A strategy to develop fast RP-HPLC methods using monolithic silica columns

Since the appearance of monolithic silica, much work has been done describing the properties of monolithic silica columns. Meanwhile the transferability of analytical methods from conventional to monolithic silica columns has been intensively investigated [1-5]. RP HPLC method development strategies for conventional columns should be updated or scaled to meet the higher performing monolithic column technology. Because of the high permeability of monolithic silica columns it should be possible to decrease the time for method development by applying high isocratic flow rates. Here we suggest a clear strategy for method development using monolithic columns. The strategy will be applicable for various sample compositions, e. g., acidic, basic, or neutral. The applicability of monolithic columns for especially complex separations of basic mixtures without the need of using a highly basic mobile phase that harms the column will be pointed out in this work. This work will describe in detail the actual method development process. For better understanding of our strategy, the influence of flow rate, column length, mobile phase composition, pH, and temperature will be discussed. Details about the application of a flow program will be mentioned.     

Titania-coated monolithic silica as separation medium for high performance liquid chromatography of phosphorus-containing compounds

A method of preparing titania-coated monolithic silica stationary phase has been developed to achieve liquid chromatographic separation of phosphorus-containing compounds, which have recently been attracting increasing attention in biochemical research. The titania-coated silica columns exhibited efficient separation with low pressure drop, which is a typical feature of monolithic structures, and also possessed phospho-selectivity, which is a unique property of the titania surface. The material characteristics of titania-coated monolithic silica were examined, and then resin-clad columns were applied to the HPLC analysis of phosphorylated compounds. Highly efficient separation of phosphorylated substances indicated that the novel titania-coated monolithic silica column will find applications as a useful tool in the field of biochemistry, especially in post-genomic analyses.     

Fast HPLC for quality control of Harpagophytum procumbens by using a monolithic silica column: method transfer from conventional particle-based silica column

The applicability of a monolithic C18-bonded silica column for the rapid HPLC separation of ingredients in medicinal plants and their phytopharmaceutical preparations has been evaluated in the author's laboratory. In this presentation, an existing method for the determination of the iridoid glycoside harpagoside in Harpagophytum procumbens (Devil's Claw) was successfully transferred from a conventional particle-based C18 silica column to a monolithic silica column. The very high porosity of the stationary phase allows chromatography with a much lower backpressure than on conventional columns. Therefore, the flow rate could be easily increased from 0.8 mL/min (particle-based column) to 5 mL/min (monolithic column) and the run-time reduced from 30 to 5 min (that is a reduction about 85% !), without losing any chromatographic resolution of the compound of interest. The amount of harpagoside was measured with the original method on a conventional particle-based silica column and on the adapted method on a monolithic silica column. The statistical mean t-test showed no significant differences of the variances and the means indicating that the fast HPLC method is an acceptable alternative. The shorter analysis time makes the method very valuable for commercial quality control of Harpagophytum extracts and its pharmaceutical preparations.     

A New Monolithic‐Type HPLC Column For Fast Separations

The application of a new silica‐based, monolithic‐type HPLC‐column for fast separations is presented. The column is prepared according to a new sol‐gel process, which is based on the hydrolysis and polycondensation of alkoxysilanes in the presence of water soluble polymers. The method leads to “rods” made of a single piece of porous silica with a defined pore structure, i. e. macro‐ and mesopores. The main feature of silica rod columns is a higher total porosity, about 15% higher than of conventional particulate HPLC columns. The resulting column pressure drop is therefore much lower, allowing operation at higher flow rates including flow gradients. Consequently, HPLC analysis can be performed much faster, as it is demonstrated by various applications.     

Chiral Monolithic Silica-Based HPLC Columns for Enantiomeric Separation and Determination: Functionalization of Chiral Selector and Recognition of Selector-Selectand Interaction

This review draws attention to the use of chiral monolithic silica HPLC columns for the enantiomeric separation and determination of chiral compounds. Properties and advantages of monolithic silica HPLC columns are also highlighted in comparison to conventional particle-packed, fused-core, and sub-2-µm HPLC columns. Nano-LC capillary monolithic silica columns as well as polymeric-based and hybrid-based monolithic columns are also demonstrated to show good enantioresolution abilities. Methods for introducing the chiral selector into the monolithic silica column in the form of mobile phase additive, by encapsulation and surface coating, or by covalent functionalization are described. The application of molecular modeling methods to elucidate the selector–selectand interaction is discussed. An application for enantiomeric impurity determination is also considered.     

