Analysis of polysaccharide pyrolysate of red algae by capillary gas chromatography-mass spectrometry

Journal of Analytical and Applied Pyrolysis, 8 (1985) 333-347

333

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

ANALYSIS OF POLYSACCHARIDE PYROLYSATE BY CAPILLARY GAS CHROMATOGRAPHY-MASS SPECTROMETRY *

OF RED ALGAE

R.J. HELLEUR and E.R. HAYES * Department of Chemistry, Acadia University, Wolfuille, NS BOP 1X0 (Canada)

W.D. JAMIESON ** and J.S. CRAIGIE Atlantic Research Laboratory, National Research Council of Canada, 1411 Oxford St., Halifax, NS B3H 321 (Canada)

SUMMARY The volatile pyrolysates of galactan sulfates have been examined by capillary gas chromatography-mass spectrometry. Electron impact and chemical ionization techniques have been employed to determine the molecular structures of major pyrolysis products. Agarose, well-characterized agars, carrageenan and model saccharides having mono-O-methyl and sulfate ester functional groups have been pyrolyzed. Pyrolytic pathways of some of the galactans are discussed. Results show that initial glycosidic bond cleavage occurs followed by formation of unique pyrolysis products, e.g., anhydrosugars, characteristic of the individual polymeric units. The pyrolytic patterns of the saccharides change markedly with increasing amounts of sulfate substituents.

INTRODUCTION

Pyrolysis-gas chromatography (Py-GC) has become a useful technique to identify and characterize synthetic polymers and biopolymers [l]. The formation of specific patterns of degradation products and their relative amounts present in the chromatogram (in this case, pyrogram) can provide structural information about the parent molecule. Often, the identification of unique peaks. in the pyrogram can lead to elucidation of the structure of the macromolecule. Examining the molecular structure of the pyrolysis products also gives clues to the nature of pyrolytic pathways. * Issued as NRCC No. 23811. ** Marine Analytical Chemistry Standards. 0165-2370/85/$03.30

0 1985 Elsevier Science Publishers B.V.

334

The pyrolysates of cellulose and related compounds have been examined extensively because of their importance in foodstuffs and textiles. The identification of the major pyrolysis products has led to a clearer understanding of the thermal degradation pattern of the glucans [l]. Other important classes of polysaccharides, e.g., galactans, mucopolysaccharides, etc., have not been as well studied by analytical pyrolysis. Recent pyrolytic studies of different classes of polysaccharides have used pyrolysis-mass spectrometry (Py-MS) to examine the relationship between structure and the pyrolysate mass spectrum [2,3]. Distinct advantages of Py-MS are rapid analysis and direct chemical information on the structure of the polymer. Structural assignments of mass peaks in Py-MS of carbohydrates have been made on the basis of identification of pyrolysis products based on GC-MS data. A recent study has used Py-GC-MS to interpret Py-mass spectra of amylose [4]. On pyrolysis of carbohydrates, structural isomers are usually formed. GC-MS analysis of carbohydrate pyrolysates can usually distinguish between the structural isomers and is capable of determining their relative distribution. Algal polysaccharides have great commercial potential. Although they have some common structural similarities with their terrestrial counterparts, many algal polysaccharides have distinctive structural features. The galactan sulfates, a class of polysaccharides of red algae, are one example. These B

A

Leoa

I

I-

OH

OR

A-corrogeenon

R’S03

k-corrogeenon

R:H

I -corrogeenon

R:SOi

or H

I

An

Fig. 1. Structure of the repeating disaccharide agarose and A-, I- and K-carrageenans.

