ANALYTICAL CHEMISTRY
S50 After the unit is adjusted initially, it usually requires no further attention. Each day’s start-up consists simply in brin@n the boiler to a boil sufficiently long to bring the cooling water in to a boil. Then the flow of cooling water is started. A second pinch clamp may be used so the flow setting is not disturbed from day to day. The still in this laboratory operating a t 980 watts puts out 1.45 liters of water per hour. When starting up initially or after a long shut-down, the author operates the still for a t least a day, discarding the product, without cooling water in condenser D so as to leach the glass condensing surfaces.
8
RESULTS
Efficiency. While it is desirable to obtain as high an efficiency as possible, this is true only within the limits that the construction of the still should be kept as simple as possible. Therefore, no lagging of the boiler or any other part of the apparatus was employed. However, by utilizing the heat of condensation in the heat exchanger, this heat can be conserved by bringing the feed water from the temperature of the water mains to nearly the boiling point. In a calibration run the still was consuming 980 watts of electrical power. If this heat were used solely t o ~+uppIyheat of vaporization of water a t its boiling point, then the theoretical product output of the still would be 1.56 liters of water per hour. The measured rate of product output was 1.45 liters per hour, giving a power efficiency of 92.9%. This figure was sufficiently high that no further attempts were made to increase it. Purity. Early in t!ie development of the still a sample was compared to a sample of glass-distilled conductivity water. The conductance values for the air-equilibrated samples were identical. In the past the author has also utilized the dithizone (3) test for freedom from metal ions. While this test gives a positive level of contamination in the products of conventional triple distillation, no such positive test could be obtained with the product of the described still. Indeed, even when the feed water was contaminated with 1000 p.p.m. of copper as the sulfate, the tests for metal contamination were still negative. Therefore, a more sensitive test was required. To this end the boiler containing 3.5 liters of water was contaminated with 5.2 y of cobalt chloride containing 3.2 X 108 counts per minute of cobalt80. Three liters of product were collected, nonradioactive car-
rier cobalt was added, and the cobalt was recovered and assayed for radioactivity ( I ) . Only 41 counts above background were found. This is a purification of 10’ over the feed water, and of the product water represents a contamination of 1 part in with cobalt ion. Frequently one requires water not only free of metal ions, but also of other contaminants, such a? phenols and ammonia. As a test of the system, the feed water was deliberately contaminated with 1000 p.p.m. of phenol and ammonium chloride by means of injection from a motor-driven syringe into the feed water entering the heat exchanger. The reagent addition unit, B, was employed to mix with the feed water a solution of 0.5N with respect to both potassium permanganate and sulfuric acid to act as trapping agents. The feed rate of these reagents was 50 ml. per hour (water feed, 4.22 liters per hour). Visual observation indicated that all of the permanganate Oxidation occurred in the heat exchanger. The ammonia present in the feed water was determined by Kessler’s reagent, and in control tests was sensitive to better than 1 p.p,m. The test on the product was negative. Phenol was tested for by a modification of the method of Vorce (4, 6), which was also determined to be sensitive to 1 p.p.m This test on the product was also negative. Inasmuch as this type of purity was not the main concern, no further development nor extension of these tests was carried on. However, it appears that this still, perhaps with slight modification, is capable of producing efficiently large amounts of metal-free mater which is also free of the other major types of contamination. The still is self-sterilizing and hence should provide an excellent source of pyrogen-free water. LITERATURE CITED
(1) Ballentine, R., and Burford, D., unpublished manuscript. (2) Holmes, F. E., ANAL.CHEX.,21, 1286 (1949). (3) Stout, P. R., and Amon, D. I., A m . J . Botany, 26, 144 (1939). (4) Voroe, L. R., Ind. Eng. Chem., 17, 751 (1925). (5) Yoe, J. H., “Photometric Chemical Analysis, Vol. I, Colorimetry,” p. 429, New York, John Wiley & Sons, 1928. RECEIVED for review April 6, 19.53. Accepted October 13, 1953. Contribution No. 44 from the McCollum-Pratt Institute. This work was supported i n part b y a contract, AT(30-1)-933,between the Atomic Energy Commission and the Johns Hopkins University.
