Improved analysis by liquid chromatography of iso-alpha acids

This paper shows that the liquid chromatography (LC) methods so far described in the literature for iso-alpha acids analysis are unsatisfactory. The main reasons for this are the failure of the advocated eluent systems to completely suppress trace metal activity of commercial LC columns and the lack of a stable reference iso-alpha acid. The trace metal problem can be solved by using a column packing material specifically designed for “Hop Acid” analysis and by adding ethylenediaminetetraacetate (EDTA) to the eluent. The reference problem is more difficult, but possibly a formulation of the iso-alpha acids in their acid form may be the answer. Chromatographic conditions changing the elution pattern of the individual iso-alpha acids are studied. This leads to better control of their co-elution in the analytical procedure.     

HPLC analysis of flavonoids and phenolic acids and aldehydes inEucalyptus spp.

Summary Standards of the polyphenols occurring in wood, bark and leaf extracts ofEucalyptus spp. (i.e. flavonoids and phenolic acids and aldehydes) have been analyzed by HPLC using reversed phase columns, gradient elution and diode-array detection. The conditions used are reported.     

Potentilla erecta (L.) rhizomes as a source of phenolic acids

The aim of this study was to evaluate the content of major phenolic acids from Potentilla erecta rhiozomes. Water and ethanol-water mixture was used for extraction of these compounds. The extracts were also evaluated for the quantification of total phenolic content and the antioxidant capacity. The contents of phenolic acids and resulting antioxidant activities are dependent on the nature of extracting solvent due to the presence of different antioxidant compounds. Results showed that P. erecta rhiozomes contained high amount of gallic and p-HBA acids. The contents of chlorogenic and protocatechuic acids in the extracts of Potentilla species have not been reported yet. The results suggested that the extracts could be used as the active cosmetics ingredients and nutraceuticals.     

Determination of Organic Acids in Ground Water by Liquid Chromatography/Atmospheric Pressure Chemical Ionization/Mass Spectrometry

ABSTRACT

Current methods of determining organic acids in ground water are labor-intensive, time-consuming and require a large volume of sample (100 milliliter to 1.0 liter). This paper reports a new method developed to determine aliphatic, alicyclic, and aromatic acids in ground water using liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC/APCI/MS). This method was shown to be fast (less than 1 hour), effective, and reproducible, requiring only 1.0 mL of ground-water sample. Ground water was pH-adjusted, filtered through 0.45 μm filters and directly injected into the LC. A binary solvent system consisting of 40 mM of aqueous ammonium acetate and methanol and a C18 column were used for chromatographical separation. The APCI was operated under negative ionization mode. Selected ion monitoring (SIM) was used for detection and quantitation of the analytes. This method was applied to the analysis of organic acids in ground-water samples collected from an aquifer contaminated with JP-4 fuel hydrocarbons at Wurtsmith Air Force Base in Oscoda, Michigan. Aromatic acids identified in the contaminated ground water include o-, m-toluic acids (2- and 3-methylbenzoic acids), 2,6-dimethylbenzoic acid, 2,3,5- and 2,4,6-trimethylbenzoic acids and two additional trimethylbenzoic acids with unknown location of methylation. The detection of aromatic acids in groundwater from the KC-135 site provided evidence for in situ microbial degradation of hydrocarbons occurring in the aquifer.     

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.

Microdetermination of Bile Acids by High-Performance Liquid Chromatography Equipped with a High Sensitive Conductance Detector

Abstract

Conductimetric detection of bile acids in reversed phase high-performance liquid chromatography is described. The solvent system of the mixture of water and organic solvent containing small amount of basic salts such as ammonium carbonate is found useable by removing the cation with the cation exchange column inserted between the ODS column and the conductance detector. Thus, a few ng of tauro-and glyco-conjugated bile acids can be detected without tedious derivatization and hydrolysis.     

Identification of very long chain fatty acids by atmospheric pressure chemical ionization liquid chromatography‐mass spectroscopy from green alga Chlorella kessleri

A method is described for the enrichment of very long chain fatty acids (VLCFAs) from total fatty acids of heterotrophically cultivated green freshwater alga Chlorella kessleri and their identification as picolinyl esters by means of liquid chromatography‐mass spectrometry with atmospheric pressure chemical ionization (LC‐MS with APCI). The method is based on the use of preparative reversed phase HPLC of hundred‐milligram amounts and their subsequent identification by microbore APCI LC‐MS. A combination of these two techniques was used to identify unusual VLCFAs up to C₄₇, both saturated and monounsaturated, with two positional isomers (ω‐9 and ω‐26).     

