Determining Phenols in Dilute Solutions Notes on the Gibbs Method A. W. BESHGETOOR, L. M. GREENE, AND V. A. SlENGER The Dow Chemical Company, Midland, Mich.
from below, The lower end is next dmwn out, the delivery tip is put OR, then the condenser tip is sealed to the condenser at E.
Some suggestions concemins the performance of the Gibbs method are given, .dealing especially with tho removal of interfering sobstances and the best conditions for color development. Tho colors produced by certain phenol derivatives are listed, and an extraction method for the analysis of very dilute solutions is presented.
ibhs method for determining phenols (f) haa been lahoratoriea, including that of The Dow Chemical Company. Nevertheless some workers have encountered difficulties; Buswell and Dunlop (4). for example, prefer to estimate phenol hy means of its ultraviolet absorption. However, since the Gibbs procedure is capable of greater sennsitivity and requires less expensive equipment, it will probably remain in common use. The present paper is intended to he supplementary to the procedure given in (1). The suhjecte discussed include notes on the distillation apparatus, interfering substances, best conditions for color development, and a procedure for determining phenol in more dilute solutions. Although many substituted phenols produce colors in the Gihhs method, relatively few give the normal blue color of ordinary phenol and in those cases the sensitivity is wnally less. Baylis (8) notes that p-cresol shows no color while 0-cresol, like phenol, gives B blue. It is generally true that substitution in the para position reduces the sensitixty considerably, while ortho substitution with hydrocarbon groups has less effect and with halogen atoms shifts the color toward green. The colors produced by several phenols are shown in Table I, with concentration ranges which yield colors of about the proper intensity for comparison in 100-ml. Nessler tubes. All these compounds are volatile with steam and those which give colors with the I Gibbs reagent will interfere in the phenol 'i determination. A
INlERFERlNG SUBSTANCES
In the analysis of industrial wastes, off-colors from the desired blue are sometimes obtained. Green-blues may indicate that part of the phenol contains halogen in the ortho position, possibly &s a result of partial chlorination. They may also be produced by sulfides, which according t o the &mount present can yield off-shades varying through yellow-greens to pink. Williams (9) suggested removing sulfides with lead oxide or carbonate, while Renzoni (6) recommended the use of freshly precipitated cadmium carhanate. The authors have used copper sulfate, adding a small volume of strong copper sulfate solution to the measured sample and filtering into the distilling flask.
F used G successfully in a number of
sulfuric acid may be substituted for phosphoric? if aniline or othkr weak hsses are present. Tho entire 500 ml. of distillate are then returned to the distillation flask after the latter hits been washed,
x
OI
Flgure 1.
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Battery of Six Stills
ANALYTICAL EDITION
November, 1944 2 grams of fairly coarse marble chips are
0.5, 1.0, 2.0,3.0, 5.0, 7.0, and 10 parts per billion are prepared from standard phenol solution 1.8 ( I , p. 247), and phenol-free water. These should have the same volume as the samole. One milliliter of the standard phenol solution diluted to I liter corre-
added, and the second distillation is carried out. Calcium carbonate furnishes sufficient alkalinity t o retain salicylic acid, but not enough to interfere with the distillation of phenol.
