Simple chamber for temperature-controlled planar chromatography

This article describes a construction of a simple developing device designed for temperature control of thin-layer chromatographic plates. The plates can be developed by the ascending technique under temperature gradient or non-gradient conditions. Saturated or unsaturated chamber conditions can be easily selected. The effects that give rise to pseudo-non-linear Van't Hoff plots, e.g. a temperature irregularity inside the chamber or heat evolving during solvent adsorption near the migrating front of the mobile phase are minimized. The preliminary temperature-retention studies show that the device is suitable for temperatures ranging from -20 to 60 degrees C. Using a binary mobile phase mixture (methanol-water, 70:30, v/v) the velocity of the mobile phase front on the HPTLC RP-18W plates at different temperatures was investigated. Under these conditions the retention profiles of four natural estrogens (estetrol, estriol, 17beta-estradiol and estrone) were examined. The application of the described device for temperature-retention studies is also discussed.     

Chemical stability of midazolam injection by high performance liquid chromatography

The chemical stability of midazolam hydrochloride injection, undiluted or diluted with dextrose sterile solution, was studied at different storage conditions by LC. The study was performed at room temperature (23 +/- 2 degrees C) under light exposure and light protection, +8 +/- 1 degrees C and -20 +/- 0.5 degrees C, in glass and plastic containers over 14 days with midazolam hydrochloride injection, undiluted or diluted with 5% dextrose sterile solution. Chromatographic separation was carried out on a RP-18(e) column, using a mobile phase consisting of ACN-phosphate buffer (pH 3.3; 0.1 M) (30:70 v/v) at a flow rate of 1.0 mL/min and UV detection at 220 nm. The concentrations of all samples remained greater than 90% of the original concentration. The chromatographic assay exhibited an adequate linearity (r(2) >0.999), selectivity, precision (RSD <3.1), and accuracy (recoveries from 100.46 to 101.40%). Injectable midazolam hydrochloride was chemically stable in all conditions that were studied.     

Numerical determination of the adsorption isotherms of tryptophan at different temperatures and mobile phase compositions

Single-component adsorption isotherm data were acquired by frontal analysis (FA) for tryptophan on a C(18)-Kromasil packed column, using acetonitrile-water solutions of various compositions (2.5, 5, and 7.5% ACN+1% acetic acid) and at five different temperatures between 25 and 65 degrees C. The adsorption isotherm model accounting best for these data is the bi-Moreau model, showing that two types of adsorption sites coexist on the surface and that strong adsorbate-adsorbate interactions take place. Large concentration band profiles of tryptophan were obtained for the three mobile phase compositions, at five different temperatures and the best values of the adsorption isotherm coefficients were determined by the inverse method (IM) of chromatography. The advantages and drawbacks of using the FA and the IM for determining the coefficients of the adsorption isotherm of tryptophan under the experimental conditions selected are discussed. The results of the FA and IM measurements are in good agreement. Both indicate that the retention time of tryptophan decreases rapidly with increasing acetonitrile concentration in the mobile phase as well as the saturation capacities of the two types of adsorption sites, with the highest values of the two saturation capacities being found for the lowest ACN content and the lowest temperature. The adsorption constant on the low-energy sites decreases with increasing acetonitrile content and temperature. In contrast, the adsorption constant on the high-energy sites increases with increasing ACN content of the mobile phase but decreases with increasing temperature. The solute-solute interaction parameters for the low and the high-energy adsorption sites increase rapidly with increasing ACN concentration in the mobile phase and with increasing temperature.     

