Anal. Chem. 1990, 62, 2275-2283
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Determination of Carbon-Centered Radicals in Aqueous Solution by Liquid Chromatography with Fluorescence Detection David J. Kieber and Neil V. Blough* Chemistry Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543
A slmpte method to detect subnanomdar to micromolar levels of photochemically generated carbon-centered radicals in aqueous solutions has been developed and optlmlred. Thls method Is based on the efficient trapping of radicals by a watergduMe amlno nltroxlde, followed by derlvatlzatlon of the trapped products with fluorescamlne to produce hlghly fluorescent adducts. These adducts can be separated by reversed-phase hlgh-performance liquid chromatography and detected fiuorometricaliy. The fluorescent derivatives are stable over a period of days. The detectlon Ilmlt, primarily determlned by reagent lntederences, ranged from 0.3 to 1 nM per analyte for a 500-pL lnlectlon at a slgnal-to-noise ratio of two. The preclslon of the method for the determinatlon of adduct concentratlons In the 1-10 nM range varied from 2.4 to 8.4% relatlve standard deviation ( n = 6). A direct comparison with electron paramagnetic resonance spectroscopyhpin trapplng Illustrates the advantages of our technique. One Important feature of the method Is that It permits the simultaneous detection of an array of radicals, as demsnstrated through the study of the photochemlcai production of radlcals In a varlety of natural water samples and In Suwannee River fulvlc acid.
INTRODUCTION The lack of methods with which to detect and identify very low concentrations of free radicals in condensed phases presents a major hurdle to our understanding of the impact of these highly reactive species on numerous chemical ( I , 21, environmental (3), biological (4, 51, and toxicological (6) processes. Direct detection of radicals by electron paramagnetic resonance (EPR) or optical spectroscopies is generally not possible at the commonly encountered low, steady-state levels of these species. More often, radicals are detected indirectly by employing radicaI traps or scavengers which react rapidly with transient radicals to form stable or persistent products. The accumulation of products (or loss of trap) thus acts to integrate the radical “signal”. Spin trapping is one of the most often used indirect methods of radical detection (7,8). In this method, radicals are scavenged by nitrones or nitroso compounds (spin traps) to produce persistent nitroxyl radicals (spin adducts); the pattern and magnitude of the hyperfine splittings obtained from the EPR spectrum of the spin adduct can be used in some instances to identify the structure of the trapped radical. While often successfully employed, this approach is subject to a number of limitations and potential artifacts, including (1) spin adduct instability ( e l l ) ,( 2 ) the artifactual production of spin adducts (12),( 3 ) the need for high concentrations of spin traps to scavenge some radicals due to low rate constants for the trapping reaction ( l 3 ) ,and (4) the inability to resolve
* To whom correspondence should be addressed.
mixtures of structurally similar spin adducts by EPR because of indistinguishable g values and/or hyperfine splitting constants (7-9). Better selectivity may be achieved through use of high-performance liquid chromatography (HPLC) combined with EPR detection to separate and identify individual spin adducts (14,15). However, spin adduct instability can make extraction, separation, and structural identification difficult, if not infeasible, in many cases. As compared to spin traps, the stable di-tert-alkyl nitroxides offer some distinct advantages as free radical traps. First, rate constants for reaction of nitroxides with radicals (- 108-109 M-’ s-l (16-18)) are generally at least an order of magnitude higher than those for spin traps (- 106-10’ M-* ~ ’ ( 1 9 ) )Thus . for an equivalent trapping efficiency, much lower concentrations of nitroxides can be employed. Second, the reaction of nitroxides with most carbon-centered radicals leads to the formation of stable alkoxyamine products, allowing for the isolation and identification of an important class of radical intermediates (18). Third, while the aliphatic nitroxides do not react with peroxy radicals (20, 2 l ) , superoxide and the hydroxyl radical can be detected through the addition of appropriate compounds (22, 23). Fourth, the sensitivity of radical detection can be substantially improved by covalently linking the nitroxide to a fluorophore (24-26). When coupled to the nitroxide, the fluorescence yield of the fluorophore is very low owing to efficient intramolecular quenching of the fluorophore by the nitroxide. However, conversion of the nitroxide to a diamagnetic product through radical (or redox) reactions produces greatly enhanced fluorescence that can be used as a very sensitive measure of radical scavenging. On the basis of this property of enhanced fluorescence, we recently developed a highly sensitive HPLC method for the determination of photochemically generated carbon-centered radicals in aqueous solution (27). In this method, a watersoluble amino nitroxide (3-(aminomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyfree radical, 3-AMP) is used to trap carbon-centered radicals (Figure I). Products are subsequently reacted with fluorescamine to produce highly fluorescent alkoxyamine- and hydroxylamine-fluorescamine adducts (AF1 and HF1 adducts, respectively). Adducts are separated by reversed-phase HPLC and detected fluorometrically (27). Here we present results on the optimization of this method for the detection of very low levels of carbon-centered radicals in aqueous media. Advantages and drawbacks of this technique and potential applications of this approach are discussed.
