Anal. Chem. 1982, 5 4 , 939-942
939
Derivatization of Secondary Amino Acids with 7-Nitro-4-benzofurazanyl Ethers Lars Johnson, Soren 'Lagerkvlst, and Peter Llndroth Deparfmenlt of Analytical and Marine Chemistty, Chalmers University of Technology and University of Gijteborg, 5-4 12 96 Goteborg, Sweden
Martln Ahnoff * and Kerstln Martinsson Depaltment of Analytical Chemistry, AB Hassle, 5-43 1 83 Miindal, Sweden
Flve 7-nltro-4-benzofurmzanyl ethers have been Synthesized and their water solubllitles and reactivities have been determlned. 4-Methoxy-7-nltrobenzofurazan (NBD-OCH,) has a solublllty slmllar to that of 4-chloro-7-nltrobenzofurazan (NBD-CI) but reacts -3.5 tlmes faster wlth hydroxyprollne. Hydroxyprollne reacts quantltatlvely wlth NBD-OCH, In acetonltrlle-water, while conslderable losses are seen when NBD-CI Is used In this; medlum. 4-(2-Hydroxyethoxy)-7nltrobenzofuraran Is 16 tlmes more soluble In water than NBD-CI and therefore permits complete derlvatlzatlon of hydroxyproline In aqueous solutions wlthout any organic solvent. I t Is shown that hlgh sensltlvlty analytical methods (pmol/mL range) can be designed by comblnlng derlvatlzatlon in a pure aqueous medlum wlth Injection of large volumes of sample onto a reversed-phase liquid chromatographlc column.
The reactivity of nitro-substituted aromatic ethers is documented as well as their use in organic synthesis (1-3). We here report on the use of one type of aromatic ethers as derivatizing agents for amines, namely, ethers of 7-nitrobenzofurazan. It was previously demonstrated ( 4 ) that 4-chloro7-nitrobenzofurazan (NBD-Cl), when reacting in a methanol/buffer medium, forms 4-methoxy-7-nitrobenzofurazan (NBD-OCH3) which itself reacts with amines. In this work it is shown that, apart from NBD-OCH3, other NBD ethers can be used as reagents for the determination of amines. 4-(2-Hydroxyethoxy)-7-nitrobenzofuraztm(NBD-OCHzCH2OH) and NBD-OCH, have been selected for a closer examination arid comparison with NBD-C1. For this purpose the different reactions involved, shown in the general reaction scheme iin Figure 1, have been studied.
EXPERIMENTAL SECTION Synthesis of 4-Methoxy-7-nitrobenzofurazan(NBDOCH,). PBD-OCH, was synthesized from NBD-C1 and CH30Na in CH3011 as described previously (4). Synthesis of 4-Ethoxy-7-nitrobenzofurazan (NBD-OCHzCH3).NBD-OCH2CH3was prepared by dropwise addition of 0.22 g (5.!5 mmol) of NaOH in 20 mL of CzH50H to 1.0 g (5.0 mmol) of NBD-C1 in 80 mL of CzH50H. After being stirred overnight the dark colorecl solution was acidified with HC1, diluted with water, and extracted with CHZCl2.The extract was dried and the solvent evaporated. The product was purified on a silica gel column (eluant CHZCl2)and recrystallized from CHCICClz to yield 0 40 g (38%): mp 76-80 "C (Buchi 510, 2 "C/min); lH NMR (Varian EM 390,90 MHz, CDC1,) 6 8.7 (d, J = 9 Hz, 1H), 6.8 (d, J := 9 Hz, 1 H), 4.6 (9, J = 7 Hz, 2 H), 1.7 (t, J 3 7 Hz, 3 H).It should be noted. that when the amount of NaOH in the procedure above was doubled, the synthesis unexpectedly gave 5-ethoxy-4-nitrobenzofurazan as the major product. Synthesis of 4-(2-Hydroxyethoxy)-7-nitrobenzofurazan (N€3D-OCHzCHz0H). k 0.40-g portion (10 mmol) of NaOH was dissolved in 20 mL of ethylene glycol and added to a suspension
