Thin-layer chromatography plate spotting

Spot 3 pi of each of the amino acid standards along the origin. As with the thin-layer chromatography plate, spot each sample 1 /u.1 at a time. Indicate the identity of each sample spot below the origin using a soft lead pencil. 

Occasionally use several drops to spot, develop, and visualize a thin layer chromatography plate. Although thin layer is very similar to column, you should read up on it as 1 do not have time to go into the complete operation. 

In our laboratory crude preparations of aphantoxins and anatoxin-a(s) are extracted similarly except at the final stages of purification (Fig. 2). A Bio-gel P-2 column (2.2 x 80 cm) is used for aphantoxins gel filtration and a Sephadex G-15 (2.6 x 42 cm) column for ana-toxin-(s). Both toxins are eluted with 0.1 M acetic acid at 1.5 ml/ min. Fractions of aphantoxins from Bio-gel P-2 run are spotted on thin-layer chromatography plates (Silica gel-60, EM reagents) and developed according to Buckley et al. (1976) (31). The Rf values for the aphantoxins, saxitoxin and neosaxitoxin standards (Table 1) indicates that two of the aphantoxins (i.e. I and II) are similar to saxitoxin and neosaxitoxin. 

Obtain a silica-gel thin-layer chromatography plate that has been activated in a 100°C oven for 5 to 10 min. On either side of the plate, make a small scratch in the silica matrix about 2 cm from the bottom of the plate. This will mark the position of the origin, along which the samples will be spotted. Do not mark on the plate with pencil. 

D-enantiomers of amino acids have been frequently reported in various tissues of diverse organisms. A simple and rapid method of separating optical isomers of amino acids on a reverse-phase thin layer chromatography plate is described. Amino acids, derivatized with l-fluoro-2,4-dinitrophenyl-5-L-alanine amide, were spotted onto a reverse-phase thin layer chromatography plate. Acetonitrile in triethylamine-phosphate buffer was used as the developer. 

A methanol extract of the polyurethane is applied to a thin-layer chromatography plate and developed with a mixture of chloroform, ethylacetate, ethanol, glacial acetic acid (120 33 20 7 v.v). Fluran spray reagent reveals the two diaminotoluene isomers. The intensity of the spots are evaluated (500 nm excitation 340 nm emission) using a spectrofluorimeter coupled to a thin film scanner and photomultiplier microphotometer. The method is calibrated against standard solutions of the two diaminotoluene isomers. 

Analyses in the pg range, e.g., in water and oil, can be carried out with thin-layer chromatography [244]. After separation on the thin-layer plates, then by Dragendorff reagent, colored spots are measured with the help of a spectral photometer at 525 nm. 

Two-dimensional thin-layer chromatography. This method is used to verify the presence of terminal 5-sultones, terminal unsaturated y-sultone, and terminal 2-chloro-y-sultone, if they are detected in the gas chromatographic determination. After extraction of the neutral oil from the AOS sample, the neutral oil is made up volumetrically to at least a 10% solution in hexane. Of this solution 3 pi is spotted onto a silica gel TLC plate together with standard sultones. It is twice developed with 2-propyl ether and then after turning the plate 90° twice developed with 60/40 n-butyl chloride/methylene chloride. The... 

Dichloromethane extraction of culture broth, thin layer chromatography of the extract, and visualization with 5% vanillin/sulfuric acid spray is effective for detecting cycloheximide in culture broth. Cycloheximide applied to TLC plates in amounts as low as 1 yg/spot will produce visible color with the vanillin spray. 

Although the interest in, and application of layer chromatography has historically resulted from the development of PC, it was soon replaced by thin-layer chromatography (TLC). In PC, only one stationary phase matrix is available (cellulose), at variance to TLC (silica, polyamide, ion-exchange resins, cellulose). Using a silica-gel plate, separation of a sample can be accomplished in approximately 1 h as compared with many hours on paper. The plate size is much smaller than the necessary paper size. Also, more samples can be spotted... 

Caffeine was extracted from ficw varieties of roasted coffee beans and was determined in parallel by (1) measurement of spot area after thin layer chromatography on silica gel GF plates (development with chloroform/ cyclohexane/glacial acetic acid, 8 2 1, visualization in UV light), and (2) Kjeldahl N determination. Caffeine contents by (1) and (2), respectively, in the five varieties analyzed were (percent in DM) Santos lave 0, 1.10, and 1.12 Java Robusta 3, 1.19, and 1.22 Camerun Robusta 2, 1.16, and 1.19 Mocca 2, 1.21, and 1.26 Guatemala 0, 1.18, and 1.20. (1) is considered slightly less accurate than (2) but rather easier and more rapid.21... 

The theoretical work that exploited the advantages of the multidimensional separation format appears to have been developed much later than the original experimental work. One of the earliest studies was conducted by Connors (1974), who assumed that the distribution of spots on a two-dimensional thin-layer chromatography (2DTLC) plate could be modeled using a Poisson distribution of data on each retention axis. He then constructed equations that related the number of chromatographic systems needed to resolve a specific number of compounds. One... 

Examine the sample by thin-layer chromatography, using silica gel G R as the coating substance. Dissolve 10 mg of the substance to be examined in 4 mL of water R as a test solution, and dissolve 10 mg of penicillamine reference substance in 4 mL of water R as a reference solution. Apply 2 pL of each solution separately to the plate. Develop over a path of 10 cm using a mixture of 18 volumes of glacial acetic acid R, 18 volumes of water R, and 72 volumes of butanol R. Dry the plate at 100-105 °C for 5-10 min, and expose to iodine vapor for 5-10 min. The principal spot in the chromatogram obtained with the test solution is similar in position, color, and size to the principal spot in the chromatogram obtained with the reference. 

Analytical thin layer chromatography (TLC) was conducted on 10 x 2.5-cm precoated glass plates (silica gel GF, 0.25-mm thickness, Analtech), eluted with 10% ethyl acetate in hexane, and visualized with both UV (254 nm) and aqueous 50% sulfuric add spray/heating. The carbene complex moves as an orange spot on TLC the reaction is complete when this spot is no longer visible. 

Thin-layer chromatography does not provide quantitative information of the highest precision and accuracy. Linear relationships between the mass of a substance and the logarithm or square-root of the spot area can sometimes be established under very closely controlled conditions. The optical absorbance of a spot determined by reflectance measurements can be similarly related to mass, or the substances can be scraped from the plate and dissolved in a suitable solvent for a spectrometric determination. The main difficulties with area and density measurements lie in defining the boundaries of spots and controlling chromogenic reactions in a reproducible manner. Relative precision can be as good as 1-2% but is more usually 5-10%. 

The identification of bromocriptine mesilate in the dosage form can be carried out by thin layer chromatography using Merck plates with dichloromethane/methanol/formic acid 78 20 2 (v/v/v) and subsequent uv-visualization at 254 and 360 nm. Using this method, it is important to only air-dry the spot after application to the plate, since more vigorous evaporation of the solvent will give rise to artifacts (32). 

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