Adsorption in columns packed with porous adsorbent particles having partially fractal structures

A mathematical model is constructed and solved that could describe the dynamic behavior of the adsorption of a solute of interest in single and stratified columns packed with partially fractal porous adsorbent particles. The results show that a stratified column bed whose length is the same as that of a single column bed, provides larger breakthrough times and a higher dynamic utilization of the adsorptive capacity of the particles than those obtained from the single column bed, and the superior performance of the stratified bed becomes especially more important when the superficial velocity of the flowing fluid stream in the column is increased to accommodate increases in the system throughput. This occurs because the stratified column bed provides larger average external and intraparticle mass transfer and adsorption rates per unit length of packed column. It is also shown that increases in the total number of recursions of the fractal and the ratio of the radii between larger and smaller microspheres that make up the partially fractal particles, increase the intraparticle mass transfer and adsorption rates and lead to larger breakthrough times and dynamic utilization of the adsorptive capacity of the particles. The results of this work indicate that highly efficient adsorption separations could be realized through the use of a stratified column comprised from a practically reasonable number of sections packed with partially fractal porous adsorbent particles having reasonably large (i) total number of recursions of the fractal and (ii) ratio of the radii between larger and smaller microspheres from which the partially fractal particles are made from. It is important to mention here that the physical concepts and modeling approaches presented in this work could be, after a few modifications of the model, applied in studying the dynamic behavior of chemical catalysis and biocatalysis in reactor beds packed with partially fractal porous catalyst particles.     

The dynamic behavior of a stratified column bed packed with porous adsorbent particles having partially fractal structures and a nonuniform ligand density distribution

The dynamic behavior of adsorption in a single column and in stratified column beds packed with porous adsorbent particles having partially fractal structures is studied when all columns have the same total length and the spatial ligand density distribution in the porous microspheres from which the porous adsorbent particles are made, is either uniform or nonuniform and such that the concentration of the immobilized ligands (active sites) increases monotonically from the center of the microspheres to their outer surface. The total number of immobilized ligands in the porous adsorbent particles has the same value whether the spatial ligand density distribution is uniform or nonuniform. The results in this study clearly show that for a given value of the superficial velocity of the flowing fluid stream in the column (for a given value of throughput) the breakthrough time is significantly increased when the radius of the microspheres is decreased, the total number of sections of the stratified column bed is increased, and the spatial ligand density distribution employed in the microspheres is nonuniform. Furthermore, when the superficial velocity of the flowing fluid stream in the column is increased (throughput is increased) the effect that (i) the reduction in the radius of the microspheres and (ii) the increase in the number of sections of the stratified column bed have on providing robust and effective dynamic adsorptive capacity and smaller reductions on the breakthrough time is substantially larger than that realized through the use of the nonuniform ligand density distribution. Similar trends are also observed in the dynamic behavior of adsorption in the systems studied here when the value of the concentration of the adsorbate in the flowing fluid stream entering the column (inlet concentration) has such a high magnitude that the value of the equilibrium concentration of the adsorbate in the adsorbed phase determined from the equilibrium Langmuir isotherm that would correspond to the inlet concentration of the adsorbate in the flowing fluid stream is, for all practical purposes, at its saturation limit.     

A comparative study of large volume injection techniques for microbore columns in HPLC

Summary This paper focuses attention on the potentially larger signal-to-noise ratios produced by microbore columns in comparison with conventional columns. The increased chromatographic signals by the application of microbore columns are due to the lower chromatographic dilution of elution profiles which are proportional to the square of the column inner radius. Generally less than 1μl sample should be injected into microbore systems to obtain the full benefit of the column performance. However, since more sample can be loaded on conventional columns compared to microbore columns the advantage of improved signal-to-noise ratio can only be realised in situations where very little sample is available. To inject more than 1μl sample, at the same time avoiding extra band-broadening effects, suitable injection techniques must be available. In this study three injection methods for microbore systems that meet this condition, are studied and compared.     

