tracted into chloroform and then into
E.P.4. The phosphorimetric determination of aspirin in serum or plasma has several advantages over the use of the double salicylate method (16). The phosphorimetric method requires less than 10 minutes per analysis, is precise and accurate, and is nearly free of interference of constituents normally present in serum or plasma. The presence of certain drugs including the sulfa drugs, phenacetin, and some alkaloids as atropine and homatropine would cause some interference. Such interferences might be eliminated by the use of selective excitation of aspirin or by the use of phosphoroscopic resolution which would involve measurement of the longlived aspirin phosphorescence about 1 or 2 seconds after the start of its decay, or by a combination of the two methods in conjunction 11 ith the proper selection of emission navelength. It should be possible to cut down the volumes of all reagents in an analysis so that 0.1 ml. of serum or plaqma or less could be analyzed rather than the 0.4 ml. used in the study. The application of phosphorimetric
analysis to the analysis of various organic constituents in the areas of biology, medicine, food, plants, and water pollution seems possible. Phosphorimetric analysis is reasonably sensitive and accurate but the greatest advantages lie in the speed and selectivity of analysis. ACKNOWLEDGMENT
The authors thank J. Henry of the Clinical Laboratory of the J. Hillis hIiller Health Center for obtaining the blood serum and plasma used in this study. LITERATURE CITED
(1) American Instrument Co., Inc., Bulle-
tin KO. 2334, Silver Spring, Md., 1960. (2) Chen, 11.IT., “Bibliography of Phosphorescent Molecules,” pamphlet American Instrument Co., Inc., Silver Spring, hId. (3) Forster, T , “Fluoreszenz Organischer T’erbindungen,” Chap. 12, T’andenhoeck and Ruprecht, Gottingen, 1951. (4) Freed, S., Salmre, W.,Sczence 28, 1311 (19.58) (5) Hiers, R. ,J , Britt, R. D., Jr., Wefitworth, W. E , ANAL.CHEW 29, 202 (1957); (6) Lewis, G. Y., Kasha, M., J . Am. Chem. SOC.66, 2100 (1944).
( 7 ) Locket, S.,
“Clinical Toxicology,” p. 661, The C. V. illosby Co., St. Louis, 1957. (8) McGlynn, S. P., Neely, B. T., Neely, C., Anal. Chim. Acta 28, 472 (1963). (9) Satelson, S., “Microtechniques of Clinical Chemistry,” p. 332, Charles C Thomas, Springfield, Ill., 1967. (10) Parker, C. A., Hatchard, C. G., Analyst 87,664 (1962). (11) Parker, C. A., Hatchard, C. G., Trans. Far. Soc. 57, 1894 (1961). (12) Parker, C. A., Rees, W. T., Analyst 85, 587 (1960). (13) Pringsheim, P., “Fluorescence and Phosphorescence,” pp. 290, 434, Interscience Publishers, Inc., New York, 1949. (14) Seiverd, C. E., “Chemistry for Medical Technologists,” pp. 343, 344, The C. V. Mosby Co., St. Louis, 1958. (15) Smith, J. H., J . Pharm. Pharrnacol. 3, 409 (1957). (16) Udenfriend, S., “Fluorescence Assay in Biology and Medicine,” Chap. 4 and 6, Academic Press, Sew York, 1962. (17) West, W., “Fluorescence and Phosphorescence,” Chap. V I of “Chemical Applications of Spectroscopy,” W. JTest, ed., pp. 540-58, Volume IX, Interscience Publishers, Inc., New York, 1956. (18) White, C. E., Ho, V., Keimer, E. Q., ANAL.CHEM.32,438 (1960). RECEIVEDfor review January 31, 1963. Accepted June 27, 1963.
