of Dowex 50-X2, 0.9 cm. in diameter, used for this purpose can be operated with a pump a t flow rates close to 30 ml. per hour (6). Schram and Lombaert (11) have developed a scintillation counter which permits measurement and recording of the concentration of carbon-14 or sulfur35 in a flowing stream without evaporation of the effluent. Their counting cell is designed to be inserted between a column and a fraction collector, but it could be interposed ahead of the stopcock manifold in the present instrument, to provide a continuous record of radioactivity in each of the peaks measured by the ninhydrin reaction. ACKNOWLEDGMENT
The authors wish to express their appreciation to C. H . W. Hirs of this laboratory and Kenneth Woods of Cornel1 Medical Center, who have built and operated recorders according to this design, and have given the valuable benefit of their experience with the instrument. They also are indebted to Herbert Jaffe for the spectroscopic data on the filters, to Nils Jernberg and Carl R. Ti-
den of the Instrument Shop for expert advice and shop work on the photometer, and to Kerstin Johansson for skillful technical assistance. It is a pleasure to acknowledge the correspondence of D. H. Simmonds of Melbourne concerning the details of the instrument which he has designed to render the discontinuous type of procedure automatic (12). The authors appreciate the suggestion of E. G. Pickels and J. E. Miller, Spinco Division, Beckman Instruments, Inc., regarding the "dot-counting" method of measuring the width of a peak, and w e indebted to them for permission to describe the procedure. The procedure for the accurate measurement of the rate of flow was suggested by A. J. P. LMartin, to whom the authors are much indebted. LITERATURE CITED
(1) Hill, R. L., Smith, E. L., J . Biol. Chem. 228, 577 (1957). (2) Hirs, C. H. W., Stein, W. H., Moore, S., IUPAC Symposium on Protein Structure, Wiley, Kew York, in press. (3) Hirs, C. H. W., Stein, W.H., Moore, S., J . Biol. Chem. 211, 941 (1954). (4) Zbid., 221, 151 (1956).
Moore, S., Spackman, D. H., Stein, W. H.. ANAL. CHEM.. , 30., 1185 (1958).' i\Toore, S., Stein, W. H., J . Biol. Chem. 176, 367 (1948). Zbid., 192, 663 (1951). Zbid., 211, 893 (1954). Zbid., p. 907. Schram, E., Dustin, J. P., Moore, S., Bign-ood, E. J., Anal. Chim.Acta 9, 149 (1953). Schram, E., Lombaert, R., Zbid., 17, 417 (1957). Simmonds, D. H., ANAL.CHEY.30, 1043 (1958). Smith, E. L., Spackman, D. H., J . Biol.Chem. 212.271 11955). Soupart, P., Mobre. S.. Bimood. E. J., Zbid,, 206, 699 ((954); Spackman, .D. H., Stein, W. I3., Moore, S., Fed eration Proc. 15, 3'58 (1956): Stein. W. H.. J . Biol. Chem. 201. 45 (1953). ' Stein, W. H., Moore, S., Zbid., 211, 915 (1964). Tallan, H. H., Moore, S., Stein, W. H., Ibid., 211, 927 (1954). Zbid., 230, 707 (1958). '
RECEIVED for review February 28, 1958. Accepted May 15, 1958. Presented in art before the Federation of American iocieties for Experimental Biology, Atlantic City, N. J., April 1956. The second and third instruments of this type were constructed with the aid of a grant from The rational Science Foundation.
Separation of Isomeric Polyphenyls by Adsorption Chromatography MAX HELLMAN, ROY L. ALEXANDER, Jr., and CHARLES F. COYLE National Bureau o f Standards, Washington 25, D. C.
b The chromatographic behavior of selected polyphenyls was studied to develop a method for separating mixtures of these compounds. A variety of adsorbents such as alumina, fuller's earth, silica gel, and different grades of charcoal were used. The separations of known mixtures were studied by the elution technique and elution curves were drawn. Results obtained with several adsorbent-eluent systems are compared. Some separation occurred in most cases, with the alumina-iso-octane combination providing maximum over-all effectiveness in separation and recovery. Some charcoals give better separation - but material recovery is poor. O n the basis of the results obtained, the behavior of mixtures of higher polyphenyls is predicted.
