24
ANALYTICAL EDITION
Vol. 3. No. 1
Determination of Copper in Dairy Products'*' H. T. Gebhardt and H. H. Sommer DEPARTMENT OB DAIRYHUSBANDRY, UNIVERSITY OF WISCONSIN, MADISON, WIS.
A new method for the determination of copper in biological materials is more N THE past, copper has foodstuffs and biological material has been devised. recent. It is based on the been considered toxic or It was found that the method of Elvehjem and Lindow color reaction given by copper undesirable in foods, but could be shortened considerably, the separation of with potassium thiocyanate today it is generally known copper sulfide improved, and the danger of losses in and pyridine, which is very that copper is an essential transfers minimized, by shaking out the precipitated specific, and the color in the constituent of most plant and copper sulfide with chloroform instead of separating it chloroform solution is proporanimal material, and that i t by filtering. It was found that the ashing temperature tional to the a m o u n t of plays an important role in life and time used may be the source of large errors in copcopper present. processes. This has been defiper determinations. The ashing temperature should nitely shown by the recent Observations Leading to not exceed 565" C. for 3 to 4 hours. work by Hart and eo-workers Modified Method Detailed directions for the procedure, embodying (6) on the function of minute precautions as to ashing conditions, and the modificaThe method of Elvehjem amounts of copper in foods in tion mentioned, are given. The detailed method as and Lindow was tentatively the cure of nutritional anemia. outlined gives results that agree closely with those selected mainly because the This recent work has opened obtained by the original Elvehjem-Lindow method, reagents were available. an important field for further when the latter is used with the necessary precautions The accuracy of themethod studies of the functions of this as to ashing conditions. was tested by making a large element in life processes, and number of recovery determiits distribution in living manations; but the results were disappointing. Not more than 55 terial. However, in the manufacture of many foodstuffs, it is to 70 per cent of the added copper was recovered, even when still desirable to keep the copper content a t a minimum for the directions of the method were followed in detail. To find the cause of the losses of copper during the protechnical, if not legal, reasons. This is especially true in the case of dairy products, where the detrimental effect cedure, efforts were made to induce more complete and of copper has long been known, but not fully appreciated rapid flocculation of the copper sulfide, but no improvements until the recent work of Guthrie (4). A method for deter- in the percentage of recovery were attained. Removing the mining the copper content of foodstuffs is therefore of general iron and phosphate in the manner of Supplee and Bellis (addition of an excess of ammonium hydroxide to the ash and current interest. The method given in this paper was developed in connec- solution and filtering) and making the colorimetric determination with experimental work to determine the solubility tion directly on the concentrated filtrate did not give the deof copper in milk. However, the details evolved are appli- sired recovery, thus indicating that copper was lost a t some cable to the analysis of other foodstuffs and biological mate- other step in the procedure. When the copper was added to the milk ash solution, rerials. The method as given is a modification of the Elvehjem-Lindow (3) method for the determination of copper in covery .was greatly improved. This finding, together with biological materials, which in turn was based on the method of the observation that any attempt to obtain a whiter ash Biazzo (8) for the copper determination in canned vegetables. by using higher ashing temperatures and longer times was followed by lower results, called for improvement of the Methods Previously Used ashing procedure. I n further determinations the ashing temperature was Recent work on the distribution of copper in foodstuffs and biological materials has become possible only through the confined to a range of between 510" and 565' C. Using this development of the colorimetric methods which permit the temperature, ashing was completed in about 3 to 4 hours. estimation of minute amounts of copper in relatively small Under these conditions results given in Table I were obtained. samples. Previous to this, electrolytic methods had been Table I-Copper D e t e r m i n a t i o n s in S k i m Milk w i t h a n d w i t h o u t Added Copper Using Ashing Temperature of 510° to 5 6 5 O C. used most frequently, but for samples containing mere traces of copper these methods were unsatisfactory because of the COLORIMETER ADDED copREADINGS COPPER COPPER large samples required to obtain a weighable deposit, and yL;INDICATED RecovPEB ADDED Standarda Unknown BRED because of the other ash