Solutes in gels

Various techniques are available for determining the effective diffusivity of solute in gel (Itamunoala, 1988). One of the most reliable techniques is the thin-disk method which uses a diffusion cell with two compartments divided by a thin gel. Each compartment contains a well-stirred solution with different solute concentrations. Effective diffusivity can be calculated from the mass flux verses time measurement (Hannoun and Stephanopoulos, 1986). A few typical values of effective diffusivities are listed in Table 3.2. 

Peptide In-solution In-gel % Recovery Peptide In-solution PVDF % Recovery... 

In order to compare peptide recovery from proteolysis in solution, in gel and on PVDF membrane, we digested the same amount of BSA (2.5 ig each, 35 pmol). Figure 1 shows chromatograms of peptides from Lys-C digestion of reduced and denatured BSA without alkylation (A), or with the addition of either iodoacetamide (B), or 4-vinylpyridine (C). As was demonstrated before (Stone et al. 1989), it is clear that BSA was not efficiently digested, as indicated by the low intensity of the peptide peaks when it was not alkylated. On the other hand, alkylated BSA, either by iodoacetamide (B) or 4-vinylpyridine (C), were digested much better, as indicated by the higher intensity of the peaks and the number of peaks. 

We should stress here, as was pointed before (Stone et al. 1989), that whichever digestion approach is used—in solution, in gel, or on membrane—reduction of cystine without subsequent alkylation results in incomplete digestion leading to peptide loss. The difference is especially great for proteins such as BSA which contains 6% cysteine residues. Thus, for best peptide yield from unknown proteins, we recommend alkylation before resolving the proteins by SDS-PAGE or HPLC and before digestion. 

Hydrated with modified porcine trypsin lOng/L in 50 mM NH4HCO3 solution In-gel digestion at 37 °C overnight... 

Protein detection Protein quantitation in solution in gels, in CE and 2DE, and on blots SYTO, SYBR, fluorescamine and o-phthaldialdehyde, novel dyes BODIPY, NanoOrange, CBQCA and SYPRO protein gel stains In addition to the conventional dyes for protein staining (Coomassie Blue, colloidal gold), many novel fluorescent stains have been developed that allow highly sensitive protein quantitation in solution and in gels, particularly the SYPRO protein gel stains. 36... 

A solution of 2,3-dimethylindole (145 g, 1 mol) in dry dioxan containing hydroquinone (100 mg) was treated with JV,JV,JV-trimethylbenzylammonium ethoxide (5 ml of a 40% solution in MeOH) and warmed to 35 C. Freshly distilled acrylonitrile (150 ml, 2.5 mol) was added at a rate such that the temperature did not rise above 40°C. The solution was then stirred overnight and diluted with 10% aq. acetic acid (11). The solution was extracted with CH Clj and the extract was washed with water and dried (MgS04). extract was then mixed with silica gel (800 g) and the solvent removed in vacuo. The silica was placed in a Soxhlet extractor and extracted with cyclohexane. The extract deposited the product as colourless needles (125 g, 63% yield). 

Potassium hydride (1 eq.) was washed with hexanes and suspended in anhydrous ether at 0°C. 7-Bromoindole was added as a solution in ether. After 15 min, the solution was cooled to — 78°C and t-butyllithium (2 eq.) which had been precooled to — 78°C was added by cannula. A white precipitate formed. After 10 min DMF (2 eq.) was added as a solution in ether. The reaction mixture was allowed to warm slowly to room temperature and when reaction was complete (TLC) the suspension was poured into cold 1 M H3PO4. The product was extracted with EtOAc and the extract washed with sat. NaHCOj and dried (MgS04). The product was obtained by evaporation of the solvent and purified by chromatography on silica gel (61% yield). 

A rationalization of the complex behavior of pectins in solutions and gels with respect to their stmcture, solvation, and the presence of ions and other saccharides has been presented (123). The solution and sorption properties of gum tragacanth and the pectin isolated from the roots of Hibiscus mani/)ot F (Tororoaoi) contributes to their use in specialty paper manufacture (124—126). 

In gel-forming processes, the reactive aluminosibcate gel is first formed into a pellet which reacts with sodium aluminate solution and caustic solution. The 2eobte crysta11i2es in situ within an essentiaby self-bonded pellet, or as a component in an unconverted amorphous matrix. 

Molded polyamide surfaces can be hardened by grafting with Ai,Ai-diallylacrylamide [3085-68-5] monomer under exposure to electron beam (159). AijAZ-DiaHyltartardiamide [58477-85-3] is a cross-linking agent for acrylamide reversible gels in electrophoresis. Such gels can be dissolved by a dilute periodic acid solution in order to recover protein fractions. 

Highly acidic/alkaline moieties may also cause permanent chemical alterations in the adsorbent (aluminas are sensitive to acid solutions, siUca gels are stre —12 range. 

Significant amounts of comonomer also reduce the abiUty of the polymer to form lamellar crystals from solution. In some cases, the polymer merely gels the solution as it precipitates rather than forming distinct crystals. At somewhat higher VDC content, it may precipitate in the form of aggregated, ill-defined particles and clusters. 

In early hterature, the molecular weights of PVDC and VDC copolymers were characterized by the absolute viscosity of a 2 wt % solution in (9-dichlorobenzene at 140°C. The exact correlation between this viscosity value and molecular weight is not known. Gel-permeation chromatography is the... 

Cyclodextrin stationary phases utilize cyclodextrins bound to a soHd support in such a way that the cyclodextrin is free to interact with solutes in solution. These bonded phases consist of cyclodextrin molecules linked to siUca gel by specific nonhydrolytic silane linkages (5,6). This stable cyclodextrin bonded phase is sold commercially under the trade name Cyclobond (Advanced Separation Technologies, Whippany, New Jersey). The vast majority of all reported hplc separations on CD-bonded phases utilize this media which was also the first chiral stationary phase (csp) developed for use in the reversed-phase mode. 

In general, derivatization increases solution and gel clarity, reduces the tendency to gel, improves water binding, increases freeze—thaw stabiHty, reduces the gelatinization temperature, increases peak viscosity, and reduces the tendency to retrograde. Combinations of substitutions are used to obtain desiredproperti.es for specific appHcations. 

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