Particle packed mixed-mode chromatographic stationary phase for the separation of peptide in liquid chromatography.

A particle-based stationary phase has been prepared for the separation of five synthetic peptides and a mixture containing tryptic digest of cytochrome C in liquid chromatography. Particles originating from silica monolith were differentially sedimented to obtain 1-2 µm particles. A stationary phase was achieved by the coating of poly(styrene-methacrylic acid-N-phenylacrylamide) copolymer onto the particles via reversible addition-fragmentation chain transfer polymerization reaction. Stainless steel column (30 cm long and 1 mm internal diameter) was packed with stationary phase. Very high separation efficiency (ca. 351 000 plates/m) was achieved for five commercial peptides with a percent relative standard deviation of less than 1%. Protocol for the synthesis and modification of silica monolith particles has been well optimized with a good reproducibility both in particle and pore size. The column resolved about 21 peptide components from a mixture containing tryptic digest of cytochrome C, under the elution conditions of acetonitrile/15 mM ammonium format (65/35 v/v%) with a flow rate of 28 µL/min.

Styrene-N-phenylacrylamide co-polymer modified silica monolith particles with an optimized mixing ratio of monomers as a new stationary phase for the separation of peptides in high performance liquid chromatography.

A stationary phase was prepared by chemical derivatization of the support particles with a layer of copolymer composed of styrene and N-phenyl acrylamide. Silica monolith particles of ca. 2.6 µm (volume-based average) have been prepared as the support particles by sol-gel reaction followed by differential sedimentation. The particles were reacted with 3-chloropropyl trimethoxysilane followed by sodium diethyldithiocarbamate to introduce an initiator moiety. Then, the copolymer layer was immobilized via reversible addition-fragmentation transfer polymerization. The resultant phase was packed in glass-lined stainless-steel micro-columns (1 x 150 mm) and evaluated for the separation of a mixture composed of five peptides (Trp-Gly, Thr-Tyr-Ser, angiotensin I, isotocin and bradykinin). The effect of monomer mixing ratio (styrene versus N-phenyl acrylamide) on the chromatographic separation efficiency of the stationary phase was examined. A number of theoretical plates (N) as high as 33 600 plates/column (224 000 plates/m, 4.46 µm plate height) was achieved using the column packed with the optimized stationary phase. The column-to-column reproducibility based on three columns packed with three different batches of stationary phase was found satisfactory in separation efficiency, retention factor, and asymmetry factor.

An optimized mixed-mode stationary phase based on silica monolith particles for the separation of peptides and proteins in high-performance liquid chromatography.

A phase with both hydrophobic and hydrophilic functionalities has been synthesized by modification of ground silica monolith particles with C18 and 1-[3-(trimethoxysilyl)propyl] urea ligands. A series of phases was prepared by changing the ratio of the two ligands to determine the optimal ratio in view of separation efficiency. The resultant optimized stationary phase was packed in narrow-bore glass-lined stainless-steel columns (1 × 300 mm and 2.1 × 100 mm) and used for the separation of synthetic peptides and proteins. The average numbers of theoretical plates (N) of 52 100/column (174 000/m, 5.75 µm plate height) and 35 500/column (118 000/m, 8.47 µm plate height) were achieved with the 300 mm column at a flow rate of 25 µL/min (0.86 mm/s) in 60:40 v/v acetonitrile/30 mM aqueous ammonium formate for the mixture of peptides (Thr-Tyr-Ser, Val-Ala-Pro-Gly, angiotensin I, isotocin, and bradykinin) and for the mixture of proteins (myoglobin, human serum albumin, and insulin), respectively. Fast analysis of the peptides and proteins was also carried out at a flow rate of 0.9 mL/min (6.88 mm/s) with the 100 mm column and all the analytes were eluted within 2 min with good separation efficiency.

Open tubular capillary column of 50 µm internal diameter with a very high separation efficiency for the separation of peptides in CEC and LC.

