Semimicro Size Exclusion Chromatography For Oligomers. I. Column Packing Procedure

Abstract

Polystyrene gels of a particle diameter 10 ± 2 μm for the use in oligomer separation were packed into 1.5 mm i.d. × 25 cm length columns by the balanced density slurry-packing technique under a constant flow rate of 500 μL/min. The slurry solvent was a mixture of toluene and chloroform (50.5/49.5, v/v). The example of the number of theoretical plates (N) of these columns was 8600 plates/25 cm (HETP = 29.1 μm) at flow rate of 40 μL/min by injecting 1 μL of 0.5% benzene solution. Sixteen columns were connected and the overall value of N was 103000 plates/4 m. A typical example of oligomer separation was demonstrated. A constant-flow technique is preferable to a constant-pressure technique. When two or three column blanks were packed together, the columns located at the outlet of the packer-column assembly had higher values of N. Optimum flow rate of the slurry solvent when three column blanks were packed together lay between 400 and 500 μL/min. The packing efficiency, that is, the probability of getting valid columns was about 60%. Viscous slurry solvents were not effective to get efficient columns. To pack gels in the less swollen state gave sometimes efficient columns. Pressure monitoring in progress of packing was very effective to foresee the column efficiency.     

Large-Scale Preparative Counter-Current Chromatography with a Coil Planet Centrifuge

Abstract

Development of the large-scale preparative countercurrent chromatographic schemes has been continued by increasing the diameter of the separation column. A 0.55 cm i.d. FEP tube was coaxially coiled around the holder (7.5 cm, 10 cm or 15 cm in diameter) of a horizontal flow-through coil planet centrifuge (15 cm revolutional radius). Performance of each column was evaluated on the separation of dinitrophenyl amino acid samples with a two-phase solvent system composed of chloroform, acetic acid, and 0.1N hydrochloric acid (2:2:1) by using both aqueous and nonaqueous phases as the mobile phase. Experiments with the short preliminary columns (114 ml capacity) revealed that the hydrodynamic distribution of the two solvent phases was sensitively affected by the helical diameter of the column. However, by choosing the proper elution mode of the mobile phase, satisfactory results were obtained with the helical diameters of 7.5 cm and 15 cm at a high flow rate of 500 ml/h under a moderate revolutional speed of 300 rpm. With the long coiled columns (750 ml capacity), the preparative capability of the present scheme was successfully demonstrated on separations of the 1g-quantity sample mixture under optimized operational conditions. Overall results indicated that the sample-loading capacity of the present scheme can be further increased by the use of longer and/or larger-diameter columns.     

Comparison of nonporous silica-based ion exchangers and monolithic ion exchangers in separations of inorganic anions

Low capacity anion exchangers for IC have been prepared by modification of nonporous uniformed silica MICRA microbeads and by modification of the organic polymeric monolithic matrixes prepared in situ in quarz capillary. Due to the small particle size (1.5 microm) high-performance adsorbents were prepared allowing to obtain up to 190,000 tp/m. However, the column possesses a very high back-pressure and can be used in a short length up to 50 mm only to meet the requirements of conventional chromatographic equipment. An analysis of a test mixture of seven anions was completed within 3 min with a back column pressure of about 350 bar (HETP of about 5.5 microm, where HETP is the height equivalent to the theoretical plate). Monolithic capillary columns provide lower efficiency per column unit length than MICRA columns; however, they can be used at a longer length because of their low flow resistance. Monolithic column of ca. 40 cm length has workable pressure below 10 bar and allows separation of a five anions test mixture within less than 10 min. A better efficiency of monolithic column (HETP approximately 75 microm) can be achieved at reduced flow rates when the analysis time is not a critical parameter.     

