Liquid Chromatography Mobile Phase Research Articles

Nebulizing conditions of pneumatic electrospray ionization significantly influence electrolyte effects on compound measurement

Composition of mobile phase can greatly influence the success of electrospray ionization (ESI)-interfaced liquid chromatography-mass spectrometry analysis. To investigate the relationship between formic-acid-based modification of mobile phase and ESI nebulizing conditions, an API 4000 ESI source and a TSQ Quantum one were compared under the same chromatographic conditions. Ginkgo terpene lactones and flavonols were measured in plasma, which involved using ascorbic acid to circumvent cross-interference between the analytes. ESI responses to using formic acid included changes in signal intensity, matrix effect, and upper limit of quantification. Significant disparities in the responses were observed between the two ESI sources, suggesting that the use of electrolyte modifier in liquid chromatography mobile phase and the pneumatic nebulization for ESI should be properly balanced to accomplish optimal ESI-based analysis. The distribution of unpaired ions toward the surface of the initial droplet was assumed to be an important step in the pneumatic ESI process. When using the electrolyte in mobile phase, a too fast droplet reduction by rapid-heating-assisted pneumatic nebulization could negatively decrease the time available for the unpaired ions to migrate from droplet interior to its surface. Ascorbic acid was identified as a major interfering substance for the bioanalytical assay; the interference mechanism might be associated with hindering the unpaired analyte ions from distributing toward the droplet surface rather than outcompeting the analyte ions for the limited excess charge on droplets surface. The current work extends the knowledge base of pneumatic ESI, which has implication for optimal use of the ESI-interfaced liquid chromatography-mass spectrometry technique.

Polar aprotic modifiers for chromatographic separation and back-exchange reduction for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.

Hydrogen/deuterium exchange monitored by mass spectrometry is an important non-perturbing tool to study protein structure and protein–protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast high-performance liquid chromatography (HPLC) and supercritical fluid chromatography have been shown to decrease back-exchange. Here, we show that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent.

Determination of sinigrin, sinalbin, allyl- and benzyl isothiocyanates by RP-HPLC in mustard powder extracts

A reversed phase-high performance liquid chromatography method was developed to quantify sinigrin, sinalbin, allyl isothiocyanate and benzyl isothiocyanate present in aqueous and freeze-dried yellow and Oriental (brown) mustard extract samples using two pre-treatment methods (autoclaving, boiling) to prevent degradation by myrosinase. The lowest detection limits for sinigrin and sinalbin were 0.05 mg/L and for allyl- and benzyl isothiocyanate were 2 mg/L. The methods developed make it possible to quantify both the glucosinolates (sinigrin, and sinalbin) and their hydrolysis products (allyl- and benzyl isothiocyanate) with the same mobile phase, and only require adjustment of the wavelength and a change in the ratio of the high performance liquid chromatography mobile phase solvents (tetrabutylammonium hydrogen sulphate and acetonitrile). The use of a single method yielded accurate and rapid results for the four compounds (sinigrin, sinalbin, allyl- and benzyl isothiocyanate). Autoclaving of both yellow and brown mustard powder before glucosinolate extraction did not consistently improve the amount of sinalbin and sinigrin recovered over boiling treatments because the thermal stability of myrosinase proved problematic in glucosinolate recovery. Nonetheless, the highest extract yields found were 4.06 g/100 g for sinigrin and 2.57 g/100 g for sinalbin, respectively, which represented over 94 g/100 g extract yield of sinigrin from the Oriental mustard powder.

Infrared Spectroscopy and Molecular Simulations of a Polymeric Sorbent and Its Enantioselective Interactions with Benzoin Enantiomers

