Preparation of Samples for Microbiological Determination of Riboflavin F. M. STRONG
AND
L. E. CARPENTER,College of Agriculture, University of Wisconsin, Madison, Wis.
T
HE Snell and Strong microbiological assay for riboflavin (IO) has been employed successfully by many laboratories on a large variety of biological materials. It is generally accepted that the values so determined compare favorably wit,h those obtained b y animal and physicochemical methods. However, it has been recognized that the microbiological method gives higher results than other methods on certain types of material, notably cereals and cereal products. I t has been re orted (1, 9) that the starch component of cereals, or some mistance associated with it, is responsible for the high values obtained by the microbiological method. According t o Scott, Randall, and Hessel (9) the stimulatory substance is destroyed by digestion with taka-diastase, while Andrews, Boyd, and Terry ( 1 ) found that taka-diastase digestion alone did not suffice to avoid the stimulating effect. Bauernfeind, Sotier, and Boruff (a) reported that stimulatory substances were removed from food materials by extraction with organic solvents and demonstrated that a number of known fat-soluble compounds are c a p able of stimulating the test organism, Lactobacillus cusei. An ether-soluble fraction has also been obtained from blood which stimulates the growth of the organism, and a substance is present in alkali-hydrolyzed liver which inhibits it (7). This paper describes a comparative study of modified procedures proposed for the riboflavin assay, a study of the effect of pure fatty acids, glycerides, and other organic materials on the test organism, and a simple and effective procedure which has been developed in this laboratory for determining riboflavin in cereal products and other materials.
Experimental The microbiological assays were carried out as originally described (IO),except that modifications were made in the preparation of the samples for assay. [A minor alteration in the preparation of the yeast su plement was also introduced-viz., the first filtration after aBding the basic lead acetate was omitted. Instead, the mixture was a t once made alkaline with ammonia, and was then filtered. This procedure is much less laborious. Entirely satisfactory yeast supplements have also been p r e pared from whole autolyzed yeast (Difco Laboratories, Detroit), rather than from yeast extract as originally recommended (IO). These supplements have been used a t a level equivalent to 20 mg. of the whole autolyzed yeast per tube.] PROCEDURE 1, DIRECT. A weighed sample containing approximately 10 micrograms of riboflavin was suspended in 50 cc. of 0.1 N hydrochloric acid, the suspension was autoclaved for 15 minutes at 1 kg. per sq. cm. (15 pounds per sq. inch) pressure,
and cooled, 1 cc. of phosphate buffer (pH 7.0) was added, and the mixture was adjusted to pH 6.6 to 6.8 with N sodium hydroxide. It was then diluted to 100 cc., and aliquots were added to the assay tubes. PROCEDURE 2, FILTRATION. The autoclaved suspension obtained as above was adjusted to H 4.5 (or until a heavy precipitate appeared) with 2.5 N sogum acetate solution, and the volume made to 100 cc. The mixture was filtered by pouring repeatedly through a fluted, KO.40 Whatman filter paper until a clear filtrate resulted. A 50-cc. aliquot of the filtrate was adjusted to pH 6.6 to 6.8, and diluted to 100 cc. for assay. In the case of a few samples it was found advantageous to filter a second time after the pH had been adjusted to 6.6 to 6.8 (cf. Table VIII). PROCEDURE 3, FILTRATION AND ETHER EXTRACTION.This was the same as procedure 2, except that the 50-cc. aliquot of the first filtrate was shaken out three times with 30-cc. portions of ether. Without removing dissolved ether the aqueous phase was adjusted to pH 6.6 to 6.8, and diluted to 100 cc., and aliquots were added ‘directly to the assay tubes. Scow PROCEDURE. This was carried out exactly according to published directions (9). ANDREWPROCEDURE. This also was carried out as published (1).
