1886
Anal. Chem. 1983, 55, 1886-1893
Quenched Room Temperature Phosphorescence Detection for Flow Injection Analysis and Liquid Chromatography J. J. Donkerbroek, A. C. Veltkamp, C. Gooijer,* N. H. Velthorst, and R. W. Frei Department of General and Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
The potentlal of quenched room temperature phosphorescence In llqulds (RTPL) Is demonstrated as a detection technlque. The method Is based on quenchlng of the phosphorescence of blacetyl by sultable analytes. Groups of compounds such as chloroanlllnes, amines, heterocyclic nltrogen compounds, sulfur organics, chlorophenols, other aromatic and allphatlc hydroxy compounds, and several lnorganlc Ions have been tested. For Ideal (dlffuslon controlled) quenchlng processes, detectlon llmlts (LODs) on the order of lo-’ to lo-’ M can be obtained, which are comparable to sensltlzed RTPL and only less favorable than dlrect fluorescence detection. The llnearlty of response can be extended to 2 to 3 orders by using electronlc slgnal Inversion techniques. The application potentlal of quenched RTPL as a detection technlque In flow InJectlonanalysls ( F I A ) or hlghperformance liquid chromatography (HPLC) has been demonstrated wlth a group of sulfur organics.
In recent papers (1,Z) we have shown that the room-temperature phosphorescence of biacetyl in normal (deoxygenated) liquid solutions (to be denoted as RTPL) can be successfully uitlized for detection in flow injection analysis and liquid chromatography. Most examples presented until now are based on sensitized RTPL of biacetyl; the analyte is excited, subsequently its triplet-state energy is transferred to biacetyl, and the phosphorescence of biacetyl is monitored. Since it is well-known that in liquid solutions phosphorescence intensities can be strongly quenched by traces of impurities present in the solution (3,4), it can be expected that the sensitivity of quenched RTPL detection will be of analytical interest for selected compounds with quenching properties. In this paper, the attention is therefore focused on the quenching of the direct RTPL of biacetyl by suitable analytes. In a recent HPLC study on polychloronaphthalenes (Z), we have already indicated that quenched RTPL detection can provide useful information in addition to sensitized RTPL detection. For these compounds, quenching is observed if the triplet state energy of the analyte is lower than that of biacetyl, so that quenched RTPL can be considered as complementary to sensitized RTPL. However, quenched RTPL of biacetyl is not limited to analytes with low lying triplet states. Other quenching mechanisms may also be important, such as electron transfer and hydrogen abstraction (5). In the literature, the quenching of fluorescence has also been introduced as a detection method in HPLC, i.e., by the group of Winefordner (6,7).However, it should be realized that this detection principle is essentially different from the quenched RTPL method described here. Our detection is based on a “dynamic quenching process”; i.e., the analyte (acting as a quencher) reacts with excited biacetyl thus prohibiting phosphorescence emission. Hence, the sensitivity inherent to the quenched RTPL method must be attributed to the biacetyl triplet lifetime in liquid solutions (e.g., in acetonitrile
about 5 X loe4s (8)). Dynamic quenching of fluorescence is far less effective since the lifetime of the lowest excited singlet states is 3 to 5 decades shorter than the triplet state lifetime mentioned above. Therefore the fluorescence quenching principle used by Winefordner et al. is based on a different concept, Le., on the static quenching as a result of the interaction between the fluorophor and the analyte in their ground states and/or on the pre- and postfilter effects caused by the analyte. In this paper, a screening test of the quenched RTPL detection method is presented for a variety of organic compounds and for a number of inorganic ions. It is shown that the sensitivity of the method for a particular compound is directly related to the bimolecular rate constant of the quenching reaction. As will be seen in the theoretical section, the decrease of the RTPL signal of biacetyl is not linearly dependent on the analyte concentration. It will be demonstrated that inversion of the decreased signal can eliminate this problem. Finally, the applicability of the quenched RTPL detector is illustrated by means of liquid chromatograms for some thiourea derivatives. THEORETICAL ASPECTS The direct RTPL intensity of biacetyl, I(dir), is given by (8)
I(dir) = IBabsOBiscdBp
(1)
where IBabs is the rate of light absorption by biacetyl (the excitation wavelength ABex is usually around 420 nm). OBisc is the intersystem crossing or triplet formation efficiency of biacetyl and OBP its phosphorescence efficiency in the liquid solution at hand. The word direct is incorporated to emphasize the difference with sensitized RTPL, previously denoted as Z(sens). In the absence of quencher, OBp = kBprBo,so that eq 1can be written as I(dir) = IBabsdBisckBp7Bo
(2)
Here kBpis the rate constant of the phosphorescence process (about 100 s-l) and T~~ the triplet lifetime of biacetyl. In deoxygenated and purified solvents T~~ ranges from lo-* to 10-3 s (9). If an amount of quencher is introduced, the triplet lifetime of biacetyl reduces to T~ where (rB)-l= (rBo)-’ -I- k ~ [ & ] (3)
k , is the bimolecular rate constant of the quenching reaction and [Q] the concentration of quencher. As a result I(dir) decreases to I’(dir) given by I’(dir) = IBabsOBisckBp{ ( TBo)-l
he[&] 1-l
(4)
From a combination of eq 2 and 4 it is obvious that the decrease of the biacetyl phosphorescence intensity, AI,does not depend linearly on [Q], i.e.
