Pyrolysis-Gas Chromatography for the Analysis of Proteins - American

Chapter 4

Pyrolysis-Gas Chromatography for the Analysis of Proteins: With Emphasis on Forages James B. Reeves, III, and Barry A. Francis

Downloaded by CORNELL UNIV on October 18, 2016 | http://pubs.acs.org Publication Date: June 29, 1998 | doi: 10.1021/bk-1998-0707.ch004

Nutrient conservation and Metabolism Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Building 200, Room 124, BARC East, Beltsville, MD, 20705

Pyrolysis-gas chromatography has been shown to be very useful i n the analysis of forages, particularly for the lignin and carbohydrate fractions. However, the same efforts have not shown a similar abundance o f information on the proteins present, even for forages containing almost 30% protein. Despite the fact that pyrolysis o f isolated proteins produces an abundance o f information, only small amounts o f phenylacetonitrile, indole, methylindole, a methylphenol, dimethyl- or ethylpyrrole, and methanethiol are generally reported for forages. Reexamination o f the subject indicates that many more products are produced, but have been missed due to the low levels found, or because many do not contain nitrogen and may have been assumed to originate from other sources, such as lignin. In conclusion, results indicate that more information on proteins is present i n pyrograms o f forages than has been recognized.

Ruminants, such as sheep and dairy cows, are often fed diets containing large amounts of lignocellulosic materials in the form o f forages such as legumes (alfalfa, clovers, etc..) or grasses (orchardgrass, tall fescue, etc.). While ruminants, unlike humans and many other animals, are able to digest much o f the lignocellulosic material present i n these forages, they still, like humans and other animals, require a source o f nitrogen for building proteins, nucleic acids, etc.. Thus, the composition o f animal feedstuffs is extremely important both with respect to the nutritional well being o f the animal and to the farmer trying to make a profit. Because of the importance of forage composition to animal nutrition, forages have been extensively studied in efforts to improve animal performance by improving the nutritional value o f their feedstuffs. The same can be said for the assay procedures used to determine forage composition.

American Chemical Society. © 1 9 9 8 American Chemical Society Libfafy 1155 15th St., N.W. Washington. D.C. 20036

Stankiewicz and van Bergen; Nitrogen-Containing Macromolecules in the Bio- and Geosphere ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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In addition, the role o f nitrogen i n animal diets, of which protein is the principle source, has taken on an importance beyond the nutritional well-being o f the animal, due to the role o f animal wastes i n pollution of water sources, ground water, streams, etc. (7). Considerable research is being carried out on various components o f the "feed to animal to waste" cycle shown i n F i g . 1. A s can be seen, organic forms o f nitrogen, o f which protein is the primary fraction, occupy a prominent position i n this cycle. Considerable efforts are now being expended on understanding the various pathways and final disposition o f the organic nitrogen fractions i n the feed and manure. For example, at present, the rate and degree to which organic nitrogen i n manure is converted to the inorganic forms useable by plants is largely unknown, with estimates ranging from 0 to 50% being converted on a time scale o f a few months (Personal communications). W i t h forages comprising a large fraction o f both the feed used for growing animals and the subsequent manure produced, it is very important to be able to determine their composition and contribution to the various pathways i n the cycle shown. Due to the need for rapid and practical tests, most o f the analyses carried out on the feeds and wastes involved i n animal nutrition are based on empirical methods using extractive techniques, such as the extraction o f fiber with neutral or acidic solutions o f detergent (2). These measures have then been related to the nutritional status o f ruminants (3). Similarly, the protein i n forages is determined as total nitrogen, either by combustion (4) or by digestion (5) and the total nitrogen is converted to protein by the use o f conversion factors (5). While some efforts have been made to study forage components i n a more detailed fashion, such as the use o f nitrobenzene oxidation to determine lignin composition (6), such efforts are hampered by the complex nature o f the materials i n question and the need for different methodologies for each o f the individual components present (proteins, lignins, soluble carbohydrate monomers and polymers, insoluble carbohydrates, waxes, etc., 7). It is easy to see how useful a single procedure capable o f simultaneously determining all or many o f the various components would be. While near-infrared reflectance spectroscopy has been extensively investigated for determining forage composition and is capable o f determining many different components simultaneously, it functions by relating spectral information to other procedures, such as fiber determinations, i n a correlative manner (#). The resulting relationships are then used to determine the composition o f other similar samples for which only spectra are available. While this procedure is extremely useful where large numbers o f similar samples are to be assayed, the need for developing calibrations for each component o f interest and the required number o f samples for such development make it less than ideal for research studies, where sample sets are often small and vary greatly i n composition from one set to the next. Pyrolysis-Gas Chromatography-Mass Spectrometry ( P Y - G C M S ) would appear to be potentially an extremely useful tool for providing more detailed qualitative information on the composition o f forages and wastes, without the need to conduct a multitude o f different assays to obtain information on all the different components present. Indeed, it would allow the use o f a single method for the study o f the fate o f the nitrogen fraction o f forages as they pass from the feed, through the animal, and



