V O L U M E 20, N O , 4, A P R I L 1 9 4 8 (13) Sandstedt, R. M., Kneen. E., and Blish, M .J., Ibid., 16,712-23 ,,n2n\
353 (17) Windiach, W., and Kolbaoh, P., Wochchr. Rrau.. 38. 149-51
(1921). (18)Ibid.. 42, 1 3 9 4 2 (1925). (19) Wohlgemuth, J., Biochern. Z., 9,I-g (1908).
(16) Wnderkofler, L. A,, Severson. G. M., Goeiing, K. 3.. and Chrie tensen, L. M., Cereal Chem.. 24,l-22 (1947).
R ~ o e r v s oJune 30, 1947. Presented before the Fermentation Section. Division of Agrioultural and Food Chemistry, st the 111th Meeting of the AMERICAN C n e m c ~SOCIETY, ~ Atlantic City, N. .I.
Determination of Amino Nitrogen in Corn
Sirlln
BARRETT L. SCALLET Research Laboratory, Anheuser-Busch, Inc., S I . Loriis, Mo. In the m a n u f a c t u r e of c o r n sirup very small a m o u n t s of a m i n o n i t r o g e n cont r i b u t e considerably t o f o r m a t i o n of undesirable yellow color. The present m e t h o d shows that a modified Van Slyke m e t h o d for amino n i t r o g e n c a n be applied to oorn sirup w i t h good results. A 100-ec. reaction vessel is combined w i t h a 3-ee. m e a s u r i n g buret, p e r m i t t i n g the use of 25-ml. samples. Sugars in the solution have an i n h i b i t i n g effeot on the reaction, b u t this can be overcome by proper selection of the q u a n t i t i e s of reagents. D e t e r m i n a t i o n of amino nitrogen w i t h satisfactory precision is possible i n the range from 10 t o 40 p.p.m.
0.
NE of the greatest problems in the manufacture of corn
srup 1s the development of color in process solutions and in the finished sirup. A portion of this color is formed by the condensation of amino acids and sugars (9, 6, 8). Determination of amino nitrogen in the corn sirup is of value in controlling color formation, but, the small quantities involved (as low as 10 p.p.m.) are difficult to measure by ordinary m-s. Colorimetric methods such as Folin's (3) are strongly affected by the presence of sugars. An additional diffioulty is the selection of a standard for cornparirison; each amino aoid gives a different degree of color with the reagent. A modification of the Van Slyke amino nitrogen method gives good results on corn sirup. APPARATUS
Harrsl (4)combined a mama reaction vessel of a new type with a micro measuring buret, permitting the estimation of very mmll concentrstions af amino acids by the use of a large sample. With a few changes, the Harral apparatus has been used bere.
In Figure 1, A is the reaction vessel, of 100-cc. capacity. B is a 3-ee. measurine buret eraduated in 0.01 cc. and having a reservqir bulb of about &cc. cabscity. F is the leveling bulb f i r the buret; E is the leveling battle for the reaction chamber. C is a threeway stopcock leading through stopoock D to B or directly to H , t,he inlet, i is~ the ..~ . .t,nhe. ~ ~ T~ . air tran nrotectine A . Thc lower end of the reaction vessel is surroundid by B h e a h g element consisting of a closely fitting copper cylinder wrapped with resistance wire surrounded by asbestos. The ourrent is regulated by a rheostat (not shown) and switch S. ~~~~
~
REAGENTS
Saturated sodium nitrite was made by dissolving 80 grams of sodium nitrite sticks in 100 ml. of hot water, cooling, and decanting. The alkaline permanganate was made by dissolving 50 grams of potassium permanganate and 25 grams of sodium hydroxide, and making them up t.o 1liter. The amino acids used as standardr were obtained from the Eastmsn Research Laboratories. &Leucine was used without further purification. d-Glutamic acid was recryst,allizcd from water. Its melting point was then found to be 199-200" C. The reported value is 199" C. (0These acids were dried for 1 hour at 100" C. and 29-inch Vacuum, cooled, weighed, dissolved, and made up to volume. Aliquots were used for the determinations. PROCEDURE
Figure 1. Modified Amino Nitrogen Apparatus
After d l air in the apparatus is replaced by mercury, the sample and acetic acid are drawn into the reaction chamber, where they are shaken and heated until suhstmtially air-free. The air is removed; sodium nitrite is drawn in and mixed with the solution by shaking. The reaction is allowed to proceed for 30 minutes, after which the mixture of nitrogen and nitric oxide is removed to the measuring buret. The reaction chamber is then cleaned, and alkaline permanganate is introduced and made airfree. The gases me farced back into the reaction chamber, where the nitric oxide is rapidly absorbed, leaving only nitrogen, which is then forced back into the gas buret and measured. The st,apcocks are carefully greased and the system is rinsed thoroughly with water by letting