0003-2700/83/0355-1886$01.50/00 1983 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
A linear detector based on quenching can be obtained by measuring the reciprocal 'of the partially quenched biacetyl phosphorescence intensity, in other words by measuring 1'(dir)-l, which is given by
Equation 6 implies that plotting of I'(dir)-l vs. [Q]will deliver a straight line with a slope proportional to kQ and an intercept equal to the inverted intensity of the unquenched signal. It is noted that the dope, in contrast to the intercept, does not depend on T ~which ~ , is known to be influenced significantly by the choice of the solvent (9). Nevertheless, the sensitivity of the method is dependent on the solvent via T~~ since the noise on the base signal, 1 (dir)-l, depends on this parameter. This point will be considered below. For the dynamic system, a linear response of the detector as a function of [&I is predicted. Therefore an electronic signal inverter should be introduced between the spectrometer and the recorder. EXPERIMENTAL SECTION Apparatus. A description of the experimental setup including the deoxygenation procedure for the batch experiments ( 1 0 , I I ) and the chromatographic system ( I ) has been given. The latter was extended with a multiplier/divider (M/D) system, inserted between the Kontron SFM 22 spectrometer and the recorder. The M/D system was borrowed from the PEN (Provinciaal Electriciteits bedrijf Noord-Holland, Velsen, The Netherlands). It consists of a power supply type 19-601 A (Elliott Process Automation Nederland N.V.), EL millivolt preamplifier type 19-101A (Elliott), and a multiplier/divider type 19-302 (Elliott). The preamplifier transforms the spectrometer output E,, (0-100 mV) to the input potential E,, (0.5-10.0 V) of the M/D; the minimum value of 0.5 V for E, is introduced to protect the M/D against division by zero. The M/D transforms E , to Eout,the potential being registered by the recorder, according to
where the parameters a, (i = 1 to 4) are dimensionless and the potentials E, are variable. In the present setup the parameter choice is as follows: a. = 1.00, al = 0.05,a2E2= u4E4= 0 V, El = E, = 10.0 V, which leads t o Eout = 5.0 (Ein)-'V. Determination of the k QValues. The bimolecul.ar rate constants of quenching, kq,, were determined from Stern-Volmer plots, based on the following equation:
M biacetyl These plots were obtained as follows: a 1.0 X solution was deoxygenated for 8 min and the corresponding Z(dir was measured (excitation wavelength 420 nm, emission wavelength 516 nm). Subsequently, variable amounts of the quenching analyte were added with a hypodermic syringe (10 fiL) (Hamilton, Bonaduz, Switzerland). The solutions were deoxygenated again and Z'(dir) was determined. For each analyte, at least five measurements were carried out, thus varying Z(dir)/Z'(dir) from 1 to 4;the correlation coefficients were larger than 0.995. The determination of kQ from the slope of the Stern-Volmer plots requires the knowledge of T ~ For ~ the . different solvents, these lifetimes were estimated from the ratio of the phosphorescence and fluorescence intensities measured at their respective maxima, as described elsewhere (11). As a reference,the value of 5 X s for T~~ in hexane corresponding t o a ratio of 18 as reported by Turro (12) was used. Operation of the Detector in the Dynamic System. In the continuous-flow system, the biacetyl present in the eluent is excited at 420 nm and its phosphorescence detected at 516 nin. The background emission as recorded before deoxygenation, when no phosphorescence is present, is substracted from the phos-
1887
