Hydrodynamic chromatography of macromolecules using polymer monolithic columns

The selectivity window of size-based separations of macromolecules was tailored by tuning the macropore size of polymer monolithic columns. Monolithic materials with pore sizes ranging between 75 nm and 1.2 μm were prepared in situ in large I.D. columns. The dominant separation mechanism was hydrodynamic chromatography in the flow-through pores. The calibration curves for synthetic polymers matched with the elution behavior by HDC separations in packed columns with 'analyte-to-pore' aspect ratios (λ) up to 0.2. For large-macropore monoliths, a deviation in retention behavior was observed for small polystyrene polymers (M(r)<20 kDa), which may be explained by a combined HDC-SEC mechanism for λ<0.02. The availability of monoliths with very narrow pore sizes allowed investigation of separations at high λ values. For high-molecular weight polymers (M(r)>300,000 Da) confined in narrow channels, the separation strongly depended on flow rate. Flow-rate dependent elution behavior was evaluated by calculation of Deborah numbers and confirmed to be outside the scope of classic shear deformation or slalom chromatography. Shear-induced forces acting on the periphery of coiled polymers in solution may be responsible for flow-rate dependent elution.     

Achieving rapid low-pressure ion chromatography separations on short silica-based monolithic columns

Short silica-based monolithic columns (0.5-1 cm) are coated with the surfactant didodecyldimethylammonium bromide (DDAB) and used for fast ion exchange separations of small inorganic anions. Sources of extra-column band broadening are assessed and minimized to obtain separations of seven analytes (iodate, chloride, nitrate, bromide, nitrite, phosphate, sulphate) in two minutes at 2 mL/min. Eluents used are 6 and 9 mM 4-cyanophenol at pH 7.3-7.4 or 5 mM 4-hydroxybenzoic acid at pH 5.6. DDAB coating stability is improved by 15-fold by the addition of a DDAB coated pre-column before the injection valve. Separations are obtained using a low-pressure glass syringe.     

Rapid separation of inorganic anions by capillary ion chromatography using monolithic silica columns modified with dilauryldimethylammonium ion.

The rapid separation of inorganic anions was determined by capillary ion chromatography using monolithic silica capillary columns modified with dilauryldimethylammonium bromide. The stability of the modified stationary phase was satisfactory owing to a strong hydrophobic interaction between the lauryl groups of the reagent, even if the eluent did not contain dilauryldimethylammonium ion. Bromide in seawater samples could be determined by the present system. The repeatability of a retention time of bromide for six successive measurements was around 1.8% when a 500 mM sodium chloride aqueous solution was used as the eluent. Seawater samples were directly injected onto the prepared column without any interference of matrix ions, because an aqueous solution of high-concentration sodium chloride could be used as the eluent. Bromide in seawater samples could be determined within 2 min.     

Rapid determination of bromide in seawater samples by capillary ion chromatography using monolithic silica columns modified with cetyltrimethylammonium ion

Monolithic silica capillary columns dynamically modified with quaternary ammonium ions were evaluated for the determination of bromide in seawater samples. A quaternary ammonium ion such as cetyltrimethylammonium ion was dynamically introduced onto monolithic silica surfaces. The first layer of the modifier was introduced by electrostatic interaction, whereas the second layer was introduced by hydrophobic interaction. The latter layer worked as the anion-exchange sites. The modified monolithic silica capillary columns could be used for rapid separation of inorganic anions. Separation of authentic mixture of five anions was achieved within a few minutes. The addition of small amount of the modifier in the eluent improved the repeatability of the retention time. Seawater samples could be directly injected onto the prepared capillary columns, and bromide could be determined to be 63 mg/L.     

Double gradient ion chromatography using short monolithic columns modified with a long chained zwitterionic carboxybetaine surfactant

The rapid separation of inorganic anions on short monolithic columns permanently coated with a long chained zwitterionic carboxybetaine-type surfactant is shown. The surfactant, N-dodecyl-N,N-(dimethylammonio)undecanoate (DDMAU), was used to coat 2.5, 5.0 and 10 cm long reversed-phase silica monoliths, resulting in a permanent zwitterionic exchange surface when used with aqueous based eluents. The unique structure of the surfactant results in a charge double layer structure on the surface of the stationary phase, with strong internal anionic and weak external cationic exchange groups. The dissociation of the weak external carboxylic acid group acts to shield the inner anionic exchange site, resulting in substantial effective capacity changes with eluent pH. Utilising this effect with the application of an eluent pH gradient, simultaneously combined with eluent flow-rate gradients, very rapid simultaneous separations of both weakly retained anions and strongly retained polarisable anions was possible, with up to 10-fold decreases in overall run times. Coating stability and retention times under isocratic and isofluentic eluent conditions were shown to be reproducible over >450 repeat injections, with peak efficiency values averaging 29,000 N/m for the 2.5 cm column and 42,000 N/m for the 10 cm monolithic column, again under isocratic elution conditions.     

