HPLC-Isocratic Study of the Separation and Determination of Phenolphthalein and Its Metabolite, Phenolphthalein-Glucuronic Acid, as Markers of Enterohepatic Circulation

An isocratic high-performance liquid chromatographic method for separation and determination of phenolphthalein and its metabolite, phenolphthalein-glucuronide, using bromocresol purple as an internal standard is described. The method uses a mobile phase of 50 mM phosphate buffer (pH 7.7)-methanol (52.5:47.5, v/v), a 3-μm reversed-phase C₁₈ column (50 × 4.6-mm i.d.), a flow rate of 1 ml/min, and UV detector wavelength of 230 nm. The most important variables that can affect the retention of these compounds (i.e., organic modifier concentration, buffer concentration, and pH) were systematically studied. Two different retention orders were observed, depending on buffer concentration and pH. The effects on retention of the addition of triethylamine or acetic acid to the mobile phase are also discussed. This method has been developed for future application to the determination of phenolphthalein and phenolphthalein-glucuronide in biological fluids such as plasma, bile, and urine of rats within a study involving a new model for enterohepatic recirculation and pharmacokinetics.     

Retention of ionisable compounds on high-performance liquid chromatography XVIII: pH variation in mobile phases containing formic acid,piperazine, tris,boric acid or carbonate as buffering systems and acetonitrile as organic modifier

In the present work dissociation constants of commonly used buffering species, formic acid, piperazine, tris(hydroxymethyl)–aminomethane, boric acid and carbonate, have been determined for several acetonitrile–water mixtures. From these pK_a values a previous model has been successfully evaluated to estimate pH values in acetonitrile–aqueous buffer mobile phases from the aqueous pH and concentration of the above mentioned buffers up to 60% of acetonitrile, and aqueous buffer concentrations between 0.005 (0.001 mol L⁻¹ for formic acid–formate) and 0.1 mol L⁻¹. The relationships derived for the presently studied buffers, together with those established for previously considered buffering systems, allow a general prediction of the pH variation of the most commonly used HPLC buffers when the composition of the acetonitrile–water mobile phase changes during the chromatographic process, such as in gradient elution. Thus, they are an interesting tool that can be easily implemented in general retention models to predict retention of acid–base analytes and optimize chromatographic separations.     

Effect of Buffer Constituents on Retention and Separation in Achiral and Chiral HPLC Systems with β-Cyclodextrin-Based Stationary Phase

In HPLC, cyclodextrin-based stationary phases allow separation of both achiral and chiral analytes. Complexation of analytes into the cyclodextrin cavity is a very important part of the interaction mechanism. However, possible complexation of buffer constituents must be also considered. This fact should not be overlooked in method development procedures. In this study, β-cyclodextrin-based stationary phase (CYCLOBOND I 2000) was used, and two different buffers, namely aqueous solutions composed of lithium hydroxide/benzoic acid 10.0/24.5 mM, pH 4.00 and lithium hydroxide/acetic acid 9.9/60.0 mM, pH 4.01 of the same ionic strength were compared. Methanol was added as organic modifier in a methanol/buffer volume ratio 40/60. The linear free energy relationship method was used for evaluation of interactions contributing to chromatographic performance of the separation system. Strong complexation of benzoic acid with cyclodextrin was obvious from the results. This effect was clearly reflected in reduced retention and restricted (enantio) separation ability of the separation system containing benzoic acid in the buffer. On the other hand, complexing buffer constituents of mobile phases can offer a worthy alternative, to less “green”organic solvents, for reduction of the analysis time.

     

Mixed-mode reversed-phase and ion-exchange separations of cationic analytes on polybutadiene-coated zirconia

The retention and selectivity of the chromatographic separation of basic (cationic) analytes on a polybutadiene-coated zirconia (PBD-ZrO2) stationary phase have been studied in greater detail than in previous studies. These separations are strongly influenced by the chemistry of the accessible surface of zirconia. In the presence of buffers which contain hard Lewis bases (e.g., phosphate, fluoride, carboxylic acids) zirconia's surface becomes negatively charged due to adsorption of the buffer anion at the hard Lewis acid sites. Consequently, under most conditions (e.g., neutral pH), cationic analytes undergo both hydrophobic and cation-exchange interactions. This mixed-mode retention process generally leads to greater retention factors for cations relative to those on silica-based reversed phases despite the lower surface areas of the zirconia phase, but, more importantly, adsorption of hard Lewis bases can be used to control the chromatographic selectivity for cationic analytes on these zirconia-based stationary phases. In contrast to our prior work, here we show that when mixed-mode retention takes place, both retention and selectivity are easily adjusted by changing the type of hard Lewis base buffer anion, the type of buffer counter-ion (e.g., sodium, potassium, ammonium), the pH, and the ionic strength of the eluent as well as the type and amount of organic modifier.     

