Adsorption mechanism in RPLC. Effect of the nature of the organic modifier

The adsorption isotherms of phenol and caffeine were acquired by frontal analysis on two different adsorbents, Kromasil-C18 and Discovery-C18, with two different mobile phases, aqueous solutions of methanol (MeOH/H2O = 40/60 and 30/70, v/v) and aqueous solutions of acetonitrile (MeCN/H2O = 30/70 and 20/80, v/v). The adsorption isotherms are always strictly convex upward in methanol/water solutions. The calculations of the adsorption energy distribution confirm that the adsorption data for phenol are best modeled with the bi-Langmuir and the tri-Langmuir isotherm models for Kromasil-C18 and Discovery-C18, respectively. Because its molecule is larger and excluded from the deepest sites buried in the bonded layer, the adsorption data of caffeine follow bi-Langmuir isotherm model behavior on both adsorbents. In contrast, with acetonitrile/water solutions, the adsorption data of both phenol and caffeine deviate far less from linear behavior. They were best modeled by the sum of a Langmuir and a BET isotherm models. The Langmuir term represents the adsorption of the analyte on the high-energy sites located within the C18 layers and the BET term its adsorption on the low-energy sites and its accumulation in an adsorbed multilayer system of acetonitrile on the bonded alkyl chains. The formation of a complex adsorbed phase containing up to four layers of acetonitrile (with a thickness of 3.4 A each) was confirmed by the excess adsorption isotherm data measured for acetonitrile on Discovery-C18. A simple interpretation of this change in the isotherm curvature at high concentrations when methanol is replaced with acetonitrile as the organic modifier is proposed, based on the structure of the interface between the C18 chains and the bulk mobile phase. This new model accounts for all the experimental observations.     

Heterogeneity of the adsorption mechanism of low molecular weight compounds in reversed-phase liquid chromatography

The retention mechanism in RPLC mode was investigated based on the acquisition of adsorption isotherm data by frontal analysis measurements and their modeling. This work is a review of the results of four years of adsorption data measurements. The data were acquired on a wide variety of brands of C18-silica columns (from Akzo Nobel, Bishoff, Hypersil, Merck, Phenomenex, Supelco, Vydac, and Waters) with several low molecular weight compounds such as phenol (94 g/mol), caffeine (194 g/mol), tryptophan (204 g/mol), sodium 2-naphthalenesulfonate (235 g/mol), and propranololium chloride (295 g/mol). The mobile phase was a mixture of methanol and water at variable composition. The adsorption isotherms were all convex upward (langmuirian), and the degree of heterogeneity of the adsorption system was determined from the calculation of the adsorption energy distribution using the expectation-maximization method. The adsorption isotherm parameters (number of types of adsorption sites, surface concentration of each type of site, and difference between the adsorption energies E(i) - E(j) on sites i and j), obtained from the mathematical fit of the adsorption data to the appropriate multi-Langmuir adsorption isotherm model, were analyzed and compared. The results allow the drawing of general conclusions regarding the relationships between the size of the analyte and the adsorption properties (saturation capacities, adsorption energies) characterizing the retention mechanism in RPLC mode for neutral, anionic, and cationic compounds.     

Effect of the surface heterogeneity of the stationary phase on the range of concentrations for linear chromatography

The range of sample sizes within which linear chromatographic behavior is achieved in a column depends on the surface heterogeneity of the RPLC adsorbents. Two widely different commercial adsorbents were tested, the end-capped XTerra-C18 and the non-end-capped Resolve-C18. Adsorption isotherm data of caffeine were acquired by frontal analysis. These data were modeled and used to calculate the adsorption energy distribution (AED). This double analysis informs on the degree of surface heterogeneity. The best adsorption isotherm models are the bi-Langmuir and the tetra-Langmuir isotherms for XTerra and Resolve, respectively. Their respective AEDs are bimodal and quadrimodal distributions. This interpretation of the results and the actual presence of a low density of high-energy adsorption sites on Resolve-C18 were validated by measuring the dependence of the peak retention times on the size of caffeine samples (20-microL volume, concentrations 10, 1, 0.1, 1 x 10(-2), 1 x 10(-3), 1 x 10(-4), and 1 x 10(-5) g/L). The experimental chromatograms agree closely with the band profiles calculated from the best isotherms. On Resolve-C18, the retention time decreases by 40% when the sample concentration is increased from 1 x 10(-5) to 10 g/L. The decrease is only 10% for Xterra-C18 under the same conditions. The upper limit for linear behavior is 1 x 10(-4) g/L for the former adsorbent and 0.01 g/L for the latter. The presence of a few high-energy adsorption sites on Resolve-C18, with an adsorption energy 20 kJ/mol larger than that of the low-energy sites while the same difference on Xterra is only 5 kJ/mol, explains this difference. The existence of adsorption sites with a very high energy for certain compounds affects the reproducibility of their retention times and a rapid loss of efficiency in a sample size range within which linear behavior is incorrectly anticipated.     

