Influence Of Organic Modifiers On The Retention Characteristics Of Graphitized Carbon Column

Abstract

The retention time of α-(1,1,3,3-tetramethylbutyl)phenyl ethylene oxide oligomer surfactants was determined on a porous graphitized carbon column (PGC) in eluents containing acetonitrile, methanol, ethanol, 2-propanol, tetrahydrofuran, dioxane and 2-ethoxyethanol as organic modifier. Linear relationships were calculated between the logarithm of the capacity factor and the concentration of organic modifier in the eluent. Ethylene oxide oligomers were well separated on the PGC column in acetonitrile-water eluent mixtures. Oligomers were not separated in eluents containing 2-propanol, tetrahydrofuran. dioxane and 2-ethoxyethanol as organic modifier. This result was explained by the supposition that the bulky organic modifiers occupy the active adsorption centers on the PGC surface decreasing in this manner the separation efficiency of the support.     

Application of silica-based hyper-crosslinked sulfonate-modified reversed stationary phases for separating highly hydrophilic basic compounds

The separation and determination of hydrophilic basic compounds are of great importance in many fields including clinical and biological research, pharmaceutical development and forensic analysis. However, the most widely used analytical separation technique in these disciplines, reversed-phase liquid chromatography (RPLC), usually does not provide sufficient retention for several important classes of highly hydrophilic basic compounds including catecholamines, many drug metabolites and many drugs of abuse. Commonly eluents having little or no organic modifier and/or strong ion pairing agents must be used to achieve sufficient retention and separation. Use of highly aqueous eluents can lead to column failure by dewetting, resulting in poor retention, low selectivity and irreproducibility and slow recovery of performance. The use of a strong ion pairing agent to increase retention renders the separation incompatible with mass spectrometric detection and complicates preparative separations. This paper describes the successful applications of a novel type of silica-based, hyper-crosslinked, sulfonate-modified reversed stationary phase, denoted as (-)SO(3)-HC-C(8)-L, for the separation of highly hydrophilic cations and related compounds by a hydrophobically assisted cation-exchange mechanism. Compared to conventional reversed-phases, the (-)SO(3)-HC-C(8)-L phase showed significantly improved retention and separation selectivity for hydrophilic amines. Concurrently, due to the presence of both cation-exchange and reversed-phase retention mechanisms and the high acid stability of hyper-crosslinked phases, the separation can be optimized by changing the type or concentration of ionic additive or organic modifier, and by varying the column temperature. In addition, gradients generated by programming the concentration of either the ionic additive or the organic modifier can be applied to reduce the analysis time without compromising resolution. Furthermore, remarkably different chromatographic selectivities, especially toward cationic solutes, were observed upon comparing the (-)SO(3)-HC-C(8)-L phase with conventional reversed-phases. We believe that the combination of these two types of stationary phases will be very useful in two-dimensional liquid chromatography.     

Towards compound-independent calibration for organic compounds using online isotope dilution mass spectrometry

Isotope dilution mass spectrometry (IDMS) can be considered a primary measurement method directly traceable to the International System of Units (SI). This measurement technique is increasingly employed in routine laboratories, owing to its unequalled analytical performance, precision and ease of accreditation. Unfortunately, for the adequate application of IDMS, several isotopically labelled standards, corresponding to the compounds of interest, are required. Additionally, when the enriched isotope is continuously added after a chromatographic separation, and an elemental ion source is used, it allows quantification of the different analytes being eluted from the column without requiring specific standards for each compound (online IDMS). In this article, we discuss how the traditional applicability of online IDMS for elemental speciation can be dramatically expanded by using carbon isotope tracers, oxidation or combustion reactions and a conventional molecular ion source. With such a strategy every carbon-containing compound being eluted from a chromatography system can be quantified without the need for specific standards as long as quantitative combustion/oxidation and complete elution occur. So far, only gas chromatography–combustion–mass spectrometry applications have been described, but recent results indicate the great possibilities of extending this novel approach to the quantification of organic compounds after separation by liquid chromatography.     