phorescence signal. Sublsequently the resulting signal is expanded to 100 mV and transformed by the M/D system, giving the base line. The passing of an analyte through the flow cell leads to ;I partial quenching, in other words, to a decrease of E,, and #a positive signal after the inversion. Reagents. The source and purification of biacetyl and the azeotropic acetonitrileJwater mixture have been given ( I ) . The demineralized water was distilled before mixing. Benzene (Baker, Deventer, The Netherlands) and hexane, HPLC quality (Baker), were applied without further purification. The investigated analytes were used as supplied 3,4-dichloroaniline,thiourea, glucose, the inorganic salts, triethylamine, aniline, and pentachlorophenol by Merck, Darmstadt, G.F.R.; the other chloroanilines and furthermore trihexylamine, tribenzylamine, and hydroquinone by Fluka, Buchs, Switzerland; the other chlorophenols and furthermore p-aminobenzoic acid, sulfanilamide, indole, 8-hydro2,4-dinitrophenol, quinoline, 2-mercapto-l-methylimidazole, phenol, and barbituric acid by Aldrich, Beerse, Belgium; a-naphthylamine, pyridine, isoquinoline, @naphthol,and resorcinol by Baker, Phillipsburg, NJ; indole-3-propionic acid and ethyl indole-3-acetate by Chemical Lab. Co., Cleveland, OH; promethazine, thioridazine, sulforidazine, the lanatosides A and C, ad digitoxigenin by Sandoz, Basel, Switzerland; streptomycin by Glaxo, Barnard Castle, G.B.; tryptophan by Sigma Chemical Co., St. Louis, MO; methionine by B.D.H. Chemical, G.B.; thiuram, thiobenzamide, thioacelmilidine, and thiohydantoin by the Health Protection Branch, Ottawa, Canada; and ethylenethiourea biy Riedel de Haen, Hannover, G.F.R. RESULTS AND DISCUSSION Bimolecular Quenching Rate Constants. As will be obvious from eq 6, the sensitivity of the quenched RTPL detection method for a particular analyte is proportional to the value of the associated 12,. An estimation of the detection limits can be made as follows: (a) If the direct biacetyl phosphorescence signal, for instance, in the azeotropic acetonitrile/water mixture, a t 5116 nm is set equal to Eq, = 100.0 mV, it is found experimentally that the RMS noise, inainly due to the lamp, is about 0.2 mV. (b) Consequently, the minimum analyte concentration which can be detected causes a decrease of E,, from 100.0 to 99.4 mV, if the detection limit is determined a t a signal to noise rat0 of 3 to 1. (c) Substituting these results in eq 8 and taking for convenience kQ = 1 X log M-l s-l and T~~ = 6 X 10" s reveals that LOD = 1 X lo-@M. Thus, an estimation of the LOD for a particular analyte can be readily obtained, since under tlhe above assumptions
LOD (in M) = 10/kQ (in M-' s-l)
(9)
These considerations imply that the quenched RTPL method will be of interest for analytes quenching the biacetyl phosphorescence with rate constants lo7 to lo9 M-' s-l depending on the group of compounds tested. Therefore, the determination of kQvalues for a large number of analytes is important as a screening technique to examine the analytical potentiial of the detection method. It is expected that analytes with triplet energies distinctly lower than the triplet energy of biacetyl produce diffusion controlled quenching via energy transfer (13). Hence, these analytes will be sensitively detectable by recording the quenched RTPL of biacetyl. However, in the following sections, the attention is mainly focused on compounds with higher triplet energies than biacetyl, illustrating that for quenching other mechanisms can also be operative. We have determined kQ values for a great variety of compounds, mostly in the azeotropic acetonitrile/water mixture, utilizing eq 8; see Tables I to VIII. The results will be discussed in the following sections. Screening Program. Amines. In Table I, k , values For a number of chloroanilines are presented, together with the estimated order of the LOD; see eq 9.
