2160
Anal. Chem. 1984, 56, 2160-2165
(Figure 1). The color development rate is slow when water ratio is 30% or below, and in neat organic solvents negligible color is produced.
Registry No. Acetic anhydride, 108-24-7;succinic anhydride, 108-30-5; propionic anhydride, 123-62-6; butyric anhydride, 106-31-0;maleic anhydride, 108-31-6;phthalic anhydride, 85-44-9; benzoyl chloride, 98-88-4;acetyl chloride, 75-36-5;cinnamic anhydride, 538-56-7;camphoric anhydride, 76-32-4;4-aminophenol, 123-30-8;sulfanilamide,63-74-1; 2-iodylbenzoic acid, 64297-64-9. LITERATURE CITED
Figure 2. Photometric titration curves of mixtures of N-acyi-4aminophenois: (A) acetic (1.58 mg) and phthalic anhydride (1.48 mg); (B) succinic (3.52 mg) and maleic anhydride (1.86 mg); (C) propionic (3.25 mg) and maleic anhydride (3.43 mg); (D) acetic (2.70 mg) and camphoric anhydride (6.66 mg).
(1) Siggia, S.; Hanna, J. G. "Quantitative Organic Analysis via Functional Groups", 4th ed.; Wlley-Interscience: New York, 1979; pp 175, 223-243. (2) Johnson, J. B.; Funk, 0. L. Anal. Chem. 1955, 27, 1464. (3) Ruch, J. E. Anal. Chem. 1975, 47,2057. (4) Critchfield, F. E.; Johnson, J. B. Anal. Chem. 1958, 28,430. (5) Siggia, S.;Hanna, J. G. Anal. Chem. 1951, 23, 1717. (6) Goddu, R. F.; LeBlanc, N. F.; Wright, C. M. Anal. Chem. 1955, 27, 1251. (7) Verma, K. K.; Gupta, A. K. Anal. Chem. 1982, 5 4 , 249. (8) Banerjee, A,; Banerjee, G. C.; Bhattacharya, S.: Banerjee, S.;Samadar, H. J . Indian Chem. SOC. 1981, 58,606. (9) Berka, A.; Vulterin, J.; Zyka, J. "Newer Redox Titrants"; Pergamon: Oxford, 1965; p 37. (10) Lucchesi, C. A,; Kao, L. W.; Young, G. A,; Chang, H. M. Anal. Chem. 1974, 4 6 , 1331.
is found to be critically dependent on the proportion of ethanol, methanol, acetone, or anhydrous acetic acid used as solvent for the acylation reaction, there always being observed a hyperchromic effect with an optimum shift with acetone
RECEIVED for review February 8,1984. Accepted May 1,1984. Thanks are due to the Council of Scientific and Industrial Research, New Delhi, for a Junior Research Fellowship to P.T.
mL OF 2-IODOXYBENZOATE
Comparison of Analytical Pyrolysis Techniques in the Characterization of Chitin Arie van der Kaaden* and Jaap J. Boon F.0.M.-Institute for Atomic and Molecular Physics, Kruislaan 407, 1098SJ Amsterdam, T h e Netherlands Jan W. de Leeuw, Frits de Lange, and P. J. Wijnand Schuyl Department of Chemistry and Chemical Engineering, Delft University of Technology, Delft, T h e Netherlands Hans-R. Schulten and Ute Bahr Fachhochschule Fresenius, Dambachtal20, 6200 Wiesbaden, Federal Republic of Germany The results of various analytical pyrolysls techniques wlth on-llne and off-llne detectlon have been examlned wlth respect to characteristlclty and contents of Information, using chltln [poly-( 1+4)+3-~-2-acet amldo-2-deoxyglucopyranose] as a model polymer. Curie-polnt pyrolysis-low-energy electron impact mass spectrometry (cPy-EIMS), using normal and preheated telescopic glass tube sample holders, was applied, as well as Curle-point pyrolysis-gas chromatography/mass spectrometry, with electron Impact lonlration and chemlcal Ionization (cPy-GC/EIMS, cPy-GC/CIMS). Mlcro-oven pyrolysis-high-resolution fleld lonlzatlon mass spectrometry (Py-FIMS) was used. I n addltlon, milligram-scale pyrolysls combined wlth off-llne gas chromatographlc and gas chromatographlc/mass spectrometric detectlon was carried out. Chemlcal characterlzatlon of the pyrolysls products Indicates various anhydro-2-aceiamldo-2-deoxyglucosesubstances as the most representative for the chltln structural constltuents. I n this respect Py-FIMS and cPy-GCMS yleld patterns whlch characterize best the chltln structure.