A specially designed long open tubular capillary column (50 µm internal diameter and 112 cm effective length) was prepared by fabrication of a thin three-component co-polymer layer on the inner surface of silica capillary. A pretreated silica capillary was reacted with 4-(chloromethyl)phenyl isocyanate in the presence of dibutyltin dichloride as catalyst followed by sodium diethyl dithiocarbamate. Then a thin polymer layer was made on the inner surface of capillary by reversible addition-fragmentation transfer polymerization of styrene, N-phenylacrylamide, and methacrylic acid. A carefully adjusted formulation of reaction mixture and elaborated procedures were adopted to secure formation of the co-polymer layer of enhanced separation performance. The co-polymer immobilized open tubular capillary column was used for the separation of a synthetic mixture of five peptides and excellent separation efficiency (over 1.7 million per column) was obtained in the capillary electrochromatography mode. Such excellent separation efficiencies of ca. 1 m column have not been obtained in the isocratic elution mode so far. The column was also used for separation of the peptides in the liquid chromatography mode to show very good separation efficiency (average 286 700 per column).

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.

Disposable microcolumns with welded metal frits.

This study reports the preparation of disposable microcolumns with welded metal frits for the first time. First, the bottom of glass-lined stainless-steel tubing of 30 cm length, 1.6 mm od, and 0.5 mm id was welded with a stainless-steel screen frit of 1.6 mm diameter. A micro-welding machine was used for this. Next, the column was connected to a slurry packer and packed with porous silica particles. Then, the inlet of the column was carefully welded with another frit. The column was tested for separation of a test mix composed of phenol, 2-nitrophenol, acetophenone, aceanilide, and benzamide. Another column of the same physical dimension was also prepared with frits that were not welded to the column. The chromatographic performances of the two groups of columns (welded frits versus non-welded frits) were examined. The columns of welded frits showed ca. 18% better separation efficiency (number of theoretical plates) than those of non-welded frits.

Open tubular capillary electrochromatography with an N-phenylacrylamide-styrene copolymer-based stationary phase for the separation of anomers of glucose and structural isomers of maltotriose.

A ligand with a terminal halogen (4-chloromethylphenyl isocyanate) was chemically bound on the inner surface of pretreated silica capillary with 50 µm internal diameter and 58 cm total and 50 cm effective length in the presence of dibutyl tin dichloride as a catalyst through isocyanate-hydroxyl reaction. Attachment of initiator (sodium diethyl dithiocarbamate) to the bound ligand was carried out and followed by in situ polymerization. Reversible addition-fragmentation chain transfer polymerization was used for the immobilization of N-phenylacrylamide-styrene copolymer on the inner surface of capillary column. The resultant open tubular column showed excellent separation performance for derivatized saccharide isomers in capillary electrochromatography. D-Glucose was separated into α- and ß-anomers while five structural isomers were separated for derivatized maltotriose with separation efficiency above one million theoretical plates per meter. The effects of pH and acetonitrile composition on the electrochromatographic performance of the derivatized saccharides were studied and the optimized elution condition was found to be 90:10 v/v% acetonitrile/30 mM sodium acetate at pH 6.6. UV absorption at 214 nm was used as detection mode in open tubular capillary electrochromatography separations.

Organic monolith frits encased in polyether ether ketone tubing with improved durability for liquid chromatography.

This study introduces a preparation method for polymer-encased monolith frits with improved durability for liquid chromatography columns. The inner surface of the polyether ether ketone tubing is pretreated with sulfuric acid in the presence of catalysts (vanadium oxide and sodium sulfate). The tubing was rinsed with water and acetone, flushed with nitrogen, and treated with glycidyl methacrylate. After washing, the monolith reaction mixture composed of lauryl methacrylate, ethylene glycol dimethacrylate, initiator, and porogenic solvent was filled in the tubing and subjected to in situ polymerization. The tubing was cut into thin slices and used as frits for microcolumns. To check their durability, the frit slices were placed in a vial and a heavy impact was applied on the vial by a vortex mixer for various periods. The frits made in the presence of catalysts were found to be more durable than those made without catalysts. Furthermore, when the monolith-incorporated tubing was used as a chromatography column, the column prepared in the presence of catalysts resulted in a better separation efficiency. The separation performance of the columns installed with the polyether ether ketone encased monolith frits was comparable to that of the columns installed with the commercial stainless-steel screen frits.

Open tubular capillary column for the separation of cytochrome C tryptic digest in capillary electrochromatography.

