Anal. Chem. 1990, 62, 1519-1522
different. (dO/df)f, is greater for A1 than for Au electrodes a t each frequency; the opposite is true for f, - f,.
CONCLUSIONS The network analyzer method completely characterizes a quartz crystal sensor for all conditions of the crystal. This method replaces the oscillator method, which is unsatisfactory for two reasons. First, only one quantity that characterizes the crystal is measured, and so the electrical characterization is incomplete. More importantly, however, the oscillator method does not function in all circumstances. There is no oscillation when the maximum phase of the quartz crystal is less than zero. This is the case, for example, when viscosity of the liquid in contact with the crystal exceeds a certain value. Modified Sauerbrey expressions fail to explain the experimental measurements of series resonant frequency, f,, as a function of viscosity. The basic reason is that these equations do not take into account the dissipation of electrical energy in the quartz crystal. In the liquid phase, the energy dissipation is due principally to the flow of acoustic waves into the liquid, which depends on the properties of the liquidcrystal interface, as well as the bulk properties of the liquid. Registry No. AI, 7429-90-5; Au, 7440-57-5;quartz, 1480860-7.
LITERATURE CITED (1) Sauerbrey, G. 2.Phys. 1959, 155, 206-212. (2) Sauerbrey, G. Z. Phys. 1984, 778, 457-471. (3) McCallum, J. J. Analyst 1989, 7 74, 1173-1 189.
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(4) Fox, C. G.;Alder, J. F. Analyst 1989, 774, 997-1003. (5) Gullbault. G. G. I n Lu, C.. Czanderna, A. W., Eds. Applications offiezoelechic Quartz Crystal Microbalances: Vol. 7 of Methods and phenomena, Their Applications to Science and Technology, Elsevier: New York, 1984; p 251. (6) Thompson, M.; Dhaliwal, G. K.: Arthur, C. L.; Calabrese, G. S. I€€€ Trans. Ultrason. Ferroeiec. Freq. Con*. 1987, UFFC-34, 127-135. (7) Muramatsu, H.; Dicks, J. M.: Tamiya, E.; Karube, I.Anal. Chem. 1987, 59, 2760-2763. (8) Ebersole, R. C.; Ward, M. D. J. Am. Chem. Soc. 1988, 770,
8623-8628. (9) Davis, K.A.; Leary, T. R. Anal. Chem. 1989, 67, 1227-1230. (10) Nomura, T.; Nagamune, T. Anal. Chim. Acta 1983, 755, 231-234.
(11) Nomura, T.; Watanabe, M.; West, T. S. Anal. Chim. Acta 1985, 175,
107-1 16. (12) Bruckenstein, S.: Shay. M. Elecfrochim. Acta 1985, 30,1295-1300. (13) Nomura, T.; Okuhara, M. Anal. Chim. Acta 1982, 142, 281-284. (14) Yao, S.-2.: Mo, 2.-H. Anal. Chim. Acta 1987, 793, 97-105. (15) Thompson, M.; Arthur, C. L.; Dhaliwal, G. K. Anal. Chem. 1988. 58, 1206- 1209. (16) Muramatsu, H.; Tamiya, E.; Karube, I.Anal. Chem. 1988, 6 0 , 2 142-2 146. (17) Muramatsu, H.; Tamlya. E.: Suzuki, M.; Karube, I. Anal. Chim. Acta
1988. 275, 91-98. (18) Weast, R. C., Ed. CRC Handbook of Chemistry and Physics, 61th ed.; CRC Press: B o a Raton, FL, 1980-81. (19) Cady. W. G. Piezoeiectrlcity; Dover: New York, 1964. (20) Bottom, V. E. Infroduction to Quartz Crystal Unir &sign; Van Nostrand Reinhold: New York, 1962. (21) Kanazawa, K. K.; Gordon 11, J. G. Anal. Chlm. Acta 1985, 775, 99-105.
