(8) Redfield, R. R., Barron, E. S., Arch. Biochem. & Biophys. 35, 443
(11) Ven Horst, Sr. H., Carstens, Y., J . Chem. Educ. 31,576 (1984). (12) Wellington, E. F., Can. J . Chem. 30, 581 (1952). (13) Ibid., 31, 484 (1953).
(1952). (9) Thompson, J. F., Zacharius, R. hf., Steward, F. C., Plant Physiol. 26, 375 (1951). (10) Underwood, J. C., Rockland, L. B., ANAL.CHEM.26, 1553 (1954).
Accepted January 26, 1957. Supported by Grant DRG-334 (T) from the Damon Runyon Memorial Fund for Cancer Research. Presented a t the Section of Inorganic and Physical Chemistry, Iowa Academy of Scienre Meeting, Grinnell, Iowa, April 21, 1956.
RECEIVEDfor review May 16, 1956.
Chromatography of Organic Acidic Compounds on Multibuffered Paper MORTON SCHMALL, E. G. WOLLISH, REM0 COLARUSSO, C. W. KELLER, and E. G. E. SHAFER Analyfical Research laborafory, Hoffmunn-la Roche Inc., Nufley, N. 1.
b
Acidic organic compounds have been separated by a paper chromatographic technique similar to one applied to organic bases. Many acidic compounds form a salt a t a particular pH. level with alkaline buffers, applied in sequence of their ascending pH on a filter paper strip in marked zones. Upon equilibration, chloroform is used as the single mobile phase for descending chromatography. As the stronger acidic compounds are often immobilized at lower pH levels than the more weakly acidic ones, it was possible to separate benzoic acid, several barbiturates, and some phenolic compounds from each other. These compounds could b e visualized on the paper under short wave ultraviolet light.
literature of paper chromatography, including paper chromatography of acidic compounds, has been covered by Block, Durrum, and Zweig HE
( I ) , Lederer (4, Turba (IO),and Hais and LIacek ( 3 ) . The most recent developments were reviewed by Strain and Sat0 (9). While filter papers completely impregnated with buffers have been widely used for the separation of compounds of related structure, the technique of multibuffered paper Chromatography has been developed for the separation of organic basic compounds. The procedure, employed for the separation of acidic compounds, was somewhat similar to the one described for organic bases ('7).
PROCEDURE
The paper was prepared by application of buffers at varied p H levels in marked zones (7). For the separation of acidic compounds, Clark and Lubs buffers were applied at 0.2 p H intervals for the p H range of 7.8 to 9.4, while Soerensen buffers were used for the p H range 9.8 to 12.6, spaced at 0.3 p H intervals. A solution of the sample, equivalent to about 15 7,was placed a t the starting line and the strip was introduced into the chromatographic chamber containing water at the bottom and, in addition, a n open beaker with chloroform. To hasten saturation with vapor, the chamber was partially lined with filter paper which dipped into the water. It was allowed to come to equilibrium overnight. Chloroform, as the single mobile phase, was then placed in the solvent trough for descending chromatography. When the solvent front had moved down almost to the end of the paper, which usually required about 3 hours, the strip was removed and air-dried. It was inspected under short wave ultra-
METHOD
Apparatus. Usual equipment for descending paper chromatography. Short-wave hIineralight (maximum emission at 2553 A.). Reagents. Chloroform, reagent grade. Clark and Lubs bufkers, double strength ( 2 ) . 3IacIlvaine buffers, double strength ( 5 ) . Soerensen buffers, double strength (8).
Table I.
pH of Immobilization of Barbiturates
pH Range
of Buffered
Compound Barbituric acid Isopropylbarbituric acid Barbital Phenobarbital Allylbarbituric acid Diallylbarbituric acid Probarbital Aprobarbital Butabarbital C yclopal sec-Bromallylbarbituric acid Amobarbital Pentobarbital Secobarbital Hexobarbital
Paper 8,3-12.6 Unbuffered 8.3-12.6 Unbuffered 7.8- 9 . 4 7.8- 9 . 4 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6 9.8-12.6
pH of
Immobilization 8.3 8:3
912 9.2 10.2 10.2 10.5
R/ 0 0 0 0
0.7 0.75 0.3 0.3 0.35
11.o
0.45 0.6
11.7 11.7 12.2 12.5 12.5 12.5 Past 12.6
0.6 0.7 0.75 0.8
0.8 0.95
VOL. 29,
NO. 5 , MAY 1957
791
violet light, where compounds such as barbiturates, benzoic acid, or esters of p-hydroxybenzoic acid appear as dark spots on light background. The spots were circled with pencil, to mark them for photographic reproduction. Hexylresorcinol was rendered visible bv spraying with amino reagent [IO0 m i . of 4-methoxy-2.nitroaniline dissolved in 25 ml. of glacial acetic acid and diluted t o 50 ml. with sulfuric acid, 10% w./v. A mixture of equal parts of this reagent was sprayed with 0.2'% aqueous sodium nitrite solution. This was followed by a spray of 5% sodium carbonate solution (6)1.