of 1.0 g (5.0 mmol) of NBD-Cl in ethylene glycol. The reaction mixture was stirred for 1h and then acidified with HCl, diluted with water, and extracted with ethyl acetate. The extract was dried and the solvent evaporated. The product was purified on a silica gel column (eluant CHC13:CH3COCH39:l) and recrystallized from CHC13to yield 0.60 g (53%): mp 87-89 "C (Buchi 510,2 "C/min); lH NMR (CD,OD) 6 8.8 (d, J = 9 Hz, 1H), '7.1 (d, J = 9 Hz, 1 H), 4.6 (t,J = 5 Hz, 2 H), 4.1 (t,J = 5 Hz, 2 13). Synthesis of 4-(2,3-Dihydroxypropoxy)-7-nitrobenzofurazan (NBD-OCHzCH(OH)CHzOH). NBD-OCHzCH(OH)CHzOHwas prepared by adding 2.0 g (10 mmol) of NBD-Cl to a solution of 0.80 g (20 mmol) of NaOH in 100 mL of glycerol and thereafter following the procedure for NBD-OCHzCHzOIH. The product was purified on a silica gel column (eluant CHzClZ:CH3COCH3l:l),redissolved in CHzC12,and filtered and the solvent evaporated to leave a yellow oil that slowly crystallizied. The crystals were filtered off from diisopropyl ether to yield 0.40 g (16%): mp 80 OC (Buchi 510,2 "C/min); 'H NMR (CD30D) 6 8.8 (d, J = 9 Hz, 1H), 7.1 (d, J = 9 Hz, 1 H), 4.5 (m, 2 H), 4.2 (m, 1 H), 3.8 (d, J = 5 Hz, 2 H). Synthesis of 4-Phenoxy-7-Nitrobenzofurazan(NBD-0CsH5). A 0.20-g (5.0 mmol) portion of NaOH and 0.47 g (5.0 mmol) of phenol were dissolved in 20 mL of CH3CN, 1.0 g (5.0 m o l ) of Nl3D-C1 was added and the mixture stirred for 1h. Af'ter the solvent had been evaporated, the product was purified on a silica gel column (eluant CHC13:CH30H9:l) and recrystallized from CzH50Hto yield 0.40 g (31%): mp 114-119 "C (Buchi 510, 1OC/min); lH NMR (CDCl,) 6 8.6 (d, J = 9 Hz, 1H), 7.8-7.3 (m, 5 H), 6.7 (d, J = 9 Hz, 1 H). Synthesis of 1-(7-Nitro-4-benzofurazanyl)-4-hydroxy-~proline (NBD-Hyp). NBD-Hyp was prepared by adding NBD-Cl in CH30H to NaHCO, and 4-hydroxy-~-prolinein water, as described previously (4). Synthesis of 1-(7-Nitr0-4-benzofurazanyl)-~-alanine (NBD-Ala). A 1.25-g (15 mmol) portion of NaHC0, and 0.45 g (5.0 mmol) of rJ-alaninewere dissolved in 10 mL of HzO. A 1.0-g (5.0 mmol) portion of NBD-C1 in 40 mL of CH,OH was added. After 1 h at 55 "C the reaction mixture was acidified to pH 1.5 with 2 mol/L HCl(6 mL). Most of the solvent was evaporated and water added. The precipitate was filtered and recrystallized from a solution of HaHC0, in HzO (0.5 g in 10 mL) by adding 2 mol/L HCl(6 mL) to yield 0.80 g (63%): mp 157-160 "C (Biichi 510,2 "C/min); 'H NMR (CD30D) 6 8.6 (d, J = 9 Hz, 1 H) 6.4 ( d , J = 9 Hz, 1 H), 4.8 (4, J = 7 Hz, 1H), 1.7 (d, J = 7 Hz, 3 'H). Synthesis of 7-Nitro-4-benzofurazanol(NBD-OH). NBD-OH was prepared by demethylating NBD-OCH, in aqueous NaOH as described previously (4). Measurement of Reaction Rates. All reactions were performed at 50 OC in borate buffer solutions. Continuous measurements were carried out in a Perkin-Elmer 550 spectrophotometer. Diluted samples of reaction mixtures were measured with an Aminco-Bowman spectrophotofluorometer. Chromatographic separation and quantitation of reaction components were carried out on a Varian 5000 liquid chromatograph equipped with a Nucleosil C-18 5 wm column (200 mm X 5.0 mm i.d.) and UV-VIS and fluorescence detectors (Schoeffel 770 and 970). Reaction mixtures were injected by use of a 1O-fiLloop injector and eluted with a mixture of acetonitrile (40%) and 0.05 mol/L phosphate buffer (pH 1.9). For the quantitation of reaction
0003-2700/82/0354-0930$01.25/0 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
940
NO,
I
OR
R'NR"
\
NO2
k
6
Figure 1. Reactions of NBD ethers.
