Study on conical columns with different conical angles for semi-preparative liquid chromatography

The dynamic flow profiles and separation performances in conically shaped preparative liquid chromatographic columns (inlet i.d. larger than outlet i.d.) with three different angles (7, 10 and 15) were studied and compared with cylindrical column of the same length and internal volume. The shapes of dynamic flow profiles were studied by on-column visualization method. The transparent chromatographic columns made of polymethyl methacrylate (PMMA), packed with C18 bonded silica, were immerged into a cubic pool filled with glycerol to eliminate the cylindrical and conical lens effect. The flow profiles of colored iodine solution in the columns were observed clearly using cyclohexane as mobile phase since the refractive indices of C18, column wall and the mobile phase are very close. In the conical column of 15 degrees (20-7 mm i.d.) the mobile phase in the central region migrated slower than in wall region as it moved toward the column outlet, while in the conical column of 7 degrees (17-11 mm i.d.) the mobile phase in the central region migrated faster than in wall region just like in cylindrical column. We found that a plug-like flow profile was generated in the conical column of 10 degrees (18-9 mm i.d.) during the whole migration process. A carmine and brilliant blue mixture was used as a probe to test the separation ability of the columns. The resolutions of the two compounds on the conical column of 7, 10, 15 and on the cylindrical column were 0.6, 1.57, 1.29 and 0.8, respectively.     

Instrumental considerations for the effective operation of short,highly efficient fused-core columns. Investigation of performance at high flow rates and elevated temperatures

Fused core or superficially porous columns offer the advantages of higher efficiency compared with totally porous columns of the same particle size, but similar operating pressures. However, their performance may be adversely affected by extra-column effects that become more significant as the column efficiency increases, and as the diameter of the column is reduced. In this study, we show that 10 cm × 0.46 cm fused-core columns can be used on modified conventional instruments (“microbore systems”) without serious loss in performance, and this approach is at present likely to yield superior results compared with use of 0.21 cm columns (of identical efficiency) on current UHPLC instruments that have minimised extra-column volume. Furthermore, the true efficiency of commercial narrow-bore fused-core columns appears to be reduced compared with those of conventional bore, which may be due to packing difficulties for the former type. The fused-core columns in general gave excellent performance, showing no evidence of an upturn in the Knox plots at high flow velocities and elevated temperatures. Careful control of experimental conditions is necessary to ensure accurate data for these plots.     

The optimization of capacity and efficiency when coupling fused silica open tubular columns in gas chromatography

Summary There are a number of parameters which have to be chosen depending on the analysis being done in gas chromatography. While the choice of stationary phase material is based on the solutes to be separated, the thickness is dependent on the concentration and the volatility of the components to be analyzed. This study undertakes a coupled column phase ratio optimization by connecting a short piece of a particular column prior to a normal length of an analytical column. Various columns of different dimensions (phase ratio), but of the same stationary phase material (methyl silicone), are coupled together by a deactivated glass press-fit connector, and the efficiency and capacity are measured. The coupling of fused silica open tubular columns is optimized in efficiency by matching or decreasing the phase ratio of successive columns. Capacity optimization is accomplished by increasing the phase ratio of consecutive columns. Capacity and efficiency optimization are opposing each other; therefore, if some efficiency can be sacrificed a substantial increase in capacity is possible.     

Study of conical columns with 10 degrees opening angle for preparative liquid chromatography

The efficiency and sample capacity of conical liquid chromatographic columns with 10 degrees opening angle were studied at different ratio of cross section areas of inlet to outlet (A(in)/A(out)) and column dimension. As the A(in)/A(out) ratio changed from 4 to 2.25, the reduced plate height (h) was reduced and the h value decreased 12% when the column dimension was scaled up proportionally because of relatively smaller dead volume on both end of the column. Compared to cylindrical columns having corresponding lengths and volumes, the conical columns with 10 degrees opening angle were superior in column efficiency, resolution and the maximum peak concentration at column outlet; the loadability of conical column was improved 30-40% on injection volume and 50-60% on sample mass, respectively, over cylindrical columns.     