Retention factors, k(R) and k(S), and enantioselectivities, S ≡ k(R)/k(S), of amylose tris[(S)-α-methylbenzylcarbamate] (AS) sorbent for benzoin (B) enantiomers were measured for various isopropyl alcohol (IPA)/n-hexane compositions of the high-performance liquid chromatography (HPLC) mobile phase. Novel data for pure n-hexane show that k(R) = 106, k(S) = 49.6, and S = 2.13. With some IPA from 0.5 to 10 vol %, with S = 1.8-1.4, the retention factors were smaller. Infrared spectra showed evidence of substantial hydrogen bonding (H-bonding) interactions in the pure polymer phase and additional H-bonding interactions between AS and benzoin. Density functional theory (DFT) was used to model the chain-chain and chain-benzoin H-bonding and other interactions. DFT was also used to predict fairly well the IR wavenumber shifts caused by the H-bonds. DFT simulations of IR bands of NH and C═O allowed for the first time the predictions of relative intensities and relative populations of H-bonding strengths. Molecular dynamics (MD) simulations were used to model a single 12-mer polymer chain. MD simulations predicted the existence of various potentially enantioselective cavities, two of which are sufficiently large to accommodate a benzoin molecule. Then "docking" studies of benzoin in AS with MD, Monte Carlo (MC), and MC/MD simulations were done to probe the AS-B interactions. The observed enantioselectivities are predicted to be primarily due to two H-bonds, of the kind AS CO···HO (R)-benzoin and AS NH···OC (R)-benzoin, and two π-π (phenyl-phenyl) interactions for (R)-benzoin and one H-bond, of type AS CO···HO (S)-benzoin, and one π-π interaction for (S)-benzoin. The MC/MD predictions are consistent with the HPLC and IR results.

Determination of endocrine-disrupting compounds in water by carbon nanotubes solid-phase microextraction fiber coupled online with high performance liquid chromatography

The commercial solid phase microextraction (SPME) fibers are not stable enough in organic solvent and tend to swell and strip off from the silica fiber in the high performance liquid chromatography (HPLC) mobile phase, and therefore the application of SPME coupled online with HPLC is limited. In this study, an SPME fiber coated with single walled carbon nanotubes (SWCNTs), prepared by means of electrophoretic deposition, was coupled on line to HPLC for the determination of four endocrine-disrupting compounds, i.e. bisphenol A (BPA), estrone (E1), 17α-ethynylestradiol (EE2) and octylphenol (OP), in aqueous samples. The results showed that the SWCNTs coating on the prepared fiber did not swell and strip off from the platinum fiber throughout the experiment, thus indicating a high resistance to the HPLC mobile phase, the mixture of water and acetonitrile. The SWCNTs fiber had similar (for OP) or higher (for BPA, EE2 and E1) extraction efficiencies than the commonly used polyacrylate fiber, and had a lifetime of more than 120 operation times. Under the optimized conditions, the linearity of the proposed method was 1.0–30.0μg/L for BPA and OP and 3.0–90.0μg/L for E1 and EE2. The limits of detection (LODs; S/N=3) and limits of quantification (LOQs; S/N=10) of the method were 0.32–0.52μg/L and 1.06–1.72μg/L, respectively. Repeatability for one fiber (n=3) was in the range of 1.3–7.1% and fiber-to-fiber reproducibility (n=3) was in the range of 1.6–8.4%. The proposed method was successfully applied for the analysis of spiked tap water and seawater samples with recoveries from 81.8 to 97.3%.

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation

Background Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Methods In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100 mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC). Results An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37 °C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl 2, MgCl 2 and ZnSO 4, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V max and lowest K m values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors. General significance Substrate specificity, V max and K m values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis.

Properties of 2,4-dinitrophenyl derivatives of amino acids as analytical forms for high-performance liquid chromatography

Dissociation constants of 2,4-dinitrophenyl derivatives of α-amino acids in micellar solutions of sodium dodecyl sulfate used as a micellar mobile phase in reversed-phase liquid chromatography were determined. The method of micellar liquid chromatography was used to determine the composition of polypeptide fractions of animal origin.

Development and Validation of an SPE-HPLC-FL Method for the Determination of Anatoxin-a in Water and Trout (Oncorhincus mykiss)

Anatoxin-a is a neurotoxic alkaloid which is produced by various cyanobacteria genera and it is highly toxic to animals. Anatoxin-a risk evaluation is not fully determined yet. Therefore, it is imperative to implement detection and quantification methods in the various possible matrices. The recent worldwide shortage of acetonitrile, as well as environmental reasons, demand an urgent alternative to this high-performance liquid chromatography (HPLC) mobile phase solvent. This work describes an internal study that validated the measuring method of anatoxin-a in water and lyophilized trout matter by means of HPLC with fluorimetric detection (FLD) utilizing methanol as mobile phase.

