1626
Anal. Chem. 1988, 60, 1626-1629
Isotope Dilution Liquid Chromatography Sir: Isotope dilution analysis (IDA) has been historically used in situations where separation of an analyte from its matrix is difficult or imprecise (1). A requirement of IDA is that analyte mass be either measured or inferred for the determination of specific activity or its equivalent. Thus, two measurements are necessary: one of activity and one of mass, and the latter requirement frequently limits the sensitivity potentially available from IDA. Methods to circumvent the problem have been described ( I ) , but these are usually subject to other constraints. For example, in substoichiometric IDA, equal amounts of analyte are isolated and counted before and after dilution, but the isolation of exactly equal quantities of material is usually difficult. This paper describes the principle of a new technique that couples IDA to high-performance liquid chromatography (HPLC). I t removes the need for a direct mass measurement and thereby greatly improves the sensitivity of IDA while retaining its high selectivity. Viewed from the chromatographic perspective, it offers a sensitive, analyte-specific procedure for indirect detection. EXPERIMENTAL SECTION The chromatographic system consisted of a Varian 5560 instrument coupled to a Radiomatic Flo-One BD flow scintillation counter containing a 250-pL solid scintillator cell, a HewlettPackard 3390A integrator, and a 2.1 x 30 mm pCls Brownlee column. Injections were made through a Rheodyne 7125 valve fitted with a 10-pL loop. [I4C]Benzeneof specific activity 60 mCi/mmol was obtained from New England Nuclear. The mobile phase (10 vol % methanol in water) was saturated with toluene by stirring for 48 h. Most of the excess toluene was removed after phase separation, but a film was allowed to remain over the aqueous solution to ensure continued saturation. For retention measurements, the [14C]benzenetracer was added to the sample, which was either a solution of benzene or a blank. For the isotope dilution experiments, the activity was added to the eluent rather than to the sample. The toluene-saturated solution was spiked with [14C]benzeneand pumped through the system until the base line reached a constant maximum. For experiments where measurements were made as a function of toluene saturation, the saturated solution was diluted at the pump with a corresponding toluene-free solution. Analytes were prepared in 50 vol % methanol in water. Injection of a blank led to a weak signal at the void volume of the system, corresponding to solubilization of [14C]benzene. RESULTS AND DISCUSSION Principle. Consider an HPLC system where the eluent is spiked with a radiolabeled derivative of the analyte (An) to be determined. The detector is tuned to the tracer, An*, and at equilibrium, the isotope An* is distributed evenly through the stationary phase of the column, as shown by the bold line in Figure 1A. When the analyte (An) to be determined is injected (as represented by the dashed curve in Figure lA), it is superimposed over the equilibrium An* layer. It is assumed that An and An* are chromatographically identical and that the mass of An in the injected band on the column greatly exceeds the corresponding mass of An*. The relative proportion of An* has been exaggerated in Figure 1 for the sake of clarity. Assume, for the moment, that conditions can be arranged such that retention of An increases with increasing mass. Since the concentration of injected An on the stationary phase is many times greater than that of An* already present, the An band is retained longer than the An* present on either side of it. As a result, the An* immediately following the band continuously moves into it, as shown in Figure 2. This An* equilibrates with the An in the band, and isotope dilution 0003-2700/88/0380-1626$01.50/0
occurs. Now, since the masa of An greatly exceeds that of An* within the band, the material leaving the band contains more An than An*. As a result, the band is progressively enriched in An*; i.e. the An is "washed" out. The above scenario only applies to a very narrow band whose isotopic distribution is homogeneous. I t is assumed that when the incoming An* is diluted with material in the band, the isotopic composition within the band changes evenly. In practice, most bands are too wide to be homogeneously diluted. Thus, the trailing edge of the band where the An* enters will be enriched in An*, and the leading edge where the An exits will be correspondingly depleted of isotope. Figure 1A illustrates the relative proportion of An and An* on the column shortly after injection of An. In Figure 1B the band has progressed through the column, and isotopic enrichment-depletion as described above has occurred within the band. When the An*-depleted region at the leading edge of the band enters the detector, it will induce a negative signal, which will swing positive as the enriched trailing zone moves through. The peak-to-trough intensity will be a measure of An mass. One way to