Characterization of a microparticulate strong anion-exchanger in the HPLC of acidic drugs

The use of Waters Spherisorb S5SAX for the HPLC of acidic compounds, including a number of non-steroidal anti-inflammatory drugs (NSAIDs), has been investigated. Adequate retention, separation, and peak efficiency and symmetry were obtained for most analytes on a 250 x 4.6 mm i.d. column using methanol containing ammonium perchlorate (10 mmol L(-1), pH 6.7 or pH 8.3) as eluent. The results of changes in (i) eluent pH (constant ionic strength); (ii) eluent ionic strength (constant pH); and (iii) adding water to the eluent (constant pH) were consistent with a retention mechanism dominated by ion-exchange with the bonded strong anion-exchange (SAX) moieties. However, there were some unexpected observations, including (i) a general decrease in retention at eluent pH values above 7.7; (ii) a marked increase in retention on adding 1% (v/v) water to the eluent; (iii) a subsequent marked decrease in retention on adding 5% (v/v) or more water; and (iv) decreased column activity with time. These observations may be due to (i) interaction between the charged SAX moieties and ionised surface silanols (with ionization increasing at higher eluent pH values) and (ii) influence of the solvation of silanols, analytes, SAX moiety, and counter-ion varying with both pH and water content. Nevertheless, the factors influencing separation of individual NSAIDs remain unclear especially as no relation between log k and pKa exists for these compounds. Hydrophobic interactions are unlikely to be important since basic and neutral compounds were hardly retained. Ease of accessibility of the counter-ion to the SAX moiety for analyte displacement may be a factor.     

High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. I. Factors influencing retention, peak shape and detector response

The use of silica columns together with non-aqueous ionic eluents provides a stable yet flexible system for the high-performance liquid chromatographic analysis of basic drugs. At constant ionic strength, eluent pH influences retention via ionisation of surface silanols and protonation of basic analytes, pKa values indicating the pH of maximum retention. At constant pH, retention is proportional to the reciprocal of the eluent ionic strength for fully protonated analytes and quaternary ammonium compounds. The addition of water up to 10% (v/v) has little effect on retention if the protonation of the analytes is unaffected. Thus, it is likely that retention is mediated primarily via cation exchange with surface silanols. However, additional factors must play a part with compounds such as morphine which give tailing peaks at acidic or neutral eluent pHs.     

Optimisation and characterisation of silica-based reversed-phase liquid chromatographic systems for the analysis of basic pharmaceuticals

Reversed-phase liquid chromatography using silica-based columns is successfully applied in many separations. However, also some drawbacks exist, i.e. the analysis of basic compounds is often hampered by ionic interaction of the basic analytes with residual silanols present on the silica surface, which results in asymmetrical peaks and irreproducible retention. In this review, options to optimise the LC analysis of basic pharmaceutical compounds are discussed, i.e. eluent optimisation (pH, silanol blockers) and stationary phase optimisation (development of new columns with minimised ionic interactions). The applicability of empirical based, thermodynamically based and test methods based on a retention model to characterise silica-based reversed phase stationary phases, as well as the influence of the eluent composition on the LC analysis of basic substances is described. Finally, the applicability of chemometrical techniques in column classification is shown.     

The application of retention indices using the alkylarylketone scale to the separation of the barbiturates by HPLC. I. The effect of the eluent

Summary In order to determine the applicability of retention indices, based on the alkylarylketone scale, as the basis of a reproducible method of reporting retentions, the separation of ten barbiturates and a set of column test compounds were examined on an octadecylsilyl bonded silica (ODS-Hypersil) column with methanol-buffer pH 8.5 as eluent. The effects on the capacity factors and retention indices, on changing the eluent composition, pH, ionic strength and temperature, showed that the retention indices of the barbiturates were much less susceptible to minor changes in the eluent than the capacity factors. For non-ionised compounds the retention indices were virtually independent of the experimental conditions. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Retention behaviour of acidic, neutral and basic drugs on a CN column using phosphate buffers in the mobile phase

