Sephadex thin layers

Sephadex thin layer chromatography and bioautography were combined for the identification of antibioticsIdentity tests for antibiotics sensitivity discs were developed. Several thin layer chromatography methods for identification of penicillin are reportedl58-152, A simple and rapid polyamide chromatography of penicillins is described S. (See Section 6.2.4 for additional systems). 

To obtain reliable chromatograms in the final step of the determination of the analytes by LC or GC, it is important to remove interfering signals resulting from coelution of other compounds. To this end, a variety of techniques are applied for cleanup of the sample extract. The most effective procedures for sample cleanup for PAH measurements are partitioning between M, N-dimethylformamide/water/cyclo-hexane and LC on silica and on Sephadex LH 20. Other cleanup procedures include LC on alumina or XAD-2 and preparative thin-layer chromatography. 

Delincee and Radola100 used a commercial preparation, as well as fresh tomatoes, for the preparation, purification, and characterization of tomato pectinesterase. The tomatoes were pressed and then homogenized directly with ammonium sulfate at 70% saturation. The precipitate obtained was extracted with 0.3 M phosphate and repeatedly salted out with ammonium sulfate, and the product was separated on a column of Sephadex G-75. The pattern of separation was similar to that in preceding work.50,97 A detailed study of the size properties of pectinesterase was conducted by gel-filtration and sedimentation analysis.100 By column and thin-layer gel-filtration on Sephadex G-75, the approximate molecular weight of a number of preparations of tomato pectinesterase was determined, values of 24,000 and 27,000 being obtained. A possible interaction of the... 

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. 

The purification of glutamine cyclotransferase from papaya latex has been carried out by Messer and Ottesen (116). A batch procedure was used for the removal of impurities by passage of a papaya latex extract through a thin layer of carboxymethyl-Sephadex the active protein was separated by selective elution. Additional purification was achieved by chromatography on a column of carboxymethyl-Sephadex and by gel filtration on Sephadex G-100. The purified enzyme was homogeneous by the criteria of paper electrophoresis, ultracentrifugation, and gel filtration on Sephadex G-100 columns and chromatography on carboxy-methyl- or DEAE-Sephadex. The electrophoretic behavior of the enzyme indicates that it is a basic protein with an isoelectric point near... 

Figure 1 shows the scheme for the preparation of purified lipid A from endotoxin. S. typhimurium G30/C21 was extracted by the method of Galanos t aK (24) and submitted to one of two different conditions of hydrolysis (a) 0.1 N HC1 [in methanol-water (1 1, v/v)], 100 °C, 45 min, to yield the crude monophosphoryl lipid A (nontoxic), and (b) 0.02 M sodium acetate, pH 4.5, 100 °C for 30 min (two cycles) to yield the crude diphosphoryl lipid A (toxic). The 0.1 N HC1 hydrolysis product was fractionated on a Sephadex LH-20 column (23). Each of these fractions was then separated by preparative thin layer chromatography (TLC) on silica gel H (500 ym), with the solvent system chloroform-methanol-waterconcentrated ammonium hydroxide (50 25 4 2, v/v) as previously described (23) to yield TLC fractions 1-7 and 1-9 respectively. 

The neutral lipid fraction from the DEAE-Sephadex A-50 column was combined with the lower phase obtained after Folch partition of the total lipid extract and the combined lipids dried. To the same flask, 10Q ml of 0.6 M NaOH in methanol was added. The mixture was incubated at 37°C for 5 hours. Five volumes of acetone were then added and stored overnight at 4°C. The precipitate was collected by centrifugation at 4°C and dissolved in C M (4 1, v/v). After application to the column (2.0 x 25 cm), the column was washed with chloroform. Neutral glycolipids were then eluted with tetrahydrofuran H2O (10 1). Fractions containing neutral glycosphingolipids were pooled and their glycolipid content examined by thin-layer chromatography. 

Glycolipid analysis. Gangliosides were extracted with chloroform-methanol (2 1), purified on Sephadex G-25 columns and separated into individual species by thin-layer chromatography as described previously (12). 

Labelled Gm2 and gm1 were purified by Sephadex G-25 and thin-layer chomatography as described previously (12,13). ... 

Peak from Sephadex LH-20 column chromatography (see Fig. 4.6) Polyamide thin-layer chromatographic method (3-dimensional) Silica gel thin-layer chromatographic method0 (one-dimensional )... 

Following adjustment to pH 6.0, the solution is applied to a SP-Sephadex C-25 column in the sodium form. Amino acids are then eluted with 0.2 M citrate phosphate buffer, pH 8.0, and the effluent evaporated to dryness at 50 °C. The residue is dissolved in 0.1 N hydrochloric acid and applied to the amino acid analyser. Amino acids are separated by passing 0.2 M, pH 8 sodium citrate solution down the column. The S-methylmethionine content can then be obtained from the chromatogram, as illustrated in Fig. 8.1. The results obtained agree reasonably well with those obtained by thin-layer chromatography [13]. 

In this equation, 8 is a differential parameter developed from Sephadex and polyamide thin-layer chromatography, and ARm(3) and ARm(6) are the values for the substituents at the 3 and 6 positions, respectively. The number of compounds is sufficiently large so that the correlation could be accepted statistically. Although Equation 31 can be used to predict... 

Gas Chromatography-Mass Spectrometry. Prepare methoxime derivatives by heating the extract from the thin-layer chromatographic plate with 100 xl of an 8% solution of methoxyamine hydrochloride in dry pyridine at 60° for 30 minutes, and evaporate in a rotary film evaporator. Silylate by dissolving the residue in 100 Lll of chloroform, adding 100 Lll of A,0-bis(trimethylsilyl)acetamide and 20 Lll of trimethylchlorosilane, then seal, and heat for 2 hours at 60°. When only 5a-estrane-3P,17a-diol is to be confirmed the methoxime formation can be omitted. Prepare a 2-cm column of Sephadex LH-20 in a Pasteur pipette with a cotton wool plug, using a... 

The tripeptide was purified by Sephadex GIO filtration followed by silica-gel chromatography. The pure material did not react with ninhydrin because of the presence of N -terminal pyroglutamic acid. After silica-gel thin layer chromatography in several solvent systems, the product gave a single spot when detected with Pauly s reagent with overall yield of 13%. 

P. Fasella, A. Giartosio, and C. Turano, Applications of thin-layer chromatography on Sephadex to the study of proteins, in Thin-Layer Chromatography (G. B. Marini-Bettolo, ed.), Elsevier, Amsterdam, 1964, pp. 205-211. 

Fig. 3. Thin-layer chromatography in superfine Sephadex G-200 on microscope slides, followed by diffusion against monospecific antisera to identify proteins as indicated.

Thin-layer chromatography of isolated serum proteins on a weakly cross-linked dextran gel (Sephadex G-200) has been reported to give Rf values linearly related to the logarithms of the molecular weights of the proteins (M28). 

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