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Workplace monitoring of oximes using 2,4-dinitrophenylhydrazine-coated silica gel cartridges and liquid chromatography
Analytica Chimica Acta 388 (1999) 181±186
Workplace monitoring of oximes using 2,4-dinitrophenylhydrazinecoated silica gel cartridges and liquid chromatography Christine Kempter, Andrea BuÈldt, Uwe Karst* WestfaÈlische Wilhelms-UniversitaÈt MuÈnster, Anorganisch-Chemisches Institut, Abteilung Analytische Chemie, Wilhelm-Klemm-Str. 8, MuÈnster D-48149, Germany Received 22 September 1998; received in revised form 20 January 1999; accepted 25 January 1999
Abstract A liquid chromatographic (LC) method for the determination of oximes in air samples using pre-column derivatization with a hydrazine reagent has been developed. Air samples are drawn over a 2,4-dinitrophenylhydrazine (DNPH)-coated silica gel cartridge to form the corresponding hydrazones. The acid coating of the tubes and the ¯ow rate of the air sample have been selected considering the chemical characteristics of the oximes to avoid Beckmann rearrangement or incomplete recovery. The hydrazones are separated by LC with UV/Vis detection at 365 nm. Calibration is performed externally using the stable hydrazone standards. The limit of quanti®cation is 0.5 pmol absolute for 2-butanone oxime (corresponding to 15.0 ng/l in a 2 l air sample) and cyclohexanone oxime (corresponding to 19.5 ng/l in a 2 l air sample), respectively. Real samples from industrial workplaces have successfully been analysed using this method. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Liquid chromatography (LC); Oximes; 2,4-Dinitrophenylhydrazin (DNPH); Air samples; Workplace monitoring
1. Introduction Oximes are structural analogues of carbonyl compounds. They are formed in a reaction of aldehydes and ketones with hydroxylamine according to the following equation:
2-Butanone oxime (methyl ethyl ketoxime, MEKO) is *Corresponding author. Tel.: +49-251-8333-182; fax: +49-2518333-169; e-mail: [email protected]
an important solvent and additive in the polymer and coatings industries. MEKO is primarily used as an antiskinning agent in alkyd paints. It is also used as blocking agent for urethane polymers, and as corrosion inhibitor in boilers. It is released in small amounts from sealants containing MEKO-based oxime silanes during curing. MEKO is listed in chapter III A 2 of the German MAK list [1] as a substance which has been identi®ed as possible carcinogen in animal tests. The conditions of these tests are considered to be comparable to the exposition at workplaces. No of®cial threshold value is therefore listed. However, several industrial enterprises use the MEKO concentration of 9.0 mg/l in air as internal threshold value. Cyclohexanone oxime (CHO) is used in large amounts as intermediate in the formation of poly-
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(99)00128-2
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C. Kempter et al. / Analytica Chimica Acta 388 (1999) 181±186
amides. In the presence of mineral acids, a rearrangement of the oxime to e-caprolactame is observed (Beckmann rearrangement). The lactame is then polymerized to polyamides:
performed externally using the stable solid hydrazones which may easily be synthesized according to literature methods [13±15]. Due to the analogy between the oxime function and the carbonyl function in aldehydes and ketones, the goal of the present work was to investigate the suitability to determine oximes as well using the DNPH method. 2. Experimental 2.1. Chemicals
As the oximes are volatile, a signi®cant concentration in the gas phase at industrial workplaces may be expected. Therefore, the concentration of the oximes in workplace air should be determined. Only few methods have been described for this purpose. The only method of widespread acceptance in industry is the gas chromatographic analysis with ¯ame ionization detection after trapping the analytes on solid sorbents. However, as oximes are reducing agents, their subsequent decomposition on the sorbent could be suspected at least in the presence of strong oxidants. Hydrazine reagents have been used for a long time for the determination of aldehydes and ketones in liquid and air samples [2±12]. Among these, 2,4dinitrophenylhydrazine (DNPH) has established as the most popular reagent. DNPH reacts with aldehydes and ketones in acidic media under formation of the respective hydrazones:
These are separated by liquid chromatography (LC) and detected spectroscopically by UV/Vis in most cases [2±8]. Mass spectrometric detection has also been performed in recent years [9,10]. Different air sampling technologies have been used in combination with the DNPH method, including impingers [2,3], test tubes ®lled with reagent-coated sorbents [4±6], and passive sampling devices [7,8]. Calibration is
All chemicals were purchased from Aldrich Chemie (Steinheim, Germany) in the highest quality available. Sulphuric acid was Merck (Darmstadt, Germany) analytical grade. Acetonitrile for HPLC was Merck gradient grade. Quartz wool was obtained from Fleischhacker (Schwerte, Germany). 2.2. Synthesis DNPhydrazones. The derivatives were prepared according to a procedure published by Allen [13] and Brady [14] for the synthesis of DNPH derivatives: 100 mg 2,4-dinitrophenylhydrazine (5.110ÿ4 mol) were dissolved in 0.5 ml sulphuric acid and a mixture of 0.7 ml water and 2.5 ml ethanol. A 50% molar excess of the aldehyde, ketone or oxime, respectively (7.710ÿ4 mol) was added as 10±20% solution in ethanol. The hydrazones precipitated as reddish crystalline material. The precipitate was ®ltered off and washed ®rst with a 5 mass% aqueous solution of sodium bicarbonate until no further development of carbon dioxide was observed according to Behforouz et al. [15]. Excess bicarbonate was removed by an additional washing step with distilled water. If necessary, the products were recrystallized from ethanol. The derivatives were fully characterized by means of NMR, IR and UV/Vis spectroscopy, elementary analysis and melting point. 2.3. Preparation of the DNPH sampling tubes A 0.2 mol/l DNPH solution was prepared by adding 400 mg of DNPH to 100 ml of concentrated sulphuric acid in 10 ml dimethylformamide. Two kinds of sampling tubes were used:
C. Kempter et al. / Analytica Chimica Acta 388 (1999) 181±186
Sampling tubes A. The sampling tubes were purchased as special order (part no. 854 122) from Supelco (Deisenhofen, Germany): cartridge lengths 7.4 cm, ®lled with 350 mg silica gel (chromatographic quality); particle size 150±250 mm, 60/100 mesh. Sampling tubes B. These sampling tubes were purchased as SPE cartridges from ICT HandelsGmbH (Bad Homburg, Germany): cartridge lengths 7.4 cm, ®lled with 200 mg RP-18 silica gel (chromatographic quality); particle size 50 mm, pore width Ê. 60 A All tubes were conditioned ®rst with 570 ml of dimethylformamide. Afterwards, the silica gel was coated with DNPH by seeping 570 ml of the reagent solution through the tubes. Subsequently, the material was dried in a nitrogen stream. 2.4. Air sampling procedure and analysis Air sampling was performed using a personal air sampler pump (Buck I.H. Pump) from A.P. Buck (Orlando, FL, USA) with a corresponding calibrator, also from A.P. Buck. For all measurements, a collecting tube and a controlling tube (``back-up tube'') were connected in series to identify incomplete recovery on the collecting tube. The sampling rate was 0.2 l/min. During sampling of the industrial workplace air, the Time (min) c (CH3CN) (%) Flow rate (ml/min)
0 30 1.5
1.1 30
6.5 42
tubes were located in a typical workers position close to a reaction vessel for 10 min. The simulated air sampling with a de®ned amount of the analyte was performed by pipetting 50 ml of the solution of formaldehyde, propanal, 2-butanone oxime and cyclohexanone oxime in acetonitrile on quartz wool that was placed in front of the collecting layer. Afterwards, a constant air stream was pumped through the tube for 10 min. After sampling, the tubes were eluted with 10 ml acetonitrile. 10 ml of this solution were injected into the LC system.