Detection of Steroidal Pseudosapogenins by Infrared Spectroscopy ALMA L. HAYDEN, PHYLLIS B. SMELTZER, and IRVING SCHEER National lnstitute of Arthritis and Metabolic Diseases, National Institutes of Health, Department o f Health, Education, and W e l f a r e , Bethesda 14, Md.
been reported 3, 4,8, that the spiroketal side chain Ispectrum in steroidal sapogenins has a characteristic infrared absorption in the fingerprint region. This absorption, which is T HAS
(1,
HO-
9)
distinctive for sapogenins of the normal and is0 series, is absent in other steroids and in sapogenin derivatives lacking the spiroketal structure. The results obtained in this laboratory from an infrared spectroscopic study of five steroidal pseudosapogenins and some of their 0-acyl derivatives agree in part with this earlier work. I n the present investigation it has been found that the pseudosapogenins (Table I) possess a band of moderate intensity (under the conditions given in the experimental section, the observed absorption is 20 to 50’%) near 1695 cm.-l and do not exhibit strong absorption in the 1000- to SOO-cm.-l region. The absorption near 1695 cm.-l is significant in that it is absent in the original sapogenins, dihydrosapogenins, and products resulting from oxidation or reduction a t the CzO-Cz2 double bond in the pseudosapogenin structure (I). The presence of this band near 1695 cm.-’ and the absence of strong absorption in the 1000- to SOO-cm.-l region may be used as
B
means for the detec-
‘ % E d tionof pseudosapogenins. Identification of an individual pseudosapoyenin is made in the usual manner by comparison of its specAtrum with reference spectra. Because the spectra of chloroform solutions of the side chain isomeric pseuHO dosapogenins-e.g., pseudosarsasapoI genin and pseudosmilagenin-are very similar, positive identification can be made most advantageously by comparing the spectra of their Nujol suspensions in the fingerprint region. The compounds were studied in carbon disulfide and chloroform solutions and as Nujol suspensions or liquid smears. The chloroform spectra of the pseudosapogenins are greatly affected by the solvent purity. Commercial chloroform or distilled chloroform which may have undergone partial decomposition on standing can cause rapid transformations (within 12 minutes) of
A&?
1
‘ l i
kv
~
I
V O L U M E 26, NO. 3, M A R C H 1 9 5 4 the pseudosapogenins during spectral analysis as shown by complete changes in the spectra including the disappearance of the moderate band near 1695 cm.-' Owing to the difficulty encountered with this solvent, it was purified and stabilized prior t o use. EXPERI3IENTAL
All measurements were made with a Perkin-Elmer Model 21 double-beam spectrophotometer with sodium chloride prism and cells. One millimeter macro- and microcells were used for
6ot
40i& 20
551 carbon disulfide solutions (ca. 15 mg. per ml.) of the diacetates. Five-tenths-millimeter macro- and microcells were utilized for chloroform solutions (ca. 30 mg. per ml.) of the pseudosapogenin alcohols and the 3,5-dinitrobenzoates. Spectra of the Sujol suspensions and the liquid smears were obtained in the usual way. Measurements were made from 5000 to 800 cm. -I in chloroform and from 5000 to 660 cm.-' in carbon disulfide, in Nujol, and in the liquid smears. However, only tracings of the 2000- to 1400-cm. -' region are reproduced below. Atmospheric water vapor and carbon dioxide were used for calibration in this region. Band positions are accurate within 1 5 cm.-' with somewhat less accuracy for broad bands. The carbon disulfide was purified by distillation over phosphorus pentoxide. The commercial chloroform was shaken with one tenth its volume of concentrated sulfuric acid; the organic layer was washed with water, 3,V sodium hydroxide solution, and again with water, and was dried overnight over calcium chloride. The dry solvent was distilled a t atmospheric pressure and the distillate was stabilized by adding 0.1 to 0.5% absolute ethyl alcohol. RESULTS
With the exception of pqeudohecogenin, all of the pseudosapogenins and their esters exhibit moderate absorption a t 1701 t o 1692 cm. -'in chloroform and carbon disulfide solutions (Figure 1, Table I). The diacetates chow this band in addition to the usual ester carbonyl absorption. With the 3,5-dinitrobenzoates in chloroform, this former band appears as a shoulder on the side of the strong carbonyl absorption; in Nujol suspensions this shoulder is resolved into a distinct band (1706 to 1704 cm.-') on the side of the carbonyl doublet Additional evidence that the physical condition of the sample has an effect on the observed poqition of this significant band is shown by a comparison of the spectra of a solution and a Sujol suspension of pseudohecogenin. In chloroform solution the strong keto-carbonyl band (1704 em.-') obqcures the absorption near 1695 cm.-'; in Kujol, this latter absorption is detected as a shoulder a t 1692 to 1680 cm.-' on the side of the carbonyl band. This observed difference in spectral resolution in chloroform and Sujol may be the result of broadening and shifting of the carbonyl band in the solvent and displacement of the 1695-cm -1 band in the crystalline state. Frequency shifts have been observed previously ( 7 ) on comparison of the spectra of other steroids in solution and solid states. The weak band which i.; seen near 1620 to 1600 cm.-l in the chloroform spectra of the pqeudoqapogenins is not detected in the spectra of the pseudosapogeriin esters nor in Sujol suspensionq of the alcohol^. This band has been seen in the chloroform spectra of some sapogeninQ and dihydroqapogenins examined in thip laboratory. This absorption may indicate the presence of water absorbed by the dry chloroform during preparation of the solutions.
