Wheat digalactosyldiacylglycerol molecular species profiling using porous graphitic carbon stationary phase

The potential of porous graphitic carbon stationary phase (PGC) was assessed for the separation of molecular species of digalactosyldiacylglycerol (DGDG). Detection was by an evaporative light scattering detector (ELSD). A conventional optimization strategy allowed definition of a quaternary non-aqueous mobile phase and separation of 9 wheat DGDG molecular species with isocratic elution: methanol/toluene/tetrahydrofuran/chloroform 64.3/21.5/13.7/0.5 v/v with 0.1% of triethylamine and a stoichiometric amount of formic acid. The molecular species were identified by LC/MS. The chromatographic behavior of DGDG on PGC was then compared to previous studies. The addition of a carbon double bond on the alkyl chain decreased the retention. This contribution was less important when the number of unsaturations increased in the alkyl chain. The consequence of this retention behavior with PGC was an elution order of molecular species which did not agree with the partition number as observed with C18 grafted stationary phases.     

The pH Influence on the HPLC-Retention of Chemotherapeutic Fluoroquinolone Derivatives

Abstract

The gyrase inhibitor fluoroquinolone derivatives were investigated by RP-HPLC methods in C₁₈/methanol-phosphate buffer system. Considering the amphoteric feature of the substances it was reasonable to study the relationship between their chromatographic behaviour vs. pH of the mobil phase. The formation of k¹ values was characterized by a maximum curve, and the retention maxima could be found close to or at the pH values of isoelectric point of the compounds. This may be explained by the presence of the zwitter ionic or the unionized form of the derivatives at this pH value.

By addition of ion-pairing (IP) agent (cetrimide or hexanesulfonic acid) in different concentration to the eluent a definite k' value increase (formation of a characteristic saturation type curve) was observed indicating an ion-pairing mechanism.

In case of IPHPLC significant correlation was found between the octanol-water partition coefficients measured at the isoelectric point or at pH 7. 4, and the retention values of the compounds.     

A systematic study of the desorption of different ion-pairing reagents on reversed phase thin-layers

Summary The absolute concentrations and the concentration distribution of different cationic ion-pairing reagents, such as tetramethylammonium bromide, tetrabutylammonium bromide, cetyltrimethylammonium bromide and trioctylmethylammonium chloride were studied after chromatographic development on physically and chemically bonded reversed-phase layers. The combination of the chromatographic conditions involved three variations. Untreated layers were chromatographed with mobile phases containing the ion-pairing reagent. Several layers were treated with the ion-pairing reagent prior to the chromatographic run, and methanol-water mixtures were used as mobile phase. In a third set of experiments both the layer and the mobile phase contained the ion-pairing reagent. The chromatographic behaviour of acid, neutral and basic model compounds were also studied. For the determination of different reagents remaining on the layer after the chromatographic run, spectrophotometric, potentiometric methods and capillary electrophoresis were used. The performance of the analytical methods was evaluated. Results obtained for the absolute concentrations and distribution of the reagents and the retention data were compared to those obtained on silica gel layers. A discussion of the retention mechanism is given. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

High-performance liquid chromatography of some basic drugs on a n-octadecylphosphonic acid modified magnesia-zirconia stationary phase

The high-performance liquid chromatographic behavior of some basic drugs was studied on a n-octadecylphosphonic acid modified magnesia-zirconia (C18PZM) stationary phase. The effect of mobile phase variables such as methanol content, ionic strength, and pH on their chromatographic behavior was investigated. The retention mechanism of basic drugs on the stationary phase was elucidated. The results indicate that both hydrophobic and cation-exchange interactions contribute to solute retention under most chromatographic conditions. The inherent Br?nsted-acid sites and also the adsorbed Lewis base anionic buffer constituents on accessible ZM surface Lewis acid sites play a role in the retention of ionized solutes by cation-exchange interaction. However, especially at high mobile phase pH, the retention of basic drugs depends mainly on hydrophobic interactions between solutes and support. Separations of the basic drugs on the C18PZM phase by a predominantly reversed-phase retention mode were very promising. The mixed-mode retention feature on this phase, as a result of the adsorbed Lewis base anionic buffer constituents acting as sites for cation-exchange, could also be very useful, e.g. for enhancing the chromatographic selectivity of such analytes. The C18PZM seems to be an excellent alternative to silica-based reversed-phase stationary phase for the separation of strongly basic solutes.     

High-performance liquid chromatographic determination of sotalol in plasma. I. Application to the disposition of sotalol enantiomers in humans.

