Novel approach for fritless capillary electrochromatography

At present, the main limitation for the further adoption of capillary electrochromatography (CEC) in the (routine) laboratory is caused by the lack of reproducible and stable columns. The main source of column instability is concentrated in the frits needed to retain the packed bed inside the CEC capillary. The sintering process used to prepare the frits can be rather problematic and irreproducible, particularly for small stationary phase particles and wide column diameters. Since the (surface) composition of the frits is different from the bulk stationary phase packing, different electroosmotic flow (EOF) velocities are generated. This effect is assumed to be primarily responsible for rapid column destruction. In this contribution, a novel approach for the preparation of fritless CEC capillaries is presented and evaluated. Using 5 microm Hypersil ODS particles, separation efficiencies in the range of 130,000-200,000 plates/m were obtained. In a 100 microm inner diameter packed column, electrical currents up to 50 microA could be tolerated without negative effects such as bubble formation. The prepared CEC columns were found to be stable and could easily be operated continuously for several days without column damage. An additional advantage of the proposed tapering approach is that application of pressure on the in- and outlet vial during separation was not required to prevent bubble formation.     

Enantiomeric separation of amino acids and nonprotein amino acids using a particle-loaded monolithic column

A solution is prepared of 5 microm silica particles modified with (S)-N-3,5-dinitrobenzoyl-1-naphthylglycine (particle 1) or (S)-N-3,5-dinitrophenylaminocarbonyl-valine (particle 2) suspended in liquid tetraethylorthosilicate, ethanol, and aqueous hydrochloric acid. This solution is injected under pressure into a 30 cm long, 75 microm inner diameter capillary column and heated for 1 h at 120 degrees C after which the modified particles are embedded in a monolithic column of sol gel. The packed column measures approximately 15 cm from the inlet to the window used to view the laser-induced fluorescence. Thirteen different amino acids and three nonprotein amino acids are derivatized with the fluorogenic reagent 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) before injection onto the column for capillary electrochromatographic separation. The enantiomeric separation of the monolithic column packed with particle 1 results in a resolution ranging from 1.14 to 4.45, whereas that packed with particle 2 results in a resolution ranging from 0.79 to 1.17. On the basis of resolution and amount of chiral packing material the enantiomeric separation obtained by capillary electrochromatography is judged to be superior to that obtained previously with high performance liquid chromatography (HPLC).     

Capillary electrochromatography without external pressure assistance. Use of packed columns with a monolithic inlet frit

A fused silica capillary column was packed with RP(18) silica stationary phase entrapping the particles between two frits obtained by two different procedures. The inlet frit consisted of a short organic polymer made via a thermopolymerization process while the outlet frit was prepared by sintering the octadecylsilica (ODS) material. The packed column was employed in capillary electrochromatography (CEC) experiments for the separation of three selected test compounds. Retention time and separation efficiency were evaluated. Results were compared with those ones obtained with a packed capillary containing the same stationary phase entrapped between two sinterized frits. The novel packed column exhibited comparable separation efficiency and resolution with the traditional one. However, it allowed experiments without pressure support during the runs with no bubble formation.     

New particle-loaded monoliths for chiral capillary electrochromatographic separation

Fritless particle-loaded monoliths for chiral capillary electrochromatographic (CEC) separation were prepared. Silica particles containing a chiral selector are suspended in a monomer solution, which is drawn into the capillary followed by in situ polymerization. Thereby the silica-based particles containing the chiral selector are embedded in a nonchiral continuous bed. This kind of chiral stationary phase is inexpensive, easy, and reproducible to prepare and circumvents the preparation of frits. As a model, teicoplanin aglycone as chiral selector bonded to 3 microm silica particles was used. The applicability of this approach is demonstrated by means of the chiral separation of aliphatic and aromatic amino acids and dipeptides. As a further application, the chiral selector ristocetin A bonded to 3 microm silica particles was used for the enantiomeric separation of chiral alpha-hydroxy acids. Since alpha-hydroxy acids migrate toward the anode, a cationic charge-providing agent was copolymerized with the matrix. This served to reverse the direction of the electroosmatic flow (EOF).     

