Repeatability of monolithic HPLC columns while using a flow program

Fast HPLC methods are becoming more and more important. Using monolithic HPLC columns for fast separations, a flow program can be applied for further decrease in the total run time. An interesting issue was whether the flow program affects repeatability. The investigated method was a generic assay for the oral antidiabetic drugs glibenclamide and glimepiride in the presence of two of their degradation products. A flow program ranging from 5.0 to 9.9 mL/min had been set up to decrease the run time to approximately 1.7 min. Within-day RSD% (n = 40) for both retention times and peak areas were less than 1%. At flow rates higher than 7 mL/min, repeatability was impaired to some extent. It became mainly noticeable through the day-to-day precision (n = 60) which showed RSD% up to 2%. However, further investigations indicated that this was rather related to pump inefficiency at high flow rates than to the flow program as such. Presuming the use of appropriate equipment, qualified for high flow rates, the application of a flow program for shortening the run time is absolutely reasonable and does not affect repeatability.     

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.     

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.     

Preparation and application of hydrophilic monolithic columns

Hydrophilic interaction chromatography (HILIC) has experienced increasing attention in recent years. Much research has been carried out in the area of HILIC separation mechanisms, column techniques and applications. Because of their good permeability, low resistance to mass transfer and easy preparation within capillaries, hydrophilic monolithic columns represent a trend among novel HILIC column techniques. This review attempts to present an overview of the preparation and applications of HILIC monolithic columns carried out in the past decade. The separation mechanism of various hydrophilic monolithic stationary phases is also reviewed.     

Reversed phase monolithic analytical columns for the determination of HA1 subunit of influenza virus haemagglutinin

Monoliths are chromatographic stationary phases, which were specially designed for efficient purification of large biomolecules, like proteins, viruses and DNA. In this work, the small scale monolithic butyl (C4) and styrene-divinyl benzene (SDVB) columns were applied for reversed phase analyses of various degraded influenza viruses. The binding of the HA1 subunit of haemagglutinin to the monolithic columns was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Western blot. The working linear range was determined as 1.60 × 10¹⁰ viral particles/mL to at least 1.64 × 10¹¹ viral particles/mL, the limit of detection was found to be 2.56 × 10⁹ virus particles/mL and the limit of quantification was 5.12 × 10⁹ virus particles/mL. The analytical HPLC method developed with the H1N1 virus was also applicable for the analytics of the HA1 subunit of H3N2 influenza virus and the influenza B virus.     

Preparation and application of monolithic columns with narrow immobilized pH gradients

Monolith with immobilized pH gradient (M-IPG) is a novel separation matrix for amphoteric substances, such as peptides and proteins. To improve the properties of the newly designed column, efforts were made to optimize the preparation procedure, including the concentrations of the monomers, glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA), as well as the kinds of porogens, which led to a monolith with improved permeability, uniformity and continuity. In addition, different diamines, including ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane and 1,5-diaminopentane, were subjected to aminate the polymer, and the last two exhibited excellent reactivity with epoxy on the polymer surface, which could obviously make the immobilization of pH gradient facilitated. Under the optimal conditions, a simple method to prepare M-IPG column with narrow pH gradient was developed with commercial carrier ampholytes (CAs) solution. Such columns were applied into the analysis of proteins and peptides, and showed the improved resolution compared to the traditional one with a wide pH distribution.     

Rapid and high-resolution analysis of geometric polyprenol homologues by connected octadecylsilylated monolithic silica columns in high-performance liquid chromatography

A Chromolith Performance octadecylsilyl (ODS) monolithic silica column (Merck) was compared with a conventional microparticulate ODS-bonded silica column in the high-performance liquid chromatography separation of natural polyprenols. A system comprising two connected monolithic columns afforded an equivalent separation at half the analysis time of the conventional method. Furthermore, ten connected columns achieved a tremendously high-resolution separation, in which the complicated series of homologous polyprenols with geometric isomerism were fully separated.     