Table II. pH of Immobilization of Benzoic Acid and Phenolic Compounds pH Range Compound Benzoic acid Methylparaben
of Buffered
PI! of Immobilriation
Paper
5 . 2 - 6.9
9.8-12.6 Unbuffered
.-~,.~~.~"~.. 9.8-12.6 Unbuffered
Pmm.lna-ohan ~
Hexylresorcinol
g.ai2.6 Unbuffered
EXPERIMENTAI
Barbiturates (as the free acids), benzoic acid, and phenolic compounds were dissolved in either chloroform or ethyl alcohol and the equivalent of about 15 y mas chromatographed as described. In Table I the p H values are listed at which each one of the barbiturates was immobilized. When chloroform is used as the mobile phase, barbituric acid and isopropylbarbituric acid will not move on the paper. Some of the barbiturates, such as barbital and phenobarbital, could not be separated on multibuffered paper, a.s they move t o the same pH. In Table 11, acidic compounds such as benzoic acid and several phenolictype compounds and their R, values are listed. MacIlvaine buffer, double strength, was applied a t p H 0.2 intervals for the chromatography of benzoic acid, which was immobilized at p H 6.3. As is evident from Table 11, phenolic-type compounds were not affected by the buffers on the paper. For example, methylparaben, when applied to apaper, buffered a t 9.8 to 12.6, stopped a t a p H of 11.6 with an R, value of 0.7. When chromatographed on an unbuffered paper, it moved again to the same R,. Propylparaben and hexylresorcinol behaved in a similar manner. It appears, therefore, that the movement of these phenolic-type compounds is dependent solely upon the solvent system. Controlled Separation of AcidicTppe Compounds. Attempts were made t o separate combinations of known mixtures of some acidic compounds from each other by buffering a smell area of t h e paper at a selected p H value, so as t o stop the movement of one of t h e components, allowing the other one t o move down the strip. I n Figure 1, strip 1, such a controlled separation between benzoic acid and aprobarbital is shown. As benzoic acid is immobilized at any pH above 6.3, a 3-cm. wide upper zone on the paper was buffered at p H 9.8. As aprobarp H above 10.2, bital will stop at any . . a center portion of the paper was buffered at p H 12.5, to stop its move792
.
ANALYTICAL CHEMISTRY
Figure 1. Separation of benzoic acid, barbiturates, a n d phenols 1. Benzoic acid-aprobitrbital 2. Benzoic acid-aprobmhital 3. Benzoic .w5did-phenobarbital-hexy1resorcinol 4. Benzoic ~cid-barbital-methylparaben-prop?ilparaben 5. Benzoic acid-disllglharbituric acid-pentobarbital
Figure 2. Separation compounds
of
barbiturates from phenolic
10. PTob~~bital-methllpitraben-prop).lparsben
11. Probarbital-hexylresorcinol
ment. T h e n a mixture of these compounds was placed on such a strip, benzoic acid stopped at the p H 9.8 area, with aprobarbital moving to the pH 12.5 zone. An even wider separation of these compounds could he effected by using the same buffers as in strip 1, hut placing the p H 12.5 zone farther down the paper (strip 2). Phenobarbital, benzoic acid, and hexylresorcinol were easily separated by buffering an upper zone at pH 6.9, a central zone at p H 11.0, and leaving the remainder of the paper unbuffered.
Three bands were observed, with benzoic acid at the first buffered zone, phenobarbital at the second, and hexylresorcinol moving down into the unbuffered area. Strip 4 was prepared in the same manner as strip 3. Benzoic acid stopped a t the top, barbital moved to the p H 11.0 zone, and methylparaben (R, 0.7) and propylparaben (R, 0.9) moved to the unbuffered portion of the paper. In strip 5 an upper zone was buffered a t pH 9.8 and a central one a t pH 11.7. Benzoic acid moved to the first zone, diallylharbituric acid to the second, and
I
I
I
I
I
I
Figure 3.