Effect of temperature on the adsorption behavior of tryptophan in reversed-phase liquid chromatography

Single-component adsorption isotherm data of l-tryptophan on a C(18)-bonded silica column were acquired by frontal analysis (FA), with aqueous mobile phases containing 5% of acetonitrile at five different temperatures between 23 and 62 degrees C. The non-linear fitting of these data provided the bi-Moreau model for all temperatures as the best isotherm model. The inverse method (IM) was used to derive the parameters at these temperatures from the parameters of the 25 degrees C isotherm. The adsorption constants and the saturation capacities of the low and high-energy sites decreases by increasing the temperature, while the adsorbate-adsorbate parameters of both sites increase. An excellent agreement was found between the experimental and calculated overloaded band profiles at all the temperatures used. The breakthrough curves obtained and the overloaded band profiles obtained were found to have different shapes according to the range of concentration studied and the temperatures. At low concentration 0.05-0.5 g/L the breakthrough curves and the overloaded band profiles have a front shock and diffuse rear, which indicates langmuirian behavior, but at intermediate 1-2 g/L and high concentration 8 g/L they start to have diffuse fronts and shocks at the rear or more than one shock at the rear which indicates non-langmuirian behavior. At 23 degrees C the isotherm has another langmuirian part, which appears at high concentration. The behavior of the breakthrough curves is explained by the shape of the isotherm in which all of the isotherms have a langmuirian part (the isotherm is concave upward) and an antilangmuirian part (the isotherm is concave downward). The temperature affected the breakthrough curves by decreasing the time of the appearance of the fronts for all concentration ranges studied, and by decreasing the time difference between the highest concentration and lowest concentration of the fronts, especially the low concentration range at 0.5 g/L. The fronts of the breakthrough curves at high concentration seems to be the most affected by temperature.     

The adsorption mechanism of nortryptiline on C18-bonded Discovery

The adsorption isotherms of an ionizable compound, nortriptyline, were accurately measured by frontal analysis (FA) on a C(18)-Discovery column, first without buffer (in an aqueous solution of acetonitrile at 15%, v/v of ACN), then with a buffer (in 28%, v/v ACN solution). The buffers were aqueous solutions containing 20 mM of formic acid or a phosphate buffer at pH 2.70. The linear range of the isotherm could not be reached with the non-buffered mobile phase using a dynamic range larger than 40,000 (from 1.2 x 10(-3) g/L to 50 g/L). With a 20 mM buffer in the liquid phase, the isotherm is linear for concentrations of nortriptyline inferior to 10(-3) g/L (or 3 micromol/L). The adsorption energy distribution (AED) was calculated to determine the heterogeneity of the adsorption process. AED and FA were consistent and lead to a trimodal distribution. A tri-Moreau and a tri-Langmuir isotherm models accounted the best for the adsorption of nortriptyline without and with buffer, respectively. The nature of the buffer affects significantly the middle-energy sites while the properties of the lowest and highest of the three types of energy sites are almost unchanged. The desorption profiles of nortriptyline show some anomalies in relation with the formation of a complex multilayer adsorbed phase of acetonitrile whose excess isotherm was measured by the minor disturbance method. The C(18)-Discovery column has about the same total saturation capacity, around 200 g of nortriptyline per liter of adsorbent (or 116 mg/g), with or without buffer. About 98-99% of the available surface consists in low energy sites. The coexistence of these different types of sites on the surface solves the McCalley's enigma, that the column efficiency begins to drop rapidly when the analyte concentration reaches values that are almost one hundred times lower than those that could be predicted from the isotherm data acquired under the same experimental conditions. Due to the presence of some relatively rare high energy sites, the largest part of the saturation capacity is not practically useful.     

Effect of the ionic strength of salts on retention and overloading behavior of ionizable compounds in reversed-phase liquid chromatography I. XTerra-C18

The influence of the salt concentration (potassium chloride) on the retention and overloading behavior of the propranolol cation (R'-NH2+ -R) on an XTerra-C18 column, in a methanol:water solution, was investigated. The adsorption isotherm data were first determined by frontal analysis (FA) for a mobile phase without salt (25% methanol, v/v). It was shown that the adsorption energy distribution calculated from these raw adsorption data is bimodal and that the isotherm model that best accounts for these data is the bi-Moreau model. Assuming that the addition of a salt into the mobile phase changes the numerical values of the parameters of the isotherm model, not its mathematical form, we used the inverse method (IM) of chromatography to determine the isotherm with seven salt concentrations in the mobile phase (40% methanol, v/v; 0, 0.002, 0.005, 0.01, 0.05, 0.1 and 0.2 M). The saturation capacities of the model increase, q(s,1) by a factor two and q(s,2) by a factor four, with increasing salt concentration in the range studied while the adsorption constant b1 increases four times and b2 decreases four times. Adsorbate-adsorbate interactions vanish in the presence of salt, consistent with results obtained previously on a C18-Kromasil column. Finally, besides the ionic strength of the solution, the size, valence, and nature of the salt ions affect the thermodynamic as well as the mass transfer kinetics of the adsorption mechanism of propranolol on the XTerra column.     

