Anal. Chem. 1988, 60, 1096-1102
1096
(18) Gianz, P.; Korner, B.;Findenegg, G. H. Adsorpt. Sci. Techno/. 1984, I , 183. (19) Chester, T. L.; Innis, D. P. M C CC,J . Hlgb Resolut. Cbromfogr. Cbromfogr. Commun. 1085, 8 , 561-566. (20) Chirnside, G. c.; Pope, c. G. J . phvs. &ern, 68, 1064. 2377-2379, (21) Kern, H.; Rybinski, W. v.; Findenegg, G. H. J . Co//o/dInferfaceSci.
1077, 59, 301-307. (22) Serplnet, J. J . Cbromtogr. 1073. 7 7 , 289-298.
R
~
~ for E review D September 2,1987. Accepted February
1, 1988.
Multimode Separation of Polycyclic Aromatic Compounds by Size Exclusion Chromatography with Poly(diviny1benzene) Arthur L. Lafleur* and Mary J. Wornat Environmental Health Sciences Center, Department of Chemical Engineering and Energy Laboratory, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
The separatlon of polycycHc aromatlc compounds (PAC) by size exclusion chromatography (SEC) with a poly(dlvlnyC benzene) column and dlchloromethane mobk phase was found to be governed by both slze-dependent and nonslze effects. The elutlon of nonplanar PAC substltuted with alkyl, phenyl, or ektron-wtthdrawing groups was govemed by both effects but generally proceeded In order of decreaslng molecular welght. On the other hand, planar PAC demonstrated a pronounced nonslze effect whose strength varled Inversely with IonIzaUon potential. PAC separated Into two ba8lC groups: planar and nonplanar. I n addnton, planar PAC eluted In three groups: (1) cataannelated PAC, (2) simple perlcondensed PAC, and (3) hlghly perlcondensed PAC.
Size exclusion chromatography (SEC) has been used extensively for the separation and characterization of polycyclic aromatic compounds (PAC) (1)especially in fossil-fuel-derived products such as petroleum asphaltenes (2)and coal-derived materials (3)in addition to its more common use for the characterization of polymers ( 4 , 5 ) . The great utility of the method, at least in the case of common polymers, lies in the fact that for a particular column and mobile phase combination, the elution volume is related to molecular size or to some size-related parameter such as hydrodynamic volume, in a simple and straightforward manner (6, 7). However, for fossil fuel products and other samples composed primarily of' PAC, the relationship between elution volume and molecular size is often complex, and for some samples, a satisfactory correlation cannot be made. The principal reason for this situation is the fact that for the types of gels currently in use, a number of factors other than molecular size govern the separation of PAC (8-16). Although a common goal in size exclusion chromatography is the elimination of nonsize effects, their influence can be beneficial. For example, multimode SEC has been useful for the characterization of petroleum (8,IO);for the determination of alcohols in gasoline (17), and for obtaining difficult separations (18,19). Also, a pronounced nonsize effect often observed with pericondensed PAC has proved useful for the characterization of petroleum crudes (20,21).Furthermore, with the Sephadex gels, chromatographic conditions have often been altered to exaggerate a specific nonsize effect such as adsorption or hydrogen bonding to obtain a desired separation
(22-24). In a recent study of the pyrolysis of bituminous coal, we utilized PAC nonsize effects with poly(diviny1benzene) and
dichloromethane to separate unsubstituted PAC from alkyl-substituted species (25). We used this method to monitor the pyrolytic formation of unsubetituted PAC from alkylated precursors. In this study we have examined the elution behavior of model compounds with the PDVB/CH2C12 system in order to shed light on the separation mechanism and to determine retention behavior for different classes of PAC.
EXPERIMENTAL SECTION Apparatus. The high-performance liquid chromatographic system used in this study consisted of a Perkin-Elmer Series 4 quaternary solvent delivery system coupled to a Model LC-85B variablewavelength detector with a 1.4-pL flow cell. The output signal was fed to a Chromatographics I1 data acquisition system running on a Model 3600 data station. Sample injection was performed by using a Rheodyne injector with either a 6-pL or a 100-pL loop. A microswitch on the injectors actuated the data system to ensure reproducible start times. All units were obtained from Perkin-Elmer Corp., Analytical Instruments, Norwalk, CT. The SEC column was 50 cm in length and 1.0 cm in diameter. It was packed with 500-AJordi-Gel poly(diviny1benzene)material (Jordi Associates, Inc., Bellingham, MA). Chemicals and Reagents. The reference standards used in this work are listed in Tables I-IV with Chemical Abstracts Service Registry Numbers and suppliers. The dichloromethane was Caledon-AmericanHPLC grade obtained from American Bioanalytical, Natick, MA. Procedure. Solutions of reference compounds were prepared in dichloromethane. Depending on concentration, either 6 or 100 pL was injected on the HPLC. The mobile phase was dichloromethane and the flow rate was 1.50 mL/min. Prior to each set of analyses, the column was conditioned for 40 min and a reference standard was injected to ensure reproducible performance. Retention times were measured by the Chromatographics I1 data system.
