Elution Capacity of Organic Substances in Solution-Adsorption Chromatography M. JEROME SABACKY, LEE
B. JONES,
HARLAN D. FRAME, Jr., and HAROLD
H. STRAIN
Argonne National laboratory, Argonne, 111.
b The effectiveness of various solvents and solutions as eluting agents in solution-adsorption chromatography has been investigated using a standard magnesia column and p-carotene as sorption indicator. The distribution of several eluting agents relative to the solvent and the indicator carotene was also determined. Curves for R value of the carotene vs. concentration of eluting agent are presented. The eluting power of the organic solvents tested apparently is not consistently related to their dipole moments or dielectric constants. The eluting power of a particular structural group varies with its location in the molecule, as in isomeric substances, and with the nature of the molecule in which it occurs, as in a homologous series. Certain substances behave as eluting agents at low concentration and as displacing agents at higher concentration. The observations indicate a complex elution mechanism involving two dynamic equilibria: a competition for the sorbent surface among the eluting agent, the species to be separated, and the solvent; and, a competition among these substances for the newly-formed adsorption layer.
I
N SOLUTION-ADSORPTIOS chromatog-
raphy, the solvent system plays several interrelated roles. On the basis of formal relationships, i t dissolves the substances to be separated; it exhibits differential solution affinity for these substances; and through sorption by the adsorbent, it displaces the substances to be separated (3, 4). With a given sorbent, the solvent system determines the relative rate ( R value) a t which substances migrate through adsorptive paper or chromatographic columns (1,S, 4,8,12-19). Numerous experimental observations indicate that the effect of single solvents and solutions on sorbability is incompletely understood. For example, the classification of solvents with respect to their eluting power is subject to great variation. Specifically, there is one elution series of solvents for lipides on alumina ( 1 7 ) , another series for various substances on magnesia (14), and yet another for carotene adsorbed on hydrated lime ( I ) . I n one series of sol306
ANALYTICAL CHEMISTRY
vents, the sorbability and eluting pon er are related to dielectric constant ( 7 ) , and, in others, to dipole moment (3, 4, 18), but there are numerous exceptions (3, 12) * The adsorption affinity of organic substances and, presumably, their eluting power, increase with the presence of polar groups (Z-4,8,14). Numerous examples are available among the steroids, carotenoids, and chlorophylls (3, 4, 8, 11-17). The sorbability of these substances, as indicated by the chromatographic sequence, varies unpredictably with variation of the solvent (8, 12, 13'1. With variation of sorbents and solutes as 1%-ell as solvents, enormous variation of sorbability has been encountered (19). Even with similar sorbents and sorbed substances, there arc some significant irregularities in the elution effects of various solvents. For carotenoids adsorbed on magnesia, acetone and anhydrous ether are poorer eluting agents than benzene and toluene (13, l 4 ) , but for other systems, the reverse has been reported (I, 17). Aklthough methanol is usually regarded as a strong eluting agent, it has been reported as a relatively poor eluting agent for carotene adsorbed on lime ( 1 , 8 ) . The proposed relationship between the elution capacity of solvents and their dielectric constants (3, 4, 7 ) has some conspicuous exceptions. .icetone, having a dielectric constant greater than that of 2-butanol, should be more sorbed than the alcohol and, therefore, a better eluting agent ( 7 ) . Although this conclusion appears to be supported by one elution series ( I ) , i t is not supported by observations on the use of these solvents for the formation of chromatograms, as in the separation of a- and p-carotene (13, f 4 ) . Additional exceptions are found with many isomeric substances, because the position, the number, and polarity of the substituent groups are of greater influence than the dielectric constant of the molecule (3, 4,8, 12). These irregularities are especially conspicuous when the comparisons are based upon the dipole moment (3,12). To explore some of the ramifications of the eluting capacity, we have restudied the effect of various substances upon the elution of B-carotene adsorbed
in columns of activated magnesia. The action of single solvents and of solutions of various eluting substances r a s investigated. Separate measurements of the distribution of the eluting agents in the magnesia columns Ivere also made, and the eluting poner of thc solvents n-as compared with their dielectric constants and dipole moments. The sorptive capacity of the activated magnesia varies greatly n ith its treatment. Therefore, special care n-as taken to obtain comparable results n ith each series of solvents. EXPERIMENTAL
