T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
May, 1917
T =
+
L-0.j
I.
z X Per cent Fat
4 is applied to milk kept a t 37-40' C. for one and a half hours or until no further change in specific gravity occurs. DEPARTMEXT O F AGRICULTURE BUREAU OF CHEMISTRY
WASHINGTON, D. C.
B y PHILIPADOLPH KOBER Received December 15, 1916
For sometime we have been trying t o develop further, methods for the study and analysis of proteins. In connection with this work, which will involve the scheme of Van Slyke, we have first attempted t o improve and standardize some parts of the copper method' for estimating amino acids. Our improvements are: (I) A simpler method for making standard copper solutions; (11) a method for making a stock suspension of cupric hydroxide which keeps for months, and (111) a method for making and keeping iodometric reagents; i. e., saturated potassium iodide solutions, containing starch and acetic acid. METHOD FOR MAKING
shaking every 15 min. the cupric sulfate is practically dehydrated and is obtained perfectly white; the apparatus is then removed from the oil bath without interrupting the aeration and the oil, while t h e tube is still hot, carefully and completely wiped off with a towel. Then, when cool, the paraffined stopper is quickly inserted and t h e tube placed in the desiccator until it is ready for weighing. By using apparatus with ground glass joints, which was not available t o us for this investigation, more accurate results can undoubtedly be obtained. If care is taken the tube may be heated without an oil bath, by putting i t over a Bunsen burner, so as to maintain i t just below visible red heat a t about 400' C. The following results were obtained: '
IMPROVEMENTS IN THE COPPER METHOD FOR ESTIMATING AMINO ACIDS
I-THE
501
STANDARD
G. CUSOI Taken 1.1846 1.0515 1.1044 1.0695
G. Cu
G. Cu
(Electrolysis) 0.4706 0.4173 0.4374 0.4247
Expected 0.4720 0,4190 0.4390 0,4250
Ratio Cu Found t o Theory (%) 99.7 99.6 99.6 99.7
This method of dehydrating cupric sulfate, as the work of T. W. Richards' indicates, may not give suffi-
COPPER
SOLUTIONS
I n the original paper* no specific directions were given for making an iodometric standard, the directions in any standard text-book having been considered sufficient. None of the methods given in these books, however, are very convenient nor are the solutions stable. Standardization of copper solutions by electrolysis, which is of course most accurate, is decidedly unsuitable for biological laboratories, nor is i t a particularly short method unless special apparatus involving rotating cathodes is used. The weighing-out of a suitable amount of a pure copper salt was thought to be the simplest procedure. Cupric sulfate is t h e purest and cheapest source of copper on the market and the only disadvantage encountered in using it for standard solutions is its uncertain water content and, therefore, unknown copper content. This disadvantage may be practically overcome by dehydrating the salt, as shown below, in a very simple and inexpensive apparatus.3 About I lb. of "parowax" is melted in a liter beaker or other suitable container and used in a hood as an oil bath a t 24j' t o 260' C. 4 test tube (ISmm. inside diameter, length I j 5 mm.) is cleaned and dried a t 105 to 110' C.,and weighed with a paraffined cork stopper. Allowing the stopper t o remain in the desiccator, 1.77 g. of monohydrate cupric sulfate4 are put into the test-tube and connected with the aerating tubes as shown in Fig. I . Upon heating for z or 3 hrs. with 1 Kober and Sugiura. J . Am. Chem. SOL.,35 (1913), 1546; applied t o soils by Potter and Snyder, Ibid.. 37 (1915). 2219; THIS JOURNAL, 7 (1916). 1049; applied to study of Lecithin, by IvIacArthur. J . A m . Chem. SOL.,36 (1915). 2397. 2 J . Am. Chcm. SOC., 36 (1913). 1546. * For working out this method and for all the analytical work, I am indebted to Mr. Walther Eberlein of this laboratory. CuSOi 5Hx0 heated t o 105 to 110' C. for several hours is converted to:monohydrate CuSOd H30,having only a light blue tint.
