Anal. Chem. 1003, 55, 270-275
270
Isocratic Nonaqueous Reversed-Phase Liquid Chromatography of Carotenoids H. J. C. F. Nells and A. P. De Leenheer’ Laboratorla voor Medlsche Blochemle en voor Klinlsche Analyse, Rijksuniverslteit Gent, Harelbekestraat 72, 8-9000 Gent, Belgium
Nonaqueous reversed-phase chromatography (NARP) Is shown to be a useful approach for the separatlon of complex carotenold mlxtures. The rationale behlnd the NARP concept Is the beneficlal effect of enhanced sample solublllty In totally organlc eluents, on chromatographlc efflclency, recovery, sample capacity, and column IHetlme. Advantage was taken of the unlque propertles of a hlghly retentlve reversed-phase materlal to chromatograph a mixture of nlne carotenolds In a 16-32 mln run, without the need for gradlent elution. Eluent strength and selectlvlty were optlmlzed by using &carotene, echlnenone, and two vltamln A derlvatlves as test substances. Resolution of polar, substltuted derlvatlves was greatly affected by the presence of methanol. NARP-based systems have been successfully applled to carotenoid proflllng In blologlcal materials of human, anlmal, and vegetable orlgln.
The majority of studies on carotenoids have been concerned so far with the isolation and identification of these pigments in plants (1) and lower animals (2). Recent evidence suggests that some carotenoids, in addition to their capability to act as vitamin A precursors (provitamin A), may be involved in other biochemical processes in higher animals and humans as well. For example, a protective effect of @-caroteneagainst cancer has been postulated (3). This hypothesis, though still somewhat speculative, has not failed to arouse new interest in the fate of carotenoids in living organisms. Yet, analytical developments in this area have not kept pace with this revival of interest. Although modern liquid chromatography (HPLC) is gradually emerging as a method of choice for carotenoid separation, no systematic study of its merits and shortcomings has yet been conducted. Rather, a diversity of HPLC systems has been devised for specific applications, often of limited scope, such as the determination of p-carotene and/or some selected derivatives in pizza ( 4 ) , oil, margarine, and milk (5-9), orange and citrus juice (10-14), tomatoes (15),and tobacco leaves (16,17). HPLC has proven particularly useful for photosynthetic pigment profiling in algae and higher plants (18-28). One paper deals with the determination of carotenoids in samples of geochemical interest (29). No reports on carotenoid patterns in higher animals (e.g., blood serum) have appeared as yet. ”Total carotene” levels are still determined spectrophotometrically as part of a diagnostic test in clinical laboratories. A number of straight-phase HPLC systems, based on silica (9, 16, 17, 20-22, 28, 29), alumina (4, IO),or magnesia (11)) have been reported. Silica is of particular benefit for the separation of cis-trans isomers and diastereoisomers (28) but fails to distinguish between positional isomers such as a-and @-carotene(28,30). This support has also been suspected to catalyze carotenoid degradation (18). In keeping with their general popularity, reversed-phase (RP) materials are likely to supersede their straight-phase counterparts for carotenoid chromatography in the near future. A drawback of several “conventional” R P materials however is their relatively low retentivity toward the more polar derivatives (xanthophylls). 0003-2700/83/0355-0270$0 1.50/0
As a result, substantial amounts of water have to be included in the eluent to ensure sufficient solute retention (12-14,18, 19,23,24,26,27,31,32). This may have a deteriorating effect on peak shape and even cause partial solute precipitation on the column. In fact, in order to cover the whole range of polar and nonpolar carotenoids, many systems employ gradient elution (18, 19, 23-27, 31, 32). A few years ago, the concept of nonaqueous reversed-phase chromatography (NARP) on highly retentive packing materials was recommended as a useful approach for the chromatography of nonpolar compounds (33). The commercial Zorbax ODS material (Du Pont, Wilmington, DE) is a unique support for this purpose. General advantages of NARP include optimal sample solubility and hence minimal risk for solute precipitation on the column as well as increased sample capacity, excellent chromatographic efficiency, and prolonged column lifetime. NARP has found only limited application, e.g., for the separation of glycerides (34), hydrocarbons (33)) vitamin K analogues (35,36), polystyrene (33,and fat-soluble vitamin standards, including vitamin A and @-carotene(33), 38). Although nonaqueous eluents, in conjunction with other reversed-phase packing materials, have been occasionally used for the separation of the least polar, unsubstituted carotenes (15,30), the potential of the unique NARP approach has never been exploited for the differentiation of complex carotenoid mixtures, particularly not in biological materials. This paper presents a systematic investigation into the use of NARP on Zorbax ODS for the chromatography of carotenoids as well as some vitamin A derivatives. It is the objective of this study to demonstrate the superiority of NARP over conventional R P chromatography with semiaqueous mobile phases in terms of selectivity, sample solubility, column stability, chromatographic convenience (no gradient), and application to biological materials of human, animal, and vegetable origin.