RECEIVED for review January 25,1990. Accepted March 29, 1990. Support for this work from the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged.
High-Performance Liquid Chromatography/Chemical Ionization Mass Spectrometric Analysis of Pyrolysates of Amylose and Cellulose Peter W. Arisz,* James A. Lomax, and Jaap J. Boon Unit for Mass Spectrometry of Macromolecular Systems, FOM-Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands I n-source pyrolysis chemlcal lonlzatlon mass spectrometry (PyCIMS) of polysaccharldes shows a number of Ion series that correspond to anhydroollgosaccharldes and ollgosaccharides wkh attached sugar rlng-cleavage fragments. To conflrm that these observed Ion serles are produced upon pyrolysis and not by cluster formatlon or fragmentation In the Ion source, off-line Curle polnt pyrolysates of amylose and cellulose were prepared, and thelr condensates were per-0benzoylated. The desorptlon chemkal lonlzatlon (DCI) mass spectra of the pyrolysates show lon serles that correspond to those In the PyCI mass spectra, taklng Into account mass changes upon derlvatlzatlon. Chromatographlc separatlon (high-performance llquld chromatography (HPLC)) of these derlvatlzed pyrolysates lndlcates a range of lsomerlc products correspondlng to each of the major Ions found In the DCI mass spectra. The fact that the retentlon tlmes of the main peaks In the dlmer and the trlmer region of the amylose and cellulose pyrolysates In the chromatograms are qulte dmerent conflrms that the conformation of the glycosldlc bonds Is preserved upon pyrolysis. There are strong indications that pyrolysates of polysaccharides contain anhydrooligosaccharides. The presence of anhydrocellobiose (cellobiosan) in pyrolysates of cellulose
has been reported by Radlein et al. (1): between 6% and 15% cellobiosan (dimer) was found in the syrup, and the presence of higher oligosaccharides was indicated. Studies on larger polysaccharide fragments have been carried out by using in-source pyrolysis mass spectrometry (PyMS), usually with positive ion chemical ionization with ammonia (2-5). In-source PyMS is a rapid method, and almost no sample pretreatment is required. The most characteristic ion series found correspond to anhydrooligosaccharides, but other series, corresponding to (anhydro)oligosaccharides with attached ring-cleavage fragments, are also present ( 2 , 4 ) . The spectra contain information a t the submonomer, monomer, and oligomer levels and are of analytical interest because of the structural insights they could provide. However, it is still possible that the observed ion series are recombination products of the anhydro monomer, which is the most abundant pyrolysis product (6). Such synthetic oligomers would contain a mixture of linkages (7),but these would not be distinguished by conventional MS, which is not sensitive to isomeric differences. To obtain information about the nature of the monomers and their mode of linkage, chromatographic methods such as gas chromatography (GC), supercritical fluid chromatography (SFC),or high-performance liquid chromatography (HPLC) must be used in combination with MS.
