Ternary Mobile Phase Research Articles

Liquid chromatography at critical conditions in ternary mobile phases: Gradient elution along the critical line

In ternary mobile phases consisting of acetone, methanol, and water, the retention of PEG on reversed-phase columns is independent on molar mass at certain compositions of the mobile phase. Along this critical adsorption line, the retention of polypropylene glycol varies quite strongly, which can be utilized in the separation of block copolymers. Gradient elution along the critical line allows a baseline separation of all oligomers in polypropylene glycol up to approximately 25 propylene oxide units. The same resolution can be achieved in the separation of ethylene oxide-propylene oxide block copolymers, regardless of the length of the ethylene oxide block.

Combined Effect of Temperature and Ternary Mobile Phase Composition on the Retention in Ternary Isocratic and Gradient Elution RP-LC under Isothermal Conditions. Application to the Retention Prediction of Four Macrolide Antibiotics

Retention models considering simultaneously ternary mobile phase organic contents and column temperature (T) were developed by a direct combination of equations expressing separately the dependence of the retention upon each of these factors. Thus, a combination of a linear dependence of the logarithm of the solute retention factor, ln k (T), against 1/T, i.e. ln k (T) = a + b/T, and of either a linear or a quadratic dependence of ln k upon the compositions of a binary mobile phase, gave two different expressions for the logarithm of the solute retention factor in terms of both temperature and organic contents in the ternary mobile phase. The effectiveness of the above models was tested in the prediction of isothermal retention of a mixture of four macrolide antibiotics under ternary isocratic and gradient elution conditions using mobile phases modified by methanol and acetonitrile. The limiting case of using ternary eluents with constant ratio of the concentrations of the two organic modifiers was also tested.

Evaluation of a Vancomycin-Based LC Column in Enantiomeric Separation of Atenolol: Method Development, Repeatability Study and Enantiomeric Impurity Determination

An LC method was developed and validated for the enantioselective separation and enantiomeric impurity quantitation of atenolol. Separation of the atenolol enantiomers on the Chirobiotic V2 (150 mm × 4.6 mm, 5 μm) column was best achieved using a ternary mobile phase of methanol–acetonitrile-triethylamine acetate 0.5% (w/v), pH 4.5 in a ratio of (45:50:5; v/v/v). Good resolution value of R s = 3 was obtained at a flow rate of 1 mL min−1 within a total run time of less than 40 min. Peak identification was achieved using the standard reference of individual enantiomers. The peak of the impurity was eluted in front of the peak of the main enantiomer. Detection was performed by UV at 226 nm. Within and between day’s repeatabilities for both retention time and peak area were investigated at three concentration levels and found to be low. The method was also found to be efficient for the determination of atenolol enantiomeric impurity. An impurity quantitation level of (R)-atenolol down to 0.08% relative to the main enantiomer (S)-atenolol was found possible.

Adsorption interaction parameter of polyethers in ternary mobile phases: The critical adsorption line

It is shown that in LC of polymers, the interaction parameter in ternary mobile phases can be described by a plane, which is determined by the dependencies in binary mobile phases. Instead of a critical adsorption point, critical conditions are observed along a straight line of composition between the two critical points in binary mobile phases. Consequently, a separation of block copolymers under critical conditions for one block by an adsorption mechanism for the other block can be achieved in ternary mobile phases of different compositions, which allows an adjustment of the retention of the adsorbing block.

Standardization of Ayurvedic formulations containingAloe veraby quantification of a marker compound

HPTLC has been used for quantitative analysis of aloin, as marker compound, in commercial Ayurvedic preparations, in the solid dosage form, containing Aloe vera Linn as major herbal ingredient. Chromatography was performed on aluminum plates coated with 0.2-mm layers of silica gel 60 F 254 , with ethyl acetate-methanol-water 10:1.4:1 ( v/v ) as ternary mobile phase after saturation at 30 ± 4°C for 5–7 min. The development distance was 70 mm. Aloin was quantified at 360 nm, its wave length of maximum absorbance. The limits of detection and quantification were 10 and 20 ng per band, respectively. Regression analysis of the calibration data revealed a good linear relationship ( r = 0.9998) between peak area response and concentration in the range 20 to 100 ng per band. Instrumental precision and the repeatability of the method were 0.44% and 0.89% (CV), respectively. The accuracy of the method, determined by measurement of recovery at three different levels, was in the range 98.13 to 99.75%. These results ar...

