High pH instability of quaternary ammonium surfactant coatings in capillary electrophoresis.

The two-tailed cationic surfactant dioctadecyldimethyl ammonium bromide (DODAB) produces semi-permanent coatings that yield strongly reversed electroosmotic flow (EOF), for example -0.31 ± 0.01 cm2 kV-1 s-1 at pH 3.5. Moreover, these coatings are easy to prepare, regenerable, cost effective, and yield high efficiency (520 000-900 000 plates per m) separations of cationic proteins over many runs under acidic (pH 3.5) conditions. Given the quaternary amine functionality of DODAB, we were surprised to observe that DODAB coatings become unstable at pH > 7. At pH 7.2, the EOF of a DODAB coated capillary drifted from reversed to cathodic over only 5 runs, and protein separations became severely compromised. By pH 12, no EOF reversal was observed. Electrophoretic and mass spectrometric studies demonstrate that the coating decomposition involves a surface conversion of the quaternary amine in DODAB to a variety of products, although the exact mechanism remains elusive. Regardless, the results herein demonstrate that semi-permanent coatings based on cationic two-tailed surfactants such as DODAB are limited to separations using acidic buffers.

Peak distortion effects in analytical ion chromatography.

The elution profile of chromatographic peaks provides fundamental understanding of the processes that occur in the mobile phase and the stationary phase. Major advances have been made in the column chemistry and suppressor technology in ion chromatography (IC) to handle a variety of sample matrices and ions. However, if the samples contain high concentrations of matrix ions, the overloaded peak elution profile is distorted. Consequently, the trace peaks shift their positions in the chromatogram in a manner that depends on the peak shape of the overloading analyte. In this work, the peak shapes in IC are examined from a fundamental perspective. Three commercial IC columns AS16, AS18, and AS23 were studied with borate, hydroxide and carbonate as suppressible eluents. Monovalent ions (chloride, bromide, and nitrate) are used as model analytes under analytical (0.1 mM) to overload conditions (10-500 mM). Both peak fronting and tailing are observed. On the basis of competitive Langmuir isotherms, if the eluent anion is more strongly retained than the analyte ion on an ion exchanger, the analyte peak is fronting. If the eluent is more weakly retained on the stationary phase, the analyte peak always tails under overload conditions regardless of the stationary phase capacity. If the charge of the analyte and eluent anions are different (e.g., Br(-) vs CO3(2-)), the analyte peak shapes depend on the eluent concentration in a more complex pattern. It was shown that there are interesting similarities with peak distortions due to strongly retained mobile phase components in other modes of liquid chromatography.

Carboxylate modified porous graphitic carbon: a new class of hydrophilic interaction liquid chromatography phases.

Stationary phases for hydrophilic interaction liquid chromatography (HILIC) are predominantly based on silica and polymer supports. We present porous graphitic carbon particles with covalently attached carboxylic acid groups (carboxylate-PGC) as a new HILIC stationary phase. PGC particles were modified by adsorbing the diazonium salt of 4-aminobenzoic acid onto the PGC, followed by reduction of the adsorbed salt with sodium borohydride. The newly developed carboxylate-PGC phase exhibits different selectivity than that of 35 HPLC columns, including bare silica, zwitterionic, amine, reversed, and unmodified PGC phases. Carboxylate-PGC is stable from pH 2.0 to 12.6, yielding reproducible retention even at pH 12.6. Characterization of the new phase is presented by X-ray photoelectron spectroscopy, thermogravimetry, zeta potentials, and elemental analysis. The chromatographic performance of carboxylate-PGC as a HILIC phase is illustrated by separations of carboxylic acids, nucleotides, phenols, and amino acids.

Ion chromatography on carbon clad zirconia modified by diazonium chemistry and functionalized latex particles.

This work explores the potential of 3 µm carbon coated zirconia particles as a stationary phase for ion chromatography for the separation of organic acids and inorganic ions. A 4-phenylsulfonic acid functionality is introduced onto the carbon surface by reducing 4-phenylsulfonic acid diazonium chloride with borohydride in the presence of carbon clad zirconia particles. The elemental sulfur analysis gave 132 µeq-SO(3)H/g carbon clad zirconia and 2% S atomic concentration by XPS analysis. The -SO(3)(-) groups serve as electrostatic anchors for latex nanoparticles bearing quaternary triethylamine functional groups. The agglomeration step in 5 × 0.4 cm i.d. columns converts the packed particles into an anion exchanger. The breakthrough curves with nitrate indicate a capacity of 3 µeq/column. Separation of common organic acids and inorganic ions using carbonate eluent and suppressed conductivity detection yield plate heights (H) of 0.023-0.05 mm.

