Peak Asymmetry Factor Research Articles

Comparison of 624-type Capillary Columns II

The present paper is the continuation of our "equivalent column" study. 624-type columns were investigated in a capillary gas chromatograph with a flame ionization detector (FID). In the preceding work, we used a homemade test mixture containing mainly acidic type compounds, which we complemented in the current study with additional basic test compounds to give a more precise description of the behaviour of the stationary phases. The columns had the same dimensions (30 m × 0.32 mm × 1.0 μm; length, internal diameter and film thickness, respectively) but were purchased from different manufacturers or the same manufacturer but from different batches. In addition, also their pre-life was different. These circumstances influence the condition of the stationary phase, and therefore their separation ability. These factors imply the necessity of testing the column from time to time. To distinguish between the separation ability of the columns, we compared the chromatograms, the retention times and retention order of the molecules, as well as peak asymmetry factors. Besides these parameters, quantitative determination is also performed based on the results of separation. We used the effective carbon number (ECN) and the limit of quantitation (LoQ) to demonstrate these differences which are caused by the different stationary phase conditions.

Automatic peak detection algorithm based on continuous wavelet transform for complex chromatograms from multi-detector micro-scale gas chromatographs

Peak detection for chromatograms, including the detection of peak retention times, peak start locations, and peak end locations, is an important processing step for extracting peak information that is used for chemical recognition. Compared to benchtop gas chromatographs, the chromatograms generated by microscale gas chromatographs (µGCs) often contain higher noise levels, peak overlap, peak asymmetry, and both positive and negative chromatographic peaks, increasing the challenges for peak detection. This paper reports an automatic peak detection algorithm based on continuous wavelet transform (CWT) for chromatograms generated by multi-detector µGCs. The relationship between chemical retention time and peak width is leveraged to differentiate chromatographic peaks from noise and baseline drift. Special features in the CWT coefficients are leveraged to detect peak overlap and asymmetry. For certain detectors that may generate positive and negative chromatographic peaks, the peaks cannot be independently detected reliably, but the peak information can be well extracted using peak information generated by other in-line single-polarity detectors. The implemented algorithm provided a true positive rate of 97.2 % and false discovery rate of 7.8 % for chromatograms generated by a µGC with three integrated detectors, two capacitive and one photoionization. The chromatograms included complex scenarios with positive and negative chromatographic peaks, up to five consecutive overlapping peaks, peak asymmetry factor up to 24, and signal-to-noise ratios spanning 9-2800.

Non-Line-of-Sight Localization of Passive UHF RFID Tags in Smart Storage Systems

The UHF radio-frequency identification (RFID) has gained growing attention for tagged object localization in smart storage systems. Due to Non-Line-Of-Sight (NLOS) condition, it is challenging to accurately locate the position of tags inside closed spaces. In this paper, we propose a precise and cost-effective solution for tagged object localization in closed spaces, using only received signal strength (RSS) information. We establish a RSS profile for each tag and discover some important features of RSS profiles including uniqueness, time-variation, column-dependence and waveform-similarity. Based on these features, we propose a reference-free RSS-profile (RFRP) localization scheme. The advantage of our propose scheme is to accurately localize multiple tags in closed spaces by overcoming the challenges including the lack of pre-deployed reference tags, NLOS propagation, multi-path propagation and coupling effect. The RFRP scheme first roughly estimates tags’ coordinates based on Peak Asymmetry Factor, then acquires reference-tag substitutes through the similarity of RSS sequences. Subsequently, our scheme refines the relative positions of all tags by these substitutes. Finally all tags’ absolute positions are estimated through a RSS-ranging model. Extensive experiment results demonstrate that our approach can achieve high ordering accuracy and localization accuracy for the tags inside closed spaces.

