Mercuric acetate adducts

There has been no lack of attempts to convert lipids into derivatives which could be chromatographically fractionated and from which the original substances could be subsequently recovered. Acetoxymercuri-methoxy adducts are particularly easily accessible derivatives, being formed by reaction of unsaturated compoimds with a solution of mercuric acetate in methanol containing a little acetic acid. This addition reaction takes place quantitatively at room temperature [68]. ci -Com-pounds react 10—20 times faster than fm i -isomers [68]. The imsaturated compounds can be recovered by treating their adducts with hydrochloric acid, without cis-trans isomerisation or rearrangement of the double bond occurring [68]. 

Saturated lipids and the mercuric acetate adducts of unsaturated lipids of the same compound class can be separated by TLC. In the same way the adducts can be fractionated according to the number of acetoxy-mercuri-methoxy groups. This is illustrated in Fig. 144 for the example of the methyl esters of long chain fatty acids. 

The adducts of methyl chaidmoograte and of the esters of other cyclic acids show chromatographic behaviour which is markedly different from that of the corresponding purely aliphatic esters [124]. 

Mixtures of different classes of lipids can also be fractionated in accordance with the degree of unsaturation of their components, using TLC of the mercuric acetate adducts, provided that these are of not too varied polarity. The chromatographic behaviour of the adducts of, e. g.. 

Both partition and adsorption effects play a part in the chromatographic separation of mercuric acetate adducts. 

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C 77.38%, H 7.58%, N 15.04%. Found in coal tar and in bone oil. Synthesis (40-50% yield) from cyclohexyl-amine with excess ammonia and zinc chloride at 350 Nordt, PB Report 704 (1941). Prepn from ethylene -mercuric acetate adduct and ammonia water (70% yield) Gumboldt, Feichtinger. Z. Naturforsch. 4b, 123 (1949). Review of the various methods of condensing aldehydes, ketones, ethylene, butadiene, etc., with ammonia F. Brody, P. R. Ruby in A. Weissberger, 77te chemistry of Heterocyclic Compounds, vol. 14, part I (New York, i960) pp 99-589. Contains 1851 references. 

Linoleic acid or ester can be obtained from any linoleic-rich oil which has less than 1% of linolenic acid (Section 3.2.5) by the following steps (a) urea fractionation to reduce the combined content of saturated and oleic acids in the mother liquor to <5%, (b) repeated low-temperature crystallization from acetone at — 75 C until the precipitate contains >90% linoleic acid, (c) further urea fractionation to reduce the combined content of saturated and oleic acid to below 0.8%, and (d) low-temperature crystallization at —75 to give pure acid (>99%) which is finally eluted from a column of silica. The presence of linolenic acid at levels initially above 1% renders the purification more difficult. Further purification by repeated anaerobic low-temperature crystallization from acetonitrile has been described (Arudi et al., 1983). Christie (1982) claims to have obtained pure methyl linoleate from safflower oil by partitioning the mercuric acetate adducts between 5% aqueous methanol and hexane in separating funnels. 

Chromatography of mercuric acetate adducts. Unsaturated lipids form acetoxymercuri-methoxy compounds with methanolic mercuric acetate. These mercuric acetate adducts of unsaturated compounds must be prepared before chromatography in contrast to the silver complexes which are formed on the silver nitrate-containing adsorbent during chromatography. 

Fig. 144. Fractionation of a lipid class by TLC of the mercuric acetate adducts of its unsaturated components [119]. Adsorbent sihca gel G solvents 1 petrol ether (BP 60—70° C) diethyl ether (80 + 20) 2 n-propanol-acetic acid (100 + 1) times of run 1 1.5—2h 2 3—4h visuahsation s-diphenylcarbazone in ethanol, then exposure to iodine vapour amounts about 20 jjig of each ester, 50—100 jxg of the adducts, a methyl stearate 6 methyl oleate c methyl linoleate d methyl linolenate e—j mercuric acetate adducts of c methyl esters of Cig-acids from CJdoreUa pyrenoi-dosa f methyl esters of Cig-acids from Chlorella g methyl esters of all acids from Chlorella h methyl oleate i methyl linoleate j methyl linolenate k mercuric acetate...

The mercuric acetate adducts of unsaturated lipids are clear, colourless oils. They are applied to thin layers in chloroform solution. 

Adsorbents. Silica gel G is suitable for fractionating mercuric acetate adducts of lipids [119]. The adducts of highly unsaturated lipids are chromatographed on layers of silica gel G + kieselguhr G (30 + 70) [211,212]. 