Surfactant bilayer coatings in narrow-bore capillaries in capillary electrophoresis.

The cationic surfactants didodecyldimethylammonium bromide (DDAB) and dioctadecyldimethyl-ammonium bromide (DODAB) have previously been shown to form semi-permanent coatings that effectively prevent adsorption of cationic proteins in fused silica capillaries with inner diameters of 25-75 µm. This paper investigates the impact that narrower capillary diameters (≤25 µm) have on the stability of surfactant bilayer coatings and the efficiency of separations of model cationic proteins and neurotransmitters. Using a DODAB-coated 5 µm i.d. capillary 210 consecutive protein separations (1050 min) were performed without recoating the capillary between runs. Separation efficiencies of 1,400,000-2,000,000 plates per m (340,000-430,000 plates) were obtained. Migration time reproducibilites of 6.8% RSD were observed for 300 injections performed over a 30 day period without any regeneration of the coating. Neurotransmitters were separated with efficiencies ranging from 470,000-610,000 plates per m (110,000-140,000 plates) in a 5 µm capillary.

Agglomerated silica monolithic column for hydrophilic interaction LC.

Hydrophilic interaction LC (HILIC) has gained wide acceptance in recent years due to its ability to retain and separate polar compounds such as pharmaceuticals. Most commercial HILIC phases are particle based, which limit the speed with which HILIC separations can be performed. Herein, agglomerated silica monolithic columns are prepared by electrostatically attaching polyionic latex particles onto a silica monolith by simply flushing a suspension of the ionic latex through a silica monolith. Such phases retain the high efficiency and permeability of the native silica monolith, while the agglomerated phase is easy to introduce and provides excellent mass transfer. High %ACN in the mobile phase dramatically increases the efficiency and retention, consistent with HILIC behavior. Test analytes such as benzoates, nucleotides and amino acids are separated with plate heights of 25-110 microm. The high permeability of monoliths allows HILIC separations to be performed with baseline resolution in less than 15 s. Electrostatic repulsion-hydrophilic liquid interaction chromatographic retention behavior of the latex-coated monoliths is verified using amino acids as test analytes.

Covalently modified graphitic carbon-based stationary phases for anion chromatography.

Carbon-clad zirconia particles have been converted into ion-exchange media by in situ diazonium generation and thermal deposition. The surfaces prepared possess either a permanently charged anion-exchange site or a weak anion-exchange site. Surface modification optimization experiments were performed both on planar carbon surfaces and on non-porous 2 microm and porous 3 microm carbon-based particles. Modification by traditional electrochemical and thermal deposition were compared. Surface modification with the tertiary amine functionality, N,N-dimethyl-p-phenylenediamine, yielded a capacity of 6.5 microequiv./column, stable retention for >33,000 column volumes and retention reproducibility of <2% RSD. A quaternary amine functionality (strong base exchanger) was achieved by reaction of the tertiary amine phase with methyl iodide. Utilizing short columns (35 x 4 mm i.d.) mixtures of common inorganic anions were separated with efficiencies of 21,000 plates/m.

Effect of nature of electrolytes on retention and selectivity in hydrophilic interaction liquid chromatography.

This work investigates the selectivity and retention changes on four classes of HILIC columns upon addition of 1-20 mM electrolyte. The effect of four electrolytes (Na+Cl-, Na+ClO4-, Na+PF6-, and Na+CF3CO2-) were tested on neutral, cationic, anionic, and zwitterionic analytes under HILIC conditions (70-90% ACN). These electrolytes alter the retention and selectivity on silica, zwitterionic and diol columns through ion exchange and ionic screening mechanisms. Neutral analytes were unaffected by addition of 1-20 mM electrolyte, indicating minimal change to the retentivity of the water layer. Cationic and anionic analytes increase and decrease in retention, respectively (Na+PF6- ≈ Na+ClO4- > Na+CF3CO2- > Na+Cl-). Chaotropic electrolytes accumulate at the mobile phase/water layer interface, enhancing the ion exchange and ionic screening effects of the mobile phase electrolyte. Altering the buffer cation (Li+, Na+, K+) caused small but statistically significant changes in retention.

