Reversed-phase Stationary Phases Research Articles

Temperature dependence of retention in reversed-phase liquid chromatography. 1. Stationary-phase considerations.

The retention mechanism in reversed-phase liquid chromatography (RPLC) has been investigated by examining the temperature dependence of retention, with emphasis on the role of the stationary phase in the retention process. Both chromatographic temperature studies and differential scanning calorimetry were used to examine the role of alkyl chain bonding density on the retention mechanism in RPLC. Phase transitions of reversed-phase stationary phases were observed at bonding densities greater than 2.84 mumol/m2. Thermodynamic constants for the transfer of a solute from the mobile phase to the stationary phase (delta H degrees and delta S degrees) were calculated for low bonding density columns, and comparison of these values to previously reported values for the partitioning of a nonpolar solute from the bulk organic liquid to water indicated that the chromatographic retention process is not well-modeled by bulk-phase oil-water partitioning processes. In addition, this data showed that the entropic contribution to retention becomes more significant with respect to the enthalpic contribution as the stationary-phase bonding density is increased, providing additional support that partitioning, rather than adsorption, is the relevant model of retention.

Cation-exchange liquid chromatography of choline and acetylcholine on free shielded silanols of silica-based reversed-phase stationary phases

Free anionic functions present on the surface of reversed-phase packing materials were used for the selective cation-exchange preconcentration and separation of the neurotransmitters choline and acetylcholine from a biological matrix. The cation-exchange behaviour of different reversed-phase packing materials in the neat aqueous mobile phase, the properties of an end-capped column, the dependence of capacity factors and peak shape on the concentration of counter ions, ionic strength, pH and the addition of acetonitrile and optimum conditions for enzymatic conversion of solutes to hydrogen peroxide were studied. The studied reversed-phase columns exhibit better pH stability and longer lifetimes than normal silica-based cation exchangers. Acetylcholine is an effective and sensitive test sample for the measurement of adsorption on silica support. A large sample volume was injected onto a precolumn inserted instead of an injection valve and after injection the solutes were focused and separated on an analytical column with a mobile phase containing tetramethylammonium perchlorate as the counter ion.

Preparative liquid chromatography with analytical separation quality : Internal injection/displacement reversed-phase high-performance liquid chromatography

A method for the enrichment and separation of compounds, called interval injection/displacement reversed-phase high-performance liquid chromatography (HPLC), was applied to several analytical and preparative separations. In principle, the surface of a reversed-phase stationary phase, equilibrated with a weak mobile phase, is “coated” with compounds by stepwise injection of small samples. Distinct time intervals between injections allow the stationary phase to re-equilibrate. In this way, sample enrichment can be achieved in ranges comparable to those in overload elution or displacement HPLC. Sample fractionation proceeds in a similar manner to conventional displacement modes. Also under full mass load conditions, a quality of separation similar to that in analytical elution HPLC is achieved. The results obtained indicate that a very large number of samples can be applied to conventionally sized columns without overload problems, the full mass capacity of a column can be utilized and the method is useful for enriching and separating compounds with a wide range of polarities. Interval injection/displacement reversed-phase HPLC was successfully applied to complex mixtures of natural products and enzyme assay mixtures. Further, this technique is likely to be useful for the analysis of trace compounds and quality control of chemicals. The experiments reported were performed on highly unstable natural products (thiophenic and benzofuran compounds) from Tagetes plants.

Relationships between nucleotide incorporation rates and molecular parameters obtained by molecular modelling and chromatography

Deoxyuridine derivatives play an important role in pharmaceutical chemistry as they are potential antiviral and antitumour agents. Their pharmacological activity depends on their ability to incorporate into DNA in their triphosphate forms. High-performance liquid chromatographic (HPLC) retention behaviour of a series of 5-alkyl, alkenyl and alkynyl substituted deoxyuridine derivatives were investigated on reversed-phase stationary phase using various mixtures of methanol and water as mobile phases. The slope and the intercept values of the linear relationships between the logarithmic capacity ratio (log k') values and methanol concentration have been calculated. Non-polar, non-polar unsaturated and polar surface areas, surface energies, dipole moments, van der Waals radii of the derivatives have been calculated on the bases of molecular mechanics by PC Model approach. The correlation study of the above-mentioned parameters revealed that hydrophobic and hydrophilic surface areas and the electronic effects of the substituents determine not only the retention behaviour of the derivatives but also their incorporation rate into DNA in their triphosphate forms.

