Overloaded Band Profiles Research Articles

Competitive adsorption equilibrium model with continuous temperature dependent parameters for naringenin enantiomers on Chiralpak AD column

Determination of competitive adsorption equilibrium model with continuous temperature dependent parameters is important for the design and optimization of a chromatographic separation process operated under non-isothermal conditions. In this study, linear pulse experiments were first carried to determine the parameters of transport-dispersive model and their temperature dependences in the range of 283–313K. Overloaded band profiles of naringenin enantiomers on a Chiralpak AD column were acquired under various temperatures. Three of them were first separately fitted using Langmuir, linear-Langmuir and bi-Langmuir isotherm models substituted into the transport-dispersive column model. The comparison showed that bi-Langmuir model captures more details of the experimental results. This model was then extended with three extra parameters accounting for adsorption heat effects and used to simultaneously fit the band profiles at three temperatures.

Hydrophilic interaction chromatography: A promising alternative to reversed-phase liquid chromatography systems for the purification of small protonated bases.

The overloaded band profiles of the protonated species of propranolol and amitriptyline were recorded under acidic conditions on four classes of stationary phases including a conventional silica/organic hybrid material in reversed-phase liquid chromatography mode (BEH-C18), an electrostatic repulsion reversed-phase liquid chromatography C18 column (BEH-C18+), a poly(styrene-divinylbenzene) monolithic column, and a hydrophilic interaction chromatography stationary phase (underivatized BEH). The same amounts of protonated bases per unit volume of stationary phase were injected in each column (16, 47, and 141 μg/cm(3)). The performance of the propranolol/amitriptyline purification was assessed on the basis of the asymmetry of the recorded band profiles and on the selectivity factor achieved. The results show that the separation performed under reversed-phase liquid chromatography like conditions (with BEH-C18, BEH-C18+, and polymer monolith materials) provide the largest selectivity factors due to the difference in the hydrophobic character of the two compounds. However, they also provide the most distorted overloaded band profiles due to a too small loading capacity. Remarkably, symmetric band profiles were observed with the hydrophilic interaction chromatography column. The larger loading capacity of the hydrophilic interaction chromatography column is due to the accumulation of the protonated bases into the diffuse water layer formed at the surface of the polar adsorbent. This work encourages purifying ionizable compounds on hydrophilic interaction chromatography columns rather than on reversed-phase liquid chromatography columns.

Determination of Competitive Adsorption Isotherm of the Etodolac Enantiomers Using Inverse Method

The competitive adsorption isotherm of etodolac enantiomers on a cellulose carbamates-based chiral stationary phase (Chiralcel OD) at 25°C was determined in this study by the inverse method (IM). An equilibrium dispersive (ED) model combined with bi-Langmuir adsorption model was used in predicting the elution profiles. The adsorption isotherm model with 5 parameters was determined by fitting the two overloaded band profiles. The non-dominated sorting genetic algorithm (NSGA-II) and Levenberg-Marquardt algorithm (LMA) were crucial tools and utilities in this process. Suitable model parameters have been obtained and the experimental results were in good agreement with the model predictions.

Separation of peptides and intact proteins by electrostatic repulsion reversed phase liquid chromatography

A new brand of BEH-C18 hybrid particles chemically bonded to a leash carrying an amine group permits the implementation of electrostatic repulsive interactions chromatography. Using columns packed with this material, the influence of the concentration of positive charges bonded to the BEH-C18 surface on the overloaded band profiles of a few positively charged peptides and proteins was investigated in the gradient elution mode. Three columns packed with endcapped BEH-C18 particles bonded with three different surface-charge densities (LOW, MEDIUM and HIGH) were used and compared with those provided by a column packed with non-doped, endcapped BEH-C18 particles. The surface concentrations of fixed charges in the LOW, MEDIUM and HIGH columns were estimated at 0.029, 0.050, and 0.064μmol/m2, for example, about two orders of magnitude smaller than the surface density of bonded C18 chains (2.1μmol/m2). Three different mobile phase additives (0.1% v/v of trifluoro-acetic, phosphoric, and formic acid) were used to optimize the purification levels of proteins under different loading conditions. The weak ion-pairing ions (formate and phosphate) generate smaller retention but broader, more fronting band profiles than those eluted with a stronger ion-pairing ion (trifluoroactate). This effect is worse in the presence of fixed charges at the surface of the BEH-C18 particles. This was explained by an enhanced anti-Langmuirian adsorption behavior of the charged proteins in the presence of fixed surface charges. As the protein concentration increases in the bulk, so does the internal ionic strength, the electrostatic repulsive interactions weaken, and retention increases. Band fronting is mostly eliminated by replacing weak ion-pairing acids with TFA with which the adsorption isotherm remains weakly langmuirian. Faster but still complete gradient separation of insulin and myoglobin were achieved with the HIGH column than with the reference neutral column, despite a measurable loss in selectivity.

