TLC silica gel

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 4 pi is spotted onto a silica gel TLC plate, together with terminal 5-sultone standard in the range 0.4-4 pg (equivalent to 0.1-1% sultone in the neutral oil). It is twice developed in a chamber saturated with 2-propyl ether. The solvent is completely evaporated and the spots visualized by vapor phase sulfuric acid charring using the technique described by Martin and Allen [139]. Humidity is not critical (10-30% is optimum) and activation of the plates has not been found necessary, but it might be required under conditions of high humidity. The level of sultone can be estimated by visual comparison with the standards or by the use of a densitomer. 

Analyses for the Saxitoxins. Early methods for analysis of the saxitoxins evolved from those used for toxin isolation and purification. The principal landmarks in the development of preparative separation techniques for the saxitoxins were 1) the employment of carboxylate cation exchange resins by Schantz et al. (82) 2) the use of the polyacrylamide gel Bio-Gel P2 by Buckley and by Shimizu (5,78) and 3) the development by Buckley of an effective TLC system, including a new solvent mixture and a new visualization technique (83). The solvent mixture, designated by Buckley as "E", remains the best for general resolution of the saxitoxins. The visualization method, oxidation of the saxitoxins on silica gel TLC plates to fluorescent degradation products with hydrogen peroxide and heat, is an adaptation of the Bates and Rapoport fluorescence assay for saxitoxin in solution. Curiously, while peroxide oxidation in solution provides little or no response for the N-l-hydroxy saxitoxins, peroxide spray on TLC plates is a sensitive test for all saxitoxin derivatives with the C-12 gemdiol intact. 

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

FIGURE 10.4 Silica gel 60 TLC pattern of commercially available Bj2 reagents, hydroxo-cobalamin and dicyanocobinamide. The concentrated solution (4 pi) was spotted on the silica gel TLC sheet and developed with 2-propanol/NH40H(28%)/water (7 1 2, v/v) at room temperature in the dark. 1 — hydroxocobalamin, 2 — dicyanocobinamide. 

FIGURE 10.5 Elution profile on OH-B12 treated by microwave heating for 6 min during silica gel 60 column chromatography. Fifty milliliters of the treated OH-B12 solution (5 mmol/1) was evaporated to dryness and dissolved in a small amount of w-butanol/2-pro-panol/water (10 7 10, v/v) as a solvent. The concentrated solution was put on a column (1.4 X 15.0 cm) of silica gel 60 equilibrated with the same solvent and eluted with the same solvent in the dark. The eluate was collected at 4.0 ml with a fraction collector. Fractions I to V were pooled, evaporated to dryness, dissolved with a small amount of distilled water, and analyzed with silica gel TLC. Inset represents the mobile pattern of the OH-B12 degradation products of fractions I to V on the TLC plate. Data are typical, taken from one of five experiments. (Reprinted with permission from Watanabe, F. et al., J. Agric. Food Chem., 46, 5177-5180, 1998. Copyright (1998) American Chemical Society.)... 

FIGURE 10.13 The TLC profiles of labeled peaks isolated from [U- C]ascorbic-acid-modified calf lens protein obtained from Bio-Gel P-2 chromatography. Peaks 2 to 7 were spotted on a preparative silica gel TLC plate and developed with ethanol/ammonia (7 3, v/v). The fluorescence in each lane was detected by irradiation with a Wood s lamp at 360 nm, and the pattern of radioactivity was determined by scanning the plate with AMBIS imaging system. (Reprinted with permission from Cheng, R. et al., Biochim. Biophys. Acta, 1537, 14-26, 2001. Copyright (2001) Elsevier.)... 

FIGURE 12.4 (A) Diagrammatic representation of the separation of major simple lipid classes on silica gel TLC — solvent system hexane diethylether formic acid (80 20 2) (CE = cholesteryl esters, WE = wax esters, HC = hydrocarbon, EEA = free fatty acids, TG = triacylglycerol, CHO = cholesterol, DG = diacylglycerol, PL = phospholipids and other complex lipids). (B) Diagrammatic representation of the separation of major phospholipids on silica gel TLC — solvent sytem chloroform methanol water (70 30 3) (PA = phosphatidic acid, PE = phosphatidylethanolamine, PS = phosphatidylserine, PC = phosphatidylcholine, SPM = sphingomyelin, LPC = Lysophosphatidylcholine). 