EXPERIMENTAL SECTION Chemicals. Boric acid, ketones, 5,5-dimethylpynoline 1-oxide (DMPO), and 3-AMP were purchased from Aldrich. Sodium dithionite was obtained from Fluka. Amino acids, a-keto acids, nucleotide bases, buffer salts, and fluorescamine were from Sigma. All chemicals were of the highest purity available and, except where noted, were used without further purification. Distilledin-glass grade solvents were obtained from Burdick and Jackson.
0003-2700/90/0362-2275$02.50/00 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990
I OR Figure 1. Reaction scheme employed to trap carbon-centered radicals
in aqueous solution. The 3-AMP reacts rapidly with carbon-centered radicals (R') to form stable alkoxyamines or, for reducing radicals, the hydroxylamine (R = H). The DMPO was purified with activated charcoal (28). A 5 mM stock solution of fluorescamine in acetonitrile was prepared daily and stored in the dark at 20 "C. Stock solutions of purified 3-AMP (vide infra) in sodium borate buffer (pH 8.1, 0.2 M) were stored at 4 OC. Water used in all experiments was from a Millipore Milli Q system. Suwannee River fulvic acid was obtained from the U.S. Geological Survey, Denver, and used as received. A stock solution (15 mg/L) was prepared by dissolving the fulvic acid into 0.2 M borate buffer (pH 8.1). The sodium acetate buffer used in the HPLC mobile phase was prepared by adding glacial acetic acid to Milli Q water and adjusting the pH with sodium hydroxide pellets. Carbonate buffer was prepared by adjusting the pH of a 50 mM potassium bicarbonate solution with concentrated HCl. Purification of 3-AMP. The %AMP was purified by a solid phase extraction procedure, which employed a Waters CI8Sep-Pak (SP) to remove traces of diamagnetic amine impurities that produced a fluorescent background upon coupling with fluorescamine. The SP was rinsed sequentially with 20 mL of water, 10 mL of methanol, and 30 mL of water. A 2-mL aliquot of 3-AMP in water (- 10 mM) was then loaded onto the SP and rinsed with 24 mL of water. The 3-AMP was eluted with 5 mL of 20% methanol in water and the first 4 mL was saved. This entire procedure was repeated until 10-20 mL of eluate was collected. The combined eluate was roto-evaporated to dryness at 50 "C. The 3-AMP was redissolved in 0.2 M borate buffer (pH 8.1) and its purity determined by HPLC. This extraction procedure removed most of the contaminants that eluted after the 3-AMPfluorescamine peak. However, hydrophilic interferences such as the hydroxylamine were not removed. The concentration of the stock 3-AMP solution ( 10 mM) was determined on diluted samples by comparing the area obtained by double integration of the EPR signal with that obtained from a standard curve of area versus concentration determined for aqueous solutions of 3-carboxy-2,2,5,5-tetramethyl-lpyrrolidinyloxy free radical (Aldrich). Apparatus. The HPLC consisted of an Eldex Model B-100-S single piston pump followed by a Gilson Model 811B dynamic mixer, a 0-5oOo psi pressure gauge (C and H Sales),a Valco Model ClOW injection valve, and a RCM 8 X 10 cm Waters radial compression module containing a 0.5 X 10 cm Nova-PAK column with 4-pm reversed-phase (C18)packing; 0.5-pm filters were placed after the pump and injector. A Hitachi Model FlOOO fluorescence detector (Tokyo) set at 390 nm (excitation, 15 nm band-pass) and 480 nm (emission, 15 nm band-pass) was connected to an ELAB PC-based data collection system (OMS Tech). Unless noted, HPLC injections were through a 100-pL sample loop. In some cases, as a comparison, samples were also injected into the chromatograph via a 0.2 X 5.5 cm i.d. enrichment column (Upchurch) with 40-pm CU packing. The mobile phase composition was controlled by an ELAB low-pressure gradient system. Chromatographic separations were at room temperature and at a flow rate of 1.3 mL/min. The mobile phase used for isocratic elutions consisted of 35% sodium acetate (50 mM, pH 4.0)/65% methanol (v/v). For standard gradient elutions, the mobile phase composition was (A) 60% sodium acetate (50 mM, pH 4.0)/40% methanol (v/v) and (B) methanol, and the gradient used was 0% B for 5 min, 0 to 35% B from 5 to 10 min, 35 to 65% B from 10 to 26 min, 65 to 0% B in 1min, and 0% B for 13 min. The gradient used for the separation of amino acid or natural water samples was 0 to 48% B in 15 min, isocratic at 48% B for 4 min, 48 to 0% B in 10 min, N
