Analysis of wax and resin components from Minnesota peat bog

International Journal of Coal Geology, 8 (1987) 99-109

99

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

A n a l y s i s of Wax and Resin Components from M i n n e s o t a Peat Bog DURGA KUMARI

Center for Environmental Studies, Division of Science and Mathematics, Bemidji State University, Bemidji, M N 56601, U.S.A. (Revised and accepted for publication August 7, 1986)

ABSTRACT Kumari, D., 1987. Analysis of wax and resin components from Minnesota peat bog. In: D.J. Boron (Editor), Peat: Geochemistry, Research and Utilization. Int. J. Coal Geol., 8: 99-109. A peat sample from a Minnesota (St. Louis County) bog was examined for waxy and resin components by extraction with petroleum ether (35-60°), chloroform and ethyl acetate. The waxy solids were separated from the resins by crystallization from methanol at 0 ° C. A sample of wax was saponified with 0.5 N NaOH for 4 h. Wax, resin, unsaponified and saponified fractions were analyzed with a Hewlett-Packard Model No. 5995 MS/GC/DA system equipped with a fusedsilica capillary column, internally coated with SE-30. All fractions contained C21 through Cz7 alkanes, n-alcohols, straight-chain fatty acids, phenolic acids, polynuclear aromatic hydrocarbons and triterpenoids. Many of the compounds isolated and identified have not been previously reported in peat analyses.

INTRODUCTION

Peat is a valuable raw material for the chemical industry. Various nonfuel uses have been sought for it. Raw peat can be used as such, in the manufacture of carbon papers, or it can be processed further by bleaching with chromic acid, or by esterifying. Other potential uses include impregnation of papers and fabrics, polishing agents, manufacture of color pastes and records, casting molds, emulsifying agents (Fuchsman, 1980), etc. The bitumen (crude peat wax) fraction that can be extracted from peat with organic solvents has been a special object of interest. On the basis of solubility, the bitumen fraction can be divided into waxes, resinous substances, and asphaltenes. The properties of peat wax resemble those of montan wax extracted from brown coal and it can be used for the same purposes as montan wax. The present world-wide consumption of montan wax is estimated at about 40,000 tons per a n n u m and in the U.S.A. about 5000 tons per annum. Interest in peat waxes exists among

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100 U.S. chemical companies which already sell wax-containing formulations as surface coatings and industrial lubricants. Finnish, Soviet and Israeli peats have been analyzed by many investigators (Brenner et al., 1978; Ketola et al., 1979; Fuchsman, 1980; Ekman and Ketola, 1981 ), but no detailed analyses of waxes and resins from Minnesota have been reported. The present report deals with the analysis of wax and resin components of Minnesota peat from St. Louis County bog. EXPERIMENTAL

Material The peat material was taken from St. Louis County, Minnesota and is derived from mosses of the genus Sphagnum. It also contains remains of ericaceous shrubs, forbs and sedges. It contains approximately 95% moisture.

Extraction of peat An air-dried sample, containing about 15% moisture was ground in a laboratory Wiley mill, using 20-mesh screen. The milled air-dried peat ( 700 g) was successively extracted with petroleum ether (35-60 ° ), chloroform and ethyl acetate. The extracts were reduced to dryness with a rotary evaporator. The total bitumen (sum of individual extracts) was 27.940 g.

Separation of wax Evaporated petroleum ether ( 35-60 ° ) and chloroform extracts were heated with methanol and filtered. The hot methanol-insoluble matter (asphaltene) amounted to 5.749 g. The filtrates were kept at 0°C for about 24 h and the developed precipitate was filtered, washed with methanol and dried, yielding 11.056 g of wax. The resin (6.245 g) was obtained by evaporation of the mother liquor.