Effects of the surface concentration of fixed charges in C18-bonded stationary phases on the adsorption process and on the preparative chromatography of small ionizable compounds

The effects of the surface concentration of positive charges attached to the surface of research BEH-C18 hybrid particles on the overloaded band profiles and the adsorption isotherms of a neutral (caffeine) and a positively charged (nortryptilinium hydrochloride) compounds were measured and investigated. The inverse method (IM) of chromatography was used to determine the isotherm parameters. Three columns were packed with endcapped BEH-C18 particles doped with three different charge densities on their surfaces (LOW, MEDIUM and HIGH). Two other columns packed with unbonded, non-endcapped, and endcapped BEH-C18 particles served as standard reference materials. Minor disturbance method (MDM) experiments were conducted with acetonitrile/water mixtures in order to assess qualitatively the surface densities of the fixed positive charges. A more quantitative approach based on the solution of the linearized Poisson–Boltzmann equation and the decrease of the experimental Henry constant was also applied. The results show that the surface concentrations of the fixed charges in the LOW, MEDIUM and HIGH columns were 0.029, 0.050, and 0.064μmol/m2, e.g., close to two orders of magnitude smaller than the surface density of bonded C18 chains (2.1μmol/m2).The adsorption isotherm of the ionizable compound nortryptilinium onto the BEH-C18 columns is consistent with a two-sites adsorption model. The density of the high energy sites correlates directly to the total amount of the fixed charges and isolated silanols amidst the C18-bonded chains. The amount of low energy sites reflects the specific surface area of the adsorbent. The binding constants on the high- and low-energy adsorption sites are respectively ten and two times lower on the HIGH column than on the reference endcapped column. The active sites are closer to the adsorbent surface than the weak adsorption sites. Finally, a higher production rate of ionizable compounds can be achieved in preparative chromatography with the charge doped than with the reference RPLC-C18 columns. For the same analysis time, larger recovery yields are observed despite a slight loss in selectivity due to non-selective repulsive electrostatic interactions.

Comparison of volume and concentration overloadings in preparative enantio-separations by supercritical fluid chromatography

The adsorption isotherms of both naproxen enantiomers were measured on a (R,R)-Whelk-O1 chiral adsorbent from a mobile phase made of liquid carbon dioxide and methanol, using frontal analysis. These isotherms were used to model the competitive adsorption behavior of a racemic mixture of naproxen enantiomers. Their overloaded elution band profiles were calculated numerically using these isotherm data. The injected volume and the injected concentration of the samples of racemic mixture were carried out simultaneously, using two objective functions. The maximization of the production rate and of the product of the recovery yield and the production rate were performed. Based on the optimum points obtained, the two objective functions were compared for several recovery yield constraints in a range of injected volumes and concentrations. The consequences of choosing a separation design focused on the degree of band overlapping are discussed based on the results of these optimizations.

Effect of the pH and the ionic strength on overloaded band profiles of weak bases onto neutral and charged surface hybrid stationary phases in reversed-phase liquid chromatography

