Anal. Chem. 1888, 58, 2060-2072
Data Processing. An H P 3392A integrator was used to process the gas chromatograph signal. When the system was used a t sea, the data report from the integrator was transmitted to an HP 85B computer, which performed intermediate concentration calculations and displayed a real time running plot of the Be data vs. depth. This procedure was extremely useful, enabling rapid identification of samples with suspect values and allowing immediate reinjection of the extracts. Full listings of both the integrator and computer programs are available from the authors. CONCLUSION The method outlined above is part of a continuing program aimed a t developing and adapting analytical techniques for trace element determinations in natural waters. The techniques are developed to allow geochemical cycles to be delineated for elements where atomic absorption lacks sensitivity. For elements where other methods are available, ECD-GC of volatile chelates provides considerable advantages both in terms of sensitivity, lowering sample volume requirements and the associated collection problems, and suitability for use at sea. Currently the range of elements that have been determined a t sea with this approach is Se (10)by 4-nitro-ophenylenediamine and Be, Al, and Cr by Htfa. The last two will be reported later. ACKNOWLEDGMENT We wish to thank the Chief Scientist, Dean Roemmich, and the entire complement of the R.V. Thomas G. Thompson for both the opportunity to participate in, and their help throughout, the trans-Pacific cruise. Ed Boyle provided helpful comments on the paper as well as many useful sug-
2089
gestions during the dark days inevitably encountered during this kind of work. We wish to thank K. W. Michael Siu and one anonymous reviewer for their helpful comments. Registry No. HtFa, 367-57-7; H20, 7732-18-5; Be, 7440-41-7. LITERATURE CITED (1) Arnold, J. R. S c h c e , 1958, 724. 584. (2) Merrlll, J. R.; Lyden, E. F. X.; Honda, M.; Arnold, J. R. Geochlm. Cosd i n . Acta 1960, 78, 108-129. (3) Ross, W. D.; Sievers. R. E. Taknta 1988, 75, 67-94. (4) Elsentraut, K. J.; West, D. J.; Sievers, R. E. Anal. Chem. 1971, 43, 2003-2007. (5) Rlsby, T. H., Field, L. R.; Yang, F. J.; Cram, S. P. Anal. Chem. 1982, 54, 410R-428R. (6) Measwes, C. 1.; Edmond, J. M. Nature (London) 1982, 297. 51-53. (7) Measures, C. 1.; Edmond, J. M. €a& Planet. Scl. Len. 1983, 66, 101-1 IO. (8) Monaghan, M. C.; Klein, J.; Mkldleton, R. €OS. Trans. Am. Osophys. U . 1985, 4 , 1111. (9) Measures, c. I.; &ant. B.; Khadem, M.; Lee, D. S.; Edmond, J. M. Earth &net. Scl. Lett. 1984, 7 7 , 1-12. (IO) Measures, C. I.; Buton, J. D. Anal. Chim. Acta 1980, 720. 177-186. (11) Wentworth, W. E.; Becker, R. S.; Tung, R. J . Pbys. Chem. 1987, 77, 1652-1665. .._. ~ . (12) OBrlen, R. J.; Dumdei, B. E.; Hummel, S. V.; Yost, R. A. Anal. Chem. 1884. 58. 1329-1335. (13) Hynes, M: J.; O’Regan, B. D. J . Chem. Soc.,Dalton Trans. 1979, 162-166. (14) Hynes, M. J.; O’Regan, B. D. J . Chem. Soc., Dalton Trans. 1980, 7-13. (15) Hyes, M. J.; O’Regan, B. D. J . Chem. Soc., Dalton Trans. 1980, 1502-1510.
RECEIVED for review February 18,1986.
Accepted April 14, 1986. The Office of Naval Regearch generously supported the development and implementation of this technique. The cruise upon which the field data were colleded was supported by the National Science Foundation.
