ANALYTICAL CHEMISTRY
1402 Table I .
Hydrogen Numbers for Some Unsaturated Acids and Esters
Sa1nple
Found
Hydrogen S u m b e r Average Calculated
115.0 116.5 116.9 115.; 115 8 115.2 116., 115.3 115.1 ll5,4
115.9"
110.1
209. n 297.4
298.7
206.5
hIethi.1 linoleate
1 4 7 . '2 147.0 146.0
146.9
147.1
9,11-Iainoleic acid
140.4 141.2
140.8
140.2
10,1?-1,inoleir acid
140.4 139.3
140.0
140.2
146.4 147.0 146,G 184.2 183.6
146.7
147.2
R1uttii.l linoleate
(alkali i-omerizd)
10- IIendccenainide
Standard deviation
183.9
183.3
The confidence limits for the mean (115.9 =t0.5) calculated from the maleic acid analyses brackets the theoretical value and shows that there is no significant inherent error in the method. I n general, the values are 0.2 to 0.5% below those calculated, which means that slightly more than the theoretical amount of hydrogen is consumed during the reaction. This indicates that there is a slight loss of hydrogen by diffusion during the analysis. Further evidence is that the hydrogen volume slowly but continuously diminishes if the apparatus is allowed to stand for several hours. After more data are obtained, it may be possible to correct for this apparent loss by subtracting a blank from the volume of hydrogen used, but probably this will not be greater than 0.01 nil. The apparatus has been used only in a room in which the temperature is kept under close control. If such a room is not available, it would be advisable to house the apparatus in a cabinet, in which a relatively uniform temperature can be maintained. LITERATURE CITED
(1) Johns, I. B., and Seiferle, E. J., IXD. ENG.CHEM.,ANAL.ED.,13, 841 (1941).
Prater, A. N., Ibid., 12, 184 (1940). Prater, A. N., and Haagen-Smit, A . J., Ibid., 12, 705 (1940). Weygand, C., and Werner, A , J . prakt. Chem., 149, 330 (1937). ( 5 ) Zaugg, H. E., and Lauer, W.-M., A x ~ ~CHEM., L. 20, 1022 (1948). (2) (3) (4)
= 0.6.
shown by the standard deviation of 0.6 for the ten values for maleic acid. The accuracy of the method is indicated by the close agreement between the calculated and found valucs in Table I.
RECEIVEDApril 5 , 1949. Presented a t the Meeting-in-Miniature of t h e Philadelphia Section, AVERICASCHEVICALSOCIETY,Philadelphia, Pa., January 1949.
Simplified Partition Chromatographic Procedures Resolution of Sulfonamides, Sulfones, and Their Metabolic Products f r o m Biological Materials WILL1431 €1. LOSGENECKER' iVational I n s t i t u t e s of Health, Bethesdu 1 4 , M d , Apparatus suitable for partition chromatography, on narrow filter-paper strips or strings of various materials, using both ascending and descending solvent flows, is described. The feasibility of using these smaller columns and the resulting advantages are set forth. The use of these apparatus for the resolution of a sulfonamide mixture from the plasma of a chick fed a mixture of sulfonamides in a proprietary preparation is reported. The use of benzene-water and benzene-cyclohexane-water to resolve p,p'-sulfonyldianiline, related compounds, and metabolic products from solutions and biological fluids is reported.
P
ARTITIOS chromatography on filtcr paper is a relatively new technique. Although it was originally used by Consden, Gordon, and Martin ( 4 ) for the separation of amino acids, it has subsequently been employed for the separation of minute amounts of a wide variety of materials such as peptides, organic acids, sugars, antibiotics, anthocyanins, creatine, vitamin B6, flavine nucleotides, sulfonamides, and other substances. The literature on many applications is reviewed by Consdcn ( 3 ) . In this paper details are given for the construction of two types of apparatus which have given satisfactory separations of mixtures of sulfonamides and sulfones from aqueous solutions and biological fluids. Cse of either type of apparatus effectively permits the conservation of solvents, reagents, filter paper, and to a degree, test substances. Provision is made in one of the pieces of equipment for chromatographing labile compounds. The width of the 1 Present address, Experimental Station, E. I. du Pont de Nemours & Company, Wilmington, Del.