Adsorption mechanisms and effect of temperature in reversed-phase liquid chromatography. meaning of the classical Van't Hoff plot in chromatography

The effect of temperature on the adsorption and retention behaviors of a low molecular weight compound (phenol) on a C18-bonded silica column (C18-Sunfire, Waters) from aqueous solutions of methanol (20%) or acetonitrile (15%) was investigated. The results of the measurements were interpreted successively on the basis of the linear (i.e., overall retention factors) and the nonlinear (i.e., adsorption isotherms, surface heterogeneity, saturation capacities, and equilibrium constants) chromatographic methods. The confrontation of these two approaches confirmed the impossibility of a sound physical interpretation of the conventional Van't Hoff plot. The classical linear chromatography theory assumes that retention is determined by the equilibrium thermodynamics of analytes between a homogeneous stationary phase and a homogeneous mobile phase (although there may be two or several types of interactions). From values of the experimental retention factors in a temperature interval and estimates of the activity coefficients at infinite dilution in the same temperature interval provided by the UNIFAC group contribution method, evidence is provided that such a retention model cannot hold. The classical Van't Hoff plot appears meaningless and its linear behavior a mere accident. Results from nonlinear chromatography confirm these conclusions and provide explanations. The retention factors seem to fulfill the Van't Hoff equation, not the Henry constants corresponding to the different types of adsorption sites. The saturation capacities and the adsorption energies are clearly temperature dependent. The temperature dependence of these characteristics of the different assorption sites are different in aqueous methanol and acetonitrile solutions.     

咖啡因分子印迹酚醛吸附树脂的分子识别特性

合成了咖啡因印迹酚醛吸附树脂,采用静态和动态吸附法考察了分子识别特性。Scatchard分析表明,咖啡因印迹酚醛吸附树脂具有特定的高亲和性结合位点,其离解常数和最大吸附量为Kd1=26.8 mg/L,Qmax1=13.7 mg/g,低亲和性结合位点的离解常数和最大吸附量为Kd2=114 mg/L,Qmax2=28.5 mg/g。在动态条件下,浓度为400 mg/L的咖啡因溶液以3BV/h的流速流经树脂床时,在第26BV出现穿透;用1 mol/L的盐酸以1.5 BV/h的流速进行洗脱,在4BV的洗脱率达到97.5%。     

General HETP equation for the study of mass-transfer mechanisms in RPLC

Classical HETP equations including the Van Deemter and the Knox equations, are semiempirical, approximate equations that provide apparent mass-transfer coefficients with little sound physical justifications. The conventional A and B coefficients are revisited, the former through the use of the fundamental theory of eddy diffusion due to Giddings, the latter by taking into account the intraparticle diffusion (pore and surface diffusion). Our work confirms that eddy diffusion originated from three different sources in RPLC: trans-channel, short-range interchannel, and long-range interchannel velocity biases. Accordingly, the eddy diffusion term is given by the ratio of two third-degree polynomials. Finally, the C term is the sum of two terms corresponding to the resistance to mass transfer due to diffusion through the external stationary film of liquid phase surrounding the silica particles and to the classical resistances to mass transfer due to diffusion through the silica particles. It is easily related to the physical characteristics of the phenomena involved. Experimental HETP data were derived from moment analysis for phenol on a C(18)-Sunfire column, with a mixture of acetonitrile and water as the mobile phase (15/85, v/v). The linear interstitial velocity ranged between 0.027 cm/s and 4.7 mL/min, and six temperature (21, 36, 45, 55, 67, and 77 degrees C) were applied successively. The HETP equation obtained was tested to study the mass-transfer mechanism. An excellent agreement was found between the experimental and theoretical HETP. The model allows the precise calculation of the activation energy for surface diffusion (E(S) = 31.3 kJ/mol) and the coefficient beta that relates the restriction energy for molecular diffusion on the C(18)-bonded surface to the isosteric heat of adsorption Q(st) (beta = 0.80).     