Comparison of separation power of ultra performance liquid chromatography and comprehensive two-dimensional liquid chromatography in the separation of phenolic compounds in beverages

In this study, 1-D and 2-D liquid chromatographic systems, namely, conventional HPLC, UPLC, HPLC x HPLC and HPLC x UPLC systems were developed and evaluated for the separation of phenolic acids in wine and juices. In the LC x LC studies, the first dimension separation was based on RPLC and the second dimension was performed with ion-pair chromatography. Three different columns, namely two short columns packed with either 2.5 or 1.7 microm particles and a monolithic column, were tested for the fast second dimension separation. The best results were obtained when the monolithic column was applied for the second dimension separation. The peak capacities for comprehensive 2-D systems varied from 330 to 616.     

The influence of major components on the direct chromatographic recovery of a protein from transgenic milk

This work presents a systematic evaluation of the influence of lipids and casein on the performance of a chromatographic capture step for the recovery of a target protein from transgenic milk. Lactoperoxidase (LPO) was spiked at concentrations typical of those to be expected for transgenic proteins in commercial bovine milk and the dynamic adsorption of LPO to fixed beds of SP Sepharose FF studied in frontal analysis experiments. By removing successively selected components from whole milk, their individual influence on the dynamic adsorption behaviour of LPO could be studied. A mathematical model, fitted to the breakthrough curves of LPO, provided a quantitative measure of parameters describing mass transfer and adsorption in the column. A significant reduction in column capacity for LPO in the presence of milk or whey was recorded, which could be attributed to competing adsorption of alkaline earth metal ions to the cation exchange resin. While the high concentrations of lipids present in whole milk did strongly reduce the column permeability, no significant influence of either casein or low concentrations of lipids on the hydraulic properties of columns or on the adsorption of LPO could be detected. The results indicate that chromatography, which forms an essential part of all current large-scale processes for the recovery of proteins from transgenic milk, could potentially be moved further upstream. Alternatively, existing operations for the removal of lipid and casein could be re-designed so as to maximise product yields. This suggests that significant product losses during current pre-chromatography milk purification could be reduced or potentially even avoided.     

Homogeneous gels for capillary electrochromatography

Homogeneous gels represent a new type of (electro)chromatographic media possessing unique separation properties unmatched with any other chromatographic beds. It is important to emphasize that they principally differ from continuous beds, polymer rods (better known as monoliths), which are particulate separation media with pores permitting hydrodynamic flow through the columns. Monoliths, thus, are more similar to beds conventionally packed with beads, although the particles building up monolithic columns are usually smaller in size (few submicometers) and covalently linked together. Consequently, homogeneous gels deserve better the term "monoliths" having a non-particulate structure formed by crosslinked free polymer chains (according to a dictionary a monolith is a non-modularized column). The goals of this minireview are to clarify the position of homogeneous gels among the separation media (including polymer solutions), to explain and to exemplify their outstanding (electro)chromatographic properties. This review gives hopefully a complete list of references to homogeneous gels developed for capillary electrochromatography.     

Large-bore coated columns for sampling and concentration of organic volatiles in air, headspace and water analysis

Large-bore coated (LBC) columns were used as sampling and concentrating traps in analyses for traces of organic volatiles in air and water. This simple technique utilizes long metal columns thinly coated with SE-30 for direct trapping of the organics. The sample is simply passed through the LBC column; the trapped organics are then thermally desorbed onto a conventional porous polymer pre-column or onto a second LBC column. If desired, this can be shorter or narrower bore than the initial LBC sampling column. The sample is finally desorbed onto the gas chromatographic column for analysis. Multiple transfers between LBC columns are possible, with increased concentration at each transfer, resulting in a "concentration pump" effect. The technique offers the advantages of great simplicity, efficiency and ease of sample transfer. Samples are obtained with low back-pressure and minimal interfering artifacts. The system shows almost complete imperturbability to moisture. Indifference to moisture and the low back-pressure enable direct sampling of very large volumes of air and even breath. Direct sampling of aqueous systems was also possible. The latter area was not fully investigated but offers potential for water pollution analysis and in direct examination of biological fluids and aqueous flavor extracts where heat sensitivity is a problem. With LBC columns the sampling and concentration sequence exposes the substances sought to no more drastic conditions than those they will be subjected to in the process of gas chromatographic analysis.     

