water. Our most successful method for adding known amounts of water was the addition of crystalline BaC12 2H20. As can be seen from Table 11, the water acted as a monobasic acid. Of course, the data in Table 11 have been corrected for the water initially present in the solution. Urea. Although urea is reported to be a dibasic acid in ammonia (9)) its mononegative conjugate base reacts so slowly with the ammoniated electron that only one end point was observed in our titrations (See Table 11). Diphenylamine. As expected, diphenylamine acted as a weak monobasic acid. Unsuccessful Titrations. Benzamide, which gave an end point in the neighborhood of 2.5 meq/mole, undoubtedly underwent reduction. I t has been reported (11) that benz(11) H. Smith, “Organic Reactions in Liquid Ammonia,” Interscience, New York, N. Y . , 1963, p 219.
amide reacts in ammonia with more than two equivalents of sodium, giving, inter aka, benzyl alcohol and dihydrobenzamide. N o meaningful results were obtained from titrations of heptasulfur imide and thiosemicarbazide; these compounds probably underwent reduction. Triphenylmethane gave no end point, it apparently reacts too slowly with the ammoniacal electron. Attempts were made to titrate solutions of AgI and PbIz in hopes of observing end points corresponding to the formation of AgNH2, Ag(NH&-, etc. However, completely erratic potentials were measured. In each case, dark precipitates formed; in the case of the Agl titration, this precipitate appeared to be metallic silver.
RECEIVED for review October 5 , 1970. Accepted January 7, 1971. This work was supported by the U. S. Atomic Energy Commission.
Adsorption Elution Chromatography of Alkylbenzenes on Alumina Milan Popl, JiH Mosteck?, Vladimir Dolansk?, and Merislav Kurai Department of Synthetic Fuel and Petroleum, Institute of Chemical Technology, Technickci 1905, Praha 6, Czechoslovakia A description is given of the separation of monoalkylbenzenes by means of adsorption elution chromatography using alumina-n-pentane and aluminamethylcyclohexane systems. Special interest was devoted to the effect of the structure of the alkyl group, the length of the side chain, branching, etc. Detection of solutes in the column eluate was by means of a UV spectrophotometer with a flow-through quartz cell. The possibility of predicting retention volumes for a given chromatographic system was studied and compared with the experimental results. A large effect on the decrease of retention volume was observed for alkylbenzenes with the alkyl chain symmetrically branched at the a-carbon atom to the ring. A good separability is shown for mixtures of n-alkylbenzenes and those branched at the a-carbon atom to the ring.
matic hydrocarbons. It has merely been stated that the steric hindrance to adsorption is markedly increased by a n alkyl group branched at the a-carbon to the ring. This hindrance increases as a n alkyl group gets bulkier. This paper concerns the effect of a n alkyl group-its length and structure-on retention volumes of alkylbenzenes. Particular interest has been devoted to CI1 alkylbenzenes, which were not studied until now. The possibility of different structures of these compounds enabled us t o investigate the effect of an alkyl group o n the behavior of the aromatic nucleus. The use of two solvents, pentane as compared with cyclohexane, ought partially to explain the differences between results reported by Snyder ( I ) and Klemm ( 4 ) .
THEEFFECr of a n alkyl substituent on the adsorbability of aromatic hydrocarbons has been studied by several workers (1-4) and found to be generally small. It was further observed that the adsorbability increases with the number of methyl groups. In the case of aromatic hydrocarbons containing only one alkyl group with different numbers of carbon atoms, the results were ambiguous. Snyder ( I ) has found for the alumina-n-pentane system that retention volumes of n-alkylbenzenes and n-alkylnaphthalenes increase with increasing length of the alkyl chain. On other hand, Klemm (4) observed that for the alumina-cyclohexane (aluminacyclohexane tert-butylbenzene) system, the adsorbability of aromatic hydrocarbons with alkyl groups containing three o r more carbon atoms was less than the adsorbability of the parent compound. Relatively little attention has been given to the effect of the structure of a n alkyl group on the adsorbability of alkylaro-