Ionic liquids monolithic columns for protein separation in capillary electrochromatography

A series of ionic liquids (ILs) monolithic capillary columns based on 1-vinyl-3-octylimidazolium (ViOcIm⁺) were prepared by two approaches (“one-pot” approach and “anion-exchange” approach). The effects of different anions (bromide, Br⁻; tetrafluoroborate, BF₄⁻; hexafluorophosphate, PF₆⁻; and bis-trifluoromethanesulfonylimide, NTf₂⁻) on chromatography performance of all the resulting columns were investigated systematically under capillary electrochromatography (CEC) mode. The results indicated that all these columns could generate a stable reversed electroosmotic flow (EOF) over a wide pH range from 2.0 to 12.0. For the columns prepared by “one-pot” approach, the EOF decreased in the order of ViOcIm⁺Br⁻ > ViOcIm⁺BF₄⁻ > ViOcIm⁺PF₆⁻ > ViOcIm⁺NTf₂⁻ under the same CEC conditions; the ViOcIm⁺Br⁻ based column exhibited highest column efficiencies for the test small molecules; the ViOcIm⁺NTf₂⁻ based column possessed the strongest retention for aromatic hydrocarbons; and baseline separation of four standard proteins was achieved on ViOcIm⁺NTf₂⁻ based column corresponding to the highest column efficiency of 479 000 N m⁻¹ for cytochrome c (Cyt c). These results indicated that the property of ILs based columns could be tuned successfully by changing anions, which gave these columns potential to separate both small molecules and macro biomolecules.     

十八烷基键合硅胶整体柱的制备及其色谱性能评价

选用十八烷基二甲基氯硅烷作为硅烷化试剂,制备十八烷基反相键合硅胶整体柱,并用元素分析进行了表征。以苯、甲苯、联苯、萘、菲混合物作为测试溶质,在以甲醇和水为二元流动相的反相色谱条件下评价了该键合整体柱的色谱性能,考察了该整体柱适用的pH范围,以及柱压降、柱效与流速的关系。结果表明,该硅胶整体柱键合效果良好,具有较好的反相色谱性能,且在pH 2~8之间稳定性好,柱压降、柱效受流速影响较小,对5种心血管系统用药可以达到快速、有效的分离。     

Purification of genomic DNA by short monolithic columns

The isolation and purification of nucleic acids is essential for many procedures in molecular biology. After showing that bacterial and eukaryotic genomic DNA can be specifically bound to the CIM DEAE monolithic column, this characteristic was exploited in development of a simple and fast chromatographic procedure for isolation and purification of genomic DNA from cell lysates that does not include the usage of toxic organic solutions. The purity and the quality of the isolate as well as the duration of the procedure was similar to other chromatographic methods used today for isolation of genomic DNA, but the initial sample volume was not restricted.     

Fast HPLC method for the determination of glimepiride, glibenclamide, and related substances using monolithic column and flow program

This work presents a fast method for the simultaneous separation and determination of glimepiride, glibenclamide, and two related substances by RP LC. The separation was performed on a Chromolith Performance (RP-18e, 100 mm x 4.6 mm) column. As mobile phase, a mixture of phosphate buffer pH 3, 7.4 mM, and ACN (55:45 v/v) was used. Column oven temperature was set to 30 degrees C. The total chromatographic run time was 80 s. This was achieved using a flow program from 5 to 9.9 mL/min. Precisions of the interday and the intraday assay for both retention times and peak areas for the four analyzed compounds were less than 1.2%. The method showed good linearity and recovery. The short analysis time makes the method very valuable for quality control and stability testing of drugs and their pharmaceutical preparations.     

Ionic liquids-assisted fabrication of silica-based monolithic columns

The mesopores of a monolithic silica column are very important and useful for chromatographic separation since they can offer sufficiently large surface area. In this paper, a novel method with the assistance of an ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate ([bmin]BF₄)) was developed for the preparation of a C18-modified monolithic silica column for the first time, in which, the through pores and mesopores were formed simultaneously during the sol-gel reaction. The method is effective to simplify the preparation process of the silica-based monolithic columns. The factors influencing the sol-gel process, including the content of methanol and pH, were studied. The chromatographic performance of the prepared monolithic column was evaluated by the separation of alkylbenzenes.     

