Contributions to reversed-phase column selectivity: III. Column hydrogen-bond basicity.

Column selectivity in reversed-phase chromatography (RPC) can be described in terms of the hydrophobic-subtraction model, which recognizes five solute-column interactions that together determine solute retention and column selectivity: hydrophobic, steric, hydrogen bonding of an acceptor solute (i.e., a hydrogen-bond base) by a stationary-phase donor group (i.e., a silanol), hydrogen bonding of a donor solute (e.g., a carboxylic acid) by a stationary-phase acceptor group, and ionic. Of these five interactions, hydrogen bonding between donor solutes (acids) and stationary-phase acceptor groups is the least well understood; the present study aims at resolving this uncertainty, so far as possible. Previous work suggests that there are three distinct stationary-phase sites for hydrogen-bond interaction with carboxylic acids, which we will refer to as column basicity I, II, and III. All RPC columns exhibit a selective retention of carboxylic acids (column basicity I) in varying degree. This now appears to involve an interaction of the solute with a pair of vicinal silanols in the stationary phase. For some type-A columns, an additional basic site (column basicity II) is similar to that for column basicity I in primarily affecting the retention of carboxylic acids. The latter site appears to be associated with metal contamination of the silica. Finally, for embedded-polar-group (EPG) columns, the polar group can serve as a proton acceptor (column basicity III) for acids, phenols, and other donor solutes.

Nonionic micellar liquid chromatography coupled to immobilized enzyme reactors.

Immobilized enzyme reactors are used as post-column reactors to modify the detectability of analytes. An immobilized amino acid oxidase reactor was prepared and coupled to an immobilized peroxidase reactor to detect low level of amino acids by fluorescence of the homovanilic dimer produced. A cholesterol oxidase reactor was prepared to detect cholesterol and metabolites by 241 nm UV absorbance of the enone produced. The preparation of the porous glass beads with the immobilized enzymes is described. Micellar liquid chromatography is used with non-ionic micellar phases to separate the amino acids or cholesterol derivatives. It is demonstrated that the non ionic Brij 35 micellar phases are very gentle for the enzyme activity allowing the reactor activity to remain at a higher level and for a much longer time than with hydro-organic classical chromatographic mobile phases or aqueous buffers. The coupling of nonionic micellar phases with enzymatic detection gave limits of detection of 32 pmol (4.8 ng injected) of methionine and 50 pmol (19 ng injected) of 20alpha-hydroxy cholesterol. The immobilized enzyme reactors could be used continuously for a week without losing their activity. It is shown that the low efficiency obtained with micellar liquid chromatography is compensated by the possibility offered by the technique to easily adjust selectivity.

Linear solvation energy relationships of mixed micelles of sodium dodecyl sulfate and decanol: towards a better model of octanol/water partitioning.

We show that we can alter the mechanism of micelle/water partitioning by the addition of decanol as a co-surfactant to an SDS micellar solution. Linear solvation energy relationship (LSER) studies indicate that as we increase the amount of decanol added to sodium dodecyl sulfate solution, the hydrogen bond donating ability of the aqueous phase increases and the cavity term of the micellar phase increases. We obtain a better correlation with octanol/water partitioning using the mixed micelle system compared to normal micelle solution. Choosing the appropriate micelle marker is very important. Significant changes in the LSER equations can occur if a different compound is used as the micelle marker.

Polyoxyethylene alkyl ether nonionic surfactants: physicochemical properties and use for cholesterol determination in food.

Polyoxyethylene alkyl ethers, C(n)E(m), are nonionic surfactants made of an alkyl chain with n methylene groups and a hydrophilic part with m oxyethylene units. C(n)E(m) nonionic surfactants are very useful in chemical analysis. The commercially available products are often a mixture of several C(n)E(m) molecules with different m values. Pure C(n)E(m) surfactants are now available. The physicochemical parameters: critical micelle concentration (c.m.c.), molar volume, density, cloud-point temperature and hydrophile-lipophile balance value for pure C(n)E(m) surfactants were collected from the literature. Regression analyses were carried out on the data. They showed that strong correlations existed between the structure of the molecule (n and m values) and its physicochemical properties. General equations linking the c.m.c., molar volume, density and cloud-point temperature of the C(n)E(m) surfactants and their structure (n and m values) are proposed and discussed. The use of these surfactants in chemical analysis is illustrated by the determination of cholesterol in egg yolk. Cholesterol was separated from the bulk yolk by cloud-point extraction using the C(12)E(10) surfactant. It was quantitated using micellar liquid chromatography. The C(12)E(23) surfactant was used to prepare the micellar mobile phase that allowed the separation of cholesterol and the use of an enzymatic detector.