The retention behaviour of a set of sixteen more or less drugs on a CN column with methanol-phosphate buffer as eluent was studied. The influence of the volume percentage of methanol, pH and the ionic strength on the capacity factors (k') of the drugs was determined using an experimental design consisting of three pH values (3, 5 and 7), four ionic strengths (0.025, 0.05, 0.075 and 0.1) and three volume percentages of methanol (10, 30 and 50%). The selected drugs were basic, acidic and neutral compounds with different polarity properties. The number of carbon atoms, considered here as reflecting the hydrophobic characteristics of the solutes, varied from 7 to 25. The influence on the retention of drugs on a CN column of the different parameters studied was evaluated. The volume percentage of organic modifier and the pH are the most important factors. A change in ionic strength is important only when large molecules are chromatographed. As the interaction between pH, ionic strength and volume percentage of methanol is small, optimization of the three parameters separately is possible.     

离子色谱-直接电导检测法分析哌啶离子液体阳离子

建立了离子色谱-直接电导检测法分析N-甲基,乙基哌啶(［MEPi］+)、N-甲基,丙基哌啶(［MPPi］+)和N-甲基,丁基哌啶(［MBPi］+) 3种哌啶离子液体阳离子的方法。采用磺酸型阳离子交换色谱柱,以乙二胺-柠檬酸-乙腈为流动相。考察了流动相组成及色谱柱温度对哌啶阳离子保留的影响,并讨论了保留规律。结果表明,哌啶阳离子的保留是一个放热过程,即哌啶阳离子的保留时间随着色谱柱温度的升高而缩短,且哌啶阳离子同系物的保留符合碳数规律。在以0.2 mmol/L乙二胺-0.3 mmol/L柠檬酸-3%(v/v)乙腈(pH 4.4)为流动相、流速1.0 mL/min、柱温30 ℃条件下,［MEPi］+、［MPPi］+和［MBPi］+3种哌啶阳离子可以在7 min内分离,检出限(信噪比为3)分别为0.14、0.20和0.56 mg/L,峰面积的相对标准偏差(n=5)在1.2%以下。将此方法应用于分析实验室合成的哌啶离子液体样品,加标回收率在97.6%与105.1%之间。本方法准确、可靠、快速,具有较好的实用性。     

反相硅胶整体柱离子对色谱法快速测定吡啶离子液体阳离子

建立了整体柱离子对色谱-紫外检测法梯度淋洗快速分离测定4种吡啶离子液体阳离子的方法。分离采用C18反相硅胶整体柱,以离子对试剂(用柠檬酸调节pH值)-乙腈为淋洗液,并采用多级梯度洗脱程序。实验考察了色谱柱、离子对试剂、乙腈浓度、色谱柱温度及流速对吡啶阳离子保留的影响,并讨论了其保留规律。咪唑阳离子的保留符合碳数规律。最佳色谱条件是:在流速3.0 mL/min,柱温30℃下,以1.0 mmol/L庚烷磺酸钠(pH 4.0)(A)+乙腈(B)为淋洗液进行梯度洗脱。淋洗梯度为0～2.0 min,10%B;2.0～2.5 min,10%～15%B;2.5～4.0 min,15%B;4.0～4.5 min,15%～20%B;4.5～10.0 min,20%B。在此条件下,4种吡啶阳离子可在7 min内基线分离。所测阳离子的检出限(S/N=3)为0.05～0.17 mg/L;峰面积的相对标准偏差(n=5)小于0.6%。将本方法用于实验室合成的离子液体样品和污水样品的分析,加标回收率在95.7%～99.0%之间。本方法准确、快速,具有较好的实用性。     

Retention of alkali,alkaline earth and transition metals on an itaconic acid cation-exchange column. Eluent pH,ionic strength and temperature effects upon selectivity

The unusual selectivity of a methylene succinic (itaconic) acid modified polymeric column was investigated for the separation of alkali, alkaline earth, transition and heavy metals employing non-chelating inorganic eluents. The retention of selected metal ions on the column was investigated with simple HNO3 eluents and eluents prepared from KNO3 and KCl salts of varying pH (adjusted using HNO3). From these studies both the effect of eluent ionic strength and pH upon retention was evaluated for the itaconic acid stationary phase. The results obtained showed that despite slow exchange kinetics causing poor efficiencies, acceptable baseline separations of selected alkaline earth and transitions could be obtained under optimum conditions (the baseline separation of Mg(II), Ca(II), Mn(II), Cd(II), Zn(II) and Co(II) was possible using a 15 mM KNO3-5 mM KCl eluent at pH 3.50 in under 25 min). The use of an simple ionic strength step gradient was shown that facilitated the addition of Pb(II) to the above group of metal ions. An investigation into the effect of temperature upon peak efficiency and retention showed increased column temperature could be used to improve the resolution of closely eluting metal ions such as Ca(II) and Sr(II) and Ca(II) and Mn(II).     