Fast HPLC method for the determination of glimepiride, glibenclamide, and related substances using monolithic column and flow program

This work presents a fast method for the simultaneous separation and determination of glimepiride, glibenclamide, and two related substances by RP LC. The separation was performed on a Chromolith Performance (RP-18e, 100 mm x 4.6 mm) column. As mobile phase, a mixture of phosphate buffer pH 3, 7.4 mM, and ACN (55:45 v/v) was used. Column oven temperature was set to 30 degrees C. The total chromatographic run time was 80 s. This was achieved using a flow program from 5 to 9.9 mL/min. Precisions of the interday and the intraday assay for both retention times and peak areas for the four analyzed compounds were less than 1.2%. The method showed good linearity and recovery. The short analysis time makes the method very valuable for quality control and stability testing of drugs and their pharmaceutical preparations.     

Thermodynamic analysis of the heterogenous binding sites of molecularly imprinted polymers

The thermodynamic interactions of two polymers, one Fmoc-L-Trp-imprinted (MIP), the other one an unimprinted reference (NIP), with the two Fmoc-tryptophan enantiomers were studied by frontal analysis, which allows accurate measurements of the adsorption isotherms. These isotherms were acquired at temperatures of 40, 50, 60, and 70 degrees C, for sample concentrations ranging between 0.005 and 40 mM. The mobile phase used was acetonitrile with one percent acetic acid as an organic modifier. Within the measured concentration ranges, the tri-Langmuir isotherm model accounts best for the isotherm data of both enantiomers on the MIP, the bi-Langmuir model for the isotherm data of Fmoc-L-Trp on the NIP. These isotherm models were selected using three independent processes: statistical tests on the results from regression of the isotherm data to different isotherm models; calculation of the affinity energy distribution from the raw isotherm data; comparison of the experimental and the calculated band profiles. The isotherm parameters obtained from these best selected isotherm models showed that the enantiomeric selectivity does not change significantly with temperature, while the affinity of the substrates for both the MIP and the NIP decrease considerably with increasing temperatures. These temperature effects on the binding performance of the MIP were clarified by considering the thermodynamic functions (i.e., the standard molar Gibbs free energy, the standard molar entropy of adsorption, and the standard molar enthalpy of adsorption) for each identified type of adsorption sites, derived from the Van't Hoff equation. This showed that the entropy of transfer of Fmoc-L-Trp from the mobile to the MIP stationary phase is the dominant driving force for the selective adsorption of Fmoc-L-Trp onto the enantioselective binding sites. This entropy does not change significantly with increasing temperatures from 40 to 70 degrees C.     

Analysis of tricyclic antidepressant drugs in plasma by means of solid-phase microextraction-liquid chromatography-mass spectrometry

Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 microm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 degrees C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 degrees C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96-98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm x 2.1 mm, 5 microm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plasma.     

Determination of azadirachtin and fatty acid methyl esters of Azadirachta indica seeds by HPLC and GLC

A simple and economical method has been developed to estimate the azadirachtin content and fatty acid composition of neem kernels. Neem kernels are crushed and soaked overnight in ethanol. The extract obtained is analysed by HPLC after filtering through a 0.22 micro m membrane. The peaks are separated using acetonitrile-water (40:60) 1 mL min(-1) as the mobile phase on an RP-18 column and monitored at 214 nm. For the determination of fatty acid composition, the fatty acids are directly transmethylated in the kernel powder by heating with methanol-acetyl chloride-benzene (20:1:4, v/v) for 1 h in a water bath. The fatty acid methyl esters (FAMEs) obtained are extracted in hexane and analysed using GLC. The separation of the FAMEs is achieved using an RH-Wax column using temperature programming, 170-200 degrees C at 2 degrees min(-1). The peaks are detected using an FID. Both the methods do not require any clean up or defatting of seeds. This results in faster, easier and more economical sample preparation.     