constituents which freqclently interfered with the copper deposition. The colorimetric methods which have been developed 12.27 0.041 91.0 1 250 0.0 5.00 12.88 0.039 5.00 250 0.0 to overcome these difficulties have been used in analyses to 9.98 0.130 13.00 250 0.lb determine the copper content of raw milk and the amount 9.87 0.132 13.00 250 O.1b 2.90 10.60 0.028(0.0235)d 96.656 2 250 0.0 of copper dissolved by milk from dairy equipment. The 3.45 11.63 0.030 (0.0262)d 250 0.0 potassium ethylxanthate method was adapted to the deter10.00 8.27 0.121 210 0.1C mination of copper in milk by Supplee and Bellis (9) and this a Color standard 0.1 mg. copper in 5 cc. chloroform. b Copper added to ash solution. adaptation was used by King and Etael (6), Quam and coc Copper added to skim milk before ashing. workers (7), and, in combination with the electrolytic method, d Figured to basis of 210-cc. milk sample. by Rice and Miscall (8). The colorimetric method developed The addition of 0.1 mg. copper to the ash solution of by Elvehjem and Lindow for the determination of copper in sample 1 increased the result of the analysis from 0.040 * Received March 28,1930;revised paper received September 24,1930. to 0.131 mg., or an increase of 0.091 mg. This represents a 9 Published with the permission of the Director of the Wisconsin Agrirecovery of the added copper of 91 per cent. In sample 2 cultural Experiment Station.
I
INDUSTRIAL AND ENGINEEIZING CHEMISTRY
January 15,1931
the addition of 0.1 mg. copper to the skim milk before ashing increased the result of the analysis from 0.02435 to 0.1210 mg. copper, or an increase of 0.09665 mg. This represents a recovery of added copper of 96.65 per cent. Theseresults indicate that the ashing procedure used had not caused the loss of any copper. The possibility of losing copper in ashing and the suitability of a low ashing temperature is further illustrated in Table 11. Table 11-Effect WHOLE COPMILK^ PER USED ADDED
CG.
Mg.
250 250 250 250
0.0 0.1 0.0
0.1
of Ashing Conditions on Copper Determinations ASH-
ASH-
ING
ING
COLORIMETER copREADINGS PER
TEMP. TIME Standardb C. 700-840 700-840
Hrs.
I
Mm.
Unknown
Mm.
rlDnED
COPPER
IND1-
I
CATED
BRED
Mg.
%
510
510
The same milk was used throughout. b The color standard contained 0.1 mg. copper in 5 cc. chloroform. a
From the results in this table it is clear that a large fraction of the copper may be lost in ashing due to high ashing temperature or excessive time. The low percentage recovery of added copper, even where an ashing temperature of 510" C. was used, is due to the prolonged ashing, the results differing from those of Table I where the ashing time was 3 to 4 hours. It is probable that part of this loss is caused by the copper fusing with the silica dishes that were used, and part to volatilization. With the importance of the ashing temperature and time recognized and carefully controlled, it was now decided to compare the original method of Elvehjem and Lindow with the modified form of this method, the modification consisting, as already stated, in the removal of the interfering iron and phosphate in the manner of Supplee and Bellis. The comparison of the results obtained by the two methods showed good agreement in a reasonably large number of experiments. On the average the original Elvehjem-Lindow method gave slightly higher results. I n the study to which this work on methods was incidental, both methods have been used, and both served quite well for the routine determinations, provided the precautions a5 to ashing temperature and time were observed. However, both methods require considerable time. The desire to shorten the time b l l y led to the use of chloroform to shake out the copper sulfide precipitated from the ash solution instead of allowing it to stand to flocculate, and then filtering. Chloroform has been used for removing the colloidal copper sulfide to facilitate the reading of the end point in the volumetric titration of copper with ammonium sulfide (1). It was found that this procedure could be applied advantageously instead of filtering for the removal of the copper sulfide precipitate from the milk ash solution. A considerable saving of time is effected and, as will be shown, there is no sacrifice in accuracy, Method for Determination of Copper in Milk
A 250-cc. portion of the sample is carefully evaporated over a low flame in a quartz evaporating dish. The addition of 10 to 15 drops of glacial acetic acid prevents film formation and foaming, and thus hastens evaporation. After evaporation of most of the moisture, the drying is completed in an oven a t 150" to 200" C. When the sample has been thoroughly dried, as indicated by the caramelized or even charred appearance, it is transferred to an electric ashing furnace, the temperature of which should not exceed 565" C. The use of a pyrometer so that the ashing may be conducted a t a known temperature is advisable. At this temperature ashing is usually complete in 3 to 4 hours, and a grayish white ash is obtained.
