Normal-phase Systems Research Articles

Thermodynamic analysis of adsorption and retention behaviors in normal-phase liquid chromatography

This study investigated the adsorption mechanisms in a normal-phase system using a cyano-based stationary phase as the sorbent. The minor disturbance method was used to measure the adsorption isotherms of acetone and alcohols with various structures. Excluding data in pure n-hexane revealed that the adsorption behaviors on cyano sites were well described by the Langmuir model. The adsorption equilibrium constants, ranging from 8.86 to 11.15 at 25 °C, showed no significant differences across alcohol structures and decreased with increasing temperature. The saturation adsorption concentration decreased with increasing alcohol molecule size, with branched-chain alcohols showing a lower saturation adsorption amount compared to straight-chain alcohols. The standard state adsorption enthalpies and entropies calculated from the equilibrium constants for various alcohols ranged from −29 to −22 kJ/mol and −78 to −55 J/K·mol, respectively, showing enthalpy-entropy compensation. A discrepancy was observed between these adsorption enthalpies and those obtained from the retention factors of alcohols using pure n-hexane as the mobile phase. This discrepancy may result from the affinity energy distribution of the adsorbent. In pure n-hexane, the adsorption behaviors of adsorbates were considerably affected by high-affinity sites. Moreover, acetone and these alcohol molecules were used as solvent modifiers to investigate the relationship between the retention factor, modifier concentration, and temperature for various solutes with distinct functional groups. The retention curves were converted to enthalpic curves using the van't Hoff equation. A theoretical model was proposed to describe the relationship between the van't Hoff enthalpy change and mobile phase composition. The proposed model effectively described the enthalpic curves, indicating that the enthalpy change follows a saturation curve with increasing modifier concentration. This trend is primarily due to competitive adsorption and complexation behaviors between the solute and modifier molecules.

Application High-Performance Thin-Layer Chromatography with Controlled Eluent Velocity to Determine Fingerprints of Various Poplar Species Buds Extracts: Optimization of Operating Variables

The study used the prototype device enabling the development of isocratic and gradient thin-layer chromatograms with controlled eluent velocity to obtain fingerprints of extracts from buds of various poplar species. This equipment can deliver an eluent to virtually any place on the adsorbent layer of the chromatographic plate. Moreover, the composition of a mobile phase can be easily modified even when the chromatogram is developing. This paper shows the effect of various operating variables of the prototype in a normal phase system, e.g., eluent velocity and composition, on the chromatographic behavior of the mixture. The prototype’s results were compared to conventional TLC development in horizontal DS chambers. The UV light (366 nm) was used to detect the solute zones and document both systems. The results prove that the prototype device has great potential in plant material standardization. The mobile phase gradient improves the separation of the bands of strongly retained solutes. Additionally, this fact does not enhance the blurring of low-retained bands.

Separation and determination of the group-type composition of modern base and lubricating oils with a wide range of polarity, especially emitted to the environment

Lubricating oils are composed of base oils (>85% v/v) and enriching additives (<15% v/v). Three types of base oils may be distinguished: 1) traditional bases (obtained by low-volatile fractions from crude oil distillation refining), 2) synthetic bases (mainly poly-alpha-olefins, sometimes esters, especially succinic acid esters), 3) bases of natural origin (especially obtained from refined plant oils). The bases of natural origin are the only ones recommended for application when lubricating oil may be emitted to the environment (e.g. when the machine with an open cutting system is used). Group-type separation and analysis of group-type composition of base and lubricating oils are of significant importance in quality control and environmental monitoring. Due to the potentially wide range of polarity of the components of base and lubricating oils, group- type separation becomes a difficult separation problem. It is also a serious analytical problem due to the considerable diversity of physicochemical properties. The authors propose a new procedure for the separation and determination of the group-type composition of base and lubricating oils using thin-layer liquid chromatography in normal phase systems (abr. NP-TLC) on silica gel plates impregnated with berberine salt/in the coupling of thin-layer chromatography with flame ionization detection (abr. TLC-FID). A new, effective procedure of TLC plates impregnation with berberine sulphate was presented. The proposed procedure ensures the visualization of all groups of base oils. Extensive experimental research showed that a 2-step development procedure with application of n-hexane up to 100% height of development +15 min and further n-hexane: isopropanol: tri-fluoroacetic acid 96.25: 3: 0.75 (v: v: v) up to 75% height of development is advantageous for the group-type separation, both in TLC-FID and TLC.