Basic hydrolysis A sample of wax (5 g) was saponified with hot 0.5 N NaOH in 1:5 methanol:water, for 4 h. After cooling the solution was filtered, the solids washed with distilled water. The combined filtrates and washings were extracted with ethyl acetate. The combined solids and ethyl acetate extracts yield 4.350 g of unsaponified matter. The remaining aqueous portion was acidified with 2 N HC1 to precipitate acids. The precipitated acids were extracted with ethyl acetate, washed with distilled water and dried over sodium sulphate, yielding 0.706 g of saponified wax acids.

101

Chromatography A glass column of 2" diameter and 30" long was packed with a petroleum ether slurry of 250 g silica gel. Peat wax (6 g) was added to the column and the chromatogram developed with petroleum ether. The elution of the column was continued, using increasing polarity of solvents and 500-ml fractions were collected and evaporated. The resulting chromatogram showed four major fractions according to thin-layer chromatography. The unsaponified waxes and the saponified wax acids were also fractionated by silica gel column chromatography. The total resin was also separated on a silica gel column into a number of fractions by successive elution with petroleum ether, followed by solvents of increasing polarity. TLC of wax fractions using silica gel and developments in petroleum etherbenzene (50:50) revealed distinct spots for oxygenated and nonoxygenated compounds. Spots were made visible by spraying with an aqueous solution of Rhodamine B or by using an iodine chamber. The polar resin fractions were developed in CHC13-MeOH (90:10), and the spots were exposed to iodine vapor.

Gas chromatography The acidic constituents of the wax acid and the resins were methylated with ethereal diazomethane. Hydroxy compounds were converted to their trimethylsilyl (TMSi) ethers by addition of BSTFA-TMCS (bis- (trimethylsilyltrifluoroacetamide-trimethylchlorosilane) (TMSi 1%) and heated at 60°C for 15 min. The monomers were separated by gas chromatography (GC) equipped with a flame ionization detector (FID) on a fused-silica capillary column (50 mX0.32 mm I.D. Altech) coated with a SE-30 liquid film and on a second column ( 25 m X 0.30 mm I.D. Altech) coated with a SE-54 liquid film. The GC oven was programmed for 130-290 °C at 4 ° C/min (in a few tests the range was 100-310°C at 8°/min).

Mass spectra All the compounds previously separated by chromatography were analyzed with a Hewlett-Packard Model No. 5995 MS/GC/DA system equipped with a fused-silica capillary column, internally coated with SE-30. Triterpenoids obtained as crystals from the column chromatograph fraction, and recrystallized from CHC13-MeOH were run with a direct probe at 70 eV.

General methods All melting points are uncorrected. Infrared spectra were obtained with solid samples dispersed in KBr pellets, using a Beckman IR-33 spectrophotometer.

102 TABLE 1 Amounts of wax, resin and asphaltene from peat (700 g) by successiveextraction Solvent

Extract

Wax

Resin

Asphaltene

Pet. ether {35-60° ) Chloroform Ethyl acetate

7.710 g 15.340 g 4.890 g

5.870 g 5.186 g *

1.840 g 4.405 g *

-5.749 g *

• Not determined. UV measurements were made in ethanol. 1H N M R was determined at 200 MHz on a Bruker WH-400 spectrometer. 13C N M R was determined at 100.62 MHz on a Bruker WH-400 spectrometer. Spectra were run in CDC13 solution containing 1% T M S as internal reference. Chemical shifts are in ppm (3). RESULTS AND DISCUSSION In the present work, the 700 g milled peat was successively extracted with petroleum ether (35-60 ° ), chloroform and ethyl acetate. The a m o u n t of wax, resin and asphaltene are shown in Table 1. The ethyl acetate extract was not further fractionated. It appeared to contain some fatty acids and chlorophyll by using G C / M S and UV data. From Table 1 it appears t h a t the petroleum ether extract contains predominantly peat wax. However, much of the wax remained unextracted. The subsequent use of chloroform, a much less specific solvent, removed most of the remaining wax. In the course of column chromatography, a number of fractions were collected. From the concentrated solution of the first three fractions in petroleum ether, silvery white flake crystals (m.p. 55-57 ° C) separated on standing. These were filtered and washed. The infrared spectrum, using a potassium bromide disc, appeared to be t h a t of an alkane hydrocarbon. Fractions 4 to 12 deposited a white material which was filtered off and washed. It was not very crystalline. The infrared spectrum indicated t h a t it was an ester; it had a m.p. of 75 °C. Subsequent fractions also yielded solids with a m.p. of 75 ° C, however, these appeared to be n-alcohols from the infrared spectrum. Solids from fractions 20-25 had a m.p. of 63-64°C; they had an infrared spectrum typical of long chain fatty acids. The acids were converted to methyl esters by reaction with excess diazomethane. The unsaponified solid and ethyl acetate extractives yielded a complex mixture of compounds t h a t was further purified on column chromatography. The fractions were separated into alkane hydrocarbons, n-alcohols and polynuclear aromatics; triterpenoids were also detected. Saponified waxes contained fatty acids and phenolic acids.

