Modified Van Slyke Method for Microquantitative Determination of Aliphatic Amino Groups ALLEN S. HUSSEY'
AND
JOHN E. 41.4URER
Northwestern University, Eranston, I l l .
HE Van Slyke procedure for the determination of aliphatic Tamino groups on a micro scale has been modified by the use of carbon dioxide as a sweep gas and a chromic acid-alkaline permanganate absorption train to remove oxides of nitrogen. The Van Slyke procedure ( 4 )for aliphatic amino nitrogen analysis uses the oxides of nitrogen generated by the action of acids on sodium nitrite t o sweep air from the apparatus. The nitrogen subsequently generated from the sample by the reaction RXH2
+ HOSO
--j
ROH
+ N, + H20
is swept by excess oxides of nitrogen into a modified Hempel pipet where the oxides are absorbed by alkaline permanganate. The residual nitrogen is measured in a special gas buret. Since high blank values (0.20 to0.30 ml.) areobtained by this procedure, a sample large enough to liberate more than 2 ml. of nitrogen must be used to ensure adequate precision. Errors are introduced because of the difficulty in ensuring complete exclusion of air, complete transfer of nitrogen to the measuring buret, and ?omplete removal of the oxides of nitrogen and because of the fouling of the gas buret by manganese dioxide. These errors reduce the precision of the determinations.
IG
of the reagents and of the aqueous or dilute acid solution of the sample as well as the draining and rinsing of the flask without its removal from the train. The chromic acid absorber (Figure 3) is filled to within 3 cm. of the top with a saturated solution of potassium dichromate in concentrated sulfuric acid. The alkaline permanganate absorber is filled to the top of the spiral (Figure 4) with a solution containing 10 grams of potassium permanganate dissolved in 100 ml. of saturated sodium bicarbonate solution. The micronitrometer and rubber connectors used are those described for the Dumas nitrogen microanalysis train ( 2 ) . PROCEDURE
The train, with freshly filled absorber tubes, must be swept out with carbon dioxide for 30 to 60 minutes in order to remove dissolved air. This is accomplished in the following way: Stopcocks B, C, and D are closed, and stopcock A is opened for a moment to place the system under a slight pressure of carbon dioxide. With A closed, D , E, and F a r e opened in that order to vent the system to air a t F . Stopcock A is then adjusted so that a slow stream of carbon dioxide passes through the train. The nitrometer is filled by opening stopcock G and raising the leveling bulb. The complete removal of air from the system is indicated by the formation of microbubbles when F is adjusted to divert the gas stream to the nitrometer. Bfter having been swept free of dissolved air, the absorbers remain nearly free of nitrogen and oxygen if D , E, and F are kept closed when the apparatus is not in use. When microbubbles are obtained, the system is tested for leaks by closing stopcock A and lowering the leveling bulb of the nitrometer. A leak is indicated by a continuing stream of bubbles into the nitrometer. zmm M /P\ TUBING
CHROMIC
REACTION
AB SORPTION TUBE
ALKALINE
I I K
FL AS K
PERMANGANATE ABSORPTION TUBE
Figure 1. Modified Van Slyke .4mino Nitrogen Apparatus I n the present modification, carbon dioxide from a dry ire generator is used to sweep air from the reaction flask, the reagents, and the chromic acid-alkaline permanganate absorption train. The nitrogen, liberated after the addition of the sample, and the oxides of nitrogen are swept through the absorption train with carbon dioxide. The absorbers remove the oxides of nitrogen. The nitrogen and the carbon dioxide pass t o a micronitrometrr where the nitrogen is collected over 50% potassium hydroxide as in the Dumas microdetermination of nitrogen ( 2 ) . The chromic acid absorber removes the bulk of the excess oxides of nitrogen and greatly prolongs the life of the permanganate absorber. The entire procedure requirea 20 minutes, excluding sample preparation time. The blank values are consistent and small (0.020 to 0.030 ml. 1. APPARATUS
The apparatus is shown in Figures 1 to 4. Finely powdered dry ice packed in a 1-quart Dewar flask provided with a Hershberg valve ( 1 ) serves as a convenient source of carbon dioxide. One filling lasts for 6 days. Three hours after filling, the carbon dioxide blank for a 5-minute sweep should be 0.002 to 0.005 nil. The design of the reaction flask (Figure 2) permits the addition
*
Present address, The Carwin Co., K o r t h Haven, Conn.