RESULTS AND DISCUSSION We obtained an initial calibration curve for the column using polystyrenes, phthalates, and nitromethane, compounds proven to elute with a true size-exclusion mechanism on other columns (1,4,5,26,27). We added other nitroalkanes and an alkylated azaarene to the calibration set because their elution volumes fell exactly on the regression line. The calibration curve is shown in Figure 1 and the compounds are listed in Table I. A linear regression calculation yielded the following relationship between elution volume (V,) in milliliters and log MW:
V, = 30.40 - 4.40 log MW
(1)
The correlation coefficient was 4.999, indicating a highly
0003-2700/88/0380-1096$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
1097
Table I. Reference Compounds for Column Calibration Primary Calibration Series
MW
compound or standard
source’
CAS no.
polystyrene standard polystyrene standard polystyrene standard polystyrene standard polystyrene standard polystyrene standard polystyrene standard polystyrene standard polystyrene standard N,”-bis[2,5-di-tert-butylpheny1]-3,4,9,10perylenedicarboximide (C5zHwNz04) di-n-octyl phthalate di-n-hexyl phthalate di-n-butyl phthalate diethyl phthalate dimethyl phthalate 1-nitrohexane 1-nitropentane 1-nitropropane nitroethane nitromethane
PL PL PL PL PL MN MN MN MN AC
9003-53-6 9003-53-6 9003-53-6 9003-53-6 9003-53-6 9003-53-6 9003-53-6 9003-53-6 9003-53-6 83054-80-2
2.95 X 1.84 X 1.13 X 6.80 x 3.45 x 1.70 x 7.60 X 3.55 x 1.77 x 767.0
cs cs cs cs cs
AC AC AC AC AC
117-84-0 84-75-3 84-74-2 84-66-2 131-11-3 646-14-0 628-05-7 108-03-2 79-24-3 75-52-5
390.6 334.0 278.3 222.2 194.1 131.2 117.2 89.1 75.1 61.0
18.95 19.34 19.70 20.15 20.54 21.06 21.17 21.72 22.04 22.68
-0.04 +0.05 +0.06 +0.07 +0.21 -0.02 -0.12 -0.09 -0.11 +0.14
dichloromethane-dz
AC
1665-00-5
87.0
24.14
+2.27
[Vel
lo6 lo6
9.24 9.48 9.72 10.42 10.61 11.78 13.11 14.67 16.20 17.73
lo6 104 104 104
los
103 103
A Vcai
+0.18
+o.oo
-0.21 -0.11 +0.09 +0.03
Chemical suppliers: (AC) Aldrich Chemical Co. Milwaukee, WI; (CS) Chem Services, Inc., Chester, PA; (MN) Machery-Nagel; (PL) Polymer Labs polystyrene standards obtained from American Bioanalytical, Natick, MA. [VJ, elution volume. CDeviationfrom value calculated by using eq 1. (I
rigorous thermodynamic evidence. A parameter based on the difference between volumes predicted from this equation [ V d ]and measured elution volumes [Vel was designated A V d It served to quantify the deviation from size-dependent elution. It is defined as Avcal
t 9
12
15
21
18
E l u t i o n Volume
24
(mL)
Figure 1. Callbration curve for POVB/CH,CI,. Reference compounds are listed in Table I: (0) polystyrenes, (V)CS2H5,N,0,, (+) alkyl phthalates, ( 0 )nitroalkanes.