Zwitterionic ion chromatography with carboxybetaine surfactant-coated particle packed and monolithic type columns

Both particle packed (25 cm x 0.46 cm I.D. SUPELCOSIL 5 microm C18) and monolithic type (10 cm x 0.46 cm I.D. Merck Chromolith Performance C18) reversed-phase substrates were dynamically coated with a carboxybetaine type zwitterionic surfactant ((dodecyldimethyl-amino) acetic acid) and investigated as stationary phases for use in zwitterionic ion chromatography (ZIC). Investigations into eluent concentration and pH were carried out using KCl eluents containing 0.2 mM of the carboxybetaine surfactant to stabilise the column coatings. It was found that eluent concentration decreased anion retention whilst simultaneously increasing peak efficiencies, which may be due to the dissociation of intra- and inter-molecular salts of the carboxybetaine surfactant under higher ionic strength conditions. The Effect of eluent pH was an increase in anion retention with decreased eluent pH due to the increased protonation of the weak acid terminal group of the carboxybetaine, causing both a relative increase in the positive charge of the stationary phase and less repulsion of the anions by the dissociated weak acid group. The carboxybetaine-coated monolithic phase was applied to rapid anion separations using elevated flow rates and flow rate gradients.     

Preparing titania aerogel monolithic chromatography columns using supercritical carbon dioxide

The search for a method to fabricate monolithic inorganic columns has attracted significant recent attention due to their unique ability in separation applications of various biomolecules. Silica and polymer based monolithic columns have been prepared, but titania and other metal oxide monoliths have been elusive, primarily due to their fragility. This article describes a new approach for preparing nanostructured titania based columns, which offer better performance over conventional particle packed columns for separating a wide variety of biomolecules including phosphopeptides. TiO₂ monolithic aerogels were synthesized in separation columns using in situ sol‐gel reactions in supercritical carbon dioxide (scCO₂) followed by calcination, and compared to those prepared in heptanes. The characterization results show that scCO₂ is a better solvent for the sol‐gel reactions, providing lower shrinkage with the anatase TiO₂ monolith composed of nanofibers with very high surface areas. The monolithic columns show the ability to isolate phosphopeptides with little flow resistance compared to conventional titania particle based microcolumns.     

The synthesis and study of monolithic capillary columns for ion chromatography of anions

The physicochemical and chromatographic parameters of ion-exchange monolithic matrices and capillary columns on their basis were measured. A method was suggested for producing high-efficiency ionexchange monolithic capillary columns with the monolith structure optimized for efficient selective separation of anions in ion chromatography. The influence of the conditions of synthesis of monoliths on their porosity, permeability, the structure of the monolith layer, and the impedance of monolithic columns on their basis was investigated. Original Russian Text ? A.A. Kurganov, A.A. Korolev, E.N. Viktorova, A.Yu. Kanat’eva, 2009, published in Zhurnal Fizicheskoi Khimii, 2009, Vol. 83, No. 2, pp. 375–379.     

Fast ion chromatography using short anion exchange columns

A fast ion chromatographic system is described which uses shorter column lengths and compares various eluent profiles in order to maximise the performance without sacrificing the chromatographic resolution. Both isocratic and gradient elution profiles were considered to find the most efficient mode of separation. The separation and determination of seven target anions (chloride, chlorate, nitrate, chromate, sulfate, thiocyanate and perchlorate) was achieved using a short (4 mm ID, 50 mm long) column packed with Dionex AS20 high-capacity anion exchange material. A hydroxide eluent was used at an initial concentration of 25 mM (at a flow-rate of 1.0 mL/min) and two performance maxima were found. The maximum efficiency occurred at a normalised gradient ramp rate of 5 mM/t₀, resulting in a peak capacity of 16, while the fastest separation (<3 min) occurred at a normalised ramp rate of 30 mM/t₀. The retention time, peak width and resolution using the different eluent profiles on varying column lengths is also compared. Further investigations in this study determined that the highest peak capacity separation under gradient conditions could be approximated using an isocratic separation. The advantage of using this novel approach to approximate the maximum efficiency separation removes the need for column re-equilibration that is required for gradient elution resulting in faster analyses and enhanced sample throughput, with benefits in particular for multidimensional chromatography.     