Controlled separation of barbiturates
12. Phenoberbitabaorobasbit~l
13. DitLllylbsrbituriE acid-secobarbital 14. Diallylbsrbituric xid-aprobarbitd 15. Allylbarbituric acid-butaharhital 16. Barbituric acid-barbital 17. Barlituric rtcid-probarbital-eyelopal
0
(3
I
1
I
1 ,
a
a i
I I
Figure 4.
Separation on completely buffered p a p e r
18. 19. Butabarbital-cyclopal 20. Isopropylbarbituric acid-allylbarbituric itoid-aprobarbital-metbyp~ylon
21. Amabarbit~l-hexobsit~l
22. .4mobsrbit~l-seobromsllylbarbitu~icmid 23. seoRromallylhsrbituric acid-hexobmhitsl
pentobarbital went down almost with the solvent front (R,0.9). Because the movement of some of the barbiturates is affected hy buffers, while the movement of phenolic compounds is impeded, a controlled separation between both tvnes ". could he achieved. All strins in Fieure 2 were DreDared by bufferhg a sm& upper zone with a p H 12.6 buffer, leaving the remainder unbuffered. As can he seen from strips 6 to 11, all barbiturates wereimmobilized a t the buffered zone, with the phenolic compounds moving to their respective R, values, as shown in Table 11. As phenobarbital is stopped at pH 9.2 and aprobarbital is immohilized at p H 10.2, a wide separation between these compounds could he accomplished, as shown in strip 12, Figure 3, which was prepared by buffering an upper zone at p H 10.0 to stop the phenobarbital and a Iomr one a t pH 11.7 to halt aprobarhital. Strips 13 through 17 exemplify five other examples of such controlled separations. In strip 13 the upper zone was buffered a t p H 11.7; the remainder was unbuffered. Strip 14 had an upper portion a t p H 10.3 and a lower one at pH 12.6. In strip 15 the upper zone was buffered a t p H 10.8 and the lower at pH 12.5. Strip 16 had an upper portion a t pH 8.3 and a middle portion a t pH 10.0. Strip 17 had a central zone a t pH 10.8 and a lower one a t p H 12.6. As several of the barbiturates (Table I) could not be separated from each other on multibuffered paper, it was thought that buffering of the entire paper at a pH slightly below that a t which these barbiturates are immobilized, might allow separations. I n Strip 18, Figure 4, the entire paper mas buffered at p H 10.8. The R, value for probarbital was found to he 0.25, while that for butabarbital was 0.5. Strip 19 was buffered at a pH 11.7. The R, value of butabarbital was 0.1, and that of cyclopal was 0.5. I n strip 20, buffered at pH 10.2, the separation of three barbiturates of related structure from eadh other, as well as Noludar (iitethyprylon, 3,3diethyl - 5 methyl - 2,4 - piperidinedione)-a sedative of entirely different configuration-can he seen. Isopropylbarbituric acid (R, O), allylbarbituric acid (R, 0.2), aprobarbital (allylisopropyl barbituric acid) (R, 0.45), and Noludar (R, 0.9). Strips 21,22, and 23 were all buffered a t pH 12.2. Amobarbital had an R, of 0.6 in all three strips, the secondary bromallylbarbituric acid showed an R, of 0.5, while the R, of hexobarbital was 0.9. The illustrations present examples of the type of separations that can be accomplished by the technique of multi-
-
VOL. 29, NO. 5, MAY 1957
-
793
buffered papers. Many other controlled separations can be carried out (Tables I and 11). ACKNOWLEDGMENT
The authors are indebted to Carmine Auricchio for the photographic work. LITERATURE CITED
(1) Block, R. J., Durrum, E. L., Zweig,
G., "Manual of Paper Chromatography and Paper Electrophoresis," Academic Press, New York, 1955.
The System
2 Clark, W. M., Lubs, H. A., J . Bacteriol2, 1, 109, 191 (1917). ( 3 ) Hais, I., Macek, K., "Chromatografia papirova," Xakladatelstvi Ceskoslovensk6 Akademie, Prague, 1954. (4) Lederer, E., Lederer, M., "Chromatography," Elsevier, Houston, Tex., 1953. ( 5 ) MacIlvaine, T. S., J . Biol. Chem. 49, 183 (1921). (6) Schmall, Morton, Pifer, C. W., Wollish, E. G., ANAL. CHEX 25, 1486 (1953). ( 7 ) Schmall, Morton, Wollish, E. G., Shafer, E. G. E., Zbid., 28, 1373 (1956).
