Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation

Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7 μm average particle size were assessed for the fast and sensitive analysis of porphyrins in clinical materials. The fastest separation was achieved on an Agilent Poroshell C(18) column (2.7 μm particle size, 50 × 4.6 mm i.d.), followed by a Thermo Hypersil Gold C(18) column (1.9 μm particle size, 50 × 2.1 mm i.d.) and the Thermo Hypersil BDS C(18) column (2.4 μm particle size, 100 × 2.1 mm i.d.). All columns required a mobile phase containing 1 m ammonium acetate buffer, pH 5.16, with a mixture of acetonitrile and methanol as the organic modifiers for optimum resolution of the type I and III isomers, particularly for uroporphyrin I and III isomers. All UHPLC columns were suitable and superior to conventional HPLC columns packed with 5 μm average particle size materials for clinical sample analysis.     

Use of small diameter WCOT columns for ultra-high resolution GC/MS

For clinical and environmental analyses utilizing capillary gas chromatography/mass spectrometry (GC/MS), increased sensitivity and speed of analysis are highly desirable. These performance advantages are realized using a WCOT column of 100 μm i.d. as compared to the more conventional 200 μm i.d. capillary columns. The improved sensitivity of capillary direct GC/MS with the 100 μm i.d. column for the confirmation of drugs of abuse will be demonstrated. For environmental analysis, the superior efficiency and resolution of the 100 μm i.d. column can be employed for the separation of priority pollutants. This approach is more amenable to capillary direct GC/MS providing a more effective interface to the mass spectrometer. As a result improved sensitivity and a considerable decrease in analysis time is achieved over that obtained with the larger diameter environmental specialty phase columns.     

Analysis of tocopherols in margarine by on-line HPLC–HRGC coupling

Tocopherol analysis in margarine is usually carried out by HPLC after saponification of the sample and extraction of the vitamin compounds; these steps consume both time and solvents. In this paper we propose an on-line HPLC–HRGC coupling method, which allows us to simplify the preparation of the analytical sample. The sample of margarine is solubilized in hexane in an ultrasonic bath in the dark; the filtered solution is then injected into the liquid chromatograph using a normal phase microbore column eluted with hexane–isopropanol 99.8:0.2. The α-tocopherol, which is eluted with some wax esters, is transferred on-line to the gas chromatograph, using a loop-type interface with the concurrent eluent evaporation and solvent vapor exit, thus it is separated from interfering compounds and determined using an Alltech RSL 300 column (22 m × 0.25 μm i.d., 0.2 μm film thickness). The β, γ, and δ–tocopherols are determined in the same LC run, using fluorimetric detection. The analysis was carried out in 50 min.     

Free release static coating of glass capillary columns; rapid coating at lower temperatures and elevated pressures

The coating speed upon static coating of glass capillary columans was evaluated in terms of inner diameter and length of the column, viscosity and pressure of solvent vapor, etc. From the equation obtained it can be shown that a smaller diameter of a microbore column restricts solvent vapor transfer to the orifice of the column drastically. To compensate for this restriction, a higher pressure at the meniscus is needed. As an alternative to using a higher coating temperature, application of more volatile solvents such as n-butane and isobutane is proposed. Several glass capillary columns (130 μm i.d.) were coated with SE-54 dissolved inpentane-acetone mixed with n-butane or isobutane and the column performances were evaluated. Selection of these solvents permitted free release static coating of ca. 100 μm columns at lower or even ambient temperatures and they were equally suitable as commonly applied solvents (e.g. pentane) to coat highly efficient columns.     

Cyclodextrin derivatives in GC separation of racemic mixtures of volatiles – part XII: Thick-film wide-bore columns for enantiomer GC preparation

Preliminary results on thick-film wide-bore (0.53mm i.d.) columns for GC preparation of pure enantiomers are described. In particular the loading capacity for several racemates of a 3μm, 30% 2,6-di-O-methyl-3-O-Pentyl-β-CD/OV1701 column and of a 2 μm, 30% 2,3-di-O-acetyl-6-O-dimethyl-t-butylyl-β-CD/PS-086 collumn were determined. Consideration is also given to the relation-ship between resolution values of two enantiomers on anayltical columns and their loading capacity and scaling-up to the corresponding micropreparative columns.     