Effect of the endcapping of reversed-phase high-performance liquid chromatography adsorbents on the adsorption isotherm

The retention mechanisms of n-propylbenzoate, 4-t ert-butylphenol, and caffeine on the endcapped Symmetry-C(18) and the non-endcapped Resolve-C(18) are compared. The adsorption isotherms were measured by frontal analysis (FA), using as the mobile phase mixtures of methanol or acetonitrile and water of various compositions. The isotherm data were modeled and the adsorption energy distributions calculated. The surface heterogeneity increases faster with decreasing methanol concentration on the non-endcapped than on the endcapped adsorbent. For instance, for methanol concentrations exceeding 30% (v/v), the adsorption of caffeine is accounted for by assuming three and two different types of adsorption sites on Resolve-C(18) and Symmetry-C(18), respectively. This is explained by the effect of the mobile phase composition on the structure of the C(18)-bonded layer. The bare surface of bonded silica appears more accessible to solute molecules at high water contents in the mobile phase. On the other hand, replacing methanol by a stronger organic modifier like acetonitrile dampens the differences between non-endcapped and endcapped stationary phase and decreases the degree of surface heterogeneity of the adsorbent. For instance, at acetonitrile concentrations exceeding 20%, the surface appears nearly homogeneous for the adsorption of caffeine.     

Characterization of the acidity of residual silanol groups in microparticulate and monolithic reversed-phase columns

The residual silanol acidity and activity of several microparticulate and monolithic C18 columns has been measured from the retention of LiNO3 in the columns with a methanol/buffer (1 mM in Na+) (60:40 v/v) mobile phase buffered to different pH values. For Luna C18 (2) and LiChrospher RP-18 columns, at least two different types of silanols with different acidity for each packing, were observed. Purospher RP-18e and Chromolith RP-18e packings present evidence of some active silanols only at pH values close to their basic pH stability limit or higher. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MSC18 and Underivatized XTerra. A modification of an equation previously proposed has been applied to all columns studied and the results obtained have been used to classify the columns according to their silanol acidity and activity. The method allows the prediction of the extent of the silanol activity of the columns studied at a particular mobile phase pH.     

Effect of the mobile phase composition on the isotherm parameters and the high concentration band profiles in reversed-phase liquid chromatography

Single-component adsorption isotherm data were acquired by frontal analysis for phenol on a C18-Kromasil packed column, under reversed-phase liquid chromatography conditions, using various methanol-water solutions (30-60%, v/v, methanol). The isotherm model accounting best for these data was the biLangmuir model. With increasing methanol content, the two saturation capacities decrease, particularly that of the high-energy sites, the adsorption constant of the low-energy sites decreases significantly and that of the high-energy sites decreases strongly. These results allow a quantitative investigation of the properties of the high-energy sites (which are not necessarily the so-called active sites), a feature rarely discussed yet. The band profiles calculated with the numerical values of the isotherm model parameters derived by fitting the frontal analysis data to the model and using the equilibrium-dispersive model agree very well with the experimental band profiles in the whole concentration range.     