(8) Soerensen, S. P. L., Compt. rend. trav. lab. Carlsberg 8,41 (1909). (9) Strain, H., Sato, T. R., ANAL. CHEM.28, 687 (1956). (10) Turba, F., "Chromatographische
Methoden in der Protein-Chemie, einschliesslich verwandter Methoden wie Gegenstromverteilung, Papier-Ionophorese," Springer, Berlin, Gottingen, Heidelberg, 1954.
RECEIVED for review October 20, 1956. Accepted December 13, 1956. Division of Analytical Chemistry, 130th Meeting, ilCS, Atlantic City, N. J., September 1956.
Na pht haIe ne-T hia na pht hene
S. V. R. MASTRANGELO and R. W. DORNTE Barreff Division, Allied Chemical & Dye Corp., Glenolden, Pa.
A solid solutions treatment for calorimetric melting point data was verified for the system naphthalenethianaphthene. The solid solutions phase diagram, including liquidus and solidus lines, was also determined. Theoretical analysis permitted an accurate prediction of the solid-liquid equilibrium. The heat of fusion was determined as a function of composition. The use of these data for the evaluation of purity of naphthalene i s presented.
T
of freezing point a s a specification for the evaluation of purity depends upon knowledge of the character of the impurities present and their behavior in solution with the major component. The formationof solid solutions is 3 serious deviation of a system from the results predictable by the ordinary form of the melting point equation : HE USE
Log,,Nl
= 2,00000
'
- 2.30209 A% 1 1 + BAL( (1)
where iYl to
At
A
3
=
- ti
c.
mole per cent purity =
freezing point depression,
AH A degrees-'
RTO2
where AH,
heat of fusion of the major component, calories per mole To= freezing or melting point of 100.0 mole % major component, O K.
794
=
ANALYTICAL CHEMISTRY
( L W ~ )=~difference in molar heat capacity between liquid and solid at To The system naphthalene-thianaphthene was described in the liquidus region ( 2 ) , and the only reference to solid solution formation (8) gave no specific information in the solidus equilibrium region. The present investigation used both the calorimetric ( 1 ) and the freezing curve met'- ods to determine the equilibrium regions bounded by the liquidus and solidus curves. Application of the analytical treatment (6) of calorimetric-melting point data for a dilute impurity forming solid solutions n a s verified for this system. EXPERIMENTAL
Apparatus and Procedure. An adiabatic calorimeter was used ( 5 ) . The method of Aston, Cinnes, and Fink ( I ) for plotting energy input vs. equilibrium melting temperatures was used t o determine the liquidus and solidus temperature. Cooling curves were plotted a t two concentrations so t h a t the equilibrium points were approached from both sides. The cooling curve method employed an air-jacketed test tube and stirrer, and an KBS-calibrated, short-range thermometer to define the liquidus line over short composition intervals. Materials. Refined, liquid naphthalene (Barrett Division. Allied Chemical & Dye Corp.) was further purified by slow freezing in a n insulated Dewar flask, discarding the central core. This procedure was repeated four times in successively smaller Dewar flasks. The thianaphthene (Jefferson Chemical Co., Inc.) was purified by frac-
tional distillation. Its freezing Doint v a s 31.40" C. Molecular Sieve, 4ii powder (Linde Air Products Co.) was used to drv samples; 2 grams were required f i r every 50 grams of sample. "
1
SOLID SOLUTIONS TREATMENT T O NAPHTHALENE
A calorimetric purity determination was carried out (5) on a 36-gram sample of the purified naphthalene. Equilibrium temperature was plotted against the reciprocal fraction melted (Figure 1). The melting point obtained by extrapolating the best straight line through these points was 80.081' C., while the derived melting point of 100.00 mole % naphthalene was only 80.193" C. Other reported naphthalene freezing points are 80.290" C. ( 7 ) and 80.287' C. (4. Figure 1 indicates the presence of solid solutions. The treatment of Rlastrangelo and Dornte (6) was applied
RECIPROCAL fRACTION MELTED,+
Figure 1 . Equilibrium melting curve for naphthalene sample