Simultaneous Determination of Carbon, Hydrogen, and Nitrogen at the DecimiIIigram Level KEllCHlRO HOZUMP and WOLFGANG J. KIRSTEN Institute for Medical Chemistry, University o f Uppsala, Uppsala, Sweden
b A simple, fast, accurate, and reliable method for the simultaneous determination of carbon, hydrogen, and nitrogen in organic compounds a t the decimilligram level is based on the dry combustion of the sample in a sealed tube and a volumetric determination of the combustion gases. The fact that all operations, including combustion as well as gas measurements, are carried out in the same small tube with a minimum of reagents contributes to accuracy and reliability. Standard deviations of 24 analyses of 15 compounds containing fluorine, chlorine, bromine, iodine, sulfur, phosphorus, and potassium were: carbon 0.2 1 %,hydrogen 0.20%, and nitrogen 0.1 2% (only seven samples analyzed contained nitrogen).
I
THE course of several year, a method for the simultaneous determination of carbon, hydrogen, and nitrogen in decimilligram samples of organic compounds was worked out in this laboratory (3, 4,6 , 7 , 9, IO). The method was based upon a sealed-tube
N
1522
ANALYTICAL CHEMISTRY
combustion and a combination of manometric and gas volumetric measurements for the resulting carbon dioxide, water, and nitrogen. During further investigations, a new method for measuring small gas volumes, based upon weighing of mercury which replaced the gas formed, mas worked out, which allowed the measurement of micro amounts of gas without transfer from the combustion vessel. It was first applied to the ultramicrodetermination of nitrogen ( g ) , and later to the decimilligram level ( 1 ) . Further applications were also reported: It was used for the analysis of aqueous solutions, its sensitivity u-as increased by measuring a n expanded nitrogen gas volume, and the same principle of measurement was extended to the determination of other elements (8). The first method for the simultaneous determination of carbon, hydrogen, and nitrogen mentioned above has been used routinely in this laboratory for several years. The demand for such analyses increased considerably, however, and as it was impossible t o run a
sufficient number of analyses using the tedious measuring procedure, it appeared very desirable to try to apply the new measuring method to the simultaneous decimilligram determination of carbon, hydrogen, and nitrogen, in the hope of obtaining a faster and simpler method without loss of accuracy and reliability. PRINCIPLE OF METHOD
The sample is inserted into a narrow tube of Siipremax or Pyrex 1720 glass, closed a t one end, together with a small amount of metallic copper and a combustion catalyst. The open end of the tube is then drawn o u t to a long cnpillary. ‘The tube is swept free from air with pure oy-gen. sealed off. and heated to 950” C:. in a furnace. The ,
Figure 5. Removal of mercury and main portion of potassium hydroxide
1524
Figure 7. Accessories for weighing and introducing sample into combustion tube 70-mg. platinum scoop 2 X 2 X 6 mm. 5. Platinum boat, 2 X 2 X 8 mm.
MI.
A.
AD. A€.
C.
Combustion tube a s supported b y rack Enclosed nitrogen in the capillary Remaining potassium hydroxide AF. Fine polyethylene tubing AG. Potassium hydroxide meniscus
ANALYTICAL CHEMISTRY
+
Boat-handling forceps (surgical excision tongs); stem 1.2 X 400 mm.; head diameter, when closed, 2 mm.
Figure 9. Marking tube for measurement of COZ
Figure 8. Sweeping and sealing off combustion tube 1.
2.
3. 4.
5.
Filled combustiontube H. Saniple K. Catalyst 1. Hemicylinder of copper wire gauze M. Combustion tube Combuslion tube with sweeping E. Stainless steel capillary N. Coribustion tube capillary F. Splbtype furnace Cooled combustiontube withsweeping 0. Coiitainer of dry ice Sealing combustion tube capillary Combustion tube prepared for combustion
small diamond wheel, about 3 mm. in diameter, which is mounted on the motor of a dentist's drill. 11 similar roughening of the glass can also be produced with fresh, fine sandpaper, but great care must be exercised in this technique to prevent breakage. COMBUSTION. With the combustion tube held in a horizontal position again, gentle vibration is applied to bring all particles of the sampla-catalyst mixture back down from the fine tip a t the end of the tubcs. Several combustion tubes, prepared in this rranner, are then placed horizontally in the combustion furnace and kept at 760" C. for 90 minutes. The furna:e is turned off, but the tubes are not taken out before the temnerature of the furnace is well below 500' C. A fine, thin line (X,Figure 3) is drawn with a shar;x hard graphite pencil (Faber Castello 10 H)-on the roughened glass surface at the middle of every combustion tube, perpendicularly to the axis of the tube. It is marked with a crossing line to avoid confusion aTith the If2 COZ mark, which is to be drawn later on. The tubes are then placed in the gas diffusion furnace (Figure 2), which is held a t 500' C. The cooling cuffs are placed upon the capillaries and filled with liquid nitrogen. They are kept in this position for 10 minutes, during which the liquid nitrogen is replenished as quickly a9 it boils away. All water and carbon dioxide from the sample are thus frozen out in the capillary. MEASUREMENT.Ir: the meantime, the mercury reservoir (Figure 3), held in its support, is filled with pure mercury to a height of !iC mm. Next, a combustion tube is removed from the
+
+ NZ
AN. Upright image cathetometer AO. Combustion tube AP. Ring of black marking ink AQ. Frosted glass surface on front wall of A 0 AR. Glossy white background AS. Cathetometer image showing pencil point, black ring, and AT.
