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Ultramicrodetermination of nitrogen in organic compounds
MICROCHEMICALJOURNAL
21, 445-457 (1976)
Ultramicrodetermination of Nitrogen Compounds
in Organic
VII. Determination of Total Nitrogen in Dilute Aqueous Solution by Sealed-Tube Method KEIKICHI
MIYAHARA
AND
TOMO
TAKAOKA
Shionogi Research Laboratory, Shionogi & Co., Ltd., Fukushima-ku, Osaka, 553 Japan Received
June
14, 1976
To determine traces of nitrogen in dilute aqueous solution, three methods have been generally used (4). The first method involves extractive percolation of the liquid sample through sulfuric acid on a carrier, followed by Kjeldahl digestion of the nitrogen-containing concentrate 2,141; the second is oxy-hydrogen combustion, whereby the nitrogen compound of the liquid is initially converted into nitrogen oxides, and later into ammonia by Devarda reduction (1,3) ; and the third is catalytic hydrogenation, followed, by microcoulometric titration of the ammonia formed (9,15). In these cases, the final reduction product is ammonia. The conventional Dumas method is limited to samples containing at least 30 ppm of nitrogen, determined by Oita modification of the Coleman Nitrogen Analyzer (13). Kirsten and Hozumi employed a sealed tube containing a mg/ml of organic compounds to determine the nitrogen contents of organic compounds in aqueous solution (9). We are convinced that after a few modifications of this method, dilute aqueous solutions with nitrogen levels from 1 to 70 ppm can be analyzed. The method was simple and required no special apparatus, and the results agreed reasonably well with known values. APPARATUS
AND REAGENTS
Electrolytic Oxygen Generator This apparatus was the same as that used in a previous study. (10). Combustion Tube The tube was prepared, except for the closed end, as in the previous study.’ One end of the tube widened out into a ball of 2-ml capacity with a fine tip for receiving the solution, as shown in Fig. la.
445 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form resewed.
446
MIYAHARA
AND
TAKAOKA
Nitrometer
A suitable nitrometer, described previously1 (12), was used according to the volume of produced nitrogen. Silica
Wool
The wool was warmed with fuming nitric acid and rinsed with water until the rinsing was free of nitric acid, then dried. Next, the wool was heated in a current of air for 3 hr at 750-800°C and kept in a clean bottle with a stopper. Diluted
Sulfuric Acid
A drop of G.R. sulfuric acid was added to 1000 ml of distilled water. Copper
Wire Gauze
Copper wire gauze of 50 mesh was cut into small pieces of 10 x 10 mm* and formed into rolls 3.5 mm in diameter. The roll was treated as described elsewhere (5). Copper
Wire
Copper wire (gauge, 45-B.S. ; 100 mg) was formed into a ball which could be introduced into the combustion tube and treated in a manner similar to copper wire gauze. METHODS
OF ANALYSIS
Sampling
A sample solution (0.5- 1.O ml.) was measured into the combustion tube by plain pipet or by weighing the amount in the tube. It was freeze-dried or evaporated under reduced pressure (see Fig. 1). To complete freezedrying in l-2 hr, the formation of a frozen l-mm layer of the solution was necessary. In this case, the freezing was done as follows. The position of the widened ball of the combustion tube holding a solution was immersed into the freezing mixture of acetone-dry ice and the tube was rotated by hand or an electric drill. The frozen layer formed a great circle on the spherical internal surface, as shown in Fig. la. Figures lb and lc are cases of evaporation under reduced pressure. The times required to evaporate 1 ml of water at 2 mm Hg were 95 and 65 min in cases a and b, respectively. In case c, quantitative drying frequently could not be carried out by bumping. Thus, freeze-drying is highly recommended to desiccate liquid samples. Many liquid samples can be treated if the apparatus has polyconnecting tubes, which are employed in biological applications (7). Procedure
As shown in Figs. 2a-2e, the sample was desiccated to a dry powder capable of being restored to its original state, then burned in a sealed tube with pure oxygen and a small amount of copper wire gauze. Nitrogen was 1 See “Ultramicrodetermination
of nitrogen
in organic
compounds.
V.”
DETERMINATION
a FIG.
1 Freeze-drying
OF TOTAL
b
(a) and evaporating
447
NITROGEN
c under
reduced
pressure
(b, c).
collected above 50% potassium hydroxide, and its volume was measured with the nitrometer as described previously (12,12). In the case of freezedrying, the sample became a light powder and could be lost by suction between the primary and secondary drying stages. To prevent this, silica wool or copper wire was used in the combustion tube (see Figs. 3a and 3b). The ball of copper wool frequently fell into the widened ball of the combustion tube when the drying apparatus was shaken. Therefore, the combustion tube was constricted on this side of the widened ball (see Fig. 3b). An alternative method of adding a drop of diluted sulfuric acid to the sample solution was also tried (see Fig. 3~). In this case, the sample remained damp even after drying. Blank Test Table 1, columns A show the blank values with 1 ml of water, a 5-mm layer of silica wool, and copper wire gauze; columns B show the blank values with 1 ml of water and 100 mg of copper wire, which were used to reduce nitrogen oxides and absorb excess oxygen; and columns C show them with 1 ml of water, a drop of diluted sulfuric acid, and copper wire gauze. The water had been passed through an ion exchange resin. We found that the values of B and C were about one-third that of A. Calculation Nitrogen present in the sample was calculated as previously described.’