Stability of surfactant coated columns for ion chromatography.

Ionic surfactant coatings are a popular means to convert reversed-phase columns into ion-exchange phases with adjustable ion-exchange capacity and selectivity. However, the perceived lack of stability of surfactant coatings has hindered their use for routine separations. Coating conditions (acetonitrile content, ionic strength, surfactant concentration and temperature) were varied to determine their effect on coating stability. Under all coating conditions, cetyltrimethylammonium (CTAB) coated columns exhibited an initial decrease in ion retention. However, after the initial 1 L flush, both retention times and efficiency remained stable for >or=3000 column volumes. Greatest column stability and control of column capacity are achieved if the surfactant in the coating solution is below its critical micelle concentration.

Hydrophilic interaction chromatography of nucleotides and their pathway intermediates on titania.

Nucleotides and their pathway intermediates play important roles in all living species. They are essential cellular components in energy transfer, metabolic regulatory processes and biosynthesis. Titania (TiO(2)) has strong Lewis acid sites which have an affinity for the strongly electronegative phosphonate group of nucleotides. Herein a bare titania column (150 mm x 4.6 mm I.D., 3 microm) with UV detection at 254 nm was used for the separation of a set of nucleotides (AMP, ADP, ATP, UMP, UDP, UTP, GMP, GDP, GTP, CMP and CTP) and their intermediates (NAD, NADH, UDP-Glu and UDP-GluNAc). Addition of phosphate to the eluent suppresses the ligand-exchange interactions with the titania surface such that hydrophilic interaction chromatography (HILIC) separations may be performed. Increasing the %ACN resulted in increasing retention and efficiency (up to 13,000, 9500 and 4500 plates/m for AMP, ADP and ATP, respectively). The effects of pH, buffer concentration and other eluent anions (fluoride and acetate) were also studied. Fifteen nucleotides and their intermediates were separated in 26 min (R(minimum)>1.3) using an one-step gradient.

Selective preconcentration of chemical warfare agent degradation products using a zirconia preconcentration column.

Zirconia (ZrO(2)) has strong Lewis acid sites which have an affinity for the strongly electronegative phosphonate group of organo-phosphates. To investigate whether this affinity can be used for selective preconcentration, the retention of methyl, ethyl, and propylphosphonic acid (MPA, EPA and PPA) and inorganic anion matrix components on ZrO(2) was investigated. Only organo-phosphates and sulfate exhibited retention on zirconia. After preconcentration, the retained species were eluted from ZrO(2) by 0.75 mM Na(2)CO(3), and separated by a Dionex Ionpac AS11 anion-exchange column (250 mm x 2 mm I.D.) and a Hypercarb RPLC column (50 mm x 4.6 mm I.D., 3 microm) in series followed by suppressed conductivity detection. Calibration curve showed a linear response for MPA, EPA and PPA in the range of 0.01 microM to 1 microM (R(2)>0.9989). Detection limits after preconcentration of a 10 mL sample were 0.16, 0.19 and 0.16 microg/L for MPA, EPA, and PPA, respectively.

Non-covalent capillary coatings for protein separations in capillary electrophoresis.

Adsorption of proteins, particularly basic proteins onto fused silica capillaries severely degrades capillary electrophoretic performance. This review provides a synopsis of the fundamentals underlying protein adsorption and its impact on CE performance. The efficacy of small molecule background electrolyte additives, surfactants, physically adsorbed polymers (dynamic and static), and successive multiple ionic-polymer layer coatings are evaluated using a number of performance metrics. Peak efficiency and migration time reproducibility are used as measures of reversible protein adsorption, while protein recovery, electroosmotic flow reproducibility and step changes in the baseline are used as indicators of irreversible protein adsorption.

Linear solvation energy relationship (LSER) characterization of the normal phase retention mechanism on hypercrosslinked polystyrenes.

