552
ANALYTICAL CHEMISTRY
Table I.
(2) Jones, R. S . , Huniphries, P., Packard, E., and Dobriner, K . , J . Am. Chem.
Band Positions" of Pseudosapogenins and Pseudosapogenin Esters between 2000 and 1400 Cm.-l CHCI3
Compound
CBZ
1695
...
Suc., 72, 86
(1953). (4) Jones, R. S , Kataenellenbogen, E., and Dobriner, K., Natl. Fleseavh Coiciicil Pub., NRC 2929 (195.1)
nitrobenzoatec (1730), 1701-1693sh (1745 and 1727), 1704 Pseudosinilagenin C 1695 169.5 Pseudosmilagenin diacetarec ... Smear (17421, 1695sh Pseudosmilagenin di-3,s-dinitrobenzoatee ( 1 i33), 1701-1692sh (1736 and 1718), 1706 Pseudotigogenin lG98 ... I695 I'seudotigogenin diacetate ... Pseudodiosgenin 1695 1693 Pseudodiusgenin diacetate Pseudohecogenin (iio41 ( i m ) , 1692-1689~11 a Only carbonyl absorption and the significant band a t 1706-1689 cm. -1 are listed. b Values in parentheses are those of ester or ketone carbonyls. C The preparation and properties of these previously unreported compounds will be described in a forthcoming publication by Scheer. Kostic, and RIoaettig.
~
_
_
_
LITERATURE CITED
(1) Eddy,
c. R., Wall, AI. E., and Kiumpp Scott, ll.,AN.AL.C m w . ,
2 5 , 2 6 6 (1053).
(1950).
( 3 ) Jones, K. S . , Katzenrllenhogen, E., and Dohriner, X., Ibid.. 7 5 , 158
S u j o l or Sinear 1692 Sinear (1739-1724), 1692sh
(5) Randall, H. AI., Fowler, R. G., Fuson, S . ,and Dangl, J. R., "Infrared Determination of Organic Structures," p. .5, New Ynrk. I). Van Nostrand Co., Inc., 1919. (6) Rosenkranta, H., and Gut, >I., H e h . Chim. Acta, 36, 1000 (1953). (7) Rosenkrantz, H., and Zablow, L., ANAL.CHEV..2 5 . 1025 (1953). ( S ) Wall, 31. E., Eddy,'C. R.,'lIchennan, 11. L.. and Klumpp, 31 E., Ibid.,
_ _ _ _ ~ _ ~ ~ _
24, 1337 (19,52). (9) Wall, 11. E., Kridcr, 31. lI,,Rothman, E. S.,and J . B i d . Chem., 198, 533 (1952). R E C E I V EfD o r review October 10, 1953.
Eddy, C. R.,
Accepted .January 8, 1954.
Paper Chromatography of Some Substituted Naphthoquinones THOMAS SPROSTON, JR., and E. G. BASSETT University o f Vermont, Burlington, V t .
is pronounced interest in naphthoquinones because of Ttheir. biological activity, chemistry, wide natural occurrence, HERE
and commercial use as fungicides, antimalarial$, and antihemorrhagics (4). Paper chromatographic techniques have been applied satisfactorily to the isolation and identification of naphthoquinones and some benaoquinones. Procedures follow standard methods, particularly those used by Bate-Smith ( 1 ) and Gage ( 2 ) . An exhaustive study \vas made of solvents and chromogenic sprays to establish accurate R, and color values for many different 1,4naphthoquinones and benzoquinones. I t was impossible to chromatograph a homologous series of substituted quinones because of their unavailability. R, values were markedly influenced by the presence or absence of OH, CHa, OCH3, nnd SH2 radicals.