448
MIYAHARA
sample
silica /
AND
TAKAOKA
0.7 to 0.75cm we.11 0.6 to o..amm 1 1 (a) t
wool
em 2n.l
(b)
(d)
FIG.
2 Sweeping
and sealing
of the combustion
water(l
tube and collecting
of nitrogen.
ml) I
a copper silica
gauze
roll
wool
copper
wire
(100
mg j I
b \
copper
,O.OZ%
wire
H,S04
sohil
ml I I
C
2ml
- ‘Copper
FIG. 3 Blank test: (a) with frozen water, frozen water and copper wire; (c) with frozen
gauze
roll
silica wool, and a copper gauze roll; (b) with 0.02% H?SO, solution and a copper gauze roll.
DETERMINATION
OF TOTAL
TABLE BLANK
1
VALUES
Aa
B”
Ca
PI
pm (w/v)
4
mm (w/v)
0.55
0.14
0.16
0.46 0.30 0.45 0.33
0.10 0.12 0.11
0.11 0.14 0.12
5
0.48 0.40 0.26 0.39 0.28
0.13
Mean
0.36
0.42
0.12
Experiment 1 2 3 4
449
NITROGEN
4
wm (WM
0.11 0.10
0.13 0.12
0.14
0.16
0.15
0.10 0.14
0.12 0.16
0.14
0.12
0.14
cLA, B, and C: The data correspond to Fig. 3a, b, and c, respectively.
RESULTS AND DISCUSSION
Table 2 shows the results for aqueous solutions of methionine and picric acid with 35-70 ppm nitrogen; silica wool was used to prevent loss of sample. The results calculated from the sample solution taken by weighing agreed fairly well with those from the sampling by plain pipet. We found that sampling of dilute solutions of this level by plain pipet was satisfactory. The standard deviation of relative error was 0.982% for the results. With silica wool, we frequently needed to float a nitrogen microbubble at the position of the silica wool using ultrasonic apparatus. Table 3 shows the results for aqueous solutions of several organic compounds containing less than 10 ppm nitrogen with a drop of diluted sulfuric .acid in the sample solution. The results obtained by this method had a standard deviation of relative error of 3.78%. Table 4 shows the results for aqueous solutions of several organic compounds containing less than 10 ppm nitrogen and copper wire packing. The results obtained by this method had a standard deviation of relative error 1.38%. Comparison of the results obtained between the Kjeldahl and the sealed tube methods on nitrogen contents of various drinks are shown in Tables 5-8. In the Kjeldahl method, the sample size was 0.5 ml, and the results obtained were calculated as the value of 1 part in 50 or 100 of N found. In the sealed-tube method, the samples were diluted to 50 or 100 times with water because they contained much carbohydrate, which was very difficult to bum out. The results obtained by our method, using a drop of added diluted sulfuric acid, agreed very closely with those obtained by the Kjeldahl method. However, the results obtained using copper wire were slightly low by comparison. This seemed to be caused by loss of sample through the copper wire. Thus, using a drop of diluted sulfuric acid to prevent loss due to flying is recommended.
Picric
acid
Methionine
Sample
OF NITROGEN
a Mean of relative error: -0.54% * Sample solution was weighed. r Sampled by plain pipet.
8
7
6
5
4
3
2
1
Experiment
ANALYSES
TABLE
2
(-0.53%).
383.6 (383.4) 383.6 (383.4) 332.5 (333.7) 332.9 (333.7)
Standard
370.2b (372.3)c 374.5 (372.3) 374.5 (372.3) 372.0 (372.3)
Sample in l-ml solution (CLg)
deviation
26.7
26.6
24.6
24.5
24.6
24.5
24.4
24.4
Temperature (“(3
of relative
error:
0.982%
(1.059%).
768.8
52.2
60.2
59.9
768.8
51.9
70.0
769.3
70.0
35.0
35.1
35.1
35.1
Found N @pm)
70.4 (70.3) 70.4 (70.3) 61.0 (61.2) 61.1 (61.2)
34.8 (35.0) 35.2 (35.0) 35.2 (35.0) 34.9 (35.0)
N
NITROGEN
Calculated (wm)
35 TO 70 ppm
769.3
752.0
752.0
752.0
Pressure (mm Hg)
CONTAINING ROLL)~
769.3
60.3
60.3
30.1
30.9
30.9
30.9
N2 volume (ELI)
IN Ayueous SOLWION OF SEVERAL ORGANIC COMPOUNDS (PACKING: SILICA WOOL AND A COPPER GAUZE
-0.57 (-0.43) -0.57 (-0.43) -2.30 (-2.61) -1.47 (-1.63)
(0
(0.29) -0.28 (0.29) 0.29
0.86 (0.29) -0.28
Relative error (%I
)
ii tl F
5
d
E
z
j: 2
R 0
IN
25.6
45.0
45.0
Gly-Gly
24.6 24.8
7.90
7.82
5.82 5.87
n Mean of relative error: -5.36%. Standard deviation of relative error: 3.78%. * Analytical value with solid sample.