103 TABLE 2 Major wax compounds occurring in Minnesota peat Compound Alkanehydrocarbons Heneicosane Docosane Tricosane Tetracosane Pentacosane Hexacosane Heptacosane Alcohols (TMS) l-Hexadecanol 1-Heptadecanol l-Octadecanol l-Nonadecanol l-Eicosanol 1-Heneicosanol l-Docosanol 1-Tricosanol 1-Tetracosanol 1-Pentacosanol l-Hexacosanol

Carbon chain

Molecular ion (M + )

21 22 23 24 25 26 27

296 310 324 338 352 366 380

16 17 18 19 20 21 22 23 24 25 26

314 328 342 356 370 384 398 412 426 440 454

Compound

Carbon chain

Molecular ion (M ~)

Alcohols ( T M S ) (continued) 1-Heptacosanol 27 l-Octacosanol 28 l-Nonacosanol 29 1-Triacontanol 30

468 482 496 510

Fatty acids (methyl ester) Pentadecanoic 15 Hexadecanoic 16 Heptadecanoic 17 Octadecanoic 18 Nonadecanoic 19 Eicosanoic 20 Heneicosanoic 21 Docosanoic 22 Tricosanoic 23 Tetracosanoic 24 Pentacosanoic 25 Hexacosanoic 26 Heptacosanoic 27

256 270 284 298 312 326 340 354 368 382 396 410 424

T h e resin was c o l u m n - c h r o m a t o g r a p h e d a n d t h e c h r o m a t o g r a m d e v e l o p e d w i t h p e t r o l e u m ether. T h e s o l v e n t p o l a r i t y was i n c r e a s e d gradually, a n u m b e r of t h e f r a c t i o n s collected were s e p a r a t e d a c c o r d i n g to t h e n a t u r e of c o m p o u n d s , e.g., t r i t e r p e n o i d h y d r o c a r b o n s , polycyclic a r o m a t i c h y d r o c a r b o n s , n - a l k a n o l s , o t h e r t r i t e r p e n o i d s , a n d n - f a t t y acids. A p o r t i o n of t h e e t h y l a c e t a t e e x t r a c t of p e a t was m e t h y l a t e d w i t h e t h e r e a l d i a z o m e t h a n e a n d a n a l y z e d by G C / M S . T h e m o n o m e r i c w a x y a n d resin c o m p o u n d s fall into t h e following c h e m i c a l classes: n - a l k a n e s , n - a l c o h o l s , s a t u r a t e d f a t t y acids, p h e n o l i c acids, p o l y n u clear a r o m a t i c h y d r o c a r b o n s a n d t r i t e r p e n o i d s . T h e m a j o r wax c o m p o u n d s o c c u r r i n g in t h i s M i n n e s o t a p e a t s a m p l e are s h o w n in T a b l e 2. n-Alkanes

A l k a n e h y d r o c a r b o n s were p r e s e n t in wax, u n s a p o n i f i e d wax a n d resin fractions. T h e earlier f r a c t i o n s of c o l u m n c h r o m a t o g r a p h y c o n t a i n e d p r e d o m i n a n t l y a l k a n e h y d r o c a r b o n s . ( I R b a n d s ( K B r ) 2920, 2840, 1450, 1370, 1165, 1102 a n d 985 c m 1)

104

GC/MS analysis indicated the presence of alkane hydrocarbons in the range of C~1 through C27.

