Evaluation and application of liquid chromatographic columns coated with 'intelligent' ligands: (I) acylcarnitine column

Unique stationary phases of octadecylsilica (ODS) coated with acylcarnitines have been developed for liquid chromatographic columns. The ODS column coated with acylcarnitine was readily prepared by recycling the solution containing acylcarnitine through an ODS column in a closed loop. Acylcarnitine was adsorbed on the ODS surfaces by hydrophobic interaction between the acyl group of acylcarnitine and the octadecyl group of the ODS phases. The ODS column coated with stearoylcarnitine (CN-18 column) was the most stable among the four columns coated with acylcarnitines of various acyl chain lengths (decanoylcarnitine, lauroylcarnitine, myristoylcarnitine, and stearoylcarnitine) under the condition of delivery of the mobile phase, indicating that adsorption of acylcarnitine on the ODS surfaces depended on the length of acyl chains. The CN-18 column was usable for delivering the mobile phase contained less than 20% (v/v) acetonitrile, retaining almost the same separation efficiency as the intact ODS column. The retention behavior of ionic solutes on the CN-18 column could be explained by both ionic and electrostatic interactions between the solutes and the stationary phase. The CN-18 column enabled efficient separation of inorganic anions, nicotinic acids, amino acids, and nucleotides. The chiral ODS column coated with enantiomer of stearoylcarnitine, L-stearoylcarnitine (L-CN-18 column) could achieve direct enantiomeric separation of DL-tryptophan, alpha-methyl-DL-tryptophan and DL-3-indolelactic acid using 100% water as the mobile phase. The L-CN-18 column could also separate enantiomers of amino acids and alpha-hydroxycarboxylic acids by ligand-exchange chromatographic mode using a mobile phase containing copper(II) ion. The chiral recognition is discussed for enantiomeric separation on the L-CN-18 column.     

Characterization of dynamically prepared phospholipid-modified reversed-phase columns.

We have modified a reversed-phase (RP8) column by passing through it an aqueous solution of phosphatidylcholine-based liposomes. The phospholipids from the liposomes adsorb onto the octyl chain of the stationary phase, thus altering the nature of the stationary phase and of the chromatographic interactions. The properties of the phospholipid-modified column were investigated using solutes belonging to several chemical classes. We found that the retention factors of negatively and positively charged solutes decreased as the amount of phospholipid in the column was increased. For the solutes studied here the extent of the decrease was smaller for the positive solutes. With neutral solutes, the retention factors of some (benzenediols) increased markedly while with others (ketones) the retention factors decreased. The selectivities between the various solutes on the phospholipid-modified column were different than on the original reversed-phase column. The retention behavior of the solutes can be explained in terms of (1) effective shielding of the hydrophobic part of the stationary phase by the polar head groups of the phospholipids and (2) hydrogen bond formation between the solutes and the carbonyl oxygens as well as the non-ester phosphate oxygens in the polar head groups of the phospholipids.     

稠环芳烃在烷基膦酸改性锆镁复合氧化物固定相上的反相液相色谱保留行为

 以稠环芳烃为探针,考察了烷基膦酸改性锆镁复合氧化物材料的反相色谱性能。研究了稠环芳烃类化合物的结构与其保留值的关系,比较了烷基膦酸改性锆镁复合氧化物固定相和十八烷基键合硅胶ZorbaxODS对稠环芳烃异构体的选择性,并对可能的保留机理进行了讨论。以甲醇-水(体积比为75∶25)为流动相,在烷基膦酸改性锆镁复合氧化物固定相上分离了8种稠环芳烃类化合物。     

稠环芳烃在烷基膦酸改性锆镁复合氧化物固定相上的反相液相色谱保留行为

以稠环芳烃为探针 ,考察了烷基膦酸改性锆镁复合氧化物材料的反相色谱性能。研究了稠环芳烃类化合物的结构与其保留值的关系 ,比较了烷基膦酸改性锆镁复合氧化物固定相和十八烷基键合硅胶 Zorbax ODS对稠环芳烃异构体的选择性 ,并对可能的保留机理进行了讨论。以甲醇 -水 (体积比为 75∶ 2 5)为流动相 ,在烷基膦酸改性锆镁复合氧化物固定相上分离了 8种稠环芳烃类化合物     

Noncovalent immobilized artificial membrane chromatography, an improved method for describing peptide-lipid bilayer interactions.