and isocratic at 0% B for 10 min, with (A) 55% 50 mM potassium carbonate (pH 7.0)/45% methanol (v/v), and (B) methanol. A Bruker/IBM ER 2OOD-SRC EPR spectrometer was employed to obtain spin concentration. Samples were drawn into 50-pL calibrated capillaries, which were then sealed at the top and bottom and placed within standard 3 mm i.d. quartz EPR tubes. Standard instrument settings were as follows: frequency, 9.77 GHz; power, 10 mW; and modulation amplitude, 1.6 G. Nitroxide loss was calculated from the decrease in peak amplitudes, since line widths were identical for all samples. Excitation and emission spectra were collected with a SLMAminco SPF-500 spectrofluorometer using a 2 nm band-pass for both the excitation and emission wavelengths. Samples were thermostated at 20 "C. Spectra were corrected for instrumental response and integrated with correction factors and software provided by the manufacturer with some modifications (25). Irradiation Conditions. Alkoxyamines and the hydroxylamine were generated by the photolysis of ketones or a-keto acids in the presence of micromolar concentrations of 3-AMP in 0.2 M, pH 8.1 borate buffer (hereafter denoted as standard buffer); natural water samples and the fulvic acid (buffered to pH 8.1) were treated in a similar fashion. Samples in a 1-cm quartz cell were irradiated with a 300-W xenon lamp (Varian Model PS300.1). The light was first filtered through 22 cm of Milli Q water and then passed through either a 2275 nm Pyrex filter (50% transmission at 305 nm; ketones, a-keto acids) or a 1295 nm Pyrex filter (50% transmission at 335 nm; natural water samples and fulvic acids). Unless noted, the light intensity at the cell as determined with a YSI Model 65A radiometer was 140 and 160 mW/cm2 with the 295- and 275-nm filter, respectively. Prior to irradiation, samples were deoxygenated for 5 min with Nz (99.999% purity, Union Carbide) or Ar (99.995% purity) that had been passed through an oxygen trap (Alltech). The cell headspace was continuously purged with either Nz or Ar during sample irradiation. Derivatization Procedure. The standard procedure entailed the addition of a 200-pL aliquot of the stock fluorescamine solution to 1mL of sample within an all-Teflon vial, which was then stirred vigorously on a vortex mixer. The reaction time was 1 min at room temperature and at pH 8.1. The derivatization procedure was optimized with respect to the fluorescamine concentration and reaction pH. The dependence of the fluorophore yield on fluorescamine concentration was determined for the alkoxyamine of the acetyl radical formed by the photolysis of 330 pM pyruvate in standard buffer containing 5.1,14.5,or 65.3 pM 3-AMP. The alkoxyamine concentration was held constant at 0.7 pM while the fluorescamine concentration was varied from 6 to 1200 pM, depending on the concentration of 3-AMP in the solution. The dependence of fluorophore yield on fluorescamine concentration was also determined for the alkoxyamine derivatives of radicals 4 through 9 (Table I) in the presence of 5.1 pM 3-AMP. The dependence of fluorophore yields on reaction pH was similarly determined for the alkoxyamines of radicals 4 through 12 (Table I). The standard buffer containing 10 pM 3-AMP and 3-pentanone, 3-methyl-2300-400 pM 3,3-dimethyl-2-butanone, butanone, a-ketovalerate, a-ketocaproate, acetone, and P-phenylpyruvate was irradiated for 10 min to generate alkoxyamines in the 0.1-0.5 pM range. Aliquots of this solution were adjusted to a pH between 5.1 and 11.0 by addition of a strong borate buffer of the appropriate pH. The pH-adjusted solutions were then derivatized by the standard procedure, and fluorophore yields were determined by HPLC. To determine the stability of hydroxylamiie, it was fmt formed by the substoichiometric addition of dithionite to a deoxygenated solution of 3-AMP (33 pM) in standard buffer at 20 "C. This solution was stored under aerated or deaerated conditions and aliquots were withdrawn periodically to determine the hydroxylamine concentration following derivatization. The stability of the HFI adduct was also determined by measuring its loss upon storage under aerated conditions in standard buffer containing excess fluorescamine. To determine the stability of the AFl adducts, the alkoxyamines were first formed by irradiating deoxygenated standard buffer containing 9.3 pM 3-AMP and 300-500 pM 3-methyl-2-butanone, 3,3-dimethyl-2-butanone, 3pentanone, a-ketovalerate,a-ketoadipate, a-ketocaproate, acetone,