Fabrication and Characterization of a Reversed-Phase/Strong Cation Exchange Stationary Phase Gradient

Continuous stationary phase gradients for liquid chromatography (LC) have been recently shown to be a promising method of altering selectivity. In this work, we present the first multicomponent continuous stationary phase gradient for separations involving both reversed-phase (RP) and strong cation exchange (SCX) mechanisms. These columns are fabricated using a two-step methodology based on controlled rate infusion (CRI). First, destructive CRI creates a gradient of excess silanol groups along a uniform C18 column. Next, these columns are infused with 3-mercaptopropyltrimethoxysilane (MPTMOS), which bonds to the excess silanol groups. The terminal thiols of the MPTMOS ligands are oxidized with H2O2 to create the sulfonate functionality (SO3−) needed for SCX separations. The success of the modification procedure is characterized by thermogravimetric analysis and X-ray photoelectron spectroscopy. The stability of the C18-SO3− gradients were found to have less than 5 % retention loss and the column-to-column reproducibility had a relative standard deviation under 9 %. The peak asymmetry factors for seven biogenic amines were found to be between 1.03 ± 0.04 to 1.30 ± 0.02, which illustrates minimal peak tailing due to poor packing and residual silanol groups. Characterization of the gradient columns using an isocratic mobile phase showed a unique elution order compared to a uniform C18 and SO3− columns. At lower counterion concentrations, more than 48 % of the overall retention on the gradient stationary phase is due to a SCX mechanism. Meanwhile, the RP mechanism was shown to predominate at higher counterion concentrations on the gradient columns (SCX retention contribution less than 40 %). Coupling the stationary phase gradient to a salt gradient in the mobile phase showed that the gradient phase provides a unique, intermediate selectivity to the uniform C18 and SO3− columns. Under an ACN mobile phase gradient, a significant increase (p < 0.003) in the retention times of three biogenic amines (15 – 16 %) was observed when the multicomponent gradient was oriented to have a high SO3− ligand density near the detector. This work serves as a proof-of-concept design for a multicomponent stationary phase gradient to continue fundamental studies into retention and encourage novel applications.

Quality by Design oriented development of hydrophilic interaction liquid chromatography method for the analysis of amitriptyline and its impurities

This paper presents integration of Quality by Design concept in the development of hydrophilic interactions liquid chromatographic methods for analysis of amitriptyline and its impurities (A, B, C, and F). This is the first time that HILIC method for amitriptyline and its impurities is developed. Using QbD concept, it is possible to design a robust method and incorporate quality directly into its development. QbD concept in combination of Design of Experiments methodology (DoE) enables creation of well-defined design space. In this study, for method optimization a Box-Behnken design was used to test the effect of acetonitrile content, buffer concentration and pH of water phase on critical system responses such as retention factor of impurity A, resolution between impurity B and impurity C, amitriptyline peak asymmetry factor and retention time of last eluted impurity F. The defined mathematical models and Monte Carlo simulations were used to identify the design space. For robustness testing, fractional factorial design was applied. Optimal chromatographic conditions were the analytical column ZORBAX NH2 (250 mm x 4.6 mm, 5 μm particle size); mobile phase consisted of acetonitrile-water phase (60 mM ammonium acetate, pH adjusted to 4.5 with glacial acetic acid) (92.5:7.5 v/v); column temperature 30 °C, mobile phase flow rate 1 mL min−1, wavelength of detection 254 nm. Finally, method was fully validated and applicability of the method in tablet analysis was confirmed.

Effect of the Mobile Phase Composition on Selectivity in Supercritical Fluid Chromatography in the Separation of Salbutamol Enantiomers

Three chiral chromatography stationary phases based on amylose and cellulose were tested for the supercritical fluid chromatography separation of antiasthmatic drug salbutamol enantiomers. A cellulose tris- 3-chloro-4-methylphenylcarbamate stationary phase provided the highest selectivity for this separation. The optimization of mobile phase composition allowed us to maximize the selectivity coefficient and to minimize the peak asymmetry factors. A successful salbutamol enantiomer separation is achieved at moderate modifier fraction (10–15 vol %) in the mobile phase on this column. Amine additives are necessary to suppress peak tailing. Among three tested amines isopropylamine gives the most symmetrical peaks along with the highest enantioselectivity.