Apparatus. Adsorption tubes (1 cm. i.d. by 24.5 cm.) Fere packed with a mixture of adsorptive magnesia (Sea Sorb 43, Fisher Adsorptive Magnesia) and heat-treated siliceous earth (Celite 545, Johns-Nanville) (1 to 2 ) . Small portions of the dry, adsorptive mixture were pressed into the tubes with a wooden dowel to form a column 20 c ~ n .tall containing 8.5 i 0.3 grams of the sorptive mixture. These columns lvere operated with suction, from a water aspirator, which rras regulated Kith an adjustable bypass so that the pressure was aliout one half atmosphere. Procedure. For carotene solutions in petroleum ether (b.p. 20" to 40" C.), 10 mg. of p-carotene were added to 100 ml. of solvent (ca. 0.0002M), and the mixture \vas stirred until the carotene dissolved. For solutions of eluting agents, the quantity of the eluting agent required for a 0 . l M solution plus 10 mg. of $-carotene n-ere added to 100 nil. of petroleum ether. This mixture IVXS qtirred with a magnetic stirrer until the carotene dissolved. A portion of the solution was employed for the 0.LU adsorption t,est. The remainder n-as treated with enough eluting agent t,o bring the concentration to about 0.2JL. In this way, concehtrations were increased to 1.11, if permitted by solubility. The resulting decrease in carotene concentration nas insignificant. The solutions vere poured into the columns until the solvent front reached the bottom. The temperature Fas 24' i 2" C. The R value of t'he carotene was then recorded. For locating the eluting substances relative to carotene, the columns were examined in various ways. With fluorescent eluting agents, the zones were located in ultraviolet light. With
nonfluorescent substances, the columns with the adsorbed carotene were washed with petroleum ether, and successive effluent fractions usually 5 ml., were collected and analyzed. With nonvolatile eluting agents, the petroleum ether was evaporated from these fractions using a current of air, and the residues were weighed. With volatile hydroxy compounds, such as methanol] the presence of the eluting agent in t h e effluent fractions was determined by infrared analysis.
o f Adsorbent-
-Top
Gorotene ----adsorbed solution
(0) (Y)
-
concentroted (0)
\
Alcohol
Fron
-
RESULTS
Appearance of
Columns.
When sorbed from petroleum ether alone, the carotene formed a very narrow zone (1 t o 2 mm.) at t h e t o p of t h e column. K i t h certain other solvents, t h e carotene usually formed a uniform zone varying from t h e narrow one observed with petroleum ether to the very long or wide ones observed when the carotene n as not sorbed. When eluting agents a ere addcd to the carotene solution, the appearance ot the carotcnc zone varied with the eluting agent and with its concentration. K i t h substances of weak elution capacity, such as benzene, the carotene formed a T'ery uniform zone, and the eluting agent cxtwded from the top of the column through the carotene zone and nixnrly to the solvent front (Figure 1, A ) . K i t h substances of moderate elution capacity employed a t low concentration. the carotene usually formed a uniform zone like those obtained with single solvents, but tvith the mild eluting agents a t higher concentration, the carotene yielded a n irregular zone with a narron, deep orange region belon the principal yellon zone (Figure 1, 13). -it these higher concentrations, the eluting agent did not extend below the narrow, concentrated carotene zone K i t h strong eluting ngmts, most of the carotme f o r n i d a sloirly migrating] concentrated zone. d t all concentrations tested. tliesc Strong eluting agents never extended below the narrowv,intense pigmcnt zone. Th(s significance of the R value of carotene must vary n ith the appearance of the columns. I n columns with wide, uniform zones of the adsorbed carotene, the R value should provide a measure of the elution capacity of the solvent. But with narro\v zones belon a very lightly colored section, the R value of th(3 carotene indicates the R value of the eluting agent. The carotene itself may be virtually nonsorbcd in the zone of the eluting agcnt but very strongly sorbed by the magnesia just below the front of thc eluting agent. Yery strong eluting agcnts may produce carotene R values lcss than those yielded Ly substances of moderate elution capacity. Indeed, for substances that displace carotene, the smaller the R value, the greater the sorbability and the eluting
Hyd rocor bon
Petroleum Ethe
Front
Front
I I
B Figure 1. Distribution of carotene in columns of activated magnesia A. Uniform distribution from solutions in petroleum ether plus aromatic hydrocarbons E. Irregular distribution from solutions in petroleum ether plus alcohols (V, yellow; 0, orange)
power. For substances that migrate past carotene, the smaller the R value, the weaker the sorbability and the eluting power. Activity of Magnesia. T h e adsorption capacity of t h e magnesia varied greatly with t h e conditions t o which i t was exposed. For example, its adsorption capacity for carotene, adsorbed from 0.1M methanol in petroleum ether, was reduced t o a small fraction of the initial activity (from R = 0.16 to R = 0.66) when a shallow layer (about 0.5 cm.) was permitted to stand overnight in an airconditioned room. For this reason, the magnesia was mised Rith the Celite in stoppered bottles, and the misture was preserved in screw-cap bottles.