0003-2700/90/0362-1519$02.50/0 0 1990 American Chemical Society
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Pyrolysis can be coupled with M S in a number of ways including in-source pyrolysis using a desorption chemical ionization (DCI) probe (PyCIMS) and on-line and off-line pyrolysis utilizing an appropriate chromatographic separation (GC, SFC, or HPLC). In the latter case, there is the opportunity to modify chromatographic properties by derivatization of the pyrolysate. On-line pyrolysis GC/MS is a widely used technique for investigations on pyrolysates of polysaccharides (6,8-10), but only volatile pyrolysis products can be determined with this technique and oligosaccharides or anhydrosaccharides with attached ring-cleavage fragments have not been seen. Off-line pyrolysis derivatization GC/MS has been used to examine pyrolysis products as their tetramethylsilane (TMS) derivatives (I). However, as with on-line methods, GC is limited in its ability t o handle oligosaccharides. Direct liquid chromatographic separation of pyrolysis products is problematic, since pyrolysates contain a wide range of compounds of different polarities and molecular weights. Another problem is the absence of sensitive methods for the detection of underivatized anhydrooligosaccharides. Introduction of chromophores by perbenzoylation of the hydroxy groups ( 1 1 , 12) facilitates highly sensitive UV detection and allows some selective cleanup of the oligomeric fraction. The reduced polarity of the derivatized sample also leads to improved chromatographic resolution. This paper deals with the generation of off-line Curie point pyrolysates, the perbenzoylation of the condensates, their HPLC separation, and M S of the products larger than 1,6anhydro-6-D-glucopyranose (levoglucosan). The procedures are demonstrated by the analysis of cellulose and amylose pyrolysates. EXPERIMENTAL SECTION Materials. Solid-phase extraction columns (CI8coating, 1-mL capacity) and acetonitrile (HPLC grade) were obtained from J. T. Baker Chemical Co. Amylose (potato, MW > 150000),benzoic anhydride, 4-(dimethylamino)pyridine,and pyridine were purchased from Janssen Chimica. Cellulose (microcrystalline Avicel, MW 32 400) and glucose were obtained from Merck, and levoglucosan was obtained from Carl Roth KG. Curie Point Pyrolysis. Cellulose or amylose (5 pL of a 1% (w/v) aqueous suspension) was placed on a wire with a Curie point of 510 "C. The wire was placed in a glass liner and dried under rotation in vacuo. After drying, the glass liner was flushed for 1 min with argon, directly followed by pyrolysis a t atmospheric pressure. Pyrolysis was achieved by inductive heating of the wire in an electromagnetic field generated by a Fischer Curiepunkt Pyrolyse high-frequency generator (1.1MHz) for 4 s. The heating rate of the wire in a gaseous atmosphere was estimated to be about 200 OC/s. The condensate that formed on the walls of the glass liner was collected. Per-0-benzoylation. For benzoylation, a reagent was prepared containg 10% (w/v) benzoic anhydride and 5% (w/v) 4-(dimethy1amino)pyridinein dry pyridine. The condensates on the walls of the glass liners were washed out with 100 pL of reagent, maintaining a contact time of 5 min for dissolution. The condensates of five pyrolysates were pooled, another 500 pL of reagent was added, and the mixture was allowed to stand overnight at 37 "C. Following the benzoylation, the sample was diluted with 9 volumes of water and applied to a CI8 solid-phase extraction column. The column was washed with 3 mL of 10% (v/v) aqueous pyridine, followed by 3 mL of water. After drying of the column, the benzoylated pyrolysates were eluted with 1 mL of acetonitrile. Reversed-Phase Chromatography. The HPLC equipment consisted of a CM 4000 gradient pump, an AlOOO auto injector (Rheodyne Model 7126 fitted with a 20-pL loop), and a SM 4000 variable-wavelength detector from LDC/Milton Roy. A LiChroCART system with a 250 X 4 mm RP-18 column (5-rm spherical particles) from Merck was used. The detector was connected to a Nelson Analytical 760 Series interface, and the data were processed on an Olivetti M28 personal computer.
-
-.
A
a
-
B A
,tC
,am
40Z
W
4%
ecc
Figure 1. PyCI mass spectra of amylose (A) and cellulose (B).