ANALYTICAL CHEMISTRY, VOL. 62,NO. 21, NOVEMBER
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Table I. List of Primary Radicals ( R ) Expected from the Norrish Type I Photochemical Cleavage of a-Keto Acids (at pH > pK,) and Ketones in Aqueous Solution' precursor
R'
8-hydroxypyruvate
(1)cop'(2) H,C(OH)CO (1) coz(3) -O,C(CH,),CO (1) COZ'T (4) CH3CO (1) COz'(5) CH3CHzC0 (1)CO2*(7) CH3(CHz)zCO (1) (8) CHs(CHJ3CO
CY-
ketoadipate
pyruvate cy-
ketobutyrate
cy-
ketovalerate
cy-ketocaproate
coz-
precursor
R'
P-phenylpyruvate acetone 2-butanone 3-pentanone 3-methyl-2-butanone 3,3-dimethyl-2-butanone
'The C0,'- radical reduce 3-AMP t the hydroxylamine. and P-phenylpyruvate. Fluorescamine was then added to form the AFl adducts, which ranged in concentration from 0.2 to 1.3 pM. This solution was stored in the dark at 4 "C, and 100-pL aliquots were analyzed by HPLC periodically over a period of 4 weeks. Prior to sample analysis by HPLC, the fluorometer was calibrated with a standard solution of quinine sulfate in 0.1 N HzS04. Apparent Fluorescence Quantum Yield Determinations. A solution of 15.1 pM 3-AMP in standard buffer was derivatized according to the standard procedure, yielding the fluorescamine derivative of 3-AMP in 17% acetonitrile/83% borate buffer (v/v). The apparent fluorescence quantum yield of the 3-AMPfluorescamine adduct was determined before and after its reduction with dithionite (vide infra). Quantum yields were calculated with respect to quinine sulfate as previously described (25).
Dithionite Titrations. The 3-AMP-fluorescamine adduct was reduced to the corresponding HF1 adduct by the stepwise addition of a deoxygenated solution of dithionite in standard buffer. After each dithionite addition, an EPR spectrum and fluorescence excitation and emission spectra were recorded. Complete reduction of 3-AMP-fluorescamine to the HF1 adduct was verified by EPR. Determination of Adduct Concentration. The standard buffer containing a ketone or a-keto acid (300-400 pM) and 7.3 pM 3-AMP was irradiated for specific time intervals ranging from 0.5 to 25 min to produce differing levels of 3-AMP loss and adduct formation. Adduct concentrations were calibrated by comparing the loss of 3-AMP as monitored by EPR with the increase in fluorescence area of the HPLC peaks correspondingto the adducts that formed (27). Once alkoxyamine concentrationswere determined as described above, they were used as standards to quantify adduct concentrations in the picomolar to nanomolar range (where calibration by EPR was not possible). Calibration curves in the nanomolar range were generated by dilution of the micromolar standards and plotting fluorescence area as a function of adduct concentration. Comparison to Spin Trapping. Standard buffer containing 500 pM pyruvate and either 500 pM DMPO or 50 pM 3-AMP was irradiated under deoxygenated conditions for 2-18 min. For samples containing DMPO, aliquots were withdrawn with 50-pL calibrated capillaries and the acquisition of the EPR spectrum for each aliquot was initiated 40 s after irradiation. EPR settings were as follows: frequency, 9.77 GHz; power, 10 mW; modulation amplitude, 1.0 G; time constant, 0.16 s; scan range, 100 G; and scan time, 200 s. Spin adduct concentrations were obtained by the same procedure employed for the standardization of 3-AMP. Samples containing 3-AMP were derivatized according to the standard procedure and separated by isocratic elution. Sampling Procedure. Natural water samples were collected in 2-L acid-washed reagent bottles with Teflon-lined caps. Prior to sample collection, glassware was washed overnight in 10% HNOBand then copiously rinsed with water and sample. After collection, samples were filtered through 0.22-pm Nylon 66 filters (MicronSep) and stored at 4 O C until analyzed. Immediately prior to an irradiation, the pH of the natural water sample was adjusted
300
400
500
600
700
WAVELENGTH (nm) Flgure 2. Anaerobic reduction of the 3-AMP-fluorescamine adduct to the HFI adduct by dithionite. Diionite was added in stepwise additbns increasing in concentration from 1 to 8. The EPR spectra show the spin loss of the paramagnetic 3-AMP-fluorescamine adduct upon reduction (A) while the excitation and emission spectra (B) show the concomitant increase in fluorescence yield due to hydroxylamine
formation. to pH -8 by a small addition of 0.2 M borate (pH 8.1).