Fritless capillary electrochromatography

The preparation of packed capillaries with stable frits of good quality can be a hurdle to obtain efficient separations in capillary electrochromatography (CEC). Especially with particles smaller than 3 microm, frit preparation is cumbersome. Highly efficient separations using packed capillaries without frits are presented. Under appropriate CEC conditions the particles were retained by electrophoretic attraction towards the anode by a tapered capillary inlet, without the need of a frit at the outlet end. Such fritless capillaries, packed with 1.5 microm nonporous reversed-phase particles, allowed separations with efficiencies of more than 500,000 plates/m. Once the capillaries were conditioned properly, more than 100 separations could be performed with good repeatability. With respect to separation efficiency, fritless capillaries packed with 3 microm particles were comparable with standard CEC capillaries with frits. Examples of separations of steroids, a pesticide and its by-products, and cardiac glycosides under various CEC conditions are shown.     

Nonaqueous electrochromatography on C30 columns: separation of retinyl esters.

A nonaqueous packed capillary electrochromatographic reversed-phase method for separation of retinyl esters has been developed. The retinyl esters all-trans-retinyl acetate, palmitate, heptadecanoate, stearate, oleoate, and linoleoate were separated on a 180 microm ID column packed with 5 microm C30 particles with a mobile phase consisting of 2.5 mM lithium acetate in N,N-dimethylformamide-methanol (99:1, v/v). With this mobile phase, the electroosmotic flow was 0.8 mm/s at 650 V/cm and 40 degrees C, and the separation was completed in less than 30 min on 30 cm columns. To obtain electrostable frits of the hydrophobic C30 material both the preparation of the frits and the conditioning of the column prior to electroconditioning were of importance. Selectivity changes were introduced by replacing up to 70% v/v of the N,N-dimethylformamide by acetonitrile. The increase in the amount of acetonitrile was, however, accompanied by a significant increase in analysis time. Liver extracts obtained from arctic seal were analyzed.     

A Simple Method to Prepare On‐Capillary Frits for Micro‐Column Separation

Sintering stainless steel powders was initially used to prepare the inlet frit in fused silica capillaries. The use of such inlet frits and outlet frits sintered by the stationary phase itself in the capillary to retain C₁₈ particles was demonstrated to withstand the long exposure, up to a high pressure of 60 MPa, for packing and the prepare column was stable and robust enough to do the continuous chromatographic operations. Characterization of the inlet and outlet frits by scanning electron micrography showed the fused metal particles formed a porous network in the capillary inlet and the homogenous separation beddings were obtained by slurry packing.     

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.     

Polyether ether ketone encased monolith frits made of polyether ether ketone tubing with a 0.25 mm opening resulting in an improved separation performance in liquid chromatography

Tiny polyether ether ketone encased monolith frits have been prepared by modified catalytic sulfonation of the inner surface of polyether ether tubing (1.6 mm od, 0.25 mm id) followed by modified formation of organic monolith and cutting of the tubing into slices. The frit was placed below the central hole of the column outlet union and supported by a combination of a silica capillary (0.365 mm od, 0.05 mm id) and a polyether ether ketone sleeve (1.6 mm od, 0.38 mm id) tightened with a nut and a ferrule when the column was packed to prevent sinking of the frit element into the union hole (0.25 mm opening) otherwise. The column packed this way with the frits investigated in this study has shown better separation performance owing to the reduced frit volume in comparison to the column packed with a commercial stainless‐steel screen frit. This study establishes the strategy of disposable microcolumns in which cheap disposable frits are used whenever the column is re‐packed to yield columns of even better chromatographic performance than the columns with commercial frits.     

Microcolumns with self-assembled particle frits for proteomics

LC-MS-MS experiments in proteomics are usually performed with packed microcolumns employing frits or outlets smaller than the particle diameter to retain the packing material. We have developed packed microcolumns using self-assembled particles (SAPs) as frits that are smaller than the size of the outlet. A five to one ratio of outlet size to particle diameter appears to be the upper maximum. In these situations the particles assembled into an arch over the outlet like the stones in a stone bridge. When 3 microm particles were packed into a tapered column with an 8 microm outlet, two particles bridged the outlet with 0.3 pl dead volume and perfect success rate. In peptide analysis by LC-MS, the peak width at half height was normally less than 6 s, compared to 12 s without SAPs. The LC-MS-MS system provided 37% sequence coverage (21 matched peptides) for a tryptically-digested sample of 10 fmol bovine serum albumin. We also describe application of the SAP principle to make disposable pipette tip columns with short pieces of fused-silica capillary as the outlet.     