E
COLOR DEVELOPMENT AND COMPARISON
A
Diffioulties in the method may freD quently he traced to the instability of 2,6-dibromoquinonechloroimide. It has been found advisable to purohase this compound in brown glass battles containing only 1 gram each. These are kept closed until needed; after a bottle is opened the material in i t has occasionally been found to decompose within a week or two. The precautions given in (I, p. 248), concerning the preperetion and use C of solutions of this reagent, should be carefully observed. The authors employ the itlcohalic solution (1.61) exclusively. D Since the dye produoed by reaction of the reagent with phenol is a n oxidation- ,cigvrO p, reduction indicator as well as an acid-base indicator (4,it is essential that the pH and the oxidation potential be maintained arithin the specified limits. It is a good practice to add copper sulfate solution to all samples and standards (1). Solutions in the Nessler tubes will turn pink if exposed to snnlightaftertheaddition ofreagent. Forthebestresultsitisneoessary to keep the tubes in the dark during color development. This is conveniently done by keeping them in a box similar to the one illustrated in Figure 3. After 4 hours or longer, the comparison is made in a Fisher Nessler tube support or similar rack, by fluorescent light, The reaction between phenols and 2,6dibromoquinoneohloroimide proceeds slowly and the color continues t o develop for a long period. On this account the samples and standard should be prepared at. the same time. I n any attempt to determine phenols with s photoelectric colorimeter (8),the standard curve should he prepared with allowance of a definite period for color development a t a specified temperature. The same conditions should he adhered to carefully in the analysis of B sample. ANALYSIS OF MORE DILUTE SOLUllONS
Various methods for concentrating dilute phenol solutions have heen proposed, involving evaporation of an alkaline solution (7), distillation (S), or extraction of the free phenol with ether (6). A procedure based on developing the color in a larger volume of sample and subsequently extracting the colored compound can also he used. This procedure multiplies the sensitivity of the Gibbs method by about 10 and increases the stability of the color. The reagents are the same as for the usual Gibbs method, with the further inclusion of 1 t o 4 hvdrocblario acid, chloroform. ~~~~~
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695
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Add about 200 ml. of fineiv Dowdered activated carbon t o
off as neeaed. I n the absence of substances which interfere in the ordinary Gibbs method, 1liter of the neutral sample may be rim directly; otherwise a sinele or double distillstion is necessary. A liter of the water is disfilled and the entire amount of phenoiis considered t o be recovered when 900 ml. have been collected in the first distillation, or 850 ml. in the second. Standards containing 0,
dibromoquinon&hlaroimide reamnt. Mix well and allow 'to stand in dark place overnight.Transfer to a large separatory funnel, add 5 ml. of 1 t o 4 hvdrachlorie acid and 18 ml. of chloroform. mix well. allow to 8;Damte. and draw off the chloroform laver &to a 50-mL Nessler
potassium hydroxido, invert a few times, and compare the colors.
It is barely possible to detect 0.25 part of phenol per billion, provided that interferences are absent and the standard water is pure. The detection of 0.5 pert is rather easy with phenol, which gives a blue-green color. The blank may'be slightly yellow because of the presence of excess reagent, the intensity of the yellow color being greater with older reagent. With o-chloroTable I. Colon of Several Phenols in tho Gibbr Test Phenol Ordinary p-Chloro *-Bromo o-chloro o-Bro"W
2.4-D/ohloro 2 5-Dlehloro
2:B-Diohloro
Triollloro Tribromo o-Phenyl 2-Chloro-6-phenyl 4-Chloro-0-phenyl p-Phenyl p-le,t-B"Q.l
Color BLW Blue
BlW Green-blue Green-blue Green-blue Green-blue Green. , Insensltlve insensitive Blue DlW Green-hlue Insensitive Insensitive
Concentration Range, Parts per Billion 6100 20-400 25-500 10-150
10-2oc
4&8CO
30-600 30-600
..
1o:ioo IO-zoo 10-2oc
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INDUSTRIAL AND ENGINEERING CHEMISTRY
phenol the color is much the same as with phenol, but the test is about one half as sensitive. The color from pchlorophenol is more toward the blue, and the sensitivity is only about one fourth that of phenol. LITERATURE CITED
Am. Puhlic Health Assoc. and Am. Water Works Aesoc., New York, “8tandard Methods of Water Analysis”, 8th ed., 1936. (2) Baylis, J. R.,J. Am. Water Works Assoc., 19, 604 (1929). (1)
Vol. 16, No. 11
(3) Baylis, J. R.,Water Works and Sewage, 79,343 (1932). 14) Buswell, A. D., and Dunlop, E. c.,“Estimation Of Phenols h Water by Mems of Ultraviolet Absorption Spectra”, presented at 103rd Meeting of AMERICAN CHEMICAL SOCIETY, Memphie, Tenn. (5) Cohen, B., Gibbs, H. D., and Clark, W. M., Pub. Hculth Hepta., 39,381, 804 (1924). (6) Rensoni, L. S., J. Am. Water Work8 Assoc., 32, 1035 (1940) (7) Theriault, E.J., IND. ENO.CHEM.,21, 343 (1929). (8) Turker, I. W., J. Assoc. Oficial A g r . Chem., 25, 779 (1942! (9) Williams, R. D., IND. ENQ.CHEM.,19, 530 (1927).