Reproducibility of low and high concentration data in reversed-phase liquid chromatography. II. Overloaded band profiles on Chromolith-C18

Single-component adsorption isotherm data were acquired by frontal analysis (FA) for six low molecular weight compounds (phenol, aniline, caffeine, o-toluidine, p-toluidine and propylbenzoate) on one Chromolith-C18 column (#30, Merck, Darmstadt, Germany), using different methanol:water solutions (composition between 60/40 and 15/85 v/v, depending on the solute) as the mobile phase. These data were modeled for best agreement between the experimental data points and the adsorption isotherm model. The adsorption-energy distributions were also derived and used for the selection of the best isotherm model. Widely different models were obtained for these six compounds, four being convex upward (i.e., Langmuirian) and two having at least one inflection point. Overloaded band profiles corresponding to two different sample sizes (a low and a high loading factor) were recorded on six monolithic columns (#30-35) belonging to the same manufactured lot. These experimental band profiles were compared to the profiles calculated from the isotherm measured by FA on the first column, using the equilibrium-dispersive (ED) model of chromatography. For four of the six columns (#30, #32, #33, and #35), the reproducibility was better than 5 and 2.5% for the low and the high concentration profiles, respectively. On the other two columns (#31 and #34), the bands showed significant and systematic retention time shifts for all six compounds (with nearly identical band shapes), the relative adsorption being between 6 and 15% stronger on column #31 or between 2 and 7% lower on column #34. These differences seem to be correlated with the differences in the total porosities of these columns, which differ by 3% from columns #31 to #34, the higher porosity column giving the stronger adsorption.     

Accuracy and precision of adsorption isotherm parameters measured by dynamic HPLC methods

The fluctuations of the column temperature, the composition and the flow rate of the mobile phase affect the accuracy and precision of the adsorption isotherm parameters measured by dynamic HPLC methods. Experimental data were acquired by frontal analysis (FA) for phenol in equilibrium between C18-bonded Symmetry and a methanol:water mixture (20:80, v/v), at 303 K and a flow rate of 1 mL/min. The fluctuations of the experimental parameters were 0.1 K for the temperature, 0.1% for the mobile phase composition and 0.001 mL/min for the flow rate. The best isotherm model was shown to be the tri-Langmuir isotherm. Random errors were calculated and shown to agree with experimental results. Overloaded band profiles of phenol were acquired at low (sample size, 100 microL, concentration 3 g/L) and high (same sample size, concentration 60 g/L) loadings, at seven temperatures (298, 300, 302, 303, 304, 306, and 308 K), for seven mobile phase compositions (methanol 16, 18, 19, 20, 21, 22, and 24%), and with seven mobile phase flow rates (0.95, 0.97, 0.99, 1.00, 1.01, 1.03, and 1.05 mL/min), always keeping two experimental parameters at the values selected for the FA runs. Assuming that the isotherm model stays the same, the inverse method (IM) was used to derive the isotherm parameters in each case. Temperature affects the equilibrium constants according to Van't Hoff law. A temperature change of 1 K around 303 K causes a relative variation of 1.5% of the high-energy adsorption constant b3 and of 0.6% of the saturation capacity q3. The isotherm parameters are very sensitive to the mobile phase composition, especially the highest energy mode. Both adsorption constants b2 and b3 follow the linear strength solvent model (LSSM). A methanol volume fraction change of 1% causes a relative decrease of 3.2 and 5.0% of b2 and b3, respectively and a 2% decrease of the saturation capacity q3. Finally, flow rate changes affect only the saturation capacities. A flow rate change of 1 % causes a 2% change in the saturation capacity parameters.     

Characterization of the acidity of residual silanol groups in immobilized artificial membranes

Residual silanol acidity and activity of one immobilized artificial membrane (IAM) column have been measured from the retention of LiNO(3) in the column with a methanol/buffer (1mM in Na(+)) (60:40, v/v) mobile phase buffered to different pH values. Just one type of silanol with pssK(a)=7.61 (near the pH limit recommended by the manufacturer) was found, although these silanols show large activity. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MS C18, underivatized XTerra, Lichrospher 100 RP-18, Purospher RP-18e, Luna C18 (2) and Chromolith Perfomance RP-18e, showing that the IAM column is similar to Luna C18 and Symmetry C18 in terms of silica quality, as measured by Li(+) retention. A warning about the use of IAM columns for emulation of biological systems at physiological pH 7 is given because the ionized silanols may contribute to the retention of some drugs at this pH.     