Application of chromatographic retention data in an investigation of a quantitative structure—nucleotide incorporation rate relationship

A series of 5-alkyl-, 5-alkenyl- and 5-alkynyl-substituted deoxyuridines and their triphosphate derivatives were synthesized and studied in DNA polymerase reactions. The initial rate of incorporation of the derivatives catalysed by Klenow fragment DNA polymerase enzyme ( E. coli) was measured. Calf thymus DNA and synthetic poly (dA—dT) served as templates. The rate values were expressed as a percentage relative to the incorporation rate of natural substrate dTTP. The high-performance liquid chromatographic (HPLC) retention behaviours of the nucleoside derivatives were investigated on silica and reversed-phase stationary phases using various mixtures of ethyl acetate—methanol and methanol—water, as respectively, mobile phases. According to the results of principal component analysis, the HPLC retention data describe the hydrophobic properties of the compounds. The inclusion complex stability constants of the derivatives with cyclodextrins determined by reversed-phase thin-layer chromatography served as a measure of the steric properties of the subtituents. The electronic properties of the 5-substituents were characterized by the Swain-Lupton inductive and resonance parameters. The results of the stepwise linear regression analysis of the nucleotide incorporation rate data and the above-mentioned physico-chemical data revealed the importance of the electronic, steric and hydrophobic properties of the substituents in the DNA polymerase reactions. The importance of the steric parameter was more significant when the poly (dA—dT) template was used instead of the random base sequence template (calf thymus DNA).

Retention characteristics of CN, NH 2 and diol precoated high-performance thin-layer chromatographic plates in the adsorption and reversed-phase separation of some benzodiazepine derivatives

The retentions of eighteen benzodiazepine (BZD) derivatives on CN, diol and NH 2 high-performance thin-layer chromatographic layers was determined using eight adsorption (carbon tetrachloride-ethyl acetate) and five reversed-phase (water-methanol) eluent systems. In most instances a linear correlation was found between the R M value of BZD and the ethyl acetate or methanol concentration in the eluent, which allows the calculation of the optimum eluent composition. In aqueous eluents the lipophilicity of BZD determines the retention, that is, the plates behave as real reversed-phase stationary phases. The differences in the reversed-phase retentions of BZD were lower than in adsorption chromatography, advocating the application of adsorption chromatography for their separation. The low retention capacity and elongated spot shape make the NH 2 plate unsuitable for the reversed-phase separation of BZD without further modification of the composition of the mobile phase. As chlordiazepoxide and medazepam form distorted spots on CN and diol plates, for the best separation of these BZD, NH 2 plates in the adsorption separation mode are recommended.

ChemInform Abstract: The Molecular Mechanism of Retention in Reversed‐Phase Liquid Chromatography

The term “reversed-phase” chromatography seems at first inappropriate for what is by far the most popular mode of modern liquid chromatography. Estimates of the popularity of the technique range from 57% of all analytical chromatography1 to as high as 80-90% .* The term itself can be traced to Howard and Martin in 1950.3 In attempting the separation of long-chain fatty acids they realized that the “normal” mode of chromatography, using a polar stationary phase and nonpolar mobile phase, would not work, as the hydrophobic compounds had too little retention to effect a separation. They were able to treat Kieselguhr with dimethyldichlorosilane vapor and then coat this hydrophobic support with a nonpolar liquid stationary phase. Both the polarity of the phases and the respective elution order of solutes were reversed from traditional chromatographic systems, and they christened the technique “reversed-phase” partition chromatography. The popularity of reversed-phase liquid chromatography (RPLC), as practiced today, can be attributed to the development of chemically stable, microparticulate bonded phases that provide rapid mass transfer and a high degree of reproducibility. Attempts to utilize liquid-liquid chromatography, with a liquid stationary phase physically coated on an inert support, were rapidly abandoned with the introduction of commercially available bonded phases. Interesting perspectives on the early development of bonded phases for modern liquid chromatography can be found in a book devoted to the history of liquid chromatography.* H. A. Laitinen, in an editorial in Analytical Chemistry, described the seven ages of an analytical method from the birth of an idea to the ultimate replacement of the method by newer technique^.^ The fourth phase he described as “...detailed studies of principle and mechanisms are pursued with the aid of improved instrumentation. This represents the stage at which the method matures as an accepted procedure in competition and cooperation with other approaches. This stage represents the crest of analytical research as distinguished from instrumentation research.” This most clearly describes the present status of reversedphase liquid chromatography. Many methods of investigation are being brought to bear on the problem of understanding the molecular mechanism of retention of RPLC. These range from spectroscopic studies, including UV-visible, IR, NMR, fluorescence, and others, to thermal methods, to neutron scattering, to chromatographic methods themselves. Experimental studies alone are not enough. The cooperation of theorists and experimentalists is leading to dramatic advances in the understanding of the retention process. The goal of this review is to critically assess the current understanding of retention of small molecules in reversed-phase chromatography from both a theoretical and experimental perspective. We first discuss the synthetic methodologies for the preparation of reversed-phase stationary phases and deal next with the partitioning processes and stationary-phase structural details.