Some compounds from the rhizome of Zingiber montanum

Ngai say vang has the scientific name Zingiber montanum or synonym Zingiber cassumunar Roxb. belongs to the Ginger family (Zingiberaceae). The tree is native to India and suitable for the humid tropical climate of Asian countries. In Vietnam, this species is found in Binh Dinh, An Giang and Soc Trang provinces, with local names such as Gung dai, Gung tia, and Ngai say vang. In traditional medicine, the rhizomes of Z. montanum have been often used in remedies that treat rheumatism, muscle pain, stomach irritation, laxative, anti-diarrheal, colic and mosquito repellent. By combining column chromatography (CC) and thin-layer chromatography (TLC, PTLC) techniques on normal phase and reversed-phase systems, five pure compounds were isolated from the fraction H of the rhizomes of Z. montanum. Structures of the compounds were elucidated by analyzing nuclear magnetic resonance spectroscopy, HRESI-MS mass spectrometry and comparison with references. All five compounds have been two diphenylbutenoids – phlain III (1) and phlain V (2), two stigmastanes – 6b-hydroxystigmast-4-en-3-one (3) and stigmast-4-en-3,6-dione (4), and a simple phenolic compound – vanillin (5). This study has added to the database of the structural chemistry of natural compounds, especially the phenylbutenoid compounds from Z. montanum. This is the first time that compounds 3-5 have been found in the rhizomes of Z. montanum.

First-order quantum phase transitions as condensations in the space of states

We demonstrate that a large class of first-order quantum phase transitions, namely, transitions in which the ground state energy per particle is continuous but its first order derivative has a jump discontinuity, can be described as a condensation in the space of states. Given a system having Hamiltonian H = K + gV, where K and V are two non commuting operators acting on the space of states , we may always write where is the subspace spanned by the eigenstates of V with minimal eigenvalue and . If, in the thermodynamic limit, M cond/M → 0, where M and M cond are, respectively, the dimensions of and , the above decomposition of becomes effective, in the sense that the ground state energy per particle of the system, ϵ, coincides with the smaller between ϵ cond and ϵ norm, the ground state energies per particle of the system restricted to the subspaces and , respectively: ϵ = min{ϵ cond, ϵ norm}. It may then happen that, as a function of the parameter g, the energies ϵ cond and ϵ norm cross at g = g c. In this case, a first-order quantum phase transition takes place between a condensed phase (system restricted to the small subspace ) and a normal phase (system spread over the large subspace ). Since, in the thermodynamic limit, M cond/M → 0, the confinement into is actually a condensation in which the system falls into a ground state orthogonal to that of the normal phase, something reminiscent of Anderson’s orthogonality catastrophe (Anderson 1967 Phys. Rev. Lett. 18 1049). The outlined mechanism is tested on a variety of benchmark lattice models, including spin systems, free fermions with non uniform fields, interacting fermions and interacting hard-core bosons.

Estimating Limits of Detection and Quantification of Ibuprofen by TLC-Densitometry at Different Chromatographic Conditions