opaque mercury meniscus against white background. Meniscus and tip of pen are seen distinctly only after individual focusing Hard graphite pencil
diffusion furnace and its lower end, after a minute's cooling, is cautiously pushed into the mercury, as illustrated in Figure 3, whereupon the fine tip beneath the surface of the mercury is broken off against the bottom of the vessel. After the break, mercury . is drawn into the tube and begins to rise slowly. After the meniscus has reached the middle of the tube or slightly above, the cooling cuff is lifted off. To make sure that there has been no break between the mercury column within the tube and the pool without, each tube is drawn up slightly for observation of the moving meniscus. If the nieniscus does not move readily, the hole in the tube is widened by further turning and pressing the tube downward, or, if necessary, by means of a bent needle. After the meniscus has stopped moving, a black ring, about 3 nini. wide. is painted in glass marking ink around each tube a t a point about 0.5 to 1 mm. above the top of the meniscus. The tubes are now tied to the stem of the mercury reservoir by pieces of narrow rubber band, but are kept from it by rubber rings which serve as separators (Figure 3). The area of frosted glass should point outward. The entire support assembly with vessel and tubes is next transferred to a constant temperature room a t 25' C. (If there is a significant temperature difference between the laboratory and the constant temperature room, painting of the black ring should be done there instead.) After at least 20 minutes, the entire support assembly with vessel and tubes is rocked gently a few times, to make sure that the mercury menireus i n each tube has risen to its true equilibrium
level. The frosted glass surface in front of the mercury meniscus is slightly wetted with paraffin oil to make it transparent. -4piece of white paper or other white surface is placed behind the support and tubes. By means of the hard graphite pencil a thin, horizontal mark is made on the frosted glass a t the position of the top of the meniscus, as shown in Figure 9, the marking operation being followed by observation through a cathetometer. For such work it is convenient to have an arm rest near the support assembly, since it takes a steady hand to mark accurately. When all of the tubes have been marked, the correctness of each marking is checked once more thtough the cathetometer, and its elevation is noted. The elevation of the outer mercury level in the vessel is next determined with the cathetometer, in order to obtain the difference in height, hl, between the meniscus in each tube and the reference level of the pool. The rubber rings on the stem of the mercury reservoir and the pieces of rubber band holding the tubes are now replaced by a ring of light copper wire encircling the upper capillary sections of the combustion tubes. Then the mercury reservoir and its tubes are lowered down inside of an upright ground joint air condenser serving as a reflux for boiling Decalin (Figure 4). An accurate immersion thermometer (not shown in the figure) is also used inside the condenser for a boiling point determination. The outside wall of the condenser has been marked off in a linear millimeter scale, with graduation marks that go about 2/3 around. After a 20-minute reflux the distances of each meniscus from the middle line x,Z, and distance3 of each meniscus from the mercury level in the reservoir, lh, are measured using the scale on the condenser. The barometric pressure is again taken and the boiling point of the Decalin is taken. The reservoir with the tubes is now VOL. 35,
NO. 10,
SEPTEMBER 1963
1525