Chromatographic silanol activity test procedures: the quest for a universal test.

Reversed-phase chromatography is the most used and the most studied method of modern liquid chromatography. There is yet no ideal support available for preparing reversed-phase stationary phases, but the vast majority have historically been and are still prepared on microparticulate silica. The silica surface has a number of properties which make it attractive for derivatization, including easily controlled particle size and porosity and mechanical stability. There are several types of surface silanols which have their own unique properties that affect both chemical derivatization reactions and adsorptive interactions with solutes. The relative distribution of these different types of silanols may affect the characteristics of silica-based stationary phases more than the absolute number of surface silanol groups. The relative importance of each of these different types of silanols has not yet been unambiguously established. Free or isolated silanols, internally hydrogen-bonded vicinal silanols, and geminal silanols all have been implicated as the primary reaction and adsorption sites. There are many different synthetic schemes that have been used to block the remaining silanols, and "deactivated" phases are very popular. Unfortunately, there is still no universally agreed upon method to measure the accessibility or interaction of these silanols with solute molecules. Many tests have been proposed, focusing mainly on chromatographic probe molecules, but different tests run on the same column will often show different interactions. We will briefly review the surface chemistry of silica and focus on the multitude of tests that have been proposed. Our focal point will be silanol activity test; other aspects of column performance will not be included. Where possible, comparisons among the methods will be made.

Gradient elution techniques for capillary electrochromatography.

Capillary electrochromatography (CEC) is a rapidly maturing technique, but still in need of further instrumental development and in need of unique applications that are not possible by traditional pressure-driven LC. We review the development of gradient elution schemes for CEC, beginning with pH gradients initially developed for capillary electrophoresis. Step gradients are the most easily instrumentally implemented, but provide less flexibility in separation than continuous gradients. Pressure-assisted CEC is easily adapted to gradient elution schemes, but does not offer the advantages of very high column efficiency provided by totally electro-driven mobile phases. The development of flow-injection interfaces allows a true solvent gradient to be generated by micro-LC pumps, with the mobile phase drawn into the separation capillary by pure electroosmotic flow. While requiring both a CEC instrument and a traditional pump or pumps capable of generating the gradient, this method offers advantages of greatly reduced column handling, prolonging column lifetimes, and allows simple autosampling. We also discuss voltage gradients, which provide a mobile phase velocity gradient.

Characterization of C18-bonded liquid chromatographic stationary phases by Raman spectroscopy: the effect of mobile phase composition.

Raman spectroscopy is used to examine the effect of mobile phase composition on the orientation of octadecyl-bonded silica-based reversed-phase liquid chromatographic (RPLC) stationary phase ligands. The effect of ligand bonding density is also investigated. The present experimental set-up utilizes a direct, noninvasive, on-column approach to examine the solvent dependent conformational behavior of the bonded ligands under flow-rate and back pressure conditions similar to those used during conventional RPLC measurements. Neat, single-component, mobile phase solvents including water, acetonitrile, methanol and chloroform are used to investigate the hypothesized collapsing and extension of stationary phase ligands with changes in mobile phase composition. No evidence of phase collapse was observed upon changing the mobile phase composition from an organic to an aqueous content. Also, Raman spectroscopic measurements allowed the differentiation between associated and free acetonitrile solvent.

Characterization of C18-bonded liquid chromatographic stationary phases by Raman spectroscopy: the effect of temperature.