1888
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 198
Table I. b Values and Order of the Estimated Limits of Detection (%ODs) for Several Chloroanilines in the Azeotropic Acetonitrile/Water (83.7:16.3, v/v) Mixture compound aniline (An) 3-ClAn 4-ClAn 2,4431,An 2,5-C1,An 2,6-C1 An 3,4-C1*An 3,5-C12An 2,3,4-C13An 2,4,5-C13An 2,4,6-C13An 2,3,4,5-C14An 2,3, 5,6-C14An
k Q , M-'
order of LOD, M
S-'
1.8 x 109 4.4 x 108 1.8 x 109 2.7 X l o 8 1.1x 108 1.4 X l o 8 4.7 x 108 1.1 x 108 1.2 x l o 8 1.2 x 108 8.0 X l o 7 9 x lo7 4.9 x 107
10 -8-10 -9 10 -7-10- 8 1 0- 8 - 1 0 - 9 10- 7 - 1 0- 8
io - 7 - 1 0 - 8 10- 7-10-8 10-7-10-8
10 - 7 - 1 0 - 8
i o - 7-10-8 i o - 7-10-8 10-6-10-7 10 -6-1 0 - 7 10 -6-10 - 7
I
Table 11. h~ Values and Order of the Estimated LODs for Some Amines in the Azeotropic Acetonitrile/Water Mixture compound 2-nitroaniline 4-nitroaniline 4-aminobenzoic acid sulfanilamide a-naphthylamine triethylamine trihex ylamine tri benzylamine
kg,M-' 4.9 2.6 1.4 2.0
Table 111. k Q Values and Order of the Estimated LODs for Some Heterocyclic Aromatic Nitrogen Compounds in the Azeotropic Acetonitrile/Water Mixture
5-l
x 109 x 109 x 109 x lo8
1.6 X 10"
3.7 x l o 8 3.0 X l o 8 7.2 x 107
order of LOD, M 10 -8-10-9 IO -8-10 -9 10-8-10-9 10-7-10-8 ~0'9-~0'10 10-7-10-8
10 -'-lo-*
1 0- 6 - 1 0
-7
Turro and Engel (5)have reported that effective quenching based on electron transfer is observed for aliphatic and aromatic amines with ionization potential below 8 eV. This is in accordance with the high kQ value we found for aniline whose ionization potential is about 7.7 eV (5). Presumably, the ionization potential and thus the associated kQ changes with the degree of chlorination. It is obvious from Table I that for the analysis of mixtures of chloroanilines quenched RTPL detection can be very useful. In Table 11,the values for some other amines are collected. The results are very promising, especially the LODs expected for the alkylamines, which are not easily detectable via UVabsorption techniques. The high kQ values obtained for 2and 4-nitroaniline are rather unexpected in view of their high ionization potential, i.e., 8.7 and 8.85 eV, respectively. This leads to the conclusion that another quenching mechanism must play a role. Generally, we conclude that amines have interesting rate constants of biacetyl phosphorescence quenching, presumably via electron transfer. This implies that quenching will be inhibited in acidic solutions where protonation of the amino group occurs, as we have observed indeed. Therfore, as a rule, quenched RTPL detection of these compounds will not be applicable in acidic media. However, from a selectivity point of view, it will be interesting to make use of differences in protonation constants, applying pH variations. Nitrogen-Containing Heterocyclic Compounds. In Table 111, the kQ values for some heterocyclic aromatics are presented. I t can be seen that the compounds containing a pyridine-type nitrogen are poor quenchers, with the exception of 8-hydroxyquinoline where probably the OH group plays the dominant role. Contrary to this, the compounds with a pyrrole-type nitrogen quench rapidly and will therefore be well detectable. These results are not unexpected in view of the difference of 1.1eV between the ionization potentials (IP) of pyridine and pyrrole (14).