T
HE SEPARATION of mixtures of aromatic compounds by chromatographic techniques has found extensive use in recent years, particularly in the
1206
ANALYTICAL CHEMISTRY
analysis of heavy oils and tars. However, nearly all of the work done in the field deals with the separation of monoand polynuclear aromatics. The group of aromatic compounds known as polyphenyls, which correspond to the general formula, CeH.5(C6H4),CeH6,has been almost entirely neglected. Only one paper in the recent literature (6) has dealt with the attempted separation and identification of mixtures of polyphenyls by paper chromatography. Recent interest in biphenyl and related compounds as potential high temperature lubricants and nuclear reactor coolantmoderators (3) has created a need for a systematic method of analysis of mixtures resulting from the thermal or radiative degradation of these materials. Consequently, this study was undertaken with the aim of obtaining some information concerning the behavior of polyphenyl mixtures on a variety of chromatographic adsorbents. No attempt was made to find ideal conditions for a specific separation, but rather
to establish definite trends which would enable one to choose the proper system for a given mixture. REAGENTS
Adsorbents.
Alumina, Fisher Scientific Co., absorption grade, 80 to 200 mesh. Reactivated by heating a t 200" C. Fuller's earth, Floridin Co., Florex X X F . This was sifted and 100- t o 200-mesh size was selected. Reactivated by heating a t 160' C. Silica gel, Davison Chemical Co., Code 912, 28 t o 200 mesh. Pittsburgh charcoal, Pittsburgh Coke and Chemical Co., 8 to 30 mesh. Coconut charcoal, National Carbon co. Bone char, Baugh & Sons Co. The above charcoals were ground and 40 to 60 mesh was employed. Each charcoal was heated a t 110' C. for several hours prior to usage. Deactivation of alumina, silica, and Pittsburgh and coconut charcoal was performed by mixing the desired amount of deactivation agent (usually water) with the adsorbent in a glass-stoppered
L 2
,
6C-
-
t
Figure 1. Separation m-terphenyl
of
FRACTIONS
mixture of equal parts of
0-
Concentrafion, 200 mg. per 20 ml.; 10-cc. fractions o-Terphenyl - - - - m-Terphenyl Solvents: A , B. Iso-octane C, D,E, F. Cyclohexane G, H. Chloroform hdsorbents: A , C. Alumina, 4.0 grams (20 to 1) B , D. Fuller's earth, 4.0 grams (20 to 1) E, G. Coconut charcoal, 2.0 grams (10 to 1) F , H. Pittsburgh charcoal, 2.0 grams
and
Figure 2. Separation of mixture of equal parts m- qua te r p h eny I
bottle and shaking the mixture on a horizontal shaker for at least 6 hours.
CHROMATOGRAPHIC SEP-
and
t o 1) F , H. Pittsburgh charcoal, 1.0 gram (10
m-Quinquephenyl and m-sexiphenyl were prepared in this laboratory (1).