Estimation of Catechin in Ayurvedic Oil Formulations Containing Acacia catechu

A sensitive, simple, rapid, and efficient HPTLC method was developed and validated for the analysis of catechin in marketed Ayurvedic oil formulations containing Acacia catechu. Chromatography of methanolic-0.1% formic acid (7:3, v/v) extracts of these formulations was performed on silica gel 60 F254 aluminum-backed TLC plates of 0.2 mm layer thickness. The plate was developed up to 85 mm with the ternary-mobile phase chloroform-acetone-0.1% formic acid (7.7 + 1.5 + 0.8%, v/v/v) at 22 +/- 2 degrees C with 20 min of chamber saturation. The system produced compact spots of catechin at an Rf value of 0.36. The marker, catechin, was quantified at its maximum absorbance of 296 nm. The limit of detection and quantitation values were 6 and 20 ng/spot, respectively. The linear regression analysis data for the calibration plot showed a good linear relationship with a correlation coefficient of 0.9993 in the concentration range of 200-1200 ng/spot for catechin with respect to peak area. Repeatability of the method was 0.88% RSD. Recovery values from 97 to 102% indicate excellent accuracy of the method. The developed HPTLC method is accurate, precise, and cost-effective, and it can be successfully applied for the determination of catechin in marketed Ayurvedic oil formulations containing Acacia catechu.

Quantification of β-Sitosterol from Mucuna pruriens by TLC

Mucuna pruriens Linn. one of the popular and important medicinal plants of India is a constituent of more than 200 indigenous drug formulations. β-Sitosterol is one of the most prevalent phytosterols which is ubiquitous throughout the plant kingdom. A sensitive, selective and precise thin-layer chromatographic method has been developed and validated for the analysis of β-sitosterol in Mucuna pruriens roots. Separation and quantification was achieved by TLC using ternary mobile phase of toluene: chloroform; methanol (4:4:1 v/v) (RF 0.55) on precoated silica gel 60F254 aluminium plates and densitometric determination was carried out after derivatization with anisaldehyde-sulphuric acid reagent in reflection/absorption mode at 527 nm. The calibration curve was linear in the concentration range of 100–600 ng spot−1. The method was validated for precision, repeatability and accuracy. The proposed method was found to be simple, precise, specific, sensitive and accurate for the quantification of β-sitosterol.

TLC Determination of Betulinic Acid from Nymphodies macrospermum: A New Botanical Source for Tagara

The accepted botanical source of Tagara, an ayurvedic drug is Valeriana jatamansi Jones. In South India, a drug by the name of Granthika Tagara is used as Tagara in several therapeutic preparations. Currently, no analytical procedures appear to be available for quality control purposes. A sensitive, selective and precise thin-layer chromatographic method has been developed and validated for the analysis of betulinic acid in Nymphoides macrospermum. Separation and quantification was achieved by TLC using ternary mobile phase of hexane:ethyl acetate:acetic acid (7:3:0.03, v/v) (RF 0.60) on precoated silica gel 60F254 aluminium plates and densitometric determination was carried out after derivatization with anisaldehyde-sulphuric acid reagent in the reflection/absorption mode at 540 nm. The calibration curve was linear in the concentration range of 100–600 ng spot−1. The method was validated for precision, repeatability and accuracy. The proposed method was found to be simple, precise, specific, sensitive and accurate for the quantification of betulinic acid. This is the first TLC report for the identification and quantification of betulinic acid in N. macrospermum and may be useful for the routine quality control of Granthika Tagara.

