Anal. Chem. 1987, 59, 1834-1841
1834
which were accessible with the nitrogen laser (8). We still possess standards for 16 of those compounds, and of those 16, 1 2 were conclusively identified by the two-point lifetime technique. With the aid of new standards, three additional PAH were found. It is interesting to note that there were many other compounds in our standard set that were not identified in the sample by either method. That is, no compound listed as clearly absent by the diode-array technique was subsequently found by using the ratio method. The agreement between the two different techniques is extremely encouraging. Work is now in progress to expand the application of the new method to other systems.
LITERATURE CITED (1) Seitz, W. R.; Frei, R. W . CRC Crit. Rev. Anal. Chem. 1880, 18(14), 367-405.
(2) Richardson, J. H.; Larson, K. M.; Haugen, G. R.; Johnson, D. C.; Clarkson, J. E. Anal. Chim. Acta 1880, 116, 407-411. (3) Imasaka. T.; Ishibashi, K.; Ishibashi, N. Anal. Chim. Acta 1982, 142, 1-12. (4) Desiiets, D. J.; Coburn. J. T.; Lantrip. D. A.; Kissinger, P. T.; Lytie, F. E. Anal. Chem. 1988, 58, 1123-1128. (5) Lytle, F. E. Photochem. Photobiol. 1973, 17, 75-78. (6) Sander, L. C.; Wise, S. A. In Advances in Chromatography; Giddings, J. C., Grushka. E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1986; Voi. 25, pp 139-218. (7) Wise, S. A.; Bonnet, W. J.; May, W. E. I n Polynuclear Aromatic Hydrocarbons: Chemistry and Siological Effects; Bjorseth, A,, Dennis, A. J.. Eds.; Battelle Press: Columbus, OH, 1980; pp 791-806. (8)Desiiets, D. J.; Kissinger. P. T.; Lytle, F. E.; Horne, M. A,; Ludwiczak, M. S.; Jacko, R. B. Environ. Sci. Techno/. 1984, 18, 386-391.
RECEIVED for review August 7 , 1986. Accepted April 6, 1987. This work was supported in part by the American Cancer Society, the Indiana and the National Science Foundation, Grant CHE-8320158.
Photochemical Amplifier for Liquid Chromatography Based on Singlet Oxygen Sensitization Curtis L. Shellum and John W. Birks*
Department of Chemistry and Biochemistry and Cooperative Institute for Research i n Environmental Sciences ( C I R E S ) ,Campus Box 449, University of Colorado, Boulder, Colorado 80309
A postcolumn photochemical reaction scheme designed to enhance the detectability of UV-absorbing compounds has been coupled to high-performance liquid chromatography (HPLC). Specifically, the method detects members of the large class of organic compounds termed “singlet oxygen sensitizers”. These compounds transfer excitation energy to ground-state oxygen, forming the excited singlet species, O,( -let oxygen in turn reacts with a substituted furan such as 2,54lmethylfuran (DMF) or 2,l-diphenylfuran (DPF), and UV absorption or fluorescence is used to detect either the loss of reactant (DMF or DPF) or appearance of a product. The reaction sequence is photocatalytic In nature, resulting in a large chemical ampllflcation of the signal. Detection h i t s are Improved by 1 to 2 orders of magnitude for a wide variety of UV-absorbing compounds. Discussed in this report are the theory and characterizationof the detectlon system as well as its application to several classes of compounds including polycyclic aromatic hydrocarbons, substituted anthracenes, anthraquinones, and polychlorinated biphenyls (PCBs).
‘4).
Sufficient detection sensitivity is not currently achievable for many types of molecules separated and analyzed by HPLC. Particularly significant are UV-absorbing organic molecules having low or negligible fluorescence quantum yields as a result of high quantum efficiencies for singlet-to-triplet intersystem crossing. At low temperatures or in organized media such compounds are often phosphorescent. The excited triplet to ground singlet state transition is spin forbidden, however, yielding an inherently long triplet state lifetime of typically to 10 s (1). More rapid deactivation processes prevent phosphorescence under the usual conditions in HPLC of room temperature and the presence of dissolved oxygen. Oxygen quenches the excited triplet state at a diffusion-controlled rate
-
and is present at a level of M in most solvents ( 2 , 3 ) . Phosphorescence has been used for detection in HPLC, however, under conditions of virtually complete oxygen removal. The approaches include micelle-stabilized room-temperature phosphorescence ( 4 ) ,sensitized room temperature phosphorescence (5),and phosphorescence quenching (6). The necessity of rigorous oxygen removal is a disadvantage of all of these detection methods. Furthermore, these approaches have not resulted in substantially improved detection limits as compared to conventional UV absorption. This paper describes an approach that uses to advantage the triplet quenching ability of molecular oxygen. Groundis promoted to an excited singlet state, state oxygen, 02(3C.J, either Oz(l&+) or O#$) (2). O#.&,+) decays to the metastable 02(lAg) state within s (7). The OZ(lAg)species is involved in a variety of photochemical oxidations, some of the most rapid of which occur with substituted furans such as 2,5-dimethylfuran (DMF) and 2,E~diphenylfuran(DPF). Figure 1presents absorption spectra of four furans including DMF and DPF. These compounds may serve as “singletoxygen traps”. The analytical signal may be based on a decrease in concentration of the furan or an increase in concentration of the oxidation product, as measured by a spectroscopic property such as UV absorption or fluorescence. A large sensitivity advantage results in that the analyte molecule may absorb light many times and produce large numbers of singlet oxygen molecules during its residence time in a postcolumn photochemical reactor. As a result, detection limits for a wide variety of UV-absorbing compounds may be improved by between 1and 2 orders of magnitude by using this photochemical reaction scheme. The only modification to a standard HPLC apparatus is the insertion of a photochemical reactor between the analytical column and the detector. The compound serving as a singlet oxygen trap (substituted furan) is spiked into the mobile phase at a concentration typically in the range to M. The
0003-2700/87/0359-1834$01.50/0 0 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
1835
Table I. Lifetimes of Singlet Oxygen in Various Solvents
solvent
T,
HZO CHBOH CZHSOH (CJ&)zCO
W a v e l e n g t h , nrn
analytes separated by the chromatographic column enter a photochemical reactor having a residence time of 1-3 min where reactions induced by a 254-nm, low-pressure Hg lamp take place. Either a positive or negative peak is produced at the detector, depending on whether the appearance of a product or the loss of a reactant is being monitored.
