SPECTROSCOPIC METHODS FOR VOCs DETERMINATION

Transcription

1 SPECTROSCOPIC METHODS FOR VOCs DETERMINATION Monica Culea 1, O.Cozar 1, Cora Craciun 1, R.Fechete 2, Andreea Iordache 1, E.Culea 2 1 Faculty of Physics, Babes-Bolyai University of Cluj-Napoca, Cluj- Napoca, Romania 2 Physics Department, Technical University of Cluj-Napoca, Cluj- Napoca, Romania Abstract Qualitative and quantitative methods for measuring volatile organic compounds (VOCs) by using GC/MS. IR or UV-VIS analysis were developed. Different extraction methods have been used. In the quantitative work, validation of the method in the presence of a proper internal standard was necessary. The trace level VOCs were analysed in the selected ion monitoring (SIM) mode. The methods are simple and rapid, quantitation being precise and accurate for the studied compounds. The methods were compared with other spectroscopi techniques for rapid diagnosis. The main goal was to develop non invasive, rapid and low cost spectroscopic methods. 1. Introduction In the last years the spectroscopic methods are very much used in the diagnosis field, by using new biomarkers test. Quantitative analyses of semi-volatile or volatile organic compounds (VOCs) biomarkers from biological fluids [1-3] were analysed by gas chromatography coupled with mass spectrometry (GC-MS), IR and UV-VIS spectroscopy for the diagnosis of some diseases. The GC-MS spectroscopy is a very precise but a laborious and expensive method, while other methods as IR and UV-Vis spectroscopies are lower precise but very rapid and cheep. Due to this fact the use of IR and UV-Vis spectroscopies for the diagnosis of different diseases by body fluid and breath air becomes of great interest in the last years [4-5]. The aim of the present work was to perform a comparative study by using different spectroscopic techniques, GC-MS, IR and UV-VIS for analyzing urine samples, with no pretreatment of the samples, in order to observe the possibility to use these techniques for the rapid diagnosis of one of the important metabolic disorder diseases, the diabetes. The main goal was to develop non invasive, rapid and low cost spectroscopic methods. 2. Experimental The separation of the compounds was made on a capillary column Rtx-5MS was of 30 m length x 0.25 mm, 0.25µm film thickness, in the temperature programs: 50 o C (1 min), then 20 o C/ min to 310 o C, by using the selected ion monitoring mode (SIM). The flow rate of helium carrier gas was of 1 ml/min. A Trace DSQ ThermoFinnigan quadruple mass spectrometer in the EI mode coupled with a Trace GC was used. The qualitative analysis was carried out in the mass range a.m.u. The following conditions were followed: transfer line temperature 250 o C, injector temperature 200 o C; ion source temperature 250 o C; Splitter: 10:1. Electron energy was 70eV and emission current, 100µA. The blood samples were obtained from patients and voluntiers from the Pediatric Clinic III Cluj-Napoca. Written informed consents were obtained from each subject parent prior to this study. Corresponding author: eugen.culea@phys.utcluj.ro 1

2 The IR spectra were registered using a Jasco 6200 FT/IR equipment with Fourier transform. The accumulation of several IR spectra recorded for the same sample was used in order to improve the signal/ noise ratio. The accumulation of 64 spectra for the same sample permitted an increase by 8 times of the signal/ noise ratio. The liquid samples were measured in the range of cm -1, using a special sample holder with 2 KBr windows with a good transmission for the investigated IR spectra. The measured samples were drops extended between the 2 windows covering an area of about 8 mm in diameter. The UV-Vis spectra were registered using a CAMSPEC M501 single beam UV-VIS spectrometer. A quartz cell with a geometric way of 1 cm was used as sample holder.the amount of the measured samples was about 800 ml. The IR and UV-Vis spectra were recorded at the room temperature 3. Results and discussion GC-MS measurements. The quantitation of acetone was folloed by using the headspace extraction method and the GC-MS analysis. VOC s were injected from the headpsace of 20 ml screw cap vials, with 10 ml urine and 10 µg halothane, the internal standard, after heating at 50 o C for 30 or 45 minutes. The SIM-GC/MS mode was used for acetone determination, by measuring the molecular ions m/z 58 for the analyte and for the internal standard the molecular ion m/z 198 and the fragment ion m/z 117. The regression curves obtained in the range of 0-50 µg/ml for acetone, by using the concentration of 1, 5, 7, 10, 30, 50 µ/ml acetone, were y=0.062 x+0.02 (t= 30 min) with a coefficient of regression of and respectively y=0.063 x (t = 45min), r=0.99. Fig. 1 presents the separation chromatograms for acetone and halothane, in the Sim mode, measured by GC-MS method. C:\Xcalibur\...\CEEX\acetonahal30min70ug 05/05/ :14:26 PM RT: RelativeAbundance RelativeAbundance Time (min) NL: 1.87E5 TIC F: MS acet onahal3 0min10ug NL: 1.90E5 TIC F: MS acet onahal3 0min70ug acetonahal30min70ug # 231 RT: 2.35 AV: 1 NL: 1.28E5 T: + c SIM ms [ , , ] RelativeAbundance m/z 2