grade iso-octane, cyEXPERIMENTAL PROCEDURES clohexane, chloroform, and ether were Weight ratios of adsorbent to the further purified by passage through mixture t o be separated ranged from silica gel before use. SPECTROSCOPIC MEASUREMENTS.5 to 1 to 50 to 1. The charcoal adsorbents required ratios between 5 to 1 Spectroscopic grade iso-octane, cycloand 15 to 1. The diameter of the hexane, a n d chloroform were used t o column was selected to give a heightdilute t h e samples t o t h e desired conto-diameter ratio between 5 to 1 and centrations for absorption measure10 to 1. Prior to each run the columns ments. These solvents could be reused were wetted with the first eluent. after removal of the dissolved polySamples were collected in 10-cc. fracphenyls. For iso-octane and cyclohextions except for a few runs, where 5-cc. ane this was accomplished by passage fractions were collected. Each fracthrough si1ic.a gel; for chloroform, by tion was diluted to a desired concentradistillation. tion with the appropriate solvent and Polyphenyls. Biphenyl, 0-,m-, and its absorption measured at preselected p-terphenyl were Eastman Co. chemwave lengths on a Beckman Model icals purified b y recrystallization. DU spectrophotometer. The weights o-Quaterphenyl was purchased from of material in each fraction were calt h e Organic Specialties Co. and reculated by a procedure used for quancrystallized. titative spectrophotometric analysis of m-Quaterphenyl was prepared in two- or three-component mixtures (4). this laboratory by t h e method of These measurements were preceded Woods and Reed (7'). by calibrations which consisted of the m,p-Quaterphenyl was obtained from determination of specific absorbances G. F. Woods of the University of hlaryfor each compound a t two or three land (S). C.P.
of 0-
Concentration, 100 mg. per 20 ml.; 10-cc. fractions o-Quaterphenyl _ _ - - m-Quaterphenyl Solvents: A , B. Iso-octane C, D,E, P. Cyclohexane GI H . Chloroform Adsorbents: A , C. Blumina, 2.0 grams (20 to 1) E , D. Fuller's earth, 2.0 grams (20 to 1) E, G. Coconut charcoal, 1.0 gram (10
(10 to 1)
Solvents.
6
H
FRACTIONS
ARATIONS.
4
,
t o 1)
wave lengths. The wave lengths selected were the absorption maxima for each compound in the appropriate solvent. RESULTS
Figure 1 presents a comparison of the separation of 0- and m-terphenyl in three different solvents and on different adsorbents. I n each case some separation occurs. As shonm in graphs E, F , G, and H , the charcoals give nearly complete separation; however, the recovery of material is poor. Addition of better eluents such as chloroform or benzene improves the recovery, but not beyond 70 to 80%. Alumina or fuller's earth gives excellent recovery and fair separation, particularly when iso-octane is used as the solvent (graphs A and B ) . I n addition to the adsorbents indicated here, others were used which gave unsatisfactory results. Silica gel retains the materials completely with isooctane or cyclohexane as eluent. Chloroform elutes all of the material VOL. 30, NO. 7, JULY 1958
1207
rapidly without separation. Silica deactivated with 8% water effected some separation with iso-octane as eluent, but was considerably poorer than alumina or fuller's earth. Other charcoals also tried were bone char and Darco (3-60 mixed with celite. Keither gave separations comparable to those shown by Pittsburgh or coconut charcoals. I n Figure 2 are presented results obtained from separations of 0- and m-quaterphenyls in the same adsorbent-eluent systems. The behavior of these mixtures is similar to the corresponding terphenyls, but a sharper separation is apparent on alumina and fuller's earth. The separation of three-component mixtures of terphenyls and quaterphenyls is shown in Figures 3 and 4, respectively. The compounds containing para links are strongly adsorbed and, therefore, more easily separated. Elution of these compounds from alumina is possible with a more effective eluent such as chloroform or ether, but in the case of charcoal very little recovery could be effected. Numerous runs were also made in which the ratio of adsorbent to solute was varied. The general effect observed was that a n increase in the relative amount of adsorbent broadened the elution