Table
I.
R Values. T h e R values of pcarotene dissolved in dissimilar solvents are presented in Table I along with dielectric constants and dipole moments (8, 6, 9, IO). All alcohols, pyridine, tetrahydrofuran, aromatic ethers, esters, and ketones, primary and secondary amines, aromatic hydrocarbons] and various nitro compounds eluted the carotene completely ( R = 1 ) . The R values of carotene dissolved in petroleum ether plus various eluting agents are shon-n in Figures 2 to 12. Substances of similar composition and structure are grouped together, permitting a convenient comparison of molecular structure, eluting capacity a t various molecular concentrations, and dielectric constants. Additional substances tested are reported in connection with the comments on each figure. K i t h aromatic hydrocarbons (Figure 2 ) , the curve for toluene was nearly identical with that for benzene. T h a t for mesityltne was barely above that for D-limonene. The slight solubility of phenanthrene limited the determination to a single solution. With all these eluents, the carotene formed TI ide, uniform zones, and as shon-n by analyses with napthalene, the hydrocarbons extended belon the carotene zonc . Curves for chlorobenzenes arc' much alike. The p-dichlorobenzene ( D b p = 2.41) yielded a curve identical with that shown for thp nieta isomer in Figure 3. Chloroform (&o = 4.9) yielded a curve very similar to that of chlorobenzene. As all these chloro compounds produced uniform carotene zones, the chloro derivatives are prcsumed to have advanced ahead of the carotene as shown by analyses of the effluent when p-dichlorobenzene n-as used as the eluent. Curves for various ethers and etherlike compounds, as pyridine (6), are presented in Figure 4. The curve for diethyl ether (D200= 4.3) !vas identical with that for benzene (Figure 2 ) , thus indicating very weak elution properties. Tetrahydrofuran eyhibited
Effect of Various Solvents on the
R
Value of /%Carotene
Solvent Ra D(0 C.)* p X 10'' ( " K.)' Petroleum ether (b.p. 20-40") 0.02 n-Hexane 0 .os 1.89(20) O( 337-484) 0.03 1.92(20) O( 348-501 ) n-Heptane 0 ... 0.03 1.99(20) Decane 0 ... 2,2,4Trimethylpentane 0.03 Cyclohexane 0.04 2.02(20) Carbon tetrachloride 0.11 2.24(20) 0( 296-368) 0.23 4.34(20) 1.15(289-455) Diethyl ether O( 325-489) Carbon disulfide 0.30 2.64(20) 0.48 2.42(25) 0.66(373-453) Triethylamine 2,88(301-455) Acetone 0.84 20.7(2 5 ) 1 .oo 2.28(20) O( 326-480) Benzene a R value of solution containing 10 mg. p-carotene per 100 ml. Dielectric constant of solvents from reference tables ( 3 , 6, 9, 10). e Dipole moment, p X lo1*e.s.u., from tables (5, 6, 9, 10).
