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.     

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 (C₁₈) 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.     

Determination of column selectivity toward polycyclic aromatic hydrocarbons

An empirical test is described for the evaluation of column selectivity in reversed-phase liquid chromatography. Using a test mixture of three polycyclic aromatic hydrocarbons (PAH), overall column selectivity toward PAH was assessed for over 20 different commercial C₁₈ columns. Retention behavior was correlated to phase type (i.e., monomeric and polymeric surface modification chemistry) for custom synthesized phases. A classification scheme is proposed in which commercial C₁₈ columns are grouped into three classes based on retention behavior: monomeric-like, polymeric-like, and intermediate phase selectivity toward PAH. Correlation of retention behavior of the test mixture with the separation of PAH mixtures and with more general column properties (e.g., phase thickness) is discussed.     

Separation and identification of higher fullerenes in soot extract by liquid chromatography-mass spectrometry

Summary Two-step liquid chromatographic separation (LC) has been applied to soot extract and the identification of higher fullerenes has been accomplished by LC-MS measurements using an ESI interface. The first separation step is preparative-scale LC using a 50 mm i.d. column packed with monomeric octadecylisilica (ODS) because elution is mainly controlled by relative molecular mass. 39 batches of five fractions each were collected and then as the second separation step each fraction was analysed by analytical-scale LC using a conventional column of a polymeric ODS phase which can elute fullerenes according to shape and structure. This stationary phase can also separate many isomers of higher fullerenes, consequently the existence of several higher fullerenes larger than C₈₆ has been confirmed and their UV-Vis spectra were obtained by the photodiode array detection system coupled to the analytical LC.     

Modification of Selectivity in Reversed-Phase Liquid Chromatography of Polycyclic Aromatic Hydrocarbons Using Mixed Stationary Phases

Abstract

Monomeric and polymeric C₁₈ materials provide significantly different selectivities for polycyclic aromatic hydrocarbons (PAH) in reversed-phase liquid chromatography. Selectivity factors vary in a regular manner with respect to surface concentration of C₁₈ groups on different C₁₈ columns. In this study, we investigated the feasibility of “customizing” a C₁₈ column to provide an intermediate selectivity by mixing 5-μm polymeric C₁₈ material from two different lots with high and low C₁₈ surface concentrations. Polymeric C₁₈ materials from different production lots were mixed in ratios of 30/70, 50/50, and 70/30 (w/w). Selectivity factors for these columns were found to be similar to those predicted by the linear addition of the selectivities of the two individual phases. The PAH selectivities on these mixed columns were also found to be comparable to data obtained from coupled short columns of appropriate lengths each containing one of these different C₁₈ materials. These studies indicate that columns of specific selectivity can be prepared by either mixing two different C₁₈ materials or by coupling columns containing each of these different phases. The use of mixed phase columns is illustrated for the analysis of a fraction containing five condensed ring PAH isomers (molecular weight 278) isolated from an air particulate sample.     

Automated clean-up, separation and detection of polycyclic aromatic hydrocarbons in particulate matter extracts from urban dust and diesel standard reference materials using a 2D-LC/2D-GC system

A multidimensional, on-line coupled liquid chromatographic/gas chromatographic system was developed for the quantification of polycyclic aromatic hydrocarbons (PAHs). A two-dimensional liquid chromatographic system (2D-liquid chromatography (LC)), with three columns having different selectivities, was connected on-line to a two-dimensional gas chromatographic system (2D-gas chromatography (GC)). Samples were cleaned up by combining normal elution and column back-flush of the LC columns to selectively remove matrix constituents and isolate well-defined, PAH enriched fractions. Using this system, the sequential removal of polar, mono/diaromatic, olefinic and alkane compounds from crude extracts was achieved. The LC/GC coupling was performed using a fused silica transfer line into a programmable temperature vaporizer (PTV) GC injector. Using the PTV in the solvent vent mode, excess solvent was removed and the enriched PAH sample extract was injected into the GC. The 2D-GC setup consisted of two capillary columns with different stationary phase selectivities. Heart-cutting of selected PAH compounds in the first GC column (first dimension) and transfer of these to the second GC column (second dimension) increased the baseline resolutions of closely eluting PAHs. The on-line system was validated using the standard reference materials SRM 1649a (urban dust) and SRM 1975 (diesel particulate extract). The PAH concentrations measured were comparable to the certified values and the fully automated LC/GC system performed the clean-up, separation and detection of PAHs in 16 extracts in less than 24 h. The multidimensional, on-line 2D-LC/2D-GC system eliminated manual handling of the sample extracts and minimised the risk of sample loss and contamination, while increasing accuracy and precision.