EXPERIMENTAL SECTION Liquid Chromatography (HPLC). The liquid chromatograph used was equipped with a constant flow pump (Model 5020, Varian Associates, Walnut Creek, CA), a Valco CV-6-UHPa-N6O sample valve with a 50-rL loop (Valco Instrument Co., Houston, TX), and a Varichrom variable wavelength detector (Varian Associates, Walnut Creek, CA), set at 450 nm. Two types of stainless steel columns, filled with 7 - ~ m Zorbax ODS (Du Pont, Wilmington, DE) were used. One wm prepacked (25 X 0.46 cm) and purchased from Du Pont, whereas the other (15 x 0.32 cm) was home-packed under conditions described elsewhere (39). Mobile phases consisted of mixtures of analytical grade acetonitrile and dichloromethane, methanol, tetrahydrofuran, ethyl acetate,chloroform or diisopropyl ether. The columns were operated at ambient temperature and flow rates ranged from 1 to 2 mL/min. Experiments designed to optimize the eluent composition were conducted on the 15 X 0.32 cm column, which is characterized by faster equilibration. The eluent composition was varied and capacity ratios k’ (k’ = ( t -~t o ) / t o , where t R and to are the retention times of the compound of interest and an unretained peak, respectively) of two selected carotenoids (pcarotene and echinenone) were determined. In addition,two more test substances, i.e., retinol and retinyl propionate were included. The latter proved to be good model compounds to substitute for 0 1883 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983 271
Table I. Comparison of Two Reversed-Phase Materials for the Chromatography of Retinol and p-Carotene
eluent composition
a
capacity ratios ( h ’ ) on Ultrasphere ODS on Zorbax ODS retinol p-carotene retinol p-carotene
methanol methanol: water (97:3, v/v) acetonitrile
0.86
acetonitri1e:dichloromethane(85:15, v/v) acetonitrile :dichloromethane (75 :25, v/v)
0.80
1.49 1.46 0.51
11.20a 39a >40 13.70 5.90
0.90
> 25
2.46 1.40
12.20 5.1 0
0.89
Distorted peak.
some polar carotenoids that were of short supply. Final separations, under optimized conditions, of complex mixtures were carried out on the commercial 25 X 0.46 cm column. Nonaqueous reversed-phase chromatography was also compared to conventional chromatographyon a 15 X 0.46 cm Ultrasphere ODS column (Altex, Berkeley, CA). Reference Samples. at-Carotene, @-carotene,and lycopene were purchased from Sigma (St. Louis, MO). Lutein and zea xanthin came from SarsynBx (Merignac, France) and retinol from Fluka (Buchs, Switzerland). Retinyl propionate was obtained from AEC (Commentry, France)l. All other substances, including a* taxanthin, canthaxanthin, &cryptoxanthin, echinenone, and torulene were gifts from Hoffmann-La Roche (Basle,Switzerland). Stock solutions were prepared in the chromatographic solvent and stored at -20 O C , protected from light. Structural formulas of carotenoids studied are presented in Figure 1. Extraction of Carotenoids from Biological Samples. Serum samples (0.5 mL) of human volunteers were extracted with 3 mL of n-hexane. The organic layer was isolated and evaporated to dryness under reduced pressure and the residue reconstituted with 200 pL of chromatographic solvent. A 50-pL aliquot was injected on the column. Samples of adult brine shrimp Artemia were homogenized in the chromatographic solvent with a Potter-Elvehjem homogenizer. After centrifugation, 5O-kL aliquots of the supernatant were directly injected on the column. Alternatively, homogenization was carried out in acetone. Plant materials, e.g., the blue-green alga Spirulinu, were treated identically as the Artemia samples.