Formation of iron complexes from trifluoroacetic acid based liquid chromatography mobile phases as interference ions in liquid chromatography/electrospray ionization mass spectrometric analysis

Two unexpected singly charged ions at m/z 1103 and 944 have been observed in mass spectra obtained from electrospray ionization mass spectrometric analysis of liquid chromatography effluents with mobile phases containing trifluoroacetic acid (TFA) that severely interfered with sample analysis. Accurate mass measurement and tandem mass spectrometry studies revealed that these two ions are composed of three components; clusters of trifluoroacetic acid, clusters of mass 159 and iron. Formation of these ions is inhibited by removing TFA from the mobile phases and using formic acid in its place, replacing the stainless steel union with a titanium union or by adding a small blank fused‐silica capillary column between the chromatography column and the electrospray tip via a stainless steel union without any adverse effects to chromatographic separation, peak broadening or peptide identifications. Copyright © 2011 John Wiley & Sons, Ltd.

Chromatographic studies of unusual on-column degradations of aniline compounds on XBridge Shield RP18 column in high pH aqueous mobile phase

This paper reports unusual on-column degradations of aniline compounds on Waters XBridge Shield RP18 column when ammonium hydroxide in water and acetonitrile were used as mobile phases in liquid chromatography. The change of the level of on-column degradation of a model compound (Compound 1) with time was observed in the first fifteen injections when started at 60 °C. During a subsequent cooling program from 60 °C to 10 °C with a 10 °C interval, the levels of the degradation products of Compound 1 changed with the change of temperature and reached a maximum at 40 °C. The on-column degradation of Compound 1 was observed when started at 10 °C in the first injection, however, the magnitude of the change of the level of on-column degradation of Compound 1 with time in the first fifteen injections was much smaller than that at 60 °C. During a subsequent heating program from 10 to 60 °C with a 10 °C interval, the levels of the degradation products of Compound 1 increased with the increase in temperature but without a maximum. The change of the degradation product levels of this model compound in the heating process is not super-imposable with that in the cooling process, which demonstrates the degree of the degradation also depends on the heating or cooling process. Column history studies demonstrated that the on-column degradation of Compound 1 changed dramatically on the used columns at both starting temperatures while the dependency of heating and cooling processes on on-column degradation still existed. The unusual on-column degradation of Compound 1 on the used columns can be regenerated in a very similar fashion with an acetic acid column-wash procedure, but is not identical to that on the new column. Similar degradations of other commercially available aniline compounds were also observed with this high pH aqueous mobile phase system.

Applications of Nano-Baskets of Calixarenes in Chromatography

Nano-baskets of calixarenes have been subject to extensive research in the construction of liquid chromatographic phases, extractants, transporters, electrode ionophores, and optical and electrochemical sensors over the past 4 decades. There has long been interest in calixarene-based liquid chromatographic phases. Owing to the recent rapid growth in the number of publications on calixarene-based liquid chromatographic phases, this review paper focuses on their different applications in the main fields of molecular and ionic species as well as liquid chromatographic mobile and stationary phases. Although the recent reports have focused on the optimization and application of one kind or a unique group of calixarenic mobile or stationary phases, this review is a collection and comparison of a variety of research data dealing with the synthesis, preparation and behavior of calixarene-based liquid chromatographic phases.

Influence of temperature and pressure on the preferential adsorption of component of hydroorganic mobile phase in liquid chromatography

The excess isotherms of methanol and acetonitrile were measured on the series of C18 bonded phases. The measurements were done using the minor disturbance method. The goal of our work was to determine the influence of the temperature on the adsorption of two commonly used solvents. The influence on the mobile phase flow rate on the both organic solvent adsorption was also investigated. The effect of these two parameters was compared on the octadecyl packed columns with different coverage density and on the monolithic Chromolith column. Adsorption of both solvents decreases with the increase of the temperature. The increase of the pressure increases adsorption of methanol but decreases adsorption of acetonitrile.