i t in at H and driving i t back and forth between A aud B by means of leveling bulb E. The water is famed out at H and mercury is made to fill the reaction vessel, the gas buret, the connecting capillary, and the entrance capillary, H . Tbe sample (25 ml.) is drawn in at H from a small beaker, which is washed twice m t h 5-ml. portions of water; these are drawn in also, followed by 4 ml. of glacial aoet,icacid and finally by 1 ml. of water from small graduated tubes. The total volume is always kept. at 40 ml. With C closed bulb E is lowered until the solution is surrounded by the heating element, leaving a vacuum space above t,he solution. The current is turned on and the solut,ion is warmed and shaken. After a few minutes E is raised and the removed air shows up as a bubble above the solution. This is forced out the
354
ANALYTICAL CHEMISTRY
Table I. Reaction Time Min. 10 30 30 30 45
Effect of Reaction Time on Recovery of Amino Nitrogen from Glutamic Acid Nitrogen Taken
Nitrogen Found
Recovery
Mg. 0,880 1.760 0.880 1.596
i%lQ.
70
0.657 1.680 0,848 1.520 1.517
74.3 95.5 96.0 95.3 95.0
l.5Q6
stopcock and the process is repeat,ed until only a very small bubble of air esists above the solution. This takes 20 to 30 minutes. The solution is allowed to cool to about room temperature and the sodium nitrite solution (4 ml.) is drawn in from a graduated tube, care being taken to admit no air. The nitrite in the capillary is not washed in. The solutions are shaken until thoroughly mixed, then bulb E ia left at t,he upper level and the reaction is allowed to go on for 30 minutes. The gas is then forced over into B by adjust.ing C and D and raising R. Cis closed and E is lowered; more gas is shaken out and passed over into B; this is repeated until very litt'le gas comes out of the solution. C is then turned SO that the solution is removed through H . A is cleaned by rinsing with t8hreeportions of water, and 8 ml. of permanganate solution are drawn in and freed from air. When all the air is out, A and B are connected and E is lowered. The gas is drawn over into A and absorbed by shaking. When no more gas will dissolve, A and B are connected again and the permanganate is forced up to stopcock C, which is then closed while the permanganate is boiled out. C is again opened and E raised, forcing permanganate through the capillary t'o D. By careful manipulation the hole in D can be filled n-ith permanganate without allowing any to come through into B. D is then closed and the mercury in level bulb F is brought to the same level as that in B. The volume of gas, thc temperature, and the barometer reading are taken. A blank must always be run on the reagents. CALCULATIONS
St'andard tables ('7) give the weight of amino nitrogen corresponding to 1 cc. of nitrogen gas at 11' to 30" C. and 728 to 772 mm. pressure. I t is only necessary to multiply the figure obtained from the table by the volume of nitrogen found to get the weight of amino nitrogen in the sample. EXPERIMENTAL
Optimum reaction time was determined for a solution of glutamic acid, using 2 ml. of saturated sodium nitrite and 4 ml. of glacial acetic acid. The results (Table I) showed that a 10minute reaction period was too short'and a 45-minute period unnecessarily long. A standard reaction time of 30 minutes was decided upon, and further trials were made to improve the determination. A solution of &leucine, run under conditions similar t o those for glutamic acid, yielded only the same amount of nitrogen (Table 11). These results indicated that the incompleteness of reaction was not due to the amino acid, but to the conditions of analysis. I t was recognized that the presence of corn sirup in the reaction mixture would probably have an inhibiting effect on the reaction. In order to evaluate this effect, determinations were made on corn sirup and a mixture of corn sirup with standard glutamic acid solution under the same conditions. Corn sirup in the mixture reduced the recovery of amino nitrogen from 95 t o 69% (TableII). Accordingly, the acid addition was increased to 10 and then 15 ml., with corresponding increases in recovery to 97 and 92%. At the highest acid concentration a heavy white material, probably a mercury salt, appeared and seemed to interfere with the reaction. The acid concentration was reduced to its original value and the nitrite addition was increased to 4 ml. Under these conditions the reaction went to completion even in the presence of corn sirup. The slightly high results were probably due to a very slight amount of air not removed from the sample solution a t the beginning of the analysis.
