Good Peak Symmetry Research Articles

Analysis of Basic Drugs: A Comparison of Two Different Strong Cation Exchange-Modified LC Packings

Evidence in the literature of systematic investigation of retention mechanisms on strong cation-exchange (SCX) modified silica LC packings is lacking. In this study, the retention behaviour of selected basic drugs using a propylbenzenesulfonic acid-modified packing (Nucleosil 100SA) were compared, under similar conditions, with previous findings from a propylsulfonic acid-modified packing (Waters Spherisorb S5SCX). Eluent ionic strength, apparent pH, and solvent composition were investigated. Good efficiency (70,000 plates m−1) and peak symmetry (<1.5) were observed using the Nucleosil 100SA packing. The use of different alkyl ‘spacer’ moieties in each packing influenced retention, with hydrophobic partitioning of analytes more evident for the Nucleosil packing compared with Spherisorb, particularly when water-containing eluents were used. On Nucleosil 100SA, non ion-exchange interactions accounted for up to 61% of retention. Despite some correlation with analyte LogP on Nucleosil 100SA, and the environment surrounding basic nitrogen moieties, retention on both SCX packings was unrelated to analyte pKa.

Novel fabrication of on-column capillary inlet frits through flame induced sintering of stainless steel particles

A novel fritting technology was introduced for the fused-silica capillary. The technique involved sintering of stainless steel (SS) particles at the tip of capillary through flame heating. A simple butane gas based welding torch was used for sintering the SS particles. The new fritting technique, flame induced sintering of SS particles (FIS/SSP), was applied for making frits with different inlet diameters (75 μm, 100 μm, 250 μm and 530 μm). The changes in morphologies of SS particles during sintering process were identified by scanning electron microscopy (SEM). Frits with the length of 0.5–1 mm and capillaries with inner diameter about 50–100 μm were fabricated through suitable selection of experimental conditions (size of SS particles and heating mode). The frits prepared by FIS/SSP technique exhibited adequate separation properties and mechanical strength. Columns packed with C 18 particles were stable with these frits in a few important chromatographic operations. Frits prepared by FIS/SSP technique was used in three typical separation modes namely, capillary electrochromatography (CEC), p-assisted CEC (p-CEC) and low pressure liquid chromatography (LPLC). Importantly, no bubble formation was noticed with the frit over a period of one week. A good peak symmetry and high efficiency for separation were obtained using pressure-assisted CEC, p-CEC and low pressure-driven separation modes.

Fast and Sensitive New High Performance Liquid Chromatography Laser Induced Fluorescence (HPLC-LIF) Method for Quinine. Comparative Study in Soft Drinks

A simple, quick, and accurate new method for the determination of quinine (6′-methoxycinchonan-9-ol) in soft drinks is presented. The analysis is carried out using high performance liquid chromatography (HPLC), coupled laser induced fluorescence (LIF) that consisted of a 325 nm He-Cd laser and a ZETALIF detector. The chromatographic separation was performed on a Phenomenex Synergi Fusion-Reversed Phase (RP) column and allows good peak shape and symmetry in less than 1.5 min. A calibration curve ranging from 1 to 100 ng/mL was shown to be linear with a correlation coefficient (R) of 0.9999. The limit of detection of quinine was 3.2 pg on the column. The method was applied to the analysis of several beverages (n = 43) containing quinine, whose analysis required minimum pretreatment before direct injection, and can therefore be used for quality control in comparison to the classical methods. Data obtained from different commercial beverages containing quinine show no homogeneous concentration of this compound. This article describes, for the first time, the successful application of HPLC coupled LIF detection for quinine determination in common beverages.

Application of phase optimized liquid chromatography to oligopeptide separations

Stationary phase optimized liquid chromatography (POPLC) has been applied to the separation of oligopeptides. The retention factors and theoretical plate numbers of 13 peptides were determined on five different stationary phases. Based on these values, an optimal stationary phase composition of 250 mm total length consisting of 3 segments of 20 mm octadecyl silica, 10 mm phenyl silica and 220 mm embedded polar octadecyl silica was calculated by the optimizer software. Good agreement between the calculated and experimental chromatograms was observed. In order to achieve short analysis time and baseline separation of all peptides gradient elution and optimization of the column temperature were performed. The optimized mobile phase conditions consisted of (A) acetonitrile and (B) 0.025 M aqueous sodium phosphate buffer, pH 3.0, operated at 10% A (0–12 min) followed by 10–50% A (12–32 min) at a flow rate of 0.5 mL min −1 and column temperature of 35 °C. Using these conditions all peptides could be separated in less than 30 min with good resolution and peak symmetry.

