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

In a companion paper, we describe the influence of the concentration and the nature of salts dissolved in the mobile phase (methanol:water, 40:60, v/v) on the adsorption behavior of propranolol (R'-NH2+ -R, Cl-) on XTerra-C18. The same experiments were repeated on a Symmetry-C18 column to compare the adsorption mechanisms of this ionic compound on these two very different RPLC systems. Frontal analysis (FA) measurements were first carried out to determine the best isotherm model accounting for the adsorption behavior of propranolol hydrochloride on Symmetry with a mobile phase without salt (and only 25% methanol to compensate for the low retention in the absence of salt). The adsorption data were best modeled by the bi-Moreau model. Large concentration band profiles of propranolol were recorded with mobile phases having increasing KCl concentrations (0, 0.002, 0.005, 0.01, 0.05, 0.1 and 0.2 M) and the best values of the isotherm coefficients were determined by the inverse method (IM) of chromatography. The general effect of a dissociated salt in the mobile phase was the same as the one observed earlier with XTerra-C18. Increasing the salt concentration increases the two saturation capacities of the adsorbent and the adsorption constant on the low-energy sites. The adsorption constant on the high-energy sites decreases and the adsorbate-adsorbate interactions tend to vanish with increasing salt concentration of the mobile phase. The saturation capacities decrease with increasing radius of the monovalent cation (Na+, K+, Cs+, etc.). Using sulfate as a bivalent anion (Na2SO4) affects markedly the adsorption equilibrium: the saturation capacities are drastically reduced, the high-energy sites nearly disappear while the adsorption constant and the adsorbate-adsorbate interactions on the low-energy sites increase strongly. The complexity of the thermodynamics in solution might explain the different influences of these salts on the adsorption behavior.     

Effect of the ionic strength of the solution and the nature of its ions on the adsorption mechanism of ionic species in RPLC III. Equilibrium isotherms and overloaded band profiles on Kromasil-C18

In two companion papers, we have described the influence of the concentration and the nature of completely dissociated salts dissolved in the mobile phase (methanol:water, 40:60, v/v) on the adsorption behavior of propranolol (R'-NH2+-R, Cl-) on XTerra-C18 and on Symmetry-C18. The same experiments were repeated on a Kromasil-C18 column to compare the adsorption behavior of this ionic compound on these three different RPLC systems. The adsorption data of propranolol hydrochloride were first measured by frontal analysis (FA) using a mobile phase without salt. These data fit best to the Bi-Moreau model. Large concentration band profiles of propranolol were recorded with mobile phases containing increasing KCl concentrations (0, 0.002, 0.005, 0.01, 0.05, 0.1 and 0.2 M) and the best values of the isotherm coefficients were determined using the numerical solution of the inverse problem of chromatography. The general effect of a dissociated salt in the mobile phase was the same as the one observed earlier with XTerra-C18 and Symmetry-C18. However, obvious differences were observed for the shape of the band profiles recorded at low column loading (1.5 g/L, 250 microL injected). A long shoulder is visible at all salt concentrations and the band broadening is maximum at low salt concentrations. A slow mass transfer kinetics on the high-energy sites of the bi-Moreau model might explain this original shape. Five other salts (NaCl, CsCl, KNO3, CaCl2 and Na2SO4) were also used at the same ionic strength (J = 0.2 M). As many different band profiles were observed, suggesting that specific solute-salt interactions take place in the adsorbed phase.     

Effect of the flow rate on the measurement of adsorption data by dynamic frontal analysis

The adsorption data of propyl benzoate were acquired by frontal analysis (FA) on a Symmetry-C18 column, using a mixture of methanol (65%, v/v) and water as the mobile phase, at three different flow rates, 0.5, 1.0 and 2.0 mL/min. The exact flow rates Fv were measured by collecting the mobile phase in volumetric glasses (deltaFv / Fv < or = 0.2%). The extra-column volumes and the column hold-up volume were accurately measured at each flow rate by tracer injections. The detailed effect of the flow rate on the value of the amount adsorbed was investigated. The best isotherm model accounting for the adsorption data was the same BET isotherm model at all three flow rates. Only slight differences (always less than 5%) were found between the three different sets of isotherm parameters (saturation capacity, q(s), equilibrium constant on the adsorbent, b(s) and equilibrium constant on successive layers of propyl benzoate, bL). The reproducibility of the same isotherm parameters measured by the inverse method (IM) is less satisfactory, leading to R.S.D.s of up to 10%. A flow rate increase is systematically accompanied by a slight increase of the amount adsorbed. This phenomenon is consistent with the influence of the pressure on the equilibrium constant of adsorption due to the difference between the partial molar volumes of the solute and the adsorbate. The larger average pressure along the column that is required to achieve a larger flow rate causes a larger amount of solute to be adsorbed on the column at equilibrium. This result comforts the high sensitivity and versatility of the FA method for isotherm determination under any kind of situation.     

