Sieving matrix solutions

Nowadays, the sieving matrices most employed in CSE are polymer solutions that under suitable conditions may form a transient mesh or sieving matrix that provide the size-based separation of charged biopolymers. The polymer solutions can be formulated with linear acrylamide and N-substituted acrylamide polymers, cellulose derivatives, polyethylene oxide, and its copolymers or with a variety of polymers, such as polyvinylpyrrolidone (PVP), polyethylene oxide (PEO), and hydroxypropyl cellulose(HPC), which do not necessitate the preventive coating of the capillary wall due to their ability to dynamically coat the inner surface of the capillary, resulting in suppressed EOE and sample interactions with the capillary wall. 

Free-solution (gel-free) CE separation has been used in the detection of a target gene sequence amplified using the CPT reaction (see Chapter 9, section 9.2.1.7 for details). The intact chimeric oligonucleotide probe (fluorescein-labeled at the 5 end and biotin-labeled at the 3 end) was separated from a cleaved probe (only fluorescein-labeled at the 5 end). Owing to the presence of biotin in the intact probe, it migrated later than the cleaved probe, even in the absence of a gel sieving matrix [606]. 

Various sieving matrices for DNA fragment separation in a glass chip were evaluated. It was found the performance of HEC was comparable to PDMA, but was superior to polyacrylamide and PEO [611]. Hydroxylpropylcellulose (HPC), instead of HEC or HPMC, was used as a sieving matrix to achieve better DNA separations. In addition, the lower viscosity of HPC allows for more concentrated solution to be used without filling problems. CGE separation of DNA for analyzing mutations associated with Duchenne muscular dystrophy (DMD) was demonstrated [612],... 

In electrophoresis, protein variants are separated due to differences in their mobility in the presence of an electrical field. Traditionally, electrophoresis has been performed using a stationary slab gel. The most common electrophoretic technique is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this technique, a polyacrylamide gel provides a sieving matrix for the protein solution. The protein is mixed with an SDS solution prior to loading onto the gel. By coating the protein with SDS, the charge to mass ratio becomes uniform between proteins. This allows proteins to be separated solely on molecular weight in the presence of the electrical field. The protein/SDS solution is loaded onto the gel which is... 

In PVA-coated capillaries it was possible to separate at pH 2.5 the standards of poly-2-vinylpyridinium hydrochloride (p(2-VPy)) in the molecular mass range between 1500 and 1,730,000 g mol-1 with dextran T70 as sieving matrix [20]. An example is shown in Fig. 4, where a 5% solution of dextran T70 has been used. The efficiency of the monomolecular basic marker 4-aminopyridine is excellent, demonstrating the exclusion of secondary adsorptive effects at the capillary surface. Hence, the broad peaks of the polymeric standards are due to their polydispersity. As in CE the width of the peaks depends on their migration velocity through the detection window, no direct comparison of broadness of the individual peaks and analyte polydispersity is possible. However, for each individual peak the methods applied in SEC for calculation of the different molecular mass averages can be applied. 

This chapter discussed several aspects of DNA analysis by CE and microchip technologies using polymer solutions as the sieving matrix. Analysis of DNA is a multivariate system by nature and both the separation and the result of the separation are suitable to a large number of chemometric tools. 

Although their free solution behaviors are similar, flexible molecules (like DNA and denatured proteins) exhibit dramatically different behavior in a sieving matrix. Once the size of the pores in the gel becomes small relative to the radius of gyration of the polyelectrolyte chain, the polyelectrolyte chain must uncoil in order to move through the gel. Although the uncoiling process is entropically unfavorable (since it reduces the number of available conformations for the chain), the entropy loss is offset by the reduction in the electrical potential energy as the chain moves in the field. 

CMS particles (<25 pm) have been fabricated and characterized. CMS particles were treated in PVP kollidone 15 (PVP K-15) sizing bath solution (1-10 wt% in isopropanol) prior to embedment into the matrix solution to enhance matrix-sieve inteifacial adhesion i.e., PVP K-15 powder was dissolved in isopropanol to produce dilute solution with the concentration of 1-10 wt% PVP. A predetermined amount of CMS particles was added to the PVP solution and stirred at 30 rpm for 1 h. The sized CMS cake was rinsed with isopropanol to remove unadsorbed PVP before further drying in a vacuum oven at 50 °C for 24 h. 

Fibers coated with the matrix solution were carried out simultaneously along with fiber winding. The matrix solution is composed of sieved salt particles dispersed in a Hydrothane(D/DMAC (Dimethylacetamide) solution. After winding, the impregnate fibers were removed from the machine and inserted in a glass cylinder containing ethyl alcohol to remove the DM AC. Once the DMAC was removed the composite structure is immersed in water to leach out the salt. 

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