FRONT
VIEW
Figure 2.
RlGblT SIDE VlEW
Reaction Flask
For the determination of the blank or the analysis of a sample, stopcocks B and C are closed and D is turned to divert carbon dioxide to the air. Stopcock A is adjusted so that a slow stream of gas passes through the reaction flask. A 6-ml. portion of a 307, sodium nitrite solution (prepared with boiled, distilled water and allowed to stand 24 hours before use) and 3 ml. of boiled, glacial acetic acid are added through stopcock C. The inlet tube is rinsed with 1 ml. of boiled, distilled water, and stopcock C is closed in such a way that the bore of the stopcock and the tube leading to the reaction flask are kept completely filled with water. This is necessary to prevent the introduction of air when the solution to be analyzed is added later. The reaction flask is 1642
V O L U M E 2 4 , NO. 10, O C T O B E R 1 9 5 2 shaken vigorously by hand for 15 seconds and allowed to stand for 3 minutes before a second shaking. During this interval and for an additional 3-minute period, the reaction flask is swept with a slow stream of carbon dioxide which is vented a t stopcock D. The gas stream is now diverted through the train into the nitrometer, and within 4 minutes (10 minutes from the time the reagents were added) only microbubbles should be visible. The reaction flask, reagents, and absorption train are now completely free of air.
mm. C A P
1643
The reaction flask is now shaken vigorously for 15 seconds and allowed to stand for 5 minutes before a second shaking. A 5minute sweep with a slow stream of carbon dioxide (2 to 4 bubbles per second through the bubble counter) gives a blank of 0.020 to 0.030 ml. of gas in the nitrometer or carries over all of the nitrogen evolved from the analytical sample. For optimum precision, the sample should be of sufficient size to liberate 0.4 to 1.2 ml. of nitrogen. A piece of No. 10 or 15 gage iron wire about 1 cm. long placed on top of the mercury in the nitrometer can be manipulated by means of a strong magnet so as to dislodge small bubbles of gas which adhere to the surface of the mercury or to dislodge bubbles of gas or liquid trapped in the graduated portion of the nitrometer ( 3 ) .
Table I.
Determination of Amino Nitrogen in . h i i n o Acids
Compound &Alanine arginine mono. hydrochloride
20-22 nm. 0.D.
Figure 3.
Chromic Acid Absorption Tube
Stopcock D is opened to vent the gas stream to the air, and the nitrometer is rinsed and refilled However, before a blank determination is made, a small amount of air is introduced into the nitrometer, and the volume is accurately determined and recorded. This is necessary in order to read the extremely small volume of the blank by difference. Immediately after stopcock A is closed, the solution to be analyzed is added through stopcock C‘ in such a way that no air is introduced. For a blank determination 2 ml. of boiled, distilled water is added; for an anal& 1 ml. of the solution of the sample in boiled, distilled water or in boiled, dilute ac4d is added; and the inlet tube is rinsed with 1 ml. of boiled, distilled water. Immediately after the addition of the mater, stopcock D is turned $0 that the gas stream passes through the train into t h r nitrometrr.
2 m m CAP STOPCOCK
20-22 m
Wt. of Vo!. Sample, of 3 2 , Mg. MI, 2.63 0.773 0.765 0.777 3.23
L-Glutamic acid
3.23
L-Leucine
3.22
m-Serine
2.96
DL-Threonine
2.82
2.74
0.391 0.399 0.396 0.415 0 396 0.388 0.41fi 0.575 0 568 0.617 0.625 0.626 0.653 0,726 0.716 0.617 0.624 0.611 0.621 0.617 0.625
Vol. of Blank,
%N
% N Theory 15.72
%N Range 15.6-15 8
6.2 6.4 6.4 6.6 6.4 6.3 6.6
6.65
6.55-6.7
Y.5
9.52
9.4-9 6
10.68
10.5-10 Y
13.33
13.2-13 4
11.76
11 5-12.0
11.96
11.8-12.1
Found 0.031 15.7 0.030 15.6 0,030 15.9 111.