Polycyclic aromatic hydrocarbon 13C/12C ratio measurement in petroleum and marine sediments application to standard reference materials and a sediment suspected of contamination from the Erika oil spill

This paper describes a simple and rapid sample preparation procedure allowing to measure the stable carbon isotopic composition of polycyclic aromatic hydrocarbons (PAHs) in petroleum and in sediments. The aromatic fraction is first purified and isolated on alumina and silica micro-columns. A high-performance liquid chromatography fractionation allows one then to isolate each aromatic family in order to limit coelutions between PAHs. Moreover, this purification step reduces the importance of the unresolved complex mixture which otherwise contribute to the GC-isotope ratio MS background signal. The application of this analytical procedure has allowed one to determined PAH isotopic composition in a reference material crude oil (SRM 1582) and a marine sediment (SRM 1944) with good reproducibility as uncertainties between three independent assays performed were lower than 0.5 per thousand. This analytical procedure has then been successfully applied to confirm the contamination of a sediment by the petroleum product spilled by the Erika tanker after its wreck on 12 December 1999 close to the Atlantic Coast of France.     

Separation of polycyclic aromatic hydrocarbons with a C60 bonded silica phase in microcolumn liquid chromatography

A chemically bonded C₆₀ silica phase was synthesized as a stationary phase for liquid chromatography (LC) and its retention behavior evaluated for various polycyclic aromatic hydrocarbons (PAHs) using microcolumn LC. The results indicate that the C₆₀ bonded phase offers selectivity different from that of octadecylsilica (ODS) bonded phases in the separation of isomeric PAHs. With the C₆₀ phase, PAH molecules having a partial structure similar to that of the C₆₀ molecule, e.g. triphenylene and perylene, were retained longer than with ordinary ODS stationary phases. The results also show that good correlation exists between the retention data with this C₆₀ bonded phase and with C₆₀ itself as the stationary phase.     

Development and validation of a liquid chromatographic method for the determination of hydroxymethylfurfural and alpha-ketoglutaric acid in human plasma

Hydroxymethylfurfural (HMF) and alpha-ketoglutaric acid (KG) have been recently investigated as potential cancer cell damaging agents. We herein report for the first time a validated quantitative assay for their simultaneous determination in human plasma which is amenable to be applied in the future screening of the target compounds in human probands in order to properly design a targeted chemotherapeutic regimen for certain types of malignant tumors.A simple liquid chromatographic method in conjunction to derivatization after a two-step optimized solid phase clean-up procedure is described. The method is based on the reaction of HMF and KG with 2-nitrophenylhydrazine or 2,4-dinitrophenylhydrazine in an aqueous environment. Reaction conditions were studied with respect to pH, reagent volume, reaction temperature and time. Exact testing of such parameters beside careful selection of the mobile phase composition rendered feasible the quantification of the chemically significantly differing analytes along a single chromatographic run. The formed derivatives could be separated isocratically by reversed-phase LC on a C₈-column. Detection in the UV and in the visible range is possible. Results showed good recovery and reproducibility with detection limits (S/N = 3) down to 2 picomoles analyte on column. Resolution of the syn and anti geometric isomers of the HMF and KG derivatives is possible. The isomeric ratio in relation to the reaction pH is discussed.     