VOL. 34, NO. 3, MARCH 1962
307
0.4
I
0.3
1
c 0.5
0.5
Naphthalene
0.4 Diphenylmethane
cc
K
0.3 r d- Limonene D20e 2 36
0 .I
0.1
0.2
I
I
I
I
I
I
I
0.8
0.9
-
Benzene I
n "
0
I
I
1
I
Figure 2.
I
0.2 0.3 0.4 0.5 0.6 0.7 0.8
0.1
"
"
"
"
1.0
Effect of aromatic hydrocarbons on R values for carotene
weak elution properties ( R = 0.11 a t 0.39X and 0.25 a t 0.77Ji), especially as compared to pyridine. At the lowest concentration (O.IM), the aromatic ethers provided uniform carotene zones. Separate tests revealed the p-dimethoxybenzene below the carotene zone. At higher concentration (0.431 for p-dimethosybenzene), the ethers displaced much of the carotene and remained behind the narrow carotene zone. Curves for various nitro compounds, Figures 5 and 6, indicate great variation in elution capacity. The solubility limited the concentration ranges for p-nitrotoluene and 2-nitro-2-methylpropane. The curve for I-nitropropane (Dao0 = 23.2) was below that of nitroethane (Figure 6) a t the lower concentrations but was equal to i t a t the two highest concentrations. The curve = 25.5) was for 2-nitropropane significantly lower than the curve for the 1-nitro isomer. The 2-nitro-2methylpropane (nitrotertiarybutane) exhibited remarkably weak elution capacity. At the higher concentrations, the nitrotoluenes, nitrobenzene, nitroethane, and nitromethane produced rotene irregular distribution of the
/
0.9
1-
0.8
0.6 y r i d i n e D2g.' 12.5
0.6
0.4 0.5
I
K
0.5
1
2-
1 -
Dirnethoxybenzene
-
i
-
I
0.1
0
0.2
0.3
0.4
I
MOLARITY
Figure 4.
I
I
I
I
0.5 0.6 0.7 0.8 0.9 OF
1.0
ANALYTICAL CHEMISTRY
-
D20. = 27.4
// /
t
p//
0.4 L
0.3
/I
I 0.2 0.1
I ,-I
t dl
0
0.1
ELUENT
Effect of aromatic ethers on R values for carotene
1.0
1
23.8 \
p-Nitrotoluene Dae. = 22.2
0.7
0.7 ELUENT
0.6
cohols is shon-n by Figures 9 and 10. n-.imyl alcohol (Dpja = 13.9) yielded a curve nearly identical n ith that for n-butyl alcohol, Figure 9. Isoamyl alcohol (Dzp = 15.3) provided a curve identical with that for the isobutyl alcohol, Figure 10. With each of the alcohols, the carotene was concentrated into a narrow zone. With methanol, no hydrovy compounds were found below the carotene zone as measured bs; infrared absorption analysis. The methanol appeared with the carotene when the latter was washed through the column, 1-Heladecanol, determined by evaporation of the solvent, also follon-ed the carotene into the percolate. Primary amines. the nitrogen analogs of alcohols, and tertiary amines, the analogs of ethers (a), exhibit great variation of the elution capacity as indicated by Figure 11. Of these amines, only aniline caused the carotene to form a narrow zone. Xitriles, the nitrogen analogs of the carboxylic acid anhydrides (a), also exhibited great variation in their elution properties, Figure 12. At higher
/ K t i N - & D2y
l
0.3 0.4 0.5 M O L A R I T Y OF
Effect of aromatic chlorocompounds on R values for carotene
Figure 3.
in the columns. The other nitro compounds produced uniform zones. Curves for a series of ketones are presented in Figure 7 . The curve for methyl ethyl ketone (Dzija = 18.5) was identical with that for acetone. The aliphatic ketones produced wide, uniform zones of the carotene. hcetophenone a t concentrations of 0.1 and 0.2V produced a uniform carotene zone. At concentrations of 0.4 to l . O M , the acctophrnone yielded a narrow carotene zone. Cinnamalacetophenone, saturated a t less than O.O52f, produced a narrow carotene zone. As shown by the fluorescence, this ketone extended from the narrorr carotene zone to the top of the column. Benzalacetophenone, also less than 0.05.V, produced a similar effect. The elution capacity of various esters is summarized in Figure 8. Except for dimethyl phthalate, the esters produced uniform carotene zones. The isomers of dimethyl phthalate were not sufficiently soluble in petroleum ether. The elution capacity of various homologous and isomeric aliphatic al-
rn;Dimethokyban\ene
'
0.7 Ir = I
0
OF E L U E N T
MOLARITY
308
I
..-
0.2
0.3
I
Figure 5.