For the collection of HPLC fractions, the perbenzoylated pyrolysates were evaporated under nitrogen and redissolved in 100 pL of acetonitrile. The column was eluted a t 0.5 mL/min with water-acetonitrile (1:3) for 12 min and then with a linear gradient to 100% acetonitrile over 40 rnin and a further 15 rnin a t the final composition. Fractions were collected manually. The benzoylated compounds were detected at their maximum absorbance (230 nm). Mass Spectrometry. The mass spectrometer used was a JEOL DX-303 double-focusing mass spectrometer equipped with a JEOL DCI unit and a JEOL DA-5000 data system. The samples were deposited on a platinum wire (0.1-mm thickness) that was resistively heated at a rate of 2.0 A/min to a maximum current of 0.8 A. The heating rate was estimated at 32 OC/s, and the final temperature was about 790 "C. The evaporated or pyrolyzed sample was ionized under ammonia chemical ionization conditions at an ammonia pressure of 20 Pa (7.10-4Pa above the diffusion pump). The ion source was kept at a temperature of 180 OC. The accelerating voltage was 2.2 kV, the scan time was 0.8 s from m/z 50 to 2000, the postaccelerating voltage was -10 kV, and the resolution was set at 2000. .4n amount of 2 pL of the perbenzoylated pyrolysates was deposited on the DCI probe. The HPLC fractions were evaporated to dryness under nitrogen, redissolved in 10 pL of acetonitrile, and deposited on the probe.
RESULTS A N D DISCUSSION Comparison of the In-Source P y C I Mass Spectra of Amylose and Cellulose and the DCI M a s s Spectra of Their Perbenzoylated Pyrolysates. Figure 1 shows the PyCI mass spectra from amylose and cellulose. The general composition of the ion series can be described as [(162), X + NH4]+. T h e main series is given for X = 0, which represents anhydro(oligo)saccharides, series A according to the Coates-Wilkins notation (13, 14), which will be used in this paper. T h e ring-cleavage product series, for which X = 60 (F) and X = 42 (D), are also prominent features. The amylose and cellulose spectra are very similar. They cannot easily be distinguished from each other because their pyrolysates consist of closely related series of isomers that the mass spectrometer cannot differentiate. Figure 2 shows the DCI mass spectra of perbenzoylated Curie point pyrolysis condensates of amylose and cellulose. Taking the mass shifts due to derivatization into account, the pyrolysates show a great similarity to the original PyCI mass spectra. Table I lists the main mass peaks in the spectra of the derivatized pyrolysates together with the mass peaks of the probable underivatized parent ions and the number of derivative groups added. The main series of anhydro-
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
Table 11. Retention Times of Perbenzoylated Amylose and Cellulose Curie Point Pyrolysis Products
R I t
9 b d
, , 600
R
I t
,
600
.
I
a00
1000
1200
11 1‘ 6r
80
60
P
1521
b
la00
1600
niz
B ‘
F
F
1130
579
d
I 400
600
800
!000
I
4 1200
1400
1600
n/z
Flgure 2. D C I mass spectra of the per-0-benzoylated Curie point pyrolysates of amylose (A) and cellulose (B).
Table I. Suggested Correlation of the Number of Hydroxy Groups of the Curie Point Pyrolysis Products of Amylose and Cellulose mass ( r n l z ) native benzoylated material compound (M-NH4+) (M’*NH4+) 180 198 222 240 342 384 402 504 546 564
492 718 534 656 966 1008 1130 1440 1482 1604
no. of derivatized OH groups (R) 3 5 3 4 6 6 7 9 9 10
saccharide ions, series A (mlz 180,342,504, etc.), is found in the derivatized pyrolysates as major ions at mlz 492,966, and 1440 (series A’). The derivatized tetramer (mlz 1914) is not observed in the DCI spectrum possibly due to pyrolysis of this compound during desorption from the DCI probe: fragmentation of the benzoyl derivatives was found to be a significant process above about mlz 1600. Equivalents in the spectra of the derivatized pyrolysates are also found for the other ion series in the original (in-source pyrolysis) spectra, which represent anhydrosaccharides with attached ring-cleavage fragments. The most abundant series (F) a t mlz 240,402,564, etc. (A C2H4O2), corresponds to the derivatized series mlz 656, 1130, and 1604 (F’ = A’ + C2H302R,where R = COC6H5). This indicates an original ring fragment with the formula CZH4O2 where both oxygen atoms are part of derivatizable OH groups. The original ion series (D) a t mlz 222, 384, 546, etc. (A + CzH20),is reflected by two series in the derivatized off-line pyrolysates. In one, the ring fragment contains no derivatizable group. This produces ions with one less benzoyl group compared to the A’ series, since the fragment is attached to an otherwise derivatizable hydroxy group on the oligosaccharide: D1’ = A’ - R CzH30(mlz 430,904, and 1378). The second series contains a derivatizable group in the fragment giving ions (D2’) a t m/z 534,1008, and 1482 (A’ + CzHzO).