Peak spreading in linear gradient elution chromatography with a thin monolithic disk

The peak spreading of DNAs of various sizes [12-mer, 20-mer, 50-mer and 95-mer poly(T)] in linear gradient elution (LGE) chromatography with a thin monolithic disk was investigated by using our method developed for determining HETP in LGE. Electrostatic interaction-based chromatography mode (ion-exchange chromatography, IEC) was used. Polymer-based monolithic disks of two different sizes (12 mm diameter, 3mm thickness and 0.34 mL; 5.2 mm diameter, 4.95 mm thickness and 0.105 mL) having anion-exchange groups were employed. For comparison, a 15-μm porous bead IEC column (Resource Q, 6.4mm diameter, 30 mm height and 0.97 mL) was also used. The peak width did not change with the flow velocity for the monolithic disks where as it became wider with increasing velocity. For the monolithic disks the peak width normalized with the column bed volume was well-correlated with the distribution coefficient at the peak position K(R). HETP values were constant (ca. 0.003-0.005 cm) when K(R)>5. Much higher HETP values which are flow-rate dependent were obtained for the porous bead chromatography. It is possible to obtain 50-100 plates for the 3mm monolithic disk. This results in very sharp elution peaks (standard deviation/bed volume=0.15) even for stepwise elution chromatography, where the peak width is similar to that for LGE of a very steep gradient slope.     

Separation of15N by chemical exchange in NO, NO2−HNO3 system under pressure

The basic isotopic exchange reaction is responsible for the separation of¹⁵N in the Nitrox system that between gaseous nitrogen oxides and aqueous nitric acid with a single stage separation factor α=1.055 for 10M nitric acid, at 25°C and atmospheric pressure takes place. In order to know what happens in¹⁵N separation at higher pressure, when the isotopic transport between two phases is improved, a stainless steel laboratory experimental plant with a 1000 mm long × 18 mm i.d. column, packed with triangular wire springs 1.8×1.8×0.2 mm², was utilised. At 1.5 atm (absolute), and 2.36 ml·cm⁻²·min⁻¹ flow rate HETP was 7% smaller than at atmospheric pressure and 1.5 times smaller flow rate. HETP at 3.14 ml·cm⁻²·min⁻¹ flow rate and 1.8 atm is practically equal with that obtained at atmospheric pressure and 2 times smaller flow rate. The operation of the¹⁵N separation plant at 1.8 atm (absolute), instead of atmospheric pressure, will permit doubling of the 10M nitric acid flow rate and of¹⁵N production of the given column.     

Hydrodynamic chromatography for the size classification of micron and sub-micron sized packing materials

Hydrodynamic chromatography (HDC) was used as a size classification and purification method for porous bridged ethyl hybrid (BEH) packing materials (particles) in the micron to sub-micron range. Using packed column HDC, a batch of particles with size 0.76 ± 0.26 μm was fractionated to yield classified material of 1.05 ± 0.16 μm, reducing the relative standard deviation from 33% to 15%. Subsequent chromatographic evaluation of this packing material showed significant improvement in column performance and decrease in flow resistance over the unclassified material. Comparing a column packed with the classified versus non-classified material, the effective flow resistance of the two columns was decreased by 58% and the minimum HETP for the packing material was improved from 4 to 2.5 μm.     

On the optimization of the solid core radius of superficially porous particles for finite adsorption rate

Packed chromatographic columns with the superficially porous particles (porous shell particles) guarantee higher efficiency. The theoretical equation of the Height Equivalent to a Theoretical Plate (HETP), for columns packed with spherical superficially porous particles, was used for the analysis of the column efficiency for finite rate of adsorption-desorption process. The HETP equation was calculated by the application of the moment analysis to elution peaks evaluated with the General Rate (GR) model. The optimal solid core radius for maximum column efficiency was estimated for a wide spectrum of internal and external mass transfer resistances, adsorption kinetic rate and axial dispersion. The separation power of the shell adsorbent for two component mixture, in analytical and preparative chromatography, was discussed. The conditions of the equivalence between the solutions of the General Rate model with slow adsorption kinetic and the Lumped Kinetic Model (LKM) or the Equilibrium Dispersive (ED) model were formulated.     