units in the idealized

polymers

representing

335

galactans are usually variants of either agar or carrageenan. A common, simplifying structural feature of these biopolymers is the backbone, a linear chain of alternating p-1,3- (A unit) and a-1,4- (B unit) linked galactopyranose units. In practice, the galactan structures are complex and highly variable. In the agar families, as idealized in agarose (Fig. l), the B unit is replaced by 3,6-anhydro-r_-galactopyranose residues in the repeating biose unit. The agarose structure is frequently masked in that D-galactose residues (A unit) may be 6-0-methylated, pyruvated, or may carry sulfate ester substituents at position 2, 4 or 6. Similarly, the anhydrosugar may be methylated, sulfated or replaced by its biological precursor, L-galactose 6-sulfate. In some agarophytes, 4-0-methyl-galactopyranose residues are present. Agars thus may be regarded as a family of galactans differing in the variety, extent and relative proportions of substituted galactan units [5]. The carrageenans differ chemically from agars in that the idealized backbone is much more heavily sulfated and does not contain L-residues. Types of carrageenans differ in the degree and site of sulfate esterification and anhydrogalactose content. In K- and I-carrageenans (Fig. 1) 3,6-anhydro-Dgalactopyranose units replace the B units of the basic structures. The present study has attempted to identify major pyrolysis products of agars and carrageenans by GC-MS to better understand the pyrolysis of the galactan sulfates. The identification of distinctive pyrolysis products in the pyrograms of these algal polysaccharides has greatly assisted in the interpretation of pyrograms of other agars and carrageenans and of different algal samples [6]. EXPERIMENTAL

Samples Methyl-/3-D-galactopyranoside, 6-O-methyl-D-galactopyranose and glucopyranose 6-sulfate were purchased from Sigma (St. Louis, MO, U.S.A.) and agarose (Cat. No. 747331), electrophoresis grade, from J.T. Baker (Phillipsburg, NJ, U.S.A.). 4-0-Methyl-L-galactopyranose was isolated from the hydrolysate of the agar from Gracilaria tikvahiae. Agar MP44 and l-carrageenan were extracted from Gracilaria tikvahiae and Eucheuma spinosum, respectively, using established methods [7]. The composition of agar MP44 was 1.79 (6-0-Me-galactose), 0.23 (4-0-Me-galactose), 1.20 (galactose), 2.52 (3,6-anhydro-galactose) and 0.39 (SO,Na) pmol/mg [8]. Agar from G. eucheumoides was a gift from Drs. C. Ji and W. Yaphe (Institute of Oceanology, Qingdao and McGill University, Montreal, Canada). Pyrolysis-gas

chromatography

Pyrograms were obtained using a Chemical Data Systems Pyroprobe 120 equipped with a platinum coil probe, interfaced to a Hewlett-Packard 5880A

336

gas chromatograph equipped with a flame ionization detector. The interface-gas chromatograph inlet connection was well insulated to prevent condensation of the gaseous pyrolysates. A solid sample (0.15-0.20 mg, except 0.35-0.40 mg for glucose 6-sulfate and l-carrageenan) was placed on top of a quartz wool plug located halfway along a thin quartz tube. The sample was always placed at the center of the coil filament. The probe was inserted into the interface (2OO”C), the interface purged with the helium carrier gas for 30 s at 50 ml/mm and the sample then pyrolysed to a final temperature of 750°C ramp off, for a 10-s interval. The pyrolysate was swept from the interface into the GC inlet at a total helium flow-rate of 13.5 ml/min. The split ratio was 9 : 1. The purge flow was turned back on after 30 s. The pyrolysate was chromatographed on a fused silica capillary column [J &W, DB-1701 (bonded phase), 1.0 pm thickness, 30 m X 0.329 mm]. Temperature program settings were as follows: initial temperature 50°C hold 5 min; increase S”C/min until 240°C hold 7 min. Other settings: inlet and detector temperature 250°C; carrier gas linear velocity 24 cm/s; chart speed 1.0 cm/min. Pyrolysis-gas

chromatography-mass

spectrometry

The conditions of pyrolysis and chromatography were identical to those given above. The gas chromatograph was a Finnigan Model 9610. The column outlet was led directly into the ion source region of a Finnigan MAT 4500 quadrupole mass spectrometer. This GC-MS system was used with an INCOS data system. Mass spectrometric detection was carried out with 70-eV electron impact ionization, interface temperature 250°C and ion source temperature 120°C. Chemical ionization was done with isobutane (0.6 Torr instrument reading) as the reagent gas. Reference compounds

All reference compounds were obtained commercially. Measurement of retention times of reference compounds was carried out using the injection port adapter on the pyroprobe interface and identical interface-gas chromatograph settings as above with the exception that the 30-s purge was omitted. One ~1 of an aqueous solution of the reference compounds was injected.

