Infrared Spectra of 3-Pheny l-2-t hiohydanto ins of Amino Acids AGNES EPP Nafional Research Council, Prairie Regional laboratory, Saskatoon, Sask., Canada
b Infrared
absorption spectra are described characterizing the 3-phenyl2-thiohydantoins, the 3-(phenyl-p-azophenyl)-2-thiohydantoins, and the 3-0nitrophenyl-2-thiohydantoinsof amino acids. The spectra of some preparations deviate from the characteristic phenylthiohydantoin absorption pattern. For these compounds the openchain thiocarbamyl structure is assumed. The spectra are highly characteristic and serve to identify the amino acids from which they are derived.
Spectra were recorded with a PerkinElmer Model 21 infrared spectrophotometer equipped with a rock salt prism. The regions from 4000 to 3800 and from 2600 to 1800 cm.-' were always free from absorption bands and are therefore omitted from the figures.
T
1770 to 1740 crn.-'
absorption spectra of 3-phenyl-2-thiohydantoins (1,a) have been described previously, as well as their use in identifying amino acids ( 7 ) . The p-azophenyl-and o-nitro-substituted 3-phenyl-2-thiohydantoins (1,b and c) have been synthesized subsequently (8) for the same purpose as well as for color chromatography. This work deals with the infrared spectra of these compounds. Included are the spectra of thiocarbamyl derivatives (11) of some amino acids obtained instead of the 3-(phenyl-p-azophenyl)-2-thiohydantoins, where ring closure could not he achieved. HE
INFRARED
\3/
N I
DISCUSSION
Phenylthiohydantoin Ring. The following assignments were given to the absorption bands characterizing the different groups of the 3-phenyl-2thiohydantoin ring (7):
1600 cm.-' 1425 to 1400 cm.-' 3300 to 3150 crn.-"
vibrations Phenyl group vibrations C=S vibration6
Ring C-=O
NH
1530 t o 1500 ~ r n . - ~ ]
These data hold for most of the nitro- and azophenyl-substituted phenylthiohydantoins studied and are in good agreement with those given by Randall and coworkers (Q),who examined a number of cyclic diacylimide-type compounds including thiohydantoins. These authors state that a band near 1750 cm.-' is characteristic for the ring carbonyl a t the 4 position in the
I1
R.' I
where R = side chain of amino acid for a, R' =
0
EXPERIMENTAL
Samples were dissolved in ethyl alcohol and mounted as potassium bromide windoivs in the manner described previously ( 7 ) . Such spectra showed better resolution than those of the compounds which were mounted as solids in the potassium bromide window.
thiohydantoin ring, although values as low as 1720 cm.-' are also given, and a second band in the region from 1600 to 1450 cm.-' is assigned to the thioureide ion. This latter band undoubtedly is of the same origin as that assigned above to the N-H deformation vibration a t 1530 to 1500 cm.-l, because it is absent from the spectra of the phenylthiohydantoins of proline and hydroxyproline, where fused ring structures are assumed, All phenylthiohydantoins show absorption in the region from 1425 to 1400 cm.-' ( 7 ) . A band of medium intensity has also been found in this region for the present substituted phen-
ylthiohydantoins and is given by related model compounds such as thiourea, acetylthiourea. 2-thiohydantoin, and 5-methyl-2-thiohydantoin. On the other hand, for all phenylthiohydantoins studied a strong absorption is present a t 1200 cm.-'; this has been designated as the C-S stretching vibration by hlecke and lfecke (6) for a series of cyclic thiourea derivatives. This latter band shifts, as noted below, when thc thiohydantoin ring is opened. Thc band a t 1425 to 1400 cm.-I, however, does not shift noticeably upon ring opening and therefore may not be caused by C=S stretching vibrations as supposed earlier. Amino Acid Side Chain. I n addition to the bands given by the phenylthiohydantoin ring, the infrared spectra of the compounds studied contain bands which are caused by various groups in the amino acid side chain Some of these are quite specific and aid in the identifiration of the particular compound. The derivatives of glutamic and aspartic acids, for example, have a carboxyl group in the side chain. In the solid state these compounds give a strong band a t 1725 to 1700 cm.-' I n solution, however, the band combines with that of the ring carbonyl a t 1770 to 1740 cm.-l to form one broad absorption centered around 1750 cni.-'. The glutamine and asparagine derivatives have an amide group in the side chain which is easily recognized by its amide I band a t 1670 to 1650 cm.-'. The amide I1 band is not resolved for this series of compounds. It probably coincides with either the benzene ring absorption a t 1600 cm.-l or with the N H band a t 1530 to 1500 cm.-l. The NH2 group of a primary amide would be expected to cause two N H stretching bands in the region from 3500 to 3200 cm.-'. Only the 3-phenyl-2-thiohydantoin and the 3-(phenyl-p-azophenyl) 2-thiohydantoin derivatives of asparagine show two sharp bands near 3400 and 3200 cm.+, respectively. For the rest of the compounds containing amide groups these bands are resolved poorly, if at all, from the wide general absorption extending from 3500 to 3000 cni.-*. Extensive hydrogen bonding undoubtedly causes this phenomenon. VOL. 29, NO. 9 , SEPTEMBER 1957