Overloading study of bases using polymeric RP-HPLC columns as an aid to rationalization of overloading on silica-ODS phases

The separation of ionized bases by reversed-phase liquid chromatography with alkyl silica columns often leads to severely tailed bands that are highly detrimental. Band shape and its dependence on sample mass are notably different when mobile-phase pH is changed, and this behavior has not been previously explained. Ionized silanols present in the stationary phase have been credited with a role in determining peak shape. In the present study, separations on two different polymer columns were compared with those previously obtained on alkyl silica phases. Because silanols are absent from polymer columns, this comparison enabled us to assess the role of silanols in separations on alkyl silica phases and to offer an explanation of why band shape changes with sample size and mobile-phase pH for both polymer and silica-based phases.     

Analysis of films prepared by plasma polymerization of acetylene in a D.C. magnetron

The complex dielectric constant of amorphous hydrogenated carbon films was determined by reflection and transmission measurements in the photon energy range from 0.5 to 6.54 eV. The results, determined by the application of a sum rule, showed the presence of 0.47 π-bonded electrons per carbon atom in the films. Electron energy loss spectroscopy was carried out which confirmed the presence of π-bonded electrons and gave a value of the combined σ and π plasmon peak at 23 eV, too low to be consistent with the presence of a distinct phase containing diamond microcrystals or random tetrahedral bonding. A model is presented in which threefold coordinated sheets are linked by tetrahedrally bonded carbon atoms.     

A (-)-menthyl bonded silica phase for chiral separations: synthesis and solid state NMR characterization

A new (-)-menthyl bonded silica phase has been prepared by hydrosilation of a hydride silica intermediate. The hydride silica intermediate was synthesized by the reaction of a monoalkoxysilane (CH(3))(2)SiH(OEt) with silica gel, yielding a relatively high surface coverage (4.4 μmol/m(2)) of SiH groups. This intermediate was then used successfully in the preparation of a monomeric (-)-menthyl bonded silica phase. The bonded phase produced has been used for the chromatographic separation of enantiomers in a reversed phase mode (chiral separations). Solid state (13)C and (29)Si CP-MAS NMR spectroscopy and DRIFT spectroscopy provides valuable information on the structure of the different surface species formed on silica after modification. The surface coverage of the hydride silica intermediate and of the final bonded silica phase produced are also determined. It is found that this modification procedure can exclusively produce a monomeric coverage of SiH groups on the silica surface and can further produce a final monomeric bonded organic silica phase for the separation of enantiomers.     

The retention of heterocyclics by siliceous frameworks Part I The role of the heterocyclic

Flow microcalorimetry has been used to probe acid-base interactions between five-membered-ring heterocyclics and thermally pre-treated, porous silica. The adsorbates (1-methylpyrrolidin-2-one, pyridine, pyrrolidine, pyrrole, 2-methylthiophene, 2-octyl-4-isothiazolin-3-one, 4,5-dichloro-2-octyl-4-isothiazolin-3-one and 2-cyclopentenone,) varied in basicity, polarity and -character. The amounts of the adsorbates retained by the silica were determined, along with enthalpy of adsorption (ranging from –5.5 kJ mol^–1 to –57.8 kJ mol^–1) and enthalpy of desorption (ranging from 5.6 kJ mol^–1 to 26.1 kJ mol^–1). For the majority of the adsorbates the enthalpy of adsorption is consistent with hydrogen bonding to isolated silanols. Although increasing basicity enhanced the adsorption enthalpy and hence the strength of associations, desorption was inhibited when a carbonyl, or unsaturated carbonyl, group was adjacent to the active basic centre. Bulky electron-withdrawing agents (chlorine atoms) substituted at the double bond of the unsaturated carbonyl reduced the adsorption considerably. This was attributed to steric hinderance restricting the proximity of the basic groups with the active silanol sites.     