This study represents the first time that both the mobile phase composition and the temperature are simultaneously controlled to examine silica-bonded octadecylsilyl (C18) ligands spectroscopically at typical liquid chromatographic (LC) mobile phase flow-rates and back-pressures. Raman spectroscopy is used to characterize the behavior of the C18 bonded ligands equilibrated at temperatures from 45 to 2 degrees C in neat, single-component, mobile phase solvents including: water, acetonitrile, methanol, and chloroform. In addition, the effect of stationary phase ligand bonding density is examined by using two different monomeric reversed-phase liquid chromatographic (RPLC) stationary phases, a 2.34 and a 3.52 micromol m(-2) Microporasil C18 stationary phase, under identical conditions. The direct, on-column, spectroscopic analysis used in this study allows direct evaluation of the temperature-dependent behavior of the bonded C18 ligands. The temperature-dependent ordering of the stationary phase ligands is examined to determine if the ligands undergo a phase transition from a less-ordered "liquid-like" state at higher temperatures to a more-ordered "solid-like" state at lower temperatures. A discrete phase transition was not observed, but rather a continual ordering as temperature was lowered.

pH effects on micelle-water partitioning determined by micellar electrokinetic chromatography.

In many biological and environmental situations, the pH of aqueous media varies and differences in solute partitioning may result. However, the majority of biopartitioning and hydrophobicity studies conducted have been at pH 7. Using migration factors measured by micellar electrokinetic chromatography, we have determined pH effects on micelle-water partitioning for 19 compounds. We develop an improvement to the migration factor equation and the corrected migration factor for aniline shows a definite increase as pH decreases. The corrected migration factor was constant for the rest of the compounds over the pH ranged studied. We also investigated five micelle markers and determined that decanophenone is the best micelle marker to date. Decanophenone has a strong chromophore, detectable at all pH levels, and is easy to dissolve in the mobile phase.

Gradient elution electrochromatography with a flow-injection analysis interface.

A flow-injection analysis-capillary electrochromatography interface is used for gradient elution capillary electrochromatography giving purely electroosmotic flow through the analytical column. Solvent gradients were generated with a micro-LC system connected to the interface. Injections were carried out on-line using an inert rotary LC valve controlled by an electric actuator. Gradient shape was measured from acetonitrile (5% acetone)-water (50:50, v/v) to (100:0) in open tubular experiments. When compared to conventional instrumentation, peak tailing and peak width increased slightly using the interface. A test mixture of nine solutes was evaluated in isocratic and gradient elution modes. Using the interface, a gradient of MeCN-water (60:40) to (90:10) provided baseline separation of all nine solutes in under 18 min with good band spacing. Reproducibility of retention times in eight replicate injections was found to be better than 2% R.S.D. for all solutes. This interface also allows use of autoinjectors and dramatically lessens movement of the packed column, improving column lifetime.

Behavior and use of nonaqueous media without supporting electrolyte in capillary electrophoresis and capillary electrochromatography.

Five nonaqueous solvents (acetonitrile, methanol, N,N-dimethylformamide, dimethyl sulfoxide, formamide) and deionized water were investigated for their ability to support electroosmotic flow (EOF) without electrolytic additives. In general, flow was found to be equal to or greater than flow with typical CE buffer systems. The magnitude of EOF was determined for each solvent by open tubular capillary electrophoresis (CE) and related to viscosity (eta), dielectric constant (epsilon), and the ratio of dielectric constant to viscosity (eta/epsilon). Zeta potentials (zeta) were derived indirectly from flow data and tabulated. Comparisons of flow behavior and zeta were made between pure solvents and conventional CE buffers, and questions of equilibrium and reproducibility were addressed. Similar experiments were performed using hydroorganic mobile phases (ACN/water, MeOH/water) across the complete compositional range (100% water-100% organic), with flow characteristics and zeta reported for each mobile phase system. Packed capillary columns (5-microns ODS) were evaluated for flow and retention stability under capillary electrochromatographic (CEC) conditions. A separation of 11 polycyclic aromatic hydrocarbons was performed in under 13 min by CEC with an ACN/water mobile phase. Reduced plate heights (h) were calculated between 2.5 and 3.0 for solutes with capacity factors (k') up to 4.5 for the most retained solute.

Characterization of liquid chromatographic stationary phases by Raman spectroscopy. Effect of ligand type.

