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(18) Van der Maeden, F. P. 6.; Biemand, M. E. F.; Janssen, P. C. G. M. J. Chromatogr. 1978, 149, 539-552. (19) Otsuki, A,; Shiraishi, H. Anal. Chem. 1979, 5 1 , 2329-2332. (20) Allen, M. C.; Linder, D. E. J. A m . Oil Chem. SOC. 1981, 58. 950-957. (21) McClure, J. D. J. Am. 011 Chem. SOC. 1982, 5 9 , 364-373. (22) Bogatzki, B. F.; Lippmann, H. Acta Polym. 1083, 3 4 , 219-223. (23) Ahel, M.; Giger, W. Anal. Chem. 1985, 5 7 , 1577-1583. (24) Ahei, M.; Giger, W.; Molnar-Kubica, E.; Schaffner, C. I n "Analysis of
Organic Water Pollutants"; Angeiettl, G., Bjmseth, A,, Eds.; Reidel Publishing Co.: Dordrecht, Holland; 1984; pp 280-288. (25) OECD "Guidelines for Testing of Chemicals"; Organization for Economic Co-operation and Development, Parls, 1981. (26) Grob, K.; Grob, G.; Bium, W.; Walter, W. J. Chromatogr. 1982, 2 4 4 , 197-208.
(27) Schick, M. J. "Nonionlc Surfactants"; Marcel Dekker: New York, 1967; Voi. 1 Surfactant Science Series. (26) Giger, W.; Ahei, M.; Schaffner, C. I n "Analysis of Organic Water
Pollutants"; Angeietti. G., Bjmseth, A., Eds.; Reidel Publishing Co.: Dordrecht, Holland, 1984; pp 91-109. (29) Eadsforth, C. V.; Moser, P. Chemosphere 1984, I.?, 1459-1475.
RECEIVED for review March 5,1985. Accepted June 14, 1985. This project was supported in part by the Swiss National Science Foundation (Nationales Forschungsprogramm 7 D, research project on "Organic Contaminants in Sewage Sludge").
General Indirect Detection of Nonelectrolytes in Liquid Chromatography by Solubility Enhancement Sujit Banerjee
Safety and Environmental Protection Division, Brookhaven National Laboratory, Upton, New York 11973
The moblle phase in a reversed-phase liquld chromatographic system Is saturated with a lipophilic addltive of low moblllty In the system. The column is equliibrated with the amended mobile phase, and the effluent Is monitored for a property characteristic of the additive. When an analyte enters the system, It alters the local solvent strength of the mobile phase and increases the solublllty of the addltlve. The presence of the analyte Is therefore indlrectly signaled by the enhanced concentration of the additive. The technique Is Illustrated by the spectrophotometric detectlon of methylene chiorlde and ethyl acetate at 260 nm, where both compounds are transparent.
When an analyte elutes from a liquid chromatographic column, it alters the local composition of the mobile phase. If this compositional change is measured by monitoring a suitable component of the medium, then a means to indirectly quantitate the analyte results. The technique has been most often applied to the detection of ions, where a suitable property of the counterion can be monitored. In ion chromatography the counterion typically derives from the buffer (1,2),whereas in ion-pair work, it usually forms a part of the ion interaction reagent (3, 4). Extension of indirect detection to nonelectrolytes is more difficult, since the convenience of a responsive counterion is lost. Gnanasambandan and Freiser (5)devised a system where the complexation between aliphatic alcohols and methylene blue was used to indirectly detect the former. The transparent alcohols led to a signal by altering the distribution of the dye between the stationary and the mobile phase. Bobbitt and Yeung (6) have presented another approach where the optical activity of the mobile phase was amended by the analyte. The present paper describes a method where an analyte induced change in the solvent strength of the mobile phase is used to indirectly detect the analyte.
EXPERIMENTAL SECTION The chromatographic assembly consisted of a Varian 5560 instrument coupled to a Varian UV 200 detector and a Hew-
lett-Packard 3390A integrator. Injections were made with a Rheodyne 7125 valve fitted with a 10-pL loop. A 150 x 4 mm Micropak MCH-N-Cap5 (reversed-phasepCIs) column completed the system. The methanol and water used were of HPLC quality. All other compounds were of reagent grade and were used as received. The mobile phase was saturated with toluene by stirring the two liquids together for 36 h and then allowing the phases to separate. A layer of toluene was maintained over the mobile phase to ensure continued saturation of the latter. Analyte solutions were prepared in toluene-free mixtures of methanol and water of the same composition used in the mobile phase.