Comparison of normal-phase and reversed-phase gravity-flow column methods for provitamin A determination

Summary Considering the high cost of HPLC, the use of preparative C₁₈ packing material as stationary phase in classical gravity-flow columns is proposed as an alternative for developing countries. In the present study, three methods were compared, one utilizing a normal-phase MgO: HyfloSupercel column, developed with increasing concentration of acetone in petroleum ether and two utilizing C₁₈ reversed-phase columns developed with acetonitrile-methanol-chloroform (RP-C₁₈ method) or with decreasing concentration of water in acetone (modified RP-C₁₈ method). Percent recoveries of -carotene from the columns were 98, 96 and 98% and of the entire analysis (-carotene added to kale) 92, 89 and 92%, for the normal phase, RP-C₁₈ and modified RP-C₁₈ methods respectively. No significant difference was observed in the provitamin A contents (exclusively -carotene) of two leafy vegetables determined by the modified reversed-phase and normal-phase methods; however, significantly lower values were obtained by the RP-C₁₈ method. The situation became more complicated when samples containing other provitamins were analyzed. For squash both types of stationary phase could separate - and -carotene; however, for tomato and red-fleshed papaya, part of lycopene remained mixed with -carotene in both reversed-phase columns. Although -cryptoxanthin was separated in the modified RP column, it was also mixed with lycopene in the RP column. For all samples, the normal-phase column demonstrated much better separation.     

Evaluation of Pentaproline-Based Chiral Stationary Phase by LC

A pentaproline-based chiral stationary phase was prepared and the selectivity of the column was evaluated with 194 racemic compounds in three mobile phase modes: normal-phase mode, polar organic mode and reversed-phase mode. 94 racemates out of 194 were separated and the normal-phase mode proved to be the separation mode of broadest applicability. The column is stable in all common organic solvents and no degradation in column performance was observed in any mode even after more than 1,000 injections. A brief sample loading test was performed on the 250 mm × 4.6 mm column and 13.2 mg of α-methyl-9-anthracenemethanol was baseline separated. Retention behavior in the normal-phase mode and the effect of analyte structure on retention and enantioselectivity are discussed.

     

Determination of vitamins in food-matrix Standard Reference Materials

In recent years, the National Institute of Standards and Technology (NIST) has developed several food-matrix Standard Reference Materials (SRMs) characterized for vitamins and other organic nutrients. NIST uses several "modes" for assignment of analyte concentrations in SRMs, one of which includes the use of data provided by collaborating laboratories. Certification modes and liquid chromatographic methods that were used by NIST for value assignment of vitamin concentrations in recently introduced food-matrix SRMs are described in this paper. These materials and methods include vitamins D and E in coconut oil (SRM 1563) by gravimetry and multi-dimensional liquid chromatography (LC); vitamins A, E, and several B vitamins by reversed-phase LC and vitamin C by ion-exchange chromatography in infant formula (SRM 1846); and carotenoids and vitamins A and E by reversed-phase liquid chromatography in a baby food composite (SRM 2383).     

Normal-phase high performance liquid chromatographic separation and characterization of short- and long-chain triacylglycerols

Summary Short- and long-chain triacylglycerols (SLCT) are a family of lipids prepared by chemical or enzymatic interesterification of triacetin, tripropionin and/or tributyrin, and long-chain (C_16!18) hydrogenated vegetable oils. In this study, a normal-phase cyanopropyl high-performance liquid chromatographic (HPLC) method was developed for the separation and quantification of SLCT. The method is capable of separating SLCT mixtures, free fatty acids and the neutral lipid classes of saturated long-chain triacylglycerols, diacylglycerols and monoacylglycerols. To characterize the specific SLCT classes, a normal-phase HPLC procedure using a non-modified silica column was developed to separate the SLCT into individual isomers based on total carbon number and position of fatty acids on the glycerol backbone. Online coupling with a mass detector (LC/MS) allowed the identification of the individual triacylglycerol structures.     

Preparative isolation and dual column high-performance liquid chromatography of ginkgolic acids from Ginkgo biloba

A chromatographic procedure for the preparative isolation of six different 6-alkylsalicylic acids (syn. ginkgolic acids) with as alkyl substituents C13:0, C15:0, C15:1, C17:1, C17:2 and, tentatively C17:3 from Ginkgo biloba leaves was developed. The procedure consisted of a combination of normal-phase, reversed-phase and argentation chromatography. The compounds were characterised by means of UV, ¹H-NMR and ¹³C-NMR spectroscopy, and mass spectrometry after silylation. A 15 cm C₁₈ RP-HPLC column connected in series with a 20 cm silver(I) loaded cation exchanger HPLC column in combination with the solvent methanol–water (93:7) acidified with 0.1% formic acid was capable of separating the ginkgolic acids C13:0, C15:1, C17:2, C15:0 and C17:1 within 21 min on an analytical scale. The separation is based on a combination of reversed-phase mechanisms and double bond complexation. Detection took place by UV at 311 nm. The separation is a good starting point for the development of a quantitative procedure for the five major ginkgolic acids in Ginkgo leaves and standardised extracts.     