THEORY The light-initiated reaction sequence can be outlined as follows:
A
+ hv -% lA*
3A*
kd
(3)
A
(4)
(5) furan (F)
+ 02(lAg)
+
product (P)
(6)
In reaction 1,the analyte A absorbs a photon and is promoted to an excited singlet state. The first-order rate constant for this process, kabs, is given by kabs
=
slAOA
dX
(7)
where uAis the absorption cross section of the analyte a t where wavelength X in units of cm2 molecule-l (2303eA/N~ is the molar absorption coefficient and NA is Avagadro's number) and I , is the photon flux in units of photons cm-2 s-' at wavelength A. Following absorption, a molecule typically fluoresces, returns to the ground state by internal conversion, or undergoes intersystem crossing to its lowest triplet state. The latter process is illustrated by reaction 2 and occurs with a quantum efficiency q$sc. In reaction 3 the excited triplet analyte molecule, 3A*, is quenched to the ground state by with a diffusion limited rate molecular oxygen, 02(3&-), constant k,. A product of this energy exchange is singlet oxygen, O2('Ag). The energy requirement for 3A* is that it be a t least 22.5 kcal mol-' above the ground state (8). This requirement is met by nearly all organic molecules. For those compounds having lowest triplet states with energies greater than 37.5 kcal mol-', the singlet oxygen species 02(l&+) is usually formed as an intermediate in reaction 3. In solution this species is quenched to 02(1$)in 110-'O s (7).A competing
CHC13 CSZ C6F6
cc1,
CFC13
30 60
200 600 700 1000
process for deactivation of 3A* is intersystem crossing to the ground state, reaction 4; however, in a solution saturated with oxygen, reaction 4 is lo2 to lo3 times slower (2). As seen in Table I (9), the lifetime of singlet oxygen in the solvents commonly used in reversed-phase HPLC is relatively short due to a radiationless decay to the ground state, reaction 5. In our detection scheme, a substituted furan (F) competes with this deactivation by reacting with 02('A,) according to reaction 6 to form an oxidized product (P). Values of k,, for various acceptors under a range of conditions have been reported (9, IO). Detection may be based on either the decrease in concentration of the furan (-A[F]) or the increase in the oxidation product (Alp]), using a conventional UV absorption, fluorescence, or other appropriate detector. The success of detection utilizing this reaction sequence depends on the relative rates of the above reactions. An additional consideration arises if the furan molecule absorbs some of the excitation light. It also can induce singlet oxygen formation, leading to undesirable self-photooxidation. The rate of furan oxidation and of product formation can be derived by considering the contributions from each of reactions 1-6 above and including the effect from self-photooxidation. If one applies the steady-state assumption to singlet oxygen (a very good assumption since the lifetime of singlet oxygen is measured in microseconds, and the residence time in the reactor is tens to hundreds of seconds) and assumes that reaction 4 is slow relative to reaction 3 (2), the result is -d[PI =--dt
d[FI dt
kabs,AIAlhsc,A
k,,[F]
kox
7 , IrS
CHSCN
2 7 12 17 24 26
CsH12 CsH6
Flgure 1. Absorption spectra of 2-methylfuran, 2,5dimethylfuran, 2,5diphenylfuran,and 1,3-diphenyIisobenzofuran in acetonitrile.
solvent
Ps
+1
+ habs,F[Fl&c,F
/ ~ k,,[F]
+1
)
/ ~
(8)
This equation has been written with two terms, for illustrative purposes. The f i s t term is the rate of photooxidation resulting from the absorption of light by the analyte, and the second term is an analogous one which must be included if the furan itself absorbs light in the reactor. Self-photooxidation via the second term reduces the furan concentration and thereby also reduces the signal arising from the first term. Also, fluctuation in the degree of self-oxidation due to variation in lamp intensity, variation in the reactor residence time, etc. has the potential to contribute to the detection noise. Clearly, it is advantageous to maximize term 1 and minimize term 2. The rate constant for reaction of singlet oxygen with the furan, KO,, affects both terms equally, as does the singlet oxygen lifetime, T. Clearly, the most effective means of enhancing term 1relative to term 2 is by choice of excitation wavelength for a given singlet oxygen trap molecule. Equation 8 yields analytic solutions upon integration in the ~ 1 / >> ~ k,,[F]. In this paper the limits of k,,[F] >> 1 / and furan, F, is either 2,5-diphenylfuran or 2,5-dimethylfuran. On the basis of the range of reported rate constants and the concentrations used in our work, we cannot state with certainty that either condition is rigorously met when using DMF or DPF. However, we estimate that 1 / >~k,[F] for DPF and k,,[F] > 1 / for ~ DMF. Integration of eq 8 when k,,[F] = 1 / ~ leads to a very complex rate law which will not be discussed here.