1283
The derivatives of arginine also absorb around 1660 cm?, but this band can be distinguished from the amide I band by its greater intensity and width. It extends well into the 1650 to 1600 cm.-'-region, so that the benzene ring absorption, which is well resolved for all other compounds studied, appears only as a small shoulder a t the side of this -C=NH band. Lieber and coworkers ( 5 ) also have observed a band for a number of other guanidine derivatives in this region. It mould be expected that an OH group in the side chain would absorb a t slightly higher wave numbers than the ring NH group in the region from 3500 to 3000 cm.-l. This was observed for 3-phenyl-2-thiohydantoin and the 3 - (phenyl - p - azophenyl) - 2 - thiohydantoin of threonine, which show sharp bands a t 3500 cm.-' and a t 3200 cm.-'. For all other OH-containing compounds, except the hydroxyproline derivatives, hydrogen bonding causes the OH group to absorb a t lower wave numbers, which results in a common OH and NH band extending in most cases from 3500 to 3100 cm.-l. The hydroxyproline derivatives have no N H group and therefore exhibit the normal OH band a t 3500 cm.-l. Definite assignments were not attempted for other groups of amino acid side chains because the absorption bands are either weak or coincide with other major bands. N - (Phenyl-p- azophenyl) - thiocarbamyl Derivatives. As has been pointed out, ring closure was not accomplished with some of the azophenyl derivatives, the products being thiocarbamates rather than thiohydantoins (8). Chemical analyses and spectroscopic studies indicated that this occurred with derivatives of glycine and proline. It was noted also that the derivatives of serine, phenylalanine, and glutamine differ from the main group of 3-(phenyl-p-azophenyl)2-thiohydantoins in their ultraviolet absorption properties. Infrared studies show that the latter compounds possess absorption properties similar to those of derivatives of glycine and proline, thus indicating the open chain thiocarbamyl structure. This follows from the fact that when the phenylthiohydantoin ring is assumed to have been formed, the ring carbonyl band occurs above 1725 cm.-' (in most cases near 1750 cm.-1), but otherwise between 1725 and 1700 cm.-l, the absorption range for saturated carboxylic acids. Also, the C=O stretching vibration for the known A'-phenylthiocarbamate of glycine, whose spectrum was examined for reference purposes, occurs between 1725 and 1700 cm.-l The lysine derivative has its major C=O absorption peak a t 1740 cm.-l, but shows a shoulder a t 1700 cm.-l This suggests 1284
ANALYTICAL CHEMISTRY
W
0
a
t-
t 5
a z t(L b-
z "0 w (L
a
v
i
I 4
1
5 O
r
L 3800
U 3400
L 3000
L
,1
1
2600 1800
I
' 1600
1400
,200
low
7
aoo
WAVE NUMBER ( c m - l )
Figure 1. Infrared spectra of N-(phenyl-p-azophenyl)thiocarbamates derived from amino acids
1. &Glycine 2 . dl-Serine 3. &Glutamine
4. dl-Phenylalanine 5. &Proline
a mixture of the ring and the open chain structures. As mentioned above the C=S band near 1200 cm.-l is also shifted on ring opening. None of the compounds presumed to be thiocarbamyl derivatives absorb strongly here. For the glycine derivative only one band occurs a t 1250 cm.-l, and the N-(phenyl-pazopheny1)-thiocarbamates of serine, phenylalanine, proline, and glutamine have somewhat intensified absorptions near 1250 and 1150 cm.-l The spectrum of the 3-(phenyl-pazophenyl)-2-thiohydantoin of proline would not be expected to possess any NH bands if the ring were closed. However, there are definite absorptions in the region from 3300 to 3100 cm.-l as well as the 1530 cm.-'-region. This serves as an additional proof of a thiocarbamyl structure in the proline derivative. 3-(Phenyl-p-azophenyl)-2-Thiohy-
dantoins. The spectra of these amino acid derivatives in the range from 4000 to 900 cm.-l do not show absorption characteristics which strikingly distinguish them from the unsubstituted 3-phenyl-2-thiohydantoins. The new group in the compounds is the -N=N- bond which gives rise to only weak absorption bands near 1600 cm.-1, in the same region as the benzene ring. However, the 1600 cm.-'band for most of the unsubstituted phenylthiohydantoins is sharp and narrow, but it is rounded and somewhat broadened a t the base with the phenylazophenylthiohydantoins. The derivatives of methionine, valine, and isoleucine show a definite shoulder a t 1585 to 1580 cm.-' caused by the -X=Xvibration. Le FBvre, 0'Dwyer, and Werner ( 4 ) found a second band for diazo compounds a t 1406 =t14 cm.-1, which also seemed to be characteristic for the class of compounds. For
I
P
I
W
4 z
t I (I)
4 I-
+ 0 W Y cc
a
0 3800
,
3400
/
I
3000
,
I
2600 i e o o
,
1 1600
,
l l 1400
1
1200
1
I
,
1000
l
l
BOO
W A V E NUMBER Icrn-1)
Figure 2.
