Separation and Detection of Cyanamide and Its Derivatives and Determination of Urea by Paper Chromatography J. E. MILKS'
and
R. H. JANES
North American Cyanamid, Ltd., Welland Plant, Niagara Falls, Ontario, Canada
Khatman 10. 3 filter paper, but this JTas partly coirected b! using Whatman No. 3321hf. The discovery of these combinations of solvents was fortunate because of the desirability to use neutral solvents, since the substances under investigation were subject to chemical reaction in an acid or basic medium. Reagents. Alkaline ferricyanide-nitroprusside (FCKP), ammoniacal silver nitrate ( AmAg), p-dimethylaminobenzaldehyde (DAB), and 2,6-dichloroquinonechloroimide (DCC) were prepared according t o the procedures of Berry and others (2, 31. Sullivan's reagent, 1,2-naphthoquinone-4-sodium sulfonate (SQS) (If), for detecting guanidine on paper chromatograms, wa* adapted from the procedure of Bertrand and Myers (4)for determining guanidine in eluates from ion exchange resins. Reparation and Use of Reagents. All solvents and reagents for spraying chromatograms were analytical reagent grade. REAGESTFERRICYANIDE-KITROPRUSSIDE. Equal volumes of 10% sodium hydroxide, 10% sodium nitroprusside, and 10% potassium ferricyanide were mixed and diluted with 3 volumes of distilled water. Allowed to stand for 20 minutes, the dark brown solution turned pale yellow and the reagent was ready for use. This reagent was unstable a t room temperature, but could be kept for 2 or 3 weeks in the cold without deterioration. Colored spots occurred immediately after spraying with the reagent. REAGENTAMMONIACAL SILVERNITRATE. Equal volumes of 0.1.V silver nitrate and 5 5 ammonium hydroxide from previously prepared stock solutions were mixed. After spraying with this reagent, some areas appeared immediately and further development was obtained by heating the chromatogram a t 100' C. for 10 minutes. REAGENTP-DIMETHYLAMINOBENZALDEHYDE. TWO grams Of p-dimethylaminobenxaldehyde were dissolved in 100 ml. of 1.2A-
Cyanamide and its derivatives which were formed in the manufacture of dicj andiamide were chromatographed with 1-butanol-ethyl alcohol-water (4 :1 :1) and with methyl ethll ketone-petroleum ether-water (9:4:3) on Whatman No. 3 and No. 3MM filter paper. Sixteen spots were detected on the chromatograms; cyanourea, biguanide, melamine, three salts of guanidine, urea, guanylthiourea, thiocyanate, thiourea, dicyandiamide, and cyanamide were characterized. Quantitative analysis for urea gave a recovery of 957'.
D
URIKG the manufacture of dicyandiamide from cyanamide,
by-product formation occurs from the reaction of these substances with water or with impurities introduced into the reaction mixture from the raw material. In a continuous process, these by-products and their decomposition products are allowed t o remain in the system, and further reaction may occur slowly R ith cyanamide and diryandiamide to consume further amounts of both substances. Much effort has been devoted to the development of methods of analysis of the compounds likely to be formed (6, f4),but these procedures as well as unpublished modifications of them have yielded results of questionable accuracy. The use of paper chromatography as an aid in studying the nature and quantities of these compounds in solutions of unknown composition has not been reported, although the development of urea, guanidine, and guanidine derivatives on paper chromatograms has been described (1-3, 10, 12). When the work reported here n-as neaily complete, it came to the authors' attention that Hubener and others ( 7 ) had quantitatively determined urea on paper chromatograms. Measurements of urea involved the addition of p-diniethylaminobenzaldehyde t o the chromatogram to form a yellow complex which was eluted with pyridine and measured spectrophotometrically a t 449 mp. This procedure was modified recently by Bode and Ludwig (5).