Cyclodextrin Derivatives in GC Separation of Racemic Mixtures of Volatiles. Part XIV: Some Applications of Thick-Film Wide-Bore Columns to Enantiomer GC Micropreparation

Representative examples of preparative GC isolation of pure enantiomers in amounts ranging from 0.5 to 2 mg, with thick-film wide-bore open tubular columns coated with cyclodextrin (CD) diluted in polysiloxanes, are described. Two 25 m×0.53 mm i.d. columns, coated with 3 μm of 30% 2,6-di-O-methyl-3-O-pentyl-β-CD/OV-1701 and 2 μm of 30% 2,3-di-O-acetyl-6-O-tert- butyldimethylsilyl-β-CD/PS-086 as stationary phases, were used. Methyl 3-hydroxyhexanoate, methyl 2-methylbutanoate, δ-hexalactone, δ-octalactone, and γ-decalactone (the latter from a nature-identical mango aroma), were submitted to automated micropreparative GC. Average yields of about 30% were achieved when amounts above 20 μg/μl were injected, and an enantiomeric purity above 90% was obtained.     

Large Volume Injection in Fast Gas Chromatography with On‐Column Injector

In this work a fast gas chromatography set‐up with on‐column injection was optimized and evaluated with a model mixture of C₈–C₂₈ n‐alkanes. Usual injection volumes when using narrow‐bore (e. g., 0.1 mm i.d.) analytical columns are ca. 0.1 μL. The presented configuration allows introduction of 10–30‐fold larger sample volumes without any distortion of peak shapes. In the set‐up a normal‐bore retention gap (1 m×0.32 mm i. d.) was coupled to a narrow‐bore (4.8 m×0.1 mm i. d.×0.4 μm film thickness) analytical column using a low dead volume column connector. The effects of the experimental conditions such as inlet pressure, sample volume, initial injection temperature, and oven temperature on a peak focusing are discussed. H‐u curves for helium and hydrogen are used to compare their suitability for high speed gas chromatography and to show the dependence of separation efficiency on the carrier gas velocity at high inlet pressures. In the fast gas chromatography system a baseline separation of C₁₀–C₂₈ n‐alkanes was achieved in less than 3 minutes.     

Dual-column GC analysis of mediterranean fish for ten organochlorine pesticides and sixty two chlorobiphenyls

The simultaneous analysis of α-HCH, β-HCH, γ-HCH, HCB, p,p′-DDD, p,p′-DDT, p,p′-DDE, o,p′-DDT, mirex, dieldrin and 62 chlorobiphenyl congeners on two parallel capillary GC columns of different polarity is described for nine Mediterranean fish species. Ten commercially available columns with stationary phases completely characterized in respect of their PCB elution patterns were considered for dual-column GC-ECD analysis. The combination of a 60 m × 0.25 mm i.d. column coated with a 0.25 μm film of 50% diphenyl dimethylsiloxane and a series combination of a 25 m × 0.25 mm i.d. column coated with a 0.25 μm film of 5% diphenyl dimethylsiloxane with a 25 m × 0.22 mm i.d. column coated with a 0.10 μm film of 1, 10-dicarba-closo-dodecarborane dimethylpolysiloxane furnished the highest number of separated chlorobiphenyl congeners (104). The dual-column GC system performed with high stability and reproducibility over a broad concentration range (1–3000 ng/g lipid) of the organochlorine compounds in the investigated fish.     

Automated at-column injection into narrow bore capillary GC columns

An injector designed for automatic direct liquid injection into narrow bore capillary GC columns has been constructed and evaluated. The tip of the syringe needle is aligned with, and positioned close to, the column entrance in a small, pressurized cavity: when the sample is dispensed it is immediately forced into the column by the action of the surrounding carrier gas. A standard autosampler equipped with a standard stainless steel syringe needle was utilized for at-column sample transfer into 100 μm i.d. columns. RSD values for n-alkanes were between 0.1 and 0.3% for relative area counts and approximately 1% for absolute area counts.     