183
Chem Station 845x ± biochemical UV/VIS-system ± was used. 2.6. UV/Vis absorption measurements All UV/Vis measurements were performed with a concentration of 2.810ÿ5 mol/l of 2-butanone DNPhydrazone and 2.710ÿ5 mol/l of cylohexanone DNPhydrazone in acetonitrile. The spectra were recorded in the range from 200 to 560 nm. 2.7. LC instumentation and analysis A liquid chromatograph consisting of the following components was used: two LC-10AS pumps (Shimadzu, Duisburg, Germany), SPD-10AV variable wavelength detector (Shimadzu), SIL-10A autosampler (Shimadzu), Class LC-10 Version 1.4 software (Shimadzu), and CBM-10A controller unit (Shimadzu). The injection volume was 10 ml. The column material was Deltabond AK (Keystone Scienti®c, Bellefonte, PA, USA): particle size, 5 mm; pore size, Ê ; column dimensions, 150 mm4.6 mm. 300 A For separation, a binary gradient consisting of acetonitrile and water was chosen with the following pro®le: 9.5 100
10.0 100
12.0 30
13.0 (stop) 30
3. Results and discussion Oximes react with DNPH in analogy to aldehydes and ketones forming the corresponding hydrazones as stated below. Here, MEKO is selected as example for the reaction:
2.5. Photometer The HP 8453 diode array spectrophotometer (Hewlett-Packard, Waldbronn, Germany) with software HP
Identity of the reaction products with the hydrazones formed from aldehydes or ketones with DNPH
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C. Kempter et al. / Analytica Chimica Acta 388 (1999) 181±186
Fig. 1. UV/Vis spectrum of 2-butanone 2,4-DNPhydrazone.
was con®rmed by synthesizing and characterizing the derivatives. Characterization was performed using 1 H and 13 C NMR, IR and UV/Vis spectroscopy, mass spectrometry, elemental analysis and melting point analysis. The UV/Vis spectra of both 2-butanone 2,4DNPhydrazone and cyclohexanone 2,4-DNPhydrazone are very similar. The spectrum of 2-butanone 2,4-DNPhydrazone is presented in Fig. 1. 365 nm was selected as suitable wavelength for LC analysis in both cases. Air sampling was performed using the active sampling technology with two test tubes in series as collecting tube and back-up tube. First attempts, however, to determine MEKO and CHO by using selfprepared test tubes according to [5] and commercially available test tubes failed due to very low recovery rates. Surprisingly, no breakthroughs were detected in these cases on the back-up tubes. As good recoveries were obtained with the respective ketones, the loss of analyte must be due to the formation of amides from the analytes. This type of reaction is known as Beckmann rearrangement and is shown above for the formation of e-caprolactame from CHO. In case of
MEKO, an aliphatic amide is formed instead of the cyclic derivative. For this reason, a sampling technology using less acid on the solid sorbents had to be developed. It is known from literature that the addition of acids greatly enhances the reaction between aldehydes or ketones and DNPH. Therefore, a compromise had to be found to achieve high reaction rates and high ¯ow rates on the one hand, and to avoid the rearrangement on the other hand. The preparation on the respective tubes is presented in Section 2. Two different types of tubes were used, both being ®lled with chemically unmodi®ed silica. To compare the recovery rates to those of compounds known to react almost quantitatively with DNPH, recovery experiments were carried out with MEKO, CHO, formaldehyde and propanal. A solution containing known amounts of all four analytes was pipetted on a quartz wool plug in front of the test tube to simulate real samples. An air stream was pumped over the quartz wool until complete dryness. A ¯ow rate of 200 ml/min was selected as the best recoveries were obtained this way. The recoveries for the four different carbonyls and oximes on the two sorbents are listed in Table 1. No breakthroughs into the back-up tubes were observed for any of the analytes. In case of MEKO, CHO and propanal, the blanks were below the detection limit. For formaldehyde, a signi®cant blank was obtained. The data for formaldehyde in Table 1 are corrected for the blank. The limits of detection for both oximes are 0.2 pmol absolute, limits of quanti®cation are 0.5 pmol absolute, and a linear range of the calibration curve is observed from 0.5 pmol to 50 nmol absolute. This correlates to oxime concentrations in a 2 l sample of 15.0 to 150.0 mg/l for MEKO and 19.5 to 195.0 mg/l for CHO. Multiple injection of a sample with a concentration of 450 ng/l MEKO in an air