Capillary electrophoresis time-of-flight mass spectrometry of an opium powder

Summary Intensive work has been invested in recent years to evaluate the performance of capillary electrophoresis (CE) in forensic analysis. Tremendous progress has also been achieved in interfacing CE to sensitive and specific detection systems such as the mass spectrometer (MS). We have recently developed an electrospray time-of-flight mass spectrometer (ESI-TOFMS) for use as a detector for fast and efficient liquid phase separations. In the present paper we investigated ESI-TOFMS for the analysis of an opium powder. Both continuous infusion and CE were studied for direct sample introduction into the TOFMS and mixture separation, respectively. CEMS analysis of the opium was performed in a citrate buffer, using aqueous or mixed aqueous/organic eluents. Low fmol detection was achieved.     

Elemental speciation by liquid chromatography-inductively coupled plasma mass spectrometry with direct injection nebulization.

A new version of the direct injection nebulizer (DIN) is used to interface liquid chromatographic (LC) separations with element-selective detection using inductively coupled plasma mass spectrometry (ICP-MS). The DIN injects all of the sample into the ICP and has a dead volume of less than 1 microliter. Charged species of arsenic and tin are separated as ion pairs on a micro-scale (1 mm i.d.), packed, reversed-phase column. Detection limits are 0.2-0.6 pg for arsenic and 8-10 pg for tin. For methanol + water eluents, the signal is highest at 25% methanol and stays within 25% of this maximum as the methanol fraction is varied from 20 to 80%. Compared with LC-ICP-MS with conventional nebulizers, the absolute detection limits and chromatographic resolution are substantially superior, and the dependence of analyte signal on solvent composition is somewhat less severe with the DIN.     

Liquid chromatography techniques for characterizing poly(Vinyl Alcohol)

Poly(vinyl alcohol) (PVOH) samples may contain several heterogeneities requiring the development of chromatographic techniques for characterization. Size exclusion separations have been carried out using a number of aqueous eluents, incorporating electrolyte, or electrolyte/organic modifier, or surfactant. The most favourable molecular size separation was obtained using 0.25% w/v sodium lauryl sulfate as eluent. Reasonable values for molecular weights of PVOH samples have been determined. Compositional distributions in copolymer systems can be assessed using high-performance liquid chromatography employing a reversed-phase separation mechanism. For poly(vinyl alcohol), gradient elution with water/tetrahydrofuran (THF) with a wide pore polystyrene-based packing produced separations dependent on degree of hydrolysis and sequence length distribution. The elution results were verified with a column packed with non-porous beads. Partially hydrolysed PVOH samples appeared to have a broad distribution of composition.     

Determination of Atenolol in Human Plasma by Pseudo Reversed Phase Liquid Chromatography-Tandem Mass Spectrometry

Previous HPLC determination of atenolol on reversed-phase packings has often required a mobile phase containing three components: organic modifier, buffer and ion-pairing reagent or organic amine. In addition to the complexity of the eluents employed, alkyl sulphonates and organic amines in the mobile phase can reduce the life of silica-based bonded columns. A new simple, rapid and sensitive method—pseudo reversed-phase liquid chromatography/tandem mass spectrometry has been developed for the analysis of atenolol in human plasma using bare silica as the stationary phase coupled with a simple mobile phase consisted of 5% acetonitrile and 95% formate buffer. The optimization of separation is fast and easy. The assay was validated for the concentration range 1–100 ng mL^?1 with a detection limit of 1 ng mL^?1. Moreover, the silica column was durable with the mainly aqueous eluents. No obvious loss in performance was observed for 30,000 column volumes of eluent.     