Probing topography and tailing for commercial stationary phases using AFM, FT-IR, and HPLC

Atomic force microscopy was used to study surface characteristics of three chromatographic silica products: Agilent Zorbax SB300, Waters Symmetry 300, and Merck Chromolith. Each is modified with a monomeric C18 monolayer. Both topographic and adhesive force measurements were made for each product. Topographical images revealed that all three materials are as smooth as glass on the scale of 100 nm and below. Adhesive forces for all three materials were much lower and much more uniform than for chemically modified fused silica. FT-IR spectra for all three materials showed a low abundance of isolated silanols, thus explaining the low adhesion. Chromatograms of a cationic dye, 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), ranging in concentration from 1 to 300 microM were obtained for each column. All three materials exhibited classic nonlinear tailing; the Zorbax exhibited fronting as well. Chromatographic simulations were performed for the Symmetry and Chromolith products to determine the number of strong adsorption sites. The AFM, FT-IR, and HPLC were all consistent in indicating that the Chromolith material had half as many strong adsorption sites as the Symmetry material. The Zorbax material exhibited a number of isolated silanols that was comparable to the other materials, yet its adhesive force suggested a less adsorptive material, and its chromatographic performance suggested a more adsorptive material. Its topography is discussed as a possible reason for its anomalous chromatographic behavior.     

Rationalization of retention and overloading behavior of basic compounds in reversed-phase HPLC using low ionic strength buffers suitable for mass spectrometric detection

The retention and overloading behavior of some basic (and acidic) compounds has been studied on different RP-HPLC columns in buffers of varying ionic strength. Anomalous retention patterns of acids and bases were found on one phase in low-pH, volatile buffers such as formic acid, favored for mass spectrometric analysis. Unusual retention compared to that in higher ionic strength phosphate buffers is attributed to the presence of positively charged sites existing on this phase at low pH. Overloading of bases as well as acids is shown to be a function of mobile-phase ionic strength. This result is a logical consequence of previous suggestions that mutual repulsion of ions held on the hydrophobic surface of the stationary phase, rather than overload of silanols, is largely responsible for overloading on pure silica RP columns. Thus, overloading occurs much more readily in low ionic strength formic acid buffers. Appreciable loss of efficiency can occur in such buffers when only 50 ng of some bases is analyzed on a standard-sized column.     

Separation of peptides from myoglobin enzymatic digests by RPLC. Influence of the mobile-phase composition and the pressure on the retention and separation

The influence of the mobile-phase composition and the pressure on the chromatographic separation of the peptides from the enzymatic digest of myoglobin was studied under linear conditions. The retention behavior of these tryptic peptides was measured under isocratic conditions with different mobile-phase compositions, ranging from 9 to 28% (v/v) acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid. The effect of the pressure was studied by analyzing the separation of the tryptic peptides under different average column pressures between 14 and 220 bar, at 13, 20, and 26% (v/v) acetonitrile. The differences between the partial molar volumes of these peptides in the stationary and mobile phases were derived from these results. All the measurements were performed on a 10-cm-long C18-bonded, end-capped monolithic column. The results obtained illustrate the highly complicated behavior of the complex peptide mixtures afforded by tryptic digestion. The capacity factors of the analyzed peptides do not depend linearly on the acetonitrile concentration but follow exactly a quadratic relationship. The adsorption changes of partial molar volumes are in good agreement with other literature data. The consequences of the influence of the average column pressure (hence of the flow rate) on the column phase ratio and on the retention factors of the peptides are discussed. The retention pattern of the complex mixture is affected by both the mobile-phase composition and the pressure, and the resolution of certain peptide pairs is so much affected by the pressure that inversions in the elution order of some pairs are observed.     