Reversed-phase high-performance liquid chromatography coupled to ultraviolet and electrospray time-of-flight mass spectrometry on-line detection for the separation of eight tetracyclines in honey samples

Eight antibiotics, chlortetracycline, demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline and rolitetracycline, were separated and quantified in Spanish honey extracts of different floral origin using a commercial RP-C18 HPLC column and two different on-line detectors (diode array and electrospray time-of-flight mass spectrometry (ESI-TOF-MS) systems). Operating a linear gradient at a flow of 2 ml min −1 the HPLC separation of the eight antibiotics was obtained within 10 min with good peak symmetry and an acceptable resolution (2.1) for the critical band pair rolitetracycline and oxytetracycline. Values of the numbers of theoretical plates ( N) were comprised between 2328 and 19 448 while the limits of detection in honey were within 0.02–1.03 μg kg −1 in the case of UV detection and 0.05–0.76 μg kg −1 for ESI-TOF-MS detection (operating in negative mode). A recovery study was carried out by preparing some quality control samples at four levels of concentration (10, 25, 50 and 100 μg kg −1) and percentages between 72% and 98% were attained.

Simultaneous Determination of Vincristine, Vinblastine, Catharanthine, and Vindoline in Leaves of Catharanthus roseus by High-Performance Liquid Chromatography

A simple reversed-phase liquid chromatographic method is developed for the simultaneous quantitation of the anticancerous drugs vincristine, vinblastine, and their precursors catharanthine and vindoline using a Merck Chromolith Performance reversed-phase high-performance liquid chromatography column. A better resolution is obtained in comparison with available particulate-type C18 columns. The column provides good reproducibility and peak symmetry. Chromatography is carried isocratically with a mobile phase of acetonitrile-0.1M phosphate buffer containing 0.5% glacial acetic acid (21:79, v/v; pH 3.5) at a flow rate of 1.2 mL/min and UV detection at 254 nm. Parameters such as linearity, limits of quantitation (LOQ) and detection (LOD), precision, accuracy, recovery, and robustness are studied. The method is selective and linear for alkaloid concentration in the range 0.25 microg-25 microg/mL. The LOQ and LOD are 25, 46, 56, and 32 microg/mL and 8, 14, 18, and 10 microg/mL, respectively. The results of accuracy studies are good. Values for coefficient of variation are 2.50, 1.82, 1.33, and 1.13, respectively. The percent recovery of the alkaloids was found to be 96%, 97%, 98%, and 98%, respectively. Peak purity and homogeneity of these compounds in plant extract is studied using a photodiode-array detector. This simple and rapid method of analysis is applied for the determination of these alkaloids in a large number of leaf extracts of Catharanthus roseus..

Simultaneous Determination of Clobutinol Together with Some Anti‐inflammatory Drugs in Urine by HPLC

An isocratic high performance liquid chromatography method is described for simultaneous determination of clobutinol hydrochloride together with some anti‐inflammatory drugs, such as diclofenac, meloxicam, and nimesulide in urine. For the development and optimization of the system, three different buffers containing ammonium acetate, tetraethylammonium hydrogen sulfate (THAS), and tetrabutylammonium hydrogen sulfate (THBS) were investigated, because it has been proven that different salts added in the mobile phases considerably affect solute retention and selectivity. The effect of salt content in the aqueous portion of the mobile phase together with pH over a wide range, was investigated. A response surface method based on non‐linear multiple regression analysis was employed to illustrate the changes in k′ values as a function of a range of pH values and different salts contents. On the basis of the chromatographic behavior of clobutinol together with the other drugs determined, optimum chromatographic conditions were achieved with good peak symmetry, reasonable retention time, and noticeable separation. The intra‐ and inter‐day accuracy and precision at low, medium, and high concentration(s) for the compounds were in the range %error 5.40–11.50 and %RSD 1.76–5.06, respectively.