0.2
I
I
I
I
1
1
0.3 0.4 0.5
0.6 0.7 0.8
MOLARITY
ELUENT
OF
I
0.9
1.0
Effect of nitrotoluenes on R values for carotene
,
0.8 r
I
I
1
1
0.5
-
i
T
'
0.6
IIT
0.4
1
i
Dee.; 39.4
1
i
I
0.3
-
1
0.2 r 0.1
2- N i t r o - 2 - M e t h y l propane
1
i
Acetophenone
0
07 -
K1l 0.6
DzO.= 10.3
0.2
0.1 00
I
0.1
0
Figure 6.
0.2
0.3 0.4 0.5
0.6 0.7 0.8 0.9 .1.0
MOLARITY
ELUENT
OF
Effect of nitro compounds on carotene
R
values for
conccntrations, both acetonitrile and bmzonitrile produced irregular distribution of the carotene in the columns. The elution effect of N-methylacetamide was very small, R = 0.02 a t 0.1 and 0.2-$1. The effect of N,N-dimethylacetamide was relatively large, 0.40, 0.50, and 0.58 at0.1,0.2, and 0.4M. iit the highest concentration, the dimethylacetamide caused the carotene to form a narrow zone.
0.1
Figure 7.
The elution of carotene sorbed on activated magnesia is a complex phenomenon. -4s shown by Table I, certain nonpolar solvents, such as petroleum ether, permit the strongest adsorption (or weakest elution) of the carotene. Weakly polar substances, such as benzene, increase the elution of the carotene. Ether and acetone appear as weaker eluting solvents than benzene, in support of the series reported by Strain (14) and contrary to the conclusions of Lederer and Lederer (8). In solution in petroleum ether, however, benzene and ether, Figure 2 , show nearly idrntical elution properties. Acetone in dilute solution is also virtually identi-
0.2
0.3
0.4 0.5
I
I
I
I
I
Dimethvl P h t h a l a t e
i1
1
1
D20.
5
0.8 0.9
1.0
R
values for carotene
Alcohol]
-
0.7 0.6 0:
0.5 Methyl Alcohol
0.4
Ethyl Acetate
a 0.3
0.7
ELUENT
',,Proby1
Hexodeconol - I
1
-
0.6 -
0.4
I
21.4
solution are weaker eluting agents than the same substances as pure liquids. Compare benzene in Table I and Figure 2, acetone in Table I and Figure 7 . Elution capacity increases with ring formation and with unsaturation, Table I, Figure 2. I n a series such as benzene, napthalene, and phenanthrene, the elution capacity increases with the number of condensed rings, and in this series, it increases rapidly n ith increasing dielectric constant. But n ith a modification of structure without condensed rings as in diphenylmethane, the relation between elution capacity and the dielectric constant is irregular. With chlorobenzenes, Figure 3, the elution capacity varies with the number of chlorine atoms and with their positions in the molecule. Comparison of carbon tetrachloride in Table I and the chlorobenzenes in Figure 3 s h o w that substituted chlorine per se has little effect upon the elution capacity. It is the molecular unit formed by the substitution of the chlorine that determines the elution properties. With the isomeric dichlorobenzenes, there is no
0.8 1
0.6
M O L A R I T Y OF
I 1
i
1
1
Effect of ketones on
cal with benzene, but a t higher concentration, it is a slightly better eluting agent, Figures 2 and 7 . As Bickoff ( 1 ) compared these two substances a t the same percentage concentration, and as the acetone has a smaller molecular weight than benzene, his molecular concentration of acetone was greater than his molecular concentration of benzene by nearly a third. I t is clear from Figures 2 to 12 that the elution capacity is a function of concentration. Moreover, the distribution of the carotene in the columns also varies with concentration of the eluent. There is, therefore, no simple basis and no preferred concentration for comparison of the eluting capacity of various substances in solution. V i t h the dissimilar solvents in Table I, the elution capacity is not systematically related to dielectric constant or dipole moment. Even with many similar compounds, as shown in the figures, there is also no obvious relation among these three properties. As expected, substances in dilute
DISCUSSION
Dzoe I
6.4
0.3
Ethyl Propionate
t
1
0. I 0
D2y =
0
0.1
0.2
0.3
0. I
5.2
0.4 0.5 0.6 0.7 0.8 0.9
M O L A R I T Y OF E L U E N T
Figure 8.