Reversed-Phase Liquid Chromatographic Separation of p-tert-Butylphenol-Formaldehyde Linear and Cyclic Oligomers F. John Ludwig, Sr.,* a n d A. Gibbes Bailie, Jr. Petrolite Corporation, 369 Marshall Avenue, St. Louis, Missouri 63119
Linear condensates have been separated from cyclic com-
ponents, calixarenes, of either ackl-catalyzed or base-catalyzed p - t o t f ~ o n n a ~ y resins d e by b w pressue semipreparative column chromatography on silica gel. The Isolation of callx[4, 5, 6, 7, and Illarenes in amounts of 3 to 6% from acld-catalyzedresins was accompkhd for the fkst the. Up to 35 linear oligomers of either acld-catalyzed or baseeataiyzed resins were resolved on a C,, revemd-phase HPLC cokmn uslng gradlent eluHon with mlxtues d methanol, ethyl acetate, and acetic acid.
In our previous paper, we described a low-pressure, semipreparative scale procedure to separate those cyclic condensates that are known as calixarenes from the linear condensates that are produced in base-catalyzed reactions of p-alkylphenols with formaldehyde (1). We also described HPLC methods to separate either the dimer through heptamer linear oligomers or calix[4, 5, 6, 7, and 8larenes and oxacalix[l]arene. The use of calixarenes as enzyme mimics, ion carriers, and selective complexing agents is being studied a t 0003-2700/88/0358-2069$01.50/0
Washington University, St. Louis, MO (2). Our methods were developed to assist these comprehesive studies. Our initial purpose was to measure the total amounts of calixarenes and linear condensates and to determine the amounts of each calixarene oligomer. Now we have developed a HPLC procedure to separate the linear condensate fractions of either base-catalyzed or acidcatalyzed resins. Resolution of linear oligomers that contained from 3 to about 35 methylene-bridged phenolic nuclei was achieved. These analyses are required to deduce the mechanisms of either base-catalyzed or acid-catalyzed p-alkylphenol-formaldehyde condensations. The formation of calixarenes during acid-catalyzed condensation of p-alkylphenols and formaldehyde has been disputed for more than 40 years (3).Consequently we were surprised when, using our previously reported TLC method (11,we detected small amounts of calix[4,5,6,7,and 8larenes in an acid-catalyzed reaction product of p-tert-butylphenol with formaldehyde. When a number of other acid-catalyzed condensates were analyzed by TLC and HPLC, small amounts,e.g., 3-6%, of calixarenes were detected also. Thus, any mechanism that is proposed for the acid-catalyzed p0 ID88 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 9, AUGUST 1986
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Table I. Measured Peak Area Percents and Known Weight Percents of a Mixture of Linear Condensates and Calixarenes of p -tert-Butylphenol and Formaldehyde 70
peak area compound
%"
std dev
linear dimer linear trimer linear tetramer linear pentamer linear heptamer calix[4]arene calix[ 5larene calix[6]arene calix[7]arene calix[81arene
22.0b 19.0 3.6 8.8 4.5 4.8 4.3 11.0 6.8 14.0
2.8d 0.77 0.15 0.19 0.18 0.65 0.31 0.15 0.19 1.5e
difference, peak area % wt '70 from wt % 18.2 18.2 2.38c 9.16 5.30' 7.98 4.58 14.6 7.98 11.7
+20.9 +4.40 +51.3 -3.90 -15.1 -39.8 -6.10 -24.7
-14.8 +19.7
a Mean of seven replicate determinations. May include solvent contribution to peak area. Calculated from HPLC estimated amounts of tetramer and heptamer in impure sample. dSolvent component co-elutes with dimer. e Integration of broad peak is less reproducible.
6
lI
0
5
10
15
20
25
30
35
40
45
TME, MNVTES
Flgwe 1. Chromatogram of known mixture of p-fed-butylphenolformaldehyde linear and cyclic condensates (1) linear trimer, (2) linear tetramer, (3) linear pentamer, (4) llnear heptamer, (5) callx[4]arene, (6) calix[6]arene, (7) callx[7]arene, (8)callx[5]arene, and (9) calix-
[8]arene.
alkylphenol-formaldehyde condensation reaction must contain a route to explain formation of calixarenes as minor components. Gutsche and co-workers are continuing their studies of the mechanism of p-alkylphenol-formaldehyde condensations and will report their conclusions in future publications.
