777
V O L U M E 26, NO. 4, A P R I L 1 9 5 4 Table I. Colors of Steroids Tested with Iron Reagent Compound Tested Cholesterol Cholestanol Dehydroisoandrosterone Isoandrosterone 11ethyl-As-36-hydroxyetiocholenate As-Pregnenolone A b 18-Preenenolone acetate Testosterone A6-Androstene-l7,8-acetory-3-ethylenecyclic hemithioketal Cholesterol acetate Cholesterol oleate Cortisone Kryptogenin acetate 7-Dehydrodiosgenin acetate Stigmasterol Sitosterol A4~~-22-Isospirostadiene-3-one Cholesteryl is0 methyl ether Estradiol Ergosterol acetate Pregnane-3B-01-20-one 17-Methyl-androstan-3B-17,9-diol 17-Methyl-A~-androsten-3~-17B-diol
Color Formed Purple Yellow Red Yellow Green Reddish-purple Brownish-oranee Pale yellow
-
Blue-violet Purple Purple Pale yellow Pink Purple Purple Blue-violet Yellow Red-violet Pale yellow Brownish Yellow Yellow Purple
PROCEDURE
Pipet 1.0ml. of steroid solution into a test tube. Add 2.0 ml. of glacial acetic acid. Pipet in 2.0 ml. of the color reagent by carefully allowing it to flow down the side of the test tube, thus producing two layers. Record the colors of the several rings ormed for aid in qualitative analysis and then strike the bottom of the tube sharply while holding it a t the top between the thumb and forefinger to effect mixing. The color of the solution formed may be used as a qualitative test or the measurement of the absorbance a t the proper wave length ma be used as a quantitative test for the A6 sterols such as c%olesterol, pregnenolone, stigmasterol, sitosterol, and others. The colors are stable and show no change over a period of several hours. DISCUSSION AND RESULTS
The compounds tested were several saturated and unsaturated steroids where the latter included A’, A‘J7, A6, A6,’, and unsaturated steroids. The colors all appear almost immediately after mixing, and Table I shows the colors obtained with the
available compounds which xere reacted with the iron reagent. The A d and the A4r7 steroids give a brownish-yellow or purple ring, while only a thin yellow ring appears a t the interface of the two liquids with the saturated compounds. The AsJ the A%’, and the steroids give a multicolored ring (usually 3 to 4 colors) which may include any combination of purple, green, yellow, red, pink, orange, violet, and brown. Most A6 compounds such as cholesterol (12) and A6 pregnenolone produced linear curves up to 100 p.p.m. obeying Beer’s law over the entire range. The authors used a Coleman Jr. spectrophotonieter for measurements. This preliminary work was carried out with a large variety of pure compounds but the work in progress ie directed toward more specific determinations. ACKNOWLEDGMENT
The authors wish to express their appreciation to the Shering Corp., Bloomfield, S. J., and to Carl Djerassi, Department of Chemistry, Wayne University, Detroit, Mich., for their liberal donations of the steroids used in this investigation. LITERATURE CITED
(1) Burchard, H., Chenz. Zentr., 61 (l),25 (1890). (2) Fieser, L. F.,“Chemistry of Natural Products Related to Phenanthrene,” pp. 11 1-86, New York, Reinhold Publishing Corp., 1936. (3) Gilman, Henry, “Organic Chemistry, An Advanced Treatise,” 2nd ed., pp. 1341-532,New York, John Wiley & Sons, 1948. (4) Lange, W., Folzenlogen, R. G., and Kolp, D. G . , J . Am, Chem. SOC.,71, 1733 (1949). (5) Liebermann, C., Ber., 18, 1803 (1885). (6) Salkowski, Hoppe-Seyler’s 2.physiol. Chem., 57, 523 (1908). (7) Schaltegger, H., H e h . Chim.Acta, 29,285(1946). ( 8 ) Scherer, Ibid,, 22, 1329 (1939). (9) Teuber, H., ANAL.CHEM.,24, 1494 (1952). (10) Titchugaev, L., and Gastev, A., Ber., 42,4631 (1910). (11) Woker, G., and Antener, I., Helv. Chim. Acta, 22, 1309 (1939); 22, 511 (1939). (12) Zlatkis, A,, Zak, B., and Boyle, A. J., J. Lab. Clin. Med., 41,486 (1953). RECEIVED for review September 8, 1953. -4ccepted January 6 , 1954. Presented before the Division of Biological Chemistry at the 123rd Meeting of the AMERICAX C H E \ I I C ~SOCIETY, L Los Angeles, Calif., March 1953
Kjeldahl Method as Applied to Determination of Nitrogen in Nitrates WILLIAM E. DICKINSON F. S. /?oyster Guano Co.,
T
Atlanta, Ga.
