Countercurrent Distribution of Some Aromatic Hydrocarbons YI-CHUNG CHANG' and ROBERT D. WOTRING Low-Temperature Tar laboratory, Branch o f Bituminous Coal, Bureau of Mines, U . S. Department of the Interior, Morgantown, W. Va.
The suitability of the system $,@'oxydipropionitrile and iso-octane for countercurrent distribution of mixtures of aromatic hydrocarbons i s demonstrated. The partition coefficients of 24 aromatic hydrocarbons are presented; values vary from 0.52 to 14, depending upon the size of the alkyl side chains. An illustration i s given of the countercurrent distribution of a synthetic mixture of naphthalene, 1 methylnaphthalene, and Tetralin. A plot of the concentration ratio of two adjacent tubes as a function of the tube number and the number of transfers i s used to determine consistency of the data, homogeneity of the samples, and partition coefficients. A formula i s derived for locating accurately the position of peaks of a concentration distribution curve. The technique may be useful for separating and characterizing components of petroleum fractions and of oils derived from coal tars.
-
I
t o find new markets for coal. the bureau is conducting a n intensive research program on characterizing and upgrading tars resulting from low-temperature carbonization of coal. -4s part of the program on characterization, it has been necessary to develop nen- (or to modify existing) analytical techniques for separating and identifying compounds in complex organic mixtures. Countercurrent distribution is used widely t o resolve difficultly separable organic mi.rtnres (4, 6, 7 ) . I t s use in separating aromatic hj-drocarbons has been somewhat hindered by the lack of suitable solvents. During recent years, however, dipropionitriles hal-e been made available that selectively dissolw aromatic hydrocarbons and are virtually immiscible with saturated hydrocarbons (1, 8-10). Thus, a paraffin and a dipropionitrile should be a suitablc pair of solvents for countercurrent distribution of mixtures of aromatic hydrocarbons. To denionstrate the usefulness of such a system, the resolving power of N AN ATTENPT
1 Present address, Research Center, U. S. Steel Corp., Monroeville, Pa.
P,P'-oxydipropionitrile and iso-octane was studied. Partition coefficients for 24 aromatic hydrocarbons were determined and a countercurrent distribution of a synthetic mixture of naphthalene, 1-methylnaphthalene, and Tetralin was made to illustrate the performance of solvents. The technique described here may be applicable to the separation and identification of mixtures of aromatic hydrocarbons present in certain petroleum fractions and neutral oils derived from coal tars. CHEMICALS
Benzene, toluene (Fisher, ACS grade), 0-xylene, m-xylene, p-xylene, cumene, n-propylbenzene, naphthalene, n-butylbenzene, sec - butylbenzene, pentamethylbenzene, p-tert-butyltoluene, biphenyl, acenaphthene, mesitylene, indan, indene, cymene (Eastman Kodak Co., white label), 1-methylnaphthalene, Tetralin (1,2,3,4-tetrahydronaphthalene, American Petroleum Institute), tetrahydroacenaphthene, 2 - ethylnaphthalene, 1,3,5 - triethylbenzene (Aldrich Chemical Co.), phenanthrene (Fisher. Durified). and is0 - octane (Fisher: sljectro grade). P,P'-Oxydipropionitrile (CSCH2CH2OCH,CH,CK;). - - ,, Durchased from the American Cyanamid Co., was a strawcolored liquid of 93.5970 purity, based on nitrogen. Its physical properties are given by the supplier (1). This material was washed with spectro-grade iso-octane before use. PROCEDURE
Determination of Partition Coefficients. Two procedures were used for determining t h e partition coefficients.
PROCEDURE 1. Standard solutions of the aromatic hydrocarbons were prepared in iso-octane in concentrations ranging from 0.1 t o 2%. Ten milliliters of each solution was equilibrated with an equal volume of P,P'-oxydipropionitrile a t 25" rt 0.5" C. The iso-octane layer was washed twice with 20-ml. portions of distilled water to remove traces of dissolved oxydipropionitrile that might interfere with subsequent spectrophotometric analysis. The iso-octane phase was analyzed by ultraviolet spectrophotometry ( 6 ) , and the amount of solute in this phase was
Table 1. Partition Coefficients for Some Aromatic Hydrocarbons between Iso-octane and P,P'-Oxydipropionitrile at 25' C.