linear inverse relationship. Reproducibility was better than 1.5%. For this study we followed the traditional SEC criterion that demonstration of the above linear relationship between V , and log MW defines size-dependent elution and that compounds falling on the calibration curve are considered to elute with a size-dependent mechanism (4,5). However, recent work has shown that pure, entropycontrolled size exclusion occurs in far fewer cases than previously thought, especially for small molecules (26-30), and that a completely satisfactory elucidation of the SEC mechanism has yet to be demonstrated. So, in the light of these studies, the classical definition of size-dependent elution will have to be considered somewhat arbitrary in the absence of
=
Ve -
Veal
(2)
Since the standard error of the estimate for the calibration data set is 0.15 mL, deviations greater than 2 or 3 times this value indicate the onset of nonideal behavior. The elution volume of the deuteriated mobile phase, in this case, CD2C12,has been proposed as a marker for nonexclusion effects (26,27). For our system, this volume is 24.14 mL ( u = 0.3). Compounds eluting later than this volume show definite nonsize behavior. PAC Retention Behavior. The elution behaviors of different classes of PAC were investigated to elucidate the relationship between structural features and retention mechanism. Two basic groups were studied: (1) unsubstituted polycyclic aromatic compounds including both planar and nonplanar species, examples of which are found in Table 11, and (2) substituted polycyclic aromatic compounds including PAC with functional groups, found in Tables I11 and IV. Compounds were further classified into cataannelated or pericondensed PAC, two basic types descriptive of polycyclic ring structure. These two classes of PAC can demonstrate markedly different retention behavior as evidenced by the demonstration of the pericondensation effect mentioned earlier (20, 21). Elution volumes and AVd results were obtained for aIl PAC listed in Tables 11-IV. Using V, for CDzClz as a nonsize marker made it clear that virtually all planar PAC demonstrated nonsize elution with this system. Nonplanar PAC eluted earlier than CD2C12;however, none of their elution volumes fell exactly on the calibration line. A reason often proposed for this observation is the fact that nonplanar PAC are cyclic compounds with structures more compact than those of the calibration compounds, so they will elute later than predicted by a MW-based calibration curve (10,31-35). Nevertheless, evidence is lacking that any general
1098
ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
Table 11. Unsubstituted Polycyclic Aromatic Hydrocarbons reference standard
sourcea
MW
[Vel*
A Vealc
78.1 128.2 152.2 178.2 178.2 202.3 228.3 228.3 228.3 228.3 252.3 278.4 278.4
24.02 24.00 23.93 24.02 23.99 23.98 23.96 24.12 24.02 23.93 24.00 24.14 24.30
+1.95 +2.88 +3.13 +3.53 +3.50 +3.73 +3.94 +4.10 +4.00 +3.91 +4.17 +4.34 +4.66
24.77 24.80 25.28 24.90 25.14 25.13
+4.52 +4.76 +5.45 +5.07 +5.66 +6.41
CAS no.
(a) Planar, Predominantly Cataannelated PAC clawd H-PAC/CATA benzene naphthalene acenaphthylene anthracene phenanthrene flouranthene benz[a]anthracene chrysene naphthacene triphenylene benzo[k]fluoranthene benzo[b]chrysene picene
CA AC AC AC AC AC AC AC AC AC
cs co co
71-43-2 91-20-3 208-96-8 120-12-7 85-01-8 206-44-0 56-55-3 218-01-9 92-24-0 217-59-4 207-08-9 214-17-5 213-46-7
(b) Planar, Predominantly Pericondensed PAC class: H-PAC/PERI-A PYene cyclopenta[cd]pyrene perylene benzo[a]pyrene dibenzo[a,i]pyrene decacyclene class: H-PAC/PERI-B benzo[ghi]perylene anthanthrene coronene diindenoperylene
AC SN AC AC
129-00-0 27208-37-3 195-65-0 50-32-8
AC
191-48-0
202.3 226.3 252.3 252.3 302.4 450.5
AC AN AC AC
191-24-2 191-26-4 191-07-1 188-94-2
276.3 276.3 300.4 400.5
25.74 26.27 26.69 26.80
+6.08 +6.61 +7.19 +7.85
154.2 166.2 180.3 190.3 216.3 228.3 254.3
23.60 23.03 22.50 23.81 23.11 23.63 20.78
+2.83 +2.40 +2.03 +3.44 +2.99 +3.60 +0.97
cs
(c) Nonplanar PAC class: X-PAC acenaphthene fluorene 9J0-dihydroanthracene 4H-cyclopenta[denphenanthrene benzo[blfluorene benzo[c]phenanthrene triptycene
AC
cs cs
AC AC AC
cs
83-32-9 86-73-7 613-31-0 203-64-5 243-17-4 195-19-7 477-75-8
Chemical suppliers: (AC) Aldrich Chemical Co., Milwaukee, WI; (AN) Analabs Div., Foxboro Analytical; (CA) Caledon-American, American Bioanalytical, Natick, MA; (CO)Columbia Organic Chemical; (CS) Chem Servies, Inc., Chester, PA; (LS)Lancaster Synthesis, Inc.; (SN) Synthetic chemical. *[Vel,elution volume. cDeviation from value calculated using eq 1. dSeparation class: discussed in the text, refer to Figures 2-4. class of PAC eluted with a true size-dependent mechanism. Compounds with AV, values of 3.0 mL or greater generally eluted later than the CDzClz elution volume. The data strongly suggest that these PAC elute with a mainly nonsize mechanism. Those giving values of less than 3.0 mL appear to elute with a multimode mechanism whose nonsize component is expected to vary with the magnitude of AVd. PAC Separations. After the data were processed graphically for the compounds in Tables 11-TV, it became evident that PAC separated into two major groups as illustrated in Figure 2. The vertical line dividing the two groups corresponds to the lower 95% confidence limit for the elution volume of CD2C12. The first group, designated X-PAC, consisted of PAC incorporating alkyl or phenyl substituents. The second group, designated H-PAC, consisted of planar, unsubstituted PAC. Further analysis showed that H-PAC eluted in three distinct clusters as shown in Figure 3: (1) cataannelated species, divided into either nonplanar or planar types; (2) simple pericondensed PAC (A in Figure 3); (3) highly pericondensed PAC (B in Figure 3). One remarkable characteristic of the planar cataannelated PAC was the fact that their elution volumes were very nearly identical. The set of 13 PAC gave a relative standard deviation for V, of only 0.27%. The mean elution volume for these compounds is 24.01 mL, a value which is not statisticaly different from the 24.14 mL result for CD2C12.
