Compounds In High-performance Liquid Chromatography Research Articles

Separation of Methanolic Leaf Extracts of Three Rutaceous Plants by Capillary Electrophoresis and High-Performance Liquid Chromatography Methods

Pharmacognosy Research,2023,15,2,392-398.DOI:10.5530/pres.15.2.042Published:February 2023Type:Original Article Authors:Chakravarthi Guntupalli, Narender Malothu, Ankarao Areti, Buchi N Nalluri, Praveen Sivadasu, and Narayanarao Alla Author(s) affiliations:Chakravarthi Guntupalli*, Narender Malothu, Ankarao Areti, Buchi N Nalluri, Praveen Sivadasu, Narayanarao Alla K L College of Pharmacy, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Guntur, Andhra Pradesh, INDIA. Abstract:Introduction: In the phytochemical analysis of various multi component mixtures, Capillary electrophoresis (CE) method was considered as the best alternative tool due to high efficiency resolution separations, low solvent consumption and less maintenance costs. Materials and Methods: This study uses CE and high-performance liquid chromatography (HPLC) methods to evaluate the major components of methanolic leaf extract of Rutaceous plants. And also, to assess the purity of the components in isolated fractions and most importantly for the qualitative identification of the individual components of extracts based upon the relative retention time and UV profile. Results: The separation of the compounds in HPLC was attained by using ACE-5-C18 (250 x 4.6 mm) with a flow rate of 1.5 ml/min, with the UV detection at 254 nm and CE was equipped with a UV diode-array detector and bare, fused silica capillaries (56 cm 50 μm) with micro extended light path were used for the separations. Conclusion: Our study reveals that CE technique is a simple, economical and convenient method and shown high-resolution for multi-components found in plant extracts compared with HPLC profiles. Keywords:Capillary Electrophoresis, HPLC, Relative Retention Time, Rutaceae.View:PDF (425.61 KB)

Synthesis of Large-Pore Silica Microspheres Using Dodecylamine as a Catalyst, Template and Porogen Agent

This paper reports a strategy combing a modified Stober method and a post-hydrothermal treatment for the fabricating of silica microspheres with large pores. Dodecylamine (DDA), used as a catalyst, template and porogen agent, plays an important role in controlling both the particle morphology and the pore expansion. By adjusting the concentration of DDA (20, 40 and 80 mmol/L) and the temperature of hydrothermal treatment (80–140 °C), the pore size ranging from 3.5 to 16 nm could be obtained. The synthesized sub-2 µm silica spheres were modified with dimethyloctadecylchlorosilane (C18) and successfully applied for the separation of aromatic compounds in high-performance liquid chromatography (HPLC). The silica may be a good candidate as HPLC packing materials.

Preparation and Application of Aza-15-crown-5-capped Methylcalix[4]resorcinarene-Bonded Silica Particles for Use as Chiral Stationary Phase in HPLC

Aza-15-crown-5-capped (3-(C-methylcalix[4]resorcinarene)-2-hydroxypropoxy)-propylsilyl (MCR-HP)-appended silica particles (15C5-MCR-HPS), a new type of substituted C-methylcalix[4]resorcinarene (MCR)-bonded chiral stationary phase (CSP) for high-performance liquid chromatography (HPLC), have been synthesized by reaction of bromoacetate-substituted MCR-HPS with excessive aza-15-crown-5 in anhydrous acetonitrile in the presence of potassium carbonate. The bonded phase 15C5-MCR-HPS is characterized by elemental analysis and further evaluated by separating several disubstituted benzenes and some chiral drug compounds in HPLC. The new CSP has a chiral selector with two recognition sites: 15-crown-5 and MCR-HP, which can provide cooperative multiple interactions with solutes to enhance chiral recognition and to improve chromatographic separation. The chromatographic evaluation results show that 15C5-MCR-HPS has excellent selectivity for the separation of aromatic positional isomers and enantiomers of some chiral compounds under multiple-mode mobile phase conditions including normal phase, reversed phase, and polar organic mobile phase conditions.