高效液相色谱整体柱在药物分离分析中的应用进展

本文对高效液相整体柱在药物分离分析方面的应用进行了综述.主要介绍了以烷氧基硅烷为主要原料,采用溶胶-凝胶法制备的硅胶整体柱,由于其具有微米级通孔结构和大的比表面积,他们在高效、快速分离小分子物质方面得到广泛地应用.对于聚合物整体柱,主要介绍了包括分子印迹聚合物在内的有机聚合物整体柱在药物分离、生物样品的处理等方面的应用.     

Practical aspects of fast HPLC separations for pharmaceutical process development using monolithic columns

A large number of samples can be generated during pharmaceutical process development. Fast separation for these samples is usually challenging due to the complexity of sample matrix, which requires high efficiency as well as high speed. Monolithic columns (E. Merck, Germany) were investigated as a possible tool for reducing separation time in reversed-phase HPLC without significantly sacrificing efficiency or resolution. Both van Deemter plots and separations of alkyl benzenes and in-process samples showed that monolithic columns were suitable for fast separations without significantly compromising resolution. Practical parameters including the pressure drop, retention factor, selectivity, and tailing factor of monolithic columns (Chromolith type) were compared to those of conventional YMC 150 mm × 4.6 mm (3-μm particles) and 250 mm × 4.6 mm (5-μm particles) packed columns. The batch-to-batch reproducibility of the 100 mm × 4.6 mm Chromolith columns from five randomly ordered batches was also compared to the 250 mm × 4.6 mm YMC particle-packed columns. Fast and efficient separations of complicated process samples including crude drug substances, reaction mixtures, and crystallized mother liquors were demonstrated for both monolithic columns and conventional packed columns. The analysis times were decreased by three to seven times on the coupled monolithic columns, while maintaining the comparable resolution to typical 5-μm particle-packed 250 mm × 4.6 mm columns.     

S-citalopram imprinted monolithic columns for capillary electrochromatography enantioseparations

In this study, the molecular imprinting method was used to separate enantiomeric forms of chiral antidepressant drug, R,S-citalopram (R,S-CIT) in aqueous solution by CEC system combining the advantages of capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). For that, an amino acid-based molecularly imprinted monolithic capillary column was designed and used as a stationary phase for selective separation of S-citalopram (S-CIT) for the first time. S-CIT was selectively separated from the aqueous solution containing the other enantiomeric form of R-CIT, which is the same in size and shape as the template molecule. Morphology of the molecularly imprinted (MIP S-CIT) and non-imprinted (NIP S-CIT) monolithic capillary columns was observed by scanning electron microscopy. Imprinting efficiency of MIP S-CIT monolithic capillary column used for selective S-CIT separation was verified by comparing with NIP S-CIT and calculated imprinting factor (I.F:1.81) proved the high selectivity of the MIP S-CIT for S-CIT. Cavities formed for S-CIT form enabled selective (α = 2.08) separation of the target molecule from the other enantiomeric R-CIT form. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 7.68 × 10⁻⁶ m²/Vs for R,S-CIT at pH 7.0 10 mM PB and 50% ACN ratio. The performance of both MIP S-CIT and NIP S-CIT columns was estimated by repeating the R,S-CIT separations with intra-batch and inter-batch studies for reproducibility of retention times of R,S-CITs. Estimated RSD values that are lower than 2% suggest that the monolithic columns separate R,S-CIT enantiomers without losing separation efficiency.     

Repeatedly usable immobilized pH gradient in a monolithic capillary column

Glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) were used to synthesize a monolithic capillary column containing reactive epoxy groups. Glutaraldehyde was introduced and linked to the monolith after a process of amination. An aqueous solution of commercial carrier ampholytes (CAs, Ampholine) was focused in such a polymer column. The primary amino groups of CAs reacted with glutaraldehyde along the capillary. CAs were immobilized at different positions in the column according to their isoelectric points (pI), resulting in a monolithic immobilized pH gradient (M-IPG). Isoelectric focusing (IEF) was performed without CAs in such an M-IPG column. Due to the covalent attachment of the CAs this M-IPG can be repeatedly used after its preparation. Good stability, linearity, and reproducibility were obtained.     