This work reports on the effects of the solution pH and its ionic strength on the overloaded band profiles and the parameters of the adsorption isotherms of nortriptylinium hydrochloride on the bridge ethylene hybrid (BEH) and the charged surface hybrid (CSH) C18-bonded columns. The mobile phases used were mixtures of acetonitrile and water buffered with hydrogenophosphate, formate, acetate, and dihydrogenophosphate buffers. The results show that the adsorption behavior of this protonated base onto the BEH-C18 column depends barely on the mobile phase pH and is slightly affected by its ionic strength. From both physical and statistical viewpoints, the Linear-Langmuir model is the most relevant adsorption isotherm. According to the inverse method of chromatography, this model is consistent with weak dispersive interactions taking place onto the C18-bonded chains and some strong ion–dipole interactions with residual silanols. In contrast, adsorption on the CSH-C18 column depends on the applied WSpH, e.g., on the degree of ionization of the amine groups tethered to the CSH surface. For WSpH<3, the electrostatically modified Langmuir model (EML) is acceptable because analyte molecules cannot access and interact with any active sites due to the electrostatic repulsion by the positively charged adsorbent surface. At WSpH>7.0, the Linear-bi-Langmuir model describes best the weak adsorption of the protonated base molecules onto the C18 chains and their strong adsorption onto the residual silanols and neutral amine groups.

Effect of the ionic strength on the adsorption process of an ionic surfactant onto a C18-bonded charged surface hybrid stationary phase at low pH

The adsorption behavior of nortriptylinium hydrochloride was investigated in detail on a new C18-bonded charged surface hybrid stationary phase (CSH-C18, Waters) at a constant WSpH of 2.60 (phosphate buffer dissolved in an acetonitrile/water mixture, 34/66, v/v) in order to promote strong repulsive electrostatic interactions between the surface of the packing material and the analyte molecules. This new stationary phase was recently designed to reduce the peak tailing of ionizable compounds in RPLC. Overloaded band profiles were recorded over a broad range of column loading factors (0.03–2.18%) and eluent ionic strengths (5–50mM). The experimental data were compared to those predicted by some electrostatically modified adsorption isotherms, including the Langmuir and the Moreau isotherms. For small loading factors (<0.1%), the electrostatic repulsion definitely plays a role on the adsorption behavior and the retention factors increase with increasing ionic strength due to the expected diminution of the Debye length through the bulk solution. Still this effect could not be consistently described by either of the above two electrostatic isotherm models over the whole range of ionic strengths. For higher loading factors (>0.1%), the compound was mostly adsorbed as a neutral species and the adsorption behavior was best represented by an apparent Langmuir–Moreau isotherm model.

Deformations of overloaded bands under pH-stable conditions in reversed phase chromatography

It has recently been demonstrated, using mathematical models, how peculiar overloaded band profiles of basic compounds are due to the local pH in the column when using low capacity buffers. In this study, overloaded peak shapes resulting after injection of carefully pH matched samples close to the p K a of the chosen solute are investigated primarily on two columns; one hybrid silica C18 column (Kromasil Eternity) and one purely polymeric column (PLRP-S), the latter lacking C18 ligands. It was found that distorted peaks of the basic test compound appear even though there is no difference in pH between the injected sample solution and the eluent; the previous explanation to why these effects occur is based on a pH mismatch. Thus, the unusual band shapes are not due to an initial pH difference. Furthermore, it was observed that the effect does not appear on polymeric columns without C18 ligands, but only on columns with C18 ligands, independently of the base matrix (silica, hybrid silica, polymeric).

The adsorption of Naproxen enantiomers on the chiral stationary phase Whelk-O1 under reversed-phase conditions: The effect of buffer composition

The adsorption of the Naproxen enantiomers on the chiral stationary phase ( S,S)-Whelk-O1 from methanol–water 80:20 (v/v) solutions modified with the addition of acetic acid or an acetic acid–sodium acetate buffer was studied using elution chromatography. Adsorption was found to be best accounted for by a two-site model assuming different retention mechanisms for the two enantiomers. Under experimental conditions causing a considerable degree of solute dissociation, strong distortion of overloaded band profiles is observed. This phenomenon is explained by the superimposition of the adsorption and the dissociation equilibria. The effect of the buffer composition on the retention is discussed and the results compared with previous ones obtained with the same system. The proposed model explains all the principal features of the adsorption of Naproxen on Whelk-O1 that were found earlier. Moreover this model applies well in a wider range of buffer concentrations, encompassing both the eluents in which solute dissociation is suppressed and those in which dissociation is significant.