EXPERIMENTAL
+
(1) L. R. Snyder, J. Chromatogr., 6, 22 (1961). (2) N. Kucharczyk, J. Fohl, and J. Vymetal, ibid., 11, 55 (1963). (3) P. A. Estep, E. E. Childers, J. J. Kovach, and C. Karr, ANAL. CHEM., 38, 1847 (1966). (4) L. H. Klemm, D. S. W. Chia, C . E. Klopfenstein, and K. B. Desai, J. Chrontatogr., 30,476 (1967). 518
ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
Adsorbent. Alumina, Woelm Eschwege Neutral. Prior to use, it was calcined a t 400 “C and then deactivated by addition of 0.5% wt of water. The surface of calcined alumina was 90 m2/g. Column. Length 1 m, inside diameter 4 mm. Into the column filled with 15 g of dry adsorbent was introduced 0.5-1 mg of sample. Eluent flow rate was constant in all cases at 70 ml/hour. Ahead of the column was placed a guard column with active silica gel. Detector. UV Spectrophotometer SP 800 B (Pye Unicam, Ltd.) with flow-through cell, pathlength 5 mm, effective volume 0.64 ml, working at 260 nm. Eluents. n-Pentane and methylcyclohexane were first rectified and further purified by passage over a silica gel column. The purity was checked by means of a spectrophotometer. HANDLING OF DATA
Observed retention volumes, R’, were converted to equivalent retention volumes, R ” (mlig), i.e., the eluent volume required to elute the band center from the dry column divided by the weight of an adsorbent. The equivalent retention
volumes were calculated from Equation 1 :
Table I. Group Adsorption Energies Q o i and Secondary Effects q o j on Alumina (7)
where R' is retention volume in milliliters of sample required to elute the band center from a prewet column, Vois the free bed volume in milliliters, and W is the weight of adsorbent in grams. The values of relative retention volumes, R*, related to benzene, are defined as Ro of the substituted compound divided by that of benzene. The experimental values of R" were compared with values of Ro2j2calculated by Snyder ( I , 5). Retention volume of a sample can be calculated from Equation 2: log Ro2h= log V,
+ a(S0 - As€') + Aeas
(2)
where V , is the adsorbent surface volume, a is the adsorbent activity, So is the sample adsorption activity, A , is the molecular area of adsorbed molecule, E" is the solvent strength parameter, and Aeas is the term of secondary adsorption effect. F o r n-pentane as solvent, E" is defined as zero, and the term Aeas can be ignored. Equation 2 then gives: log
Roth =
log V ,
+ aso
(3)
I n the case of alkylbenzenes and the system alumina-n-pentane, the term So can be rewritten as follows:
(4) where Qoi is the dimensionless free energy of adsorption of 3
a sample group i and Cq",includes any interaction between two groups i a n d j in the sample molecule. Substituting this expression for So into Equation 3 gives:
Further procedure consists of calculation of the parameters Vu and a from R" values determined for several compounds with the known value So. In this work for calculation of a and log V,,, the experimental values of R" for benzene, n-amylbenzene, and n-decylbenzene have been used. It has been found that a = 1.7289 and log V , = -3.174. Snyder ( I ) also reported Q o Z and q",,values which enable calculation of Roih values for a given alkylbenzene by insertion into Equation 4. I n Table I some values of Q o Z and q o j are presented. If a n eluent other than n-pentane is used, R o l hvalues are calculated according to Equation 2, including the term A s € " . Values of A , were tabulated (6) for a great number of compounds as well as E" values for some solvents (5, 7). F o r methylcyclohexane as eluent, the term Aeas can be neglected. RESULTS AND DISCUSSION
Data given in Tables 11-V show considerable differences for the alumina-n-pentane system among individual types of monoalkylbenzenes. Differences between values R o,h and R" are given in Tables 11-VI1 as the term ACnlcd = ( R o t hR") X 100. The series of monoalkylbenzenes with straight chain alkyls is presented in Table 11. Comparison with cor( 5 ) L. R. Snyder, "Principles of Adsorption Chromatography." Marcel Dekker, New York, N. Y . . 1968. (6) L. R. Snyder and H. D. Warren. J . Ckromntogr., 15, 344 (1964). (7) L. R. Snyder, ibid.,16, 55 (1964).