Ibuprofen is one of the best-known nonsteroidal anti-inflammatory and analgesic drugs. Following the previous work, the current study is focused on estimating the effect of different chromatographic conditions on the sensitivity of thin-layer chromatography in combination with UV densitometry, i.e., the detection and quantification of ibuprofen in a wide range of its concentrations including the lowest limits of detection (LOD) and quantification (LOQ). For this purpose, a reliable and easy-to-use calculation procedure for LOD and LOQ determination is presented in this work. In addition, the impact of type plates and mobile phase composition on the LOD and LOQ, respectively, of this active substance is accurately described. The results of detection and the quantification level of ibuprofen obtained under applied chromatographic conditions confirmed the utility of silica gel plates as well as silica gel bonded phases (i.e., reversed-phase (RP) plates) in the thin-layer chromatography (TLC)-densitometric analysis of ibuprofen at a low level, i.e., from several nanograms (below one microgram) to a few micrograms/spot. Among all chromatographic systems tested, the best are those consisting of silica gel 60F254 plates with concentrating zone (1.05583) and the mixture of n-hexane:ethyl acetate:glacial acetic acid in ratio 36:12:2 (v/v/v) as well as RP plates, i.e., RP-18F254 aluminum plates developed with methanol:water in volume composition 40:10 (v/v). These chromatographic systems allowed quantifying ibuprofen in the amount of 0.229 µg/spot and 0.228 µg/spot, which is less than 1 µg/spot. It can be stated that different chromatographic systems in combination with UV densitometric scanning at 224 nm proposed in this work can be successfully applied for the cost-effective and sensitive determination of ibuprofen as a widely used drug component as well as a residual in domestic wastewater. It was found that the modification of silica gel as well as the layer thickness of unmodified or modified silica gel 60 can influence the quality of chromatograms and the detection/quantification of ibuprofen in both normal phase (NP) and RP systems. Therefore, to obtain the best possible LOD and LOQ values of ibuprofen with precoated layers, suitable mobile phase and chromatographic plates are required.

A Rapid and Simple TLC-Densitometric Method for Assay of Clobetasol Propionate in Topical Solution.

A rapid, simple to use and low-cost thin-layer chromatographic procedure in normal phase system with densitometric detection at 246 nm was carefully validated according to the International Conference on Harmonisation (ICH) guidelines for assay of clobetasol propionate in topical solution containing clobetasol propionate in quantity 0.50 mg/mL. The adopted thin-layer chromatographic (TLC)-densitometric procedure could effectively separate clobetasol propionate from its related compound, namely clobetasol. It is linear for clobetasol propionate in the range of 0.188 ÷ 5 µg/spot. The limit of detection (LOD) and limit of quantification (LOQ) value is 0.061 and 0.186 µg/spot, respectively. Accuracy of proposed procedure was evaluated by recovery test. The mean recovery of studied clobetasol propionate ranges from 98.7 to 101.0%. The coefficient of variation (CV, %) obtained during intra-day and inter-day studies, which was less than 2% (0.40 ÷ 1.17%), confirms the precision of described method. The assay value of clobetasol propionate is consistent with the pharmacopoeial requirements. In conclusion, it can be suitable as a simple and economic procedure for routine quality control laboratories of clobetasol propionate in topical solution.

Can RP-8 plates be used in normal-phase systems? A chemometric approach to estimate the elution strength of popular solvents

During our previous studies, we discovered that reversed-phase thin-layer chromatography (RP-TLC) plates can behave in typical normal-phase manner, which is not widely known nor used in the literature. Therefore, we decided to investigate this behavior more comprehensively on RP-8 plates. We used 35 model compounds, and performed chemometric screening mixture design approach for 20 popular solvents. This gave us the possibility to estimate separate retention effect for each solvent being the measure of average solvent strength. The results were compared to the previous study done on silica gel. It can be concluded that RP-8 plates can be used in typical normal-phase systems and their behavior does not differ substantially from silica gel plates.

Development and Validation of Chiral HPLC Method for Quantification of 3-(S)-Quinuclidinol in Pharmaceutically Important Precursor, 3-(R)-Quinuclidinol, by Precolumn Derivatization.

A sensitive and specific high-performance liquid chromatography (HPLC) method for the separation and quantification of 3-(S)-quinuclidinol in 3-(R)-quinuclidinol, precursor for active pharmaceutical ingredients (solifenacin, revatropate and talsaclidine), has been developed and validated by precolumn derivatization. Several chiral columns were tested in a normal phase system. Excellent enantioseparation with the resolution more than 11.4 was achieved on Chiralpak IC column using isocratic mobile phase consisting of n-hexane, ethanol, 2-propanol and diethylamine (80:8:12:0.4, v/v). The detection was carried out using UV detector at 230 nm. The influence of mobile phase composition, namely organic modifiers, additives, aliphatic alkanes and water content in mobile phase, on retention and enantioseparation was studied. Validation of the developed method including specificity, system suitability, limit of detection, limit of quantification, linearity, repeatability, intermediate precision, accuracy and solution stability was performed according to the International Conference on Harmonization guidelines. The advantage of the method is a good HPLC enantioseparation by precolumn derivatization using reaction mixture as sample, simpler UV detection, short analysis time (<30 min), and therefore this method is suitable option for routine quantification of 3-(S)-quinuclidinol in 3-(R)-quinuclidinol.