compound pyridine isoquinoline 8-hydroxyquinoline indole indole-3-propionicacid tryptophan ethylindole 3-acetate
k ~M-',
3x
S-'
lo6
lo+
10-8-10-9
10-7-10-8 10- 8 - 1 0-9 10 -7-1 0- 8 10-'-10-8
Besides it should be mentioned that isoquinoline, though not detectable by quenched RTPL, appears to be a good sensitizer of biacetyl phosphorescence so that it can be detected by utilizing the RTPL detector in the sensitized mode. Sulfur-Containing Compounds. Table IV shows that many sulfur-containing compounds being of pharmaceutical and biological interest (15,16)have high kQ values and thus good quenching properties for detection by quenched RTPL. The determination of the k g values of sulforidazine is somewhat complicated because its native fluorescence partly compensates for the decrease of the biacetyl phosphorescence. Therefore, only a lower limit for its kQ is given. Also for these compounds, the electron transfer mechanisms may be a t least partly responsible for the quenching. This is obvious for the three phenothiazines which have I P values below 7.5 eV (17). The ionization potentials of the thioureas are somewhat higher (for thiourea, IP = 8.5 eV (15)). Here probably, the lone pair electrons of the sulfur play an important role in the quenching process. This is supported by the observaton that methionine has a considerable quenching ability, whereas methionine sulfoxide has been reported not to quench a t all (18). Aromatic Hydroxy Compounds. In Table V, the kQ values of a series of chlorophenols are assembled. For the parent compound we found, in line with Turro (5),a significant difference between the solvents tested. Turro ascribed this dependence to a quenching mechanism based on reversible hydrogen abstraction by the biacetyl triplet. In our opinion, the k~ values obtained for the chlorophenols suggest that in polar solvents another quenching mechanism can also be operative. It is noted that most of the highly chlorinated compounds have high kQ values in acetonitrile/ water. These compounds probably have low pKa values; e.g., the pK, of pentachlorophenol is around 5.0 (19),implying that in acetonitrile/water this phenol is dissociated almost quantitatively. Hence, the quenching observed comes from the phenolate anion which might be able to induce a rapid quenching via electron transfer. This hypothesis is supported M H3P04solution, the k , value by the fact that in a 2 X for pentachlorophenol is nearly 2 orders of magnitude smaller. Therefore, we conclude that phenolates are well detectable by quenched RTPL. This indicates that also for the other chlorophenols much higher kQ values will be attainable by increasing the pH of the solution. Unfortunately in solutions with a pH larger than 8.5 the biacetyl phosphorescence intensity appears to be strongly reduced. Nevertheless, the pH dependence of the k~ values for the chlorophenols is interesting for selectivity purposes. Table VI shows the results obtained for some other aromatic hydroxy compounds. As expected in view of its pKa value (14), the influence of the pH on the kQ value of 2,4-dinitrophenol indicates that also for this compound, the anion plays an important role. Barbituric acid (pK, = 4.0 (15))probably also quenches via its anion. Contrary to this, for /3-naphthol and resorcinol, the pK, values are such (14) that anion formation seems unlikely. For
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983 1889 -.___ Table IV. k Q Values and Order of the Estimated LODs for a Number of Sulfur Containing Compounds in the Azeotropic AcetonitrileJWater Mixture or Water compound order of name formula solvent LOD,M
mixture
4.3 x 109
10-8-10-9
mi :< t ure
7.8
x
109
10-8-10-9
mixture
>2.4
X
lo9
10-8-10-9
mixture
2.7 X
lo8
10- '-10
thio benzamide
mixture
8.6 X 10'
thioacetanilidine
mixture
4.5 x
lo8
10-7-10-8
ethylenethiourea (ETU)
mixture wiater
8.4 X 7.5 x
lo8 lo8
10- 7-1 0 - 8 10- 7-1 0 - 8
thiohydantoin (THT)
water
3.2 X
lo8
10-7-10-8
water
6.3 X 10'
10 -7-10
mixture
8.1 x 107
10-6 -1 0 -'
mixture water
6.3 X
promethiazine
NJ
thioridazine
/ CH3 R2=
- S -CH3
sulforidazine
CH3
0
H 3c\
thiuram H,C'
thiourea (TU)
methionine
,CH3
N
-- c -s -s - c -N /I 'CH, 11 S
S
S
II
H2N-
HQOC -C
H
I
C -NHz
H?