Spherical ordered mesoporous silicas and silica monoliths as stationary phases for liquid chromatography

Ordered mesoporous silicas such as micelle-templated silicas (MTS) feature unique textural properties in addition to their high surface area (approximately 1000 m2/g): narrow mesopore size distributions and controlled pore connectivity. These characteristics are highly relevant to chromatographic applications for resistance to mass transfer, which has never been studied in chromatography because of the absence of model materials such as MTS. Their synthesis is based on unique self-assembly processes between surfactants and silica. In order to take advantage of the perfectly adjustable texture of MTS in chromatographic applications, their particle morphology has to be tailored at the micrometer scale. We developed a synthesis strategy to control the particle morphology of MTS using the concept of pseudomorphic transformation. Pseudomorphism was recognized in the mineral world to gain a mineral that presents a morphology not related to its crystallographic symmetry group. Pseudomorphic transformations have been applied to amorphous spherical silica particles usually used in chromatography as stationary phases to produce MTS with the same morphology, using alkaline solution to dissolve progressively and locally silica and reprecipitate it around surfactant micelles into ordered MTS structures. Spherical beads of MTS with hexagonal and cubic symmetries have been synthesized and successfully used in HPLC in fast separation processes. MTS with a highly connected structure (cubic symmetry), uniform pores with a diameter larger than 6 nm in the form of particles of 5 microm could compete with monolithic silica columns. Monolithic columns are receiving strong interest and represent a milestone in the area of fast separation. Their synthesis is a sol-gel process based on phase separation between silica and water, which is assisted by the presence of polymers. The control of the synthesis of monolithic silica has been systematically explored. Because of unresolved yet cladding problems to evaluate the resulting macromonoliths in HPLC, micromonoliths were synthesized into fused-silica capillaries and evaluated by nano-LC and CEC. Only CEC allows to gain high column efficiencies in fast separation processes. Capillary silica monolithic columns represent attractive alternatives for miniaturization processes (lab-on-a chip) using CEC.     

Properties of monolithic silica columns for HPLC.

Monolithic silica columns and their use in high peak-capacity HPLC separations are reviewed. Monolithic silica columns can potentially provide higher overall performance than particle-packed columns based on the variable external porosity and variable through-pore size/skeleton size ratios. The high permeability of monolithic silica columns resulting from the high porosity is shown to be advantageous to generate large numbers of theoretical plates with long capillary columns. High permeability together with the high stability of the network structures of silica allows their use in high-speed separations required for a second-dimension column in two dimensional HPLC. Disadvantages of monolithic silica columns are also described.     

Synthesis of spherical porous silicas in the micron and submicron size range: challenges and opportunities for miniaturized high-resolution chromatographic and electrokinetic separations

Classical silica technology has reached its limit with respect to an ultimate minimum particle size of about 2 microm in diameter. Here, a novel process is presented which allows one to synthesize porous silica beads and control their particle diameter in situ, within the range of 0.2-2.0 microm. As a result, no sizing is required and losses of silica are avoided. Furthermore, the process enables one to control in situ the pore structural parameters and the surface chemistry of the silica beads. Even though surface funtionalized silicas made according to this process can principally be applied in fast HPLC the column pressure drop will be high even for short columns. In addition, the column efficiency, expressed in terms of the theoretical plate height is about H-2d(p) in the best case and limited by the A and C term of the Van Deemter equation. In other words the gain in total plate number when using 1-2 microm silica beads in short columns is minimal as compared to longer columns packed with 5 microm particles. Capillary electrochromatography (CEC) as a hybrid method enables the application of micron size as well as submicron size particles. This consequently enhances column efficiency by a factor of 5-10 when compared to HPLC. The use of short CEC columns packed with submicron size silicas provides the basis for fast and efficient miniaturized systems. The most significant feature of CEC as compared to HPLC is that the former allows one to resolve polar and ionic analytes in a single run. An alternative method for miniaturization is capillary electrophoresis (CE) which generates extremely high efficiencies combined with fast analysis. Its application, however, is limited to ionic substances.     