filter paper is narromr than that previously reported (6, 6, 11) or even recommended by some (S, 9). However, strips of this width are more easily handled, particularly when wet with solvents, yet are adaptable to the same types of separations described foi paper of the more conventional size. The narrow strips have an additional advantage over wide strips because they may be used with simply constructed, all-glass apparatus. The compounds studied are localized in bands which may occupy a smaller area than spots. It has been found possible to do partition chromatography in one dimension not only on narrow strips of filter paper, but also on strips or strings of materials other than filter paper: plain and mcrcerised cotton, glass wool, and thin asbestos paper. These materials may be considered to form internally supported chromatographic columns of small cross section. They are as much columns as glass tubes filled with silica gel or starch, and in many cases will accomplish the same separations but on a micro scale.
1403
V O L U M E 21, N O . 1 1 , N O V E M B E R 1 9 4 9 APPARATUS
Type I. Dwices for Ascending PAPER. Solvent Columns. FILTER The apparatus for filter paper in which the solvents travel upward by capillary forces is shown in Figure 1.
Two or more rimless test tubes, wiLh the lips adjacent to the pouring spout, are attached with cellulose taoe to a 1-liter eraduated cylindei. Wicks ai filter paper carry the individual solvents in the solvent cornhinetion from the external reservoirs, so farmed, dawn the inner Walls oi the cylinder from which the solvents evaporate to maintain a saturated atmosphere. A straightened, larw-size psper cli is skewered through a No. 12 corf and au ointment jar lid or inverted No. 14 rubber stopper. One half inch (1.24 cm.) of the clip is then bent against the cork and the part above the stopper is fashioned into a hank. Midway between upper and lower edges of the cork 10 to 12 pins are inserted and their heads snipped off. The pins hold the strips of filter paper (Whatman No. 1, 3 t o 6 mm. X 34 cm., machine cut, with 2-mm. holes punched 6 nun. from either end). Laug-Levy pipets ( 1 ) calibrated t o deliver 0.003 to 0.01 ml. are used t o apply measFigure 1. A p p a r a t u s ured volumes of test solutions t o for Ascending Filterthe filter paper ahout 2 to 4 cm. Paper Chromatoabove the level to which the paper grams will hang in the solvents. The cylinder is filled to the 100-ml. mark with the solvent combination, and the papers, with weights attached for tautness, are suspended in the oylinder until the solvents front (observed as boundarv of visible damoeninx) has"%soendedt o the bias or-slows to a halt. Either event may occur in 3 to 48 hours. the time beine somewhat 'dependent on &e particular solvent combination. STRINGS.To employ partiion chromatography nu king, the two essentials are 2 keep the strings taut m d aeparated from each other. Thus, using a 1-liter, graduated cylinder with attached tubes as for filter-paper columns, one merely inserts a rack on which a continuous length of string (making 20 to 30 "columns") has been
Figure 2. Apparatus for Descending-Solvent Chromatographic Columns
20- to 2&m. rod (6'to 8 mm. in diameter) with each end fused to the oenter of an edgenotched, concave disk. The strings may be ordinary mereerieed cotton, white wrapping cord rinsed in boiling water, or glass twine. The test solutions are applied a t a point an the columns 2.5 em. above the level to which they will stand in the solvents combination. Because the strings of mercerized cotton present a smaller cross section than the strips of filter paper, smaller amounts of solutes in the test solutions must he used.
Otherwise the bands oi substances whose Rr values are in a tiarrow range will overlap. movement of baud __ I" ( 4 ) = movement of advancing front of liquid Lumns.