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RUMINANT ANIMALS

MILK, MEAT, WOOL, ETC.

(Cows, sheep, etc.)

MANURE (Feces and U r i n e )

Organic Ν

+ Inorganic Ν

(undigested feed,

(ammonia, nitrates)

bacteria, sloughed cells)

STORAGE ATMOSPHERIC LOSS OF NH,

(partial conversion o f organic to inorganic N )

SOIL APPLICATION INORGANIC Ν ORGANIC Ν — CROPS (corn, silage, etc.)

Figure 1. Pathways for distribution o f nitrogen i n production o f ruminants.


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subsequently to manure, etc.. (Fig. 1), while also providing information on the fate o f the non-nitrogen components (carbohydrates, lignin, etc..). However, while P Y - G C M S has been applied to the study o f forages by many researchers (9-13), it would appear to have a serious weakness when it comes to the nitrogen fraction. The primary form o f nitrogen i n forages is protein, and very few products are reported i n the literature with respect to nitrogen and forages. The objective o f this report is to reexamine this problem i n more detail.


Methods A l l spectra presented were produced using a Chemical Data System Pyroprobe model 2000 equipped with an AS-2500 Pyrolysis Autosampler (Chemical Data System Inc, Oxford P A , U S A ) . Pyrolysis was carried out at 600° C for 10 sec using sample aliquots o f approximately 0.5 mg i n quartz tubes. The pyrolyzate was swept directly into a Finnigan M A T G C Q gas chromatograph coupled to a Finnigan M A T G C Q ion trap mass spectrometer (Finnigan M A T , San Jose C A , U S A ) . The gas chromatographic column was a D B - 5 m s ( J & W Scientific, Inc, Folsom, C A , U S A ) (30 m χ 0.25 m m i.d., 0.25 μιτι film thickness) operated from 50 to 300° C at 5 °C/min holding the initial temperature for 10 min. The injector temperature was 300° C . The P Y / G C interface temperature was 200° C . Carrier gas (He) was held at a constant velocity o f 40 cm/sec. with the split vent flow set to 100 ml/min. The G C / M S transfer line temperature was 300° C and the ion source temperature was 200° C . Mass spectra were obtained by electron impact at 70 e V from 10 or 40 to 650 m/z (1 scan/sec). Peak identification (Table 1) was based on mass spectral interpretation and published libraries o f mass spectra o f lignocellulose pyrolyzates (9-13). When using tetramethylammonium hydroxide ( T M A H ) the sample to be pyrolyzed was placed i n the pyrolysis tube and approximately 5 μΐ o f T M A H solution (25% by wt. in water) added. Finally all samples were finely ground using a W I G - L - B U G single ball m i l l (Crescent Dental M F G , C o . Lyons, I L , U S A ) . Pyrolysis Products Reported in the Literature for Forage Proteins Previous efforts by the authors and results i n the literature have reported only a few products as originating from the pyrolysis o f the protein fraction o f forages (9-13). Those being methanethiol from the sulfur containing amino acids, indole and methylindole from tryptophan, 4-methylphenol from tyrosine, phenylacetonitrile from phenylalanine, and derivatives o f pyrrole from proline. However, even for samples containing a reasonable amount o f protein (i.e, red clover hay with a protein content o f 14.5%) several o f these products were, i f found at all, present i n relatively small amounts, compared with other products produced from lignin or carbohydrates. In the case o f methanethiol, resolution from the other low molecular weight products produced is also a problem, although the use o f a different G C column or temperature program could probably be used to counteract this problem. Considering that there are 20 amino acids commonly found i n proteins, and for