Two high-performance liquid chromatographic analytical methods have been developed for the measurement of dl-sotalol or d-sotalol and l-sotalol in plasma, using dl-atenolol as internal standard. Quantitation of dl-sotalol was carried out, following solid-phase extraction, on a 5-microns C18 reversed-phase column, with a mobile phase containing acetonitrile, ion-pairing reagent and distilled water, using ultraviolet detection at 235 nm. Quantitation of d-sotalol and l-sotalol was based on derivatisation with the chiral agent S-(-)-alpha-methylbenzyl isocyanate, followed by chromatographic separation on a 3-microns C18 reversed-phase column, with a mobile phase containing methanol, glacial acetic acid and distilled water, with fluorimetric detection at 220 nm excitation and 300 nm emission. A preliminary application of the latter method suggests that the disposition of sotalol in humans is not enantioselective.     

High-performance liquid chromatography of sulfonamides and quinolones on p-tert-butyl-calix[6]arene-bonded silica gel stationary phase

The high-performance liquid chromatographic behavior of some sulfonamides and quinolones was studied on a p-tert-butyl-calix[6]arene-bonded silica gel stationary phase. The effect of mobile phase variables such as methanol content, ionic strength and pH on their chromatographic behavior was investigated. The retention behavior of sulfonamides on the stationary phase was compared with that on both Zorbax C₁₈-bonded silica gel and γ-(ethylenediamino)propyltriethoxylsilane-bonded silica gel (diamino-bonded phase). The retention mechanism of sulfonamides and quinolones on the stationary phase was also discussed. The results indicate that the stationary phase behaves as a reversed-phase packing and its separation selectivity is much better than that of not only Zorbax C₁₈ phase but also diamino-bonded phase. Some sulfonamides and quinolones were separated on the stationary phase, but the separation of sulfonamides is far more successful.     

Regeneration of tetrabutylammonium ion-pairing reagent distribution in a gradient elution of reversed phase ion-pair chromatography

The regeneration of ion-pairing reagent distribution on liquid chromatography columns after gradient elution has been well recognized as the cause for long column equilibration time, a major drawback associated with gradient elution reverse phase ion-pair chromatography. To date, the majority of studies have focused on optimizing the separation conditions to shorten the equilibration time. There is limited understanding of the ion-pairing reagent distribution process between the mobile phase and stationary phase in the course of gradient elution, and subsequent column re-equilibration. The focus of this work is to gain a better understanding of this process. An ion-pair chromatographic system, equipped with a YMC ODS C(18) column and a mobile phase containing tetrabutylammonium (TBA) hydroxide as the ion-pairing reagent, was used in the study. The TBA distribution profile was established by measuring its concentration in the eluent fractions collected during the gradient cycle using different column equilibration times with an ion chromatographic method. Furthermore, the analyte retention time was evaluated as the function of the column equilibration time and TBA concentration in the mobile phase. The column equilibration and its impact on the method robustness will also be discussed.     

Studies on the chromatographic behavior of nucleosides and bases on p-tert-butyl-calix[8]arene-bonded silica gel stationary phase by HPLC

The chromatographic behavior of some nucleosides, pyrimidines and purines on a new p-tert-butyl-calix[8]arene-bonded silica gel stationary phase (CABS) were studied by high performance liquid chromatography. Their retention behavior on CABS were compared with those on ODS. The influence of mobile phase variables, such as methanol content, pH and ionic strength on the retention behavior were studied. Some nucleosides, pyrimidines and purines on CABS were successfully separated. The results show that the calix[8]arene-bonded phase exhibits high selectivities for the above analytes in high aqueous mobile phases. According to the chromatographic data, it is indicated that hydrophobic interaction, hydrogen-bonding interaction, and dipole-dipole interaction are mainly responsible for the retention behavior. In addition, in some extent, the vertical stacking action of the analytes on CABS can also change the retention behavior. CABS was superior to ODS in the routine fast separation of nucleosides and bases.     

A simple model describing the retention behavior of octreotide and its glycosylated derivatives in reversed phase HPLC

Summary The retention behavior of octreotide, a somatostatin analogue, and its glycosylated derivatives containing different numbers of glucose units has been studied by reversed phase HPLC. A retention model was developed by correlating the logarithm of the retention factor with the hydrophilic-lipophilic balance of the analyte. Linear functions could be derived for all the separation systems investigated. The slopes of the straight lines were a measure of the selectivity of the chromatographic system and enabled calculation of increments for the saccharide groups in different eluent systems. The highest increment was found using trifluoroacetic acid (TFA) as ion pairing agent. The model was extended to substitution of the same peptide with hydrophobic groups such as acetyl and alkyl. Straight lines were again obtained. The influence of the different eluent systems upon peak shape and retention is also discussed. Owing to the strong peak tailing a dual retention mechanism consisting of hydrophobic and silanophilic interactions was assumed. It was shown that addition of quaternary ammonium compounds to mask the surface silanols of the stationary phase reduced both the peak tailing and the retention of the peptides.     