Automated instrumental method for on-line fraction analysis and peak deconvolution in gradient multicomponent overloaded high performance liquid chromatography

An automated method for deconvolution of overloaded band profiles in gradient elution is described. The instrumental set-up consists of a pseudo-bidimensional HPLC system, where overloaded band profiles generated in the first direction are sampled on the second one. The method, previously employed under isocratic conditions, has been now extended to gradient elution, where one has to face the problem of band compression (and possibly band interference) during gradient, which decreases the time-window available along the first direction for sampling the overloaded profiles. The effect of the gradient steepness on the problem of defining a minimum number of sampling points to reconstruct single component bands from overloaded profiles is investigated. This approach is particularly useful in the framework of using inverse method (IM) to determine adsorption isotherm parameters for preparative gradient elution. In fact, it allows for the gathering of the information necessary to run IM calculations with minimum efford, also in cases where the individual component forming the mixture have different UV spectra.

Simultaneous measurement of and overloaded band profiles of small peptides when insufficiently buffered mobile phases are used in preparative liquid chromatography

We recorded simultaneously the (W)(S)pH and the elution band profiles of phenylalanine-alanine (Phe-Ala, (W)(S)pK(a,1)=3.6, (W)(S)pK(a,2)=7.5) on a column packed with C(18)-bonded organic (ethyl)/inorganic (silica) hybrid porous particles (BEH), eluted with a series of buffered methanol-water mobile phases (20/80, v/v). Using different buffers, the (W)(S)pHs of the mobile phases was set to values between 2.5 and 9.8. The ionic strength of the mobile phases was kept constant at 20 mM. The injected solutions were prepared with Phe-Ala as its zwitterionic species. Analytical to overloaded sample sizes were used, under strongly or insufficiently buffered conditions. With insufficiently buffered mobile phases, the experimental results show significant variations of (W)(S)pH during band elution, which proves a significant change of the concentration ratio of the different sample species during elution. This result confirms and validates our earlier general adsorption isotherm model of acido-basic compounds in RPLC. The (W)(S)pH record shows also the system peak of the buffer component, consecutive to sample injection. The phosphate II and carbonate II ions are adsorbed and may compete with the peptide for adsorption onto the active sites of BEH-C(18). This suggests refinements of the general adsorption isotherm model, depending on the nature of the buffer components, including competitive adsorption of the sample and the buffer species.

Characteristics of the adsorption mechanism of acido-basic compounds with two p [formula omitted] in reversed-phase liquid chromatography

Overloaded band profiles of phthalic acid, nicotinic acid, and sodium nicotinate eluted with a methanol-aqueous buffer solution were recorded at the exit of a 150 mm × 4.6 mm column packed with 3.5 μ m X-Bridge-C 18 porous particles. The pH W S s of the mobile phase was adjusted at different values, with addition of hydrochloric acid ( pH W S ∼ 1.37), phosphate ( pH W S ∼ 2.01, 2.85, and 7.32), formate ( pH W S ∼ 3.61), acetate ( pH W S ∼ 4.41 and 5.15), or succinate buffers ( pH W S ∼ 6.11). The ionic strengths of all the buffers were fixed at 0.1 M by adding the necessary amount of potassium chloride when necessary. The compositions of the solutions at equilibrium were solved by using the extended Debye–Hückel theory, which estimates the activity coefficients of the ions in the bulk phase. The adsorption of the samples onto X-Bridge-C 18 assumed a simple bi-Langmuir isotherm for the neutral species (or zwitterion) and a Langmuir isotherm for the ionizable species. Competition for adsorption between the neutral and the ionic species was neglected. The band profiles were calculated based on the equilibrium-dispersive model of chromatography. An excellent agreement was observed between the experimental and the calculated band profiles for all buffer solutions. This work validates the adsorption mechanism assumed in earlier work for neutral and ionizable species. It is consistent with the neutral species adsorbing weakly (due to dispersive interactions) onto and/or into the C 18-bonded layer (large number of sites) while the singly charged species adsorb strongly onto the few residual accessible silanol groups (due to charge–dipole interactions). Doubly charged species are not retained and are probably excluded from the pores of the hydrophobic stationary phase.