Procedure
Pro-
cedure
Benzene Toluene o-Xylene m-Xylene p-Xylene Mesitylene n-Propylbenzene Cumene Cymene sec-Butylbenzene n-Butyl-
2 13 2 3 2 6 3 9 4 1 2 0 6 0 7 7 2 8
82
benzene 8 7 Pentamethllbenzene 4 8 p-ferf-Butyl- 9 5 toluene l,S,S-Trlethylbenzene 14
2
Saphthnlene 1-Alethylnaphthalene
2-Ethylnaphthalene Biphenyl Indene Indan Tetralin -1renaphthene
0 77 1
5
2 1 0 2 2
3 0 98 2 9
0 77
Tctra-
hydroacenaphthene 13 Phenanthrene 0 52
calculated. This amount was subtracted from the total amount known to be present in both phases to yield the amount of solute in the nitrile phase. The partition coefficient was calculated as K = concentration in iso-octane concentration in oxydipropionitric (1)
PROCEDURE 2. A small amount (0.1 to 0.2 gram) of the aromatic compound was dissolved in 10 ml. of &P'-oxydipropionitrile. This solution was extracted successively with two IO-ml. portions of iso-octane a t 25' f 0.5' C. Traces of nitrile were removed from iso-octane layers of the two extractions by washing twice with distilled water. The ultraviolet absorbance of each iso-octane layer was measured a t a certain wave length. From the absorbances the partition coefficient was calculated (5) as K = ( A , - A?)/A> (2) where AI and A 2 are absorbances at the selected wave length of the first and second iso-octane layers. Partition coefficients of 24 aromatic VOL. 31, NO. 9, SEPTEMBER 1959
1501
1.0
I
1
1
I
I
I
1
1
I
I
-
-
X BENZENE
SERIES [HEPTANE]
A BENZENE S E R I E S 1150-OCTANE] 0 NAPHTHALENE
S E R I E S (HEPTANE)
-
C NAPHTHALENE SERIES (150-OCTANE) 0 PHENANTHRENE
2
0 NUMBER
32
24
16
TUBE
40
48
56
OF
CARBON
3 ATOMS
SERIES
4
IN
SIDE
1150-OCTANE1
5
6
CHAINS
Figure 3. Correlation between partition coefficient and a romaticity
NUMBER
Figure 1 . Resolution of absorbance curve into components
C
I
Y
Y
x
x
x
> 3
o
2 2
for concentrations of 2.0, 1.0, 0.5, and 0.1%. Similarly, for naphthalene, 1methylnaphthalene, 2 - ethylnaphthalene, and 1,3,5-triethylbenzene, values obtained a t concentrations of approximately 0.1 and 2% were not significantly different, indicating linearity of concentration distribution curves over this range. This condition has been assumed to be fulfilled by the other compounds.
N
w
> 3
v
> 3
v
1 2 13
2 0 I 1 12
I 8
I O
I1
-+
+ I'
6 09 I O
\
5 4 0 8 0 9
I 2 07 0 8
I O 06 07
0 8 05 06
06 0 4 0 5 C U R V E I 01 CURVE 2 0 3 CURVE 3 I
03 07
3
05 1 1 5
07 15
7
09 I9 9
( r t i)/[n-r)
Figure 2. Determination of partition coefficients
compounds are presented in Table I. Both procedures yielded consistent data. These values are valid for concentrations from 0.1 to 2%. This conclusion is based on data obtained for five compounds listed in this table: For Tetralin, values for the iso-octane phase were 2.93, 3.01, 2.86, and 2.89 1502
ANALYTICAL CHEMISTRY
Countercurrent Distribution. -4 mixture of 0.0787 gram of naphthalene, 0.1150 gram of l-methylnaphthalene, and 0.8211 gram of Tetralin was distributed countercurrently between oxydipropionitrile and iso-octane a t 25' i 2' C. in a Craig machine having 60 glass transfer tubes. The experiment was carried out according t o t h e procedure described by Craig and others, for which concentration distribution followed the binomial expansion law (4, 6, 7 , 11). A cycle consisted of 400 seconds for mixing (50 inversions), 720 seconds for settling, 20 seconds for decantation, and 20 seconds for transfer. Sixty transfers were made. It is understood that laboratories having machines equipped with a large number of transfer tubes can make the separations reported here much more selective. Samples of the iso-octane phase in each tube were collected, washed twice with water to remove traces of the nitrile, and scanned from 220 to 340 mfi with a Beckman DK-2 recording spectrophotometer. To obtain satisfactory spectra, samples of higher concentrations were diluted with iso-
octane. From a spectrum of the original synthetic mixture, wave lengths where appreciable absorption occurred were selected for analysis; the three compounds absorb strongly at 265 and 275 mp. A t each of these two wave lengths, absorbance values were obtained for all samples. An absorbance distribution curve for 275 mk was prepared by plotting absorbance, calculated to correspond to one tenth of the concentration of the original samples, as a function of tube number as shown in Figure 1.