Linear solvation energy relationships (LSERs) were applied to retention on hypercrosslinked polystyrene on silica (HC-Tol) to elucidate the type and relative importance of molecular interactions between model solutes and the HC-Tol stationary phase. Classical amino phase and another hypercrosslinked phase (5-HGN) were used as reference columns. On both the HC-Tol and amino, polar interactions predominate and contribute to retention. Solute volume V has no impact on retention on the amino column, while V has a slightly negative influence on retention for the HC-Tol column. The differences in coefficient v between the amino and the HC-Tol columns might explain why the HC-Tol is capable of group-type separations. 5-HGN phase has smaller a and b values compared to HC-Tol, which means that 5-HGN is not as basic or acidic in terms of hydrogen bonds as is HC-Tol. This suggests that the hydrogen bonding character of the HC-Tol phase arises from its silica substrate.

Surfactant coated graphitic carbon based stationary phases for anion-exchange chromatography.

Separations of common inorganic anions were carried out on three different surfactant coated media using carbonate/bicarbonate eluents with suppressed conductivity detection. Graphitic carbon columns (porous graphitic carbon and carbon-clad zirconia) packed with 3 microm particles have been converted into anion-exchange stationary phases by equilibration with the cationic surfactants: didodecyldimethylammonium bromide (DDAB); cetyltrimethylammonium bromide (CTAB); and cetylpyridinium chloride (CPC). Additionally, an ethylene-bridged silica column was studied with CPC coatings. Porous graphitic carbon (PGC) columns coated with DDAB exhibited pressure increases and loss of resolution at higher capacities. CPC coatings on PGC exhibited better repeatability and efficiencies of 5.0 x 10(4)plates/m. However, CPC coatings exhibited a 15% loss in retention factor with <1.2 x 10(3) column volumes on PGC. Conversely, the ethylene-bridged silica column showed complete failure in less than 8 h of use. As with PGC, carbon-clad zirconia coated with CPC showed an initial loss of capacity, but thereafter was stable for more than 1.7 x 10(3) column volumes (t(r) RSD<2%).

Ion chromatography on a latex-coated silica monolith column.

A silica monolith column (Merck Chromolith, 100 mm x 4.6 mm) has been coated with Dionex AS9-SC latex nanoparticles to convert the column into an anion-exchange stationary phase. For comparison purposes, a reversed-phase silica monolith was also converted into an anion-exchange column by coating with the cationic surfactant didodecyldimethylammonium bromide (DDAB). Separations of common inorganic anions were carried out using 7.5 or 5.0 mM 4-hydroxybenzoic acid at pH 7.0 along with suppressed conductivity detection. Direct comparisons were then made between the two columns in terms of selectivity, efficiency and stability. The latex-coated column was on average 50% more efficient than the DDAB-coated column. A 10% decrease in retention times was observed on the DDAB column over 11 h of continuous eluent flow, while the latex coating exhibited <1% change in retention even after 2.5 months of periodic use.

Linear solvation energy relationships in normal phase chromatography based on gradient separations.

Coupling the modified Soczewiñski model and one gradient run, a gradient method was developed to build a linear solvation energy relationship (LSER) for normal phase chromatography. The gradient method was tested on dinitroanilinopropyl (DNAP) and silica columns with hexane/dichloromethane (DCM) mobile phases. LSER models built based on the gradient separation agree with those derived from a series of isocratic separations. Both models have similar LSER coefficients and comparable goodness of fit, but the LSER model based on gradient separation required fewer trial and error experiments.

Highly efficient protein separations in capillary electrophoresis using a supported bilayer/diblock copolymer coating.

A surfactant/polymer wall coating consisting of the doubly chained cationic surfactant dimethyldioctadecylammonium bromide (DODAB) and polyoxyethylene (POE) 40 stearate is investigated. The coating is formed by simply rinsing a capillary with a solution containing DODAB and POE 40 stearate. The resultant coating is semi-permanent--demonstrating stable electroosmotic flow (EOF) even after a 60 min high pressure rinse with buffer. The EOF (-0.45+/-(0.23) x 10(-4) cm(2) V(-1) s(-1) at pH 7.4) is suppressed by more than a factor of ten compared to that observed for DODAB alone. Model protein mixtures were separated over a pH range of 3-10 with efficiencies of up to greater than 1 million plates/m for the basic proteins cytochrome c, lysozyme, ribonuclease A and alpha-lactalbumin, and the acidic proteins insulin chain A, trypsin inhibitor, and alpha-chymotrypsinogen A. Migration time reproducibility was 0.5-4.0% from run to run and 0.6-4.3% from day to day. Protein recoveries with this coating ranged from 84% to 97%.