The
This apparatus is essentially a chamber eonstrGcted of Pyrex
outward-projecting rim, and below this is a thermometer. The stem from the funnel leads into the center of the reservoir. In the solvent reservoir is fitted 8, glass ring with inward-pointing spikes (Figure 3), on which are hung paper strips or strings. Alternatively a 3-cm. length of glass tubing 30 mm. in outside diameter, to which are tied paper strips or strings, may be used. This method is illustrated in Figure 4. The strips or strings are weighted by the glass crooks. One 07 more solvent boilers are connected to the main chamber The boilers are heated by coiled resistance wire in which the current is regulitted by a variable transformer, and insulated with asbestos tape and cement. Gases may be bubbled into the solvent boilers through the capillary tubing to obtain a. solvent-saturated inert gas atmosphere. The solvent boilers are more efficient than pools of solvents or solvent-saturated wicks in bringing the atmosphere in the chamber to a saturated equilibrium and are partioularly advantageous with law-boiling solvents. SPECIAL PROCEDURES
Two-dimensional chromatography is possible under certain circumstances on the narrow filter-paper strips. Substances which have a common Rp value in one solvent combination, and which are separated from other test substances by a considerable distance of blank strip, may be rechromatographed in a second set ai solventson the remainder of the strip left after the other substances have been excised. Forexample, in butyl alcohol saturated with 3% ammonia, sulfadiazine has Figure 3. Glass Ring w i t h spikes for securing ~ i l ~ ~ , . - an Rr value of0.17 (Q),SUIPaper Strips fathiazole 0.38 (91, p,p'sulfonvldianiline (DDSI . , 1.0., and ~-(psulfanilybnilino)ethanol 1.0. To separate the two sulfones i t is necessary to shorten the strip t o within 2.5 om. less than the distance traveled by the sulfones and to rechromatograph in a second set of solvents. Benzene, saturated with water, is satisfactory for the resolution of these two sulfones. Illustrative of the types of compounds which may be separated by this procedure and apparatus a m the sulfonamides. Although Shepherd (9)has detailed a method ioor their separation from aqueous solution and urine hy paper chromatography, the experimental procedure given below is a variation of his technique which also permits the separation of sulfonamides from plasma. ZXPERIMENTAL
Resolution and Identification of Sulfonamides from Chick L be used. Lang-Levy pipets ( l ) ,0.603-ml. a n d 0:Ol-ml. ca pacity. 1-MI. pipets or medicine droppers. Guillotine paper cutter. Whatman No. 1 filter paper strips, 3 X 34 em. n t i a u r a ~ e ~ y cut strips in this size m w be readily trimmed in a millotine-tvoe
ANALYTICAL CHEMISTRY
1404
fonnmides likely to he encountered artre prepared by suspending 0.1 gram of each sulfonamide in approximately 50 ml. of water, adding sufficient concentrated ammonia. to effect solution, and dilut6g to volume with distilled water. Chromatographing Solvents. The hutyl alcohol-ammonia solution of Shepherd (9) i s prepared by adding 40 ml. of hutyl alcohol to 10 ml. of concentrated ammonia and makine to 100 ml. with distilled water. The solution i s shaken to formittemporary emulsion and poured into the cylinder. Best results with the chromatography of substances in viscid materials such as plasma have been obtained by adding 0.1 gram of nonaethylene dveol manastearate (a nonionic surface-active agent, mannfac-
EXPERIMENTAL PROCEDURE. On individual filter-paper strips
0,003- and 0.01-ml. diquots of the sulfonemidesolutions,mixtures of the sulfonamide solutions, or plasma. samples are applied. The
he chrorn&groeraphed). and left for r6 or mare h o u k or until the
hooh for treatment by the cal&develo&g agents and then mounted an graph paper. When the paper strips are dry, the p-dimethylaminobenzaldehyde is applied to representative strips as desired; a 1-ml: pipet or medicine dropper i s used to deliver a quantity sufficient to
indicate the Dosition$of &e hands. The~-N-(l-naphthyl)ethylene-
This is accomulished bv add&, hv pipet, the ace& acid solution ~
~~
~
light e x $ when vi&& An x-ray film viewer i r a oontrasting background will enable one to identify the fainter bands. If the bands are fused, it indicates that too much sulfonamide was a p plied to the strip and lower concentrations must be used.
Figure 4. Positions TakenL by Sulfadiazine (Lowest Band), Sdfatliiazole (Middle Band), andDDS (UppermostBand)inButyl Alcobol-3% A mrnonia Positions ramain constant, xh.;the= subetanoen are alone or in mix,Lure.
Kiihnau's reagent (8) is made by dissolving 1 gram of p dimethv1aminohenz;aldehvdein 100 ml. of 3% hydrochloric acid.
discolored.