Alcohols Long chain C16 to C3o homologous alcohols represented another group of major wax components identified in the gas chromatogram. Similar alkanols have previously been reported to occur in Finnish Peat (Ketola et al., 1981). The mass spectra of these alcohols as trimethylsilyl (TMSi) ethers are characterized by very intense peaks located at m/z M-15, whereas the molecular ions are almost indistinguishable. No unsaturated n-alkanols were found. (IR bands(KBr) 3310 (br), 2920, 2850, 1470, 1415, 1370, 1345, 1130, 1092, 1070, 1042, 912,858 and 718 cm -1)

Fatty acids One of the major series of homologues consisting of n-alkanoic acids were present in the last and middle fractions of column chromatography. All the C15 through C27 fatty acids were present in the wax prior to saponification. After saponification, the same fatty acids (C15-C27) were identified. The mass spectra of these long-chain acids (as methyl esters) provide facile interpretations, since different members in the series, in addition to their molecular ions, give characteristic fragment ions in the high mass region (M ÷-29, M +-31, M ÷-43) while the base peak is found at m/z 74. (IR bands (KBr) 3390 (br), 2920, 2860, 1700, 1615, 1525, 1476, 1440, 1420, 1380, 1275, 1210, 1100, 1030 and 800 cm 1)

Phenolic acids On saponification, the wax yielded several phenolic acids along with fatty acids. GC/MS of methylated phenolic acids show the presence of p-hydroxybenzoic, ferulic, caffeic, p-coumaric, vanillic, and methylhydroxybenzenecarboxylic acid. Their main fragments are shown: p-hydroxybenzoic acid 166 (M + ), 135 (M+-31), 121 (M+-45), 107 (M+-59), ferulic acid 222 (M ÷ ), 207 (M÷-15), 191 (M+-31), caffeic acid 222 (M+), 191 (M+-31), p-coumaric acid 192 (M+), 161 (M+-31), 133 (M+-59), vanillic acid 196 (M+), 181 (M÷-15), 165 (M+-31), 137 (M+-59), methylhydroxybenzenecarboxylic acid 180 (M ÷ ), 165 (M+-15), 137 (M+-43). The occurrence of these aromatic acids from peat and the peat humic matter has been demonstrated by Ishiwatari et al. (1983).

Polynuclear aromatic hydrocarbons' Polynuclear aromatic hydrocarbons were predominantly present in the earlier resin fractions along with alkane hydrocarbons and in a few fractions after

105

alkane hydrocarbons. The fractions were yellow with an intense turquoise-blue fluorescence in solution. The ultraviolet absorption spectrum of the fraction in ethyl alcohol showed peaks at 265, 302, 340, 355 sh, 366, 384, 409, 430 sh and 436 nm characteristic of the spectrum of polynuclear aromatic hydrocarbons. This was further confirmed by the characteristic color change (green--,bluish-greenoblue-,reddish-violet) on the addition of concentrated sulphuric acid to a small crystal of solid hydrocarbon. It is evident that there are polycyclic aromatic hydrocarbons present in resin fractions of peat. Polynuclear aromatic hydrocarbons were predominantly present in resin and to a lesser extent in wax and unsaponified wax fractions. Bergmann et al. (1964) and Gilliland et al. (1960) found perylene in the crude peat wax. GC/MS analysis indicated the presence of fluoranthene, pyrene (M ÷ 202 ) ; benzo(a)anthracene (M ÷ 228); benzo(b)fluoranthene, benzo(k)fluoranthene, benzo (a) pyrene and perylene ( M ÷ 252 ).