mist of 5% aqueous sodium h l d r o u d e from a compressed air sprai Pr DISCUSSION AYD RESULTS
R, values and color characteristics of the spots are listed in Table I. Rj values represent the average of five strips of each compound. For anv given compound the mavimum variation n as f O 04 R/ valup. .- -
0
EXPEKIhlEh-TAL
Standard equipment supplied by the University Apparatus Co., Berkeley, Calif., was used throughout the experiments. One-dimensional chromatograms were produced in a large Chromatocab; large glass cylinders were also wed. .ill apparatus was held a t 18' C in a constant-temperature room Synthetic 1,4-naphthoquinones A ere dissolved in absolute ethyl alcohol to make saturated solutions a t 18" C. These solutions were applied to 1-cm. disks of Whatman S o . 1 filter paper n hich were later attached to 2 X 18 inch strips of the same paper bv fixing between two slits in the strip. The center of each disk was placed 6.5 cm. from one end of the chromatographic strip. By concentrating the solution on paper disks of 1-cm. diameter, smaller and more definite spots were produced. Twodimensional chromatograms were run on 18 X 22 5 inch Whatman &-0. 1 paper sheets, as Figure 1 illustrates. Initial deposits on the paper disks were micropipetted in measured amounts, ranging from 20 to 200 pl , depending on the compound uwd, and allowed t o dry in moving air. S o advantage was observed in the buffering of papers before the solvent run. After the solvent had been allowed to descend 39 to 40 em. over a 15- t o 18-hour period, papers were removed and dried in moving air at 18" to 20" C. Many solvent mixtures and ratios of mixtures, composed of butanol, ethyl alcohol, propionic acid, acetic acid, collidine, lutidine, phenol, toluene, and water were tested for performance. T h e most satisfactory mixture was fusel oil (Eimer & Amend amyl alcohol) plus pyridine plus water ( 3 : 2 :1.5). Jeanes et al. ( 3 ) have used a similar mixture for separating simple sugars. Development of spots was accomplished by applying a fine
.--
?>OCkpOH-
I d
_
I3
09
Figure 1.
08
07
2-c -3-c
_ _ _ _ ~-
06
05
04
03
02
0
Two-Dimensional Chromatogram of Four Substituted 1,4-Naphthoquinoues
Some compounds were not pure and consequently produced several spots. I n some cases an attempt was made to purify by sublimation, but this could not be done with all impure compounds, on-ing to the limited supply. Commercial, unsubstituted 1,i-naphthoquinone which prwiously had produced four spots, v a s sublimed and subsequently showed only one spot on paper. No efforts were made to identify its contaminants. It is possible to test the questionable purity of quinones b y paper chromatography. Chromatographic spots were eluted from the paper strips after
V O L U M E 26, N O . 3, M A R C H 1 9 5 4 Table 1.
553
Rj Values of Substituted Quinones and Colors Produced by Chromogenic Spray, 5 % Aqueous Sodium Hydroxide Compound
1.4-NaphthoquinoneUnsubstituted 2-Methyl2-Methoxy2,3-Dimethoxy2-Methox~-3-h~drosv2-HydroxyZ-Methy1-3-acetyl3-Methyl-6-succino2 3-Dichloro2k!hloro-3-methoxy-
*
2-Chloro-3-ethoxy-
Fa+ +.- salt of 2-chloro-3-hydroxy.
2-Amino2-Amino-3-chloro2-Acetylamino2-Dimethylamino2-Chloro-3-dimethylamino2-Chloro-3-ethylamino-
Rf
(One-dimensional chromatograms) Colorn
0.85 0.87 0.81 0.84 0.37 0.47 0.83 0.53 0.41 0.33 0.86 0.27 0.89 0.42 0.82 0.83 0.83 0.83 0.87 0.88
OB OB 0 PUP PUP 0 PaB P Y 0 Pa0 PaRO RO Op TB
0 POB
0
OP PO
RI
Compound
1,4-Saphthoquinones 2-Chloro-3-n-decylaminoZ-(S-Acetanilido)-3-chloro2-Methylmercapto- . 2-Mercapto-3-chloro-
Z-Methyliminonaphtho(2,3)1,3-Dithiole-4,9-dione-methochloride monohydrate 1,2-Naphthoquinone Benzoquinones p-Benzoquinone p-Toluquinone p-Xyloquinone 2.5-Dichloro-p-benzoquinone
2.5-Dichloro-3,6-dicarbethoxy-a-benzoquinone 2.3,5,6-Tetrachloro-p-benzoquinone 3,4,5,6-Tetrachloro-o-benzoquinone Others 2,2,3,4.2-Pentachloro-l-ketotetrahydronaphthalene
Color PaY PaOB
0.91 0.89 0.84 0.43 0.95
Y Y
0.92 0.46 0.84
PaYB P PaY
0.87 0.85 0.97 0.28 0.93
PaR YR PaB PaT PaY
0.94 0.22 0.90
PaY PaY PaYB
0.90
PaPR
PaY
P a , pale; P, pink: B, brown: Y ,yellow: 0, orange; R , red: P u . purple. Impure compounds producing t a o spots on chromatographing.
