Paper chromatography spraying with

This material, normally supplied or stored in sealed ampoules and used as a paper chromatography spray reagent, is photo-sensitive. Exposure to UV light causes exothermic decomposition to acetone and chloromethane. Ampoules have burst because of pressure build-up after exposure to fluorescent or direct day light. Store cool and dark, and open ampoules with personal protection. The material also reacts violently with rubber [1,2], It should not be heated to above its boiling point [3], There is also a preparative hazard. 

Arsonic acids are easily detected on paper electrophoresis and chromatography by the fact that they bind Fe3+ tightly. Hence, they show up in the test of Wade and Morgan (130) for phosphates, in which the paper is sprayed with a solution of FeCl3 (1%) and sulfosalicylic acid (1%). Phosphonates and arsonates show up like phosphates as white spots on a purple background, because they remove the Fe3+ from its purple complex with the acid. For following column effluent, a test with a similar principle may be used. The sample is mixed with a buffered, acidic solution of Fe3+ and azide, and the absorbance at 450 nm is diminished by phosphates and phosphonates (52) we have since used this method successfully with arsonates. 

The peptides may be separated by electrophoresis on filter paper followed by chromatography at right angles to the direction of electrophoresis. When the paper is sprayed with ninhydrin the peptides show up as a characteristic pattern of spots which is known as the fingerprint of the protein in question (Figure 22 2). 

Hydantoin itself can be detected ia small concentrations ia the presence of other NH-containing compounds by paper chromatography followed by detection with a mercury acetate—diphenylcarba2one spray reagent. A variety of analytical reactions has been developed for 5,5-disubstituted hydantoias, due to their medicinal iaterest. These reactions are best exemplified by reference to the assays used for 5,5-diphenylhydantoiQ (73—78), most of which are based on their cycHc ureide stmcture. Identity tests iaclude the foUowiag (/) the Zwikker reaction, consisting of the formation of a colored complex on treatment with cobalt(II) salts ia the presence of an amine (2) formation of colored copper complexes and (3) precipitation on addition of silver(I) species, due to formation of iasoluble salts at N. ... 

Paper chromatography has been used to separate and detect pseudoephedrine hydrochloride from other pharmacologically active amines. Whatman No. 1 paper developed in n-butanol water 95% acetic acid (4 5 1), n-butanol toluene water 95% acetic acid (10 10 5 5), ethyl acetate water 95% acetic acid (3 3 1), or chloroform water 95% acetic acid (10 5 4) gave Rf values of 0.73, 0.35, 0.57, and 0.52 for pseudoephedrine hydrochloride respectively. Visualization of pseudoephedrine hydrochloride was done by spraying the chromatogram with 0.5% bromcresol green in methanol or 0.2% nin-hydrin in acetic acid butanol 5 95.30... 

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. 

Diagonal electrophoresis and its relative diagonal chromatography are useful for other purposes as well. After electrophoresis or chromatography is conducted in one direction, the paper or thin-layer plate may be sprayed with a reagent that will react with some components or may be irradiated with light before the separation is repeated in the second direction (Fig. 3-5).102/26°... 

Qualitative paper chromatography of the ether extractives indicated vanillin, vanillic acid, acetovanillone, and a trace of p-hydroxybenzoic acid along with many unidentified spots giving reactions with the indicator spray reagent. The ethanol extract gave most of the same spots. The water solution gave spots for oxalic acid and for several unidentified acids. 

The reaction of carbohydrates with ethylenediamine sulfate produces a stable fluorescence with excitation at 394 nm and emission at 470 nm. The reaction is specific for aldehydes and aliphatic polyhydroxyl compounds. It is used for the spray detection of carbohydrates separated by paper chromatography [109]. 

Saccharin has been detected and estimated after extraction from food samples on Whatman No. 1 filter paper (61,62). The sample in solution form, such as carbonated water can be used for paper chromatography. The spotted paper is developed in BuOH-ACOH-HpO (k0 l0 22) for l8 hours and sprayed with a solution of phthalic acid and aniline for spot development. Saccharin with an Rp 0.17 can be estimated colorimetrically after elution of the spot with 60 AcOH (62). 

Identification of SeMet after being separated from other amino acids by means of paper chromatography and electrophoresis is facilitated by spraying spots with H202 or preferably by exposure to cyanogen bromide (Shepherd and Huber, 1969). Pre-derivatization of SeMet through reaction with o-benzoquinone facilitates separation and identification in the presence of Met. Both SeMet and Met react with o-benzoquinone at pH 1 to form phenolic sulfonium- or selenonium derivatives whose UV absorption spectra differ. 

Small quantities of compounds in natural extracts are often a problem when these need to be evaluated in bioassays. Sometimes there is just not enough of the compound isolated to carry out the usual bioassay.20 Microassays have been developed10 1 to overcome this problem. Typically, a microassay is carried out on a thin-layer chromatography plate with a cellulose layer. A small droplet (1.5 pi) of the tested compound in a solvent (1-102 nmol cm-2) is then added on the plate. After the solvent has evaporated a small amount (5 pi) of sucrose solution lmoll-1 is added to the place where the compound was added. In the control the same procedure was followed on a different plate, but with the solvent alone, with no compound added. These two plates are then placed in a petri dish with the test insect species. In the past, when paper chromatography was widely used, a crude plant extract was placed on the origin of the paper and then eluted into bands. The paper was freed of solvent, sprayed with sugar solution, and used directly in a bioassay to see which parts of the paper were not eaten, and therefore of interest for further examination. 

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