Separation of geometrical isomers of {Fe[3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine]3}2+ ([Fe(PDT)3]2+) using ion-pair reversed-phase high performance liquid chromatography

A method has been developed for the separation of two geometrical isomers of the {Fe[3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine]3}2+([Fe(PDT)3]2+) using ion-pair reversed-phase high performance liquid chromatography (RP-HPLC). The effects of the chromatographic conditions, such as the content of acetonitrile and the type and concentration of the ion-pair reagents (sodium perchlorate (NaClO4) or sodium dodecyl sulfate (SDS)) in the binary mobile phase (acetonitrile and water), on the retention factor (k), resolution, and selectivity were iscussed. It was found that no matter how the ion-pair reagent was NaClO4 or SDS at different concentrations, the acetonitrile content in the mobile phase has good linear regression equations with the ln k of the two isomers. It was also observed that SDS showed more positive effect on the k of the two geometrical isomers than that of NaClO4. Moreover, the separation of the two isomers in the ternary mobile phase (acetonitrile, methanol and water) was developed. The chromatographic conditions, including the content of the organic modifier (acetonitrile and methanol) and the type and concentration of the ion-pair reagents (SDS and NaClO4), were optimized. Two geometrical isomers were rapidly and successfully separated under the optimized conditions, which used acetonitrile/methanol/water (20: 50: 30, v/v/v) as the mobile phase and 60 mmol/L NaClO4 as the ion-pair reagent. Good linear regression equations between the peak areas and concentrations for the two isomers were obtained. The detection limits of the fac-isomer and mer-isomer were 4.28 and 3.44 ng/mL (S/N = 3), respectively.

Effect of decreasing column inner diameter and use of off-line two-dimensional chromatography on metabolite detection in complex mixtures

Capillary liquid chromatography coupled with electrospray ionization to a quadrupole ion trap mass spectrometer was explored as a method for the analysis of polar anionic compounds in complex metabolome mixtures. A ternary mobile phase gradient, consisting of aqueous acidic, aqueous neutral and organic phases in combination with an aqueous compatible reversed-phase stationary phase allowed metabolites with a wide range of polarities to be resolved and detected. Detection limits in the full scan mode for glycolysis and tricarboxylic acid cycle intermediates were from 0.9 to 36 fmol. Using this system, 111 ± 9 ( n = 3) metabolites were detected in Escherichia coli lysate samples. Reducing column I.D. from 50 to 25 μm increased the number of metabolites detected to 156 ± 17 ( n = 3). The improvement in number of metabolites detected was attributed to an increase in separation efficiency, an increase in sensitivity, and a decrease in adduct formation. Implementation of a second separation mode, strong anion exchange, to fractionate the sample prior to capillary RPLC increased the number of metabolites detected to 244 ± 21 ( n = 3). This improvement was attributed to the increased peak capacity which decreased co-elution of molecules enabling more sensitive detection by mass spectrometry. This system was also applied to islets of Langerhans where more significant improvements in metabolite detection were observed. In islets, 391 ± 33 small molecules were detected using the two-dimensional separation. The results demonstrate that column miniaturization and use of two-dimensional separations can yield a significant improvement in the coverage of the metabolome.

Analytical and semipreparative enantioseparation of 9-hydroxyrisperidone, the main metabolite of risperidone, using high-performance liquid chromatography and capillary electrophoresis: Validation and determination of enantiomeric purity

The HPLC semipreparative enantioseparation of 9-hydroxyrisperidone (9-OHRisp) was studied by optimizing various experimental conditions: the nature of the chiral stationary phase (CSP), mobile phase composition, temperature and analyte loading. This semipreparative enantioseparation was successfully completed using the polysaccharide Chiralcel OJ chiral stationary phase and a n-hexane/ethanol/methanol (50/35/15, v/v/v) ternary mobile phase. To assess the enantiomeric purity of both isolated isomers, three analytical methods using UV detection were developed and validated: one CE method using dual cyclodextrin mode and two HPLC methods using either the Chiralcel OJ CSP in normal-phase mode or the α-acid glycoprotein (α-AGP) CSP in reversed-phase mode. The three methods make it possible to obtain excellent enantioseparations ( R s > 3) with analysis times lower than 15 min, and the calculated limits of detection allow for the determination of minor enantiomeric impurities (0.1%). Enantiomeric purity obtained for dextrorotatory and levorotatory enantiomers was superior to 99.9% and equal to 98.9%, respectively, which proved the success of the semipreparative enantioseparation. A brief comparison of the performances of the analytical methods completes this work.