Comparison between the adsorption behaviors of an organic cation and an organic anion on several reversed-phase liquid chromatography adsorbents

Adsorption data of an organic cation (propranololium chloride) and an organic anion (sodium 1-naphthalene sulfonate) were measured by frontal analysis on two RPLC adsorbents, Symmetry-C18 and XTerra-C18, with aqueous solutions of methanol as the mobile phases. The influence of supporting neutral salts on the adsorption behavior of these two ions are compared. The Henry constants are close (H approximately 5). The four sets of isotherm data are all well accounted for using the bi-Moreau model. However, the isotherms of the two ions behave differently at high concentrations. The initial behaviors of all the isotherms are antilangmuirian but remain so in a much wider concentration range for the cation than for the anion, due to its stronger adsorbate-adsorbate interactions on the low-energy adsorption sites. The retention times of both ions increase with increasing concentration of neutral salt in the mobile phase, suggesting the formation of ion-pair complexes, with Cl- for the cation and with Na+ for the anion. The adsorbate-adsorbate interactions vanish in the presence of salt and the bi-Moreau isotherm model tends toward a bi-Langmuir model. Differences in adsorption behavior are also observed between the cation and the anion when bivalent inorganic anions and cations, respectively, are dissolved in the mobile phase. High concentration band profiles of 1-naphthalene sulfonic acid are langmuirian, except in the presence of a trivalent cation, while those of propranolol are antilangmuirian under certain conditions even with uni- or divalent cations.     

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.     

Physical origin of peak tailing on C18-bonded silica in reversed-phase liquid chromatography

Single component isotherm data of caffeine and phenol were acquired on two different stationary phases for RPLC, using a methanol/water solution (25%, v/v, methanol) as the mobile phase. The columns were the non-endcapped Waters Resolve-C18, and the Waters XTerra MS C18. Both columns exhibit similar C18 -chain densities (2.45 and 2.50 micromol/m2) and differ essentially by the nature of the underivatized solid support (a conventional, highly polar silica made from water glass, hence containing metal impurities, versus a silica-methylsilane hybrid surface with a lower density of less acidic free silanols). Thirty-two adsorption data points were acquired by FA, for caffeine, between 10(-3) and 24 g/l, a dynamic range of 24,000. Twenty-eigth adsorption data points were acquired for phenol, from 0.025 to 75 g/l, a dynamic range of 3000. The expectation-maximization procedure was used to derive the affinity energy distribution (AED) from the raw FA data points, assuming a local Langmuir isotherm. For caffeine, the AEDs converge to a bimodal and a quadrimodal distribution on XTerra MS-C18 and Resolve-C18, respectively. The values of the saturation capacity (q(s,1) approximately equal to 0.80 mol/l and q(s,2) approximately equal to 0.10 mol/l) and the adsorption constant (b1 approximately equal to 3.11/mol and b2 approximately equal to 29.1 l/mol) measured on the two columns for the lowest two energy modes 1 and 2, are comparable. These data are consistent with those previously measured on an endcapped Kromasil-C18 in a 30/70 (v/v), methanol/water solution (q(s,1) = 0.9 mol/l and q(s,2) = 0.10 mol/l, b1 = 2.4 l/mol and b2 = 16.1 l/mol). The presence of two higher energy modes on the Waters Resolve-C18 column (q(s,3) approximately equal to 0.013 mol/l and q(s,4) approximately equal to 2.6 10(-4) mol/l, b3 approximately equal to 252 l/mol and b4 = 13,200 l/mol) and the strong peak tailing of caffeine are explained by the existence of adsorption sites buried inside the C18-bonded layer. It is demonstrated that strong interactions between caffeine and the water protected bare silica surface cannot explain these high-energy sites because the retention of caffeine on an underivatized Resolve silica column is almost zero. Possible hydrogen-bond interactions between caffeine and the non-protected isolated silanol groups remaining after synthesis amidst the C18-chain network cannot explain these high energy interactions because, then, the smaller phenol molecule should exhibit similarly strong interactions with these isolated silanols on the same Resolve-C18 column and, yet, the consequences of such interactions are not observed. These sites are more consistent with the heterogeneity of the local structure of the C18-bonded layer. Regarding the adsorption of phenol, no matter whether the column is endcapped or not, its molecular interactions with the bare silica were negligible. For both columns, the best adsorption isotherm was the Bilangmuir model (with q(s,1) approximately equal to 2 and q(s,2) approximately equal to 0.67 mol/l, b1 0.61 and b2 approximately equal to 10.3 l/mol). These parameters are consistent with those measured previously on an endcapped Kromasil-C18 column under the same conditions (q(s,1) = 1.5 and q(s,2) = 0.71 mol/l, b1 = 1.4 l/mol and b2 = 11.3 l/mol). As for caffeine, the high-energy sites are definitely located within the C18-bonded layer, not on the bare surface of the adsorbent.     