Infrared spectra of 3-(phenyl-p-azophenyI)-2-thiohydantoins derived from amino acids
1 . dl-Alanine
2 . dl-Valine 3. dl-Leucine 4. &Isoleucine 5 . dl-Threonine
6. 7. 8. 9. 10.
dl-Aspartic acid 1-Asparagine Z-Glutamic acid 1-Hydroxyproline Z-Tyrosine
dl-Tryptophan dl-Methionine Z-Lysine 1-Arginine 15. 1-Histidine 11. 12. 13. 14.
VOl. 29, NO. 9, SEPTEMBER 1957
1285
the phenylthiohydantoiii derivatives this band would be overlapped by the 1400-~m.-~ absorption. A remarkably constant absorption pattern for the azophenyl-substituted thiohydantoins is found in the range from 900 to 700 cm.-' Three prominent peaks a t 855 to 840, 770 to 765, and 690 to 680 cm.-l dominate the region. Tetlow (11) found that both cis- and trans-azobenzene give the latter two bands, but did not observe a peak near 850 cm.-l There is no doubt that the 850-cm.-l absorption band is connected with the para-disubstitution of the benzene ring a t position 3 of the thiohydantoin ring. On the other hand, the strong band a t 927
cm.-' observed by Tetlow for azobenzene is not shown by any of the phenylazophenylthiohydantoins. These three peaks are present for a11 compounds studied, including the phenylazophenylthiocarbamates. Only in the spectra of the derivatives of tryptophane, tyrosine, and to a minor extent, of phenylalanine, is the regularity of the pattern somewhat obscured by additional absorption bands from the benzene ring in the amino acid side chain. 3-o-Nitrophenyl-2-hydantoins. The spectra of this group of compounds are characterized by the bands specified for the phenylthiohydantoin ring and by t w o strong bands arising from
0
0 I
3800
1 3400
I
l
3000
i
i
!
,
2600 I800
I
1600
I
1
!
1400
,
1200
l
,
,
I000
l
000
l
j
3800
W A V E N U M B E R (ern-')
Figure 3.
l 3100
.
l 3000
.
,
.
1
1
L
2600 I000
1600
1
,
I100
W A V E NUMBER
L
L
1200
L 1 1000
(srn-1)
Infrared spectra of 3-o-nitrophenyl-2-thiohydantoins derived from amino acids
1. dl-Glycine 2. dl-Valine 3. dl-Aspartic lrcid 4. 1-Asparagine
1286
asymmetrical and symmetrical valence vibrations of the NO2 group which occur a t 1530 and 1350 cm.-l, respectively. The first band emerges as a separate peak from the strong common absorption of the NH and NOz groups. The 1600-cm.-' band for these compounds is split into two components. The more intense peak is located a t 1610 cm.-l, the weaker one a t 1590 to 1585 cm.-* The shift of the 1600cm.-l band to higher values for nitrobenzenes is known and has been observed by Bellamy (1). Brown (3) also reports the benzene ring vibrations to occur a t 1612 and 1590 cm.-l for aromatic nitrocompounds. The present findings confirm BrownJs observation
ANALYTICAL CHEMISTRY
I-Glutamic acid I-Glutamine i. 1-Cystine 5. (-Tyrosine 5. 6.
9.
I-Proline
10. I-Histidine 11. I-Lysine 12. dl-Tryptophan
I
800
I
j
that these values vary independently of the U-values of the substituted groups in the benzene ring. The spectra in the region from 900 to 650 cm.-’ differ from compound to compound. In some instances a strong band between 750 and 700 cm.-l is dominant. According to Bellamy (a), this is caused by NOz deformation modes. The 850-cm.-I band observed hy Randle and Whiffen (10) for aromatic nitro compounds, and assigned to C N stretching vibrations, is present in all o-nitro-substituted phenylthiohydantoins studied. The 3-o-nitrophenyl-2thiohydantoin of proline is somewhat unusual in that it shows a shoulder a t 1710 cm.-’ as well as peaks in the KH stretching and NH deformation regions. Although the findings of the elementary analysis are in good ngreemmt with the theoretical
values for the closed-ring structure of thiohydantoins, spectral evidence suggests that the preparation used for this work is contaminated to some extent with the thiocarbamyl derivative. ACKNOWLEDGMENT
The author is indebted t o L. K. Ramachandran and %’. B. McConnell who synthesized the compounds studied and to B. D. Leddy who prepared the drawings of the infrared spectra.