Optimization of the TLC separation of seven amino acids

A ternary mobile phase for thin-layer chromatographic separation and identification of seven amino acids on microcrystalline cellulose, with ninhydrin as detection reagent, has been optimized by use of the experimental design software packages Design-Expert 6 and Statistica . When the results from each product were compared the optimum mobile phase was found to be butanol-glacial acetic acid-water 60.14:18.77:21.09. The performance of this mobile phase was confirmed experimentally.

Determination of polymethoxylated flavones in peels of selected Jamaican and Mexican citrus (Citrus spp.) cultivars by high‐performance liquid chromatography

The concentrations of the polymethoxylated flavones (PMFs) in peels of selected citrus cultivars grown in Jamaica and Mexico were determined. The PMFs were extracted from sun-dried citrus peels with reagent-grade methanol. Analyses were carried out by reverse-phase HPLC and UV detection. The column used was a C(18) 5 microm (150 x 4.6 mm) Discovery column. Elution was in the gradient mode, using a ternary mobile phase. The results showed that all the citrus cultivars used contained at least three of the six major PMFs quantified. Ortanique peel contained the highest quantity of PMFs (34,393 +/- 272 ppm), followed by tangerine (28,389 +/- 343 ppm) and Mexican sweet orange (sample 1; 21,627 +/- 494 ppm). The major PMFs, i.e. sinensetin, nobiletin, tangeretin, heptamethoxyflavone, tetramethylscutellarein and hexamethyl-o-quercetagetin, present in the peels of 20 citrus cultivars, was quantified. The results were compared with those of Florida citrus peels. A large amount of citrus peels and byproducts are produced in the Caribbean which could provide a cheap and convenient source of PMFs.

Purification and quantification of ginsenoside Rb 3 and Rc from crude extracts of caudexes and leaves of Panax notoginseng

In this work, the separation of ginsenoside Rb 3 and Rc from the crude extracts of caudexes and leaves of Panax notoginseng (CECLPN) was studied with reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic separation was achieved using a SynChropak C 18 column (at 25 °C). A satisfied separation of the compounds was obtained in less than 40 min with a flow rate of 1.0 ml/min. Chromatography was performed using ternary mobile phase composed of methanol, water and phosphoric acid, 65:33:1.4 (v/v). UV detection was accomplished at 203 nm. Ginsenoside Rb 3 and Rc found in the CECLPN constitute up to 38.25% and 26.65% of the dry powder, respectively. Ginsenoside Rb 3 and Rc were prepared by semi-preparative HPLC/ELSD using gradient elution system of methanol–water = 70:30 → 65:35 at a flow rate of 30 ml/min with a sample load of 300–400 mg. With this chromatographic condition, each individual ginsenoside Rb 3 and Rc in purity of more than 97% were gained. The products were confirmed by spectroscopic (UV, IR, ESI-MS/MS, NMR) and HPLC methods. The methods were validated for linearity, precision, recovery, limits of detection (LOD) and limits of quantification (LOQ). The results show that the proposed method appears to be an adequate method for quality control of CECLPN and a useful tool for pharmaceutical study of ginsenosides.

HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 μm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane–isopropanol with ethylene glycol, water or acetonitrile) are applied.

Optimization of the assay of naphthodianthrones in dry St John’s wort extract by reversed-phase liquid chromatography

An optimization procedure for the reversed-phase liquid chromatography (RPLC) assay of naphthodianthrones in St John's wort extract from the European Pharmacopoeia project is described. The results obtained from two screening designs showed that light exposure, recommended in the monograph as sample pretreatment, does not permit one to obtain a reproducible quantification of the main ingredients. Improvement of the method robustness implies the need to overcome the problem of light exposure, to subsequently quantify protonaphthodianthrones and to perform the separation on octadecyl (ODS)-bonded phase at optimized flow rate. The method robustness was checked by using a bifurcation sequential approach investigating the influence of 13 factors. The eluent recommended in the monograph is a ternary mixture of methanol, phosphate buffer and ethyl acetate. For the sake of simplicity, the phosphate buffer was substituted by an acetate buffer. The best composition of the ternary mixture was determined by a combined design including three mixture variables and the temperature as an independent variable. Chromatographic parameters were modelled in terms of analysis time, resolution and asymmetry. Desirability functions permit one to cope with these parameters and to determine the best compromise. The naphthodianthrones were separated on a conventional endcapped octadecyl silica gel column eluted by a ternary mobile phase at 40 degrees C in 10 min.

