Chloroform: methanol: water systems

The determination of sugars in fruits and other materials involves extraction of the sample with 80% ethanol, transfer of sugars into an aqueous phase with the aid of a chloroform—methanol—water system (64 32 24) and drying. The analysis is carried out on SE-54 or SE-52 stationary phase after silylation [424,425]. The concentrations of monosaccharides can similarly be determined in the course of a fermentation process [426] and, after hydrolysis, also in polysaccharides from cellular walls and apple tissues [427,428]. SE-30 served as a stationary phase for these analyses. 

A chloroform/methanol/water system is appropriate for the separation of an acidic fraction. If the solvent compositions are not optimized, the phospholipids and glycolipids are eluted at the solvent front or in the column contents. When we chose a suitable solvent, acidic phospholipids (phosphatidic acid, phosphatidylserine, phosphatidylinositol. 

Helting and Peterson (1972) have found that if retinyl galactosyl phosphate is incubated with microsomes from murine mastoc)1 oma, label enters a substance that behaves like protein in a chloroform-methanol-water system, but it has not been further characterised. The physiological signifi-... 

A suspected water gel is examined on a microscope slide to identify the microspheres used in slurry and emulsion explosives. The gel is directly extracted with methanol, which dissolves the amine salt, and a small amount of NH4NO3. TLC on a cellulose plate using a chloroform-methanol-water system separates the sensitizers. The plate is sprayed with ni-nhydrin and heated to visualize the amine salt. A second spray with diphenylamine in ethanol followed by long-wave UV irradiation visualizes the NFi4N03 if desired. Alternatively, the methanol extract may be evaporated to near dryness, redissolved in water, and analyzed by IC to identify the sensitizer. An intact sample can also be extracted with water using either a small homogenizer or ultrasonic agitation to disrupt the gel structure. Microspheres float on the surface and are removed for examination by SEM-EDX to characterize the manufacturer. Spot tests and IC identify ammonium, calcium, and nitrate ions in the water extract. Flake aluminum, if present, is identified as described above. 

A chloroform/methanol/water system is appropriate for the separation of an acidic fraction. If the solvent compositions are not optimized, the phospholipids and glycolipids are eluted at the solvent front or in the column contents. When we chose a suitable solvent, acidic phospholipids (phosphatidic acid, phosphatidylserine, phosphatidylinositol, lysophosphatidyl-inositol, and lysophosphatidylserine) were satisfactorily separated between the solvent front and the column contents (Fig. 2A). Samples (2 mg each) of the acidic fraction of human brain lipids was applied to TC-CCC by using the chloroform/methanol/water (5 4 3) solvent system. Aliquots of each fraction were spotted and developed by HPTLC. 

Table 2 Chloroform/methanol/water system. Chloroform Methanol...

First Chloroform-methanol-water Systems 1 and 2 are good for separ- Rouser et al. (1967) (99)... 

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). 

Two-dimensional systems are occasionally used to separate phospholipids (Figure 12.5). After the separation of phospholipids using the chloroform methanol water (65 25 4) system, the plates can be dried and turned by 90°, followed by the second development in either n-butanoFacetic acid water (60 20 20) or chloroform acetone ... 

Preparative TLC (System I, 85 15 2 chloroform/methanol /water followed by System IV, 50 50 2 hexane/ethyl ether/formic acid) of the chloroform/acetone fraction produced 43 separate bands, 33 of which had definite activity. IR spectra of these active bands were strikingly similar, exibitlng the same major features as noted for the unseparated chloroform/acetone fraction. UV spectra were also similar absorption at 275 and 220 nm, with the latter being strongest. 

Plumericin, isoplumericin, plumieride, 13-O-coumaroylplumieride and protoplumericine A were isolated from the ethyl acetate extract of P. rubra heartwood [85]. The extract (44 g) obtained by percolation was submitted to column chromatography on silica gel with light petroleum ether/ethyl acetate (1 1) and methanol as eluent to furnish four fractions (A-D). Fraction C directly afforded plumericin (1.22 g) after recrystalUzation from toluene/ethyl acetate. Fraction B was rechromatographed on a silica gel with toluene/ethyl acetate (9 1), and isoplumericin (140 mg) was isolated from fraction 2. Fraction D was also rechromatographed on a silica gel column, but with chloroform/methanol/water (90 10 0.5 to 70 30 10) as solvent system. Fraction 3 was 13-O-coumaroylplumieride (2.0 g) and fraction 7 was protoplumericine (1.6 g). Fraction 5 was subjected to Sephadex LFI20 column eluted with methanol, followed by silica gel column eluted with chloroform/ methanol/water (85 15 0.7) to furnish plumieride (120 mg). 

