V O L U M E 22, NO. 9, S E P T E M B E R 1950 sulfide, and thiophene offered no interference. When a mercaptan was added, the sulfur was consumed in forming a disulfide. The rate of consumption of the free sulfur by a mercaptan can' be followed with the polarograph. HydTogen sulfide, if present, will be removed upon bubbling.
1139 The materials of higher molecular weight appear to retain more sulfur. This is probably a kolubility effect rather than a variation in plant process. The method should be applicable to aqueous solutions as well as to hydrocarbons. LITERATURE CITED
Table 111. Elemental Sulfur Content of Gasoline Fractions and Kerosene after Doctor Sweetening (Determined by polarographic procedure) Sample s, P.P.M. 7 Light crude naphtha 10 Heavy crude naphtha Light cracked naphth;8 , high octane 4 101 High sulfur refined oil (kerosene)
APPLICATIONS
The polarographic method for elemental sulfur has been applied in connection with studies of different gasoline sweetening processes. Table 111 shows the concentration of sulfur found in various gasolines and kerosene after doctor sweetening.
(1 ) .\m. ?oc. Testing Materials, "Specification for Petroleum Spirits (Mineral Spirits)," D 235-39. (2) .Zm. SOC. Testing Materials, "Specifications for Stoddard Solvent," D 454-40. (3) Ani. 50c. Testing Materials, "Standard Method of Sampling and Testing Lacquer Solvents a n d Diluents," D 268-44. (4) Am. Soc. Testing Materials, "Standard Method of Test for Detection of Free Sulfur a n d Corrosive Sulfur Compounds in Gasoline." D 130-30. ( 5 ) Ball. ,J. Y., U. S.Bur. Mines, R e p t . Itmest. 3591 (1941). ( 6 ) Kolthoff. I. M., and Lingane, J. J., "Polarography," rev. reprint, p. 144, New York, Interscience Publishers, 1946. (7) Mapstone, G. E., IND. ENG.CHEM.,.-INAL. ED..18, 498-9 (1946). CHEM., . (8) Morris, H. E., Lacombe, R. E., and Lane, Vi. H., A N . ~ L 20, 1037-9 (1948). (9) Proske, G.. .4q7ew. Chem., A59, 121-2 (1947). (10) Wirth, C.. 111. and Strong, J. R., IND. ENG.CHEH.,ANAL.ED., 8, 344-6 (1936).
RECEIVED .4pril 20, 1950.
Copper Contamination and Ascorbic Acid loss in Waring Blendor MERTON P. LAXIDEN College of M e d i c i n e , University of V e r m o n t , B u r l i n g t o n , V t .
The Waring Blendor, which has extensive usage in food analysis, vitamin assays, and biochemical preparations, is regarded with disfavor by some workers. Loss in ascorbic acid and loss in enzyme activity during blending have been cited. Adverse results may arise from use of containers having worn chrome plating on their blending assemtiies. Copper dissolving from exposed brass parts may catalyze the loss of ascorbic acid in solutions' during blending or create false high results in the determination of the copper content of certain foodstuffs. Inhibition of the destructive effect of copper dissolved from the blending assembly on ascorbic acid is discussed.
T
HE Waring Blendor is an exceedingly popular and useful tool
for comminuting or homogenizing material in the laboratory, especially for food analysis, vitamin assays, and biochemical preparations (6). There are few reports concerning limitations on its use. The Blendor has been recommended for ascorbic acid determinations in tissues (1, 6, 8, 12), although Roe et al. (If) advise against use of any homogenizer that would introduce increased amounts of oxygen into the slurry. Stern and Bird ( I S ) found that treatment of wheat germ and mill stream suspensions in the Waring Blendor caused oxidation of sulfhydryl groups and inactivation of enzyme S.StemS.