curves for the component without showing appreciable improvement in the separation. Charcoals are more sensitive to changes in the ratio of adsorbent to solute, particularly with respect to material recovery. This can be ascribed to the strong adsorption of polyphenyls on carbon. Several methods were tried to improve the recovery of material from charcoal. Deactivation with water or sulfuric acid improved the recovery somewhat, but a portion of the meta compounds was still retained. Another method attempted was the displacement of the polyphenyls by a substance which might be more strongly adsorbed. Solutions of phenol and l-methyl-naphthalene in cyclohexane were used. The results were unsatisfactory inasmuch as the meta compounds were more strongly held than either of these. Figures 1 to 4 illustrate the separation of mixtures of isomers having the same molecular weight. Figure 5 presents studies of mixtures of polyphenyls having the same structural features but different molecular iTeights. The data in this figure r e r e plotted using absorbance values at 248 mp, which is near the maximum absorption for most m-polyphenyls. This method does not yield analyses of individual fractions, but provides an estimation of the relative compositions. Because the higher polyphenyls are strongly adsorbed, the use of deactivated adsorbents shows definite promise in this case. Also, the use of 1208
ANALYTICAL CHEMISTRY
a graded series of eluents employing mixtures of iso-octane and ether enables one to recover all of the materials gradually. Finally, a relative comparison of the adsorption of various polyphenyls can be obtained from specific retention volumes, a concept first formulated
I
/
I
/
by Tiselius ( 2 ) . The values presented in Table I are average values determined from several runs using different amounts of adsorbent. These values were all determined from mixtures rather than individual conipounds; therefore, any effects caused by the presence of a second or third
l
I
/
'
1
2o
10
l
I
I
4
CYCLOHEXANE
CHLOROFORM
1
A
-
'.--. -- -- ----_ ~
A---
2
0
--AI
6
4
----.-----
-c--
10
8
I2
FRACTION5
Figure 3. Separation of 40 to 40 to 20 mixture of p-terphenyl in cyclohexane
0-,
rn-, and
Concentration, 100 mg. per 20 ml.; 10-cc. fractions o-Terphenyl _ _ - _ m-Terphenyl -.-.- *-. p-Terphenyl A . Alumina, 4.0 grams (40 to 1) B. Pittsburgh charcoal, 1.0 gram (10 to 1)
IO
r i
-
: Ezo. IC
0
1-
CYCLOHEXANE
'CHLOROFORM
E
i
2
6
E
10
12
14
16
FRACTIONS
Figure 4. Separation of 40 to 40 to 20 mixture of 0 - , rn-, and rn,p-quaterphenyl in cyclohexane
Concentration, 100 mg. per 20 ml.; 10-cc. fractions ------o-Quaterphenyl _ _ _ _ m-Quaterphenyl - .-. - .- .m,p-Quaterphenyl A . Alumina, 4.0 grams (40 to 1) B. Pittsburgh charcoal, 1.0 gram (10 t o 1)
Terphenyl, 12.5 mg.; quaterphenyl, 12.5 mg.; quinquephenyl, 12.5 mg.; sexiphenyl, 10 mg.; in 10 cc. of iso-octane; 5-cc. fractions; 1 to 50 dilution A . Activated alumina, 4.25 grams (50 to 1) B. Deactivated alumina (1.5W water), 4.25 grams (50 to 1) C. Deactivated silica (8% .~ water). 2.55
I
A
r
I SOOCTANE --jic- ISOOCTANE
I%ETHER
6o 40
I
T
ISWCTGNE -ISOOCTANE 4% ETHER 2% ETHER
t
1SOOCThNE
50% ET'IER
I 1
L
grams (30 to 1)
compound would be included. Specific retention volumes for charcoals were not included as they were found to vary greatly with the amount of adsorbent used. The results indicate t h a t the retention is stronger with alumina than fuller's earth, and iso-octane is a poorer eluent than cyclohexane. Also the order of retention increases in the order, ortho < rneta < para, for mixtures of the same niolecular weight. Comparison of materials of different molecular neights shows t h a t the higher compounds are more strongly retained. Howeyer. o-quaterphenyl and m-terphenyl have specific retention volumes which are nearly equal in three of the four systems. This would indicate that mixtures containing isomers of terphenyl and quaterphenyl would be difficult to separate if they contain any 0-q uaterp henyl. DISCUSSION
The results indicate that the separation of isomeric polyphenyls follows a definite pattern which can be correlated with the steric configurations of the molecules. BeloTv are the structural formulas of the compounds studied here.