Chiral separation of basic compounds on a cellulose 3,5-dimethylphenylcarbamate-coated zirconia monolithin basic eluents by capillary electrochromatography

Porous zirconia monolith (ZM) modified with cellulose 3,5-dimethylphenylcarbamate (CDMPC) was used as chiral stationary phase to separate basic chiral compounds in capillary electrochromatography. The electroosmotic flow behavior of bare and CDMPC-modified zirconia monolithic (CDMPC-ZM) column was studied in ACN/phosphate buffer eluents of pH ranging from 2 to 12. The CDMPC-ZM column was evaluated by investigating the influences of pH, the type and composition of organic modifier of the eluent on enantioseparation. CEC separations at pH 9 provided the best resolutions for the analytes studied, which are better than those observed on CDMPC-modified silica monolithic columns under similar chromatographic conditions. No appreciable decline in retention and resolution factors after over 200 injections, and run-to-run and day-to-day repeatabilities of the column of less than 3% indicate the stability of the zirconia monolithic column in basic media.     

Liquid chromatographic separation of the enantiomers of beta-amino acids on a ligand exchange chiral stationary phase

A liquid chromatographic ligand exchange chiral stationary phase (CSP) derived from (S)-leucinol was applied in the separation of the enantiomers of 12 beta-amino acids. The resolution was quite successful especially for the enantiomers of beta-amino acids containing aromatic functional group in the side chain. The chromatographic resolution behaviors were dependent on the organic modifier and Cu(II) concentration in aqueous mobile phase and the column temperature.     

Analysis of low molecular mass organic acids in natural waters by ion exclusion chromatography tandem mass spectrometry

A sensitive and selective method for the analysis of aliphatic low molecular mass organic acids (LMMOAs) in natural waters is presented. The method is based on separation with ion exclusion chromatography and detection with electrospray ionization tandem mass spectrometry (LC-MS/MS). The extra selectivity gained by applying MS/MS allows for a minimum of sample preparation and the use of a sub-optimal mobile phase regarding chromatographic resolution. Instead the mobile phase, comprising aqueous formic acid with methanol as organic modifier, was mainly optimized for maximum sensitivity and long term MS stability. Detection limits for malonic, fumaric, maleic, succinic, citraconic, glutaric, malic, alpha-ketoglutaric, tartaric, shikimic, trans-aconitic, cis-aconitic, isocitric and citric acid were in the range 1-50 nM, while the detection limits for pyruvic, oxalic and lactic acid were around 250 nM for an injection volume of 100 microL. Due to their metal-chelating properties, these LMMOAs are all considered to affect the bioavailability of metals and to be involved in soil forming processes. It is thus of interest to be able to monitor their presence in natural waters, and the method developed within this work was successfully applied for the analysis of LMMOAs in soil solution and stream water samples.     

Immobilized artificial membrane chromatography coupled with atmospheric pressure ionization mass spectrometry

Liquid chromatographic separations on monolayers of cell membrane phospholipids covalently immobilized to silica particles at high molecular density is used for mimicking solute partitioning into biological membranes that generally correlates with membrane transport. This technique called immobilized artificial membrane chromatography usually employs ultraviolet (UV) detection where a single compound is analyzed in a chromatographic run limiting thereby its throughput for drug discovery applications. For coupling with atmospheric pressure ionization mass spectrometry, the phosphate-buffered saline mobile phase was replaced with one that used ammonium acetate as a volatile buffer. While atmospheric pressure chemical ionization accommodated a purely aqueous effluent, interfacing with electrospray ionization required effluent splitting and the addition of an organic modifier (5%, v/v, acetonitrile). Neuropeptide FF antagonists as early-phase drug candidates were used for the comparative evaluation of the methods. Whereas electrospray ionization produced essentially no fragment ions, several compounds involved in our study yielded low-abundance molecular ions with atmospheric pressure chemical ionization. The use of mass spectrometry yielded data that correlated well with those obtained by the method employing UV detection. Both atmospheric pressure ionization methods permitted the simultaneous determination of the k'(IAM), capacity factors and, therefore, an increased-throughput ranking of potential new leads emerged from the drug discovery process based on affinity to artificial membranes.     