Efficiency of small-bore HPLC-columns in different liquid chromatographs

Summary It is demonstrated that small-diameter (2 mm) columns can provide their full efficiency only if the instrument is specially designed for them. In general purpose commercial instruments actual column performance will only be a fraction of what the column is capable of providing, and deterioration is particularly evident with early peaks. With the more conventional 4.6-mm i.d. columns the difference is less significant although the design of the sample inlet system still plays a role. An equation is derived which permits prediction of actual column efficiency.     

Theory of chromatography for columns with a variable configuration

Theory of ion chromatography developed in our previous work was generalized for the case of columns with variable configuration. The results obtained were used for the calculation of the efficiency of chromatography on conical columns.     

Fast headspace analysis with short microcapillary columns

Summary A new method of analysis using headspace gas chromatography with microcapillary columns is proposed. Small diameter (50 μm I.D.) fused-silica capillary columns with non-extractable SE-54 and PS-255 polysiloxane stationary phases were used for the analysis of low boiling organic compounds. The small diameter columns possess the usual very high efficiency so that the method can be employed for the headspace analysis of complex mixtures. The use of short microcolumns reduces the analysis times in comparison to conventional capillary columns. Good performances were obtained in the analysis of volatile compounds in some lemon essential oil, perfumes, and water samples.     

Chromatographic separation of simulants of nerve and blister agents by combining one‐ and two‐channel columns with different stationary phases

A two‐channel gas chromatography column and a single‐channel column were made by deep reactive‐ion etching technology. The two short columns were coated with different stationary phases, and then linked without a modulator. This is to aim at increasing the sample capacity and achieving a higher separation efficiency in complex environments. The results show that the capacity of the connected column is approximately 4 and 1.5 times larger than that of the single‐ and two‐channel columns, respectively. The linked column was utilized to separate a six‐component mixture, composed of three simulants of nerve and blister agents and three interfering vapors. The results demonstrate that the combined column has a remarkably higher separation efficiency than the individual columns, and an acceptable resolution is achieved although the total length of the linked column is only 1.5 m.     

Investigation of capillary micro-packed columns composed of two segments packed with a sorbent of different particle size

Summary For the first time gas chromatographic characteristics of fused-silica capillary micro-packed columns consisting of two segments (i.e., serial columns) and packed with two different particle size fractions of the same sorbent have been investigated. The height equivalent to a theoretical plate (HETP) and the specific separation number vs. carrier gas velocity dependence has been studied. The expediency of empolying serial columns in which the segment containing the finer sorbent fraction is located near the column outlet has been theoretically and practically expalined. Serial columns with such an arrangement of the two segments show a better performance from the view point of efficiency and mass transfer coefficient.     

Characterization of High‐Pressure Liquid Chromatography Columns using Chromatographic Methods

Abstract

A great variety of columns for liquid chromatography (LC) are available in dimensions ranging from industrial scale to micro‐bore, nano‐bore, and capillary size, and on‐chip columns. The columns may be used in various liquid chromatography modes or in capillary electrochromatography, depending on the support materials and stationary phase chemistry. Every year many new column types are introduced on the market, with improved selectivity and efficiency, long lifetime, and mobile phase compatibility, intended for general use, for liquid chromatography/mass spectrometry (LC/MS) applications, proteomic research, or for the analysis of other specific sample types. Considerable improvement in pH, high‐temperature, and high‐pressure stability of new column types, together with advances in the instrumentation, enabled introduction of capillary, high‐temperature, and ultra‐high‐pressure HPLC into routine practice. Even though reversed‐phase mode is still by the most widely used in contemporary LC, applications of other separation modes (such as ion, normal‐phase, or high‐interaction liquid chromatography (HILC)) have become more frequent recently, because of unique separation selectivity for certain sample types.

Characterization of column quality is not a simple task, because a number of factors should be taken into account, that affect the selectivity, efficiency and resolution of sample separation and the reproducibility of chromatographic data. These include the type of the support, the arrangement and density of the stationary phase on the adsorbent surface, the homogeneity of the chromatographic bed, etc. Various physicochemical techniques are used for characterization of the properties of column packings however, most of them are suitable for bulk materials only and cannot be directly applied for commercial columns without damaging them. Not to destroy the columns, often precious and expensive, practicing chromatographers can apply chromatographic methods to characterize columns and evaluate their analytical suitability under real‐life conditions, where the intermolecular interactions between the analytes, the stationary phase, and the mobile phases affect the retention. The present review reports various chromatographic tests and strategies available for column evaluation.     