1
2 0
3
4
5
7
8
RESULTS AND DISCUSSION Conventional vs. Nomqueous Reversed-Phase Chromatography. The close biochemical relationship that exists between retinol and @-caroteneinitially led us to investigate reversed-phase chromatography as a possible tool for the simultaneous determination of both compounds. A comparison between a “conventional” material (Ultrasphere ODs) and Zorbax ODS revealed the latter’s superiority for this purpose. This particular material displays higher retentivity toward retinol and slightly less toward @-carotenethan does the Ultrasphere support (Table I). While on Ultrasphere ODS retinol is readily chromatographed by acetonitrile, methanol, or mixtures of methanol-water, stronger eluents, containing a nonpolar modifier, are required to yield comparable retention on Zorbax ODs. Table I shows that this phenomenon is most pronounced for acetonitrile-based eluents. The same type of eluents also yields minimal selectivity for the pair retinol/@carotene. On the conventional material, similar mobile phases result in a selectivity factor a (a = V 2 / k ‘ , )about twice as high as the one obtained on Zorbax ODs. This implies that retinol is still relatively strongly retained in conjunction with the nonaqueous eluents that are required to elute the very nonpolar @-carotene. As evidenced from Table I, Zorbax ODS thus would allow an isocrutic separation of retinol and @carotene within a reasonable time. With 20% (v/v) dichloromethane in acetonitrile, k’ values of 1.1 and 7.7, respectively, were obtained. On Ultrasphere ODS and on most other conventional reversed-phase supports, gradient elution would be necessary to bring both k’ values in the range 1-8.
9
10
Flgure 1. Structural formulas of carotenoids, arranged in order of elution from a Zorbax ODS column, as represented in Figure 5: 1, lutein; 2, zeaxanthin; 3, canthaxanthin;4, @-cryptoxanthin;5, echinenone; 6, lycopene; 7, toruiene; 8, a-carotene; 9, @-carotene;(10, astaxanthin, not chromatographable in this system).
In view of its unique ability to chromatograph two compounds of widely divergent polarity isocratically, Zorbax ODS looked very promising as a basis for an isocratic separation of the entire spectrum of carotenoids, ranging from polar xanthophylls to nonpolar carotenes. It is obvious that from the standpoint of sample solubility of nonpolar compounds, NARP is superior over conventional reversed-phase chromatography using methanol, acetonitrile, or semiaqueous eluents. Carotenoids are only sparingly soluble in those typical reversed-phase solvents and any water addition further reduces this solubility. The distorted peak and low efficiency obtained for @-caroteneusing methanol indicates poor sample solubility and possibly even partial precipitation on the column. The risk for incomplete recovery increases upon water addition, and indeed peak shape further deteri-
272
ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983
Table 11. Effect of Different Organic Modifiers on Retention ( k ' ) and Selectivity ( a ) A. Eluent Consisting of Acetonitrile and 1 8 % ( v / v ) of Organic Modifier, No Methanol Presentmodifier THF diisopropyl ether chloroform dichloromethane ethyl acetate
kIla
kIza 1.00
0.85 0.86 0.96 1.22 1.47
b
k', a
1.18
3.21 2.99 3.69 5.84 7.74
01
1.00 1.00 1.00
0.86 0.96 1.22 1.47
1.00
b
k',O
a2
7.97 6.10 5.83 8.69 14.10
2.48 2.04 1.58 1.49 1.82
B. Eluent Consisting of Acetonitrile and 1 8 % (v/v) of Organic Modifier but Containing 8% Methanol (v/v)
modifier THF diisopropyl ether chloroform dichloromethane ethyl acetate a
k ' , retinol:
C
b
kfla
k',O
0 1
0.64 0.63 0.64 0.68 0.89
0.85 0.80 0.90 0.95
1.33 1.27 1.40 1.39 1.32
k ' , retinyl propionate:
1.17
k ' , echinenone, k ' , p-carotene.