RESULTS AND DISCUSSION The chromatography of an analyte is governed primarily by the interaction between analyte, column, and mobile phase. During the period in which the analyte is contained in the mobile phase, it alters the solvent strength of the mobile phase to a small degree. If this change can be determined, then an indirect method for detecting the presence of the analyte will be at hand. One way to achieve this goal would be to saturate the system with a material of low solubility in the mobile phase and to monitor the presence of this material. Now, if the analyte is such that it enhances the solubility of the additive, then the elution of the analyte should be signaled by an increase in concentration of the additive. The phenomenon is demonstrated in Figure 1 with ethyl acetate as the analyte and toluene as the additive. Ethyl acetate can be detected at 210 nm, and Figure 1B illustrates a conventional chromatogram of this compound obtained with a reversed-phase column, and a 4:l (v/v) mixture of watermethanol, The mobile phase was then amended by saturating it with toluene, and the solution was pumped through the column until the base line stabilized at 260 nm where toluene has a nominal local maximum. Injection of ethyl acetate into this system provided the chromatogram in Figure 1A. Given that ethyl acetate is transparent at 260 nm and that the base line corresponds to a saturated solution of toluene, the observed signal can only be reasonably attributed to the solubilization of toluene by ethyl acetate. One outcome of the presence of toluene is to decrease the retention volume of ethyl acetate. The solubility of toluene
0003-2700/85/0357-2590$01.50/00 1985 American Chemical Society
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W V
z a
W
V
m
z a
m
L1:
0 v)
m
a
L1:
4
0
12
mL
Figure 1. Chromatogram of ethyl acetate (80 pg) recorded at 0.9 mL/min at A 210 nm using a 80:20 water:methanol mobile phase and B 260 nm with the mobile phase saturated with toluene. The approximate intensity of the peak in A is 0.005 AU, that in B is 0.02 AU.
in the largely aqueous mobile phase is quite small, and the bulk of the toluene in the system will be associated with the stationary phase. Hence, while toluene will affect the characteristics of both the stationary and the mobile phase, its influence on the former will be more pronounced. Furthermore, since toluene is less lipophilic than the CI8 stationary phase, it will lower the capacity factor of an analyte such as ethyl acetate and facilitate its transport through the column, as observed. Two constraints apply to the detection procedure as described. First, solvent gradient elution is impractical since it would be difficult to hold the system a t saturation while the composition of the mobile phase was varied. Second, the base line in the indirect method is noisier than that in a conventional analysis, as is evident from a comparison of the two chromatograms in Figure 1. It was found that the base line was sensitive to variations in flow rate, and this may derive from the pressure gradient which exists across the column, and which is, in turn, dependent on flow rate. The effect of pressure on solubility is insignificant for most purposes (7), but a combination of the high pressures prevalent in HPLC and the sensitivity of the detector to small changes in solubility may make the base line susceptible to pressure fluctuations. Temperature variation could also contribute to base line instability, but the importance of this factor depends on the shape of the solubility-temperature curve of the additive. Bohon and Claussen (8) showed that a number of aromatic hydrocarbons exhibit minima in their water solubility curves a t about 18 “C, where the temperature dependence of solubility is minimal. The concentration of a mobile phase component by the analyte must create a corresponding cavity in the system, which will eventually emerge as a valley in the chromatogram. However, if the retention volume of the additive is large, the valley will tend to be broad and can be effectively removed, if necessary, by methods such as series difference detection (9-1 1).
In general, one might expect the extent of solubility enhancement to increase as the lipophilicity of the additive converges with that of the analyte and diverges from that of the bulk mobile phase. Consider for example, the chromatogram in Figure 2, where ethyl acetate and methylene chloride were indirectly detected by monitoring toluene absorption at 260 nm. The two analytes were present in equal amounts (1.1 pmol), but the response of the later eluting methylene chloride
0 v)
m U
I
4
0
12
16
mL
Flgure 2. Chromatogram of ethyl acetate (99 hg) and methylene chloride (96 pg) obtained at 260 nm at a flow rate of 1.5 mL/min with a 60:40 water:methanol mobile phase saturated with toluene. The intensity of either peak is approximately 0.015 AU.
is greater, since this material is more lipophilic than ethyl acetate, and solubilizes toluene which is even more lipophilic, to a greater extent. The signal in Figure 1A is approximately 0.7% higher than the base line absorbance, and an increase of this order can be anticipated from structural considerations. Consider a system a t equilibrium containing toluene, water, and methanol, with the two latter components being present in the same proportion as the mobile phase in Figure 1. In the presence of sufficient toluene a toluene-rich organic phase will separate from a predominantly aqueous layer. The solubility of toluene in the aqueous phase is given by eq 1 (12)
where xaqand xnrgare toluene mole fractions in the aqueous and organic phases, respectively, and yaqand yorgare corresponding activity coefficients. If a small amount of ethyl acetate is introduced into the system, eq 2 follows
where x Igq is the solubility of toluene in the amended system and x &, yraq,and ylOrg are similarly defined. Equations 1and 2 can be combined to give
Now, since the amount of ethyl acetate in the system is very small, x’,,,~ == xOrgand yrnrg= yorg,eq 3 simplifies to
(4) Equation 4 will apply to a volume element of the column if it is assumed that the toluene-coated stationary phase behaves as toluene itself. The observation that toluene reduces the capacity factor of ethyl acetate, as in Figure 1,is consistent with this view. In principle, the magnitude of the observed signal can be verified through a determination of the activity coefficients in eq 4. However, these measurements would be exceedingly difficult to make at the precision required, and a practical alternative is to use a computational procedure. One such technique is the UNIFAC method described by Fredenslund et al. (13). Here, the solute and solvent are factored into their component groups, and the activity coefficient of each group is calculated. The activity coefficients
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Table I. Analyte Induced Solubilization of Some Additives % increase in solubility in presence of analyte' (water:methanol VIV)
mobile phase
80:20
60:40
toluene xyleneb cymene methylnaphthalene trimethylnaphthalene
0.13 0.17 0.16 0.19
0.10 0.15 0.13 0.15 0.19
0.24
"Calculation based o n a n 80-pg injection o f e t h y l acetate o n a column of 1100 plates as in Figure 1A. b T h e isomer is unspecified as the UNIFAC procedure does n o t distinguish between isomers.