超动态法快速制备毛细管色谱柱的研究

用超动态法快速制出了几种非极性毛细管柱,柱效、涂渍效率分别为3000～4500理论塔板/m和65～85%。研究了超动态法中涂渍压力、涂渍液浓度与容量因子的关系;考察了制柱重复性和液膜厚度均匀性;给出了涂渍一般口径和试涂大口径厚液膜毛细管柱的部分结果。     

Effect of fabrication strategy on the enantioseparation performance of β‐cyclodextrin‐functionalized polymethacrylate monoliths: A comparative evaluation

To evaluate the effect of the preparation strategy on the enantioseparation performance of β‐cyclodextrin‐functionalized monoliths, a series of β‐cyclodextrin‐functionalized organic polymeric monolithic columns were prepared through two‐step, single‐step, and one‐pot approaches, using the same cyclodextrin, linker–spacer, and crosslinker. Physicochemical characterization of the columns was carried out by determining the morphology, β‐cyclodextrin density, permeability, and chromatographic efficiency. For each type of monolithic column, the enantioresolution of 22 chiral compounds, including mandelic acid derivatives, nonsteroidal anti‐inflammatory drugs, N‐derivatized amino acids, and herbicides, was comparatively studied under optimum chromatographic conditions. The β‐cyclodextrin‐functionalized monolithic columns prepared through the one‐pot approach exhibited higher enantioresolution for most chiral compounds, and they have the advantage of good controllability and simple preparation. On the other hand, the enantioresolution obtained on columns prepared through the single‐step approach was quite unsatisfactory, and therefore the effect of using different linking spacers and crosslinkers was studied. A significant improvement of enantioresolution for 2‐chloro‐mandelic acid was obtained by using N ,N‐methylenebisacrylamide instead of ethylene dimethacrylate as the crosslinker in the single‐step preparation.     

台锥形柱尾的填充毛细管液相色谱柱

首次将台锥形柱尾用于填充毛细管液相色谱柱,并与环氧胶粘的柱尾连接方式进行比较,发现在柱内径≥０．３２ｍｍ时,前者的柱效比后者高出３０％～５０％,且明显改善柱效－流速特性。对比研究了不同内径柱（０．５３ｍｍ,０．３２ｍｍ,０．２５ｍｍ）采用两种尾接方式的塔板高度与流速的关系,对产生差异的原因进行了探讨。     

10°锥角台锥型液相色谱柱尺寸等比例放大和柱内谱带流型的可视化研究

摘要利用改进的可视化装置, 研究了10°锥角的台锥型液相色谱柱内的谱带流型与柱参数变化的关系. 将有机玻璃柱管加工成内圆台外方型的一体结构, 选择折射率一致的色谱固定相硅胶和流动相环己烷, 使整个色谱柱成为高度清晰的透明体, 能直接观察柱中彩色样品谱带的动态三维流型. 研究结果表明, 在实验条件范围内, 流动相流速对谱带流型无影响, 填料的形状和性质对塞子状流型有一定程度的影响. 比较了柱长为5cm和等比例放大后柱长为10cm锥型柱内的流型, 发现放大后的锥型柱内仍然保持塞子状流型, 总柱效等比例增加. 表明继续按比例放大成为工业规模色谱柱后仍能保持塞子状流型.     

Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro‐liquid chromatography system. Fully porous, core–shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub‐2 μm fully porous as well as the 2.7 μm core–shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56–2.69) before correction for extra‐column contribution compared to normal‐bore columns. Moreover, the influence of extra‐column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub‐2 μm fully porous particle packed column for ultra‐fast liquid chromatography.     

Studies on column size scale-up and flow profile in conical shape liquid chromatographic column of 10° by visualization method

An improved visualization device made of polymethyl methacrylate (PMMA) was used to study the 3D flow profile inside conical columns of a 10° opening angle packed with C₁₈ phase. The outside wall of the conical columns was rectangular in shape in order to improve the transparency property of the column wall and reduce the deformation of the image for better observation of the flow profiles of colored solutes inside the column. The influence of flow rate, particle size and shape on the flow profile of a colored band were studied on a 5-cm-long column and a scaled-up column of four fold in volume. It was found that the flow rates of the mobile phase had little influence on the flat flow profile of the iodine band while the properties of the stationary phase had a certain influence on them. We observed that the flow profiles of the scaled-up column were flat during the whole chromatographic process, and the efficiency and resolution of the column were also increased in accordance with theoretical prediction. The experimental results proved that the 10° conical columns can be proportionally scaled up while still keeping the flat flow profile, sample load per unit volume of packing material, and column efficiency, which are superior to the conventional column. __________ Translated from Chemical Journal of Chinese Universities, 2006, 27 (1) (in Chinese)     