If a broad peak should be present in a mixture of unknown composition, wave lengths should be selected a t equal intervals to cover a wider region, so that no constituents present in the original mixture will be missed. RESULTS
Results of the countercurrent distribution were analyzed to determine the number of constituents originally present and their degree of separation, the homogeneity of samples taken from certain tubes, and the partition coefficients. As suggested by Craig and by Golumbic (3, 7 ) , a partition coefficient can be used for identifying an unknown compound. For this purpose an absorbance distribution curve can replace a concentration distribution curve when Beer's law holds, as it did in this case. The first step in analyzing distribution curves usually is determining partition coefficients from the experimental data. Williamson and Craig (11) used two
formulas for doing this. One is r = nK/(K
+ 1)
(31
where n is the total number of transfers and r is the rth tube. Their other method is based on the fact that the ratio of the concentrations of a given solute in one of the solvents in t x o adjacent tubes is a function of the partition coefficient: K
=
[(r
+ I)/(n - r)l[(Tr+d/T7j
(4)
where T , and T , are the concentrations of solute in one solvent in the 1)th tubes. They rth and ( T plotted T , / ( T , I as a function of K a t fixed ( r l ) / ( n - r ) to obtain the partition coefficient. However, Formula 4 can be used to greater advantage by plotting T,/(T, + 1) as a function of (r l ) / ( n - T ) ; rectilinear portions of such a plot have a slope equal to 1/K. The plot not only permits use of several experimental points for determining K , thus giving a more reliable result, but it also tests the degree of separation and the consistency of the data. Consistent data produce a smooth curve. When part or all of the curve is straight, the corresponding tubes usually contain a single solute. When more than two solutes are present, the curve usually is not straight. The absorbance distribution curve in Figure 1 shows that tubes 17 to 26 probably contained a homogeneous solute. Ratios of (T,/T, + I), in this instance absorbance values, are calculated and plotted as a function of (T l ) / ( n - r) in curve 1 of Figure 2. As this curve is straight from tubes 17 to 24, a homogeneous solute is indicated; in this case, it is known to be naphthalene. The reciprocal of the slope of this straight line yields a partition coefficient of 0.77, which agrees with the previously determined value of 0.78. As an internal check on the data a t 275 mu, similar curves were prepared using the absorbance values a t 265 mp; the partition coefficient was 0.76. The distribution curve in Figure 1 indicates that the peak of the absorbance distribution curve for naphthalene lies between tubes 25 and 28. To locate the exact position of this peak, one may use
+
+
+
+
r
2 ( K n - lj/(K
+ 1)
(5)
The derivation of this formula is discussed below. When numerical values are substituted for n and K and the resulting ratio is an integer, the equal sign applies: The concentration peak will occur in tubes r and ( r 1). When the ratio is nonintegral, the “greater than” sign applies: The peak will 1). For example, occur in tube ( r since n = 60 and K = 0.77, r = 25.6. As this is a noninteger, tube 26 contains the doncentration peak. Curve 1 of Figure 2 also shows that,
+
+
beginning a t tube 25, the experimental points deviate from the straight line, indicating that another solute or solutes are present besides naphthalene. To resolve this part of the curve, theoretical absorbances for naphthalene in tubes 25 to- 36 are calculated by Formula 4, with K = 0.77. These calculated values are subtracted from the experimental curve, and the difference is plotted as a residual curve as shown in Figure 1. Values of T,/(T, + for this residual curve are again plotted as a function of (r l)/(n - r ) as shown in curve 2 of Figure 2. The part of curve 2 corresponding to tubes 25 to 35 is a straight line, indicating the presence of a homogeneous solute with a partition coefficient of 1.5. Curve 2, derived from the data a t 265 mg, also gives a partition coefficient of 1.5. Use of Formula 5 indicates that the concentration peak for the second compound (1 - methylnaphthalene) occurs in tube 36. Again, starting with tube 36, the points deviate from the straight line, indicating that yet another constituent is present. Theoretical absorbances for I-methylnaphthalene in tubes 36 to 47 are calculated and subtracted from the experimental curve, resulting in a residual curve for the third constituent as shown in Figure 1. The straightline plot is again applied as shown in curve 3 of Figure 2. All the points fall on a straight line, indicating a third homogeneous constituent. The reciprocal of the slope of this straight line is 2.9, the partition coefficient of Tetralin. The partition coefficient based on the absorbance values a t 265 mp is 2.8. The concentration peak, as determined by Formula 5, is in tube 45. Thus, the absorbance distribution curve is resolved into three parts, whose ordinates are directly proportional to the concentrations of the solutes.