1836
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
Under the conditions where 1 / >> ~ k,,[F] and the decrease in [F] resulting from reaction with the analyte is small compared to the amount of F available (Le., pseudo-first-order conditions), eq 8 may be integrated to obtain
A [ P ] = -A[F] =
where A[P] and -A[F] are the changes in concentrations of the product and the furan, respectively, exiting the reactor in the presence of an eluting analyte. This result predicts that, provided the detector responds linearly to either P or F, the signal will be linear in analyte concentration. In the case that self-photooxidation is negligible, eq 9 simplifies to
Under these conditions, the signal is expected to increase dX), ablinearly with photon flux of the lamp (kabs a JIhtX sorption coefficient of the analyte, quantum yield for intersystem crossing, rate constant for reaction with the furan, lifetime of singlet oxygen in the solvent, concentration of the furan, and concentration of the analyte. For the results presented in this paper where DPF is used in conjunction with a 254-nm lamp, self-photooxidation causes a reduction in DPF concentration by a factor of -2-3 during the residence time in the photoreactor. In this case, eq 9, which includes the effect of self-photooxidation, is required to describe the results. Equation 10 would be applicable, for example, in experiments using DPF in conjunction with a light source whose output is at wavelengths longer than 350 nm. In this case there would be no self-photooxidation, as DPF does not absorb in this wavelength region (Figure 1). As stated above, for DMF the reaction conditions most closely correspond to the situation where k,,[F] >> 1 / r in the denominator of eq 8. Also, because DMF absorbs very little excitation light at 254 nm (Figure 1) and thus undergoes a negligible amount of self-photooxidation, the second term of eq 8 is approximately zero. Integration of eq 8 with these simplifications gives the expression
which predicts a signal linear in analyte concentration. Of course, these analyses are only correct for the specified reactions and stated assumptions concerning relative concentrations of reactive species. It is possible that other reactions not given in reaction sequence 1-6 may take place. For example, reaction of the analyte with singlet oxygen or other reactive intermediates could lead to its destruction. Also, the product of the furan oxidation may itself absorb light and further amplify the photooxidation of the furan. Quinone analytes may sensitize the formation of hydrogen peroxide, and hydrogen peroxide is known to photodissociate at 254 nm to produce OH radicals (11). Also, many analytes may themselves react with singlet oxygen to yield products with altered photochemical properties. In particular, anthracene, one of the model compounds of this study, is known to react with singlet oxygen to form anthraquinone (12),another of the model compounds of this study. Thus, the photochemistry may be quite complex, but the above analysis may serve as a guide in understanding the results presented here. In this work we have chosen an excitation wavelength of 254 nm, as this wavelength is almost universally absorbed by compounds having at least one aromatic ring, and low-pressure Hg lamps are a convenient and relatively intense source of this wavelength of light. Of course, these are the same reasons that 254-nm absorption detectors are the most frequently used detectors in HPLC. Furthermore, as can be seen in Figure 1. 254-nni radiation is well wited to these studies in that the
two singlet-oxygen traps used in this study, 2,5-dimethylfuran and 2,5-diphenylfuran, have small extinction coefficients at this wavelength. The molar extinction coefficient of 2700 for DPF at 254 nm, however, does result in significant selfphotooxidation and therefore requires the use of a stable photochemical lamp. A solution filter, having a band-pass centered near 254 nm, reduces self-photooxidation from shorter and longer wavelength emission lines of the Hg lamp. Its use is required when using DPF as the singlet oxygen trap. Emission lines of Hg shorter than 254 nm also can cause self-photooxidation of DMF, which is also largely reduced by the use of a filter. In this case the filter may be commercial quartz (thickness 2 2 mm) or the same solution filter used with DPF. Also shown in Figure 1are the absorption spectra of 2-methylfuran and 1,3-diphenylisobenzofuran. The 2methylfuran has the advantage of not absorbing 254-nm radiation to any significant extent, but unfortunately, we found that the photooxidation product of this singlet oxygen trap has a very small extinction coefficient. The self-photodue to its strong aboxidation of 1,3-diphenylisobenzofuran, sorbance at 254 nm, is so large that this singlet oxygen trap can only be used in conjunction with a visible lamp. Its use is therefore limited to colored compounds such as commercial dyes, plant pigments, and certain metal coordination complexes.