Comparison of peak shapes obtained with volatile (mass spectrometry-compatible) buffers and conventional buffers in reversed-phase high-performance liquid chromatography of bases on particulate and monolithic columns

Retention factor, column efficiency and asymmetry factor were recorded for nine basic compounds on a number of RP-HPLC columns using phosphate and a variety of (MS-compatible) volatile mobile phase buffers of acid and neutral pH, in order to assess any effects of the buffer on performance. With formic or acetic acid, some phases gave partial or complete solute exclusion effects (reduced or negative k) compared with results using phosphate buffers at low pH. Despite its possible suppression of mass spectrometer sensitivity, trifluoroacetic acid was useful in enhancing retention times of relatively hydrophilic protonated bases, due to ion-pair effects. Peak shape was relatively poor on some pure silica-based ODS phases at pH 7 compared with results at acid pH. At low pH and at pH 7, ammonium and potassium phosphate gave very similar k, but the former may be preferable due to its volatile cation. Improved peak shapes, attributed to superior silanol masking effects, were obtained with ammonium phosphate at pH 7, but not at acid pH. Ammonium acetate gave acceptable peak shape at pH 7, but due to very limited buffer capacity, poor results were obtained for solutes having a pKa close to the mobile phase pH. Due to its instability, ammonium hydrogen carbonate is not a viable alternative buffer at pH 7.     

Retention of ionisable compounds on high-performance liquid chromatography XVII. Estimation of the pH variation of aqueous buffers with the change of the methanol fraction of the mobile phase

The use of methanol-aqueous buffer mobile phases in HPLC is a common election when performing chromatographic separations of ionisable analytes. The addition of methanol to the aqueous buffer to prepare such a mobile phase changes the buffer capacity and the pH of the solution. In the present work, the variation of these buffer properties is studied for acetic acid-acetate, phosphoric acid-dihydrogenphosphate-hydrogenphosphate, citric acid-dihydrogencitrate-hydrogencitrate-citrate, and ammonium-ammonia buffers. It is well established that the pH change of the buffers depends on the initial concentration and aqueous pH of the buffer, on the percentage of methanol added, and on the particular buffer used. The proposed equations allow the pH estimation of methanol-water buffered mobile phases up to 80% in volume of organic modifier from initial aqueous buffer pH and buffer concentration (before adding methanol) between 0.001 and 0.01 mol L(-1). From both the estimated pH values of the mobile phase and the estimated pKa of the ionisable analytes, it is possible to predict the degree of ionisation of the analytes and therefore, the interpretation of acid-base analytes behaviour in a particular methanol-water buffered mobile phase.     

Retention of ionisable compounds on high-performance liquid chromatography: XIX. pH variation in mobile phases containing formic acid,piperazine and tris as buffering systems and methanol as organic modifier

In previous works a model to estimate the pH of methanol–aqueous buffer mobile phases from the aqueous pH and concentration of the buffer and the fraction of organic modifier was developed. This model was successfully applied and validated for buffers prepared from ammonia, acetic, phosphoric and citric acids. In the present communication this model has been extended to formic acid, piperazine and tris(hydroxymethyl)aminomethane buffers. Prior to the modelling work, the pK_a values of the studied buffers at several methanol–water compositions were determined.     

Ion‐exchange vs reversed‐phase chromatography for separation and determination of basic psychotropic drugs

Ion exchange chromatography, an alternative to reversed‐phase (RP) chromatography, is described in this paper. We aimed to obtain optimal conditions for the separation of basic drugs because silica‐based RP stationary phases show silanol effect and make the analysis of basic analytes hardly possible. The retention, separation selectivity, symmetry of peaks and system efficiency were examined in different eluent systems containing different types of buffers at acidic pH and with the addition of organic modifiers: methanol and acetonitrile. The obtained results reveal a large influence of the salt cation used for buffer preparation and the type of organic modifier on the retention behavior of the analytes. These results were also compared with those obtained on an XBridge C₁₈ column. The obtained results demonstrated that SCX stationary phases can be successfully used as alternatives to C₁₈ stationary phases in the separation of basic compounds. The most selective and efficient chromatographic systems were applied for the quantification of some psychotropic drugs in fortified human serum samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Chromatographic evaluation using basic solutes of the silanol activity of stationary phases based on poly(methyloctylsiloxane) immobilized onto silica