Feasibility study for high-resolution multi-component separation of protein mixture using a cation-exchange cuboid packed-bed device

In recent papers we have discussed the optimization of design and operating conditions for cuboid packed-bed device for chromatographic separations. The efficiency metrics used in these studies included the number of theoretical plates per unit bed height as well as attributes of flow-through and eluted peaks. These studies were carried out using equivalent columns as benchmarks. The cuboid packed-bed devices consistently outperformed the columns in terms of the above metrics. The current study examines how well, or indeed if at all these superior efficiency metrics translate to superiority in multi-component protein separation. Cation exchange resin was examined in the current study using appropriate multi-component model protein system which was chosen with close isoelectric points to make the separation challenging. Effects of operating and experimental parameters such as flow rate, loop size and linear gradient length on separation performance were systematically investigated. Separation metrics examined included peak width, tailing factor, asymmetry factor and resolution of separated protein peaks. The results obtained showed that the cation exchange cuboid packed-bed device significantly outperformed its equivalent commercial column (e.g., the number of theoretical plates per unit bed height was 8636/m for the cuboid packed-bed device as opposed to 1480/m for the column at a flow rate of 0.5 mL/min). The difference in efficiency was particularly high at lower flow rate and when shorter gradients were employed. The results suggest that the cuboid packed-bed devices could potentially have promising application in preparative separations such as biopharmaceutical purifications.

Evaluating the solvation properties of metal-containing ionic liquids using the solvation parameter model.

Ionic liquids (IL) have been utilized as gas chromatography stationary phases due to their high thermal stability, negligible vapor pressure, wide liquid range, and the ability to solvate a range of analytes. In this study, the solvation properties of eight room temperature ILs containing various transition and rare earth metal centers [e.g., Mn(II), Co(II), Ni(II), Nd(III), Gd(III), and Dy(III)] are characterized using the Abraham solvation parameter model. These metal-containing ILs (MCILs) consist of the trihexyl(tetradecyl)phosphonium cation and functionalized acetylacetonate ligands chelated to various metals. They are used in this study as gas chromatographic stationary phases to investigate the effect of the metal centers on the separation selectivities for various analytes. In addition, two MCILs comprised of tetrachloromanganate and tris(trifluoromethylphenylacetylaceto)manganate anions were used to study the effect of chelating ligands on the selectivity of the stationary phases. Depending on the metal center and chelating ligand, significant differences in solvation properties were observed. MCILs containing Ni(II) and Mn(II) metal centers exhibited higher retention factors and higher peak asymmetry factors for amines (e.g., aniline and pyridine). Alcohols (e.g., phenol, p-cresol, 1-octanol, and 1-decanol) were strongly retained on the MCIL stationary phase containing Mn(II) and Dy(III) metal centers. This study presents a comprehensive evaluation into how the solvation properties of ILs can be varied by incorporating transition and rare earth metal centers into their structural make-up. In addition, it provides insight into how these new classes of ILs can be used for solute-specific gas chromatographic separations. Graphical abstract The solvation properties of eight metal-containing ionic liquids (MCILs) comprised of transition and rare-earth metal centers are evaluated for the first time using gas chromatography. The results reveal that metals comprising the MCILs provide unique separation selectivities for various analytes and that these materials can be exploited as stationary phases in solute-specific gas chromatographic separations.

Synthesis, characterization, and application of a novel multifunctional stationary phase for hydrophilic interaction/reversed phase mixed-mode chromatography