@I
=
k',a
k',a
2.52 2.28 2.81 3.34 4.66
6.26 5.45 5.32 6.53 10.13
k'Jk',,
a2
2.48 2.39 1.89 1.96 2.17
= k',/k',.
0 1 ~
CH20-R
where R = -H, retinol; R = -C(=O)CH,CH,, retinyl propionate.
i 15.
I
x)
XI
30
LO
I
,C*,CI,
Figure 2. Plot of k' values of p-carotene (1) and retinol (2) vs. percentage (% vlv) of dichloromethane in acetonitrile.
orated and retention time became excessively long in the semiaqueous eluent. Reduction of the chain length of the bonded phase, e.g., use of a hexyl or octyl instead of an octadecyl type material, had an unfavorable effect, since shorter chain supports required higher water concentrations in the eluent to yield sufficient retinol retention. Optimization of Eluent Strength. Binary and ternary solvent mixtures were tested as mobile phases. A typical NARP eluent consists of a polar basis, usually acetonitrile, a nonpolar modifier, e.g., dichloromethane to adjust solvent strength, and occasionally a small amount of a third solvent to optimize selectivity. The nonpolar modifier basically acts as a solubilizer. Dichloromethane unlike acetonitrile and methanol is a good solvent for carotenoids and vitamin A derivatives. Accordingly capacity ratios ( k)' decrease when solubility and solvent strength increase, as illustrated in Figure 2. Alternatively, other nonpolar solvents were tried (Table 11) but none of them was found to have advantages over dichloromethane in terms of chromatographic efficiency or selectivity (see below). Several anomalies in the relative order of eluotropic strengths were observed. Solvent polarities, as calculated for straight-phase systems, cannot be used to predict elution strength in NARP. For example, solvent classification according to the reciprocal value of P' (40) would theoretically yield the following order of eluotropic strength: diisopropyl ether > dichloromethane > tetrahydrofuran >
1
5
x)
15
20 %CtJG-
Flgure 3. Dependence of capacity ratios (k') of retinol (l), retinyl propionate (2), and the selectivity factor a (3) on percentage ( % v/v) of methanol in the eluent (20 % , v/v dlchloromethane in acetonitrile).
chloroform > ethyl acetate. Table I1 shows that this order could not be confirmed experimentally. Surprisingly, even typical reversed-phase solvents behaved in an unpredictable manner as well. Substitution of acetonitrile by methanol consistently decreased retention of all test substances (Table 11). Hence methanol acts as a stronger solvent than acetonitrile, although the latter would be expected to interact preferably with the polyene chain of carotenoids. This phenomenon has been observed in nonaqueous medium for other compounds as well (41). However, water addition seems to restore the normal order of eluotropic strength (acetonitrile > methanol) (41). Optimization of Eluent Selectivity. A binary mobile phase, consisting of acetonitrile and dichloromethane, failed to resolve the solute pair retinol-retinyl propionate (a= 1.00) (Table 11). In an attempt to improve the selectivity, dichloromethane (selectivity group V) was substituted by other organic solvents belonging to different selectivity groups (40), i.e., diisopropyl ether (group I), tetrahydrofuran (group III), ethyl acetate (group VI), or chloroform (group VII) (Table 11). Only tetrahydrofuran displayed improved selectivity characteristics (a = 1.18) and afforded partial resolution of retinol/retinyl propionate, even when added in small amount to acetonitrile-dichloromethane mixtures. The most significant selectivity enhancement, however, was obtained when methanol (selectivity group 11) was incorpo-
ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983
273
3
Y
t
+ io
15
/
rated in the eluent. As demonstrated in Table 11, this effect was independent of the nature of the nonpolar modifier. Figure 3 illustrates the efffect of methanol concentration 011 capacity ratios (12 )' of retinol and retinyl propionate, as well as on selectivity (a). The capacity ratio of retinol decreases at a faster rate than doe8 the k'value of retinyl propionate, which results in a gradual selectivity improvement for this pair when the methanol content of the eluent rises. Similarly, methanol preferably interacts with echinenone and to a lesser extent with @-carotene(Figure 4). It is attractive to speculate that hydrogen bonding underlies this selectivity enhancing effect. Retinol, unlike retinyl propionate, is a hydrogen donor and accordingly is likely to form hydrogen bonds with methanol. On the other hand, methanol can also act as a hydrogen donor and thuia interact with the ketocarotenoid echinenone but not with the unsubstituted 6-carotene. The positive effect of