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Anal. Chem. 1987, 59. 1203-1206
the organic phase with 6 M hydrochloric and 1M nitric acids, respectively (Table VI).
Application to Analysis of Strontium from Milk and Geological Samples. A sample of milk was prepared for analysis as described earlier (16). An aliquot (20 mL) of solution was taken and potassium was first removed with DB-18-crown-6 in chloroform at pH 1.0 from 1X M picric acid leaving strontium in the aqueous phase. Then strontium from the aqueous phase was extracted with 18-crown-6 in chloroform at pH 3.0. Potassium and strontium were stripped from the organic phase with 6 M hydrochloric acid and 1 M nitric acid, respectively. In triplicate analyses the amount of strontium found was 29 ppm. The standard value was 30 ppm. For the analysis of strontium from geological samples like basaltic rock (B.R.) and syanite rock (SY II), the samples were brought into solution as per the procedure described earlier (17). An aliquot of this solution was taken and potassium was extracted with DB-18-crown-6 in chloroform at pH 1.0. Then strontium from the aqueous phase was extracted with 18crown-6 a t pH 3.0. Potassium and strontium were stripped from the organic phases with 6 M hydrochloric acid and 1 M nitric acid, respectively. In triplicate analyses the amount of strontium found in basaltic rock was 1115 ppm, as against the standard value of 1100 ppm, and the amount of strontium found in syanite rock was 238 ppm, as against the standard value of 233 ppm. The proposed method for solvent extraction separation of strontium has several advantages. It is possible to separate strontium not only from alkali and alkaline-earth elements in the concentration range of 1:50 but also from commonly associated elements such as lead, mercury, thorium, and uranium in fission product mixtures. The separation of strontium from halides was possible in higher ratios (1:500). Similarly the separation of strontium from phosphate and
perchlorate was of significance as they were associated with minerals. The method is applicable to the analysis of strontium in milk and geological samples. The total time required for analysis was 2 h. The method is simple, rapid, selective, and reproducible. Registry No. Sr, 7440-24-6;18-C-6,17455-13-9;Pb, 7439-92-1; U, 7440-61-1; Th, 7440-29-1; Ba, 7440-39-3; Ca, 7440-70-2; Be, 7440-41-7; Mg, 7439-95-4; K, 7440-09-7; picric acid, 88-89-1.
LITERATURE CITED Blasius E.; Kleln, W.; Schoenon, U. J . Radioanal. Nucl. Chem. 1985, 89, 389-398. Huizhong, Y.; He Huaxne Yu Fangshe Huaxue 1984, 6 , 186-188. Alvarez, J. R. G.; Arrbas, J. S.; Sanz-Medal, A. Tec. Lab. 1985, 9 ,
426-429. Sekine, T.; Shioda, K.; Hasegawa, Y. J . Inorg. Nucl. Chem. 1979, 4 1 , 571-573. Langrock, E. J.; Langguth, H. East German patent 217 190 (CI. COlD17/00),09 Jan. 1985, Appl. 253973, 16 Aug 1983; Chem. Abstr. 1985, 103, 6290m. Bowers, C. B., Jr. Report 1984, DOE/SR/10714-TI, order No. DE 8555140 Chem. Abstr. 1985, 103, 444749. &row, I. H.; Smith, J. W.; Davis, M. W. Sep. Sci. Techno/. 1981. 16, 519-549. Gerow, 1. H.; Davis, M. W. Sep. Sci. Techno/. 1979, 14, 395-414. Takeda, Y.; Oshio, K.; Segawa, Y. Chem. Lett. 1979, 601-602. Kirnura, T.; Iwashima, K.; Shimori, T.; Hamada, T. Anal. Chem. 1979, 5 1 . 1113-1116. Kimura, T.; Iwashima, K.; Ishimori, T.; Hamaguoni, H. Chem, Lett. 1977. 563-564. Vogei, A. I. A . Text Book of Quantitative Inorganic Analysis: Longmans: London, 1975;p 553. Sax, N. I. Dangerous Properties of Industrial Materials; Van NostrandReinhold: New York, 1984;p 2230. Takeda, Y.; Kato, H. Bull Chem. SOC.Jpn. 1979, 52, 1027-1030. Gokel, G. W.; Goli, D. M.; Minganti, C.; Echegoyen, L. J . Am. Chem. Soc. 1983, 105, 6786-6788. Mohite, B. S.;Khopkar, S. M. Indian J . Chem., Sect. A 1983, 22A,
962-964. Mohite, B.