lo3
I
I
-
-
I
X-PAC
H-PAC
1
0
0
0 0 0
a0 >
0
0
0 0 0
o
0 0 0
0
18
20
22
24
26
E l u t i o n Volume (mL1
Figwe 2. Multimode separation of PAC into two basic groups of substituted (X-PAC) and unsubstituted (KPAC): (0)planar PAC: (0) alkylated PAC: (+) phenyl-substituted PAC.
Although benzo[c]phenanthrene, pointed out in Figure 2, appears to provide an exception to the coelution observed for planar cataannelated PAC; it is in fact slightly skewed (36). This example illustrates the importance of planarity as a factor in nonsize PAC elution for PDVB/CH,Cl,. Results for PAC with functional groups are illustrated in Figure 4. As in previous cases, the compounds separated into early-eluting and late-eluting groups. Early-eluting com-
ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988 Table 111. Substituted
1099
PAC
compound or standard
sourcea
CAS no.
MW
[Velb
A VCdC
92.1 190.3 142.2 156.2 156.2 156.2 192.3 192.3 206.3 240.4 242.4 256.4
23.52 21.38 23.33 22.85 23.13 22.97 23.34 23.57 22.58 20.94 21.54 23.09
+1.76 +1.01 +2.40 +2.10 +2.38 +2.99 +3.22 +2.36 +1.02 +1.63 +3.29
154.2 178.2 202.3 254.3 230.3 230.3 306.4 306.4 306.4 330.4 382.5 506.6 534.7
23.09 22.38 22.13 22.49 22.04 21.78 21.17 20.90 20.90 21.71 20.46 20.33 19.04
+2.32 +1.88 +1.88 +2.67 +2.03 +1.78 +1.71 +1.44 +1.44 +2.40 +1.43 +1.83 +0.65
(a) Alkyl-Substituted PAC X-PAC toluene n-octylbenzene 2-methylnaphthalene 2,6-dimethylnaphthalene 1,4-dimethylnaphthalene 2,3-dimethylnaphthalene 2-methylanthracene 9-methylanthracene 3,6-dimethylphenanthrene 2,6-di-tert-butylnaphthalene 1,4-dicyclohexylbenzene 7,12-dimethylbenz[a]anthracene
CA AC
cs cs cs cs cs cs
AC AC AC
cs
108-88-3 2189-60-8 91-57-6 581-42-0 571-58-4 581-40-8 613-12-7 779-02-2 1576-67-6 3905-64-4 1087-02-1 57-97-6
+2.22
(b) Phenyl-Substituted PAC class: X-PAC biphenyl diphenylacetylene 1,4-diphenylbutadiyne 9-phenylanthracene m-terphenyl p-terphenyl m-quaterphenyl p-quaterphenyl 1,3,5-triphenylbenzene 9,lO-diphenylanthracene m-quinquephenyl
cs
AC
LS
cs cs
AC AC AC AC
cs cs cs
1,3,6,84etraphenylpyrene
hexaphenylbenzene
AC
92-52-4 501-65-5 886-66-8 602-55-1 92-06-8 92-94-4 1166-18-3 135-70-6 612-71-5 1499-10-1 16716-13-5 992-04-1
Chemical suppliers: (AC) Aldrich Chemical Co., Milwaukee, WI; (CA) Caledon-American, American Bioanalytical, Natick, MA; (CS) Chem Services, Inc., Chester, PA; (LS)Lancaster Synthesis, Inc. b[Ve],elution volume. cDeviation from value calculated using eq 1. Separation class: discussed in the text, refer to Figures 2-4.