+
+
m
FIG.1
ciently accurate results for the purpose of calculating atomic weights, but the shaking from time to time in an atmosphere of dry air, may make the results slightly higher than the lowest given by Richards a t 99.9 per cent. However, with the apparatus and equipment at our disposal, we were not able t o test this out. DIRECTIONS-After weighing, the contents of the tube are dissolved, avoiding loss due t o spattering by keeping the tube covered or in a horizontal position: 1.77 grams of the monohydrate cupric sulfate will give sufficient copper when dissolved in 2 5 0 cc. of water t o make a 1/25 molar solution, which if carefully stoppered can be used for standardizing stock solutions almost indefinitely. T o guard against the formation of basic salt, due t o hydrolysis and any alkalinity of the glass, it is well to add I or z drops of concentrated sulfuric acid. METHOD F O R MAKING A PERMANEKT
11-A
SUSPESSION
O F CUPRIC HYDROXIDE
I n the original directions, cupric hydroxide had to be made fresh every day. Its preparation, because it required chopped ice, neutralization, filtration and 12.
anorg. Chem., 1 (1892). 179
T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
502
washing, consumed considerable time and attention. Furthermore, its use as a jelly made the amount taken for an analysis variable and a t times uncertain. Cupric hydroxide, as ordinarily made by neutralization of any cupric salt, is not stable, but slowly, and in most cases within 24 hrs., changes over into t h e black cupric oxide. This, owing partly t o its agglutination, is not so reactive with amino acids, particularly the conjugated (the peptides). By avoiding an excess of alkali; i. e., not adding enough completely t o precipitate the copper from its salt, a suspension of cupric hydroxide can be obtained which keeps for months, if not indefinitely, and can be pipetted easily. The following results, with glycine, show its reactivity when freshly made and after the lapse of time: Time Days 1 11 27 31 73
Per cent CuO Found to be Theoretical 99.3 100.2 99.6 99.6 100.0
Titrations
20.64 20.60 20.61 20.60
78
100.0 Average,
99.8
METHOD FOR IODIDE
KEEPING A SATURATED POTASSIUM
SOLUTION,
CONTAINING
STARCH
AND ACETIC ACID
I n the original directions potassium iodide solutions had t o be made fresh every day and titrated from time t o time, almost constantly, t o keep i t free from iodine. This production of iodine occurs apparently regardless of t h e source of the iodide. Undoubtedly some if not most of t h e iodine originated from iodates or other impurities present in the iodide but the second and subsequent yields seem not t o be due t o iodate, and not as the literature on the subject states, t o oxygen and carbon dioxide, b u t t o t h e presence of nitrites and nitrates, which with any oxygen from the solution constituents and the air continue t o decompose the potassium iodide, catalytically. By removing most if not all of these substances, 1 1
Abegg’s “Handbuch dPr Anorg. Chem.,” 11, 356. J . SOC.Chem. Ind., 37 (1882). 197.
and by decreasing the amount of available oxygen in contact with the solution, i. e., keeping it anaerobically, saturated solutions of potassium iodide, starch and acetic acid can be kept for a long time, if not indefinitely. Although the simplest and most efficient method of accomplishing this result has not been definitely decided upon yet, owing t o the long time desirable for such a stability test, the following scheme has proved practical : Four hundred grams of potassium iodide were dissolved in 4jo cc. of distilled water, and with 40 cc. of 1.0per cent “soluble” starch‘ solution, and I O cc. glacial acetic acid were placed in a 750 cc. flask or Erlenmeyer. After adding 2 . 5 cc. of M / 2 cupric sulfate solution and an inch of paraffin and mineral oil mixture ( I : 3), the solution was allowed to stand for 30 min., when the iodine was titrated with M / a sodium thiosulfate t o just the neutral point; i. e., when it becomes colorless, when the flask was fitted with an inlet tube containing strong alkali, as shown in Fig. 2 , and boiled for 30 min. This inlet tube is for the pur-
I-
D I R E C T I O N S : - ~ 2 cc. M / 2 cupric sulfate solution are diluted with water and chopped ice t o 6 liters, and a t o t o I O C. precipitated with about 175 cc. N C02-free sodium hydroxide, using I cc. phenolphthalein indicator t o test for final acidity. After filtering through paper containing some ice and washing slightly with a spray, the precipitate of cupric hydroxide is suspended in two liters of distilled water, preferably ammonia-free, allowed to settle, and the supernatant liquid poured off, This is repeated three or four times in the course of a day or so, finally suspending the precipitate in 2 5 0 cc. of water. Any ammonia in t h e water, and alkali dissolved from the glass, may interfere with its keeping indefinitely. According t o Abegg,’ who mentions a similar preparation of Tommasi,2 t h e final product is Cu(OH)2 and in the absence of salts and alkali will keep indefinitely. Its use and apparent excellent adaptability for amino acid work, however, was not discovered, so far as we are able t o learn. 111-A
Vol. 9 , No. 5
Li
FIG. 2
pose of washing the air t h a t enters the flask after boiling. I n this way we have had stock solutions keep three months or more and in certain experiments we have had a solution of potassium iodide, starch and acetic acid keep in contact with oxygen without oil for 42 days, which seems to us to indicate t h a t when certain catalysts in the air are removed, oxygen has no effect upon potassium iodide or hydriodic acid, contrary t o the statements in the literature. This phenomenon we are studying further, with t h e hope of finding a reagent that will prevent the oxidation by means of nitrites and nitrates, or better, t o remove these substances quantitatively from solution. We believe t h a t this reaction may play a r81e in many obscure phenomena, such as the deterioration of toxins and antitoxins, and other biological changes, and any practical method t h a t would prevent air from exerting an oxidizing effect, because of its nitrite and 1 Stock solutions of 1.0 per cent starch may also be preserved separately, by adding tricresol to the extent of 0.2 per cent. An experiment with a large amount of cresol-20 to 40 times more than would be used ordinarily - s h o w e d that it had no effect on the iodometric titration.