S.;Khopker, S.
M. Talanta 1985, 3 2 , 565-567.
RECEIVED for review June 24, 1986. Accepted December 29, 1986.
Coumarin Derivatizing Agents for Carboxylic Acid Detection Using Peroxyoxalate Chemiluminescence with Liquid Chromatography Mary Lynn Grayeski* and Joseph K. DeVasto
Chemistry Department, Seton Hall University, South Orange, New Jersey 07079
4-( Bromomethyi)-7-methoxycoumarln (Br-Mmc), 7 4dlethylamino)coumarln-3-carbohydrazide (DCCH), and 7-( dlethyiamino)-3-[4-( (iodoacetyl)amlno)phenyl]-4-methylcoumarln (DCIA) are evaluated as carboxylic acid derlvatlzing agents to be detected using peroxyoxalate chemllumlnescence with hlgh-performance liquid chromatography. The derivatlzatlon procedure for Br-Mmc and DCIA requires only one step as opposed to two for DCCH derlvatlzatlon. No chemliumlnescence Is observed from the Br-Mmc derivatives but detectlon limits of stralght chaln carboxylic acids derlvatlzed with D C I A and detected with peroxyoxalate chemliumlnescence are In the low femtomole range.
The detection of fluorophors by peroxyoxalate chemiluminescence was first demonstrated by Curtis and Seitz who
detected dansylated amino acids on a thin-layer chromatography (TLC) plate (I). This approach was later adapted to high-performance liquid chromatography (HPLC) detection by Kobayashi and Imai and further investigated by a number of other groups (2-7). Bis(2,4,6-trichlorophenyl) oxalate (TCPO) reacts with hydrogen peroxide to form a proposed energetic intermediate, 1,2-dioxetanedione, which transfers its energy to a fluorophor via a proposed electron transfer and excites it to the first excited singlet state followed by the emission of a photon. The reason for the interest in CL detection is that for certain fluorophors, detection limits obtained by using chemical excitation as opposed to conventional photoexcitation are lower because of the lack of problems from scattering and fluctuations of the source. Examples of fluorophors detected by using peroxyoxalate chemiluminescence are polycyclic and reduced nitropolycyclic aromatic hydrocarbons (3, 4 ) , poly-
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
cyclic aromatic amines ( 5 ) ,fluorescarnine-labeled catecholamines (6),and dansylated amino acids (7). However; not all fluorophors are more efficiently detected by chemical excitation when compared to the more conventional photoexcitation. Several characteristics of the fluorophor that contribute to efficient chemical excitation include high fluorescence efficiency, low oxidation potential, and low singlet energy (5,8, 9). To extend the applicability of peroxyoxalate chemiluminescence detection beyond the detection of only certain compounds with native fluorescence, our research efforts are focused on developing and identifying new chemiluminescent derivatizing agents that could react with a number of different functional groups. This particular study investigates tagging agents for carboxylic acids that have not previously been reported to be derivatized for detection by chemiluminescence (CL). Several fluorescent coumarin tagging agents for carboxylic acids have been reported. Dunges introduced a 4-(bromomethyl)-7-methoxycoumarin(Br-Mmc) as a suitable fluorescent tag for a wide range of aliphatic carboxylic acids (IO), and 4-(bromomethyl)-7-acetoxycoumarin(Br-Mac) was used in prostaglandin analyses (11). We report the comparison of three coumarin compounds: 4- (bromomethyl)- 7-methoxycoumarin (Br-Mmc), 7-(diethylamino)coumarin-3-carbohydrazide(DCCH), and 7-(diethylamino)-%[4-((iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA) as potential chemiluminescent derivatizing agents for carboxylic acids. EXPERIMENTAL SECTION Chemicals. Fatty acid standards, hexanoic, octanoic,nonanoic, and decanoic acids were obtained from Sigma Chemical Co. 7-(Diethylamino)coumarin-3-carbohydrazide(DCCH) and 7(diethylamino)-3-(4-( (iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA) were purchased from Molecular Probes. 