Some parameters affecting the extraction chromatographic performance of TBP impregnated macroporous XAD-4 columns for Am(III)−Eu(III) separations

TBP impregnated macroporous styrene-divinylbenzene XAD-4 polymer was employed as the stationary phase in extraction chromatography of Eu(III) and Am(III). Parameters affecting the performance (HETP) of the chromatographic column were investigated. These include the effect of flow rate, TBP loading, aqueous nitrate concentration, and particle size. One striking result obtained is that the column performance improves as the TBP loading increases.     

Nuclear magnetic resonance studies of solvent flow through chromatographic columns: effect of packing density on flow patterns

NMR (nuclear magnetic resonance) techniques have been used to measure and characterise solvent flow through chromatographic columns. NMR imaging was used to track an injection of D2O. PGSE (pulsed gradient spin echo) NMR was used to measure the flow-rate dependence of axial and transverse apparent diffusion. A combination of these two techniques (dynamic NMR imaging) gave the spatial distribution of the local velocity and apparent diffusion through a cross-section of the column. Significant column wall effects were observed and these effects were found to be highly dependent upon the column packing density. The column performance was assessed in terms of the HETP (height equivalent to a theoretical plate) determined by the NMR techniques employed.     

Influence of the degree of coverage of C18-bonded stationary phases on the mass transfer mechanism and its kinetics

The influence of the degree of coverage of a silica surface with bonded C18 alkyl chains on the mass transfer mechanism in RPLC was investigated. Five packing materials were used, prepared with the same batch of silica particles (5 microm diameter, 90 A average pore size): one column was packed with the silica derivatized by trimethylchlorosilane (TMS) (C1, 3.92 micromol/m2), and the other four with the silica first derivatized with octadecyl-dimethyl-chlorosilane (C18, 0.42, 1.01, 2.03, and 3.15 micromol/m2), and then endcapped with TMS. A solution of methanol and water (25/75, v/v) was used as the mobile phase. The experimental HETP curves were acquired for each column by measuring the first moment and the second central moment of phenol and correcting them for the influence of the temperature increase due to the heat generated by the friction of the stream against the bed. The different kinetic parameters of the mass transfer in these packed chromatographic columns were identified (longitudinal diffusion, eddy diffusion, film mass transfer, and transparticle mass transfer) and quantified by fitting the experimental data to a new general HETP equation recently derived [F. Gritti, G. Guiochon, Anal. Chem., in press (AC-060203R).]. The agreement was excellent and allowed the comparison of the kinetic parameters among the six columns used. The highest column efficiency measured at conventional or fast flow rates (>0.5 ml/min) is obtained for the most retentive column, which has a surface coverage of 2.03 micromol/m2. The smallest HETP measured is as low as 10 microm, only twice the average particle diameter dp, due to the large contribution of surface diffusion (90%) to the particle effective diffusivity. However, no significant difference was observed between the efficiencies of the columns packed with C1 and C18 derivatized silica.     

一种从蛇毒中纯化神经生长因子的新工艺

为了能从中华眼镜蛇蛇毒中简单快速地分离纯化神经生长因子（一种治疗各种神经性损伤和神经退行疾病的药物,简称NGF）,采用不同的色谱柱联用的方式对NGF的纯化工艺进行了研究。结果表明,采用DEAE Sepharose F.F.和Sephadex G 50二步柱色谱工艺可以从蛇毒中快速分离得到神经生长因子。经十二烷基硫酸钠聚丙烯酰胺凝胶电泳和反相高效液相色谱分析, 证明所得到的NGF为单一组分,相对分子质量约为 29 000。对8　d龄鸡胚背根神经节采用体外培养法,结果证明,所得NGF具有明显的促进神经纤维     

High speed liquid-solid chromatography of phenothiazines using columns packed with spherosil porous silica beads.