Magnetically immobilized frits for the preparation of packed columns used in capillary electrochromatography

Fabrication of porous frits to retain stationary phases is a critical issue in column preparation for capillary electrochromatography (CEC). In this work, porous frits were prepared by applying an external magnetic field to magnetically responsive particles placed inside a fused-silica capillary. Three batches of uniform magnetite spheres with particle diameters of 0.3, 0.4, and 0.6 μm and saturation magnetization values of 73.03, 74.41, and 77.83 emu/g, respectively, were used as frit particles and octadecyl- and phenyl-bonded silica gels were packed successfully into frit-containing capillaries. The performance of the resulting magnetically immobilized frits and packed columns was evaluated. The electroosmotic mobilities in capillaries containing outlet frit only were found to be reduced by 2–4% whereas the plate heights of an unretained marker increased by 30–50% as compared to those in open capillaries. These variations are believed to be associated with the inhomogeneities of the packed structure of the frits. The magnetically immobilized frits showed adequate mechanical strength to withstand the flow drag force, allowing separation in capillaries packed with 5-μm stationary phases up to 10–15 cm, thus rendering column efficiency and reproducibility comparable with those obtained with sintered frits. Taken together, retaining frits made of uniform magnetite particles serves as a viable alternative to sintered frits for column preparation, which offers several distinct advantages such as ease of preparation, improved durability as compared to sintered frits where the removal of the polyimide coating makes the packed column susceptible to breakage, and use of large-bore capillaries for semipreparative separations.     

Enantiomer separations by capillary electrochromatography using chiral stationary phases

The applicability of capillary electrochromatography (CEC) using packed capillary column to enantiomer separations was investigated. As chiral stationary phases, OD type packing materials of 5 and 3 microm particle diameters, originally designed for conventional high-performance liquid chromatography (HPLC) were employed. The chiral packing materials were packed by a pressurized method into a 100 microm I.D. fused-silica capillary. Several racemic enantiomers, such as acidic, neutral and basic drug components, were successfully resolved, typically by using acidic or basic solutions containing acetonitrile as mobile phases. The separation efficiencies for some enantiomers in the chiral CEC system using the 5 microm OD type packing were superior to those obtained in HPLC using chiral packings. The plate heights obtained for several enantiomers were 8-13 microm or the reduced plate height of 1.6-2.6, which indicates the high efficiency of this chiral CEC system.     

The Development of Reversed‐Phase Capillary Electrochromatography for the Separation of Steroids

This paper describes the preparation and optimization of packed capillary columns for reversed‐phase separation of steroids with CEC. The fabrication of on‐column frits is considered to be the most important step for obtaining a reproducible packed column for CEC separation. Porous silicate frits were generated in a fused‐silica capillary by heating the silica gel/sodium hydroxide solutions electrically. The optimized conditions involve silica gel (10.8%), sodium hydroxide (5.8%), and heating time (5 sec) with heating voltage (5V) for obtaining a 100‐μ end‐frit that can withstand pressure over 6000 psi. A HPLC pump was utilized to pack the 5‐μm ODS particle slurry into the capillary column. The ODS packed capillaries were then utilized for the separation of four anabolic cholesterols with a capillary electrophoresis system without pressurization of the column. The reproducibility of the packed columns was evaluated by measuring the relative standard deviations of four steroids. The relative standard deviations of migration time for column‐to‐column, day‐to‐day, and run‐to‐run are less than 7%, 2%, and 1% for four steroids, respectively.     

Pressurized gradient capillary electrochromatographic separation of eighteen amino acid derivatives

A pressurized gradient capillary electrochromatography (pCEC) instrument was developed to separate 18 amino acid derivatives. A reversed-phase C18 column (3 microm, 130 mm x 75 microm I.D.) and an acetate buffer (50 mmol/l NaAc, pH 6.4) with an ion-pair reagent (1% N,N-dimethylformamide) were used to separate derivatized amino acids from a standard solution (2 microg/ml), and the wavelength of the UV-Vis detector was 360 nm. The pressure on the capillary column was kept at approx. 70 Pa and 3 kV positive voltage was added on the outlet end of column. The effect of voltage on the eluting order of amino acids and the resolution of separation were studied, and it was found that when the voltage was higher than 3 kV, the adsorption of amino acids in the porous C18 column occurred. The effect of salt concentration, injection volume, and column length on the separation of amino acids was determined. The amino acid sample was separated by pCEC, and RSDs of the migration times of each amino acid were all less than 2.5%.     