全二维气相色谱技术及其进展

 许多分析问题的解决需要得到比一维色谱技术能提供的更高的分辨率。分离能力可通过使用多种分离技术或机理的组合来增强。此时 ,样品被分散在不同的时间维 ,最终的分辨率强烈地依赖于这些维间分离特性的差异。当它们之间没有关联 ,也即相互间正交时 ,系统可获得最高的分辨率。全二维气相色谱 (GC×GC)提供了一个真正的正交分离系统。它把分离机理不同而又互相独立的两支色谱柱以串联方式结合组成二维气相色谱。在这两支色谱柱之间装有的一个调制器起捕集再传送的作用。全二维色谱的峰容量为组成它的两支色谱柱各自峰容量的乘积。     

色谱柱选择对23种防腐剂测定结果的影响

以化妆品中23种防腐剂检测方法为例,探讨色谱柱选择对液相色谱方法测定结果的影响。参照《化妆品安全技术规范》甲基异噻唑啉酮等23个组分的检验方法,在2台不同的高效液相色谱仪上用15款不同品牌、型号的C₁₈色谱柱检测23种防腐剂,计算色谱峰的理论塔板数和分离度,对23种组分的分离效果进行分析,并应用USP (United States Pharmacopeia)数据库和PQRI (Product Quality Research Institute)数据库等2种等效色谱柱选择方法,对不同色谱柱的分离效果及等效性进行评价和预测。实验结果表明,15款色谱柱对23种防腐剂的分离效果差异显著,仅有2款色谱柱可以实现23种组分的完全分离。USP和PQRI数据库中2种等效色谱柱选择方法均无法预测出合适的等效色谱柱,对23种防腐剂的液相色谱分析参考价值均较小。色谱柱是影响23种防腐剂液相色谱法测定结果准确性的关键因素,有关实验室在应用该方法时,应考虑色谱柱选择性差异。化妆品基质复杂,如何在现有研究成果的基础上,开发色谱柱的筛选和预测评价体系,进而指导实际样品的分离是下一步研究的重点、难点。建议有关部门在制修订检测方法时,注重色谱柱的耐用性考察,完善系统适应性指标,细化色谱柱分类和增加描述信息,指导色谱柱的合理选择,从而规避由于色谱柱使用过程中选择依据缺失而导致测定结果不准确的风险。     

Increased productivity in quantitative bioanalysis using a monolithic column coupled with high-flow direct-injection liquid chromatography/tandem mass spectrometry

The feasibility of using a monolithic column as the analytical column in conjunction with high-flow direct-injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) to increase productivity for quantitative bioanalysis has been investigated using plasma samples containing a drug and its epimer metabolite. Since the chosen drug and its epimer metabolite have the same selected reaction monitoring (SRM) transitions, chromatographic baseline separation of these two compounds was required. The results obtained from this monolithic column system were directly compared with the results obtained from a previously validated assay using a conventional C18 column as the analytical column. Both systems have the same sample preparation, mobile phases and MS conditions. The eluting flow rate for the monolithic column system was 3.2 mL/min (with 4:1 splitting) and for the C18 column system was 1.2 mL/min (with 3:1 splitting). The monolithic column system had a run time of 5 min and the conventional C18 column system had a run time of 10 min. The methods on the two systems were found to be equivalent in terms of accuracy, precision, sensitivity and chromatographic separation. Without sacrificing the chromatographic separation, sensitivity, accuracy and precision of the method, the reduced run time of the monolithic column method increased the sample throughput by a factor of two.     