Group QOi Aliphatic carbon (for saturated eluents only) 0.020 Aromatic carbon 0.31 Methyl substituent on aromatic Alkyl is0 branch: Isolated from aromatic ring ... Adjacent to ring ... Naphthene ring closure ... Does not include Qci for one aliphatic carbon. . . I
4Oi
... ... 0.04" -0.045 -0.13 0.08
Table 11. Retention Data for n-Alkylbenzene (Alumina, 0.5 wt 2 H 2 0 ; eluent, rz-pentane) R" R'th &aledl Solute R* ml/g ml/g 1.00 Benzene 1.10 1.10 0 Ethylbenzene 1.17 1.29 I .29 0 rz-Propylbenzene 1.28 1.41 1.40 -1 n-Butylbenzene 1.37 1.51 1.51 0 n-Amylbenzene 1.49 1.64 1.64 0 n-Heptylbenzene 1 ,73 1,90 1.92 +1 n-Octylbenzene 1.91 2.10 2.08 -1 2.06 2.27 2.44 +7 n-Decylbenzene
x
Table 111. Retention Data for Alkylbenzenes Symmetrically Branched a t the a-Carbon Atom to the Ring (Alumina, 0.5 wt %, H 2 0 ; eluent, rz-pentane) R", Rat>,, h o d , Solute R* ml/g ml/g Benzene 1 .oo 1.10 1.10 0 Toluene 1.25 1.38 1.40 +1 Isopropyl benzene 0.91 1.00 0.83 - 17 3-Phenylpentane 0.73 0.80 0.98 +23 4-Phenylheptane 0.68 0.75 1.15 +53 5-Phenylnonane 0.65 0.72 1.34 +86
x
responding values of Snyder ( I ) shows that our increases in relative retention volumes, R*, with chain length are about double, which indicates the higher activity of the adsorbent used in these experiments. Retention volumes of straightchain monoalkylbenzenes increase with carbon number linearly up to the Cs-alkyl group. The experimental R" values are in good agreement with R o i h values calculated from contributions given in Table I. I t confirms that with known Vu and a values for the alumina-n-pentane system, R" can be predicted with sufficient precision. Toluene differs from other members of this series and is not included in Table 11. These results further show that for n-pentane as eluent, the n-alkyl group is adsorbed o n the surface of alumina in a coplanar position with the adsorbed ring also planar to the surface. I n this way the total adsorption energy of n-alkylbenzenes is increased. I n Table 111 are given results for alkylbenzenes with the alkyl group symmetrically branched at a-carbon atom t o the ring. Toluene, which has two hydrogen atoms instead of two alkyl groups at the a-carbon atom, is taken as the first member of this series. The R" values of these compounds rapidly decrease with increasing carbon number of the alkyl group up to C5. Scrutiny of Table 111 shows that the calculation of retention volumes, R ot h , from the contributions given in Table I is not reliable and experimental values of Ro are lower. Branching of a n alkyl group at the a-carbon atom to the ring represents a considerable steric hindrance t o the ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
519
Table IV. Retention Data for Monoalkylbenzenes Cll (Alumina, 0.5 wt H20; eluent, n-pentane)
Solute
R*
Benzene tz- Amylbenzene Cyclopentylbenzene 1-Phenyl-3-methylbutane 1-Phenyl-2-methylbutane Neopent ylbenzene 2-Phenyl-3-methylbutane 2-Phenylpentane rert-Amylbenzene 3-Phenylpentane
1.00 1.49 1.52 1.44 1.21 1.16 0.90 0.87 0.85 0.73
R",
Roth,
h c d ,
ml/g 1.10 1.64 1.67 1.58 1.33 1.28 0.99 0.96 0.94 0.80
mlig 1.10 1.64 2.25 1.37 1.37 1.37 0.98 0.98 0.98 0.98
?4 0 0
$35 -13 $3 $7 -1 $2
$4 +23
Table V. Retention Data for Alkylbenzenes (Alumina, 0.5 wt H 2 0 ; eluent, n-pentane) R", R'th, Acrtlod, Solute R* ml/g ml/g Benzene 1.00 1.10 1.10 0 Isobut ylbenzene 1.26 1.39 1.27 -9 tevt-Butylbenzene 0.99 1.09 0.90 -17 see-Butylbenzene 0.90 0.99 0.90 -9 Cyclohexylbenzene 1.56 1.72 2.44 +42 m-Xylene 1.65 1.50 1.78 +8 m-Diisopropylbenzene 0.73 0.80 0.63 -21
z
z
Table VI. Retention Data for n-Alkylbenzenes (Alumina, 0.5 wt H 2 0 ; eluent, methylcyclohexane) R", R"ti,, ACaicd, Solute R* ml/g ml/g Benzene 1 .00 0.87 - 12 0.99 Ethylbenzene 1.27 1.26 0.96 - 24 rz-Propylbenzene 1.19 1.18 1.03 - 13 17- Butyl benzene 1.15 1.14 1.10 -4 12- Amylbenzene 1.27 1.26 1.18 -6 rz-Heptylbenzene 1.34 1.33 1.35 +2 ri-Octylbenzene 1.38 1.37 1.45 +6 ri-Decylbenzene 1.51 1.49 1.67 +I2
z
z
Table VII. Retention Data for Alkylbenzenes Symmetrically Branched at the a-Carbon Atom to the Ring (Alumina 0.5 wt H20; eluent, methylcyclohexane)
z
Solute Benzene Toluene Isopropylbenzene 3-Phenylpentane 4-Phenylheptane 5-Phenylnonane
R*
1 .oo 1.40 0.92 0.68 0.60 0.59
R",
Roth,
ml/g 0.99 1.39 0.91 0.67 0.59 0.58
ml/g 0.87 1.06 0.61 0.70 0.81 0.93
Acalcdr
z
- 12
- 24 - 33 +5
+37 +57
adsorption of a solute on the surface of alumina. This is likely due to free rotation around the a-carbon atom-benzene ring bond, and consequently the branches of the alkyl group can retard the process of adsorption. A relatively small decrease in R" with extension of the alkyl group branches can be qualitatively understood from a steric model of 4-phenylheptane which shows that the branches of the alkyl groups can approach each other so that rotation around the C (phenyl)C(alky1) bond does not significantly increase the radius, which both are circumscribing. An alkyl group branched at the a-carbon atom cannot become coplanar with the adsorbed benzene nucleus; however, this fact does not seem to be the only reason for the observed steric hindrance. If so, one could 520
ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
Figure 1. Equivalent retention volume, R", us. number of aliphatic carbon atoms for n-alkylbenzenes and alkylbenzenes symmetrically branched at the a-carbon atom to the ring 0 +Pentane eluent A Methylcyclohexaneeluent
