Determination Used after Elution

Evaluation by measuring UV absorption has been used in many cases for substances which absorb in this region because this is often possible directly after extraction with a suitable eluent, without any other stage. Limits are set only by the absorptivity of the compound to be determined. 

Generally at least 5 ml solvent is used for extracting a spot, so as to be able to work with the normal 1 cm cuvettes. A 50 jig amount applied to the layer thus yields a concentration of 10 [xg/ml after extraction. If such a procedure is used for example for the four pharmaceuticals aminopyrine, caffeine, benzyl mandelate and phenacetin, which absorb in the UV, the extinctions at the maxima are, respectively, 0.36, 

Use of a micro-elution assembly has permitted determination of even small amounts of substances with low absorptivities, by extracting with 100 [xl of solvent [451] (cf. Table 14). A microphotometer is then necessary, however. 

Material for analysis Adsorbent and solvent Detection Method of elution and evaluation 

Methyl p-hydroxy-benzoate II. Propyl p-hydroxy-benzoate 50 + 50% mixtures Silica gel G + 2% fluorescent additive Pentane-acetic acid (88 -f- 12) UV 254 nm Micro-filtration with G 28 and G 4 glass filter rods. Determination byUV 

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Thiazinamine, prothipendyl, prochlorperazine, triflupromazine, chlorpro-thixene, fluphenazine, thiopropazate and phenothiazine can for example, be separated using the chromatographic conditions E from Table 100. Amongst others, perazine, prochlorperazine, prothipendyl, promazine, isothipendyl, pecazine, chlorpromazine, levomepromazine and diethazine can be separated under conditions G. 2- and 4-Chlorophenothiazines have been separated on silica gel layers, using petrol ether-ether (60 + 40) and determined spectrophotometrically after elution [157 a]. 

Vitamin Bg and related compoimds (Figure 10.2) were quantitatively separated by preparative TLC on silica gel H. After elution, the pyridoxic acid lactone method was employed for fluorimetric determination of the concentration of the vitamin forms involved [8]. Table 10.2 shows Revalues obtained for various forms of vitamin Bg, using several solvent systems. The solvent selected, ethyl acetate/pyridine/water (2 1 2, v/v), gave excellent separation of pyridoxamine, pyridoxic acid, and pyri-doxine together with pyridoxal. 

Acetochlor, alachlor, and metolachlor are determined in ground and surface water samples. Deuterated internal standards are added to each water sample, and analytes are extracted using an SPE column. After elution and concentration to an appropriate volume, the analytes are quantitated by GC/MS. 

Chlornitrofen in river water (1000 mL) was determined using two Sep-Pak Gig cartridges connected together (Waters), which were rinsed with 5 mL of methanol, and cleaned and conditioned with 10 mL of water. Chlornitrofen was eluted from the cartridge with 10 mL of methanol after being rinsed with 3 mL of water-methanol... 

By use of a crude preparation obtained after the cultivation of Aspergillus niger,104 pectinesterase was purified by repeated chromatography on DEAE-cellulose, using gradient elution. The homogeneity of the product was checked by free electrophoresis, sedimentation analysis, and determination of the N-terminal amino acid (phenylalanine). 

Figure 6. Affinity chromatography of EGD from Clostridium thermocellum. Nucleic acid preparation, heat treatment and ammonium sulfate precipitation (0-70%, 70-100%) were carried out as described (10). The final precipitate ( 50 mg protein), dissolved in 50 mM sodium acetate, pH 5.0, was applied (after centrifugation) on the affinity column (2 x 25 cm) (4 -aminobenzyl l-thio-/ -cellobioside coupled to Sepharose 4B) (11). Protein was monitored at 280 nm and the activity of the fractions (2 ml) determined using 2 -chloro-4 -nitrophenyl / -cellobioside (pH 6.5, 25°C) as described in the text. Elution with 10 mM G2 was started as indicated.

The quantitative assay for PBG and ALA (Bio Rad, Hercules, CA, USA) that is based on the classical method by Mauzerall and Granick may be used for determination of the porphyrin precursors. PBG is absorbed by the anion-exchange column and ALA by the cation-exchange column interferences are washed out. After elution from the column, ALA is derivatized by acetyl acetone to form a pyrrole. Both ALA and PBG are determined colorimetrically with the modified Ehrlichs reagent. Instead of this broadly used standard method ALA, but not PBG may be detected and quantified using amino acid chromatography. However, our experience has shown that this method is only valid for detecting massively increased concentrations of ALA. 

For the determination of supplemental vitamin E in infant formulas, Woollard and Blott (222) employed a radially compressed Radial-PAK cartridge. This enabled lipid material to be rapidly cleared by stepping up the mobile-phase flow rate from 2 ml/min to 10 ml/min after elution of the a-tocopheryl acetate. Fluorescence detection, using a filter-type fluorometer, allowed the indigenous a-tocopherol to be conveniently estimated, while UV absorbance detection was used to quantify the a-tocopheryl acetate. Supplemental retinyl acetate could be assayed simultaneously with either added or indigenous vitamin E using the appropriate detection mode. With the aid of a dual-monochromator spectrofluorometer, a-tocopheryl acetate and a-tocopherol could be determined simultaneously with wavelengths of 280 nm (excitation) and 335 nm (emission), but the increased selectivity eliminated detection of the vitamin A esters (233). 

A qualitative thin-layer chromatography method has been described by Kleef et al. [27] for the detection of rocuronium bromide and its metabolites in biological samples. This method was developed to confirm the identity of rocuronium bromide and its metabolites prior to their determination using HPLC coupled with fluorescence detection. In this method, the dried residue from the extraction process was dissolved in 0.05 ml of 0.01 M HC1. The stationary phase used was silicagel plates, that were developed in a mobile phase consisting of 2% solution of Nal in 2-propanol. The elution process was run for 4 h, and after the elution... 

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