Triterpenoids Triterpenoids are predominantly present in resin fraction. Minor quantities appear in the wax. These were taraxerene, hop-12-ene, hop-22 (29)-ene, neohop-12-ene, taraxerone and taraxerol. Ives and O'Neill (1958) isolated taraxerone and taraxerol from peat moss. Triterpenoid hydrocarbons were present in the first few fractions along with alkane and polycyclic hydrocarbons. IR bands(KBr) 2950, 2850, 1640, 1470, 1390, 1135, 1060, 1040 and 1000 cm 1. The MS of triterpenoid hydrocarbons showed the molecular ion M + 410. Taraxerone, long needles from CHC13-MeOH, had m.p. 240 °. INMR s 0.83, 0.92, 0.96, 1.00, 1.02, 1.06, 1.11, 1.14 (all s, CH3), 5.56 (q, H-15). 1H-NMR of taraxerone is shown in Fig. 1. The attached proton test (APT) (Shoolery, 1984 ) showed the presence of 30 C-atoms, which were assigned to eight methyl, ten methylene, and three methine groups as well as six fully substituted Catoms, one tri-substituted double bond and one carbonyl group. It is shown in Figs. 2, 3 and Table 3. Taraxerone showed the presence of keto group in the infrared spectrum. (IR bands (KBr) 2940, 2860, 1715, 1460, 1380 and 1080 cm -1) The MS of taraxerone showed molecular ion M + 424. Taraxerol, long needles from CHC13-MeOH had a m.p. of 279-283 ° C. The infrared spectrum showed the presence of hydroxyl group. (IR bands (KBr) 3480 (br), 2950, 2880, 1475, 1390, 1138, 1040 and 1000 cm -1) The MS also showed the molecular ion M ÷ 426. The fragments of triterpenoids are shown in Fig. 4. CONCLUSION

The wax and resin fractions extractable from a Minnesota peat sample by the use of organic solvents have been shown by gas chromatography, TLC, IR,

106

/

I

I . . . .

I

8

7

6

,

,

,I,

S

4.

3

0

2

Fig. 1. ~H-NMR spectrum of taraxerone at 200 MHz.

UV, MS and GC-MS to contain a wide variety of identifiable organic compounds, some of which have not been reported in the peat literature. The wax fraction contains predominantly straight-chain fatty acids (C,s-C27), alcohols (C16-C30), their esters and n-alkanes ( C21 - C27). Saponification of peat wax yielded a number of phenolic acids (p-hydroxybenzoic, ferulic, caffeic, p-coumaric, vanillic and methylhydroxybenzenecarboxylic acid). The resin fraction contained predominantly triterpenoids (taraxerene,

EXPANDED I

I

Fig. 2. A P T (attached proton test) spectrum oftaraxerone (singlets and triplets are positive and doublets and quartets are negative signals).

107

%

. . . . . . .

q . . . . . . . .

q

. . . . . . .

I . . . . . . . .

I . . . . . . . . .

i . . . . .

i . . . . . . . . .

i . . . . . . . . .

i . . . . . . . . .

i . . . . . . . . .

i . . . . . . .

i .....

Fig. 3. Expanded APT spectrum of taraxerone (singlets and triplets are positive and doublets and quartets are negative signals).

hop-21-ene, hop-22 (29)-ene, neohop-12-ene, taraxerone, and taraxerol), and polycyclic aromatic hydrocarbons ( fluoranthene, pyrene, benzo (a) anthracene, benzo (b) fluoranthene, benzo (k) fluoranthene, benzo (a) pyrene and perylene. TABLE3 ~:~Cshielding values for taraxerone (Assignment of shifts are given as ppm downfield form TMS ) -C=O C14 C,~