This study represents the first Raman spectroscopic characterization of conventional chemically-bonded liquid chromatographic (LC) stationary phases under typical flow-rate and pressure conditions. Raman spectra were obtained for amino propyl (NH2), cyano propyl (CN), phenyl (Ph), octadecyl (C18), octyl (C8), and methyl (C1) chemically-bonded silica-based stationary phases in 100% aqueous mobile phases. The present experimental set-up has allowed Raman spectra of various stationary phase ligands, present in sub-monolayer coverages on the siliceous supports, to be obtained. This study: (1) demonstrates that conventional Raman spectroscopic techniques can be used to study LC stationary phases; (2) presents the experimental set-up, conditions, and approaches utilized to obtain Raman spectra of conventional stationary phases; (3) examines the spectroscopic differences observed for a variety of different types of bonded ligands that are typically used in reversed-phase (RPLC) and normal-phase (NPLC) liquid chromatographic separations; and (4) considers other future studies that are possible with this experimental approach, including mobile phase composition and temperature studies.

Cyclohexylamine additives for enhanced peptide separations in reversed phase liquid chromatography.

While the choice of stationary phase, organic modifier, and gradient strength can have significant effects on biomolecule separations, mobile phase additives can also have a significant effect on the chromatographic selectivity, recovery, efficiency and resolution. Given the importance of stationary phase coverage, the beneficial, silanol-masking properties of amines, and the potential for selectivity modification through ion-pair interactions, cyclohexylamine was examined as a mobile phase additive and compared with triethylamine and trifluoroacetic acid. Greatly improved separation was possible when cyclohexylamine was used as compared with phosphate buffer, and cyclohexylamine did not require purification before use, while triethylamine required distillation before 'clean' chromatograms were obtained.

Informational orthogonality of two-dimensional chromatographic separations.

A qualitative informational similarity technique has been used to describe the informational orthogonality of projected two-dimensional (2-D) chromatographic separations of complex mixtures from their one-dimensional 1-D separations. The reversed-phase liquid chromatography (RPLC), supercritical fluid chromatography (SFC), gas-liquid chromatography (GLC), and micellar electrokinetic capillary chromatography (MECC) retention behavior of up to 46 solutes of varying molecular properties was studied by 2-D range-scaled retention time plots and information entropy calculations. One hundred five combinations of technique/stationary phase pairs were used to simulate the 2-D chromatographic analyses. The informational entropy of one and two dimensions, the mutual information, the synentropy or "cross information", and the informational similarity were calculated to describe the informational orthogonality. In addition, pattern descriptors were used to qualitatively describe the 2-D peak distribution. With the solutes tested, informational orthogonality, zero informational similarity, was observed with MECC-SDS/SFC-C1, MECC-SDS/SFC-Carbowax, MECC-TTAB/SFC-Carbowax, HPLC-C18/GLC-DB-5, HPLC-PBD/SFC-phenyl, SFC-Carbowax/GLC-DB5, and HPLC-phenyl/SFC-phenyl 2-D chromatographic systems. Conversely, with the solutes tested, informational nonorthogonal behavior described by range-scaled retention time plots to moderate to severe band overlap and data clustering was observed with 2-D chromatographic systems with high informational similarity and moderate to high degrees of synentropy. These results should prove useful for predicting complementary 2-D techniques as well as for choosing a second separation technique for confirmation of separation or peak purity.

Silver(I)-mediated separations by capillary zone electrophoresis and micellar electrokinetic chromatography: argentation electrophoresis.

The addition of Ag(I) to the run buffer in capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), containing sodium dodecyl sulfate, applies the principles of argentation chromatography to electrophoretic separations and is termed "argentation electrophoresis". This technique is shown to provide a complementary method to CZE and MEKC for the separation of specific types of solutes that selectively complex with Ag(I). Baseline resolution in the CZE separation of nine sulfonamides is achieved by the addition of 50 mM silver nitrate to the run buffer. Retention mechanisms in MEKC separations can also be manipulated by the addition of Ag(I) to the micellar solution. Only slight resolution of a pair of sulfonamides was achieved under normal MEKC conditions. Upon the addition of Ag(I) to the mobile phase containing SDS micelles, baseline resolution of the compounds is shown. The retention order and resolution of five sulfonamides changed significantly when 25 mM Ag(I) was added to the SDS-containing buffer. The use of Ag(I) addition in MEKC is also applied to the separation of various other compounds that show selectivity toward Ag(I) complexation, including S-containing heterocycles and vitamin D compounds. The effect of the addition of Ag(I) on the elution range in MEKC is also investigated. A steady increase in the elution range is seen upon increasing the concentration of Ag(I). With a constant percentage of organic modifier (15%), the addition of higher concentrations of silver nitrate (25-50 mM) results in elution ranges greater than 12. The results using Ag(I) as buffer additives in MEKC are also compared to studies utilizing a mixed counterion surfactant of sodium/silver dodecyl sulfate.