25
The ash is moistened with 4 cc. of dilute hydrochloric acid (1 to l), and diluted to about 100 cc. with distilled water. To bring about complete solution, the solution in the quartz evaporating dish is heated and kept near the boiling point for about 20 minutes. It is then filtered, the dish and filter carefully washed with distilled water, and the filtrate and washings collected. The undestroyed carbon together with the filter paper is returned to the quartz dish and ashed for 30 minutes; the ash is dissolved in a few drops of dilute hydrochloric acid, diluted with distilled water, and warmed, filtered, and washed as before, the filtrate and washings being added to the main ash solution. The total ash solution, or an aliquot thereof, if the copper content is expected to be high, is evaporated to about 10 cc. and transferred to a 25-cc. volumetric flask. Complete transfer is aided by adding a drop of dilute hydrochloric acid (1 to 1) to the first rinsing, and by heating this solution to boiling. After cooling to about room temperature, hydrogen sulfide is passed into the solution in the volumetric flask for 5 minutes through a capillary glass tube (a 4- or 5-mm. glass tube drawn out to a capillary of about 1 mm. outside diameter). The capillary should not be immersed in the solution until the hydrogen sulfide has been turned on, to prevent the solution from rising in the capillary; for the same reason it is advisable to remove the capillary from the solution before shutting off the hydrogen sulfide. The outside of the capillary is rinsed with 0.5 cc. of saturated hydrogen sulfide water. The flask is stoppered and set in cold water or otherwise cooled to about 10" C. To this cooled solution containing the precipitated copper sulfide, 5 cc. of chloroform, which has previously been cooled to about 10" C., are added, the flask is securely stoppered and shaken vigorously for about 1 minute. The flask is then set in cold water and the chloroform allowed to settle out. The water layer becomes perfectly clear, and the copper sulfide is now contained in the chloroform a t the bottom of the flask. The water solution is drawn off by means of a capillary tube, leaving enough to avoid the possibility of drawing out some of the chloroform, The remainder is diluted with 15 to 20 cc. of cold, saturated hydrogen sulfide water, and this is again drawn out as completely as possible. This one washing has been found sufficient in all cases; it can be omitted entirely if the aliquot does not represent more than the ash of 50 cc. of milk. The small amount of phosphate which is left in the solution in this case dissolves completely in the acetic acid added later in the colorimetric determination. Gas bubbles may make the removing of the water portion difficult if the temperature during the operations is too high. It is desirable to limit the volume of the ash solution to 10 cc. as directed above, so that after the addition of the chloroform there is ample space for thorough shaking. The capillary tube which is used to draw out the water solution should not touch the side of the neck, because a fine film of chloroform containing copper sulfide usually covers the glass. After the copper sulfide is shaken out wit'h chloroform, the water solution drawn off, and the residue rinsed as directed, the chloroform is evaporated by placing the flask in a hot water bath for 10 minutes, increasing the temperature to boiling towards the end. The residue in the flask is dissolved by adding 1 cc. of concentrated nitric acid, placing the flask in the hot water bath for 20 minutes with occasional shaking to bring the acid in contact with the entire inside surface of the flask. The solution is then neutralized with dilute sodium hydroxide (200 grams in 1 liter of solution) so that the reaction is just alkaline phenolphthalein. The neck of the flask must be rinsed carefully to remove every trace of nitric acid (also oxides of nitrogen) which would interfere with the color reaction later. The indicator should
~
ANALYTICAL EDITION
26