tetrahydrofuran may be rationalized on the same basis. As such, T H F is superior to other nonpolar modifiers but the presence of methanol overrides this advantage, as shown in Table IIB. It is not clear why other hydrogen acceptors such ai3 diisopropyl ether failed to improve selectivity. Separation of Nine Ciarotenoids (Standards). Eluents both with and without methanol were tested on their capability to separate complex carotenoid mixtures. A representative chromatogram 11sshown in Figure 5. Total chromatographic analysis time is 32 min but this can be reduced to half with minor loss in resolution by doubling the flow rate. The major improvement of NARP over conventional reversed-phase chromatography for carotenoids lies in its ability to chromatograph both polar and nonpolar derivatives isocratically. Disadvantages of gradient elution are well-known and include the need for sophisticated, expensive equipment, the often poor reproduciblility, its incompatibility with sensitive detector settings, which limits detectability in biological materials, and the need for column reequilibration between runs. As indicated in Table 111which lists capacity ratios of nine carotenoids in four differeint mobile phases, pronounced selectivity differences were encountered depending on the presence or absence of methanol. As expected, the presence of methanol shifted k' values of hydroxylated xanthophylls and to a lesser extent ketocarotenoids to much lower values, while it little affected retention of nonpolar carotenes, i.e., lycopene, torulene, a-carotene, and @-carotene.Even reversals of elution order were observed, e.g., for the pairs canthaxanthinlzeaxanthin and @-cryptoxanthinlechinenone. This typical retention behavior may serve as a useful criterion for
1 I
20 o/.CH,CH
!
0
I
,
L
,
,
,
8
12
16
K)
U
Hi;, MIN.
28
Flgure 5. Separation of Carotenoid standards on Zorbax ODS (25 X 0.46 cm). The mobile phase consisted of acetonitrlle:dichloromethane:methanol (70:20:10, v/v). Flow rate was 1 mL/min and detection was at 450 nm. Peak identification: 1, lutein; 2, zeaxanthin; 3, canthaxanthin; 4, @-cryptoxanthin;5, echinenone; 6, lycopene; 7, torulene; 8, a-carotene; 9, @-carotene.
Table 111. Capacity Ratios ( k ' ) of Carotenoids on Zorbax ODS in Different Eluents capacity ratios lutein zeaxanthin canthaxanthin p-cryptoxanthin echinenone lycopene torulene 01 -carotene P-carotene
3.9 4.7 3.3 8.1 6.9
6.2 8.0
11.5 12.5
1.3 1.4 2.0 4.5 5.3 6.3 8.2 11.4
3.6 4.2 3.0 8.9 7.8
8.5 11.3 16.2 17.6
1.2 1.4 1.9 5.6 5.0 1.5 10.2 14.7 16.3 Aceto-
12.4 a Acetonitrile:dichloromethane,80:20, v/v. nitri1e:dichloromethane :methanol, 70 :20 :10, vjv. Acetonitrile: Acetonitri1e:ethyl acetate, 80:20, v/v. ethyl acetate:methanol, 70 :20:10, v/v.
peak identification. Eluents not containing methanol yielded better resolution of zeaxanthin and lutein but at the expense of incomplete separation of lycopene and echinenone. The opposite situation was found with methanol present. Astaxanthin displayed an erratic chromatographic behavior in that this compound gave excessively asymmetric peaks and extremely low theoretical plate counts. The presence of two relatively acidic hydroxyl groups in its structure was believed to account for these phenomena. Evidence supporting this assumption was obtained from the observation that addition of organic acids, e.g., formic acid (1.5%, v/v), to the eluent restored normal peak shape and column efficiency. Applications. In order to demonstrate the applicability of the present isocratic NARP system for carotenoid separation to biological materials, we analyzed samples of human, animal, and vegetable origin. A carotenoid profile of human serum is shown in Figure 6. Major carotenoids in serum include a-carotene, @-carotene,@-cryptoxanthin,lutein, and lycopene. It is obvious that a total carotene spectrophotometric determination is not a useful indicator of the 0-carotene level. To the best of our knowledge, this is the first report on a liquid chromatographic separation of individual caro-
274
ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983
;JLiL
9501
AL50
I
1 I
2
3
I
I
Figure 6. Carotenoid profile of a human serum extract. Chromatographic condkions are given in Flgure 5. Peak aentification: 1, lutein; 2, zeaxanthin; 3, @-cryptoxanthin;4, lycopene; 5, a-carotene; 6, @carotene. 80
Figure 8. Carotenoid profile of a homogenate (in acetonitriie:dichloromethane:methanoi 70:20: 10, v/v) of a blue-green alga (Spiru/ina). Chromatographic conditions are given in Figure 5. Peak identification: 1, unknown; 2, zeaxanthin; 3, unknown; 4, @-cryptoxanthin;5, echinenone; 6, unknown; 7, @-carotene.
