Anal. Chem. 1999, 71, 4245-4249
Determination of Trace C1-C4 Aliphatic Alcohols in Aqueous Samples by 9-Fluorenylmethyl Chloroformate Derivatization and Reversed-Phase High-Performance Liquid Chromatography Gu Huang,† Guohong Deng,† Huancheng Qiao,‡ and Xianliang Zhou*,†,‡
Department of Environmental Health and Toxicology, School of Public Health, State University of New York at Albany, and Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509
A simple procedure for precolumn fluorescence derivatization of low-molecular-weight aliphatic alcohols (C1C4) with 9-fluorenylmethyl chloroformate is presented. The derivatization reaction proceeds in 1:1 (v/v) aqueous-acetonitrile solution at room temperature with a sodium phosphate buffer of pH 12.5 as a catalyst. Stable fluorescent derivatives of the alcohols are formed within 10 min. The four derivatives are separated by reversedphase high-performance liquid chromatography and detected by a fluorescence detector at an excitation wavelength of 259 nm and an emission wavelength of 311 nm. The method detection limits are 4, 40, 70, and 30 pmol for methanol, ethanol, propanol, and butanol, respectively, per 5-µL injection volume. The relative standard deviations are 3.7% for methanol at 75 pmol and 2.1, 1.5, and 2.2% for ethanol, propanol, and butanol, respectively, at 750 pmol. As a preliminary application, the method was used to determine methanol concentration in laboratory air and ethanol content in a commercial alcoholic beverage. Low-molecular-weight (LMW) aliphatic alcohols are a group of important compounds that are widely found in beverages, foods, pharmaceuticals, and biological fluids. They are used as organic solvents in laboratories and industries, and they also occur naturally in the environment. Detection of these alcohols at trace level is still a challenging subject. Gas chromatography and highperformance liquid chromatography (HPLC) with precolumn or postcolumn derivatization have been previously employed for their determination. Derivatization techniques for HPLC analysis have received special attention because they enable highly sensitive detection of these compounds by bonding a chromophore or fluorophore that results in products with strong UV absorption or fluorescence emission. In recent years, several types of fluorescent labeling reagents, such as nitrile group-containing compounds, carbonyl azides, and carbonyl chlorides, have been proposed for the derivatization of aliphatic alcohols and other hydroxy compounds.1-8 Most of these derivatization reactions * Corresponding author: (e-mail)
[email protected]; (fax) (518)-402-5085. † State University of New York at Albany. ‡ New York State Department of Health. 10.1021/ac990010m CCC: $18.00 Published on Web 09/03/1999
© 1999 American Chemical Society
proceed in organic media and at elevated temperatures of 60130 °C over a period of 30 min to several hours, sometimes under dark conditions. Extraction and preconcentration procedures are often needed before HPLC analysis. In addition, these reagents are generally very moisture-sensitive and unstable. Therefore, these methods may not be suitable for analysis automation in which rapid formation of stable derivatives under mild conditions, preferably at room temperature and in the aqueous phase, is required. In a recent report, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)9 was used for fluorescence labeling of aliphatic alcohols under mild reaction conditions. The C1-C8 alcohol derivatives were formed at 25 °C for 1 min in a basic buffer solution, and the fluorescence detection limit was in the picomolar range. However, the reagent solution was very moisture-sensitive and needed to be flushed with dry and inert gas after each use. 9-Fluorenylmethyl chloroformate (FMOC) was introduced as a precolumn derivatization fluorescence labeling reagent for HPLC analysis of amines10 and amino acids.11 It has become one of the most popular amino acid derivatization reagents.12-15 In this paper, we report a simple procedure for precolumn derivatization of LMW aliphatic alcohols (C1-C4) with FMOC to form highly fluorescent (1) Goto, G.; Komatsu, S.; Goto, N.; Nambara, T. Chem. Pharm. Bull. 1981, 29, 899-901. (2) Goto, J.; Goto, N.; Nambara, T. Chem. Pharm. Bull. 1982, 30, 4597-4599. (3) Goto, J.; Goto, N.; Shamsa, F.; Satio, M.; Komatsu, S.; Suzaki, K.; Nambara, T. Anal. Chim. Acta 1983, 147, 397-400. (4) Takadate, A.; Irikura, M.; Suehiro, T.; Fujino, H.; Goya, S. Chem. Pharm. Bull. 1985, 33, 1164-1169. (5) Yamaguchi, M.; Iwata, T.; Nakamura, M.; Chkura, Y. Anal. Chim. Acta 1987, 193, 209-217. (6) Iwata, T.; Yamaguchi, M.; Hara, S.; Nakamura, M.; Ohkura, Y. J. Chromatogr. 