Separation gels, color detection

Separation of anions as counter ions of metal-diantipyrilmethane cationic complexes and diantipyrilmethane cations on silica gel layers using acetone-0.1M HCl (4 1) or acetone-CHCl3 (3 1) has been reported (143). The separated anions were detected by 0.5% FeCl3 solution as highly colored iron(ni) diantipyrilmethane complexes, or by viewing under UV light as fluorescent zones in the case of terbium (III) diantipyrilmethane complexes. 

Note When combined with thin-layer chromatographic separation the reagent provides a specific detection method for nitrate and nitrite. The color development is often completed within a few minutes on silica gel plates. In the absence of ammonia vapor traces of oxides of nitrogen in the laboratory atmosphere can slowly cause the background to become reddish-brown. The simultaneous presence of the following ions in the chromatogram zones interferes with the detection of nitrate/nitrite I , 10J, IO4, MoO and H2PO2. 

A variety of methods are available to detect proteins separated by electrophoresis or to measure the concentration of total protein in a solution. These methods are normally based on the binding of a dye to one of the amino acids in protein, or a color reaction with an amino acid side chain. The most commonly used stains for protein detection on gels are Coomassie Brilliant Blue (98) and silver stain (99,100). These methods detect any protein residues, either in solution or on an electrophoresis gel. Their main requirement is sensitivity, not specificity. New, more sensitive dyes are being developed for the proteomic analysis of protein structure and sequence, for example Ruby Red (101). 

Immediately purify the oxidized enzyme by gel filtration using a column of Sephadex G-25. The chromatography buffer is 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2. To obtain efficient separation between the oxidized enzyme and excess periodate, the sample size applied to the column should be at a ratio of no more than 5 % sample volume to the total column volume. Collect 0.5-ml fractions and monitor for protein at 280 nm. HRP also may be detected by its absorbance at 403 nm. In oxidizing large quantities of HRP, the fraction collection process may be done visually—just pooling the colored HRP peak as it comes off the column. 

A review on TLC of rf-block elements and their connteranions discnsses types of stationary phases, mobile phases, development modes and detection and qnantitative determination techniqnes . The colored complexes Ni(ttfac)2, Co(ttfac)2, Mn(ttfac)2, Cn(ttfac)2, Fe(ttfac)3, Ce(ttfac)4, Th(ttfac)4 and U(ttfac)6 were prepared by adding a solntion of l-thenoyl-3,3,3-triflnoroacetone (ttfacH) to a solntion of the metal salts bronght to pH 7.5 with sodium acetate buffer. The complexes were separated on silica gel G TLC plates. Best results were obtained with the solvent systems butanone-xylene, acetone-cyclohexane and 4-methyl-2-pentanone-xylene . ... 

Water-soluble a-DNP-Arg, a-DNP-His, s-DNP-Lys, bis-DNP-His, O-DNP-Tyr, DNP-cysteic acid (CySOjH), and DNP-cystine (Cys)2 have been identified on silica gel plates in the n-propanol-34% ammonia (7 3 v/v) system. Although separation of DNP-Arg and s-DNP-Lys is incomplete Rf values of 0.43 and 0.44, respectively), both of them can be detected because of the color difference produced in the ninhydrin reaction. 

The 2-D TLC was successfully applied to the separation of amino acids as early as the beginning of thin-layer chromatography. Separation efficiency is, by far, best with chloroform-methanol-17% ammonium hydroxide (40 40 20, v/v), n-butanol-glacial acetic acid-water (80 20 20, v/v) in combination with phenol-water (75 25, g/g). A novel 2-D TLC method has been elaborated and found suitable for the chromatographic identification of 52 amino acids. This method is based on three 2-D TLC developments on cellulose (CMN 300 50 p) using the same solvent system 1 for the first dimension and three different systems (11-IV) of suitable properties for the second dimension. System 1 n-butanol-acetone -diethylamine-water (10 10 2 5, v/v) system 11 2-propanol-formic acid-water (40 2 10, v/v) system 111 iec-butanol-methyl ethyl ketone-dicyclohexylamine-water (10 10 2 5, v/v) and system IV phenol-water (75 25, g/g) (h- 7.5 mg Na-cyanide) with 3% ammonia. With this technique, all amino acids can be differentiated and characterized by their fixed positions and also by some color reactions. Moreover, the relative merits of cellulose and silica gel are discussed in relation to separation efficiency, reproducibility, and detection sensitivity. Two-dimensional TLC separation of a performic acid oxidized mixture of 20 protein amino acids plus p-alanine and y-amino-n-butyric acid was performed in the first direction with chloroform-methanol-ammonia (17%) (40 40 20, v/v) and in the second direction with phenol-water (75 25, g/g). Detection was performed via ninhydrin reagent spray. 

Activity stains are of great importance during the isolation, purification, and characterization of enzymes, since a particular catalytic reaction is involved and the detection of this activity leads to the unequivocal identification of the zone of interest on the electrophoresis gel. Following separation, the gel is removed from the electrophoresis apparatus and is immersed in a minimal volume of a substrate solution. Detection relies on the formation of a colored product by enzyme in the zones containing the enzyme. Examples of activity stains are given in Table 9.2. 

Electrophoretic separation on agarose gels or cellulose acetate membranes is the procedure most commonly used to demonstrate LD isoenzymes." After the isoenzymes have been separated by electrophoresis, a reaction mixture is layered over the separation medium. The mixture (typically D, L-lactate> 500mmol/L, and NAD, 13mmol/L, often dissolved in a suitable pH 8.0 buffer) is applied as a liquid or in a gel. The NADH generated over the LD zones is detected either by its fluorescence, when excited by long-wave ultraviolet light (365 nm), or by its reduction of a tetrazolium salt to form a colored formazan. 

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