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other components, such as lignin, it has been observed that even after chemical extraction of the majority of the lignin fraction by sodium chlorite (14) one can still find markers for lignin (9), the question remains as to what happens to all the products one might expect (quantitatively and qualitatively) from the pyrolysis o f proteins i n forages. The lack o f protein pyrolysis products, and the relatively low amounts found for those reported, is especially bewildering when considered i n light o f the excellent correlations found between pyrolysis products and protein concentrations i n a study o f 67 different forage type materials using a packed column (75). While identification o f the products was not carried out, correlations (R ) o f pyrolysis products and protein concentration for the 67 samples were found at the 0.94 level for single product correlations and at the 0.96 level using 3 products. Interestingly, on an individual feed basis (5 different feeds studied), the best results were achieved for the samples containing the least amount o f protein (vegetative corn and wheat plants). While it is possible that the protein concentration was determined by difference (that which was not something else must be protein), it is extremely unlikely that such high correlations would have been found i f that were the case. Another possibility is that a low molecular weight and highly volatile product or products is/are produced during the pyrolysis o f proteins, such as ammonia or a low molecular weight amine. Such products may have been detected using the packed column and flame detector, but have been missed by mass spectrometry because o f the instrument settings used. Generally, the first minute or two of the pyrogram is ignored, as well as fragments with m/z o f less than 40, to eliminate interferences from all the impurities i n the carrier gas, water i n the samples, and low molecular weight material produced by pyrolysis o f almost anything.


2

The question then remains: W h y pyrolysis o f forages does not produce more evidence o f proteins? Possibilities included: 1) Proteins i n general only produce l o w yields o f relatively few products, 2) the products are being produced, but are not being recognized as originating from proteins or are being missed entirely, and 3) forages behave differently during pyrolysis than other protein containing materials due to the way the protein is bound within the plant cell wall (16). Pyrolysis Products of Amino Acids Since proteins are polymers composed of amino acids joined by amide bonds, one might expect that analysis of the products produced by pyrolysis o f amino acids (7 7) might be useful i n searching for unidentified protein-derived pyrolysis products. However, our data from the pyrolysis of amino acids have shown that the major products produced are often not the same as those produced during the pyrolysis of proteins. This can be seen by comparing the pyrograms in F i g . 2 for the amino acids L-asparagine (A) and L cysteine (B) and the results found for gelatin i n F i g . 3. While the major product produced by pyrolysis o f L-asparagine (lH-pyrrole-2,5-dione) was found at 885 scans and for L-cysteine (2-methylthiazolidine) at 555 scans, there were no major products in the pyrogram o f gelatin i n these regions. Examination o f the mass-spectral data confirmed the absence these two products i n the gelatin pyrogram (Data not shown). Similar results are found for most amino acids when comparing results obtained with


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T a b l e l . Identification o f pyrolysis products shown in Figures 2-8. Peak# 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 38 39 40 41 42

Compound name unknown unknown unknown unknown unknown pyrazole pentanenitrile pyrrole toluene an ethylpyrrole 2-furaldehyde a methylpyrrole ethylbenzene 2-furanmethanol styrene 5-methyl-2(3H)-furanone phenol 4-methoxytoluene trans 1-propenylbenzene 3 -methyl-1,2-cyclopentanedione unknown unknown guaiacol a methylphenol unknown unknown unknown unknown phenylacetonitrile a dimethoxybenzene 4-methoxystyrene unknown 4-methylguaiacol unknown unknown unknown 4-vinylphenol 3 -phenylpropanenitrile unknown unknown 1-methylindole 4-ethylguaiacol 4