Determination of the adsorption energy distribution of neutral and charged compouds onto endcapped silica-C18 adsorbent from polar liquid phases

The adsorption energy distributions (AED) of a few neutral and ionic compounds were determined onto Gemini-C18, an endcapped silica-C18 surface, from solutions of methanol and water. The adsorption data were accurately measured using the dynamic frontal analysis method. The adsorption heterogeneity of this surface toward neutral and ionic compounds was investigated by calculating the AED using the Expectation-Maximization algorithm. The calculations converged toward a well-defined AED in all cases. Neutral compounds adsorb on two distinct types of sites (with relatively low adsorption energies) while the ionic compounds adsorb on three distinct types of sites (two having a weak and one a strong adsorption energy). The combination of these results allows the selection of a satisfactory adsorption isotherm model and the correct prediction of the overloaded band profiles of these compounds, including those of the acido-basic compounds when eluted with a buffer having a weak capacity, e.g. when the ratio of the concentrations of the neutral and ionic conjugated species in the band and the mobile phase pH change during elution.

Band profiles of reacting acido-basic compounds with water–methanol eluents at different [formula omitted] and ionic strengths in reversed-phase liquid chromatography

Overloaded band profiles of aniline (25 μ L injection of a 30.5 mM solution) eluted with a methanol-aqueous buffer solution (30/70, v/v) were recorded at the exit of a 150 mm × 4.6 mm column packed with 3.5 μ m XBridge-C 18porous particles. The pH W S of the mobile phase was adjusted with phosphate ( pH W S ∼ 2.7 and pH W S ∼ 7.5 ) or acetate buffers ( pH W S ∼ 5.3 ) of different concentrations (11, 56, and 278 mM). The elution times and profiles of the bands observed at low ionic strength were succesfully accounted for using the extended Debye–Hückel theory to estimate the activity coefficients of the ions in the bulk phase and a simple non-competitive Langmuir adsorption model, the adsorption of pure aniline or pure anilinium onto XBridge-C 18 being described by a Langmuir isotherm. The band profiles were calculated using this adsorption model and the equilibrium-dispersive model of chromatography. The calculated and the experimental band profiles are in excellent agreement at all buffer pH and ionic strength. This demonstrates that the elution times and the band profiles are controlled by the chromatographic dilution process and by the reaction of aniline with the buffer solution.

Peak shapes of acids and bases under overloaded conditions in reversed-phase liquid chromatography, with weakly buffered mobile phases of various pH: A thermodynamic interpretation

We measured overloaded band profiles for a series of nine compounds (phenol, caffeine, 3-phenyl 1-propanol, 2-phenylbutyric acid, amphetamine, aniline, benzylamine, p-toluidine, and procainamidium chloride) on columns packed with four different C 18-bonded packing materials: XTerra-C 18, Gemini-C 18, Luna-C 18(2), and Halo-C 18, using buffered methanol–water mobile phases. The pH W S of the mobile phase was increased from 2.6 to 11.3. The buffer concentration (either phosphate, acetate, or carbonate buffers) was set constant at values below the maximum concentration of the sample in the band. The influence of the surface chemistry of the packing material on the retention and the shape of the peaks was investigated. Adsorbents having a hybrid inorganic/organic structure tend to give peaks exhibiting moderate or little tailing. The retention and the shape of the band profiles can easily be interpreted at pHs W S that are well above or well below the pK W S a of the compound studied. In contrast, the peak shapes in the intermediary pH range (i.e., close to the compound pK W S a ) have rarely been studied. These shapes reveal the complexity of the competitive adsorption behavior of couples of acido-basic conjugated compounds at pHs W S that are close to their pK W S a . They also reveal the role of the buffer capacity on the resulting peak shape. With increasing pH W S , the overloaded profiles are first langmuirian (isotherm type I) at low pHs W S , they become S-shaped (isotherm type II), then anti-langmuirian (isotherm type III), S-shaped again at intermediate pHs W S , and finally return to a langmuirian shape at high pHs W S . A new general adsorption isotherm model that takes into account the dissociation equilibrium of conjugated acidic and basic species in the bulk mobile phase accounts for these transient band shapes. An excellent agreement was achieved between experimental profiles and those calculated with a two-sites adsorption isotherm model at all pHs W S . The neutral species adsorbs strongly on a first type of sites that have a high density while the ionic species adsorb preferentially on a second type of sites that have a very low density. The evolution of the peak shape when the pH W S changes from acidic to basic is well explained by the weak buffer capacity of the mobile phase used compared to the concentration of the eluted compounds.