RESULTS Fluorescence Properties of Fluorescamine Adducts. Unlike other primary amines, 3-AMP reacts with fluorescamine (29,30) to form a product with a low apparent quantum yield of fluorescence (aff u = 0.0017 f 0.0003), due to the efficient intramolecular quenching of the excited singlet state of this fluorophore by the nitroxide moiety (24,W). Reduction of the nitroxide to the diamagnetic hydroxylamine (the HF1 adduct) with dithionite leads to a fluorescence yield increase that is directly proportional to the loss of the nitroxide spin (Figure 2A), leaving the line widths of the excitation and emission spectra unaltered (Figure 2B). Complete reduction of the 3-AMP adduct to the hydroxylamine produces an 50-fold increase in fluorescence yield (@f f u = 0.080 0.001). Fluorescence quantum yields reported here are estimates, since
-
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990 1n
4
cm W
cm
z
w
Z
A 4
Z
t-
I-
z
w
0
w
m
w 0 Z w
LL 1
w
Z
W
0
q-
0
m a:
10
20
3
30
TiME (min) Flgure 3. Chromatogram showing the standard gradient separation of a mixture of AFI adducts. Peak assignments refer to the AR adducts of the radicals listed in Table I. Concentrations of the AFI adducts ranged from 69 nM (peak 9) to 528 nM (peak 4), while concentrations of the HFI (peak 1)and 3-AMP-fluorescamine (peak D) adducts were 2.5 and 520 pM, respectively. these solutions contain fluorescamine hydrolysis products with a constant, but unknown absorbance contribution. We are currently determining the fluorescence quantum yield for the purified fluorescamine derivative of 3-AMP, as well as for the fluorescamine derivatives of other amine-substituted nitroxides. Estimates of affor the AF1 adducts obtained by comparison to the HFl adduct (27) are even higher, ranging from 0.16 to 0.20. These values agree reasonably well with reported values of afin ethanol (30) for the fluorescamine adducts of various primary amines such as butylamine (af= 0.23) and benzylamine (af= 0.27). HPLC Separation of HFI and AFl Adducts. We showed previously that 3-AMP efficiently scavenges carbon-centered radicals to form alkoxyamines or the hydroxylamine. The AFl and HF1 adducts, obtained by derivatization with fluorescamine, can be separated and identified by reverse-phase HPLC using isocratic elution and fluorometric detection (27). Under the conditions reported here, we are now able to resolve and sensitively detect a significantly more complex array of AF1 adducts having substantially different capacity factors (Figure 3). Where possible, these AFl adducts were assigned directly through intercomparison of the elution patterns of adducts formed from the photolysis of individual ketones or a-keto acids (27). For example, photolysis of 3-pentanone or 2-butanone gives rise to a common product which was assigned as the ethyl radical adduct (Table I). Where direct intercomparisons were not possible, adducts were assigned on the basis of known or expected photochemistry and the chromatographic elution characteristics of the AFl adducts (e.g., polar vs nonpolar). No fluorescent products were formed when either aerated or deaerated solutions of 3-AMP alone were irradiated. When aerated solutions of ketones or a-keto acids were irradiated in the presence of low concentrations of 3-AMP (- 10 pM), no AFl or HFl products corresponding to the radicals listed in Table I were detected, consistent with an efficient scavenging of the primary radicals by oxygen to form oxygencentered radicals that are unreactive with 3-AMP (20,22). However, small amounts of an unknown species eluting near the void volume was detected. We are currently investigating the nature of this unknown. All dark controls, with and without added a-keto acids or ketones, also showed no evidence for the formation of fluorescent products. No fluorescent products were detected when solutions of the aliphatic ketones were irradiated in the presence of 3-AMP at wavelengths >350 nm.