meet the key requirement that retention increase with increasing An mass is to saturate the eluent with a suitable additive (Add). Saturated additives have previously been used in HPLC as indicators for the indirect detection of transparent analytes (2-4). In one instance (3),a water-rich eluent was saturated with p-terphenyl and pumped through the column. At equilibrium, the stationary phase was also saturated with p-terphenyl. When alkanes such as pentane and hexane were injected, they solubilized p-terphenyl from the stationary phase and could be indirectly detected at 275 nm, an absorption maximum of p-terphenyl. Consider a situation where the eluent contains additive at saturation together with a trace amount of An*. At equilibrium, the stationary phase is coated with the Add-An* mixture. It has been suggested ( 5 , 6 )that distribution into the stationary phase from a polar eluent may approximate liquid-liquid partitioning. If this view be adopted, it follows that the Add-An* coating can be considered as a homogeneous liquid layer influenced by the potential field of the stationary phase. Since An and An* are assumed to be chromatographically indistinguishable, they can be collectively defined as A; i.e. A = An + An*. The capacity factor of A is
k a = nAsp/nAel (1) where nAsPand nAelare the number of moles of A in the stationary phase and in the eluent, respectively. The mole fraction of A in the organic layer coating the stationary phase is
The molar solubility of A in the eluent, sAel, is given by
(3) where SAe1 is the solubility of pure A, i.e. in the absence of any additive, and yAsPis the activity coefficient of A in the stationary phase coating (7-10).The number of moles of A in volume V of eluent is (4) Suitable substitution of terms from eq 2-4 into eq 1 gives
0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988
I
4n
A
B
An*
Table I. Dependence of the Retention Volume of B* on Toluene Content in the Eluent and on the Mass of Injected B toluene, % of saturation
(equilibrium)
0
Column Length
VR,mL" blank
+ B*
B (54 pg)
3.38 f 0.01 0.01 4.40f 0.01 6.00 f 0.06 8.63 f 0.02 7.24 f 0.05
Figure 1. Distribution of analyte (An) on a stationary phase containing An': Initially (A) and after partial movement through the column (B).
40 60 80 100
+ B*
3.43 f 0.01 3.73 f 0.03 4.50 0.02 6.10 f 0.01 8.88 f 0.05 7.59 f 0
I 20 3.63 f
C o l u m n Length
1627
AVR, mL
0.05 0.10 0.10 0.10 0.25 0.35
*
"Average of two to three determinations; there was 50% less B* in the blank than in the sample containing B.
Column Length H
Figure 2. Mechanism of band enrlchment with An'.
Both (n~"p+ nAddsp)and 7 ~ will ~ 'be sensitive to h mass, with 7AnP being the more affected. Most hydrophobic compounds show positive deviations from Raoult's law, and will decrease as analyte mass increases. Accordingly, k' will increase with %ASP,i.e. with analyte mass. It should be noted that this situation will only apply to additives that are either liquid or at least exist as such when coated on the stationary phase. Solids tend not to interact within a mixture (9-11), and it is unlikely that a similar decrease in retention could be observed for solids. Applications. Benzene (B),["Clbenzene (B*), and toluene were used as the analyte, tracer, and additive, respectively. Two types of experiments were conducted. Where measurement of retention volume was the sole objective, the tracer was added to the sample and the eluent was free of activity. In other experiments, the isotope was a part of the eluent, as described earlier in the discussion of Figure 1. Equation 5 requires k' to increase with the amount of analyte injected and also with the amount of additive in the system. In order to verify these conditions, a blank and a solution containing 54 pg of B were each spiked with B* and injected into an eluent that was initially free of additive. The additive content in the eluent was increased progressively to saturation, and the injections were repeated after each increment. The results provided in Table I show that in the presence of toluene, the retention of B* was greater in the sample containing B. In general, retention of B* in both samples increased as the eluent was enriched in toluene. However, the final progression from 80 to 100% saturation led to an unexpected decrease in retention. It is possible that under conditions that are close to full saturation, toluene coats the RP-18 material and contributes substantially to the sorptive properties of the stationary phase. In other words, retention of B or B* occurs primarily on the toluene-coated stationary phase rather than on the stationary phase itself. It has previously been noted that a toluene coating can decrease the retentive properties of the stationary phase (2). In any event, the data in Table I establish that retention increases with increasing analyte level in the sample and, with the above
i I 0
I 5
1
I
IO
15
mL
Figure 3. Chromatogram obtained upon injection of 85 p g of B Into an eluent containing toluene at saturation and 20 nCi/mL of B'.