Table 11. Effects of Quantities of Reagents and Presence of Corn Sirup on Recovery of Added Amino Nitrogen Sample
Glacial Acetic hcid
JIZ.
Sodium Xitrite .lfL
dl-Leucine
4 4
2
Solution l a Solution 1 plus glutamic acid
4
2
4
2
Glutamic acid Solution 2 Solution 2 plus glutamic acid
10 10
2 2
10
2
Solution 3 Solution 3 plus glutamic acid Solution 4 Solution 4 plus glutamic acid Glutamic acid Solution 4 Solution 4 plus glutamic acid Solution 5 Solution 5 plus glutamic acid a
2
15
2
15
2
15
2
15
2
4
4
4 4
4
4
4
4
4
4
Nitrogen Taken Mg.
Sitrogen Found Recovery
%
1 ,544 1 ,644
.MQ. 1.439 1.462
. .
0.408
,
0.932
69
+ 0.760
0.408
.p 0.293 + 0.760 1
93.2 94.7 ,.
1.069 0,293
100.9
1.0'29
97.0
, , .
?
0.30
...
0.30
+ 0.53
0.80
95
0.21
+ 0.53
1.:6 0.25
+ 0.33
0.52
+ 0.54
0.21
..
0.68
89
1 Oi
101
0 25
..
0 79
102
0.52
..
1.06
100
These solutions wer.3 corn sirup process liquors a t about 22' BB.
DISCUSSION
The experiments show that an adaptation of the Tan Slykr amino nitrogen method can be used for the determination of very small amounts of amino nitrogen in corn sirup liquors. Although several amino acids yield high results by this method ( 5 ) ,these are either absent from or present in amounts less than 2y0 in corn proteins. Glutamic acid and leucine account for thr major portion of corn proteins, and these react normally. Ammonia nitrogen interferes, but it can be removed with Permutit before analysis. The Van Slyke manometric apparatus can also be used in the analysis, but the effect of sugars has not yet been worked out. The error in the determination is about *0.02 cc. of nitrogen p.as, which amounts to *l% when 2 cc. (40 p.p.m.) are obtained and about =t4% when 0.5 cc. (10 p.p.m.) is obtained. This is about the same precision as that of the Kjeldahl method for total nitrogen at similar concentrations. The tendency towards slightly high results was also found by Van Slyke in some cases; his results on glutamic acid were high by about 1% in the macromethod (9) and high on leucine by about 0.2% in the micromethod (IO). CONCLUSIONS
Amino nitrogen can be determined in corn sirup liquors by a modified Van Slyke method with a precision of *l% when 40 p.p.m. are present and of *4y0 when 10 p.p.m. i r e present. LITERATURE CITED (1)
Albertson, N, F., and Archer, S., J . Am. Chem. Soc., 67, 2043 I1 04.5). \ - - - - I .
ag. Chem., 21,47 (1929). (2) Ambler, J. 9.,I n d . Eng. (3) Folin, O.,J.Biol. Chen., 51,377 (1922). 527 (1931). (4) Harral, J. C., A n a l y s t , 56,527 (5) . . Kendrick. -4. B., and Hanke, M. E., JI . Biol. Chem., 117, 161 (1937). (6) Maillard, L.C.,Compt. rend., 154,66(1912). (7) Schmidt, C. L. A. (ed.), "Chemistry of the Amino Acids and Proteins," p. 202.Baltimore, C.C.Thomas, 1938. (8) Schubert, M.P.,J . B i d . Chem., 130,601 (1939). (9) Van Slyke, D. D., Ibid., 9,185(1911). (10) Ibid., 23,407 (1915).
RECEIVEDSeptember 4, 1947.