MATERIALS TESTED.The fat-soluble substances to be tested for their effect on the assay were dissolved in a small amount of ethanol, and the solution was diluted with approximately 20 volumes of water. Aliquots of the suspensions so obtained were added directly to the assay tubes. The small amount of alcohol so introduced had no detectable influence on the bacterial response. The ether-soluble fraction of cornstarch was obtained by continuous extraction of a suspension prepared by autoclaving 20 grams of commercial cornstarch for 15 minutes at 1 kg. per sq. cm. (15 pounds per sq. inch) pressure in 200 cc. of 0.1 N hydrochloric acid. Palmitic acid, stearic acid, and tristearin were Eastman products, and were used as received. The oleic acid used had been purified by low-temperature crystallization from acetone, followed by distillation in vacuo. Iodine value, 89.8; calculated, 89.9. Triolein was prepared from oleic acid purified in this manner, and was subjected to molecular distillation before use. The linoleic acid was a sample which had been fractionated at reduced pressure. The acid was peroxide-free. Iodine value, 179.5; calculated, 181.1. The oleic acid, linoleic acid, and triolein were all preserved by sealing in evacuated ampoules immediately after distillation. The lecithin tested was a sample of commercial soybean lecithin which had been rendered “oil-free” by repeated extraction with acetone in a Waring blendor.
Results
The type of difficulty encountered in applying the original, direct procedure (procedure 1) to a cereal is illustrated in Table I. The drift in the values obtained for whole wheat flour is typical of cereal products in general. The same effect is seen to a greater degree in the case of cornstarch containing a known amount of added riboflavin. It is evident that TABLEI. RIBOFLAVIN CONTENTOF CEREALSAMPLES both stimulation and inhibition of the Volume of Whole Cornstarcha Containing 5 Micrograms per Riboflavin plus bacterial response occur when the direct Suspension Wheat Gram of Added Riboflavin Ether Extract procedure is followed. The stimulating Added Flour Procedure Procedure Scott Andrews of Hydrolyzed per Tube Procedde 1 1 3 procedure procedure Cornstarchb material was evidently not removed by cc . Micrograms per gram Microgram the Scott procedure. However, procedure 3 and the Andrews procedure resulted in satisfactory assay values. The ether-soluble nature of the interfering material is shown by the data in the last column. Separate assays a Cornstarch itself contained less than 0.20 microgram of riboflavin per gram. showed that the stimulation observed b Aqueous suspension containing 6 micrograms of riboflavin and ether-soluble material from 1 gram of cornstarch. in this case was not caused by riboflavin in the ether extract itself.
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Vol. 14, No. 11
INDUSTRIAL AND ENGINEERING CHEMISTRY
110/
z w U
I
055
I
I
1
0.15
0.10
MICROGRAMS RIBOFLAVIN PER
I
I 0.20
TUBE
FIGURE 1. EFFECT OF FAITYACIDSON MICROBIOLOGICAL DETERMINATION OF PURERIBOFLAVIN Upper. acid:
Effect of palmitic acid.
1.
2.
3.
4.
Lower. acid:
Effect of stearic
1.
Ratio of riboflavin t o Dalmitio
1 to 5200 1 to 1040 1 t o 260 1 to 26 acid. Ratio of riboflavin to stearic
3.
1 to 1000 1 to 400 1 to 200
5.
1 to 5
2.
4.
20
to the fat-soluble nature of the interfering material (2,7),the effect of various known fatty substances was studied. Figure8 1 and 2 summarize the results obtained with different mixtures of pure riboflavin with various fatty acids. The effect, as indicated by the deviation of the recovery values from 100 per cent, was found to depend both on the absolute amounts of riboflavin and fatty acid present in the tube, and on bhe ratio between the two. The bacterial growth was affected very markedly at ratios of 1 to 500 or 1 to 1000. At the 0.05-microg~amlevel of riboflavin this corresponds to only 25 to 50 micrograms of the fatty acid per tube. I n general, the widest deviations from 100per cent recovery were observed at the lowest level of riboflavin tested. Oleic and stearic acids markedly stimulated the bacterial response, while palmitic acid and especially linoleic acid acted as potent inhibitors. These results corroborate, in part, the work of Bauernfeind et al. (d), who found that oleic, stearic, and palmitic acids stimulated the test organism, while linoleic acid either stimulated or inhibited, depending on the amount used. The effect of a mixture of two fatty acids was also investigated. The particular mixture tested, which consisted of oleic and linoleic acids in the ratio 1 to 0.8, gave very high recoveries when tested at a level of 3600 parts of the mixture &o one of riboflavin.