oo ON
EXPERIMEYTAL
Apparatus. Borosilicate glass chromatography jars with paper support racks and solvent assemblies, and a Chromatocab were purchased from the Schaar Co., Chicago. Reagents were sprayed on the developed chromatograms with a laboratory constructed glass atomizer. A Beckman Model D U spectrophotometer with Corex cells of 1.002-em. light path was used for the urea determinations. The slit width was kept constant at 0.16 mm. Developing Solvents. Numerous solvent mixtures consisting of various combinations of 1-butanol, methyl ethyl ketone, ethyl alcohol, water, petroleum ether, ammonia, and acetic acid were tested as the mobile phase on Whatman No. 3 filter paper. This paper was used because of its high capacity for solids. The paper was not prewashed and the chromatograms were developed over a temperature range of a t least &so C. from room temperature without any noticeable change in the resolution of the various cyanamide derivatives. The solvent which gave the best separation of most of the compounds was a solution of l-butanolethyl alcohol-mater (4: 1 : l ) . The organic layer of a mixture of methyl ethyl ketone-petroleum ether-water (9:4:3) was employed, however, to separate dicyandiamide, thiourea, and the thiocyanate ion which formed only one spot with the former solvent. Considerable streaking occurred with this solvent on
0 O
p
Figure 1 ( l e f t ) and Figure 2 ( r i g h t ) . Chromatograms of cyanamide and its derivatives Developed with 1-butanol-ethyl alcohol-water (4: 1 : 1) for 23 hours
1 Present address, Stamford Research Laboratories, American Cyanamid Co., Stamford, Conn.
846
Developed with methyl ethyl ketone-petroleum ether-water (9:4 : 3) for 30 hours
a47
V O L U M E 28, NO. 5, M A Y 1 9 5 6 hydrochloric acid. This reagent c o d d be h p t for 3 to 5 c1aj.s in the cold without deterioration. \-ellox colors appeared immediately after spraying. One gram of 2,6REAGEKT DICHLOROQUISOSECHLOROIMIDE. dichloroquinonechloroimide was dissolved in 100 ml. of absolute ethyl alcohol. Storage in the cold kept the reagent for 2 to 3 weeks. T h e colors produced- , 1 this reagent reached their maximum intensity after 2 to 3 hour8. REAGENT 1 , 2 - N A P H T H O Q r I S O S E - 4 - ~ O D I L ~SL-LFOSATE. ~I Four volumes of a freshly prepared 1yo solution of 1,2-naphthoquinone-4-sodium sulfonate and 1 volume of 41V sodium hydroxide were mixed immediately before use. After heing sprayed n ith this solution, the chromatogram was heated in an oven for 2 minutes a t 100" C. and then sprayed consecutively with a 2.5% solution of urea, concentrated hydrochloric acid, and concentrated nitric acid (density 1.421. Method. PREPARATIOX OF CHROKITOGRA~I?. Fifty niicroliters of dicyandiamide mother liquor were applied in the normal manner to an area 1 em. in diameter on the starting line of a sheet of Whatman No. 3 paper, 6 X 22.5 inches. The chromatogram was developed for 23 hours \yith a soliltion of 1-l)iitnnol-ethyl
Table I.
Compound
Rf
A
B
0 0.3
C
0.07
D
0 10
E
0.17
I'otasium cyanoureste
0.04
Biguanide
0.08
F?
0 14
0 22
c;
0 "I
0.2Y
Melaniine
0.08
0.2''
Guandurea
0.18
0 il'
Hearbonate
0 . li
I
0 34
Paper. Whatinan T o . 3 Developing solvent. S'Aution of I-butanol-ethyl alcohol-water ( 4 : 1 : 1) Sample. 0.05 nil. inother liquor .4mAg After After FCSP spraying heating DhB
+
Orange-red f
Green-blue -
0 64
.1
t ;uanidine
Streaked
carbonate
fro:n 0.22-0 33 0 77
Ii 0.35
L
0 79
0,s'' 0.35
0 80
hmmonium t liiocyanate
0.38
Thiourea
0.49
Dicyandiamide
0.51
P Cyanamide
0 '3'1 1
.on
-
1.47
0 61
1 .50
distance moved b y compound R' = distance moved b y thiourea ' Negative test. Positive test. Very positive test.
+
Blue-green
-
Light gray T
Orange
+-
-
White
Orange
White
-7-
SeZ
tc
.(\.ti;
T -
-
-
+t
Ora:?ge -c I
Light hron.:i wit!i dark brown triiii on the bottoin
+
Orange
+
Purple
++ -
Yellow t Tcllon.
Gray
-
G s a r brown
Gray
+ &
Pii r rile
+
Brown gray
-+ Rpd
Light brown
-
-
--
Red-blue +Red-hhle
-r Red-blue
-Red +-c
Red
i+
Red ++ Red
tr
-
-
-
++
-
-
-
-
-
ReJ-blue
Red
white tor)
Yellow Uellow
t
-
+ -
Red-brown Red-brown
+-
t
+-
Blue. turnrd yellow after 1 miii.
Gray-hron n +A
+
Blur
4-T
\Thltt!