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Ions (jn/zf 44,34,18,45,19,17 58,60,59,45,47,61 43,45,61,42 43,75,45,31,29,42 41,43,39,58,27 Ρ 68,41,39,28,40 Ρ 41,39,54,43,27,84 Ρ 67,39,41,40,38,28 Ρ 91,92,65,39,63,51 Ρ 80,95,78,53 Ρ 95,39,96 C 80,81,41,53,39 Ρ 91,106,65,51,77,92 Ρ? 98,41,42,53,81,39 C 104,78,103,77,51,50 Ρ 70,98,55,41 C P , C , L 94,66,39,65 122,77,121,91,107 Ρ 117,115,91,94,69,65 Ρ 112,84,41,55,69 c 80,91,53,92,123,81 Ρ 126,55,83,67,39,97 ? 109,124,81 L 107,108,77,79,80,51 P,L 84,113,28,26,85,114 Ρ ? 43,44,57,39,29,117 41,127,42,39,112,58 Ρ 139,39,54,53,110,82 Ρ 90,117,89,116 Ρ 77,138,95,123,65 ? 91,134,119,65 Ρ? 109,107,55,122,82 ? 138,123,95,67 L 42,127,142,56 Ρ ? 142,127,39,41,83,113 130,42,98,88 Ρ 120,91,65,51 L 91,65,131,92,39,51 Ρ 91,120,133,132,117,118 ? ? 109,82,84,55,129 131,130,89,103,77 Ρ 137,152,122,91,77 L

Origin Ρ A ? ?

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3

3

3

3

3


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T a b l e 1. Continued 3

P 43 unknown Ρ 44 indole L? 45 4-vinylguaiacol P 46 L-proline, l-methyl-5-οχο-, methyl ester L 47 2,6-dimethoxyphenol P 48 a dimethylindole L 49 eugenol 50 L 1,2,4-trimethoxybenzene 51 Ρ 3-methylindole Ρ 52 L-proline,5-ΟΧΟ-, methyl ester 53 a methoxybenzene acetic acid, methyl ester ? ? 54 unknown L 55 2,6-dimethoxy-4-methylphenol L 56 trans isoeugenol 57 unknown ? ? 58 unknown L 59 2,6-dimethoxy-4-vinylphenol ? 60 unknown 61 ? a dimethoxybenzoic acid, methyl ester ? 62 unknown ? 63 unknown ? 64 unknown 65 P? unknown L 66 trans 2,6-dimethoxy-4-propenylphenol 67 P? unknown ? 68 unknown L 69 acetosyringone ? 70 unknown L 71 trans coniferyl alcohol L 72 syringylacetone ? 73 unknown ? 74 unknown ? unknown 75 ? 76 unknown A 77 unknown A unknown 78 P? 79 unknown P? 80 unknown ? 81 unknown P=Protein, C=Carbohydrate, L=Lignin, A=Artifact. Masses listed in decreasing abundance. Formed in presence of tetramethylammonium hydroxide. u n c o n f i r m e d library search result. Matched with published results. Verified with authentic compound. 5

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39,54,82,139,53,108 117,90,89,63 150,135,77,107 98,41,42,70 154,65,93,139,96,111 144,145 164,77,103,149,133 125,153,168,93,110,65 130,131,77,103,51 84,41,39,28,56 121,77,91,180 125,42,98,69,139,166 168,107,153,125 164,149,77,103,131,121 101,144,45,75,88,129 91,65,104,182 180,165,137,91,77,122 107,108,136,80,53,81 165,196,79,77 170,185,153,143,128,98 98,97,140,41,42,167 94,164,136,66 156,129,199 194,119,179,77,131,151 186,93,130,103,65,38 138,123,70,151 191,196,153,138 70,97,125,44,168 137,91,124,180 167,210,123 83,111,41,68,55,154 67,95,81,41,55,123 67,81,95,41,123 94,190,134,162,106 67,55,81,41,96 43,55,87,143,74,101 70,154 70,194 87,41,55,129,73

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1 H-pyrrole-2,5-dione TIC


680

1280 Time (sec)

1880

B

2-methylthiazolidine

TIC

680

1280 Time (sec)

1880

Figure 2. Pyrogram o f L-Asparagine (A) and L-Cysteine (B) (TIC = Total ion current).