not be added too soon, because the compound formed by the destruction of phenolphthalein by strong acid or alkali dissolves in chloroform with a yellow color, and thus interferes with the colorimetric determination even if present only in traces. I n the above neutralization, the indicator may be added after the first permanent turbidity develops due to the precipitation of traces of phosphate remaining in the flask. When this point has been reached, one drop of dilute sodium hydroxide solution then usually suffices t o make the solution just alkaline to phenolphthalein. From this point in the procedure the colorimetric determination as given by Elvehjem and Lindow is followed. To the neutralized solution in the flask, containing the dissolved copper sulfide, 1 cc. of glacial acetic acid, 1 CC. of a 10 per cent solution of potassium thiocyanate, and 10 drops of pyridine are added in the order given, with slight shaking after each addition. Finally 5 cc. of chloroform, accurately measured, are added, and the volume made up to the mark with distilled water, After thorough shaking, the chloroform has taken up the green copper-thiocyanate-pyridine compound. The water portion is then removed by drawing off with a capillary tube, and the chloroform solution is used for the colorimetric comparison with a standa,rd in a suitable colorimeter. T a b l e 111-Results O b t a i n e d by New M e t h o d a n d Elvehjem-Lindow M e t h o d Using Ashing T e m p e r a t u r e s of 510'' to 565O C. a n d Ashing T i m e s of 3 t o 4 Hours
-
ELVEHJEM-LINDOW
METHOD(A)
SAMPLE
4liouot taken Copof per ash detd. soh. m of 250 alicc. quot milk
Mg.
.
Mdl. 0.864 4.016 3.616 0.500 0.480
6"
0.25
0.432
7'"
0.25
0.832
8a
0.25
0.520
10
0.25
11
0.25
0.656 0.304 0.096
1.536
12
0.40
0.070 0.700
13 14 15 16
0.25 0.25 0.016 0.02
0.094 1.504 0.073 1.168 0.051 12.750 0.047 9.400
-
copper
DEVIATION OB B PROM A
Copper detd. in aliquot
Copper
Mg.
Mg./l.
milk
0.25 0.25 0.25 0.40 0.20
0.25
NEW
METHOD (33)
\Aliquot taken Copof per ash detd. Copsoh. in per of 250 ali-
1 2 3 4 5a
9
I
Mg. 0.054 0.251 '/4 0.263 '/a 0.053 2/6 0.049 '/a 0,052 "5 0.055 l/4 0.056 1/4 0,103 l/4 0.105 114 0,067 l/q 0.078 '/4 0.043 l/4 0,044 '/a 0.020 1/4 0.021 0.103 l/4 0.110 1/4 0.072 2/s 0.036 1/6 0.104 1/4 0.066 1/4 0.047 2/m I/EQ 0.050 l/4
MgJl 0.864 4.016 4,208 0.530 0.490 0.520 0,440 0.448 0.824 0,848 0.536 0.624 0.688 0.708 0.320 0.336 1.648 1.760 0.720 0.720 1.664 1.056 11,750 10,000
0.000 0.000 +0,0370 +0.0030 $0.0025
0.000 0.000 0.592 0.030 0.025
+0.0015
0.012
+0.0005
0,004
+0.0075
0.060
$0 0025 0.040
Vol. 3, No. 1
pound dissolved in accordance with the amount of stock solution used. I n working at low room temperatures, the colorimetric comparisons are frequently seriously influenced by the tendency of the standard to become turbid. Slight warming will cause the standard to become clear, but on cooling it again becomes cloudy. This difficulty may be overcome entirely by adding 1 cc. of concentrated nitric acid in making up the standard; the solution is then neutralized to phenolphthalein with dilute sodium hydroxide before adding the glacial acetic acid, potassium thiocyanate and pyridine, and shaking out with chloroform. Comparison of New Method with Elvehjem-Lindow
Method
Before the method, as outlined above, was finally adopted for the routine analyses in the problem under investigation, the copper content of a number of samples of milk, contaminated by varying amounts of copper, was determined by both methods. These results are given in Table 111. When the results given in this table are compared, it it must be considered that minute amounts of copper are being dealt with and that the final determinations are by colorimetric comparisons. I n view of this, it may be concluded, first, that the results obtained by the two methods agree closely, and, second, that the results obtained by method B are slightly higher in the majority of cases and in the average. This slightly higher result by method B must be expected because there is obviously less opportunity for losses by transfers in method B, and because it was observed that the chloroform removed additional copper sulfide from the filtrate of the copper sulfide precipitation as used in method A. From the above, and from results of recovery determinations made with the new method, and as shown in Table IV, it is apparent that the modifications introduced in the new method do not impair the accuracy of the original method. The advantages of the new method appear to be the saving of considerable time, reduced danger of contamination and of losses in transfers, and more complete separation of the copper sulfide. T a b l e IV-Copper D e t e r m i n a t i o n s in Milk w i t h a n d w i t h o u t Added Copper Using Ashing T e m p e r a t u r e s of 510° to 565O C.