Enantiomer separation on monolithic silica HPLC columns using chemically bonded methylated and methylated/acetylated 6-O-tert-butyldimethylsilylated beta-cyclodextrin

A new 2,3-methylated 3*-monoacetylated 6-O-tert-butyldimethylsilylated beta-CD derivative was synthesized and chemically bonded onto aminopropyl derivatized monolithic silica HPLC columns. In this CD derivative, only one of seven methyl groups in 3-position was substituted by an acetyl group. Its applicability as a chiral stationary phase for HPLC was tested and compared with exclusively 2,3-methylated 6-O-tert-butyldimethylsilylated beta-CD immobilized onto aminopropyl-modified monoliths. Thirty-two chiral compounds from different chemical classes and different functionalities were tested under RP conditions. Fourteen compounds were resolved into their enantiomers by methylated 6-O-tert-butyldimethylsilylated beta-CD. By use of methylated/acetylated 6-O-tert-butyldimethylsilylated beta-CD as the chiral stationary phase 7 analytes were successfully stereodifferentiated.     

Surfactant-bound monolithic columns for separation of proteins in capillary high performance liquid chromatography

A surfactant-bound monolithic stationary phase based on the co-polymerization of 11-acrylamino-undecanoic acid (AAUA) is designed for capillary high performance liquid chromatography (HPLC). Using D-optimal design, the effect of the polymerization mixture (concentrations of monomer, crosslinker and porogens) on the chromatographic performance (resolution and analysis time) of the AAUA–EDMA monolithic column was evaluated. The polymerization mixture was optimized using three proteins as model test solutes. The D-optimal design indicates a strong dependence of chromatographic parameters on the concentration of porogens (1,4-butanediol and water) in the polymerization mixture. Optimized solutions for fast separation and high resolution separation, respectively, were obtained using the proposed multivariate optimization. Differences less than 6.8% between the predicted and the experimental values in terms of resolution and retention time indeed confirmed that the proposed approach is practical. Using the optimized column, fast separation of proteins could be obtained in 2.5 min, and a tryptic digest of myoglobin was successfully separated on the high resolution column. The physical properties (i.e., morphology, porosity and permeability) of the optimized monolithic column were thoroughly investigated. It appears that this surfactant-bound monolith may have a great potential as a new generation of capillary HPLC stationary phase.     

Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform

In the presence of methanol and n‐decanol as porogens, a partially filled capillary monolithic column was prepared by in situ reaction of glycidyl methacrylate and poly (ethylene glycol) diacrylate. Then, Pharmalyte 3–10 was immobilized on this column in order to obtain a capillary isoelectric focusing (cIEF) column with monolithic immobilized pH gradient (M‐IPG). In addition, an online self‐built platform for protein separation was established on account of the introduction of a cross‐shaped unit and two short‐off valves. In this platform, a cross‐shaped unit was not only used to join the M‐IPG column and a six‐way injection valve (1.5 μL sample loop), but also to supply a volume pool of anode buffer so that the process of injection, focusing and mobilization of samples could be sequentially performed. The short‐off valve in the tee unit or cross‐shaped unit could be used to control the direction of the fluid flow. Using this online cIEF platform and under the optimized conditions, 7‐proteins mixture could be separated and a good linear correlation between pI values and migration times was obtained by the M‐IPG column. Meanwhile, based on the online cIEF platform, human serum proteins and a mixture of Hb A and Hb A_1c have been successfully resolved with the newly developed M‐IPG column.     

Retention behaviour of beta-blockers in HPLC using a monolithic column

The chromatographic behaviour of a new HPLC-stationary phase (Chromolith RP-18e) is described for separation of beta-blockers. The effects of the different chromatographic conditions (buffer system, pH value, content of organic modifier, injection volume and flow rate) on the separation behaviour were studied. At higher flow rates the peaks seemed to be more symmetrical than at lower flow rates. The use of buffer or salt in the mobile phase for this separation is found to be very essential and the counter-anion type of this buffer or salt significantly affected the retention behaviour of beta-blockers while the cation type did not play the same important role.