RESULTS

AND DISCUSSION

A quartz sample tube was used for these pyrolysis studies because it could readily accommodate solid samples and facilitated sample handling. The

337

final pyrolysis temperature (750°C) and rapid heating rate yielded adequately reproducible pyrolyses and minimized secondary reactions, producing pyrolysis products characteristic of the initial degradation of the polymer. Due to the polar nature of carbohydrates, a great variation in the polarity of their pyrolysate was expected. A fused silica capillary column coated with a bonded medium polarity stationary phase was found suitable. The bonded stationary phase and a coating thickness of 1 pm led to excellent stability and reproducibility even after repeated high temperature programming and hundreds of sample loadings. The pyrolysates were examined using both electron impact (EI) and chemical ionization (CI). Mass spectra of carbohydrates obtained by EI give important structural information but EI usually yields molecular ions of low or even insignificant intensity [9]. CI gives molecular mass information but the mass spectra usually lack the structural differentiation produced by EI, especially when dealing with structural isomers. Hence, our study has incorporated both EI and CI (isobutane) to gain maximum information on saccharide pyrolysates, as suggested earlier [lo]. Identification

of pyrolysates

In general, the pyrolysis products were identified by matching their EI mass spectrum with a mass spectral library [ll] and, in some cases, confirmed by GC retenticn times of authentic samples. For some compounds, mass spectrometric identification was supported by reference spectra from other pyrolysis studies [12-151 as indicated in Table 1. Tentative identifications of other pyrolysis products are based on molecular ion assignment from CI-MS measurements and interpretation of mass spectra and are reasonably supported by carbohydrate mass spectrometry [9]. The following discussion of the pyrolysates from model and natural saccharides is given with emphasis on tentative identification of unique pyrolysate structures. Model saccharides The only Py-GC peaks of model saccharides analyzed were those also found in the pyrograms of agar and carrageenan. of methyl-P-D-galactopyranoside, 6-O-methyl-DTypical pyrograms galactopyranose, 4-0-methyl-L-galactopyranose and glucopyranose 6-sulfate are shown in Fig. 2. The important peaks are numbered and their identities are given in Table 1. The EI and CI mass spectra of compound 23 (Fig. 3) supports the assignment of a 1,6-anhydro-P-D-galactopyranose structure. The CI mass spectrum (Fig. 3b) exhibits a prominent m/z 163, corresponding to the protonated molecular ion (MH)+. Separated from m/z 163 by multiples of 18 mass numbers, the fragments at m/z 145, 127, and 91 arise

338

by loss of one molecule, a second molecule and finally two more molecules of water, respectively, from (MH)+. The fragment at m/z 115 evidently arises by expulsion of formaldehyde from m/z 145 [16]. Finally the EI mass spectrum (Fig. 3a) of 23 closely resembles the EI mass spectrum of 1,6anhydro-/3-D-glucopyranose (levoglucosan) [14]. The structural assignments of 21 and 22, two pyrolysis isomers arising from 6-O- and 4-0-methyl-galactopyranose respectively, are more tentative. Based on CI-MS (Fig. 4b and d) their apparent molecular weight is 176 which fits the proposed structures of monomethyl anhydrogalactoses (see Fig. 9). The CI mass spectrum of 21 (Fig. 4b) shows a strong m/z 177 (MH)+, followed by fragments at m/z 159 and 141 arising from loss of one and two molecules of water, respectively. The EI mass spectrum of 21 (Fig. 4a) exhibits extensive fragmentation, typical of underivatized carbohydrates.

TABLE

1

Compounds

identified

in the pyrolysates

of model saccharides,

and galactan

Peak Compound No.