-PER1
d
-A
0 0 Y
.c m
0
0
x 01
I o
0
0
-
0
0 0
0
Io2l 18
u
22
23
24
20
22
24
26
Elution Volume ImLI Figure 4. Multimode elution of PAC with functional groups: Reference compounds are listed in Table IV: ( 0 )nitro and cyano PAC plus aldehydes, ketones, and carboxylic acids: (+) amino compounds: (0) halogenated naphthalenes: (0)other halogenated PAC.
Elution Volume (mLi Figure 3. Separation of unsubstltuted PAC (WAC) into cataannelated structual types. Cataannelated PAC (CATA) and pericondensed (-1) fwther separate into planar and nonplanar types. Pericondensed PAC fall into two groups (A & B)with increasing degree of pericondensation.
pounds (X-PAC) consisted of PAC with electron-withdrawing substituents including carbonyl, carboxylic acid, nitro, and cyano groups. Late eluting types (H-PAC) were amino PAC and halogenated naphthalenes. Halogenated derivatives of nonplanar PAC eluted with the X-PAC; however, those of planar PAC eluted with the H-PAC. The halogenated naphthalenes, for example, gave increasing V , values with increasing halogen size. This anomalous result strongly suggests that the halogen groups contributed more
than simple bulk to the molecules. Model Compounds as Nonsize Retention Probes. In order to shed some light on the factors responsible for the nonsize retention of PAC with PDVB/CH2C12,two fundamental effects were considered: (1)size effects (shape and stereochemistry)and (2) nonsize effects (partition, adsorption, and other distribution effects). Molecular shape can contribute to anomalous PAC retention because PAC have more compact molecular shapes than the polymeric species usually used for calibration, causing them to elute later than calibration compounds of the same molecular weight. The effect of molecular shape on V, can be significant for small molecules and has been studied (10, 31-35).
1100
ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
Table IV. Halogenated and Other Functionalized PAC sourcen
compound or standard
CAS no.
MW
[Velb
A Vcd'
22.80 23.04 21.84 22.50
+2.72 +3.06 +1.95 +2.62
254.1 257.1
24.02 24.54 24.53 24.72 24.17 24.71 24.78
+3.14 +3.80 +3.86 +4.51 +4.09 +4.89 +4.99
307.1
25.55
+6.10
123.1 173.2 199.2 202.0 225.2 247.3 270.2 290.1 315.2
21.90 22.31 21.36 22.04 21.62 21.06 22.73 20.21 23.39 20.12
+0.70 +1.76 +LO8 +1.79 +1.45 +1.01 +2.86 +0.51 +3.83
cs
86-53-3 66-99-9 93-09-4 486-25-9
153.2 156.2 172.2 180.2
22.16 22.26 21.54 22.20
+1.38 +1.51 +0.98 +1.73
AC AC
947-73-9 153-78-6
193.3 181.2
24.62 24.32
+4.28 +3.86
(a) Halogenated PAC clawd X-PAC 1-(bromomethy1)naphthalene
4-bromobiphenyl 2-bromofluorene 9-bromofluorene class: H-PAC 1-fluoronaphthalene bromobenzene 1-chloronaphthalene 1-bromonaphthalene 1-bromo-4-methylnaphthalene 1-iodonaphthalene 9-bromophenanthrene class: PERI-1 1-bromopyrene
AC AC AC AC
3163-27-7 92-66-0 1133-80-8 1940-57-4
221.1
AC AC
321-38-0 108-86-1 90-13-1 90-11-9 6627-78-7 90-14-2 573-17-1
146.2 157.0 162.6 207.1
AC
AC AC AC
cc cc
233.1 245.1 245.1
221.1
(b) Nitrated PAC class: X-PAC nitrobenzene 1-nitronaphthalene 4-nitrobiphenyl 1-bromo-4-nitrobenzene 2-nitrofluorene 2-nitro-9-fluorenone 1-nitropyrene 2,7-dinitro-g-fluorenone 2- bromo-7-nitrofluorene
98-95-3 86-57-7 92-93-3 586-78-7 607-57-8 3096-52-4 5522-43- 0 31551-45-8 6638-61-5 129-79-3
AC AC AC AC AC AC AC AC AC AC
2,4,7-trinitro-9-fluorenone
( c ) Other
class: X-PAC 1-cyanonaphthalene 2-naphthaldehyde 2-naphthoic acid 9-fluorenone class: H-PAC 9-aminophenanthrene 2-aminofluorene
211.2
+0.71
Functionalized PAC
AC AC AC
Chemical suppliers: (AC) Aldrich Chemical Co., Milwaukee, WI; (CC) Cambridge Chemical Co.; (CS) Chem Services, Inc., Chester, PA. IV.1, elution volume. CDeviationfrom value calculated using ea 1. dSeuaration class: discussed in the text, refer to Figures 2-4.