HPLC Determination of Tetracyclines in Lamb Muscle Using an RP-C18 Monolithic Type Column

Four antibiotics, oxytetracycline (OTC), tetracycline (TCC), chlortetracycline (CTC) and doxycycline (DXC), were separated and quantified in lamb muscle extracts using a commercial RP-C18 monolithic HPLC column and a DAD detector. Operating an isocratic flow of 2 mL min−1 the HPLC separation of the four compounds was obtained within 6 min with a good peak symmetry and an acceptable separation factor (1.27) for the critical peak pair OTC-TCC. Values of the normalized numbers of theoretical plates (N cm−1) were comprised between 338–780 while the limits of quantitation (S/N=10) were within 12–65 μg kg−1.

Separation of twenty underivatized essential amino acids by capillary zone electrophoresis with contactless conductivity detection.

Twenty underivatized essential amino acids were separated using capillary zone electrophoresis and consequently detected with contactless conductivity detection (CCD). A simple acidic background electrolyte (BGE) containing 2.3 M acetic acid and 0.1% w/w hydroxyethylcellulose (HEC) allowed the electrophoretic separation and sensitive detection of all 20 essential amino acids in their underivatized cationic form. The addition of HEC to the BGE suppressed both, electroosmotic flow and analyte adsorption on the capillary surface resulting in an excellent migration time reproducibility and a very good analyte peak symmetry. Additionally, the HEC addition significantly reduced the noise and long-term fluctuations of the CCD baseline. The optimized electrophoretic separation method together with the CCD was proved to be a powerful technique for determination of amino acid profiles in various natural samples, like beer, yeast, urine, saliva, and herb extracts.

Application of mixed partition–adsorption systems in high-performance liquid chromatography of purines and pyrimidines

Separation of the test mixtures of some purine and pyrimidine derivatives on silicas (types A and B) in adsorption normal-phase (A-NP) and mixed partition–adsorption normal-phase (MPA-NP) mode has been studied. When the A-NP mode is used the peak shapes are unsatisfactory (especially on type A silica). At the same time MPA-NP systems show a good peak symmetry on all silica types. The findings have demonstrated that the MPA-NP mode offers a specific selectivity. This allows MPA-NP systems to supersede reversed-phase systems in some application areas.

Separation of purines and pyrimidines by normal-phase high-performance liquid chromatography using dimethyl sulfoxide in binary and ternary eluents

Chromatographic systems with a silica sorbent and mobile phases containing dimethyl sulfoxide have been studied. It has been established that the substitution of isopropanol by dimethyl sulfoxide in binary eluents results in a specific selectivity of the chromatographic system and shows an improvement of the peak shape for the solutes under study. When mobile phases consisting of hexane, isopropanol and dimethyl sulfoxide (solvents with a limited mutual solubility) are used, changes in retention characteristics and peak symmetry are caused by a transition from adsorption to partition sorption mechanism. The stationary liquid phase is generated dynamically in the pores of silica, even in the mobile phases not saturated with a polar component. If the phase ratio of the column reaches 0.1, partition dominates over adsorption and such mixed partition–adsorption (MPA) systems show very good peak symmetry for the solutes under study. The investigation has shown that dimethyl sulfoxide-containing MPA systems are applicable in analytical practice.

High-performance liquid chromatography of some purine and pyrimidine derivatives on silica in hexane–isopropanol–ethylene glycol mobile phases

An alternative approach for the separation of polar solutes in chromatographic systems consisting of unmodified silica and mobile phases containing solvents with limited mutual solubility has been developed. The investigation of ternary mobile phases consisting of hexane, isopropanol and ethylene glycol has shown that the stationary liquid phase is generated dynamically in the pores of silica, even in the mobile phase not saturated with a polar component. If the phase ratio of the column reaches 0.1 partition dominates over adsorption and such mixed partition–adsorption systems show a good column efficiency and peak symmetry of some purine and pyrimidine derivatives.