217.48 157.68 117.26

Singlets

Doublets

C4 47.61 Cs 38.94 Clo 37.61 C~:~37.76 Clv 35.82 C2o 37.81

C.~ 55.85 C9 48.89 C,s 48.77

Triplets

Quartets

C1 38.42 C~ 34.17 C~ 20.03 C7 35.18 C,, 17.51 C1~ 28.85 C16 36.74 C,9 40.72 C21 33.64 C22 33.15

C2.~26.19 Ce4 21.52 C25 14.85 C26 21.40 C27 25.61 C2s 29.92 C29 33.41 C3o 29.97

108 Taraxerene

>

m/z 204 (i00)

>

m/z 271 (36)

-CH 3

m/z 189 (30)

M + 410 (6.2)

m/z 286 (29.4)

-8-CH~

m/z 257 (16.1)

-CH~

m/z 204 (I00)

Taraxerone

m/z 189 (28.1)

M + 424 (11.9)

m/z 300 (37.5)

-8-CH~

•

Taraxerol

-CO

m/z 285 (28.1)

m/z 257 (9.9)

m/z 204 (i00)

-CH 3

m/z 287 (18.1)

-H20

m/z 269 (11.5)

-CH3"

m/z 203 (50)

>

m/z 189 (28.5)

M + 426 (4.9)

ra/z 302 (17.9)

-8-CH~

Hop-21-ene

;

m/z 191 (I00)

M + 410 (i0)

m/z 218 (15), m/z 189 (75), m/z 175 (25)

Hop-22(29)-ene

~, m/z 191 (I00)

M + 410 (12.6)

m/z 218 (37.1), m/z 204 ( 5 9 . 2 ) , m/z 189 (35.8) Neohop- 12-ene M + 410 (i0)

m/z 218 (100) _~H ~ ~CH

m/z 191 (30)

CH 3 3

m/z 175 (20)

Fig. 4. Fragments of triterpenoids, 70 eV m/z (rel. int. ). ACKNOWLEDGMENTS I t h a n k Professor C.H. F u c h s m a n and Dr. S.A. Spigarelli for critically reading and m a k i n g valuable suggestions for this paper. I am grateful to Professor H. Ageta for a kind gift of a u t h e n t i c triterpenoid samples.

REFERENCES Bergman, E.D., Ikan, R. and Kashman, J., 1964. The occurrence of perylene in Huleh peat. Israel J. Chem., 2: 171-172.

109 Brenner, S., Ikan, R., Agron, N.A. and Nissenbaum, A., 1978. Hula Valley peat: Review of chemical and geochemical aspects. Soil Sci., 125: 226-232. Ekman, R. and Ketola, M., 1981. Analysis of lipid components in peat from a Finnish Sphagnum bog. Kemia-Kemi (Finnish Chemistry), 8: 488-493. Fuchsman, C.H., 1980. Peat Industrial Chemistry and Technology. Academic Press, New York, NY, pp. 39-74. Gilliland, M.R., Howard, A.J. and Hamer, D., 1960. Polycyclic hydrocarbons in crude peat wax. Chem. Ind., pp. 1357-1358. Ishiwatari, R., H/ininen, K., Lehto, 0. and Tirronen, A., 1983. Chemical degradation and oxidation studies of Finnish peat and of peat humic fractions, qualitative analysis. Proc. Int. Peat Symp., Bemidji, MN, pp. 457-476. Ives, D.A.J. and O'Neill, A.N., 1958. The Chemistry of peat. The triterpenes of peat moss ( sphagnum). Can. J. Chem., 36: 926-930. Ketola, M., Pihlaja, K., Jalonen, J., Luomala, E., Euranto, E. and Gltikert, G., 1979. Distribution of lipids in Finnish peat at different depth levels of sampling. Kemia-Kemi (Finnish Chemistry), 6: 718-721. Ketola, M., Ekman, R. and Luomala, E., 1981. Compositional features of waxy materials in Finnish peat. Proc. Int. Peat Syrup., Bemidji, MN, pp. 221-~.37. Shoolery, J.N., 1984. Recent developments in 1:~Cand proton NMR. J. Nat. Products, 47: 226-259.