HE fact that nitrates in excess of the salicylic acid capacity in the modified Kjeldahl method give nitrogen results that are only slightly low has resulted in acceptance of unscientific practices. I n this study are recorded some developments indicating the need for corrections in current Kjeldahl methods prior to 1950, and in the Gunning method current today, as applied to materials of high-nitrate content. Incidental to this work are some previously unreported findings enabling us to deal more informatively with Kjeldahl procedure. Some applications of these developments are suggested. The modified Kjeldahl method is designed to deliver the correct percentage of nitrogen i n nitrates. Most other nitrogenbearing compounds succumb to the reduction and/or digestion features of the method. The seventh (1950) edition of the Association of Official .4gricultural Chemists’ methods for the determination of nitrogen in fertilizers by the modified Kjeldahl procedure ( 2 ) increased the salicylic acid from 1 gram per 30 ml. of sulfurir acid to 2 grams.
The Gunning variation of the method was retained without change a t 1 gram per 30 ml. of sulfuric acid. Prior to this edition, both variations of the method (1) directed, for converting the nitrates to nitrosalicylic acid: “Place 0.7 t o 3.5 grams, according to the nitrogen content of material to be analyzed, in a Kjeldahl digestion flask. Add 30 ml. of sulfuric acid containing 1 gram of salicylic acid, shake thoroughly until mixed, allow to stand a t least 30 minutes with frequent shaking. . .” If these variations of the method are applied literally to the analysis of sodium nitrate, both will give nitrogen results about 0.20% low.
.
DEFICIENCIES O F KJELDAHL 4fETHOD
Since 1 gram of salicylic acid is converted to nitrosalicylic acid by 0.6160 gram of sodium nitrate, a 0,0840-gram residue of unconverted sodium nitrate w-ill be left from a 0.7-gram sample to f o l l o ~through in the method, unless some dinitrosalicylic acid is formed. If the residue does not go into dinitrosalicylic acid, the
778
ANALYTICAL CHEMISTRY
implication of the method is that the nitrogen result should be 1.98y0 low, instead of 0.20% low. In the interest only of correct results, some analysts weigh out not more than 0.5 gram of sodium nitrate. In so doing there has been a drift from an official method giving slightly erroneous results on high-content nitrates, to a n unofficial method on the empirical level giving correct results. The situation, as described, is responsible for this study of the Kjeldahl methods. The modified Kjeldahl procedure involves three main steps i n the conversion of nitrates t o ammonia. a? follov s: 1 Solution of the nitrates in salicylic-sulfuric acid, for converting the nitrates to nitrosalicylic acid. 2. Treatment of the nitro compound with sodium thiosulfate or zinc dust to reduce the nitro group to an amino group. 3. Prolonged digestion of the amino compound with the sulfuric acid to convert the amino group to ammonium sulfate.. REDUCTIOY STUDIES
All three of these steps are essential to a quantitative coiiversion of the nitrates t o ammonia, but if the second step of the method is omitted, the conversion will be about 99% complete. As evidence of this, two 0.35-gram portions of sodium nitrate were treated according to the modified Kjeldahl procedure. with nitrogen results 16.44 and 16.48%, the average of which is very close t o the theoretical 16.48%. Concurrently with these, two other determinations, with the second step omitted, gave 16.20 and 16.28% nitrogen. Undoubtedly the heat breakdonn of the nitrosalicylic acid results in reducing substances that are able to convert the nitro group almost quantitatively to an amino group. If the 0.35-gram portion of sodium nitrate be treated according to the modified Kjeldahl procedure, but with the salicylic acid omitted, the recovery of the nitrogen as ammonia by the sodium thiosulfate will be about 75% effective. I n the heat breakdown of nitrosalicylic acid the reducing substances formed are much more effective in recovering nitrogen from the nitro group than sodium thiosulfate is in recovering nitrogen from sodium nitrate. EVIDENCE OF EXCESS NITRATES
Early in this investigation, IT hen the amounts of sodium nitrate taken were varied (in order t o note the effect on the nitrogen results), a red color developed a t times when the salicylicsulfuric acid was heated to hasten the solution of the nitrates. Thus, with 0.7-gram portions of sodium nitrate the red c-olor developed, and when the Kjeldahl cycle was completed the nitrogen results were about 0.20% low. On the other hand, u i t h 0.35gram portions no color developed, and the results Rere correct. A little sodium nitrate introduced into a sulfuric acid solution of Eastman 5-nitrosalicylic acid which had been partially reduced to aminosalicylic acid by sodium thiosulfate further confirmed the finding that nitrates produce a red coloration in a sulfuric acid solution of amino-salicylic acid. T h a t this coloration takes place in concentrated sulfuric acid makes i t very useful in following the course of the reactions of the Tiieldahl method. NO DINITROSALICYLIC ACID FORMED
When more than 0.6160 gram and less than twice that amount of sodium nitrate reacts with 1 gram of salicylic acid in sulfuric acid, a red color develops on heating to 100" C., showing thnt the sodium nitrate in excess of the 0.6160 gram required to produce mononitrosalicylic acid does not go into dinitrosalicylic acid, but goes into the second step of the method as nitrate. For 50 years chemists have been overlooking a potential warning in the Kjeldahl method when the nitrate was in excess of the salicylic acid capacity.