A novel multifunctional stationary phase based on silica gel was synthesised starting from L- isoleucine and 4-phenylbutylamine and evaluated as a hydrophilic interaction/reversed-phase mixed-mode stationary phase for high-performance liquid chromatography (HPLC). The prepared stationary phase was characterized by elemental analysis, infrared spectroscopy (IR), scanning electron microscopy (SEM), Brunauer, Emmett and Teller (BET) and solid-state 13C nuclear magnetic resonance (NMR). The mechanisms involved in the chromatographic separation are multi-interaction, including hydrophobic, π-π, hydrogen-bonding, dipole-dipole and ion-dipole interactions. Based on these interactions, successful separation could be achieved among several aromatic compounds having different polarities under both hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) condition. Nucleotides/nucleosides were separated in the HILIC mode. The effects of different separation conditions, such as pH value, mobile-phase content, column temperature, buffer concentration and flow rate, on the separation of nucleotides/nucleosides in HILIC mode were investigated. The seven nucleotides/nucleosides were separated within 22min, while six of them were separated within 10min by isocratic elution. To determine the influence of the new multifunctional stationary phase under the RP condition, a number of moderately and weakly polar and nonpolar compounds, such as 10 substituted anilines and eight substituted phenols were separated successfully under the RP condition within 14 and 15min, respectively. Additionally, nine mixtures of polar/nonpolar test compounds were simultaneously separated within 19min, while seven of them were separated within 12min, under HILIC/RP mixed-mode conditions. Chromatographic parameters, such as the retention factor and peak asymmetry factor, were calculated for all of the analytes, while the theoretical plate number was calculated for analytes separated by isocratic elution. Compared to traditional C18 and commercial HILIC columns, the new stationary phase exhibited both HILIC and RPLC performance, and the scope of analyte separation was thus enlarged.

Chromatographic behavior of selected dyes on silica and cellulose micro-TLC plates: Potential application as target substances for extraction, chromatographic, and/or microfluidic systems

ABSTRACTThe main goal of this research communication focuses on screening of chromatographic behavior of 18 colorants including amaranth, black PN, bromophenol blue, bromocresol green, bromocresol purple, bromothymol blue, carmine, dimethyl yellow, erythrosine, fluorescein, methyl red, naphthalene black, patent blue, phenol red, Sudan II, Sudan III, Sudan IV, and p-xylenol blue. They are commonly used as food and industrial colorants or sensing molecules for analytical and biomedical applications. We studied retention (RF), peak base width (Wb), and peak asymmetry factor (AS) data collected under thermostatic conditions (303 K) using silica and cellulose-coated microplates (5 × 5 cm) and simple as possible mono/binary eluents. Presented retention database may help to select the proper conditions for, for example, chromatographic separation, extraction to solid phase (e.g. bar-type extraction devices) or application of such chemicals as the internal standard substances for planar/column chromatography and/or microfluidic devices (e.g., microfluidic paper-based analytical devices—μPADs). Furthermore, the microplates peak capacity (maximum number of resolvable spots, n) was calculated and discussed. As the second goal, we presented results of literature concerning dyes quantification in complex samples. We compared latest methodologies involving simple spectrophotometric analysis and more sophisticated protocols like high-throughput separation/detection procedures based on high-performance planar/liquid chromatography, capillary electrophoresis and microfluidic devices.

QbD-driven development and validation of an efficient bioanalytical UPLC method for estimation of olmesartan medoxomil

ABSTRACTThe present studies describe quality by design-based development of bioanalytical ultra performance liquid chromatography method of olmesartan medoxomil. Initially, method objectives were defined and critical analytical attributes (CAAs) earmarked. Method optimization was conducted using a central composite design for optimizing mobile phase ratio and injection volume as the critical method parameters (CMPs) identified from risk assessment and factor screening studies, and evaluated for their influence on peak area, theoretical plates, and asymmetry factor as CAAs. Chromatographic separation was achieved using acetonitrile:water solvent system containing 0.1% orthophosphoric acid (54:46, v/v) as the mobile phase with UV detection at 243 nm. Further optimization of bioanalytical extraction process was accomplished using a Box–Behnken design selecting extraction time, centrifugation speed, and centrifugation time as the CMPs identified from failure mode and effect analysis, and evaluated for percent recovery, peak asymmetry, and theoretical plate count as the CAAs. Establishment of calibration curve indicated linearity between concentration range of 100 and 800 ng mL−1, excellent accuracy and precision with limit of detection and limit of quantification as 6.2 and 19.0 ng mL−1, respectively. Drug stability studies indicated mean percent recovery ranging between 92.4 and 97.3% under various stress conditions.