I
I
7
FIGURE 2. EFFECT OF F.~TTYACIDSON MICROBIOLOGICAL DETERMINATION OF PURE RIBOFLAVIN Upper. acid:
1 t o 50
In view of the above results and of previous work pointing
I
I
0.10 0.15 0.20 MICROGRAMS RIBOFLAVIN PER TUBE
0.05
Lower.
Effect of linoleic acid. 1. 2. 3. 4. Edect of oleic acid. 1. 2. 3. 4.
Ratio of riboflavin to linoleio
1 to 4000 1 to 800 1 to 400 1 to 40 Ratio of riboflavin to oleic acid: 1 to4800 1 to 960 1 to480 1 to 96
The data in Table I1 show that in contrast to the free fatty acids neutral fats had little disturbing influence on the microbiological determination of riboflavin. Lecithin gave a moderate stimulation a t high levels (Figure 3). Although the pH of the medium (6.6 to 6.8) is such that the fatty acids tested should have been present to only a very small degree in the form of soaps, it was of interest to try the effect of substances capable of lowering the surface tension in neutral solution. The synthetic wetting agents listed in Table I11 proved to be inert a t the concentrations tested. These Aerosols are sodium salts of various alkyl sulfosuccinic acid esters (S,16), In order to determine whether the effect of the unsaturated acids might be attributable to peroxides or other oxidation products, a sample of oleic acid was oxidized by bubbling air through it for 15 hours at room temperature, and then tested. Substantially the same results were secured as with highly purified oleic acid. Benzoyl peroxide at. concentrations of 40 to 800 microgram per tube also was without effect. A comparison of the different procedures as applied to various cereal samples is given in Table IV. It seems certain that the values obtained by the direct method and by the
ANALYTICAL EDITION
November 15, 1942
TABLE 11. EFFECT OF FATS ON RECOVERY OF KNOWN AMOUNTS OF RIBOFLAVIN Riboflavin Added per
Tube
Fat Added per Tube
Micrograms 0.05 0.10 0.15 0.20 0.05 0.10 0.15 0.20 0.05 0.10 0.15 0.20
200 400 600 800 40 80 120 160 4 8 12 16
-Riboflavin Maaola Wesson oil oil
RecoveredTriolein
Tristearin
%
%
%
%
115 103 94 89 121 100 91 82 108 92 97 103
112 104 99 99 106 95 96 98 112 92 98 103
97 96 101 99 99 98 102 101 120 100 100 110
132 104 110 105 99 93 99 98 108 96 100 103
TABLE111. EFFECTOF WETTINGAQENTS ON RECOVERY OF KNOWN AMOUNTSOF RIBOFLAVIN Riboflavin Added per Tube
Wetting Agent Added per Tube
Riboflavin Recovered Aerosol Aerosol Aerosol AY IB MA
MiCrOQTam8
%
%
Scott method were too high, while the other procedures gave reasonably concordant results. I n most cases procedures 2 and 3 gave essentially the same values. The recovery of added riboflavin as determined by the filtration procedure is indicated in Table V, and Table VI shows that entirely consistent results at the different dosage levels were secured by this method. The validity of procedure 2 is further attested by the data in Table VII, which demonstrate that the values so obtained agree very well with results of fluorometric and animal assays on identical samples. These data show definitely that the present method is capable of yielding reliable values for composited human diets (Table VII, sample 1). That it is also applicable to mixed feeds was demonstrated by analysis of 12 individual ingredients of a chick ration. The riboflavin content of the ration as calculated from the individual analyses was 4.6 micrograms per gram, while the average of four determinations according to procedure 2 nas 5.0 microgram per gram (Table
Iv).