Adsorption isotherms of the fullerenes C60 and C70 on a tetraphenylporphyrin-bonded silica

The single-component adsorption isotherms of the C60 (from 0 to 15 g/L) and C70 (from 0 to 8 g/L) buckminsterfullerenes on a tetraphenylporphyrin-bonded silica were acquired by frontal analysis, using a solution of toluene-1-methylnaphthalene (40:60, v/v) as the mobile phase. The best isotherm model derived from the fitting of these adsorption data was the bi-Langmuir model, a choice supported by the bimodal affinity energy distribution (AED) obtained for C60. The isotherm parameters derived from the inverse method (IM) of isotherm determination (by fitting calculated profiles to experimental overloaded band profiles of C60 and C70) are in very good agreement with those derived from the FA data. According to the isotherm parameters found by these three methods (FA, AED, IM), the tetraphenylporphyrin-bonded silica can adsorb 54 and 42 mmol/L of C60 and C70 fullerenes, respectively, a result that is consistent with the relative molecular size of these two compounds. The 20% lower surface accessibility for C70 is compensated by a three times higher equilibrium constant on the low-energy sites, giving a selectivity alpha(C70/C60) = 3.6. Large volumes (0.2, 0.8 and 1.7 mL) of mixtures of C60 (3.2 g/L) and C70 (1.3 g/L) were injected and their elution profiles compared to those calculated from the competitive bi-Langmuir model derived from the single-component isotherm data. A good agreement is obtained between calculated and experimental profiles, which supports the two-site adsorption mechanism derived from the single-component adsorption data. The measurements of the influence of the pressure on the retention of C60 and C70 demonstrate that the partial molar volumes of the two buckminsterfullerenes are 12 mL/mol larger in the stationary than in the mobile phase.     

Phase transition in an adsorption layer of a soluble surfactant at the air-water interface

In this paper we provide experimental evidence for a phase transition between a liquid- and gas-like phase occurring in an adsorption layer of a soluble surfactant at the air-water interface. The equilibrium surface tension sigma(e) versus bulk concentration sigma(e) (c) isotherm of surface chemically pure sodium 2-[4-(4-trifluoromethyl-phenylazo) phenoxy]-ethane sulfonate was measured at a temperature of 295 K up to the solubility limit of the amphiphile. The sigma(e) (c) isotherm could be fitted by Frumkin's equation of state. The lateral interaction energy is just above the limit for which Frumkin's model predicts a phase transition. The corresponding surface pressure pi versus surface area A isotherm possesses striking similarities to first-order phase transitions in the Langmuir monolayer. The fact that the difference in the two-dimensional density is only a factor of 2 indicates that the system is very close to the critical point. The surface phases were further characterized by surface second harmonic generation. The major structural difference between the two surface phases is the amphiphile's molecular orientation. A mean orientation of the amphiphile of about 80 degrees was found in the gas analogous phase, whereas a molecular tilt of 38 degrees has been identified in the liquid-like phase.     

Separation mechanism of nortriptyline and amytriptyline in RPLC

The single and the competitive equilibrium isotherms of nortriptyline and amytriptyline were acquired by frontal analysis (FA) on the C18- bonded discovery column, using a 28/72 (v/v) mixture of acetonitrile and water buffered with phosphate (20 mM, pH 2.70). The adsorption energy distributions (AED) of each compound were calculated from the raw adsorption data. Both the fitting of the adsorption data using multi-linear regression analysis and the AEDs are consistent with a trimodal isotherm model. The single-component isotherm data fit well to the tri-Langmuir isotherm model. The extension to a competitive two-component tri-Langmuir isotherm model based on the best parameters of the single-component isotherms does not account well for the breakthrough curves nor for the overloaded band profiles measured for mixtures of nortriptyline and amytriptyline. However, it was possible to derive adjusted parameters of a competitive tri-Langmuir model based on the fitting of the adsorption data obtained for these mixtures. A very good agreement was then found between the calculated and the experimental overloaded band profiles of all the mixtures injected.