Preparation and characterization of high capacity, strong cation-exchange fiber based adsorbents

Motivated by the demand for more economical capture and polishing steps in downstream processing of protein therapeutics, a novel strong cation-exchange chromatography stationary phase based on polyethylene terephthalate (PET) high surface area short-cut fibers is presented. The fiber surface is modified by grafting glycidyl methacrylate (GMA) via surface-initiated atom transfer radical polymerization (SI-ATRP) and a subsequent derivatization leading to sulfonic acid groups. The obtained cation-exchange fibers have been characterized and compared to commercially available resin and membrane based adsorbers. High volumetric static binding capacities for lysozyme (90mg/mL) and polyclonal human IgG (hIgG, 92mg/mL) were found, suggesting an efficient multi-layer binding within the grafted hydrogel layer. A packed bed of randomly orientated fibers has been tested for packing efficiency, permeability and chromatographic performance. High dynamic binding capacities for lysozyme (50mg/mL) and hIgG (54mg/mL) were found nearly independent of the bed-residence time, revealing a fast mass-transport mechanism. Height equivalent to a theoretical plate (HETP) values in the order of 0.1 cm and a peak asymmetry factor (AF) of 1.8 have been determined by tracer experiments. Additionally inverse size-exclusion chromatography (iSEC) revealed a bimodal structure within the fiber bed, consisting of larger transport channels, formed by the voidage between the fibers, and a hydrogel layer with porous properties.

Preparation of a novel ionic hybrid stationary phase by non-covalent functionalization of single-walled carbon nanotubes with amino-derivatized silica gel for fast HPLC separation of aromatic compounds

Single-walled carbon nanotubes (SWCNTs) were immobilized on spherical silica gel with a 4-μm average particle size and a 60-Å average pore size. The amino-derivatized silica gel was non-covalently coated with carboxylated SWCNTs to preserve the structure of the nanotubes and their physico–chemical properties. The novel ionic hybrid stationary phase was characterized by scanning electron microscopy (SEM), infra-red (IR) spectroscopy and elemental analysis, and then, it was used to fill an empty 150×4.6mm2 high-performance liquid chromatography (HPLC) column. Chromatographic parameters, such as the theoretical plate number, retention factor and peak asymmetry factor, and analytical parameters, such as the limit of detection (LOD), limit of quantification (LOQ), linear range, calibration equation, and R2 value, and quantitative analysis parameters were calculated for all of the analytes. Using different mobile phases, five different classes of aromatic hydrocarbons were separated in a very short analysis time of 4–8min. Furthermore, a high theoretical plate number (up to 25000) and an excellent peak asymmetry factor (1.0) were obtained. The results showed that the surface of the SWNTs had very strong interactions with aromatic groups, therefore providing high selectivity for the separation of different classes of aromatic compounds. This study indicates that SWCNTs enable the extension of the application range of the newly prepared stationary phases for the fast separation of aromatic compounds by HPLC.

Synthesis of a mixed-model stationary phase derived from glutamine for HPLC separation of structurally different biologically active compounds: HILIC and reversed-phase applications

A novel mixed-mode stationary phase was synthesised starting from N-Boc-glutamine, aniline and spherical silica gel (4µm, 60Å). The prepared stationary phase was characterized by IR and elemental analysis. The new stationary phase bears an embedded amide group into phenyl ring, highly polar a terminal amide group and non-polar groups (phenyl and alkyl groups). At first, this new mixed-mode stationary phase was used for HILIC separation of four nucleotides and five nucleosides. The effects of different separation conditions, such as pH value, mobile phase and temperature, on the separation process were investigated. The optimum separation for nucleotides was achieved using HILIC isocratic elution with aqueous mobile phase and acetonitrile with 20°C column temperature. Under these conditions, the four nucleotides could be separated and detected at 265nm within 14min. Five nucleosides were separated under HILIC isocratic elution with aqueous mobile phase containing pH=3.25 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 265nm within 14min. Chromatographic parameters as retention factor, selectivity, theoretical plate number and peak asymmetry factor were calculated for the effect of temperature and water content in mobile phase on the separation process. The new column was also tested for nucleotides and nucleosides mixture and six analytes were separated in 10min. The chromatographic behaviours of these polar analytes on the new mixed-model stationary phase were compared with those of HILIC columns under similar conditions. Further, phytohormones and phenolic compounds were separated in order to see influence of the new stationary phase in reverse phase conditions. Eleven plant phytohormones were separated within 13min using RP-HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 230 or 278nm. The best separation conditions for seven phenolic compounds was also achieved using reversed-phase HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and seven phenolic compounds could be separated and detected at 230nm within 16min.