A promising approach in assessing hydrophobic peptide-membrane interactions is the use of reversed-phase high-performance liquid chromatography. The present study describes the preparation and properties of a noncovalent immobilized artificial membrane (noncovalent IAM) stationary phase. The noncovalent IAM phase was prepared by coating the C18 chains of a reversed-phase HPLC column with the phospholipid ditetradecanoyl-sn-glycero-3-phosphocholine. Lipid coating was achieved by pumping a lipid solution in water-2-propanol through the column. The formation of a bilayer-like structure on the chromatographic surface was confirmed by calculating the phospholipid surface density of the stationary phase. The surface density was determined to be approximately 1.95 mumol m-2, which is close to that of lipid vesicles. The coating was found to be stable in chromatographic elution systems containing less than 35% of acetonitrile. Employing this new technique, we determined interaction parameters of a set of helical antibacterial magainin-2-amide peptides with pairwise substitutions of adjacent amino acids by their D-enatiomers. The results demonstrate that the chromatographic retention behavior of peptides on noncovalent IAM stationary phase shows an excellent correlation with lipid affinities to phospholipid vesicles.     

Dynamic coating ion-exchange chromatography of cations on an octadecyl-bonded silica stationary phase

It was found that common cations (Na+, NH4+, K+, Mg2+ and Ca2+) could be strongly retained on an ODS stationary phase when aqueous solutions of carboxylic acids were used as eluents. The chromatographic conditions used in this work were the same as in common cation-exchange chromatography on a cation-exchange resin and the retention behavior of the above-mentioned cations on the ODS column was quite similar to that on a cation-exchange column. The retention behavior and mechanism have been investigated using a number of carboxylic acids as eluents. The retention mechanism of the cations in these experiments was considered to be a dynamic coating ion-exchange mechanism. The carboxylic acids in the mobile phase were coated onto the surface of the ODS stationary phase and formed a dynamic carboxylic acid functional layer which could act like the functional group layer of a carboxylic group cation exchanger.     

Description of retention characteristics of calixarene-bonded stationary phases in dependence of the methanol content in the mobile phase

Calixarene-bonded stationary phases in HPLC are known to support additional interactions compared to conventional alkyl-bonded phases (π–π interactions, complex-building interactions). Thus it cannot be presumed that the same mechanisms of retention apply and that retention can be predicted in similar ways. Here 31 solutes of highly various molecular structures have been analysed at different mobile phase compositions (0–98% (v/v) methanol) in order to characterise the chromatographic behaviour of the novel stationary phases and to test the applicability of established models predicting retention factors. The influence of a change of the methanol content is discussed for non-polar, polar and ionic solutes and differences of their behaviour on the differing column types are shown. Additionally estimates about underlying retention mechanisms are given.     

Separation of Phenolic Compounds and Organic Anions Using ODS Column Coated with Meso-octamethylcalix[4]pyrrole and Water as Eluent

Octadecylsilanized silica (ODS) was coated with meso-octarnethylcalix[4]pyrrole to obtain a novel calix[4]pyrrole containing stationary phase for HPLC. Compared to ODS, the new stationary phase showed a relatively large retention and an improved separation for phenolic compounds and organic anions, using pure water as mobile phase. The results can be ascribed to the interaction between analytes and calix[4]pyrrole.     

Liposome chromatography: liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography

Liposomes have been used as a stationary phase for column chromatography with an aqueous mobile phase. They were immobilized in the pores of carrier gel beads by two methods: (A) hydrophobic ligands were coupled to the matrix of gel beads, which then were packed into a column and liposomes were applied and became associated with the ligands by hydrophobic interaction; and (B) phospholipids and detergent were dialysed in the presence of gel beads; many of the liposomes that formed in the pores of the beads were sterically immobilized by the gel matrix. Proteoliposomes containing red cell glucose transport protein in the lipid bilayers were immobilized in a column by method A. This column retained D-glucose longer than L-glucose. In contrast to L-glucose, D-glucose was transported into and out of the immobilized liposomes, causing an increased retention. Liposomes with (stearylamine)+ or (phosphatidylserine)- in their lipid bilayers were immobilized by method B and the gel beads were packed into a column. A protein of opposite charge was applied in excess. Under suitable conditions, the protein molecules became close-packed on the liposome surfaces. Ion-exchange chromatographic experiments with proteins showed that these sterically immobilized liposomes were also stable enough to be used as a stationary phase. The loss of lipids was 5-23% in the first run at high protein load and with sodium chloride gradient elution but was lower in subsequent runs. It is proposed that water-soluble molecules can be separated and their interactions with liposome surfaces studied by chromatography on immobilized liposomes in detergent-free aqueous solution. Membrane proteins can be inserted and ligands can be anchored in the lipid bilayers for chromatographic purposes.     