17 18
25.5
37.9 37.9
3.89 4.27
Glycine
3.93
25.3 25.4
6.29 6.71 6.64
1.99
1.95
2.31
2.44
3.01
2.43 2.51 2.40
N, volume (PI)
25.2
15 16
34.8 34.8 34.8
24.8 24.9 25.0
Albumin
46.3
46.3 46.3
12 13 14
IO II
Picric acid
37.6
27.4 26.1 26.8
27.5
28.3
38.6
45.0
23.7 27.5 23.6
3
ORGANIC COMPOUNDS H,SO, AND A COPPER
TABLE
Temperature (“Cl
OF SEVERAL DROP OF 1%
30.8 30.8 31.7
37.6
9
ONE
Sample in l-ml solution (Pd
(PACKING:
AQUEOUS SOLU.I.IONS
45.0
D-glycosamine hydrochloride
Methionine
Sample
NITROGEN
I 8
OF
6
5
Experiment
ANALYSES
6.82
771.1
9.17
9.07
770.5
9.43"
9.43b
7.08" 7.0gb
4.99b 4.996 4.95
4.990
4.51
8.49 8.49 8.49
2.92 2.44 2.44
2.92
3.62
2.89 2.89 2.98
-3.82 -2.76
-4.24 -3.67
-0.80
-9.62
-8.42
-9.19
-13.90 -8.24
-5.74
-7.38
-7.88
-2.74
+1.04 -4.70 -4.14
-0.35
Relative error (%I
NITROGEN
Calculated N (wm)
10 ppm
4.57
7.31 7.79 7.71
2.69 2.26 2.30
2.84
3.47
2.88 2.92 2.84
6.78
771.3
THAN
Found N (mm)
LESS
770.5
771.7 771.1
771.1
770.5 770.4 770.4
772.6 772.6
777.6
777.6
772.7
782.3 777.5 782.3
Pressure (mm Hd
CONTAINING GAUZE ROLL)~
35.7
35.7 35.7
34.4 34.4 34.4
26.7
26.8 26.7
26.1 26.2 26.3
4 COMPOUNDS WOOL)~
1.92
I.98 1.96
2.71 2.63 2.63
N, volume b.4
COPPER
(PACKING:
Temperature (“C)
ORGANIC
TABLE OF SEVERAL
(2Mean of relative error: -4.56%. Standard deviation of relative error: 1.38%.
6
5
Methionine
1 2 3 4
SOLUTION
Sample in l-ml solution (wd
IN AQUEOUS
D-glucosamine hvdrochloride
Sample
OF NITROGEN
Experiment
ANALYSES
758.8
758.8 758.8
770.3 770.3 770.3
Pressure (mm Hg)
CONTAINING
THAN
2.19
2.25 2.23
3.13 3.04 3.04
Found N (pw)
LESS
NITROGEN
2.32
2.32 2.32
26. I 26.2 26.3
Calculated N (mm)
10 ppm
-5.60
-3.02 -3.88
-3.10 -5.88 -5.88
Relative error (%I
s *
F
2
E
k
G
10 10 JO
Cu wireY
A
26.9 26.9 26.9
25.0 25.0 25.0
TABLE
5 OF K.JELDAHL
7.01 6.89 6.95
7.24 7.64 7.09
N, volume (PO
Sealed-tube method
BY MEANS
Temperature (“0
ON SAKE
a These treatments are carried out using only the sealed-tube method b Value of 1 part in 100 of found N.
10 10 10
One drop of 1% H,SO,a and Cu gauze
Packing
RESLILTS
Sample in I-ml solution (PI)
ANALYTICAL
758.4 758.4 758.4
770.0 770.0 770.0
Found N (w-d
METHODS
7.96 7.81 7.89 Mean: 7.89
8.40 8.87 8.22 Mean: 8.50
SEALED-TUBE
Pressure (mm Hg)
AND
0.5
0.5 0.5
Sample (ml)
Found N (wm)
method
842 (8.42)6 829 (8.29) 842 (8.42) Mean: 837 (8.37)
Kjeldahl
26.4 26.0 26.1
10 10 10
Cu wire0
o*b See footnotes to Table 5.
6 OF KIELDAHL
7.58 7.59 7.58
8.06 8.13 8.00
N2 volume (/.a
Sealed-tube method
28.2 28.3 28.3
Packing
TABLE BY MEANS
10 10 10
B
One drop of 1% H,SOP and Cu gauze
ON SAKE
Temperature (“C)
RESULTS
Sample in I-ml solution (/.a
ANALYTICAL
770.2 772.1 772.1
772.1 772.1 772.4
Mean:
Mean:
SEALED-TUBE
Pressure (mm Hg)
AND
8.75 8.80 8.79 8.78
9.28 9.36 9.20 9.28
Found N (wm)
METHODS
0.5
0.5
0.5 0.5
Sample 0-4
928 (9.28) 925 (9.25) 928 (9.28)
939 (9.39Y 922 (9.22)
Found N (ppm)
method
Mean:
Kjeldahl
2 F
5
E
z 2 ;
G* See footnotes
Cu wire”
to Table
One drop of 1% H,SO,” and Cu gauze
Packing
5.
RESUL-IX
TABLE
7
3.95 3.88 3.90
26.1 26.1
10 10
10
10 10 10
26.2
(“(2
10
method
OF KJELDAHL
3.96 3.87 4.18 3.98
Sealed-tube
RY MEANS
26.3 26.4 25.7 25.9
BEER
N, volume (/.a
ox
Temperature
Sample in l-ml solution (PO
ANALYTICAL
770.2 770.2 770.2
772.1 112.1 773.9 173.9
Mean:
Mean:
SEALED-TWX
Pressure (mm Hd
AND
4.53
4.58 4.49 4.51
4.64
4.59 4.48 4.87 4.63
Found (mm)
N
METHODS
0.5
0.5
0.5
0.5
Sample (ml)
Kjeldahl
Mean:
N
471 (4.71)
464 (4.64)
(4.73)
476 (4.76y 471 (y;’
(mm)
Found
method
: z
2
i; $ it
2 5
d 5 s 2:
;
w Fi
In
,”
Packing
gauze
of 1% H,SO,”
_“,,
,,?.