Solvents. The chromatograms are developed stepwise as a rule Petroleum ether (60—70° C)-diethyl ether (80 + 20) is suited to separation of saturated neutral lipids from the mercuric acetate adducts of the corresponding unsaturated compounds [119]. This solvent migrates 15 to 18 cm in 1.5—2 h on silica gel G layers. A second solvent, n-pro-panol-acetic acid (100+ 1), serves for fractionating the adducts of Upids with one, two, three or more double bonds. It migrates 12—14 cm in 3—4 h. The saturated compounds are found between the two solvent fronts (see Fig. 144). Hexane-dioxan (60 + 40) is better for separating adducts of neutral lipids which contain three, four and more double bonds, on silica gel G it migrates about 15 cm in an hour [219]. 

Mercuric acetate adducts of lipids containing more than three double bonds are fractionated on silica gel G-kieselguhr G (30 + 70), using isobutanol-formic acid-water (100 + 0.5+ 15.7) [211, 212]. The solvent should be placed in the chamber 5 hours before chromatography in order to saturate the atmosphere it migrates about 16—17 cm in ca. 5 h. 

Saturated lecithins can be separated from the mercuric acetate adducts of unsaturated lecithins, on silica gel G, using chloroform-methanol-water (70 +30 + 4) [14]. 

Methods of Detection. Mercuric acetate adducts can be visualised as violet to purple red spots on a light pink background, by spraying with a solution of s-diphenylcarbazone in 96% ethanol (or Rgt. No. 87) [119, 211]. Saturated lipids are rendered visible in UV light by spraying with 2, 7 -dichlorofluorescein (Rgt. No. 63). 

Fig. 145. Thin-layer chromatogram of the mercuric acetate adducts of the fatty acid methyl esters from the alga Chlorella pyrenoidosa (cf with Figs. 144 and 146)...

Fig. 146. Gas chromatographic separation of fatty acid methyl esters after prior separation of their mercuric acetate adducts by TLC (see Fig. 145) [119]...

The sensitivity of detection of traces as well as the accuracy of gas chromatographic analysis is augmented by coupling with TLC of the mercuric acetate adducts a more reliable identification of the fractions becomes possible. 

This corresponds to the behaviour of the mercuric acetate adducts (cf. Fig. 144) in contrast to these, however, the ozonides are only slightly more polar than the starting materials. 

Mercuric acetate adducts are best detected by heating briefly at 80 C, causing the zones to turn blue-violet. 

Figure 4.1. Derivatives of fatty acids, (a) pyrrolidide (b) picolinyl ester (c) trimethylsilylether (d) isopropylidene derivative (e) butylboronate derivative (f) mercuric acetate adduct (g) dimethyidisulfide...

The lipid sample is refluxed with a 20 % excess of the theoretical amount of mercuric acetate in methanol (2 mL per g of mercuric acetate) for 60 min. After cooling to room temperature, a volume of diethyl ether 2.6-fold that of the methanol is added to precipitate inorganic mercury salts, and the solution is filtered. The mercuric acetate adducts are obtained on evaporation of the solvent."... 

Concentrates of polyunsaturated fatty acids (as the methyl esters) can be obtained from suitable starting materials, by preparing the mercuric acetate adducts (see Chapter 4 for practical details of the preparation of adducts and regeneration of the original double bonds) and partitioning them between methanol and pentane the methanol layer retains the adducts of the more unsaturated esters which can be regenerated unchanged. For example, methyl linoleate of 95 % purity and methyl linolenate of 90 % purity have been produced on the 50-100 g scale by this method from the esters of safflower and linseed oils respectively. The method could no doubt also be adapted to the preparation of concentrates of other polyunsaturated fatty acids. 

One further procedure worth mentioning briefly here consists in preparing methoxy- or methoxyhalogeno-derivatives, via the mercuric acetate adducts, for identification by MS [611-613,834]. Oxymercuration has also been employed to convert triple bonds to keto groups, which can be located in this form or after reduction to hydroxyl derivatives with sodium borohydride [140,480],... 

Comparison of retention times of the major component with that of hexadecyl acetate suggested that it might be an unsaturated Cie acetate. A sample purified by preparative GC was examined by TLC comparison of Rf values of the sample and its mercuric acetate adduct with those of suitable standards confirmed the presence of two double bonds. Saponification and LiAlHi, reduction destroyed BAG activity, which was regained on reacetylation. Acetylation had no effect and bromination destroyed activity. Hydrogenation also destroyed activity and gave a product that cochromatographed with hexadecyl acetate. MS analysis... 

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