0.028 0.029 0.029 0.029 0.029 0.029 0.029 0 023 0 023 0.029 0.029 0.028 0.028 0,024 0.024 0.028 0.028 0 028 0.028 0.024 0.024
9.4 10.4 10.5 10.3 10.8 13.2 13.0 11.6 11.7 11.5 11 7 12.1 12.2
During the &minute interval required for the potassium hydroxide solution to drain before the volume of gas in the nitrometer can be measured, stopcock D is turned to vent the reaction flask to air. The reaction flask is drained through stopcock B and is rinsed two or three times nith boiled, distilled water. The nitrite and acetic acid solutions are then added and the second analysis is started. CALCULATIONS
r
The per cent of nitrogen in a given sample is calculated i11 the usual way for a gasometric determination of nitrogen ( 2 ) except that the factor 1/2 must be introduced, since half of the nitrogen i i derived from the nitrite solution. The observed volume of nitrogen is corrected for drainage and vapor pressure of the 50% potassium hydroxide solution by subtracting 1.1% of the ohserved volume ($), and for incomplete absorption of carbon dioxide and oxides of nitrogen by subtracting the blank value.
3 SUPPORTS ON EACH END
10-12 mm
!There V, Vb
= observed volume of nitrogen in ml. = observed volume of blank in ml.
Wty, = weight of 1 ml. of nitrogen a t the temperature and pressure of the observation in mg.
Wt,
= weight of sample in mg.
The calculation can be carried out most conveniently by using logarithms, since the logarithms of the weights of 1ml. of nitrogen a t various temperatures and pressures are given in “nitrogen reduction tables” ( 2 ) . RESULTS
Figure 4.
Alkaline Permanganate Absorption Tube
Representative analyses of several amino acids are given i n Table I. The acids analyzed were purchased from Nerck & Co., Inc., and were given no treatment prior to analysis other than
ANALYTICAL CHEMISTRY
1644 drying under vacuum. The 1% nitrogen range given in the last column of Table I is that stated on the labels of the samples. Blank values for this procedure increase gradually as the time of reaction is increased; after 30 minutes they are approximately double the 5-minute values. Consequently, the precision of the determination decreases somewhat for compounds which require more than 10 minutes for the liberation of all of their amino nitrogen under the conditions present in the reaction flask. 4CKNOWLEDGMEYI
The authors gratefully arknodedge the assistance of D.
Warren Stanger, whose comments and suggestions were especially helpful. LITERATURE CITED
(1) Hershberg, E . B., and Kellwood, G.
W.,ISD. E S G . CHEX.,ASAL. ED.,9,303 (1937). (2) Xiederl, J. B., and Niederl, V., “Micromethods of Quantitative Organic Analysis,” 2nd ed., pp, i9-99. 301-10, S e w York, John TViley &- Sons, 1942. (3) Pomatti, Renato, 1x11. ENG.CHEM.,-2x.k~.ED.,18, 63 (1946). (4) Van Slyke, D. D., J . B i d . Chem., 9, 185 (1911): 12,278 (1912).
RECEIVED for review X a y 28, 1952.
Accepted June 17, 1952.
Determination of Heavy Metals and Silica in Chromate-Treated Cooling Waters SIDYEY SUSSMAN AND IRVING L. PORTNOY r a t e r Service Laboratories, Znc., ‘Yew York 27, h’.Y .
N T H E analysis of chromate-treated cooling waters, the color
I and chemical reactivity of the chromate increase the difficulty of analyzing for certain ions. In some cases, it is possible to remove this interference by reduction of the chromate ion to chromic ion, precipitation of the hydroxide, and separation of the precipitate by filtration. This process is laborious and timeconsuming. In recent years, ion exchange resins have proved useful tools for separating various components of solutions prior to analysis ( 2 ) . I t appeared likely that ion exchange could be utilized for the separation of chromates prior to analyzing cooling waters for silica and for heavy metals. This proved correct, and the authors have been able to develop procedures which are simple, rapid, and sufficiently accurate for the ordinary requirements of water treatment control. SEPARATION OF HEAVY METALS
It is often necessary to determine corrosion products, principally iron and copper, in treated cooling waters. Removal of these heavy metals from chromate-containing samples can be effected by passing the sample through a column of acidregenerated cation exchanger. The heavy metals, retained on the euchanger, can be recovered by elution with an acid.