+
DISCUSSION
Two other dipropionitriles, @,@’-thiodipropionitrile and &@‘-iminodipropionitrile, with properties similar to @,p’oxydipropionitrile are now available (1). Iminodipropionitrile is completely miscible with water, a desirable property for removing the nitrile from the raffinate layer and for recovering the solute from the nitrile layer. Isooctane was chosen mainly because of its availability in spectro grade; other paraffins of similar quality can serve as well. However, consideration should be given to their boiling point, specific gravity, and viscosity: If the boiling point is too low, excessive loss by evaporation will result; if the viscosity and specific gravity are too high, settling of the layers of the mixture will be slow. Only two significant figures have been
used for K , because concentrations in the iso-octane layers are accurate only to i 2 % and most of the compounds used were not absolutely pure. Partition coefficients between oxydipropionitrile and n-heptane of some of these compounds have been published (1). For the same compound, the partition coefficient between oxydipropionitrile and iso-octane is smaller than that between oxydipropionitrile and n-heptane. Hence n-heptane is a better solvent than iso-octane for aromatic hydrocarbons, especially for Tetralin with a partition coefficient of 8.4 for n-heptane as compared with 2.9 for iso-octane. Considering alkyl aromatic hydrocarbons to consist of two parts, an aromatic portion and a paraffinic portion of one or more side chains, one finds that partition coefficients generally increase rapidly with the total number of carbon atoms in the chains. With benzene and naphthalene as the first members for the mono- and dinuclear series, partition coefficients are plotted as a function of size of side chain in Figure 3. The data for the benzene and naphthalene series in n-heptane were taken from the literature (1). Although the points are somewhat scattered, the trend is unmistakable. Formula 5 has been introduced for locating the peaks of the concentration distribution curve. The concentration of solute in tube r can be obtained from the binomial expansion of the partition coefficient (5, 7, 11): T n , = n!/r!(n-
T)’
1/(K
+ 1)“K‘
(6)
When this formula is applied to a countercurrent distribution machine having discrete tubes, K may have any positive value, but n and T must be positive integers. A corollary of this consideration is that, for a set of n and r, there is a certain range of values for K in order for the concentration peak to fall on a fixed tube. The relationship between the location of the peak (maximum T?),n, r, and K can be obtained by first deducing the range of values that K must have if the peak is to fall on a certain tube when r and n are given. To determine the maximum value for T,, magnitude of T , for all the tubes from tube 0 to tube r (tubes are numbered as 0, 1, 2, . . . T ) must be compared. This can be done by using Formula 4 for concentration of adjacent tubes: For the maximum value to be T , (or for equal maxima of T , and T , + I), T , L T , + and (r l ) / ( n - T ) L K . For the peak to occur in tube 0, Tomust be greater than TI. Setting r = 0, one gets (0 l)/(n - 0) = l / n ; K must thus be smaller than l/n. When K = l / n , To= T1. The concentration peak, instead of occurring in one tube,
+
+
VOL. 31, NO. 9 , SEPTEMBER 1959
1503
is spread equally over two tubes, 0 and 1 in this case. When K is greater than
l/n, the peak occurs in tube 1. Similarly, if the peak is to occur in tube 1, K must be smaller than (1 l ) / ( n - 1) = 2 / ( n - 1) but greater than l / n , etc. I n general. for the peak to fall on tube r, K must have values between r l ( n T 1) and (r l ) / ( n - r). Using the upper limit alone for simplicity, one may derive Formula 5 as follows:
+
+
+
+ I)/(n - 2 K ( r + 1) 2 K n - Kr ( K + l)r 2 Kn - 1 r L ( K n - 1 ) / ( K + 1) (1
1.)
(5)
When n becomes very large, ( K n 1) approaches Kn and Formula 5 approaches Formula 3. Unlike Formula 3, however, Formula 5 shows the exact location of the concentration peak and thus permits accurate determination of the shape of the concentration distribution curve more exactly. Only 60 transfers were used, so that the individual curves would 01-erlap consider-
ably as shown in Figure 1. Despite this condition, satisfactory resolution can be obtained by the use of Formulas 4 and 5 .
ultraviolet analysis, and compared with the theoretical peak tubes as determined from the partition coefficients using Formula 5 .