Table 1 Recoveries of MEKO, CHO, formaldehyde and propanal using different sorbents Sampling tubes A
MEKO CHO Formaldehyde Propanal
Sampling tubes B
Recovery (%)
RSD (n3) (%)
Recovery (%)
RSD (n3) (%)
95.7 92.0 95.7 95.3
1.5 4.0 0.7 2.3
92.3 92.7 92 94.3
0.9 2.8 3.9 3.6
C. Kempter et al. / Analytica Chimica Acta 388 (1999) 181±186
sample results in a RSD of 1% (n10) for the process of injection and chromatographic analysis. RSDs covering the total analytical process including sampling are listed in Table 1. It is obvious from Table 1 that the recoveries for all four analytes are signi®cantly above 90% for both types of tubes. The RSD (n3) of the measurements is in the range between 0.7% and 4.0%, thus being well acceptable for both types of tubes. For the subsequent measurements, the tubes A have been selected as the average recovery was slightly higher. The collecting tubes and back-up tubes were eluted each with 10 ml acetonitrile. The eluate was directly injected into the LC system. A binary gradient of acetonitrile/water was applied on a reversed phase C18 column to obtain baseline separation of the hydrazones from each other and from the reagent. As detection wavelength, 365 nm was selected. A chromatogram of a slightly acidi®ed DNPH solution and the hydrazones of formaldehyde, propanal, 2-butanone and cyclohexanone is presented in Fig. 2. The hydrazones are well separated. The double peak of the 2-butanone 2,4-DNPhydrazone results from the formation of the E and the Z isomers, respectively [16]. An acetone blank is visible. This is almost inevitable when performing sample preparation in a laboratory in which acetone is used for cleaning glassware. In Fig. 3, the chromatogram obtained from a sampling experiment with cyclohexanone oxime is
Fig. 2. Chromatogram of a mixture of DNPH ((A) protonated reagent, (B) free reagent), and the 2,4-dinitrophenylhydrazones of formaldehyde (C), propanal (E), 2-butanone (double peak F), and cyclohexanone (G). Peak D is the blank of acetone 2,4DNPhydrazone.
185
Fig. 3. Chromatogram from a sampling experiment with CHO. Peak A is DNPH, B the acetone 2,4-DNPhydrazone blank, and C the cyclohexanone 2,4-DNPhydrazone peak.
depicted. The absolute amount of CHO sampled here is 16 mg, thus corresponding for example to a concentration of 6.2 mg/l in a 2 l air sample. MEKO real samples in the workplace air of a polymer production plant have been investigated. In Fig. 4, the respective chromatogram is shown. Again, the characteristic double peak is observed for the reaction product of MEKO, and there is a signi®cant blank for acetone. It should be noted that the formation of the same product from the ketones and the oximes will lead to a sum parameter in case both substances are present in a sample. However, due to the advantageous properties
Fig. 4. Chromatogram of a real sample from an industrial workplace. Peak A is DNPH, B is the acetone 2,4-DNPhydrazone blank, and C the double peak of the 2-butanone 2,4-DNPhydrazone isomers.
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C. Kempter et al. / Analytica Chimica Acta 388 (1999) 181±186
of MEKO compared to 2-butanone, producers try to use MEKO with almost no 2-butanone content. Therefore, the presence of 2-butanone in this type of real samples is not likely, and interferences from the ketone should be low. Acknowledgements Financial support of parts of this work by the European Community (``Aldehydes'' project, SMT4-CT97-2190) and the Fonds der Chemischen Industrie is gratefully acknowledged. C.K. thanks the Deutsche Bundesstiftung Umwelt (OsnabruÈck, Germany) for a scholarship. References [1] Deutsche Forschungsgemeinschaft, MAK-und BAT-WerteListe 1997, Wiley±VCH, Weinheim, 1997.