The chromatographic behaviors of some basic solutes were evaluated on stationary phases based on poly(methyloctylsiloxane) immobilized onto silica (PMOS-SiO(2)). The test solutes present both hydrophobic and hydrophilic properties. Evaluations of the pH effect used 80:20 v/v methanol/buffered mobile phase over the pH range of 5-11.5 with inorganic buffers such as borate, carbonate and phosphate and with organic buffers such as citrate, tricine and triethylamine. Evaluations in acidic mobile phases used 50:50 v/v and 30:70 v/v methanol/buffer (pH 2.5; 20 mmol/L) mobile phases. The buffer concentration effect used 65:35 v/v methanol/phosphate (pH 7; 20 and 100 mmol/L) mobile phases. The results are compared with those obtained with two chemically bonded stationary phases. The immobilized phases show greater contributions from an ion-exchange mechanism than do the commercial phases. The results indicate that the silanol activity of PMOS-SiO(2) stationary phases can be adequately evaluated by using appropriate basic probes and mobile phases having different pH, using different buffers.     

Novel ultra stable silica-based stationary phases for reversed phase liquid chromatography--study of a hydrophobically assisted weak acid cation exchange phase

A mixed-mode reversed-phase/weak cation exchange (RP/WCX) phase has been developed by introducing a small amount of carboxylate functionality into a hydrophobic hyper-crosslinked (HC) platform. This silica-based HC platform was designed to form an extensive polystyrene network completely confined to the particle's surface. The fully connected polymer network prevents the loss of bonded phase, which leads to superior hydrolytic stability of the new phase when compared to conventional silica-based phases. Compared to previously introduced HC phases the added carboxylic groups impart a new weak cation exchange selectivity to the base hydrophobic HC platform. The phase thus prepared shows a mixed-mode retention mechanism, allowing for both neutral organic compounds and bases of a wide polarity range to be simultaneously separated on the same phase under the same conditions. In addition, the new phase offers the flexibility that gradients in organic modifier, pH or ionic competitors can be used to affect the separation of a wide range of solutes. Moreover, the inherent weak acid cation exchange groups allow formic and acetic acid buffers to be used as eluents thereby avoiding the mass spectrometric ionization suppression problems concomitant to the use of non-volatile additives such as strong amine modifiers (e.g. triethylamine) or salts (e.g. NaCl) to elute basic solutes from the strong cation exchange phase which was previously developed in this lab. The use of the new phase for achieving strong retention of rather hydrophilic neurotransmitters and drugs of abuse without the need for ion pairing agents is demonstrated.     

Study of the retention behavior of small polar molecules on different types of stationary phases used in hydrophilic interaction liquid chromatography

The retention behavior of a large group of analytes (35) with varied properties (pK_a and logP) was studied on eight hydrophilic interaction LC columns with different surfaces, stationary phase chemistries, and types of particles. The acetonitrile content (5–95%), buffer concentration (0.5–200 mM), and pH of the mobile phase (3.8 and 6.8) were evaluated for their effects on the retention behavior. The type of stationary phase had a significant impact on the selectivity and retention time of the tested analytes. Completely different selectivity was observed on the aminopropyl stationary phase. In this study, the influence of the buffer concentration was similar for all tested columns, except for the aminopropyl stationary phase. Increasing the buffer concentration led to decreased retention times for the basic compounds and increased retention times for the acidic compounds, while the inverse behavior was observed on the aminopropyl stationary phase. The selectivity of the individual stationary phases was evaluated at pH 3.8 and 6.8. Much lower selectivity differences between the stationary phases were observed at pH 6.8 than pH 3.8. Bare silica stationary phases were used in the comparison of the particles (fused‐core and fully porous particles of 3 and 1.7 μm) and the columns provided by different manufacturers.     

High performance liquid chromatographic analysis of dehydroepiandrosterone and its pharmaceutical tablet formulation

A rapid, sensitive and stability indicating high performance liquid chromatographic method was developed and validated for the analysis of dehydroepiandrosterone (DHEA) in pharmaceutical tablet formulation. The analysis was done on a Supelcosil C(18) column (25 cm x 4.6 mm i.d., 5 microm). The mobile phase consisted of methanol:sodium acetate buffer solution (5 g/L):acetic acid (500 mL/L), 57:42:1, v/v/v, adjusted to pH 5 at a flow rate of 1 mL/min. Detection was carried out at a wavelength of 258 nm. The polynomial regression data for the calibration curve showed good linear relationship in the concentration range of 0.2-1 mg/mL with r = 0.9996. The method was validated for precision, accuracy and recovery. The limit of detection was found to be 50 ng/ microL. The method was applied for the analysis of DHEA in its pharmaceutical tablet formulation. The effects of different buffers and alcohols on the retention of DHEA were studied and the role of acetic acid as an organic phase modifier was also investigated.     