Calibration of an Evaporative Light-Scattering Detector for the Universal Quantitative Analyses in Liquid Chromatography—Application to the Determination of Triacylglycerols in Cucurbitaceous Oils

The goal of this work is to validate a quantitative method to analyze 15 triacylglycerols (TAG) which are present in cucurbitaceous oils using a new liquid chromatography-evaporative light-scattering detector (LC-ELSD) methodology. For the majority of the TAG, the absence of chromophoric groups absorbing in a spectral zone different from those of the high performance liquid chromatography mobile phases limits the use of UV detectors. The most convenient detectors for this type of analyses are the “universal” aerosol-based detectors, and the most frequently used one among these detectors is the ELSD. TAG responses using ELSD can vary according to their structure and the analyst may not possess all the 15 different TAG standards. Therefore, the determination of these analytes in unknown samples cannot be accurate and depends on the nature of the available standards. We propose here a new quantification methodology by LC-ELSD using the standard response model A = a × mb, where a and b are numerical coefficients which are specific for each solute and both correlated. The method uses a sample dilution procedure which provides for each compound to quantify an accurate b value, and by calculation, the corresponding a value. The quantification of both coefficients a and b easily gives the direct mass determination of each TAG. Such LC-ELSD results are validated by the comparison with the fatty acid methyl ester (FAME) analysis using the conventional capillary gas chromatography-flame ionization detector that transforms non-volatile TAG in FAME after a transesterification step. To perform this validation, we compared the experimental FAME composition to the theoretical one calculated from the TAG results obtained by LC-ELSD. For each TAG, theoretical and experimental values were statistically similar, which proves and validates the relevance of the new quantification methodology proposed in this work. The concordance between the two different methods also highlights the great advantage of the direct LC-ELSD method which additionally provides more information on TAG structures.

Effect of Trace Amounts of Water in the Mobile Phase of Normal-Phase Enantioselective High-Performance Liquid Chromatography on Selectivity and Resolution of Optical Isomers

The irreproducibility of normal-phase enantioselective high-performance liquid chromatography (HPLC) could be attributed to the presence or absence of trace amounts of water in one or more components of the mobile phase. The effect of trace amounts of water on chromatographic characteristics in normal-phase enantioselective HPLC was investigated by deliberate addition of controlled, trace amounts of water into the mobile phase for the separation of T-3811ME and its undesired enantiomer. Commercial 2-propanol was pre-mixed with 2% (v/v) water and then used for preparation of the mobile phase in combination with such organic modifiers as ethanol and methanol at different ratios. The results showed up to 30% improvement in the resolution (Rs), 4% in selectivity (alpha), and 39% in efficiency (plate number N) compared to using a mobile phase prepared from neat commercial 2-propanol. Thus, the effect of trace amounts of water in the mobile phase of normal-phase enantioselective HPLC was demonstrated.

Penetration isotherms of binary mobile phases for liquid chromatography

Penetration isotherms ψ of eight binary solvent systems used as mobile phases in liquid chromatography are calculated. Dependences of penetrability coefficients of binary mixtures on the volume fraction of the more active solvent are described using modified polynomial functions, in which some empirical coefficients are changed for penetrability coefficients of pure solvents.

Simultaneous detection and quantitation of highly water-soluble herbicides in serum using ion-pair liquid chromatography–tandem mass spectrometry

We report the simultaneous screening of highly polar, water-soluble, and less-volatile herbicides, including glyphosate, glufosinate, paraquat, and diquat, in serum using liquid chromatography–mass spectrometry. The herbicides were separated by solid-phase extraction using a Strata-XC cartridge. A heptafluorobutyric acid solution was chosen as the mobile phase for ion-pair liquid chromatography. Mass spectrometry was used for analysis and was optimized for operation in the positive mode for all analytes. The serum specimens were screened for the presence of the herbicides at the following concentrations: 5 ng/mL for glyphosate, 2 ng/mL for glufosinate, 1 ng/mL for diquat, and 5 ng/mL for paraquat. This is the first report on the simultaneous detection of these compounds.