o-Terphenyl
~
i-
'
'
~
I SOOCTANE
~-
ISOOCTGNE I% ETHER ISOOCTGNE 2% ETHER
I
~
'
-
'
I
~
'
'
1
--~ I
20 FRACTlOhS
of various naphthyl cycloalkenes and polycyclic aromatics (5). However, the increase in adsorption from meta to para compounds cannot be explained solely on this basis. Resonance effects due to the increased conjugation of para compounds most likely contribute to their strong adsorbability. A simple correlation mhich would include all these factors mould be a comparison of chromatographic adsorbability and the ultraviolet spectra of polyphenyls. Table I1 lists the adsorption maxima for the terphenyls
nz-Terphenyl
and quaterphenyls studied here. It is evident from these data t h a t the adsorbability of polyphenyls of the same molecular weight increases with a shift of their ultraviolet adsorption maxima toward longer wave lengths. To substantiate this statement, holyever, a study of the remaining quaterphenyls and perhaps some higher polyphenyl would be needed. It must be further stressed that the correlation is definitely limited to compounds of the same molecular weight, as increases in molecular weight generally result in
p-Terphen yl
a
a-0-0'
m-Quaterphenyl
Although it is not possible to show it by the use of two-dimensional formulas, the ortho compounds exhibit steric hindrance to free rotation and therefore, cannot be coplanar. Meta and para isomers, however, are not hindered and thus can approach coplanarity. The results given here show that hindrance to coplanarity lowers the adsorbability. This finding has been corroborated by a recent study on the chromatographic adsorbability
I 1
t
ISOOCTANE 2% ETHER
a-0D-6 o-Quaterphenyl
ISOOCTANE - L I S O O C T A N E 1-L ETHER
m,p-Quaterpheny]
[ 1-phenyl-3- (4-xen yl) benzene]
Table 1.
Specific Retention Volumes (Cc. per gram of adsorbent)
Adsorbent Alumina Fuller's earth
Solvent Biphenyl oIso-octane ... 4.4 Cyclohexane 2.0 3.3 Iso-octane ... 2.8 Cyclohexane ., , 1.4
Terphenyls mP5.7 ~ 2 6 4.8 11.4 4.6 ... 2.8 .,.
Quaterpheny 1s 0-
7.3 4.4
4.5 3.4
m9.7 7.5 8.5 6.6
VOL. 30, NO. 7,JULY 1958
m,p-
,..
-30
...
..,
1209
Table II. Ultraviolet Absorption Maxima of Polyphenyls in Cyclohexane
Compound o-Terp henyl m-Terphenyl p-Terphen yl o-Quaterpheiiyl m-Quaterphenyl m,p-Quaterphenyl
MIL 232.5 247.5 277 229 248 265
Xrn,x,
greater adsorption on polar adsorbents. Chromatographic separation of polyphenyls depends on tiyo main factors:
structure and molecular iTeight. Because these two factors may occasionally work at cross purposes, it would be advisable to separate mixtures of polyphenyls first into mixtures of the same molecular weight before attempting a separation of isomers. LITERATURE CITED
(1) Alexander, R. L., Jr., J . Org. Chew$. 21, 1464 (1956). ( 2 ) Cassidy, H. G., “.-idsorption and Chromatography,” pp. 224-7, Interscience, Xem York, 1951.
Freund, G. A., Nucleonics 14, No. 8, 52 (1956).
Friedel, R., Orchin, M., “Ultraviolet Spectra of Aromatic Compounds,’’ pp. 29-32, Riley, New York, 1951. Klemm, L. H., Reed, D., Lind, G. D., J . Org. Chem. 22,739 (1957).
Silverman, L., Bradshaw, W.,ANAL. CHEM.27, 96 (1955). Woods, G. F., Reed, F. T., J . Am. Chem. Soc. 71, 1348 (1949). Woods, G. F., Tucker, I. W.,Ibid., 70, 3340 (1948).
RECEIVED for review Sovember 8, 1957. Accepted February 4, 1958. Work supported in part by the Naval Reactors Branch, U. 8. Xavy Bureau of Ships.