,,,,
,
0 See footnote a to Table 5. b Value of 1 part in 50 of found
Cu wird
and Cu
One drop
RESULTS
1,,“,1,
N.
.,,,,,
20
v,.,
21.0 27.3 26.3
23.3
20 20
24.9
-9
(“Cl 25.0
20
,”
COL.~
Temperature
ON Coc.4
20
20
Sample in I-ml solution (PI)
AN.~LYTICAL
/,,
method
1.06 1.08 1.12
1.28
,,,,,>
1.14
1.17
,,,,,
8 OF KJELDAHL
N, volume (111)
-,*,,
Sealed-tube
TABLE BY MEANS
,,,&
768.7 768.7 765.9
771.5
770.0
770.0
,*,,
Pressure (mm Hg)
,,il
,
Mean:
Mean:
*,/,
,,/,
1.25
1.29
1.22 1.25
1.39
1.49
1.32
,.,,,
N
METHODS
1.35
(wm)
Found
AND SEALED-TUBE
,,,”
-,,,
,,“,,,,
Mean:
0.5
0.5
0.5
Sample (ml)
Kjeldahl
,,
Y,,,,
“,”
(1.38)
68
,,,,“.
( 16;8,
(1.3O)b
65
Found (mm)
method
,,”
N
*/,
2 F 2 >
is
$ $
ST ?
/,,,”
./
DETERMINATION
OF TOTAL
NITROGEN
457
In our laboratory, we have used this method for determination of the N/P ratio of phospholipid components and the concentration of synthesized polypeptides, ranging between 5 and lo%, with no band present at about 280 nm (6). SUMMARY With a modified combustion tube having a ball of 2-ml capacity with a fine tip, our sealed-tube method was used to determine the nitrogen contents of nonvolatile organic compounds of dilute aqueous solution with nitrogen levels from 1 to 70 ppm. A drop of diluted sulfuric acid was added to the sample, which was freeze-dried, then burnt.
REFERENCES 1. Gertner, A., and Grdinic, V., Determination of total organic nitrogen in dilute aqueous solution. Acta Pharm. Jugosl. 15, 209-212 (1965). 2. Gouverneur, P., The determination of trace amounts of total nitrogen in petroleum distillates extractive percolation method. Anal. Chim. Actu 26, 212-223 (1962). 3. Gouverneur, P., Snoek, 0. J.. and Heeringa-Kommer. M. Nitrogen determination in mineral oils by means of Wickbold oxyhydrogen combustion. Anal. Chim Acta 39, 413-422 (1967). 4. Gouverneur, P., and Van De Craats, F., Recent method for determining traces of nitrogen in mineral oils. Analyst 93, 782-787 (1968). 5. Hozumi, K., and Kirsten, W. J., New Method for the ultramicrodetermination of nitrogen. And. Chem. 34, 434-435 (1962). 6. Jnoue, H., and Izumi, T., The interaction of poly(N5-(3-hydroxyproply)-L-glutamine) with solvent components in water-dioxane mixture. Biopolymers 15,797-812 (1976). 7. Institute of Medical Science, University of Tokyo, Method of freeze-drying. In “Saikingaku Jissyu Yotei (Handbook Bacteriological Study)” (Y. Tsunematsu, et nl., Eds.), revised ed., pp. 343-357. Maruzen, Tokyo, 1961. 8. Martin, R. L., Fast and sensitive method for determination of nitrogen. Selective nitrogen detector for gas chromatograph. Anal Chem. 38, 1209-1213 (1966). 9. Kirsten, W. J. and Hozumi, K., Gas-volumetric ultramicro and microdetermination of nitrogen and other elements in organic compounds. Mikrochim. Actu 1962,777-786. 10. Miyahara. K., Ultramicrodetermination of nitrogen in organic compounds. 1. Application of interpolation in the sealed tube method. Micro&em. J. 19, 416-422 (1974). Il. Miyahara, K., Ultramicrodetermination of nitrogen in organic compounds. 2. A new simple nitrometer for the sealed tube method. Microchem. J. 19, 423-428 (1974). 12. Miyahara, K., Ultramicrodetermination of nitrogen in organic compounds. 4. Thermal behavior of a gas in a nitrometer due to local variations of temperature. Microchem. J. 20, 453-461 (1975). 13. Oita, 1. J., Direct Dumas determination of 30 to 1000 parts per million of nitrogen in oils. Anal. Chem. 38, 804-805 (1966). 14. Smith, A. J.. Cooper, F. F. Jr., Rice, J. O., and Shaner, W. C. Jr., The determination of trace amounts of total nitrogen in petroleum distillates. Anal. Chim. Actu 40, 341-343 (1968). 15. Tolg, G., Beitrage zur Ultramicro- und Supurenanalyse organischer Stoffe. V. Bestimmung des Stickstoffgehaltes schwertltichtiger organischer Verbindungen mit 5-20 pg Substanz. Z. Anal. Chem. 205, 40-50 (1964).