An improved method for determination of light hydrocarbons in stabilized crude oil

Summary This paper reports a quick, and simple method for quantitative determination of C₂ to C₆ hydrocarbons in stabilized crude oil without using a back flush system. A mixture of crude oil and internal standard is injected into a GC equipped with a 6 meter length of fused silica capillary as a guard column. The light hydrocarbons are separated individually up to the last peak of the hexane group with the heavier components trapped in the guard column. The total analysis time for each sample is 15 minutes. The base line is table for up to 15 consecutive analyses. The guard column and the injector port are then reconditioned by simply heating them for one hour at 300 °C.     

The efficient analysis of neutral and highly polar pharmaceutical compounds using reversed-phase and ion-exchange electrochromatography

Summary Highly polar compounds, such as tricyclic antidepressants are very difficult to analyse by electrochromatography with conventional reversed-phase silica-based chromatography packings. At high pH (high electroosmotic flow) the test compounds were not eluted from a Spherisorb ODS-1 column, as a result of strong interactions between the analyte and residual silanol groups on the packing material. By lowering the pH of the mobile phase, whereby the highly basic test compounds become positively charged, it was possible to elute the samples but only with severe peak tailing. Because the electroosmotic flow was greatly reduced, the elution time for neutral species became prohibitively long. By use of a strong cation exchanger in place of C₁₈-silica it was found possible to resolve a series of highly basic compounds with very high efficiencies, with very little evidence of peak tailing. Plate numbers in excess of 8 million per metre were observed.     

Synthesis of Petroleum Polymeric Resin by Cationic Polymerization of the C9 Fraction

Cationic oligomerization of the C₉ fraction of liquid pyrolysis products of petroleum raw materials, catalyzed by aluminum trichloride diphenyl ether complex, was studied, and a procedure was developed for preparation of a petroleum polymeric resin and petroleum polymerizate to be used as film-forming agents in paint-and-varnish materials.     

Separation mechanism of chiral compounds in chiral stationary phase liquid chromatography

In this paper, the concept of reversed- or normal-phase chiral stationary phase liquid chromatography has been put forward according to the polar strength of mobile and stationary phases. The statistical model developed in HPLC has been used to investigate the separation mechanism of D- and L-enantiomer in chiral stationary phase liquid chromatography. It has been observed that the variation of capacity factor of enantiomers with mobile phase composition in both reversed-phase and normal-phase chiral stationary phase liquid chromatography can be described by the fundamental elution equation lnk' = a + blnC_b + _cC_b. The effect of mobile phase composition on the selectivity of enantiomers D and L in normal-phase chiral stationary phase liquid chromatography can be described by the equation lnα = Δa + ΔblnC_b, but in reversed-phase chiral stationary phase liquid chromatography the selectivity is almost independant of the mobile phase composition.     

LC-GC analysis of the aromatics in a mineral oil fraction: Batching oil for jute bags

Aromatics of a C₁₅? C₃₀ mineral oil fraction, the “batching oil” used for producing jute fabrics, were analyzed for estimating the toxicity of oil transferred from jute bags to foods. Group-type pre-separation according to ring systems occurred on a LC NH₂ column with pentane and small additions of methanol as eluent. LC fractions were characterized by LC-GC-MS and quantitated by LC-GC-FID. The oil contained 23% aromatics and 1% “polars”; some 99.5% of the aromatics were alkylated.     

Direct determination of surfactants on textiles by laser desorption—Fourier transform mass spectrometry

Laser desorption—Fourier transform mass spectrometry was utilized for the determination and quantification of mixtures of ionic surfactants, both neat and from textiles. Sodium dodecyl sulfate and the perdeuterated standard were desorbed from cotton cloth and quantified over a standard: analyte ratio range of 50. A neat C₁₀-C₁₄ sodium alkyl benzenesulfonate homolog mixture was also assayed, and selective analyte precipitation on the copper probe from which desorption occurs was found to be a significant factor in causing component discrimination.