吡啶离子液体阳离子的离子色谱法分析

建立了离子色谱-直接电导检测法分离测定3种吡啶离子液体阳离子(N-乙基吡啶、N-丁基吡啶和N-己基吡啶阳离子)的方法.采用磺酸型阳离子交换色谱柱,以乙二胺-柠檬酸-乙腈为淋洗液,考察了淋洗液种类和浓度以及色谱柱温度对离子液体阳离子保留的影响.实验发现,吡啶阳离子的保留过程是放热过程,其同系物的保留符合碳数规律.优化的色...     

Modelling gradient elution of bioactive multicomponent systems in non-linear ion-exchange chromatography

A theoretical framework for the ion-exchange behaviour of bioactive substances in non-linear ion-exchange chromatogaphy is described. The aim of the study was the creation of a model basis to support a process design for production-scale ion-exchange chromatography. The theory can be applied to a whole variety of biological substances, such as amino acids, polysaccharides, peptides and proteins and either isocratic or gradient elution can be carried out. The influence of the eluent concentration on the ion-exchange as well as on the characteristic charge was considered. Experimental measurements showed a strong non-linear ion-exchange equilibrium with a transition from a Langmuir-type to a sigmoidal isotherm at higher eluent concentrations. Hereby, the compound binds to the surface though it is not ionic. Therefore, the model considered the possibility of ion-exchange as well as adsorption. A simplified distribution of the counter-ions based on the Gouy-Chapman theory with a discrete distribution of the counter-ions was used. The theory was extended by a selectivity in the double layer to allow specific adsorption. Calculations of adsorption-elution cycles showed, in agreement with the experimental observations, the development of non-linear elution profiles with a desorption fronting. As a result, the column loading and the eluent concentration were varied. The effect of contaminants, in this case sodium ions, was investigated and included in the model. Finally, the model was extended to multicomponent systems to investigate the effect of side components on the retention behaviour. The development of the characteristic elution profiles and the effect of the column loading on the separation are discussed. Calculated concentration profiles along the column at discrete time steps were used to reveal the influence of side components and the underlying separation mechanism. The simulations provided a new insight into the phenomena involved in biochromatography and make convenient design concepts at least doubtful as the separation is in this case mainly determined by the loading step and not by the choice of the elution gradient.     

Eluent-Induced Separation of Inorganic Cations in Capillary Liquid Chromatography with Contactless Conductivity Detector

A non-suppressed contactless conductivity detector has been used as a capillary detector in a capillary ion chromatograph, combining a reversed-phase C30 column permanently modified with ionic surfactant. The C30 column (100 × 0.32 mm. id) was modified with sodium dodecyl sulfate (SDS) for the separation of inorganic cations. Monovalent cations could be separated by the proposed system, in which methanesulfonic acid (MSA) and SDS were employed as the mobile phase component, but divalent cations could not be eluted under this condition. As for the case of SDS used as the eluent, an H⁺-cation-exchange column was placed before the sample injector to convert the Na⁺ from the eluent into H⁺, and when the mixture of MSA and dodecyl sulfuric acid was used as the eluent, the retention of cations was improved and baseline separation of the cations was achieved within 23 min. The effect of the eluent composition on the retention behavior of inorganic cations was investigated. The repeatability of retention time and peak height varied from 0.39 to 0.58 and 2.21 to 3.25 % as relative standard deviation, respectively.