Preparation,evaluation, and comparison of wide bore (320 μm) and narrow bore (50 μm) cyanosilicone-coated capillary columns for gas chromatography

The preparation of wide bore (320 μm) and narrow bore (50 μm) fused silica capillary columns is described for immobilized cyanopropyl substituted silicones containing 60 and 88% substitution. The effect of high temperature deactivation with cyanopropylcyclosiloxanes was studied with a special test mixture. Curing was achieved with dicumyl peroxide or azo-tert-butane. The columns were evaluated and compared in terms of efficiency, activity, polarity, and temperature stability. Different coating methods were compared for the narrow bore columns. The activity of the 60% cyanopropyl columns that had been immobilized with dicumyl peroxide was significantly larger than for azo-tert-butane immobilized columns. The polarity of polar columns appeared to depend greatly on column temperature and is completely different for wide and narrow bore columns.     

Capillary GC on 50 micrometer I.D. Columns coated with thick films. Theory and selected practical results

The chromatographic performance of 50 μm internal diameter (i.d.) fused silica columns coated with up to 2 μm films of immobilized SE-54 (methyl phenyl (5%) silicone) is evaluated under gas chromatographic conditions. The influence of pressure drop on the plate height is discussed. In comparison to thick film 250–530 μm i.d. columns, much higher efficiencies and faster analyses are obtained. Practical examples, performed on a standard GC instrument, illustrate the features of thick film 50 μm i.d. columns.     

At-column injector for capillary GC — design and evaluation

The present paper describes constructional details and evaluations of an at-column injector for capillary GC. Injections were made via a sample loop on a 0.32 mm i.d. capillary column. Two rotary valves were employed to allow a wash of the sample loop and a backflush of the transfer line. Repeatability, calculated from absolute area counts for n-alkanes was between 0.3–1% RSD, for injected sample volumes between 5 and 100 μl. Promising results were also obtained with syringe-based injections on narrow bore (100 μ i.d.) columns. Repeatability on the basis of normalized area counts was in the order of 0.1–0.2% RSD, while solvent tailing was practically absent.     

Positional isomer differentiation of synthetic cannabinoid JWH‐081 by GC‐MS/MS

Like many new designer drugs of abuse, synthetic cannabinoids (SC) have structural or positional isomers which may or may not all be regulated under law. Differences in acute toxicity may exist between isomers which impose further burden in the fields of forensic toxicology, medicine and legislation. Isomer differentiation therefore becomes crucial from these standpoints as new designer drugs continuously emerge with just minor positional modifications to their preexisting analogs. The aim of this study was to differentiate the positional isomers of JWH‐081. Purchased standard compounds of JWH‐081 and its positional isomers were analyzed by gas chromatography‐electron ionization‐mass spectrometry (GC‐EI‐MS) first in scan mode to investigate those isomers who could be differentiated by EI scan spectra. Isomers with identical or near‐identical EI spectra were further subjected to GC‐tandem mass spectrometry (MS/MS) analysis with appropriate precursor ions. EI scan was able to distinguish 3 of the 7 isomers: 2‐methoxy, 7‐methoxy and 8‐methoxy. The remaining isomers exhibited near‐identical spectra; hence, MS/MS was performed by selecting m/z 185 and 157 as precursor ions. 3‐Methoxy and 5‐methoxy isomers produced characteristic product ions that enabled the differentiation between them. Product ion spectrum of 6‐methoxy isomer resembled that of JWH‐081; however, the relative ion intensities were clearly different from one another. The combination of EI scan and MS/MS allowed for the regioisomeric differentiation of the targeted compounds in this study. Copyright © 2015 John Wiley & Sons, Ltd.     