Rapid preliminary separation of petroleum hydrocarbons by solid-phase extraction cartridges

Small, prepacked columns are tested for fractionating petroleum hydrocarbons from crude oil and products oils. Both normal-phase (silica) and reversed-phase (C 18) stationary phases yielded fast, easy, and reproducible separations, which facilitated further analysis of the oils by gas or liquid chromatography and mass spectrometry. The different separation characteristics of the stationary phases offer great flexibility in fractionating oil, enriching special compound classes, or separating hydrocarbons from environmental matrices.

Ultrasound driven synthesis of reversed-phase stationary phases for liquid chromatography using 4-dimethylaminopyridine as acid-acceptor

A reproducible new method for synthesizing high density monomeric reversed-phase chromatographic packings based on the use of ultrasonic waves as the driving force for the bonding reaction is presented. The use of ultrasound has two distinct advantages over traditional reflux methods. It provides a reaction driving force that is independent of temperature; in addition, the power of the ultrasonic driving force can be varied using a variable-power ultrasonic probe. The use of a novel acid-acceptor, 4-dimethylaminopyridine, is also recommended in the bonding reaction due to its ease of use and the resulting high bonding densities.

Analysis of propantheline bromide and related quaternary ammonium drugs by reversed-phase, ion-pair liquid chromatography with two counterions in the eluent

The addition of appropriate concentrations of an organic amine and an alkylsulphonate to the mobile phase in reversed-phase, ion-pair liquid chromatography can introduce unique selectivity in to the chromatographic system allowing separation of complex mixtures of basic, acidic and neutral compounds. As an example, the methodology for a specific stability-indicating determination of propantheline bromide, a quaternary ammonium anticholinergic agent, on several reversedphase stationary phases, was developed. The retention mechanism was studied and it was concluded that both ion-interaction processes and ion-exchange processes were involved in the separation technique developed with two counterions of opposite charge in the eluent.

Selectivity in micellar chromatography using a zwitterionic surfactant.

This work discusses the effects of neutral zwitterionic micelles composed of N-dodecyl-N,N-dimethylammonium-3-propane-1-sulfonate (C12DAPS) on the retention of several substituted benzene compounds. The correlation of molecular properties and retention demonstrates that negative charge, hydrophobicity, and polarizability are important in governing a solute's distribution in the micelles. The selectivity of the micelles differs from that of the native reversed-phase stationary phase, especially for nitrophenol isomers and halobenzenes.

Fluorometric assay using high-pressure liquid chromatography for the microsomal metabolism of certain substituted aliphatics to 1, N6-ethenoadenine-forming metabolites

Monohaloacetaldehydes and monohalooxiranes are early oxidative metabolites of several carcinogenic haloaliphatics. Since monohaloacetaldehydes and supposedly monohalooxiranes react with adenines to form fluorescent 1, N 6-ethenoadenines, it was hypothesized that in vitro metabolic systems that produce an ethenoadenine-forming metabolite could be assayed quantitatively by trapping the metabolite in situ with an adenine and identifying it by its characteristic retention and fluorescence during HPLC. Bromoacetaldehyde was chosen as a model haloacetaldehyde to develop an assay based on this concept for measurements in a microsomal system. The optimal trapping reaction requires a postmetabolic step involving acidification and heating. Cyclic AMP was found to be a suitable adenine for the trapping reaction under these conditions. The chromatographic analysis utilizes tetrabutylammonium phosphate and a nonsilica reversed-phase stationary phase (Hamilton PRP-1). The chromatography is isocratic and allows an analysis time of less than 5 min per sample. The titration of bromoacetaldehyde in a microsomal system is affected by typically studied metabolic conditions: incubation time, pH, and protein concentration. Using this assay, the following were found to be metabolized by rat liver microsomes to ethenoadenine-forming products: 1,2-dibromoethane, 1,2-dichloroethane, cyclophosphamide, vinyl chloride, and acrylonitrile. Chloroacetone and 1,3-dichloroacetone also are fluorochromogenic without metabolism but the latter apparently forms a positively charged, nonetheno adduct. The proposed assay should be useful for in vitro metabolic studies of 1,2-dihaloethanes and mustards and has potential application for similar studies of monohalogenated ethanes, ethanols, and ethenes. The positive results with acrylonitrile suggest also that many types of substituted aliphatics may be studied with this proposed assay.