21, 445-457 (1976)
Ultramicrodetermination of Nitrogen Compounds
in Organic
VII. Determination of Total Nitrogen in Dilute Aqueous Solution by Sealed-Tube Method KEIKICHI
MIYAHARA
AND
TOMO
TAKAOKA
Shionogi Research Laboratory, Shionogi & Co., Ltd., Fukushima-ku, Osaka, 553 Japan Received
June
14, 1976
To determine traces of nitrogen in dilute aqueous solution, three methods have been generally used (4). The first method involves extractive percolation of the liquid sample through sulfuric acid on a carrier, followed by Kjeldahl digestion of the nitrogen-containing concentrate 2,141; the second is oxy-hydrogen combustion, whereby the nitrogen compound of the liquid is initially converted into nitrogen oxides, and later into ammonia by Devarda reduction (1,3) ; and the third is catalytic hydrogenation, followed, by microcoulometric titration of the ammonia formed (9,15). In these cases, the final reduction product is ammonia. The conventional Dumas method is limited to samples containing at least 30 ppm of nitrogen, determined by Oita modification of the Coleman Nitrogen Analyzer (13). Kirsten and Hozumi employed a sealed tube containing a mg/ml of organic compounds to determine the nitrogen contents of organic compounds in aqueous solution (9). We are convinced that after a few modifications of this method, dilute aqueous solutions with nitrogen levels from 1 to 70 ppm can be analyzed. The method was simple and required no special apparatus, and the results agreed reasonably well with known values. APPARATUS
AND REAGENTS
Electrolytic Oxygen Generator This apparatus was the same as that used in a previous study. (10). Combustion Tube The tube was prepared, except for the closed end, as in the previous study.’ One end of the tube widened out into a ball of 2-ml capacity with a fine tip for receiving the solution, as shown in Fig. la.
445 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form resewed.
446
MIYAHARA
AND
TAKAOKA
Nitrometer
A suitable nitrometer, described previously1 (12), was used according to the volume of produced nitrogen. Silica
Wool
The wool was warmed with fuming nitric acid and rinsed with water until the rinsing was free of nitric acid, then dried. Next, the wool was heated in a current of air for 3 hr at 750-800°C and kept in a clean bottle with a stopper. Diluted
Sulfuric Acid
A drop of G.R. sulfuric acid was added to 1000 ml of distilled water. Copper
Wire Gauze
Copper wire gauze of 50 mesh was cut into small pieces of 10 x 10 mm* and formed into rolls 3.5 mm in diameter. The roll was treated as described elsewhere (5). Copper
Wire
Copper wire (gauge, 45-B.S. ; 100 mg) was formed into a ball which could be introduced into the combustion tube and treated in a manner similar to copper wire gauze. METHODS
OF ANALYSIS
Sampling
A sample solution (0.5- 1.O ml.) was measured into the combustion tube by plain pipet or by weighing the amount in the tube. It was freeze-dried or evaporated under reduced pressure (see Fig. 1). To complete freezedrying in l-2 hr, the formation of a frozen l-mm layer of the solution was necessary. In this case, the freezing was done as follows. The position of the widened ball of the combustion tube holding a solution was immersed into the freezing mixture of acetone-dry ice and the tube was rotated by hand or an electric drill. The frozen layer formed a great circle on the spherical internal surface, as shown in Fig. la. Figures lb and lc are cases of evaporation under reduced pressure. The times required to evaporate 1 ml of water at 2 mm Hg were 95 and 65 min in cases a and b, respectively. In case c, quantitative drying frequently could not be carried out by bumping. Thus, freeze-drying is highly recommended to desiccate liquid samples. Many liquid samples can be treated if the apparatus has polyconnecting tubes, which are employed in biological applications (7). Procedure
As shown in Figs. 2a-2e, the sample was desiccated to a dry powder capable of being restored to its original state, then burned in a sealed tube with pure oxygen and a small amount of copper wire gauze. Nitrogen was 1 See “Ultramicrodetermination
of nitrogen
in organic
compounds.
V.”
DETERMINATION
a FIG.
1 Freeze-drying
OF TOTAL
b
(a) and evaporating
447
NITROGEN
c under
reduced
pressure
(b, c).
collected above 50% potassium hydroxide, and its volume was measured with the nitrometer as described previously (12,12). In the case of freezedrying, the sample became a light powder and could be lost by suction between the primary and secondary drying stages. To prevent this, silica wool or copper wire was used in the combustion tube (see Figs. 3a and 3b). The ball of copper wool frequently fell into the widened ball of the combustion tube when the drying apparatus was shaken. Therefore, the combustion tube was constricted on this side of the widened ball (see Fig. 3b). An alternative method of adding a drop of diluted sulfuric acid to the sample solution was also tried (see Fig. 3~). In this case, the sample remained damp even after drying. Blank Test Table 1, columns A show the blank values with 1 ml of water, a 5-mm layer of silica wool, and copper wire gauze; columns B show the blank values with 1 ml of water and 100 mg of copper wire, which were used to reduce nitrogen oxides and absorb excess oxygen; and columns C show them with 1 ml of water, a drop of diluted sulfuric acid, and copper wire gauze. The water had been passed through an ion exchange resin. We found that the values of B and C were about one-third that of A. Calculation Nitrogen present in the sample was calculated as previously described.’