taken from the Decalin condenser and allowed to cool, and 50% potassium hydroxide, as used in the Dumas nitrogen determination, is added to the vessel so t h a t a layer of 20 to 30 mm. is formed between the mercury and condensed Decalin. One by one, the combustion tubes are raised carefully, so that the opening comes above the mcrcury, and they are shaken slightly in a vertical direction, as shown in Figure 10. A few drops of mercury will come out, and an equal amount of potassium hydroxide solution will pass into the tube and up t o the capillary, where it absorbs the carbon dioxide and most of the water vapor. Twenty minutes after tile introduction of the potassium hydroxide, the tubes are taken out of the mercury reservoir and laid horizontally upon a rack in the 25' C. thermostated room. The open end of each tube is then enlarged somewhat by breaking out the mall of the drawn out tip with a n awl or a thick needle until a wide enough section of the tube is reached. The mercury and part of the potassium h j droxide solution are next withdrawn by suction through a polyethylene tube as shown in Figure 5, in order t o counterbalance the rising forces of the menisci of potassium hydroxide in the capillaries. After further standing for 5 minutrs a mark, NZ (Figure 6), is made with the graphite pencil on the glass tube at the top of the meniscus between potassium hydroxide and nitrogen. The barometric pressure is again read. The potassium hydroxide is then shaken out from each tube, and all of the tubes are cut off at a point about 50 inm. from the bottom end. The upper parts of the tubes are washed well with
+
+
Figure 10. hydroxide
Combustion tube before introduction of KOH Combustion tube roised to admit K O H and with absorption of C02 and HzO in process 3. Combustion tube after completed absorption
1 Lauric acid 2 Anthracene
3 Acetanilide Sulfanilic acid 5 Trifluoroacetanilide 6 o-Iodobenzoic acid 7 Benzoic acid 8 Dimer dithioacetylacetone (CinHi&) 9 Benzoic acid 10 Triiodophenol 11 Triphenylphospliine 12 Potassium biphthalate 13 Thiourea 14 p-Bromobenzoic acid 15 Lauric acid 16 Acetanilide 17 Sulfanilic acid 18 p-Fluorobenzoic acid 19 Triphenylphosphine 20 TriiodoPhenol 21 o-Iodobknzoic acid 22 Trifluoroacetanilide 23 Perfluoroperhydrop yrene 24 Perfluoroperhydropyrene 4
1526
pg.
+
CALCULATION OF RESULTS
1.
Analytical results were calculated as folloTTs:
water from a springe with a long stainless steel capillary, then with alcohol and ether. Finally they are dried by blowing air through thcm. A shsrp mark is then made on each tube with the hard graphite pencil at the point corresponding to the location of the meniscus during the stay in the boiling Decalin-Le., a t distance ll from X. The tubes are then weighed to an accuracy of 1 mg., first empty and then filled with mercury up to mark T\Tz on
Nitrogen. mg' Hg = ul. Hg = pl. S2 (at room 13.53
conditions 01er 50% KOH) = S'Hg Aqueous tension of 50% KOH at 25" C. = 7.5 mm.Hg (5) Keight of 1 ~ 1S2 . (dry) is read from a nomograph (11). p1. Kz(at room condition? and dry) =
T.Y '
VN X weight of 1 pl. K2 = sample = WN
pg. S
Analyses Carried Out with Described Method
K'eigh t of sample,
Substance
Introduction of potassium
2.
Table I.
No.
the capillary, corresponding to the earlier position of the potassium hydroxide meniscus. If the top of the mercury meniscus matches the mark, the weight of mcrcury obtained permits calculation of the volume of nitrogen previously present. The mercury is then flipped out from the tubes, and the stainless steel tip of the piston buret is introduced into each tube, which is held open end up, in a vertical position on a support stand. Carbon tetrachloride is norr delivered into the tube from the buret until the lowest part of the meniscus coincides with the second mark, Sz COz, as shown in Figure 6. The buret reading is taken. Further carbon tetrachloride is then added, until the meniscus is a t the third mark, Kl C02 H20, and the buret reading is again taken. During each addition the buret tip should not be kept too high above the mark to he reached, in order to aroid drainage errors.
Calcd.
% Carbon ______ Found Dev.
__
";. Hrdrogen
Calcd.
Found
Dev.