Chiral separation of beta-adrenergic antagonists, profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides using teicoplanin as the chiral selector in capillary liquid chromatography

Three groups of structurally diverse chiral compounds were used to study the interaction mechanism responsible for stereoselective recognition with teicoplanin as chiral selector in capillary liquid chromatography. Teicoplanin-based chiral stationary phase (CSP) was used. The effect of the variation of mobile phase composition on retention and enantioselective separation was studied. The mobile phase composition suitable for enantioresolution of the various chiral compounds differed according to the interaction forces needed for chiral recognition. Mobile phases with high buffer portion (70-90 vol.%) were preferred for separation of enantiomers of profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides that require hydrophobic interactions, inclusion and pi-pi interactions for stereoselective recognition with teicoplanin. Higher concentration triethylamine in the buffer (0.5-1.0%) increased resolution of these acids. On the other hand, H-bonding and electrostatic interactions are important in stereoselective interaction mechanism of beta-adrenergic antagonists with teicoplanin. These interaction types predominate in the reversed phase separation mode with high organic modifier content (95% methanol) and in polar organic mobile phases. For this reason beta-adrenergic antagonists were best enantioresolved in the polar organic mode. The mobile phase composed of methanol/acetic acid/triethylamine, 100/0.01/0.01 (v/v/v), provided enantioresolution values of all the studied beta-adrenergic antagonists in the range 1.1-1.9. Addition of teicoplanin to the mobile phase, which was suitable for enantioseparation of certain compounds on the CSP, was also investigated. This system was used to dispose of nonstereoselective interactions of analytes with silica gel support that often participate in the interaction with CSPs. Very low concentration of teicoplanin in the mobile phase (0.1 mM) resulted in enantioselective separation of 2,2- and 2,4-chlorophenoxypropionic acids.     

Effect of the pH, the concentration and the nature of the buffer on the adsorption mechanism of an ionic compound in reversed-phase liquid chromatography. II. Analytical and overloaded band profiles on Symmetry-C18 and Xterra-C18

In a previous report, the influence of the pH, the concentration, and the nature of the buffer on the retention and overloading behavior of propranolol (pKa = 9.45) was studied on Kromasil-C18 at 2.75 < pH < 6.75, using four buffers (phosphate, acetate, phthalate, and succinate), at three concentrations, 6, 20, and 60 mM. The results showed that the propranolol cation was eluted as an ion-pair with the buffer counter-anion. A similar study was carried out with Symmetry-C18 and Xterra-C18. Two additional buffers, formate and citrate, were also used. Propranolol elution band profiles were recorded for a small (less than 1 microg) and a large (375 microg) sample size. The results are similar to those obtained with Kromasil and confirm earlier conclusions. The buffer concentration, not its pH, controls the retention time of propranolol, in agreement with the chaotropic model. The retention factor depends also on the nature of the buffer, particularly on its valence, and on the hydrophobicity of the basic anion. With the monovalent anions HCOO- (pH 3.75), H2PO4- (pH 2.75), HOOC-Ph-COO- (pH 2.75), HOOC-CH2-CH2-COO- (pH 4.16), CH3COO- (pH 4.75) and HOOC-CHCOOH-COO- (pH 3.14), at moderate loadings, and for the two larger buffer concentrations, the band profiles are well accounted for by a simple bi-Langmuir isotherm model (no adsorbate-adsorbate interactions). By contrast, these profiles are accounted for by a bi-Moreau isotherm model (i.e., with significant adsorbate-adsorbate interactions) with the bivalent anions -OOC-Ph-COO- (pH 4.75), -OOC-CH2-CH2-COO- (pH 5.61), HPO4(2-) (pH 6.75), and HOOC-CHCOO(-)-COO- (pH 4.77) and with the trivalent anion -OOC-CHCOO(-)-COO- (pH 6.39). The best values of the isotherm parameters were determined using the inverse method. The saturation capacity and the equilibrium constant on the low-energy sites increase with increasing buffer concentration, a result consistent with the formation in the mobile phase of a hydrophobic complex between the propranolol cation and the buffer anion. With bivalent and trivalent anions, adsorbate-adsorbate interactions are strong on the low-energy sites but they remain negligible on the high-energy sites. The density of the high energy sites is lower and the equilibrium constant on the low-energy sites are both higher with the bivalent and the trivalent buffer anions than with the univalent buffer anions. These results are consistent with the formation of a 2:1 and a 3:1 propranolol-buffer complex with the bivalent and the trivalent anions, respectively.     