[2] F. Lipari, S.J. Swarin, J. Chromatogr. 247 (1982) 297. [3] D. Grosjean, K. Fung, Anal. Chem. 54 (1982) 1221. [4] R.H. Beasley, C.E. Hoffmann, M.L. Rueppel, J.W. Worley, Anal. Chem. 52 (1980) 1110. [5] W. PoÈtter, U. Karst, Anal. Chem. 68 (1996) 3354. [6] E. Goelen, M. Lambrechts, F. Geyskens, Analyst 122 (1997) 411. [7] J.O. Levin, K. Andersson, R. Lindahl, C.-A. Nilsson, Anal. Chem. 57 (1985) 1032. [8] J.O. Levin, R. Lindahl, Analyst 119 (1994) 79. [9] K.L. Olson, S.J. Swarin, J. Chromatogr. 333 (1985) 337. [10] S. KoÈlliker, M. Oehme, C. Dye, Anal. Chem. 70 (1998) 1979. [11] D.R. Rodier, L. Nondek, J.W. Birks, Environ. Sci. Technol. 27 (1993) 2814. [12] A. BuÈldt, U. Karst, Anal. Chem. 69 (1997) 3617. [13] C.F.H. Allen, J. Am. Chem. Soc. 52 (1930) 2955. [14] O.L. Brady, J. Chem. Soc. (1931) 756. [15] M. Behforouz, J.L. Bolan, M.S. Flynt, J. Org. Chem. 50 (1985) 1186. [16] N. Binding, W. MuÈller, U. Witting, Fresenius' J. Anal. Chem. 356 (1996) 315.
Workplace monitoring of oximes using 2,4-dinitrophenylhydrazinecoated silica gel cartridges and liquid chromatography Christine Kempter, Andrea BuÈldt, Uwe Karst* WestfaÈlische Wilhelms-UniversitaÈt MuÈnster, Anorganisch-Chemisches Institut, Abteilung Analytische Chemie, Wilhelm-Klemm-Str. 8, MuÈnster D-48149, Germany Received 22 September 1998; received in revised form 20 January 1999; accepted 25 January 1999
Abstract A liquid chromatographic (LC) method for the determination of oximes in air samples using pre-column derivatization with a hydrazine reagent has been developed. Air samples are drawn over a 2,4-dinitrophenylhydrazine (DNPH)-coated silica gel cartridge to form the corresponding hydrazones. The acid coating of the tubes and the ¯ow rate of the air sample have been selected considering the chemical characteristics of the oximes to avoid Beckmann rearrangement or incomplete recovery. The hydrazones are separated by LC with UV/Vis detection at 365 nm. Calibration is performed externally using the stable hydrazone standards. The limit of quanti®cation is 0.5 pmol absolute for 2-butanone oxime (corresponding to 15.0 ng/l in a 2 l air sample) and cyclohexanone oxime (corresponding to 19.5 ng/l in a 2 l air sample), respectively. Real samples from industrial workplaces have successfully been analysed using this method. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Liquid chromatography (LC); Oximes; 2,4-Dinitrophenylhydrazin (DNPH); Air samples; Workplace monitoring
1. Introduction Oximes are structural analogues of carbonyl compounds. They are formed in a reaction of aldehydes and ketones with hydroxylamine according to the following equation:
2-Butanone oxime (methyl ethyl ketoxime, MEKO) is *Corresponding author. Tel.: +49-251-8333-182; fax: +49-2518333-169; e-mail: [email protected]
an important solvent and additive in the polymer and coatings industries. MEKO is primarily used as an antiskinning agent in alkyd paints. It is also used as blocking agent for urethane polymers, and as corrosion inhibitor in boilers. It is released in small amounts from sealants containing MEKO-based oxime silanes during curing. MEKO is listed in chapter III A 2 of the German MAK list [1] as a substance which has been identi®ed as possible carcinogen in animal tests. The conditions of these tests are considered to be comparable to the exposition at workplaces. No of®cial threshold value is therefore listed. However, several industrial enterprises use the MEKO concentration of 9.0 mg/l in air as internal threshold value. Cyclohexanone oxime (CHO) is used in large amounts as intermediate in the formation of poly-
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(99)00128-2
182
C. Kempter et al. / Analytica Chimica Acta 388 (1999) 181±186
amides. In the presence of mineral acids, a rearrangement of the oxime to e-caprolactame is observed (Beckmann rearrangement). The lactame is then polymerized to polyamides:
performed externally using the stable solid hydrazones which may easily be synthesized according to literature methods [13±15]. Due to the analogy between the oxime function and the carbonyl function in aldehydes and ketones, the goal of the present work was to investigate the suitability to determine oximes as well using the DNPH method. 2. Experimental 2.1. Chemicals