EXPERIMENTAL SECTION HPLC Apparatus. The chromatographic equipment consists of a Kratos Spectroflow 400 solvent pump, a Rheodyne 7125 injector (20-pL loop), a Zorbax ODS column (25 cm X 4.6 mm) packed with 5-pm ‘2-18 particles, a Kratos Spectroflow 773 variable wavelength UV detector, and a Kratos FS 950 fluorometric detector with a medium-pressure mercury excitation source. A 326 i 11 nm band-pass filter served as the excitation filter, and the emission filter passed wavelengths longer than -370 nm. A Shimadzu C-R3A integrating recorder was used to plot chromatograms and integrate peaks. Photochemical Reactor. The photochemical reactor consists of one or two low-pressure Hg pencil lamps separated from a crocheted PTFE reactor by either a cylinder of quartz or a quartz sleeve containing a solution of 2,7-dimethyl-3,6-diazacyclohepta-2,6-diene iodate, which serves as a 254-nm band-pass filter 113). The path length through the filter is 4 mm, and the solution concentration is 0.042 g/L. Work with DPF required use of the solution filter, while for DMF the short wavelength cutoff of commercial quartz of greater than 2 mm thickness was adequate to minimize the degree of self-photooxidation induced by emission lines other than 254 nm. The construction of crocheted PTFE reactors for postcolumn photochemistry recently was described I 14). The PTFE tubing (Small Parts Inc., SST-30) has a 0.30 mm 1.d. Reactors used in this work are 5-20 m in length and provide reaction times of 0.5-3 min for flow rates in the range 0.7-1.0 mL/min. Crocheting the reactor tubing greatly reduces the degree of band broadening caused by this amount of postcolumn tubing (14). The exterior of the reactor is wrapped in aluminum foil to increase the photon flux. It was found that base-line drift was substantially reduced by immersion of the exterior of the reactor in an ice bath. The ice bath did not significantly affect peak heights or the extent of self-photooxidation but reduced low-frequency noise, apparently by stabilizing the lamp temperature and thereby stabilizing the photon flux. The effect of temperature stabilization of the effluent passing through the UV absorption cell may also be important. Reagents. 2,7-Dimethyl-3,6-diazacyclohepta-2,6-diene iodate was prepared as described by Kasha (13) and was recrystalized from methylene chloride. The substituted furans 2,5-dimethylfuran and 1,3-diphenylisobenzofuranwere obtained from Eastman Kodak, while 2-methylfuran and 2,5-diphenylfuranwere obtained from Aldrich. The solvents methanol, acetonitrile, and water were HPLC grade. RESULTS AND DISCUSSION Detection Based on Quenched Fluorescence of DPF. Many of our early investigations of detection based on singlet
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
1837
1 0 ' ~ M DPF in acetonitrile ( I ng of anthracene injected)
-9.00
rn
1
-10.00
LL
a 0
Y
E -
-11.00
1
I
1
I
50
100
150
I 200
Reactor Residence T i m e , s
Figure 3. Anthracene peak height as a function of residence time in
-12.00 0 10-4M DPF in methanal 10-4M DPF inacetonitrile
the photochemical reactor. Table 11. Effect of Substituents on Photochemical Amplification
20 40 60 80 100 120 140 160 Time, s
Figwe 2. Concentration of 2.5diphenyffuran as a function of residence time in the photochemical reactor for methanol and acetonitrile sol-
vents. oxygen sensitization made use of 2,5-diphenylfuran as the singlet oxygen trap. DPF is a highly fluorescent compound, which reacts rapidly with singlet oxygen to form the nonfluorescent product cis-dibenzoylethylene (DBE) (15) +O, ('As)0
DPF
0
cis-DBE
(12)
Thus, one potentially very sensitive means of detecting singlet oxygen sensitizers is to spike the HPLC mobile phase with DPF and monitor the quenching of a standing fluorescence upon elution of the analyte. Surprisingly, this method of detection turned out to be less sensitive than that of moniusing toring the reduction in absorbance of DPF at its,,A a UV absorption detector. Although the fluorescence quenching method did not result in detection limits that were substantially improved relative to conventional UV absorption, experiments using this system helped elucidate the general features of the photochemical system, and for this reason some results of these experiments will be described. Figure 2 plots the logarithm of the DPF concentration, as measured by fluorescence, vs. residence time in the photoM chemical reactor for an initial DPF concentration of in methanol and in acetonitrile. The negative of the slope of this plot is proportional to the reaction rate. The decay curve for D P F in methanol is seen to be linear, consistent with a first-order photochemical reaction. In acetonitrile the reaction rate increases with increasing time. This could possibly be explained by the appearance of a product that itself absorbs reactor light and adds to the rate of DPF destruction. When methanol is the solvent, however, the extent of reaction, and therefore product accumulation, is too small, even at 160-s reaction time, for an enhancement of the rate of photooxidation to become apparent. The initial rate of selfphotooxidation is acetonitrile is 4.5 times greater than in methanol. This is consistent with the fact that the lifetime of singlet oxygen in acetonitrile is 4.3 times greater than in methanol (Table I, 30 p s compared to 7 ws). Of course, other factors, such as the extinction coefficient of DPF, its quantum yield for intersystem crossing, and its rate of reaction with singlet oxygen in the two solvents, also contribute to differences in the degree of self-photooxidation.