Micro-2D-TLC separation of phenolics in some species of mint and their fingerprints on diol bonded polar stationary phase

Micro-thin-layer chromatography in two dimensional (2D-mTLC) mode in normal and reversed phase systems by use of diol bonded stationary phase was applied to make fingerprints of 11 species of Mentha genus and two finished pharmaceutical products. Nonaqueous eluents (propan-2-ol or ethyl acetate dissolved in n-heptane) were used in normal phase systems. Mixtures of acetonitrile with water were used in reversed phase chromatographic systems. Optimization of one dimensional systems was performed by determining of RF vs. composition of mobile phases dependencies for standards occurring in various species of Mentha. Most selective eluents were chosen to optimize two-dimensional systems by creating RF in normal-phase (NP) systems vs. RF in reversed-phase (RP) systems correlations. 2D-mTLC on diol polar bonded stationary phase were optimized to separate phenolic compounds and make fingerprints of examined plant materials and this method was never applied earlier in the chromatographic analysis.

Thin-layer chromatography and pressurized planar electrochromatography of amino acids in systems with silica gel and water mobile phase

Thin-layer chromatography (TLC) and pressurized planar electrochromatography (PPEC) of amino acids in normal-phase system is presented. The results have been obtained for various normal-phase high-performance thin-layer chromatography (HPTLC) and PPEC systems with the mobile phase that comprised acetonitrile in the concentration ranges 40–90% and 20–90%, respectively, and HPTLC silica gel 60 F254s plates from Merck. The data obtained show differences in separation selectivity between HPTLC and PPEC systems. The respective separation selectivities have been obtained for HPTLC and PPEC systems with the mobile phase buffer pH in the range 2.6–9.0. The retention of amino acids in HPTLC systems has demonstrated minor dependence on buffer pH, while, in analogous PPEC systems, migration distances of the solutes have shown considerable changes. The differences of separation selectivity in HPTLC and PPEC systems are interpreted in terms of solute partition in the former and solute partition and electrophoresis in ...

Analyzing hydrocarbon fractions in crude oils by two-dimensional gas chromatography/time-of-flight mass spectrometry under reversed-phase column system

Abstract Two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOFMS) is quite effective to analyze organic compounds in crude oils with high resolution and normal-phase column condition (non-polar/polar) was commonly used. Here, to improve the understanding on this method, based on Chinese oil samples, we analyze hydrocarbon fractions in the oils using GC × GC/TOFMS with reversed-phase column system (polar/non-polar) in addition to the normal-phase column system. Results show that the reversed-phase column system is more effective in separating saturated hydrocarbons than normal-phase column system. It is especially effective in generating two-dimensional spectra, because the compounds have a higher separation among different series (polarities). The reversed-phase column system is also particularly effective in separating weakly polar to non-polar saturated hydrocarbons (e.g., branched alkanes and naphthenes of low-to-medium molecular weight). It is also suitable for the isolation and identification of conventional biomarkers such as isoprenoids, steranes, and terpanes. Overall, in the GC × GC/TOFMS analysis of crude oils, the reversed-phase system has distinct advantages over the normal-phase system and specific compounds were identified in detail. Thus, this method deserves more attention. These results and understandings add new knowledge to oil geochemistry and have general implications.

Estimation of Lipophilicity of Some Polyoxygenated Steroids by the Means of Normal-Phase Thin-Layer Chromatography

Unmodified silica gel in combination with two mobile phases, acetone–n-hexane and acetonitrile–dichloromethane, was used in order to evaluate the capability of normal-phase (NP) chromatography on bare silica gel in estimation of lipophilicity of some polyoxygenated steroids. Soczewinski equation coefficients were employed as a measure of lipophilicity. The RM0 values obtained in NP-systems were correlated with those derived by extrapolation from reversed-phase (RP) systems. In addition, retention data, i.e., lipophilicity parameters determined in NP systems were compared with logP values calculated by use of several commercial computer programs. The results showed that chromatographic parameters RM0, and m obtained in NP system consisted of silica gel as stationary phase and acetone–n-hexane as mobile phase, are acceptable as the measures of lipophilicity of polyoxygenated steroids. The mechanism of retention was discussed.