1986, 362, 209-216. (7) Hamada, C.; Iwasaki, M.; Kuroda, N.; Ohkura, Y. J. Chromatogr. 1985, 341, 426-431. (8) Abdullah, I.; Haj-Yehia; Benet, L. Z. J. Chromatogr., A 1996, 724, 107-115. (9) Motte, J. C.; Windey, R.; Delafortrie, A. J. Chromatogr., A 1996, 728, 333341. (10) Moye, A. H.; Boning, A. J. Anal. Lett. 1979, 12 (B1), 25-35. (11) Einarsson, S.; Josefsson, B.; Lagerkvist, S. J. Chromatogr. 1983, 282, 609618. (12) Gustavsson, B.; Betner, I. J. Chromatogr. 1990, 507, p67-77. (13) Haynes, P. A.; Sheumack, D.; Kibby, J.; Redmond, J. W. J. Chromatogr. 1991, 540, 177-185. (14) Haynes, P. A.; Sheumack, D.; Greig, L. G.; Kinny, J.; Redmond, J. W. J. Chromatogr. 1991, 588, 107-144. (15) Ou, K.; Wilkins, M. R.; Yan, J. X.; Gooley, A. A.; Fung, Y.; Schumack, D.; Williams, K. L. J. Chromatogr., A 1996, 723, 219-225.
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derivatives followed by reversed-phase HPLC separation and fluorescence detection. EXPERIMENTAL SECTION Chemicals. 9-Fluorenylmethyl chloroformate (98%) was obtained from Aldrich and used without further purification. A 100 mM FMOC stock solution was prepared in acetonitrile and was stable for at least one month when refrigerated in the dark. HPLC grade methanol was obtained from Burdick & Jackson; HPLC grade propanol and butanol were obtained from Sigma-Aldrich. Dehydrated 200 proof ethanol was obtained from Pharmco Products. Acetonitrile (HPLC grade), sodium hydroxide, anhydrous disodium hydrogen phosphate, and sodium carbonate (A.C.S reagent) were obtained from J. T. Baker. Hydrochloric acid (A.C.S. reagent) was obtained from Fisher Scientific. Water was purified with a Millipore Milli-Q system with resistivity of g18 MΩ. Derivatization Procedure. The FMOC-alcohol derivatization proceeded in a water-acetonitrile (1:1, v/v) solution in a basic medium. A 1-mL alcohol sample in a basic aqueous medium was mixed first with 1 mL of acetonitrile, and 100 µL of 100 mM FMOC stock solution was added. The solution was shaken for 1 min and was allowed to stand for 14 min at room temperature. After derivatization, an appropriate amount of 5 N HCl was added to the solution until the final pH was in the range of 2-3. The sample solution was directly injected into the HPLC system for analysis. Several basic aqueous media at different pH levels were tested, including 0.002-20 mM NaOH solutions, 0.1-2% (10-200 mM) Na2CO3 buffers, and 0.1-0.7% (7-50 mM) phosphate buffers. The pH of the buffer solutions was measured using a pH meter (Corning model 440). HPLC Analysis. The HPLC system consists of a gradient HPLC pump (Hitachi, model L-7100), a six-port injection valve (Valco) with a 5-µL sample loop, a guard column (Upchurch) packed with C18 packing, a C18 reversed-phase column (Rainin, MicroSorb-MV), and a fluorescence detector (Hitachi, model L-7480). The mobile phases were 5 mM hydrochloric acid (A) and acetonitrile (B). The excitation wavelength was 259 nm, and the emission wavelength was 311 nm. A PeakSimple chromatography data system (SRI, model 202) installed on a 486-PC was used to collect, store, and reprocess chromatograms. Preliminary Application. This method was applied to determine the alcohol concentrations in laboratory air (Wadsworth Center Laboratory), water samples from the Hudson River, and several commercial alcoholic beverages. The laboratory air was scrubbed with deionized water at 0 °C using a fritted glass bubbler, at 1 L min-1 for 2 h. The commercial alcoholic beverages were diluted with water to a concentration within the calibration standard range. Aqueous samples were modified with the phosphate buffer before addition of derivatization reagent. RESULTS AND DISCUSSION Fluorescence Excitation and Emission. The excitation and emission spectra of FMOC and its alcohol derivatives were collected using the scanning mode of the fluorescence detector. Figure 1 shows a spectrum of 3 µM FMOC in acetonitrile. Spectra of the hydrolysis product of FMOC and the four alcohol derivatives were identical to that of FMOC with no spectral shifting observed. Maximum fluorescence responses were achieved at the excitation 4246 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
Figure 1. Excitation and fluorescence emission spectra of FMOC in acetonitrile. The spectra of FMOC, FMOC-OH, and FMOCalcohol derivatives were all identical.
