Marianna Kele

Repeatability and reproducibility of retention data and band profiles on six batches of monolithic columns

Chromatographic data were acquired for eight different mixtures, under five different sets of experimental conditions, for a total of 30 neutral, acidic and basic test compounds, on a series of six Chromolith Performance columns from Merck. These columns are made of a C18 chemically bonded silica monolith. Each column belonged to a different production batch, so the data reported here characterize their batch-to-batch reproducibility. The parameters studied in this work were the retention times, the retention and separation factors, the hydrophobic and the steric selectivities, the column efficiencies, and the tailing factors for all 30 compounds.

Study of the thermodynamics and mass transfer kinetics of two enantiomers on a polymeric imprinted stationary phase

The adsorption isotherms of d- and l-phenylalanine anilide (PA) on an l-phenylalanine anilide imprinted stationary phase have been determined using staircase frontal analysis. An aqueous buffer–organic solvent mixture has been used as mobile phase. The measurements were done at temperatures of 40, 50, 60 and 70°C for sample concentrations ranging between 5·10−4 to 1 g/l. It was found that the adsorption data fit well to both the Freundlich and the Bi-Langmuir isotherm models. Examination of the best values of the numerical coefficients of the Bi-Langmuir model shows that the site class representing the binding sites with the highest binding energy exhibits a very low saturation capacity for the non-imprinted enantiomer, indicating a high selectivity for the imprinted l-enantiomer. The low energy site class also shows some selectivity for the l-enantiomer. Mass transfer rate coefficients were obtained for each single breakthrough curve by using the transport model of chromatography. It was found that the mass transfer coefficient of l-PA increases very rapidly with the sample concentration while there is only a slight increase for the other enantiomer.

Repeatability and reproducibility of retention data and band profiles on reversed-phase liquid chromatography columns: I. Experimental protocol

Abstract A procedure is described for the determination of the short-term and long-term repeatabilities and of the column-to-column and lot-to-lot reproducibilities of the retention and profile characteristics of the peaks obtained with a number of different reversed-phase liquid chromatography (RPLC) C18-bonded silica columns of several commercial brands. Data characterizing the retention, the steric selectivity, the hydrogen bonding capacity, the hydrophobic interaction selectivity, the column efficiency and the peak asymmetry will be acquired for all the probe compounds. These include 30 neutral, acidic and basic compounds distributed into five groups to be eluted under as many different sets of chromatographic conditions. The data will be obtained using an HP 1100 liquid chromatograph. The compounds and experimental conditions selected were similar to those previously used by different authors for comparison purposes. Careful attention will be paid to minimization of external error contributions by adhering to a strict operational procedure. The precision expected will be high because preliminary results gave standard deviations of around 0.04% for the separation factors, below 0.15% for the retention times and around 1% for the column efficiency in short-term repeatability experiments performed with one column.

Influence of Thermal Annealing on the Thermodynamic and Mass-Transfer Kinetic Properties of d- and l-Phenylalanine Anilide on Imprinted Polymeric Stationary Phases.

An investigation of the material, chromatographic, thermodynamic, and kinetic properties of thermally treated (i.e., annealed) polymeric stationary phases imprinted with l-phenylalanine anilide (l-PA) was carried out. The imprinting procedure of the solid phase used in this study was the same as for the untreated imprinted stationary phase studied previously. However, after polymerization, these new stationary phases were treated at elevated temperatures (50, 120, 140, and 160 °C) for 24 h. The treatment at 120 and 140 °C led to a larger decrease in the retention of l-PA than that of d-PA. The polymer treated at 160 °C could no longer resolve the d,l-PA racemate. The heat treatments were accompanied by a decrease in swelling and an increase in density causing an increase in the density of the remaining active sites. The polymer treated at 120 °C was chosen for classical frontal analysis. The adsorption isotherms and the mass-transfer rate coefficients of d- and l-PA were derived from the experimental breakthrough curves. This study was carried out in the same temperature and concentration ranges as the previous one. A comparative discussion of the properties of the two polymeric molecular imprinted stationary phases is presented. The thermal treatment increases the saturation capacity of the packing material by one-third to one-half, reduces markedly the separation factor of the two enantiomers, and slightly accelerates their mass-transfer kinetics. There seems to be no interactions on the annealed polymer between the selective l-PA imprinted sites and the d-PA molecules.