MW Identification

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

32 72 60 60 74 82 84 96 110 126 110 94 126 140 126

m, m, m m, m m, m m, m m m, m, m m, m

158 126 144

m, t m, r m

144 144 176 176 162 176

m, m, m, m, m, m,

16 17 18 19 20 21 22 23 24

Methanol 2-Butanone Hydroxy-acetaldehyde Acetic acid 1-Hydroxy-2-propanone 2-Methylfuran 2(3H)-Furanone 2-Furaldehyde I-(2-Furanyl)-ethanone I-(3-Hydroxy-2-furanyl)-ethanone SMethyl-2-furaldehyde Phenol 2-Fury1 hydroxymethyl ketone 5-(Methoxymethyl)-2-furaldehyde 1,6-Anhydro-3,4-dideoxy-P_D-glycerohex-3-enopyranos-2-ulose 6-0-methyl-hexose 5-(hydroxymethyl)-2-furaldehyde 1,5-Anhydro-4-deoxy-D-gIycero-hexl-en-3-ulose 1-Deoxy-3,6-anhydro-lyxo-hexopyranos-2-ulose Anhydro-deoxy-galactopyranose 1,4-Anhydro-6-0-methyl-galactopyranose 1,6-Anhydro-4-0-methyl-galactopyranose 1,6-Anhydro-P-r~galactopyranose 2-O-Methyl-3,6-anhydro-galactose

Identification by m, CI and EI mass spectra; samples; t, tentative identification.

l

r, confirmed

r r r r r

r r

l

sulfates Reference spectra

mass

11 11 11 11 11 11 11 11 11 11 11 11 12

t 13,14

11 15

t t t t t t

by retention

times of authentic

339

tb)

I4 \

* 10

15

20

I 25

30

35

40

Fig. 2. Pyrograms of (a) methyl-fl-D-gdactopyranoside, (h) 6-O-methyl-D-galactopyranose, (c) 4-0-methyl+gafactopyranose and (d) glucopyranose 6-sulfate. The identities of the numbered peaks are given in Table 1.

100

lb:

WI+

(b)

50

60

ml2

Fig. 3. (a) Electron impact and (b) chemical ionization tentatively identified as 1,6-anhydro-fi-wgalactopyranose.

-XL-mass spectra of pyrolysis

product

23,

The base peak at m/z 45 reveals prominent loss of CH,OCH, from the parent molecule. Compound 22, tentatively identified as 1,6-anhydro-4-O-methylgalactoshows a mass fragmentation pattern similar to that of 1,6pyranose, anhydro-/?-D-galactopyranose, 23, with the exception of a CH,Osubstituent associated with 22. After loss of CH,OH from m/z 177 (MH)+ to give the fragment at m/z 145, the CI mass spectrum of 22 (Fig. 4d) is similar to that of the unmethylated anhydrogalactose, 23. There is similarity between the fragmentation patterns evident in the EI mass spectra of 22 (Fig. 4c) and 23 (Fig. 3a) when allowance is made for the prominent shift of 14 mass numbers associated with the mass spectrum of the monomethyl saccharide, 22. The pyrogram of glycopyranose 6-O-sulfate (Fig. 2d) and subsequent MS analysis of the pyrolysate reveal that lower molecular weight compounds are produced, where compound 15, 1,6-anhydro-3,4-dideoxy-P_D-glycero-hex-3enopyranose-2-ulose (also called “levoglucosenone”) represents the largest portion of the volatile pyrolysate. Various substituted furans constitute the bulk of the remaining pyrolysis products. Agarose Agarose consists of two building blocks, 3,6-anhydro-a+galactopyranose and ,8-D-galactopyranose (Fig. 1). Pyrolysis of agarose should result in the formation of distinctive pyrolysis products from each type of sugar residue. The pyrogram of agarose (Fig. 5a) shows the presence of 1,6-anhydrogalactopyranose, 23, as a major pyrolysis product, thus confirming galactopyranose units in the agarose structure. Peak 19 is a probable initial pyrolysis product of the 3,6-anhydro unit. Its CI mass spectrum (Fig. 6b)

341 loo-

171

’ WI+

(b)

40.

i

IOO.