In brief, the isolation scheme is as follows. First, the lipids are all extracted from the animal tissue, generally by using chloroform-methanol or chloroform-methanol-water. An increase in the polarity of the extracting system makes it possible to extract more fully the polar glycosphingolipids, especially polysialoglycolipids, but may, however, lead to contamination... 

Figure D1.6.5 Sequential latroscan TLC-FID profiles of the lipid classes extracted from the dorsal white muscle of Atlantic salmon. I, II, and III represent partial chromatograms from the three-stage development of total lipids on aChromarod Sill. The solvent systems were (I) 80 14 1 0.2 (v/v/v/v) hexane/chloroform/isopropanol/formic acid for 55 min (II) acetone for 15 min and (III) 70 30 3 (v/v/v) chloroform/methanol/water for 60 min.

Glycolipid 11, Table 1 (2) glycolipid 9, Table 11 (3) glycolipid 10, Table I. Solvent system chloroform/ methanol/ water (60/ 35/8, v/v/v). Visualization orcinol reagent. (26)... 

A solution containing 5.0 mg of N-trifluoroacetyladriamycin dissolved in 0.5 ml of anhydrous pyridine was treated with 18 microliters of valeryl chloride, which was added in small portions over a two-day period. The reaction was monitored by thin layer chromatography and when the presence of N-trifluoroacetyladriamycin could no longer be observed, the reaction mixture was diluted with 10 ml of chloroform. The chloroform solution was extracted three times with pH 4 buffer and once with pH 7 buffer. The dried chloroform solution was then evaporated under reduced pressure, and the residue was purified by preparative thin layer chromatography on silica gel G with chloroform methanol water (120 20 1 by volume) as the solvent system. The major orange-colored band was removed and washed free of silica gel with a mixture of methanol and ethyl acetate. Upon evaporation of the methanol and ethyl acetate, 2.19 mg of N-trifluoroacetyladriamycin-14-valerate was obtained. This material was idential by spectral and chromatographic comparison with samples of N-trifluoroacetyladriamycin-14-valerate prepared by earlier described methods. 

Currently, there is no doubt that the most widely used method for extraction of tissue lipids is that of Bligh and Dyer (1959). Basically, this is a modification of the Folch method and employs a careful calculation of the amount of sample (tissue) water such that the overall mixture will have a final composition of chloroform-methanol-water of 1 2 0.8 (v/v). Thus, a singlephase extract can be obtained and extraction completed very rapidly, even within minutes. Recovery of the lipid in a chloroform-rich phase can be achieved by addition of equal volumes of chloroform (under certain conditions) and water to produce a two-phase system. The lower (CHC13) phase is subsequently washed with a methanol-water (1 0.9, v/v) mixture to allow removal of a substantial amount of the nonlipid contaminant with little or no problems with interfacial fluff formation or emulsions. However, even though this is a highly efficient method, it is still advisable that one take steps... 

Using the total platelet lipid extract as described in the section entitled Isolation of Human Platelets, single-dimensional thin-layer chromatography on a 250- pm-thick silica gel G-coated plate with a solvent system of chloroform-methanol-water (65 35 7, v/v) will yield the pattern shown in Figure 3-2. 

It is best to purify the methyl esters by thin-layer chromatography of the sample on another silica gel G (250 p,m) plate using this solvent system, but without any spray reagent being used. A comparable plate is run and the methyl ester band is detected by the sulfuric acid/char reaction. Then the unsprayed plate is scraped at the methyl ester area and the silica gel is extracted with petroleum-diethyl ether (80 20, v/v) or with chloroform-methanol-water (1 2 0.8, v/v). It is always prudent to spray the latter plate (after removing the desired area by scraping) with sulfuric acid and then charring. This will validate whether the apparent removal of all of the methyl esters has been accomplished. 

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