Effect of esters on
R
0
0.1
1.0
values for carotene
0.2
0.3 0.4 0.5 MOLARITY
Figure
9.
OF
0.6 0.7 0.8 0.9
1.0
ELUENT
Effect of normal, aliphatic alcohols on R values for carotene VOL. 34, NO. 3, MARCH 1962
309
c I
0.9
1
I
I
I
I
I
I
I
I
I
/
Propyl Alcohol
"
"
'
I
N, N-Dimethylanilins D20.= 4.91
0.7 0.6
=
"
t
Aniline
J a 0.4 0'5!
I
d I
I
I
I
I
0
0.1
0.2
0.3
0.4
0.5
0.3
MOLARITY
Figure 10.
0
I
I
o.2 0.I
I
0.9
1.0
OF E L U E N T
ANALYTICAL CHEMISTRY
r1 - - 7 Benzonitri 11 020.,= 26.5
0.i
/":
01 0.1
4 I
0.2 0.3 h l 0 LA R i T Y
Figure
12.
0.4 0F
0.1
0.2
0.3 0.4 0.5 0.6 MOLARITY OF ELUENT
0.7
0.8
Figure 1 1 . Effect of amines on R values for carotene
plus acetone, Figure 7 , would not be nearly so effective as acetophenone. The elution capacity of esters, Figure 8, resembles that of ketones. Aliphatic esters are rather poor eluting agents. The aromatic ester, dimethyl phthalate, is an excellent eluting agent. Alcohols, Figures 9 and 10, are strongly sorbed by magnesia. At all concentrations, they displace completely the adsorbed carotene. The R values are those of the alcohols themselves, not the R values of the displaced carotene. From this standpoint, the sorbability of the alcohols increases with decreasing R value. Methanol is, therefore, the most sorbed alcohol, and presumably the best eluting agent among the aliphatic alcohols. For methyl, ethyl, and propyl alcohols, the sorbability increases with increasing dielectric constant, but for butyl and hexadecyl alcohols, this relationship does not apply. I t follow from the curves that the sorbability of the lower alcohols is not equivalent vith
0.9
t 0
Effect of iso-alcohols on R values for carotene
apparent relation between elution capacity, dielectric constant, or dipole moment ( p for ortho a t 445-5'23' K. = 2.52; 1 for meta a t 413' and 458' K. = 1.72; p for para a t 434' K. = 0). Diethyl ether exhibits weak elution properties. I n the elution series of Table I, it occupies the same position relative to benzene and acetone as reported before by Strain (14). Kow, however, diethyl ether appears as a slightly poorer eluting solvent than carbon disulfide. With respect to the methoxy groups, the dimethoxy benzenes exhibit remarkably great elution capacity, Figure 4. As with the chlorobenzenes, the elution capacity is determined largely by the molecular skeleton and by the functional groups. The symmetrical p-dimethoxybenzene,, presumably with no dipole moment, is just as effective an eluting agent as the less symmetrical ortho and meta isomers. None of these ethers has an enolizable or dissociable hydrogen atom to which the elution capacity may be ascribed. The nitrocompounds, all of which have high dielectric constants, Figures 5 and 6, exhibit great variation in the elution capacity. The elution properties are not directly proportional to the dielectric constant, to the presence or absence of replaceable hydrogen on the carbon atom attached to the nitro group, or to the size of the molecules. With the homologous aliphatic ketones, Figure 7 , there is a systematic increase in the elution capacity with increase in the molecular weight. Here, however, the increase in the elution capacity is inversely related to the dielectric constant. Acetophenone, the aromatic counterpart of methyl hexyl ketone, is a much better eluting agent than the aliphatic ketone. This increased elution capacity exceeds that due to the CHBC(=O)-group as it occurs in aliphatic ketones and to the phenyl group as it occurs in benzene. For illustration, benzene, Figure 2, 310