-- __ - -
- - - .-- - .. - -
~
- - -~-
- -- -
~
^i
-\ -4-
,--,& 12
J-\
I
- 6
~
c
7
-
/ 3
4
\a.
Fy-.
4
-1
I
_
I -
E ut on Yo m e m L )
Flguro 5. Effects of molecular size and configuatkn on elution volume for three isometric groups of polycyclic aromatic hydrocarbons: group 1 (11.9-12.3 A) biphenyl (I), phenanthrene (2), perylene (3), benzo[ghi]perylene (4coronene (5), fluorene (e), cyclopenta[def]phenanthrene (7); group 2 (14.3-14.6 A) 1,3,5-trlphenylbenzene (8), m-terphenyl (Q), chrysene (lo), benzo[a Ipyrene (1l), anthanthrene (12); group 3 (15.1-16.1 A) pdlcycbhexylbenzene (13), plcene (14), decacyclene (15), p-terphenyl(l6). The reference point V , Is at the elution volume for cataannelated PAC. Data points are at molecular centers.
Secondly, distribution processes such as adsorption or partitioning have also been reported to contribute to nonsize
elution of PAC (8-16). Compounds exhibiting such phenomena give elution volumes larger than predicted on the basis of molecular size. We studied the effect of molecular size on PAC retention by using model compounds with similar size but different molecular structures. The results are illustrated in Figure 5. The PAC are divided into three groups with similar molecular diameters (longest dimension across the molecule) (8). Data points are at the centers of the molecules. The vertical line labeled V , corresponds to the mean V, value for cataannelated
PAC. For other SEC systems, it has been reported that elution volumes for planar, cataannelated PAC varied either directly (12, 13, 18) or inversely (11-13, 18, 20) with molecular size, depending on the gel and solvent combination used. For PDV13/CH2C12, V, correlates with molecular dimensions for some nonplanar PAC 1 and 6 and 13 and 16 in Figure 5); however, planar PAC do not show a linear correspondence between size and retention volume. An obvious illustration of this phenomenon is the coelution of the cataannelated PAC phenanthrene (2), chrysene (lo), and picene (14), three compounds having noticeably different molecular dimensions. Another nonsize effect illustrated in Figure 5 is the pericondensation effect (20,21) mentioned earlier. For both the following series: perylene (3)-benzo[ghi]perylene (4)-coronene ( 5 ) and chrysene (10)-benzo[a]pyrene (11)-anthanthrene (12) retention volume increases with degree of pericondensation and not with molecular size.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
V,
= 4 1 12
ir
1101
-
=
2 16lIP) 9201
b+ 34
35
Elufian Volume
imLI
Flgure 6. Substkuent effects In multimode size exclusion chromatography wlth PDVB/CH,CI,: Effects of increased steric bulk: elec-
trorrdonating and electron-withdrawing groups. Reference compounds are noctylbenzene (17),nitrobenhe (la), toluene (lQ), benzene (20), bromobenzene (21), 2,6-di-tert-butyinaphthalene (22), l-nltronaphthalene (23), 2,6dlmethylnaphthalene (24), naphthalene (25), 1-bromonaphthalene (26), 1-(bromomethyi)naphthaiene (27), 1bromo-4methylnaphthalene (28), 1-iodonaphthalene (29), 9,1O d l phenylanthracene (30), 3,B-dimethylphenanthrene (31), 9-aminophenanthrene (32), g-bromophenanthrene (33), 9-phenylanthracene (34), anthracene (35), 1,3,6,8-tetraphenylpyrene (36),1-nitropyrene (37),pyrene (38),and 1-bromopyrene(39). The reference point V , Is at the elution volume for cataannelated PAC. Data points are at molecular centers. A number of mechanisms for nonsize behavior of PAC have been advanced (9,14,15). It is often proposed that a weak type of charge-transfer interaction or bonding occurs between PAC and SEC packing materials. In order to gain additional understanding of PAC nonexclusion behavior, the retention of substituted reference PAC was used as a structural and mechanistic probe. The compounds studied are listed in Table IV and illustrated in Figure 6. The location of each compound in Figure 6 is determined by its elution volume ( X axis) and its aromatic ring number (Yaxis). Again, the dotted vertical line corresponds to the elution volume for cataannelated PAC [ VJ. The elution volumes for the parent PAC (20,25, and 35 in Figure 6) are shown to be equal to V,,, except in the case of pyrene [38], which is pericondensed and elutes later than the cataannelated species. The substituent effects can be summarized as follows: (1)The addition of alkyl or phenyl groups shifted retention to lower volumes. (2) Substituting bromine or other halogen for hydrogen led to increased V, with planar PAC [21,26,29,33,and 391 and to decreased V, for nonpolar PAC [27, and the bromofluorenes]. (3) Nitro-PAC [18, 23, and 371 eluted at lower V, than the parent PAC. (4) Polycyclic aldehydes, ketones, carboxylic acids, and nitriles showed decreased V, values compared to the parent compounds while amino derivatives [32] gave increased values. The effect of bulky alkyl substituents in minimizing nonsize retention of PAC has been observed in previous studies (2, 8,12,14); however, the introduction of molecular bulk with halogen substituents led to remarkable results illustrated in Figure 6. Comparing the retention of 1-(bromomethy1)naphthalene [27] with its isomer, 1-bromo-4-methylnaphthalene [28], in Figure 6, reveals that the isomer with bromine attached directly to the ring [28] elutes much later than the one with a bromine attached to a methyl carbon [27]. This strongly suggests that when bromine is directly substituted on an aromatic moiety, an electronic effect comes into play that
61 21
1 22
23
24
25
26
27
E l u t i o n Volume ( m i l
Figure 7. Correlation of PAC elution volume with ionization potential.