Divinylbenzene-based monolithic capillary columns in capillary liquid chromatography

The effect of the conditions of synthesis of divinylbenzene-based monolithic capillary columns on their chromatographic characteristics was studied. It was demonstrated that the porosity and permeability of the column change significantly even at small deviations from the optimum conditions of polymerization of the monolith in the column. By contrast, the minimum value of HETP proved to be only slightly sensitive to the conditions of synthesis, ranging within ～10–20 μm. The conditions of polymerization of the monolith were found to produce more pronounced effect on the slope of the right branch of the van Deemter curve (parameter C), with the flattest curve being observed for columns prepared under optimum conditions. The minimum value of HETP for polymer monolithic capillary columns was found to be similar to that for silica gel monolithic capillary columns, but the latter are characterized by C values approximately an order of magnitude lower.     

Polymethacrylate monolithic columns for capillary liquid chromatography

In recent years, continuous separation media have attracted considerable attention because of the advantages they offer over packed columns. This research resulted in two useful monolithic material types, the first based on modified silica gel and the second on organic polymers. This work attempts to review advances in the development, characterization, and applications of monolithic columns based on synthetic polymers in capillary chromatography, with the main focus on monolithic beds prepared from methacrylate-ester based monomers. The polymerization conditions used in the production of polymethacrylate monolithic capillary columns are surveyed, with attention being paid to the concentrations of monomers, porogen solvents, and polymerization initiators as the system variables used to control the porous and hydrodynamic properties of the monolithic media. The simplicity of their preparation as well as the possibilities of controlling of their porous properties and surface chemistries are the main benefits of the polymer monolithic capillary columns in comparison to capillary columns packed with particulate materials. The application areas considered in this review concern mainly separations in reversed-phase chromatography, ion-exchange chromatography, hydrophobic and hydrophilic interaction modes; enzyme immobilization and sample preparation in the capillary chromatography format are also addressed.     

Full verification of the liquid exclusion-adsorption chromatography theory using monolithic capillary columns

Capillary electrochromatography (CEC) was used to separate alkyl phenol ethoxylates (APEs) as model diblock copolymers, with monolithic polymers as stationary phases. The order of elution indicate that the two polymer blocks follow distinct chromatographic modes: size-exclusion for the poly(oxyethylene) group and adsorption interaction for the alkyl part. Therefore, our experimental results were compared to the theory describing liquid exclusion-adsorption chromatography (LEAC). They were found in perfect agreement with the theory, which turned to be verified for the first time over the full range of polymer lengths.     

Applications of monolithic columns in gas chromatography and supercritical fluid chromatography

Porous monoliths are well‐known stationary phases in high‐performance liquid chromatography and capillary electrochromatography. Contrastingly, their use in other types of separation methods such as gas or supercritical fluid chromatography is limited and scarce. In particular, very few studies address the use of monolithic columns in supercritical fluid chromatography. These are limited to silica‐based monoliths and will be covered in this review together with an underlying reason for this trend. The application of monoliths in gas chromatography has received much more attention and is well documented in two reviews by Svec and Kurganov published in 2008 and 2013, respectively. The most recent studies, covered in this review, build on the previous findings and on further understanding of the influence of preparation conditions on porous properties and chromatographic performance of poly(styrene‐co‐divinylbenzene), polymethacrylate, and silica‐based monolithic columns while expanding to polymer‐based monoliths with incorporated metal organic frameworks and to vinylized hybrid silica monoliths. In addition, the potential application of porous layer open tubular monolithic columns in low‐pressure gas chromatography will be addressed.     

Efficiency of monolithic capillary columns in high-pressure gas chromatography

Monolithic sorbents for gas chromatography obtained in quartz capillaries are analyzed by means of kinetic curves (Poppe plots). The values for the time of a theoretical plate and the maximum number of plates are found to be strongly dependendent on the parameters of monolith synthesis, i.e., the relative amount of a monomer in the initial mixture, the temperature, and the polymerization time. Synthesis conditions are established using the kinetic curves, allowing the preparation of sorbents suitable for both performing high-rate analysis and achieving the most effective separation. It was shown that plotting kinetic curves on the basis of van Deemter curve data requires we allow for the compressibility of mobile phase.     