Determination of Thiamine in Pharmaceutical Preparations by Reverse Phase Liquid Chromatography Without Use of Organic Solvent

A novel green aqueous mobile phase modified with room temperature ionic liquids (RTILs) was employed in the absence of volatile organic solvents or ion-pairing reagents to analyze thiamine, a very polar compound, by reverse phase high performance liquid chromatography (RP-HPLC). Due to its strongly hydrophilic nature, thiamine was eluted near the column dead time (t0) using a mobile phase without adding RTILs or ion-pairing reagents, even if a 100% aqueous mobile phase, which has weak elution power under reverse phase conditions, was used. Thus, 1-ethyl-3-methyl-imidazolium hexafluorophosphate ([EMIM][PF6]), which has the strongest chaotropic effect, was selected as a mobile phase additive to improve retention and avoid baseline disturbances at t0. Various mobile phase parameters such as cation moiety, chaotropic anion moiety, pH and concentration of RTILs were optimized to determine thiamine at the proper retention time. Method validation was performed to assess linearity, intra- and inter-day accuracy and precision, recovery and repeatability; all results were found to be satisfactory. The developed method was also compared to the current official United States Pharmacopoeia (USP) and Korean Pharmacopoeia (KP) methods using an organic mobile phase containing an ionpairing reagent by means of evaluating various chromatographic parameters such as the capacity factor, theoretical plate number, peak asymmetry and tailing factor. The results indicated that the proposed method exhibited better efficiency of thiamine analysis than the official methods, and it was successfully applied to quantify thiamine in pharmaceutical preparations.

Polymerized phospholipid bilayers as permanent coatings for small amine separations using mixed aqueous/organic capillary zone electrophoresis

Phospholipid bilayer (SPB) coatings have been used in capillary electrophoresis to reduce the nonspecific adsorption between the capillary wall and cationic analytes. This paper describes the use of the polymerizable lipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (Diyne PC) as a permanent capillary coating. A supported phospholipid bilayer was formed on the capillary walls and polymerization was performed in situ using ultraviolet irradiation. The polymerization reaction was monitored by UV–visible absorbance spectroscopy and atomic force microscopy. The EOF of the polymerized Diyne PC coating was moderately suppressed (2.0×10−4cm2/Vs) compared to a non-polymerized Diyne PC bilayer (0.3×10−4cm2/Vs), but the stability was improved significantly. Separations of benzylamine, veratrylamine, phenylethylamine and tolyethylamine using a poly Diyne PC coated capillary yielded efficiency of 220,000–370,000 plates/m and peak asymmetry factor 0.48–1.18. Specifically, the poly(Diyne PC) coating provided improved separation resolution in NACE due to the reduced surface adsorption.

Chromatographic characterization of hydrophilic interaction liquid chromatography stationary phases: Hydrophilicity, charge effects, structural selectivity, and separation efficiency

Fourteen commercially available particle-packed columns and a monolithic column for hydrophilic interaction liquid chromatography (HILIC) were characterized in terms of the degree of hydrophilicity, the selectivity for hydrophilic–hydrophobic substituents, the selectivity for the regio and configurational differences in hydrophilic substituents, the selectivity for molecular shapes, the evaluation of electrostatic interactions, and the evaluation of the acidic–basic nature of the stationary phases using nucleoside derivatives, phenyl glucoside derivatives, xanthine derivatives, sodium p-toluenesulfonate, and trimethylphenylammonium chloride as a set of samples. Principal component analysis based on the data of retention factors could separate three clusters of the HILIC phases. The column efficiency and the peak asymmetry factors were also discussed. These data on the selectivity for partial structural differences were summarized as radar-shaped diagrams. This method of column characterization is helpful to classify HILIC stationary phases on the basis of their chromatographic properties, and to choose better columns for targets to be separated. Judging from the retention factor for uridine, these HILIC columns could be separated into two groups: strongly retentive and weakly retentive stationary phases. Among the strongly retentive stationary phases, zwitterionic and amide functionalities were found to be the most selective on the basis of partial structural differences. The hydroxyethyl-type stationary phase showed the highest retention factor, but with low separation efficiency. Weakly retentive stationary phases generally showed lower selectivity for partial structural differences.