Microassay of propranolol enantiomers and conjugates in human plasma and urine by high-performance liquid chromatography after chiral derivatization for pharmacokinetic study

A microdetermination of propranolol enantiomers and of their glucuronide and sulphate conjugates in human plasma and urine by reversed-phase HPLC after chiral derivatization is described. After extraction from 100 microliters of plasma or urine with racemic 4-methylpropranolol as internal standard (I.S.), the enantiomers are derivatized with R(+)-phenylethylisocyanate as chiral derivatization reagent. Chromatography is performed on Novapak C18 column with fluorescence detection. Glucuronide and sulphate conjugates are cleaved prior to extraction by incubating, respectively, the samples with glucuronidase-arylsulphatase and saccharic acid 1-4 lactone as specific glucuronidase inhibitor. The retention times of propranolol and I.S. enantiomer derivatives are short (tR = 5.5-6.2 min and 8.8-10.1 min, respectively). The diastereomeric derivatives are very stable and show good peak symmetry and resolutions (RS = 2 and 2.2). The use of 4-methylpropranolol as I.S. improves significantly relative standard deviations (RSD: 1.7-5.1). Sensitivity is about 1 ng ml-1 per enantiomer. The method is applied to pharmacokinetic studies of racemic propranolol in human plasma and urine. S-propranolol and its conjugates show higher concentrations than R-propranolol and its conjugates in plasma and urine.

Packed-column supercritical fluid chromatography of omeprazole and related compounds: Selection of column support with triethylamine- and methanol-modified carbon dioxide as the mobile phase

Using standardized chromatographic conditions with respect to carbon dioxide and methanol containing 1% of triethylamine as modifier, the chromatographic behaviour of omeprazole and four of its analogues was investigated on six types of chromatographic support at 40°C. Superior selectivity was obtained for omeprazole versus its anlogues with NH 2-modified silica and also good peak symmetry (LiChrosorb and Kromasil). Using the present chromatographic system it is possible to detect and separate the analogues added to omeprazole in the 0.5% (w/w) range within 10 min.

High-performance liquid chromatography of some purine and pyrimidine derivatives on silica in formamide-ethyl acetate mobile phases

The chromatographic system with a silica sorbent and formamide-ethyl acetate as the mobile phase, amongst others, has been studied. It was established that the liquid stationary phase is deposited in the pores of silica during its equilibration with the eluent. The dynamically generated liquid-liquid system showed very good column efficiency and peak symmetry for the polar solutes under study. The mechanism of sorption is mixed, involving adsorption on the silica surface and partition. The contribution of each process depends on the volume of deposited liquid phase. In favourable cases the phase ratio of the system can reach 0.3. The sensitivity of the retention values to the temperature is usually not greater than that in traditional chromatographic modes on silica, although column thermostatting sometimes may be desirable. The disadvantage of such systems is a high consumption of solvents for reequilibration after work with chemically different eluents. In routine analysis, when this disadvantage is not critical, such systems can be useful because of their unique selectivity.

Reversed-phase ion-pair liquid chromatography of the angiotensins

The isocratic reversed-phase liquid chromatography of the angiotensins and a number of their synthetic analogues is described. Complete separation of 10 out of 12 peptides was achieved through a solvent optimization strategy with a total analysis time of about 20 min. The retention behavior of the angiotensins studied was described in terms of the hydrophobic contribution of their amino acid residues; there was good correlation between predicted and experimental retention for those peptides that were retained by a common mechanism. However, because ion-pair chromatography was required for good peak symmetry, retention was substantially modulated by the presence of acidic and basic residues. The limit of detection of these peptides was 3–5 pmol by UV absorbance at 214 nm. For those peptides containing a primary amino group the detection limit was improved by two orders of magnitude by fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde/cyanide to the corresponding N-substituted 1-cyanobenz[ f]isoindole (CBI) derivatives. The contribution of the CBI ring system to retention was also investigated.

Use of silica gel with aqueous eluent for simultaneous high performance liquid chromatographic assay of disopyramide and mono-N-dealkyldisopyramide.

Disopyramide and its pharmacologically active metabolite, mono-N-dealkyldisopyramide, were determined simultaneously in serum by high performance liquid chromatography using a bare (unbonded) silica gel with aqueous eluents. p-Chlorodisopyramide was used as the internal standard. Separations of all compounds, which contain amine moiety in their chemical structures, were easily accomplished with a good peak symmetry on silica. The method is sufficiently precise, sensitive, and specific. Analytical recoveries of all compounds were greater than 96%; CVs for reproducibility were less than 6.5% for disopyramide and mono-N-dealkyldisopyramide; the lower detection limits of both analytes were 0.05 mg/L. Comparison of the present method with a fluorescence polarization immunoassay for disopyramide gives a good correlation (r = 0.992).