Spherosil, a spherical porous silica, has been investigated for use in high speed liquid-solid chromatography and compared to Lichrosorb Si-60, using three phenothiazines as test solutes. Particle size distribution for four different size ranges with nominal mean diameters of 5, 10, 20 and 40 mu m is given. Distribution is very homogeneous. Columns from 15 to 100 cm in length and 1/4 or 1/8 in.o.d. have been prepared and efficiency measured by determination of HETP with the three phenothiazines: [methylamino-3-propyl]-10-chloro-3-phenothiazine [k' = 3]; [N-methyl-N-[dimethylamino-3-propyl]amino-3-propyl]-10-chloro-3-phenothiazine [k' = 6.5]; and [dimethylamino-3-propyl]-10-chloro-3-phenothiazine-N-oxide [k' = 15]. Influence of column length and incidence of bead diameter have been studied. 1/4 in.o.d. columns are easier to fill than 1/8 in.o.d. columns for Spherosil 5 mu m and have, therefore, a greater efficiency. HETP, H, varies according to flow rate as H = DVn with 0.4 less than n less than 0.6 and according to particle size ad H = A d beta p with 1.7 less than beta less than 1.8. The best figures for H are between 0.1 or 0.2 mm for a flow rate of 1800 ml hr-1 cm-2 [k' = 15]. The separation of a mixture of 6 phenothiazines with the mobile phase, anhydrous ethyl acetate 60 V, water saturated ethyl acetate 20 V, anhydrous methanol 20 V, 33% aqueous solution of ethylamine 0.25 V, is given. Its duration is 20 min. instead of 90 min. by thin-layer chromatography.     

分子印迹整体柱快速分离烟酰胺及烟酸

以药物烟酰胺为模板分子,甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂,甲苯和正十二醇的混合溶液为致孔剂,采用原位聚合法制备了具有特定识别性能和分离能力的分子印迹聚合物,并将其作为高效液相色谱固定相,实现了模板分子与烟酸在2 min内的快速分离。在规格为50 mm×4.6 mm i.d.色谱柱上,以纯水为流动相（流速为7.0 mL/min）、操作温度为室温的色谱条件下,模板分子与烟酸的分离度达1.8。讨论了流动相中有机溶剂含量、醋酸及碱含量和流速对分离的影响。结果表明,原位聚合法制备的整体分子印迹聚合物在以纯水作流动相时对模板分子与其类似物有快速分离能力,这对于体内药物的分离富集研究具有很好的应用前景。     

Effect of moisture on the extraction efficiency of polycyclic aromatic hydrocarbons from soils under atmospheric pressure by focused microwave-assisted extraction

The performance of a large commercial chromatographic column was investigated using a short pulse of a tracer and an extension of the reverse-flow technique. This technique permits separate determination of the unavoidable irreversible microscopic processes and the reversible effects of flow maldistribution, and allows for the separation of flow maldistribution in the flow distributors from flow maldistribution inside the packed bed. This analysis was performed on a 0.44 m Millipore IsoPak column using Cellufine GC 700, cellulosic-based media with an average particle diameter of 75 microm, for the stationary phase. The column efficiency was quantified by analysis of the effluent curve from a short pulse of a 5% aqueous acetone tracer. The study examined behavior of beds of different lengths (10-24 cm) and beds packed from different slurry concentrations (10-75% v/v). The slurry-packed columns were very uniform, and no significant macroscopic flow maldistribution was observed inside the column. The observed bed plate heights conformed to the predictions of available one-dimensional continuum models. Dispersion in the flow distributors was significant, corresponding to 15-25% of the intracolumn dispersion when the full 24 cm available bed length was used and a proportionally larger increase for shorter bed lengths. Thus, the headers are shown to produce a significant increase in the observed plate height.     