Retention and selectivity of flavanones on homopolypeptide-bonded stationary phases in both normal- and reversed-phase liquid chromatography.

Three linear polymers of repeating amino acid units, or homopolypeptides, have been individually covalently bonded to microparticulate silica and evaluated for liquid chromatographic separations. The retention and selectivity of seven flavanones were investigated on these stationary phases and a structurally similar, commercially available reference stationary phase, Chiraspher. All three of the homopolypeptide stationary phases retain solutes in the normal-phase mode. The aromatic-containing homopolypeptide stationary phases also retain solutes in the reversed-phase mode. Selectivity values for the flavanones were higher in the normal-phase mode; chiral selectivity was observed for the amphiphilic homopolypeptide stationary phase in the reversed-phase mode. The retention mechanism of each stationary phase is suggested based on the chemical nature and conformation of the corresponding homopolypeptide ligand.

Arsenic speciation by micellar liquid chromatography with inductively coupled plasma mass spectrometric detection.

Four environmentally and biologically important arsenic species, dimethylarsenic acid (DMA), monomethylarsonic acid (MMA), As(III) and As(V) are separated by micellar liquid chromatography. Linear dynamic ranges for the four species are three orders of magnitude and detection limits are in the picogram range with inductively coupled plasma mass spectrometric (ICP-MS) detection. This paper discussed in detail the development of the chromatographic conditions. The micellar mobile phase, which consisted of 0.05 M cetyltrimethylammonium bromide, 10% propanol and 0.02 M borate buffer, showed good compatibility with ICP-MS. This method allowed direct injection of urine samples onto the chromatographic system without extensive pretreatment and presented no interference from chlorine in the matrix. Detection limits are comparable with other LC-ICP-MS studies. An SRM urine sample was used to demonstrate the applicability of this technique to "real-life" situations. Results indicated that DMA, MMA and As(V) were present in the urine sample.

Estimation of liquid-vapor critical loci for CO2-solvent mixtures using a peak-shape method.

Critical-mixture curves for 13 CO2-solvent binary mixtures were estimated using the peak-shape method. Mixture critical points were determined within 1 degrees C and 1 atm. The results for CO2-toluene and CO2-methanol were compared to previously reported data from high-pressure view cell studies. No more than a 3% difference was observed in the data generated by the two different techniques. A few abnormalities encountered while using the peak-shape method are also discussed.

Bioavailability estimation by reversed-phase liquid chromatography: high bonding density C-18 phases for modeling biopartitioning processes.

There have been many attempts to estimate biological activity with either 1-octanol/water partition coefficients or chromatographic retention parameters. Bulk phases may not be appropriate, however, for modeling a partitioning process in an interphase such as biological membranes. Chromatographic stationary phases can be argued as having structure similar to a membrane because of chain organization; however, the density of the grafted stationary-phase chains in commercially available stationary phases is much too low to provide a suitable model. We have previously developed a new scheme for derivatizing silica surfaces that produces stationary phases of significantly higher chain density than traditional methods. Investigation of the molecular mechanism and thermodynamics of solute partitioning into the different phases has shown that densely bonded reversed-phase stationary phases mimic partitioning to a biomembrane better than does bulk-phase octanol. Here we report chromatographic retention for pesticides, PAHs, and barbiturates using a C-18 column with high alkyl chain density, and in all cases, correlations of log k'w with bioavailability are equivalent to or better than correlations of bioavailability with the octanol/water partition coefficient.