The chromatographic interaction and separation of metal ions with 8-Quinolinol stationary phases in several aqueous eluents

Several 8-quinolinol silica gel (QSG) columns were used, with metal-uptake capacities of 10–156 μmol g^?1. Various transition and heavy metal ions were used as analytes in nitrate, sulfate, phosphate, citrate, tartrate, oxalate, phthalate, and maleate mobile phases. Metal-ion retention increased with column capacity and pH. Optimum capacity factors were obtained on columns of intermediate capacity (27 and 46 μmol g^?1). Retention times decreased with an increase in eluent buffer concentration, typically by half with a doubling of buffer. Evidence is presented for the occurence of mobile-phase complexation of analyte ions by eluent buffer species. Multiple or split peaks were often observed when the analyte solvent differed from the mobile phase. Chromatographic separation of up to six metals on the QSG columns is demonstrated in tartrate and maleate mobile phases.     

Polarographic studies on o-aminobenzenethiol

Polarographic behaviour of o-aminobenzenethiol (RSH) is studied at dme in 0.1M-HClO₄, 0.1M-NaOH,Britton-Robinson buffers of different pH values and aqueous acetic acid. A well defined single wave is obtained in 0.1M-HClO₄ and buffers of pH 2.0 at all concentrations of the depolarizer whereas in all the other buffers, a well defined single wave is obtained only if the concentration is less than 2×10^–4 M. In other cases, two waves are observed. With the increase in pH, theE _1/2 becomes more negative and finally attains a constant value. Thus the dissociation constant of sulphydryl group under the experimental conditions is 5.14. The diffusion coefficient in buffer of pH 2.0 is calculated to be 5.76×10^–6 usingIlkovi equation.The reaction is reversible and the oxidation product is a mercurous compound (RSHg) which converts quickly to (RS)₂Hg. Even in acetic acid media, well defined reversible anodic wave is obtained in all proportions of acetic acid and water.With 4 Figures     

Cellulase Activity in Different Buffering Media During Waste Paper Hydrolysis by HPLC

The activity of cellulase has traditionally been described by pH and temperature; however, the buffering medium is also an important factor, Taking plain water as a reference medium, three kinds of buffer including KH₂PO₄/K₅HPO₄, citric acid/sodium citrate, and acetic acid/sodium acetate were adopted to survey their effects on the activity of cellulase. Chromatographic assays indicated that xylose, glucose, and cellobiose were the major products and that minor products such as cellotriose and cellotetraose were present in some cases. The activities of cellulase based on glucose production showed that the phosphate buffer acted as a deactivator for cellulase and each of the two organic acid buffers acted as activators for cellulase. The concentration of activation buffer should be high to reach a high cellulase activity; however, this effect would be compensated for by the product inhibition of cellulase. The highest activity obtained was 4.16 ± 0.08 (× 10^?3) IU mg^?1 for the citric acid/sodium citrate buffer under pH 4.80, 40 °C and an agitation speed of 150 rpm.     

The Separation of Organic Analyte Cations on a Low-Capacity Cation Exchange Column Using Indirect UV Detection

Abstract

The retention of organic analyte cations on a low-capacity cation exchange column using indirect UV detection was studied. It was found that a combination of cation exchange/reversed-phase interactions affected the retention of organic analyte cations provided the analytes have both a cationic charge site and a hydrophobic center. The factors that influenced the organic analyte cation retention were: concentration of organic modifier, concentration of UV absorbing analyte, pH, and mobile phase ionic strength. Elution orders for several of the organic analytes studied on the low-capacity cation exchange column were different than those observed on silica-based strong cation exchange columns.     

Development and evaluation of new zwitterionic hydrophilic interaction liquid chromatography stationary phases based on 3-P,P-diphenylphosphonium-propylsulfonate

New zwitterionic stationary phases were synthesized by covalently bonding 3-P,P-diphenylphosphonium-propylsulfonate to silica gel. The resulting materials possess both a negatively charged sulfonate group and a positively charged quaternary phosphonium group, which means that there is no net charge over a wide pH range. The retention mechanism and chromatographic behavior of polar solutes under HILIC conditions were studied on these zwitterionic phases. Compared to the commercial ZIC-HILIC column and a bare silica gel stationary phase, the newly synthesized zwitterionic stationary phases provided greater retention, higher peak efficiency and better peak symmetry in the HILIC mode. The analytes examined included: β-blockers, nucleic acid bases and nucleosides, salicylic acid and its analogues, and water soluble vitamins. Factors, such as the type of organic modifiers, solvent composition, pH and the buffer concentration of the mobile phase, have been considered as potential variables for controlling the chromatographic retention of polar analytes.