ANALYTICAL EDITION
July 15, 1943
and to C. R . Noller of the Department of Chemistry for indicating the possible use of methylene chloride for the work presented in this paper.
Literature Cited (1) Carr, F. H., and Price, E. A.,Biochem. J., 20,497 (1926). (2) Hamm, W.S.,personal communication. (3) Hume, E, M.,and Chick, H., Medical Research Council (Britain), Spec. Report 202 (1935). (4) Koehn, C. J., and Sherman, W. C., J. Biol. Chem., 132, 527 (1940). (5) Norris, E. R., and Church, A. E., Ibid., 87,139 (1930)
439
(6) Rosenthal, J., and Erdelyi, J., Jfagyar Orvasi Arch., 35, 232 (1934). (7) Rosenthal, J., and Weltner, M., Biochem. J., 29, 1036 (1935). (8) Stansby, M. E., and Lemon, J. M., IND.ENG.CHEM.,ANAL. ED.,9,341 (1937). (9) U.S. Pharmacopoeia XII,Supplement, p. 109. (10) Wilkie, J. B., J . Assoc. Oficial Agr. Chem., 22,465 (1939). (11) Wokes, F.,and Willimott, S. G., Analyst, 52,515 (1927). THISinvestigation is incidental to a survey on the vitamin A potency of soupfin shark livers. The survey is part of a program investigating the entire California soupfin shark fishery and is conducted by the California Division of Fish and Game.
Rapid Determination of Sodium Chloride in the Presence of Protein Application to Salt-Cured Food Products W. J. DYER, Atlantic Fisheries Experimental Station, Halifax, N. S., Canada
Sodium chloride in protein-containing material and solutions is determined by direct titration with 0.0856 N silver nitrate, using dichlorofluorescein as an adsorption indicator (40 to 100 mg. of sodium chloride in a volume of 400 ml.). A 0.2 N sodium acetate-acetic acid buffer of pH 4.5 is added to the solution or minced sample and
P
ROTEIN interferes in all the usual methods for the de-
termination of chloride, and must be removed by some wet- or dry-ashing procedure. This requires considerable time and inconvenience when large numbers of routine determinations are to be carried out. Attempts to remove protein by various precipitants have not been satisfactory (6). Recently, adsorption indicators have come into common use for the direct titration of halide ions, and in relatively simple solutions the method is very much more rapid and equally as accurate as the common Volhard procedure in which the chloride is precipitated by addition of silver nitrate, and the excess silver ion titrated with thiocyanate (1, 3, 12). Dichlorofluorescein seems to be the most suitable adsorption indicator for the chloride ion in dilute solution (6, 7, 9, IO). Here also, low results are obtained in the presence of protein, apparently because of an adsorption of silver on the protein (6, 11). I n fish muscle the principal proteins have isoelectric points a t about p H 5 and 6 (8), and it was reasoned that this adsorption should be a t a minimum, and negligible in amount, a t a hydrogen-ion concentration near the isoelectric point of the protein micelles. Muscle suspensions and extracts were titrated in sodium acetate and phosphate buffer solutions over the range pH 3 to 9. Theoretical results were obtained between about pH 4 and 4.8 in the presence of protein, while in pure salt solution the indicator was satisfactory between p H 4 and 7. Results have been checked on salt fish, canned fish, fish meals, bacon, etc., and on pickles and brines. The optimum
allowed to stand or boiled and cooled. The salt is completely extracted from the sample and it is shown that silver is not adsorbed by the protein present at this hydrogen-ion concentration. Results obtained agree closely with the common Volhard method of digestion with nitric acid in the presence of silver nitrate.
p H range was from 4.3 to 4.8 in each case, and the method as finally developed was found to be entirely satisfactory from an analytical standpoint. I n the author's experience, the use of organic solvents as sometimes recommended did not result in any improvement over the above method.
Reagents Dichlorofluorescein indicator, 0.1 per cent in 50 per cent ethanol. Acetate buffer, equal volumes of 0.2 N sodium acetate and 0.2 N acetic acid, adjusted to pH 4.5 (*0.2) if necessary. Silver nitrate, 14.52 grams in 1000 ml. of solution standardized against pure dry sodium chloride, 1 ml. = 5 mg. of NaCl. Procedure PICKLESAKD SOLUTIONS.To an aliquot of solution containing about 40 to 100 mg. of sodium chloride, add approximately 20 ml. of acetate buffer solution, and dilute t o about 400 ml. with distilled water. Add 10 drops of indicator and titrate with the silver nitrate solution, tchile slowly swirling the flask, until a pinkish red coloration appears throughout the solution. Titration must be fairly rapid, since the adsorption of the dye considerably increases the light-sensitivity of the colloidal silver chloride, thereby rapidly increasing the rate of aging and decreasing the sharpness of the end point. If the pickle contains much protein, a much sharper end point will be obtained if the solution is boiled for a minute or more after addition of the buffer and cooled t o 20" C. before dilution and titration. SALT FISH AND OTHERCUREDFOODPRODUCTS. A wellcomminuted sample is mixed with 25 ml. of buffer, and diluted to about 75 ml. with water. The mixture is boiled for a few minutes, and cooled. The whole may then be diluted to 400 ml. and titrated as before, or the solution may be made to 100 ml. and an aliquot of the supernatant liquid used for the titra-
INDUSTRIAL AND ENGINEERING CHEMISTRY
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tion. The weight of sample used will depend on the salt content and degree of subdivision of the sample. If it is desirable to use a large sample for the sake of uniformity in sampling, the titration of an aliquot containing 40 t o 100 mg. is the preferable procedure. Either fresh or dried samples may be used, so that moisture and salt analyses are often conveniently made on the same sample. If a 0.5-gram sample is used, as is convenient with salt fish, the percentage salt content is read directly on the buret, 1 ml. being equivalent to 1 per cent of sodium chloride in the original material.
