Application of Convective Interaction Media (CIM) disk monolithic columns for fast separation and monitoring of organic acids

The separation of organic acids on the anion-exchange monolithic support, commercially available as Convective Interaction Media (CIM), is presented in this study. It is demonstrated that citric, isocitric, pyruvic, fumaric, malic, and alpha-ketoglutaric acid can be successfully separated using a CIM monolithic column of suitable user-adjustable length. The effect of the mobile phase composition on the separation is investigated. CIM monolithic columns of adjustable length from 3 to 18 mm are compared regarding the resolution and the back pressure. It is shown that the CIM monolithic column of 12 mm in length enables a good separation of all six organic acids within 3 min and exhibits a linear dependence of back pressure versus flow rate. The resolution and the dynamic binding capacity are found to be flow-unaffected. A filtrated sample of bioprocess supernatant is analyzed without previous pretreatment, which indicates the possibility of online monitoring of small molecules during the bioprocess using CIM monolithic columns.     

Separation of pectin methylesterase isoenzymes from tomato fruits using short monolithic columns

One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.1.1) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.     

Potential for the speciation of Zn using fast protein liquid chromatography (FPLC) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection

Analytical procedures were developed for the speciation of Zn using fast protein liquid chromatography (FPLC), flame atomic absorption spectrometry (FAAS) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection. The investigation was performed on synthetic solutions (2 microg cm-3 Zn) of hydrated Zn2+ species and Zn complexes with citrate, oxalate and EDTA (ligand-to-Zn molar ratio 100:1) over a pH range from 5.4 to 7.4. It was found that Zn interacts with various buffers and the careful adjustment of the pH with diluted solutions of KOH is, therefore, required. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange column, applying an aqueous 1 mol dm(-3) NH4NO3 linear gradient elution over 15 min, at a flow rate of 1.0 cm3 min(-1). The separated Zn species were determined in 1.0 cm3 eluate fractions "off line" by FAAS. Speciation of Zn was also performed on a weak anion-exchange CIM DEAE fast monolithic disc by applying an aqueous 0.4 mol dm(-3) NH4NO3 linear gradient elution over 7.5 min, at a flow rate of 2.0 cm3 min(-1) and determination of the separated Zn species in 1.0 cm3 eluate fractions "off line" by FAAS. Zn-binding ligands in separated fractions were also characterized by electrospray (ES)-MS-MS analysis. The CIM DEAE disc was found to be more efficient in the separation of negatively charged Zn complexes than the Mono Q FPLC column. On the CIM DEAE disc Zn-citrate was separated from both Zn-oxalate and from Zn-EDTA. All these species were also separated from hydrated Zn2+, which was eluted with the solvent front. This method has an advantage over commonly used analytical techniques for the speciation of Zn which are only able to distinguish between labile and strong Zn complexes. Good repeatability of the measurements (RSD 2-4%), tested for six parallel determinations (2 microg cm(-3) Zn) of Zn-EDTA, Zn-citrate and Zn-oxalate was found at a pH of 6.4 on a CIM DAEA disc. The limit of detection (3s) for the separated Zn species was 10 ng cm(-3). The proposed analytical procedure was applied to the speciation of Zn in aqueous soil extracts and industrial waste water from a lead and zinc mining area.     

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of approximately 25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18相似文献   

Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilization of antibodies

Convective interaction media (CIM; BIA Separations) monoliths are attractive stationary phases for use in affinity chromatography because they enable fast affinity binding, which is a consequence of convectively enhanced mass transport. This work focuses on the development of novel CIM hydrazide (HZ) monoliths for the oriented immobilization of antibodies. Adipic acid dihydrazide (AADH) was covalently bound to CIM epoxy monoliths to gain hydrazide groups on the monolith surface. Two different antibodies were afterwards immobilized to hydrazide functionalized monolithic columns and prepared columns were tested for their selectivity. One column was further tested for the dynamic binding capacity.     

阴阳离子交换色谱串联分离纯化苦瓜籽核糖体失活蛋白

将阴阳离子交换色谱柱在灌注色谱工作站上串联,使苦瓜籽粗提液中未被阴离子色谱柱吸附的蛋白质直接在阳离子色谱柱上吸附,用盐线性梯度洗脱从阳离子色谱柱上分离出两个具有抗真菌活性的蛋白。两个抗真菌蛋白经鉴定都达到了电泳纯,相对分子质量均为30000左右,N-端氨基酸序列分别为DVSFRLSGADPRSYGMFI与DVNFDLSTATAK,表明所得到的两种蛋白为苦瓜籽中的2个Ⅰ型核糖体失活蛋白α-苦瓜素(α-MMC) 和β-苦瓜素(β-MMC)。抗菌实验表明,α-MMC对香蕉枯萎菌和果腐霉菌均具有较强的抑制作用,β     