Adsorptive removal of phenol from aqueous phase by using a porous acrylic ester polymer

The removal of phenol from aqueous solution was examined by using a porous acrylic ester polymer (Amberlite XAD-7) as an adsorbent. Favorable phenol adsorption was observed at acidic solution pH and further increase of solution pH results in a marked decrease of adsorption capacity, and the coexisting inorganic salt NaCl exerts positive effect on the adsorption process. Adsorption isotherms of phenol were linearly correlated and found to be well represented by either the Langmuir or Freundlich isotherm model. Thermodynamic parameters such as changes in the enthalpy (DeltaH), entropy (DeltaS) and free energy (DeltaG) indicate that phenol adsorption onto XAD-7 is an exothermic and spontaneous process in nature, and lower ambient temperature results in more favorable adsorption. Kinetic experiments at different initial solute concentrations were investigated and the pseudo-second-order kinetic model was successfully represented the kinetic data. Additionally, the column adsorption result showed that a complete removal of phenol from aqueous phase can be achieved by XAD-7 beads and the exhausted adsorbent was amenable to an entire regeneration by using ethanol as the regenerant. More interestingly, relatively more volume of hot water in place of ethanol can also achieve a similar result for repeated use of the adsorbent.     

Micromachined GC columns for fast separation of organophosphonate and organosulfur compounds

This article demonstrates how to prepare microfabricated columns (microcolumns) for organophosphonate and organosulfur compound separation that rival the performance of commercial capillary columns. Approximately 16,500 theoretical plates were generated with a 3 m long OV-5-coated microcolumn with a 0.25 microm phase thickness using helium as the carrier gas at 20 cm/s. Key to the advance was the development of deactivation procedures appropriate for silicon microcolumns with Pyrex tops. Active sites in a silicon-Pyrex microcolumn cause peak tailing and unwanted adsorption. Experimentally, we found that organosilicon hydride deactivation lowers adsorption activity in microcolumns more than silazane and silane treatments. But without further treatment, the phosphonate peaks continue to tail after the coating process. We found that heat treatment with pinacolyl methylphosphonic acid (PMP) eliminated the phosphonate peak tailing. In contrast, conventional resilylation employing N, O-bis(trimethylsilyl)acetamide, hexamethyldisilazane, and 1-(trimethylsilyl)imidazole does not eliminate peak tailing. Column activity tests show that the PMP treatment also improves the peaks for 2,6-dimethyl aniline, 1-octanol, and 1-decanol implying a decrease in the column's hydrogen bonding sites with the PMP treatment. FT-IR analysis shows that exposure to PMP forms a bond to the stationary phase that deactivates the active sites responsible for organophosphonate peak tailing.     

In situ adsorption studies at silica/solution interfaces by attenuated total internal reflection fourier transform infrared spectroscopy: examination of adsorption models in normal-phase liquid chromatography

ATR-FT-IR spectroscopy was employed to the study the adsorption of ethyl acetate and 2-propanol to the surface of thin silica sol-gel films in contact with n-heptane solutions. In situ vibrational spectra of silica-adsorbed species provided information regarding the mechanisms of solute retention and elution in normal-phase chromatography. Previous normal-phase chromatographic studies of ethyl acetate adsorption revealed nonlinear isotherms which were explained by both bilayer and adsorbate delocalization models. Infrared spectra of ethyl acetate at the silica surface versus concentration showed that nonlinear adsorption can be attributed to site heterogeneity, where adsorption to free silanols and surface-adsorbed water can be distinguished. Least-squares modeling of the data produced resolved spectra for the two sites and adsorption equilibrium constants that differed by about an order of magnitude. Adsorption of 2-propanol was best modeled by a single Langmuir isotherm showing no significant difference in adsorption energy for the two sites; 2-propanol was shown to easily displace ethyl acetate from the silica surface. Ethyl acetate could also displace 2-propanol from the silica, and least-squares modeling again revealed two-adsorbed-component spectra for ethyl acetate that were indistinguishable from spectra obtained when ethyl acetate adsorbed directly onto the surface.     

Effect of grooves on adsorption of RGD tripeptide onto rutile TiO2 (110) surface

Molecular dynamics (MD) simulations have been performed to investigate the adsorption mechanism of Arg-Gly-Asp (RGD) tripeptide onto perfect and grooved rutile TiO₂ (110) surfaces, respectively. The simulation results suggest that RGD tripeptide can strongly adsorb onto TiO₂ surface through specified Ti coordination sites. Analysis of adsorption energy, mean-squared displacements and radial distribution functions indicates that the adsorption of RGD onto grooved surface is more stable and rapid than that onto the perfect surface, with the adsorption energy around −331.59 kcal/mol. And among the chosen groove surfaces, adsorption energies, adsorption speeds and adsorption depths of RGD onto the surfaces increase evidently with the extension of groove dimensions. For both perfect and grooved surfaces, once bonded to the surfaces by interactions of carboxyl groups or carbonyl groups with nearby surface Ti atoms, RGD tripeptides show a reasonable propensity to remain there and undergo relatively limited hinge-bending motions.     