Comparative study on the liquid chromatographic enantioseparation of cyclic β‐amino acids and the related cyclic β‐aminohydroxamic acids on Cinchona alkaloid‐based zwitterionic chiral stationary phases

The enantiomeric pairs of cis and trans stereoisomers of cyclic β‐aminohydroxamic acids and their related cis and trans cyclic β‐amino acids containing two chiral centers were directly separated on four structurally related chiral stationary phases derived from quinine and quinidine modified with (R,R)‐ and (S,S)‐aminocyclohexanesulfonic acids. Applying these zwitterionic ion‐exchangers as chiral selectors, the effects of the composition of the bulk solvent, the acid and base additives, the structures of the analytes, and temperature on the enantioresolution were investigated. To study the effects of temperature and obtain thermodynamic parameters, experiments were carried out at constant mobile phase compositions in the temperature range 5–50°C. The differences in the changes in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°) were calculated from the linear van't Hoff plots derived from the ln α versus 1/T curves in the studied temperature range. Results thus obtained indicated enthalpy‐driven separations in all cases. The sequence of elution of the enantiomers was determined and found to be reversed when ZWIX(–)™ was changed to ZWIX(+)™ or ZWIX(–A) to ZWIX(+A).     

Is Multidimensional High Performance Liquid Chromatography (HPLC) an Alternative in Protein Analysis to 2D Gel Electrophoresis?

The interactive modes of High Performance Liquid Chromatography (HPLC) of proteins provide a platform for the construction of a multidimensional HPLC system coupled to mass spectrometry. We present a system composed of both anion and cation exchanger columns, in the first dimension, and n‐octadecyl bonded 1.5 μm nonporous silica columns in the second dimension. Both columns are operated under gradient conditions. A system suitability test with standard proteins showed that the total analysis can be performed within about 20 minutes. The fractions taken from the ion exchanger column are directly analyzed within one minute on the reversed phase column at a high flow rate. Two reversed phase columns are applied and operated alternatively: while the first column performs the separation within one minute, the analytes leaving the first dimension are enriched in an on‐column focusing mode on top of the second column. The sample clean‐up and enrichment is performed on a novel type of restricted access cation exchanger column with internal sulfonic acid groups and external diol groups. The columns exhibit a molecular weight exclusion limit for globular proteins of about 15 kDa. Our next studies will be directed towards the analysis of proteins and peptides from extracts of fibroblasts.     

Fully automated reagent peak‐free liquid chromatography fluorescence analysis of highly polar carboxylic acids using a column‐switching system and fluorous scavenging derivatization

In this study, we combined a column‐switching system with a fluorous scavenging derivatization method to develop a fully automated reagent peak‐free LC fluorescence detection protocol for the analysis of highly polar carboxylic acids. In this method, highly polar carboxylic acids were derivatized with fluorescent 1‐pyrenemethylamine in the presence of 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide and 1‐hydroxy‐1H‐benzotriazole. Residual excess of the unreacted reagent was tagged with 2‐(perfluorooctyl)ethyl isocyanate and then removed selectively using a fluorous column‐switching system placed in front of an analytical reversed‐phase column. The signal of the fluorous‐tagged unreacted reagent was completely absent in the resulting chromatograms; therefore, it did not interfere with the quantification of each acid especially those eluted before 20 min. The detection limits (S/N = 3) for the examined acids were in the range from 4.0 to 22 fmol per injection. We have applied this method to comparative analysis of highly polar carboxylic acids in urine samples obtained from diabetes mellitus type‐II model mice and their control.     