多羟基化合物键合亲水色谱固定相的制备及其分离性能

用硅胶与氨丙基三甲氧基硅烷反应,再与δ-葡萄糖酸内酯反应,制备了一种多羟基化合物键合的新型亲水色谱固定相。以水-有机溶剂(乙醇、乙腈、四氢呋喃)为流动相,通过改变流动相中有机溶剂的种类及浓度、缓冲盐浓度和柱温,考察了该固定相对6种强极性中药组分的保留行为和保留机理。当水的比例在0～40%(v/v)范围时,溶质的保留随着流动相中水的比例的增大而减小,属于典型的亲水色谱分离模式;而当流动相中水的比例在0～100%(v/v)范围内变化时,溶质的保留随着水的比例变化呈“U”形曲线,属于亲水色谱和反相色谱的混合保留机理。缓冲盐的浓度和pH效应说明,所选用的中药组分与所制备的固定相间还存在弱的静电作用。该固定相对6种中药组分以及丹参注射液具有良好的分离性能,表明其在强极性中药有效成分的分离及其他强极性物质的分离分析中具有一定的应用前景。     

Thermally-treated clay as a stationary phase in liquid chromatography

Spray-dried, spherical synthetic hectorite particles have been thermally-treated at 500 degrees C for 16 h and used as adsorbent materials in reversed-phase liquid chromatography. The retention of a 22 mono and disubstituted aromatic compounds was evaluated to study the retention mechanisms on the clay mineral. The retention of solutes on the thermally-treated clays was markedly different than that measured on octadecylsilica (ODS) columns under identical conditions, but remarkably similar to retention characteristics of the same solutes on porous graphitic carbon columns. The clay columns exhibit an enhanced selectivity over the ODS column in separation of nitroaromatic positional isomers. Under identical mobile phase compositions, a selectivity, alpha, of 7.15 between ortho- and para-dinitrobenzene isomers was measured on the clay column compared to a alpha of 1.04 on the ODS column.     

Insights into the retention mechanism on an octadecylsiloxane-bonded silica stationary phase (HyPURITY C18) in reversed-phase liquid chromatography

Plots of the retention factor against mobile phase composition were used to organize a varied group of solutes into three categories according to their retention mechanism on an octadecylsiloxane-bonded silica stationary phase HyPURITY C18 with methanol-water and acetonitrile-water mobile phase compositions containing 10-70% (v/v) organic solvent. The solutes in category 1 could be fit to a general retention model, Eq. (2), and exhibited normal retention behavior for the full composition range. The solutes in category 2 exhibited normal retention behavior at high organic solvent composition with a discontinuity at low organic solvent compositions. The solutes in category 3 exhibited a pronounced step or plateau in the middle region of the retention plots with a retention mechanism similar to category 1 solutes at mobile phase compositions after the discontinuity and a different retention mechanism before the discontinuity. Selecting solutes and appropriate composition ranges from the three categories where a single retention mechanism was operative allowed modeling of the experimental retention factors using the solvation parameter model. These models were then used to predict retention factors for solutes not included in the models. The overwhelming number of residual values [log k (experimental) - log k (model predicted)] were negative and could be explained by contributions from steric repulsion, defined as the inability of the solute to insert itself fully into the stationary phase because of its bulkiness (i.e., volume and/or shape). Steric repulsion is shown to strongly depend on the mobile phase composition and was more significant for mobile phases with a low volume fraction of organic solvent in general and for mobile phases containing methanol rather than acetonitrile. For mobile phases containing less than about 20 % (v/v) organic solvent the mobile phase was unable to completely wet the stationary phase resulting in a significant change in the phase ratio and for acetonitrile (but less so methanol) changes in the solvation environment indicated by a discontinuity in the system maps.     

Properties and Applications of Mixed Packing Capillary Electrochromatography

Mixed packing capillary electrochromatography (MP CEC) with the stationary phase comprising a physical mixture of strong cation exchange (SCX) phase and octadecysilyl (ODS) phase was developed. With the existence of a sulfonic acid group on the surface of SCX, not only could the electroosmotic flow (EOF) remain high at low pH, but also the hydrophilicity of the stationary phase was increased greatly, leading to broad adaptable ranges of both pH and organic modifier concentration in the mobile phase. At the same time, with the coexistence of C₁₈ on the surface of ODS, both the retention and the resolution of samples were improved. Accordingly, MP CEC combined the advantages of both SCX and ODS columns. Effects of operation parameters on EOF and the capacity factors of solutes as well as the retention mechanism of such a column were studied systematically. In addition, MP CEC columns were used in the analysis of strong polar solutes as well as for the high speed separation of acidic, basic, and neutral compounds in a single run.     