TABLEI. RECOVERY OF ADDEDSODIUM CHLORIDE Sample Fresh cod, 0.5 gram Light salt cod, 0.5 gram Hard salt cod, 0.5 gram
Recovery of Added NaCl
h’aC1 Added
NaC! Found
MU.
MQ.
%
0 25
0.5a 25.3 51.0 60.0 85.2 109.5 138.0 97.5 122.0 170.3
...
50 0
25 50 75
0 25 75
101 101
...
101 99 100 .
.
I
98
97
Volhard method. I
Vol. 15, No. 7
It is indeed shown that between p H 4 and 5 a range probably slightly more acid than the isoelectric point of the known proteins present, little or no adsorption of silver or of chloride occurs. I n solutions containing considerable soluble protein, such as those from fish meal where the salt content is low and consequently a high dilution cannot be used, a p H of 4.2 t o 4.8 gives results agreeing with the Volhard method. A buffer of p H 4.5 was accordingly selected for routine use. This indicates that cod and haddock muscle has an apparent isoelectric point near p H 4.5. Other work on the salting of cod muscle (4) has also shown an apparent isoelectric point between p H 4.0 and 4.5. Further study of the proteins present is needed, although the isoelectric point does not necessarily coincide with the point of minimal acid and base-binding capacity ( 6 ) . Recoveries of added sodium chloride are shown in Table I and agreement of results with those of the standard Volhard method in Table 11, on samples of salt cod, fish meal, and bacon. The fish meal samples were analyzed by boiling a suspension of 5 grams of meal in 100 ml. of buffer and water, then adding 50 mg. of sodium chloride (standard solution), to a 20-ml. aliquot of the suspension. Otherwise the salt content was too low, and insufficient colloidal silver chloride was formed to allow easy observation of the end point.
Results a n d Discussion
A series of titrations was conducted on pure sodium chloride solutions and on extracts of salt fish and of fish meal with sodium chloride added, over a range of p H 3 to 8 in sodium acetate and phosphate buffers. The results are shown in Figure 1.
-
TABLE11. COMPARISON OF ADSORPTIONINDICATOR AND VOLHARD
a
id
14
t t I ”
IO
4
1
A
a
5
6
7
8
PH
FIGURE 1. IKFLUEKCE OF pH
ON
Sodium Chloride Found Adsorption indicator Volhard
Sample Hard salt cod, 0.5 gram Light salt cod, 0.5 gram Bacon, 5 grams Fish meal, cod Fish meal, herring
20
TITRATIOS ENDPOINT
1 ml. of AgNOa s o h . = 5 mg. of NaCl A . Standard solution of sodium chloride. Titer = 11.60 ml. 1 gram of fresh cod muscle. B. Standard solution of sodium chloride Titer = 11.80 ml. extract of 1 gram of fish C. Standard sqlution of sodium chloride meal. Titer = 17.60 ml. Dotted lines show indefinite end points-approximate limits shown.
+ +
At acidities below p H 4 the indicator showed no color change, and above p H 7.5 the change became indefinite, an orange-yellow or yellow color being obtained. I n the presence of protein the end point becomes indefinite above approximately p H 6, a t an acidity just below the point where an increased adsorption of silver occurs. The amount of silver and dye ions adsorbed by the protein increases as the solution is made more alkaline, and thus no definite end point is obtained in this range.
METHODS %
%
19.8 (*0.2)= 11.3 (==0.2)= 1.30 (*O.O3)G 3.00, 2.95 0.75, 0.70
19.9 (*0.3)0 11.2 (*0.2)0 1.31 (+0.03)= 2.93, 2 . 9 1 0.72, 0.73
Average of 4 determinations.
Recovery of added salt is very satisfactory, and the results agree with the standard method. Complete estraction of salt from a minced sample by the buffer solution was obtained. Boiling for 2 to 5 minutes, folloir.ed by cooling to room temperature, gave the same results 2 s extracting for approximately 3 hours a t room temperature, and the end point was considerably more satisfactory. Decreasing the estraction time to less than 3 hours a t room temperature resulted in incomplete extraction, as found by comparison with the wet-digestion methods. The presence of fat or oil in fish meal or meats had no influence on the results. The method has been in continuous use for more than a year in the research and routine analyses of salt fish and other fishery products.
Literature Cited Assoc. Official Agr. Chem., Official and T e n t a t i v e Methods of Analysis, 5 t h ed., p p . 134, 318 (1940). Callow, E. H., Biochem. J., 23, 648 (1929). . 24, 631 (1941). Deal, E. C., J . Assoc. Oficial A ~ TChem., D y e r , W. J., unpublished results, 1942. Edsall, J. T., J . Biol. Chem., 89, 289 (1930). Kolthoff. I. &I., Chem. Rev., 16,87 (1935). Kolthoff, I. hl., Lauer, TT. bl., and Sunde, C. J., J . Am. Chern. Soo., 51, 3273 (1929). Logan, J. F., Contrib. Can. Biol. Fisheries, 6, 3 (1930). Rose, C. F. M., Biochem. J., 30, 1140 (1936). Saifer, A , , a n d K o r n b l u m , M., J . Biol. Chem., 112,117 (1935). Sendroy, J., Ibid., 120, 441 (1937). Treadwell, F. P., a n d H a l l , W. T., “Analytical Chemistry”, Vol. 11, 8 t h ed., p. 664, New York, J o h n Wiley & Sons, 1935.