A Validated TLC-Densitometric Method for the Determination of Mesterolone in Bulk Material and in Tablets.

Mesterolone is a synthetic androgenic steroid indicating a weak anabolic activity. A new, simple in use, and economical TLC-densitometric method in normal phase system (NP-TLC) has been developed and validated for the identification and quantitative determination of mesterolone in bulk drug and in tablet formulation. NP-TLC analysis was performed on aluminium plates precoated with silica gel 60F254 as the stationary phase using chloroform-acetone (40 : 10, v/v) as mobile phase. Densitometric analysis was carried out at λ = 745 nm after staining with phosphomolybdic acid. These conditions were found to give visible (dark blue) spot and sharp peak, respectively, for mesterolone at R F 0.75 ± 0.02 and enabled satisfactory separation of mesterolone from its related substance (potential impurity). The proposed NP-TLC-densitometric method was validated for specificity, linearity, precision, accuracy, robustness, and sensitivity according to ICH guideline and other validation requirements. The limit of detection (LOD) and limit of quantification (LOQ) were 61.0 ng·spot−1 and 184.0 ng·spot−1, respectively. The percent content of mesterolone in marketed tablet formulation was found to be 99.40% of label claim. The developed TLC-densitometric method can be successfully used in quality control of mesterolone in bulk material and also tablet formulation.

A Comparison of Normal and Reversed Phase Columns in Oil Analysis by Comprehensive Two-dimensional Gas Chromatography With Time-of-flight Mass Spectrometry

Four typical crude oil samples collected from the Junggar Basin of northwest China were analyzed by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) under different experimental conditions, which include normal phase column system (nonpolar × polar) and reversed phase column system (polar × nonpolar). A comparative study was conducted on the general chromatogram features, and chromatogram features of saturate and aromatic hydrocarbons. Analytical results show that the normal phase (NP) column system has higher resolution for the identification of high molecular weight polycyoalkanes and polycyclic aromatic hydrocarbons, while the reverse phase (RP) column system has higher separation on the saturate hydrocarbons, especially on medium-low molecular weight iso-alkanes and cycloalkanes. In addition, the RP system can also meet the separation requirements of aromatic compounds, by the extraction of diverse organic compounds by characteristic ions. Therefore, the RP system has special advantages in the analysis of medium-low molecular weight hydrocarbon compounds in crude oil, and can be applied more widely in the future besides the presently common use of the NP system.

A Validated Quantification of Benzocaine in Lozenges Using TLC and a Flatbed Scanner

We present a video-densitometric quantification method for benzocaine in lozenges. The quantification is based on a derivatisation reaction with 4-dimethylaminobenzaldehyde. Measurements were carried out using a 16-bit flatbed scanner. Benzocaine was separated to a distance of 50 mm in a vertical developing chamber without vapour saturation. We present an RP-18 phase separation on a cyanopropyl plate (Merck, Darmstadt, Germany) using water, CH3CN, dioxane, ethanol, and NH3 (25 %) (8 + 2 + 1 + 1 + 0.05, v/v) as the mobile phase. We also separated benzocaine in a normal phase system on silica gel 60 LiChrospere® plates (Merck, Darmstadt, Germany) with the mobile phase MTBE/cyclohexane (1 + 1, v/v). The calibration functions for benzocaine in both separations were linear in the range from 1 to 1,000 ng per spot. The range of linearity covers two magnitudes of power because the Kubelka–Munk expression was used for data transformation. In the cyanopropyl-system, the benzocaine amount was quantified as 242.5 ± 18.2 ng in a spot or 6.86 ± 0.52 mg in a single lozenge. The amount of 7.0 mg benzocaine per lozenge was labelled. The combined uncertainty of sample and calibration measurements was statistically calculated using a significance level of α = 0.05 to a total relative uncertainty of 7.49 %. The separation method is inexpensive, fast and reliable.