Study of a monolithic silica capillary column coated with poly(octadecyl methacrylate) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds

The performance of a monolithic silica capillary column coated with poly(octadecyl methacrylate) (ODM column) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds was studied, and the results were compared to those obtained by using a monolithic silica capillary column modified with octadecylsilyl-(N,N-diethylamino)silane (ODS column). Benzene and naphthalene derivatives, polycyclic aromatic hydrocarbons (PAHs), steroids, alkyl phthalates, and tocopherol homologues were used as test samples. In general, compounds with aromatic character, rigid and planar structures, and lower length-to-breadth ratios (more compacted structures) seem to have more preference for the polymer coated stationary phase (ODM). Compounds with acidic character have also a higher retention on ODM columns because of the presence of ester groups in the stationary phase. The polymer coated column allowed the separation of some PAHs, alkyl phthalates, steroids, and of beta- and gamma-tocopherol isomers which cannot be separated under the same conditions on ODS columns, while keeping similar column efficiency. These results allowed to suggest ODM columns as a good alternative to conventional ODS columns for reversed-phase liquid chromatography.     

Characterization of some functionalized RP-HPLC phases by the use of linear solvation energy relationships

The linear solvation energy relationship equation developed by Abraham and coworkers was applied to the retention factors k of a series of 20 polar solutes on four chemically different RP-HPLC phases. Three of them were specially synthesized and are functionalized with ether, phenylsulfide or phenylsulfoxide groups. Their retention properties are compared with those of a nonpolar octadecylsiloxane (ODS) phase. The phase properties r, the excess molar refraction; s, the dipolarity; a and b, the hydrogen-bond basicity and acidity; and v, the cavity factor show significant differences on the four phases and are used here to suggest a classification of stationary phases based on the type of interactions that are important for the retention. The hydrophilic system properties r, s, a and b are the reason for different elution orders of a set of solutes on the four phases. The intrinsic hydrophobicity of the system, the v/A ratio (A is the surface coverage in μmol/m²), shows a dependence on the mobile phase composition as do the normalized phase properties r/v, s/v, a/v and b/v. Averaging the constants over a large span of mobile phase composition should be done very carefully. The LSER model is used to predict the elution order on the stationary phases for five phenols which show coelution on ODS. On the phenylsulfide phase they are resolved. Received: 3 December 1998 / Revised: 1 February 1999 / Accepted: 8 February 1999     

Column selectivity in reversed-phase liquid chromatography III. The physico-chemical basis of selectivity

Reversed-phase liquid chromatography (RP-LC) retention data for 23 additional solutes have been acquired to further test and evaluate a general relationship from part I: log alpha = log (k/kref) = eta'H(i) + sigma'S(ii) beta'S(iii) + alpha'B(iv) +kappa'C(v) The physico-chemical origin of terms i-v above is examined here by comparing values of (a) the solute parameters of Eq. (1) (eta', sigma', etc.) vs. solute molecular structure, and (b) the column parameters (H, S, etc.) vs. column properties (ligand length and concentration, pore diameter, end-capping). We conclude that terms i-v correspond, respectively, to hydrophobic (i), steric (ii), hydrogen bonding (iii, iv) and ionic (v) interactions between solute and stationary phase. While steric interaction (term ii) is superficially similar to what previously has been defined as "shape selectivity", the role of the solute and column in determining steric selectivity (term ii) appears more complex than previously proposed for "shape selectivity". Similarly, what has previously been called hydrogen bonding between donor solutes and an acceptor group in the stationary phase (term iv) is very likely an oversimplification.     

Preparation and characterization of phosphatidylcholine‐coated zirconia–magnesia stationary phase for artificial membrane chromatography

Immobilized artificial membrane chromatography stationary phase was prepared by coating soybean phosphatidylcholine (PC) on zirconia–magnesia micro‐particles. The stability and chromatographic properties were investigated and compared with the PC‐coated silica chromatography stationary phase prepared by the same method. PC‐coated zirconia–magnesia chromatography stationary phase was more stable than the silica especially on resisting organic solvents. Hydrophobic action was the main factor for the retention of analyte on the new artificial membrane chromatography stationary phase, and electrostatic interaction had some contribution to retention. In addition, the special interaction between analyte and matrix affected retention greatly. Basic solutes were appropriate to be analyzed on PC‐coated zirconia–magnesia stationary phase and acidic solutes were appropriate to be done on the silica one. Hence, the two different matrices artificial membrane stationary phases were perfectly complementary.     