3 Fig. 1 The separation chromagrams for the standard of 10µg/ml and the standard of 70 µg/ml (the first peak, 2.03 min) and halothane (2.35 min), the internal standard. The ions selected for the intenal standard are presented in the mass spectrum. Table 1. Acetone values for patients and controls by GC-MS Patient acetone (µg /ml) control control UV-Vis spectroscopy measurements. Some standard samples were prepared by solving known amounts of acetone in distilled water in order to determine quantitatively the acetone content of urine samples. The concentrations of the standard samples ranges from 0% to 2% acetone. UV-Vis survey spectra were recorded for the standard samples in the nm wavelength range. An UV-Vis spectrum characteristic of the standard samples is presented in Fig.2. Fig. 2. The UV-Vis survey absorption spectrum characteristic of a standard sample. 3 spectral region were evidenced by the analysis of the UV-Vis spectra obtained for the standard samples: 1) the nm range that show an intense absorption band assigned to the presence of acetone, 2. the nm region where the absorption is neglectible and 3. the nm region where a single broad absorption band assigned to the water occurs. The detail spectra corresponding to the nm range were recorded for all the samples in order to permit a more precise analysis of the data. The UV-Vis measurements performed on the standard samples, namely the intensity of the absorption bands from 250 nm associated with the acetone content of the samples, permitted to obtain the regression curve which was used for quantitative determination of acetone in urine. Urine samples were prepared for measurements by solving 1% urine in distilled water. A characteristic UV-Vis detail spectrum of a urine sample is presented in Fig.3. 3

4 Fig. 3. UV-Vis detail absorption spectrum ( nm) characteristic of a urine sample. The absorption band characteristic of acetone that appears in the UV-Vis spectra of urine samples is broader that that appearing in the spectra of the standard samples and is located at higher wavelengths. This may be explained by the fact that the absorption band from 250 nm appearing in the urine sample UV-Vis spectra is due not to pure acetone but to ketonic bodies. UV-Vis absorption spectra were recorded for patients and control. The values obtained for the intensity of the absorption band from 250 nm are shown in Table 1 in order to be compaired with the values determined for the acetone content of their urine (measured by GC- MS) and the values measured for their glicemia (from blood samples). Table 2. The glycemia (determined from blood samples), the intensity of the absorption band at 250 nm (from UV-VIS spectra of urine) and the acetone content (from GC-MS measurements on urine samples) for patients and control Sample Glycemia (mg/dl) Intensity of the 250 nm absorption band (%) Acetone content (µg/ml) Patient Patient Patient Patient Control A simple examination of the data in table 2 shows that the intensity of the absorption band from 250 nm is invers proportional to the glicemia values. The good agreement between the UV-Vis spectroscopic data and the measured glicemia values suggests that the UV-Vis spectroscopic technique may be used for the rapid diagnosis of the diabetes. IR spectroscopy measurements. IR absorption spectra of acetone were recorded for the cm -1 range. Due to the volatility of the acetone the IR spectra modify their features with respect to time. Fig.4 shows the IR spectra of acetone for different intervals of time. 4

5 Fig. 4. IR absorption spectra of acetone for different time intervals (t=2, 5, 20 and 23 minutes). The marked with 1 absorption bands in Fig.4 are located around 3000 cm -1 and are assigned to the skeleton vibrations of the acetone molecule, while the important absorption bands marked with 2, 3, 4 and 5 in Fig.4, located at cm -1, are assigned to vibrations of the structural groups from the acetone molecule [5, 6]. IR spectra of human urine were recorded for a group of patients. The characteristic vibrations from ~1220 cm -1, ~1360 cm -1 and ~1423 cm -1 that appear both in the IR spectra of the acetone and in the IR spectra of the urine samples were considered as an indicator of the acetone content of the samples and were compared with the acetone content of the samples determined by GC-MS (see Table 3). Table 3. Acetone content of urine measured by GC-MS and intensity of the IR absorption bands from ~1220 cm -1, ~1360 cm -1 and ~1423 cm -1 of urine samples. Sample Acetone content (by GC-MS) (µg/ml) Intensity of the IR absorption bands from ~1220 cm -1, ~1360 cm -1 and ~1423 cm -1 a.u. Pacient 1 7 1, 1, 2 Pacient , 1, 1 Pacient , 1, 2 Pacient , 1, 1 Pacient , 1, 1 Pacient 6 1, 2, 4 Pacient , 2, 4 The analysis of the data presented in Table 3 do not permit a reasonable correlation between the GC-MS and IR data. This fact could be due to the high volatility of the acetone. Further IR measurements performed in more appropriate conditions are necessary in order to obtain the correlation of the GCMS and IR data. 4. Conclusions The comparative study by GC-MS and the IR and UV-VIS spectroscopic techniques was performed on no pre-treated urine samples in order to observe the possibility to use the 5

6 spectroscopic techniques for the rapid diagnosis of the diabetes. Ketonic bodies from urine samples were measured in order to diagnose the diabetes. The UV-Vis measurements performed on urine samples seems to offer a good hope concerning the use of this technique for the rapid diagnosis of diabetes. References [1] M. Phillips, R.N. Cataneo, T. Cheema, J. Greenberg, Clin Chim Acta. vol.344, 1-2 [2] C. Deng, N. Li, X. Wang, X. Zhang, J. Zeng, Rapid Commun Mass Spectrom. vol.19, 5 (2005) 647. (2004) 189. [3] R.J. Delfino, H Gong, WS Linn, Y Hu, ED Pellizzari, J Expo Anal Environ Epidemiol. vol.13, 5 (2003) 348. [4] G.Hosafci, O.Klein, G.Oremek, W.Mantele, Anal.&Bioanal.Chem., 387, 5 (2007) [5] O.Laakso, M.Haapala, T.Pennanen, T.Kuitunen, J.J.Himberg, J.Forensic Sci. 52, 4 (2007)