Red ? n standing
Red-brown
t
T
Yelluw
7
Light brown
Yellow
Red-brown
c-
-
-
White
Magepta +r Magenta
Tellow i f Yellow
-+
f t
Yellow
++ Yellow
T
++
Gray-brJwn
Lfagenta
++
~-l?lluw
Purple
++ Green turned
+T
-
++ Red-blur
-
+++ Blue, turned
YellJw
t-
leilow +f
I
-
i+
-
4-CT
T +
Blue-green
-
-
--
-
Red
-
Yellow
+
-
T
-
Purple
+
-
-
T +
+Dark brown witli
Light brown
-
-
Pusple
-
XQS After final sprayinq
-
Tello w
Light, orang? -+ Orange
f
ilfrer heating
Purple
-
-7
+
Purple-gray
Faint orange t
T
Pur 1; I ,
DCC
JTello w
Orange-red
yellow af rer 1 inin.
1 03
+
Grty Wljite
yellow after 1 niin.
1 00
+
Light gray T
-
0r.i:iq
Guanylthiourea
Tables I and I1 list R , and K , values of thc spots arid the colors developed with the reagents. R c values (distance a compound moved relative to thiourea) were of greater aid than R , values in characterizing the compounds. as only a partial separation had occurred n-hpn the solvent front had reached the bottom.
Comparison of Constituents in Dicyandiamide Mother Liquor with Zuthentic Cyanamide Derivatives
R1 0.00
Urea
alcohol-water (4: 1: 1) by the descending method, and after dr>,irig in a well-ventilated fume hood, the chromatogram was sprayed with the desired reagent. Development on Whatman No. 3SIRI paper with the organic layer of a mixture of methyl ethyl k e t o n petroleum ether-water 19:4:3) required only 5.5 hours for t h r solvent front to reach the bottom of the paper, but for good resolution the chromatogram was developed for 30 hours. One spray alone did not reveal the position and size of all the spots developed with 1-butanol-ethyl alcohol-water and the chromatogram illustrated by Figure 1 was prepared from the combined information derived by spraying chromatograms v i t h ammoniacal silver nitrate, 1,2-dichloroquinonechloroimide,and alkaline ferricyanide-nitroprusside. The lat,ter reagent WRS used to spray the chromatogram illustrated by Figure 2.
Slight gray Slight gray
+t
Orange-red
++ Orange-red
+
Yello iy + Yellow
+- + +
a Compound P and cyanamide gave orange-red color after spraying with alkaline solution of 1.2-naphthaquinone-4-sodium sulfonate. duced with other compounds.
No oolors were pro-
ANALYTICAL CHEMISTRY
848
Table 11. Rt Values of Constituents in Dicyandiamide Mother Liquor Developed with Organic Layer of a Mixture of Methyl Ethyl Ketone-Petroleum Ether-Water (9:4 :3)
Compound Unknown
0.00
Unknown
0.02
Rt
K (urea)
0.15
31 (thiocyanate and
0.30
another compound) 0 (dicyandiamide)
’i (thiourea)
Paper. Whatman No. 3 h I h I . Sample. 0.02 ml. mother liquor AmAg After .4fter FCNP spraying heating
++ Orange ++ Red I
Rzd
+ Buff
++ Magenta ++ Blue, center
0.66 1.00
QUANTITATIVE DETERMINATION OF UREA. Approximately 6 mg. of urea in a weighed aliquot of the solution (0.3 t o 0.4 gram) were distributed over 10 spots on the starting line of a sheet of Whatman No. 3 filter paper, 13 X 22.5 inches. Three guide strips, one of which was located a t each end of the chromatogram and one in the middle, were spotted with the solution and the chromatogram was developed for 30 hours with I-butanol-ethyl alcohol-water (4:1: 1). After drying, the guide strips were sprayed with alkaline ferricyanide-nitroprusside to locate the urea, and the urea-containing zones in the unsprayed sections of the chromatogram were cut out and then cut diagonally in half to form two triangles. These papers were hung vertically between glass rods above a large funnel fitted into a 25-ml. volumetric flask and washed with about 22 ml. of water. The eluate was then made up to volume.