-~
'6 Xitrogcn ________
Calcd.
Found
Dev.
406.0 412.3 499.4 456.4 506.15 441.0 408.5
71.95 94.34 71.09 41.63 50.80 33.90 68.80
71.94 94.21 71.09 42.03 B0.89 33.99 68.89
-0.01 -0.13 0.00 +0.40 +0.09 +0.09 4-0.09
12.08 ci.66 6.71 4.07 3.20 2.03 4.95
11.84 5.52 6.43 4.32 3.24 2.22 LO6
-0.24 -0.44 -0.28 +0.25 +0.04 $0.19 +0.11
0.00 0.00 10.37 8.09 7 41 0.00 000
0 00 0.00 10.30 7.97 7 40 0 00 000
0 00 0.00 -0.07 -0 12 -0.01 0 00 0.00
415 6 502 8 4,53.9 519 0 . _. 370.6 382.5 378.7 438,9 403.5 476.0 389.3 364.6 458.3 397.3 431.5 626.9
45 41 68 80 I5 30 82 42 47 10 15 78 41 76 71.95 71 09 41 63 60 00 82 42 15 30 33 90 50 80 28 00
45 67 69.13 15.56 82.66 47.00 16.03 41.89 71.73 71.01 41.96 60.01 82 29 15.16 34.12 50.92 28.36
+0 26 4-0 33 +0.26 t O 24 -0.10 $0.25 + O . 13 -0.22 -0 08 +o 33 +o. 01 -0.13 -0.14 +o 22 +0.12 + O . 36
6.01 4.95 0.64 5.76 2.80 5.30 2.50 12,08 6.71 4.07 3.60 5.76 0.64 2.03 3.20
0.00
6 22 4 96 0.53 5.76 2.86 5.64 2.68 11.94 7.01 4.07 3.87 6 s9 0.75 2.09 3.27 0.00
+0.21 $0.01 -0 11 0 00 + O . 06 +0.34 $0.18 -0.14 0.30 0.00 + O . 27 +o. 13 +o. 11 +0.06 +0.07 0.00
0 00 0 00 0 00 0 00 0 00 36 SI 0.00 0 00 10 37 8 09 0 00 0 00 0.00 0 00 7 41 0 00
0 00 0 00 0.00 0 00 0.00 36.73 0.00 0.00 10.21 5.23 0 00 0.00 0.00 0.00 7.26 0.00
0 00 0 00 0.00 0 00 0.00 -0.08 0.00 0.00 -0.16 +o 14 0.00 0.00 0.00 0.00 -0.15 0 00
731,7
28 00
27.80
-0.20
0.00
0.00
0.00
0 00
0.00
0.00
ANALYTICAL CHEMlSTRY
+
in
A blank of 0.3
p g . of nitrogen
is
subtracted. Carbon. Vapor pressure of H2O at 25’ C. = 23.76 mm. H g PtOt,1 i n t u b s = barometer - ( h ~ 2 mni. capillary depression) I’co? + x2 = Ptotal in tube - 23.76 nim.
+
=
P I
T’co~ + x2 = 1st volume of CC14from buret pl. (C02 S*)8.0rr. = vcoz + 5 2 corr. t o bargmetr. mess. =
+
V(C PI.
+N)
coz = V(C + N) - VN
The weight of nitrogen. which would occupy -the vo:ume fil. COZ is read from the nomograph, pg. XC. p g . C = pg. Kc X 0.4314 = p g . C in sample = W C -1 blank of 1.4 pg;. of carbon is suhtracted. V
I
Hydrogen. h2 X 0.97 = 122: = height of H g column corrected for decrease in density of Hg at Decalin b. p. P , total i n tube = l)?rom. - (ho 2 mm. cap. depression) Pz = P C O , + KZ + 1 1 2 0 l’coz + N2 + HzO = 2nd volume of CC1, from buret PI. (COz Ez HiO)Oorr. = VcoZ+ xZ+ H ~ Ocorrected from P2 to barometric press. and room temp. pl. HzO = pl. (‘202 Kz Hz0)cm.