As the oximes are volatile, a signi®cant concentration in the gas phase at industrial workplaces may be expected. Therefore, the concentration of the oximes in workplace air should be determined. Only few methods have been described for this purpose. The only method of widespread acceptance in industry is the gas chromatographic analysis with ¯ame ionization detection after trapping the analytes on solid sorbents. However, as oximes are reducing agents, their subsequent decomposition on the sorbent could be suspected at least in the presence of strong oxidants. Hydrazine reagents have been used for a long time for the determination of aldehydes and ketones in liquid and air samples [2±12]. Among these, 2,4dinitrophenylhydrazine (DNPH) has established as the most popular reagent. DNPH reacts with aldehydes and ketones in acidic media under formation of the respective hydrazones:
These are separated by liquid chromatography (LC) and detected spectroscopically by UV/Vis in most cases [2±8]. Mass spectrometric detection has also been performed in recent years [9,10]. Different air sampling technologies have been used in combination with the DNPH method, including impingers [2,3], test tubes ®lled with reagent-coated sorbents [4±6], and passive sampling devices [7,8]. Calibration is
All chemicals were purchased from Aldrich Chemie (Steinheim, Germany) in the highest quality available. Sulphuric acid was Merck (Darmstadt, Germany) analytical grade. Acetonitrile for HPLC was Merck gradient grade. Quartz wool was obtained from Fleischhacker (Schwerte, Germany). 2.2. Synthesis DNPhydrazones. The derivatives were prepared according to a procedure published by Allen [13] and Brady [14] for the synthesis of DNPH derivatives: 100 mg 2,4-dinitrophenylhydrazine (5.110ÿ4 mol) were dissolved in 0.5 ml sulphuric acid and a mixture of 0.7 ml water and 2.5 ml ethanol. A 50% molar excess of the aldehyde, ketone or oxime, respectively (7.710ÿ4 mol) was added as 10±20% solution in ethanol. The hydrazones precipitated as reddish crystalline material. The precipitate was ®ltered off and washed ®rst with a 5 mass% aqueous solution of sodium bicarbonate until no further development of carbon dioxide was observed according to Behforouz et al. [15]. Excess bicarbonate was removed by an additional washing step with distilled water. If necessary, the products were recrystallized from ethanol. The derivatives were fully characterized by means of NMR, IR and UV/Vis spectroscopy, elementary analysis and melting point. 2.3. Preparation of the DNPH sampling tubes A 0.2 mol/l DNPH solution was prepared by adding 400 mg of DNPH to 100 ml of concentrated sulphuric acid in 10 ml dimethylformamide. Two kinds of sampling tubes were used:
C. Kempter et al. / Analytica Chimica Acta 388 (1999) 181±186
Sampling tubes A. The sampling tubes were purchased as special order (part no. 854 122) from Supelco (Deisenhofen, Germany): cartridge lengths 7.4 cm, ®lled with 350 mg silica gel (chromatographic quality); particle size 150±250 mm, 60/100 mesh. Sampling tubes B. These sampling tubes were purchased as SPE cartridges from ICT HandelsGmbH (Bad Homburg, Germany): cartridge lengths 7.4 cm, ®lled with 200 mg RP-18 silica gel (chromatographic quality); particle size 50 mm, pore width Ê. 60 A All tubes were conditioned ®rst with 570 ml of dimethylformamide. Afterwards, the silica gel was coated with DNPH by seeping 570 ml of the reagent solution through the tubes. Subsequently, the material was dried in a nitrogen stream. 2.4. Air sampling procedure and analysis Air sampling was performed using a personal air sampler pump (Buck I.H. Pump) from A.P. Buck (Orlando, FL, USA) with a corresponding calibrator, also from A.P. Buck. For all measurements, a collecting tube and a controlling tube (``back-up tube'') were connected in series to identify incomplete recovery on the collecting tube. The sampling rate was 0.2 l/min. During sampling of the industrial workplace air, the Time (min) c (CH3CN) (%) Flow rate (ml/min)
0 30 1.5
1.1 30
6.5 42
tubes were located in a typical workers position close to a reaction vessel for 10 min. The simulated air sampling with a de®ned amount of the analyte was performed by pipetting 50 ml of the solution of formaldehyde, propanal, 2-butanone oxime and cyclohexanone oxime in acetonitrile on quartz wool that was placed in front of the collecting layer. Afterwards, a constant air stream was pumped through the tube for 10 min. After sampling, the tubes were eluted with 10 ml acetonitrile. 10 ml of this solution were injected into the LC system.