A silica capillary of 50 µm internal diameter and 500 mm length (416 mm effective length) was chemically modified with 4-(trifluoromethoxy) phenyl isocyanate in the presence of dibutyl tin dichloride as catalyst. Sodium diethyl dithiocarbamate was reacted with the terminal halogen of the bound ligand to incorporate the initiator moiety, and in situ polymerization was performed using a monomer mixture of styrene, N-phenylacrylamide, and methacrylic acid. The resultant open tubular capillary column immobilized with the copolymer layer was used for the separation of tryptic digest of cytochrome C in capillary electrochromatography. The sample was well eluted and separated into many components. The elution patterns of tryptic digest of cytochrome C were studied with respect to pH and water content in the mobile phase. This preliminary study demonstrates that open tubular capillary electrochromatography columns with a modified copolymer layer composed of proper nonpolar and polar units fabricated by reversible addition-fragmentation transfer polymerization can be useful as separation media for proteomic analysis.

Fritting techniques in chromatography.

It is surprising that there has been no devoted review article for frits and relevant studies so far despite the long history of packed columns and the use of frits in them. This review was activated for such a reason. Both separate frits and in situ permanent frits have been covered since the appearance of primitive frits. The in situ fritting methods such as the formation of organic monoliths, sol-gel technology, sintering, fritless techniques such as tapered tip and capillary restrictors, and miscellaneous fritting techniques including magnetically trapped frits and single particle frits are introduced and discussed. In addition, frit-related studies and patents are also introduced. Finally, some conclusive comments on the choice of fritting technique in different situations and future perspectives are given.

C18-bound porous silica monolith particles as a low-cost high-performance liquid chromatography stationary phase with an excellent chromatographic performance.

Ground porous silica monolith particles with an average particle size of 2.34 µm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol-gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end-capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185,000/m) and chromatographic resolution were achieved when the C18 -bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless-steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 µL/min. The column was also evaluated for fast analysis at a flow rate of 100 µL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60,000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C18 modification in addition to partial-monolithic architecture has resulted in a useful stationary phase (C18 -bound silica monolith particles) of low production cost showing excellent chromatographic performance.

Molecular imprinted polymers for separation science: a review of reviews.

Molecular imprinted polymer is an artificial receptor made by imprinting molecules of a template in a polymer matrix followed by removing the template molecules via thorough washing to give the permanent template grooves. They show favored affinity to the template molecule compared to other molecules, and this property is the basic driving force for such diverse application of this techniques. Such techniques have been increasingly employed in a wide scope of applications such as chromatography, sample pretreatment, purification, catalysts, sensors, and drug delivery, etc., mostly in bioanalytical areas. A major part of them is related to development of new stationary phases and their application in chromatography and sample pretreatment. Embodiments of molecular imprinted polymer materials have been carried out in a variety of forms such as irregularly ground particles, regular spherical particles, nanoparticles, monoliths in a stainless steel or capillary column, open tubular layers in capillaries, surface attached thin layers, membranes, and composites, etc. There have been numerous review articles on molecular imprinted polymer issues. In this special review, the reviews in recent ca. 10 years will be categorized into several subgroups according to specified topics in separation science, and each review in each subgroup will be introduced in the order of date with brief summaries and comments on new developments and different scopes of prospects. Brief summaries of each categories and conclusive future perspectives are also given.

Synthesis of monolith silica anchored graphene oxide composite with enhanced adsorption capacities for carbofuran and imidacloprid.

Highly efficient adsorbent was prepared for the removal of carbofuran and imidacloprid pesticides from wastewater. The silica monolith anchored graphene oxide composite was synthesized by the modified Fischer esterification protocol. The composite showed improved adsorption capacity for the removal of pesticides from wastewater. Graphene oxide was synthesized using the modified Hummer's method, while the silica monolith was prepared via sol-gel method. The composite was characterized via X-ray diffraction, Fourier transform infra-red, Brunauer Emmett and Teller (BET/BJH) analysis, zeta potential, and FESEM imaging. Different adsorption parameters such as pH, contact time, adsorbate and adsorbent concentration, and temperature were optimized for the adsorption of pesticides. The equilibrium and kinetic models were applied to the adsorption process of the pesticides. Qe of the composite as found to be 342.46 mg g-1 for imidacloprid and 37.15 mg g-1 for carbofuran. The adsorption process followed the pseudo 2nd order kinetic model for carbofuran (R2~0.9971) and imidacloprid (R2~0.9967). The Freundlich isotherm best fitted to the adsorption data of the pesticides with R2 value of 0.9956 for carbofuran and 0.95 for imidacloprid. The resultant adsorbent/composite material came out with very good results for the removal of pesticides.

