Efecto de la solución amortiguadora de pH en la selectividad cromatográfica de compuestos ácido-base

Es bien sabido que el pH de la fase móvil es una variable fundamental en la separación de sustancias ionizables en cromatografía de líquidos, ya que de él depende el grado de ionización de los analitos presentes en la mezcla problema. En el desarrollo de métodos analíticos se requiere pues, ejercer un control eficiente sobre el pH mediante la elección de un tampón adecuado. En cromatografía de líquidos en fase inversa, suelen usarse solucio- nes hidro-orgánicas tamponadas como fases móviles. Habitualmente éstas suelen prepararse añadiendo el volumen adecuado de un disolvente orgánico, típicamente acetonitrilo o metanol, sobre una solución acuosa tamponada. Sin embargo, fases móviles del mismo contenido en modificador orgánico y preparadas a partir de soluciones acuosas del mismo pH pero que contienen ácidos distintos como reguladores del pH, pueden presentar valores de pH significativamente diferentes

Buffer Considerations for LC and LC-MS

In this article, the buffer capacity concept is revisited, particularly concerning its behavior in hydroorganic mobile phases. The buffer capacity of a polyprotic acid, or a mixture of monoprotic acids, depends upon the concentration of each weak acid-conjugate base pair, and the pH of its maximum value mainly fits to the acid-base pKA, but it is shifted to a certain degree according to the ionic strength of the buffered solution. Consequently, when an organic solvent is added to an aqueous buffer to prepare a particular mobile phase, the buffer capacity of the hydroorganic mixture is reduced due to the dilution effect, and the maximum buffer capacity is shifted to lower or higher pH values according to the nature of the buffering acid-base pair

Volume and composition of semi-adsorbed stationary phases in hydrophilic interaction liquid chromatography. Comparison of water adsorption in common stationary phases and eluents.

Pycnometric and homologous series retention methods are used to determine the volume and mean composition of the water-rich layers partially adsorbed on the surface of several hydrophilic interaction liquid chromatography (HILIC) column fillings with acetonitrile-water and methanol-water as eluents. The findings obtained in this work confirm earlier studies using direct methods for measuring the stationary phase water content performed by Jandera's and Irgum's research groups. Water is preferentially adsorbed on the surface of the HILIC bonded phase in hydroorganic eluents containing more than 40% acetonitrile or 70% methanol, and a gradient of several water-rich transition layers between the polar bonded phase and the poorly polar bulk mobile phase is formed. These layers of reduced mobility act as HILIC stationary phases, retaining polar solutes. The volume of these layers and concentration of adsorbed water is much larger for acetonitrile-water than for methanol-water mobile phases. In hydroorganic eluents with less than 20-30% acetonitrile or 40% methanol the amount of preferentially adsorbed water is very small, and the observed retention behavior is close to the one in reversed-phase liquid chromatography (RPLC). In eluents with intermediate acetonitrile-water or methanol-water compositions a mixed HILIC-RPLC behavior is presented. Comparison of several HILIC columns shows that the highest water enrichment in the HILIC retention region for acetonitrile-water mobile phases is observed for zwitterionic and aminopropyl bonded phases, followed in minor grade for diol and polyvinyl alcohol functionalizations. Pentafluorophenyl bonded phase, usually considered a HILIC column, does not show significant water adsorption, nor HILIC retention

Chasing the elusive hold-up time from an LFER approach

A homologous series approach derived from the Abraham's solvation model was developed for the determination of hold-up times. Firstly, it was tested from reversed-phase liquid chromatography data obtained in the literature involving several series of homologues, followed by its application in a polymeric zwitterionic HILIC column using two different homologous series (n-alkyl benzenes and n-alkyl phenones). Acetonitrile and methanol were selected as organic modifiers in a composition range between 80% and 100% in volume. Results obtained for both series were consistent, and hold-up times were found to be strongly dependent on the water content and the organic modifier nature of the mobile phase

Lipophilicity determination of acidic compounds: MEEKC as a reliable high-throughput methodology

In the present study apressure-assisted MEEKC method with reversed-polarity using a conventional CE instrument with UV detection and uncoated fused silica capillariesis validated as a high-throughput methodology for the lipophilicity determination of the neutral species of acidic compounds(pKa> 3.5).After the calibration of the system with four standard compoundsof known log Po/w, mass distribution ratios (log kMEEKC) of new molecules can be directly converted into log Po/wvalues by means of a simple linear equation (log Po/w=a·log kMEEKC+b). The method was internally and externally validated for a log Po/wrange between-1.54 and4.75, with higheraccuraciesthan conventional liquid chromatographic methods

HILIC characterization: estimation of phase volumes and composition for a zwitterionic column