The data in Table VI11 indicate that on certain types of material the original direct method yields reliable values, while many others must be handled by the modified procedure.
Discussion
911
stance which stimulates an organism a t one concentration may prove inhibitory or even toxic at somewhat higher concentrations. The fact that a ratio of 960 parts of oleic acid to one of riboflavin gave a greater stimulation than either smaller or larger amounts (Figure 2, lower) is probably to be explained on this basis. The only other reports known to the authors in which oleic acid has been shown to influence bacterial growth are those of Bauernfeind et al. with Lactobacillus casei (2) and of Mueller with the diphtheria bacillus (4). I n the latter case it was also found that there wzts an optimum concentration of the acid, and the effective quantities were of the same order of magnitude as those found to be active toward Lactobacillus casei. Further evidence that the drift is attributable to :atty acids was secured in carrying out the experiments summarized in Figures 1 to 3. I n working with these mixtures, which contained only pure chemicals and were entirely free from “solids”, typical drifts similar to those encountered in actual assays were very frequently observed. Since starch yields about 0.6 per cent of fatty acids on hydrolysis (6), cereal samples in the amounts required for the determination would supply such acids in sufficient quantities to account for the observed effects. Likewise the stimulatory and inhibitory substances found in blood and in alkaline hydrolyzed liver (7), although not yet identified, could well be oleic acid and linoleic acid, respectively. From the information now available it would seem that the preparation of cereals and certain other samples for microbiological riboflavin assay should consist of two main steps, (1) a hydrolysis, and (2) a procedure designed to remove fat-
TABLE IV.
DETERMINATION OF RIBOFLAVIN IN CEREALPRODUCTS BY VARIOUSPROCEDURES
Sample
20
1
Procedure Used 3 Scott
Andrews
Micrograms of riboflavin per gram Whole wheat flour Whole wheat bread Chick ration Wheat bran Farm feed 1 Farm feed 2 0
2.5 2.7 6.7 6.0 6.5 5.8
1.3 1.8 5.0 3.6 3.2 3.2
1.9 4.6 3.3 2.9 3.2
1.2 3.3 7.8
0.90 1.6 4.3
6.1 5.8
3.6 3,s
Average of a t least three assays.
TABLE V. RIBOFLAVIN RECOVERED BY PROCEDURE 2 Sample Chick ration Wheat bran Whole wheat flour Whole wheat bread Farm feed 1 Alfalfa meal Dried whey Fish meal Meat s c r m s
Riboflavin Riboflavin Content Added 5.0 3.6 1.2 1.9 3.2 10 25 6.8 4.3
Riboflavin Found Recovery
Micrograms p e r gram 5.0 10.1 2.5 1.0 2.0 2.5 10 20 6.7 5.0
5.9 2.3 3.9 5.4 20 45 12.5 9.2
% 102 92 110 100 88
I n the light of the results reported in the present paper inn ~ . . 100 and in previous publications, it is probable that the “drift” 85 98 frequently encountered in the microbiological determination of riboflavin is caused mainly by free fatty acids in TABLEVI. COMPARISON OF INDIVIDUAL ASSAY DATA FOR the suspensions being tested. This CEREAL SAMPLES ASSAYED BY PROCEDURES 1 AND 2 view is consistent with the fact that vo+e or the Lextracted solids from some types SuspenChick Ration Wheat Bran Whole Wheat Bread Farm Feed 1 Of cause effects in % ;d Procedure Procedure Procedure Procedure Procedure Procedure Procedure Procedure the assay (g), while other solids, such per Tube 1 2 1 2 1 2 1 2 c c. Micrograms of riboflavin per gram as pape; pulp, ground glass, and the 1 8.8 5.2 8.1 3.9 3.1 2.2 7.1 ... like, areinert ( 1 , d , IO). 9.1 5.3 1 7.3 4.2 3.1 2.0 8.0 ... The stimulation and inhibition which 2 6.1 5.0 6.1 4.0 2.6 2.0 6.7 3.3 2 6.0 5.2 6.6 3.6 2.6 1.8 6.5 3.4 operate to produce the observed drift 3 5.3 5.0 5.6 3.5 2.3 2.0 5.9 2.8 3 5.2 4.9 5.9 3.5 2.2 1.9 5.6 3.4 may obviously be caused by one and ... 1.8 4.8 2.8 4 4.8 5.1 5.1 3.5 4 4.8 4.8 4.1 3.6 ... 1.8 5.3 3.3 the same substance (cf. Figures 1 to 3). It is common knowledge that a sub-