Synthesis, column packing and liquid chromatography of molecularly imprinted polymers for the acid black 1, acid black 210, and acid Brown 703 dyes.

Molecularly imprinted polymers have been synthesized for the acid black 1, acid black 210, and acid brown 703 dyes using methacrylic acid, ethylene glycol, and azobisisobutyronitrile as the monomer, cross-linker, and initiator, respectively, in the ratio of 1 : 10 : 44 (template:monomer:cross-linker). The MIPs were used for the selective removal of their corresponding dyes. The selective nature of the MIPs towards their respective dyes was confirmed by a homemade liquid chromatography system. The resultant polymer materials were packed in a stainless steel column and checked for the separation of mixtures of dyes in liquid chromatography. The dyes complementary in structure to the imprinted cavities in the MIPs had long retention times, showing the highly selective nature of the MIPs. The pH, quantity of the MIPs, time, and concentration of the dyes were optimized for the highly efficient removal of the newly synthesized MIP adsorbents in batch adsorption studies. First-order, second-order, and intra-particle diffusion models were applied to all the three MIP-based adsorbents for their kinetic investigations towards the dyes. All the three MIPs selectively absorbed their target template molecule in the presence of four other template dyes having closely related structures with % RSD < 4% for the three batch experiments. The synthesized MIPs were characterized by FTIR, SEM imaging and liquid chromatography. FTIR results strongly confirmed the presence of hydrogen bonding interactions (600-900) between the template and the individual monomers present in the unwashed MIPs. Liquid chromatography revealed the highly selective nature of the MIPs towards their template molecules. The synthesized polymeric substances possess excellent thermal, chemical, and mechanical stability and can be reused several hundred times. The MIPs were applied in the removal of dyes from spiked water samples (river water, tap water and distilled water) where the % removal of the dyes by their corresponding MIPs was greater than 90%.

Analysis of phospholipids using an open-tubular capillary column with a monolithic layer of molecularly imprinted polymer in capillary electrochromatography-electrospray ionization-tandem mass spectrometry.

In this study, an open-tubular capillary electrochromatography (OT-CEC) column with a monolithic layer of molecularly imprinted polymer (MIP) based on methacrylic acid, ethylene glycol dimethacrylate, and 4-styrenesulfonic acid was utilized for the simultaneous separation and characterization of phospholipid (PL) molecular structures by interfacing with electrospray ionization-tandem mass spectrometry (ESI-MS-MS). Introducing an MIP-based monolith along with charged species at the OT column made it possible to separate PL molecules based on differences in head groups and acyl chain lengths in CEC. For the interface of OT-CEC with ESI-MS-MS, a simple nanospray interface utilizing a sheath flow was developed and the resulting OT-CEC-ESI-MS-MS was able to separate PL standards (phosphatidylserines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acid, and lysophosphatidylglycerols). The developed method was applied to human urinary lipid extracts, and resulted in the separation and structural identification of 18 molecules by data-dependent collision-induced dissociation.

Preparation of open tubular molecule imprinted polymer capillary columns with various templates by a generalized procedure and their chiral and non-chiral separation performance in CEC.

A generalized preparation procedure of open tubular (OT) molecule imprinted polymer (MIP) columns is proposed for a number of templates with acidic functionality such as profen drugs and others. The template (S-enantiomer) was mixed with methacrylic acid, ethylene glycol dimethacrylate and 4-styrenesulfonic acid, dissolved in a porogen mixture of ACN/2-propanol (9/1), and incubated in a pretreated and silanized fused silica capillary by the thermal non-covalent polymerization procedure. The whole preparation procedure was exactly the same for all the MIP capillaries except for the selection of template. Nevertheless, the morphologies of the MIP layers were markedly variant depending upon the choice of template. The separation efficiency of each OT-MIP column for chiral separation of R- and S-enantiomers was examined and tuned to obtain the best separation efficiency by changing the chromatographic parameters such as eluent composition and pH. Different optimized conditions were obtained for different OT-MIP columns. Nevertheless, a unified eluent could be used to obtain still quite satisfactory results. Non-chiral separation of the MIP columns were also examined in the unified eluent with two sets of test mixtures, that is, a mixture of alkylbenzenes and a mixture of small polar solutes. The chiral and non-chiral separation of this study resulted in very good separation efficiencies. This work is the very first study for the generalization of preparation of OT-MIP columns for a number of templates.