Quantitative work on biological samples is currently in progress.
ACKNOWLEDGMENT The authors are indebted to G. Brubacher, A. Jenni, F. Leuenberger, and D. Hornig, all from Hoffmann-La Roche, Basle, Switzerland, for supplying carotenoids and to the Artemia Reference Center, State University of Gent, Belgium, for supplying Artemia and Spirulinu samples.
2 L
Registry No. @-Carotene,7235-40-7;Zorbax ODs, 69345-17-1; acetonitrile, 75-05-8; dichloromethane, 75-09-2; methanol, 67-56-1; tetrahydrofuran, 109-99-9;echinenone, 432-68-8; retinol, 68-26-8; retinyl propionate, 7069-42-3; a-carotene, 7488-99-5;lycopene, 502-65-8; lutein, 127-40-2; xeaxanthin, 144-68-3; astaxanthin, 472-61-7; canthaxanthin, 514-78-3; @-cryptoxanthin, 472-70-8; torulene, 547-23-9. 0
L
8
12
I6
20
24
28 MIN.
Carotenoid profile of an acetone homogenate of adult Artemia. Chromatographic conditions are given in Figure 5. Peak identification: 1, zeaxanthin; 2, canthaxanthin; 3, @-cryptoxanthin;4, echinenone; 5, &carotene.
LITERATURE CITED
Figure 7.
tenoids in blood. The present system, in conjunction with a simple hexane extraction, provides a useful basis for an assay of various carotenoids. In case there is a need for a simultaneous determination of retinol and @-carotene,this can be accomplished as well, provided the detection wavelength is altered during the run. Marine organisms and algae are very rich carotenoid sources. Figures 7 and 8 represent chromatographic patterns of extracts of adult brine shrimp (Arternia)and of a blue-green alga (Spirulina), respectively. Peak identification is still tentative, i.e., based on cochromatography with reference standards but not further confirmed by spectral data. At this point the same column has been in use for approximately 1 year, without appreciable loss in efficiency, despite the injection of hundreds of biological extracts. It was noted however that k ’values of carotenoids slowly decreased over this time period, as can be inferred from a comparison of Figures 5-8. This decrease was most pronounced during the first months and gradually leveled off. Whether this phenomenon is typical for NARP or would apply to “conventional” reversed-phase chromatography on this column as well remains to be established.