counteracts the steric effect introduced by the atom's bulk. Since halogens can be either electron withdrawing or electron donating (37), we studied the effect of other electron-interacting groups to give us additional information about the nature of the electronic effect that causes PAC nonsize effects in this system. In contrast with the halogens; nitro derivatives, which are strongly electron withdrawing, gave consistently lower elution volumes than parent molecules for all types of PAC. PAC with other electron-withdrawing groups such as cyano, carboxylic acid, or carbonyl (aldehydes + ketones) also showed decreased elution volumes, while PAC with electron-donating amino substituents showed increased elution volumes. Therefore, the data strongly suggest that halogen substituents act as electron-donatinggroups and it is the increased electron density in the aromatic ring that leads to the pronounced nonsize effect observed for halogenated derivatives of planar PAC. The evidence described strongly suggests that some type of electronic interaction is occurring between the eluates and the PDVB packing. Furthermore, it appears that the packing material is an electron acceptor (Lewis acid) while the PAC are electron donors (Lewis bases). Consistent with this hypothesis is the fact that PAC made electron deficient (through the addition of nitro groups for example) show a markedly reduced nonsize effect. Since increased electron density was implicated as a causative factor in the nonsize retention of planar PAC, an attempt was made to correlate their elution volumes with molecular parameters that reflect electron density. These parameters included the following: energies of highest occupied molecular orbitals, polarographic reduction potentials, UV band energies, and several reactivity indicators (38);resonance energy (15,23); total number of A electrons (13);number of bonding a electrons (9, 13); ionization potential (13); and number of a-bonding carbon atoms (9). Of the parameters investigated, only the ionization potential gave an acceptable correlation with V,. The data set included the following 13 PAC, listed in order of increasing V,: diphenylacetylene, fluorene, biphenyl, 2-methylnaphthalene, benzo[c]phenanthrene, phenanthrene, naphthalene, anthracene, chrysene, pyrene, perylene, benzo[ghi]perylene, and anthanthrene. The curve is illustrated in Figure 7. A linear regression analysis resulted in a correlation coefficient of -0.920 and the following equation:
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 11, JUNE 1, 1988
V , = 41.12 - 2.16[IP] Values for the ionization potential (IP), measured in electron volts (eV) and defined as the minimum energy required to remove an electron from a molecule or atom, were obtained from a U.S. National Bureau of Standards data compendium
(39). Ionization potential measurements were not used for predicting retention for halogenated PAC, because the I P measurements in these compounds reflect the removal of an electron from the halogen atom and not from the aromatic a-electron system involved in bonding. The data show that the nonsize effect for planar PAC increases with decreasing ionization potential. In other words, the more loosely bound the electrons, the stronger the PAC bind to the gel material. This result is consistent with the chargetransfer bonding mechanism hypothesized as the cause for PAC nonsize retention in size exclusion chromatography (13) because the strength of charge-transfer bonding depends strongly on the ionization potential of the electron donor (38). One nonsize retention model consistent with these data involves the planar PAC as electron donors and the PDVB column as the acceptor, forming a donor-acceptor pair. The later-eluting compounds are the better electron donors, as evidenced by their lower ionization potentials. Examples are anthanthrene, pyrene, and perylene, which which are highly pericondensed PAC. This observation is in accord with previous results for these PAC types (20,21). With this model, it can also be seen that perturbation of donor-acceptor interaction either by bulky substituents or by electron-withdrawing groups will lead to decreased bond strength and diminished nonsize effects. The donor-acceptor model accounts for the basic differences in PAC elution behavior that we observed with PDVB/CH2C12 and sheds light on some PAC nonsize effects; however, a comprehensive model for multimode size exclusion chromatography will require much additional work.