+
+
peak
retention time, min
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
13.70 15.72 16.95 17.43 18.77 19.86 21.46 23.23 29.08 29.74 30.37 31.17 31.63 32.73 33.80 35.23 35.93 36.57 36.68 37.07 38.01 40.25 40.73 41.37 42.03 42.67 43.00 44.12 44.83 45.53 46.20 47.98 49.13 49.23 50.53 51.20 52.90
mass (rn/z)(M’.NH4+) 492 456 420 656 656 656 656
no signif mass above 400 718 718 718 718 1024 + 966 1024 966 966 966 966 966 1130 966 966 1130 1130 1130 1130 1130 1130 1130 1130 1130 1440 1440 1440 1440 1440 1604 1482
+
typea A’, levoglucosan U U F‘ F’ F’ F’ U a-glucopyranose P-glucopyranose hexose hexose U/Af U A‘, cellobiosan? A‘ A‘ A’ A‘/F‘ A‘ A‘, maltosan? F’ F’ F‘
F F’ F’ F’ F’ F’ A‘, cellotriosan? A’ A’, maltotriosan? A‘ A’ F’ D2‘
A‘ = anhydrosaccharide, F’ = ring-cleavage product corresponding to A + 60, D2’ = ring-cleavage pfoduct corresponding to derivatizable A + 42. U = unknown.
The peak m / z 718 in the pyrolysate spectra is consistent with a completely derivatized hexose. The peak mlz 579 is a known fragment produced within the ion source from oligosaccharide ions (15). Levoglucosan is also present in the M.H+ form, which gives mlz 475. No equivalent derivatized compounds are found for the ions a t mlz 270,288,306,324,348,366,426,486,648, and 810 in the PyCI mass spectra of the native amylose and cellulose. Possibly some of these ions are formed in the ion source by dehydration or by the formation of clusters. Also lower molecular weight components in the pyrolysis condensates would be inefficiently collected and, since they contain fewer derivatizable groups (6),would be likely to be lost during sample workup. HPLC Chromatograms. Figure 3 shows the separation of the derivatized pyrolysates by HPLC. The retention times and measured masses for the major peaks are listed in Table 11. In the monomer region the same peaks are found in the two chromatograms (peaks 1-10) though the amounts vary, whereas in the dimer and trimer regions large differences are found, reflecting the difference of the internal glycosidic bonds. Both chromatograms contain peaks of perbenzoylated cy- and (3-glucose (peaks 9 and 10). These could be rehydration products from levoglucosan. The largest peak (peak 1) in each chromatogram has the same retention time as levoglucosan and gives an appropriate
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
techniques such as NMR and IR spectroscopy and tandem
MS.
B
Figure 3. HPLC chromatograms of the per-O-benzoyhted Curie point pyrolysates of amylose (A) and cellulose (B). The retention times and the measured masses of the numbered peaks are listed in Table 11.