TIC-

680

1280 Time (sec)

1880

Figure 3. Pyrogram o f gelatin with major peaks labeled as listed i n Table 1 (TIC = Total ion current).


55 free amino acids to those obtain with proteins, and are due to the differences between free amino acids and the chemically bound forms found i n proteins. When free, amino acids have the form: NHrCHtRO-COOH but i n proteins they are bound as amide linked peptides i n the form: ^-CHiRO-CONH-CHCR^-CONH-CHiR^-CO.... NH-CH(R )COOH where R are various aliphatic and aromatic moieties. It is easy to see why the products from proteins and amino acids could be different, with deamination and decarboxylation being more likely for amino acids, although some o f the products such as indole and methanethiol are found for both materials (18). While the products resulting from amino acids might also be produced from free amino acids found i n forages, the small amounts o f free amino acids present means that such products would, at best, be minor contributors to forage pyrograms. x


( 1 , 2 , 3 X )

Pyrolysis of Isolated Proteins Pyrolysis o f isolated proteins o f both plant (commercial soy powder) and non-plant origin (commercial gelatin) demonstrated that pyrolysis of proteins is not a problem i n and o f itself. A s shown i n F i g . 3., the pyrolysis o f purified gelatin produces a multitude o f products. These products are not unique to animal derived proteins as shown by the many similar products found (Fig. 4) for the pyrolysis of soybean extract (83% protein, 13% carbohydrate, 3% fat). Previously (18), we reported that while some o f these products were also found i n an alfalfa sample which contained 29.8% protein (Fig. 5), examination o f the red clover hay pyrograms did not find any additional protein products. A s part of this effort, the results for both the alfalfa (Fig. 5) and the red clover hays (Fig. 6) were carefully reexamined. It was found that more o f the protein-derived products were present i n both pyrograms that previously reported by either the authors (18) or others (10-12), with 16 protein-derived products found for the alfalfa hay and 13 for the red clover hay. These results indicate that at least part of the explanation for the question of why reports on pyrolysis o f forages do not show more protein-derived products, is simply a failure to recognize the products for what they are. A s shown i n F i g . 6, forage pyrograms can be roughly divided into three regions: 1) The early scans (labeled matrix) dominated by low molecular products, such as acetic anhydride and carbon dioxide, which originate from a variety o f sources, 2) a region dominated by products from carbohydrates, and 3) a region dominated by phenolic products from lignin. Note, however, that no single area contains products originating from proteins (13). A few o f the products (those eluting i n the first two minutes) have not been reported because, due to the multitude o f low molecular weight products produced during pyrolysis o f almost any biological material, this early part o f the pyrogram is generally ignored. One o f these early products (Peak #1) appears to be a volatile, low molecular weight compound containing an amine group. This product was found by examining mass spectra for fragments with masses down to 10, something generally not



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η

'

'

680

1

1

'

'

1280 Time (sec)

'

Γ

1880

Figure 4. Pyrogram o f soybean powder (protein content = 83%) with major peaks labeled as listed in Table 1 (TIC = Total ion current)

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'

1280 ' Time (sec)

1880

Figure 5. Pyrogram o f an alfalfa hay, protein content = 29.8%, with major peaks labeled as listed in Table 1 (TIC = Total ion current).



