Column assay

Bayard, R., Bama, L., Mahjoub, B., Gourdon, R. (1998) Investigation of naphthalene sorption in soils and soil fractions using batch and column assays. Environ. Toxicol. Chem. 17, 2383-2390. 

Most kits for column assays contain reagents to remove this labile component. 

The above assays are summarized in Tables 9.1 and 9.2 for OBPs and PBPs where binding dissociation constants are available. Interestingly, the dissociation constants differ by an order of magnitude, depending on whether the assay is a vial adsorption/mini column assay, or a fluorescence assay. We believe this discrepancy may be the result of a dose effect attributed to the different ligand PBP ratios used in binding assays (see below). 

Esposito A., Del Borghi A., and Veglio F., Investigation of naphthalene sulphonate compounds sorption in a soil artificially contaminated using batch and column assays. Waste Manage., 22, 937-943, 2002. 

The column assay is indispensable during the introduction of a binding assay, in spite of the complicated setup and handling of the columns and the expensive gel filtration material. If a tissue extract shows no binding, the possible causes are countless and difficult to discern. Among these are the possibilities that the binding assay does not work, the binding protein is... 

The ligand does not penetrate into the gel pores. This is the case when the ligand incorporates into high-molecular buffer components (e.g., small hydrophobic ligands in TRITON micelles). This error source can be eliminated by a simple experiment if you perform the column assay without binding protein, the applied radioactivity must remain in the column. 

As for the binding assay if a radioactive ligand is available, try the filter assay first. It s the fastest and its applicability usually only depends on whether or not the ligand sticks to the filter. You should be able to answer this question after a few experiments. If the filter test does not work, use the column assay. If that is not possible (e.g., because ligand and binding protein are about equal in size), you can employ PEG precipitation or the methods from Petrenko et al. (1990) or Scheer and Meldolesi (1985). 

Hydrochloric acid [7647-01-0], which is formed as by-product from unreacted chloroacetic acid, is fed into an absorption column. After the addition of acid and alcohol is complete, the mixture is heated at reflux for 6—8 h, whereby the intermediate malonic acid ester monoamide is hydroly2ed to a dialkyl malonate. The pure ester is obtained from the mixture of cmde esters by extraction with ben2ene [71-43-2], toluene [108-88-3], or xylene [1330-20-7]. The organic phase is washed with dilute sodium hydroxide [1310-73-2] to remove small amounts of the monoester. The diester is then separated from solvent by distillation at atmospheric pressure, and the malonic ester obtained by redistillation under vacuum as a colorless Hquid with a minimum assay of 99%. The aqueous phase contains considerable amounts of mineral acid and salts and must be treated before being fed to the waste treatment plant. The process is suitable for both the dimethyl and diethyl esters. The yield based on sodium chloroacetate is 75—85%. Various low molecular mass hydrocarbons, some of them partially chlorinated, are formed as by-products. Although a relatively simple plant is sufficient for the reaction itself, a si2eable investment is required for treatment of the wastewater and exhaust gas. 

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

An hplc assay was developed suitable for the analysis of enantiomers of ketoprofen (KT), a 2-arylpropionic acid nonsteroidal antiinflammatory dmg (NSAID), in plasma and urine (59). Following the addition of racemic fenprofen as internal standard (IS), plasma containing the KT enantiomers and IS was extracted by Hquid-Hquid extraction at an acidic pH. After evaporation of the organic layer, the dmg and IS were reconstituted in the mobile phase and injected onto the hplc column. The enantiomers were separated at ambient temperature on a commercially available 250 x 4.6 mm amylose carbamate-packed chiral column (chiral AD) with hexane—isopropyl alcohol—trifluoroacetic acid (80 19.9 0.1) as the mobile phase pumped at 1.0 mL/min. The enantiomers of KT were quantified by uv detection with the wavelength set at 254 nm. The assay allows direct quantitation of KT enantiomers in clinical studies in human plasma and urine after adrninistration of therapeutic doses. 

Spectrophotometric deterrnination at 550 nm is relatively insensitive and is useful for the deterrnination of vitamin B 2 in high potency products such as premixes. Thin-layer chromatography and open-column chromatography have been appHed to both the direct assay of cobalamins and to the fractionation and removal of interfering substances from sample extracts prior to microbiological or radioassay. Atomic absorption spectrophotometry of cobalt has been proposed for the deterrnination of vitamin B 2 in dry feeds. Chemical methods based on the estimation of cyanide or the presence of 5,6-dimethylben2irnida2ole in the vitamin B 2 molecule have not been widely used. 

Hplc techniques are used to routinely separate and quantify less volatile compounds. The hplc columns used to affect this separation are selected based on the constituents of interest. They are typically reverse phase or anion exchange in nature. The constituents routinely assayed in this type of analysis are those high in molecular weight or low in volatility. Specific compounds of interest include wood sugars, vanillin, and tannin complexes. The most common types of hplc detectors employed in the analysis of distilled spirits are the refractive index detector and the ultraviolet detector. Additionally, the recent introduction of the photodiode array detector is making a significant impact in the analysis of distilled spirits. 

The assay of ethyleneamines is usually done by gas chromatography. Compared to packed columns, in which severe tailing is often encountered due to the high polarity of the ethyleneamines, capillary columns provide better component separation and quantification. Typically, amines can be analyzed using fused siUca capillary columns with dimethyl silicones, substituted dimethyl silicones or PEG Compound 20 M as the stationary phase (150). 

MLC enables to analyse drugs and active phamiaceutical substances without using special column and lai ge quantity of organic solvents. So, from the point of view of pharmaceutical analysis ecology and green chemistry conception, assay with MLC using will be better than conventional reversed-phase chromatography. 

The purity of the 2-cyclohexenone may be assayed by gas chromatography on an 8 mm. x 215 cm. column heated to 125° and packed with di-(2-ethylhexyl) sebacate suspended on ground firebrick. This method of analysis indicates that the 3-cyclo-hexenone in the product amounts to no more than 3%. The fore-run from this fractional distillation contains substantial amounts of 2-cyclohexenone accompanied by ether, ethanol, and minor amounts of other lower-boiling impurities. Additional quantities of pure 2-cyclohexenone can be recovered by redistillation of this fore-run. The preparation of 2-cyclohexenone has been run on twice the scale described with no loss in yield. The ultraviolet spectrum of an ethanol solution of the 2-cyclohexenone obtained has a maximum at 226 m/i (s = 10,400). 

In the development of a SE-HPLC method the variables that may be manipulated and optimized are the column (matrix type, particle and pore size, and physical dimension), buffer system (type and ionic strength), pH, and solubility additives (e.g., organic solvents, detergents). Once a column and mobile phase system have been selected the system parameters of protein load (amount of material and volume) and flow rate should also be optimized. A beneficial approach to the development of a SE-HPLC method is to optimize the multiple variables by the use of statistical experimental design. Also, information about the physical and chemical properties such as pH or ionic strength, solubility, and especially conditions that promote aggregation can be applied to the development of a SE-HPLC assay. Typical problems encountered during the development of a SE-HPLC assay are protein insolubility and column stationary phase... 

C. L. Hsu and R. R. Walters, Assay of the enantiomers of ibutilide and artilide using solid-phase extraction, derivatization and achhal-cliiral column-switcliing high-performance liquid cliromatography , J. Chromatogr. B 667 115-128 (1995). 

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