+
Yellow
-
++
Yellow
+
Red on standing
+t White
-
+
Brown-gray center, gray ring
turned yellow in 1 minute after spraying
A comparison of the mobilities and color reactions of the constituents of the mother liquor with those of authentic cyanamide derivatives indicated that compounds E, F, G, K, L, &I, N, 0, and P in Figures 1 and 2 and Tables I and I1 were cyanourea, biguanide, melamine, urea, guanylthiourea, thiocyanate, thiourea, dicyandiamide, and cyanamide, or salts thereof. The spots listed in Table I1 as unknown were those compounds which had low mobilities in the methyl ethyl ketone-petroleum etherwater solvent and did not separate in the manner illustrated by Figure 1. Compounds H, I, J, guanidine carbonate, and guanidine nitrate gave a positive test with Sullivan’s reagent, which indicated that the three unknowns were probably different salts of guanidine. Compound J could not be identified as guanidine nitrate, although this salt had the similar R t value of 0.65, since no nitrate ions were introduced into the system. One of the salts was presumed to be guanidine carbonate, however, even though this compound formed a long streak from the starting line, and another was thought t o be guanidine cyanate. The presence of cyanate ions in the mother liquor was indicated from the number of equivalents of conjugate acids with pK, values between 4 and 5 . All of these acids could not be accounted for by melamine and cyanourea (8).
DAB
+ White + White + Gray + Gray
Yellow
photometer a t 420 mp, The urea concentration was determined from a calibration curve obtained x i t h standard urea solutions. Beer’s law was obeyed, with an absorptivity a t 420 mp of 3.96. Table I11 shows that urea solutions varying in concentration from 1 to 2.3% could be analyzed by paper chromatography with a recovery of 95%.
Table IV.
Determination of Urea i n Presence of Other Cyanamide Derivatives % Urea Sample
Chromatographic method
Dicyandiamide mother liquor
1.81 1.79
1.81 1.86 1.82
1.93 1.96
1,95 1.97
1.31 1.38
1.39 1.40
1.80 1.79
1.70 1.66
1.67 1.74
1.63 1.67
Cyanamide solution
Precipitation 1 method
When a chromatogram of the constituents in commercial dicyandiamide liquors was sprayed with the p-dimethylaminobenzaldehyde reagent, yellow areas developed with the cyanoureate ion, melamine, urea, thiourea, and cyanamide. Only urea and cyanamide, however, formed a complex which showed any appreciable absorption a t 420 mp. Table IV gives the results obtained for the determination of urea in a number of liquors by the chromatographic method and by a procedure that involved the removal of cyanamide and thiourea with silver nitrate prior to adding the reagents for spectroscopic measurements. The data obtained by chromatography were corrected on the basis of the per cent recovery shown in Table 111.
Table 111. Analysis of Standard Urea Solutions % ’ Urea
Recovery,
Found
Tlieoretical
0.92 1,69 2.18
0 97 1.76 2.29
7c
95
96 95.5
A 10-ml. aliquot of the eluate was mixed with 10 ml. of a solution of p-dimethylaminobenxaldehyde ( I S ) , diluted t o 25 ml. with distilled water, and after standing for 20 minutes the intensity of the yellow solution was measured with a Beckman DU spectro-
DISCUSSION
Paper chromatography has been useful for elucidating the nature of the principal compounds which were present in solutions in various parts of the dicyandiamide process, and an estimate of the number of compounds in trace amounts has been obtained. The method should also be suitable for detecting and identifying cyanamide and its derivatives in other sourccs. Because a large number of compounds can be formed theoretically from cyanamide, the possibility remains, however, that other compounds were present which did not give a color reaction with
V O L U M E 28, NS). 5, M A Y 1 9 5 6 the reagents used in this work. Indeed, from an inspection of the contour of biguanide, it appeared that a compound was present between cyanourea and biguanide. With the exception of dicyandiamide, urea, and thiourea, the other compounds were acids and bases and it is possible that a compound could be located in more than one spot in order to preserve electrical neutrality of the spots. Compounds H, I, and J were characterized in part as the guanidinium ion, but the identity of the anions was not determined with certainty. The nature of compound B, which appeared from its color reactions to contain sulfur and which \Vas not thiourea, guanylthiourea, or thiocyanate, is open t o speculation. An ultraviolet absorption spectrum of an aqueous eluate of this zone gave a band at 236 mp which is the wave length maximum reported for thiourea ( 9 ) , indicating the presence of a closely related molecule. Only the areas corresponding t o the guanidinium salts underwent the Sullivan reaction. However, an orange color was formed b - cyanamide when an aqueous solution of 1,2-naphthaquinone-4sodium sulfonate and alkali was sprayed on the chromatogram and this color disappeared in successive stages of the Sullivan reaction. These two reactions suggest specific methods of analysis for cyanamide and guanidine in miltures of related compounds.
