Columns direct spectrophotometric

One milligram of microsomal protein is added to 0.1 M potassium phosphate buffer (pH 7.4) containing 50 mM NaF, 10 mM dithiothreitol, 1 mM EDTA, 20% glycerol (v/v), 150 iM 5-cholestene-3/3, 7a-diol, and 0.915% CHAPS. The reaction is initiated by 1 mM NAD+ to give a final reaction volume of 1.0 mL. After incubation at 37°C for 5 minutes, the reaction is terminated by adding 2 mL of 95% ethanol. An internal recovery standard, 4-cholesten-3-one (3 fig in methanol) is also added. The steroid products are extracted into 5 mL of petroleum ether (repeated twice). After the ether has been removed at 40°C under a stream of nitrogen, the products are dissolved in 100 fxL of mobile phase and 20 ju.L is injected into the column. The amount of product formed is linear with protein (to 1.5 mg) and with time (up to 10 min, 1 mg protein). The assay is much more sensitive than the direct spectrophotometric assay, and it avoids the use of thin-layer chromatography and radioisotopes described in other methods. 

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. 

The United States Pharmacopoeia 23 [11] and Indonesian Pharmacopoeia IV [9] describe the assay of benzoic acid and salicylic acid in ointments. Two chromatographic columns (20 x 2.5 cm) are used to effect the separation. One transfers a mixture of 1 g siliceous earth and 0.5 mL diluted phosphoric acid (3 in 10) to the first column (A), then packs above this a mixture of 4 g siliceous earth and ferric chloride-urea reagent. A mixture of 4 g siliceous earth and 2mL of sodium bicarbonate solution (1 in 12) is packed into the second column (B). For analysis, column A is mounted directly above column B. The sample solution is inserted onto column A, allowed to pass into the column, and then washed with 2-40 mL of chloroform. Benzoic acid can be eluted from column B by using a 3 in 100 solution of glacial acetic acid in chloroform. The benzoic acid content then can be determined by a spectrophotometric method such as that described earlier (section 4.5). 

J. C. B. Fernandes, G. Olivera Neto, and L. T. Kubota, Use of column with modified silica for interfering retention in a FIA spectrophotometric methods for direct determination of vitamin C in medicine, Analytica chimica Acta 366, 11-22 (1998). 

The discussion of UV-VIS detectors for use in ion chromatography is divided into two parts (a) the direct monitoring of column effluents and (b) post-column derivati-zation with subsequent spectrophotometric measurement. 

Besides the spectrophotometric detectors based on absorbance or fluorescence of UV/visible radiation, and used when the mobile phase does not absorb appreciably, another mode of detection exists based upon electrolyte conductivity. Thus, at the outlet of the column, the conductance (the inverse of the resistance) of the mobile phase is measured between two microelectrodes. The measuring cell should be of a very small volume (approx. 2pL). The difficulty is to recognize in the total signal the part due to ions or ionic substances present in the sample. In order to do direct measurements, the ionic charge of the mobile phase has to be as low as possible and the measuring cell requires strict temperature control to within 0.01 °C because of the high dependence of conductance on temperature ( 5%/°C). 

The ultraviolet assay of tablets which contain only Isopropamlde as the active ingredient Is the most direct method employed (lif). Twenty tablets (5 mg/ tablet) are placed In a 250 ml. volumetric flask, disintegrated In about 150 ml. of water, and diluted to volume with water. The solution is filtered through Whatman No. 1 filter paper (l5),50.0 ml. of the filtrate percolated through an anion exchange column (16), and the column rinsed thoroughly with water. All eluates are collected in a 100 ml. volumetric flask and diluted to volume with water. The ultraviolet absorption spectrum of this solution Is compared with that of a known standard of Isopropamlde under the same spectrophotometric conditions and the assay value per tablet calculated. 

FI manifolds for column separation and preconcentration in spectrophotometry are diverse, and there is hardly one which may be considered typical. However, the reader may refer to the manifold used in the determination of boron just mentioned (63]. Another interesting contribution by Novikov et al.[ll] combined ion-exchange column preconcentration with on-line solvent extraction followed by spectrophotometric detection. The eluate from the column preconcentration was released into an on-line liquid-liquid extraction system. An advantage of this approach is that interferences from Schlieren effects are avoided, since the eluate does not flow directly to the detector. Selectivity and sensitivity are also enhanced due to the combination of two separation procedures. The system has been used successfully for the determination of lead in alloys, soil leachates and sea water. 

Although applications for column separation or preconcentration systems coupled to chemiluminescence determinations are few, published reports show no particular difficulties in such applications, except for the requirement of an adjustment of the chemical conditions of the eluate to suit the chemiluminescence reaction. Interferences due to refractive index effects are not likely to occur, owing to the often used spiral shape of the chemiluminescence flow-cell and to the fact that light emission is measured perpendicular to the direction of the flow. Therefore, column washing is usually not as important as for spectrophotometric applications, so that time-based sample loading manifolds such as those used for flame AAS may be used to advantage for improving the concentration, efficiencies. 

With the incorporation of CIS-modified silica material to the flow scheme for the preconcentration of the imine product formed in the oxidative reaction between phenol and 4-AAP two detection variants have been used, namely (1) spectrophotometric measurement of the methanolic eluent containing the concentrated dye or (2) direct at-column optosensing of the retained reaction product followed by methanol elution. The LOD obtained for measurement of the absorbance in the eluent and on-column optosensing are 11 pg/1 and 0.4 pg/1, respectively [185]. 

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