2
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INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 14, No. 11
soluble interfering substances. The TABLEVII. RIBOFLAVIN CONTENTOF VARIOUS MATERIALS hydrolysis should be sufficiently drastic (Determined by the microbiological, fluorometric, and rat assay methods) to break up various combined forms of Riboflavin riboflavin and thus reduce the problem No. Sample Description Analyst Method of Assay Content to the determination of a single comMicrograms/g. pound. Although the bacterial method 3licrobidogica1, procedure 2 Fresh mixed diet A 4.35 IIicrobinlogical, procedure 2 4.7 A Dried mixed diet has been shown to measure the riboB \IicroSiological, procedure 2 4.1 C Microbiological, procedure 2 4.9 flavin contained in at least some of these Fluorometric C 4.8 combined forms (6),others may exist Fluorometric D 4.2 R a t growth E 4.8 which are not so measured. FurtherMicrobiological, procedure 2 A 17 Rumen soluble8 Fluorometric C 17 more, riboflavin itself is less likely to AIicrobiolngical, procedure 2 A Whole wheat flour 1.3 be lost in subsequent precipitations, C Fluorometric 1.1 hIicrobiologica1, procedure 2 Whole wheat bread 1.8 than, for example, riboflavin phosFluorometric !i 1.5 A Microbiological, procedure 2 62 Dried yeast phate, which has been shown to have Fluorometric C 59 a greater affinity for at least one proa Calculated on dry basis. tein than does free riboflavin (8), and may well behave similarly toward others. TABLEVIII. RIBOFLAVIN CONTENT OF VARIOUS MATERIALS The removal of interfering substances (Determined by original and modified procedures) from the hydrolyzate can be accomRiboflavin Content Riboflavin Content plished successfully in most cases by Procedure Procedure Procedure Procedure a careful filtration. Ether extraction Sample 1 2 Sample 1 2 Micrograms per gram Micrograms p4r gram has been found equally effective, but 6.0" 3.6 0.25 0.3 Wheat bran Grapefruit juice is usually complicated by emulsion Dried whey 6.6 6.6 24 25 American cheese formation if much solid matter is presB. Y. feed 66 65 Whole milk 2.10 1.7 Whole oats 3.70 1.7 ent. I n any event it is time-conTomato juice 0.40 0.3 Meat scraps 5.75 4.3) Hamburg 3.95 1.3 Fish meal 7.85 6.8b suming in routine work, and has been Ice cream 7.7 2.0 Soy bean meal 6.80 3.3 used as an adjunct to filtratJion in Yellow corn 2.25 1.3 Corn gluten 3.4. 1.7 meal only a few cases where the filtration Whole barley 2 . 9 ' 1.6 Dehydrated 11. 10 5.7" 3.1 alfalfa meal Wheat gray shorts alone proved inadequate. Extraction of the original material with a fat sola Significant drift in individual values observed in these assays. vent could obviously not be substib A second filtration was employed after neutralization of extracts. tuted for the above precautions on account of the formation of additional free fatty acids on hydrolysis. Such ment with the results of independent assays by other methods extraction is desirable, however, in the case of high-fat on the same samples. materials such as cheese, fish meal, and the like. The stimulations and inhibitions studied in this paper tended to be greatest at the 0 05-microgram level of riboflavin. This observation emphasizes the desirability of calculating the riboflavin content of unknowns from tubes for which the titration values fall above this level on the standard curve. It has been the authors' experience that results from tubes containing less than 0.05 microgram of riboflavin are practically worthless, and it is the practice in this laboratory to exclude all such values from the calculation of