Determination of pesticides in vegetables using large-volume injection column liquid chromatography-electrospray tandem mass spectrometry

Direct injection of a large volume (900 microl) of a sample extract onto a liquid chromatographic (LC) column, LC separation and electrospray tandem mass spectrometric detection were used for the quantitative analysis of a wide polarity range of pesticides in carrots and potatoes. Rapid sample preparation involved extraction of a small amount of sample (2 g) with a small volume of organic solvent (3 ml), clean-up over a filter and dilution of the organic extract with the aqueous LC eluent. The extraction efficiency for the selected pesticides was studied using methanol, acetone and acetonitrile as solvents. Evaluation of the performance of the overall method, using extraction with acetonitrile and detection in the selected-reaction-monitoring mode, showed excellent linearity in the range of 2-100 microg/kg with limits of detection of 0.5-2 microg/kg for both types of vegetable. With relative standard deviations of the MS peak area measurements of less than 6.5% (n=8) the repeatability of the method was fully satisfactory.     

A new concept for isotope ratio monitoring liquid chromatography/mass spectrometry

A new interface for the on-line coupling of a liquid chromatograph to a stable isotope ratio mass spectrometer has been developed and tested. The interface is usable for (13)C/(12)C determination of organic compounds, allowing measurement of small changes in (13)C abundance in individual analyte species. All of the carbon in each analyte is quantitatively converted into CO(2) while the analyte is still dissolved in the aqueous liquid phase. This is accomplished by an oxidizing agent such as ammonium peroxodisulfate. The CO(2) is separated from the liquid phase and transferred to the mass spectrometer. It is shown that the whole integrated process does not introduce isotope fractionation. The measured carbon isotope ratios are accurate and reproducible. The sensitivity of the complete system allows isotope ratio determination down to 400 ng of compound on-column. By-passing the high-performance liquid chromatography (HPLC) separation allows bulk isotopic analysis with substantially lower sample amounts than those required by conventional elemental analyzers. The results of the first applications to amino acids, carbohydrates, and drugs, eluted from various types of HPLC columns, are presented. The wide range of chromatographic methods enables the analysis of compounds never before amenable to isotope ratio mass spectrometry techniques and may lead to the development of many new assays.     

Validation of a gas chromatography/mass spectrometry method for the quantification of aerosolized Jet Propellant 8

The retention and the separation of the enantiomers of 1-phenylpropanol (1PP), 2-phenylpropanol (2PP), and 3-chloro-1-phenylpropanol (3CPP) on silica-bonded quinidine carbamate under normal phase HPLC conditions were investigated. A relatively high selectivity of the stationary phase for 3CPP and 1PP (alpha approximately 1.07-1.09) was achieved with eluents containing ethyl acetate as the polar modifier. These mobile phases were examined in detail. Based on the set of chromatographic and thermodynamic data collected, conclusions regarding the mechanism of enantioselectivity and the structure of the selector chiral center are made.     

Quantitative Analysts of 5-Hydroxytryptamtne in Biological Material by High Perfornance Liquid Chromatography and Electrochmical Detection

Abstract

A quantitative method for the analysis of 5-hydroxytryptamine in biological material is described. The method is based on high performance liquid chromatography (HPLC) with electrochemical detection. A simple purification on a weakly acidic ion exchange resin prior to the analysis gives quite clean samples and permits concentration of diluted samples. The chromatographic separation is performed on a reverse phase column with organic modifier added to an aqueous eluent. With this analytical system 25 pg of 5-hydroxytryptamine can be detected.     

The effect of chromatographic conditions on the separation of selected alkaloids in RP-HPTLC

Selected alkaloid standards were chromatographed on C18 W layers using various aqueous eluents containing an organic modifier and pH 3 buffer to suppress silanol ionization or an organic modifier and pH 8 buffer to suppress alkaloid ionization. Anionic ion pairs such as sodium dodecyl sulfate, octane-1-sulfonic acid sodium salt, pentane-1-sulfonic acid sodium salt, and bis(2-ethylhexyl)ortho-phosphoric acid are used to improve peak shape, efficiency, and selectivity. Amines (e.g., diethylamine, triethylamine, and tetrabutylamonium chloride) are incorporated into mobile phases to block surface silanols. The effect of chromatographic conditions on the separation of the investigated alkaloids is analyzed by the comparison of particular densitograms, asymmetry factor, or theoretical plate number. The best efficiency, peak symmetry, and separation selectivity of the investigated compounds is obtained through the addition of amine (especially diethylamine) to the mobile phases.     