A number of analytical pyrolysis techniques are presently
available (1). Preference largely depends on practical considerations: availability of instrumentation, information contents, time of analysis, sample capacity, possibilities for automatic sample handling, and data processing. The techniques differ especially with respect to the pyrolysis conditions applied and the detection of the volatile pyrolyzate. The pyrolysis conditions vary from microgram to milligram sample quantities, vacuum to inert atmospheric reaction conditions, and heating rates varying from OC/ms to OC/s. On-line and off-line analysis of the pyrolyzate is possible. Mass spectrometry is the most commonly used detection system, but is performed in several modes, viz. electron impact ionization (EI), chemical ionization (CI), field ionization (FI),and field desorption (FD). Gas chromatographic separation of the pyrolyzate, previous to mass spectrometric analysis, is also often applied. The recent developments in pyrolysis-mass spectrometry have been reviewed (2). The application of Curie-point pyrolysis-gas chromatography/mass spectrometry has been described in a number of reports (3-5). The aim of analytical pyrolysis methods is to obtain fingerprints of the original material, which can be compared with patterns of reference substances or model compounds. The
0 1984 American Chemical Society 0003-2700/84/0356-2160$01.50/0
ANALYTICAL CHEMISTRY, VOL. 56, NO. 12, OCTOBER 1984
analytical value of such fingerprints is enlarged if the identification of specific pyrolysis products, as represented by their molecular ions or electron impact induced fragmentation pattern, leads to structural elucidation of the material analyzed or to quantitative detection of specific sample components. The major objective of this study is to compare the various analytical pyrolysis techniques with respect to their ability to yield characteristic fingerprints and the structural information content of these patterns. This comparison was made for one model compound: chitin. Chitin is of interest as a model polymer in analytical pyrolysis studies. The polymer is substantially composed of (1+4)-glycosidically linked /3-2-acetamido-2-deoxyglucopyranose (GlcNAc). Its known structure, its insolubility in many solvents, and its particular resistance against chemical treatments make chitin a suitable representative for the type of materials for which analytical pyrolysis is the most appropriate analysis technique. In addition, its pyrolytic behavior differs from that of carbohydrates composed of neutral sugars, because of the presence of the N-acetyl function at C-2 of GlcNAcp. Theiefore the thermal decomposition of chitin niay sewe as an aid to trace nitrogen-containing sugar moieties in biopolymers, such as peptidoglycans and glycopeptides. The analysis of chitin as a raw or derivatized material also gains more intbrest. Novel technological applications of chitinlike materials hdve been proposed (6, 7). This means that methods have to be developed to characterize this raw material, since the properties of chitin preparations vary with source and method of isolation. Furthermore there will be the need for quality control of the modified products. In this respect analy;tical pyrolysis could become a valuable technique in the arsenal of.methods to be applied, An example in this field is the use of pyrolysis-mass spectrometry (Py-MS) to determine the degree of N-deacetylation of chitin (8). Finally the pyrolytic behavior of chitin is of importance because of its use as a tobacco extender (9) and more generally by its contribution to the aroma of roasted foods (6). In this study chitin was investigated by using Curie-point pyrolysis-mass spectrometry/EI-mode (cPy-EIMS), Curiepoint pyroljisis-gas chromatography mass spectrometry/EIand CI-modes (cPy-GC/MS), and micro-oven pyrolysis mass spectrometrykF1-mode (Py-FIMS), which all combine pyrolysis with on-line detection. Off-line pyrolysis combined with EIMS and G%MS was also performed. The fingerprints of chitin, obtAin6d by the various analytical pyrolysis techniques, and the relationship between the structure of the pyrolyzate components and the GlcNAcp units that constitute the chitin polymer will be discussed. The results of the different techniques will be evaluated with respect to their characteristicity and content of information.