assume that one branch of a n alkyl group would be coplanar while the other protruded into the bulk of the mobile phase, and for unsymmetrically branched alkyl groups, it would be possible to suppose that the longer branch is adsorbed. However, the observation in Table IV including all saturated CI1-monoalkylbenzenes shows that the low values of R" for symmetrically branched alkyl groups cannot be explained only by adsorption of a part of the alkyl groups. If it could, the value of R" for tert-amylbenzene should have been equal to that for 3-phenylpentane and the corresponding value for 2-phenylpentane should have been higher, which evidently is not the case. The next results in Table IV show that the crucial factor influencing the adsorption is the position of the carbon atom with regard to branching of the chain. The branching at the P-carbon atom has no substantial effect for decreasing of R" values, even for such structures as neopentyl groups. Cyclopentylbenzene has the same retention volume as namylbenzene, which is in contradiction with the results of Snyder ( I ) , who reported that alkylation by a naphthene ring markedly increases retention volumes of alkylbenzenes. This result confirms the R" value for cyclohexylbenzene, which lies between R" values for n-amylbenzene and n-heptylbenzene, as shown in Table V. The values of R" for butylbenzenes given in Table V confirm the effect of alkyl chain branching at the a-carbon atom. The example of the pair m-xylenem-diisopropylbenzene shows that this effect on the decrease of R" values applies also for dialkylbenzenes. The results obtained for the system alumina-methylcyclohexane are given in Tables VI and VII. These results demonstrate first of all that the use of methylcyclohexane as a stronger solvent substantially decreases the differences among retention volumes of individual n-alkylbenzenes. Thus the inconsistencies in the values reported by Klemm ( 4 ) and those measured by Snyder ( I ) can be partially explained. The results in Table VI show that all n-alkylbenzenes in the range C8-Cll have approximately the same values of R" and slightly increasing values of R" for longer alkyl chains. From data presented in Table VI1 it is evident that for npentane as eluent, retention volumes of alkylbenzenes symmetrically branched at the a-carbon atom also decrease
with alkyl group size. This decrease, expressed in the values of relative retention volumes, R*,is practically the same for both eluents. The values of equivalent retention volumes, R O , for methylcyclohexane as eluent are lower, which is in accordance with the higher solvent strength in comparison with n-pentane. I n Figure 1 are plotted the values of log R o for n-alkylbenzenes and alkylbenzenes symmetrically branched at the a-carbon atom against the number of aliphatic carbon atoms for the two eluents, n-pentane and methylcyclohexane. The R“ values for the rest of the alkylbenzenes which were measured lie between the curve for n-alkylbenzenes and that for alkylbenzenes symmetrically branched at the a-carbon atom. Figure 1 shows very good separation of both types of alkylbenzenes for alkyl chains containing five or more atoms. It is evident that for methylcyclohexane as eluent, both curves approach one another closely, and the separation of alkylbenzenes of different structure becomes more difficult when using a more polar solvent. By means of relations suggested by Snyder ( I , 5 ) for calculation of theoretical retention volumes, Ro for methylcyclohexane, the value eo = 0.01 was used, offering the best results for the series of n-alkylbenzenes. The calculated Rothvalues (see Tables VI and VII) show poorer agreement of experimental and theoretical data for methylcyclohexane than for n-pentane as eluent. However, the results given in Table VI1 confirm conclusions drawn from Table 111,
namely that for alkylbenzenes symmetrically branched at the a-carbon atom, the calculated values are always substantially higher than experimental ones. It might be noted that the method for calculating retention volumes suggested by Snyder ( I , 5) gives very good results in most cases, especially for the system alumina-n-pentane. It seems possible to achieve a good correlation of experimental data by this method, whereas other data such as equivalent retention volumes, R O , or relative retention volumes, R*, depend so much on the sort of alumina and its activity that without the specification of alumina by means of a and V, values, they give only information about the order of eluted solutes. Substantial deviations were observed for alkyl groups symmetrically branched at the a-carbon atom to the ring. In this case the experimental R o values were lower than corresponding R o I hvalues calculated by means of the mentioned method. It seems to be proved that alkyls of this type represent a considerable steric hindrance to adsorption of a n aromatic nucleus. Another discrepancy has been found for cycloalkyl groups, where Ro values were practically identical to those for corresponding n-alkyl groups. These results are in contrast to those of Snyder ( I ) , who found substantially higher values. In this case, probably, a different q o j value for a naphthene ring closure is required. RECEIVED for review October 29, 1970. Accepted January 12, 1971.