Material Deposited along Path of Chromatographed Sugar Spot CHARLES K. HORDE1 and GEORGE N. KOWKABANY The Catholic University o f America, Washington, D. C.
,The amount of material distributed along the path of a sugar spot chromatographed on paper has been estimated by means of a radioassay method. Some of the material along the path of the spot appears to be the sugar originally chromatographed and is in excess of the amount predicted b y a Gaussian distribution. Adsorptive forces competing with the liquidliquid distribution process may account for the distribution of material along the path of the spot.
T
much evidence that a liquid-liquid distribution process is not the only mechanism operative in paper chromatography. Adsorption of a solute by the cellulose n q - also be involved, as !vel1 as ion exchange phenomena. One avenue of approach to the mechanism of the chroniatogram lies in determining quantitatively the distribution, along the path of the chromatographed spot, of the substance being chromatographed-Le., the material between the spot and the point of initial application of the spot. The object of this investigation was to determine, by use of carbon-14-labeled carbohydrates and radioassay, the amount of material distributed along the path of the spot. The liquid-liquid distribution concept has been supported by good agreement of distribution coefficients calculated from Rr values and those experimentally determined ( 5 ) . Benson et al. HERE IS
Present address, Cathedral Preparatory School, Erie, Pa. 12 10
ANALYTICAL CHEMISTRY
showed this for glucose ( 2 ) . Calculations of R, and similar factors also support the liquid-liquid mechanism (1). On the other hand, Moore and Stein have given evidence of adsorptive phenomena (11). Other workers have resolved racemic mixtures on the paper chromatogram without use of asymmetric solvents (4, 7 ) . Some type of interaction with the asymmetric cellulose fibers must be involved. Thompson and Steward, who used quantitative colorimetric procedures on the eluate, were unable to recover amino acids completely from the chromatogram. The loss appeared to be proportional to the distance moved by the spot ( I S ) . Proportionality of loss to distance traveled agreed with the findings of Koiwod (14) for glycine, but conflicted with the work of Fotvden (6). However, all agreed that appreciable losses of amino acid occurred during the paper chromatography. Carbon-14-labeled sugars were used in this investigation for the following reasons: Similarity of structure between cellulose and the sugars used may enhance any adsorption occurring. Amino acids react with ions in the paper-e.g., copper-to form salts with R, values different from those of the acids ( 5 ) . Further, it is extremely difficult, if not impossible, to prepare an ion-free filter paper, Such complications are less likely to occur with sugars. R2dionssay techniques are much more sensitive than the usual revelation of spots by color reactions. Color tests may detect as little as 1 y of a sugar, while radioassay techniques may detect
as little as a small fraction of a microgram (@, depending on the specific activity of the sugar used. EXPERIMENTAL
General Method. Whatman KO. 1 paper, cut in t h e machine direction, was spotted with a 1% solution of glucose-l-C14 or arabinose-l-Cl4 (specific activity about 1 pc. per mg.), then developed by the descending method (6). The sugars were obtained from H. S. Isbell, National Bureau of Standards. The paper was conditioned for 1 hour before development with the solvent developer (the upper layer of n-butyl alcohol, acetic acid, and water, 4 to 1 to 5 by volume). After development the chromatogram was air dried. Guide strips from the outer edges of the chromatogram mere cut off and sprayed with aniline hydrogen phthalate (12) to locate the sugar spots. The remainder of the chromatogram was sectioned from the region a t the point of initial application of the spot to just beyond the location of the spot. Each piece of the chromatogram was then assayed in a gas-flow, windowless, Geiger-?rIidler counter (Nuclear Instrument and Chemical Corp., Xodel D468). Standard deviation, u, was determined for each assay (9). Counting Efficiency. T o check the efficiency of t h e radioassay method, 1 pl. each of maltose, glucose, and arabinose mas chromatographed separately and total activity of each chromatogram measured. Total counts per minute above background total disintegrations per minute calculated x 100 = countingefficiency Counting efficiency varied from 11 to