running in the standard solvent, but before spray development. These eluates and the corresponding solutions prior to running on paper were compared on a Beekman Model D U spectrophotometer. The absorption curves produced mere superimposable in the four cases tried, indicating that no change occurred during the solvent run. This method would be of great aid in the isolation and identification of naturally occurring naphthoquinones. I n working with the above procedure, i t was found that flavonoid and phenolic substances were also chromatographed and developed. However, other solvents and chromogenic sprays are better suited for the isolation and identification of such compounds (1, 2 ) . Spectrophotometric absorption curves from eluate samples of chromatographic spots will serve as a means for preliminary classification of the compound producing the spot.
ACKNOWLEDGMENT
The authors are grateful to the Naugatuck Chemical Division,
U. S. Rubber Co., for supplying the majority of the quinones studied. This research was supported in part by a grant from the Sational Science Foundation, Washington, D. C. LITERATURE CITED
(1) Bate-Smith, E. C., and Westall, R. G., Biochem. et Biophys. Acta, 4, 427 (1950). ( 2 ) Gage, D., and Wender, S. H., ASIL. CHEY.,23, 1582 (1951). (3) Jeanes, .4llene, Wise, C. S., and Dimler, R. J . , Ibid., 23, 415
(1951). (4) LIcSew, G. L., and Burchfield, H. P., Contrib. Boyce Thompson Inst., 16, 357 (1951). RECEITE D for review J u n e 11, 1953.
Accepted September 17, 1953.
Flame Photometric Determination of Phosphate WILLIAM A. DIPPEL, CLARK E. BRICKER, and N. HOWELL FURMAN Department o f Chemistry, Princeton University, Princeton,
EVERAL
extensive discussions of flame photometric inter-
S ferences have appeared in the literature 8). Although cations, anions, and organic substances have been investigated, (1-6,
the cationic interferences have been studied most thoroughly. Probably the only useful generalizations regarding these interferences are that both their directions and magnitudes are dependent upon the instrument used and that the interferences are minimized a t high dilutions. In making a more extensive study of anion interferences, it was found, in agreement Kith the work of Parks et al. ( 6 ) , that phosphate had a pronounced inhibiting effect on the flame intensity of potassium and a lesser inhibiting effect on sodium. I t s effects on calcium or magnesium are most striking, however, since a plot of emission intensity against concentration of phosphate a t a constant calcium or magnesium concentration exhibits a reversal (Figures 1 and 2). &4tthe lower phosphate concentrations, the emission intensity of calcium varies linearly and inversely with the phosphate Concentration. At higher phosphate con-
N. J. centrations, the emission intensity passes through a minimum and then increases until finally enhancement occurs a t phosphate concentrations exceeding approximately 1X for magnesium or 1.5.11for calcium. The linear relationship between phosphate concentration and emission intensity has been made the basis for the quantitative measure of phosphate in the concentration range between 0.005 and 0.012.11 phosphoric acid. Rieman and Helrich ( 7 ) employed an ion exchange column to remove interfering cations prior to a p H titration of the phosphoric acid present in a sample of phosphate rock. Likewise, the procedure described here employs a cationic exchange resin on the hydrogen cycle to remove all calcium and other cations present in the sample. The phosphate passes through the column and is collected in the effluent solution. After a known amount of a standard calcium solution is added, the intensity of the calcium flame is measured on the flame photometer. The weight of phosphorus pentoxide present is obtained from a calibration curve previously prepared from a series
