Thin-layer chromatography spot detection

Separation of amino acids and their identification in different mixtures are frequent tasks encountered in biochemistry. Thin layer chromatography is a fast, simple, and inexpensive approach to attain this goal. Because some of the components are UV-inactive, other methods, such as vibrational spectroscopy, should be applied for detection and identification. Comparative study based on Raman spectroscopy of thin layer chromatography spots of some weak Raman scatterers (essential amino acids) was carried out using four different visible and near-infrared laser radiation wavelengths 532, 633,785, and 1064 nm. The best results were obtained with simple silica gel plates. 

Thin-Layer Chromatography. Chiral stationary phases have been used less extensively in tic as in high performance Hquid chromatography (hplc). This may, in large part, be due to lack of avakabiHty. The cost of many chiral selectors, as well as the accessibiHty and success of chiral additives, may have inhibited widespread commerciali2ation. Usually, nondestmctive visuali2ation of the sample spots in tic is accompHshed using iodine vapor, uv or fluorescence. However, the presence of the chiral selector in the stationary phase can mask the analyte and interfere with detection (43). 

Chemical stabiUty studies are monitored by siUca gel thin-layer chromatography (dc) or by high performance Hquid chromatography (hplc) using a reverse-phase C g coated column (24). Hplc peaks or dc spots are visualized by thek uv absorption at 245 nm the tic spots can also be detected by ceric sulfate or phosphomolybdic acid staining. 

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. 

Most of the reactions described in the following chapters were monitored by Thin Layer Chromatography (TLC) using plastic TLC plates coated with a thin layer of Merck 60 F254 silica gel. The products were detected by using an ultraviolet lamp or the TLC plates were treated with p-anisaldehyde reagent, prepared as explained below, and then heated to 120 °C to stain the spots. After visualization and measurement, the Rf values were recorded. 

Methylacrylate and ethylenediamine were obtained from the Merck. Jeffamine T-3000 was purchased from Texaco Chemical Company. Other chemicals were used as obtained from the Fluka without further purification unless otherwise noted. Solvents were dried and distilled according to literature procedures prior to use. Reactions were controlled by thin layer chromatography (TLC) on silica gel 60 F254 and spots were detected either by UV-visible light or by charging with vapor. 

Muscarine is detected by high-performance thin-layer chromatography (HPTLC), with a limit of detection of 50 ng (Stijve, 1981). Using Dragendorff reagent, muscarine appears as an orange spot. 

Thin Layer Chromatography (TLC). Commercially available (Whatman LK6D, linear-K) silica gel (40S) plates of 250 ym thickness were used. These were developed in chloroform acetone formic acid (80 19 1). Spots were detected by UV light and iodine vapor. 

Diphenylacetyl-l,3-indandione-l-hydrazone, first prepared by Braun and Mosher [143], reacts readily with carbonyl compounds such as aldehydes and ketones in weakly acidic media. The derivatives are separated by paper or thin-layer chromatography [144]. The derivatives are excited at 400-415 nm and emit at ca. 525 nm. The limit of detection is 2 ng per spot. 

I. Thin-layer chromatography Mangalan et al. [53] used of an HPTLC method for the detection and determination of omeprazole in plasma levels. Plasma was extracted three times with dichloromethane at pH 6.5-7 and the combined extracts were evaporated to dryness at 60 °C. The residue was dissolved in dichloromethane and the solution was analyzed by TLC on aluminium-packed plates precoated with Silica gel 60 F254 with the upper organic layer of butanol-ammonium hydroxide-water (14 1 15) as mobile phase. The spots were observed by fluorescence quenching under UV light illumination at 280 nm the total area of each... 

A very useful identification tool is the combination of GC and thin-layer chromatography (TLC). The first work on combined GC-TLC appears to have been by Janak [59]. The GC column effluent is split into two streams, one of which enters the detector and the other, led via a heated conduit, impinges on the chromatographic thin layer carried by a moving plate. The GC fractions sampled in this way are subsequently developed and the TLC spots detected in the usual manner. The result is a kind of two-dimensional thin-... 

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