Heterogeneity of the surface energy on unused C18-chromolith adsorbents in reversed-phase liquid chromatography

Single-component adsorption isotherm data were acquired by frontal analysis (FA) for phenol and caffeine on a new C18-Chromolith column (Merck, Darmstadt, Germany), using a water-rich mobile phase (methanol/water, 15/85, v/v). These data were modeled for best agreement between the experimental data points and the adsorption isotherm model. The adsorption-energy distributions, based on the expectation-maximization (EM) procedure, were also derived and used for the selection of the best isotherm model. The adsorption energy distributions (AEDs) for phenol and caffeine converged toward a trimodal and a quadrimodal distribution, respectively. Energy distributions with more than two modes had not been reported before for the adsorption of these compounds on packed columns. The third high energy mode observed for both phenol and caffeine seems to be specific of the surface of the monolithic column while the first and second low energy modes have the same physical origin as the two modes detected on packed columns. These results suggest significant differences between the structures of the porous silica in these different materials.     

Adsorption-desorption isotherm hysteresis of phenol on a C18-bonded surface

Single component adsorption and desorption isotherms of phenol were measured on a high-efficiency Kromasil-C18 column (N = 15000 theoretical plates) with pure water as the mobile phase. Adsorption isotherm data were acquired by frontal analysis (FA) for seven plateau concentrations distributed over the whole accessible range of phenol concentration in pure water (5, 10, 15, 20, 25, 40, and 60 g/l). Desorption isotherm data were derived from the corresponding rear boundaries, using frontal analysis by characteristic points (FACP). A strong adsorption hysteresis was observed. The adsorption of phenol is apparently modeled by a S-shaped isotherm of the first kind while the desorption isotherm is described by a convex upward isotherm. The adsorption breakthrough curves could not be modeled correctly using the adsorption isotherm because of a strong dependence of the accessible free column volume on the phenol concentration in the mobile phase. It seems that retention in water depends on the extent to which the surface is wetted by the mobile phase, extent which is a function of the phenol concentration, and of the local pressure rate, which varies along the column, and on the initial state of the column. By contrast, the desorption profiles agree well with those calculated with the desorption isotherms using the ideal model, due to the high column efficiency. The isotherm model accounting best for the desorption isotherm data and the desorption profiles is the bi-Langmuir model. Its coefficients were calculated using appropriate weights in the fitting procedure. The evolution of the bi-Langmuir isotherm parameters with the initial equilibrium plateau concentration of phenol is discussed. The FACP results reported here are fully consistent with the adsorption data of phenol previously reported and measured by FA with various aqueous solutions of methanol as the mobile phase. They provide a general, empirical adsorption model of phenol that is valid between 0 and 65% of methanol in water.     