Detection and quantification of ppb level potassium in biological samples in the presence of high sodium by ion chromatographic method

Employing a silica gel column modified with carboxyl groups and an eluent consisting of 4.0 mM tartaric acid and 0.75 mM dipicolinic acid in 2% acetone, an otherwise difficult quantification of K + at ppb level in presence of 6000 ppm NaCl was achieved by incorporating 0.75 mM 18-crown-6 ether in the mobile phase and subtracting the blank NaCl signal from each chromatogram. Optimized analytical conditions were established in terms of relative standard deviation (%) of retention time, peak area and calibration equations, and also by peak asymmetry factor. The net efflux of K + into the gastric lumen under in vitro conditions of acid secretion was investigated in Ussing chamber model. The effects of the physiological secretagogue histamine and the antisecretory agents cimetidine, omeprazole and SCH28080 were studied. The decline of K + efflux in presence of cimetidine, and the rise of the same in the presence of omeprazole and SCH28080 were conspicuously discernible, thereby validating the usefulness of ion chromatography based K + quantification method under biological experimental conditions.

Optimization of the separation of ionizable compounds in micellar electrokinetic chromatography by simultaneous change of pH and SDS concentration

A method to optimize the separation in micellar EKC (MEKC) of mixtures of acidic compounds as a function of two parameters, pH and concentration of sodium dodecyl sulfate, has been developed. The method considers the prediction of the retention time and the shape of the peaks. The retention time is predicted from the retention factor model and the peak shape by a polynomically modified Gaussian function that considers peak width, asymmetry factor, and height. An algorithm to calculate the global resolution of the separation at any experimental pH and [SDS] has been applied. This algorithm provides a 3-D resolution map to easily detect the areas in which resolution for the separation of the compounds is maximum. Initial experiments to fit the models have been performed with a set of ten phenolic compounds with different hydrophobicities and pK(a) values, and therefore, expected to behave in a different way with changes of pH and surfactant concentration. The experiments encompassed a pH range from 6.7 to 11.1, and a sodium dodecyl sulfate concentration range from 40 to 80 mM. Through the proposed methodology, chromatograms have been simulated at different pH and [SDS] very accurately. Furthermore, the resolution at any experimental point within the studied ranges have been also calculated, giving an optimum resolution value at pH 6.7 and [SDS] = 72 mM.

Optimization of binary porogen solvent composition for preparation of butyl methacrylate monoliths in capillary liquid chromatography

Butyl methacrylate monolithic columns in 320 μm i.d. fused silica capillaries for reversed-phase capillary liquid chromatography were prepared by radical polymerization initiated thermally with azobisisobutyronitrile (AIBN). Polymerization mixture contained butyl methacrylate (BMA) as the function monomer and ethylene dimethacrylate (EDMA) as the crosslinking agent with 1,4-butanediol and 1-propanol as a binary porogen solvent. Ratio of 1,4-butanediol to 1-propanol in the porogen solvent was optimized regarding the monolithic column efficiency and performance. Total porosity, column permeability, separation impedance, Walters hydrophobicity index, retention factors, peak asymmetry factors, height equivalents to a theoretical plate and peak resolutions were used for characterization of the prepared monolithic columns. The polymerization mixture consisting of 17.8% of BMA, 21.8% of EDMA, 18.0% of 1,4-butanediol, 42.0% of 1-propanol and 0.4% AIBN generated monolithic columns of the best performance having a sufficient permeability and the lowest separation impedance. It was also demonstrated that monolithic columns of this composition exhibited good preparation reproducibility and an excellent pressure resistance when applied in capillary liquid chromatography.