849 ACKNOWLEDGMENT
The authors wish t o acknowledge the assistance of S. C. Blodgett. This work was part of the development program of North American Cyanamid, Ltd. LITERATURE CITED
(1) Adachi, S., Kagaku 23, 582 (1953). (2) Berry, H. K., Biochemical Institute Studies IV, pp. 88-92, Univ. Texas Publ. 5109, Austin, Tex., May 1, 1951. (3) Berry, H. K., Sutton, H. E., Cain, L., Berry, J. S., I b i d . , pp. 22-55. (4) Bertrand, AI., Myers, J. L., Can. J . Chem. 31, 1252 (1953). ( 5 ) Bode, F., Ludwig, E. M., Schweiz. med. Wochschr. 84,629 (1955). (6) Bourjol, S., Teindas, LIrs., Mbm. poudres 31, 51 (1949). (7) Hiibener, H. J., Bode, F., Mollat, H. J., Wehner, M., H o p p e Seyler’s 2. physiol. C h e m . 290, 136 (1952). (8) Kennerly, G. W., unpublished data. (9) Alaaon, S. F., J . Chem. SOC.(London) 1954, 2071. (10) Roche, J., Van Thioai, S . ,Hatt, J. L., Btochem. et B i o p h y s . Acta 14,1,71 (1954). (11) Sullivan, 11.X., Proc. Soc. Exptl.B i d . M e d . 33, 106 (1935). (12) Tuppy, H., Monatsh. Chem. 84,342 (1953). (13) Watt, G. W., Chrisp. J. D., -$SAL. CHEM.26, 452 (1954). (14) Williams, H. E . , “Cyanogen Compounds,” Edward Arnold & Co., London, 1948. RECEIVED for review July 14, 1955.
Accepted February 17, 1956.
Rapid Paper Chromatography of Carbohydrates and Related Compounds H. T. GORDON, WAYNE THQRNBURG, and L. N. WERUM Department of Entomology and Parasitology, University of California, Berkeley 4, Calif-, and California Packing Corp., €meryville, Calif.
This method is useful for the rapid separation and tentative identification of carbohydrates and related compounds in biological fluids and extracts. No desalting or other purification is required. Interference by inorganic salts is prevented by a simple “overspotting” technique using pyridinium sulfate. Ascending onedimensional chromatograms are completed in 2 hours. Spots of carbohydrates, polyhydric alcohols, aldonic and uronic acids, nucleosides, phosphate esters, and other derivatives are semiquantitatively detected by improved specific color reagents. R j values are highly reproducible; this makes possible an analysis of some of the causes of variation in R j , especially the systematic variations due to interference by inorganic ions and to varying loads. The method is not suitable for the complete separation of structurally similar carbohydrates or of a large number of carbohydrates and closely related compounds on one chromatogram; it is designed for the rapid identification of a small number of carbohydratesin complex biological systems containing considerable quantities of many other materials.
T
HE basic objective of the work reported in this paper has been to develop a rapid method of separation and tentative
identification of carbohydrates and related compounds in biological fluids without preliminary purification. This research was instigated by the discovery that an isopropyl alcoholpyridine-water-acetic acid solvent, used previously for paper chromatography of alkali and alkaline earth cations ( 5 ) ,has the useful property of moving sugars, polyols, uronic acids, purine,
and pyrimidine ribosides, and their phosphate esters all within the Rj range of 0.1 to 0 9. The solvent tends to separate carbohydrates and their derivatives into groups of similar structure, all moving within a narrow Rj range-e.g. pentoses have an R! of 0.70 to 0.77, and hexuronic acids have an R j of 0.33 to 0.38. This is useful for a preliminary classification of unknowns; more specialized solvents, with higher resolving power, can then be used to separate and identify individual compounds. The solvent has other useful properties. Inorganic salts normally do not interfere with the movement of organic compounds on the chromatogram, and desalting is usually unnecessary. The low viscosity of the solvent allows it to ascend rapidly, and an ascending one-dimensional chromatogram can be completed in 2 hours. The high volatility of the solvent makes possible complete drying of the chromatogram in 15 minutes. The solvent power is high, and loads of several hundred micrograms of most carbohydrates move as compact spots, without streaking. The commonly occurring inorganic ions (potassium, sodium, calcium, and magnesium) present in the unknown solution are well resolved and can be easily identified. A second objective has been to study the systematic variation of spot size and R/ with the quantity of substance spotted on the chromatogram. If paper chromatography is done with great care, R, values are reproducible to f 0 . 0 1 ; the variation of R f with load can then be clearly demonstrated, especially for ions (6). Such “load effects,” together Kith interference effects due t o other substances, are important causes of the R/ fluctuations which have led many workers to run known standards simultaneously with unknowns, and to replace RJ by R, (the ratio of the distance traveled by the substance to the distance traveled by glucose).