Preparation and Enantioseparation Characteristics of a Novel β-Cyclodextrin Derivative Chiral Stationary Phase in High-Performance Liquid Chromatography

A novel cyclodextrin (CD) derivative mono(6(A)-N-allylamino-6(A)-deoxy)per-3-chlorine-4-methyl-phenylcarbamoylated-β-CD was synthesized and chemically immobilized onto the surface of γ-mercaptopropyl-functionalized silica gel step by step. The products were all purified and characterized and then got a substance with definitional structure. This chiral stationary phase (CSP) of 3-chlorine-4-methyl-phenylcarbamoylated-β-CD bonded on silica gel exhibited excellent separation capability for several chiral compounds in high-performance liquid chromatography. In the enantiomeric separations of five racemates with a mixture of methanol and aqueous KH2PO4 buffer as the mobile phase, this CSP presented good chiral recognition performance.

Recent advances in development and application of derivatization reagents having a benzofurazan structure: a brief overview

Chemical derivatization is often used to improve the separation efficiency and to enhance the detectability of the target compounds in high-performance liquid chromatography and capillary electrophoresis. The derivatization reagents having a benzofurazan (2,1,3-benzoxadiazole) structure are one of the most often used reagent for this purpose. In this paper, the recent advances in the development and the application of benzofurazan derivatization reagents are reviewed.

Diaterebic Acid Acetate and Diaterpenylic Acid Acetate: Atmospheric Tracers for Secondary Organic Aerosol Formation from 1,8-Cineole Oxidation

Detailed organic speciation of summer time PM10 collected in Melbourne, Australia, indicated the presence of numerous monoterpene oxidation products that have previously been reported in the literature. In addition, two highly oxygenated compounds with molecular formulas C9H14O6 (MW 218) and C10H16O6 (MW 232), previously unreported, were detected during a period associated with high temperatures and bushfire smoke. These two compounds were also present in laboratory-produced secondary organic aerosol (SOA) through the reaction of OH radicals with 1,8-cineole (eucalyptol), which is emitted by Eucalyptus trees. The retention times and mass spectral behavior of the highly oxygenated compounds in high-performance liquid chromatography (LC) coupled to electrospray ionization-time-of-flight mass spectrometry (MS) in parallel to ion trap MS of agree perfectly between the ambient samples and the laboratory-produced SOA samples, suggesting that 1,8-cineole is the precursor of the highly oxygenated compounds. The proposed structure of the compound with molecular formula C10H16O6 was confirmed by synthesis of a reference compound. The two novel compounds were identified as diaterebic acid acetate (2-[1-(acetyloxy)-1-methylethyl]succinic acid, C9H14O6) and diaterpenylic acid acetate (3-[1-(acetyloxy)-1-methylethyl]glutaric acid, C10H16O6) based on the consideration of reaction mechanisms, the structure of a reference compound, and the interpretation of mass spectral data. Depending on the experimental conditions, the SOA yields determined in chamber experiments ranged between 16 and 20% for approximately 25 ppb of hydrocarbon consumed. The concentrations of these compounds were as high as 50 ng m(-3) during the summertime in Melbourne. This study demonstrates the importance and influence of local vegetation patterns on SOA chemical composition.

Suppression effects of carbonate on the interaction between stainless steel and phosphate groups of phosphate compounds in high-performance liquid chromatography and electrospray ionization mass spectrometry

We examined the suppression methodology of the interaction between phosphate compounds, such as nucleotides, and the stainless steel surfaces of high-performance liquid chromatography and electrospray ionization mass spectrometry (HPLC/ESI-MS) equipment in an effort to prevent the tailing of peaks seen in HPLC chromatograms of phosphate compounds. Addition of carbonate (CO 3 2−) to mobile phase was highly effective in suppressing the interaction of phosphate compounds derived from a complexation between phosphate groups and metal ions that exist on a stainless steel surface in a mechanism similar to Fe(III)- and Cr(III)-immobilized metal affinity chromatography (IMAC). Addition of ammonium hydrogen carbonate to mobile phase achieved a simple and reliable HPLC/ESI-MS analysis of mono-, di-, and triphosphate compounds (six nucleotides) without peak tailing due to the interaction between stainless steel surfaces and phosphate groups. Moreover, ammonium hydrogen carbonate buffer, a volatile buffer with good buffering capacity at neutral pH, does not compromise the stability of silica-based HPLC columns, decreases in sensitivity, ion source pollution, clogging of the ESI interface, and/or ion suppression in HPLC/ESI-MS.

Application of chemiluminescence for the detection of peroxy compounds in high-performance liquid chromatography.