High-performance liquid chromatographic analysis of bile acids in hamster bile

A high-performance liquid chromatographic procedure has been developed for the determination of pentamidine concentrations in serum samples. A microbore, reversed-phase column was used with a mobile phase consisting of methanol and water with sodium heptanesulfonate and triethylamine as modifiers. Pentamidine could be extracted from serum only by the addition of an ion-pairing agent, di(2-ethylhexyl) phosphoric acid, to the chloroform used for extraction. The method can be used to reliably detect levels as low as 5 ng/ml. The pentamidine concentration in the serum of eleven patients 24 h after their tenth daily dose of pentamidine averaged 60 +/- 34 ng/ml.     

High-Performance Liquid Chromatography of some Bases and Nucleosides on Alkylphosphonate-Modified Magnesia-Zirconia Column

ABSTRACT

The high-performance liquid chromatographic behavior of some bases and nucleosides was studied on a new reversed-phase stationary phase, alkylphosphonate-modified magnesia-zirconia. The effect of mobile phase variable such as methanol content, ionic strength and pH on their behavior was investigated. It was found that the retention behavior of the bases and nucleosides on the new stationary phase is similar to that on ODS stationary phase. The retention mechanism on the new stationary phase was also discussed. The separation of some bases and nucleosides was accomplished on the new stationary phase.     

Enhancement of selectivity in reversed-phase liquid chromatography

In an effort to gain insight into the relationship between stationary phase solvation and selectivity, the use of short- and medium-chained-length alcohols (methanol, n-propanol, n-butanol, and n-pentanol) as mobile phase modifiers in reversed-phase liquid chromatography (RPLC) was investigated to determine their impact on chromatographic selectivity. A wide range of mobile phase compositions was evaluated because of the large effect exerted by solvent strength on selectivity. Employing a set of six vanillin compounds as retention probes, evidence is presented to support the view that an increase in the hydrophobicity of the organic modifier used in RPLC can increase the selectivity of the C18 alkyl bonded phase while simultaneously decreasing the retention time of the eluting solutes. Thus, we are presented with an interesting paradox: higher selectivity and shorter retention times, which can be attributed to changes in either solvent selectivity and/or stationary phase solvation by the organic modifier.     

An ion-pairing liquid chromatography/tandem mass spectrometric method for the determination of cytarabine in mouse plasma

An ion-pairing high-performance liquid chromatographic (IP-HPLC) system interfaced with an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) with minimal sample preparation was developed for the determination of cytarabine (ara-C), a very hydrophilic anticancer drug, in mouse plasma. A conventional reversed-phase chromatographic column in combination with two ion-pairing reagents was adapted for retention and separation of ara-C from the endogenous interferences in mouse plasma. The effects of the experimental conditions such as the fraction of ion-pairing reagents and organic solvents in the mobile phase on the chromatographic performance and the ionization efficiency of ara-C were investigated. The potential of ionization suppression resulting from the endogenous biological materials on the IP-HPLC/MS/MS method was evaluated using the post-column infusion technique. Furthermore, the feasibility of the proposed IP-HPLC/MS/MS procedure for analysis of ara-C in the mouse plasma was demonstrated by comparison with those obtained by the porous graphite carbon column (PGC) HPLC/MS/MS method.     

梯度加压毛细管电色谱分离蛋白质

 以1.5 μm无孔硅胶颗粒(non-porous silica,NPS)为固定相,采用电压和压力联合驱动流动相,用反相梯度加压毛细管电色谱(p-CEC)在7.5 min内实现了核糖核酸酶A、细胞色素C、溶菌酶和肌红蛋白等4种蛋白质的快速、高效的分离。比较了梯度加压毛细管电色谱和微柱液相色谱(μ-HPLC)分离蛋白质的结果,同时考察了固定相、离子对试剂三氟醋酸(TFA)浓度和电压等条件对梯度加压毛细管电色谱分离蛋白质的影响。结果表明,梯度p-CEC可以通过调节电压精细调节带电溶质的保留,提高分离选择性,缩短分离时间,得到较高的柱效。该方法在蛋白质分离分析及蛋白质组学的研究中具有很大的应用潜力，为高效快速地分离蛋白质开辟了新的途径。     

Combined effects of mobile phase composition and temperature on the retention of homologous and polar test compounds on polydentate C8 column

Combined effects of temperature and mobile phase on the reversed phase chromatographic behavior of alkylbenzenes and simple substituted benzenes were investigated on a Blaze C₈ polydentate silica-based column, showing improved resistance against hydrolytic breakdown at temperatures higher than 60 °C, in comparison to silica-based stationary phases with single attachment sites. For better insight into the retention mechanism on polydentate columns, we determined the enthalpy and entropy of the transfer of the test compounds from the mobile to the stationary phase. The enthalpic contribution dominated the retention at 80% or lower concentrations of methanol in the mobile phase. Entropic effects are more significant in 90% methanol and in acetonitrile–water mobile phases. Anomalies in the effects of mobile phase on the enthalpy of retention of benzene, methylbenzene and polar benzene derivatives were observed, in comparison to regular change in enthalpy and entropy of adsorption with changing concentration of organic solvent and the alkyl length for higher alkylbenzenes. The temperature and the mobile phase effects on the retention are practically independent of each other and – to first approximation – can be described by a simple model equation, which can be used for optimization of separation conditions.     