Repeatability and reproducibility of high concentration data in reversed-phase liquid chromatography. I. Overloaded band profiles on Kromasil-C18

Single-component adsorption-isotherm data were acquired by frontal analysis (FA) for six low-molecular-mass compounds (phenol, aniline, caffeine, theophylline, ethylbenzene and propranolol) on one Kromasil-C18 column, using water-methanol solutions (between 70:30 and 20:80, v/v) as the mobile phase. Propranolol data were also acquired using an acetate buffer (0.2 M) instead of water. The data were modeled for best agreement between calculated and experimental overloaded band profiles. The adsorption energy distribution was also derived and used for the selection of the best isotherm model. Widely different isotherm models were found to model best the data obtained for these compounds, convex upward (i.e. Langmuirian), convex downward (i.e. anti-Langmuirian), and S-shaped isotherms. Using the same sample size for all columns (loading factor, Lf approximately 10%), overloaded band profiles were recorded on four different columns packed with the same batch of Kromasil-C18 and five other columns packed with different batches of Kromasil-C18. These experimental band profiles were compared to the profile calculated from the isotherm measured by FA on the first column. The repeatability as well as the column-to-column and the batch-to-batch reproducibilities of the band profiles are better than 4%.     

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.     

Comparison of the adsorption mechanisms of pyridine in hydrophilic interaction chromatography and in reversed-phase aqueous liquid chromatography

The adsorption isotherms of pyridine were measured by frontal analysis (FA) on a column packed with shell particles of neat porous silica (Halo), using water–acetonitrile mixtures as the mobile phase at 295 K. The isotherm data were measured for pyridine concentrations covering a dynamic range of four millions. The degree of heterogeneity of the surface was characterized by the adsorption energy distribution (AED) function calculated from the raw adsorption data, using the expectation-maximization (EM) procedure. The results showed that two different retention mechanisms dominate in Per aqueous liquid chromatography (PALC) at low acetonitrile concentrations and in hydrophilic interaction chromatography (HILIC) at high acetonitrile concentrations. In the PALC mode, the adsorption mechanism of pyridine on the silica surface is controlled by hydrophobic interactions that take place on very few and ultra-active adsorption sites, which might be pores on the irregular and rugose surface of the porous silica particles. The surface is seriously heterogeneous, with up to five distinct adsorption sites and five different energy peaks on the AED of the packing material. In contrast, in the HILIC mode, the adsorption behavior is quasi-homogeneous and pyridine retention is governed by its adsorption onto free silanol groups. For intermediate mobile phase compositions, the siloxane and the silanol groups are both significantly saturated with acetonitrile and water, respectively, causing a minimum of the retention factor of pyridine on the Halo column.     

Comparison of the adsorption equilibrium of a few low-molecular mass compounds on a monolithic and a packed column in reversed-phase liquid chromatography

Adsorption isotherm data were acquired by frontal analysis for several low-molecular mass compounds (3-phenyl 1-propanol, 4-tert.-butylphenol, butylbenzene, and butyl benzoate) on a classical packed column and a monolithic column using methanol-water RP-HPLC conditions. These columns have similar characteristics (C18-bonded silica, close specific surface areas and bonding densities). In each case, the isotherm model best accounting for the data was the same on both columns. The solute polarity determines the class of this model. For the two -OH compounds it was a Langmuirian adsorption isotherm. The hydrocarbon data were best modeled by an anti-Langmuir convex-downward isotherm model. The adsorption data for the aromatic ester exhibited a nearly linear behavior, depending on the methanol concentration of the mobile phase. A slightly convex downward isotherm was obtained at high methanol concentrations while the best fitting was obtained with a liquid-solid extended multilayer B.E.T. isotherm model at low concentrations. The validation of these models is discussed in detail. In all cases, similar values of the adsorption-desorption constants were found, underlining the closeness of the adsorption energies on both columns. By contrast, the adsorption capacity of the monolithic column was found to be approximately 1.4 greater than that of the packed column in spite of the close values of the surface areas of the silica in both columns.     

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.     

Comparison of Adsorption Isotherms of Methylxanthines on C18 Column

A comparison of the adsorption isotherms of caffeine, theophylline and theobromine and the competitive adsorption of the three compounds on a C 18 column were investigated. The experimental parameters of the equilibrium isotherms were estimated by linear and nonlinear regression analyses. The linear equation as a function of the adsorption concentration of the single compound in its solution and the competitive adsorption of a single compound in a mixed solution were then determined. The adsorption equilibrium data were then correlated to the linear, Langmuir, Freundlich, Langmuir-Freundlich and stoichiometric displacement theory for adsorption(SDT-A) isotherm models. The mixed compounds of the three compounds were competitively adsorbed on the C 18 particles. The expression of stoichiometric displacement theory for adsorption was found to be more suitable for adsorption of methylxanthines on a C 18 column.     