High speed capillary GC on 10 m × 100 μm i.d. FSOT Columns

The chromatographic performance of 10 m × 100 μm i.d. capillary columns, coated with different stationary phases, was studied. TZ-ū plots were measured at different temperature program rates. The polarity and selectivity are strongly influenced by the temperature program rate. The analysis time depends on the capacity ratio and the selectivity factor; stationary phase selection thus is an important aspect in high speed capillary GC. A number of applications is presented and the applicability of split injection is discussed.     

Kinetic investigation of the relationship between the efficiency of columns and their diameter

Many brands of packing materials made of fine particles are now available in both conventional (4.6 mm i.d.) and narrow-bore (2.1 mm i.d.) columns. It is a general observation that the efficiency of the former tends to be markedly higher than that of the latter. This report provides a detailed illustration of the characteristics of this enigma. The corrected reduced plate heights of three brands of columns packed with shell particles in 4.6 and 2.1 mm I.D. columns were measured. The brands were the 1.7 and 2.6 μm Kinetex-C(18) (Phenomenex, Torrance, CA, USA), the 2.7 μm Poroshell120-C(18) (Agilent Technologies, New Castle, DE, USA), and the 2.7 μm Halo-C(18) (Advanced Material Technologies, Wilmington, DE, USA). The extra-column contributions were minimized by optimizing the configuration of the instrument (injection volume <1.0 μL, 115 μm needle seat capillary, 80 μm connecting tubes, no heat exchanger, 0.8 μL detection cell). The correct peak variances were derived from the numerical integration of the first and second order moments of the experimental band profiles. These experimental results confirm that the kinetic performance of narrow-bore columns is inferior to that of conventional columns for all three brands of shell particles. We demonstrate that this difference is accounted for by a contribution to the column HETP of the long-range eddy diffusion term that is larger in the 2.1 than in the 4.6 mm I.D. columns. While the associated relative velocity biases are of comparable magnitude in both types of columns, the characteristic radial diffusion lengths are of the order of 100 and 40 μm in the wall regions of narrow-bore and conventional columns, respectively.     

Rapid determination of alkylphenols in aqueous samples by in situ acetylation and microwave-assisted headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry

A rapid and solvent‐free procedure for the determination of 4‐tert‐octylphenol and 4‐nonylphenol isomers in aqueous samples is described. The method involves in‐situ acetylation and microwave‐assisted headspace solid‐phase microextraction prior to their determination using gas chromatography–ion trap mass spectrometry operated in the selected ion storage mode. The dual experimental protocols to evaluate the effects of various derivatization and extraction parameters were investigated and the conditions optimized. Under optimized conditions, 300 μL of acetic anhydride mixed with 1 g of potassium hydrogencarbonate and 2 g of sodium chloride in a 20 mL aqueous sample were efficiently extracted by a 65 μm polydimethylsiloxane‐divinylbenzene fiber that was located in the headspace when the system was microwave irradiated at 80 W for 5 min. The limits of quantitation were 5 and 50 ng/L for 4‐tert‐octylphenol and 4‐nonylphenol isomers, respectively. The precision for these analytes, as indicated by relative standard deviations, were less than 8% for both intra‐ and inter‐day analysis. Accuracy, expressed as the mean extraction recovery, was between 74 to 88%. A standard addition method was used to quantitate 4‐tert‐octylphenol and 4‐nonylphenol isomers, and the concentrations ranged from 120 to 930 ng/L in various environmental water samples.     

Determination of eflornithine enantiomers in plasma by precolumn derivatization with o‐phthalaldehyde‐N‐acetyl‐l‐cysteine and liquid chromatography with UV detection

A bioanalytical method for indirect determination of eflornithine enantiomers in 75 μL human plasma has been developed and validated. l ‐ and d ‐eflornithine were derivatized with o‐phthalaldehyde and N‐acetyl‐L‐cysteine to generate diastereomers which were separated on two serially connected Chromolith Performance columns (RP‐18e 100 × 4.6 mm i.d.) by a isocratic flow followed by a gradient flow for elution of endogenous compounds. The diastereomers were detected with UV (340 nm). The between‐day precisions for L‐ and D‐eflornithine in plasma were 8.4 and 2.3% at 3 μm , 4.0 and 5.1% at 400 μm , and 2.0 and 3.7% at 1000 μm . The lower limit of quantification was determined to be 1.5 μm , at which precision was 14.9 and 9.9% for L‐ and D‐eflornithine, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Structural transformation among the aggregate forms of bacteriochlorophyll c as determined by electronic-absorption and NMR spectroscopies: dependence on the stereoisomeric configuration and on the bulkiness of the 8-C side chain

