Effects of pressure and frictional heating on protein separation using monolithic columns in reversed-phase chromatography (original) (raw)
Related papers
Analytical Chemistry, 2007
Building upon the micromachined column idea proposed by the group of Regnier in 1998, we report on the first high-resolution reversed-phase separations in micromachined pillar array columns under pressure-driven LC conditions. A three component mixture could be separated in 3 s using arrays of nonporous silicon pillars with a diameter of approximately 4.3 µm and an external porosity of 55%. Under slightly retained component conditions (retention factor k′ ) 0.65-1.2), plate heights of about H ) 4 µm were obtained at a mobile phase velocity around u ) 0.5 mm/s. In reduced terms, such plate heights are as low as h min ) 1. Also, since the flow resistance of the column is much smaller than in a packed column (mainly because of the higher external porosity of the pillar array), the separation impedance of the array was as small as E ) 150, i.e., of the same order as the best currently existing monolithic columns. At pH ) 3, yielding very low retention factors (k′ ) 0.13 and 0.23), plate heights as low as H ) 2 µm were realized, yielding a separation of the three component mixture with an efficiency of N ) 4000-5000 plates over a column length of 1 cm. At higher retention factors, significantly larger plate heights were obtained. More experimental work is needed to investigate this more in depth. The study is completed with a discussion of the performance limits of the pillar array column concept in the frame of the current state-of-the-art in microfabrication precision.
Journal of Chromatography A, 2014
The increase of the operating pressure in Liquid Chromatography, has been one of the crucial steps toward faster and more efficient separations. In the present contribution, it was investigated if the pressure limits for narrow-bore columns (2.1 mm ID) could be increased beyond those of commercially available (1300 bar) instrumentation without performance loss. Whereas previous studies applying pressures higher than 2000 bar were limited to the use of columns with a diameter smaller or equal to 1 mm, it is a difficult feat to expand this to 2.1 mm ID given that viscous-heating effects increase according to the fifth power of the column radius. A prototype LC setup was realized, allowing to operate at pressures up to 2600 bar (260 MPa) for large separation volumes (>5 mL). The performance of an in-house-built injector was compared at 800 bar to commercially available injectors, yielding equal performance but twice the maximum pressure rating. The performance of (coupled) custom columns packed with fully porous and superficially porous particles were assessed at ultra-high-pressure conditions. Increasing the inlet pressure from 800 to 2400 bar and scaling the column length proportionally (from 150 mm to 450 mm), resulted in the theoretically expected linear increase in plate count from 20,000 to 59,000. A maximum plate number of 81,000 was realized using a 600 mm long (coupled) column at 2600 bar. Viscous-heating effects were diminished by insulating coupled columns and applying an intermediate-cooling strategy in a forced-air oven.
Column Selection for the Reversed-Phase Separation of Proteins
The ability to analyze amino acid derivatives and small peptides by reversed phase HPLC has led to an active search of column types and mobile phase conditions for the fast separation of larger peptides and proteins. After the earliest publications (in 1977 and 1978) on protein separations using silica gel based alkyl bonded phases, one has seen an exponential growth in the number of scientific papers in this field, but only a modest increase in the number of useful types of stationary phases.
Journal of Chromatography A, 2008
The review highlights the fundamentals and the most prominent achievements in the field of high-performance liquid chromatography (HPLC) column development over a period of nearly 50 years. After a short introduction on the structure and function of HPLC columns, the first part treats the major steps and processes in the manufacture of a particle packed column: synthesis and control of particle morphology, sizing and size analysis, packing procedures and performance characterization. The next section is devoted to three subjects, which reflect the recent development and the main future directions of packed columns: minimum particle size of packing, totally porous vs. core/shell particles and column miniaturization. In the last section an analysis is given on an alternative to packed columns-monolithic columns, which have gained considerable attraction. The challenges are: improved packing design based on modeling and simulation for targeted applications, and enhanced robustness and reproducibility of monolithic columns. In the field of miniaturization, particularly in chip-based nano-LC systems, monoliths offer a great potential for the separation of complex mixtures e.g. in life science.