17

(d)

Fig. 4. Mass spectra of pyrolysis products 21, tentatively identified as 1,4-anhydro-6-0methyl-galactopyranose, (a) electron impact and (b) chemical ionization, and 22, tentatively identified as 1,6-anhydro-4-0-methyl-galactopyranose (c) electron impact and (d) chemical ionization.

shows a strong (MH)+ at m/z 145 along with its unprotonated molecular ion m/z 144. The fragment ion at m/z 127 indicates loss of a water molecule from (MH)+. The formation of m/t 85 in both the CI and EI mass spectra (Fig. 6) can arise from expulsion of a hydroxymethyl ketone group (-59 mass numbers) from the molecular ion m/z 144. The loss of a hydroxymethyl ketone group from 19 can come about through a mechanism similar to the pyrolytic formation of compound 13 proposed in Fig. 9. Compound 13, identified as 2-fury1 hydroxymethyl ketone, is thought to be another major pyrolysis product of the 3,6-anhydro unit in agarose. It may be formed through further dehydration of pyrolysate 19 as proposed in

342

23

_I5

5

10

10

15

15

Fig. 5. Pyrograms of (a) agarose are given in Table 1.

20 Time

(mid

Time

(mini

20

25

30

35

40

25

30

35

40

and (b) agar MP44. The identities

of the numbered

peaks

Fig. 9. This substituted furan has been identified in the pyrolysate of other sugars [4,12,17] but was present in small amounts. In contrast, the structural isomer of compound 13, S(hydroxymethyl)-2-furaldehyde, is a more common pyrolysis product of hexopyranoses [20]. In total, six furan-containing compounds have been identified in the volatile pyrolysate of agarose. Agars The pyrogram of an agar preparation from Gracilaria tikvahiae (agar MP44), which contains both 4-O- and 6-0-methyl-galactopyranose residues and a significant amount of sulfate ester substituents, is shown in Fig. 5b. The following peak assignments relating to the structural units of the agar can be made from pyrolysates identified from the model saccharides and agarose: galactopyranose (peak 23), 4-0-methyl-galactopyranose (peak 22), 6-0-methyl-galactopyranose (peak 21), and 3,6-anhydro-galactopyranose (peaks 13 and 19). In addition to these distinctive products, new peaks

343 I45

IOO-

57

(0)

.

’ INH+

(b)

5044

31

I

69

e5

I#+

I27

144 95 I

116 .*dJ I20

t mlz

99 140

100

I,

,

150

200

m/z

Fig. 6. (a) Electron impact and (b) chemical ionization mass spectra of pyrolysis tentatively identified as I-deoxy-3,6-anhydro-lyxo-hexopyranos-2-ulose.

product

19,

appeared in the middle to high end of the pyrogram. Compound 20, with an apparent molecular weight of 144, is likely a dehydration product of 1,6anhydrogalactose, 23. Compounds 16 and 14 are formed from 6-O-methylgalactopyranose units as a result of dehydration through loss of one and two molecules of water, respectively. The identity of compound 14 is likely a stable substituted furan structure. It is interesting to note that in a previous study involving pyrolysis field ionization-MS of agarose [3], mass peaks at m/z 140 and 158 were present in the field ionization mass spectrum. It is possible that the agarose preparation used in their Py-MS experiment contained substantial amounts of 6-0-methyl-galactopyranose residues. Chemical analysis of some commercial agarose preparations has indicated the presence of methylated galactose residues [18]. It was of interest to examine the pyrolysate of an agar which contains higher amounts of sulfate ester and also has another type of methylated saccharide unit. Agar from Gracilaria eucheumoides is known to have these structural qualities in that many of the 3,6-anhydro-galactose units are 2-0-methylated [18,19]. Its pyrogram (Fig. 7b) reveals that “levoglucosenone”, 15, is a major pyrolysis product, that the amount of products associated with 3,6-anhydro-galactose units, 13 and 19, is small, and that a new, substantial peak, 24, is present. The CI mass spectrum of 24 (Fig. 8a) shows (MH)’ at m/z 177, which, from its low intensity, appears to be rather unstable. The CI mass spectrum exhibits a base peak at m/z 159 from loss of one molecule of water. The fragment at m/z 145 results from the loss of methanol from. the parent ion (MH)+. Subsequent loss of water from m/z 145 would give ion m/z 127. The EI mass spectrum of compounds 24 (Fig. 8a) resembles a fragmentation pattern of a methyl anhydrosugar (compare to Fig. 4~). Compound 24 is tentatively identified as 2-O-methyl-3,6-anhydrogalactose.

