A weak ion-exchange grafted methacrylate monolith was prepared by grafting a methacrylate monolith with glycidyl methacrylate and subsequently modifying the epoxy groups with diethylamine. The ...thickness of the grafted layer was determined by measuring permeability and found to be approximately 90
nm. The effects of different buffer solutions on the pressure drop were examined and indicated the influence of pH on the permeability of the grafted monolith. Protein separation and binding capacity (BC) were found to be flow-unaffected up to a linear velocity of 280
cm/h. A comparison of the BC for the non-grafted and grafted monolith was performed using β-lactoglobulin, bovine serum albumin (BSA), thyroglobulin, and plasmid DNA (pDNA). It was found that the grafted monolith exhibited 2- to 3.5-fold higher capacities (as compared to non-grafted monoliths) in all cases reaching values of 105, 80, 71, and 17
mg/ml, respectively. It was determined that the maximum pDNA capacity was reached using 0.1
M NaCl in the loading buffer. Recovery was comparable and no degradation of the supercoiled pDNA form was detected. Protein
z-factors were equal for the non-grafted and grafted monolith indicating that the same number of binding sites are available although elution from the grafted monolith occurred at higher ionic strengths. The grafted monolith exhibited lower efficiency than the non-grafted ones. However, the baseline separation of pDNA from RNA and other impurities was achieved from a real sample.
New non-destructive method for characterization of ion exchange chromatographic columns based on transient pH formed by a step change in ionic strength of buffer solutions was examined. The method ...was used to distinguish between cation and anion or weak and strong ion exchange chromatographic supports and to determine the capacity of the chromatographic resins. The general scheme to distinguish between most commonly used types of ion exchange chromatographic columns was proposed. The duration of pH transient was shown to be linearly proportional to the total ionic capacity and was used to estimate protein dynamic binding capacity of the resin. The effects of pH, concentration and temperature on transient pH duration were examined.
Chemical and chromatographic stability of methacrylate-based monolithic columns bearing 3-
N,
N-diethylamino-2-hydroxypropyl (DEAE) and quarternary amine (QA) groups was studied. The leakage products ...from both monolithic columns were determined and the leakage of amines has been quantified in alkali solutions. Monolithic columns bearing QA functional groups being exposed to 1
M sodium hydroxide solution for up to 3 months caused reduction of ion-exchange groups for approximately 12%, while for DEAE monolithic columns was only around 3% in 1 year. In 0.1
M NaOH and 20% ethanol degradation was significantly lower. The main leaking compound from DEAE monolith was found to be 3-(diethylamino)-1,2-propanediol and 2,3-dihydroxypropyltrimethylammonium salt for QA monolith. During repeated 50 cleaning-in-place (CIP) cycles, no changes in chromatographic properties were detected.
Chromatographic monoliths exhibit several properties like flow‐unaffected resolution and dynamic binding capacity, low pressure drop and high capacity for extremely large molecules, which are ...advantageous to the purification of large biomolecules such as proteins, DNA or even viruses. Recently, large‐volume monolithic columns were introduced enabling the monoliths to be incorporated into industrial downstream processes. Due to the monolithic structure, however, conventional methods for resin characterization cannot be applied to monoliths, which limit their usage under cGMP conditions. In this article, a methodology of how to overcome this problem and to determine the most important parameters is presented. Several fast and noninvasive methods for monolith characterization are introduced. Their usage enables accurate determination of monolith pore size distribution and specific surface area, uniformity of the monolith as well as type and density of the active groups before or during monolith usage.
Chromatographic monoliths nowadays are used in all chromatographic fields, also for industrial applications. Monitoring and control of the monolith properties require the development of new, noninvasive techniques. The methods presented enable the accurate determination of monolith pore size distribution and specific surface area, uniformity of the monolith as well as type and density of the active groups before or during monolith usage.