4-(Bromomethyl)-7-methoxycoumarin(Br-Mmc) was available from Regis Chemical Co. All derivatizing agents including thionyl chloride and dibenzo-18-crown-6ether (Aldrich Chemical Co.) and HPLC grade solvents including acetone, acetonitrile, chloroform, and methanol (Fisher Scientific and J. T. Baker) were used as received. Bis(2,4,6-trichlorophenyl)oxalate (TCPO) was received from A. Mohan and recrystallized in ethyl acetate. Derivatization Procedures. The literature procedure (12) for derivatizing Br-Mmc to carboxylic acids in aprotic solvents was used with the following modification: Instead of being refluxed for 25 min, the reaction solution was prepared in acetone and placed in a sealed vial and heated to 55 "C for 1 h with periodic shaking. Each derivatized sample was decanted from the insoluble potassium carbonate and injected into the HPLC. This procedure was also followed with the DCIA reagent; however, acetonitrile was used in the reaction solvent and it was heated to 70 O C for 70 min. For the derivatization of octanoic acid with DCCH, octanoyl chloride was first prepared by adding approximately 100 pL of octanoic acid and 5 pL of dimethylformamide (DMF) to a large excess (ca. 6 mL) of thionyl chloride in a reaction vessel with a drying tube attached. Reactants were heated at 60 O C for 1 h and the excess thionyl chloride was removed in a dry nitrogen stream. A solution of DCCH was prepared in chloroform (3.5 mg/mL) with an equimolar quantity of triethylamine. Octanoyl chloride was added to the DCCH solution in a sealed vial with the derivatizing reagent in a 1/1.2 mol excess. The vial was shaken for 1 min and solution evaporated to dryness with nitrogen. The residue was dissolved in acetone and injected into the HPLC. HPLC Apparatus-Br-Mmc and DCCH Experiments. A Gilson Model 302 solvent pump, a Rheodyne Model 7125 injector with a 20-pL fixed sample loop, and a Kratm FS 970 detector with a 5-pL flow cell were used with a 5-pm, C8 Alltech Econosphere (25 cm X 4.6 mm) column. For CL measurements, a Kratos URS 051 postcolumn reactor with a pulse dampening filter (no. 2500-0567) was added. A 3-cm piece of narrow bore tubing joined the pulse dampener to a Valco 1-pL "T" which mixed the chem-
Table I. Absorbance and Fluorescence Wavelengths for Coumarin Tags derivatizing agent Br-Mmc DCCH DCIA
,,,A absorption
nm emission
325
390
415 375
480 470
iluminescent reagents with the chromatographic eluent. Solutions of 1.0 M hydrogen peroxide in acetonitrile and 4.45 mM TCPO in ethyl acetate were used as postcolumn reagents with the flow rates optimized at 1.0 mL/min each using a simplex procedure. The chromatographic eluent ranged from 70 to 80% acetonitrile/4 mM sodium phosphate buffer a t apparent pH 7.5. HPLC Apparatus-DCIA Experiments. Modificationswere made with the HPLC apparatus to reduce postcolumn pump pulsation and improve CL detection levels of the DCIA derivatives. A manometric module (Model 802) was added to the Gilson solvent pump and two Lazar pneumatic solvent pumps (Model LPP-297) replaced the Kratos URS 051 postcolumn reactor. Each postcolumn pump was connected to a Valco 1-yL mixing Tee with 55 cm of 0.009-in. stainless steel tubing. This tubing provided the necessary back pressure (ca. 10 psi) for precise