Figure 2. Scheme of reactions of FMOC with an alcohol and with water.
wavelengths of 206 and 259 nm. Further experiments showed noisier baselines at an excitation wavelength of 206 nm than at 259 nm, and therefore, the latter was used. A maximum fluorescent emission was observed at 311 nm. Derivatization pH. The reactions of FMOC with alcohols and water are shown in Figure 2. Alcohols are less reactive toward FMOC than are amino acids because the OH group has less nucleophilic reactivity than the NH2 group. Basic media may facilitate the derivatization reaction by enhancing the nucleophilic reactivity of alcohols. Several types of basic media were tested in this study, including sodium hydroxide, carbonate buffers, and phosphate buffers. Derivatization was first studied using NaOH as a catalyst in the pH range of 11-13. Maximal derivatization yields were achieved around initial pH of 12 (∼10 mM NaOH). Because significant amount of HCl was released from both reactions (Figure 2) of several millimolar FMOC reagent, the pH of the derivatization medium decreased significantly as the reaction proceeded. The derivatization reaction was therefore retarded at the early reaction stage, resulting in low derivatization yields. When higher initial pH was used, decomposition of the alcohol
Figure 3. Effect of pH on derivatization yields. The 1:1 wateracetonitrile derivatization medium contained 0.5% phosphate buffer, 5 mM FMOC, 20 µM C1 (square) and 150 µM C2 (triangle). Similar effects were observed but not shown for C3 and C4 alcohols.
derivatives occurred rapidly and lowered the final derivatization yield. Obviously, a buffer solution with a sufficiently high concentration is needed to maintain an optimal derivatization pH. Carbonate buffer at concentrations of 0.1-2% (10-200 mM) was first tested. However, the pH range between 10 and 11.8 maintained by this buffer was not adequate to achieve satisfactory derivatization yields, as suggested by a steady increase in derivative yield with pH. Phosphate buffer was then used and found to be the best choice. The effect of pH on the derivatization reaction was investigated in the pH range of 11-13, as was maintained by 0.6% (42 mM) phosphate buffer solutions (Figure 3). The maximum derivatization yields were achieved at pH 12.5. Below pH 12.5, the derivatization reaction was facilitated by base catalysis, resulting in higher yields with increasing pH. Above pH 12.5, however, hydrolysis of derivatives became significant and lowered the apparent yield. In addition, a significant amount of FMOC reagent remained unhydrolyzed at pH lower than 12.5, with its large peak interfering with the adjacent methanol derivative peak and causing a higher and noisier chromatogram baseline. This pH was therefore used in subsequent experiments. Experiments were carried out to determine the concentration needed to maintain the desirable pH of the derivatization medium. Figure 4 shows the effect of the phosphate concentration in aqueous phase on the fluorescence response of each alcohol derivative. Buffer at a concentration of e0.3% (21 mM) did not have sufficient capacity to maintain the desirable pH. When the phosphate concentration was higher than 0.7% (50 mM), it precipitated in the derivatization medium containing 50% acetonitrile. Therefore, a 0.6% phosphate buffer solution at pH 12.5 was chosen for alcohol derivatization. Reagent Concentration. The effect of FMOC concentration on the derivatization reaction was also studied within the range of 1-9 mM (see Figure 5). Derivative yields increased with FMOC concentration, and so did the method sensitivity initially when FMOC concentration was e5 mM. However, when the FMOC concentration was >7 mM, the HPLC column became overloaded, and FMOC-OH and FMOC peaks tailed so severely that the adjacent analyte peaks, i.e., methanol and propanol, respectively, could not be sufficiently separated from them. In addition, the C1
Figure 4. Effect of phosphate buffer concentration on derivatization yields. The results were obtained under the same conditions as in Figure 3, except with pH at 12.5 and varying phosphate concentration between 0.1 and 0.7%. Symbols: squares for C1 and triangles for C2. Similar effects were observed but not shown for C3 and C4 alcohols.