exception, also with increasing additive loading in the eluent, in accordance with eq 5. In contrast, when fluorobenzene, m-xylene, or 1,2,4-trichlorobenene was used as the additive in place of toluene, the retention of B* increased in the presence of saturated additive, but it remained essentially the same both in the blank and in the sample containing B. In other words, the retention of B* was independent of the amount of B in the sample, and k' was dependent on nAdJP but apparently not on nAsP. The conflicting behavior between toluene and the other additives can be reconciled if the properties of the analyteadditive mixture are considered. Owing to the structural similarity between B and toluene, their sorption characteristics will also be similar,and they will tend to compete for the same sites on the stationary phase. The activity coefficient of B in the organic layer will, therefore, be concentration-dependent, and k A' will vary with analyte mass. In essence, the process will follow the displacement model for retention. The other additives will sorb more strongly than B. As a result, ~ A ' Pand, in turn, kA ' will be less dependent on changes in the mass of B. The situation will correspond to the partition model for retention, where solutes tend to behave independently of one another. This decrease in analyte-additive interaction with increasing structural divergence of the two species is common in indirect detection (3, 12-15).
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 15, AUGUST 1, 1988 5Cr----
-
--
--
is considered to take place in n discrete cycles, each of which involves a volume of eluent equal to the volume of sample injected, Vs, then the fraction of An (fAn)remaining in the band after the first cycle will be
-
mAn
fAn
=
mAn + m*el
where mh is the mass of An injected and m*,, is the mass of An* contained in volume Vs (pL) of eluent. After n cycles I
(7)
t
30 I
-.
/
Since mAn>> m*,,, eq 7 can be simplified to mAn
-
I - 2-_. i
30
60
20
90 PJ
Figure 4.
Response curve of B under the conditions described for
Figure 3. Table 11. Dependence of Signal Intensity (23)” on Activity in the Eluent re1 activity
re1 intens (H)
1 2
1 1.8 2.8 4 4.9
3 4
5 a
From injection of 49 ~g of benzene.
As discussed in the preceding section, a derivative-like signal is expected if the tracer is contained in the eluent and if k f A depends on analyte mass. When B* was added to an eluent saturated with toluene and B was injected, signals such as that shown in Figure 3 were obtained. Response, as measured by the peak-to-trough height, H, was linear, as demonstrated in Figure 4. In principle, eq 5 predicts a nonlinear response; however, since nAddsP>> nABPand the response curve covers a relatively small mass range, it is not surprising that the nonlinear effect is minimal. More seriously, since k varies with the amount of analyte injected, peak broadening will occur with increasing analyte mass and may also contribute to a nonlinear response. This trend is not apparent in Figure 4, very probably because the differences in retention are quite small. Importantly, a signal for B was not observed when fluorobenzene, m-xylene, or 1,2,4-trichlorobenzene was used as the additive, with B* added to the eluent. This is consistent with the earlier observation that the retention of benzene was independent of its mass in the presence of these additives. Since B* cannot migrate into the analyte zone, enrichment cannot occur. The signal in Figure 3 essentially originates from a reorganization of radioactivity in the band, and its intensity should be proportional to the amount of activity involved, i.e. to the concentration of B* in the eluent. A constant amount of B was injected into eluents containing varying levels of activity, and the resulting data are presented in Table 11. As expected, the intensity-activity profile is linear. The injected An moves a t a slower rate than the An* elsewhere on the column, and the An is continuously “extracted” out of the injected band with a volume of eluent equal to the difference in retention volume between An in the band and the equilibrium An* elsewhere on the column. An expression for the degree of extraction can be obtained if it is assumed that band dilution does not occur. If the extraction
f ~ = n
mh
+ nm*el
