Factors affecting the reversed-phase liquid chromatographic separation of polycyclic aromatic hydrocarbon isomers

Reversed-phase liquid chromatography (LC) on C₁₈ stationary phases provides excellent selectivity for the separation of polycyclic aromatic hydrocarbons (PAH). Recent studies have shown that several factors affect selectivity for the LC separation of PAH including phase type (monomeric or polymeric), pore diameter and surface area of the silica substrate, and surface density of the C₁₈ ligands. In this paper the separation of eleven PAH isomers of molecular weight 278 is used to further illustrate the effect of stationary phase characteristics and shape of the solute (length-to-breadth ratio, L/B) on retention and selectivity. Only polymeric C₁₈ phases with a high C₁₈ surface coverage provided separation of all eleven isomers and the elution order of these isomers generally followed increasing L/B values. The effect of solute nonplanarity on reversed-phase LC retention was investigated on both monomeric and polymeric phases using a series of planar and nonplanar PAH pairs. For each solute pair, the nonplanar solute eluted earlier than the planar solute, the largest selectivity factors being observed on the C₁₈ phase with the highest percent carbon load. Based on these studies, a model is proposed to describe the retention of PAH on polymeric C₁₈ phases.     

Column-induced selectivity in separation of polynuclear aromatic hydrocarbons by reversed-phase,high-performance liquid chromatography

Summary Differences in retention time and relative elution order were observed when a standard mixture of 11 PAH was injected on three C-18 columns from different manufacturers under equivalent conditions.Working on a grant from the Swedish Natural Science Research Council.     

Comprehensive two-dimensional high-performance liquid chromatography for the separation of polycyclic aromatic hydrocarbons

A comprehensive two-dimensional HPLC system for the separation of polycyclic aromatic hydrocarbons was developed using a pentabromobenzyl column as the first dimension and two short monolithic C18 columns as the second dimension. The primary column and two secondary columns were coupled by a 10-port 2-position valve. The effluent from the first dimension was repetitively injected into the second dimension every 12 s. Due to its resolution, this technique is a powerful tool for the separation of polycyclic aromatic hydrocarbons in a complex matrix such as environmental samples.     

Gradient reversed-phase high-performance liquid chromatographic separation of naturally occurring retinoids

A gradient reversed-phase high-performance liquid chromatographic technique is described for the facile separation and quantitation of the naturally occurring retinoids: retinoic acid, retinol, and retinyl esters. An octadecylsilane column (Waters mu Bondapak C18) is used, with gradient elution from methanol--water (80:20) (solvent A) to 70% or 100% methanol--tetrahydrofuran (50:50) (solvent B) at 2.0 ml/min; detection is by absorbance at 325 nm. Analysis can be completed, with return to starting conditions, in 25-30 min. The method is inherently flexible: retinyl esters can be eluted as a group, with little resolution, by gradient to 100% solvent B, or mostly resolved by gradient to 70% solvent B; separation of retinoids more polar than retinoic acid can be achieved by use of greater proportions of water in solvent A. The separation of vitamin A compounds from extracts of human, rat, and pig liver and from rat kidney by this technique is described.     

Modern state of high-performance liquid chromatography of polycyclic aromatic hydrocarbons

The review is devoted to the recent studies in the field of high-performance liquid chromatography (HPLC) of polycyclic aromatic hydrocarbons (PAHs). The existing certified techniques, developments of synthesis of new sorbents for separating PAH isomers, and study of the retention mechanism are discussed. It also considers the modern methods of extraction and concentration of PAHs, mainly from environmental objects, which are compatible with subsequent identification by HPLC, as well as the necessity of analytical control of contamination of the environment, food sources, and food supplies with PAHs.     

Selective gas chromatographic separation of polycyclic aromatic hydrocarbons on Bentone 34

Summary Using modified Bentone 34 as a thin layer on glass beads in a short micropacked column at maximum working temperature reasonable retention times of polycyclic aromatic hydrocarbons were achieved up to perylene. Some differences in the elution order were observed as compared with other sorbents.     