Monolithic columns in high-performance liquid chromatography

Monolithic media have been used for various niche applications in gas or liquid chromatography for a long time. Only recently did they acquire a major importance in high-performance column liquid chromatography (HPLC). The advent of monolithic silica standard- and narrow-bore columns and of several families of polymer-based monolithic columns has considerably changed the HPLC field, particularly in the area of narrow-bore columns. The origin of the concept, the differences between their characteristics and those of traditional packed columns, their advantages and drawbacks, the methods of preparation of monoliths of different forms, and the current status of the field are reviewed. The actual and potential performance of monolithic columns are compared with those of packed columns. Monolithic columns have considerable advantages, which makes them most useful in many applications of liquid chromatography. They are extremely permeable and offer a high efficiency that decreases slowly with increasing flow velocity.     

Applications of silica-based monolithic HPLC columns

The recent invention and successive commercial introduction of monolithic silica columns has motivated many scientists from both academia and industry to study their use in HPLC. The first paper on monolithic silica columns appeared in 1996. Currently about 200 papers have been published relating to applications and characterization of monolithic silica columns, including monolithic capillaries. This review attempts to give an overview covering various aspects of this new column type in the field of high throughput analysis of drugs and metabolites, chiral separations, analysis of pollutants and food-relevant compounds, as well as in bioanalytical separations such as in proteomics. Some of the applications are described in greater detail. The numerous publications dealing with the physicochemical and chromatographic characterization of monolithic silica columns are briefly summarized.     

Simple 2D-HPLC using a monolithic silica column for peptide separation

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.     

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.     

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.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Thermo-responsive monolithic materials

One of the recent major improvements of HPLC was the introduction of monolithic silica columns, which have the advantage of faster separation and lower back pressure as compared to common silica beads. Here, we present an interesting alternative to such reversed-phase monolithic columns by a convenient coupling route of a thermo-responsive polymer to hydrophilic silica monoliths. Poly(N-isopropylacrylamide) (PNIPAM) was polymerized in solution via a reversible addition fragmentation chain transfer (RAFT) polymerization technique and coupled then in situ onto an amino-modified silica monolithic column. These columns were compared with RP-18 monolithic columns in the separation of steroids under isocratic condition in aqueous mobile phase. Separation is optimized just by changing the temperature, instead of changing the mobile phase composition.     

Comments on the Implementation of a Simple Peak Capacity Optimization Procedure and Comparison of Poly(styrene–divinylbenzene) and RP-18 Silica Monolithic Capillary Columns of 5-cm for the Analysis of Protein Digests

For the analysis of protein digests, the peak capacity in reversed-phase liquid chromatography is of paramount importance. A univariate method to maximize the peak capacity as developed by Wang et al. (Anal Chem 78:3406–3416, 20) has been applied and tested for a monolithic RP-18 silica capillary column. In their method, using model peptides representing a bovine serum albumin digest, the gradient time and temperature are kept constant while the flow rate and eluent strength are varied. Despite our criticism on the fixed starting conditions, a long gradient time leading to an unnecessary long analysis time and a high temperature leading to possible degradation products in the chromatogram, and the peak capacity as the only optimization parameter this fast and simple optimization strategy turns out to be applicable to capillary monolithic columns. Furthermore, the influence of the peak capacity on a second optimization parameter, the MS protein identification score, is examined. The procedure is also used to enhance the performance of two popular types of monolithic capillary LC columns (silica-C18 and poly(styrene–divinylbenzene)) of the same length for the analysis of protein digests. Comparison of both columns show that the calculated chromatographic parameters, like productivity and peak capacity, and identification score for both columns are about the same. For a more complicated nine-protein digest the performance of the silica monolith is slightly better.     