compound
peak height/A264
9-nitroanthracene 2-chloroanthracene anthracene 9-anthracenecarboxylicacid 2-aminoanthracene 2-methylanthracene
1.5 1.2 1.0 0.66 0.59 0.37
As shown in Figure 3, the signal resulting from quenched DPF fluorescence increases linearly with reaction time up to about 150 s in acetonitrile when anthracene is the analyte and M. Similar behavior for an initial DPF concentration of is found when using methanol as the solvent. Beyond about 150 s, peak heights level off, and additional time spent in the photoreactor beyond about 3 min provides little enhancement of the signal, while band broadening continues to increase. Note that at infinite reaction time, all DPF would be destroyed as a result of self-photooxidation whether or not an analyte is present, and there would be no analyte peak. Therefore, we expect that peak heights would begin to decline after sufficient reaction time. This is confirmed by results presented in the next section where DPF is monitored by use of UV absorbance. We also note that the optimal reaction time may be decreased to about 75-100 s by using two Hg pencil lamps, as in many of the experiments described below. As a further test of our understanding of this reaction system, the relative responses of equal molar solutions of a group of substituted anthracenes were obtained in a mobile M DPF in acetonitrile and for a photoreactor phase of residence time of 60 s using one lamp. Their relative absorbances a t 254 nm (the photoexcitation wavelength) were also measured in a Cary 219 spectrophotometer. Ratioing the peak heights for DPF fluorescence quenching to their relative absorbances allows one to factor out the effects of kabs on the signals obtained. These ratios were then normalized to the result for anthracene and are given in Table 11. According to the reaction mechanism discussed earlier, these ratios should only differ as a result of differences in the quantum yields for intersystem crossing, $isc. In Table 11, it is seen that the substituted anthracenes, in general, show the expected trend. Progression down the table is expected to be in the order of larger to smaller dkC.There are only two flaws in this trend. The order of amino- and methylanthracene is reversed from that expected, although misplacement is slight. Their fluorescence quantum yields are very similar. 9Anthracenecarboxylic acid is also out of place. This compound is expected to lie between 9-nitroanthracene and 2-chloroanthracene. The reason for this discrepancy is unknown. However, this and other experiments described here indicate that the sensitivity toward a particular compound depends
1838
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987 IC
I .o +
c
08
.-0
r”
0.8’
x 0
a u Q,
u c
O6
.0
0
0.4,
n L
Z
0
2 a
0.6
? 0 . l
04
0 0
I - Nitronaphtholene Anthraquinone
0 4,4
40
02
60
80
- Dichlwobiphenyl 100
120
140
160
Reaction T i m e , sec
/ L
l
l
I
I
I
l
/
I
/
l
l
l
l
/
l
l
l
l
0 220 240 260 280 300 320 340 360 380 4 0 0
Wavelength, nm M solution of DPF in acetonitrile Figure 4. Spectrum of a 3 X after bulk irradiation in a cuvette. Exposure times are 0, 30, 60, 90, 130, 170, and 210 s in order of decreasing absorbance at 320 nm.
not only on that compounds photophysical properties but also on the potential of that compound to undergo photooxidation itself. The altered analyte molecules will most likely have a different extinction coefficient at the excitation wavelength and may either slow down or speed up the rate of singlet oxygen production. The DPF concentration was varied between lo4 and M for a reaction time of 80 s to determine the optimal concentration in fluorescence quenching. Anthracene (33 ng) served as the analyte for the optimization. A concentration M was found to give the best signal-to-noise ratio for of both acetonitrile and methanol solvents. However, this optimum was very broad. For example, in acetonitrile the signal-to-noise ratio did not vary by more than 20% in the range to 10” M. Furthermore, the detection limit for anthracene (20 pg) was not significantly different in methanol and acetonitrile solvents. For this and several other polycyclic aromatic hydrocarbons, detection limits for DPF fluorescence quenching were a factor of 2 to 6 better than those that could be achieved by optimized ‘JV absorption. However, PAH are more sensitively detected by fluorescence than by either UV absorption or the photochemical method of DPF fluorescence quenching. The detection limits for several anthraquinone derivatives were found to be a factor of 2 to 3 poorer using DPF fluorescence quenching as compared to UV absorption. Detection Based on DPF Photobleaching. The change M solution of 2,5in the absorption spectrum of a 3 x diphenylfuran in acetonitrile upon irradiation in a cuvette by a low-pressure Hg lamp is shown in Figure 4. These spectral changes, resulting from self-photooxidation, are the same as those caused by analyte-sensitized photooxidation. There is an isosbestic point at 270 nm where the solution absorbance does not change, apparently because the reaction product has the same extinction coefficient as DPF. There is a decrease in absorbance a t the A, of D P F at 320 nm and an increase in absorbance due to product appearance near 245 nm with increasing reaction time. Thus, analytes may be detected by monitoring absorbance at either of these wavelengths. However, the largest absorbance changes occur at the DPF A,, and detection based on photobleaching of DPF at 320 nm was
Figure 5. Chromatographic peak heights as a function of time for detection of analytes by DPF photobleachlng at 320 nm; mass injected, 0.25 ng of anthraquinone, 1.3 ng of 1-nitronaphthalene,and 2.8 ng of 4,4’dichlorobiphenyl. Mobile phase is lo4 M D f f in 95% acetonitrile.