Simultaneous determination of lidocaine and its principal metabolites by liquid chromatography on silica gel, with aqueous eluent.

We describe the simultaneous determination of lidocaine and its pharmacologically active metabolites, monoethylglycinexylidide and glycinexylidide, in plasma by "high-performance" liquid-chromatography. By use of a bare ( unbonded ) silica gel with aqueous eluents, separations of organic amines such as lidocaine and its metabolites, which are very difficult and have a poor peak symmetry on bonded reversed-phase packings, were easily accomplished with a good peak symmetry. The method is sufficiently precise, sensitive, and specific. Analytical recoveries of all compounds were greater than 90%; CVs for reproducibility were less than 5% for all compounds; the lower detection limits were 0.1 mg/L or less. This method can be used to monitor the concentrations of these compounds in plasma and to prevent the concentration-related side-effect(s).

The role of bonded phase composition on the ligand-exchange chromatography of dansyl- D, L-amino acids

This paper examines the role of bonded phase composition in the ligand-exchange chromatographic separation of dansyl amino acids (Dns- D, L-AAs). As the bonded optically active chelate, we have synthesized N-ϖ-(dimethylsiloxyl)undecanoyl- L-valine and as the metal, we used Cu(II). Bonded phases, where the chelate coverage is 2.5 μmol/m 2, have been compared with mixed or diluted phases, in which the contribution of the bonded chelate to the total coverage is less than 0.2 μmol/m 2. Using a decyl group as diluent, it has been shown that capacity factors and enantio selectivities of Dns- D, L-AAs are much higher on the diluted phases relative to the concentrated phase, in spite of the fact that the amount of copper loaded on the latter column is roughly 40 times greater. This result has been interpreted in terms of the formation of his complexes between the metal ion and the bonded phase chelate, i. e., 2:1 chelate to metal, in the case of the concentrated phase. For the diluted phases, the mono complex, i.e., 1:1 metal to chelate, is the predominant form. Not only does the diluted phase yield improved separation, but it is in principle a simpler system to manipulate and optimize. We have been able to develop a simple model to analyze the chromatographic behavior on diluted phases relative to the concen trated phase. Based on this model, we have compared four diluents: a polyether, n- butyl, n-decyl and n-eicosyl ligands. By normalizing for the amount of copper loaded on the stationary phase, it is shown that the apparent complexation constants in crease from the ether to the C 20 phase. Moreover, due to differences in the character of the side chains of the amino acids, additional selectivity is observed between the different solutes. This selectivity is useful in separation applications. In addition, it is shown that the diluted bonded phases yield high performance, approximately 4000 plates for 15 cm columns, with good peak symmetry. Also included is a detailed discussion of the characterization of the bonded phases along with its optical purity.

Applications of high-speed data acquisition for semiconductor device yield analysis, Part I : John M. Charles and Mikkel W. Lantz. Solid St. Technol., 119 (March 1982)

A novel fritting technology was introduced for the fused-silica capillary. The technique involved sintering of stainless steel (SS) particles at the tip of capillary through flame heating. A simple butane gas based welding torch was used for sintering the SS particles. The new fritting technique, flame induced sintering of SS particles (FIS/SSP), was applied for making frits with different inlet diameters (75 μm, 100 μm, 250 μm and 530 μm). The changes in morphologies of SS particles during sintering process were identified by scanning electron microscopy (SEM). Frits with the length of 0.5–1 mm and capillaries with inner diameter about 50–100 μm were fabricated through suitable selection of experimental conditions (size of SS particles and heating mode). The frits prepared by FIS/SSP technique exhibited adequate separation properties and mechanical strength. Columns packed with C18 particles were stable with these frits in a few important chromatographic operations. Frits prepared by FIS/SSP technique was used in three typical separation modes namely, capillary electrochromatography (CEC), p-assisted CEC (p-CEC) and low pressure liquid chromatography (LPLC). Importantly, no bubble formation was noticed with the frit over a period of one week. A good peak symmetry and high efficiency for separation were obtained using pressure-assisted CEC, p-CEC and low pressure-driven separation modes.