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

Characterisation of metal-chelate methacrylate monoliths

Monoliths are attractive stationary phases for purification of large biomolecules like proteins because of their flow-unaffected properties. Isolation of histidine containing proteins to high purity can be efficiently performed using metal-chelate interactions within a single chromatographic step. In this work, we investigated properties of commercial metal-chelate methacrylate monoliths-Convective Interaction Media (CIM). Analytical CIM disk monolithic columns and CIM 8 ml monolithic columns were used for purification of tumor necrosis factor-alpha (TNF-alpha) analog LK-801 and green fluorescence protein with 6 histidine tag (GFP-6His). In both cases, purity over 90% was achieved. Dynamic binding capacity at 10% of breakthrough was around 17-18 mg/ml for LK-801 and around 30 mg/ml for GFP-6His. Adsorption isotherm revealed that the maximal capacity is achieved at protein concentration above 60 microg/ml. Dynamic binding capacity and resolution were found to be flow unaffected.     

Improvement of proteome coverage using hydrophobic monolithic columns in shotgun proteome analysis

Monolithic columns are widely used in shotgun proteome analysis. However, it is difficult to increase the separation capability and proteome coverage by using conventionally organic polymer-based monolithic column due to the difficulty of controlling homogeneity of the overall pore structure (both pores and microglobules), which leads to relatively low column efficiency. Therefore, we studied the effect of constitute and percentage of porogenic solvent, functional monomer, column length, and separation gradient on the peak capacity and proteome coverage by methacrylate-based reversed phase monolithic columns. It was demonstrated that the porous property of the hydrophobic monolith, which was mainly determined by the porogenic solvent, was crucial to the proteome coverage when similar methacrylate monomer was utilized and a ternary porogenic solvent was adopted to prepare C12 monolithic column with relatively homogeneous overall pore structure. It was also shown that high proteome coverage could be reliably obtained with online multidimensional separation using totally monolithic columns system with the length of analytical column at 85 cm and reversed phase separation gradient at 210 min.     

聚二乙烯苯整体柱的制备及其在气相色谱分析中的应用

初步探讨了毛细管整体柱的制备方法及其在气相色谱分析中的应用。以液相色谱用毛细管整体柱作为研究基础,通过改变甲苯和十二醇的比例,使整体柱适用于气相色谱分析。通过二乙烯苯与键合在管壁上的3-(异丁烯酰氯)丙基三甲氧基硅烷(TMP)键合以及其自身的聚合,获得具有牢固结构、良好机械强度的整体柱。将其用于混合溶剂的分析和白酒标样的分析,可直接分析水中低碳醇。与现有的商品柱进行比较,结果表明所制备的整体柱均优于用以对照的商品色谱柱,其中在混合溶剂的分析中,醇类、酯类、酮类和芳烃类的峰形均优于用于比较的多孔层开口管(PLOT)柱;在白酒标样分析中,使得乙醛、甲醇、乙酸乙酯的色谱峰能够分开,比现有的聚乙二醇(PEG-20M)柱的分析方法更为便捷。     

Pressure drop in CIM disk monolithic columns

Pressure drop analysis in commercial CIM disk monolithic columns is presented. Experimental measurements of pressure drop are compared to hydrodynamic models usually employed for prediction of pressure drop in packed beds, e.g. free surface model and capillary model applying hydraulic radius concept. However, the comparison between pressure drop in monolith and adequate packed bed give unexpected results. Pressure drop in a CIM disk monolithic column is approximately 50% lower than in an adequate packed bed of spheres having the same hydraulic radius as CIM disk monolith; meaning they both have the same porosity and the same specific surface area. This phenomenon seems to be a consequence of the monolithic porous structure which is quite different in terms of the pore size distribution and parallel pore nonuniformity compared to the one in conventional packed beds. The number of self-similar levels for the CIM monoliths was estimated to be between 1.03 and 2.75.     

Reduced-bore monolithic silica column modified with C8-TEOS for reversed-phase electrochromatography

Monolithic silica columns of 2.7 mm ID were prepared and derivatized with C8-TEOS and TEOS by on-column sol-gel reaction. These C8 large diameter monolithic silica columns gave 21 000 theoretical plates for aromatic hydrocarbons in 60% acetonitrile and 40% Tris-HCI buffer. The surface areas as well as the separation reproducibility were improved on coating by the sol-gel approach. Joule heating was greatly reduced by using monolithic columns to which fine quartz sand had been added during column preparation. Since this is a preliminary investigation on a monolithic column with such a large inner diameter, the separation efficiency was not so high as that presently achieved in normal capillary electrochromatography (CEC). However, use of the columns improved sample loadability and concentration detectability of electrochromatography, and semi-preparative separations could be performed.     