Single-step approach to beta-cyclodextrin-bonded silica as monolithic stationary phases for CEC

A novel single-step sol-gel approach for the preparation of beta-CD-bonded silica monolithic electrochromatographic columns is established. The porous silica networks were fabricated inside fused-silica capillaries using sol-gel processing of tetramethoxysilane and an organfunctional silicon alkoxide that contains beta-CD. Scanning electron micrographs and nitrogen adsorption-desorption data showed that these functional monolithic columns have double pores structures with micrometer-size co-continuous through-pores and silica skeletons with open mesopores. The beta-CD monolithic columns have successfully been applied to the separation of several neutral and negatively charged isomers by CEC. The column performance was evaluated by using positional isomers of naphthalenedisulfonic acid as model compounds. A plate height of less than 10 mum for the first eluted isomer of naphthalenedisulfonic acid was obtained at an optimal flow rate (0.47 mm/s) of the mobile phase. Moreover, the columns have been proved to be stable for more than 100 runs during 3 months period and show reasonable column reproducibility.     

Use of partially porous column as second dimension in comprehensive two-dimensional system for analysis of polyphenolic antioxidants

In the present work, a comprehensive LC system using a microbore HPLC column in the first dimension and a partially porous column in the second dimension was developed and applied to the separation of polyphenolic components in a red wine sample. The performance of the partially porous short column (3.0 cm) was compared to that of a monolithic column, of comparable dimensions. The results obtained demonstrated the possibility to use partially porous columns to obtain fast analyses, using high flow rates, under repetitive gradient conditions and with very brief reconditioning times. A conventional HPLC system was used since the backpressure generated by the shell-packed column, even at very high flow rates, was well within the operational limits. The use of an increased column temperature (60 degrees C) allowed a further pressure-drop decrease, with no stationary phase degradation, or loss in column performance.     

干凝胶无机整体柱的制备及评价

以硅酸钾为硅源,甲酰胺为催化剂在毛细管内原位聚合形成干凝胶柱,制备了一系列致密度不同的整体柱。该法在柱制备及高温干燥过程中不会发生柱床断裂和塌陷现象,此特点明显优于以烷氧基硅烷为前驱体制备无机整体柱的方法。考察了不同模数硅酸钾对整体柱柱床结构的影响,用扫描电镜(SEM)和氮吸附法对整体柱结构进行了表征,考察了整体柱柱压与流速的关系;对整体柱进行十八烷基修饰后,测定了反相整体柱(C18整体柱)对蒽的柱效,通过考察甲苯在该柱上的突破曲线,获得了其对甲苯的柱容量。结果显示该整体柱柱床刚性好,在高温、高柱压、高流速时柱床能保持其物理结构的稳定性,对蒽的柱效达到41400理论塔板/m,对甲苯的柱容量为61 ng。     

Characterization of some physical and chromatographic properties of monolithic poly(styrene-co-divinylbenzene) columns

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200 microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. Important chromatographic features of the synthesized columns were characterized and critically compared to the properties of columns packed with micropellicular, octadecylated poly(styrene-co-divinylbenzene) (PS-DVB-C18) particles. The permeability of a 60 mm long monolithic column was slightly higher than that of an equally dimensioned column packed with PS-DVB-C18 beads and was invariant up to at least 250 bar column inlet pressure, indicating the high-pressure stability of the monolithic columns. Interestingly, monolithic columns showed a 3.6 times better separation efficiency for oligonucleotides than granular columns. To study differences of the molecular diffusion processes between granular and monolithic columns, Van Deemter plots were measured. Due to the favorable pore structure of monolithic columns all kind of diffusional band broadening was reduced two to five times. Using inverse size-exclusion chromatography a total porosity of 70% was determined, which consisted of internodule porosity (20%) and internal porosity (50%). The observed fast mass transfer and the resulting high separation efficiency suggested that the surface of the monolithic stationary phase is rather rough and does not feature real pores accessible to macromolecular analytes such as polypeptides or oligonucleotides. The maximum analytical loading capacity of monolithic columns for oligonucleotides was found to be in the region of 500 fmol, which compared well to the loading capacity of the granular columns. Batch-to-batch reproducibility proved to be better with granular stationary phases compared to monolithic stationary phase, in which each column bed is the result of a unique column preparation process.