Influence of the pH on the behavior of an imprinted polymeric stationary phase — supporting evidence for a binding site model

The equilibrium isotherms of the two enantiomers of phenylalanine anilide (PA) were measured by conventional frontal analysis at three different pH on a thermally-treated imprinted stationary phase selective for the L enantiomer. The first of these pH (buffer pH=3.0, pH(app)=4.0) is well below the apparent pKa (6.4) of the two solutes, the second (buffer pH=5.8, pH(app)=7.0) slightly below this pKa, and the third (buffer pH=7.0, pH(app)=8.3) well above it. The experimental data were fitted to several isotherm models. The best estimates of the parameters of these models are reported and discussed. The corresponding isotherms are compared with the experimental ones. The contributions of the enantioselective and nonselective interactions could be separated. The results obtained show that the saturation capacity is always smaller for D-PA than for L-PA, the template. The analytical separation is best at pH=3.0 because of a good separation factor (2.82) and short retention times. A good compromise between the resolution and the saturation capacity is obtained at pH=5.8, for which the best preparative separation is found. Both analytical and preparative results are poor at pH=7.0 because the separation factor is low (1.32). At this pH, the isotherm remains nearly linear in the whole concentration range accessible to measurements. The number of nonselective sites increases with increasing mobile phase pH slightly faster than the number of selective sites, suggesting different pKa ranges for the two type of sites. Moreover, the binding energy and the homogeneity of the selective sites decreases with increasing pH. These results agree with a binding site model involving more than one carboxylic acid group, providing charge complementarity and hydrogen bond donors for binding of L-PA.

Repeatability and reproducibility of retention data and band profiles on reversed-phase liquid chromatography columns. III. Results obtained with Kromasil C18 columns

The reproducibility of the retention data and the band profiles was investigated with Kromasil C18 columns (silica-based monomeric type reversed-phase packing material). High precision data were obtained and statistically compared among five columns from the same batch (column-to-column reproducibility) and six columns, one from each of six different batches (batch-to-batch reproducibility). These data were acquired under five different sets of chromatographic conditions, for a group of 30 neutral, acidic and basic compounds selected as probes following an experimental protocol previously described. Data characterizing the retention time, the retention factor, the separation factor, the column efficiency and the peak asymmetry for the different probe compounds are reported. Factors describing the silica surface interaction with the selected probe compounds, such as the hydrophobic interaction selectivity, the steric selectivity, and the separation factors of basic compounds at different pH values were also determined. The influence of the underlying silica on these data and correlations between the chromatographic and physico-chemical properties of the different batches are discussed.

Identification of the factors that influence the reproducibility of chromatographic retention data.

Principal component analysis was used to identify the parameters that influence the column-to-column and batch-to-batch reproducibility of retention times and retention factors measured on Symmetry C18, Kromasil C18, Luna C18 (2) and Vydac RP C18, all reversed-phase silica columns. We devised a procedure that allows the determination of the differences in column volume and packing density between two columns, provided that these columns are packed with identical stationary phases (i.e., phases that originate from the same batch). Principal component analysis of the retention times confirmed that the column-to-column variations of the column volume and the total porosity of the bed are the factors that influence the reproducibility of the retention times, the column volume being the major factor. For the fluctuations of the retention factors, the column phase ratios (or the bed porosities) and some specific, secondary retention mechanisms are responsible. All the C18 columns investigated proved to behave in a very similar fashion. Two principal components were always sufficient to characterize the variations of either the retention times or the retention factors.

Optimization of the high-performance liquid chromatographic separation of fullerenes using 1-methylnaphthalene as the mobile phase on a tetraphenylporphyrin-silica stationary phase

Abstract The separation of C60 and C70 was studied on four different stationary phases. The performance of three commercial products, Buckyclutchers, PYE and PBB and of a new phase, immobilized tetraphenylporphyrin on silica, were investigated. The influence of the surface chemistry on this separation is discussed. On all four phases, good resolution is obtained with pure toluene as the mobile phase. With some of them, this result is obtained with stronger eluents, mixtures of toluene and 1-methylnaphthalene. This allows the injection of much more concentrated and larger samples than on conventional systems and gives considerably improved production rates of purified compounds. The optimization of the temperature, the flow velocity, and the mobile phase composition for the separation of C60 and C70 on the tetraphenylporphyrin bonded phase is discussed. The overloading of this stationary phase was studied empirically in mixtures of toluene and 1-methylnaphthalene. The advantage and properties of this latter solvent are illustrated. Fractions of C60 with 98.5% purity and C70 with a purity better than 99%% were prepared with near total recovery yield from 5 mg samples.

Comparison of the repeatability of quantitative data measured in high-performance liquid chromatography with UV and atmospheric pressure chemical ionization mass spectrometric detection

A detailed comparison of the repeatability of the retention times, the peak efficiencies and the peak areas of a dozen probe compounds achieved in HPLC, using either HPLC-UV or HPLC-MS for detection purpose, is reported. Three groups of conventional analytes, each one separated under a different set of experimental conditions, were selected for this study. Most of the compounds are basic, the other ones being neutral. The repeatabilities of the retention times do not exhibit any influence of the mode of detection. However, the repeatabilities of the peak areas and the column efficiencies are generally (although not always) better in HPLC-UV than that in HPLC-MS. On average, the precision for the UV peak area detection was 2.5% versus 6.8% for MS detection. Experimental results show that the response factor of the UV detector is more constant than that of the MS detector, probably because the HPLC flow-rate was sufficiently stable. The results obtained in the different tests are discussed.