Band splitting in overloaded isocratic elution chromatography II. New competitive adsorption isotherms

The equations of two new binary competitive isotherms models are derived. The first of these models assumes that the isotherms of the two pure, single compounds have distinct monolayer capacities. Its derivation is based on kinetic arguments. The ideal adsorbed solution (IAS) framework was applied to derive the second model that is a thermodynamically consistent competitive isotherm. This second model predicts the competitive adsorption isotherm behavior of a mixture of two compounds that have single-component adsorption behavior following a BET and/or a Langmuir isotherms. Both models apply well to the binary adsorption of ethylbenzoate and 4-tert.-butylphenol on a Kromasil-C18 column (with methanol-water, 62:38, v/v, as the mobile phase). The best single-solute adsorption isotherms of these two compounds are the liquid-solid extended multilayer BET and the Langmuir isotherms, respectively. The kinetic and thermodynamic new competitive models were compared, regarding the accuracy of their prediction of the elution band profiles of mixtures of these two compounds. A better agreement between experimental and calculated profiles was observed with the kinetic model. The IAS model failed because the behavior of the ethylbenzoate/4-tert.-butylphenol adsorbed phase mixture is probably non-ideal. The most striking result is the qualitative prediction by these models of the peak splitting of 4-tert.-butylphenol during its elution in presence of ethylbenzoate.     

Modeling of overloaded gradient elution of nociceptin/orphanin FQ in reversed-phase liquid chromatography

The Reversed-phase (RP) gradient elution chromatography of nociceptin/orphanin FQ (N/OFQ), a neuropeptide with many biological effects, has been modeled under linear and non-linear conditions. In order to do this, the chromatographic behavior has been studied under both linear and nonliner conditions under isocratic mode at different mobile phase compositions--ranging from 16 to 19% (v/v) acetonitrile (ACN) in aqueous trifluoracetic acid (TFA) 0.1% (v/v)-on a C-8 column. Although the range of mobile phase compositions investigated was quite narrow, the retention factor of this relatively small polypeptide (N/OFQ is a heptadecapeptide) has been found to change by more than 400%. In these conditions, gradient operation resulted thus to be the optimum approach for non-linear elution. As the available amount of N/OFQ was extremely reduced (only a few milligrams), the adsorption isotherms of the peptide, at the different mobile phase compositions examined, have been measured through the so-called inverse method (IM) on a 5 cm long column. The adsorption data at different mobile phase compositions have been fitted to several models of adsorption. The dependence of the isotherm parameters on the mobile phase composition was modeled by using the linear solvent strength (LSS) model and a generalized Langmuir isotherm that includes the mobile phase composition dependence. The overloaded gradient separation of N/OFQ has been modeled by numerically solving the equilibrium-dispersive (ED) model of chromatography under a selected gradient elution mode, on the basis of the previously determined generalized Langmuir isotherm. The agreement between theoretical calculations and experimental overloaded band profiles appeared reasonably accurate.     

前沿分析法研究对-羟基苯甲酸印迹整体柱的热力学行为

采用矩形前沿分析法对原位聚合的对-羟基苯甲酸印迹整体柱的热力学吸附等温线进行了测定. 印迹整体柱的吸附等温线是分别以乙腈、甲醇、 四氢呋喃和含有体积分数分别为1%, 3%, 5%和7%乙酸的乙腈为流动相以及在以甲醇为流动相时柱温分别为20, 40和50 ℃的条件下测定的. 吸附等温线表明, 印迹整体柱对模板分子的吸附能力比其结构类似物(邻-羟基苯甲酸)的吸附能力强. 用双Langmuir方程对不同条件下得到的实验数据进行拟合, 得到模板分子和邻-羟基苯甲酸在印迹整体柱各种吸附位点上的饱和吸附量和键合常数, 结果表明, 流动相中乙酸含量、有机溶剂的性质和柱温对模板分子容量因子的影响比对邻-羟基苯甲酸的大, 造成印迹聚合物的选择性随条件的变化而发生了明显的改变.