Application of reversed-phase high-performance liquid chromatography for the separation of deuterium and hydrogen analogs of aromatic hydrocarbons

Substitution of hydrogen by deuterium in aromatic hydrocarbons can alter various properties significantly. Benzene, toluene, naphthalene, anthracene, phenanthrene, biphenyl and durene are separated from their deuterium analogs by reversed-phase (C₁₈ liquid chromatography with acetonitrile/water mobile phases and ultraviolet detection. Deuterium compounds always elute before the hydrogen analog; possible explanations are given. The elution pattern and retention times of anthracene and phenanthrene were unchanged when D₂O replaced H₂O as the mobile phase component.     

On-site solid phase extraction and coupled-column high-performance liquid chromatographic determination of six polycyclic aromatic hydrocarbons in water

A high-performance liquid chromatographic method for the determination of fluoranthene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, benzo(g,h,i)perylene and indeno(1,2,3-cd)pyrene in water is presented. The analytical procedure consists of on-site solid-phase extraction of the analytes on a selective sample column using a new device, transport and integration of the loaded sample column into a HPLC-system and coupled-column HPLC-analysis. The method allows reliable quantification of the PAHs at the low ng/l-level in particle-free as well as in particle-containing surface water.Dedicated to Professor Dr. Dr. h.c. mult. J. F. K. Huber on the occasion of his 70th birthday     

Determination of polycyclic aromatic hydrocarbons by high-performance liquid chromatography-particle beam-mass spectrometry

In this article, we report a high-performance liquid chromatography-particle beam-mass spectrometric (HPLC-PB-MS) method for the determination of polycyclic aromatic hydrocarbons (PAHs). The PB interface consists of a concentric ultrasonic nebulizer with temperature-controlled desolvation chamber and a three-stage momentum separator. The HPLCPB-MS method showed greater sensitivity for PAHs with molecular weights above 178 than for those PAHs with molecular weights below 178. The percent relative standard deviations for the determination of 0.5 ng chrysene, 1.0 ng dibenzo[a,h]anthracene, 1.0 ng benzo[g,h,i]perylene, and 2.5 ng coronene were 20%, 2.5%, 13.7%, and 6%, respectively. The detection limits at signal/noise = 3 were 0.2 ng for chrysene, 1.0 ng for dibenzo[a,h]anthracene, 0.5 ng for benzo[g,h,i]perylene, and 1.5 ng for coronene.     

Optimization of micellar liquid chromatographic separation of polycyclic aromatic hydrocarbons with the addition of second organic additive

The micellar liquid chromatographic (MLC) separations of polycyclic aromatic hydrocarbons (PAHs) were optimized for three micellar systems, cetyltrimethylammonium chloride (CTAC), dodecyltrimethylammonium chloride (DTAC), and sodium dodecylsulfate (SDS), with 1-pentanol as the only organic additive. A difference in the separation was observed between CTAC and SDS/DTAC. Under each optimized separation conditions, CTAC-modified mobile phase provides the least desirable separation, which is attributed to its longer carbon tail (C16 vs. C12). In addition to 1-pentanol, the main organic additive, a second organic additive (3% 1-propanol) in the micelle-modified mobile phase was found to enhance the resolution of PAH chromatographic peaks. However, the extent of the enhancement varies for the different micellar systems, with the greatest resolution improvement seen for CTAC, and little effect for shorter-tail SDS and DTAC. This study shows the potential use of second organic additive (1-propanol), to the main nonpolar additive (1-pentanol), in facilitating the MLC separation of larger nonpolar compounds.     

Thermodynamic and kinetic characterization of nitrogen-containing polycyclic aromatic hydrocarbons in reversed-phase liquid chromatography

A series of five nitrogen-containing polycyclic aromatic hydrocarbons (NPAHs) was studied on polymeric octadecylsilica using methanol and acetonitrile as the mobile phase. The thermodynamic and kinetic behavior was examined as a function of ring number, annelation structure, and position of the nitrogen atom. The retention factors for the NPAHs are smaller than those for the parent PAHs in methanol, while the converse is true in acetonitrile. The changes in molar enthalpy are relatively comparable in both mobile phases with 1-aminopyrene having values of -5.0 +/- 0.2 kcal/mol in methanol and -6.3 +/- 0.7 kcal/mol in acetonitrile (1 cal = 4.184 J). However, the rate constants from mobile to stationary phase (k(sm)) and from stationary to mobile phase (k(ms)) demonstrate large differences as a function of mobile phase. For example, the rate constants k(ms) for 1-aminopyrene and 4-azapyrene are 675 and 62 s(-1), respectively, in methanol at 303 K. In contrast, the same solutes demonstrate rate constants of 3.47 and 3.9 x 10(-3) s(-1), respectively, in acetonitrile. The activation energies for transfer from mobile phase to transition state (deltaE(double dagger(m)) and from stationary phase to transition state (deltaE(double dagger(s)) also differ as a function of mobile phase. For example, the activation energies deltaE(double dagger(s)), for 1-aminopyrene are 21 and approximately 0 kcal/mol, whereas those for 4-azapyrene are 19 and 23 kcal/mol, in methanol and acetonitrile, respectively. Based on these thermodynamic and kinetic results, the relative contributions from the partition and adsorption mechanisms are discussed.     