ASSESSING E4CH STEP
FYhen duplicate 1,3258-gram portions of Eastman 5-nitrosalicylic acid were treated according to the modified Kjeldahl procedure, the weights of nitrogen recovered a ere 0.1008 and 0.1015 gram, the average of which was very close to the theoretical 0.1015 gram. Since this material represents the product of the first step of the Kjeldahl method. this determination started a t the second step of the method. \There sodium thiosulfate i* added. There was, of course, no salicylic acid in the sulfuric. acid used in this digestion In order to reproduce the conditions obtaining nheri 0.7 gram of sodium nitrate is treated nith 1 gram of salicylic acid (bThich produces 1.3258 grams of nitrosaliq lic acid and a 0.0840-gram reaidue of sodium nitrate) the determination above was repeated. but to the 13258 grams of nitrosalicylic acid was added 0.0840 gram of sodium nitrate (which contributed an additional 0.0139 gram of nitrogen toxard the whole). The nitrogen recovered totaled 0.113i gram instead of the 0.1154 gram started with. shoning that the combined effect of the breakdown products of the nitrodicylic acid and the sodium thiosulfate enables the method to recover as ammonia about 90% of small residues of nitrate still in the Kjeldahl cycle a t the second step of the method. It follows that when 0.7 gram of sodium nitrate is treated with 1 gram of salicylic acid according to the modified Kjeldahl procedure, the nitrogen recovered Rill consist of that in the 0.6160 gram reacted to nitrosalicylic acid, plus 90% of that in the 0.0840-gram residue following through from the first step of the method. The two sources will contribute, respectively, 0.1015 and 0.0125 gram of nitrogen for a total of 0.1140 gram, as against the 0.1154 gram present in 0.7 gram of sodium nitrate. This recovery is the equivalent of finding 16.28% nitrogen in a sodium nitrate containing 16.487,. CHOICE OF METHODS AYD APPLICATIOY OF DEVELOPMENTS
A sp:ite of methods for nitrogen i n nitrates, both in the tests and i n the literature, attests previous efforts to find better conditions. I n the writer's hands, Arnd's alloy, in a simple distillation of the nitrate from 3 solution containing magnesium chloride, x a s found to give results about 0.257, loa. Devarda's alloy. from a very dilute solution of caustic, gives theoretical results (3) but the distillation is far from simple. If a nitrogen method is required nhich is almost univeIsallv applicable, this may be had in the modified Iijeldahl method (2). The Gunning variation (2) of the method may be made equally applicahle by increasing the salicvlic acid from 1 to 2 grams per 30 nil. of sulfuric acid, or by reducing the factor weight from 0 . i to 0.35 gram. If a method is required which is applicable to nitrates only (and most other inorganic nitrogen compounds). it is evident that the Kjeldahl method may be considerably simplified and the nvrk expedited uithout prejudice to the results. The method may be modified as follo\i s: Weigh 0.7 gram of a nitrate or other inorganic nitrogen compound into a Kjeldahl flask, add 30 ml. of sulfuric acid containing 2 grams of salicylic acid, and with the flask held in an upright position, rotate the contents in the tip of a Bunsen burner or other heat source for about 15 seconds 01 until very hot to the touch and all nitrates are in solution. To the still hot contents of the flask add 3 to 4 grams of anhvdrous sodium thiowlfate and rotate the contents vigorous]! and up the sides of the flask to include all of the nitrogen compound. Place the flask at once over a full flame or other heat source for 10 minutes or more, add 15 grams of anhvdrous sodium sulfate or potassium sulfate, and digest vigorously for 1 to 2 hours. Cool, dilute t o half the flask capacity, and add about 0.05 gram of 20-mesh zinc and caustic soda or potash to strong alkalinity. For a sodium nitrate distill into 17 ml of 0 5 V acid, for a potassium nitrate into 15 ml. of arid, and for an ammonium nitrate into 35 nil. of acid, all diluted with 50 ml. of water. Back-titrate mith 0.1.V caustic, using nieth) 1 red or cochineal indicator.