Adsorption mechanism of acids and bases in reversed-phase liquid chromatography in weak buffered mobile phases designed for liquid chromatography/mass spectrometry

The overloaded band profiles of five acido-basic compounds were measured, using weakly buffered mobile phases. Low buffer concentrations were selected to provide a better understanding of the band profiles recorded in LC/MS analyses, which are often carried out at low buffer concentrations. In this work, 10 μ L samples of a 50 mM probe solution were injected into C 18 -bonded columns using a series of five buffered mobile phases at pH W S between 2 and 12. The retention times and the shapes of the bands were analyzed based on thermodynamic arguments. A new adsorption model that takes into account the simultaneous adsorption of the acidic and the basic species onto the endcapped adsorbent, predicts accurately the complex experimental profiles recorded. The adsorption mechanism of acido-basic compounds onto RPLC phases seems to be consistent with the following microscopic model. No matter whether the acid or the base is the neutral or the basic species, the neutral species adsorbs onto a large number of weak adsorption sites (their saturation capacity is several tens g/L and their equilibrium constant of the order of 0.1 L/g). In contrast, the ionic species adsorbs strongly onto fewer active sites (their saturation capacity is about 1 g/L and their equilibrium constant of the order of a few L/g). From a microscopic point of view and in agreement with the adsorption isotherm of the compound measured by frontal analysis (FA) and with the results of Monte-Carlo calculations performed by Schure et al., the first type of adsorption sites are most likely located in between C 18 -bonded chains and the second type of adsorption sites are located deeper in contact with the silica surface. The injected concentration (50 mM) was too low to probe the weakest adsorption sites (saturation capacity of a few hundreds g/L with an equilibrium constant of one hundredth of L/g) that are located at the very interface between the C 18 -bonded layer and the bulk phase.

Determination of competitive isotherms of enantiomers by a hybrid inverse method using overloaded band profiles and the periodic state of the simulated moving-bed process

A procedure for determination of adsorption isotherms in simulated moving-bed (SMB) chromatography is presented. The parameters of a prescribed adsorption isotherm model and rate constants are derived using a hybrid inverse method, which incorporates overloaded band profiles of the racemic mixture and breakthrough data from a single frontal experiment. The latter are included to reduce the uncertainty on the estimated saturation capacity, due to the dilution of the chromatograms with respect to the injected concentrations. The adsorption isotherm model is coupled with an axially dispersed flow model with finite mass-transfer rate to describe the experimental band profiles. The numerical constants of the isotherm model are tuned so that the calculated and measured band profiles match as much as possible. The accuracy of the isotherm model is then checked against the cyclic steady state (CSS) of the target SMB process, which is readily and cheaply obtained experimentally on a single-column set-up. This experiment is as expensive and time consuming as just a few breakthrough experiments. If necessary, the isotherm parameters are adjusted by applying the inverse method to the experimental CSS concentration profile. The method is successfully applied to determine the adsorption isotherms of Trögers base enantiomers on Chiralpak AD/methanol system. The results indicate that the proposed inverse method offers a reliable and quick approach to determine the competitive adsorption isotherms for a specific SMB separation.

Instrumental method for automated on-line fraction analysis and peak deconvolution in multicomponent-overloaded high-performance liquid chromatography

A bi-dimensional (non-orthogonal) chromatographic system made of two columns of different length (longer along the first direction and shorter on the second) but packed with identical (or similar) materials has been employed to perform automated on-line fraction analysis and peak deconvolution of multicomponent overloaded band profiles. Multicomponent overloaded peaks, eluted from the first column, are sampled through a remotely-controlled switching valve and fractions are injected, under analytical conditions, directly into the second direction. This set-up serves a twofold purpose. First of all, it has been possible to use linear calibration curves for detector calibration (DC). Secondly, since single component peaks were resolved on the second direction, individual concentration profiles have been obtained beneath the envelop of overlapping bands. All the information necessary to operate the bi-dimensional system can be achieved by analyzing the chromatographic behavior of the mixture on the shorter column under linear conditions. Therefore, solutions of unknown concentration can be prepared and their composition reconstructed with a simple chromatographic run. Two- and three-component overloaded mixtures have been employed to validate the system.

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 °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 °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 °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.