EXPERIMENTAL RESULTS. Each sulfonamide is found to migrate the same distance whether alone, in q n e o u s mixture, or in plasma. When fluids from uudrngged animals are used, no bands are produced with either color-developing reagent. Sulfadiazine and sulfamerazine, because their Rr values are so close (0.17 and 0.30, respectively, 9 ) , tend to form overlapping hands unless very small quantities are used. However, if the aliquot contains ahout 0.25 miorogram of these drugs, satisfactory rcsolution may he accomplished. Resolution and Identification of Sulfones and Sulfonamides. Junge and Smith (7) have reported a synergistic action of sulfadiazine and N-propyl-p,p'-sulfonyldianiline against tuberculosis in guinea pigs. I n anticipation of a need by clinicians to separate these two classes of compounds, mixtures of sulfones and sulfonamides have been chromatographed. It has been found, using butyl alcohol-30/, ammonia, that p,p'-sulfouyldianiline (DDS), N-propyl-p,p'-disulfonyldianiline, and ~-(psulf~nilylanilino)ethanol all migrate to a greater distance than sulfathiazole, which in turn migrates farther than sulfamerazine and sulfadiazine. The sulfones precipitate in sharp, well-defined hands in hutyl alcohol-3% ammonia, hut are coincident at the same level. Figure 4 is illustrative of the behavior of sulfadiazino, sulfathiaeole, and DDS. However, the sulfones may he resolved by a solvent conhination composed of equal parts of benzene and cyclohexane ssturatod with water. The 8-(p-sulf~nilyltLnilino)ethanolrem%i.ins stationary, tho propyl compound migrates the total distance of the solvent excursion, and DDS migrates an intermediate d i p tance. The mono-"?-substituted sulfones and DDS give a pale rust color with nitrous acid, purple r i t h Bratton and Marshall reagents, and yellow to orange with p-dimethylsminohenzaldehyde. It has been found that urine, plasma, and ethyl acetate extracts of plasma of rahhits fed DDS may he chromatographod using the identical procedure for pure solutions. These fluids yield one and sometimes two hands (lo) in addition to the band characteristic of the parent compound in benzene-water. When DDS is added to urine and plasma from undruggcd rahhits, only me hand is discernible.
PREPARATION OF SAMPLE.A 40-gram chick is fed about one DISCUSSION
heart k o a test tube containing potassium oxalate and centrifuged. The plasma, separated from the cells, is used.
The two general types of apparatus and methods described in this paper can be adapted to accomplish most of the separations which have been claimed for partition chromatography. Both
V O L U M E 2 1 , N O . 11, N O V E M B E R 1 9 4 9 types are timesaving compared to apparatus handling only one or two paper strips, inasmuch as up to 20 runs may be made a t one time in the same apparatus. The narrow strips of filt,er paper or strings possess the following advantages over the wide sheets of paper: They may be mounted on graph paper to facilitate plotting
RF values.
Cotton and glass string have strength enough to resist tearing when wet. Glass string will not disintegrate in the presence of reagents that would destroy paper. The free use of many filter-paper strips, which is facilitated by the simplicity of these procedures, often obviates the need for two-dimensional chromatography. The same effect is obtained by selecting, for each substance in a mixture, a solvent combination that isolates an individual compound from its fellows and combining the results from several chromatograms into a completed analysis. Uniformit>- of solvent excursion in any singlc run compares favorably with that obtained on wide strips of filter paper, so that the prediction of location of bands of colorless substances may be confidently made after one of two strips run simultaneously has h e n treated with color-developing agents. Appropriate sections of the second strip may then be cut and treated for quantitative analysis of the test substances. Partition chroiiiatography on filter paper is a most convenient method for st,udying the metabolic fate of sulfones and sulfonamides, and possibly many other drugs as well. It is a guide to isolation procedures (20) and can reveal the presence of unsuspected metabolites. The behavior (Rp values) of a compound in vnrious solvent combinations serves to characterize it, being pre-
1405 cise and supplementary to the more widely used physical constants such as melting and boiling points, absorption spectra, and indexes of refraction and rotation. ACKNOWLEDG1.IENT
The author acknowledges the counsel and suggestions of M.
X. Sullivan, Edward Y.Josephson, Francis L. Schmehl, G. Robert Coatney, 11. I. Smith, and Ernest L. Jackson. Arthur Robbins assembled the glass~vareshown in Figure 2 . drew the illustrations.