     

Eluent-Induced Separation of Inorganic Cations in Capillary Liquid Chromatography with Contactless Conductivity Detector

A non-suppressed contactless conductivity detector has been used as a capillary detector in a capillary ion chromatograph, combining a reversed-phase C30 column permanently modified with ionic surfactant. The C30 column (100 × 0.32 mm. id) was modified with sodium dodecyl sulfate (SDS) for the separation of inorganic cations. Monovalent cations could be separated by the proposed system, in which methanesulfonic acid (MSA) and SDS were employed as the mobile phase component, but divalent cations could not be eluted under this condition. As for the case of SDS used as the eluent, an H⁺-cation-exchange column was placed before the sample injector to convert the Na⁺ from the eluent into H⁺, and when the mixture of MSA and dodecyl sulfuric acid was used as the eluent, the retention of cations was improved and baseline separation of the cations was achieved within 23 min. The effect of the eluent composition on the retention behavior of inorganic cations was investigated. The repeatability of retention time and peak height varied from 0.39 to 0.58 and 2.21 to 3.25 % as relative standard deviation, respectively.     

Retention behaviour of strong acid anions in ion-exclusion chromatography on sulfonate and carboxylate stationary phases

Some factors influencing the retention of strong-acid anions on ion-exclusion columns were investigated using columns with sulfonate and carboxylate functional groups. The nature of the functional group on the resin, the eluent pH and the eluent ionic strength all significantly affected the retention and separation of these analytes. Retention was observed for all strong-acid anions over the eluent pH range 2.2-5.7 and increased with both decreasing eluent pH and increasing eluent ionic strength. Some separation of strong-acid anions was possible when using a resin with carboxylate functional groups. It has also been demonstrated that strong-acid anions are poor markers of column void volume for ion-exclusion chromatography. A more accurate value was obtained using the neutral polymeric material dextran blue. When using eluents of low ionic strength, poor or fronted peak shapes were observed. A mechanism for these observations is proposed that relates the shape to ionic strength changes across the peak. A system peak was encountered under most experimental conditions. The properties of this peak are discussed and a cause for the system peak postulated.     

A study of the mechanisms involved in the separation of metal ions with a mixed-bed stationary phase

Summary The optimization of chromatographic methods for the determination of metal species require an understanding of the mechanisms involved. In this work, the separation of Cd, Co, Cu, Fe(II/III), Mn, Pb and Zn using a mixed bed column (IonPac CS5A) and a cation-exchange column (IonPac CS2) is studied as a function of mobile phase composition. The type and concentration of complexing agent and of ionic strength modificators were evaluated. The charge of analytes were calculated using the classical ion exchange approach to highlight the effect of eluent composition on retention. The comparative study enabled us to identify an optimal eluent composition for the separation of the nine metal species.     

Anion-exchange chromatography on short reversed-phase columns modified with amphoteric (N-dodecyl-N,N-dimethylammonio)alcanoates

Short reversed-phase columns (50 mm x 4.6 mm Gemini C(18)) were dynamically coated with carboxybetaines of the general structure, C(12)H(25)N(+)(CH(3))(2)(CH(2))(n)COOH, namely (N-dodecyl-N,N-dimethylammonio)undecanoate, DDMAU (n=10) and (N-dodecyl-N,N-dimethylammonio)butyrate, DDMAB (n=3), and investigated for the separation of inorganic anions in ion chromatography. The role of the ionic strength of coating surfactant solutions on their adsorption and resultant column capacity was studied. The retention of inorganic anions was investigated with different eluents at various concentrations and pH. Interestingly, no retention for anions was found with pure water as the eluent, but the addition of small amounts of electrolytes, up to 0.1 mM, caused a sharp increase in the retention of analytes. The effect of increasing anion retention with an increase in eluent cation charge was also observed. Based on this effect a new cation charge gradient concept was proposed and applied to the separation of a standard mixture of anions.     

Regularities of retention of benzoic acids on microdispersed detonation nanodiamonds in water-methanol mobile phases

The dependences of the retention of benzoic acids on microdispersed sintered detonation nanodiamond (MSDN) on the concentration of the organic solvent in the eluent and the temperature of the chromatographic column under conditions of high-performance liquid chromatography (HPLC) are investigated. It is found that in the investigated range of methanol concentrations, the acids are retained by different mechanisms: at methanol contents of the eluent lower than 85%, retention decreases with increasing methanol concentration and increases at higher concentrations of the organic solvent. It is shown that retention of benzoic acids on MSDN under these conditions depends on the dissociation constant of the investigated substances. A comparison is made between the properties of MSDN and analogous properties of porous graphitic carbon.     

