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.
Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate ...monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20
mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.
•G-quadruplex separation and purification using ion-exchange chromatography•Method optimization to control and enhance the G-quadruplex formation•Native mass spectrometry was utilized to identify the ...intact G-quadruplex•Ion-pairing denaturing method to confirm the single-stranded oligonucleotide•Comparison between monolithic CIM-QA and particle-based TSKgel SuperQ-5PW columns
The current study investigates a method for purification of the G-quadruplex secondary structure, naturally formed by a guanine-rich 21-mer oligonucleotide strand using a monolithic convective interaction media-quaternary amine (CIM-QA) column under ion-exchange conditions. The monolithic support was initially evaluated on a preparative scale against a highly efficient TSKgel SuperQ-5PW ion-exchange support designed for oligonucleotide purification. The CIM analogue demonstrated clear advantages over the particle-based support on the basis of rapid separation times, while also affording high purity of the G-quadruplex. Various parameters were investigated including the type of mobile phase anion, cation, pH and injection load to induce and control quadruplex formation, as well as enhance chromatographic separation and final purity. Potassium afforded the most prominent quadruplex formation, yet sodium allowed for the highest resolution and purity to be achieved with a 30 mg injection on an 8 ml CIM-QA monolithic column. This method was applied to purify in excess of 300 mg of the quadruplex, with excellent retention time precision of under 1% RSD. Native mass spectrometry was utilized to confirm the identity of the intact G-quadruplex under non-denaturing conditions, while ion-pairing reversed-phase methods confirmed the presence of the single-stranded oligonucleotide in high purity (92%) under denaturing conditions.
The key advantage of the purification method enables isolation of the G-quadruplex in its native state on a milli-gram scale, allowing structural characterization to further our knowledge of its role and function. The G-quadruplex can also be subsequently denaturated at elevated temperature causing single strand formation if additional reactions are to be pursued, such as annealing to form a duplex, and evaluation in in vitro or in vivo studies.