ACKNOWLEDGMENT We thank Johan Lugtenberg of the Gorlaeus Laboratory, University of Leiden, for furnishing the synthetic reference sample of cyclopenta[cd]pyrene. We also thank Peter Monchamp for assistance in maintaining the chromatographic system and Howard Jordi for helpful suggestions. Registry No. Benzene, 71-43-2; naphthalene, 91-20-3; acenaphthylene, 208-96-8; anthracene, 120-12-7; phenanthrene, 8501-8; fluoranthene, 206-460; benz[a]anthracene, 56-55-3; chrysene, 218-01-9; naphthacene, 92-24-0; triphenylene, 217-59-4; benzo[klfluoranthene, 207-08-9; benzo[b]chrysene, 214-17-5; picene, 213-46-7; pyrene, 129-00-0; cyclopenta[cd]pyrene, 27208-37-3; perylene, 198-55-0;benzo[a]pyrene, 50-32-8; dibenzo[a,i]pyrene, 189-55-9; decacyclene, 191-48-0; benzo[ghi]perylene, 191-24-2; anthanthrene, 191-26-4;coronene, 191-07-1;diindenoperylene, 188-94-3; acenaphthene, 83-32-9; fluorene, 86-13-7; 9,lO-dihydroanthracene, 613-31-0; 4H-cyclopenta[deflphenanthrene, 203-64-5; benzo[b]fluorene, 243-17-4; benzo[c]phenanthrene, 195-19-7;triptycene, 477-75-8; toluene, 108-88-3; n-octylbenzene, 2189-60-8; 2-methylnaphthalene, 91-57-6; 2,6-dimethylnaphthalene, 581-42-0; 1,4-dimethylnaphthalene,571-58-4;2,3dimethylnaphthalene, 581-40-8; 2-methylanthracene, 613-12-7; 9-methylanthracene, 779-02-2; 3,6-dimethylphenanthrene,15763905-64-4;l,4-dicyclohexyl67-6; 2,6-di-tert-butylnaphthalene, benzene, 1087-02-1; 7,12-dimethylbenz[a]anthracene,57-97-6; biphenyl, 92-52-4; diphenylacetylene, 501-65-5; 1,4-diphenylbutadiyne, 886-66-8; 9-phenylanthracene, 602-55-1; rn-terphenyl, 92-06-8; p-terphenyl, 92-94-4; rn-quaterphenyl, 1166-18-3; pquaterphenyl, 135-70-6;1,3,5-triphenylbenzene, 612-71-5;9,lOdiphenylanthracene, 1499-10-1;rn-quinquephenyl, 16716-13-5; 1,3,6,84etraphenylpyrene,13638-82-9;hexaphenylbenzene, 992-
04-1; 1-(bromomethyl)naphthalene,3163-27-7;4-bromobiphenyl, 92-66-0; 2-bromofluorene,1133-80-8; 9-bromofluorene,1940-57-4; 1-fluoronaphthalene, 321-38-0; bromobenzene, 108-86-1; 1chloronaphthalene, 90-13-1; 1-bromonaphthalene, 90-11-9; 1bromo-4-methylnaphthalene, 6627-78-7;1-iodonaphthalene, 9014-2; 9-bromophenanthrene, 573-17-1; nitrobenzene, 98-95-3; 1-nitronaphthalene, 86-57-7;4-nitrobiphenyl, 92-93-3; l-bromo4-nitrobenzene, 586-78-7; 2-nitrofluorene, 607-57-8; 2-nitro-9fluorenone, 3096-52-4; 1-nitropyrene, 5522-43-0; 2,7-dinitro-9fluorenone, 31551-45-8; 2-bromo-7-nitrofluorene, 6638-61-5; 2,4,7-trinitro-9-fluorene, 129-79-3;1-cyanonaphthalene, 86-53-3; 2-naphthaldehyde,66-99-9;2-naphthoicacid, 93-09-4;g-fluorenone, 486-25-9; 9-aminophenanthrene, 947-73-9; 2-aminofluorene, 153-78-6; poly(divinylbenzene), 9003-69-4; 1-bromopyrene, 1714-29-0.
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RECEIVED for review May 18,1987. Accepted February 2,1988. This investigation was supported by National Institute of Environmental Health Sciences Center Grant EHS-5P30ES02109-08 and Program Grant EHS-5P01-ES01640-09.