高压离子交换排代法富集锂同位素(Ⅱ)——HETP计算和富集条件

本文根据“精馏平衡塔板型”富集同位素理论提出了富集同位素时计算HETP的方法,寻找出富集和实验参数之间的关系,并进行了讨论;表明Glueckanf塔板理论和Snyder公式适用于简单排代机制的轻同位素富集过程。     

Enhanced performance of expanded bed chromatography on rigid superporous adsorbent matrix

Rigid spherical macroporous adsorbent beads with surface hydroxyl groups were prepared by cross-linking of cellulose. These beads had diameter in the range 100-200 microm and a mean pore size of about 3 microm with about 60% pore volume. The matrix (bulk density approximately 1600 kg m(-3)) could be expanded into a stable bed and used for protein chromatography. Chromatographic runs were performed on a 10 mm diameter column under non-retaining and retaining conditions on the prepared matrix (called Celbeads) and performance of the runs was measured in terms of the height equivalent to a theoretical plate (HETP). The HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents, i.e. increasing and levelling with velocity. Unimpaired performance of the matrix at increasing flow-rates permitted expanded bed elution of adsorbed solutes without loss of efficiency in terms of purification factor and product concentration. As a model system, Celbeads was used to purify lactate dehydrogenase from porcine muscle homogenate by dye-affinity chromatography. The prepared matrix provided about 100 theoretical plates per meter for the enzyme system at a linear flow velocity of 1.27 cm x min(-1) in an expanded bed elution mode, and gave enzyme yields of 100% with a purification factor of 31 using an optimized procedure. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance.     

Optimization of high-performance liquid chromatographic parameters for the determination of capsaicinoid compounds using the simplex method.

A high-performance liquid chromatographic method was developed for the analysis of capsaicinoid compounds, the pungent principles of capsicum fruits. A sequential simplex method was applied to optimize the chromatographic response function used to assess the quality of separation by varying the chromatographic parameters. The separation was achieved in 11 min using a C-8 column of 15-cm length and 4.6 mm diameter using a UV detector. A flow rate of 1.15 ml min(-1) at a column temperature of 43.5 degrees C using 63.7% methanol in water gave the most efficient separation. The method was found to be suitable for the determination of the major capsaicinoid compounds in the capsicum samples.     

Etudes theoriques et pratiques sur la realisation et le fonctionnement de colonnes preparatives de diametre moyen. Modification de la structure interne de ces colonnes

The dependence of the efficiency and productivity of preparative-scale gas-chromatographic columns on various parameters is discussed. The minimum HETP is experimentally shown to increase almost proportionally to the column diameter in the range 1–5 cm. The HETP also increases with a power of the sample amount which depends on the quality of the column packing and can be as low as 1.2. High-diffusivity carrier gases (H₂ and He) arc shown to allow a three-fold increase of columns productivity.The productivity (amount of sample separated/unit time) increases with the column diameter. Given a fixed volume of stationary phase and a known mixture, the column diameter which will accept the largest sample can be calculated. The performance of a preparative unit is limited more by the loadability of columns than by loss of efficiency witli increasing diameters. The performances of preparative columns can be improved by putting rods parallel to the column axis regularly spaced in the packing. The column can be heated internally cither for programmed temperature separations or to allow thermal equilibrium to be reached rapidly.     

加芯毛细管填充柱的理论研究

 从速率方程和柱压降两个方面对加芯毛细管填充柱的柱效和渗透性进行了探讨，并详细讨论了柱直径、芯直径、芯根数和柱压降的关系。综合考虑柱压降、板高和拉制柱子时颗粒在柱内的镶嵌状况可知最佳柱型是３芯的毛细管填充柱。     

Study on Lithium Isotope Enrichment by Band Displacement Process

Based on the equilibrium plate model of the distillation type,a set of theoretical equations has been deduced for the isotope's stationary band,and a new method of calculating the height equivalent to a theoretical plate (HETP) used for isotope enrichment was suggested.Moreover,the quantitative relations between the total enrichment and the experimental parameters (concentration of eluant,flow rate,length of band and displaced distance) were established.The expression of enrichment variation,while approaching the stationary state,was corrected and demonstrated experimentally.By the use of that expression,the semicontinuous isotope enrichment process was successfully estimated