&fay, 1917
T H E J O C R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
nitrate content, might find numerous and valuable applications. CONCISE
DESCRIPTIOX
OF
AMINO
ACID
ESTIMATION
BY M E A S S O F THE COPPER METHOD SOLUTIONS A N D A P P A R A T U S REQUIRED-( I)
A ‘/z5 molar sodium thiosulfate solution, containing 0.993 g. Na2Sz03 5 HzO in a liter of COz-free water, standardized against a M / 2 j cupric sulfate solution ( 2 ) made as just described under I. (3) A suspension of cupric hydroxide as described under 11. (4) Iodimetric reagents as described under 111. ( 5 ) Glacial acetic. (6) A “buffer” solution as described in previous paper is made as follows: Sodium hydroxide solution is made free from carbonate, according t o the suggestion of Sorensen, as follows: 250 g. of sodium hydroxide are dissolved into 2 j o cc. of water and placed in a flask furnished with a soda-lime tube. After cooling and allowing the solution t o .settle, the clear, saturated supernatant solution which contains no ’ c a r b o n a t e sodium carbonate being insoluble in saturated sodium hydroxide-is diluted with sufficient boiled distilled water’ t o make a normal solution: 0 . 2 gram-molecules of boric acid (12,404 g.) are dissolved with distilled water and I O O cc. of normal sodium hydroxide a n d made up t o a liter. This solution is designated as “sodium borate” and, if kept free from carbon dioxide, can be preserved indefinitely. Three volumes of “sodium borate” mixed with one volume of 0 . 1 N hydrochloric acid, we have found thus far to be a suitable “buffer” solution. ( 7 ) so cc. graduated flasks. (8) Pipettes, burettes. (9) Glass funnels ( 2 - 2 . j in,). ( I O ) Filter paper: 1 1 cm. No. 590, S. & S., or equivalent. ( 1 1 ) I jo cc. Erlenmeyers for filtering.
+
DIRECTIOKS FOR 3fAKIXG ESTIMATIOKS O F THE TOTAL A N I S 0 ACID NITROGEK (A) INCLUDING T H E POLYPEPTIDE
AMINO NITROGES-
All solutions t o be tested for amino acids should be neutralized or made slightly alkaline to phenolphthalein. Insoluble substances may be dissolved with t h e aid of 0 . 1 N NaOH, using not more than 5-6 cc. For the factors given in this paper t h e solution should not contain more than 0.02; g. of amino acids in 2 5 cc. The volume after neutralization should be made t o about 2 5 cc. and plqced in a jo cc. graduated flask, so t h a t , when stoppered, carbon dioxide from the air can. t o some extent, be avoided. Twenty cc. of “buffer” solution are now added and 2 t o 3 cc. of cupric hydroxide suspension are introduced and the mixture vigorously shaken for about a minute. If the cupric hydroxide has not completely dissolT-ed, and, therefore, is in excess, the solution, after bringing t o room temperature, is made up t o the mark2 and shaken from two to three minutes longer. The mixture is now filtered through a good dry filter paper (S. & S. No. j 9 0 , 1 1 cm. in diameter is satisfactory). The filtrate contains all of the soluble complexes while the residue contains the insoluble Complexes and the excess of Cu(0H)z. 1 As
a rule ordinary distilled water in all these solutions is suitable but boiled distilled water is preferable. * T h e amount of cupric hydroxide, which should not be more than 0.060 g.. will produce an error in the volume of solution of not more than 0.05 per cent, which is in most cases negligible.