High performance liquid chromatography of anthraquinones: analysis of plant and insect extracts and dyed textiles

Separation and quantitative determination of anthraquinones is achieved by high performance liquid chromatography in mixtures of methanol, water and formic acid on a reversed-phase stationary phase. The products are identified by retention time and, more accurately, by standard additions and UV-visible spectroscopy. This methodology has been applied to extracts of plant roots and insects, commonly used in earlier times as the source of red dyestuffs for dyeing textiles. Quantitative evaluation of the anthraquinone derivatives present in ancient red dyes was earned out after acid hydrolysis of 0·2 to 2·0mg of textile fibre. Due to the great sensitivity of the method, important minor constituents, such as kermesic acid in cochineal, can be detected.

Effect of the chain length of chemically bonded phases on the retention of substituted benzene derivatives in reversed-phase liquid chromatography

The capacity factors of substituted benzene derivatives on bonded phases of various chain lengths in the reversed-phase mode have been measured and correlation of retention data between four stationary phases has been investigated by linear regression analysis. As a result, a difference in the retention mechanism between phases of shorter chain length and octadecylsilica was observed. In addition, it is also apparent that the multicombination treatment between retention data and descriptors such as π, HA, HD and σ promises the possibility of predicting the retention of substituted benzene derivatives on various reversed-phase stationary phases.

Elution characteristics of some solutes used to measure the column void time on reversed-phase stationary phases in micro-HPLC

The elution characteristics of some solutes commonly used to measure column void time on three reversedphase stationary phases in micro-HPLC are described. The elution characteristics of sodium nitrie, sodium nitrate, deuterium oxide, acetone and N,N-dimethylformamide were determined by using styrene-divinylbenzene copolymer, octylsilica and octadecylsilica columns with aqueous methanol and/or acetonitrile eluents. The results indicate that sodium nitrite and sodium nitrate are suitable solutes in measuring column void time, and that the elution behavior of deuterium oxide is almost similar to that of both sodium salts but it appears as two peaks on a C-18 column. One of the two peaks is confirmed as “vacancy” by the use of the micro-HPLC/IR direct monitoring system.

Constituents of Schizandra chinensis Baill. XIII. Quantitative analysis of lignans in the fruits of Schizandra chinensis Baill. by high performance liquid chromatography

Lignans in the fruits of Schizandra (syn. Schisandra MICHX.) chinensis BAILL. (Schisandraceae) were quantitatively analysed by high performance liquid chromatography using a reversed-phase stationary phase. It was found that the commercially available samples considered to be collected in central and northern parts in Korea or China contained schizandrin (I), gomisin N (IX) and gomisin A (IV) as main components, while those in the samples collected in Japan were schizandrin (I) and deoxyscizandrin (VII). Seasonal variations of lignan contents in the fruits of S. chinensis collected in Nagano prefecture were also investigated. It became clear that all of the lignans were produced in the seed with its formation and that the contents gradually decreased in the same constituent ratio.

Utilization of micro-HPLC for the direct measurement of the partition coefficients needed in studying the quantitative structure — activity relationships

Utilization of micro-HPLC is described for the direct measurement of the partition coefficients needed in studying the quantitative structure — activity relationships (QSAR). The method involves analyzing the solutes in acetonitrile — water as the mobile phase on commercially available reversed-phase stationary phases.

高速液体クロマトグラフィーによるローヤルゼリー中の10-ヒドロキシ-2-デセン酸の定量

A method for the quantitative determination of 10-hydroxy-2-decenoic acid (10-HDA) in royal jelly was establishd by high-performance liquid chromatography (HPLC) by use of a reversed-phase stationary phase. The 10-HDA extracted with CH2Cl2 from royal jelly was determined by HPLC under the following operating conditions : column, LiChrosorb RP-18 ; mobile phase, 55% methanol in water (pH 2.5) ; monitoring, ultraviolet absorption at 205 nm : internal standard, suberic acid ; temperature, 50°C. This method was simpler and more rapid than gas chromatographic method. It was successfully applied to the determination of 10-HDA contents in raw royal jelly, granules and tablets containing royal jelly.

Separation of ergot alkaloids by reversed-phase liquid chromatography

Reversed-phase of liquid chromatography with isocratic solvent elution is proposed for the separation of ergot alkaloids with a wide range of polarities. The separation properties of ergot alkaloids was studied on Lichrosorb RP-2, RP-8, RP-18 and Spherisorb ODS columns. It has been shown that the reversed-phase stationary phase on silica gel has a marked influence on the separation of ergot alkaloids. Lichrosorb RP-8 and RP-18 have the best separation properties for the complete range of ergot alkaloids with great differences in polarity, while Lichrosorb RP-2 and Spherisorb ODS are more convenient for the separation of polar ergot alkaloids. This analytical method represents a new possibility for the analysis of ergot alkaloids.