V O L U M E 26, NO. 4, A P R I L 1 9 5 4
179
DISCUSSION OF DEVELOPMENTS
No loss of nitrogen need he feared from heating the contcnts of the flask in the first step of the method when no organic matter is present. The nitrates react a8 Soon as they are in solution, and nitrosalicylic acid is very stable to heat. The solution may he heated to the boiling paint before adding the mdium thiosulfate and the loss of nitrogen will be only about 0.10%. The method suffers from the same interfering substances as thc elassiea1 Kjddahl method. Chlorides present with the nitrate will cause a, slight loss of nitrogen as NOCL. Starch and tobacco and most other organic compounds present with the nitrate develop reducing substances on heating in sulfuric acid before the nitrate is in solution, and these reducing substmces apparently take oxygen from the NO, group in its transition stage from the nitrate to the salicylic acid, with some lass of nitrogen. If these organic compounds arc d d e d after tbc nitrate is in soh-
tion, thc nitrogen found on completing Lhe Kjeldahl cyde will be the sum of thatin the nitrate plus that in the organic compound. The results reported here Yeprosent work on a reagent grade of sodium nitrate which passed a 14-mesh sieva but w&8retained on a 3O-mesh, and wa8 dried a t 110' C. This greatly facilitated manipulation and minimized errors due to absorption of moisture, adherence to brushes, etc., and made a very acceptable standard in Iijeldahl work. Treating 0.7 gram of this material by the proposed method gave results ranging from 16.40 to 16.48% nitrogen, and left little to be desired as to precision and accuracy. LITERATURE CITED
(1) Assoc. Offiic. h g r . Chemists, "Official and Tentative ,Methods of Analysis," 6th ed.. BP. 27-8. 1945. (2) Asaoc. Offio. Agr. Chemists, "Official Methods of Analysis,'. 7th d
Differential Photometric Detection in Couloi EDWARD N. WISE', PAUL W. GILLES, and CHARLES A. REYNOLDS, JR. Department of Chemistry, University of Kansas, Lawrence, Kan.
E
Xl'I?IlIME,NTS in the coulometric titration of acid and base1 with . photometric detection of the end point were complicated by the formation of bubbles during titration ( d ) . These bubbles were formed by the generating electrode and produced a density in the solution x-hich the photometric detection section of the automnt,ie titration apparatus \,-as unable to distinguish from a change in dcnsity due to a ehenge in the color of the indicator employed. It was found possible to titrate automatically those systems in which the end point was characterized by a change in the indicator which produced an increase in the absorbance of t.he solution with several false terminations of the titration to allow the bubbles to leave the solution. Howover, if an indicator r e r e used which caused a decrease in absorbance at the end point, the absorbance due to the formation of bubbles compensated for the I
Present address, University of Arizona. Tucson. A
decrease in density due to the color chango of thi a variable overtitration inevitably occurred.
To overcome the adverse effect of the absorbah, 1.10Ay8 the formation of bubbles, a differential 07 ratio-detecting photometxic unit u m designed and constructed. The principle on vhich this unit opcrates is as follows: Light from a tungsten lamp
IISV.
h
Figure 2. Cireuil Diagram for Differential Photorn, Deteotion Unit G . 0.01 sf., m i c a 6. 0.25 pf.. 400 volts
G. 0.1 "1%400 *olta C,. 4 pf.. 200 ro1ts 4 pf., 2w volts , ma., 6.3volts, 6. 115 ~ o l t s30 R,. m,wo ohms, 2 w a t t s Rz. 22 megohms, 0.5 w a t t R I , 14. 0.25 megohm, 2 watts Xi. 56 Ow ohms, 0.5 w a t t RI. 68'000 ohma, 0.5 w a t t R,. 10~000ohms, 1watt Ra. 5000 o h m s , 10 w a f t s R.. 20,000 ohms, 4 watts SI. Double-pole, double-throw switch TI, Tz. No. 929 TI. No.12AX7 Td. No. 6AS5
e..
Figure 1. Differential Phototube Assembly
passes through the solution being titxated and is separated by a heam splitter into two beams of equal intensities a t right anglcs to each other. Each beam passes through a tube, which may contain a colored solution t o serve as an optical filter, and terminates on the photooathode of a photoelectric tube.