Determination of the Nutritive Value of the Proteins
of Food Products H. H. MITCHELL, Animal Nutrition Division, University of Illinois, Urbana, 111. The nutritive value of food protein cannot b e accurately assessed lrom 4 determination of the amino acid content, nor from animal feeding experiments that fail to credit the protein with all its functions in the body. A study of the nitrogen economy of the animal when fed the protein to b e tested under certain essential conditions Is capable of giving 4 complete and reasonably satisfactory picture of protein utilization in the body, in digestion as well as in metabolism, The net protein value combines all this information in one @sure. Illustrations of application of the method to problems ot the storage 4nd processing of foods are given.
TvaryHE
protein content of food products has an uncertain significance for their evaluation in nutrition, because proteins widely in the extent of their digestion in the alimentary canal, and even more so in the extent to which the end products of their digestion, largely amino acids, are available for those functions i n the body peculiar t o dietary protein. These functions are bewildering in their complexity and in their involvement in life processes, but quantitatively the construction of new protein material in growth and the maintenance of the nitrogenous integrity of the tissues already formed are by far the most important. The differences that exist in the value to the animal body of the proteins in food products, the extent to which food proteins supplement each other’s amino acid deficiencies when combined into diets, particularly the best method of supplementing the proteins of white flour, and the effect of storag? and commercial p r o m i n g on the protein value of food products in nutrition are all problems of importance to food technologists. How can these problems be most effectively tackled in the chemical or the biochemical laboratory? At the present time, three methods are being used for these purposes: ( a ) determination of the amino acid contents of foods or food proteins, (b) measurement of the ratio of gain in weight to protein intake of growing rats subsisting on rations in which the protein content is the only factor limiting growth, and (c) measurement of the gain in nitrogen to the bodies of growing rats resulting from the consumption of diets complete in all respects but protein. The latter method can be extended to mature, pregnant, or lactating animals, and to the human subject. This paper considers briefly the advan tages and disadvantages of each method for the purposes for which they are being employed. AMINO ACJD ANALYUS OF PROTEINS AND FOODS
The basis upon which this method rests is that the nutritive quality of food proteins is determined entirely, or largely, by their content of amino acids, particularly of those nine or ten
amino acids commonly classed as dietary essentials on the evidence of Rose’s well-known experiments on growing rats. Undoubtedly the content of a food in those amino acids that the body cannot synthesize from ordinary dietary components seta an upper limit to its usefulness in serving the biological functions of dietary protein; and to this extent the basis is sound. The amino acid analysis of the proteins in a food product Ly chemical methods is laborious and the various analyses for individual amino acids are not of equal precision; those for leucine and valine in particular leave much to be desired ( 4 ) . The microbiological methods suggested for determining the amino acid contents of food products are still in the experimental stage. However, it is not too much to hope that in the near future satisfactory methods, chemical or microbiological in nature, will be available for all the amino acids commonly believed to be dietary essentials. I t should be emphasized, however, that satisfactory analyses for all these essential amino acids must be a t hand before any one food product can be evaluated; otherwise, the possibility exists that the amino acid, or acids, for which’ no analysis is available may be, or include, the amino acid limiting the nutritive value of the contained proteins. When the ultimate goal of amino acid analysis of food proteins is attained, it will be possible from the data secured on many foods to predict which are the best in supplying the needs of the body for the essential amino acids, in so far as these needs are known, and in particular to predict which protein mixtures in individual foods will correct best each other’s deficiencies in e% sential amino acids. But these predictions cannot be expressed quantitatively nor can they be made with any great assurance, because the chemical picture of food proteins is inevitably altered and distorted by biological factors before a true picture of nutritional quality is secured. The disturbing biological factors that impair the usefulness in practical nutrition of a knowledge of the amino acid contents of foods are as follows: The digestibility of food proteins iu the alimentary tract IS largely independent of amino acid composition, and may be determined, as Mendcl and Fine showed many years ago (16-19) by the constituents of foods other than protein. The di eetive apparatus of the animal, though remarkably efficient unler the most favorable conditions in reducing dietary protein to its ultimate building stones, may be impeded and frustrated in its operation by those nonprotein constituents of food that successfully resist its attack (the cellulosic and hemicellulosic components in particular), while preventing complete acceea of the proteolytic enzymes to their pro er substrates. The indigestible residue of rood protein may not be represent. tive in its amino acid constitution of the food from which it wae derived. This may be inferred from the known differences in the ease with which different peptide linkages are split by the digestive enzymes. An instance in point was reported by Jon- and