Preparation of low flow-resistant methacrylate-based monolithic stationary phases of different hydrophobicity and the application to rapid reversed-phase liquid chromatographic separation of alkylbenzenes at high flow rate and elevated temperature

Low flow-resistant alkyl methacrylate-based monolithic stationary phases of different hydrophobicity were constructed for reversed-phase capillary liquid chromatography by thermally initiated radical polymerization of respective methacrylate ester monomer with different alkyl chain (C2, C4, C6, C12, C18) and ethylene glycol dimethacrylate (EDMA) in a 250 microm i.d. fused silica capillary. The hydrophobicity was basically controlled by changing the length and/or the density of the alkyl-chain, while the composition and the ratio of porogenic solvent were adjusted to obtain highly permeable rigid monoliths with adequate column efficiency. Among the prepared monolithic stationary phases, C18-methacrylate monoliths polymerized from a binary porogenic solvent of isoamyl alcohol and 1,4-buthandiol exhibited the most promising performance in terms of hydraulic resistance and column efficiency. The pressure drops of 20-cm long monolithic columns were below approximately 0.4 MPa at a normal linear velocity of 1mm/s (a flow rate of 3 microL/min), and the numbers of theoretical plates for alkylbenzenes mostly exceeded 3000 plates/20 cm. The produced monolithic columns had good mechanical strength for high pressure and temperature, and could be properly operated even at a temperature of 80 degrees C and at a pressure of at least 33 MPa. At 80 degrees C, the theoretical plate numbers reached 6000 plates/20 cm because of the enhanced mass transfer. Due to the novel hydraulic resistance and mechanical strength, the separation time could be reduced 120-fold simply by raising the flow rate and column temperature.     

Preparation and characterization of alkylated polymethacrylate monolithic columns for micro-HPLC of proteins

Protein separations were carried out by micro-high performance liquid chromatography (micro-HPLC) with surface alkylated monolithic columns, which were prepared by in situ copolymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) in the presence of dodecanol and toluene as porogens. First, glycidyl groups at the surface of the porous monolith were hydrolyzed with sulfuric acid. The hydroxyl groups thus formed were then reacted with n-alkyl chloride to form alkylated stationary phase. Separation performance for proteins on columns with C18 and C8 stationary phases was compared. The results showed that a poly(GMA-EGDMA) support derivatized with octadecyl moieties could achieve much better resolution than one with octyl groups. A protein mixture was separated with the octadecylated poly-(GMA-EGDMA) monolithic column, and the effluent peaks were collected and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The physical properties of the monolithic columns such as column morphology, surface area, mesopore size distribution, and column permeability were further characterized by scanning electron microscopy (SEM), multipoint BET nitrogen adsorption/desorption, and Darcy's law, respectively.     

Enhanced capabilities of separation in Sequential Injection Chromatography--fused-core particle column and its comparison with narrow-bore monolithic column

In the Sequential Injection Chromatography (SIC) only monolithic columns for chromatographic separations have been used so far. This article presents the first use of fused-core particle packed column in an attempt to extend of the chromatographic capabilities of the SIC system. A new fused-core particle column (2.7 μm) Ascentis^® Express C18 (Supelco™ Analytical) 30 mm × 4.6 mm brings high separation efficiency within flow rates and pressures comparable to monolithic column Chromolith^® Performance RP-18e 100-3 (Merck^®) 100 mm × 3 mm. Both columns matches the conditions of the commercially produced SIC system - SIChrom™ (8-port high-pressure selection valve and medium-pressure Sapphire™ syringe pump with 4 mL reservoir - maximal work pressure 1000 PSI) (FIAlab^®, USA). The system was tested by the separation of four estrogens with similar structure and an internal standard - ethylparaben. The mobile phase composed of acetonitrile/water (40/60 (v/v)) was pumped isocratic at flow rate 0.48 mL min⁻¹. Spectrophotometric detection was performed at wavelength of 225 nm and injected volume of sample solutions was 10 μL. The chromatographic characteristics of both columns were compared. Obtained results and conclusions have shown that both fused-core particle column and longer narrow shaped monolithic column bring benefits into the SIC method.     

阴离子交换整体柱对蛋白质的分离与纯化

分别用乙二胺、二乙胺、三乙胺将自制的以甲基丙烯酸缩水甘油酯(GMA)为单体、乙二醇二甲基丙烯酸酯(EDMA)为交联剂的整体柱修饰为弱、强阴离子交换整体柱。考察了该整体柱的性能,选择出分离蛋白质(牛血清白蛋白、溶菌酶和谷胱甘肽)的最佳实验条件,并在最佳分离条件下考察了这些蛋白质在整体柱上的色谱行为和该整体柱对纤维素降解酶的分离纯化情况。实验结果表明,该整体柱性能良好,可以实现对纤维素降解酶的快速分离与纯化。同时,实验也证明采用梯度洗脱可以实现对某些蛋白质的分离纯化。     