(1) Goodwin, T. W. “The Biochemistry of the Carotenolds, Volume I, Plants”; Chapman and Hall: London, 1980. (2) Simpson, K. L.; Katayama, T.; Chlcester, C. 0. I n “Carotenoids as Colorants and Vitamln A Precursors”; Bauernfeind, Ed.; Academic Press: New York, 1981;pp 463-538. (3) Peto, R.; Doll, R.; Buckley, J. D.; SDorn, M. B. Nature (London) 1978. 290, 201-208. (4) Kamel, B. S.; Bueno, M. Lebensm.-Wiss. Techno/. 1980, 13, 134-137. (5) Landen, W. 0.; Eltenmiller, R. R. J. Assoc. Off. Anal. Chem. 1979, 62,283-289. (6) Thompson, J. N.; Maxwell, W. B. J. Assoc. Off. Anal. Chem. 1977, 60,766-771. (7) Maruyama, T.; Ushigusa, T.; Kanematsu, H.; Nliya, I.; Imamura, M. Shokuhln Eiselgaku Zasshi 1977, 18, 487-492. Chem. Abstr. 1978, 88, 188253d. (8) Mankel, A. Dtsch. Lebensm.-Rundsch. 1979, 7 5 , 77-85. (9) Thompson, J. N.; Hatina, 0 . ; Maxwell, W. B. J. Assoc. Off. Anal. Chem. 1980, 6 3 , 894-898. (10) Reeder, S. K.; Park, G. L. J. Assoc. Off. Anal. Chem. 1975, 5 8 , 595-598. (11) Stewart, I. J. Assoc. Off. Anal. Chem. 1977, 6 0 , 132-136. (12) Benk, F.; Treiber, H.;Bergmann, R. Riechst. Aromen, Koerperflegem. 1978, 10, 216-221. (13) Caiabro, G.;Micali, 0.;Curro, P. Essenze Derlv. Agrum. 1978, 4 8 , 359-367. Chem. Abstr. 1980, 93,61951. (14) Calabro, G.; Mlcali, G.; Curro, P. Attl-Conv. Naz. Olii Essenz. Sul Derlv. Agrum. 1978, 7 , 171-179. Chem. Abstr. 1980, 93, 168194m. (15) Zakaria, M.; Simpson, K.; Brown, P. R.; Krstulovic, A. J. Chromatogr. 1979, 176, 109-117. (16) Schlotzhauer, W. S. rob. Scl. 1978, 22, 44-45. 1171 De Jona. D. W.: Woodlief. W. G. J. Auric. Food Chem. 1978. 26. 1281- 1588. (18) Braumann, T.; Grimme, L. H. Eiochlm. Eiophys. Acta 1981, 6 3 7 , 8-17. .
1
I
’
Anal. Chem. 1983, 55,275-280 (19) Eskins, K.; Scholfield, C. R.; Dutton, H. J. J. Chromatogr. 1977, 135, 217-220. (20) Abaychi, J. K.; Riley, J. P. Anal. Chlm. Acta 1979, 707, 1-11, (21) Stransky, H. 2.Naturforsch., C 1978, 33C,836-840. (22) Iriyama, K.; Yoshiura, IM.; Shirakl, M. J . Chromatogr. 1978, 754, 302-305. (23) Braumann, T.; Grirnme, L. H. J. Chromatogr. 1979, 770, 264-268. (24) Braumann, T.; Mahro, B.; Grimrne, L. H. Ber. Dtsch. Bot. Ges. 1978, 97, S. 583-587. (25) Eskins. K.; Harris, L. Photochem. Photoblol. 1981, 33, 131-133. (26) Esklns, K.; Dutton, H. J. Anal. Chem. 1979, 57, 1885-1888. (27) Davies, D.; Holdsworth, 13. S. J . Llq. Chromatogr. 1980, 3, 123-132. (28) Flksdahl, A.; Mortensen, J. T.; LiaaenJensen, S. J . Chromatogr. 1978, 757, 111-117. (29) Hajlbrahim. S. K.; Tlbbens, P. J. C.; Watts, C. D.;Maxwell, J. R.; Eglinton, G.; Colin, H.; Gulochon, G. Anal. Chem. 1978, 50, 549-553. (30) Pfander, H.; Schurtenberger, H.; Meyer, V. R. Chhla 1980, 34, 179-180. (31) Matus, Z.; Baranyal, M.; ‘T6th, 0.; Szabolcs, J. Chromatographla 1981, 74, 337-340.
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Taylor, R. F.; Ikawa, M. I n “Methods in Enzymology”; McCormick, Wrlght, Eds.; Academic Press: New York, 1980; Voi. 67, pp 233-281. Parris, N. A. J. Chromatogr. 1070, 157, 161-170. Parris, N. A. J . Chromatogr. 1978, 149, 815-824. Haroon, Y.; Shearer, M. J.; Barkhan, P. J . Chromatogr. 1980, 200, 293-299. (38) Haroon, Y.; Shearer, M. J.; Barkhan, P. J. Chromatogr. 1981, 206, 333-342. (37) Armstrong, D. W.; Bul, K. H. Anal. Chem. 1982, 54, 708-708. (38) Landen, W. 0. J. Chromatogr. 1981, 271, 155-159. (39) De Leenheer, A. P.; Nelis, H. J. C. F. J. Pharm. Scl. 1979, 68, 1527- 1529. (40) Snyder, L. R. J. Chromatogr. Scl. 1978, 76, 223-234. (41) Colin, H., personal comrnunicatlon.