A methodology for the estimation of the different phase volumes in HILIC is presented. For a ZIC-HILIC column the mobile phase volume (hold-up volume) is determined in several acetonitrile- and methanol-water compositions by a Linear Free Energy Relationships (LFER) homologous series approach involving n-alkyl-benzenes, -phenones, and -ketones. We demonstrate that the column works as a HILIC column when the mobile phase contains high and medium proportions of methanol or acetonitrile. However, for acetonitrile contents below 20%, or 40% for methanol, same column works in RPLC. In between, a mixed HILIC-RPLC behavior is observed, and solutes of low molecular volume are retained as in HILIC mode, but the largest ones show RPLC retention. From the homologous series retention data and pycnometric measurements involving the pure organic solvents and their mixtures with water, the mean solvent composition of the water-rich transition layers between column functionalization and the bulk mobile phase, which act as stationary phase, is estimated. Finally, the phase ratio between stationary and mobile phases is also estimated for each eluent composition, allowing the calculation of the corresponding stationary phase volumes. All volumes are strongly dependent on the water content in the eluent, especially when acetonitrile is selected as mobile phase constituent. In HILIC mode, when the water content in the hydroorganic mobile phase increases, the volumes of mobile phase decrease, but the volumes of stationary phase (mainly the water layer adsorbed onto the bonded-phase and the water-enriched interface) increase. However, at high water concentrations, where the column works in RPLC mode, the mobile phase volume increases and the stationary phase (which is now the bonded zwitterion) volume decreases when increasing the water percentage in the mobile phase

High-throughput log Po/w determination from UHPLC measurements: revisiting the chromatographic hydrophobicity index

A fast and accurate lipophilicity determination is fundamental in the drug discovery process, as long as it is a relevant property in the absorption, distribution, metabolism, excretion and toxicity (ADMET) of a potential drug substance. In the present work, different models based on chromatographic retention values for a large set of compounds and some of their molecular descriptors (calculated by ACD/Labs or CODESSA programs) have been examined in order to establish reliable equations for log Po/w determination from fast chromatographic hydrophobicity index (CHI) measurements. This appears to be a very interesting high-throughput methodology for screening purposes, since CHI values can be measured by UHPLC in very short runs (<4 min) and molecular descriptors can be easily computed from the structure of any compound. The selected final descriptors were Abraham's hydrogen-bond acidity (A) and excess molar refraction (E) from ACD/Labs, and hydrogen-bond acidity HDCA-1/TMSA and HOMO-LUMO polarizability descriptors from CODESSA software. The proposed equations allow an accurate determination of log Po/w with standard errors in the range of 0.4 units

Characterization of hydrophilic interaction liquid chromatography retention by a linear free energy relationship. Comparison to reversed- and normal-phase retentions.

The Abraham solvation parameter model, a linear free energy relationship (LFER) approach, has been used to characterize a polymeric zwitterionic (sulfobetaine) column in HILIC mode. When acetonitrile (MeCN) is used in the preparation of mobile phases the main solute characteristics affecting the chromatographic behavior of analytes are the molecular size and the hydrogen-bonding (both acidity and basicity) interactions. The former property is more favorable in the acetonitrile-rich mobile phase, reducing thus the retention, but the latter reveals a higher affinity for the water layer adsorbed on the stationary phase, enhancing retention. However, if the aprotic acetonitrile is replaced by methanol, a hydrogen-bond acidic solvent, solute hydrogen-bond basicity does not contribute any more to retention, quite the opposite. Thus, a slightly different selectivity is observed in methanol/water than in acetonitrile/water. Normal-phase mode and HILIC-MeCN share the same main factors affecting retention. For reversed-phase and immobilized artificial membrane (IAM) chromatography, the solute molecular size increase retention because of the lower amount of energy required in the formation of a cavity in the solvated stationary phase. On the contrary, the analyte hydrogen-bond basicity favors interactions with the hydroorganic mobile phase and reduces retention. The determined parameters justify the reversed selectivity commonly observed in HILIC in reference to reversed-phase. In most instances, the least retained solutes in reversed-phase are the most retained in HILIC

Retention-pH profiles of acids and bases in hydrophilic interaction liquid chromatography

The high proportion of acetonitrile used in many HILIC mobile phases significantly changes the acid-base properties of pH buffers and analytes foreseen from available data in water. In this paper, the recommended stability pH range for chromatographic columns is examined with various acetonitrile water mixtures, resulting in a significant broadening in the operational pH window with the content of organic solvent. Additionally, the challenge of buffer selection in HILIC is also addressed. Commonly used ammonium acetate shrinks its pH buffering range in acetonitrile-rich mobile phases due to variations in the dissociation constants of the buffer constituents (acetic acid and ammonium). Thus, other organic acids such as formic acid, TFA, and succinimide have been studied as buffers in order to fully cover the pH range of use of the column. Also the retention-pH profiles of several acids and bases have been studied in 80% and 90% acetonitrile using the proposed buffers and their behavior compared to that obtained with buffers prepared from oxalic acid, pyrrolidine, and triethylamine. The latter two show additional interactions in 80% acetonitrile that distort the expected retention-pH profiles of acid analytes, but not the ones of bases. In 90% acetonitrile the profiles are affected by significant additional solute-buffer interactions that might be caused by ion pairing, homo- and heteroassociation in this low ion solvating medium