D
0
I
0
Dt1
LI
L
0
5
10
TIME (min) Figure 4. Effect of added methanol on the photolysis of acetone. Chromatograms A through D show the isocratic separation of the fluorescamine adducts produced by irradiation (L275 nm) of the standard buffer containing 330 pM acetone and 17 pM 3-AMP in the presence and absence of methand: (A) acetone and 3-AMP with no methanol added, (6)acetone and 3-AMP with 0.5 M methanol, (C) acetone and 3-AMP with 6.5 M methanol, and (D) 3-AMP and 6.5 M methanol with no acetone added. Peak assignments refer to the AFI adducts of the radicals listed in Table I; peak D is the fluorescamine derivative of 3-AMP. Under isocratic conditions, species 1 and D coeluted. As a simple additional test of the method, we examined the influence of an added hydrogen atom donor, methanol, on the photolysis of an aqueous solution of acetone. The photolysis of acetone proceeds through a Norrish type I cleavage from the excited triplet state to form the acetyl and methyl radicals
-
3[CH3COCH3] CH3C0 + CH3
(1)
In the presence of sufficient methanol, however, photoreduction of acetone to produce a-hydroxy radicals should occur (311
3[CH3COCH3]+ CH30H
-
CH,COHCH3 + CH,OH (2)
Because a-hydroxy radicals reduce the nitroxide to the hydroxylamine (32,331, a change in products was anticipated. Consistent with expectations, the AFl adducts of the methyl and acetyl radicals decreased with increasing methanol concentrations concomitant with an increase in the concentration of the HFl adduct (Figure 4). The light control containing methanol and 3-AMP, but no acetone, showed no appreciable production of the HF1 or AFl adducts. Optimization of the Derivatization Procedure. Because fluorescamine rapidly hydrolyzes in aqueous solution to form nonfluorescent products, an excess of fluorescamine is required for optimal fluorophore formation (30). As shown in Figure 5,with decreasing 3-AMP concentrations, a higher molar ratio of fluorescamine to 3-AMP was needed to produce maximal fluorophore yields for the alkoxyamine derivative of the acetyl radical (0.7 pM).At the highest 3-AMP concentration tested, a 1.5-fold molar excess of fluorescamine was sufficient for maximal fluorescence, while at least an %fold molar excess of fluorescamine was required for optimal yields at the lowest 3-AMP concentration tested (5.1pM). This same dependence was observed at 5.1 pM 3-AMP for the alkoxyamine derivatives of radicals 4 through 9 (Table I). Thus, with increasing concentrations of 3-AMP, a lower molar ratio of fluorescamine/& is needed to obtain optimal AF1 and HF1 yields.
ANALYTICAL CHEMISTRY, VOL. 62, NO. 21, NOVEMBER 1, 1990 w
I?! w
2279
'oofF=7 [3-AMP]
0
5.1 pM
3
G
*-A
14.5 uM
2 X
3
25
G
0
n
o
0
5
10
15
20
0 3
5
6
1
PH
[ FLUORESCAMINE]/[ 3-AMPI Figure 5. Dependence of the percentage of the maximum fluorescence of the AFi adduct of the acetyl radical (0.7 pM) on the molar ratio of fluorescamine to 3-AMP, determined for three concentrations of 3-AMP. Error bars denote u ( n = 3).
Fluorophore yields were highest and independent of pH in the range 7.5-9.5 for the reaction of fluorescamine with the alkoxyamines of radicals 4 through 12 (Table I). Outside this range, the yields decreased for all alkoxyamines tested. For example, at pH 5.1, yields were 35 to 55% less than the maximum. At pH >lo, yields decreased by 10 to 30% for all alkoxyamines, presumably owing to an increased rate of fluorescamine hydrolysis (30). The observed pH optimal is similar to that measured for amino acids which show optimal fluorophore formation between pH 8 and 9 (30). However, one important difference is that the alkoxyamines are significantly more reactive toward fluorescamine at pH