+ C U + ++CUR + 2 H + CUR + 2 H + +HlR + CU”
H2R
minute followed by a distilled water rinse a t the same flow rate until the effluent became light pink-yellow to methyl orange indicator. The tube was now fully regenerated. This operation is necessary only when utilizing new ion exchange resin. In normal use, the acid elution of the heavy metals from the ion exchanger also regenerates the resin. To carry out a separation, a 50-ml. water sample was passed through the bed a t 10 ml. per minute. Distilled water was then introduced until the effluent was no longer yellow, and the effluent to this point was discarded. Twenty-five milliliters of 15% hydrochloric acid were then passed through the exchanger a t 10 ml. per minute followed by a 50 m1.-portion of distilled water to flush the acid through the ion exchanger. The effluents of both acid and rinse portions were combined. One milliliter of concentrated nitric acid was added to the combined effluent prior to evaporating to dryness. The residue was treated with 2 ml. of 3 to 2 hydrochloric acid and warmed to bring it into solution. Distilled water was added. The solution was boiled, cooled, and made up to 50 ml. This solution was used for determining the heavy metals by the usual procedures (1). With the small portions of the water handled, it was not necessary to backwash the exchanger bed for each sample. However, it is desirable to backwash with distilled water from time to time in order to prevent the accumulation of a dirt layer a t the top of the exchanger bed.
Table I.
(1)
Effect of Separation Procedure on Iron and Copper Content of Cooling Waters
(2)
Small amounts of organic matter, presumably leached from the exchanger, tend to interfere with the normal analytical determinations for the heavy metals in the acid eluate. The interference can be removed by evaporating the effluent solution to dryness, with addition of a small amount of nitric acid or other oxidizing agent. The residue is then dissolved with hydrcchloric acid and diluted as required for the usual analyses for the heavy metals. Apparatus and Reagents. A borosilicate glass tube 17 mm. in inside diameter X 300 mm. long was used as the exchange tube. This was equipped with a thin pad of glass wool to support the ion exchange resin, a small diameter outlet tube, and an S-trap t o prevent draining of the liquid in the tube below the upper surface of the ion exchanger. A screw clamp, inserted in a rubber section between the bottom of the ion exchange tube and the outlet of the S-trap, permitted regulation of the flow rate. An enlarged diameter section a t the top of the exchanger tube or a funnel set in a stopper inserted in top of the tube provided storage capacity for feed solutions. The cation exchange resin used in this work was Dowex-50 (Nalcite HCR, National Aluminate Corp., Chicago). This was used in the usual small spheres furnished for water conditioning. Presumably any other sulfonated polystyrene-type cation exchange resin could be used equally well for this separation. Procedure. Twenty-five milliliters of 15% hydrochloric acid were passed through the ion exchange tube a t 10 to 12 ml. per
Iron
Copper
Original Sample, P . P . X . Fe 0 8 9.5 10.5 11.5 12 13 13.5 14.5 17 23 31 P.P.M. c u
After Separation of 500 P.P.M. NalCrOl, P.P.M. Fe 0 8 9: 10.5 11.5 12 11 13.6 10.5 13.5 19 29.6 P . P . M . Cu
n
n
6.7
0.9 2.4 4.2
2.2
4.2
Experimental Results. Typical experimental data are presented in Table I. In these examples, aater samples containing varying concentrations of iron and copper \\*ere analyzed by the usual procedures ( I ) , and sodium chromate was added to additional portions of the same samples to a 500 p.p.m. concentration. These chromate-containing samples were then passed through the ion exchange separation described above and analyzed for the heavy metals. Agreement between the results obtained with the original samples and those obtained after the