SUGGESTED APPLICATION T O LOW-TEMPERA.TURE TARS
LITERATURE CITED
The neutral oil fraction of the tar, freed of acids and bases, is fractionally distilled into narrow boiling range fractions under mild, noncracking conditions. Each fraction is separated into paraffins and naphthenes, olefins. and aromatic hydrocarbons by displacement development chromatography as described by Chang and Karr ( 2 ) . The aromatic hydrocarbon fractions are then subjected to additional fractionation by countercurrent distribution in the Craig machine by the technique described. The ultraviolet spectra of some of the fractions from the machine will indicate the presence of certain aromatic hydrocarbons. The numbers of the Craig machine tubes with the highest concentrations of these hydrocarbons are determined by quantitative
(2) Chang, T.-C. L., Karr, Clarence. Jr., .4hstracts of PaDers. 135th lleetinc. .4CS, Boston. l f a i s , .Ipril 1959 (3) Craig, L. C., J . Bzol. Chem 150, 33
-
-_
119&7) ’ \ - ”
( 4 ) Ibzd., 155, 519 (1944). ( 5 ) Craig, L. C., Craig, D., Chap. “Eutraction and Distribution.’’ “Technin tie of Organic Chemistry,” 1-01. iII, Interscience, Sew York, 1950. (6) Friedel, R. A , Orchin, hi., “Ultra-
violet Spectra of >\romatic Compounds,” p. 4, Wiley, New Pork, 1951. (i) , . Golnmbic,. C.,, ANAL. CHEM.23, 1310 (19513. (8) McKinnis, A. C., E. S. Patent 2,441,827 (May 18, 1948). (9) Saunders, K. TV.,I n d . Eng. Chem. 43, 121 (1951). (10) Skinner, D. -4.)Ibz‘d., 47, 222 (1955). (11) Williamson, B., Craig, L. C.. J . Biol. Chem. 168, 687 (1947).
RECEIVED for review Kovember 3. 1958. .Iccepted May 8, 1959.
Ion Exchange Chromatography of Amino Acids Semiautomatic Method of Operation with Cationic Exchange Resin Columns PAUL B. HAMILTON and ROBERTA A. ANDERSON Alfred 1. du Ponf lnstitufe o f the Nemours Foundation, Wilmington
b The amino acids usually found in a protein hydrolyzate are separated chromatographically in 38 hours by using a single column and a conventional fraction collector with automatically controlled changes of temperature and buffer solutions. Good resolution of the amino acids with sharp peaks and quantitative recoveries are obtained with 0.636 X 100 cm. columns packed with 20- to 40-micron particles of an 8.5% crosslinked cationic exchange resin. Developing buffers are pumped through the column, under pressures of approximately 7 5 p.s.i., at 39.3 ml. per sq. cm. per hour. This allows a time schedule that fits regular working hours. Operation at 7 9 ml. per sq. cm. of resin bed per hour under pressures of 175 to 200 p.s.i. and a collection time of 1 8 1 / 2hours is equally satisfactory, but with a collector rack of limited capacity unusual working hours are unavoidable. Separation of the basic amino acids on the same 1504
ANALYTICAL CHEMISTRY
99, Del.
columns is described. Results expected with a large preparative type column are indicated.
I
THE chromatography of amino acids on 8 to 8.5% cross-linked sulfonated polystyrene cationic exchange resins, Moore and Stein (9) utilized a n 0.9 X 100 cm. column developed a t a flow rate of 6 ml. per sq. cm. of resin bed per hour to separate acidic and neutral amino acids and an 0.9 X 15 cm. column to separate the basic amino acids. These latter acids were eluted quantitatively with buffers of low alkalinity. Recoveries of the amino acids usually encountered in protein hydrolysates averaged 100 3%. It was found in this laboratory that a greater number of amino acids could be resolved on a 15-em. column when it was developed mainly with pH 5.00 citrate buffers (5,4). Later Moore and Stein (IO) employed a single 0.9 X 150 cm. column, packed with 47, N
*
cross-linked resin and operated bet-iveen pH 3.1 and 5.1, but the concentrated buffer used to elute the basic amino acids contracted the column, which made repouring necessary after each analysis. This prompted the attempt to find conditions for satisfactory resolution of the amino acids on an 8% crosslinked resin and repeated use of the column. The demonstration by Spackman, Moore, and Stein (14) that flow could be increased to over 47 ml. per sq. em. per hour, without impairment of resolution with resins of finer mesh than those used previously, made it mandatory to extend the present investigation. The need for finer mesh resins led to the development of a hydraulic technique for the separation of particles of any particular size from the mixed population of commercial resins (1) and the demonstration that good resolution could be obtained with 100-cm. columns operated a t 39.3 ml. per sq. cm. per hour, packed with pulverized resin