Radioisotope Derivative Procedure for Determination of Epinephrine or Norepinephrine W. J. Blaedel and T. J. Anderson Department of Chemistry, University of’ Wisconsin, Madison,
Wis.53706
Epinephrine (E) and norepinephrine (NE) are quantitatively and stoichiometrically converted to labelled iodoaminochromes with high specific activity l25I. Inactive iodoaminochrome of the sought-for catecholamine is added, and the activity purified and isolated by column chromatography. E is determinable in the 0.01-1-pM range, and NE in the 0.1-1-pM range, relative precision being 1-2% at midrange. After an alumina adsorption step, the procedure is applicable to the determination of NE in urine. E cannot be determined in urine because of interference by dopamine.
Survey of Methods. There has been much interest for over fifty years in the analysis of epinephrine (E) and/or norepinephrine (NE) (Figure 1) in biological samples ( I ) . Current interest in NE analysis centers around NE’S function as a neurotransmitter in the sympathetic nervous system and its role in brain function and emotional states (2-6). (1) K. Engelman, B. Portnoy, and W. Lovenberg, Amer. J. Med. Sci., 225, 259 (1968). (2) M. Vogt, Brit. J. Pharmacol., 37, 325 (1969). (3) “Second Symposium on Catecholamines,” G. H. Acheson, Ed., The Williams and Wilkins Co., Baltimore, Md., 1966. (4) U. S. von Euler, “Noradrenaline,” Charles C Thomas, Springfield, Ill., 1966. (5) J. R. Crout, Anesthesiology, 29, 661 (1968). (6) J. H. Biel, Aldrichimica Acta, 1, 15 (1968).
Urinary levels of E can be taken as a gauge of the biological activity of the adrenal medulla (5), and greatly increased levels of N E or both compounds indicate a pheochromocytoma, a tumor of the pheochrome system (7). Until recently, the only chemical methods of analysis with sufficient sensitivity and specificity to analyze body fluids for E (0-88nM in urine and about 1 n M in plasma) and for NE (88-310nM in urine and 1-3nM in plasma) have been the ethylenediamine fluorescence method (ED method) and the “trihydroxyindole” fluorescence method (THI method) (8). The E D method is unable to accommodate urine samples because of interference by dopamine (DA), but many THI methods for urinary E or NE exist. Analysis of plasma samples has generally involved using both methods near the limits of their sensitivities ( I , 9, IO), although recent modifications have apparently improved the performance of the THI method (11). Differentiation (7) R. Straus and M. Wurm, Amer. J. Clin. Path., 34, 403 (1960). (8) W. M. Manger, 0. S. Steinsland, G. G. Nahas, K. G. Wakim, and S. Dufton, Clin. Clzem., 15,1101 (1969). (9) S. Udenfriend, “Fluorescence Assay in Biology and Medicine,” Academic Press, New York, N. Y . ,1962 p 149. (10) L. Martin and C. Harrison, A d . Biochem., 25,529 (1968). (11) J. O’Hanlon, Jr., H. C. Campuzano, and S. M. Horvath, Anal. Biocliem., 34, 568 (1970). ANALYTICAL CHEMISTRY, VOL. 43, NO. 4, APRIL 1971
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