Chromatography dipping

Technologies for immobilizing proteins on surfaces that have been developed over the last 25 years are particularly critical to the continued development of biosensors. There a dozens of techniques for activating surfaces so as to be able to covalently bond proteins while maintaining their biological activity. These methods have been used routinely for manufacturing adsorbents for affinity chromatography, dip-stick type analytical methods, e.g., immunoassays, and immobilized enzymes for commercial catalysts. 

Such a dipping apparatus can also be employed with advantage for applying substances to preserve or intensify fluorescence after chromatography or derivati-zation is complete (cf. Section 3.2.7.3). 

Analytical thin-layer chromatography was performed on E. Merck silica gel 60 F254 plates (0.25 mm) and compounds were visualized by dipping the plates in a cerium sulfate-ammonium molybdate solution followed by heating. 

The concept of SPME was first introduced by Belardi and Pawliszyn in 1989. A fiber (usually fused silica) which has been coated on the outside with a suitable polymer sorbent (e.g., polydimethylsiloxane) is dipped into the headspace above the sample or directly into the liquid sample. The pesticides are partitioned from the sample into the sorbent and an equilibrium between the gas or liquid and the sorbent is established. The analytes are thermally desorbed in a GC injector or liquid desorbed in a liquid chromatography (LC) injector. The autosampler has to be specially modified for SPME but otherwise the technique is simple to use, rapid, inexpensive and solvent free. Optimization of the procedure will involve the correct choice of phase, extraction time, ionic strength of the extraction step, temperature and the time and temperature of the desorption step. According to the chemical characteristics of the pesticides determined, the extraction efficiency is often influenced by the sample matrix and pH. 

Figure 11.22 represents a paper electrophoresis apparatus. The soaked cellulose sheet is sandwiched between two horizontal glass plates with the ends dipped into vessels containing more electrolyte solution. The electrodes are also dipped into these vessels, as shown. The sample is spotted in the center of the sheet, and the oppositely charged ions then have room to migrate in opposite directions on the sheet. Qualitative analysis is performed much as with paper chromatography, by comparing the distances the... 

Reagents used for the visualisation of amino acids on the dried chromatogram may be applied either by spraying or dipping. Those commonly used produce intensely coloured bands with approximately 20 nmol of each amino acid for paper chromatography and 5 nmol for thin-layer separations, although smaller amounts can be detected. 

The synthesis of the Saa-peptide conjugates in solution follows standard solution-phase synthesis using, for example, IIDQt117 or EDC as the coupling agent. The reaction is checked by TLC (ninhydrin and/or 10% H2S04 in MeOH dip). The product is usually purified by flash chromatography. 

The silylene precursor, 2,2-bis(2,6-diisopropylphenyl)hexamethyltrisilane (5), was pho-tolyzed with a low-pressure mercury lamp in a toluene solution of C7012. The adduct 12 obtained (equation 2) contains two isomers of (Dip)2SiC7o (12a and 12b) which were separated by flash chromatography on silica gel. FAB mass spectrometry of 12 displays a peak for adduct 12 at 1190-1194 as well as for C70 at 840-843 which arises from the loss of diarylsilylene. The UV-vis absorption spectra of 12 are virtually identical to those of C70 with bands at 333, 381 and 471 nm. AMI molecular orbital calculations... 

This approach has been used extensively for amino acid analysis using low-pressure ion-exchange chromatography and post-column ninhydrin reaction. Spraying, dipping and vapour-treatment techniques are well known as post-separation reactions in TLC, but these are considered only briefly since the majority of them are not quantitative. While the problems of pre-separation techniques are quite similar for TLC and HPLC, they differ considerably for post-separation reactions. 

Method. The amino acid derivatives are prepared by adding disyl chloride (1 mg/ml in acetone) to an equal volume of a solution of the amino acid (ca. 5 10 4 jumoles/ml in 0.1 M sodium bicarbonate solution). The reaction is allowed to proceed for 3 h at room temperature. The solvent is evaporated and the residue is dissolved in 1 ml of acetone-methanol (1 1) for application to a thin-layer plate of silica gel. A number of solvent systems used for the separation of amino acid derivatives is given in Table 4.14. After chromatography, the plates are dried at 10S °C for S min, cooled to room temperature and dipped in a solution of sodium ethoxide (S g of sodium per 100 ml of 96% ethanol). The plate is observed immediately under UV light at 365 nm. The amino acid derivatives appear yellow-green. 

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