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Rapid Formation of Polymer Frits in Fused Silica Capillaries Using Spatially defined Thermal Free-Radical Initiated Polymerization.

ABSTRACT: Column preparation in capillary chromatography commonly relies upon the generation of on-column porous frits. Here, we report a simple, robust and low-cost approach for preparing polymer frits on-column, in a rapid and spatially controlled manner using thermal free-radical initiated polymerization. In this approach, a simple, temperature-controlled heating apparatus is positioned adjacent to a 100 μm i.d. fused-silica capillary for a defined duration. Frits were synthesized in 3-(trimethoxysilyl)propyl methacrylate modified capillaries using a monomer solution of 2,2-azobisisobutyronitrile, glycidyl methacrylate, ethylene glycol dimethacrylate, and decanol. Frit length and stability were investigated as a function of polymerization time and temperature. Frit length was easily controlled via a combination of polymerization time and temperature and position was readily controlled using a simple mechanical placement jig. Thermal initiated frits were stable throughout column packing and did not require removal of the capillary polyimide coating. The thermal initiation approach offers higher throughput, with polymerization times of < 2 min compared to ≥ 30 min for UV-initiated polymerization and significantly reduces the cost, enabling broader access to on-column frit technology for a variety of capillary separation applications.

SUBMITTER: Sandy KE

PROVIDER: S-EPMC8313020 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Proteomic analysis using capillary zone electrophoresis (CZE) typically is performed with linear polyacrylamide (LPA) coated capillaries. These capillaries both minimize the adsorption of peptides and proteins to the inner wall of the capillary and decrease electroosmosis, which increases the separation capacity. LPA coating protocols were first reported by Hjerten in 1985. Conventional LPA production is based on the use of tetramethylethylenediamine (TEMED) to catalyze the free-radical polymerization that couples acrylamide to a capillary wall that has been pretreated with ?-methacryloxypropyltrimethoxysilane. The treated capillary is filled with a mixture of monomer, TEMED, and ammonium persulfate; free radical polymerization forms the LPA coating. Over many years, we have observed significant variation in the electroosmotic properties of commercial LPA coated capillaries both along the capillary length and between lots. We believe this variation is due to differences in the time between initiation of the reaction and the filling of the capillary. Here, we report a simple method for the generation of very stable and reproducible coatings. In this protocol, the monomer mixture and an ammonium persulfate initiator are introduced into the capillary without TEMED initiator. The mixture is stable and does not begin polymerization at room temperature. The filled capillary is then heated in a water bath to initiate polymerization in a well-controlled manner. A mixture of four standard proteins was used to evaluate the coating performance. Compared with commercialized LPA capillaries, our LPA capillaries generate much better separation performance and superior protein peak shape in CZE analysis. We also analyzed an intact antibody (MW 150K) by CZE-MS with the new LPA capillary in triplicate runs. The intact antibody generated a Gaussian-shaped electrophoresis peak with 1.2% relative standard deviation in migration time and 8.5% in base peak intensity. An automated CZE-MS system was used to generate 97 successive separations of a BSA tryptic digest over a 145-h period. Separation efficiency averaged over 100,000 theoretical plates across this period with no systematic variation. The LPA coating protocol had excellent batch-to-batch reproducibility with relative standard deviation in migration time<7%, and in separation window<1%.

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Rapid Formation of Polymer Frits in Fused Silica Capillaries Using Spatially defined Thermal Free-Radical Initiated Polymerization.

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