Determination of zearalenone and its metabolites in endometrial cancer by coupled separation techniques

This study presents a selective method of isolation of zearalenone (ZON) and its metabolite, α-zearalenol (α-ZOL), in neoplastically changed human tissue by accelerated solvent and ultrasonic extractions using a mixture of acetonitrile/water (84/16% v/v) as the extraction solvent. Extraction effectiveness was determined through the selection of parameters (composition of the solvent mixture, temperature, pressure, number of cycles) with tissue contamination at the level of nanograms per gram. The produced acetonitrile/water extracts were purified, and analytes were enriched in columns packed with homemade molecularly imprinted polymers. Purified extracts were determined by liquid chromatography (LC) coupled with different detection systems (diode array detection - DAD and mass spectrometry - MS) involving the Ascentis RP-Amide as a stationary phase and gradient elution. The combination of UE-MISPE-LC (ultrasonic extraction - molecularly imprinted solid-phase extraction - liquid chromatography) produced high (R ≈ 95–98%) and repeatable (RSD < 3%) recovery values for ZON and α-ZOL.     

Liquid Chromatography of Synthetic Polymers under Limiting Conditions of Insolubility III

Summary Performance was evaluated of silica based commercial monolithic rod-like columns in liquid chromatography of synthetic polymers under limiting conditions of enthalpic interactions (LC LC). LC LC employs the barrier effect of the pore permeating and therefore slowly eluting small molecules toward the pore excluded, fast eluting macromolecules. Phase separation (precipitation) barrier action was applied in present study. The barrier was created either by the narrow pulse of an appropriate nonsolvent injected into the column just before the sample solution (LC LC of insolubility – LC LCI) or by the eluent itself. In the latter case, the polymer sample was dissolved and injected in a good solvent (LC LC of solubility – LC LCS). In LC LCI, polymer species cannot break thru the nonsolvent zone while in LC LCS they cannot enter eluent, which is their precipitant. Therefore, polymer species keep moving in the zone of their original solvent. Macromolecules eluting under the LC LC mechanism leave the column in the retention volume (V_R) roughly corresponding to V_R of the low molar mass substances and can be efficiently separated from the polymer species non-hindered by the barrier action. The known advantages of monoliths were confirmed. From the point of view of LC LCI and LC LCS the most important quality of monolithic columns represents their excellent permeability, which allows both working at high flow rates and injecting very high (in the range of 5%) sample concentrations. Monolithic column tolerate also extremely high molar mass samples (M>10,000 kg · mol⁻¹). On the other hand, the mesopores (separation pores) of the tested monoliths exhibited rather small volume and wide size distribution. These shortcomings partially impair the permeability advantage of monoliths because in order to obtain high LC LC separation selectivity a tandem of several monolithic columns must be applied. Presence of large mesopores also reduces applicability of monolithic columns for molar masses below about 50 kg · mol⁻¹ because V_Rs of polymers eluted behind the barrier are similar to that of freely eluting species. The non- negligible break-thru phenomenon was observed for the very high polymer molar masses largely eluting behind the barrier. It is assumed that the fraction of very large mesopores present in the monoliths or association/microphase separation of macromolecules may be responsible for this phenomenon. This is why the presently marketed SiO₂ monolithic columns are mainly suitable for the fast purification of the LC LC eluting macromolecules from the polymeric admixtures non-hindered by the barrier-forming liquid. Still, monolithic columns have large potential in the LC LCI and LC LCS procedures provided size (effective diameter) of the mesopores can be reduced and their volume increased.     

Sensitive GC/MS determination of 15 isomers of chlorobenzoic acids in accelerated solvent extracts of soils historically contaminated with PCBs and validation of the entire method

Chlorobenzoic acids (CBAs) are the major metabolite of aerobic bacterial degradation of polychlorinated biphenyls (PCBs). A rapid and simple simultaneous derivatisation method has been developed for gas chromatography-mass spectrometry determination in historically PCB-contaminated soils for 15 isomers of mono-, di-, tri-, tetra-, and pentachlorobenzoic acids in CBA mixtures. Two derivatisation agents (diazomethane and methyl chloroformate) and various conditions were evaluated (temperature, time, solvents, catalysts) in terms of efficiency. The optimised derivatisation method with diazomethane and 1% methanol running 1 hour at 5°C was used for derivatisation of extracts of soils and river sediment from historically PCB-contaminated sites; the extracts were prepared using accelerated solvent extraction by a previously described method. Methylated CBAs were separated by gas chromatography using a system with two different common columns, DB-5 and DB-200, in series-coupled (tandem) arrangement and detected by EI-MS. A clean-up with a gel permeation chromatography was carried out to remove soil interfering matrix compounds as well as major portion of PCBs. The limits of quantification ranged between 1 and 10 ng g^?1 of individual CBA in the soil. The procedure was applied to various soil samples from Lhenice (Czech Republic) highly contaminated with PCBs. CBAs were found in all tested soils and also in the river sediment. The most contaminated soil contained all CBAs representatives under the study with a total concentration of 3.1 µg g^?1 of dry soil.     