~
DISCUSSION
I -Jw
1 9
L - A - - I --JL--1900 1700 1500 1900 1700 1500 1900 1700 1500
WAVENUMBER
Figure 1. A. B. C. D. E. F. 6. H. I.
(CM:')
Infrared Spectra
Pseudosarsasapogenin i n CHCla Pseudosarsasapogenin diacetate i n CSs Pseudosarsasapogenin di-3,Sdinitrobenzoate i n Nujol Pseudosmilagenin i n CHCla Pseudosmilagenin diacetate i n CSn Pseudosmilagenin di-3,5-dinitrobenzoate i n Nujol Pseudotigogenin in CHCla Pseudotigogenin diaeetate i n CSn Pseudohecogenin i n CHClt J . Pseudodiosgenin i n CHCla K . Pseudodioagenin diacetate in CSs L. Pseudohecogenin i n Nujol
The frequency and intensity of the band a t 1706 to 1689 cm.-l in these compounds are higher than have been reported previously ( 2 , 5 ) for isolated carbon-carbon double bonds. Randall group and co-authors ( 5 ) have reported that when a -C=Cis so situated that a nitrogen or an oxygen atom enters strongly into the same mode of vibration, there is sufficient change of dipole moment to produce an intense band. Recently (6) the effects of an adjacent oxygen atom on the double bond frequency and band intenqity in some enol lactones and enol acetates have been reported. I t is possible that the presence of the ether linkage a t C16-C22 in the pseudosapogenins might increase the frequencyand band intensity of the C20-C22 double bond. Nevertheless, the unequivocal asPignment of this band must await further investigation. ACKNOWLEDGMEXT
The authors are greatly indebted to Erich Mosettig for his advice and criticism throughout the course of this work.
552 Table I.
ANALYTICAL CHEMISTRY (2) Jones, R . S . , Huniphries, P., P a c k a r d , E., a n d Dobriner, K . , J . Am. Chem.
Band Positions" of Pseudosapogenins and Pseudosapogenin Esters between 2000 and 1400 Cm.-l Compound
CHCI3
CBZ
1695
...
Suc., 72, 86 (1950).
( 3 ) Jones, K. S . , Katzenrllenhogen, E., a n d Dohriner, X., Ibid.. 75, 158
Sujol or Sinear 1692 Sinear (1739-1724), 1692sh
(1953).
(4) Jones, R . S , Kataenellenbogen, E., a n d D o b r i n e r , 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
c.
(1) Eddy, R., Wall, AI. E., a n d K i u m p p S c o t t , 2 5 , 2 6 6 (1053).
ll.,AN.AL.C m w . ,
(5) R a n d a l l , H. AI., Fowler, R . G., Fuson, S . ,a n d Dangl, J. R., "Infrared Determination of Organic Structures," p. .5, New Ynrk. I). Van N o s t r a n d Co., Inc., 1919. (6) R o s e n k r a n t a , H., a n d Gut, >I., H e h . Chim. Acta, 36, 1000 (1953). (7) R o s e n k r a n t z , H., a n d Zablow, L., ANAL.CHEV..25. 1025 (1953). ( S ) Wall, 31. E., Eddy,'C. R . , ' l I c h e n n a n , 11. L.. a n d K l u m p p , 31 E., Ibid.,
_ _ _ _ ~ _ ~ ~ _
24, 1337 (19,52). (9) Wall, 11. E., Kridcr, 31. lI,,R o t h m a n , E. S.,and E d d y , C. R . , J . B i d . Chem., 198, 533 (1952).
R E C E I V EfD o r review October 10, 1953.
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 hldroude 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 at 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. The 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. In 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