Fluorescence imaging of frit effects in capillary separations

The gradient of five dansylated amino acids in a capillary-based separation system commonly used in capillary liquid chromatography (LC) or capillary electrochromatography (CEC) was followed and examined in detail using a detection method based on laser-induced fluorescence imaging. The detection system consisted of an XeCl excimer laser and an image-intensified charge-coupled device (CCD) camera. Fluorescence intensity profiles along the capillary column were displayed and continuously updated on a computer screen. The detector system enabled the separation dynamics in the column to be monitored. The experiments were focused on the course of events, especially at interfaces. The processes occurring at the beginning of the reversed-phase packing material were studied as well as at the transition from stationary phase to the outlet frit and the open tubular area. Striking differences in signal intensity and separation efficiency were revealed depending on where on the column detection was performed. Furthermore, adsorption of the analytes on the frits was observed.     

Recent innovations in enantiomer separation by electrochromatography utilizing modified cyclodextrins as stationary phases.

Enantiomer separation by electrochromatography employing modified cyclodextrins as stationary phases is performed in two ways. (i) Polysiloxane-linked permethylated beta-cyclodextrin (Chirasil-Dex 1) or related selectors are coated and immobilized onto the inner surface of a capillary column. Enantiomer separation is performed in the open tube and the method is referred to as open-tubular capillary electrochromatography (o-CEC). (ii) Silica-linked native beta-cyclodextrin, permethylated beta-cyclodextrin (Chira-Dex 2) or hydroxypropyl-beta-cyclodextrin are filled into a capillary column and the bed is secured by two frits. Enantiomer separation is performed in a packed column and the method is referred to as packed capillary electrochromatography (p-CEC). In a unified instrumental approach, method (i) as well as method (ii) can be operated both in the electro- and pressure-driven modes (o-CEC vs. open-tubular liquid chromatography (o-LC) and p-CEC vs. p-LC). It is demonstrated that the electro-driven variant affords higher efficiencies at comparable elution times. Employing a single open-tubular column coated with Chirasil-Dex 1, a unified enantioselective approach can be realized in which the same selectand is separated using all existing chromatographic modes for enantiomers, i.e., gas chromatography (GC), super-critical fluid chromatography (SFC), o-LC and o-CEC. As the chiral selector is utilized as a stationary phase, an additional chiral selector may be added to the mobile phase. In the resulting dual chiral recognition systems, enhancement of enantioselectivity (matched case) or compensation of enantioselectivity (mismatched case) may be observed. The overall enantioselectivity is dependent on the sense of enantioselectivity of the selectors chosen and their influence on the electrophoretic and electroosmotic migration of the enantiomers of a selectand.     

Robust open tubular layer of S-ketoprofen imprinted polymer for chiral LC separation

This study is about the preparation of an open tubular capillary column of molecularly imprinted polymer (MIP) and its application to chiral separation by microLC. A non-covalent in-situ molecular imprinting polymerization protocol was used to synthesize the S-ketoprofen MIP. A special procedure was employed to secure formation of an open tubular and rigid MIP layer in a silica capillary of 100 microm id. The capillary was filled with the reaction mixture, sealed, and placed in a water bath at 50 degrees C for 3 h. Then it was flushed with a 0.5 MPa nitrogen flow for 5 min, and was again placed in the water bath for 2 h to complete MIP formation. Methacrylic acid (MAA) has been known to be an inefficient functional monomer in preparation of MIP of an acid molecule. However, MAA was used with ethylene glycol dimethacrylate in preparation of the S-ketoprofen MIP in this study. The open tubular structure and the microLC mode of separation enabled free optimization without any restriction, thus a very good resolution (R=4.7) of ketoprofen enantiomers was achieved when a mobile phase composed of 30% acetonitrile and 70% acetate buffer at pH 4.5 was used with 5 mbar inlet pressure. This may be partially attributed to the open tubular structure of our MIP, enabling low column back-pressure and free optimization of eluent composition, as well as to the small capillary dimensions. Our MIP capillary column also showed some versatility in chiral separation, thus a good chiral separation was observed for naproxen, ibuprofen, and fenoprofen enantiomers.     