Separation of Proteins on Polar Bonded Phases by Hydrophobic Interaction Chromatography

Abstract

Hydrophobic interaction chromatography (HIC) with a polar bonded phase (“Acetamide”) developped for size exclusion chromatography (SEC) is described. Retention of proteins depends on the surface area of the stationary phase, the pH and ionic strength of the eluent. For efficient separation the pore diameter should be 25 nm or more. The surface area should be large to achieve retention even at low ionic strength. Separation is only possible with a gradient from high to low ionic strength. Gradient volumes of 10 empty column volumes with column lengths above 15 cm are recommended. Selectivity can be optimized via pH adjustment. The advantage of this column packing is its applicability for two different separation modes: SEC and HIC.     

Application of retention indices based on the alkylarylketone scale to the separation of the local anaesthetic drugs by high-performance liquid chromatography

The separation of the local anaesthetic drugs by reversed-phase high-performance liquid chromatography using a Hypersil ODS column has been examined in order to identify critical factors in the establishment of a retention database for interlaboratory comparisons. An eluent of methanol-aqueous orthophosphoric acid (15:85, v/v) (pH 2.5) containing 0.7% hexylamine has been used. The effects of small changes in the eluent composition, pH, temperature and the use of different column packing materials on the retentions of the analytes have been studied. The use of capacity factors, relative capacity factors and retention indices based on the alkylarylketone scale have been compared.     

Separation of acidic compounds by strong anion-exchange capillary electrochromatography

Separation of the acidic compounds in the ion-exchange capillary electrochromatography (IE-CEC) with strong anion-exchange packing as the stationary phase was studied. It was observed that the electroosmotic flow (EOF) in strong anion-exchange CEC moderately changed with increase of the eluent ionic strength and decrease of the eluent pH, but the acetonitrile concentration in the eluent had almost no effect on the EOF. The EOF in strong anion-exchange CEC with eluent of low pH value was much larger than that in RP-CEC with Spherisorb-ODS as the stationary phase. The retention of acidic compounds on the strong anion-exchange packing was relatively weak due to only partial ionization of them, and both chromatographic and electrophoretic processes contributed to separation. It was observed that the retention values of acidic compounds decreased with the increase of phosphate buffer and acetonitrile concentration in the eluent as well as the decrease of the applied voltage, and even the acidic compounds could elute before the void time. These factors also made an important contribution to the separation selectivity for tested acidic compounds, which could be separated rapidly with high column efficiency of more than 220000 plates/m under the optimized separation conditions.     

Fast determination of tetrafluoroborate by high-performance liquid chromatography using a monolithic column

Fast determination of tetrafluoroborate by high-performance liquid chromatography using a silica-based monolithic column and direct conductivity detection was carried out. Chromatographic separation was performed on a Chromolith Speed ROD RP-18e column (50 mm x 4.6 mm i.d.) with tetrabutylammonium hydroxide (TBA-OH)+ phthalic acid as eluent. The effects of eluent concentration, eluent pH, column temperature and flow rate on retention time of tetrafluoroborate were investigated. The optimized chromatographic conditions for determination of tetrafluoroborate were using 0.5mM TBA-OH + 0.31 mM phthalic acid (pH 5.5) as eluent, column temperature of 30 degrees C and flow rate of 6.0 mL/min. Retention time of tetrafluoroborate was less than 1min under the conditions. Common anions (Cl(-), Br(-), NO3(-) and SO4(2-)) did not interfere with the determination of tetrafluoroborate. Detection limit (S/N = 2) for tetrafluoroborate was 1.4 mg/L. The linear range of calibration curve between peak area and the concentration of tetrafluoroborate was from 1.4 to 100.0 mg/L. The reproducibility was 0.09% and 1.8% (n = 5) relative standard deviation (RSD) for retention time and peak area, respectively. The method has been applied to the determination of tetrafluoroborate in ionic liquids. Recoveries of tetrafluoroborate after spiking were 98.2-101.5%.