448
MIYAHARA
sample
silica /
AND
TAKAOKA
0.7 to 0.75cm we.11 0.6 to o..amm 1 1 (a) t
wool
em 2n.l
(b)
(d)
FIG.
2 Sweeping
and sealing
of the combustion
water(l
tube and collecting
of nitrogen.
ml) I
a copper silica
gauze
roll
wool
copper
wire
(100
mg j I
b \
copper
,O.OZ%
wire
H,S04
sohil
ml I I
C
2ml
- ‘Copper
FIG. 3 Blank test: (a) with frozen water, frozen water and copper wire; (c) with frozen
gauze
roll
silica wool, and a copper gauze roll; (b) with 0.02% H?SO, solution and a copper gauze roll.
DETERMINATION
OF TOTAL
TABLE BLANK
1
VALUES
Aa
B”
Ca
PI
pm (w/v)
4
mm (w/v)
0.55
0.14
0.16
0.46 0.30 0.45 0.33
0.10 0.12 0.11
0.11 0.14 0.12
5
0.48 0.40 0.26 0.39 0.28
0.13
Mean
0.36
0.42
0.12
Experiment 1 2 3 4
449
NITROGEN
4
wm (WM
0.11 0.10
0.13 0.12
0.14
0.16
0.15
0.10 0.14
0.12 0.16
0.14
0.12
0.14
cLA, B, and C: The data correspond to Fig. 3a, b, and c, respectively.
RESULTS AND DISCUSSION
Table 2 shows the results for aqueous solutions of methionine and picric acid with 35-70 ppm nitrogen; silica wool was used to prevent loss of sample. The results calculated from the sample solution taken by weighing agreed fairly well with those from the sampling by plain pipet. We found that sampling of dilute solutions of this level by plain pipet was satisfactory. The standard deviation of relative error was 0.982% for the results. With silica wool, we frequently needed to float a nitrogen microbubble at the position of the silica wool using ultrasonic apparatus. Table 3 shows the results for aqueous solutions of several organic compounds containing less than 10 ppm nitrogen with a drop of diluted sulfuric .acid in the sample solution. The results obtained by this method had a standard deviation of relative error of 3.78%. Table 4 shows the results for aqueous solutions of several organic compounds containing less than 10 ppm nitrogen and copper wire packing. The results obtained by this method had a standard deviation of relative error 1.38%. Comparison of the results obtained between the Kjeldahl and the sealed tube methods on nitrogen contents of various drinks are shown in Tables 5-8. In the Kjeldahl method, the sample size was 0.5 ml, and the results obtained were calculated as the value of 1 part in 50 or 100 of N found. In the sealed-tube method, the samples were diluted to 50 or 100 times with water because they contained much carbohydrate, which was very difficult to bum out. The results obtained by our method, using a drop of added diluted sulfuric acid, agreed very closely with those obtained by the Kjeldahl method. However, the results obtained using copper wire were slightly low by comparison. This seemed to be caused by loss of sample through the copper wire. Thus, using a drop of diluted sulfuric acid to prevent loss due to flying is recommended.
Picric
acid
Methionine
Sample
OF NITROGEN
a Mean of relative error: -0.54% * Sample solution was weighed. r Sampled by plain pipet.
8
7
6
5
4
3
2
1
Experiment
ANALYSES
TABLE
2
(-0.53%).
383.6 (383.4) 383.6 (383.4) 332.5 (333.7) 332.9 (333.7)
Standard
370.2b (372.3)c 374.5 (372.3) 374.5 (372.3) 372.0 (372.3)
Sample in l-ml solution (CLg)
deviation
26.7
26.6
24.6
24.5
24.6
24.5
24.4
24.4
Temperature (“(3
of relative
error:
0.982%
(1.059%).
768.8
52.2
60.2
59.9
768.8
51.9
70.0
769.3
70.0
35.0
35.1
35.1
35.1
Found N @pm)
70.4 (70.3) 70.4 (70.3) 61.0 (61.2) 61.1 (61.2)
34.8 (35.0) 35.2 (35.0) 35.2 (35.0) 34.9 (35.0)
N
NITROGEN
Calculated (wm)
35 TO 70 ppm
769.3
752.0
752.0
752.0
Pressure (mm Hg)
CONTAINING ROLL)~
769.3
60.3
60.3
30.1
30.9
30.9
30.9
N2 volume (ELI)
IN Ayueous SOLWION OF SEVERAL ORGANIC COMPOUNDS (PACKING: SILICA WOOL AND A COPPER GAUZE
-0.57 (-0.43) -0.57 (-0.43) -2.30 (-2.61) -1.47 (-1.63)
(0
(0.29) -0.28 (0.29) 0.29
0.86 (0.29) -0.28
Relative error (%I
)
ii tl F
5
d
E
z
j: 2
R 0
IN
25.6
45.0
45.0
Gly-Gly
24.6 24.8
7.90
7.82
5.82 5.87
n Mean of relative error: -5.36%. Standard deviation of relative error: 3.78%. * Analytical value with solid sample.