EXPERIMENTAL SECTION Reagents and Standards. The methanol Distilled-in-Glass solvent was purchased from American Burdick and Jackson. ACS Reagent-Grade ethyl acetate and acetic acid were purchased from Fisher Scientific. If opened repeatedly in atmospheres of high humidity, the ethyl acetate may undergo hydrolysis forming additional acetic acid in the eluting solvent mixture and changing retention times. p-tert-Butylcalix[4,5,6,7, and 81arenes were synthesized and characterized by Gutsche's research group ( 2 ) . Syntheses and analyses of linear dimer, trimer, tetramer, pentamer, and heptamer p-tert-butylphenol-formaldehydecondensates were described previously (I). Synthesis of the base-catalyzed p-tert-butylphenol-formaldehyde condensate whose linear fraction is shown in Figure 2 below was done by use of the method of Buriks et al. (4). Synthesis of the acid-catalyzed p-tert-butylphenol-formaldehyde condensate whose cyclic and linear fractions are shown in Figures 3 and 4 below was done by using the procedure de-
scribed by DeGroote and Keiser (5). Flash Column Chromatography Procedure. Universal Adsorbents, Inc., silica gel, 32-63 pm for chromatography with elevated pressure, Catalog Number 02824, was used to separate calixarenes from linear condensate by use of the method described previously ( I ) . HPLC Procedures. ( a ) Equipment and Columns. The instrument consisted of two Waters Associates Model M-6000 pumps, a Waters Model 660 solvent programmer, Waters U6K injector, Kratos Model 770 variable wavelength UV detector, and Texas Instruments Servo/Riter I1 recorder. A Hewlett-Packard 3357 Laboratory Automation System was used to measure peak retention times and areas. The 250 X 4 mm 5-km LiChrosorb RP-18 Hibar column was purchased from EM Reagents, Catalog Number 50333. A Brownlee MPLC guard cartridge of 5-pm CI8 reversed-phase packing was installed between the injector and Hibar column. ( b ) Operating Conditions. The gradient elution was done as follows: (1)Initial solvent composition was 98.9% methanol, 1.0% ethyl acetate, and 0.1% acetic acid. (2) Final solvent composition was 40.0% methanol, 59.9% ethyl acetate, and 0.1% acetic acid. A linear gradient over a 60-min interval was used. The flow rate was 1.0 mL/min. The absorbanceswere measured at 287 nm with a 0 . 2 absorbancerange. Typically, a 4O-pL aliquot of a 2 mg/mL ethyl acetate solution was injected.
RESULTS AND DISCUSSION A known mixture of p-tert-butylphenol-formaldehyde linear dimer, trimer, tetramer, pentamer, and heptamer and calix[4, 5, 6,7, and 8larenes was separated on a 250 X 4 mm 5-km RP-18 Hibar HPLC column. The chromatogram is shown in Figure 1. Since the solvent gradient was optimized to resolve linear condensates containing up to about 35 phenolic units, the resolution of the linear dimer through tetramer is not as good as in a solvent of lower initial ethyl acetate content. The rather broad peak shape of calix[8]arene may result from an interaction with the reversed-phase packing. The other peaks appear fairly symmetrical. The elution volume of calix[5]arene is anomalous. Previously, an anomalous retention time and response factor of calix[5]arene was observed on 250 X 4-mm 10-pm LiChrosorb SI 60 Silica Gel Hibar HPLC columns (1). Infrared OH stretching frequencies and space models indicate that calix[51arene differs from calix[4,5,6,7, and 8larenes in orientation of groups and intramolecular hydrogen band strength (2). A comparison of the weight percent of each component in the known mixture with its measured peak area percent is presented in Table I. Peak area percents differed from weight percents by f 2 5 % for eight of the ten components. The
2071
ANALYTICAL CHEMISTRY. VOL. 58. NO. 9, AUGUST 1986
b
6
lo
tu
io
,
26
30
36
40
46
60
68
60
-66
70
1
76
TYE. Y W m l
Figure 2. Chromatogram of linear fraction of a base-catalyzed p fert-butylphenol-formaldehyde condensate. 10
0