344

Carrageenan The pyrolysate of an L-carrageenan sample is described because the carrageenan represents a galactan which contains sulfate ester groups on both the galacto and 3,6-anhydro units (Fig. 1). The A- and K-carrageenans gave similar pyrolysis products [6]. The pyrogram of L-carrageenan (Fig. 7b) reveals that “levoglucosenone”, 15, represents the major volatile pyrolysis product of carrageenans. Smaller amounts of 2-fury1 hydroxymethyl ketone, 5-(hydroxymethyl)-2-furaldehyde, and the two structural isomers, 19 and 20, were also identified. Two new pyrolysates of galactan sulfates were detected in the pyrolysis of carrageenan: 1,5-anhydro-4-deoxy-D-glycero-hex-1-en-3-ulose, compound 18, and phenol, compound 12. The former product has been identified in the pyrolysate of glucose [15].

24 0

1 r-

5

10

15

20

25

30

35

40

Time lminl

15

(b)

_I 5

8

13 19

20

10

7

18

ii 11

15

L!_

II

JLLJ 10

20

25

30

35

40

Time (mini

Fig. 7. Pyrograms of (a) agar from Gracilaria eucheumoides, identities of the numbered peaks are given in Table 1.

and (b) c-carrageenan.

The

345 100

IS!

(b)

50

I?7

WI+

40

60

60

100

I20

140

I60

I60

Wh,l

Fig. 8. (a) Electron impact and (b) chemical ionization tentatively identified as 2-O-methyl-3,6-anhydro-galactose.

Formation

of pyrolysates

100

15l rnll

L200

mass spectra of pyrolysis

product

24,

of galactan sulfates

Pyrolysis pathways and structures of several key pyrolysis products arising from different saccharide units of galactan sulfates are suggested in Fig. 9. Depolymerization of the biopolymer, either through thermal rupture of the glycosidic linkages or as a result of transglycosylation reactions, is the main event during initial stages of pyrolysis. The formation of anhydrosugars, i.e., 21, 22, and 23, appears to be the primary pyrolytic pathway of methylated and unmethylated hexose units in agars. The 3,6-anhydro-galactose unit appears to form a 3,6-anhydro-keto intermediate, 19, which subsequently, through rearrangement and dehydration, forms a stable furan derivative, 13. The five-membered structure of a furan already exists in the 3,6-anhydrogalacto unit. Usually 3,6-anhydrohexoses readily form their 1,4-3,6-dianhydro structures as observed in the case of 3,6-anhydro-glucose [17,21]. Because of the stereochemistry at C-4, the formation of a dianhydro structure is not possible in the case of galactopyranose. The prominence of pyrolysates arising from 3,6-anhydro and methylated saccharide units of agarose and agar MP44 can be explained by their lower volatility compared to that from galactopyranose units. The presence, of sulfate ester groups in agars affects the pyrolytic pathways by catalyzing the formation of a greater number of structural isomers through dehydration, i.e., compounds 14, 16, 17, and 20. Increased amounts of sulfate ester substituents in galactans, i.e., agar from G. eucheumoides and carrageenans, result in more char formation in pyrolysis tubes, in lesser amounts of small molecular weight pyrolysis products [compare chromatographic front of the pyrogram of agarose (Fig. 5a) and that of t-carrageenan (Fig. 7b)] and in the production of “levoglucosenone”, 15, as the major

346 polymer

unit

CHZ--0 0

HO >

OH It;l

OH galocto CH,OR c

>

J-

0

R’O

OH 3

4--

,O

CHSOQ

bH

6-0-Me-golocto

IR=CH3,

4-0-Me-golocto

lR=H,

CH2-0

CHZOCHS OQ

OH

OH

R’=H)

-21

R”Ct13)

-22 0

_o@

(l~wcHzoH

, Hoa

0

0

reorrongeme; 0

3.6-onhydro-L-golocio

sulfated

unit

-

dehydrotton

Fig. 9. Some proposed

pyrolytic

pathways

for the formation

of key pyrolysis

products.

