pH values indicated that log k'_w increased with the basicity of the mobile phase.

The influence of the eluent pH on the capacity factor of 2-amino-2-oxazolines was related to their pKa's values determined by a potentiometric method.

Since log k' is considered as a valuable indice of the lipophilicity, the determined values will be used for quantitative structure-activity relationship studies of these new class of structurally related compounds     

The study of the lipophilicity of alpha-(4-phenylpiperazin-1-yl)-gamma-phthalimidobutyramides using chromatographic and computational methods

The lipophilicity of the series of alpha-(4-phenylpiperazin-1-yl)-gamma-phthalimido-butyramides (1-8) has been investigated. Several methods, like reversed-phase thin-layer chromatography and high-performance liquid chromatography using reversed-phase RP18 and IAM.DD2 columns, were applied to determine RMO, log k0 and log k(0IAM) factors. The RP-TLC investigations were performed in mixtures of acetone-water and acetonitrile-water. For RP-HPLC method mixtures of acetonitrile, water and 0.01% TFA were used as the mobile phases while for IAM.DD2 investigations mixtures of acetonitrile and water were applied. The partition coefficients of compounds (1-8) were also calculated with the Pallas and CAChe programs. All the obtained data, both from experimental methods and computational calculations, were compared and a suitable conclusion was reached.     

Liquid chromatography on unmodified and modified Spheron gels III. Phenolic compounds

Summary Chromatographic behaviour of phenolic compounds is studied on the ethyleneglycol methacrylate gel Spheron and on ion-exchangers produced by the chemical modification of this gels (cation exchanger Spheron S and anion exchanger Spheron DEAE) as compared to octadecyl silica. The hydrophobic effects obviously predominate in the retention mechanism on Spherons in aqueous methanolic mobile phases, but a selectivity differring from the behaviour on octadecyl silica was found for a number of phenolic compounds This is due to interactions with the functional groups in the unmodified and modified Spheron materials and may be utilized for the separation of phenols by liquid chromatography.     

Reversed-phase HPLC separation of Δ;2 and Δ;3 isomers of 7-ADCA and cephalexin monohydrate

Summary High-performance liquid chromatography was applied for the separation and determination of the Δ² and Δ³ isomers of 7-aminodeacetoxycephalosporanic acid (7·ADCA) and cephalexin monohydrate in their mixtures. Separation was performed on a column containing bonded octadecyl silica phase. The effects of pH (in the range of 3.0–7.6) on the investigated compounds were studied. Two organic modifiers, acetonitrile and methanol, were used to improve and accelerate separation. The applied procedure was very reliable for quantitative determination. Excellent correlation with the data of microbiological assay was found. The procedure was very convenient even when other impurities were present in the sample. This is an advantage with respect to microbiological assays.     

Preparation of 4‐butylaniline‐bonded silica gel for the solid‐phase extraction of flavone glycosides

To extract flavone glycosides efficiently, a new extraction material based on 4‐butylaniline‐bonded silica gel was prepared using a two‐step grafting method including a ring‐opening reaction and synchronous hydrolysis. Preparation of the silica‐based material was easily achieved under mild conditions, and the material was characterized by Fourier transform infrared spectroscopy, elemental analysis, and scanning electron microscopy. The material was used in solid‐phase extraction, and the extraction can be performed in neutral conditions without regard to ionic strength. Selectivity tests of 14 compounds on the extraction cartridge showed that the material has a high affinity to flavone glycosides in contrast to octadecyl silica, and the extraction yields for four flavone glycosides were found to be >93%. Selectivity tests further reveal that the adsorption on its surface is likely attributed to multiple interactions, including hydrophobic interactions, π–π interactions, and hydrogen bonding. To explore the applicability of 4‐butylaniline‐bonded silica gel, naringin and hesperidin from Simotang oral liquid were extracted, and the extraction yields were >90%, which is distinguished from <28% on octadecyl silica cartridge.     

HPLC retention behavior on hydride-based stationary phases

Two stationary phases attached to a silica hydride surface, cholesterol and bidentate C18, are investigated with a number of pharmaceutically related compounds in order to illustrate the various retention mechanisms that are possible for these bonded materials. The test solutes range from hydrophilic to hydrophobic based on log P (octanol/water partition coefficient) and pKa values. The mobile phases consist of acidified (formic and perchloric acid) water/methanol or water/ACN mixtures. Of particular interest are the high organic content mobile phase compositions where the retention would increase if the bonded material was operating in the aqueous normal phase (ANP) mode. Plots of retention factor (k) versus mobile phase composition are used to elucidate the retention mechanism. A number of examples are presented where solutes are retained based on RP, ANP, or dual retention mechanisms. The silica hydride-based stationary phases can also retain compounds in the organic normal phase.     

大黄蒽醌衍生物在杯[8]芳烃键合固定相上色谱行为的研究

研究了药用掌叶大黄中5种蒽醌衍生物在对-叔丁基杯[8]芳烃硅胶键合固定相上的高效液相色谱行为,并与ODS固定相进行了比较。研究发现这类化合物与杯[8]芳烃固定相之间存在多种相互作用,除疏水作用外,分离过程中还存在与ODS不同的色谱分离机制。杯芳烃键合相与溶质之间的氢键作用、包容络合作用改变了杯芳烃固定相对它们的选择性。