Table 111. Detection Limit Comparisons for Diphenylfuran Absorbance Quenching enhance-
compound
optimized UV absorbance
anthracene 45 pg (253 nm) anthraquinone 90 pg (250 nm) 1-nitronaphthalene 160 pg (243 nm) quinoline 900 pg (234 nm) biphenyl 130 pg (260 nm) 4,4’-dichlorobiphenyl 90 pg (270 nm)
ment factor 30X 1lX
44x 22x 6.5X 9x
found to give the lowest limits of detection. To detect analytes in HPLC via this decreased absorbance at 320 nm, the HPLC mobile phase was spiked with DPF at a concentration typically in the range (0.5-1.0) X lo4 M and the absorbance at 320 nm monitored. The large background absorbance, typically in the range 0.5-1.0 AU was electronically subtracted by use of the zero offset feature of the UV detector, and the leads carrying the signal to the integrating recorder were reversed so that the chromatographic peaks were positive. The effect of reaction time on peak height was determined for three model compounds in 95% acetonitrile, using two photochemical lamps. The results are plotted in Figure 5. The optimal time spent in the photochemical reactor was found to fall in the range 1-2 min, depending on the analyte and solvent conditions. Longer reaction times resulted in decreases in the signal and can be attributed to the depletion of DPF by self-photooxidation as mentioned in the previous section; the signal will decrease to zero a t very long reaction times. Detection limits for DPF photobleaching were determined for six model compounds in HPLC with 95% acetonitrile as the solvent. For these measurements, the HPLC flow rate was 0.7 mL/min, the photochemical reaction time was 75 s using two lamps, and the DPF concentration was lo4 M. Here limits of detection are defined as the mass of injected analyk that results in a peak having a height equal to three times the peak-to-peak noise of the base line. The results are summarized in Table 111. Detection limits range from 1.5 pg for anthracene to 40 pg for quinoline. Of course, detection limits are dependent on the quality of the UV absorption detector used. For this reason, detection limits were also determined for these compounds by direct UV absorption at their indi-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987 1O,0OOE
Table IV. Effect of Solvent Composition on DPF Photobleaching
anthracene anthraquinone
1-nitronaphthalene quinoline
biphenyl 4,4’-dichlorobiphenyl
1.0 1.0 1.0
1.0 1.0 1.0
0.98 0.68 1.1 1.1 0.80 0.70
1.6 0.71 1.3 1.3
I
I I I I I I I
1000
I L
c
0.68 1.0
I
I l l l l l l l
I
I
I11111(
I
I 1 1 1 1 1 1 /
-
relative peak area 5% H20 25% HzO 50% HzO
compound
,
1839
/
/
Y
/
Y
0
vidual A,, using the same detector. Table I11 compares the detection limits obtained by the two methods and gives the factors by which the detection limits are improved over direct UV absorption by use of the photochemical reaction. Sensitivity enhancement factors range from 6.5 for biphenyl to 44 for 1-nitronaphthalene. Because this detection system is designed for chromatography where the solvent conditions must be varied to optimize separation, we examined the effect of solvent composition on the sensitivity of DPF photobleaching. It is difficult to predict the overall effect of solvent composition on the reaction sequence 1-6, because altering the solvent can at the same time enhance and reduce the rates of individual reactions contributing to DPF photooxidation. For example, increasing the water:acetonitrile ratio would decrease the lifetime of singlet oxygen, thereby slowing the reaction sequence, while increasing the rate of reaction of D P F with singlet oxygen, as it has been found that singlet oxygen reacts faster with substituted furans in more polar solvents (10, 16). In fact, all of the relevant reactions are expected to be somewhat solvent dependent. Because water is used to increase retention in reversedphase HPLC, we chose to compare the signals produced by analytes in acetonitrile containing 5%, 25%, and 50% water by volume using flow injection analysis (no column). The results are presented in Table IV. Because the flow dynamics in the knitted reactor were such that the flow injection peaks broadened when going to higher percentages of water, peak areas rather than heights were used to compare the extents of D P F photooxidation. Although both increases and decreases in signal occurred, the signal did not change by more than a factor of 1.7 when the water content was varied from 5% to 50% by volume. As expected, these data cannot be explained by the effect of singlet oxygen lifetime alone. In that case, the peak areas would have correlated with the expression
1 --f _ met
rH,O
+ -1 - f
(13)
rAcN
where f is the volume fraction of water. This expression predicts that the peak area would decrease by factors of 2.6 and 4.7, respectively, in going from 5% water to 25% and 50% water. The much weaker dependence of the signal on solvent composition than is predicted by consideration of singlet oxygen lifetime alone confirms that reaction 6 and possibly other reactions in the sequence 1-6 are enhanced in the presence of water. These results suggest that this reaction system may be used in chromatography under a wide range of solvent conditions. The above analysis is based on peak areas. Detection sensitivity, as measured by peak heights, will not correlate exactly with the data in Table IV, as the extent of peak broadening by the crocheted reactor was found to increase with increasing water concentration. Furthermore, band broadening by the column is also a function of solvent composition.
a“
1 1 1 1 1 1 1 1 1
lo-*
1 1 1 1 1 1 1 I l
I 1 1 1 1 1 1 1 I
10-9
10-8
l1lllllll 10-7
Mass i n j e c t e d , g Figure 6. Chromatographic peak heights as a function of quantity
injected for anthracene, anthraquinone, 1-nitronaphthalene,and 4,4’dichlorobiphenyl. Mobile phase is M DPF in 95% acetonitrile. Reactor residence time is 75 s. Working curves for detection of the model compounds in chromatography are shown in Figure 6. Conditions are the same as for the detection limit determinations. On a log-log plot, the working curves are sigmoidal in shape. At lower masses injected, the sensitivity increases with increasing mass of analyte. However, a t sufficiently high concentration the sensitivity begins to decrease with mass of analyte injected. The deviations from linearity a t higher concentrations can be attributed to depletion of DPF; i.e., at high analyte conM DPF is not sufficient to maintain the centrations kinetics as pseudo first order in DPF. However, at analyte levels much above 1 ng, there is no advantage of using a photochemical amplifier anyway. We are not certain of the explanation of the supralinearity observed a t low analyte concentrations. T o better illustrate the advantage of using this photochemical amplification technique in chromatography, we obtained chromatograms of a four-component mixture by using UV absorption a t 254 nm and using DPF photobleaching a t 320 nm. The two chromatograms are compared in Figure 7. In both cases the mobile phase was 95% acetonitrile and the flow rate was 0.7 mL/min. The photochemical reaction conditions again were 75-s reaction time using two lamps and M. The masses of the analytes a DPF concentration of injected were all in the subnanogram range and were chosen so that the peaks would be very near the detection limits for direct UV absorption. The advantage of the photochemical amplifier, as shown in Figure 7, is quite striking. Detection Based on DMF Product Absorbance. DMF has the advantage over DPF of having no significant absorbance at 254 nm. However, some self-photooxidation does occur for this singlet oxygen trap as well, apparently due to atomic emission lines of Hg a t shorter wavelengths that are not completely eliminated by either the quartz filter or the solution filter. Still, the extent of self-photooxidation is much reduced as compared to DPF. The usable spectral change that occurs upon irradiation of a solution of DMF is the appearance of a UV-absorbing product in the wavelength region 240-320 nm. This is illustrated in Figure 8 where a M DMF solution in 95% acetonitrile is irradiated for varying lengths of time. One can see that the photooxidation product can best be detected by setting a UV detector to a wavelength in the region of 250-290 nm. For the work reported here the signal
1840
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
200
220
240
260
200
300
320
Wavelength, nm
Figure 8. Absorbance of a M solution of DMF in 95% acetonitrile after bulk irradiation in a cuvette. Exposure times are 0, 30,60, 120, 180,and 240 s in order of increasing absorbance.