17 18
25.5
37.9 37.9
3.89 4.27
Glycine
3.93
25.3 25.4
6.29 6.71 6.64
1.99
1.95
2.31
2.44
3.01
2.43 2.51 2.40
N, volume (PI)
25.2
15 16
34.8 34.8 34.8
24.8 24.9 25.0
Albumin
46.3
46.3 46.3
12 13 14
IO II
Picric acid
37.6
27.4 26.1 26.8
27.5
28.3
38.6
45.0
23.7 27.5 23.6
3
ORGANIC COMPOUNDS H,SO, AND A COPPER
TABLE
Temperature (“Cl
OF SEVERAL DROP OF 1%
30.8 30.8 31.7
37.6
9
ONE
Sample in l-ml solution (Pd
(PACKING:
AQUEOUS SOLU.I.IONS
45.0
D-glycosamine hydrochloride
Methionine
Sample
NITROGEN
I 8
OF
6
5
Experiment
ANALYSES
6.82
771.1
9.17
9.07
770.5
9.43"
9.43b
7.08" 7.0gb
4.99b 4.996 4.95
4.990
4.51
8.49 8.49 8.49
2.92 2.44 2.44
2.92
3.62
2.89 2.89 2.98
-3.82 -2.76
-4.24 -3.67
-0.80
-9.62
-8.42
-9.19
-13.90 -8.24
-5.74
-7.38
-7.88
-2.74
+1.04 -4.70 -4.14
-0.35
Relative error (%I
NITROGEN
Calculated N (wm)
10 ppm
4.57
7.31 7.79 7.71
2.69 2.26 2.30
2.84
3.47
2.88 2.92 2.84
6.78
771.3
THAN
Found N (mm)
LESS
770.5
771.7 771.1
771.1
770.5 770.4 770.4
772.6 772.6
777.6
777.6
772.7
782.3 777.5 782.3
Pressure (mm Hd
CONTAINING GAUZE ROLL)~
35.7
35.7 35.7
34.4 34.4 34.4
26.7
26.8 26.7
26.1 26.2 26.3
4 COMPOUNDS WOOL)~
1.92
I.98 1.96
2.71 2.63 2.63
N, volume b.4
COPPER
(PACKING:
Temperature (“C)
ORGANIC
TABLE OF SEVERAL
(2Mean of relative error: -4.56%. Standard deviation of relative error: 1.38%.
6
5
Methionine
1 2 3 4
SOLUTION
Sample in l-ml solution (wd
IN AQUEOUS
D-glucosamine hvdrochloride
Sample
OF NITROGEN
Experiment
ANALYSES
758.8
758.8 758.8
770.3 770.3 770.3
Pressure (mm Hg)
CONTAINING
THAN
2.19
2.25 2.23
3.13 3.04 3.04
Found N (pw)
LESS
NITROGEN
2.32
2.32 2.32
26. I 26.2 26.3
Calculated N (mm)
10 ppm
-5.60
-3.02 -3.88
-3.10 -5.88 -5.88
Relative error (%I
s *
F
2
E
k
G
10 10 JO
Cu wireY
A
26.9 26.9 26.9
25.0 25.0 25.0
TABLE
5 OF K.JELDAHL
7.01 6.89 6.95
7.24 7.64 7.09
N, volume (PO
Sealed-tube method
BY MEANS
Temperature (“0
ON SAKE
a These treatments are carried out using only the sealed-tube method b Value of 1 part in 100 of found N.
10 10 10
One drop of 1% H,SO,a and Cu gauze
Packing
RESLILTS
Sample in I-ml solution (PI)
ANALYTICAL
758.4 758.4 758.4
770.0 770.0 770.0
Found N (w-d
METHODS
7.96 7.81 7.89 Mean: 7.89
8.40 8.87 8.22 Mean: 8.50
SEALED-TUBE
Pressure (mm Hg)
AND
0.5
0.5 0.5
Sample (ml)
Found N (wm)
method
842 (8.42)6 829 (8.29) 842 (8.42) Mean: 837 (8.37)
Kjeldahl
26.4 26.0 26.1
10 10 10
Cu wire0
o*b See footnotes to Table 5.
6 OF KIELDAHL
7.58 7.59 7.58
8.06 8.13 8.00
N2 volume (/.a
Sealed-tube method
28.2 28.3 28.3
Packing
TABLE BY MEANS
10 10 10
B
One drop of 1% H,SOP and Cu gauze
ON SAKE
Temperature (“C)
RESULTS
Sample in I-ml solution (/.a
ANALYTICAL
770.2 772.1 772.1
772.1 772.1 772.4
Mean:
Mean:
SEALED-TUBE
Pressure (mm Hg)
AND
8.75 8.80 8.79 8.78
9.28 9.36 9.20 9.28
Found N (wm)
METHODS
0.5
0.5
0.5 0.5
Sample 0-4
928 (9.28) 925 (9.25) 928 (9.28)
939 (9.39Y 922 (9.22)
Found N (ppm)
method
Mean:
Kjeldahl
2 F
5
E
z 2 ;
G* See footnotes
Cu wire”
to Table
One drop of 1% H,SO,” and Cu gauze
Packing
5.
RESUL-IX
TABLE
7
3.95 3.88 3.90
26.1 26.1
10 10
10
10 10 10
26.2
(“(2
10
method
OF KJELDAHL
3.96 3.87 4.18 3.98
Sealed-tube
RY MEANS
26.3 26.4 25.7 25.9
BEER
N, volume (/.a
ox
Temperature
Sample in l-ml solution (PO
ANALYTICAL
770.2 770.2 770.2
772.1 112.1 773.9 173.9
Mean:
Mean:
SEALED-TWX
Pressure (mm Hd
AND
4.53
4.58 4.49 4.51
4.64
4.59 4.48 4.87 4.63
Found (mm)
N
METHODS
0.5
0.5
0.5
0.5
Sample (ml)
Kjeldahl
Mean:
N
471 (4.71)
464 (4.64)
(4.73)
476 (4.76y 471 (y;’
(mm)
Found
method
: z
2
i; $ it
2 5
d 5 s 2:
;
w Fi
In
,”
Packing
gauze
of 1% H,SO,”
_“,,
,,?.