Development of a fritless packed column for capillary electrochromatography-mass spectrometry

A novel procedure was developed for the fabrication of a fritless packed column for the coupling of capillary electrochromatography (CEC) to mass spectrometry (MS). The process involved the formation of internal tapers on two separate columns. Once the internal tapers are formed and the columns are packed, the untapered ends of each column were joined together by a commercially available connector. Several advantages of the fritless columns are described. First, the design used here eventually eliminates the need for any frits thus reducing the possibility of bubble formation seen with fritted packed columns. In addition, this is the first report in which the internal tapers are formed at both the inlet and outlet column ends making the fritless CEC-MS column more robust compared to only one report with externally tapered counterparts. Second, a comparison of internally tapered single frit packed CEC-MS (previously developed in our laboratory) column versus fritless CEC-MS column reported here shows that the latter provides better efficiency, suggesting no dead volume with equally good sensitivity and chiral resolution of (±)-aminoglutethimide. The fritless column procedure is universal and was used to prepare a series of columns with a variety of commercially available packing material (mixed mode strong cation exchange, SCX; mixed mode strong anion exchange, SAX; C-18) for the separation and MS detection of short chain non-chromophoric polar amines, long chain nonchromophic anionic surfactant as well as oligomers of non-chromophoric non-ionic surfactants, respectively. The fritless columns showed good intra-day repeatability and inter-day reproducibility of retention times, chiral and achiral resolutions and peak areas. Very satisfactory column-to-column and operator-to-operator reproducibility was demonstrated.     

Monolithic silica-based capillary column with strong chiral cation-exchange type surface modification for enantioselective non-aqueous capillary electrochromatography

A silica-based monolithic stationary phase prepared by the sol-gel process in a 100 microm I.D. fused-silica (FS) capillary has been modified chemically with 3-mercaptopropyl trimethoxysilane followed by immobilization of a strong cation-exchange (SCX) type chiral selector, (S)-N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutane phosphonic acid, by radical addition reaction onto the reactive sulfhydryl surface. After a fine-tuning of the mobile phase composition, the enantioselective capillary column was evaluated for the separation of various chiral basic drugs by enantioselective non-aqueous capillary electrochromatography (CEC), in comparison to capillary column analogs packed with 3.5 microm silica particles having attached the same selector. The performance of the monolithic silica column was further compared to corresponding polymethacrylate-based organic polymer monoliths. The study indicated that strong counter-ions such as 2-aminobutanol or N,N,N',N'-tetramethylethylenediamine are needed, although they reduce the electroosmotic flow velocity and separation factors in comparison to less efficient counter-ions, in order to allow the elution of the oppositely charged solutes in the ion-exchange retention mode within reasonable run time and as sharp zones. In contrast, weak counter-ions such as N,N-diisopropylethylamine (Huenig base) provided stronger electroosmotic flow and much better separation factors, but relatively poor peak efficiencies. Overall, with the chemically functionalized monolithic silica column the high quality separations of packed column analogs could be approximated, with regards to both separation factors and peak performances. On the other hand, the monolithic capillary column certainly outperformed the packed column in terms of system robustness under capillary electrochromatography conditions and showed excellent column longevity. The enantioselective strong cation-exchange-type monolithic silica column performed also well in comparison to the organic polymer monolith.     

Chemically modified chiral monolithic silica column prepared by a sol-gel process for enantiomeric separation by micro high-performance liquid chromatography

In this work a new type of chiral monolith silica column was developed for the chiral separation by micro high-performance liquid chromatography (micro-HPLC). The chiral monolith column with a continuous skeleton and a large through-pore structure was prepared inside a capillary of 100 microm I.D. by a sol-gel process, and chemically modified with chiral selectors, such as L-phenylalaninamide, L-alaninamide and L-prolinamide, on the surface of the monolithic silica column. Based on the principle of ligand exchange, these chiral monolithic columns were successfully used for the separation of dansyl amino acid enantiomers, as well as hydroxy acid enantiomers by micro-HPLC. The chromatographic conditions, the enantioselectivity and the performance of columns are discussed.