For the last entry in Table I, the difference in retention volume between the blank and the sample containing B is 0.35 mL, from which n is approximately 0.35 mL/ Vs,or 35. If this value is applied with eq 8 to the chromatogram in Figure 3, then fAn= 0.9999, Le. only about 0.01% isotopic enrichment occurred. Greater enrichment will, of course, improve sensitivity. The detector used contained a heterogenous flow cell with the scintillation immobilized on a solid support. It was designed primarily for qualitative applications, and its noise level was quite high, as seen from the chromatogram in Figure 3. Also, its cell volume at 250 MLwas too large to be compatible with a 2 X 30 mm column, and extensive band dilution occurred. Accordingly, this study was only intended to establish the principle of the technique and demonstrate its feasibility, not to probe the limits of detection. An expression for the detection limit can be obtained from consideration of the counting statistics. The term nrn*el is the accumulated mass of An* within the band. If its maximum value a t the point of elution is m*b (where the subscript b represents band), then eq 8 becomes m*bfAn 1- fAn
mh = The background count rate (cpm) in the eluent is given by
B = 2.2CTSAVG (10) where CT is An* concentration GM), SAis the specific activity of An* (MuCilpmol), V , is detector cell volume (ILL),and E is counting efficiency (16). If the minimum detectable enrichment (m*b)minof activity in the band is proportional to twice the uncertainty in background, then
where T is counting time (min). The detection limit can be expressed as
by combining eq 9 and 11. Since m*el = (1 x 10*)CTVs, eq 12 becomes
It is emphasized that the above equation does not allow for band dilution, which will significantly reduce sensitivity. High efficiency detectors with low volume cells and connectors are commercially unavailable, and the question of sensitivity cannot be experimentally addressed at present.
Anal. Chem. 1988, 60, 1629-1631
Nevertheless, the achievement of low micromole sensitivity with a detector designed for qualitative use is very promising, and a suitably configured instrument is currently being assembled. It was anticipated that the selectivity of the method would be excellent, since the tracer in the eluent can only be diluted with a species that is chromatographically identical with it. To confirm this expectation, a solution of gasoline was spiked with B, dissolved in 50% aqueous methanol, and injected into the B*/toluene system used above. Except for a weak signal at the void volume corresponding to the injection solvent, the only signal observed was that of benzene. Similarly, a solution of rn-xylene spiked with B also gave rise to only the benzene signal. In summary, a difference between the retention of injected An and that of equilibrium An* on the column is used to induce isotope dilution. In this study, solubility was used as the basis for modifying retention. In principle, other means could be used; e.g. if the column element containing the An band is kept at a different temperature than the rest of the column, a similar effect should occur. The method is preliminary in its present form, but it appears to capture the important attributes of IDA and HPLC. While it is known to be based on fine differences in solute transport, the details of the interaction between analyte, additive, and the stationary phase are not fully understood. The technique is very selective, but the full sensitivity potentially available from it has not been realized, since a truly compatible detector has yet to be constructed.
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LITERATURE CITED (1) Wang, C. H.; Willie, D. L.;Loveland, W. D. Radiotracer Methodology in the Siological, Envifonmental, and Physical Sciences ; Prentice-Hall: Englewood Cliffs, NJ, 1975; pp 381-388. (2) Banerjee, S.Anal. Chem. 1985, 57, 2590-2592. (3) Banerjee, S.;Castrogivanni, M. A. J. Chromatogr. 1987, 396, 169-1 75. (4) Banerjee, S. US. patent 4 734 377. (5) Martire, D. E.; Boehm, R. E. J . fhys. Chem. 1983, 87, 1045-1062. (6) Jaroniec, M.; Martire, D. E. J . Chromatogr. 1986, 351, 1-16. (7) Tewari, Y. B.; Martire, D. E.; Wasik, S. P.; Miller, M. M. J. Solution Chem. 1982, 1 1 , 435-445. (8) Burris, D. R.; MacIntyre, W. G. Environ. Toxicol. Chem. 1985, 4, 371-377. (9) Banerjee, S. Environ. Sci. Techno/. 1984, 18, 587-591. (10) Yalkowsky, S.H.; Banerjee, S.Estimation of the Water Solubilities of Organic Compounds; Marcel Dekker, in press. (11) Eganhouse, R. P.; Calder, J. A. Geochim. Cosmochim. Acta 1976, 555-561. (12) Denkert, M.; Hackzell, M. L.; Schili, G.; Sjogren, E. J. Chromatogr. 1981, 218, 31-43. (13) Parkin, J. E. J. Chromatogr. 1984, 267,457-461. (14) Vigh, G.; Leitold, A. J. Chromatogr. 1984, 312, 345-356. (15) Takeuchi, T.; Ishii, D. J. Chromatogr. 1987, 393, 419-425. (16) Banerjee, S.;Steimers, J. R. Anal. Chem. 1985, 57, 1476-1477.