the final assay results. A question raised by the present paper concerns the accuracy of riboflavin values previously obtained by the original microbiological method. The agreement which has been obsgrved repeatedly between such values and those secured in other ways, together with the direct comparisons shown in MICROGRAMS RIBOFLAVIN PER T U8E Table VIII, makes it appear probable that on many samplt>s, FIGURE 3. E F F E C OF ~ LECITHINON MICROBIOLOQICAL especially those of relatively high potency, the older values ESTIMATION OF PURE RIBOFLAVIN are substantially correct. Results previously obtained on Ratio of riboflavin t o lecithin: cereals, mixed feeds, composited diets, fish meals, and certain 1. 1 to 8000 other materials by the direct method are very likely too high. 2. 1 t o 800 In the future such materials should be assayed by a suitably 3. 1 t o 80 modified procedure. The authors are unable a t present to offer any procedure for determining riboflavin in blood which is an improvement over the method previously suggested (IS). In view of the above considerations, procedure 3 is recomThe possibility of avoiding interference from fatty m a t e mended as being the most generally applicable method, while rials by incorporating them in the basal medium has been procedure 2 is regarded as being suitable for most materials, considered, but this procedure appears inadvisable. I n the notably cereals and cereal products. The validity of these first place, the effects of various fatty acids differ not only in procedures is supported by (a) lower and more reasonable degree but in kind (oleic vs. linoleic), so that it would probresults on mixed feeds and composited diet samples, which ably be difficult to compensate for both at the same time. by the direct procedure have often appeared to contain twice Secondly, it has been the authors' experience that it is difas much riboflavin, or more, than the sum of the constituents; ficult to reproduce closely the effect of a given fatty acid. ( b ) the absence of drift at different dosage levels; (c) satisThis may be attributable to variations in the physical statefactory recovery of added riboflavin; and (d) good agree-
ANALYTICAL EDITION
November 15, 1942
e. g., degree of subdivision-of the suspensions tested. Finally, the fundamentally different solubility characteristics of the fatty acids and the B-vitamins offer a ready means of separation. Interference of the type here described has also been encountered, and often to an even greater degree ( 7 ) , in the microbiological determination of pantothenic acid (12). Similar preliminary treatment of the samples appears to offer a satisfactory solution (15). The nicotinic acid assay (11), on the other hand, is but little disturbed by fatty acids (14).
Summary A study has been made of the substances present in cereals and other biological materials which interfere with the determination of riboflavin by the microbiological method. It has been shown that the interference is probably due to small amounts of free fatty acids, and methods for avoiding their effect have been investigated. A procedure for the preliminary treatment of the sample has been worked out. The interfering substances are thereby removed, and reliable values for riboflavin are obtained on such materials as cereals and mixed diets which previously have been difficult to assay by the microbiological method.
Acknowledgment The authors wish to express their appreciation to F. W. Quackenbush for samples of oleic acid, linoleic acid, triolein, and lecithin; to the American Cyanamid and Chemical
913
Corporation for samples of the Aerosols AY, IB, and MA, and to John S. Andrews, Howard J. Cannon, and Miss Jean Collard for carrying out several of the analyses reported in this paper.