+O 0015 0.024 $0,0110
0.168
$0.0020
0.020
+0.0100 -0,0070 0,0040 +0.0030
0.160 0.120 1.000 0.600
-
500 cc. milk had been used for ashing. b Sample was turbid and colorimetric readings less exact. This accounts for wide discrepancy in results. With amounts of copper as in Samples 2 and 3, turbidity is likely to develop in 6-cc. chloroform solution. Exact point a t which this occurs varies with temperature and salts present.
SAM-MILK PLE ASHED
'
L:$ED TO
READINGS
cc. 1 2 3
250 250 250 260 250 250 250 250 250 250
Mg. 0.00 0.10 0.10 0.00 0.15 0.20 0.00 0.10 0.10 0.05
per
mg
Mg.
Mm. 10.0 10.0 10.0
0.05 0.10 0.10 0.10
COPPER
Standard
MILK
0
The standard for comparison is made from a stock solution made by dissolving 0.3928 gram of pure crystalline copper sulfate, CuSOc5H~0,in distilled water and diluting to 1 liter. This stock solution contains 0.1 mg. of copper in each cubic centimeter. I n making up the standard for comparison, enough of this stock solution should be used so that the standard contains approximately the same amount of copper as the unknown. If the copper content of the unknown is from 0.05 to 0.15 mg., take 1 cc. of the stock solution, add 1 cc. of glacial acetic acid, 1 cc. of 10 per cent potassium thiocyanate solution, and 10 drops of pyridine, and proceed as already described for the unknown sample. The standard for comparison will finally consist of 5 cc. chloroform with an amount of copper-thiocyanate-pyridine com-
COLORIMETER
COP-
INDI-
1; :*
12.22' 7.34
0.03
CATED
ADDED COPPER
RECOVERED
0.0409 0.0143 0.1487 0.2062 0.0264 0.1177 0,1195 0.0758
89.6 96.0 91.3 93.1 98.8
Literature Cited (1) Beckhurts, H., "Die Methoden der Massanalyse," p. 875, Vieweg, Braunschweig, 1913. (2) Biazzo, R., Ann. chim. applicala, 16, 2 (1926). (3) Elvehjem, C. A., and Lindow, C. W., J . B i d . Chem., 81,435 (1929). (4) Guthrie, E. S., unpublished paper. (5) Hart, E. B., Steenbock, H., Wadell, J., and Elvehjem, C. A., J. B i d . Chem., 77, 797 (1928). (6) King, C. G., and Etzel, G., IND.ENO.CHEM.,19, 1004 (1927). (7) Quam, G. N., Soloman, E. I., and Hellwig, A,, Ibid., 20, 930 (1928). (8) Rice, F. E,, and Miscall, J., J. Dairy Sci., 6, 261 (1923). (9) Supplee, G. C., and Bellis, J., I b i d . , 6, 455 (1922).