Polymethacrylate monolithic and hybrid particle-monolithic columns for reversed-phase and hydrophilic interaction capillary liquid chromatography

We prepared hybrid particle-monolithic polymethacrylate columns for micro-HPLC by in situ polymerization in fused silica capillaries pre-packed with 3–5 μm C₁₈ and aminopropyl silica bonded particles, using polymerization mixtures based on laurylmethacrylate–ethylene dimethacrylate (co)polymers for the reversed-phase (RP) mode and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl) zwitterionic (co)polymers for the hydrophilic interaction (HILIC) mode. The hybrid particle-monolithic columns showed reduced porosity and hold-up volumes, approximately 2–2.5 times lower in comparison to the pure monolithic columns prepared in the whole volume of empty capillaries. The elution volumes of sample compounds are also generally lower in comparison to packed or pure monolithic columns. The efficiency and permeability of the hybrid columns are intermediate in between the properties of the reference pure monolithic and particle-packed columns. The chemistries of the embedded solid particles and of the interparticle monolithic moiety in the hybrid capillary columns contribute to the retention to various degrees, affecting the selectivity of separation. Some hybrid columns provided improved separations of proteins in comparison to the reference particle-packed columns in the reversed-phase mode. Zwitterionic hybrid particle-monolithic columns show dual mode retention HILIC/RP behaviour depending on the composition of the mobile phase and allow separations of polar compounds such as phenolic acids in the HILIC mode at lower concentrations of acetonitrile and, often in shorter analysis time in comparison to particle-packed and full-volume monolithic columns.     

Column selectivity in reversed-phase liquid chromatography. VII. Cyanopropyl columns

Eleven cyanopropyl ("cyano") columns were characterized by means of a relationship developed originally for alkyl-silica columns. Compared to type-B alkyl-silica columns (i.e., made from pure silica), cyano columns are much less hydrophobic (smaller H), less sterically restricted (smaller S*), and have lower hydrogen-bond acidity (smaller A). Because sample retention is generally much weaker on cyano versus other columns (e.g., C8, C18), a change to a cyano column usually requires a significantly weaker mobile phase in order to maintain comparable values of k for both columns. For this reason, practical comparisons of selectivity between cyano and other columns (i.e., involving different mobile phases for each column) must take into account possible changes in separation due to the change in mobile phase, as well as change in the column.     

Ensuring repeatability and robustness of poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) HPLC monolithic columns of 3 mm id through covalent bonding to the column wall

Two different methods to reinforce the poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) HPLC monolithic columns of 3 mm id in a glass column reservoir were studied: composite columns with polymeric particles in the monolith and surface treatment of the reservoir wall. Of the two methods used to counter the mechanical instability and formation of flow channels (composite columns and column wall surface treatment), we demonstrated that proper column wall surface treatment was sufficient to solve both problems. Our study also indicated that no surface treatment is efficient, and of the methods studied silanization in acidified ethanol solution and constant renewal of the reaction mixture (dynamic mode) proved to be the most effective. As a result of this study, we have been able to prepare repeatable and durable methacrylate HPLC columns with good efficiencies.     

不锈钢宽口径填充毛细管液相色谱柱的制备及评价

设计了一种零死体积的柱尾连接方式和一种能使匀浆填料均匀进入色谱柱管内的锥形储料池。详述了以反相 ＯＤＳ为固定相,制备高效稳定的不锈钢宽口径（０．８ ｍｍＩ．Ｄ．）填充毛细管液相色谱柱的方法。系统地考察了填装方向、加压溶剂、匀浆溶剂、匀浆浓度和填装压力等不同因素对柱效的影响。在４０ＭＰａ压强和优化条件下,可重复制得柱效达理论塔板数为７．５×１０４／ｍ。对所测试的样品,色谱峰不对称因子为１．０３～１．１０。