Parallel, fluorous open-tubular chromatography using microstructured fibers

Although commonly used in gas chromatography, open-tubular columns for liquid chromatography have seen their development hindered by a number of factors both theoretical and practical. Requiring small diameters, great lengths and specialized detection systems to achieve a proper chromatographic response, columns of this sort have largely been ignored despite the highly desirable column performance an optimized system would provide. Here, we introduce the use of microstructured fibers (MSFs) as a platform for the development of multiplexed open-tubular liquid chromatography (OTLC) columns. The multiple, parallel silica channels presented by the MSF act as a promising substrate for an OTLC column, as they have diameters near the ideal range for interactions (1–3 μm), minimize flow-induced backpressure through their many uniform paths, and increase the loading capacity compared to a single capillary channel of similar size. Additionally, with outer diameters comparable to regular fused silica capillaries, MSFs can easily be employed in conventional chromatographic systems, eliminating the need for specialized equipment. Finally, MSF columns of this type can be functionalized using silane coupling techniques to allow the introduction of a wide variety of stationary phase chemistries. While in this report we explore the potential and limitations of fluorine-functionalized MSFs as OTLC columns, other stationary phase materials could easily be substituted by choosing appropriate silanization reagents. Particular attention here will be paid to the physical and performance characteristics of the fabricated columns, as well as avenues for their improvement and implementation.     

三步柱色谱法从江浙蝮蛇蛇毒中分离纯化类凝血酶

建立了一种用CM Sepharose CL-6B阳离子交换、DEAE Sepharose Fast Flow阴离子交换和Sephadex G-75凝胶排阻三步柱色谱从江浙蝮蛇蛇毒中分离纯化类凝血酶的方法。在实验室小柱分离方案的基础上,对该纯化工艺进行了放大。当上样量达实验室小柱的25倍时,所得类凝血酶的质量指标与实验室小柱基本一致。采用该法所得的蝮蛇类凝血酶经Shim-pack Diol-300高效凝胶排阻柱测得其相对分子质量约为33500,用Shim-pack VP-ODS反相色谱柱检测其纯度约为96%。从江浙粗蛇毒中提取类凝血酶时,类凝血酶的总质量收率约为0.3%,总活性收率约为64%,比活可达2000 U/mg。     

Comparison of different types of stationary phases for the analysis of soy isoflavones by HPLC

Nowadays, there are new technologies in high-performance liquid chromatography columns available enabling faster and more efficient separations. In this work, we compared three different types of columns for the analysis of main soy isoflavones. The evaluated columns were a conventional reverse phase particle column, a fused-core particle column, and a monolithic column. The comparison was in terms of chromatographic parameters such as resolution, asymmetry, number of theoretical plates, variability of retention time, and peak width. The lower column pressure was provided by the monolithic column, although lower chromatographic performance was achieved. Conventional and fused-core particle columns presented similar pressure. Results also indicate that direct transfer between particle and monolithic columns is not possible requiring adjustment of conditions and a different method optimization strategy. The best chromatographic performance and separation speed were observed for the fused-core particle column. Also, the effect of sample solvent on the separation and peak shape was evaluated and indicated that monolithic column is the most affected especially when using higher concentrations of acetonitrile or ethanol. Sample solvent that showed the lowest effect on the chromatographic performance of the columns was methanol. Overall evaluation of methanol and acetonitrile as mobile phase for the separation of isoflavones indicated higher chromatographic performance of acetonitrile, although methanol may be an attractive alternative. Using acetonitrile as mobile phase resulted in faster, higher resolution, narrower, and more symmetric peaks than methanol with all columns. It also generated the lower column pressure and flatter pressure profile due to mobile phase changes, and therefore, it presents a higher potential to be explored for the development of faster separation methods.     

Comparison of two gas–liquid chromatograph columns for the analysis of fatty acids in ruminant meat

Two gas–liquid chromatograph capillary columns for the analysis of fatty acids (FA) in ruminant fat are compared. Those columns are the CP-Sil 88 of 100 m long with a highly polar stationary phase and the Omegawax 250 of 30 m long with a stationary phase of intermediate polarity. Fatty acid methyl ester (FAME) patterns of branched-chain, cis and trans octadecenoate isomers, as well as conjugated and non-conjugated 18:2 and 18:3 isomers are fairly different between columns, even though most of the FAME could be separated on either column. However, the CP-Sil 88 showed better resolution of 18:1 isomers than Omegawax 250. The analysis of 96 samples of ruminant meat fat in both chromatographic systems showed that averages obtained for total FA content and for most of the individual FA did not differ between columns. Moreover, regression analysis of Omegawax and CP-Sil 88 data is highly correlated. Quantitative differences between chromatographic systems were detected for samples containing more than 66 mg fatty acids per gram of muscle dry matter.     