Direct analysis of plasma samples for drug discovery compounds using mixed-function column liquid chromatography tandem mass spectrometry

A sensitive, efficient, high throughput, direct injection bioanalytical method based on a single column and high-performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) was developed for pharmacokinetic analysis of early drug discovery compounds in plasma samples. After mixing with a working solution containing an internal standard each plasma sample was directly injected into a polymer-coated mixed-function column for sample cleanup, enrichment and chromatographic separation. The stationary phase incorporates hydrophilic polyoxyethylene groups and hydrophobic groups to the polymer-coated silica. This allows proteins and macromolecules to pass through the column due to restricted access to the surface of the packing while retaining the drug molecules on the bonded hydrophobic phase. The analytes retained in the column with a largely aqueous liquid mobile phase were then chemically separated by switching to a strong organic mobile phase. The column effluent was diverted from waste to the mass spectrometer for analyte detection. Within 200 plasma sample injections the response ratio (analyte vs. internal standard, %CV = 4.6) and the retention times for analyte and internal standard were found consistent and no column deterioration was observed. The recoveries of test compound in various plasma samples were greater than 90%. The total analysis time was 相似文献   

Reversed phase liquid chromatography of some benzimidazole derivatives

Summary The retention behaviour of a series of benzimidazole derivatives has been studied as a function of the water content of aqueous methanol and aqueous acetonitrile eluents. The relationship between the retention constant (log k) and the pH of the aqueous phase was linear, with slope values depending on the composition of the aqueous phase, the molecular structure of the compound, and the type of C-18 bonded stationary phase. The type of organic modifier significantly affected the shape of the relationship between log k and the volume fraction of organic modifier in the mobile phase.     

Liquid chromatography-mass spectrometry analysis of enzyme-hydrolysed carboxymethylcellulose for investigation of enzyme selectivity and substituent pattern

A series of celloendoglucanases: Bacillus agaradhaerens Cel 5a, Humicola insolens Cel 5a, H. insolens Cel 7b, H. insolens Cel 45a, Trichoderma reesei Cel 7b, and T. reesei Cel 45a were used to hydrolyse carboxymethylcellulose (CMC) and the hydrolysis products were investigated with a novel liquid chromatography-mass spectrometry (LC-MS) method. Separation was achieved using a graphitised carbon chromatographic column which allowed the use of electrospay compatible eluents. Analysis of the compounds produced during enzyme hydrolysis of CMC is used to understand enzyme selectivities and substitution pattern of CMC. Conventional high-performance anion-exchange chromatography (HPAEC)-pulsed amperometric detection (PAD), size-exclusion chromatography (SEC)-refractive index (RI) detection, and reducing end analysis are also used to analyse enzyme-hydrolysed CMC. The LC-MS method presented allows for a more detailed investigation of hydrolysis products, which facilitates characterisation of both enzymes and substrates.     

The Use of Proton Nuclear Magnetic Resonance Spectroscopy for the Determin- ation of pK a Values in Aqueous/Organic Solutions for Basic Analytes Employed in Column Characterisation Protocols

Proton nuclear magnetic spectroscopy has been used to measure pK _a values of two basic analytes in a range of aqueous/organic eluents. The results support the hypothesis that the poor correlation in terms of ion exchange capacity with values greater than pH 7 may be attributed to the difference in the ionisation of the silica surface and not differences in ionisation of the basic analytes under the differing chromatographic conditions (i.e. temperature, type and amount of modifier) employed in two commonly used HPLC stationary phase characterisation procedures (Tanaka and Snyder).