I
0.6 0.1 0.8
I
1
I
0.5
0.6
0.7
0.8
E L U E NT
Effect of nitriles on R values for carotene
respect to the molar concentration of the hydroxyl group alone. With the higher alcohols, propyl to hexadecanol, the hydroxyl group exerts a more equivalent sorptive action. These observations explain the anomalous position of methanol in the elution series reported by Bickoff ( I ) and accepted by Lederer and Lederer (8). Bickoff has, in effect, reported the R value of the alcohol itself, not the R value of carotene in the presence of the alcohol. The effect of amines upon the adsorption of carotene, Figure 11, varies from that shown by the tertiary aliphatic amines, the ether analogs ( 5 ) , to that shown by the primary and secondary aliphatic amines, the alcohol analogs ( 5 ) . The butyl amines, especially a t low concentration, are poorer eluting agents than the butyl alcohols. Dimethylaniline is a slightly poorer eluting agent than the analogous dimethoxybenzenes. I t is widely believed that sorbability and elution capacity are related to the presence of labile hydrogen atoms. For example, alcohols are better eluents than ketones and ethers. Primary amines are more sorbed than the secondary amines, which are more sorbed than the tertiary amines (8). But there appear to be a number of esceptions to this principle. Nitriles, A T , X-dimethylaniline, dimethyl phthalate, phenanthrene, pyridine, aromatic ethers, and many of the nitro compounds, all without labile hydrogen, are excellent eluting agents. Aromatic compounds are much better eluting agents than the analogous aliphatic compounds. With some of these compounds, as chlorobenzrne and acetophenone, the elution capacity is greater than would be expected by summation of the elution capacity of the units forming the molecule. The effect shown in Figure 1, B reveals the conditions when carotene in the wide zone migrates faster than the eluting agent. It does not reveal
the R value of the carotene in the zone of the eluting substance, which may vary from t h a t of the eluting substance to 1. It may be established in separate experiments in which solvent plus eluting substance are added t o the column before addition of the solution of carotene plus eluting substance. At first sight, Figure 1 appears to indicate a marked distinction between eluting agents-those t h a t migrate faster than the carotene-and displacing agents-those t h a t displace the carotene and follow the displaced zone through the column. B u t the results n ith certain ethers, nitro compounds, and ketoncs show that the transition from rlution to displacement may vary n i t h concentration as well as n i t h the nature of the eluting substance. For the rlution effect, R for carotene in the presence of the eluting agent must be less than R of the eluting agent. For the displacement effect, R for carotene in the presence of the displacement a g n t must be greater than R of the displacing agent. With respect to concrntration and structure, there is a gradual transition from eluting substances to displacing substances, and no special selective elution properties may be attributed to one in preference to the other.