Figure 5. Effect of FMOC concentration on derivative yields and blank. The results were obtained for 30 µM µM C1 (triangle) and reagent blank (circle) in a 0.5% phosphate buffer at pH 12.5. Similar effects were observed for the other three compounds.
and C2 blanks increased with FMOC concentration, and a longer derivatization time was required to allow adequate hydrolysis of the FMOC reagent at higher concentration. Therefore, 5 mM FMOC concentration was chosen for alcohol derivatization. The linear relationship between the HPLC signal and FMOC concentration (Figure 5) suggests that the alcohols in the samples were far from being quantitatively converted to their fluorescent derivatives, due to the fast hydrolysis consuming most of the FMOC reagent during derivatization. However, the low yields should not prevent the method from being a sensitive and quantitative technique for alcohols, since the yield is highly reproducible under specified derivatization conditions (see below). Derivatization time. Figure 6 shows the fluorescence response of the derivatives at different derivatization times. The reaction reached its maximum plateau in ∼10 min, and the derivatives were quite stable for at least 20 min afterward but gradually decomposed due to hydrolysis. To stop the decomposition of alcohol derivatives and minimize further hydrolysis of Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
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Table 1. Chromatographic Gradient Profile for the Separation of Alcohol Derivativesa time, min
acetonitrile, %
5 mM HCl, %
time, min
acetonitrile, %
5 mM HCl, %
0 4 8
48 51 63
52 49 37
12 12.1 15
70 100 100
30 0 0
a
The flow rate was 1.5 mL min-1.
Figure 6. Effect of derivatization time on yields. The results were obtained under the same conditions as in Figure 3, except with pH at 12.5. Symbols: squares for C1 and triangles for C2. Similar effects were observed but not shown for C3 and C4 alcohols.
Figure 7. Stability of alcohol derivatives after acidification. The results were obtained under the same conditions as in Figure 3, except with pH at 12.5. Symbols: squares for C1 and triangles for C2. Similar effects were observed but not shown for C3 and C4 alcohols.
FMOC during HPLC separation, the reaction solution was acidified to a final pH of 2-3 with 22 µL of 5 N hydrochloric acid after derivatization completion. The acidification could also prevent damage to the HPLC column by injection of a basic solution. Figure 7 shows the stability of alcohol derivatives after acidification. The four alcohol derivatives were found to be stable for at least 1 h at room temperature. HPLC Separation. Many gradient programs were investigated to ensure satisfactory HPLC separation within the shortest time. The final program is summarized in Table 1. HCl (5 mM) was used instead of water as the weaker mobile phase to minimize FMOC hydrolysis during HPLC separation, resulting in a lower and more stable chromatogram baseline. Figure 8a shows a chromatogram of four alcohol derivatives (C1-C4) obtained with the proposed procedure. All four alcohol derivatives were well separated without interference from the FMOC-OH and FMOC peaks. Calibration, Reproducibility, and Detection Limits. Figure 9 shows the calibration curves for methanol (C1), ethanol (C2), 4248 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999
Figure 8. Chromatograms of (a) an alcohol standard solution containing 15 µM C1 and 200 µM C2-C4; (b) a blank; (c) a laboratory air sample 34 µM C1 was found in the scrubbing solution, corresponding to a gas-phase concentration of ∼100 ppbv; and (d) a commercial beer diluted by a factor of 2000 with water.