High throughput screening of active pharmaceutical ingredients by UPLC

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.     

Determination of the porosities of monolithic columns by inverse size-exclusion chromatography

Retention data of polystyrene samples of narrow molecular size distribution and known average molecular mass were measured on several monolithic columns (Chromolith Performance, Merck) and one conventional packed column (Luna C18, Phenomenex) by size-exclusion chromatography. These data were used to determine the external, the internal, and the total porosities of these columns. These data provided also information on the pore-size distribution of the adsorbent medium. The external and the total porosities of these columns are much higher than those of conventional packed columns. The results illustrate the profound changes brought by monolithic columns to the balance of the hydrodynamic and the mass transfer kinetic properties of chromatographic columns. Classical methods of comparison between column performance must be re-evaluated.     

Study of a monolithic silica capillary column coated with poly(octadecyl methacrylate) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds

The performance of a monolithic silica capillary column coated with poly(octadecyl methacrylate) (ODM column) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds was studied, and the results were compared to those obtained by using a monolithic silica capillary column modified with octadecylsilyl-(N,N-diethylamino)silane (ODS column). Benzene and naphthalene derivatives, polycyclic aromatic hydrocarbons (PAHs), steroids, alkyl phthalates, and tocopherol homologues were used as test samples. In general, compounds with aromatic character, rigid and planar structures, and lower length-to-breadth ratios (more compacted structures) seem to have more preference for the polymer coated stationary phase (ODM). Compounds with acidic character have also a higher retention on ODM columns because of the presence of ester groups in the stationary phase. The polymer coated column allowed the separation of some PAHs, alkyl phthalates, steroids, and of beta- and gamma-tocopherol isomers which cannot be separated under the same conditions on ODS columns, while keeping similar column efficiency. These results allowed to suggest ODM columns as a good alternative to conventional ODS columns for reversed-phase liquid chromatography.     

Microbore liquid chromatographic determination of cadralazine and cephalexin in plasma with large-volume injection

The application of microbore systems (15 cm X 1 mm I.D. columns filled with Nucleosil C18, 5 microns particle size) to the determination of cephalexin and cadralazine in plasma was investigated. Factors such as mobile phase flow-rate, detector flow-cell volume and injection volume were examined with regard to the needs of routine drug analysis. Mobile phase flow-rates of 50-60 microliters/min were used. A flow cell with an optical path length of 6 mm and an intermediary volume (2.4 microliters) was selected for UV detection in order to obtain sufficient sensitivity. Large volumes of non-eluting solvent containing the drug were injected on the column. The addition of an ion-pairing reagent to samples containing cephalexin and cefroxadin prior to the injection was found to improve the chromatographic performance. The blood sample size required for analysis with microbore columns was smaller than that with conventional columns. The analysis time was similar and the limit of quantitation was also similar, provided that large sample volumes were injected on the microbore column.     

Shrinking-core modeling of binary chromatographic breakthrough

Most chromatographic processes involve separation of two or more species, so development of a simple, accurate multicomponent chromatographic model can be valuable for improving process efficiency and yield. We consider the case of breakthrough chromatography, which has been considered in great depth for single-component modeling but to a much more limited degree for multicomponent breakthrough. We use the shrinking core model, which provides a reasonable approximation of particle uptake for proteins under strong binding conditions. Analytical column solutions for single-component systems are extended here to predict binary breakthrough chromatographic behavior for conditions under which the external transport resistance is negligible. Analytical results for the location and profile of displacement effects and expected breakthrough curves are derived for limiting cases. More generally, straightforward numerical results have also been obtained through simultaneous solution of a set of simple ordinary differential equations. Exploration of the model parameter space yields results consistent with theoretical expectations. Additionally, both analytical and numerical predictions compare favorably with experimental column breakthrough data for lysozyme-cytochrome c mixtures on the strong cation exchanger SP Sepharose FF. Especially significant is the ability of the model to predict experimentally observed displacement profiles of the more weakly adsorbed species (in this case cytochrome c). The ability to model displacement behavior using simple analytical and numerical techniques is a significant improvement over current methods.