Journal of Chromatography A, 2020
Column selection often centers on the identification of a stationary phase that increases resolution for a certain class of compounds. While gains in resolution are most affected by selectivity of the stationary phase or modifications of the mobile phase, enhancements can still be made with an intentional selection of the packing material's microstructure. Unrestricted mass transfer into the particle's porous structure minimizes band broadening associated with hindered access to stationary phase. Increased efficiency, especially when operating above the optimal flow rates, can be gained if the pore size is significantly larger than the solvated analyte. Less studied are the effects of reduced access to pores due to physical hindrance and its impact on retention. This article explores the relationship between pore size and reversed phase retention and specifically looks at a series of particle architectures with reversed phase and size exclusion modes to study retention associated with access to stationary phase surface area.
Journal of Chemical Technology & Biotechnology, 2017
BACKGROUND: Poorly packed chromatography columns are known to reduce drastically the column efficiency and produce broader peaks. Controlled bed compression has been suggested to be a useful approach for solving this problem. Here the relationship between column efficiency and resolution of protein separation are examined when preparative chromatography media were compressed using mechanical and hydrodynamic methods. Sepharose CL-6B, an agarose based size exclusion media was examined at bench and pilot scale. The asymmetry and height equivalent of a theoretical plate (HETP) was determined by using 2% v/v acetone, whereas the void volume and intraparticle porosity (p) were estimated by using blue dextran. A protein mixture of ovalbumin (chicken), bovine serum albumin (BSA) and '-globulin (bovine) with molecular weights of 44, 67 and 158 kDa, respectively, were used as a 'model' separation challenge. RESULTS: Mechanical compression achieved a reduction in plate height for the column with a concomitant improvement in asymmetry. Furthermore, the theoretical plate height decreased significantly with mechanical compression resulting in a 40% improvement in purity compared with uncompressed columns at the most extreme conditions of compression used. CONCLUSION: The results suggest that the mechanical bed compression of Sepharose CL-6B can be used to improve the resolution of protein separation.
Effect of Pressure, Particle Size, and Time on Optimizing Performance in Liquid Chromatography
Analytical Chemistry, 2009
Although the principles of optimization of HPLC have a long history starting with the work of Giddings in the 1960s, and continuing with work by Knox and Guiochon extending into the 1990s we continue to see statements that flatly contradict theory. A prominent example is the notion that optimum 'performance', as measured by plate count, is always obtained by operating conventional length columns (e.g., 5 to 15 cm) at eluent velocities corresponding to the minimum plate height in the van Deemter curve. In the last decade the introduction of 'Poppe plots' by Poppe and 'kinetic plots' by Desmet and others has simplified the selection of 'optimum' conditions, but it is evident that many workers are not entirely comfortable with this framework. Here we derive a set of simple, yet accurate equations that allow rapid calculation of the column length and eluent velocity that will give either the maximum plate count in a given time, or a given plate count in the shortest time. Equations are developed for the optimum column length, eluent velocity and thus plate count for both the case when particle size is pre-selected, and when particle size is optimized along with eluent velocity and column length. Although both of these situations have been previously considered the implications of the resulting equations have not been previously made explicit. Lack of full understanding of the consequences of the differences between these two cases is very important and responsible for many erroneous conclusions. The simple closed-form equations that result from this work complement the graphical, iterative approaches of Poppe and Desmet; the resulting compact framework allows practitioners to rapidly and effectively find the operating parameters needed to achieve a specific separation goal in the shortest time, and to compare emerging technologies (e.g., high pressure, high temperature, and different particle types) in terms of their impact on achievable plate counts and speeds in HPLC. A web-based calculator based on the equations presented here is now available (http://homepages.gac.edu/\~dstoll/calculators/optimize.html). "The fastest possible analysis with any column design will be achieved with a column operated at the maximum possible pressure drop and having a length as to give the plate number necessary to perform the desired separation." 1
Proceedings of the Estonian Academy of Sciences, 2015
The separation of high-molecular compounds is very difficult, if possible at all, under isocratic conditions. For this gradient elution is needed. The theory of gradient elution for small molecules is well established; however, its applications to reversed-phase gradient separations of biopolymers are not straightforward because of specific problems, such as slow diffusion, limited accessibility of the stationary phase for larger molecules, or possible sample conformation changes during the elution. We used high performance liquid chromatography to investigate the reversed-phase chromatographic behaviour of 14 proteins. The first step was the determination of the experimental data, and then these data were used to predict gradient retention times. A water-organic solvent-trifluoroacetic acid system was used to examine the influence of experimental parameters. The chromatographic results from four C18-chain-length supports were comparable.