Table I. Kinetic and Physicochemical Data for Some 7-Nitrobenzofurazans
-c1
-0 CH,CH, -OCH,CH,OH -OCH,CH(OH)CH,OH -OC,H,
-OH -Ala -Pro -HYP
rate con- solubilityb stant,('s-l in water, capacity' mol-' L mmol L-I factor h' 1.2 4.5 4.8 0.073 0.017 1.8
9
6
7
8
9
PH
Flgure 2. Reaction rates for NBD-OCH,CH,OH at different pH: (a) reaction wlth hydroxy+-proline; (b) hydrolysis. Reaction medium: 0.1 moi/L borate buffer containing 25% CH,CN.
0-
-0 CH
8
PH
@o
substituent
7
1.3 1.2
6.3 4.7
0.78
1.8
20 120 0.089
1.6 0.9 22 1.3 1.7 2.3 0.6
a For the reaction with Hyp, measured by spectrophotometry at 50 "C in a mixture containing 0.1 mmol/L borate buffer (pH 9.5), 25% CH,CN, 0.1 mmol/L reagent and excess of amine. b At room temperature. Retention measured on a Nucleosil (2-18 5 pm column eluted with a mixture of CH,CN (40%)and phosphate buffer (0.05 mol/L, pH 1.9).
components, reference solutions of reagents as well as the NBD derivatives of alanine (NBD-Ala) and hydroxyproline (NBD-Hyp) were used. All aqueous solutions of amine derivatives were protected from light as far as possible in order to avoid decomposition (4).
RESULTS AND DISCUSSION Synthesis of 7-Nitro-4-benzofurazanylEthers. lH NMR was used to distinguish between 7-nitro-4-benzofurazanyl ethers and 4-nitro-5-benzofurazanyl ethers. The procedures described in the Experimental Section all resulted in the intended 4,7-substituted benzofurazans. lH NMR data for 4-(2-hydroxyethoxy)-7-nitrobenzofurazanreported by Ah-Kow et al. (5) differ from our data. They also reported a pKa value (6.86) for this substance dissolved in water, which is significantly lower than ours (7.85). The synthesis of both 4,7- and 4,5-substituted benzofurazans from 4-chloro-7nitrobenzofurazan will be the subject of a separate report. Preliminary Investigation of Five NBD Ethers. The second-order rate constants for the reaction between a secondary amino acid, hydroxy-L-proline (Hyp), and NBD-OCH3, NBD-OCHZCHS, NBD-OCHZCHZOH, NBD-OCHzCH(0H)CHzOH, NBD-OC6H5 and NBD-C1 were measured with a spectrophotometer using 0.1 mmol/L reagent and 10-100 times excess of amino acid in CH&N/borate buffer, pH 9.5. As can be seen in Table I the nonhydroxylated NBD ethers react faster than NBD-C1 while the hydroxylated ones react slower. Table I also shows the solubilities of the NBD reagents in pure water as well as chromatographic retention data for these and some NBD derivatives on a reversed-phase system. Two of the NBD ethers, NBD-OCH3 with high reactivity and NBD-OCHzCHzOHwith high solubility, were selected for a more thorough investigation.
Table 11. Rate Constants at 5 0 "C for 7-Nitrobenzofurazans in Aqueous Solution reaction with Hyp substituent
-c1
-OCH, -OCH,CH,OH -HYP
pH
hydrolysis hx
lo5,s-1
9.5 9.5 8.5
5(' 27(' 0.85('
9.5 8.5 7.5
5.5b
h, s-'
mol-' L 0.91c
3-03' 0.13'
l.lb
0.25b
a Both reagent and formed NBD-OH were measured by liquid chromatography (LC). NBD-Hyp was ineasured by LC. NBD-Hyp was measured by spectrofluorometry.