propanol (C3), and butanol (C4) determination. The concentrations of standards were in the range of 0-30 µM for C1 and 0-300 µM for C2-C4, respectively. Injection volume was 5 µL. The calibration curves demonstrated good linear relationships between alcohol concentrations and HPLC responses, with correlation coefficients of between 0.997 and 0.999 (Figure 9). The method reproducibility was examined by preparing and measuring seven samples containing 15 µM C1 and 150 µM C2, C3, and C4. The relative standard deviations (RSDs) of the peak areas and retention times are shown in Table 2. The RSDs of peak areas were 3.7, 2.1, 1.5, and 2.2%, and the RSDs of retention time were 0.56, 0.30, 0.26, and 0.21%, respectively, for the four alcohols. Lower detection limits of 4, 40, 70, and 30 pmol were calculated for C1, C2, C3, and C4 alcohols, respectively, based on 3 times of the standard deviations of seven samples with alcohol concentrations close to the blank values. For an injection volume of 5 µL, these correspond to lower concentration detection limits
Figure 9. Calibration curves for (a) methanol (r2 ) 0.999), and (b) ethanol (triangle, r2 ) 0.997), propanol (cross, r2 ) 0.998), and butanol (diamond, r2 ) 0.998). Table 2. Summary of Chromatographic Retention Times, Reproducibility, and Detection Limits
alcohola
retention time min RSD,b %
methanol ethanol propanol butanol
7.28 8.87 10.68 12.60
0.56 0.30 0.26 0.21
peak area RSD,b % 3.7 2.1 1.5 2.2
detection limitb,c pmol µMd 4 40 70 30
0.8 8.0 14.0 6.0
a The amount of standard methanol was 75 pmol; the other alcohols were 750 pmol. b n ) 7. c Detection limits were based on a signal-tonoise ratio of 3. dPer 5-µL injection.
of 0.8, 8, 14, and 6 µM for C1, C2, C3, and C4 alcohols, respectively. The smaller steric effect and stronger nucleophilic reactivity to FMOC of methanol results in a higher derivative yield (Figure 9) and thus a lower detection limit compared to the other three alcohols. The detection limit for propanol is higher than C2 and C4 due to its peak being adjacent to the FMOC reagent peak. Comparisons with Existing Methods. There are several precolumn or postcolumn derivatization/HPLC methods available in the literature for the determination of aliphatic alcohols and
hydroxyl compounds.1-9 However, most of these techniques involved in derivatization in organic phases and at elevated temperatures over extended periods of time and thus not suitable for aqueous samples. One recent HPLC/fluorescence technique deployed AQC as the derivatizing agent for the LMW aliphatic alcohols in aqueous solutions.9 While a straightforward derivatization procedure was involved, the technique provided only limited detection sensitivity, i.e., with a lower detection limit of g90 pmol for C1-C4 alcohols, corresponding to 18 µM for a 5-µL injection. Compared to these techniques, our method offers the advantages of high sensitivity and a simple derivatization/analysis procedure for aqueous samples. The mild derivatization condition and high derivative stability enable the measurement to be automated using an autosampler capable of sample preparation. Preliminary Application. Figure 8 illustrates some preliminary applications of the method to analyze alcohol contents in a laboratory room air sample (Figure 8c) and in a commercial beer (Figure 8d). A methanol concentration of up to ∼100 ppbv was found in the laboratory air during the period when it was frequently used as a solvent. The beer sample contained 5.0 ( 0.1% (n ) 4) ethanol, in good agreement with the nominal value of 5.0% from the manufacturer. No detectable methanol was found, but a significant amount of propanol (∼1%) and a trace amount of butanol were present in the beer (Figure 8d). Alcohol levels in the Hudson River water sample (chromatogram not shown) were all below the method detection limits, i.e., e0.8, 8, 14, and 6 µM for C1, C2, C3, and C4 alcohols, respectively. No interference from other compounds occurred. CONCLUSIONS This study has shown that FMOC is a good fluorescence labeling reagent for the determination of low-molecule-weight aliphatic alcohols. With a simple derivatization procedure followed by reversed-phase HPLC separation and fluorescence detection, this method is able to measure methanol, ethanol, propanol, and butanol in aqueous samples at picomole detection limits. The mild derivatization condition and high derivative stability enable the measurement to be automated using an autosampler capable of sample preparation. The detection limits may be further lowered if derivatization proceeds in an organic solvent to minimize FMOC hydrolysis and to obtain higher alcohol derivative yields. ACKNOWLEDGMENT We thank Kevin Civerolo and Arlene North for their helpful comments. This research was supported by National Science Foundation Grant ATM-9615748.
Received for review January 8, 1999. Accepted July 21, 1999. AC990010M
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