From these observations, one may envision a n elution mechanism in which the eluting agent and the solvent compete with carotene for the active surface of the magnesia and in which sorbed eluting agent and the dissolved eluting agent have attractive forces for carotene. Only with very strongly sorbed substances such as the alcohols, is virtually all the carotene displaced from the magnesia. The differences in the shape of the curves for different eluents indicate that the detailed elution mechanism varies from eluent t o eluent and, with many substances, from one concentration to another. LITERATURE CITED
(1) Bickoff, E. M., ANAL.CHEM.20, 51
11948). (2) Brockman, H., Volpers, F., Chem. Bey. 82,95 (1949). (3) Cassidy, H. G., “Adsorption and Chromatography,” Interscience, New York, 1951. (4)Cassidy, H. G,.: “Fundamentals of Chromatography, Interscience, Sew York, 1957. (5) Franklin, E. C., “The Nitrogen System of Compounds,” Reinhold, New York, 1935. (6) Hartshorn, L., Milligan, A. G., “International Critical Tables.” Vol. 6,81, 1929. (7) Jacques, J., Mathieu, J. P., Bull. SOC. chzm. France 1946, 94. I - -
- - I
(8) Lederer, E., Lederer, M., “Chromatography: a Review of Principles and Applications,” Elsevier, Kew York, 1953. (9) Margott, A. A., Buckley, F., “Table of Dielectric Constants and Electric Dipole Moments,” National Bureau of Standards Circular 537, June 25, 1953. (IO) Margott, A. A., Smith, E. R., “Table of Dielectric Constants of Pure Liquids,” National Bureau of Standards Circular 514, August 10, 1951. (11) Steiger, M., Reichstein, T., Helv. Chim. Acta 21,546 (1938). (12) Strain, H. H., ANAL. CHEM. 33, 1733 (1961). (13) Strain, H. H., “Chloroplast Pigments and Chromatographic Analysis,” 32nd Annual Priestley Lectures, The Pennsylvania State University, University Park, Pa., 1958. (14) Strain, H. H., “Chromatographic Adsorption Analysis,” Interscience, New Yprk, 1942. (15) Strain, H. H., IND.ENG. CHEM., AKAL.ED. 18, 605 (1946). (16) Strain, H. H., J . Am. Chem. SOC. 70,588 (1948). (17) Trappe, W., Biochem. 2. 305, 150 (1940); 306, 316 (1940); 307, 97 (1941 ~~- ~’i._ , (18) Williams, J. W., J . Am. Chem. SOC.
50,2350 (1928). (19) Williams, R. J. P., Hagdahl, L., Tiselius, A., Arkiv. Kemi 7 , 1 (1!354).
RECEIVED for review October 12, 1961. Accepted January 8, 1962. Based on work performed under the auspices of the U. S. rltomic Energy Commission.
Identification of Fluorenones in Wilmington Petroleum by Gas-Liquid Chromatography and Spectrometry D. R. LATHAM, C. R. FERRIN, and J. S. BALL Laramie Petroleum Research Center, Bureau of Mines,
b Alkyl-substituted fluorenones have been identified from Wilmington, Calif., petroleum b y API Research Project 52 and are the first aromatic ketones found in petroleum. A fraction boiling from 320” to 330” C. was reacted with sodium amide in liquid ammonia, The regenerated precipitate was separated into fractions b y gas-liquid chromatography. A low-voltage mass spectrum of one of these fractions showed the molecular weights of C1to Ca-substituted fluorenones and indicated one of them had the molecular formula of C16H120. An infrared spectrum of the fraction showed the presence of a carbonyl group. An ultraviolet spectrum of the fraction was compared with the standard spectra of fluorenone and 4-methylfiuorenone, to establish definitely the class of compound.
A
U. S. Department of the
LKYL-SUBSTITUTED
Interior, Laramie, Wyo.
FLUORENONES
have been identified in a Wilmington, Calif., crude oil by a combination of mass, infrared, and ultraviolet spectrometry on a sample obtained by distillation, chemical extraction, and gaaliquid chromatography. Fluorenones are the first aromatic ketones to be identified from petroleum. This identification was made by American Petroleum Institute Research Project 52b (Nitrogen Compounds in Petroleum) when a reagent used to extract nitrogen compounds also extracted fluorenones. Oxygen compounds in petroleum have received less study than sulfur and nitrogen compounds because they have caused fewer problems during refining operations. A review (1) has shown fatty acids, naphthenic acids, and phenol to be present in petroleum. In addition, Barton, Carruthers, and
Overton (2) identified a triterpenoid lactone from petroleum and Lochte and Littmann (9) reported the identification of several aliphatic ketones from a gas well condensate. EXPERIMENTAL
A 21-barrel sample of Wilmington, Calif., crude oil was obtained from the field for the use of various API fundamental research projects. This sample was protected from oxidation by blanketing with specially purified nitrogen gas containing less than 2 p.p.m. of oxygen. All subsequent handling of t h e fractions from this oil has been under this same protection. A portion of this oil was deasphaltened with pentane (8) and vacuum distilled (7’) t o obtain a fraction boiling from 320’ to 330” C. at a pressure of 760 mm. of Hg. A low-voltage mass spectrum of this fraction is shown in panel I of Figure 1. VOL. 34, NO. 3, MARCH 1962
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