-C-C
H2
-S -CH,
NH2
2-mercapto-1-methylimidazole(MMZ) \
I
3.2 X
loy lo8
10- 7-1 0 -6
1890
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983 50
Table V. kQ Values and Order of the Estimated LODs for a Series of Chlorophenols in the Azeotropic Acetonitrile/Water Mixture or Hexane compound
solvent
phenol (P)
mixture hexane 4-C1P mixture 2,3-C1,P mixture 2,4-C12P mixture mixture (pH 1 hexane 2,5-C1,P mixture 2,641 ,P mixture 3,4-C1,P mixture 2,3,6-C13P mixture 3,4,5-C13P mixture 2,3,5,6-C14P mixture C1,P mixture mixture (t2 x M H3P04)
kQ ,
M - 1 s-l
4 x 107 1.8 X 10’ 2 x 107 3.6 x 107 3.9 x 107 3.5 x 107 1.4 X 10, 5.1 x 107
2.1 x
lo8
8.0 X 10’ 1.1 x 109 5.1 x 107 2.3 x 109 4.8 x 109 7 x lo7
order of LOD, M 10 - 6 - 1 0-7 10 - 7 - 1 0 - 8 10 -6-1 0-7 10-6-10- 7 10-6-10- 7 10-~-10-7 10-7-1 0 - 8 10-6-10- 7 10 - 7 - 1 0 - 8 10 -6-10 - 7 10-8-10-9 1 0 -6-10- 7 10 - 8 - 1 0 - 9
solvent
MkIQ;-l
(V)
20
10
001
order of LOD, M
mixture 1.0 x 109 10-8-10-9 mixture ( t l 0 - j M 3.8 x l o 7 10-6-10-7 H3P04) P-naphthol mixture 6.8 X l o 7 10‘6-10-7 resorcinol mixture 3.4 x 107 10-6-10-7 hydroquinone mixture 8.9 x 107 10-6-10-7 barbituric acid water 6.8 X 1 0 , 10-7-10-8 hydroquinone, electron transfer may be responsible, which is realistic in view of the well-known hydroquinone-quinone equilibrium. Aliphatic Hydroxy Compounds. In Table VII, the h, values of some OH-containing aliphatic compounds are listed. These values are on the average lower than those reported for many other groups of compounds. However, if one considers that many of the aliphatic hydroxy compounds (sugars, steroids, antibiotics, etc.) have very poor UV absorption properties, it would be worthwhile to pursue their investigaton by quenched RTPL. The predicted detection limits for glucose, streptomycin, and lanatoside C, for example, are still considerably more attractive than for alternative detection techniques based on refractive index or UV absorption a t wavelengths shorter than 220 nm. An additional advantage could be the added selectivity of the quenched RTPL method. Inorganic Ions. In Table VIII, the quenching rate constants for a number of inorganic ions are assembled. Interesting results are obtained for NOz- (NO3- does not quench strongly), SCN-, S2032-, and Sn2+. In the literature, comparable h, values have been reported for I- and SeCN- in water (20). These ions will be detectable quite sensitively by quenched RTPL. It is not simple to explain the results in terms of polarographic redox potentials and ionization potentials as postulated by Bortolus (20) and Weller (22). As an example, SO-: has poor quenching properties, in spite of its low standard electrode potentials of -1.12 and -0.58 V (22), reported for the two redox reactions below:
2So3’-
30 Eout
10 -6-10-7
2,4-dinitrophenol
2S032-
1
1 0- 8 - 1 0 - 9
Table VI. h Values and Order of the LODs for Some Aromatic Hyl%roxyCompounds in the Azeotropic Acetonitrile/Water Mixture or Water compound
40
+ 2 H 2 0 + 2e- - Sz042-+ 4 0 H i3Hz0 + 4e- - S2o3’+ 60H-
(10) (11)
Although the choice of systems tested in this section has been rather arbitrary, one can conclude that the quenched
005
010
-
0 15 E~; ( rnv )-I
Figure 1. Output signal E,, of the M/D system in V vs. the reciprocal of the spectrometer output in mV-’.
RTPL mode can be interesting for detection of many ionic species in ion chromatography where detection by other methods can be problematic. Quenched RTPL f o r Continuous-Flow Detection. Signal Inversion. In the previous section, it was shown that the quenched RTPL method can be of considerable interest to analytical chemistry. According to eq 6, a linear response will be obtained if the output of the spectrometer is inverted. Hence for a continuous-flow system, it is appropriate to perform an electronic inversion of the spectrometer output (E3,),which is proportional to I’(dir). For the construction of such an inverting instrument the following points have to be considered: (i) In order to avoid additional electronic band broadening effects, its response time must be considerably smaller than the detector response time. (ii) The instrument must be protected against very small detector outputs, since division by zero may damage the electronics of the M / D system. Unfortunately, such a protection leads to nonlinearity for strongly quenched signals. Hence a compromise is necessary. (iii) The spectrometer output must be corrected for background emission, including fluorescence and scattering. This can be realized by means of the “blank compensation” of the spectrometer output. A detailed description of the instrument used here