In the ease of very immatnre organie matter, or when the main research aim is to investigate polar fractions, a different analytical scheme may be applied (Figure 15.3). Prior to the fractionation, total concentrated extracts are treated with 14% BF3 in methanol or diazomethane in ether to esterify free carboxylic acids, and then they are snbjected to silica gel TLC using methylene chloride or a mixture of... 

A variety of bisindolylnitroethanes [58] has been obtained in high yields (70-86%) by reacting 3-(2-nitrovinyl)indole with indole and 1- or 2-alkylindoles on silica gel (TLC-grade) under irradiation (Scheme 8.38). 

There are much better parallels to HPLC TLC or column chromatography. Vary the eluents in these techniques and you get widely different results. With a normal-phase silica-based column, you can get results similar to those from silica gel TLC plates. 

Take up each dye residue in one or two drops of ammonia solution (prepared by diluting 1 mL of concentrated ammonium hydroxide to 100 mL). Using open-ended capillary tubes, spot each color onto both a cellulose TLC plate and a silica gel TLC plate at about 3 cm from the bottom edge. 

The crude residue is applied to the column head using a minimum of dichloromethane. The submitters use flash-grade (230-400 mesh) silica gel purchased from E. Merck and a column 10 cm in diameter. TLC values for 2 are Rf = 0.18 (hexane acetone = 3 1) and for 3 (see Note 10) are Rf = 0.08 (hexane acetone = 3 1), employing Whatman K6F silica gel TLC plates 60 A. In a typical purification, collecting 200-mL fractions, 2 would elute in fractions 9-18 and 3 in fractions 20-30. The checkers found that immediate purification of the crude residue was necessary. Yields decreased dramatically with time between isolation and purification. Furthermore, the activity (related to the degree of dehydration) of the silica gel greatly affected yields. Only half the amount of silica mentioned above was used by the checkers to get the reported yields. When the full amount was used, the yield decreased to 40-50%. 

Merck 60 F254 silica gel TLC plates Merck 60/230 00 and 60/40-63 mesh silica heating block shaker... 

Although (Z)-hinokiresinol is stable during silica gel TLC separation, it was isomerized into the (ii)-form after storage for 3 years in a freezer at -20°C (Yamamura, Suzuki, Hattori, Umezawa, unpublished). In addition, the ( )-isomer is rather unstable under slightly acidic conditions (such as old deutrochloroform for NMR measurements) (Umezawa, unpublished). Hence, we established a system for enantiomeric analysis of hinokiresinols by converting them into the corresponding... 

Reaction progress can be followed most accurately by silica gel TLC and GLC analysis. In TLC analysis, one sees the disappearance of (1-ethenyl)-... 

Silica gel TLC shows one major spot at the baseline (20% ethyl... 

The steroids below are spotted onto a silica gel TLC plate. The plate Is developed in methylene chloride/ether/methanol/water (77 15 8 1.2) and under UV light has the appearance shown in Figure 13.4. From your knowledge of the polarity of organic molecules, match the steroids to the spots on the TLC plate shown in Figure 13.4. 

Dorzolamide ean be chromatographed using silica gel TLC plates, and a mobile phase of 80 20 1 methylene chloride methanol ammonia. Visualization can be effected by either viewing the developed plate under ultraviolet light, or by exposing the developed plate to iodine vapor. The R, of dorzolamide is approximately 0.62. 

The mixture was stirred under hydrogen (ca. 1 atm) overnight at room temperature. The reaction was monitored by silica gel TLC (eluent n-hexane-ethyl acetate, 19 1). The substrate and the product (UV active) having Rf values of 0.26 and 0.34, respectively, were visuahzed by 10% ethanolic molybdophosphoric acid dip. 

Urinary creatinine is determined on a Cobas Bio Analyser using a commercial modification of the Jaffe acid-picrate method (Wako, Neuss, Germany). Urine samples corresponding to 20 pg creatinine are applied directly as a thin streak in a stream of warm air, 1.5 cm from the lower edge of a 20x20 cm pre-coated silica gel TLC plate. Reference standards (10 pg) are used at a concentration of 1 pg/pl. 

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