Use of Zn-pyrophosphatase in the high-performance liquid chromatographic analysis of cell extracts containing 32P-labelled inositol phosphates.

A simple method is described for eliminating the interference of pyrophosphate and pyrophosphorylated nucleosides in the high-performance liquid chromatographic determination of inositol 1,3,4-triphosphate and inositol 1,4,5-triphosphate of 32P-labelled extracts of cells. Treatment of the extract with pyrophosphatase, but substituting Zn2+ for Mg2+ as the cofactor, converts all nucleoside triphosphates and pyrophosphate to their di- and monoesters. Such change shifts their position in the elution profile, allowing a clear identification and quantification of the inositol phosphates. Typical overall recoveries near 80% or higher of added markers.     

Laterally attached liquid-crystalline polymers as stationary phases in reversed-phase high-performance liquid chromatography. III. Effect of the local anisotropic order on the separation of polycyclic aromatic hydrocarbons

Specific stationary phases based upon non-liquid-crystalline polymers, liquid-crystalline molecules and side-on fixed liquid-crystalline polymers (SO-LCP) have been synthesized for use as silica modified stationary phases in high-performance liquid chromatography (HPLC). The mesogenic side group of the SO-LCP was composed of three phenyl ring benzoate type with terminal alkoxy chains and was laterally linked to a polysiloxane backbone via an alkyl ester spacer arm. This study demonstrated that the shape recognition of stationary phases based upon SO-LCP towards the length-to-breath ratio (L/B) was strongly connected to the existence of a local liquid-crystalline order into the pores of silica gel, warranting the interest of the collective organization of mesomorphic materials in liquid chromatography. Furthermore, the chromatographic performances depended on the kind of anisotropic order and it was more advantageous to use smectic side-on liquid-crystalline polymer than nematic and obviously non-liquid-crystalline ones. Finally, for a series of polymers having the same mesomorphism, the larger the temperature stability range of the mesophase, the more pronounced the local order effect and the higher the shape recognition.     

Determination of polycyclic aromatic hydrocarbons in Diesel soot by high-performance liquid chromatography

Summary The described identification and determination of polycyclic aromatic hydrocarbons (PAH) in Diesel soot is based on a class-fractionation using an open Al₂O₃-column and a following separation on a reversed-phase HPLC-column with post-chromatographic derivatization. The optimized analysis has been applied to the determination of PAH in soots from different engines such as locomotives and motor vehicles by variation of the fuel additives and different Diesel/water emulsions. The results obtained by locomotives show that a special Diesel/water emulsion emits a minor amount of mutagenic and cancerogenic polycyclic aromatic hydrocarbons, whereas the emission of the comparatively harmless compounds like phenanthrene and benzo(h)quinoline increases.     

Chemically bonded phases for the reversed-phase high-performance liquid chromatographic separation of basic substances

A chemically bonded phase with a peptide group (PB) for reversed-phase high-performance liquid chromatography (HPLC) is described. This packing was prepared by a two-stage modification of the surface of silica gel with mono- and trifunctional 3-aminopropylsilane and then with an appropriate derivative of a fatty acid. Packings prepared in this way were compared with standard C18 materials used in HPLC. Surface characteristics of the packings before and after chemical modification were determined by different physico-chemical methods, e.g., porosimetry, elemental analysis, 13C and 29Si cross-polarization magic angle spinning NMR and HPLC. Chromatographic properties of these packings were evaluated by comparison between log k' of one phase and log k' of a second phase for substances with different chemical natures. The PB packing was found to be especially useful for the separation of basic substances.