Retention of ionizable compounds in reversed-phase liquid chromatography. Effect of the ionic strength of the mobile phase and the nature of the salts used on the overloading behavior

The retention mechanism of the protonated cation in propranolol chloride on C18-Xterra was investigated using mobile phases of various compositions. Accurate adsorption data were measured by frontal analysis, with a mixture of methanol and water (25% methanol), with no salt, as the mobile phase. The experimental isotherm has at least two inflection points, at concentrations of about 0.2 and 6.0 g/L, respectively. This precludes the modeling of these data with a simple convex-upward isotherm (e.g., Langmuir). The adsorption energy distribution or relationship between the number of sites on the adsorbent surface and the energy of adsorption on these sites was calculated by assuming Moreau isotherm behavior (S-shaped isotherm). This model has never been applied to describe the surface heterogeneity of any RPLC adsorbent. The calculation converged toward a bimodal energy distribution. Accordingly, the bi-Moreau model is the simplest theoretical model accounting for the adsorption data of propranolol from a mobile phase without salt. The complex-overloaded band profiles of propranolol measured in the presence of increasing concentrations of a supporting salt (KCl) in the mobile phase demonstrate that the same isotherm model applies also under these conditions, as was merely assumed in a previous work. The elution band profiles of propranolol calculated with the bi-Moreau isotherm model for solutions of salts of different natures (CaCl2, CsCl, Na2SO4) in the same mobile phase agree very well with the experimental band profiles.     

Single-walled carbon nanotubes used as stationary phase in GC

Single-walled carbon nanotubes (SWNTs) have high surface area, high adsorption ability, and nanoscale interactions. In this study, capillary columns including SWNTs, ionic liquid (IL), and IL + SWNTs for GC were prepared. The separation results showed that SWNTs possessed a wide selectivity toward alkanes, alcohols, aromatic compounds, and ketones, and a SWNT capillary column was a very useful GC column for the separation of gas samples. Coating the IL stationary phase on the SWNT capillary column, the SWNTs were able to improve chromatographic characteristic of ionic liquid. Comparing the IL coated on three graphite carbon black capillary columns, which were prepared by dynamic coating, static coating, and chemical bonding the Carbopack C with on SWNTs capillary column, the capacity factors were much higher on the SWNT column. The SEM showed that SWNTs could be bonded to the inner surface of capillary tubing, and most of them were linked end-to-end to form a layer of network structure of skeletons resulting in a high surface area, which increased the interactions between stationary phase and analytes. This is the first single-wall carbon nanotubes bonded to the fused-silica capillary tubing. In the first approach, SWNTs assist ionic liquid with enhanced chromatographic characteristic in GC. This work indicates that SWNTs make it possible to extend the application range on the newly prepared chromatographic stationary phases for GC.     

棉秸秆活性炭吸附水溶性有机污染物研究

以棉花秸秆为原材料,采用"炭化-活化"工艺制备了高活性活性炭。研究了活性炭对苯酚、苯胺和苯甲酸等3种芳香族有机污染物的吸附过程动力学及热力学;测定了不同温度下该活性炭对3种污染物的吸附等温线。研究结果表明棉秸秆活性炭对苯酚、苯胺和苯甲酸的吸附过程均符合Lagergren二级吸附动力学方程;表观吸附活化能分别为Ea(苯酚)=15.91kJ/mol、Ea(苯甲酸)=12.56kJ/mol、Ea(苯胺)=11.16kJ/mol;吸附过程为自发的放热熵减过程;吸附等温模型符合Freundlich等温式;棉秸秆活性炭对苯酚、苯胺和苯甲酸的静态饱和吸附容量分别为450、321和298mg/g。棉秸秆活性炭制备简便、成本低廉,对水溶性有机污染物去除效果较好,可用于芳香族有机污染物的吸附治理。