503
An aliquot portion ( z j c ~ . )of the filtrate is then taken and after acidification with I t o 2 cc. of glacial acetic acid, 5 cc. of potassium iodide-starch solution are added and the solution titrated with 0.004 N thiosulfate solution. Every cc. of thiosulfate solution is an equivalent t o 0.0003184 g. of cupric oxide or 0 . 0 0 0 1 1 2 0 g. of amino acid nitrogen, or, I cc. of 0.001 N thiosulfate solution is equivalent t o 0.0000280 g. of amino acid nitrogen. When calculated in terms of peptide amino nitrogen, these values should be halved. (B)
EXCLUDING
POLYPEPTIDE
AMINO
NITROGEN-If
the free amino acids are t o be determined alone, t h e procedure for making the copper complexes is almost identical with t h a t described for the total amino nitrogen. The only change necessary is t o add t o 2 5 cc. of the filtrate, instead of directly titrating iodometrically, j cc. of 0.360 N Ba(OH)z, allow the mixture to stand for I S minutes in a stoppered Erlenmeyer flask and then filter. This precipitates a definite fraction (87 per cent) of the copper from t h e amino acid complexes (see table of precipitabilities).’ After washing the precipitate of cupric hydroxide, it is transferred with the filter paper t o the Erlenmeyer flask and the hydroxide is dissolved in I O cc. of I O per cent acetic acid (warming if necessary). After adding the potassium iodide-starch solution, the copper is titrated with 0.004 N thiosulfate solution, every cubic centimeter being equivalent t o o.ooo112 g. amino acid nitrogen, divided by the proper precipitability. On acidifying with acetic acid and concentrating t o about I j cc. the filtrate can then be titrated iodometrically. The amino acid nitrogen of the filtrate plus t h a t of the precipitate will give, if the polypeptides have five or less conjugated amino acids, the total amino acid nitrogen. If polypeptides or peptones of six or more conjugated amino acids are present they will prevent the precipitation of the free amino acid copper. Therefore, the increase in the amount of copper dissolved after total hydrolysis, alone, will give the information as t o the amount of polypeptide present. KOparticular method of hydrolysis has been tried, but it is probable that the method used by Levenel and Van Slyke will serve the purpose. FOR
SUBSTANCES
WITH
QUITE
INSOLUBLE
COPPER
SALTS-For substances with slightly soluble copper complexes, the technic just described is suitable. Where the copper complexes crystallize out, as is the case when leucine, normal-amino-caproic acid, phenyl glycine, or cistine is present and are filtered off with the excess of cupric hydroxide, it is necessary to separate the two precipitates. Very satisfactory reagents for this purpose are the bicarbonates of sodium and potassium, which will dissolve the excess of cupric hydroxide without appreciably disturbing the comple~es.~ The filter paper4 containing the insoluble complexes Am. Chem.. SOC.,SS (1913), 1551-7. 12 (1912), 304. a See Kober and Sugiura, J . Biol. Chem., 13 (1912). 13. 4 S. & S. No. 590 paper 7 cm. in diameter is preferable when it is necessary to determine the insoluble complex, as it is easier to handle than the 1 1 cm. paper. 1J.
* J . Biol. Chem.