The possibility of applying post-column reaction and chemiluminescence to determine organic peroxy compounds by RP-HPLC was investigated. Conditions of qualitative and quantitative analyses have been established. The applicability of the method has been demonstrated for a series of compounds representative of the most important groups of peroxy-type compounds, that is, hydroperoxides, dialkyl peroxides, diacyl peroxides, peroxyesters, and peroxyacids.

Retention of ionizable compounds in high-performance liquid chromatography: IX. Modelling retention in reversed-phase liquid chromatography as a function of pH and solvent composition with acetonitrile–water mobile phases

The influence of pH and solvent composition of acetonitrile–water mobile phases on the retention of acids and bases on a polymeric stationary phase is studied. Very good relationships between retention and mobile phase pH are obtained if the pH is measured in the proper pH scale. The fit of retention to pH for a particular solvent composition provides the p K a values of the equilibria between the different acid–base species and the retention parameters of these species at this solvent composition. Several models are tested that relate these parameters to solvent composition and properties in order to propose a general model to predict retention for any mobile phase pH and composition.

Retention and chiral recognition mechanism of organo-phosphorus compounds in high-performance liquid chromatography

Retention and chiral recognition mechanism of organo-phosphorus compounds in high-performance liquid chromatography

Retention and chiral recognition mechanism of organo-phosphorus compounds in high-performance liquid chromatography

In normal phase condition, a series of chiral phosphorus organic compounds have been separated by high-performance liquid chromatography. In order to study the retention and chiral recognition mechanism, the method of quantitative structure-enantioselectivity retention relationships (QSERRs) has been investigated from the quantitative equations established between the chromatographic retention of enantiomers and their molecular descriptors of physicochemical properties. The results show that on the Pirkle-type chiral stationary phase (CSP) of Sumichiral OA4700, it is the parameter of LUMO that gives the most contribution to the chromatographic retention of O-ethyl O-(substituted) phenyl N-isopropyl phosphoroamidothioates resulting from the interaction of hydrogen bond and (or) π-π interaction. Meanwhile, the chiral recognition is formed from the contribution of logP and LUMO.

Retention behavior of adenine compounds in HPLC with chloroacetaldehyde for fluorescence detection on ODS treated monodispersed—Spherical particle silicas

In this work, a simple system for the simultaneous fluorescence detection of adenine compounds in mouse liver has been developed using high-performance liquid chromatography (HPLC) with ODS-treated monodispersed spherical particle silicas (MDSi).

Effect of temperature and flow-rate on analysis of basic compounds in high-performance liquid chromatography using a reversed-phase column

The peak shape and retention of some basic probes together with a neutral reference compound were investigated as a function of temperature and flow-rate using a reversed-phase HPLC column at both pH 3.0 and pH 7.0. The retention of bases often showed an anomalous increase with temperature; retention mechanisms are complex as shown by studies of the effect of buffer cation concentration on retention. Considerable improvements in column efficiency for bases may result from operation at elevated temperature. Improvements did not seem attributable either to incidental changes in the retention factor, or (in this particular study where low sample masses were utilised) to the influence of sample load. The optimum flow-rate for highest efficiency is generally lower for basic compounds than neutrals, and due to the steepness of the Van Deemter curves obtained, high flow-rates appear to be particularly detrimental in the chromatography of basic compounds.

Study of phenformin metabolism in rat liver microsomes by HPLC, CE and on-line HPLC-electrospray ionization mass spectrometry.

Methods for the analysis of phenformin and its metabolite by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLC-ESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4-hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and K(m) was 4 microM.

Influence of organic solvent modifier and solvent strenght on peak shape of some basic compounds in high-performance liquid chromatography using a reversed-phase column

Comparative column effiency and peak assymmetry measurements were made for 16 pyridine derivatives using approximately isoeluotropic mixtures of methanol, acetonitrile and tetrahydrofuran (THF) in combination with phosphate buffer at pH 7.0 on a RP-HPLC column suitable for the analysis of basic compounds. On average, THF and methanol gave significantly better peak shape than acetonitrile, although considerable variations in behaviour for the different derivatives were noted. Methanolic mobile phases yielded straight line plots of log k′ vs. percentage of modifier; nevertheless the increase in k′ with decreasing methanol concentration is accompanied by a deterioration in peak shape which varies in extent depending on the nature of the probe compound.