is presented in the Experimental Section. The output of the as a function of E,;l is shown in Figure M / D system (Eout) 1. The plot is linear for E,, values ranging from 100 to 30 mV; the latter value is reached if 70% of the original biacetyl phosphorescence is quenched by the analyte. In the flow system for all the compounds being tested, a linear range of about 2 decades was found. This strongly suggests that the linearity of the detector system is determined by the quality of the inverting instrument used. As expected for small values of Ea,, which are reached for high concentrations of Q, the threshold potential of 0.5 V leads to deviations from linearity. It must therefore be possible to extend the linear range of the quenched RTPL detection method by reducing this threshold potential. Sensitivity. Other points to be discussed are the influences of the concentration and the lifetime of biacetyl in the mobile phase on the sensitivity of the quenched RTPL method. Their effects on Eout,the signal intensity, which is proportional to
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
1891
Table VII. kQ Values and Order of Estimated LODs for Some Aliphatic Hydroxy Compounds in the Azeotropk Acetonitrile/Water Mixture compound formula
name
R , = H; R, = G-A-D-Da R , = OH;R, = G-A-D-D R, = H;R, = H S
lanatoside A lanatoside C digitoxigenin streptomycin
h,a, M - I s-l
6.1 x 4.0 X 4.7 x 3.2 x
107 lo8 107 107
order of LOD, M
10 - 6 -10- 7 10-7-10-8 10-6-10-’ 10-6-10-7
CHzOH
glucose
4.0 x 107
HOQOH
10-6-10-7
OH
a
G = glucose; A = acetyldigitoxose; D = digitoxose
OH
OCOCH,
A
D
Table VIII. ka Values and Order of Estimated LODs for Several Inorganic Ions in the Azeotropic Acetonitrile/Water Mixture ion NO,-
NO,SCN CN-
so,2so;-
s,o,z-
Fez+(H,PO,) Fea+ Fe3+ Sn2+ Sn4+
Cr3+
k g , M-l s-’ 1 . 3 x 109
10-5 10-7-104 10-’-10-8 10-6-10-7
< i o 7
1 . 2 x 109
10- 8 - 1 0
5.8 x 107
10-6-10 - 7
< 10’
-9
1ldir-l are obvious from eq 4 and 6. However, their influences on the noise of the base line need some further comment. We denote the height of the recorded base line as Eoout and its (RMS) noise as N(out) and furthermore the height of the noninverted unquenched signal as EO,, and its noise as N(1). N(out) is determined by N(1) and by the noise contribution of the M/D system. We have found that the latter is negligible. Hence N(out) is exclusively determined by the corre-
lation between Eoorlt and Eoapwhich can be written in the differential form (:12)
Since E O , is proportional to I(dir) and Eoout is proportional to I(dir)-P, the differential term in eq 12 is proportional to I(dir)-z. Futhermore, we have found that, within the experimental accuracy, N(1) is proportional to I(dir), a result in line with Winefordner et al. (23). Therefore the conclusion must be that N(out) is proportional to I(dir)-l. Rewriting eq 6 to I’(dir)-l = I(dir)-l + ksI(dir)-l~Bo[Q] (13) directly shows that the peak height, which is proportional to I’(dir)-l- I(dir)-I, depends also linearly on I(dir)-l so that the sensitivity determined by the signal to noise ratio is independent from I(dir). This implies that the sensitivty of the quenched RTPL detection method cannot be improved by increasing the biacetyl concentration since its effect on the signal is compensated by its effect on the noise. On the contrary, the sensitivity depends linearly on rB0,the triplet lifetime of biacetyl in the eluent applied; in solvents wherein T~~ is short, the sensitivity of the quenched RTPL metlhod will be relatively low. This will be obvious since I(dir) is proportional to T ~ , ) see , eq 2, and therefore N(out) is pro-
1892
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
Table IX. Chromatographic Data and Comparative Detection Limits for Some Sulfur Compounds Selected from Table IV
compound
k Q , M - ' s-'
k'
TU THT ETU MMZ
6.3 X l o 8 3.2 X l o 8 7.5 x l o 8 1.9 x 109
1.07 2.8 3.8 10.7
plug inj. Pd(I1) calceine LOD,a ng LOD,b ng 0.5 3.7 1.5
1.3
DME detec. LOD,Cng
glassy carbon LOD,d ng
7
0.15
16e
1.3e
7 8 0.8
a Calculated from actual chromatograms, to determined with NaNO,; column length, 11 cm; RP-18; d, = 5 pm; eluent, Plug injection data from ref 24; carrier stream, phosphate buffer water + 1.0 x l o + M biacetyl; flow rate, 1 mL/min. pH 7 . Chromatographic data from ref 25; column length, 28 cm; RP-8; d, = 10 Fm; eluent, 1%(v/v) methanol in water + 0.1 M KNO, i0.02 M " 0 , ; flow rate, 0.9 mL/min. Chromatographic data from ref 25; conditions as in footnote c except: eluent, 1%(v/v) methanol in water + 0.01 M KNO, i5 X M "0,; flow rate, 1.2 mL/min. e It can be estimated from ref 25 that k ' is about 2.