~~
0
2 4 6 8 Time, min
Flgure 7. Comparison of chromatograms obtained by direct UV absorption at 254 nm (lower trace) and by DPF photobleaching at 320 nm (upper trace): (peak 1) 0.32 ng of 1-nitronaphthalene, (2) 0.16 ng of antt-:Aquinone, (3)0.072 ng of anthracene, (4) 0.70 ng of 4,4'-dichlorobiphenyl. Chromatographic conditions are given in the text.
Table V. Effect of Solvent Composition on DMF Product Formation relative peak area compound anthraquinone I-nitronaphthalene quinoline biphenyl
5%
HzO
1.0
1.0 1.0 1.0
25%
HZ0
50%
1.7 0.83 0.82 1.7
HzO
1.3 0.59
1.1 1.9
a t 280 nm was monitored; however, inspection of Figure 8 suggests that somewhat greater sensitivity may be obtained a t shorter wavelengths. The DMF system was characterized in a manner similar to that of DPF. The effect of mobile phase composition and limits of detection will be discussed here. Table V lists the relative peak areas for four model compounds detected in acetonitrile containing 5%, 25%, and 50% water by volume. Although the signal changes due to increasing water are somewhat different from those found for DPF (Table IV), again the variation is much less than that expected based only on consideration of the singlet oxygen lifetime. As for the DPF
system, DMF can be used in chromatography over a wide range of solvent composition. The detection limits for the same six model compounds examined by DPF photobleaching were obtained by monitoring product appearance using DMF as the singlet oxygen trap. Table VI summarizes the detection limits obtained in both 95% acetonitrile and 95% methanol using a concentration of DMF of M and a photochemical reaction time of 120 s with two lamps. Again, we have compared these detection limits with those for optimized UV absorption using the same UV detector and have calculated the sensitivity enhancement factors. These enhancement factors are best in acetonitrile and range from 3.3 for biphenyl to 60 for anthraquinone. Except for anthraquinone, which is detected with a sensitivity more than 5 times better by this method, comparison of Tables I11 and VI indicates better sensitivity when using the DPF photobleaching technique. The principal reason for the enhanced sensitivity using DPF photobleaching relative to DMF product formation is the difference in extinction coefficients of the species monitored. Each DPF molecule oxidized results in a decrease in absorbance proportional to its extinction coefficient a t 320 nm of 30 000 L M-' cm-', while each DMF molecule oxidized only causes an increase in absorbance of a product with an extinction coefficient of G O O L M-' cm-' at 280 nm. However, because DMF has a larger rate constant for reaction with singlet oxygen (9, IO),can be used a t higher concentrations (since a wavelength in a region of nonabsorbance is monitored), and undergoes a much smaller degree of self-photooxidation, there is probably greater potential for improving the DMF reaction system than the DPF system. For example, our experiments have shown a nearly linear correlation between intensity of excitation and resulting signal, suggesting the future use of a more intense photochemical lamp in the DMF system. For DPF, however, the lamp intensity is already high enough to cause a decrease in DPF concentration by a factor of 2 to 3 from self-photooxidation alone. For this reason,
Table VI. Detection Limit Comparisons for Dimethylfuran Product Absorbance acetonitrile compound
optimized UV absorbance
anthracene anthraquinone I-nitronaphthalene quinoline biphenyl 4,4'-dichlorobiphenyl
45 pg (253 nm) 90 pg (250 nm) 160 p g (243 nm) 900 pg (234 nm) 130 pg (260 nm) 90 pg (270 nm)
absorbance at 280 nm 10 Pg 1.5 Pg 10 Pg
45 Pg 40 Pg 20 DE
methanol
enhancement factor
absorbance at 280 nm
4.5x
20 Pg
60X 16X 20x 3.3x 4.5x
5.0 Pg 50 Pg 300 Pg 50 Pg 60
VE
enhancement factor 2.3X 18.X 3.2X 3.0X 2.6X 1.5X
Anal. Chem. 1007, 59, 1841-1843
further increases in lamp intensity are not expected to significantly improve limits of detection. Detection in Complex Mixtures. The principal advantage of applying the photochemical amplifier reported here to the analysis of complex mixtures is that of sensitivity enhancement. The selectivity of detection is also altered, as the degree of photochemical amplification is dependent on such physical properties of the analyte as its extinction coefficient and quantum yield for intersystem crossing. In general, the selectivity for nonfluorescent compounds is enhanced relative to fluorescent compounds and the selectivity toward compounds that are strong UV absorbers is further enhanced over those that are weak UV absorbers. In complex mixtures where coelution of peaks is common, the response is expected to be additive in the low concentration range where this photochemical amplification scheme is advantageous. In order for a coeluting analyte to cause a negative interference by quenching or reacting with singlet oxygen, its concentration must be comparable to that of the singlet oxygen trap molecule (lo4 to M) and its rate constant for reaction be comparably large. Very few compounds react with singlet oxygen with rate constants that are within an order of magnitude of that for DMF and DPF (17). If one assumes an order of magnitude less reaction rate constant and considering that the analyk concentration is diluted by about a factor of -50 within the HPLC column, the mass of the interfering compound would need to be in the range 100-1000 Mg to cause a significant reduction in sensitivity toward an analyte of interest.