flow rate control of the CL reagents. The CL solution concentrations and flow rates remained the same as those previously described. A Fisher Resolvex 10-pm C8 column (25 cm X 4.6 mm) plus a guard column (3 cm X 4.6 mm) packed with 30-pm C18 material was used in replacement of the Alltech Econosphere column. Also a 25-yL flow cell was used in the Kratos FS 970 detector. TLC Conditions. The DCCH-octanoic acid derivative was isolated from the reaction mixture on an Analtech, Inc., 250-ym silica gel TLC plate using 3% methanol in chloroform. The amide derivative at the solvent front was scraped from the glass plate, transferred to a vial, and sonicated in methanol. This solution was filtered and injected into the HPLC. Spectroscopic Measurements. Absorption spectra were taken with a Varian 2200 UV-Vis spectrophotometer and fluorescence spectra were determined with a Varian SF 330 instrument. R E S U L T S A N D DISCUSSION Fluorophor. Fluorophors to be used as HPLC derivatizing agents must satisfy several requirements including (1)negligible effect on separation of the analytes and (2) sensitive detection a t low levels. Intial choice of fluorophors to be investigated was based on potentially sensitive detection by
CL. Previous investigations on the nature of fluorophors most efficiently excited by CL indicate that low oxidation potentials, low singlet energies (long wavelength emitters), and high fluorescence efficiencies contribute to efficient chemical excitation (5,8, 9, 13,14). These results are consistent with the proposed mechanism that involves an electron transfer from the fluorophor to the intermediate which ultimately transfers the electron back to the fluorophor during the excitation process. The coumarin compounds to be investigated were chosen on this basis (Figure 1). The electron-donating amine functional group on DCCH and DCIA would contribute to lowering the oxidation potential and singlet energy as well as increasing the fluorescence efficiency ( 5 8 ) . Since the emission wavelength of the fluorophor is a general indication of relative singlet energies, a comparison was made of the spectral maxima values of the coumarin compounds (Table I). These results indicate that addition of the amine should contribute to increased chemical excitation efficiency. Attempts to synthesize a coumarin compound similar to Br-Mmc with a 7-dimethylamino functional group resulted in an impure product, so investigation was restricted to the three commercially available coumarins. Application t o HPLC. 4-(Bromomethyl)-7-methoxycoumarin. Hexanoic, octanoic, nonanoic, and decanoic acids
ANALYTICAL CHEMISTRY, VOL. 59,
NO. 8, APRIL
15, 1987
1205
11 .01uA
ET-
JKXNHNH2 I
ET
7-Dierhylaminocoumarin-3-c.rbohydrlride
(DCCH)
0
5 TIME (minj
10
Flgure 3. DCIA fluorescence detection: eluent, 80120 acetonitrile/ phosphate buffer, 4 mM, pH 7.5; flow rate, 1.5 rnL1min; detector, excitation 254 nm, emisslon 418 nm long pass fitter, PMT voltage 1000 V, 0.1 FA full scale; (1) hexanoic acid 370 fmol, (2) octanoic acid 290 fmol, (3) nonanoic acM 260 fmol; (4) decanoic acid 350 fmol.
ET
~ 7 - D i e t h y l a m i n o - 3 - ( - 4 - i o d o a c e t y l ~ i ~ ~ ~ h ~ ~ y l ~(DCIA) 4~~th~l~~~ari~
Figure 1. Coumarin derivatizing reagents. .01 uA
I
I
.02 u A
I
0
Y 5 TIME (min,)
1
I 1
10
Flgure 2. Br-Mmc fluorescence detection: eluent, 70130 acetonitrile/phosphate buffer, 4 mM, pH 7.5; flow rate, 1.2 mL/min; detector, excitation 325 nm, emission 389 nm long pass filter, PMT voltage 1140 V, 0.1 pA full scale; (1) hexanoic acid 7 pmol, (2) octanoic acid 9 pmol, (3) nonanoic acid 8 pmol, (4) decanoic acid 11 pmol.