Sujit Banerjee Safety and Environmental Protection Division Brookhaven National Laboratory Upton, New York 11973 RECEIVED for review August 7,1987. Accepted March 25,1988. Research was carried out under the auspices of the U S . Department of Energy under Contract No. DE-ACOZ76CH00016.
Laser-Enhanced Ionization Spectrometry in a T-Furnace Sir: This communication reports on results obtained by laser-enhanced ionization (LEI) spectrometry with a modified electrothermal atomizer a t atmospheric pressure. In this T-shaped furnace the atomization of an analyte is performed inside the graphite tube, while the excitation of the produced atoms, subsequent ionization, and detection are carried out in an external cavity connected to the tube. Two elements, Mn and Sr, have been detected by using LEI spectroscopy with this new arrangement. Extremely high sensitivities were obtained for both elements. Laser-enhanced ionization spectrometry is an ultrasensitive method for detecting atoms or molecules in an ionizing medium. Two-color LEI is based on the enhanced rate of ionization which follows when the analyte element is selectively excited by light from two dye lasers tuned to appropriate transitions. This enhanced rate of ionization is detected as a current increase by applying a voltage across the interaction region. LEI spectroscopy in flames has been extensively used for detection of a wide range of elements and an impressive potential of this method for trace element analysis has been demonstrated (1-3). In atomic absorption spectrometry (US)-today the most widely used analytical method for trace element analysis-the transition from a flame to an electrothermal atomizer led to highly improved detection limits for many elements. The main advantages of a graphite furnace as an atomizer compared to a flame are the longer residence time for the atoms in the interaction region, possibilities for microanalysis, and a lower molecular background. For the same reasons the graphite furnace was introduced to LEI. Earlier reports on 0003-2700/88/0360-1629$0 1.50/0
LEI in graphite furnace (4-7), where both atomization and detection of an analyte were performed inside the graphite tube, have shown extremely high sensitivities for a number of elements. However, severe interferences occurred which originated from electrons thermally emitted from the heated graphite tube and the electrode, making it impossible to measure, for example, Sr, which atomizes rather slowly (7). The basic idea of the new construction of an electrothermal atomizer presented here is to spatially separate the atomization volume from the detection volume and thus overcome some of the problems occurring in earlier constructions.
EXPERIMENTAL SECTION A schematic view of the experimental equipment is shown in Figure 1. An excimer pump laser (Lambda Physik EMG 102) simultaneously pumped two dye lasers (Lambda Physik EMG 2002). The dyes used were Coumarin 153, producing light in the 522-600 nm region, and Coumarin 47, producing light in the 440-484 nm region. For Mn the output from Coumarin 153 was frequency doubled by a KDP crystal. The laser pulse duration was 20 ns and the repetition rate was 25 Hz. The graphite furnace, in which the samples of 50-wL water solutions were inserted, was a commercial Perkin-Elmer (HGA-72) Model. The graphite furnace has an inner diameter of 9 mm and is 50 mm in length. The current corresponding to the maximum temperature of 2900 K was 500 A. Argon at atmospheric pressure was used as a protective gas. It was partly flowing around the graphite tube and partly fed through one end of the tube to drive the atoms toward the interaction region. The total flow of Ar was about 6 L/min. The temperatures and times used for drying and charring were those recommended for AAS measurements in a commercial graphite furnace. Maximum atomization tem0 1988 American Chemical Society