maximum percent difference was +51% for the linear tetramer, possibly reflecting the uncertainty in its purity. Furthermore, the peak area percents and weight percenta did not differ in a systematic manner. Hence, the peak area percents can be used only as crude approximations of the weight percents in studies of the effects of reaction conditions, e.g., temperature, solvent, catalyst, etc., upon product composition. A base-catalyzed p-tert-butylphenol-formaldehyderesin was separated into a calixarene fraction and a linear condensate fraction by low-pressure, semipreparative column chromatography on silica gel. The linear fraction was separated with the C18 reversed-phase HPLC column into the components that are shown in Figure 2. The retention times of four of the peaks that eluted in the first 10 min of the run coincided with the retention times of the linear trimer, tetramer, pentamer, and heptamer. Since no known higher molecular weight linear condensates were available to us, we can only presume that the major peaks that eluted between about 10 and 50 min originate from a series of methylenebridged linear condensates containing up to about 20 phenolic units. Weaker peaks possibly from a second or third homologous series were seen between the major peaks, e.g., at about 8,12,14,17,25,etc. min, or as partially resolved satellite peaks, e.g., at about 29,33,35,38,etc. min. Since condensates with terminal o-hydroxymethyl groups are suspected to be intermediates in the base-catalyzed reactions, some of the minor peaks could have that type of structure (2,3). More work, e.g., preparative liquid chromatography and synthesis, is necessary for identification of the minor components in the linear condensate fractions. Previously, using a silica gel HPLC column with an eluent of 99.4% chloroform-6% ethanol, we were able to separate only the linear dimer through heptamer condensates (I). Now, using the C18reversed-phase HPLC column, we have been able to separate about 33 components in the linear fraction of a base-catalyzedp-tert-butylphenol-formaldehydecondensate.
20
30
50
40
TIME, MHVTES
Figure 3. Chromatogram of callxarene fraction of an acid-catalyzed p 4ert -butylphenol-formaldehyde condensate.
Table 11. Retention Times and Area Precents of the Linear Condensates of an Acid-Catalyzedp - tert -Butylphenol and Formaldehyde Reaction Product retention time, min
peak area
3.45" 3.976 4.74c 5.8ad 7.50e 9.79 10.99 12.88 14.23 16.07 19.44 22.81 24.44 25.88 27.32
3.1 3.4 4.4 5.8 6.6 6.6 0.44 5.6 0.20 5.9 5.4 5.0 0.14 4.6 0.12
%
retention time, min
peak area 90
retention time, min
28.68 31.20 32.14 33.49 35.58 37.44 38.40 39.14 40.71 42.16 43.50 44.70 45.84 46.88
4.7 3.9 0.42 3.5 3.5 3.0 0.49 2.7 2.5 2.3 2.0 1.8 1.6 1.4
47.86 48.77 49.63 50.45 51.21 51.94 52.63 53.28 53.90 54.49 55.04 55.57 56.05 56.54
ODimer. Trimer. Tetramer.
peak area 90
1.2 1.1
0.97 0.85 0.74 0.65 0.55 0.46 0.40 0.34 0.28 0.24 0.20 0.16
Pentamer. e Heptamer.
This demonstrates that the composition of these resins is considerably more complex than previous studies have indicated (2, 3). An acid-catalyzed p -tert-butylphenol-formaldehydereaction mixture was separated into a calixarene fraction and a linear condensate fraction by column chromatography. The calixarene fraction in each of two separations amounted to about 3.5% of the solid reaction product. We had not expected to find calixarenes in acid-catalyzed p-alkylphenolformaldehyde condensations based on studies reported be-
Anal. Chem. 1906, 58, 2072-2077
2072
p-tert-butylphenol-formaldehydelinear condensatesare given in Table 11. The maximum area percent at 6.58 min corresponds to the p-tert-butylphenol-formaldehydelinear hexamer. Future syntheses are planned to obtain pure compounds and to determine the effect of reaction conditions upon the amount of calixarenes produced and the distribution of linear condensates in either base-catalyzed or acid-catalyzed p-alkylphenol-formaldehyde condensations. The availability of additional linear condensates of known structure would make feasible the development of a quantitative HPLC analytical procedure.