were derivatized with Br-Mmc and, although the products were readily detected by fluorescence (Figure 2), no chemiluminescence was observed for these derivatives. The unlabeled peaks in Figure 2 represent unreacted Br-Mmc and derivatization byproducts. Since the samples were not prepared in the eluent, a negative peak resulted near the void volume. The lack of a CL signal from the Br-Mmc derivatives is to be expected based on the absorption and emission wavelengths. This result suggests that the singlet excitation energy for Br-Mmc exceeds the maximum energy of 105 kcal/mol produced by the peroxyoxalate reaction (9). 7-(Diethylamino)coumarin-3-carbohydrazide. The derivatization of DCCH to a carboxylic acid required conversion of the acid to acid chloride using thionyl chloride before reaction with the carbohydrazide group. Preliminary studies included the reaction of octanoic acid with DCCH which was chromatographed and monitored with fluorescence detection. When the DCCH reaction mixture was injected, the peak from the unreacted DCCH tailed for more than an hour under the conditions used (70/30 CH3CN/4 mM Na2HP04pH 7.5). Conceivably a gradient could be used to eliminate this problem but a gradient former was not available. T o evaluate the chemiluminescent characteristics of this coumarin compound, the amide derivative of DCCH was isolated by preparative TLC. The chromatogram of the product was free of underivatized DCCH. Although CL was observed from this product,
;.i
0
5
TIME (min.)
Flgure 4. DCIA chemiluminescencedetection: detector source off, PMT voltage 1400 V, 0.2 p A full scale; all other parameters are the same as those given in Figure 3.
detection limits were not determined because of the difficulty in quantitative transfer from TLC plates. Instead, further work was pursued with a second amine-containing coumarin (DCIA). 7-(Diethylamino)-3-[4-((iodoacetyl)arnino)phenyl]-4methylcoumarin. The iodoacetyl-containing aminocoumarin was reacted with the same four carboxylic acids as Br-Mmc and the chromatograms were monitored by fluorescence (Figure 3) and chemiluminescence (Figure 4). A comparison of the fluorescence-detected derivatives of Br-Mmc and DCIA shows the increased fluorescence efficiency and corresponding improved detectability of the amine-containing compounds. It should be noted that although a 25-pL flow cell was used in the DCIA experiments (vs. a 5-pL flow cell with Br-Mmc) the enhancement is still significantly independent of the increased cell size because no improvement in signal-to-noise
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Table 11. Detectability and Precision for Fluorescence and Chemiluminescence of Br-Mmc and DCIA Derivatives" carboxylic acid
Br-Mmc FL, pmol (%RSD)b
Br-Mmc CIA, pmol (%RSD)'
hexanoic octanoic nonanoic decanoic
I (5.2) 9 (3.0) 8 (4.6) 11 (7.9)
ND ND ND ND
DCIA FL, fmol (%RSD)" 370 290 260 350
(6.5) (2.7) (7.5) (9.3)
DCIA CL, fmol (%RSD)' 74 (2.6) 58 (10.1) 52 (10.7) 70 (4.3)
"Detector response time, 3.0 s; CL, chemiluminescence; FL, fluorescence. bDuplicate injections, nonanoic acid peak, S I N = 4. "D, not detectable, no CL observed. dQuadrupicate injections, nonanoic acid peak S I N = 8. 'Quadruplicate injections, nonanoic acid peak S I N = 4.
ratio was observed for fluorescence measurements when using the larger cell. The equivalent signal-to-noise ratio for both cells is due to the fact that the fluorescent background increases equally with the signal as cell size was increased. The DCIA chromatograms also indicate that, unlike the DCCH reagent, the unreacted DCIA peaks do not interfere with the separation of the acid analytes and elute prior to the main peaks of interest. Chemiluminescence is also observed with the DCIA derivatives. On comparison of the chemiluminescence and fluorescence chromatograms where identical injection volumes were made of the same sample, a 3-fold improvement in analyte detectability was observed when using chemiluminescence. This gain is due to the fact that the photomultiplier tube is operated at a higher voltage (1400 V) without the source. Detector Linearity. Linearity of the DCIA fluorescence and chemiluminescence detector response was determined by monitoring the change in peak height with increasing sample mass of the nonanoic acid derivative. Fluorescence linearity was measured over a sample range of 260 fmol to 13 pmol. The resulting least-squares equation was y = 279x + 19 and was statistically determined to be linear a t an F value significance level of 0.95. Chemiluminescence detector linearity was evaluated over a sample range of 52 fmol to 2.6 pmol. The resulting curve was also determined to be linear, with a least-squares equation of y = 189x 1.3. Comparison of Fluorescence with Chemiluminescence Detectability and Precision. Comparisons of Br-Mmc and DCIA fluorescence and chemiluminescence detectability and precision levels (Table 11) show that improvement in fluorescence efficiency afforded by the electron-donating amino group in DCIA improves its fluorescence detectability by more than an order of magnitude when compared to BrMmc. A 5-fold increase in detectability was also observed when comparing chemiluminescence with fluorescence detection of the DCIA derivatives. It should be noted that these values are for actual injected concentrations and that the chemiluminescence signal-to-noise ratio was half that of the fluorescence. This improvement is observed because (1)amino-substituted fluorophors are efficiently excited as discussed previously (3)and (2) the detector can be operated at higher voltages to improve photon collection efficiency.