183
Chem Station 845x ± biochemical UV/VIS-system ± was used. 2.6. UV/Vis absorption measurements All UV/Vis measurements were performed with a concentration of 2.810ÿ5 mol/l of 2-butanone DNPhydrazone and 2.710ÿ5 mol/l of cylohexanone DNPhydrazone in acetonitrile. The spectra were recorded in the range from 200 to 560 nm. 2.7. LC instumentation and analysis A liquid chromatograph consisting of the following components was used: two LC-10AS pumps (Shimadzu, Duisburg, Germany), SPD-10AV variable wavelength detector (Shimadzu), SIL-10A autosampler (Shimadzu), Class LC-10 Version 1.4 software (Shimadzu), and CBM-10A controller unit (Shimadzu). The injection volume was 10 ml. The column material was Deltabond AK (Keystone Scienti®c, Bellefonte, PA, USA): particle size, 5 mm; pore size, Ê ; column dimensions, 150 mm4.6 mm. 300 A For separation, a binary gradient consisting of acetonitrile and water was chosen with the following pro®le: 9.5 100
10.0 100
12.0 30
13.0 (stop) 30
3. Results and discussion Oximes react with DNPH in analogy to aldehydes and ketones forming the corresponding hydrazones as stated below. Here, MEKO is selected as example for the reaction:
2.5. Photometer The HP 8453 diode array spectrophotometer (Hewlett-Packard, Waldbronn, Germany) with software HP
Identity of the reaction products with the hydrazones formed from aldehydes or ketones with DNPH
184
C. Kempter et al. / Analytica Chimica Acta 388 (1999) 181±186
Fig. 1. UV/Vis spectrum of 2-butanone 2,4-DNPhydrazone.
was con®rmed by synthesizing and characterizing the derivatives. Characterization was performed using 1 H and 13 C NMR, IR and UV/Vis spectroscopy, mass spectrometry, elemental analysis and melting point analysis. The UV/Vis spectra of both 2-butanone 2,4DNPhydrazone and cyclohexanone 2,4-DNPhydrazone are very similar. The spectrum of 2-butanone 2,4-DNPhydrazone is presented in Fig. 1. 365 nm was selected as suitable wavelength for LC analysis in both cases. Air sampling was performed using the active sampling technology with two test tubes in series as collecting tube and back-up tube. First attempts, however, to determine MEKO and CHO by using selfprepared test tubes according to [5] and commercially available test tubes failed due to very low recovery rates. Surprisingly, no breakthroughs were detected in these cases on the back-up tubes. As good recoveries were obtained with the respective ketones, the loss of analyte must be due to the formation of amides from the analytes. This type of reaction is known as Beckmann rearrangement and is shown above for the formation of e-caprolactame from CHO. In case of
MEKO, an aliphatic amide is formed instead of the cyclic derivative. For this reason, a sampling technology using less acid on the solid sorbents had to be developed. It is known from literature that the addition of acids greatly enhances the reaction between aldehydes or ketones and DNPH. Therefore, a compromise had to be found to achieve high reaction rates and high ¯ow rates on the one hand, and to avoid the rearrangement on the other hand. The preparation on the respective tubes is presented in Section 2. Two different types of tubes were used, both being ®lled with chemically unmodi®ed silica. To compare the recovery rates to those of compounds known to react almost quantitatively with DNPH, recovery experiments were carried out with MEKO, CHO, formaldehyde and propanal. A solution containing known amounts of all four analytes was pipetted on a quartz wool plug in front of the test tube to simulate real samples. An air stream was pumped over the quartz wool until complete dryness. A ¯ow rate of 200 ml/min was selected as the best recoveries were obtained this way. The recoveries for the four different carbonyls and oximes on the two sorbents are listed in Table 1. No breakthroughs into the back-up tubes were observed for any of the analytes. In case of MEKO, CHO and propanal, the blanks were below the detection limit. For formaldehyde, a signi®cant blank was obtained. The data for formaldehyde in Table 1 are corrected for the blank. The limits of detection for both oximes are 0.2 pmol absolute, limits of quanti®cation are 0.5 pmol absolute, and a linear range of the calibration curve is observed from 0.5 pmol to 50 nmol absolute. This correlates to oxime concentrations in a 2 l sample of 15.0 to 150.0 mg/l for MEKO and 19.5 to 195.0 mg/l for CHO. Multiple injection of a sample with a concentration of 450 ng/l MEKO in an air