Loading capacities of monolithic capillary columns in gas chromatography

The loading capacities of monolithic capillary columns based on silica gel and divinylbenzene are studied for two carrier gases, CO₂ and N₂. It is shown that the efficiency of the column is more sensitive to the overload of the column than the retention time of the sorbate is, especially for the CO₂ carrier gas. It is established that the loading capacity of a monolithic column based on silica gel decreases significantly in going from N₂ to CO₂. For columns based on divinylbenzene, the loading capacity is found to be virtually the same for both carrier gases. For monolithic columns, the loading capacity per one meter of column length is found to be 10 and more times higher than that for a standard open capillary column.     

Ion chromatography for determination of nitrite and nitrate in seawater using monolithic ODS columns

A fast and highly sensitive ion chromatographic method using monolithic ODS columns was developed for the determination of nitrite (NO2-) and nitrate (NO3-) in seawater. Two monolithic ODS columns (50 mm x 4.6 mm i.d. + 100 mm x 4.6 mm i.d.) connected in series were coated and equilibrated with 5 mM cetyltrimethylammonium chloride (CTAC) aqueous solution. The column efficiency with 0.5 M NaCl as the mobile phase did not decrease in spite of the increase in flow rate of the mobile phase. Thus, good chromatograms were obtained within 3 minutes for NO2- and NO3 in artificial seawater without interferences by coexisting ions. The detection limit (S/N = 3) with UV detection at 225 nm was 0.8 and 1.6 microg/L for NO2- and NO3-, respectively. The characteristics of the monolithic CTA(+)-coated ODS columns were discussed. The present method was successfully applied to the fast and sensitive determination of NO2- and NO3- in real seawater samples.     

Methacrylate monolithic columns for capillary liquid chromatography polymerized using ammonium peroxodisulfate as initiator

Four methacrylate ester‐based monolithic columns for capillary liquid chromatography (CLC) were prepared by radical polymerization with ammonium peroxodisulfate (3 columns) and by thermal initiation (1 column). The polymerization mixture consisted of butyl methacrylate (BMA) and ethylene glycol dimethacrylate (EDMA), propan‐1‐ol, butane‐1,4‐diol, water, and ammonium peroxodisulfate as initiator. It was necessary to add N,N,N′,N ′‐tetramethylethylenediamine (TEMED) to the polymerization mixture to activate the reaction. The amount of initiator and activator was optimized to attain quantitative polymerization. The reproducibility of three columns prepared at ambient temperature was studied. The most efficient column with HETP of 29 μm for uracil was compared to the monolithic column prepared by thermal initiation with α,α′‐azobisisobutyronitrile (AIBN). The efficiencies of all the test columns were characterized by van Deemter curves. Their total porosities were calculated from the retention time of uracil. Walters indices of hydrophobicity (HI) were calculated from the retention factors of anthracene and benzene. The columns prepared by both methods are comparable in their selectivities and efficiencies. They show the same characteristics because their total porosities and Walters indices of hydrophobicity are consistent. However, the preparation of monoliths using ammonium peroxodisulfate was less demanding, because polymerization was possible at ambient temperature.     

Developments in suppressor technology for inorganic ion analysis by ion chromatography using conductivity detection

A review is presented detailing the development and use of suppression devices for the conductimetric detection of inorganic ions by ion chromatography (IC). An overview of the general response equation for conductivity detection is also given. Topics of discussion include the role and function of suppressors, the development of early suppressors including packed column and membrane devices from 1975 to 1990 and the subsequent progression towards present day commercially available suppressors and recent innovations. Post-suppression devices for signal enhancement are also discussed.     

Preparation of monolithic silica columns for high-performance liquid chromatography

Preparation methods of monolithic silica columns for HPLC including the surface modification were reviewed. Chemical modification methods recently reported to obtain stationary phases for reversed-phase (RP), chiral, ion-exchange, and hydrophilic interaction chromatography (HILIC) separations were discussed. Recent results related to preparation methods of monolithic silica were also covered. The characteristics and properties of silica monoliths and some applications of monolithic silica columns for different analytical and bioanalytical fields will be commented.