5 04
T H E JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY
and the cupric hydroxide is transferred to a IOO cc. Erlenmeyer flask, and by means of a stirring rod the paper in the flask is unfolded so that the residue is on top, and so t h a t it can be rubbed with a rod. Five cc. of 0.1 N HC1 are then put into the graduated flask in which t h e complexes were originally made, so t h a t the residue clinging t o the sides of the flask is dissolved. This solution is then added to the filter paper in the flask and boiled gently. Another portion ( 5 cc.) of 0.1 N HC1 is used in exactly the same way. Finally the graduated flask is washed with 6 to I O cc. of water and this added t o the complexes in the Erlenmeyer flask. After the residue is dissolved the volume of the solution in the flask is made up t o about 2 0 cc. and 2 g. of powdered potassium bicarbonate are then added as follows: a little (0.2-0.3 g.) of the bicarbonate is first added with a spatula and the solution shaken. This precipitates the insoluble complexes and the excess of cupric hydroxide or carbonate again. After standing one t o two minutes t h e remainder of the bicarbonate is added. On shaking two t o three minutes the excess of cupric hydroxide or carbonate is completely dissolved and insoluble complexes are then filtered through paper (S. & S. No. 590, 7 cm. in diameter) and washed with a little water. On returning t h e filter paper t o the Erlenmeyer (also washed), adding I O cc. I O per cent acetic acid’ and heating until the copper of the complexes is dissolved, t h e solution can be titrated iodometrically. As a table of precipitabilities in the original paper shows, a correction for solubility in KHCOa is necessary. This technic gives good results with most of the insoluble complexes, but with leucine the technic described in a former paper2 is preferable; this technic required t h e washing of the first residue with small amounts of I O per cent KHCOs until the filtrates gave no appreciable tests for copper: then the insoluble complexes were dissolved in I O per cent acetic acid and titrated. It may be possible, by using an amino acid like glycine which gives a soluble complex, t o replace this bicarbonate solution and reduce t h e solubility corrections appreciably. As may be seen from the table, I t o 2 mg. of lgucine and CYStine may be determined as soluble complexes. SUMMARY
The following improvements in t h e copper method for estimating amino acids are described: I-A simple method for dehydrating and weighing cupric sulfate suitable for making standard copper solutions. 11-A stock suspension of cupric hydroxide, which is very sensitive in its reaction with amino acids, and keeps for months. 111-A method for making and keeping saturated solutions of potassium iodide, containing starch and acetic acid. DIVISION OF LABORATORIES A N D RESEARCH NEWY O R K STATE DEPARTM~NT OF HEALTH ALBANY 1 The cystine complex dissolves only slowly in acetic acid. matters a few cc. of N/10 HCl may be added. 1 J. Biol. Chem., 18 (1912). 4.
To expedite
Vol. 9 , No. 5
A STUDY OF THE DETERMINATION OF POTASH CHIEFLY CONCERNED WITH THE LINDOGLADDING METHOD By P. I,. HIBBARD Received December 1, 1916
This study was undertaken for the purpose of obtaining a more thorough and exact knowledge of t h e principles involved in t h e determination of potash according to the Lindol-Gladding2 Method as practiced by the Association of Official Agricultural Chemists*29’a0* Although no part of the original plan for the study of potash determination, some work was done on the Perchlorate Method and is here reported for t h e sake of completeness. As opportunity offered, the work has from time t o time extended over many months. The writer feels t h a t results obtained are of sufficient value t o warrant this presentation. THE LINDO-GLADDIKG
METHOD I N BRIEF
Ten grams of the material are boiled in a 500 cc. flask with 300 cc. water for 30 min. T o the hot solution ammonia and ammonium oxalate are added in excess to remove Fe, Ca, Pod, etc. The solution is cooled, volume made t o 500 cc., filtered, and an aliquot of 50 cc. evaporated in a platinum dish. During evaporation, excess of H & 0 4 is added, then the residue is slowly heated, finally to full red, so t h a t t h e residue is white. The salts are dissolved in water, a little HC1 added, and an excess of HzPtCl8 solution; the mixture is evaporated to paste. The residue is washed free of soluble platinum with 80 per cent alcohol, then sodium and magnesium salts, etc., are removed by washing with 2 0 per cent NH4Cl solution and this is removed by the 80 per cent alcohol. The precipit a t e is dried and weighed as KzPtCla. MAKING SOLUTION O F MATERIAL TO B E TESTED
The volume of water used in making solution is not important with many substances, provided it remains within 50 per cent of the official requirement. Ten grams of an ordinary fertilizer boiled with I 50, 300 and 450 cc. gave 4.78 t o 4.83 per cent K2O as an average of four determinations on each solution, the variation on each being as great as those between the different solutions. When the substance contains much soluble phosphoric acid or other material which gives a heavy precipitate with ammonia, the greater the dilution t h e more accurate the result in general, because of t h e occlusion of potash by gelatinous precipitates. If the volume in which precipitation takes place is larger there is a smaller proportion of the potash in t h e precipitate. By making the dilution I in 1000 instead of the usual I O in 500, correct results were obtained as shown under “Soluble Phosphates” below. Vigor of boiling causes no perceptible variation in result. Portions heated on a Steam bath, boiled gently over a low flame, or boiled vigorously, gave results within the usual limits of error. Time of boiling: Five, ten, thirty or sixty min. boiling gave nearly identical results.
*
Numbers refer to corresponding numbers in “Bibliography.” PP. 496 and 7.