Divergent results and conclusions in work involving use of the Raring Blendor in some instsnces may be traced to the indiscriminate use of containers. This study was initiated as a result of discrepant data on the ascorbic acid content of identical solutions blended in different containers. Inspection of a container in which significant losses of ascorbic acid occurred showed that
the chrome plate on parts of the metal blending assembly was worn, exposing the brass undersurface. Brass contains a high percentage of copper, which is' a known catalyst for the oxidation of ascorbic acid ( $ 3 , 7 ) . This paper shows that blending ascorbic acid solutions in different contajners resulted in widely varying losses in ascrobic acid, which can be attributed to the dissolution of copper during the blending procedure, EXPERIMENTAL PROCEDURE AND DISCUSSIO'Y
Two hundred milliliter portions of a solution of ascorbic acid (20 micrograms per ml.) in 5% metaphosphoric acid (HPOI) initially a t room temperature (23" C.), were blended for 3 minutes in triplicate in each container, The average t e p erature rise in the solutions a t the end of the blending was 8.7 with a low of 6.0" C. and a high of 12.0" C. The small differences in temperature rise were not reflected in the results. Ascorbic acid was determined by the 2,4-dinitrophenylhydrazine method ( 11 ) on aliquots of all blended solutions and nonblended controls. The copper content was determined by a dithizone method not subject to interference by other metals (9). The results from triplicate runs with a single Waring Blendor container were not in agreement where the loss in the first blending was high. Instead, these triplicate runs seemed to form a pattern in which there was progressive lowering of ascorbic acid loss with the two succeeding blendings. This phenomenon of lowered ascorbic acid loss with successive blendings was further investigated. Using only one container (No. 6 ) nine consecutive 3minute blendings of 200-ml. portions of ascorbic acid (20 micrograms per ml.) in 5% metaphosphoric acid were carried out. The ascorbic acid and copper conterlts were determined in each solution immediately after blending, as well as in an unblended control. This experiment was then repeated in the same container scrupulously cleaned, after an interval in which the container had general laboratory use in blending various materials.
8.
The values determined for ascorbic acid loss and copper concentration in all experiments are shown in Table I, and the correla-
ANALYTICAL CHEMISTRY
1140 Table I. Loss of Ascorbic Acid in Different Blendor Containers [Ascorbic acid (20r/ml.) in 5% HPOI solutions blended for 3 minutes1 Appearance of Blending Assembly
Blendor Container
No. 1 Cu p p . m ASk"'lost,'% No. 2 Cu,p.p.m. ASAlost, %
Bright plating Bright plating
'
4
5
.. ..
.0.02 1.2
..
0.89 5.0
0.43 3.0
0.54 2.5
,.
2.66 5.0
0.21 3.0
0.08 3.5
.
C U ,p.p.m. 1.50 ASA lost, yo 1 3 . 0 NO.5 Cu, p.p.m. 4.00 ASAlost, % 2 1 . 0 No. 6 Cu, p.p.m. 6.96 ASAlost, % 36.5 Cu,p.p.m. 12.80 AS.410st,% 5 2 . 1 Cu,p.p.m. 8.27 .4SA loat, % 4 5 . 6
0.74 13.0
1.36 11.0
..
Dull plating
SO.4
a
3
0.24 2.0
SO. 3 Cu,p.p.m.$
A S A l o s t , yo
Plating worn, brassexposed
2
0.0 1.5
Plating somewhat dull
Plating worn, brassexposed
Blending Run 1
2.48 0.88 19.0 13.0
.,
6
7
8
9
, ,
.. ..
.. ..
, .
,
.
..
..
,.
..
.. ..
.. ..
,,
, .
..
..
..
..
.. ..
.
..
..
,
.
.,
..
..
..
..
..
..
. ..
..
..
..
.
.. .. ..
,,
..
.. ,.
..
3.'44 1.60 .. ., 22.5 13.0 2.68 3.04 i . 4 8 0'.80 i,'12 14.9 17.6 9.4 7.1 7.9 2.20 1.86 1.28 2.64 11.6 10.1 13.'6 7 . 8 1 4 . 9
Reduced ascorbic acid.
tion between the amount of copper dissolved during blending and the percentage loss of ascorbic acid is shown in Figure 1. The relationship is expressed by the straight-line equation y = 0.0221 x - 0.051. The coefficient of correlation between copper concentration and per cent ascorbic acid lost in this case is 0.97; a perfect correlation coefficient is 1.0. When tested, the correlation coefficient0.97 was found to be highiy significant. When the values for copper concentration and ascorbic acid loss that were obtained with solutions blended in a single container, No. 6, are plotted (not shown), similar results are obtained. The straightline equation is y = 0.0223 X -0.064 and the correlation coefficient is 0.98. Actually, adding increasing amounts of copper (as cupric sulfate) to ascorbic acid solutions resulted in losses of ascorbic acid during blending in a container of excellent condition (KO.1) that were almost identical to those found in solutions containing dissolved copper which originated from blending assemblies having worn plate. The results make it evident that, under the conditions of t.hese experiments, copper dissolved from the blending assembly is responsible for the loss of ascorbic acid in the solutions blended. The extent of loss of ascorbic acid correlates rather well with the physical appearance of the blending assembly (Table I). Solutions in contact with well plated blending assemblies showed small losses in ascorbic acid, whereas with blending assemblies having worn plating or exposed brass parts losses were considerably higher. However, the loss of ascorbic acid, as well rn the amount of dissolved copper, decreases and finally remains fairly constant with consecutive blendings in the same container. No attempt was made to study the mechanism for the progressively smaller amount of copper dissolved with successive blendings. Possibly an insoluble copper metaphosphate forms and deposits at worn surfaces of the plated metal parts of the blending assembly and retards further dissolving of copper. I t appears that a blending assembly in which copper causes large ascorbic acid losses could be improved by preblending with a metaphosphoric acid solution. However, such a procedure would seem inadvisable unless i t results in very low losses of ascorbic acid. There is the podsibility that about 1Id.Of a solution could come in contact with the sealed bronze bearing (90% copper, 10% tin) of the blendine asaemblv when the blades are not in motion (24). For this reason an experiment was carried out to determine whether allowing ascorbic acid (20 micrograms per ml.) in 5% metaphosphoric acid solution to stand in a container of excellent condition (No, 2) for increasing lengths of time, immediately followed by a %minute blending, would result in increased loss in
..