CEC separation of insect oostatic peptides using a strong-cation-exchange stationary phase

The separation of several insect oostatic peptides (IOPs) was achieved by using CEC with a strong-cation-exchange (SCX) stationary phase in the fused-silica capillary column of 75 microm id. The effect of organic modifier, ionic strength, buffer pH, applied voltage, and temperature on peptides' resolution was evaluated. Baseline separation of the studied IOPs was achieved using a mobile phase containing 100 mM pH 2.3 sodium phosphate buffer/water/ACN (10:20:70 v/v/v). In order to reduce the analysis time, experiments were performed in the short side mode where the stationary phase was packed for 7 cm only. The selection of the experimental parameters strongly influenced the retention time, resolution, and retention factor. An acidic pH was selected in order to positively charge the analyzed peptides, the pI's of which are about 3 in water buffer solutions. A good selectivity and resolution was achieved at pH <2.8; at higher pH the three parameters decreased due to reduced or even zero charge of peptides. The increase in the ionic strength of the buffer present in the mobile phase caused a decrease in retention factor for all the studied compounds due to the decreased interaction between analytes and stationary phase. Raising the ACN concentration in the mobile phase in the range 40-80% v/v caused an increase in both retention factor, retention time, and resolution due to the hydrophilic interactions of IOPs with free silanols and sulfonic groups of the stationary phase.     

Comparison of the chromatography of octadecyl silane bonded silica and polybutadiene-coated zirconia phases based on a diverse set of cationic drugs

In this study, we compare the separation of basic drugs on several octadecyl silane bonded silica (ODS) phases and a polybutadiene-coated zirconia (PBD-ZrO2) phase. The retention characteristics were investigated in detail using a variety of cationic drugs as probe solutes. The ODS phases were selected to cover a relatively wide range in silanol activity and were studied with ammonium phosphate eluents at pH 3.0 and 6.0. Compared to any of the ODS phases, the PBD-ZrO2 phase showed very significant differences in selectivities towards these drugs. Due to the presence of both reversed-phase and ion-exchange interactions between the stationary phase and the basic analyte on ODS and PBD-ZrO2, mixed-mode retention takes place to some extent on both types of phases. However, very large differences in the relative contributions from ion-exchange and reversed-phase interactions on the two types of phases led to quite different selectivities. When phosphate is present in the eluent and adsorbs on the surface, the PBD-ZrO2 phase takes on a high negative charge over a wide pH range due to phosphate adsorption on its surface. On ODS phases, ion-exchange interactions result from the interactions between protonated basic compounds and ionized residual silanol groups. Since the pH of the eluent influences the charge state of the silanol groups, the ion-exchange interactions vary in strength depending on pH. At pH 6.0, the ion-exchange interactions are strong. However, at pH 3.0 the ion-exchange interactions on ODS are significantly smaller because the silanol groups are less dissociated at the lower pH. Thus, not only are the selectivities of the ODS and PBD-ZrO2 phases different but quite different trends in retention are observed on these two types of phases as the pH of the eluent is varied. More importantly, by using the large set of "real" basic analytes we show the extreme complexity of the chromatographic processes on the reversed stationary phases. Both the test condition and solute property influence the column performance. Therefore, use of only one or two probe solutes is not sufficient for column ranking.     

Reversed-phase liquid chromatographic separation of antiretroviral drugs on a monolithic column using ionic liquids as mobile phase additives

The use of 3-methylimidazolium cation-based ionic liquids (ILs) was evaluated as mobile phase additives for separation of antiretroviral drugs on a monolithic column by RP-HPLC. Separation of eight commonly used antiretroviral drugs was achieved on a Chromolith Flash, RP-18e column (25 × 4.6 mm, porous material) using water (pH 4.0 adjusted with acetic acid)/methanol v/v as a mobile phase containing ILs in a gradient elution mode. The effects of concentrations of ILs on retention, resolution and peak shape were studied and a regression equation correlating the interactions between stationary phase and the ILs was established. The retention of all the drugs was decreased notably by using 1-butyl-3-methylimidazolium tetrafluoroborate, while 1-ethyl-3-methylimidazolium methylsulfate reduced gradient drift drastically when compared to triethylamine.