molecular ion ( m / z 492) with MS. Further large peaks are also found with mass peaks appropriate for anhydrodisaccharides (peaks 15 and 21) and anhydrotrisaccharides (peaks 31 and 33). The retention times of these major peaks are quite different in the two chromatograms, suggesting that the configuration of glycosidic linkages are preserved in the oligosaccharides although no authentic standards of cellobiosan, maltosan, etc., were available for defiitive assignment. Each chromatogram contains a number of small peaks that MS showed to be isomeric with the anhydrosaccharides. These could be produced by random resynthesis but could also be produced by dehydration in different positions and perhaps by inversion a t the nonreducing C-4 position during pyrolytic bond cleavage. The GC separation and identification of several smaller isomeric unsaturated products from cellulose (6)supports the idea that dehydration can occur in different ways. A number of peaks (peaks 4-7 and 22-30) were identified that correspond to the F' series of ions. The different isomeric peaks could again correspond to variation in the position of dehydration, as in the anhydrosaccharide peaks, but there are additional possibilities involving the structure and linkage position (C-1 or C-4) of the ring fragment. It is also possible that some of these compounds consist of the same fragment as that in the D series but are attached to a reducing saccharide rather than to an anhydrosaccharide. Such moieties have been proposed to account for ions found on laser desorption of MS of polysaccharides (14). The full identification of these peaks will require the application of additional
CONCLUSIONS Anhydrooligosaccharidesand oligosaccharideswith attached ring-cleavage fragments have been isolated from perbenzoylated condensates of Curie point pyrolysates. This confirms that the o b s e ~ e dion series in the PyCI mass spectra are genuine pyrolysis products. A separation of these derivatized pyrolysates by HPLC gives information about the presence of isomers. It is found that most mass peaks in the DCI mass spectra are generated from more than one isomeric compound, which cannot be separated by MS. This fact makes PyMS/MS studies much more problematical. The preservation of the conformation of glycosidic bonds in the oligosaccharides on pyrolysis is demonstrated by the different retention times of the compounds in the dimer and the trimer region of the amylose and cellulose pyrolysates. ACKNOWLEDGMENT We thank G. B. Eijkel for helpful comments and discussion. LITERATURE CITED Radlein, D.; Grinshpun, A.; Piskorz, J.; Scott, D. S. J . Anal. Appl. PyfOlySiS 1987, 12, 39-49. Pouwels, A. D.; Eijkel, G. B.; Arisz, P. W.; Boon, J. J. J. Anal. Appl. Pyrolysis 1989, 15, 71-84. Helleur, R. J.; Guevremont, R. J. Anal. Appl. Pyrolysis 1989, 15, 85-95. Tas, A. C.; Kerkenaar, A.; LaVos, G. F.; van der Greef, J. J . Anal. Appl. Pyrolysis 1989, 15, 55-70. Scheijen, M. A.; Boon, J. J. Rap& Commun. Mass Spectrom. 1989, 3 , 238-240. Pouwels, A. D.; Eijkel, G. B.; Boon, J. J. J . Anal. Appl. Pyrowsis 1989, 14, 237-280. Goldstein. I. J.; Hullar, T. L. A&. Carbohydr. Chem. 1966, 21, 43 1-5 12. Helleur, R . J.; Hayes, E. R.; Craigie, J. S.; McLachlan, J. L. J . Anal. Appl. PyrO!YSiS 1985, 8 , 349-357. Helleur, R. J. J. Anal. Appl. Fpo/~& 1987, 1 1 , 297-311. van der Kaaden, A.; Boon, J. J.; de Leeuw. J. W.; de Lange, F.; Wijnand Schuyl, P. J.; Schulten, H.-R.; Bahr, U. Anal. Chem. 1984, 56, 2160-2164. Daniel, P. F. Methods Enzymol. 1987, 138, 94- 116. Daniel, P. F.; De Feudis, D. F.; Lott, 1. T.; McCluer, R . H. Carbohydr. Res. 1981, 97, 161-180. Coates, M. L.; Wilkins, C. L. Anal. Chem. 1987, 59, 197-200. Lam, 2.; Comisarow, M. B.; Dutton, G. G. S. Anal. Chem. 1988, 60, 2304-2306. Thompson, R. M.; Cory, D. A. Biomed. Mass Spectrom. 1979, 6 , 117-123.
RECEIVED for review November 15,1989. Accepted April 3, 1990. This work is part of the research program of Fundamenteel Onderzoek der Materie (FOM) with financial support from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO).