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done for pyrolysis o f forage materials. It appears that this product is an amine or amide, rather than ammonia, but exact identification has not been possible due to the presence o f many other low molecular weight products i n the same region o f the pyrogram. Other products, such as styrene and toluene, do not contain nitrogen and thus have either been ignored by the forage community or assumed, perhaps like other products, to originate from lignin, although other researchers have reported them as originating from protein (19). However, these findings do not completely account for difficulties found when examining pyrolysates of forages for evidence of proteins. The alfalfa sample contained an unusually high protein content (29.8%) due to collection at a very early stage o f growth, and yet only 16 out o f 21 possible products (Table 1) were found. Normally alfalfa hay is harvested when the plants are more mature and thus has a protein content closer to 15% than 30%. Even for this high protein alfalfa, several o f the products were present at low levels. For the red clover hay (14.5% protein), still fewer products were found, 14, and at even lower levels. Without a mass spectrometer, detection o f many o f these products would be impossible due to the small amounts present relative to the products generated from the non-protein components, indicating that the relative yield o f products from proteins, as compared to the lignin and carbohydrate fractions o f forages, may be a problem, and it is not that they have just been missed or ignored. The fact that the products originating from proteins are not found i n one region on the pyrogram (Fig. 6) also complicates their determination. Pyrolysis of Proteins in Other Materials and Possible Matrix Effects in Forages W h i l e the detection o f nitrogen compounds from the pyrolysis o f forages may be a problem, this does not appear to be a problem for the pyrolysis o f other materials. For example, it has been reported that up to 90% o f the nitrogen fraction i n soils is volatilized by pyrolysis resulting i n many G C separable compounds (20, 21). W o r k by Stankiewicz and co-workers (19) showed similar results for proteins i n invertebrate cuticles, with 44 compounds designated as originating from proteins. W h i l e some o f these, such as vinylphenol, may also originate from lignin i n forages and thus would not be distinctive markers for proteins, most were nitrogen-containing compounds for which this would not apply. While van de Meent and co-workers (22), using synthetic constructed mixtures, reported the quantitative yield o f total products from protein to be less than for carbohydrates or lignin, with relative responses o f 1.00, 0.31 and 0.65, for carbohydrates, protein, and lignin respectively, even trace amounts o f lignin i n fractions extracted for their carbohydrates are easily detected (77). Thus, the products from a sample with a 14.5% protein content, such as the red clover hay shown in F i g . 6, should be easily detected, even with a relative yield o f half that of lignin. Therefore, it appears that, at least for forages, the relative yield can be considerably less i n natural samples. One possible explanation for the lower than expected yield o f protein pyrolysis products i n the pyrograms o f forages is that the pyrolysis products produced i n the presence o f the other forage components are either not the same, due to secondary


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reactions, as those produced when a pure protein is pyrolyzed (matrix effects), or are produced i n lower amounts. Such reactions were reported by van de Meent and coworkers (22) for pyrolysis o f mixtures o f albumin, dextran and lignosulphonic acid. V a n der Kaaden and co-workers (23) also reported that the presence o f various salts during pyrolysis significantly changed the pyrogram o f amylose. Forages and byproducts can contain significant amounts o f ash (1.3% of the red clover hay) which might play a similar role. A s forage plants grow, the relative composition and morphological structure o f the plant changes. W i t h increasing maturity the relative amount o f lignin i n forages tends to increase, i n order to give structural support to the plant, and the relative amount of protein decreases (24). Also, there appears to be an intimate structural relationship between lignin and protein i n the various layers of plant cell walls, with protein being necessary for lignin disposition (16). Thus, one can imagine that with increasing forage maturity, the protein present becomes more and more intimately bound with lignin and other components o f the cell wall, thus increasing the likelihood o f matrix effects on product yield and possibly composition. Further efforts using a variety of samples w i l l be needed to determine i f this is indeed the case. Preliminary Results Using Tetramethylammonium Hydroxide Tetramethylammonium hydroxide is often used i n conjunction with pyrolysis (often referred to as "thermally assisted hydrolysis and methylation gas chromatography" to obtain products not produced by pyrolysis without T M A H . For example, pyrolysis generally results i n decarboxylation o f carboxylic acids. W i t h T M A H , the carboxylic acid is methylated, and thus the acid group can be retained (12, 25). In addition, as implied i n the name "thermally assisted hydrolysis", the nature o f the cleaving process is different (hydrolysis versus thermal cracking for pyrolysis without T M A H ) . The results for the soybean protein extract (pyrogram without T M A H shown i n F i g . 4) and the red clover hay (pyrogram without T M A H shown i n F i g . 6) using T M A H are shown in Figs. 7 and 8, respectively. A s can be easily seen by comparing the results with T M A H (Figs. 7 and 8) with those without T M A H (Figs. 4 and 6), the products produced by the two different procedures are quite different in nature, as indicated by the differing retention times. Initial efforts indicate that the use o f T M A H may be beneficial i n obtaining more information on the proteins present i n forages. Results indicate that both pyrolysis and thermally assisted methylation occurred, with both new products and also many of those produced during pyrolysis without T M A H found. A t a minimum, seven additional protein derived products were found i n the red clover pyrogram when T M A H was used, which were also found i n similar pyrograms o f gelatin and soybean protein extract. M o r e may be present, but confirmation w i l l require further work. Summary Pyrolysis-gas chromatography, with and without mass spectrometry, has been shown to be very useful i n the analysis o f forages, particularly for analysis o f the lignin and