2
10 8 6 4 2 0 + tR(min)
Figure 2. Quenched RTPL chromatogram of some thiourea derivatives: eluent, water 1.0 X M biacetyl; column RP-18, 12 cm, d , = 5 wm; flow rate, 1 mL/min; observed t , ( = t o ) for NaNO, (peak 1) = 45 s;injected amounts, thiourea 41 ng (peak Z),thiohydantoin 63 ng (peak 3), ethylenethiourea 55 ng (peak 4), methimazole 62 ng.
+
portional to ( T ~ ~ ) - ' However, . E,, - Eoout is independent of T~~ because in the product I(dir)-'TBo, see eq 13, the lifetime plays no role. Application in LC. As an example in Figure 2 a chromatogram detected by quenched RTPL is depicted. The mixture consists of a number of thiourea derivatives selected from Table IV. The quantities injected are 20 pL of a 2.7 X M solution, for NaNOz used to determine to,20 MLof a 3 X lo4 M solution. The corresponding LODs derived from the chromatograms are given in Table IX. It is interesting to compare them with the LODs obtained in our laboratory for plug injections by means of detection based on ligand exchange with Pd(I1)calceine (24). It needs no further explanation that the present direct method in which the analytes have not to be modified chemically compares quite well. Also chromatographic results of measurements performed at our laboratory with a dropping mercury (DME) electrode and with a solid-state glassy carbon detector are incorporated in Table IX (25). It is obvious that for the particular compounds our detector is comparable with the glassy carbon detector and distinctly more sensitive than the DME electrode. This again demonstrates the sensitivity of the quenched phosphorescence detector. As to selectivity, the potential of quenched RTPL detection for the analysis of traces in complex matrices is subject to further investigation. CONCLUSIONS In this study, we have shown that phosphorescence quenching in the liquid phase under room temperature conditions has interesting possibilities for many groups of compounds which may have inherently poor detection properties with other techniques. Quenched RTPL can be complementary to sensitized RTPL since it is applicable for compounds for which the latter method cannot be applied.
On the other hand, a screening of the method reveals that it is not only applicable for the detection of compounds with triplet energies lower than for biacetyl but also for compounds like aromatic and aliphatic amines, for pyrrole-type nitrogen compounds, several sulfur-containing pharmaceuticals, hydroxy compounds, and inorganic ions. The poor linearity of the signal E,, to concentration relationship (calibration curves) can be improved by using electronic signal inversion techniques. These can be relatively simple and low cost; they do not add much to the design of RTPL detectors. The same detector design can be applied for both quenched and sensitized RTPL. Further studies will be needed to understand more about the mechanisms of quenched RTPL and to be able to make full use of its application potential. Investigations with RTPL detection for analysis of traces in complex environmental and biological matrices are already performed. ACKNOWLEDGMENT
J. Veltkamp from the P.E.N. is to be thanked for making the M / D system available to us. J. F. Lawrence, Health Protection Branch, Ottawa, Ontario, Canada, Glaxo Ltd., and Sandoz Ltd. are acknowledged for the gift of model compounds. Registry No. An, 62-53-3;3-ClAn, 108-42-9;4-ClAn, 106-47-8; 2,4-ClzAn,554-00-7;2,5-CIzAn,95-82-9; 2,6-C12An,608-31-1;3,4C12An,95-76-1; 3,5-C12An,626-43-7; 2,3,4-C13An,634-67-3;2,4,5C13An, 636-30-6; 2,4,6-C13An,634-93-5; 2,3,4,5-C14An,634-83-3; 2,3,5,6-CI4An,3481-20-7; ETU, 96-45-7; THT, 503-87-7;TU, 6256-6; MMZ, 60-56-0; P, 108-95-2;4-C1P, 106-48-9;2,3-C&P,57624-9; 2,4-clzP, 120-83-2; 2,5-ClzP, 583-78-8; 2,6-ClzP, 87-65-0; 3,4-ClzP, 95-77-2; 2,3,6-C13P, 933-75-5; 3,4,5-C13P, 609-19-8; 2,3,5,6-C14P, 935-95-5; ClbP, 87-86-5; NOz-, 14797-65-0; NO