Mary King Boucher
LITERATURE CITED
B e s s e i . 0. A . . Lowry, 0. H., a n d B r o c k , 51.J., J . Biol. Chem., 164, 321 (1940). B i a t t o n , d.C., a n d M a r s h a l l , E. K., Jr.,Ibid., 128, 537 (1939). . , Consden, Ii., Suture. 162, 359 (1948). Consden. It., G o r d o n , A . H . , a n d M a r t i n , A. J. P., Biochem. J . ,
38,224 (1944). Goodall. 1:. I:.. aiid Levi, 1..1.,Analyst, 72, 277 (1947). H o r n e , It. E.. arid Pollard, -1.L., J . Bact., 55, 230 (1948). J u n g e , J. I f . . a n d S m i t h , SI. I.,J . Phurmacol. Esptl. Therap., 92,
352 (1948). K u h n a u , W. IT., Klin. Wochschr.. 17, 116 (1938). S h e p h e r d . R. G., -2merican C y a n a m i d Co., B o u n d B1,ook, S . J., personal cornrnunication. T., a n d Longenecker, S m i t h , SI. I., J a c k s o n , E. L., C h a n g , I-, IT. H . , Proc. Soc. Esptl. Biol. . l i e d . . 71, 23 (1949). Urbach. K. 'i a. nd , Giscafre, L., I b i d . , 68, 430 (19481. RECEIVED March 18, 1049. Presented in part before the Washington Section, AXERXCASCHEVICAL SOCIETY,October 14, 1948. Adapted in part from a thesis presented in partial fulfillment of the requirements for the degree of master of science, Georgeto\vn Cniversity, 1949.
Submerged Bulblet Methods in Microchemistry LEONARD K. NASH Harvard University, Cambridge, Mass. An improved method for the determination of vapor pressure and/or vapor pressure lowering in various systems is described. The value of such data in a variety of microchemical investigations is discussed, and experimental results supporting this evaluation are cited for the following measurements: vapor pressure, decomposition pressure, phase studies, solubility, purity (of solute or solvent), molecular weight (as a function of concentration), and association.
A
K E K method for the microdetermination of molecular weights, based on a measurement of the vapor pressure lowering that occurs when a known weight of the test substance is dissolved in a known weight of solvent a t the latter's boiling point has recently been described (6). In the course of a further investigation it has proved possible to simplify the determination considerably; in particular, by entirely eliminating the use of a comparison tube and interpolation. I t is then possible to achieve a generalized apparatus and technique applicable to a variety of niicrodeterminations. This paper describes the improved apparatus and technique, indicates the applications for which they are fitted, and discusses the limits of accuracy of which they are capable. APPARATUS
The apparatus now proposed is basically similar to that previously described (6); but the addition of several auxiliary devices has greatly enhanced the flexibility of the equipment. In Figure 1 a diagrammatic sketch of the apparatus is presented.
H is a constant-temperature jacket insulated with sheet asbestos. Observation ports are cut in this lagging, so that the shank and the body of bulblet L may be observed. The fabri-
cation and use of these bulblets have been fully described ( 6 ) . The bulblet is supported by a hook in a glass rod, and may be raised from, or lowered into, the manometric fluid at K by sliding the glass rod through the lubricated sleeve, F . Khen 15 to 20 ml. of a pure solvent, together with a few glass beads, are heated in H over a small free flame and under reflux fiom condenser E , the inner chamber, J , rapidly comes to a temperature which, for a given solvent, closely corresponds-according to the vapor pressure curve of the solvent-to the pressure in H . This pressure may be adjusted via pinchclamp 2; it is stabilized by the 3-liter ballast flask, D; and it may be measured with barometer A . By suitable adjustment of the pressure in this system the inner chamber and its contents can be brought t o a temperature determinable with an accuracy of a t least 0.1 C. and variable within a range from about 25 C. below the boiling point of the solvent to 5 " to 10" C. above this boiling point (if A has a working length of 1000 mm.). This range is often sufficient, for the data secured are generally precise enough to sustain a reasonably extended extrapolation. There is no need to shield the equipment from occasional drafts; and the temperature control is easy and precise over fairly long periods of time. The results are unaffected by fluctuations in barometric pressure. At K there is a 2-cm. layer of concentrated phosphoric acid, prepared by mixing phosphoric anhydride with 8570 phosphoric acid in the proportion 1 to 2 by weight. With the use of this concentrated acid the aqueous tension over the sealing medium is reduced to a satisfactorily low value-a pivotal consideration