RECEIVED for review August 23, 1982. Accepted October 25, 1982. This work was supported by the National Fund for Scientific Research (NFWO) through a grant to H.J.C.F.N.
Dynamic Coupled-Column Liquid Chromatographic Determination of Ambient Temperature Vapor Pressures of Polynuclear Aromatic Hydrocarbons W. J. Sonnefeld and W. H. Zoller Department of Chemistry, b’nlversl@ of Maryland, College Park, Maryland 20742
W. E. May” Organic Analytical Research Division, National Bureau of Standards, Building 222, A 1 13, Washington, D.C. 20234
A method Is descrlbed far the direct coupllng of a gas saturatlon system to a hlgh-performance llquld chromatograph for the detennlnatlon of the vapor pressure of organic compounds In the range of 102-10-6 Pa. The method has been used to determine the vapor pressures of selected polynuclear aromatlc hydrocarbons In the ambient temperature range between 10 and 50 OC. The vapor pressures (In pascals f standard deviation) at 25 O C as determined by this method are as follows: naphthalene, 10.4 f 0.2; naphthalene-d,, 10.4 f 0.1; acenaphthylene, (8.9 f 0.2) X IO-’; acenaphthene, (2.9 f 0.9) X IO-’; fluorene, (8.0 f 0.2) X lo-’; phenanthrene, (1.61 f 0.04) X phenanthrene-d,,, (1.92 f. 0.05) X IO-’; anthracene, (8.0 f 0.2) X lo4; fluoranthene, (1.23 f 0.07) X pyrene, (6.0 f 0.2) X benz[a].. anthracene, (2.8 f 0.1) >C These values are generally In good agreement with values extrapolated from determlnatlons made at hlgher temperatures reported In the literature.
During the past decade increased concern has developed concerning the environmental impact of anthropogenic trace organic compounds. One factor which governs the transport of organic compounds, both in the workplace and in the general environment, is the volatility of these compounds. The vapor pressure of a compound will determine, in part, the rate of evaporation from indust,rialprocesses or waste sites, as well as the tendency for the compound to adsorb on particulate matter present in the environment. Vapor pressure data can be combined with aqueous solubility data to calculate Henry’s law constants, which can be used to predict the equilibrium of dissolved organics in water with their atmospheric concentrations (I). The determination of the vapor pressures of
organic compounds in the ambient temperature range (0-50 “C) is therefore important for the development of environmental transport models as well as for the assessment of possible health hazards present in the atmosphere. While many methods have been described in the literature for the measurement of vapor pressure (2),no single method is applicable for the entire vapor pressure range of environmentally significant compounds [ lo5 to lo4 Pa (-760 to mmHg)]. Literature methods usually involved a measurement of the mass loss or gain at temperatures well above the ambient range. The gas saturation method has been shown to be applicable for compounds having vapor pressures lower than lo2Pa (1 mmHg) and is generally used in the ambient temperature range. This method was first proposed in 1845 (3) but was not used extensively until the advent of modern chromatographic analytical techniques. The gas saturation method involves the production of a saturated vapor phase by passing an inert gas through a column packed with either the pure compound of interest or with an analyte-coated inert support. The analyte is collected from a known volume of the saturated vapor using impingers, sorbents, or cryogenic traps and the amount of analyte determined by some suitable method. In the late 1960s and early 1970s, Spencer and Cliath ( 4 , 5 ) used sand and/or soil with hexane impingers for the coated support materials and subsequent collection of several pesticides. They utilized gas chromatography (GC) for the quantification of the collected analyte. Pella (6) utilized Chromosorb G (60/80 mesh) coated columns to generate saturated vapors of several explosives, charcoal adsorption traps for collection, and GC to measure the collected analyte. More recently, Westcott et al. (7) developed a “micro-scale”procedure by coating polychlorinated biphenyls (PCB’s) on 3-mm spherical glass beads
Thls article not subJectto U S . Copyright. Publlshed 1983 by the American Chernlcal Society
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