Stable isotope-coded quaternization for comparative quantification of estrogen metabolites by high-performance liquid chromatography–electrospray ionization mass spectrometry

A fast and sensitive LC–ESI-MS method is described for the comparative quantification of 16 estrogen metabolites based on the derivatization of estrogens with a novel derivatizing reagent, N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NA-NHS). The process introduces a quaternary amine to the analytes, making the analytes permanently charged regardless of the pH of the high-performance liquid chromatography (HPLC) mobile phase. This quaternization resulted in a highly efficient separation of 16 estrogen metabolites in 7 min at a detection level below 1 ng/mL. By using a deuterated derivatizing reagent (C1- d 3-NA-NHS), a complete set of deuterated standards was utilized and used as internal standards in a comparative quantification and recovery study, demonstrating acceptable results over a wide concentration range. A pooled breast cancer serum sample was analyzed using the described method, and 15 estrogens were detected in the range of 80–530 pg/mL.

Solvation of Perfluorooctane and Octane in Water, Methanol, Acetonitrile, and Aqueous Mixtures of Methanol and Acetonitrile

Molecular dynamics simulations are used to examine the local solvation structure of single octane and perfluorooctane molecules in liquid water, methanol, acetonitrile, and aqueous mixtures of methanol and acetonitrile. The motivation is to obtain baseline information about the solvation of perfluorooctane by liquids used as the mobile phase in liquid chromatography and how it differs from the solvation of octane. While octane is uniformly solvated by both water and the second component, perfluorooctane is solvated by methanol and acetonitrile with the exclusion of water from the first solvation layer when the solvent is a mixture.

Mixture design optimization of extraction and mobile phase media for fingerprint analysis of Bauhinia variegata L.

Two statistical mixture designs were used to optimize the proportions of solvents used in both the extraction medium and the reversed liquid chromatographic mobile phase to improve the quality of chromatographic fingerprints of Bauhinia variegata L extracts. For modeling, the number of peaks was used as a measure of fingerprint information. Three mobile phases, each with a chromatographic strength of two, gave good results. A methanol/water (77:23 v/v) mixture resulted in 17 peaks in the chromatographic fingerprint whereas acetonitrile/water (64.5:35.5 v/v) and methanol/acetonitrile/water (35:35:30 v/v/v) mixtures resulted in 18 and 20 peaks, respectively. The corresponding optimum solvent compositions to extract chemical substances for these three mobile phases were ethanol/acetone (25:75 v/v/v) and dichloromethane/acetone (70:30 v/v) mixtures, and pure dichloromethane, respectively. The mixture designs are useful for understanding the influence of different solvents on the strengths of the extraction medium and the mobile phase.

Influence of the presence of methyl cyclodextrins in high-performance liquid chromatography mobile phases on the separation of β-carboline alkaloids

The presence of cyclodextrins (CDs) in the mobile phase alters the chromatographic equilibria and induces a secondary chemical equilibrium associated to the chromatographic separation by HPLC. In this study the influence of the presence of CDs in the mobile phase as chemically modified β-CDs, i.e. 2,3-di- O-methyl-β-cyclodextrin (DMβ-CD) and 2,3,6-tri- O-methyl-β-cyclodextrin (TMβ-CD) on the separation of the alkaloids norharmane, harmane and harmine is described. These β-carboline alkaloids are chemically and structurally related and their quantitation by RP-HPLC is of interest due to their biological and pharmacological properties. Two stationary phases (methyl-, C 1 and octadecyl-, C 18) were employed, with methanol:buffered aqueous solution and ethanol:buffered aqueous solution as mobile phases. The role of tert-butyl alcohol as a mobile phase modifier and also as an inclusion complex stabiliser was also considered. The concentrations of DMβ-CD and TMβ-CD vary from 0 to 17 mM. The presence of increasing amounts of CDs in the mobile phase reduces the retention factor. The changes observed in the retention factor allow the determination of the alkaloid/CD apparent association constants, whose magnitude is influenced by the chemical and structural properties of the guest molecules but also by the composition of the mobile phase. Assuming a 1:1 stoichiometry for the inclusion complexes, the apparent association constants obtained were higher for norharmane and for both DMβ-CD and TMβ-CD. The strength of the complexes is higher for DMβ-CD than for TMβ-CD and this behaviour can be explained considering the steric problems associated to the permethylated-β-CDs. Besides significant differences in the magnitude of the apparent association constants were observed for the two stationary phases employed and thus can be related to the adsorption of CDs on the stationary phase. A significant reduction in the proportion of organic solvent in the mobile phase (50%) without a decrease in the selectivity or resolution of the separation is a favourable consequence of the presence of the CDs in the mobile phase.