+
+
- V ( C + r\T)
+
+ +
The weight of nitrogen which would occupy the volume pl. HzO is read from the nomograph, p g . : S H 2 0 . pg. H = p g . \ \ H ? o X 0.0719 = pg. H in sample = TVII blank of 0.8 p g . of hydrogen is subtracted. Blanks were determined by running tiilies without samples through the .?heme. DISCUSSION
Combustion Method. T h e clieniist r y of the combustion method is v>~oiitialIyt h e san-te as t h a t of the
manovolumetric CHK-determination method (IO). The combustion tube must be somewhat larger, because of the large volumes of gas measured. The excess of oxygen in the present procedure is, therefore, larger. It was necessary to carry out the combustion at 75OOC. for 90 minutes to obtain a safe absorption of all oxygen and a safe reduction of all nitrogen oxides. Experiments indicate that the reduction of nitrogen oxides first begins when almojt all free oxygen has been absorbed. Incomplete reduction of the nitrogen oxides causes low nitrogen results. It was possible to use a smaller amount of catalyst (30 mg.) instead of 50 mg. (IO), which decreases the excess of oxygen. Freezing Out. T h c tcmperaturc of 500” C. in the diffu8ion furnace gave a complete recovery of carbon dioxide from all possibly formcd carbonates. The cuffs might appear inefficient. S o troubles have, however, been experienced with them. Measurements. ‘I’he positions of tlie menisci in tlie measurement of the volumes of carbon dioxide plus water plus nitrogen are deterniincd with the mark X (Figures 3 and 9). The volume of nitrogen is determined by a simple mercury weighing. The other volumes are so large that the convenient procedure of titrating with carbon tetrachloride can be used. There is an added advantage in the use of carbon tetrachloride, because it3 meniscus (concave) is curved in the same direction as that of the mercury (convex) which originally closed off the gas volume to be measured. Earlier attempts to titrate the volumes wit’h water instead of carbon tetrachloride were abandoned, because the shape of the water meniscus was not reproducible. Carbon tetrachloride ivas finally selected from among the other liquids tried.
SUPPORTING DATA
Results of 24 analyses of 15 conipounds containing fluorine, chlorine, bromine, iodine, sulfur, phosphorus, and potassium are given in Table I. The analyses were run in direct succession. except that two unknown research qamples were analyzed between analyses 6 and 7, and t n o betneen analyses 11 and 12. Oiie analysis of anthracene was lo& betueen analyses 15 and 16, becau-e a tub? \\a. broken. Standard deviations are: for carbon 0.21%. hydrogen 0 2O7,, and nitrogen 0.12y0. ACKNOWLEDGMENT
The author. are indebted to Ludniilla S i r k and to Charlotte Alattsson for skilled t t ~ h n i r a l ab>iatance, and to A. 11. C;. llacdonald. University of Birniingham, England, for kindly supplying the samples of perfluoroperhydropyrene. LITERATURE CITED (1) Hoziimi, Keiichiro, AXAL. CHEK 35, 66h (1963).
( 2 ) Hozumi. Keiichiro, Iiirsten, W. J., I h i d . , 34, 434 (1962). (3) Kirsten, W.J.. Ibid.. 26, 1097 (1954). (4) Kirsten, K. J., Chivi. Anal. 40, 253 1 1938).
( 5 ) Kirsten, IT.J., “Comprelrensive Analytical Chemistrv,” Vol. IH, p. 447,
Elsevier, Amsterdam, 1961. Kirsten, JY. J., Mzkrochim. Acta
(6)
1956, 811. ( 7 ) Kirsten, W. J., Z. Anal. Chrm. 181, 1 (1961).
181 Kirstm.
W. .J.. Hoaumi. Keiichiro.
., Hozumi, Keiichiro,
1962, 777. J., Hozumi, Keiichiro,
Kirk. Ludmilla. 2. Snal. Chem. 191. 161 (1962). (11) Koch.
C. K., Simonson, T. R., Tashinian, W. H., ASAL. ((HEM. 21,
1133 (1949). (12) Korbl, J . Nzkrochim. d2cla 1956, 1705.
RECEIVED for rrview January 17, 1063. Accepted June 12, 1963.
VOL. 35, NO. 10, SEPTEMWR I963
1527