Acidic Behavior and Reactivity of 4-(1-Hydroxyethoxy)-7-nitrobenzofurazan.NBD-OCHzCHzOHis a weak acid, its pKa in water, determined by spectrophotometry, being 7.85 f 0.10:
&= 0
I
CH,CH,OH
NO;
o @ + " I
1
HIC-CH,
s pi ro-Meisen heimer complex
The reaction between NBD-OCHzCHzOHand Hyp has its optimum near pH 8 (Figure 2) as compared to the optimum near p H 9.5 for NBD-C1. The hydrolysis of NBD-OCHzCHzOH to 7-nitro-4-benzofurazanol (NBD-OH) proceeds a t a more or less constant rate between pH 8 and pH 9.5 which decreases at lower pH values (Figure 2). The pH dependence of the two reactions confirms the assumption that the spiroMeisenheimer complex is not reactive. Consequently, NBD-OCHzCHzOHshould be used at a pH about 8 for derivatization of amines. In the following experiments pH 8.5 was used for NBD-OCHzCHzOH while pH 9.5 was used for NBD-OCH3 and NBD-C1. Reaction Rates and Yields. Rate constants were measured for the reactions of NBD-OCH3, NBD-OCHzCH20H, and NED-Cl with hydroxy-L-proline under conditions used for analytical applications. These are listed in Table I1 together with rate constants for the hydrolysis of the reagents and the derivative. The rate of formation for NBD derivatives of amines is restricted by the solubility and reactivity of the NBD reagents, as well as by the nature of the amine. If the formation rate is too slow, then hydrolysis of the derivative and of the NBD reagent will make a quantitative yield unattainable (see Figure 1). In pure water the solubility of the reagents is limited. In one experiment NBD-C1 and NBD-OCH3 were used a t their practical maximum concentration (1mmol/L reagent solutions were added to equal volumes of Hyp solutions and a small
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
941
-OH
n
-HYP
0
30
60
90
120
150
reaction time (mid Flgure 3. Measured yields of NBD-Hyp vs. reaction time for: 4.8 mmol/L NBD-OCH2CH,0H, pH 8.5, 0 ; 0.48 mmol/L NBD-CI, pH 9.5, v; 0.48 mmol/L NBD-OCH,, pH 9.5, A;0.30 mmol/L NBD-OCH,, pH 9.5, a. Reaction medium: 0.02 mol/L borate buffer, 5 X lov5mol/L Hyp. Solid lines: theoretical yields calculated from initial concentrations and rate constants In Tabla 11.
amount of borate buffer), while NBD-OCH2CH20H was used at a 10 timles higher concentration. With NBD-OCH2CH20H a yield close to 100% was obtained, the 95% level being reached after 90 min. NBD-OCH3yields I I maximum at 88% after 50 min and NBD-C1 yields 82% after 150 min (Figure 3). Computer-simulated yield-vs.-time plots derived from the rate constants in Table I1 are included in Figure 3. The recovery of NBD-Hyp with 0.48 mmol/L NBD-OCH3 or NBD-C1 might seem satisfactory, especially as the decay is slow. However, the recovery is sensitive for variations in reagent concentration, as is illustrated in Figure 3. With 0.30 mmol/L NBD-OCH, the recovery after 50 min was 76% instead of 88%. The better yield for NBD-OCH2CH20Hin Figure 3 is mainly a function of the higher reagent concentration. If' derivatization with NBD-C1 and NBD-OCH3 is carried out a t pH 8.5 instead of pH 9.5, no significant gain in reaction yield is expected since not only the hydrolysis of reagent and NBD-Hyp but also the formation of NBD-Hyp will proceed slower. The addition of an organic solvent to the reaction medium affected both reagent solubilities and reactivities. With 5% CH3CN present, the solubilities for NBD-Cl and NBD-OCH, were roughly doubled, while the solubility of NBD-OCH2CH20H was less affected. The rate constants for Hyp reacting with NBD-C1 and NBD-OCH, (pH 9.5) and NBD-OCH2CH20H (pH 8.5) increased to 1.3, 6, and 0.6 s-l mol-' L, respectively. At their maximum concentrations both NBD-OCH, and NBD-OCH2CH20Hgave cu. 4 times faster reaction thain NBD-C1. It should be noted that not only the reactions between reagents and amino acids are promoted by the presence of an organic solvent but also the hydrolysis of reagents and amino acid derivatives. The slow reaction of the primary amino acid alanine made determination of reaction rate difficult in aqueous solutions with low content of organic solvent. In 25% CH3CN the rate constants for Ala reacting with NBD-Cl and NBD-OCH, (pH 9.5) and NBD-OCH2CH20H(pH 8.5) were 0.11,0.34, apd 0.01 s-l mol-' L. Thus the selectivity for Hyp against Ala was similar for all three reagents. When the borate buffer concentration was increased from 0.1 mol/L to 0.4 mol/L, the rate of formation of NBD-Hyp changed less than by a factor 1.5. This was also the case when 0.25 mol/L NaCl or seawater was present in the reaction mixture. Side Reactions. NBD-Cl appears to be involved in side reactions to a higher extent than NBD-OCH3. In accordance with earlier observations ( 4 ) , incomplete yields were found for NBD-Cl reacting with Hyp in a water-acetonitrile mixture. Products were formed which absorbed light at high wavelengths, 550-700 nm. Different results were obtained with
-Pro
4
'u
0
5
10
r e t e n t i o n t i m e (min)
Figure 4. Chromatogram obtained from a spiked seawater sample containing 180 pmol/mL of Hyp and Pro. Reaction conditions: 1 miL of sample, 1 mL of 10 mmoi/L NBD-OCH,CH20H in H20, plus 0.1 mL of 0.4 mol/L borate buffer, pH 8.0, were heated to 50 OC for 50 mihi; 0.8 mL was Injected onto a Nucleosil C-18 5 p m column (200 mm X 5.0 mm i.d.) and eluted with CH,CN/O.O5 mol/L phosphate buffer, pH 1.9 (30/70 v/v). Fluorescence detectlon: excitation at 475 nni, emission >550 nm.