..
..
..
.,
..
.. , .
'
:;
.
l'.iO 1.'06 8.6 6.li6.6 1.62 1.92 8.1 10.1 9 . 6
ascorbic acid. The times of standing in the nonagitating container prior to blending were 0, 0.5, 1, 2, 3, 5, 7, 10, and 15 minutes. There was no increase in the amount of ascorbic acid lost with increased time of standing of the solution. The average loss was 1.5%. The effectiveness of stannous chloride and thiourea, known inhibitors of ascorbic acid oxiclation, in lowering the ascorbic acid loss during blending of a solution in a container in poor condition (NO. 5) were tested by incorporating them into the ascorbic acid (10 micrograms per ml.) in 5% metaphosphoric acid solution before blending for 3 minutes. As shown in Table 11, 0.5% stannous chloride (higher concentrations were not used because of precipitation of stannous metaphosphate) re-
duces the loss of ascorbic acid. It probably retards oxidation by combining with molecular oxygen. Thiourea (1%) markedly reduces ascorbic acid loss by combining with copper and inhibiting its catalytic action on the oxidation of ascorbic acid, Indiscriminate use of containers may also cause trouble in the determination of trace metals in foodstuffs. In one laboratory, contamination of the food by biologically important heavy
/
12
-
II
-
10 9 -
- 51
y - 211~
a 7-
I a V
4 -
0
IO
20
30
50
40
A S C O R B I C ACID LOSS
- PERCENT
60
Figure 1. Correlation between Concentration of Copper Dissolved during Blending and Percentage Loss of Ascorbic Acid 200-ml. portions of ascorbic acid (20,i/ml.) in 5% HPO, blended for 3 minutes in various containers
Table 11. Effect of Adding Stdnnous Chloride or Thiourea to -&corbic A&idon hSs of .4scorbic Acid during Blending Ascorbic Acid Lost, % Control Control
I
Container
Controla
No. 5 (av. of 3 runs) N o 1 (BY. of 3 runs)
40 2 1 3
a
.4srorbic acid (lOy/ml,) in 5% HPOI.
+
0 . 5 % SnCh 13.8 1.2
+
1% thourea 3.0 0.0
V O L U M E 2 2 , NO. 9, S E P T E M B E R 1 9 5 0
1141
Table 111. Determination of Concentration of Copper in Canned Tomato Juice before and after Blending Tomato Juice Sample Unblended control Blended in container 2 Blended in container 6
Copper Concn., P.P.M. 1.52 2.04 8.52
rhodium-platinum, of metal parts may be desirable. Furthermore, oxidation during blending may be retarded by replacing air over the blended material with inert’gas ( f ) , removing oxygen by a chemical agent such as stannous chloride, and inhibiting the catalytic action of trace metals as in the binding of copper by thiourea. ACKNOWLEDGMENT
metals, which may be present in the metla1 blending assembly, is prevented by rhodium-platinum plating these parts (10). Use of the Waring Blendor for comminution of foods prior to the determination of their copper content has been mentionpd in the literature (4). If such a procedure is carried out with little regard for the plating of the blending assembly, false results are likely, as shown in the following brief experiment:
A determination was made on the copper content of a sample of thoroughly mixed canned tomato juice immediately removed from the can after opening. A 200-ml. portion of this same tomato juice was blended for 3 minutes in container 2 (in excellent condition) and another 200-ml. ortion was similarly treated in container 6 (in poor condition, grass exposed).
It is evident from Table I11 that a container with worn plating its metal parts can cause false high results in the copper content of a food subjected to blending during the determination.