J
--10
20
ao
40
60
60
70
TYE, Y M L O
Flgure 4. Chromatogram of linear fraction of an acid-cataiyzed ptert-butylphenol-formaldehyde condensate.
tween 1950 and 1961(6-8). The separation of the calixarene fraction into calix[4,5,6,7, and 8larenes is shown in Figure 3. There was also a major component and two or three minor components whose structures are unknown, but these did not appear to be linear condensates judging from retention times. The linear condensate fraction, which had eluted from the silica gel with an equivolume mixture of methanol, acetone, and chloroform, was separated into about 40 components on the C18reversed-phase HPLC column. Its chromatogram is shown in Figure 4. There were several very weak peaks that eluted between the major peaks a t about 5,11,15,18,21, etc. minutes. These might belong to a second homologous series of unknown structure. The retention times and area percents of the major peaks that correspond to methylene-bridged
ACKNOWLEDGMENT The authors thank C. D. Gutsche of Washington University, St. Louis, MO, and J. H. Munch of Petrolite Corp. for providing the calixarenes and p-tert-butylphenol-formaldehyde condensates used in this study. Registry No. (p-tert-Butylphenol).(formaldehyde) (copolymer), 25085-50-1;p-tert-butylphenol-formaldehydedimer, 102699-84-3;p-tert-butylphenol-formaldehydetrimer, 10269985-4; p-tert-butylphenol-formaldehydetetramer, 102699-86-5; p-tert-butylphenol-formaldehyde pentamer, 102699-87-6; p tert-butylphenol-formaldehydeheptamer, 102699-88-7; calix[4]arene derivative, 60705-62-6; calix[5]arene derivative, 8147522-1; dx[61arene derivative, 78092-53-2; &[7]arene derivative, 84161-29-5; calix[8]arene derivative, 68971-82-4. LITERATURE CITED (1) Ludwig, F. J.; Baiile, A. 0. AM/. Chem. 1984, 56. 2081-2085. (2) Gutsche, C. D. Acc. Chem. Res. 1983. 16, 161-170. (3) Gutsche, C. D. In Toplcs in Current Chemishy: Springer-Verlag: Beriln, 1984 Voi. 123, pp 6-9. (4) Bwiks, R. S.; Fauke, A. R.; Munch, J. H. US. Patent 4259464, 1981. (5) DeQroote, M.; Kelser, B. US. Patent 2499370, 1950. (6) Finn, S. R.; Lewis, G. J. J . SOC. Chem. Ind., London 1950, 69, 132- 133. (7) Hiyes,-B. T.; Hunter, R. F. J . Appi. Chem. 1958, 6 , 743. (8) Foster, H. M.; Hein, D. W. J . Org. Chem. 1961, 26, 2539-2541.
RECEIVED for review March 20,1986. Accepted April 23,1986. This work constitutes a partial fulfillment of Petrolite Corporation's commitment under National Science Foundation Industry/University Cooperative Research Grant No. CHE8216719 awarded to C. D. Gutsche of Washington University, St. Louis, MO.
Multivariate Relationships between Gas Chromatographic Retention Index and Molecular Connectivity of Mononitrated Polycyclic Aromatic Hydrocarbons Albert Robbat, Jr.,* Nicholas P. Corso, Philip J. Doherty,' and Durwood Marshall
Department of Chemistry, Tufts University, Medford, Massachusetts 02155
P-r of gas chm&o@raphic nt.nlkn Ind0x.e ( I ) was made by uae of a muttlvarlate hear rdatlarwhip between I and mX' -( dmuwrsI lXV,'XV, 5XvlOx, 1 x, and ' x ) for monoMcatad p0lycycHc aromatic hydrocarbons, on an SE-52 drtkrrcuy phme. A multkarlste hear cdbration model (Le., Inverse eatlmate) betweon predicted I and "'xt (Oxv,'x', 'xV, Ox, 'x, 2x, and ax)to predlct x estlmatcrr was used to vdldate the pr.dlct.d I . The differencenchd x values and prodktd x was approxknately 1%
.
-
0003-2700/86/0358-2072$01.50/0
The facile nitration (NOz) of polycyclic aromatic hydrocarbons (PAH) in the presence of nitrating agents and literature reporta of the mutagenic (1-3)and carcinogenic activity (4,5) of some of the nitrated PAH have produced considerable interest in the development of analytical methods for the determination of these compounds in environmentally significant samples. Recent reviews (6, 7) have summarized current state-of-the-art analytical methods. We have shown that high-resolutioncapillary gas chromatographic separation on fused silica SE-52 column has unmatched separation capability for nitrated PAH in complex environmental samples
0 1986 American Chemical Society