Influence of analyte stereochemistry and basicity on peak shape of basic compounds in high-performance liquid chromatography with reversed-phase columns, using pyridine and alkyl-substituted derivatives as probe compounds

Column efficiency, peak asymmetry and retention factor of a series of sixteen compounds comprising pyridine and some alkyl-substituted derivatives, with p K a values ranging from 5.17 to 6.74 is reported on a RP-HPLC column previously shown to be especially suitable for the analysis of basic compounds. Compound stereochemistry in close vicinity of the basic group is found to influence peak shape strongly: however, stereochemistry at more remote positions is also of some significance. The effect of p K a on peak shape is discussed in the light of measurements of the degree of protonation of the compounds in the methanolic mobile phase as well as water.

Retention Behavior of Conjugated Metabolites of Vitamin D and Related Compounds in High-Performance Liquid Chromatography

The retention behavior of sulfates or glucuronides of provitamin D, vitamin D, and 25-hydroxyvitamin D3, together with its fluorescent derivatives, are examined with reversed-phase high-performance liquid chromatography. Inclusion chromatography using cyclodextrin as a mobile-phase additive is also used for this purpose. Conventional reversed-phase high-performance liquid chromatography clearly separates the positionally isomeric conjugates of 25-hydroxyvitamin D3. The addition of the host compound to the mobile phase is effective in separating the pairs of fluorescent derivatives of vitamin-D3 and -D2 conjugates or provitamin-D3 and -D2 conjugates.

4,5-Diaminophthalhydrazide as a highly sensitive chemiluminescence derivatization reagent for α-dicarbonyl compounds in high-performance liquid chromatography

4,5-Diaminophthalhydrazide (DPH) was found to be a highly sensitive chemiluminescence derivatization reagent for α-dicarbonyl compounds in high-performance liquid chromatography. The reagent reacts with α-dicarbonyl compounds in dilute hydrochloric acid in the presence of β-mercaptoethanol to give highly chemiluminescent quinoxaline derivatives which produce chemiluminescence by reaction with hydrogen peroxide and potassium hexacyanoferrate(III). The DPH derivatives of five α-dicarbonyl compounds were separated on a reversed-phase column, TSKgel ODS-120T, with a mixture of acetonitrile and 10 m M ammonium acetate, followed by chemiluminescence detection. The detection limits are in the range 1.1–300 fmol for a 20-μl injection volume (signal-to-noise ratio 3).

Factor analysis and experimental design in high-performance liquid chromatography : XI. Factor analysis maps and chromatographic information

A progressive approach to the exploitation of chromatographic data, based on factor analysis, is presented. This approach is applied to the retention data k′ of a large series of compounds in high-performance liquid chromatography. The chromatographic information. i.e, affinity and selectivity, is extracted with help of principal component analysis (PCA) and correspondence factor analysis (CFA). The factor analysis gives rise to three factorial maps to present the chromatographic information: PCA affinity map, CFA trend analysis map and CFA distance analysis map. Examples of extraction of chromatographic information are given for the simultaneous exploitation of these maps. Possibilities and limitations of this approach are discussed.

Possibilities of enhancing the sensitivity of the determination of UV-absorbing compounds in high-performance liquid chromatography

The signal-to-noise ratio and the detection limits in high-performance liquid chromatographic (HPLC) analysis using UV detection depend on the design of the detector settings. For the best sensitivity, the detector should be operated at a wavelength as close as possible to the absorption maxiumum, with a spectral band width as narrow as possible and with a frequency of the detector signal storage between 10 to 20 data per chromatographic peak. However, variations of the detector setting parameters, except for the detection wavelength, do not affect sensitivity and the detection limits very significantly. In high-speed HPLC, computerized accumulation of several complete chromatograms from repeated runs can be used to enhance the sensitivity and to improve the detection limits. As has been verified on the example of reversed-phase chromatography of three chlorobenzenes with UV detection, this approach can be used with good accuracy to obtain detection limits below 100 ppb with an analysis time of 20–25 min. These are still considerably higher than the detection limits that can be obtained using on-line sample enrichment techniques with solid-phase extraction, but the chromatogram accumulation approach may be useful in those trace analysis problems where sample enrichment is difficult or tedious and where selective derivatization techniques or sensitive detectors are not available.