Transformation among the aggregate forms of bacteriochlorophyll (BChl) c characterized by the wavelength of the Qy absorption, i.e. the dimer (B675), B705, B720 and B745, was traced by electronic-absorption spectroscopy for each of the isomers including R[E,E], R[P,E], R[I,E], S[P,E] and S[I,E] suspended in the mixtures of methylene chloride and n-hexane. A combination of NMR spectroscopy determining the structural motifs and calculation of the shift of the Qy absorption reflecting the long-range transition dipole-transition dipole interactions among the macrocycles in the entire aggregate structures proposed the following models: B705d (B705d'), a linear array of straight (inclined) columns consisting of a pair of the piggyback dimers; B720d and B745d, an assembly of two and five shifted-inclined columns consisting of more than six piggyback dimers; and B720m and B745m, an assembly of one and two parallel stepwise stacking of approximately 30 monomers. Calculations of the steric energies rationalized two different pathways of transformations: the dimer-->B705d (B705d')-->B720d-->B745d for the R isomers; and the monomer-->(B720m)-->B745m for the S isomers. Addition of S[I,E] seems to trigger the B745d-->B745m transformation of the R isomers.     

Effect of monomer mixture composition on structure and chromatographic properties of poly(divinylbenzene-co-ethylvinylbenzene-co-2-hydroxyethyl methacrylate) monolithic rod columns for separation of small molecules

Porous poly(divinylbenzene-co-ethylvinylbenzene-co-2-hydroxyethyl methacrylate) monoliths were synthesized via thermally initiated free-radical polymerization in confines of surface-vinylized glass columns (150 mm × 3 mm i.d.) and applied to the reversed-phase separation of low-molecular-weight aromatic compounds. In order to compensate for the polymer shrinkage during the synthesis and prevent the monolith from detachment from the column wall, polymerization was conducted under nitrogen pressure. The reaction proceeded at 60°C for 22 h. 2,2'-Azo-bis-isobutironitrile was used as the initiator and 1-dodecanol was used as the porogen. A series of monoliths with different monomer ratios were obtained. All the monoliths had high specific surface areas ranging from 370 to 490 m(2)/g. In the studied range of monomer mixture compositions, the mechanical stability of the stationary phase in water/acetonitrile eluents was found to be high enough and practically insensitive to the fraction of 2-hydroxyethyl methacrylate (HEMA). Increasing the molar fraction of HEMA from 10.5% to 14.7% resulted in the decrease of column permeability by two orders of magnitude (from 1.1×10(-12) to 1.8×10(-14) m(2)) and led to weaker retention of alkylbenzenes. The higher HEMA content was shown to reduce the plate height of the columns in the separation of small molecules from 160-490 μm to 40-76 μm. This was attributed mainly to the decrease of the domain size of the monoliths leading to lower eddy dispersion and mass transfer resistance in the column.     

The use of glass and fused silica open tubular columns for the separation of structural,configurational, and optical isomers by selective complexation GC: Sec-alcohols and ketones

The use of glass and fused silica open tubular columns for selective separation of structural, configurational, and optical isomers (enantiomers) of underivatized aliphatic alcohols and ketones by complexation gas chromatography on optically active metal chelates is described. The method represents a powerful analytical tool for the precise determination of diastereomeric (d.e.) and enantiomeric (e.e.) excesses in asymmetric syntheses and in natural product characterization. The direct screening of the enantiomeric composition and absolute configuration of terpinene-4-ol, the aggregation phermone of Polygraphus poligraphus (L), is described.