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TIC

1 680

1880

1280 Time (sec)

Figure 7. Pyrogram, using tetramethylammonium hydroxide, o f soybean powder with major peaks labeled as listed i n Table 1 (protein content = 83%, T I C = Total ion current).

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680

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Figure 8. Pyrogram, using tetramethylammonium hydroxide, o f a typical red clover hay, protein content = 14.5%, with major peaks labeled as listed i n Table 1 (TIC = Total ion current).



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carbohydrate fractions. However, the literature on the pyrolysis o f forages does not report an abundance o f information (qualitatively or quantitatively) for the proteins fraction in forages, despite the fact that pyrolysis o f isolated proteins produces numerous products. Only small amounts of phenylacetonitrile, indole, methylindole, a methylphenol, methanethiol and dimethyl- or ethylpyrrole have generally been reported for forages. However, reports from the pyrolysis of isolated proteins and nonforage materials show an abundance of protein-derived products to be produced. A reexamination o f this subject indicates the problem to be two-fold i n nature: First, pyrolysis o f forages often produces relatively small quantities o f protein-derived products when compared with other components o f forages (lignin, carbohydrates, etc.), isolated proteins or other protein containing material, such as invertebrate cuticles. A n d second, it is now apparent that many of the protein-derived products i n forage pyrograms have simply not been reported, most likely due to the small amounts produced or because they do not contain nitrogen and were assumed to originate from other fractions, such as lignin. Efforts have also shown that the mass and scan cutoffs often used to avoid the compilation o f the low molecular weight products produced during the pyrolysis o f all biological materials (equivalent to eliminating the solvent front in other G C operations) results i n some protein-derived products being missed. For example, examination o f the first two minutes of pyrograms for masses down to 10 m/z found a low molecular weight compound containing an N H group. It was also found that the use o f T M A H results i n a mixture of products containing both some o f the products produced i n the absence o f T M A H and also new products from the use o f the T M A H . Initial studies indicate that using T M A H may be o f benefit to efforts to obtain more information on proteins i n forage type materials. Finally, considering the importance of proteins and other nitrogen compounds i n the evaluation of animal feeds (26), and the increasing interest i n nitrogen compounds i n the area o f animal waste disposal and water pollution (7), further research and clarification is needed on the subject of the pyrolysis o f forages and resulting protein-derived products. 2

REFERENCES 1. 2.

3. 4. 5. 6.

Ross, C. C. (Ed.), Proc. of the Seventh Inter. Sym. on Agric. and Food Processing Wastes, Amer. Soc. Agric. Engin., 1995. Goering Η. Κ.; Van Soest, P. J. Forage fiber analysis (Apparatus, reagents, procedures, and some applications). Agric. Handbook No. 379, Agric. Res. Serv., USDA, Washington, DC, 1970. Van Soest, P. J Nutritional Ecology of the Ruminant, Ο & Β Books, Inc., Corvallis, OR, 1982; pp. 230-248. A O A C Method 990.03, Official Methods of Analysis, A O A C Int., Gaithersburg, MD. A O A C Method 2.051, Official Methods of Analysis, A O A C Int., Gaithersburg, MD. Reeves, III, J. B . J. Dairy Sci. 1986 , 69, 71-76.


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8.

9. 10. 11. 12. 13. 14. 15. 16.

17. 18. 19 20. 21. 22. 23. 24. 25. 26.

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