,,,,
,
0 See footnote a to Table 5. b Value of 1 part in 50 of found
Cu wird
and Cu
One drop
RESULTS
1,,“,1,
N.
.,,,,,
20
v,.,
21.0 27.3 26.3
23.3
20 20
24.9
-9
(“Cl 25.0
20
,”
COL.~
Temperature
ON Coc.4
20
20
Sample in I-ml solution (PI)
AN.~LYTICAL
/,,
method
1.06 1.08 1.12
1.28
,,,,,>
1.14
1.17
,,,,,
8 OF KJELDAHL
N, volume (111)
-,*,,
Sealed-tube
TABLE BY MEANS
,,,&
768.7 768.7 765.9
771.5
770.0
770.0
,*,,
Pressure (mm Hg)
,,il
,
Mean:
Mean:
*,/,
,,/,
1.25
1.29
1.22 1.25
1.39
1.49
1.32
,.,,,
N
METHODS
1.35
(wm)
Found
AND SEALED-TUBE
,,,”
-,,,
,,“,,,,
Mean:
0.5
0.5
0.5
Sample (ml)
Kjeldahl
,,
Y,,,,
“,”
(1.38)
68
,,,,“.
( 16;8,
(1.3O)b
65
Found (mm)
method
,,”
N
*/,
2 F 2 >
is
$ $
ST ?
/,,,”
./
DETERMINATION
OF TOTAL
NITROGEN
457
In our laboratory, we have used this method for determination of the N/P ratio of phospholipid components and the concentration of synthesized polypeptides, ranging between 5 and lo%, with no band present at about 280 nm (6). SUMMARY With a modified combustion tube having a ball of 2-ml capacity with a fine tip, our sealed-tube method was used to determine the nitrogen contents of nonvolatile organic compounds of dilute aqueous solution with nitrogen levels from 1 to 70 ppm. A drop of diluted sulfuric acid was added to the sample, which was freeze-dried, then burnt.
REFERENCES 1. Gertner, A., and Grdinic, V., Determination of total organic nitrogen in dilute aqueous solution. Acta Pharm. Jugosl. 15, 209-212 (1965). 2. Gouverneur, P., The determination of trace amounts of total nitrogen in petroleum distillates extractive percolation method. Anal. Chim. Actu 26, 212-223 (1962). 3. Gouverneur, P., Snoek, 0. J.. and Heeringa-Kommer. M. Nitrogen determination in mineral oils by means of Wickbold oxyhydrogen combustion. Anal. Chim Acta 39, 413-422 (1967). 4. Gouverneur, P., and Van De Craats, F., Recent method for determining traces of nitrogen in mineral oils. Analyst 93, 782-787 (1968). 5. Hozumi, K., and Kirsten, W. J., New Method for the ultramicrodetermination of nitrogen. And. Chem. 34, 434-435 (1962). 6. Jnoue, H., and Izumi, T., The interaction of poly(N5-(3-hydroxyproply)-L-glutamine) with solvent components in water-dioxane mixture. Biopolymers 15,797-812 (1976). 7. Institute of Medical Science, University of Tokyo, Method of freeze-drying. In “Saikingaku Jissyu Yotei (Handbook Bacteriological Study)” (Y. Tsunematsu, et nl., Eds.), revised ed., pp. 343-357. Maruzen, Tokyo, 1961. 8. Martin, R. L., Fast and sensitive method for determination of nitrogen. Selective nitrogen detector for gas chromatograph. Anal Chem. 38, 1209-1213 (1966). 9. Kirsten, W. J. and Hozumi, K., Gas-volumetric ultramicro and microdetermination of nitrogen and other elements in organic compounds. Mikrochim. Actu 1962,777-786. 10. Miyahara. K., Ultramicrodetermination of nitrogen in organic compounds. 1. Application of interpolation in the sealed tube method. Micro&em. J. 19, 416-422 (1974). Il. Miyahara, K., Ultramicrodetermination of nitrogen in organic compounds. 2. A new simple nitrometer for the sealed tube method. Microchem. J. 19, 423-428 (1974). 12. Miyahara, K., Ultramicrodetermination of nitrogen in organic compounds. 4. Thermal behavior of a gas in a nitrometer due to local variations of temperature. Microchem. J. 20, 453-461 (1975). 13. Oita, 1. J., Direct Dumas determination of 30 to 1000 parts per million of nitrogen in oils. Anal. Chem. 38, 804-805 (1966). 14. Smith, A. J.. Cooper, F. F. Jr., Rice, J. O., and Shaner, W. C. Jr., The determination of trace amounts of total nitrogen in petroleum distillates. Anal. Chim. Actu 40, 341-343 (1968). 15. Tolg, G., Beitrage zur Ultramicro- und Supurenanalyse organischer Stoffe. V. Bestimmung des Stickstoffgehaltes schwertltichtiger organischer Verbindungen mit 5-20 pg Substanz. Z. Anal. Chem. 205, 40-50 (1964).