Chromatographic Behavior of Aromatic Diamines in Hydro-Organic, Micellar and Submicellar Reversed Phase Liquid Chromatographic Modes

The chromatographic behavior (retention, elution strength, efficiency, peak asymmetry and selectivity) of some aromatic diamines in the presence of methanol with or without anionic surfactant SDS in the four different reversed phased liquid chromatographic (RPLC) modes, i.e., hydro-organic, micellar (MLC), low submicellar (LSC) and high submicellar (HSC), was investigated. In the three surfactant-mediated modes, the surfactant monomers coat the stationary phase even up to 70 % methanol; this results in the suppression of peak tailing (by masking the silanol groups on the stationary phase). In MLC and HSC, the solute retention decreases by increasing the surfactant concentration, while this situation was reversed in LSC. In the region between MLC and HSC modes (25–50 % methanol), retention of late eluting solutes was increased by increasing methanol content which is seemingly due to disaggregation of SDS micelles. Changes in selectivity were observed after changing the concentrations of SDS and methanol, in a greater extent when concentration of SDS was changed. Among the four studied RPLC modes, HSC showed the best efficiency with nearly symmetrical peaks. Prediction of retention of solutes in HSC based on a mechanistic retention model combined with Pareto-optimality method allowed the full resolution of target diamines in practical analysis times.     

液相色谱中的一个新的表征参数Z Ⅲ.反相高效液相色谱中的生物大分子分离体系

研究了在反相高效液相色谱中的烷基醇-甲酸-水、烷基醇-三氟醋酸-水和烷基醇-磷酸盐-水三种流动相,9种生物大分子的分离体系中Z的表征。发现当蛋白质完全变性时,Z值与其分子量呈正比,而与置换剂分子大小呈反比。Z值会因流动相中离子对试剂的存在而减小,且浓度愈大,减小程度愈甚。对于在异丙醇-水溶液中的不同种类离子对试剂而言,对蛋白构象变性影响的减小顺序为44％(V/V)甲酸>0.05mol/L磷酸盐缓冲液(pH<2.5)>0.1％(V/V)三氟醋酸。     

反相离子对色谱-电感耦合等离子体质谱联用技术测定水中痕量Cr(Ⅲ)与Cr(Ⅵ)

建立了反相离子对色谱(RPIPC)与电感耦合等离子体质谱(ICP-MS)联用技术快速分离测定水中痕量Cr(Ⅲ)和Cr(Ⅵ)的方法.通过考察流动相的pH值、离子对试剂及甲醇的浓度和EDTA的添加等对不同形态铬的保留时间及分离度的影响,确定当流动相组成为2.0 mmol/L TBA,5%(V/V)甲醇,pH=5.5时,Cr(Ⅲ)与Cr(Ⅵ)可达最佳分离.ICP-MS测定时选用碰撞池技术以消除40Ar12C+与35Cl16OH+对52Cr+的谱学干扰;进样100 μL时,Cr(Ⅲ)与Cr(Ⅵ)的检出限分别为0.15 μg/L和0.16 μg/L.加标回收率在93.6%～106.2%之间; RSD<4%(n=3).以本方法分析了某市自来水、雨水及某品牌纯净水中Cr(Ⅲ)与Cr(Ⅵ)的含量,结果令人满意.     

1-萘甲膦酸改性氧化锆固定相分离芳胺类化合物的色谱性能研究

研究了一些芳胺类化合物在1-萘甲膦酸改性氧化锆固定相上的色谱行为。分别考察了流动相中甲醇含量、缓冲液pH值和离子强度等对芳胺类化合物色谱保留的影响,并对这类化合物在该固定相上的保留机理进行了探讨。研究结果表明,芳胺类化合物在该固定相上表现出反相和阳离子交换的混合保留模式。以pH 10.1的Tris-甲醇(60/40,V/V)溶液为流动相,在1-萘甲膦酸改性氧化锆固定相上成功分离了间苯二胺、邻甲苯胺、N-甲苯胺、对硝基苯胺、邻硝基苯胺和α-甲萘胺6种芳胺类化合物。