Robert J. Hurtubise Unwers~tyof Wyomtng Laram~e.Wyommg 82071
Industrial Analytical Procedure for Undergraduate Analytical Chemistry
Mixtures of Dhenolic antioxidants are used widelv in food products around the world t o inhibit oxidation of edible animal and vegetable fats and oils. They can be found in such food products as cooking oil, breakfast cereals, potato chips, candies, meat products, nuts, and rice. T h e most widelv used antioxidants are BHA (mixture of isomers, 2-tert-h&-4methoxyphenol and 3-tert-hutyl-4-methoxyphenol),B H T (2,6-di-tert-hutyl-4-methylphenol), and PG (propyl gallate). Recently a new food antioxidant T B H Q (mono-tert-butylhydraquinone) was approved for use in food products by the federal government.' It is estimated t h a t man consumes approximately 0.1 m g k g body weight daily of these antioxid a n t ~ Also, . ~ in 1967, 15.5 million pounds of antioxidants valued a t $20.5 million were used by food manufacturers in the United States.2 Because of the wides~readuse of ohenolic antioxidanh and regulations issued by 11;~:US.Food and Ihug Administration (FDA, on the level of antioxidants in food ~ r o d u c t s reliable . analytical methods are needed by food manufacturers to control the amount of antioxidants added t o food ~ r o d u c t s . In order to make undergraduates aware of such analytical methods and get first-hand experience with such methods, we have been using a method published by Eastman Chemical Products, Inc., a major producer of food antioxidants, in our Quantitative Analytical Chemistry course. This is one course in a new BS program in chemical technology a t the University of Wyoming? The method involves the determination of BHA in cooking oil and has been greeted enthusiastically by the students. In the written experiment, a brief introd&tion is included for the students and then the Eastman procedure is given. The students are required to derive the equation used to calculate % BHA. T h e procedure is reproduced helow by the courtesy of Eastman Chemical Products, Inc. ~
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Eastman Publication No. ZG-169A reproduced in part. Analvsis of Fats. Oils. and ~ssentialOils for BHA Eastman Food Laboratory Standard Procedure No. 26 Reagents 1. TENOXa BHA antioxidant 2. 100%Ethanol 3. Distilled water 4. Petroleum ether 5. 80% Ethanol (100% ethanol diluted by volume with distilled water) 6. Borax buffer (Dissolve 2.00 g NazBa07.10Hz0, C.P., in distilled water and bring to 100-mlvolume) 7. N,2,6-T reagent [Dissolve0.010 g N,2,6-trichloro-p-benzoquinoneimine (Eastman Kodak Company, organic chemical No. 2483) in 100 ml100% ethanol. Prepare fresh weekly.] Procedure A. Preparation of BHA Standard Solution 1. Prepare a TENOX BHA stock solution by dissolving 0.100 g TENOX BHA in 100 ml100% ethanol. This solutioncontains 1000 pg BHA per ml. 2. Prepare an intermediate solution by diluting a 5-ml aliquot of the stock solution to 50 ml with 100%ethanol. This solution contains 100 pg BHA per ml. 3. Preoare a standard solution bv la cine a 5-ml sliauot of the
per ml.
NOTE: Stock and intermediate solutions ore stoble for one week if kept refrigerated. Standardsolutionsshould be prepared fresh daily. B. Extraction of the Antioxidant from the Fat or Oil NOTES: I. Propyl gollote, nordihydroguoiaretie acid, and some butylated hydroxyonisole will be extracted along with the BHA if they nre present in the fat or oil. Howeuer, they will not interfere with the quantitative and qualitatiue tests. 2. For accurate results, it is necessary to include at least one untreated sample with each group of treated samples. 1. Weigh 7.5 i 0.05 g of the fat or oil into a 50-ml beaker. 2. Add 15 ml of petroleum ether to the fat or oil. 3. After the samole is thorouehlv in the oetroleum .. , disoersed . rrhrr, rrnnifer rhe dirprrsim ro a 10J-ml5epararory iunnel. Rlnw !he hraker wilh l(r ml ofperrdturn trlwond odd rhiz rlnaing lo rhi- sepnratory funnpl. I.Add I5 mlofHtrr ethanol torheseparatury lunnrl.Sropper the funnel and shnkr. for :l mm. Let the funnel stand ~ n t i l the two phases are completely separated. 5. Drain the lower or ethanolic layer through a piece of filter paper (to remove any suspendedfat globules) intoa 100-ml volumetric flask. 6. Repeat the 80% ethanol extraction four more times-each time filtering the ethanolie layer through the filter paper into the 100-mlvolumetric flask. 7. Discard the petroleum ether phase. 8. Rinse filter paper with 80% ethanol and add rinsing to the volumetric flask. Adjust to volume with 80%ethanol. C. Qualitative Analysis of the Extract 1. Place 5 ml of the extract in a 50-ml beaker. 2. Add 2 ml of the borax buffer to 5 ml of the extract. 3. Then add 1 ml of the N,2,6-T reagent. 4. The appearance of a blue color indicates the presence of BH A. D. Analysis for BHA 1. Pipet 10 ml8W ethanol into a small glass-stoppered reagent bottle of 60-ml caoacitv. This will serve as a blank. 2. Pipet three different'aliq;ots of the BHA standard solution into reagent bottles. Add enough 80% ethanol to each to give a total volume of 10 ml. 3. Pipet a suitable aliquot of each extract into additional bottles. Add enauah 80% ethanol to give a total volume of 10 ml in each bottle. 4. Assemble all bottles (steps 1through 3); add 2 ml of borax buffer, then 2 ml of N,2,6-T reagent to each bottle and mix well. .~5. Let stand 15 mi". 6. If the extracts are cloudy, add 5 ml of n-butanol and mix
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7. Measure the absorbance at 620 nm in l-cm cells, using the 8096 ethanol (blank) in the reference cell. 8. Divide the absorbance of each TENOX BHA standard bv
its corresponding concentration of BHA per aliquot. Card a t e the average of the values obtained and designate this as R ".~ . ." -
9. Calculate BHA content as follows A X 100 X 10-4 % BHA = BXMIXW
Fed. Regist., 37,25356 (1972). Branen, A. L., J. Amer. Oil Chem. Soe., 52,59 (1975). Nelson, D. A,, Holt, S. L., Archer, V. S., Hurtubise, R. J., Barden, R. E., J. CHEM. EDUC., 53,148 (1976). Volume 54. Number 10, October 1977 1 651
Where: A = Absorbance of sample at 620 nm = per agof BHA at 620 nm'calculated from standards M 1= Volume of extract taken for analysis, in ml W - = Weight of fat sample extracted, & grams
652 / Journal of Chemical Education
General Reference Anglin,Constsnee, M8hon.J.
(19561
H .and Chapman. R. A.. Agr Food Chem. &(no. 12). ,018