of the parent compounds are then constructed from those of the groups. UNIFAC calculations were made a t 25 OC as described earlier (12) with the group constants reported by Gmehling et al. (14). An ethyl acetate concentration of 2 pmol/mL was used for the calculation of x i q . This value was obtained by estimating the column efficiency to be 1100 plates from Figure lA, and then computing the maximum ethyl acetate concentration in the column effluent (15). Equation 4 showed that the solubility of toluene in the mobile phase should be higher in the presence of ethyl acetate by 0.13%. While this estimate is lower than the experimental result, it is in the right direction and of the same order of magnitude. Given the number and nature of the underlying assumptions, the result is considered to be supportive of the solubility enhancement mechanism proposed. Equation 4 can be used to assess the behavior of other additives, and the calculated solubility enhancement of some liquid additives by ethyl acetate under conditions similar to that in Figure 1A is provided in Table I. It is likely that as in the case of toluene, values for the other hydrocarbons are also underestimated by UNIFAC, and the results are intended only for comparative use. As expected, the optimum additive is one of low solubility in the mobile phase. However, a practical lower limit exists for solubility, since the period required for saturating the system increases with decreasing
solubility. Failure to saturate the system results in a loss of signal. For example, no signal whatsoever was obtained when the chromatogram in Figure 1A was developed with a mobile phase containing toluene at 90% of saturation. Under unsaturated conditions, the assumption that the behavior of the stationary phase resembles that of toluene is invalid, and eq 4 no longer applies. The situation now resembles conventional partitioning between two phases where the analyte and additive will tend to behave independently of each other (16). Finally, while UV detection was used for convenience in these illustrations, the method is applicable to most other detection systems. The range and sensitivity potentially available from the technique will be governed by the type of detector used and the interaction between analyte and additive. Present work focuses on strongly retained analytes which provide negative signals as a result of additive displacement.
LITERATURE CITED (1) Small, H.; Miller, T. E. Anal. Chem. 1982. 54, 462-469. (2) Haddad, P. R.; Heckenberg, A. L. J . Chromatogr. 1984, 300, 357-394. (3) Denkert, M.; Hackzell, L.; Schill, G.; Sjogren, E. J . Chromatogr. 1981, 218, 31-43. (4) Hackzell, L.; Rydberg, T.; Schill, G. J . Chromatogr. 1983, 282, 179-191. (5) Gnanasambandan, T.; Freiser, H. Anal. Chem. 1982, 5 4 , 1282-1285. (6) Bobbitt, D. R.; Yeung, E. S. Anal. Chem. 1984, 56, 1577-1581. (7) Hlldebrand, J. H.; Scott, R. L. "The Solubility of Non-Electrolytes", 3rd ed.; Dover: New York 1964; Chapter 17. (6) Bohon, R. L.; Claussen, W. F. J . A m . Chem. Soc. 1951, 73, 1571-1578. (9) Banerjee, S.; Pack, E. J., Jr. Anal. Chem. 1982, 5 4 , 324-326. (IO) Banerjee, S.; Pack, E. J., Jr. United States Patent 4403503, 1983. (11) Leach, R. A.; Ruzicka, J.; Harris, J. M. Anal. Chem. 1983, 5 5 , 1669-1673. (12) Banerjee, S. Environ. Sci. Techno/. 1984, 18,587-591. (13) Fredenslund, Aa; Jones, R.; Prausnitt, J. M. AIChE J . 1975, 21, 1086-1 099. (14) Gmehling, J.; Rasmussen, P.; Fredenslund Aa. Ind. Eng. Process Design Dev. 1982, 21, 118-127. (15) Guichon, G.; Colin, C. I n "Microcolumn Hlgh-Performance Liquid Chromatography"; Kucera, P., Ed.; Elsevier: New York, 1984. (16) Tewari, Y. B.; Martire, D. E.; Wasik, S. P.; Miller, M. M. J . Solution Chem. 1982, 11 435-445. ~
RECEIVED for review March 27, 1985. Accepted July 1, 1985. Research was carried out under the auspices of the U.S. Department of Energy under Contract No. DE-AC0276CH00016.