Quantification of Valerenic Acid in Valeriana jatamansi and Valeriana officinalis by HPTLC

A simple, rapid, cost-effective and accurate high performance thin layer chromatographic method has been developed for quantification of valerenic acid in Valeriana jatamansi and Valeriana officinalis which is one of the stable compounds of Valeriana officinalis and designated as a key marker compound. Valerenic acid makes substantial contribution to the sedative and spasmolytic activity of the essential oil and extract of Valeriana officinalis. Separation and quantification was achieved by HPTLC using ternary mobile phase of hexane: ethyl acetate: acetic acid (80:20:0.5 v/v) on precoated silica gel 60F254 aluminium plates and densitometric determination was carried out after derivatization with anisaldehyde-sulphuric acid reagent at 700 nm, in absorption-reflectance mode. The calibration curves were linear in the range of (500 ng–2.5 µg). This is the first HPTLC report for the identification and quantification of valerenic acid in Valeriana jatamansi and Valeriana officinalis.

Theory of Retention in Reversed Phase Liquid Chromatography with Ternary Mobile Phases

The partition model of retention is developed for reversed phase liquid chromatography with multicomponent mobile phases. Simple equations for the retention and selectivity in ternary mobile phases are derived. For the systems in which the ratio of volume fractions of organic modifiers remains fixed, new linear dependences for retention factor and selectivity are proposed. These equations are successfully used to describe experimental data found in the literature. An influence of the nature of organic solvents and proportion in which they are mixed on retention and selectivity is discussed.

Influence of mobile phase composition and thermodynamics on the normal phase chromatography of echinocandins

In the normal phase preparative HPLC of fermentation derived echinocandins, resolution of key impurities from the product of interest, pneumocandin B o, is accomplished using a ternary ethyl acetate/methanol/water mobile phase with silica gel as the sorbent. In this work, previous characterization of the system is extended to define the impact and role of water content on the separation efficiency and retention of pneumocandin B o. Experimental results indicate that column efficiency, measured using both the product of interest and small molecule tracers (compounds used for pulse tests), is good despite the use of an irregular silica and unusually high levels (greater than 6%) of water in the mobile phase. In contrast to column efficiency measurements using small molecules (MEK and toluene), measurements performed with the product itself indicate improved efficiency with increasing water content of the mobile phase. Building on these results, a scale-up/scale-down protocol was developed based on measurements of column efficiency using theoretical plate counts determined with pneumocandin B o. Since the solubility of pneumocandin B o in the ternary mobile phase is relatively low, a higher strength solvent with higher levels of methanol and water is employed for dissolution of the crude product at concentrations of up to 40 g/L. The mismatch between the high strength solvent used for the feed introduction and the mobile phase has the potential to affect column performance. The impact of this mismatch using plate count measurements with the product at both analytical and semi-preparative scales was found not to be significant. Finally, a van’t Hoff analysis was performed to characterize the thermodynamics of adsorption of pneumocandin B o on silica. The analysis shows that the adsorption process for pneumocandin B o on silica in the ternary solvent system is endothermic (Δ H ads > 0), implying that the adsorption is entropically driven. Results from an overall water balance across the column indicate significant enrichment of adsorbed water on the silica surface. These results further emphasize the importance of selective partitioning of water between the bulk mobile phase and the silica as a dominant factor in controlling retention.

Retention prediction in ternary solvent reversed-phase liquid chromatography systems based on the variation of retention with binary mobile phase composition

An extension of the treatment adopted in a recent paper [P. Nikitas, A. Pappa-Louisi, P. Agrafiotou, J. Chromatogr. A 946 (2002) 33] was used to derive expressions describing the variation of solute retention k with composition in ternary reversed phase liquid chromatography, RP-LC, solvent systems. The equation of the partition model obtained in this way for a ternary mobile phase was identical to that previously derived using the solubility parameter concept. This equation as well as two new expressions of ln k versus organic modifiers content were tested in a variety of ternary solvent systems in order to examine the possibility of predicting retention behavior of solutes under ternary solvent mixture elution conditions from known retention characteristics in binary mobile phases. It was demonstrated the superiority of both new equations derived in this paper to that previously proposed and applied to date in ternary solvent mixtures.