volatile pyrolysis product. “Levoglucosenone” has been identified as a pyrolysis product of cellulose and the presence of acid catalyst promotes its formation [22,23]. In summary, identification of the major pyrolysis products of galactan sulfates has shown that unique compounds are formed, indicative of the type and quantity of saccharide units making up the biopolymer chain. Heavily sulfated galactans do not give as complex a pyrogram. We have shown the advantages of GC-MS in characterizing the pyrolysate of carbohydrates, especially when coping with numerous structural isomers. The use of CI-MS is needed to determine the structures of carbohydrate pyrolysis products. Our results show that Py-GC may be used to routinely characterize algal polysaccharides. Some results of this use are presented in another study [6].

341 ACKNOWLEDGEMENTS

We thank Messrs D. Embree and D. Johnson for assistance. Financial support in the form of a contract, from Atlantic Research Laboratory, NRC, Canada, is gratefully acknowledged by R.J.H. and E.R.H.

REFERENCES 1 W.J. Irwin, Analytical Pyrolysis: A Comprehensive Guide, Marcel Dekker, New York, 1982. 2 H.-R. Schulten, U. Bahr, H. Wagner and H. Herman, Biomed. Mass Spectrom., 9 (1982) 115. 3 H.-R. Schulten, U. Bahr and W. Gbrtz, J. Anal. Appl. Pyrol., 3 (1981/1982) 229. 4 A. van der Kaaden, J. Haverkamp, J.J. Boon and J.W. de Leeuw, J. Anal. Appl. Pyrol., 5 (1983) 199. 5 D.A. Rees, Adv. Carbohyd. Chem. Biochem., 24 (1969) 267. 6 R.J. Helleur, E.R. Hayes, J.S. Craigie and J.L. McLachlan, J. Anal. Appl. Pyrol., 8 (1985) 349. 7 J.S. Craigie and C. Leigh, in J.A. Hellebust and J.S. Craigie (Editors), Handbook of Phycological Methods; Physiological and Biochemical Methods, Cambridge University Press, Cambridge, 1978, p. 109. 8 J.S. Craigie, Z.C. Wen and J.P. van der Meer, Bot. Mar., 27 (1984) 55. 9 J. Radford and DC. DeJongh, in G.R. Waller and O.C. Dermer (Editors), Biochemical Applications of Mass Spectrometry, Wiley-Interscience, New York, 1980, p. 255. 10 E. Lewis, E.R. Hayes, W.D. Jamieson and D.R. Budgell, Proc. 31st Annual Conference on Mass Spectrometry and Allied Topics, Boston, 1983, p. 897. 11 S.R. Heller and G.W.A. Milne, EPA/NIH Mass Spectral Data Base, Nat. Stand. Ref. Data Ser., Nat. Bur. Stand. (U.S.), 63, U.S. Department of Commerce, Washington, DC, 1978. 12 W.E. Franklin, Anal. Chem., 51 (1979) 992. 13 Y. Halpern, R. Riffer and A. Broido, J. Org. Chem., 38 (1973) 204. 14 A. Ohnishi, K. Kato and E. Takagi, Polym. J., 7 (1975) 431. 15 F. Shafizadeh, R.H. Furneaux, T.T. Stevenson and T.G. Cochran, Carbohyd. Res., 67 (1978) 433. 16 D. Horton, J.D. Wander and R.L. Foltz, Carbohyd. Res., 36 (1974) 75. 17 D. Gardiner, J. Chem. Sot., C, (1966) 1473. 18 J.S. Craigie and A. Jurgens, unpublished observation. 19 M. LaHaye and W. Yaphe, personal communication, 1984. 20 F. Shafizadeh and Y.Z. Lai, J. Org. Chem., 37, (1972) 278. 21 K. Heyns, R. Stute and H. Paulsen, Carbohydr. Res., 2 (1966) 132. 22 F. Shafizadeh and P.P.S. Chin, Carbohyd. Res., 46 (1976) 149. 23 F. Shafizadeh and P.P.S. Chin, Carbohyd. Res., 58 (1977) 79.