Transfer of gradient chromatographic methods for protein separation to Convective Interaction Media monolithic columns

Convective Interaction Media (CIM) columns are monolithic columns optimized for the separation of macromolecules. Some of them operate in the axial mode while others operate in the radial mode depending on the column size. In this work we tested the approach suggested by Yamamoto [Biotechnol. Bioeng., 48 (1995) 444] for transfer of gradient methods between columns of different size. A simplified equation for transfer was derived together with a criterion for its application. Separation was evaluated for a standard protein mixture and peroxidase enzymes present in fermentation broth. Salt and pH gradients were applied. Similar resolutions were obtained for each sample on all columns which demonstrates that the proposed approach can be successfully used for method scale-up on this type of column.     

Purification of serine hydroxymethyltransferase from Bacillus stearothermophilus with ion-exchange high-performance liquid chromatography.

The gene of serine hydroxymethyltransferase (SHMT) of a thermophilic bacterium Bacillus stearothermophilus was expressed in Escherichia coli, and SHMT was successfully purified from the crude extract of E. coli in two steps while maintaining the enzymatic activity. The purification steps involved ammonium sulphate precipitation followed by high-performance liquid chromatographic separation using the anion-exchange column Fractogel EMD DEAE-650(S). In addition to the DEAE column, three other types of anion- and cation-exchange columns were also studied for their ability to separate SHMT, and the performance of the four columns were compared.     

Efficiency enhancements in micellar liquid chromatography through selection of stationary phase and alcohol modifier

Micellar liquid chromatography (MLC) remains hindered by reduced chromatographic efficiency compared to reversed phase liquid chromatography (RPLC) using hydro-organic mobile phases. The reduced efficiency has been partially explained by the adsorption of surfactant monomers onto the stationary phase, resulting in a slow mass transfer of the analyte within the interfacial region of the mobile phase and stationary phase. Using an array of 12 columns, the effects of various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, superficially porous and perfluorinated, were evaluated to determine their impact on efficiency in MLC. Additionally, each stationary phase was evaluated using 1-propanol and 1-butanol as separate micellar mobile phase alcohol additives, with several columns also evaluated using 1-pentanol. A simplified equation for calculation of A' and C' terms from reduced plate height (h) versus reduced velocity (nu) plots was used to compare the efficiency data obtained with the different columns and mobile phases. Analyte diffusion coefficients needed for the h versus nu plots were determined by the Taylor-Aris dispersion technique. The use of a short alkyl chain, wide-pore silica column, specifically, Nucleosil C4, 1000A, was shown to have the most improved efficiency when using a micellar mobile phase compared to a hydro-organic mobile phase for all columns evaluated. The use of 1-propanol was also shown to provide improved efficiency over 1-butanol or 1-pentanol in most cases. In a second series of experiments, column temperatures were varied from 40 to 70 degrees C to determine the effect of temperature on efficiency for a subset of the stationary phases. Efficiency improvements ranging from 9% for a Chromegabond C8 column to 58% for a Zorbax ODS column were observed over the temperature range. Based on these observed improvements, higher column temperatures may often yield significant gains in column efficiency, assuming the column is thermally stable.     

Thermoresponsive polymer brush on monolithic-silica-rod for the high-speed separation of bioactive compounds

Poly(N-isopropylacrylamide), one of the most utilized thermoresponsive polymers, brush-grafted monolithic-silica columns were prepared through surface-initiated atom transfer radical polymerization (ATRP) for effective thermoresponsive-chromatography matrices. ATRP initiator was grafted on monolithic silica-rod surfaces by flowing a toluene solution containing ATRP initiator into monolithic silica-rod columns. N-Isopropylacrylamide (IPAAm) monomer and CuCl/CuCl(2)/Me(6)TREN, an ATRP catalytic system, were dissolved in 2-propanol, and the reaction solution was pumped into the preprepared initiator-modified columns at 25 °C for 16 h. The constructed PIPAAm-brush structure on the monolithic silica-rod surface was confirmed by XPS, elemental analysis, SEM observation, and GPC measurement of grafted PIPAAm. The prepared monolithic silica-rod columns were also characterized by chromatographic analysis. PIPAAm-brush-modified monolithic silica-rod columns were able to separate hydrophobic steroids with a short analysis time (10 min), compared to PIPAAm-brush-modified silica-beads-packed columns, because of the horizontally limited diffusion path length of monolithic supporting materials. Additionally, diluted PIPAAm-brush monolithic silica-rod column gave a further shorting analysis time (5 min). These results indicated (1) surface-initiated ATRP constructed PIPAAm-brush structures on monolithic silica-rod surfaces and (2) PIPAAm-brush grafted monolithic silica-rod column prepared by ATRP was a promising tool for analyzing hydrophobic-bioactive compounds with a short analysis time.