Fundamental study of enantioselective HPLC separation of tapentadol enantiomers using cellulose-based chiral stationary phase in normal phase mode

A sensitive and specific high performance liquid chromatography method for the separation and determination of tapentadol enantiomers has been developed and validated. Ten different chiral columns were tested in a normal phase system. Excellent enantioseparation with the resolution more than 2.5 for all enantiomers was achieved on Chiralpak AD-H using mixture of heptane–propan-2-ol–diethylamine (980:20:1, v/v/v). The detection was carried out using fluorescence detector at excitation wavelength of 295nm and emission wavelength of 273nm. The influence of mobile phase composition, namely organic modifiers, additives, aliphatic alkanes and water content in mobile phase, on retention and enantioseparation was studied. Validation of the developed method including linearity, limit of detection, limit of quantification, precision, accuracy and selectivity was performed according to the International Conference on Harmonization guidelines. The advantage of the method is a good enantioseparation, short analysis time (less than 20min) and therefore this method is suitable for routine determination of chiral purity of (R,R)-tapentadol in enantiopure active pharmaceutical ingredient.

Revisiting thin-layer chromatography as a lipophilicity determination tool. Part II — Is silica gel a reliable adsorbent for lipophilicity estimation?

Lipophilicity is often estimated by thin-layer chromatography (TLC) as a fundamental property related to biological and pharmaceutical activity. In a recent study, we contributed some standardization to the subject by performing a comparative analysis of the different approaches in the field. This part presents an analogous study on silica, the use of which in normal-phase systems seems to be a new trend of recent times. We have compared several approaches of TLC lipophilicity determination: a single TLC run, Soczewinski-Wachtmeister equation coefficients, principal component analysis (PCA) of the retention matrix, and PARAFAC on a three-way array. All techniques were applied to 35 model solutes with simple molecules, using nine concentrations of six modifiers: acetone, dioxane, ethyl acetate, methylethylketone, propan-2-ol, and tetrahydrofuran. Comparative analysis points to several general recommendations. Ethyl acetate seems to give the best correlations with lipophilicity, and the correlations of sing...

Determination of clofentezine in medical herb extracts by chromatographic methods combined with diode array scanning densitometry

The aim of this study is to report a new procedure for extraction, cleanup and determination of clofentezine in herb extracts by ultrasound-assisted solvent extraction, SPE and multidimensional planar chromatography with diode array detector (MDPC-DAD) and/or HPLC-DAD. The application of various extraction solvents in SPE experiments conducted on octadecyl silane coupled with styrene-divinylbenzene cartridges for fractionation and purification samples has been described. Normal-phase systems were used in MDPC experiments on silica layer. The procedure described for the determination of compounds is inexpensive and can be applied to the routine analysis of analytes in plant extracts, after preliminary cleanup and concentration, e.g. by SPE. Application of MDPC-DAD and HPLC-DAD is especially useful for correct identification of components of difficult, complicated mixtures, e.g. analytes in medical herbs.

Planar chromatography of cholic acid-derivedcis-transisomeric bis-steroidal tetraoxanes

Seven pairs of cis-trans isomers of bis-steroidal tetraoxanes have been examined by both normal-phase (NP) and reversed-phase (RP) planar chromatography. Unmodified silica gel was used with ethyl acetate-toluene and ethyl acetate-petroleum ether as mobile phases in typical normal-phase systems. CN-silica with the mobile phases methanol-water and acetone-water and RP-18 silica with water-organic modifier (methanol, acetone, or dioxane) mobile phases were used as reversed-phase systems. For the RP systems a good linear correlation was established between R M values and amount ([%] ν/ν ) of organic modifier in the mobile phase (usually r > 0.99). It was found that under both NP and RP conditions cis isomers were more weakly retained than the corresponding trans isomers. The only exception to this was the chromatographic behavior of C(24) methyl esters. It was established that increasing the polarity of substituents at C(24) and C(24a) led to stronger retention in NP systems, i.e. weaker retention in RP syste...