+
CONCLUSION The amine-containing coumarins DCCH and DCIA have been derivatized to carboxylic acids and these derivatives have been shown to be efficient chemiluminescence energy acceptors. This result strongly suggests that the amine functional
group was involved in lowering the oxidation potential and singlet excitation energy while increasing the fluorescence efficiency of DCCH and DCIA relative to Br-Mmc. In terms of the time requirement for HPLC derivatization of carboxylic acids, DCCH requires a two-step derivatization which consumes more man hours than the simpler one-step procedure used with DCIA. For this application, DCCH may also require removal of the unreacted reagent for chromatography. DCCH can be reacted with carbonyl compounds in a single derivatization step and this is currently under investigation. DCIA derivatizes under similar conditions as Br-Mmc which is conventionally used for tagging carboxylic acids but can be detected at lower levels by both fluorescence and CL. A greater than 10-fold improvement in detectability is obtained by using photoexcitation and approximately a 100-fold improvement is observed with chemical excitation. Although detection levels for the DCCH derivatives were not determined, presumably they are comparable to the structurally similar DCIA. Studies using DCIA derivatized to carboxylic acid containing drugs and prostaglandins extracted from blood samples are currently under investigation.
ACKNOWLEDGMENT The authors wish to thank R. Weinberger and Kratos Analytical Instruments for support and equipment, R. Hartwick for equipment, and A. Mohan for chemicals. We also thank E. Woolf for his helpful suggestions and comments in this study.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9)
(IO) (11)
Curtis, T. G.; Seitz, W. R . J. Chromatogr. 1977, 734, 343. Kobayashi, S . ; Imai, K. Anal. Chem. 1980, 52, 424-427. Sigvardson, K. W.; Birks, J. W. Anal. Chem. 1983, 55,432-435. Sigvardson, K. W.; Birks, J. W. J. Chromatogr. 1984, 376, 507-518. Sigvardson, K. W.; Kennish, J. M.; Birks, J. W. Anal. Chem. 1984, 56, 1096-1102. Kobayashi, S.; Sekino, J.; Honda, K.; Imai, K. Anal. Biochem. 1981, 772, 99-104. Miyaguchi, K.; Honda, K.; Imai. K. J. Chromatogr. 1984, 303, 173-1 76. Grayeski, M. L.; Mann, B. "Evaluation of Peroxyoxalate Chemiluminescence Detection for Drug Analysis". Presented at the Eastern Analytical Symposium, New York, Nov. 1984. Lechtken, P.; Turro, N. J. Mol. Photochem. 1974, 6(1), 95-99. Dunges, W. Anal. Chem. 1977, 49, 442-445. Tsuchiya, H.; Hayashi, T.; Naruse, H.; Takagi, N. J. Chromatogr. 1982, 237 247-254. Lam, S.;Grushka, E. J. Chromatogr. 1978, 158, 207-214. Catherall, C. L. R.; Palmer, T. F.; Cundall, R. B. J. Chem. Soc., Faraday Trans. 2 1984, 80, 823-834. Catherall, C. L. R.; Palmer, T. F.; Cundall, R. B. J. Chem . Soc ., Fara day Trans. 2 1984, 8 0 , 837-849. ~
(12) (13) (14)
-
RECEIVED for review January 21,1986. Resubmitted January 5, 1987. Accepted January 5, 1987. This research was supported by a grant from Research Corporation.