LITERATURE CITED (1) Prlngshelm, P. Fluorescence and Phosphorescence; Intersclence: New York, 1949.
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(2) Kawaoka, K.; Khan, A. U.; Kearns, D. R. J. Chem. Phys. 1967,4 6 ,
1842. (3) Murov, S.L. hendbodc of photochemistry; Marcel Dekker: New York, 1973;p 89. (4) Weinberger, R.; Yarmchuk, P.; Cline Love, L. J. Anal. Chem. 1982, 5 4 , 1557. (5) Donkerbroek, J. J.; van Elkema Hommes, N. J. R.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. Chromatographia 1982, 15, 218. (6) Donkerbroek, J. J.; Vekkamp, A. C.; Gooljer. C.; Velthorst, N. H.; Frei. R. W. Anal. Chem. 1983,55. 1886. (7) Arnold, S. J.; Kubo, M.; Ogryzlo, E. A. A&. Chem. Ser. 1968,No. 77, 133. (8) Gollnick, K.; Franken, T.; Schade, G.; Dorhofer Ann. N . Y. Acad. Sci.
1970, 171, 89. (9) Kearns, D. R. I n Singlet Oxygen, Organic Chemistry, A Series of Monogrephs; Wasserman. H. H., Murray, R. W., Eds.; Academic: New
York, 1979;Voi. 40,p 120. (10) Young, R. H.; Wehrly, K.; Martin, R. L. J. Am. Chem. SOC.1971,93, 5774. (11) Poulsen, J. R.; Birks. J. W.; Gubitz, G.; van Zoonen, P.; Gooijer, C.; Velthorst, N. H.; Frei, R. W. J. Chromatogr. 1988,360, 371. (12) POUlsen, J. R.; Blrks. J. W., unpublished work, 1986. (13) Kasha, M. J. Opt. SOC.Am. 1948,38, 929. (14) Poulsen. J. R.; Birks, K. S.; Gandelman, M. S.;Birks, J. W. Chromatographia 1987,22, 231. (15) Krlnski, N. I.I n Singlet Oxygen, Organic Chemisby. A Series of Monographs; Wasserman, H. H., and Murry, R. W., Eds.; Academic: New York, 1979;Vol. 40,p 602. (16) Young, R. H.; Chlnh, N.; Mallon, C. Ann. N . Y. Acad. Sci. 1970, 171,
130. Frlmer, A. A., Ed.; CRC Press: Boca (17) Monroe, B. M. I n Singlet 02; Raton, FL, 1985;Volume I: Physical-Chemical Aspects, pp 177-224.
RECEIVED for review December 29,1986. Accepted March 23, 1987. This work was carried out as partial fulfillment of the requirements for a Ph.D. degree (C.L.S.) at the University of Colorado. This work was supported by a grant from U.S. Environmental Protection Agency (No. R-810717-01-0) and by Kratos Instruments. J.W.B. thanks the John Simon Guggenheim Foundation for a fellowship.
Enzymatic Determination of Carbon- 14 Labeled L-Alanine in Biological Samples Francisca Serra, Andreu Palou,* and Antoni Pons
Departament de Biologia i CiZncies de la Salut, Laboratori de Bioquimica, Uniuersitat de Les Illes Balears, and Institut d'Estudis Avancats UZB-CSZC, 07071 -Palma de Mallorca, Balears, Spain
A method for determination of L-aianine-specifk radioactlvky in Mokglcai samples is presented. This method is based on the speclflc enzymatk transformation of L-alanine to pyruvk acld hydrazone catalyzed by the enzyme L-aianlne dehydrogenase, formatlon of the pyruvic acid 2,4-dlnitrophenylhydrazonederivative, and quantitative trapping In Amberlite X A D 7 columns, followed by radloactlvity counting of the Hpophiik eluate. No Interferences from other "Clabeied materials such as D-giucose, glycerol, L-lactate, L-serine, Lglutamate, L-phenylaianine, glycine, L-leucine, and L-arglnine were observed. This Inexpensive and hlgh-speed method is appilcable to the simutlaneous determlnatlon of L-alaninespecific radioactivity for a large number of samples.
Estimation of radioactivity in certain amino acids is basic for understanding the fate of the label in a number of met0003-2700/87/0359-1841$01.50/0
abolic tracer studies, e.g., the determination of radioactivity in L-alanine in studies of gluconeogenesis (I, 2). The presence of label in amino acids can be estimated by several methods (2-8), but the specific radioactivity determination in individual amino acids-as L-alanine (1, 2)-requires tedious and expensive methods that have high variability in the mean recovery. The use of polymeric porous hydrophobic adsorbing beads for trace amounts of organic compounds from aqueous solutions is a well-documented procedure (8-11). Previously we had applied the Amberlite XAD to the estimation of individual amino acid radioactivity in plasma samples (8), ammonia and urea determination in water samples (12),enzymatic L-lactate-specific radioactivity (11)and pyruvate-specific radioactivity (10)determinations in biological samples. In this paper we present a method for L-alanine-specific radioactivity determination based on the highly specific enzymatic transformation of L-alanine to pyruvate catalyzed by 0 1987 American Chemical Society