Separation of phenolic acids in soil and plant tissue extracts by co-electroosmotic capillary electrophoresis with direct UV detection

Summary A capillary zone electrophoretic method for the analysis of phenolic acids in soil and plant extracts was developed with direct UV detection using a phosphate electrolyte solution. The electrophoretic separation required the phenolic acids to be charged at a pH above their pKa in order to achieve their migration towards the anode. Electroosmotic flow (EOF) was reversed in direction by adding tetradecyltrimethylammonium bromide (TTAB). Factors affecting the separation selectivity, including the buffer pH and EOF modifiers, were investigated systematically. Eight phenolic acids were separated and detected in 10 min using an electrolyte containing 25 mM phosphate, 0.5 mM TTAB and 15% acetonitrile (v/v) at pH of 7.20. Linear plots for the test phenolic acids were obtained in a concentration range of 0.01–1 mM with detection limits in the range of 1.0–7.0 μM. The recoveries ranged from 92.8 to 102.3% in soil and plant tissues samples spiked at 100 μM and the relative standard deviation based on the peak area were ranged 2.0 to 4.5%. The proposed method was used for the determination of phenolic acids in plant tissue and soil extracts with direct injection.     

Reversed-phase high-performance liquid chromatography of fatty acid methyl esters with particular reference to cyclopropenoic and cyclopropanoic acids

High performance liquid chromatography of saturated, monounsaturated, diunsaturated, triunsaturated, cyclopropenoic (malvalic and sterculic) and cyclopropanoic (cis-8,9-methylenehexadecanoic and dihydrosterculic) fatty acids was performed with their methyl esters. All separations were carried out with two types of reversed phase columns, the eluent consisting of an acetonitrile/water mixture. The effect of water was studied in the range 0–15%. The best separation was obtained with acetonitrile/water (85:15 v/v). Quantitative results indicated that the detection limits depended upon ultraviolet wavelength and in the present study were 4 ng of methyl sterculate and 125 ng of methyl dihydrosterculate at 195 nm.     

Synthesis and chromatographic evaluation of phenyl/tetrazole bonded stationary phase based on thiol‐epoxy ring opening reaction

A silica‐based reversed‐phase stationary phase bonding with phenyl and tetrazole groups was synthesized by thiol‐epoxy ring opening reaction. The bonded groups could not only provide hydrophobic interaction, but also π–π, hydrogen bonding, electrostatic interactions, and so on. The results of characterization with elemental analysis and solid‐state ¹³C cross‐polarization magic‐angle‐spinning NMR spectroscopy indicated the successful preparation of phenyl/tetrazole sulfoether bonded stationary phase. Chromatographic evaluation revealed that phenyl/tetrazole sulfoether bonded stationary phase behaved well under the reversed‐phase mode. The column parameters (H, S*, A, B, and C) showed different selectivity compared with some typical commercial columns, and it was validated by the separation of estrogen, ginsenoside, alkaloid samples. Based on the different selectivity between phenyl/tetrazole sulfoether bonded stationary phase and C18 columns, phenyl/tetrazole sulfoether bonded stationary phase also showed potential to construct a 2D reversed‐phase liquid chromatography system with C18. And it was verified by the separation of corydalis tuber and curcuma zedoary extracts.     

Determination of caffeoylquinic acids and flavonoids inCynara scolymus L. by high performance liquid chromatography

Summary The main phenolic compounds in dried extracts fromCynara scolymus (artichoke)—monocaffeoylquinic acids, dicaffeoylquinic acid, and flavonoids–have been separated by high-performance liquid chromatography. By use of a narrow bore C₁₈ column and an acidic mobile phase this HPLC method enabled improved separation within 31 min with significantly reduced solvent consumption compared with other methods. The method was validated to demonstrate its linearity, precision, accuracy, and robustness. Twelve commercial samples were analyzed. Monocaffeoylquinic acids were the most abundant phenolic compounds; the amounts present ranged from 0.48 to 4.24%. The amounts of dicaffeoylquinic acids and flavonoids were smaller—from 0.03 to 0.52%. The method is a good combination of efficiency and economy and should be especially useful for commercial applications.     

