Copper soaps method

Colorimetry. Copper (Duncombe 1963 Koops and Klomp 1977) or cobalt (Novak 1965) soaps of long-chain fatty acids (>C12) are soluble in chloroform and can be determined quantitatively by colorimetric determination of the extracted metal. Shipe et al. (1980B) have recently modified the original copper soap method to make it simpler, more rapid, and adaptable to automatic equipment. 

Koops, J. and Klomp, H. 1977. Rapid colorimetric determination of free fatty acids (lipol-ysis) in milk by the copper soap method. Neth. Milk Dairy J. 31, 56-74. 

Basic Protocol 1 Titrimetric Determination of Lipase Activity Basic Protocol 2 Colorimetric Assay of Lipase Activity Using the Copper Soap Method... 

COLORIMETRIC ASSAY OF LIPASE ACTIVITY USING THE COPPER SOAP METHOD... 

In the event that lipase preparations are too active to allow for facile estimation of initial rates, the enzyme can be diluted and assayed again. This is illustrated using the copper soap method where the reduced level of C. rugosa lipase addition afforded a longer period of linearity to the reaction progress curve than did the more active B. cepacia lipase (Fig. C3.1.3). 

Bynum, D. G., Senyk, G. F., and Barbano, D. M. (1984). Determination of free fatty add content of Cheddar cheese by a copper soap method. J. Dairy Sci. 67,1521-1524. 

Recently Shihabi and Bishop (93) described a refinement in the preparation of a stable substrate and demonstrated the feasibility monitoring the reaction kinetically. This procedure has been evaluated by Lifton et. al. (9 ), who found that this method correlated well (r 0.914) with the copper soap-lipase method of Dirstine. They concluded that the method was rapid (less than 5 min. per sample), accurate, precise and linear over a clinically useful range. Its simplicity allows its application as an emergency procedure. Attempts to use this assay for urine lipase activity were unsuccessful. 

Another sensitive colorimetric procedure is that of Mackenzie et al (1967), which utilizes the dye Rhodamine B to form benzene-soluble complexes with fatty acids. Nakai et al. (1970) developed a rapid, simple method for screening rancid milk based on the foregoing procedure. The test is said to detect rancid milk with an ADV above 1.2. Like the copper or cobalt soap method, the Rhodamine B reagent is also limited to the longer-chain fatty acids. Kason et al (1972) used the method employing Rhodamine 6G of Chakrabarty et al (1969) to investigate... 

Conceptually, assays for lipase activity using the colorimetric method (copper-soap procedure Basic Protocol 2) are similar to titrimetry in that liberated fatty acids are being measured however, the colorimetric method is more specific for fatty acids (Lowry and Tinsley, 1976). Quenched subsamples of emulsified acylglycerol/lipase reaction mixtures are combined with the biphasic mixture of cupric acetate/pyridine and benzene. Cupric salts of the fatty acids are formed (molar stoichiometry of fatty acid to Cu2+ of 4 2) and these soaps, which are blue in color, are partitioned into benzene to allow for quantification by measuring absorbance of the clear benzene phase at 715 nm. 

Shipe, W.F., Senyk, G.F., Fountain, K.B. 1980. Modified copper soap solvent extraction method for measuring free fatty acids in milk. J. Dairy Sci. 63, 193-198. 

Antonis (A3) has estimated phospholipids by a procedure for determining fatty acids. This technique requires a total serum extract and a phospholipid-free extract for the measurement of both total and free fatty acids, the difference between them being a measure of the phospholipid content. Free fatty acids (A4) are determined on a phospholipid-free extract by a procedure based on partitioning the fatty acids as copper soaps into chloroform, and subsequent photometric determination of the copper with diethyldithiocarbamate. Phospholipids, as well as the free fatty acids present in the total lipid-extract, are measured by the same method, since they also form a complex with copper that is soluble in chloroform. A criticism of this technique is that equal response is not given by dipalmitoyl lecithin, dipalmitoyl cephalin, or beef brain sphingomyelin. 

A semiautomated procedure for the estimation of plasma free fatty acids has been developed by Antonis (A3, A4). The technique requires the preparation of a phospholipid-free plasma lipid extract. The automated colorimetric procedure is based on the methods of Duncombe (Dl, D2), and depends on the solubility of copper soaps in chloroform with subsequent complexing of the copper with diethyldithiocarbamate. 

Copper soaps are produced with methods 1,2 a and 3. Again, stearic acid [7617-31-4] and oleic acid [10402-16-1] are the main reactants. Fungicides for ship and wood coatings, for textiles and paper are the outlets. 

In addition to the USDA standards, The Named Vegetable Oil Standard from the Codex Alimentarius Committee on Fats and Oils (Codex Alimentarius, 2006) identifies quality characteristics that oils must meet for international trade. For example, the standard states that an oil should be characteristic of the designated product and be free of foreign and rancid odor and taste. Maximum levels of matter volatile at 105°C (ISO 662 1998) (ISO, 2005) should be 0.2% m/m, insoluble purities (ISO 663 1998) (ISO, 2005) 0.05% m/m, and 0.005% m/m soap content [AOCS Cel7-95 (97)] (AOCS, 2005). Limits for metals are 1.5 mg/kg iron (Fe) in refined oils, whereas only 0.1 mg/kg of copper (Cu) is allowed in refined oils. For methods to measure Fe and Cu, use ISO 8294 1994 (ISO, 2005) or AOCS Ca 18b-91 (97) (AOCS, 2005). Limits for oil deterioration include peroxide value and acid value. Codex allows up to 10 meq/kg oil for refined oils. Acid value limits range from a low of 0.6 mg/KOH/g oil for refined oils. 

There are two other useful methods of controlling the pH of the autoclave charge the introduction of copper-ammonio hydroxides and the use of buffer salts, such as soap or chlorates, which are attacked and decomposed when the concentration of the hydracid is sufficiently great. The oxidants cannot be used to advantage, however, when the amino compound (e.g., aniline) is readily susceptible to oxidation. 

Dr, Muter 8 Method —This method may be used for the determination of glycerin in soap and soap leys, the process being based on the power of glycerin to arrest the precipitation of copper hydrate by the alkalies. The procedure is as follows —... 

There is not much to be said, regarding the analytical procedures used to make these determinations, because these are well-known. May I mention, however, that the percentage of soap is obtained by direct potentiometric titration of sucroglyceride dissolved in 2-propanol and water the free sugar is determined by the thin layer chromatographic method in comparison with a standard and the dimethylformamide content is obtained colorimetrically by the copper dimethyldithio-carbamate method. 

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