011
CONCLUSIONS
The Waring Blendor is a valuable tool; however, judgment must be exercised in its Use. The condition of the metal parts of the container should be noted a t all times. Where dissolved trace heavy metals may affect enzyme systems, vitamin assays, or the determination of trace metals, special plating, such as
The author wishes to thank Paul E. Corley and Greta Ferguson for technical assistance. LITERATURE CITED
(1) -4ssociation of Vitamin Chemists, Inc., “Methods of Vitamin Assay,” p. 150, New Pork, Interscience Publishers, 1947. (2) Barron, E. S. G., Barron, .4. G., and Klemperer, F., J. Biol. Chem., 116,563-73 (1936). (3) Barjon, E.S. G., De Meio, R. H., and Klemperer, F., Ibid.. 112, 625-40 (1935-36). and Grabenstetter, D., IND.ENQ.CHEM.,ANAL. (4) Bendix, G.H., ED.,15,649-52 (1943). (5) Davis, W. B., Ind. Eng. Chem., 34, 217-18 (1942). (6) Davis, Vi’. B., I n d . Eng. Chem., News Ed., 17,752 (1939). (7) Mawson, C.A., Biochem. J.. 29,569-79 (1935). ( 8 ) hlorell, S. A., IND.ENG.CHEM.,ANAL.ED., 13,793-4 (1941). (9) Morrison, S. L., and Paige, H. L., Ibid., 18,211-13 (1946). (10) hfoyer, E. Z.,Beach, E. F., Robinson, A., Coryell, M. N., Milier, S..Roderuck, C., Lesher, M., and Macy, I. G., J . .4m. Dietclic Assoc., 24,85-90 (1948). (11) Roe, J. H., Mills, M. B., Oesterling, M. J., and Damron, C. AI., J . Bid. Chem., 174, 201-208 (1948). (12)Satterfield, G.H., B i d . Symposia, 12,397 (1947). (13)Stern, R., and Bird, L. H., Biochem. J.,44,635-7 (1949) (14) Waring Products Corp., New York, N.Y., personal communica-
tion. RECEIVED March 13, 1950.
Analysis of Colloidal Electrolytes by Dye Titration H. B. KLEVENS University of Minnesota, S t . Paul 1, Minn. The determination of total amount of surface-active material present in a system can be obtained by titration with a suitable dye solution. Anionic soaps can best be determined with cationic dyes, and cationic soaps with anionic dyes. Total amount of surface-active components as well as the amount of each component can be determined in soap mixtures, in soap-electrolyte systems, and in soap solutions containing other materials such as hydro-
A
NUMBER of methods have been wed to measure the concentrations of soaps and detergents., These include various chemical methods ( 5 ) , a turbidimetric method ( l a ) which is a modification of one used by Preston (16) and by Hartley and Runnicles ( 4 ) ,and a direct titration of sulfonates with p-toluidine hydrochloride (14). Some of these methods have been critically evaluated recently ( I ) . For the determination of critical micelle concentration (CMC), a relatively simple method involving titration with various dyes based in part on earlier qualitative work of Hartley (3) and Sheppard and Geddes (18) has been reported recently ( 1 ) and application of this technique to various systems has been demonstrated (9, 15). This method depends essentially on the fact that a color or intensity of fluorescence of a dye solution changes markedly in the region of the critical micelle concentration(the amount of free nonmicellar soap). This makes it possible to follow these changes
carbons, polar compounds, latices, and various adsorbants if the proper experimental techniques are employed. In the case of determinations in turbid or slightly colored media, reproducible results were obtained only with dyes that show changes in fluorescence intensity. Experimentally, the most consistent data were obtained by observations made in a darkened room using a narrow beam of light falling on a thin layer of the soap solution.
spectrophotometrically or visually, for the changes in most instances are marked enough to obtain reproducibility of results to better than 2 to 3%. The usual procedure is to titrate a volume or weight of soap solution or a known amount of dry soap with a freshly prepared dye solution, the concentration of which is usually about 1 to 5 X 10-6 M . The amount of added dye sohtion a t the point of color change in the dye-soap solution is that necessary to bring the concentration of the solution to the critical micelle concentration. It has been found that, to be most effect h e , the dye used must carry adifferent charge than that of the colloidal electrolyte. For anionic soaps dyes like pinacyanol chloride or anisoline are suitable, whereas acidified indophenol, eosin, sky blue FF, and benzopurpurine can be used with cationic detergents. With those dyes which exhibit a monomer S polymer equilibrium, it has been found that the color changes a t the critical micelle concentration are much greater than the