Use of chain transfer agent attached to silica particles in preparation of polystyrene-based stationary phases.

A new stationary phase has been prepared by attaching a new chain transfer agent on silica particles followed by reversible addition-fragmentation chain transfer polymerization. Porous silica particles were reacted with 3-chloropropyltrimethoxysilane followed by sodium diethyl dithiocarbarmate. The silica particles were then allowed to undergo radical polymerization with styrene. The phase had improved ligand morphology to show better separation performance than that of the conventional C18 phase.

Gas chromatography-mass spectrometric method for the screening and quantification of illicit drugs and their metabolites in human urine using solid-phase extraction and trimethylsilyl derivatization.

A simple and rapid GC-MS method has been developed for the screening and quantification of many illicit drugs and their metabolites in human urine by using automatic SPE and trimethylsilylation. Sixty illicit drugs, including parent drugs and their metabolites that are possibly abused in Korea, can be monitored by this method. Among them, 24 popularly abused illicit drugs were selected for quantification. Very delicate optimizations were carried out in SPE, trimethylsilylation derivatization, and GC/MS to enable such remarkable achievements. Trimethylsilylated analytes were well separated within 21 min by GC-MS. In the validation results, the LOD of all the analytes were in the range of 2-75 ng/mL. The LOQ of the quantified analytes were in the range of 5-98 ng/mL. The linearity (r(2)) of the quantified analytes ranged 0.990-1.000 in each concentration range between 10 and 1000 ng/mL. The mean recoveries ranged from 62 to 126% at three different concentrations of each analyte. The inter-day and inter-person accuracies were within -13.3 approximately 14.9%, and -10.1 approximately 13.0%, respectively, and the inter-day and inter-person precisions were less than 12.9%. The method was reliable and efficient for the screening and quantification of abused illicit drugs in routine urine analysis.

Long open tubular molecule imprinted polymer capillary columns with excellent separation efficiencies in chiral and non-chiral separation by capillary electrochromatography.

Long open tubular S-ketoprofen molecule imprinted polymer capillary columns (1, 2, and 3 m) with the number of theoretical plates (N) over a million for both chrial and non-chiral CEC separation have been prepared. Chiral separation of racemic ketoprofen and non-chiral separation of other profen drugs have been achieved with excellent efficiency and good selectivity after optimization of the chromatographic conditions. The N value per column was not proportional to the effective column length although it was increased with the column length. This study has shown the outstanding prospect of the strategy to form a porous and rugged open tubular layer in a moderately long (1 m) capillary column in molecule imprinted polymer-CEC studies.

Open tubular layer of S-ofloxacin imprinted polymer fabricated in silica capillary for chiral CEC separation.

An open tubular molecule imprinted polymer (OT-MIP) capillary column has been prepared for chiral separation of ofloxacin enantiomers in CEC. The S-ofloxacin imprinted OT column was fabricated by thermally initiated non-covalent polymerization procedure inside a pretreated and silanized fused silica capillary. The template molecule was incorporated with methacrylic acid (MAA), ethylene glycol dimethacrylate (EDMA) and 4-styrenesulfonic acid (4-SSA) and dissolved in a porogen mixture of ACN/2-propanol (9:1). The separation efficiency of the 4-SSA MIP column was found quite better than that of the MIP column without 4-SSA. It has been demonstrated that our OT-MIP column can separate ofloxacin enantiomers with excellent chiral separation efficiency after tuning the various chromatographic conditions. The optimized chromatographic eluent was 85:15, v/v%, ACN/60 mM sodium acetate at pH 7. The separation efficiency and selectivity of chiral separation of this study were far better than those obtained by previous methods for chiral separation of R- and S-ofloxacin.