EXPERIMENTAL SECTION Pyrolydis-Mass Spectrometry. The Curie-point pyrolysiselectron impact mass spectra were recorded on a home-made instrument, equipped with a quadrupole mass analyzer (Balzers QMA 15O/QMG 5111, combined with ion-counting detection (10) and on an Extranuclear 5000-1 quadrupole system. In the latter instrument the expansion chamber between the pyrolysis reactor and the ion source was removed. Telescopic glass tubes were used which were heated Sesistively to approximately 175 "C, while the ferromagnetic wire, coated with sample materid, was withdrawn from the heated zone; just before pyrolysis~was accomplished the wire was pushed into the preheated zone (11). The atlalyses were performed at the following conditions: temperature rise time, 0.1 s; equilibrium temperature, 510 "C; total heating time, 0.8 s, temperature expansion chamber, 150 "C; ionization energy, 11-14 eV; mass range, 15-180 amu; scan speed, 10 scans/s; averaged spectra, 200. Pulverized (Micro-dismembrator 2, B. Braun, FRG) chitin (Polysciences, Inc., Warrington) samples were analyzed as
2161
a suspension in a sodium phosphate buffer, pH 7 ( 3 ) . Sample quantity was 10-15 pg. ELMS of the totalpyrolyzate, obtained from the milligram-scale pyrolysis of chitin, was performed by evaporation of the sample from a ferromagnetic wire, with a Curie-point temperature of 300 "C (Fischer Labortechnik, 5309 Meckenheim bei Bonn, FRG). The FI mass spectra were produced on a double-focusing Mattauch-Herzog instrument, Varian MAT 731 (12). The sample was evaporated into the field ion source from a quartz capillary tube in the direct sample introduction system. The capillary was mounted at an angle of 90" to the FI emitter and at a distance of about 20 mm from the emitter wire, The emitters used were 10-rm-diameter tungsten wires, activated with benzonitrile (13). The average needle length was 50 pm. About 1mg of chitin was used for each analysis. The sample was heated linearly from 100 to 500 "C at a heating rate of 0.4 "C/s. Electric detection was carried out by repetitive magnetic scanning, mass range of 10-250 amu; 16 spectra were computer averaged. Photographic detection was accomplished with Ilford Q 2 plates, exposure time of 7 min. Obtained resolution was 10 000 with an average mass accuracy of approximately 3 mmu for all peaks. The photographic plate was evaluated by a Gaertner comparator. Pulverized chitin was analyzed without an additional matrix. The dependence of the total ion intensity from the absolute sample amount was investigated by weighing chitin in portions of 30-350 pg, on an electronic ultramicrobalance, Mettler UM 3, possessing a reading accuracy of 0.1 pg. For each sample 100 scans were accumulated over the mass range m / z 14-300, with a heating rate of 0.25 "C/s up to a temperature of 500 "C. Pyrolysis Gas Chromatography-Mass Spectrometry. Analyses were performed on a Varian 3700 gas chromatograph, combined with a MAT44 quadrupole mass spectrometer. The pyrolysis reactor used has been described elsewhere ( 1 4 ) . Gas chromatographic separation was accomplished on a WCOT glass column, 22 m X 0.5 mm id., coated with CP-Sil5, film thickness 1.25 pm. The oven temperature program was as follows: 0 "C (initial period 5 min); rate, 3 "C/min; final temperature, 275 "C. Helium was used as the carrier gas. Mass spectrometric analyses were performed at the following conditions: electron energy, 80-eV;ion source temperature, 200 "C; mass range, 50-400 amu, cycle time, 1.3 s. Chemical ionization was carried out with isobutane. Chitin samples were analyzed as described for Py-MS. Sample quantity was 50 pg. Gas Chromatography-Mass Spectrometry. Solvent-free injection gas chromatography was accomplished by coating methanolic samples on a ferromagnetic wire with a Curie-point temperature of 300 "C (15). The sample was evaporated by a high-frequencypulse of 10 s. Chitin pyrolyzate fractions, obtained from the milligram-scale oven pyrolysis, were analyzed in this way. Oven Pyrolysis of Chitin. Milligram-scale pyrolysis of chitin was performed in a tube furnace. About 100 mg of chitin was deposited on a glass boat (1.0 x 10 cm) which was inserted in a glass tube (2.0-cmi.d.). The position of the boat could be changed by means of a glass rod which was sealed to the outer glass tube with an O-ring. Initially the sample was withdrawn from the heated reaction zone, while helium was led through for 5 min, at a flow rate of 1L/min. Afterward the boat was moved in the preheated oven part (temperature, 450 "C). The gases were condensed on a cold finger (1.5 X 10 cm, cooled by running tap water) inserted on the other side of the reaction tube. The gases, which passed the cold finger, were condensed in a liquid nitrogen cold trap. The condensate on the cold finger was dissolved in water and evaporated to dryness, with a rotatory evaporator (bath temperature