W.,O’Dwyer, &I.F., Werner, R. L., Chemistry & Indus-
( 4 ) Le FBvre, B. J.
try 1953,378. ( 5 ) Lieber, E., Levering, D. R., Patterson, L. J., ANAL. CHEM.23, 1594 (1951). (6) Mecke, Jr., and Mecke, R., Sr., Chem. Ber. 89, 343 (1956). (7) Ramachandran, L. K., Epp, A . , McConnell. W. B.. ANAL. CHEJI. 27, 1734 (1955). (8) Ramachandran, L. K., McConnell, W. B.. J . .4m. Chem. SOC.78. 12.55 (1956j. (9) Randall, H. M., Fowler, R. . G , \ -
~~
k.,
Dangl, J. R., “Infrared Determnation of Organic Structures,” pp. 14, 21-4, Van Nostrand, New York. 1949.
LITERATURE CITED
(1) Bellamy, L. J., “Infrared Spectra of Complex Molecules,” p. 60, TViley, New York, 1954. (2) Ibid., p. 253. ( 3 ) Brown, J. F., Jr., J .Im. Chein Soc. 77, 6341 (1955).
(IO) Randle,’ R. R., Whiffen, D. H., J . Chem. SOC.1952, 4153. (11) Tetlov, K. S., Research (London) 3 , 187 (1950)
RECEIVED for review January 28, 1957. Accepted April 30, 1957. Contribution No. 4392, National Research Council.
Organometallic Precipitates and Briquetting for X-Ray Spectrography JOHN E. FAGEL, Jr.l, EARL W. BALIS, and LESTER B. BRONK Research Laboratory, General Electric Co., Schenectady, N. Y.
b Briquetted organometallic precipitates in x-ray spectrography offer a powerful general method of analysis. Suitable standards are available from stock solutions. The briquetting process alone is convenient for handling some homogeneous solids like metal powders, metal turnings, and metal chips. Density of briquets should b e controlled within 5% to avoid appreciable errors in measured x-ray intensities.
X
emission spectrography is a rapid and accurate method for direct qualitative and quantitative analysis of solids. Study of the restrictions to be considered in the preparation of samples reveals the importance of briquetted organometallic precipitates as the basis of a powerful general method of analysis by x-ray emission spectrography, and that briquetting is adequate for preparation of samples from homogeneous particulate matter. The maximum size of a sample is determined hy the geometry of the spectrograph. As most x-ray spec-RAY
Present address, Lamp Wire and Phosphors Department., General Electric Co., Cleveland 17, Ohio.
trographs can accommodate pieces up to about 5 X 2.5 x 1 cm. thick, sections or fillings or turnings cut from large solid pieces must sometimes be used. Homogeneity and roughness of the surface presented for analysis are important. The incident x-ray beam covers only a limited area of the sample and penetrates it to a finite depth. The intensity of the beam varies over the area irradiated. Hence the sample volume subject to radiation should be homogeneous within each sample and between samples. Homogeneity means uniform proportions of components on a micro scale and uniform density. Pensity of the sample volume irradiated is an uncontrolled variable for particulate material. Even for homogeneous solids, roughness of the surface is important. Alloys cut on an ordinary abrasive wheel are smooth enough; such surfaces are equivalent to polished specimens for x-ray spectrography. A sample with a pebble surface like that presented by a layer of 2-mm. shot would give different results from a smooth piece, even though both pieces were homogeneous and had the same composition. Briquetting is useful for preparing homogeneous particulate matter for use in the x-ray spectrograph. Briquets are easier to handle than par-
ticulate matter. The density of the sample volume presented for irradiation and the roughness of the surface can be controlled so neither factor will affect the analysis. Comparable standards and unknowns are easily prppared. Briquets were used for x-ray emission analysis by Mortimore, Romans, and Tews (5) and by Adler and Axelrod (1, 2) who experienced difficulty in direct measurements even on finely ground samples of ores, in part because of the heterogeneous nature of ores. Mortimore and associates dissolved the ores, precipitated the components of interest along with an internal stanclard, and briquetted the precipitate. Adler and Axelrod used a special grinding process, internal standards, and briquetting. Briquetting ii important in the general method of analysis described here, where organometallic precipitates are used. It is useful for a variety of homogeneous particulate materials, including metal powders, metal turnings, and metal chips. Such materials have been pressed here into briquets 1 inch in diameter a t 100,000 pounds per square inch. The densities of such briquets are reproducible when pressure and time of pressing are controlled. Density variations less than 5% are VOL. 29, NO. 9, SEPTEMBER 1957
1287