Potential of Flame Ionization Detection Coupled On‐Line with Microcolumn Liquid Chromatography Using Aqueous Eluents and an Eluent‐Jet Interface

The potential of the on‐line coupling of microcolumn liquid chromatography (μLC) using aqueous eluents with a flame ionization detector (FID) was evaluated. An eluent‐jet interface was modified to allow the efficient introduction of the eluent into the FID. The potential of the method is demonstrated by the μLC–FID determination of lower alcohols and bis(2‐hydroxyethylthio)alkanes on porous and non‐porous stationary phases, respectively. Flow injection analysis (FIA)–FID experiments with highly polar, thermolabile, semi‐volatile and non‐volatile compounds like amino acids, organic acids, alkylphosphonic acids, and carbohydrates showed the developed configuration to be a promising approach for the detection of a wide range of analytes. Compared with a nebulization interface, the eluent‐jet interface showed 4–10 times higher peaks for citric acid. Detection limits by FIA for all compounds were in the range of 0.2–5 ng injected. With ribose as test compound, plots of peak height vs. amount injected showed good linearity (r² > 0.999) in the range of 75–12,000 μg/mL. The repeatability showed relative standard deviations of less than 5%.     

Effects of the operation parameters on Hydrophilic Interaction Liquid Chromatography separation of phenolic acids on zwitterionic monolithic capillary columns

Strongly polar phenolic acids are weakly retained and often poorly separated in reversed-phase (RP) liquid chromatography. We prepared zwitterionic polymethacrylate monolithic columns for micro-HPLC by in situ co-polymerization in fused-silica capillaries. The capillary monolithic columns prepared under optimized polymerization conditions show some similarities with the conventional particulate commercial ZIC-HILIC silica-based columns, however have higher retention and better separation selectivity under reversed-phase conditions, so that they can be employed for dual-mode HILIC-RP separations of phenolic acids on a single column. The capillary polymethacrylate monolithic sulfobetaine columns show excellent thermal stability and improved performance at temperatures 60–80 °C. The effects of the operation conditions on separation were investigated, including the type and the concentration of the organic solvent in the aqueous-organic mobile phase (acetonitrile and methanol), the ionic strength of the acetate buffer and temperature. While the retention in the RP mode decreases at higher temperatures in mobile phases with relatively low concentrations of acetonitrile, it is almost independent of temperature at HILIC conditions in highly organic mobile phases. The best separation efficiency can be achieved using relatively high acetate buffer ionic strength (20–30 mmol L⁻¹) and gradient elution with alternately increasing (HILIC mode) and decreasing (RP mode) concentration of aqueous buffer in aqueous acetonitrile. Applications of the monolithic sulfobetaine capillary columns in alternating HILIC-RP modes are demonstrated on the analysis of phenolic acids in a beer sample.     

Advanced dress-up chiral columns: New removable chiral stationary phases for enantioseparation of chiral carboxylic acids

This paper describes the preparation of new dress-up columns featuring reproducibly removable and replaceable chiral stationary phases. After synthesizing perfluroalkylated quinine and quinidine derivatives as chiral stationary phase compounds (F-CSPs), we adsorbed them reversibly onto a fluorous LC column through pumping of their solutions. Using this dress-up chiral column and fluorophobic elution of aqueous ammonium formate/MeOH mixtures, we could enantioseparate four racemic N-acetyl amino acids, dichlorprop, and sixteen fluorescent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)-derivatized amino acids. Dressing and undressing of the coated F-CSPs could be controlled by varying the fluorophilicity and fluorophobicity of the eluent. The relative standard deviations of the retention times, the retention factors, the number of theoretical plates, the enantioseparation factors, and the resolutions of each of four preparations of such dress-up columns were all less than or equal to 5.26% (from 20 repeated analyses); the reproducibilities from four different preparations were all less than or equal to 10.6%. These columns also facilitated highly sensitive and selective analyses of AQC-amino acids when detected using LC–MS/MS.