Table 1 Recoveries of MEKO, CHO, formaldehyde and propanal using different sorbents Sampling tubes A
MEKO CHO Formaldehyde Propanal
Sampling tubes B
Recovery (%)
RSD (n3) (%)
Recovery (%)
RSD (n3) (%)
95.7 92.0 95.7 95.3
1.5 4.0 0.7 2.3
92.3 92.7 92 94.3
0.9 2.8 3.9 3.6
C. Kempter et al. / Analytica Chimica Acta 388 (1999) 181±186
sample results in a RSD of 1% (n10) for the process of injection and chromatographic analysis. RSDs covering the total analytical process including sampling are listed in Table 1. It is obvious from Table 1 that the recoveries for all four analytes are signi®cantly above 90% for both types of tubes. The RSD (n3) of the measurements is in the range between 0.7% and 4.0%, thus being well acceptable for both types of tubes. For the subsequent measurements, the tubes A have been selected as the average recovery was slightly higher. The collecting tubes and back-up tubes were eluted each with 10 ml acetonitrile. The eluate was directly injected into the LC system. A binary gradient of acetonitrile/water was applied on a reversed phase C18 column to obtain baseline separation of the hydrazones from each other and from the reagent. As detection wavelength, 365 nm was selected. A chromatogram of a slightly acidi®ed DNPH solution and the hydrazones of formaldehyde, propanal, 2-butanone and cyclohexanone is presented in Fig. 2. The hydrazones are well separated. The double peak of the 2-butanone 2,4-DNPhydrazone results from the formation of the E and the Z isomers, respectively [16]. An acetone blank is visible. This is almost inevitable when performing sample preparation in a laboratory in which acetone is used for cleaning glassware. In Fig. 3, the chromatogram obtained from a sampling experiment with cyclohexanone oxime is
Fig. 2. Chromatogram of a mixture of DNPH ((A) protonated reagent, (B) free reagent), and the 2,4-dinitrophenylhydrazones of formaldehyde (C), propanal (E), 2-butanone (double peak F), and cyclohexanone (G). Peak D is the blank of acetone 2,4DNPhydrazone.
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Fig. 3. Chromatogram from a sampling experiment with CHO. Peak A is DNPH, B the acetone 2,4-DNPhydrazone blank, and C the cyclohexanone 2,4-DNPhydrazone peak.
depicted. The absolute amount of CHO sampled here is 16 mg, thus corresponding for example to a concentration of 6.2 mg/l in a 2 l air sample. MEKO real samples in the workplace air of a polymer production plant have been investigated. In Fig. 4, the respective chromatogram is shown. Again, the characteristic double peak is observed for the reaction product of MEKO, and there is a signi®cant blank for acetone. It should be noted that the formation of the same product from the ketones and the oximes will lead to a sum parameter in case both substances are present in a sample. However, due to the advantageous properties
Fig. 4. Chromatogram of a real sample from an industrial workplace. Peak A is DNPH, B is the acetone 2,4-DNPhydrazone blank, and C the double peak of the 2-butanone 2,4-DNPhydrazone isomers.
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of MEKO compared to 2-butanone, producers try to use MEKO with almost no 2-butanone content. Therefore, the presence of 2-butanone in this type of real samples is not likely, and interferences from the ketone should be low. Acknowledgements Financial support of parts of this work by the European Community (``Aldehydes'' project, SMT4-CT97-2190) and the Fonds der Chemischen Industrie is gratefully acknowledged. C.K. thanks the Deutsche Bundesstiftung Umwelt (OsnabruÈck, Germany) for a scholarship. References [1] Deutsche Forschungsgemeinschaft, MAK-und BAT-WerteListe 1997, Wiley±VCH, Weinheim, 1997.
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