acetonitrile from different bottles. With NBD-OCH3,yields above 90% were obtained. In pure aqueous buffer solutions, the NBD ethers, after sufficient time, converted quantitatively to NBD-OH. In the case of NBD-Cl, only ca. 40% of the amount hydrolyzed was found as NBD-OH. Application. Hydroxy-L-proline and L-proline added to standard seawater (Standard Seawater Service, I.A.P.S.O., Surrey, Great Britain) were converted to their NBD derivatives by mixing a 1.0-mL sample of spiked seawater with 1.0 mL of 10 mmol/L NBD-OCH2CH20Hin H 2 0 and 0.1 mL of borate buffer (pH 8.0,0.4 mol/L). After 50 min at 50 "C, 0.8 x d was injected onto a reversed-phase column, using a column packed with glass beads instead of an open tubular injector loop, and eluted with CH,CN/phosphate buffer, pH 1.9 (30/70 v/v). A chromatogram is shown in Figure 4. The amounts of Hyp and Pro in the buffered sample were 180 pmol each, part of which originated from impurities in the buffer solution. Nonspiked standard seawater contained less than 2 pmol/mL of Hyp and Pro. Owing to the absence of organic solvent in the sample, a band sharpening effect was obtained so that no loss in resolution occurred. Peak heights increased linearly with increasing injection volume up to 0.8 mL, which was just below the internal volume of the injector column. With another injector arrangement even larger volumes may be injected. On the other hand, if NBD-Cl had been used at a concentration which gives complete dervatization, then it would have been necessary to add an organic solvent, e.g., 20% acetonitrile, to the reaction mixture. A 50-100 pL sample would have been the maximal injection volume for the column used. By injection of solutions of NBD-Hyp, the following detection limits (SIN = 2) were found for different detection modes: absorption at 500 nm, 10 pmol; fluorescence with excitation at 335 nm (deuterium lamp) and emission above 470 nm, 1 pmol; fluorescence with excitation at 475 nm (wolfram lamp) and emission above 550 nm, 2 pmol. The highest selectivity was obtained with the last detector setup.
942
Anal. Chem. 1982, 5 4 , 942-946
CONCLUSION Principally, a t least four properties of NBD ethers could render them useful as alternatives to NBD-Cl in analytical applications: (1)their higher reactivity (NBD-OCH3, NBDOCH2CH3),(2) their property of forming neutral alcohols instead of HCl when reacting, (3) the higher reagent concentration obtainable by choosing an NBD ether substituted with polar groups (NBD-CH2CH20H,NBD-OCH,CH(OH)CH,OH), and (4) their lesser tendency to be involved in undesired side reactions. ACKNOWLEDGMENT We thank A. Arfwidsson and B.-A. Persson for their helpful
discussion and criticism of this work.
LITERATURE CITED (1) Miller, J. "Aromatlc Nucleophilic Substitutlon"; Elsevier: Amsterdam,
1968. (2) Orvik, J. A.; Bunnet, J. F. J . Am. Chem. SOC.1970, 92, 2417-2427. (3) Di Nunno, L.; Florlo, S.; Todesco, P. E. J Chem. Soc ., Perkin Trans. 2 1975, 14, 1469-1472. (4) Ahnoff, M.; Grundevik, I.; Arfwidsson, A.; Fonselius, J.; Persson, B.-A. Anal. Chem. 7981, 53, 485-489. (5) Ah-Kow, G.; Terrier, F.; Lessard, F. J. Org. Chem. 1978, 43, 3578-3584.
RECEIVED for review November 18,1981. Accepted February 8, 1982.
Headspace Chromatographic Determination of Water Pollutants Rein Otson* and Davld T. Wllllams Bureau of Chemical Hazards, Environmental Health Directorate, Health Protection Branch, Health and Welfare Canada, Tunney's Pasture, Ottawa, Ontario K1A OL2, Canada
A readily constructed, automated purging assembly, on-coiumn trapping, and simultaneous use of flame lonlzatlon and electrolytic conductivity detectors were applied to develop a dynamic headspace gas chromatographlc technique which was evaluated for the determination of 42 organlc pollutants in water. Detectionlimits of