Literature Cited (1) Andrews, J. S., Boyd, H . M., and Terry, D. E., IND.ENQ. CHEM.,ANAL.ED., 14,271 (1942). (2) Bauernfeind, J. C., Sotier, A. L., and Boruff, C. S., Ibid., 14, 666 (1942). (3) Caryl, C. R., IND. ENG.CHEM.,33,731 (1941). (4) Cohen, S., Snyder, J. C., and Mueller, J. H., J. Bad., 41, 581 (1941). ( 5 ) Evans, J. W., and Briggs, D. R., Cereal Chem., 18,443 (1941). (6) Feeney, R. E., and Strong, F. M., J. Bid. Chem., 133, xxxi (1940). (7) Ibid., 142, 961 (1942). (8) Kuhn, R., and Rudy, H., Ber., 69,2557 (1936). (9) Scott, M. L., Randall, F. E., and Hessel, F. H., J. B i d . Chem., 141,325 (1941). (IO) SneI1, E. E., and Strong, F. M., IND. ENQ.CHEM.,ANAL.ED., 11, 346 (1939). (11) Snell, E.E., and Wright, L. D., J. Bid. Chem., 139,675 (1941). (12) Strong, F. M., Feeney, R . E., and Earle, Ann., IXD. ENQ.CHEY., ANAL.ED., 13,566 (1941). (13) Strong, F. M., Feeney, R . E., Moore, Barbara, and Parsons. H.T., J . B i d . Chem., 137,363 (1941). (14) Strong, F. M., and Krehl, W. A., unpublished work. (15) Strong, F. M., and Neal, A. L., unpublished work. (16) Van Antwerpen, F. J., IND. ENQ.CHEM.,33,16 (1941). PRESENTEDbefore the Division of Biological Chemistry, Joint Program on Vitamins, a t the 103rd Meeting of the AXERICAN CHEMICAL SOCIETY, Memphis, Tenn. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported by a grant from t h e Difco Laboratories, Detroit, Mich.
Systematic Identification of the Common Metallic Coatings HOWARD NECHAMKIN AND ALVIN SANDERS Chemical Division, Bureau of Standards, R. H. RIacy & Co., Inc., New York, N. Y.
T
HE testing of an unknown metallic finish for identifica-
tion of the metal which has been used for plating has not, t o the authors’ knowledge, been systematized. The series of reactions developed by the authors is designed to eliminate guesswork and to provide a simple and logical procedure for establishing the identity of the metallic coating. The method applies to the following commonly used metals: chromium, tin, nickel, gold, silver, rhodium, zinc, cadmium, copper, aluminum, and lead. This procedure is particularly adapted to a laboratory which is engaged in the testing of consumer articles where merchandise like umbrella handles, cooking utensils, fountain pens and pencils, novelty jewelry, hardware, metallic toys, trays for various purposes, etc., is submitted for the purpose of identifying the metallic coating. Before testing is begun, any lacquer which may cover the surface of the metal must be removed, using a mixture of equal parts of ethanol and acetone on a piece of absorbent cotton. The cleaned area is then subjected to the series of spot tests described in this article. Each reagent is added on a separate spot, and no reactions are carried through beyond one step unless specifically directed. Because never more than one drop of reagent is added, it is recommended that the reagents be kept in a set of GO-ml. dropping bottles for convenience. I n the special cases where a spot on a filter paper must be rendered ammoniacal, the paper is held over the
mouth of a bottle of concentrated ammonium hydroxide solution for about 30 seconds. Sometimes, the reactions of the reagents with the metal surfaces are extremely slow. I n these cases it is advisable to roughen the surface by rubbing very lightly with fine emery cloth (No. 000) or sandpaper (No. 00) before adding the reagent, taking care to keep away from the base metal, since that may greatly interfere with the proper interpretation of the results. The authors have found the use of a small magnifying glass very helpful for observing reactions. I n Table I and outline, the term “colored” is used for metals which do not have the familiar “silvery” appearance of aluminum, lead, etc.
Reagents Nitric acid, equal parts of 37 per cent nitric acid and distilled water. Hydrochloric acid, 35 per cent (concentrated). Ammonium hydroxide, 28 per cent ammonia (concentrated), Sodium sulfide, 10 per cent aqueous solution. Cacotheline. Dimethylglyoxime, 0.6 gram in 50 ml. of 95 per cent ethanol. Sodium hydroxide, 10 per cent aqueous solution.
Procedure I. Colored plating. Add one drop of nitric acid. A. A blue solution is obtained: Copper, brass, or other alloys of cop er B. Action is Zlayed, or no reaction occurs: Gold
